Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations

Abstract

The study of extremophilic fungi has received manifold global attention during the past decade. Among the members belonging to the monophyletic fungal kingdoms, very few species have the capacity to survive and proliferate between the temperature range of 45–55 °C. These temperatures are considered as high temperatures while studying thermophilic and thermotolerant fungi. Earlier classification and studies were arbitrarily carried out based on their cardinal temperatures. The temperature endured by the fungi are not as high as those witnessed in bacteria and archaebacteria, adding to the very many reasons for not receiving due publicity in the past. However, drastic improvements in the methods employed for molecular fungal phylogeny and DNA-based studies has eliminated such hassles and paved the way for the elucidation of thermophily as an interesting phenomenon in fungi. Such fungal candidates have lent themselves as tools and excellent laboratory material for classical, genetic, and applied research. Their morphological, physiological, and molecular adaptations, characteristics, diversity and their role in different habitats such as soils, compost heaps, agricultural and forest debris, etc. have been reviewed and presented.KeywordsDiversityFungiHabitatsMolecular studiesThermophilicThermotolerant

Full text

SanjaySahayEditor
Extremophilic
Fungi
Ecology, Physiology and Applications

Extremophilic Fungi

Sanjay Sahay
Editor
Extremophilic Fungi
Ecology, Physiology and Applications

Editor
Sanjay Sahay
Sarojini Naidu Government Postgraduate
Girl’s (Autonomous) College
Bhopal, Madhya Pradesh, India
ISBN 978-981-16-4906-6 ISBN 978-981-16-4907-3 (eBook)
https://doi.org/10.1007/978-981-16-4907-3
#The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
Pte Ltd. 2022
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether
the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of
illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and
transmission or information storage and retrieval, electronic adaptation, computer software, or by
similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or
the editors give a warranty, expressed or implied, with respect to the material contained herein or for any
errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore

To my wife Alka Sahay and son Amogh Sahay

Preface
Physico-chemical extremities provide opportunities for life forms to evolve
mechanisms and biomolecules, helping them to thrive under such conditions.
Fungi among eukaryotes constitute dominant flora in extreme habitats. They can
thrive in extremes of temperature, pH, salinity, pressure, water activity, etc.
Although fungi have common and important ecological functions as decomposers,
symbionts, and parasites, to play to maintain the abiotic and biotic components of the
Earth’s ecosystem, their survival in extreme conditions is key to the maintenance of
these extreme parts of the Earth’s ecosystem. The current pace of climate change has
affected all ecosystems and life forms including the bare proportion of explored and
largely unexplored proportion of fungi of extreme climates. Therefore, the last few
decades have witnessed intensive efforts to explore fungi from various extreme
climates and develop techniques for the study and maintenance of extremophilic
fungi.
The biological mechanisms behind the adaptabilities of diverse extreme climatic
groups of fungi are also very attractive and interesting subjects of study. The results
unraveled several exciting physiological pathways and mechanisms, many of them
being novel ones. The novel pathways are also supplemented with novel
biomolecules that support the adaptability of extremophilic fungi. Coincidently,
these biomolecules also prove to be important for various human uses.
The objective of the book is to present updated information about various types of
extremophilic fungi for young graduates and researchers. It is organized into three
parts: (1) Basic information comprising the techniques for their isolation, identifica-
tion, and maintenance; (2) Ecology and physiology; and (3) Biotechnological
applications.
Bhopal, India Sanjay Sahay
vii

Contents
Part I Basic Information
1 Isolation, Culture, and Maintenance of Extremophilic Fungi ..... 3
Kalhoro Muhammad Talib, Jing Luhuai, Xiaoming Chen, Ali Akbar,
Ayesha Tahir, Irfana Iqbal, and Imran Ali
2 Modern Tools for the Identification of Fungi, Including Yeasts .... 33
Ayesha Tahir, Irfana Iqbal, Kalhoro Muhammad Talib, Jing Luhuai,
Xiaoming Chen, Ali Akbar, Anam Asghar, and Imran Ali
Part II Eco-physiology
3 Major Habitats and Diversity of Thermophilic Fungi ........... 55
Swapnil Chaturvedi and Indira P. Sarethy
4 Thermophilic Fungi: Habitats and Morpho-Molecular
Adaptations ........................................... 77
Regina Sharmila Dass, Joy Elvin Dhinakar, Akriti Tirkey,
Mayukhmita Ghose, and Angeline Jessika Suresh
5 Modulation of Physiological and Molecular Switches in
Thermophilic Fungi: A Brief Outlook ....................... 97
Tuyelee Das, Samapika Nandy, Abdel Rahman Al-Tawaha,
Potshangbam Nongdam, Ercan Bursal, Mahipal S. Shekhawat,
and Abhijit Dey
6 Psychrotrophic Microfungi: Major Habitats, Diversity and Living
Strategies ............................................ 111
Kanak Choudhary, Najeeb Hussain Wani, Farooq Ahmad Ahanger,
Suhaib Mohamad Malik, Vinod Chourse, Abdul Majid Khan,
and Sanjay Sahay
ixix

7 Physiology and Molecular Biology of Psychrotrophic Fungi: An
Insight ............................................... 129
Tuyelee Das, Samapika Nandy, Devendra Kumar Pandey,
Abdel Rahman Al-Tawaha, Potshangbam Nongdam, Ercan Bursal,
Mahipal S. Shekhawat, and Abhijit Dey
8 Ecology, Physiology, and Diversity of Piezophilic Fungi ......... 141
Shyamji Shukla and Harshita Shukla
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and
Radioresistant Fungi: Habitats and Their Living Strategies ...... 171
Tuyelee Das, Abdel Rahman Al-Tawaha, Devendra Kumar Pandey,
Potshangbam Nongdam, Mahipal S. Shekhawat, Abhijit Dey,
Kanak Choudhary, and Sanjay Sahay
10 Ecology and Diversity of Microaerophilic Fungi Including
Endophytes ........................................... 195
Deeksha Patil, Vishal Dawkar, and Umesh Jadhav
11 Fungi in Hypoxic Soils and Aquatic Sediments ................ 219
Irena Maček
12 Chaotolerant Fungi: An Unexplored Group of Extremophile ..... 245
Sanjay Sahay
13 Xerophilic Fungi: Physiology, Genetics and Biotechnology ....... 253
Sanhita Sarkar, Namita Ashish Singh, and Nitish Rai
Part III Applications
14 Extremophilic Enzymes: Catalytic Features and Industrial
Applications .......................................... 273
Kanak Choudhary, Mangesh Kumar Mankar, and Sanjay Sahay
15 Biotechnological Application of Extremophilic Fungi ........... 315
Aneesa Fasim, H. K. Manjushree, A. Prakruti, S. Rashmi,
V. Sindhuja, Veena S. More, K. S. Anantharaju, and Sunil S. More
16 Extremophilic Fungal Cellulases: Screening, Purification,
Catalysis, and Applications ............................... 347
Sangita Chouhan, Rajkumar Ahirwar, Tejpal Singh Parmar, Ashiq
Magrey, and Sanjay Sahay
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay,
and Applications ....................................... 367
Aneesa Fasim, A. Prakruti, H. K. Manjushree, S. Akshay,
K. Poornima, Veena S. More, and Sunil S. More
x Contents

18 Extremophilic Fungal Lipases: Screening, Purification, Assay,
and Applications ....................................... 395
J. Angelin and M. Kavitha
19 Extremophilic Fungal Proteases: Screening, Purification, Assay,
and Applications ....................................... 439
Sourav Bhattacharya and Arijit Das
20 Extremophilic Fungal Amylases: Screening, Purification, Assay,
and Applications ....................................... 465
Ragini Bodade and Krutika Lonkar
21 Extremophilic Fungi as a Source of Bioactive Molecules ......... 489
Annada Das, Kaushik Satyaprakash, and Arun Kumar Das
22 Piezophilic Fungi: Sources of Novel Natural Products with
Preclinical and Clinical Significance ........................ 523
Tuyelee Das, Puja Ray, Samapika Nandy, Abdel Rahman Al-Tawaha,
Devendra Kumar Pandey, Vijay Kumar, and Abhijit Dey
23 Biotechnological Applications of Microaerophilic Species
Including Endophytic Fungi .............................. 547
Beenish Sarfaraz, Mehwish Iqtedar, Roheena Abdullah,
and Afshan Kaleem
24 Whole Cell Application Potential of Extremophilic Fungi in
Bioremediation ........................................ 557
Sunil Bhapkar, Rushikesh Pol, Deeksha Patil, Anupama Pable,
and Umesh U. Jadhav
25 Extremophilic Fungi: Potential Applications in Sustainable
Agriculture ........................................... 581
Sanjay Sahay
26 Extremophilic Fungi for the Synthesis of Nanomolecules ........ 615
Harshita Shukla and Shyamji Shukla
27 Fungal Extremozymes: A Potential Bioresource for Green
Chemistry ............................................ 651
Imran Mohsin and Anastassios C. Papageorgiou
28 Fungal Extremozymes in Green Chemistry ................... 683
Ajay Nair, Archana S. Rao, K. Nivetha, Prakruthi Acharya,
Aneesa Fasim, Veena S. More, K. S. Anantharaju, and Sunil S. More
29 Phylogenomics, Microbiome and Morphological Insights
of Truffles: The Tale of a Sensory Stimulating Ectomycorrhizal
Filamentous Fungus .................................... 709
Mohan Das, Ananya Pal, Subhodeep Banerjee, Subhara Dey,
and Rintu Banerjee
Contents xi

Editor and Contributors
About the Editor
Sanjay Sahay, Professor of Botany, is currently serving Sarojini Naidu Government
Girls Postgraduate College, Bhopal (India). He obtained his PhD in fungal genetics
(Patna University, Patna, India), and postdoctoral research experience in yeast
molecular biology (Indian Institute of Science, Bangalore, India). He has teaching
experience of 27 years, guided 18 PhD students, published more than 30 research
papers, contributed three book chapters, and filed two patent applications in ligno-
cellulosic ethanol, one of which has been awarded. His areas of research include
psychrophilic fungi, cold-active enzymes, and lignocellulosic ethanol. He has been
awarded Biotechnology National Associateship by the Department of Science and
Technology, Government of India, and Research Award by University Grants
Commission, India.
Contributors
Roheena Abdullah Department of Biotechnology, Lahore College for Women
University, Lahore, Pakistan
Prakruthi Acharya School of Basic and Applied Sciences, Dayananda Sagar
University, Bangalore, Karnataka, India
Farooq Ahmad Ahanger Government Science and Commerce College, Bhopal,
Madhya Pradesh, India
Rajkumar Ahirwar Department of Biotechnology, Barkatullah University,
Bhopal, Madhya Pradesh, India
Ali Akbar Faculty of Life Sciences, University of Balochistan, Quetta, Pakistan
S. Akshay School of Basic and Applied Sciences, Dayananda Sagar University,
Bengaluru, Karnataka, India
xiiixiii

Imran Ali School of Life Science and Engineering, Southwest University of
Science and Technology, Mianyang, Sichuan, China
Faculty of Life Sciences, University of Balochistan, Quetta, Pakistan
Plant Biomass Utilization Research Unit, Botany Department, Chulalongkorn Uni-
versity, Bangkok, Thailand
Abdel Rahman Al-Tawaha Department of Biological Sciences, Al-Hussein Bin
Talal University, Maan, Jordon
K. S. Anantharaju Department of Chemistry, Dayananda Sagar College of Engi-
neering, Bangalore, Karnataka, India
J. Angelin School of Biosciences and Technology, Vellore Institute of Technology,
Vellore, Tamil Nadu, India
Anam Asghar Department of Zoology, Lahore College for Women University,
Lahore, Pakistan
Rintu Banerjee Agricultural and Food Engineering Department, Indian Institute of
Technology, Kharagpur, West Bengal, India
Subhodeep Banerjee Advanced Technology Development Centre, Indian Institute
of Technology, Kharagpur, West Bengal, India
Sunil Bhapkar Department of Microbiology, Savitribai Phule Pune University,
Pune, Maharashtra, India
Sourav Bhattacharya Department of Microbiology, School of Sciences, JAIN
(Deemed-to-be University), Bangalore, Karnataka, India
Ragini Bodade Department of Microbiology, Savitribai Phule Pune University,
Pune, Maharashtra, India
Ercan Bursal Department of Biochemistry, Mus Alparslan University, Muş,
Turkey
Swapnil Chaturvedi Department of Biotechnology, Jaypee Institute of Informa-
tion Technology, Noida, Uttar Pradesh, India
Xiaoming Chen School of Life Science and Engineering, Southwest University of
Science and Technology, Mianyang, Sichuan, China
Kanak Choudhary Department of Biotechnology, Barkatullah University,
Bhopal, Madhya Pradesh, India
Sangita Chouhan Department of Biotechnology, Barkatullah University, Bhopal,
Madhya Pradesh, India
Vinod Chourse Department of Biotechnology, Barkatullah University, Bhopal,
Madhya Pradesh, India
Annada Das Department of Livestock Products Technology, Faculty of Veterinary
and Animal Sciences, West Bengal University of Animal and Fishery Sciences,
Kolkata, West Bengal, India
xiv Editor and Contributors

Arijit Das Department of Microbiology, School of Sciences, JAIN (Deemed-to-be
University), Bangalore, Karnataka, India
Arun Kumar Das Eastern Regional Station, ICAR-Indian Veterinary Research
Institute, Kolkata, West Bengal, India
Mohan Das Agricultural and Food Engineering Department, Indian Institute of
Technology, Kharagpur, West Bengal, India
Regina Sharmila Dass Fungal Genetics and Mycotoxicology Laboratory, Depart-
ment of Microbiology, School of Life Sciences, Pondicherry University,
Pondicherry, India
Tuyelee Das Department of Life Science, Presidency University, Kolkata, West
Bengal, India
Vishal Dawkar MITCON Biotechnology and Pharmaceutical Technology Busi-
ness Incubator Center, Pune, Maharashtra, India
Abhijit Dey Department of Life Sciences, Presidency University, Kolkata, West
Bengal, India
Subhara Dey P. K. Sinha Centre for Bioenergy and Renewables, Indian Institute of
Technology, Kharagpur, West Bengal, India
Joy Elvin Dhinakar Department of Studies in Biochemistry, University of
Mysore, Mysore, Karnataka, India
Aneesa Fasim School of Basic and Applied Sciences, Dayananda Sagar Univer-
sity, Bengaluru, Karnataka, India
Mayukhmita Ghose Fungal Genetics and Mycotoxicology Laboratory, Depart-
ment of Microbiology, School of Life Sciences, Pondicherry University,
Pondicherry, India
Irfana Iqbal Department of Zoology, Lahore College for Women University,
Lahore, Pakistan
Mehwish Iqtedar Department of Biotechnology, Lahore College for Women Uni-
versity, Lahore, Pakistan
Umesh U. Jadhav Department of Microbiology, Savitribai Phule Pune University,
Pune, Maharashtra, India
Afshan Kaleem Department of Biotechnology, Lahore College for Women Uni-
versity, Lahore, Pakistan
M. Kavitha School of Biosciences and Technology, Vellore Institute of Technol-
ogy, Vellore, Tamil Nadu, India
Abdul Majid Khan Department of Biotechnology, Barkatullah University,
Bhopal, Madhya Pradesh, India
Editor and Contributors xv

Vijay Kumar Department of Biotechnology, Lovely Faculty of Technology and
Sciences, Lovely Professional University, Phagwara, Punjab, India
Krutika Lonkar Department of Microbiology, Savitribai Phule Pune University,
Pune, Maharashtra, India
Jing Luhuai School of Life Science and Engineering, Southwest University of
Science and Technology, Mianyang, Sichuan, China
Irena Maček Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Faculty of Mathematics, Natural Sciences and Information Technologies
(FAMNIT), University of Primorska, Koper, Slovenia
Ashiq Magrey Department of Biotechnology, Barkatullah University, Bhopal,
Madhya Pradesh, India
Suhaib Mohamad Malik Government Science and Commerce College, Bhopal,
Madhya Pradesh, India
H. K. Manjushree School of Basic and Applied Sciences, Dayananda Sagar
University, Bengaluru, Karnataka, India
Imran Mohsin Turku Bioscience Centre, University of Turku and Åbo Akademi
University, Turku, Finland
Sunil S. More School of Basic and Applied Sciences, Dayananda Sagar University,
Bengaluru, Karnataka, India
Veena S. More Department of Biotechnology, Sapthagiri College of Engineering,
Bangalore, Karnataka, India
Anuradha Mukherjee MMHS, South 24 Parganas, West Bengal, India
Ajay Nair School of Basic and Applied Sciences, Dayananda Sagar University,
Bangalore, Karnataka, India
Samapika Nandy Department of Life Science, Presidency University, Kolkata,
West Bengal, India
K. Nivetha School of Basic and Applied Sciences, Dayananda Sagar University,
Bangalore, Karnataka, India
Potshangbam Nongdam Department of Biotechnology, Manipur University,
Imphal, Manipur, India
Anupama Pable Department of Microbiology, Savitribai Phule Pune University,
Pune, Maharashtra, India
Ananya Pal P. K. Sinha Centre for Bioenergy and Renewables, Indian Institute of
Technology, Kharagpur, West Bengal, India
Devendra Kumar Pandey Department of Biotechnology, Lovely Faculty of Tech-
nology and Sciences, Lovely Professional University, Phagwara, Punjab, India
xvi Editor and Contributors

Anastassios C. Papageorgiou Turku Bioscience Centre, University of Turku and
Åbo Akademi University, Turku, Finland
Tejpal Singh Parmar Department of Biotechnology, Barkatullah University,
Bhopal, Madhya Pradesh, India
Deeksha Patil Department of Microbiology, Savitribai Phule Pune University,
Pune, Maharashtra, India
Rushikesh Pol Department of Microbiology, Savitribai Phule Pune University,
Pune, Maharashtra, India
K. Poornima School of Basic and Applied Sciences, Dayananda Sagar University,
Bengaluru, Karnataka, India
A. Prakruti School of Basic and Applied Sciences, Dayananda Sagar University,
Bengaluru, Karnataka, India
Nitish Rai Department of Biotechnology, Mohanlal Sukhadia University, Udaipur,
Rajasthan, India
Archana S. Rao School of Basic and Applied Sciences, Dayananda Sagar Univer-
sity, Bangalore, Karnataka, India
S. Rashmi School of Basic and Applied Sciences, Dayananda Sagar University,
Bengaluru, Karnataka, India
Puja Ray Department of Life Sciences, Presidency University, Kolkata, West
Bengal, India
Sanjay Sahay Department of Botany, SN Government PG College, Bhopal,
Madhya Pradesh, India
Indira P. Sarethy Department of Biotechnology, Jaypee Institute of Information
Technology, Noida, Uttar Pradesh, India
Beenish Sarfaraz Department of Biology, Lahore Garrison University, Lahore,
Pakistan
Sanhita Sarkar Department of Biotechnology, Mohanlal Sukhadia University,
Udaipur, Rajasthan, India
Kaushik Satyaprakash Department of Veterinary Public Health and Epidemiol-
ogy, Faculty of Veterinary and Animal Sciences, Banaras Hindu University,
Mirzapur, Uttar Pradesh, India
Mahipal S. Shekhawat Biotechnology Unit, Kanchi Mamunivar Government
Institute for Postgraduate Studies and Research, Puducherry, India
Harshita Shukla Department of Biotechnology, Sri Guru Tegh Bahadur Khalsa
College, Jabalpur, Madhya Pradesh, India
Shyamji Shukla Department of Biotechnology, Mata Gujri Mahila Mahavidyalaya
(Autonomous), Jabalpur, Madhya Pradesh, India
Editor and Contributors xvii

V. Sindhuja School of Basic and Applied Sciences, Dayananda Sagar University,
Bengaluru, Karnataka, India
Namita Ashish Singh Department of Microbiology, Mohanlal Sukhadia Univer-
sity, Udaipur, Rajasthan, India
Angeline Jessika Suresh Fungal Genetics and Mycotoxicology Laboratory,
Department of Microbiology, School of Life Sciences, Pondicherry University,
Pondicherry, India
Ayesha Tahir Department of Zoology, Lahore College for Women University,
Lahore, Pakistan
Kalhoro Muhammad Talib School of Life Science and Engineering, Southwest
University of Science and Technology, Mianyang, Sichuan, China
Akriti Tirkey Fungal Genetics and Mycotoxicology Laboratory, Department of
Microbiology, School of Life Sciences, Pondicherry University, Pondicherry, India
Najeeb Hussain Wani Government Science and Commerce College, Bhopal,
Madhya Pradesh, India
xviii Editor and Contributors

Part I
Basic Information

Isolation, Culture, and Maintenance
of Extremophilic Fungi 1
Kalhoro Muhammad Talib, Jing Luhuai, Xiaoming Chen, Ali Akbar,
Ayesha Tahir, Irfana Iqbal, and Imran Ali
Abstract
Extremophilic fungi are the ones that can survive in extreme available habitats,
which either can be natural or artificial. They can be divided mainly into thermo-
philic, psychrophilic, acidophilic, alkaliphilic, halophilic, and barophilic fungi.
This chapter covers two major parts including (a) general isolation techniques,
sample collection, preparation of isolation media, strain separation, cell mainte-
nance, morphological, and molecular identifications and (b) maintenance of
species, preparation of maintenance media, and preservation of extremophilic
fungi. Apart from general operation procedures, the isolation and maintenance of
extremophilic fungi needs special adjustments according to the obligate and
tolerant needs of their handlings. This chapter is expected to be a consultative
protocol resource for the students, academicians, and for industries working of
extremophilic fungi.
K. M. Talib · J. Luhuai · X. Chen
School of Life Science and Engineering, Southwest University of Science and Technology,
Mianyang, Sichuan, China
A. Akbar
Faculty of Life Sciences, University of Balochistan, Quetta, Pakistan
A. Tahir · I. Iqbal
Department of Zoology, Lahore College for Women University, Lahore, Pakistan
I. Ali (*)
School of Life Science and Engineering, Southwest University of Science and Technology,
Mianyang, Sichuan, China
Faculty of Life Sciences, University of Balochistan, Quetta, Pakistan
Plant Biomass Utilization Research Unit, Botany Department, Chulalongkorn University, Bangkok,
Thailand
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_1
3

Keywords
Fungal protocols · Fungal isolations · Culture media · Fungal preservations
1.1 Introduction
It is a miracle of nature that many kinds of microorganisms can survive in all habitats
even in hostile climatic conditions where virtually all life cannot exist (Sharma et al.
2019). These extremely harsh environments are high or low temperature, high saline,
high acid, high alkali, high pressure, and high radiation intensity (Yegin 2017).
Besides this, some exceptional environments, such as deserts barren areas, oil fields,
mining places, subversive anaerobic atmosphere, and high halogen environment are
also extreme environments. With the development of science and technology, we
find some microorganisms that can survive in these environments (Sekova et al.
2015).
Thermophilic microbes were isolated in 1860, which is the first extremophile to
be discovered. With the extensive development of thermophilic microbes research,
new strains have been found (Yegin 2017). Recently, there is an increasing trend in
the research of fungal species that inhabit extreme environments for reasons that
vary from gaining insight into the origin of life to explore new biotechnological
applications (Álvarez-Pérez et al. 2011).
Thermophile fungi have many biotechnological, industrial, pharmaceutical and
medical applications. Under heavy metal stress, some extremophile fungi have been
found to be better than bacteria in degradation and release of heavy metals, hence
recovering heavy metal polluted biomass (Jing et al. 2020). Due to safety, sensitivity
and cost-effectiveness, natural antimicrobial agents isolated from extremophile fungi
have been found to be better for the safety and preservation of food as compared with
synthetic antimicrobials (Akbar et al. 2019a,b). Industrial processes with severe
conditions (high salt concentrations, high temperatures, and high alkalinity levels)
can utilize enzymes produced by extremophile strains that pose no hazard to humans
and can be used in medical and food industries (Ali et al. 2015; Prasongsuk et al.
2018).
This chapter is intended to cover the skimmed introduction of extremophilic fungi
accompanied by the general lab techniques for the isolation and maintenance of
extremophilic fungi. It is expected that this chapter will be helpful for the
academicians and students for the consultation of protocols for handling
extremophilic fungi.
1.2 Classification of Extremophilic Fungi
Extremophiles mostly refer to microbes isolated from extreme environments. How-
ever, there are few reports on extreme fungi, but they have great potential value
(Khan et al. 2020a). Exploration of the globe intended for the discoveries of life in
4 K. M. Talib et al.

the biosphere led to the finding of the organisms in the environments, which were
hitherto considered as non-habitable; consequently, life can persist and even flourish
under extreme environmental conditions. In recent decades, extremophiles have
aroused great interest and gradually become one of the research hotspots in life
science (Ali et al. 2016b). According to the characteristics of the environment, we
divide the extremophilic fungi into thermophilic, psychrophilic, acidophilic,
alkalophilic, halophilic, barophilic, radio resistant, extreme anaerobic fungi, and so
on (Hujslováet al. 2019). These extremophilic fungi define the boundary of bio-
sphere, revealing the origin of life, and enrich the biodiversity of nature (Khan et al.
2020a,b). They are the result of life’s adaptation to the environment and contain rich
information in the evolution of life (Dumorne et al. 2017). Extremophilic fungi can
not only tolerate these extreme natural conditions but also depend on these extreme
factors for their growth. Therefore, the revelation of their diverse adaptive
mechanisms will provide a new breakthrough for the development of life science
(Ali et al. 2014b).
1.2.1 Thermophilic Fungi
Temperature is an exceptionally significant environmental variable, which plays a
key role in the existence, development, dissemination, and biodiversity of microbes
on the globe. Thermophilic fungi are adapted to grow at higher temperature at or
around 60 C and require nominal nutrients for metabolism and growth as compared
with mesophilic fungi. Quite a few research about evolution, growth pattern, respi-
ration, substrate exploitation, nutrition interest, and protein metabolism rate can
provide basic info about thermophilic fungi (Maheshwari et al. 2000).
1.2.2 Psychrophilic Fungi
Psychrophilic fungi can thrive at a temperature lower than 15 C, the highest growth
temperature is lower than 20 C, and the lowest growth temperature is below 0 C
(Singh et al. 2006). Psychrophilic fungi are mainly distributed in Arctic and Antarc-
tic polar regions, deep sea, high mountains, ice cellars, glaciers, cold storage, and
frozen soil areas (Sayed et al. 2020). The predominant extreme situation in Antarctic
is extra low temperature, unavailability of water (frozen desert) and rainfall, frequent
freeze–thaw sequences, heavy winds and high altitudes, vaporization, and UV
radioactivity (Durán et al. 2019). Cold thriving fungi have adapted and developed
distinct characteristics in evolution process, for example, enzymes, alteration in
membrane permeability, and several cell mechanisms, which permit these fungi to
develop at ultra-low temperatures as compared with mesophilic species, which
survive at adequate temperature (Robinson 2001; Hassan et al. 2016).
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 5

1.2.3 Acidophilic Fungi
Basically fungi surviving in acidic environments ought to be considered as acid
tolerant fairly instead of being acidophilic (Saroj et al. 2020), for the reason that they
can also grow in neutral or sometimes alkaline habitat, yet no obligate acidophilic
fungus is reported (Hujslováand Gryndler 2019). Normally, fungi prefer a wide
range of pH 1.0–11.0 (Chan et al. 2016) and mostly isolated from acidic habitats
such as volcanos, springs, mining areas, or acidic industrial liquid wastes (Hassan
et al. 2019).
1.2.4 Alkaliphilic Fungi
In alkaline lakes and some alkaline environments, even in some neutral
environments, alkalophilic fungi can be isolated (Horikoshi 2016). Biodiversity of
filamentous fungal species, which often grow beyond high ambient pH ranges, i.e.,
8–11, still remain mainly unexplored (Grum-Grzhimaylo et al. 2016). Alkaliphiles
constituted of two important physiological groups of fungi, alkaliphilic and
haloalkaliphilic, first one requires an alkaline pH of 9 or above for the growth and
devise an optimum growth pH of near 10; however, haloalkaliphilic fungi require
both an alkaline pH 9 and extraordinary saline environment up to 33% w/v NaCl
(Bondarenko et al. 2018).
1.2.5 Halophilic Fungi
Naturally high concentration of NaCl is often considered as hypersaline sites
regarded solar salterns or thalassohaline (Ali et al. 2018) enriched by different ions
and high ultraviolet radiation and sometimes extremes in pH value (Plemenitašet al.
2014). Their habitats entail several cellular reactions in response of high saline
tolerance (Khan et al. 2020a,b). Fungal species, which endure extreme
concentrations of NaCl, represents standard and typical physiological systems that
enable them to tolerate osmotic and salinity strain and maintain intracellular
absorptions (Bano et al. 2018).
1.2.6 Barophilic/Deep Sea Fungi
Bottom of the sea is considered the most enigmatic and uncharted extreme environ-
ment acquiring potential and attention for evaluation of fungal biodiversity and
future direction for deep-sea fungal exploration (Nagano and Nagahama 2012).
Extremophiles generally live in the deepest seafloor. They live in an environment
with a pressure of more than 1000 atmospheres, but they cannot survive under
normal pressure (Straub et al. 2017).
6 K. M. Talib et al.

1.3 Isolation of Extremophilic Fungi
For the isolation of extremophilic fungi, we usually choose to isolate extreme fungi
by creating an extreme culture environment. Because extremophiles can grow in
conditions different from ordinary microorganisms, we can use these characteristics
to isolate extremophiles (Samiullah et al. 2017). For example, ordinary fungi grow
well at 28 C, so when we change the temperature to 60 C or higher, we can isolate
thermophilic fungi (Merino et al. 2019). Similarly the halophilic fungi are isolated in
brine solutions (Khan et al. 2017).
1.3.1 Sample Collection
In addition to the general scientific method of isolating fungi from the environment,
we also need to pay attention to the selective sampling according to the extreme
environment that the extreme fungi adapt to, which can reduce the workload of the
later separation (Yanwisetpakdee et al. 2014). For example, when separating ther-
mophilic fungi, it is necessary to collect samples in places with high ambient
temperature, such as undersea fire vents or hot spring accessories (Straub et al.
2017); for samples separated for psychrophilic fungi are generally collected from the
north and south poles or high mountains (Fariha 2016); the samples of acidophilic
fungi are generally collected from the waste water of acid mines or sulfur-containing
hot springs (Hujslováet al. 2019); the alkaline fungi are generally from the water
with high pH (Ali et al. 2016b). The samples of halophilic fungi come from a wide
range of sources. Seawater, salt fields, and salted products can be used as samples for
halophilic fungi isolation (Ali et al. 2016b); deep sea samples are generally collected
for the isolation of barophilic fungi; Specimens preserved under aseptic conditions
until transported to the laboratory in thermocole box (Akbar et al. 2016). At the same
time, the collected samples should be kept in a proper way. For example, the samples
isolated from psychrophilic fungi should be kept at a lower temperature (0–4C)
(Wang et al. 2017).
1.3.2 Culture Medium
Potato dextrose agar media (PDA) is mostly used for isolation of various extreme
fungi. The formula is: potato 200 g, glucose 20 g, agar 16 g, water 1000 mL, and pH
natural (Ali et al. 2016a). Yeast–mold (YM) media is used for isolation of psychro-
philic fungi (Paola et al. 2019). The formula is: 0.3% yeast extract, 0.3% malt
extract, 0.5% peptone, 2% glucose, 2% agar, and pH 6.2 2. Sabouraud agar
media is used for isolation of acidophilic fungi (Ali et al. 2015). The formula is:
peptone 10 g, agar 20 g, glucose 40 g, and adding distilled water to volume to 1 L
(Akbar et al. 2019b). Czapek Yeast Autolysate agar media is made of: agar 15.0 g,
yeast extract 5.0 g, NaNO
3
3.0 g, K
2
HPO
4
1.0 g, KCL 0.5 g, MgSO
4
7H
2
O0.5g,
FeSO
4
7H
2
O 0.01 g, sucrose solution 100.0 mL, and pH 7.3 0.2 at 25 C. Malt
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 7

extract agar media is composed of: malt extract 30 g, mycological peptone 5 g, agar
15 g, water 1000 mL, and pH 5.4 0.2 (Szulc et al. 2017).
It is not easy to isolate acidophilic fungi. When the pH is very low, especially at
1, the required agar concentration needs to be very high to ensure the solidification of
the separation medium. But using silica gel instead of gel can increase the number of
culturable microorganisms (Hujslováet al. 2019). For the isolation of extreme fungi,
the material of culture medium also needs special attention. In general, some changes
may occur in the material of culture medium under extreme conditions, resulting in
the failure of the separation work. This is also one of the main problems we need to
solve in the future (Ali et al. 2014c).
1.3.3 Strain Separation
Fungi can be isolated by serial dilution method. The sample can be diluted to 10
1
–
10
6
with sterile water, and 0.3 mL is drawn and coated on the sterile PDA medium
plate (Makhathini-Zincume 2017). In the isolation of different extreme fungi, differ-
ent selection conditions are set according to their types. For example, when isolating
thermophilic fungi, the culture temperature should be set be at 50 C (Ali et al.
2014a); when isolating psychrophiles fungi, the temperature should be set at 4–15 C
(Tapia-Vázquez et al. 2020), but the growth time must more than 30 days; when
separating acidophiles fungi, the pH should be adjusted to 2.5 (Yu et al. 2014); when
isolating alkalophilic fungi, the pH should more then 8.0 (Hujslováet al. 2019),
usually most of them live pH is 9–10 (Merino et al. 2019); when isolating halophilic
fungi, the salt concentration in the medium should reach 2.5–5.2 mol/L; for the
isolation of barophilic fungi, the whole process needs to be carried out in a special
pressure vessel (Ali et al. 2014d).
In general, the extremophilic fungi need to be cultured in a constant temperature
incubator for 3–10 days, observed at any time during the cultivation process. When
colonies grow on the plate, they are transferred to a new medium and continue to
culture under the corresponding conditions until pure culture is obtained (Ali et al.
2013). Rifampicin (100 μgmL
1
) or chloramphenicol (100 μgmL
1
) can be added
to prevent any bacteria growth (Gray et al. 2019).
1.3.4 Cell Culture
For details its recommended to consult Durán et al. (2019). Briefly, laying a layer of
sterile cellophane (5 cm in diameter) on the medium plate and transferring the
mycelial block with least common multiple (LCM) diameter on the cellophane,
and cultivating according to the growth rate of different strains. When the cellophane
is full of hyphae, scrape off the mycelium with sterilized scalpel, and place it in a
1.5 mL centrifuge tube for DNA extraction or cryopreservation (Verma et al. 2017).
8 K. M. Talib et al.

1.4 Maintenance of Extremophilic Fungi
Naturally extremophilic fungi do not take place in pure culture; bacteria free cultures
can be maintained by growing fungi in media under ultraviolet radiation, which may
inhibit the development of bacteria with minimum or no side effect on the fungi
(Bovio et al. 2019). Pure culture techniques allow mycologists for detection, isola-
tion, identification, and quantification of records and several types of fungi from
various environments and define nutrition, chemistry, and the environmental
requirements for their growth and metabolism (Goers et al. 2014). Pure cultural
studies not only develop understanding of any natural habitat in the infected host or
environment but also help to determine the molecular structure and exploration of
new activities/compounds. Pure cultural methodology is the foundation of basic and
applied research and commercial utilization of a fungus (Jong and Birmingham
2001; Sharma et al. 2019). Understanding the basic requirements of a growth
media is a limiting factor in culturing various extremophilic fungi. Expansion of
knowledge will require wide research in this field to achieve inexpensive culture of
the noncultivable extremophiles (Tiquia-Arashiro and Grube 2019).
1.4.1 Medium for Maintenance
Extremophilic fungi pose a versatile biodiversity and several metabolic pathways, so
there is no specific and standard culture medium for their growth. Keeping in view
the basic physical conditions that influence their growth and better understanding of
nutritional requirements will provide an intelligent approach to formulate a media
and culture conditions best for their growth (Hauser 1986; Dighton 2016). Mostly
culture medium can be very common and simple, formulated with easily available
ingredients, but sometimes require some particular and proficient recipe to formulate
(Waheeda and Shyam 2017). Essential materials necessary for fungal growth and
development comprise simple sugars, like dextrose and sucrose, inorganic salts, air,
and water (Smith and Onions 1994). Mostly fungi prefer ammonium nitrate as basic
source of nitrogen, whereas others oblige organic forms of nitrogen like asparagine
or simple amino acids (Hesami et al. 2014). Several species are capable to grow and
yield spores on a simple agar medium prepared with tap water, whereas specific
fungi with definite growth features need essential amino acids, fatty acids, trace
elements, vitamins (such as pyridoxine, nicotinic acid, thiamin, biotin, inositol, and
pantothenic acid), and even sometimes blood components (Wang et al. 2014). In
some cases, combinations of different growth factors are formulated. Amino acids
are easily available by acid hydrolysis of casein and extracts of yeast cells, which are
also considered as common sources of vitamins (de Souza et al. 2015).
Culture media are classified as natural, semisynthetic, or synthetic based on their
composition and ingredients. Natural medium is created from natural materials, like
V-8 vegetable juice, lima beans, carrots, potatoes, cornmeal, onions, prunes, dung,
or soil. These ingredients are typically utilized as extracts, infusions, or decoctions.
Semisynthetic media comprise together natural components and distinct synthetic
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 9

ingredients, such as potato dextrose agar, yeast extract dextrose agar, and peptone
glucose agar (Basu et al. 2015).
Synthetic media contain identified components, and constituents in concentration
are appropriately measured. These media are basically used for biological analysis
and enzyme activity or specific biochemical research (Kahraman and Gurdal 2002).
Culture medium can be solid and liquid form. Solidified media are either natural
materials, such as root pieces, potato or carrot slices, or filament of beans, or nutrient
solutions coagulated by the adding agar or gelatinous materials (Figueira and
Hirooka 2000). Agar is a multifaceted polysaccharide mostly extracted from several
kinds of red aquatic algae. It is practically and exclusively used at a concentration of
1.5%, although for specifically acidic medium, 2% or more is prerequisite. Com-
monly, agar medium do not liquefy up to the temperature beyond 95 C and cannot
resolidify till the temperature drops lower than 40 C, whereas gelatin liquefies
simply when temperatures exceeds 30 C (Abdollahi et al. 2016). Agar media are
usually used basically in experiments conducted for isolation, identification, main-
tenance of cultures, and study of sporulation parameters. Liquid media are ideal for
biochemical research, predominantly during investigation of microbial analyses,
metabolic by-products, and metabolic insufficiencies (Hauser 1986). Constituents
are typically mixed in a specific medium and sterilized in autoclave. If agar is used in
media for solidification, then it should be heated gradually, generally up to boiling,
for melting the agar. To avoid interaction of media constituents, such as metals, that
may precipitate, the ingredients are arranged and sterilized individually before
mixture (Valente et al. 2016). The pH of medium is generally adjusted before to
sterilization, although sometimes sterile acid or alkali needs to be mixed after
sterilization to maintain the pH. When temperature sensitive combinations are
contained within media preparations, then different sterilization technique, such as
membrane filtration, is preferred (Rezali et al. 2017).
1.4.2 Culture Conditions
Extremophilic fungi are extensively vulnerable to variations in temperature, light,
pH, and aeration that affect their growth (Ali et al. 2019). Normally, most of fungi
grow well at room temperatures (Ametrano et al. 2019). However, the optimum
temperature may differ among species of a genus and even for strains of same
species. Some fungi, which can infect vegetation in frost areas or make food spoilage
in refrigerator, can tolerate low temperatures and even grow better at or below 0 C
(Hassan et al. 2016). Heat loving fungi grow adequately at preeminent temperatures
beyond 45–55 C. Agar medium where thermophiles are cultured be likely to
dehydrate quickly, and attention must be given to confirm extra humidity during
incubation. The degree of temperature for sporulation is typically narrower than for
mycelial growth (Joshi and Chettri 2019). Fungi show embellishment to some extent
in acid media. Even though mostly fungi prefer pH range from 3 to 7, however for
sporulation its limited range (Pommerville and Alcamo 2012). Light effects equally
on growth and reproduction of fungi; however, they grow similarly well in light or
10 K. M. Talib et al.

dark. Sometimes mycelial growth gets slow by incessant acquaintance to light and
also provides stimulatory or an inhibitory effect on sporulation (Jong et al. 1996).
1.4.3 Nutritional Requirements
Fungi are virtually ubiquitous, assorted, and obligatory to human life; for detailed
research and exploration of fungi, suitable culture media preparation is one of the
prerequisites. Variety of microorganisms grow and inhabit at different environments
and have diverse growth requirements, like temperature, nutrients, osmotic
conditions, and pH. Because of versatile culture requirements and media, availability
of the favorable in vitro environment, almost 99% of all extremophilic fungi, is still
not cultivable. As per limitations of fungal growth in laboratory, preparation of novel
media is a much desirable push for current microbiology (Tregoning et al. 2015).
Extremophilic culture media may be of diversified subject to the nutritive
requirements of different fungi, which require about 10 macro-elements namely C,
H, N, O, P, S, K, Ca, Fe, and Mg, which are required for synthesis of carbohydrates,
lipids, proteins, and nucleic acids and play a vital role in metabolism. Besides these
elements they require several microelements like (Mn, Zn, Co, Mo, Ni, and Cu) for
synthesis of enzymes and cofactors. These fungi also need carbon-based
compounds, which are essential growth factors (Basu et al. 2015).
1.5 Challenges in Culturing
It is important to learn the phenomena of stoutness of extremophilic fungal
applications in biotechnology, evolution, and most fascinatingly their life in extreme
environments (Tiquia-Arashiro and Grube 2019). Despite conservative culture
methods sometimes flop in sustenance of extremophilic fungi at high temperature
and pressure circumstances, fluctuation in pH occurs. Cultivation of thermophiles on
cellulose and agar plates containing Luria-Bertani broth or nutrient broth is not
convenient because these are unstable above 70 C for longer time (Tsudome et al.
2009). Solid culture media having spongy solid plate in nonfibrous cellulose is used
currently that may keep its reliability up to 260 C at 25 MPa. This technique can be
used to growth most of extremophilic fungi including acidophiles, alkaliphiles,
thermophiles, acidothermophiles, and alkalithermophiles in extreme physiochemical
environments in lab (Tsudome et al. 2009). Extremotolerant fungi can exhibit
differences in tolerance to the medium that simulates the conditions of their natural
habitation (Álvarez-Pérez et al. 2011).
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 11

1.6 Culture Media
1.6.1 Synthetic Media
1.6.1.1 Czapek’s Solution Agar
The medium contains sucrose as the main source of carbon, whereas sodium nitrate
as sole inorganic source of nitrogen, potassium chloride as essential ions, magne-
sium glycerophosphate, ferrous sulfate as cations, and potassium sulfate as buffering
agent and agar for solidification (Sun et al. 2020). Czapek’s agar medium is
recommended for the general cultivation of fungi especially from water samples
(Polyak et al. 2020). The acidity of the medium may be increased for the cultivation
of acidophilic fungi (Baird et al. 2017).
1.6.2 Saborauds Medium
Sabouraud agar or Sabouraud Dextrose agar (SDA) is a type of growth medium
containing digests of animal tissues (peptones), which provide a nutritious source of
amino acids and nitrogenous compounds for the growth of fungi and yeasts.
Dextrose is added as the energy and carbon source. Mostly it is used to cultivate
dermatophytes and some other types of fungi. The acidic pH of this medium
(pH about 5.0) inhibits the growth of bacteria but permits the growth of yeasts and
most filamentous fungi (Seirafinia et al. 2019). Additionally, chloramphenicol
and/or tetracycline may be added as broad spectrum antimicrobials to inhibit the
growth of a wide range of gram-positive and gram-negative bacteria (Widyana et al.
2019).
1.6.3 Semisynthetic Media
1.6.3.1 Potato Dextrose Agar (PDA)
Potato dextrose agar is considered as general purpose basal medium and first choice
of mycologists for culture, identification, enumeration, and maintaining stock
cultures of various type of fungi (Dutta et al. 2008; Devi and Kanwar 2017). It is
composed of dehydrated potato infusion and dextrose that stimulate and encourage
luxuriant fungal growth (Nonaka et al. 2014). Agar is added as the solidifying agent
and sometimes added sterile tartaric acid (10%) to lower the pH of this medium to
3.5 +/0.1, inhibiting bacterial growth (Bano et al. 2018). Chloramphenicol is
added to inhibit bacterial overgrowth of competing microorganisms from mixed
specimens, while permitting the selective isolation of fungi.
1.6.3.2 Modified Leonian’s Agar
Different fungi devise a complex of concealed potentials, which may not be
observed unless exposed to the appropriate environmental stimulations. This is
especially favorable culture medium formulated that mitosporic fungi produce
12 K. M. Talib et al.

pycnidia readily on it (Leonian 1924). Mostly, it is used to promote sporulation in
ascomycetes and hyphomycetes. This media contains malt extract, which provides
carbon, protein, and nutrient sources, where maltose is chief carbohydrate source,
peptone for nitrogen supply, dihydrogen potassium phosphate, and magnesium
sulfate serving as sources for the essential inorganic salts. Agar is used for solidifi-
cation of media, which is melted in a separate lot of water before it is added to the
nutrient portion (Hearle 2018).
1.6.3.3 Martin’s Rose Bengal Agar
Rose Bengal agar is recommended for the selective isolation and enumeration of
extremophilic fungi isolated from air, soil, lakes, ponds, rivers, streams,
wastewaters, and well waters (Ottow and Glathe 1968), which can initiate growth
over a wide pH range and temperature (Kanazawa and Kunito 1996). Rose Bengal
agar contains Papaic Digest of soybean meal, which provides the carbon and
nitrogen sources required for good growth of a wide variety of fungi (Halili et al.
2016). Dextrose is an energy source, monopotassium phosphate provides buffering
capability, and magnesium sulfate provides necessary trace elements (Gawai and
Mangnalikar 2018).
1.6.3.4 Yeast Extract-Peptone-Dextrose Agar (YEPD Agar)
YEPD consists of yeast extract, peptone, and glucose or dextrose for use in the
cultivation of aquatic fungi (Deka and Jha 2018). Mostly, fungi grow well on a
minimal medium containing only dextrose and salts, but addition of protein and
yeast cell extract hydrolysates allows faster growth during exponential or log-phase
growth (Kunert et al. 2019). Yeast extract supplies B-complex vitamins and it
contains all the amino acids necessary for growth (Babcock et al. 2019). Peptone
acts as the source of nitrogen, vitamins, and minerals. Dextrose serves as the carbon
source. This medium supports the growth of most heterotrophic microorganisms, but
due to their simple composition, they have been adopted as the basal media for the
routine cultivation of fungi (Vanderwolf et al. 2019).
1.6.4 Natural Media
1.6.4.1 V-8 Agar
V8 juice medium has traditionally been recommended for sporulation and the
examination of various fungal characteristics throughout the life cycle (Basu et al.
2015). This medium contains V8 juice supplemented with calcium carbonate used to
induce sexual development in many kinds of fungi. Yeast extract provides essential
growth nutrients, and L-asparagine serves as the amino acid source and glucose as
the carbohydrate source for the growth of fungi. V-8 juice is a blend of eight
vegetable juices, which supplies the trace ingredients to stimulate the growth of
fungi (Kent et al. 2008). The acidic pH of the medium favors fungal growth and
suppresses bacterial growth (Bhattacharjee and Dey 2014).
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 13

1.6.4.2 Weitzman and Silva-Hutner’s Agar
Weitzman and Silva-Hutner’s agar is selective medium and formulated to augment
sporulation in most common and important fungi (Scott et al. 1993a), also used for
molds as well. It is a good alternative for V-8 agar and used in routine culture in the
labs (Benny 2008). Most of the fungi grow better on both Weitzman and Silva-
Hutner’s and Leonian’s agar and some fail in sporulation on one or the other media
(Weitzman and Silva-Hutner 1967). This medium is enriched with organic carbon
for energy, a source of nitrogen for protein and vitamin synthesis, and several
minerals (Scott et al. 1993b). It contains alphacel cellulose powder, pablum baby
oatmeal, hunt’s tomato paste, potassium dihydrogen phosphate, magnesium sulfate,
sodium nitrate, and agar (Nugent et al. 2006).
1.6.4.3 Corn Meal Agar
Corn meal agar is a well-established, general mycological purpose medium
recommended for use in the cultivation, maintenance of fungal stock cultures, and
for the inducement of chlamydospore formation (Bharathi 2018). This is a very
simple formulation containing only cornmeal infusion and agar; however this
infusion has enough nutrients to enhance the growth of fungi (Mehta and Anupama
2016). Polysorbate 80 is a mixture of oleic esters, which, when added to the corn
meal infusion, stimulate production of chlamydospores (Saeed et al. 2018). Dextrose
provides an energy source to enhance fungal growth and chromogenesis (Kovács
and Pusztahelyi 2018).
1.7 Preservation Extremophilic Fungi
Understanding the metabolic and evolutionary patterns of microorganisms has
played a pivotal role in the development of agriculture, industry, and health sectors
(Kannojia et al. 2020). Therefore, for the ex situ conservation of the microbial
diversity, microbial culture collections also known as Biobanks or Microbial
Resource Centers remain the most important scientific infrastructure (Karaduman
et al. 2012; Paul et al. 2015; Sharma et al. 2017). The primary methods of culture
preservation are continuous growth, drying, and freezing (Paul et al. 2015).
Recently, research relating to biodiversity, classification, epidemiology, biotechnol-
ogy, biosafety, and biosecurity of extremophilic fungi has attracted the attention of
researchers. Conserved cultures of fungal species are applied by the investors related
with agricultural science, pharmacological, winery, and manufacturing for emerging
new technologies and produces for consumption and welfare of humanity (Sharma
et al. 2017). Numerous preservation techniques for fungi are given in this chapter
with special reference to extremophilic fungi, which are mostly used, easy, simple,
and cost-effective.
14 K. M. Talib et al.

1.7.1 Short-Term Preservation
Short-term preservation implicates conserving the culture for a period less than
1 year. Mostly fungus culture can be sustained throughout this retro by periodic
transfer. Continual growing approaches, where cultures are grown on agar, usually
kept for short time storing (Boundy-Mills et al. 2016). The method is modest, low in
price, and extensively used (Hu et al. 2014). Though it is time-consuming and
laborious, systematic serial transfer is a better choice for the continued use of local
trivial collections for a short period of time (up to 1 year) (Iqbal et al. 2017).
However, this method also has some disadvantages. The culture must be often
examined for drying and contamination by small insects or other microorganisms
(Cui et al. 2018). However, with the time interval, morphology and physiology of
cultured fungi may change. Particularly, after frequent transfers, spores or the ability
to infect the host may be diminished (Boundy-Mills et al. 2016). Due to these
shortcomings, this technique is usually not suitable for long-term (over a year)
preservation of culture (Fong et al. 2000). Most filamentous fungi can endure
about 1–2 years at 4 C (Caleza et al. 2017). Viable spore cultures can also be sealed
tightly and stored at a temperature 20 C, or stored at 70 C to improve survival
and increase the transfer required interval (Al-Bedak et al. 2019).
1.7.1.1 Culture Maintenance and Periodic Transfer
There is no comprehensive method for storing extremophilic fungal cultures; the
prime objective of maintenance is to be a sanctuary of viability without contamina-
tion, genomic variant, or deterioration. Particularly, the procedure targets either to
diminish the possibility of variations or exclude regular transfer by prolonging the
time period between subculture and attempts to carry cellular activity to a cessation
(Ilyas and Soeka 2019).
Most extremophilic fungi may be well-preserved by periodic serial transfer to
fresh culture media. Time interval between these transfers differs according to
physiology of fungi, medium used, and the environmental circumstances. Generally,
nominal medium is ideal for subculturing because basal metabolic rate of the fungi
and consequently extend the time within transfers. Fungal growth is often much
more rapid in nutrient rich medium, with the absorption of metabolic contents (Iqbal
et al. 2017).
Such substances may change environmental factors such as the pH and gas
process and reduce the transfer rate. Some fungi need diverse media for growth, or
they require the addition of organic molecules to the medium to retain specific
physiochemical activities. Perspective must evaluate the subculturing duration for
those organisms (Sharma et al. 2017).
At room temperature, the time span may differ from days to months. Transferring
the culture after every 3 months interval is adequate for most of fungi, though certain
type of fungi can persist viable for years (Caleza et al. 2017). Certain fungal species
in their dormant state can persist up to 10 years deprived of a nutrient resource before
becoming inviable (Richter et al. 2016). The fungi, which cannot produce particular
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 15

propagative cells, enormous fragments of mycelium from young, vigorously devel-
oping fringe areas may be transferred (Kumari and Naraian 2016; Singh 2017).
While preserving extremophilic fungi by periodic serial transfer, it should be
considered to use the tubes that can be properly sealed. Glass and plastic test tubes
and 10 mL bottles with plastic screw lids are extensively used for this purpose. Fungi
can be cultured with faintly loose caps up to sufficient growth is attained and then
tighten up to avoid desiccation (Marvanová2020).
Solid medium is desired to broth to watch contaminants which can be easily
visible. Replication of tubes should be carried out as a provision contrary to any
damage, at least till the subculturing is obtained. Pure culture should be confirmed
after each transfer, and curtailed description of traits must be guaranteed and tested
from time to time (Singh et al. 2018). Preservation in a fridge is the ideal technique
for sub cultures. Many fungi can be stored for 3–5 months interval for transfers if
appropriate precautionary measures are taken to avoid desiccation of the culture
media (Kannojia et al. 2020).
In fact, preservation of cultures by periodical transfer is risky, laborious, and little
bit expensive. Consequences of recurrent culture transfer can be contamination,
mislabeling, and assortment of strain variation. Short-term conservation and preser-
vation processes are not suggested for industrial strains because lot of glitches may
happen in these methods (Akhtar et al. 2016). The main risks are contaminants,
species changes in assortment causing loss of genetic and biological properties, and
the expenses of maintaining cultures under these conditions.
1.7.1.2 Disposable Screw-Cap Plastic Tubes
This method for conservation and maintenance of fungal cultures, widely used in
gelatinous media, has been successfully applied to the storage of mycelial forms of
fungi and has been fully described and discussed by Fennell (Fennell 1960). The
periodic transfer method, in an appropriate substrate, is commonly used and such
transfers are carried out at time intervals determined by the rate of dehydration and
by will of the researcher maintaining the collection (de Capriles et al. 1989). Medium
can dry out hastily even at 25 C and may require transfer after 3 months (Thakur
and Sandhu 2010). But reducing the storage temperature to 5 C will reduce the rate
of evaporation from the media (O’Donnell and Peterson 2013), thereby lengthening
the period of transfer to once every year. Keeping in view the periodic transfer as
tedious, lavish, and time lapsing, this method can be practiced diminishing these
disadvantages.
These cultures are maintained at required temperatures, with the cap lightly
fastened until a fungal growth has ensued (Onions 1971). Then the caps are strongly
secured, and the cultures moved to a storing temperature of 5 C. This method is
advantageous as plastic tubes are low-cost, resilient, and hygienic (Humber 2012);
the tubes are easy to use, and the cultures are easy to spot, and they remain standard
deprived of extreme drying for more than 2 years at 5 C; this technique is swift, and
the tubes occupy minimum space in storage, minimum risk of mite invasion
(Onions) and contaminants of the cultures by other fungi during storage (Smith
1971).
16 K. M. Talib et al.

1.7.2 Long-Term Preservation
1.7.2.1 Sclerotization
Fungi have multifarious regulated phenomenon for rapid adaptation of biochemical
response to produce sclerotia to compete with fungal predators, growth competitors,
and environmental stress (Calvo and Cary 2015). Some fungal species produce
squeezed mass of hardened mycelium or long time persistent resting spores, stored
with reserve food material are called sclerotia; naturally or in culture medium,
conserving these survival bodies (Wang et al. 2019), generally at 3–5C, is a decent
technique for preservation of fungal strains (Gutiérrez-Barranquero et al. 2012).
Some higher fungi becomes separated and remains dormant until a favorable
opportunity for growth is germinated successfully after years of storage (Frawley
et al. 2020).
Mostly, soil borne fungi produce sclerotia or micro-sclerotic bodies, which may
persist and viable up to many years. Protocols for induction and development of
sclerotia, spherules, and microsclerotia can be reviewed (Daniel and Baldwin 1964;
Nakasone et al. 2004). Sclerotization can also be induced by selective media
(Frisvad et al. 2014) exposing the culture to white daylight (Gutiérrez-Barranquero
et al. 2012) induced by oxidative stress (Georgiou et al. 2006), fungal antioxidant
response (Huarte-Bonnet et al. 2019), secondary metabolites (Calvo and Cary 2015),
and reactive oxygen (Liu et al. 2018). Rice straw or toothpicks can be used in culture
medium as substratum for induced production of sclerotia. A simple method to
induce production sclerotia in fungal species is described by Ainsworth and
Sussman (2013). Briefly, cut a piece of sterilized cellophane into the size of a petri
dish and place it on a disk containing 1% water agar. The rapidly growing plasmo-
dium is then transferred to cellophane and allowed to expand at night. The cello-
phane is then removed from the agar, placed in a sterile, dry petri dish, covered, and
allowed to dry for 24 h. Then, covering the petri dish to keep the sclerotia air-dried
until they are hard. The cellophane is cut into small pieces, and each is stored in its
own screw cap vial. The nasal tube is removed from the cellophane and is stored in a
vial as a substitute. Glycerol can considerably induce formation of sclerotia on
nutrition supply in sawdust; carbon source and extreme temperatures may induce
sclerotium formation (Cheng et al. 2006).
1.7.2.2 Mineral Oil Overlay
The mineral oil is a colorless and odorless petroleum by-product as alkane with
0.8 g/cm
3
density commonly known as paraffin or white oil (Kannojia et al. 2020). It
is a cost-effective and easy to handle method for culture preservation grown on agar
plates (Tariq et al. 2015). In this technique, extremophilic fungal cultures may retain
its viability for more than a few years or, in extraordinary case, more than 30 years at
room temperature or 15–20 C (Abd-Elsalam et al. 2010). This technique is suitable
in mycelial or fungi that fail to produce spores or cultures that are not acquiescent to
freezing temperatures (Okolo et al. 2017). Another advantage of this method is that
oil decreases mite invasion and contamination (Karabıçak et al. 2016). Even though
several basidiomycetes could be preserved by this technique, but the growing level
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 17

of culture slows down as with increase in storage time (Kannojia et al. 2020). Main
drawback of the oil overlay method is that the fungal culture remain growing and
making mixture of mutants that may develop during adverse environments (Lohnoo
et al. 2018), and fungi may lack capability of sporulation and decreased virulence
(Al-Bedak et al. 2019). Good quality mineral oil or liquefied paraffin is extensively
sterilized for this purpose (Goel et al. 2020).
Fungal culture fledged on agar plates are enclosed with 10 mm of high quality
mineral oil and free from any toxic substance. The complete agar superficial layer
and fungal culture must be sunken entirely in the oil (Goel et al. 2020). Test tubes
should be retained in vertical position at room temperature. Oil content in test tubes
or ampules should be checked from time to time, and additional oil may be added, if
needed (Tariq et al. 2015). Care should be taken that medium should not get dry and
float in the tube. For reclamation of a culture from oil overlay, a slight quantity of the
fungus colony should be placed on suitable medium keeping oil separated from
culture (Ajello et al. 1951). Repeated culture can help to get rid of oil from the
fungus sample (Okolo et al. 2017).
1.7.2.3 Soil or Sand
Extremophilic fungi can be conveniently and effectively preserved for long period in
dried and sterilized soil or sand (Nakasone et al. 2004). This method is used since
1918, and advantages of this technique comprises augmented prolonged viability of
the culture, decreased or exclusion of morphological variations, and the accessibility
of identical inoculum for several years (Ryan et al. 2000). This easy, economic, and
low-cost technique is suitable for extremophilic fungi (Kannojia et al. 2020). Some
fungi have been reported having morphological and physiological changes due to
dormancy and dryness for elongated period in preservation (Windels et al. 1988).
Sandy or loamy soil (20% moisture content) in 60 ml glass flasks filled to two-thirds
volume and then autoclaved at 120 C for 20 min. Then the flasks should be cooled
down and repeatedly sterilized. Sterilized, deionized distilled water DDH
2
O is added
to the culture, and fungus colony superficial layer is scratched lightly to produce
5 mL of spore or mycelial suspension. One milliliter of the suspension is added to
each flask of soil or sand (Kannojia et al. 2020). After few days of colony growth
20–25 C temperature, the flasks are topped lightly and kept in the fridge at 4 C
(Nakasone et al. 2004). For retrieval of culture, some grains of soil are scattered in to
agar medium (Jong and Birmingham 2001). Test tubes, vials, or screw cap bottles
can be used in place of glass flasks to save space (Tariq et al. 2015).
1.7.2.4 Silica Gel
Preservation in silica gel technique is widely practiced to reserve sporulating fungal
species when freeze drying or liquid nitrogen facilities are unavailable (Perkins
1977). This method was initially suggested by Perkins (Perkins 1962) who found
that sporulating fungi preserved in skimmed milk and kept on silica gel were viable
for 4–5 years (Karabıçak et al. 2016). Generally, viability of fungal strains preserved
on silica gel depends on the growth medium and fungal species (Trollope 1975).
Main advantage of silica gel storage is that all metabolic and growth process is
18 K. M. Talib et al.

prevented. Sometimes mycologists use glass beads as an alternative to silica gel
(Humber 1997). Retrieval of culture from silica gel is very convenient; just few silica
gel crystals are sprinkled on an agar plate (Okolo et al. 2017). The storage container
can be reused again for sequential samples.
Screw cap plastic tubes filled partly by 6–22 mesh size already sterilized silica
should be stored in strongly airtight containers (Perkins 1962). Fungal spores should
be in suspension with 10% dry powder of skim milk in DDH
2
O, already chilled at
4C. Silica gel should be chilled to about 4 C and kept in an ice water. Suspension
of spores then mixed in silica gel about 3/4 of the gel 0.5 mL/4 g and left in ice water
bath for half hour. Plastic tubes then kept with the loose caps at 20–25 C for one to
two weeks. Viability of spores should be checked by quaking some crystals onto
appropriate medium (Deshmukh 2003). If the culture is viable, then caps must be
tighten up, and the tubes are stored in a strongly sealed vessel at 4 C (Jong and
Birmingham 2001). For nonsporulating species of fungi glass test tubes with cotton
plug can be used to preserve mycelium and vegetative parts of fungi following the
process discussed above (Perkins 1962).
1.7.2.5 Immersion in Distilled Water
Use of distilled water for preservation of extremophilic fungi is an economical, cost-
effective, and easy method commonly used by mycologists (Tariq et al. 2015).
Actually, the water quashes morphological changes in many fungi (Lohnoo et al.
2018). The technique applied in casing fungal culture on agar medium in oil overlay
can also be possible if overlayed with sterilized deionized distilled water (Escobar
et al. 2020). On the other hand, sterile stubbles or Pasteur pipettes can be applied for
cutting disks from margins of growing culture. The culture disks are the shuffled to
screw cap plastic tubes already sterilized and containing some water (Karabıçak et al.
2016). Small vials of 1.8 mL can also be used instead of tubes to save space (Lohnoo
et al. 2018). These plastic tubes can be kept at room temperature, and screw caps
should not be fully tightened; after few days, caps should be tightened, sealed, and
stored at 4 C. Culture disks can be transferred carefully to fresh agar medium to
retrieve cultures avoiding contamination (Sakr 2018). For sporulating fungi, same
technique can be used for spore preservation with a little modification as discussed
earlier in oil overlay method (Caleza et al. 2017). A novel technique for fungal
species isolated from marine environments preserved in sterile marine water is
appraised where marine-oriented fungi can be preserved up to 5 years and retrieved
rate is 100%. This method is adapted as castellany method by using marine water
compromises the cheap and alternate approach of preservation of marine fungi
(Reddy and Vijaya 2020).
1.7.2.6 Organic Substratum
Since long time, scientists have established useful, reliable, and creative approaches
for preservation of fungal species on several organic substances such as wooden
chips, cereal grains, straws, filter paper, and insects and plant tissues. Mostly, these
methods were used for pathogenic or some particular fungi and carefully/extensively
examined on wide range of fungal species (Siwarungson et al. 2013).
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 19

1.7.2.7 Preservation in Wood
Wood inhabiting fungal species are easily and effectively preserved on wooden
chips or toothpicks as far as the culture is growing robustly. Some fungi are reported
to be preserved on wooden chips and found viable till 10 years (Nakasone et al.
2004). Although some fungi do not strongly grow on wooden chips, then the method
can be failed. Slight fragments of crude beech wood are dipped in 2% malt-extract
broth and autoclaved at 125 C for 20 min and resterilized after 24 h (Delatour
1991). Wooden chips then smashed with fungus culture in petri dish and sealed to
get colonization on wood. Fifteen days later, colonized wooden chips should be
moved to sterilized test tubes partially filled with 2% malt agar and plugged with
cotton and wrapped with aluminum foil and kept for 7 days at 4 C. A piece of wood
can be inoculated in to fresh medium for retrieval of culture (Jong and Birmingham
2001).
1.7.2.8 Preservation on Cereal Grains
Some fungal species have been reported to be preserved for more than 10 years on
grains of wheat, barley, sorghum, rye, millet, and oats (Singleton et al. 1992). Seeds
are sodden in water having chloramphenicol (250 g/mL) solved and kept for
overnight (Jong and Birmingham 2001). Next day, water is drained. Tubes with
screw cap filled with grains and sterilized in autoclaved for 1 h at 120 C are repeated
for 2 days. The grains should be inoculated with fresh fungus culture and kept for
10 days, then screw caps should be stiffened and enfolded with parafilm and stored
room temperature (Nakasone et al. 2004).
1.7.2.9 Use of Agar Strips for Preservation
Some fungi can be preserved on dried agar strips, which can be viable for 18 months
to 3–4 years. Fungi grown on suitable culture medium in petri dishes, being cut from
the growing margin of the colony, are kept in sterilized petri dishes (Jong and
Birmingham 2001). One week later at room temperature, the fragments of dry agar
can be moved in sterile vials, sealed, and dried in vacuum. Agar strips can be
inoculated on fresh medium for retrieval of culture (Nakasone et al. 2004).
1.7.2.10 Insect or Plant Tissue
Some pathogenic fungi can be preserved on host plant tissues used as a substratum,
which is used to preserve the culture (Jong and Birmingham 2001). Sometimes
viable conidia can be preserved on frozen, infested aphid cadavers (Nakasone et al.
2004).
1.7.2.11 Freezing
Common practice to preserve fungal cultures is freezing at temperatures below
20 Cto80 C in electrical freezers (Simione et al. 1991). Isolates have been
reported viable, pure, and morphologically stable when frozen over a year, and this
technique proved as safe, easy to handle, cost-effective, and reliable (Sakr 2018).
Frozen cultures in liquefied nitrogen are explained in detail in the subsequent
section. Screw cap tubes or cotton ploughed test tubes containing cultures grown
20 K. M. Talib et al.

on agar medium can be kept in deep freezers. Cultures can be kept in 10% glycerol
below 80 C temperature. PDA petri dishes inoculated with fungus cultures
attaining appropriate growth should be frozen at 16 C temperature for 1 year, and
then these cultures can be defrosted at 4 C for 24 h. Cultures can be retrieved
periodically for viability test. To minimalize the chances of variant strains in culture
circumstances, fungal isolates should be subcultured on variable culture media
(Álvarez-Pérez et al. 2011). Cultures colonized on different organic substratum,
just like cereal grains, agar strips, plant parts, and filter paper, are dried and then
frozen. Particularly, fungi growing vigorous can survive more in freezing method
and better than less vigorous species (Atlas 2010). Recurrent freezing and liquefying
should be avoided, which may significantly diminish viability of the cultures
(Imathiu et al. 2014). The cost of electricity for equipment, repairs, and power
outage are restraining element.
1.7.2.12 Lyophilization/Freeze-Drying and Desiccation
Presence of moisture in preserved samples often deteriorate the morphology and
viability and removal of water from sample by heating can also be lethal to the
samples (Tapia et al. 2020). Lyophilization is a procedure of desiccation of samples,
while freezing water is detached in dual phases: i.e., sublimation and followed by
desorption. Since freeze drying is executed at ultra-low temperatures, intended for
reduced metabolic activity, stabilization and elongate shelf life of thermos-labile
fungi and those strains or else unstable in liquid state are necessary to be dried. The
principle behind this is sublimation of water at temperature and pressure below its
tripartite, i.e., 611 Pascal and 0.0098 C. Water separation from a sample is frozen by
annealing through conduction or radiation (Bissoyi et al. 2016). Lyophilization, or
freeze-drying, is a cost-effective method of long-term preservation and applicable to
selective strains of fungi. Over increasing reputation of this method has achieved a
global attention in this research (Wei et al. 2012). However, the method was used
mainly for fungi that form frequent, comparatively minute spores. However, myce-
lium fragments of basidiomycetes were lyophilized successfully in the existence of
trehalose by Croan (2000). Frozen dried propagules of dictyostelids are preserved
effectively over 30 years by Raper (2014). The process of preservation technique
suits to several spore-bearing fungal species, which produce minute spores. Bigger
spores have a tendency to breakdown during the lyophilic procedure, if morphologi-
cal destruction affected cannot be revocable during retrieval (Berny and Hennebert
1991). Many spores of proper size may also be physically broken and destroyed by
lyophilization during the establishment of ice crystals (Nireesha et al. 2013). For this
reason, the ampoules in the beginning need a lot of sustainable spores. Quick
freezing and adding of a menstruum, which thaws ice crystals, can reduce the
progression of ice crystals (Wang 2000). There are two widely used solvents as
menstruum, i.e., skimmed milk powder and bovine serum, while other proteinic
substances can be used too (Gaidhani et al. 2015).
Material necessary for lyophilization include good quality automated vacuum
pump; vacuum gauge; vacuum manifold with stand; cold trap; hoses to connect
pump, trap, and manifold; insulated bath; oxygen-gas torch; oxygen supply; 10 cm
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 21

length of 6 mm soft glass tubes with one end heat sealed, or lyophilization ampoules;
cotton for plugging tubes; Pasteur pipettes; mechanical or electrical pipetting aid;
sterile menstruum; and permanent ink suitable for writing on glass (Nakasone et al.
2004).
Agar slants with a suitable medium, which enables vigorous growth and induce
sporulation, should be inoculated with the fungus and allowed to develop up to a
resting stage. Lyophilized fungal isolates from cultures before or after resting phase
frequently show much lower viability after lyophilization (Ellis and Roberson 1968).
Five or some extra lyophilization vials should be sterilized and tagged for instant
use. Approximately, 1.5–2.0 mL sterilized menstruum should be mixed in agar slant;
Pasteur pipette should be scraped on agar surface to make suspension of spores in
menstruum. If spores are not enough, then another agar slant can be scraped for more
dense suspension. About 200 μL spore suspension is poured in every of several
freeze drying tubes. Tubes must be gently closed with cotton plug, one open end of
the glass tube is greased by castor oil, and then tubes should be kept on a vacuum
manifold. The manifold is let down till the freeze drying tubes are kept dipped in the
dry ice and ethylene glycol bath at 40 Cto50 C temperatures up to filling of
every tube frozen. The system should be vacuumed for half hour, while the bath
reached a temperature about 0 C for annealing. The manifold is then elevated to
take out the tubes from bath. Lyophilization materials then dried at room temperature
at a pressure of the system becomes 30 millitorrs. Cooling by condensation allows
the samples freeze during the drying course (Rey 2016). Then the tubes are sealed
under vacuum using a gas-oxygen torch. After lyophilization, tubes are kept in
sequenced plastic cases or wrapped in plastic bags at 4 C temperature (Nakasone
et al. 2004). Viability of lyophilized cultures should be checked after a week. When
the culture is to be retrieved, ampules are opened, and the contents of sample are
transferred to sterilized deionized distilled water in a test tube using sterile pipette; let
the spores to rehydrate for an hour, before transferring to freshly make suitable agar
medium. Media and culture conditions specified for fungal strains should be
maintained as recommended and incubated the culture at suitable temperature. The
remnants of the suspension should be kept in refrigerator, letting for extra retrieval
effort if the first was failed. Given proper cure and conditions, mostly culture may
propagate in few days. However, some fungi may exhibit an extended lag period and
should be given more incubation time before being discarded as nonviable.
As we discussed above, the protocol of lyophilization is a classical technique, but
recently advanced technology is available for quick freeze drying of samples with
the help of automated equipment (Pisano 2019), which are more reliable, economi-
cal, and easy to use (Shanley 2017). In latest lyophilization technique, samples are
prepared in glass vials or ampules, sealed with rubbers stoppers and metal caps and
then freeze dried in an automated freeze dryer (Boundy-Mills et al. 2020).
1.7.2.13 Liquid Nitrogen/Cryopreservation
Storing with liquid nitrogen is an efficient method for preserving several
extremophilic fungi (Zucconi et al. 2012) and the fungi which are not suitable for
lyophilization (Pegg 2015). It is a little bit expensive as compared with
22 K. M. Talib et al.

lyophilization, for this reason that liquid nitrogen requires to be refilled timely
(Silber 2018). Liquid-nitrogen storage is recommended for the preservation of
extremophilic fungi (Duarte et al. 2018), except thermophiles (Iqbal et al. 2017).
Since ratio of mutation in most cultured fungal strains is likely to resemble to those
of mitotic division and high metabolism, this technique is best suitable for such fungi
because it completely renders the metabolic activity and cell division (Paul et al.
2015). This accomplishes due to temperature below extreme levels when water
crystals cannot develop and metabolic rates are about to cease that affect cellular
life (Nannou et al. 2016). Most of the fungal species can be preserved by freezing,
but this technique usually is reticent to fungi, which do not produce spores in culture
or have big fruiting structures unable to persist in lyophilization, genome banks, and
hazardous human pathogens. American type-culture collection (ATCC) stock the
seed reserves in liquid nitrogen because if supply of stock is in shortage, then
materials used for renewal may be genetically close to the novel deposit and feasible
(Boundy-Mills et al. 2020).
Keeping in view the susceptibility of living cells to freezing and melting, com-
monly two kinds of cryoprotectants are used: penetrating agents such as glycerol and
dimethyl sulfoxide (DMSO), which freely penetrate through cell membrane and
protect cells internally and externally (Homolka 2013). Glycerol and DMSO have
shown efficacy for nonpenetrating agents such as sucrose, lactose, glucose, manni-
tol, sorbitol, dextran, polyvinyl-pyrrolidone, and hydroxyethyl starch, which exert
their defensive effect externally to the cell membrane (Paul et al. 2015), even though
another penetrating agent polyethylene glycol, can be used too (Ohmasa et al. 1996).
Key advantages of liquid nitrogen preservation comprises prevention for chances
of genetic variation of samples; streamlined, convenient; no frequent tests for
pathogenicity, less probabilities of contamination; and better assertion for accessi-
bility of cultures (Humber 2012). The main drawbacks of this method is that liquid
nitrogen is expensive and should be replenish after 2 days and require more space,
and continuous observation is needed. Long-term availability of liquid nitrogen and
shipping cost is also increased (Fahy and Wowk 2015).
Fungi, which do not sporulate in culture or grow mycelium deep into the agar,
sterilized 2 mL screw cap polypropylene ampoules are filled with 0.5–1.0 mL sterile
10% glycerol. Plugs of 4 mm diameter are cut from robustly growing cultures using
a sterilized plastic straw. Quite a few plugs are placed in the vial, the cap is tighten
up, and the tube is retained directly into the vapor phase of a liquid nitrogen tank at
170 C temperature. Each vial should be labeled with a cryoresistant ink pen or
printed onto paper sticker, and then stuck to the vial, or it should be printed onto a
special cryoresistant adhesive label, which is easily available from biotechnology
supply agencies. The location of storage in the freezer must be indexed for rapid
retrieval. Frozen preparations are retrieved by removing the vials from the freezer
and rapidly thawing them in a 37 C water bath. The thawed agar plugs are placed on
appropriate agar plates. Viability of the cultures should be checked from 2 to 7 days
after storage (Prakash et al. 2013).
For suspensions of spores or mycelial fragments from cultures growing on the
surface of agar slants or plates, the colony surface is flooded with 10% glycerol or
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 23

5% DMSO and gently scraped with a pipette. Kept in liquid nitrogen as discussed
before (Escobar et al. 2020).
The mycelium of a fungus growing in liquid culture can be infused before it can
be pipetted into vials. The broth culture is fragmented for a few seconds in a sterile
mini blender and mixed with equal parts of 20% glycerol or 10% DMSO to give a
final concentration of 10% glycerol or 5% DMSO, respectively; vials are then
transferred instantaneously to liquid nitrogen vapor at 150 Cto180 C
(Nakasone et al. 2004; Lohnoo et al. 2018).
References
Abd-Elsalam KA, Yassin MA, Moslem MA, Bahkali AH, de Wit PJ, McKenzie EH, Stephenson
SL, Cai L, Hyde KD (2010) Culture collections, the new herbaria for fungal pathogens. Fungal
Divers 45(1):21–32
Abdollahi MR, NajafiS, Sarikhani H, Moosavi SS (2016) Induction and development of anther-
derived gametic embryos in cucumber (Cucumis sativus l.) by optimizing the macronutrient and
agar concentrations in culture medium. Turk J Biol 40(3):571–579
Ainsworth GC, Sussman AS (2013) The fungal population: an advanced treatise. Elsevier
Ajello L, Grant VQ, Gutzke MA (1951) Use of mineral oil in the maintenance of cultures of fungi
pathogenic for humans. AMA Arch Derm Syphilol 63(6):747–749
Akbar A, Ali I, Anal A (2016) Industrial perspectives of lactic acid bacteria for biopreservation and
food safety. J Anim Plant Sci 26:938–948
Akbar A, Sadiq MB, Ali I, Anwar M, Muhammad N, Muhammad J, Shafee M, Ullah S, Gul Z,
Qasim S (2019a) Lactococcus lactis subsp. Lactis isolated from fermented milk products and its
antimicrobial potential. CyTA J Food 17(1):214–220
Akbar A, Sadiq MB, Ali I, Muhammad N, Rehman Z, Khan MN, Muhammad J, Khan SA, Rehman
FU, Anal AK (2019b) Synthesis and antimicrobial activity of zinc oxide nanoparticles against
foodborne pathogens salmonella typhimurium and staphylococcus aureus. Biocatal Agric
Biotechnol 17:36–42
Akhtar N, Hafeez R, Ali A (2016) Viability assessment of pure cultures of mucorales preserved at
4C. Pakistan J Phytopathol 28(2):147–152
Al-Bedak OA, Sayed RM, Hassan SH (2019) A new low-cost method for long-term preservation of
filamentous fungi. Biocatal Agric Biotechnol 22:101417
Ali I, Kanhayuwa L, Rachdawong S, Rakshit SK (2013) Identification, phylogenetic analysis and
characterization of obligate halophilic fungi isolated from a man-made solar saltern in
Phetchaburi Province, Thailand. Ann Microbiol 63(3):887–895
Ali I, Akbar A, Anwar M, Yanwisetpakdee B, Prasongsuk S, Lotrakul P, Punnapayak H (2014a)
Purification and characterization of extracellular, polyextremophilic α-amylase obtained from
halophilic Engyodontium album. Iranian J Biotechnol 12(4):35–40
Ali I, Akbar A, Anwar M, Yanwisetpakdee B, Punnapayak H (2014b) Purification and characteri-
zation of extracellular, polyextremophilic α-amylase obtained from halophilic Engyodontium
album. Iranian J Biotechnol 12(4):35–40
Ali I, Akbar A, Yanwisetpakdee B, Prasongsuk S, Lotrakul P, Punnapayak H (2014c) Purification,
characterization, and potential of saline waste water remediation of a polyextremophilic
α-amylase from an obligate halophilic aspergillus gracilis. Biomed Res Int 2014:106937
Ali I, Siwarungson N, Punnapayak H, Lotrakul P, Prasongsuk S, Bankeeree W, Rakshit SK (2014d)
Screening of potential biotechnological applications from obligate halophilic fungi, isolated
from a man-made solar saltern located in Phetchaburi Province, Thailand. Pak J Botany 46:983–
988
24 K. M. Talib et al.

Ali I, Akbar A, Anwar M, Prasongsuk S, Lotrakul P, Punnapayak H (2015) Purification and
characterization of a polyextremophilic α-amylase from an obligate halophilic aspergillus
penicillioides isolate and its potential for souse with detergents. Biomed Res Int 2015:245649
Ali I, Akbar A, Aslam M, Ullah S, Anwar M, Punnapayak H, Lotrakul P, Prasongsuk S,
Yanwisetpakdee B, Permpornsakul P (2016a) Comparative study of physical factors and
microbial diversity of four man-made extreme ecosystems. Proc Natl Acad Sci India Sect B
Biol Sci 86(3):767–778
Ali I, Prasongsuk S, Akbar A, Aslam M, Lotrakul P, Punnapayak H, Rakshit SK (2016b)
Hypersaline habitats and halophilic microorganisms. MIJST 10(3):330–345
Ali FS, Akbar A, Prasongsuk S, Permpornsakul P, Yanwisetpakdee B, Lotrakul P, Punnapayak H,
Asrar M, Ali I (2018) Penicillium imranianum, a new species from the man-made solar saltern
of Phetchaburi province, Thailand. Pak J Bot 50(5):2055–2058
Ali I, Khaliq S, Sajid S, Akbar A (2019) Biotechnological applications of halophilic fungi: past,
present, and future. In: Fungi in extreme environments: ecological role and biotechnological
significance. Springer, pp 291–306
Álvarez-Pérez S, Blanco JL, Alba P, García ME (2011) Fungal growth in culture media simulating
an extreme environment. Rev Iberoam Micol 28(4):159–165
Ametrano CG, Muggia L, Grube M (2019) Extremotolerant black fungi from rocks and lichens. In:
Fungi in extreme environments: ecological role and biotechnological significance. Springer, pp
119–143
Atlas RM (2010) Handbook of microbiological media. CRC Press
Babcock T, Borden JH, Gries R, Carroll C, Lafontaine JP, Moore M, Gries G (2019) Inter-kingdom
signaling—symbiotic yeasts produce semiochemicals that attract their yellowjacket hosts.
Entomol Exp Appl 167(3):220–230
Baird RB, Eaton AD, Rice EW (eds) (2017) Standard methods for the examination of water and
wastewater, vol 2. Public Health Association
Bano A, Hussain J, Akbar A, Mehmood K, Anwar M, Hasni MS, Ullah S, Sajid S, Ali I (2018)
Biosorption of heavy metals by obligate halophilic fungi. Chemosphere 199:218–222
Basu S, Bose C, Ojha N, Das N, Das J, Pal M, Khurana S (2015) Evolution of bacterial and fungal
growth media. Bioinformation 11(4):182
Benny GL (2008) Methods used by dr. Rk Benjamin, and other mycologists, to isolate
zygomycetes. Aliso J Syst Evol Bot 26(1):37–61
Berny J-F, Hennebert G (1991) Viability and stability of yeast cells and filamentous fungus spores
during freeze-drying: effects of protectants and cooling rates. Mycologia 83(6):805–815
Bharathi R (2018) Comparison of chromogenic media with the corn meal agar for speciation of
Candida. J Pure Appl Microbiol 12(3):1617–1623
Bhattacharjee R, Dey U (2014) An overview of fungal and bacterial biopesticides to control plant
pathogens/diseases. Afr J Microbiol Res 8(17):1749–1762
Bissoyi A, Kumar A, Rizvanov AA, Nesmelov A, Gusev O, Patra PK, Bit A (2016) Recent
advances and future direction in lyophilisation and desiccation of mesenchymal stem cells.
Stem Cells Int 2016:3604203
Bondarenko S, Georgieva M, Bilanenko E (2018) Fungi inhabiting the coastal zone of lake Magadi.
Contemp Probl Ecol 11(5):439–448
Boundy-Mills KL, Glantschnig E, Roberts IN, Yurkov A, Casaregola S, Daniel HM,
Groenewald M, Turchetti B (2016) Yeast culture collections in the twenty-first century: new
opportunities and challenges. Yeast 33(7):243–260
Boundy-Mills K, McCluskey K, Elia P, Glaeser JA, Lindner DL, Nobles D Jr, Normanly J, Ochoa-
Corona FM, Scott JA, Ward TJ (2020) Preserving us microbe collections sparks future
discoveries. J Appl Microbiol 129(2):162–174
Bovio E, Garzoli L, Poli A, Luganini A, Villa P, Musumeci R, McCormack GP, Cocuzza CE,
Gribaudo G, Mehiri M (2019) Marine fungi from the sponge Grantia compressa: biodiversity,
chemodiversity, and biotechnological potential. Mar Drugs 17(4):220
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 25

Caleza V, Castillo S, Gomis M, Kamah S, López R, Garcia-Seco D, Varma A, Akdi K (2017)
Conservation strategies of new fungi samples in culture collections: Piriformospora indica
case. In: Modern tools and techniques to understand microbes. Springer, pp 431–447
Calvo AM, Cary JW (2015) Association of fungal secondary metabolism and sclerotial biology.
Front Microbiol 6:62
Chan WK, Wildeboer D, Garelick H, Purchase D (2016) A proteomic study on the responses to
arsenate stress by an acidophilic fungal strain Aidomyces acidophilus wkc1. J Biotechnol
Biomater 6(5)
Cheng XH, Guo SX, Wang CL (2006) Factors influencing formation of sclerotia in Grifola
umbellate (pers.) pilát under artificial conditions. J Integr Plant Biol 48(11):1312–1317
Croan SC (2000) Lyophilization of hypha-forming tropical wood-inhabiting basidiomycotina.
Mycologia 92(4):810–817
Cui H, Ren X, Yun L, Hou Q, Feng F, Liu H (2018) Simple and inexpensive long-term preservation
methods for Phytophthora infestans. J Microbiol Methods 152:80–85
Daniel JW, Baldwin HH (1964) Methods of culture for plasmodial Myxomycetes. In: Methods in
cell biology, vol 1. Elsevier, pp 9–41
de Capriles CH, Mata S, Middelveen M (1989) Preservation of fungi in water (castellani): 20 years.
Mycopathologia 106(2):73–79
de Souza PM, de Assis Bittencourt ML, Caprara CC, de Freitas M, de Almeida RPC, Silveira D,
Fonseca YM, Ferreira Filho EX, Pessoa Junior A, Magalhães PO (2015) A biotechnology
perspective of fungal proteases. Braz J Microbiol 46(2):337–346
Deka D, Jha DK (2018) Optimization of culture parameters for improved production of bioactive
metabolite by endophytic Geosmithia pallida (ku693285) isolated from Brucea mollis wall
ex. Kurz, an endangered medicinal plant. J Pure Appl Microbiol 12(3):1205–1213
Delatour C (1991) A very simple method for long-term storage of fungal cultures. Eur J Forest
Pathol 21(6–7):444–445
Deshmukh S (2003) The maintenance and preservation of keratinophilic fungi and related
dermatophytes. Mycoses 46(5–6):203–207
Devi S, Kanwar S (2017) Antifungal potential of hot spring water of Manikaran, Himachal Pradesh
(India). J Environ Biol 38(2):217
Dighton J (2016) Fungi in ecosystem processes, vol 31. CRC press
Duarte AWF, dos Santos JA, Vianna MV, Vieira JMF, Mallagutti VH, Inforsato FJ, Wentzel LCP,
Lario LD, Rodrigues A, Pagnocca FC (2018) Cold-adapted enzymes produced by fungi from
terrestrial and marine Antarctic environments. Crit Rev Biotechnol 38(4):600–619
Dumorne K, Camacho Cordova D, Astorga-Elo M, Renganathan P (2017) Extremozymes: a
potential source for industrial applications. J Microbiol Biotechnol 27(4):649–659. https://doi.
org/10.4014/jmb.1611.11006
Durán P, Barra PJ, Jorquera MA, Viscardi S, Fernandez C, Paz C, Mora ML, Bol R (2019)
Occurrence of soil fungi in Antarctic pristine environments. Front Bioeng Biotechnol 7:28
Dutta T, Sahoo R, Sengupta R, Ray SS, Bhattacharjee A, Ghosh S (2008) Novel cellulases from an
extremophilic filamentous fungi Penicillium citrinum: production and characterization. J Ind
Microbiol Biotechnol 35(4):275–282
Ellis J, Roberson JA (1968) Viability of fungus cultures preserved by lyophilization. Mycologia
60(2):399–405
Escobar LPB, González JPP, Orjuela M (2020) Effect of culture preservation methods in the
stability and nutritional characteristics of Pleurotus ostreatus. Asian J Microbiol Biotech
Environ Sci 22(2):359–368
Fahy GM, Wowk B (2015) Principles of cryopreservation by vitrification. In: Cryopreservation and
freeze-drying protocols. Springer, pp 21–82
Fariha H (2016) Psychrophilic and psychrotrophic fungi: a comprehensive review. Rev Environ Sci
Biotechnol 15(2):147–172
Fennell DI (1960) Conservation of fungous cultures. Bot Rev 26(1):79–141
26 K. M. Talib et al.

Figueira ELZ, Hirooka EY (2000) Culture medium for amylase production by toxigenic fungi. Braz
Arch Biol Technol 43(5):461–467
Fong Y, Anuar S, Lim H, Tham F, Sanderson F (2000) A modified filter paper technique for long-
term preservation of some fungal cultures. Mycologist 14(3):127–130
Frawley D, Greco C, Oakley B, Alhussain MM, Fleming AB, Keller NP, Bayram Ö (2020) The
tetrameric pheromone module stec-mkkb-mpkb-sted regulates asexual sporulation, sclerotia
formation and aflatoxin production in Aspergillus flavus. Cell Microbiol 22(6):e13192
Frisvad JC, Petersen LM, Lyhne EK, Larsen TO (2014) Formation of sclerotia and production of
indoloterpenes by Aspergillus niger and other species in section Nigri. PLoS One 9(4):e94857
Gaidhani KA, Harwalkar M, Bhambere D, Nirgude PS (2015) Lyophilization/freeze drying–a
review. World J Pharm Res 4(8):516–543
Gawai D, Mangnalikar S (2018) Effect of temperature and ph on growth of Atlernaria alternata,
leaf spot pathogen of soybean. Biosci Discov 9(1):162–165
Georgiou CD, Patsoukis N, Papapostolou I, Zervoudakis G (2006) Sclerotial metamorphosis in
filamentous fungi is induced by oxidative stress. Integr Comp Biol 46(6):691–712
Goel R, Soni R, Suyal DC (2020) Microbiological advancements for higher altitude agro-
ecosystems & sustainability. Springer
Goers L, Freemont P, Polizzi KM (2014) Co-culture systems and technologies: taking synthetic
biology to the next level. J R Soc Interface 11(96):20140065
Gray DA, Dugar G, Gamba P, Strahl H, Jonker MJ, Hamoen LW (2019) Extreme slow growth as
alternative strategy to survive deep starvation in bacteria. Nat Commun 10(1):1–12
Grum-Grzhimaylo AA, Georgieva ML, Bondarenko SA, Alfons J, Debets M, Bilanenko EN (2016)
On the diversity of fungi from soda soils. Fungal Divers 76(1):27
Gutiérrez-Barranquero JA, Pliego C, Bonilla N, Calderón CE, Pérez-García A, de Vicente A,
Cazorla FM (2012) Sclerotization as a long-term preservation method for Rosellinia necatrix
strains. Mycoscience 53(6):460–465
Halili F, Arboleda A, Durkee H, Taneja M, Miller D, Alawa KA, Aguilar MC, Amescua G, Flynn
HW Jr, Parel J-M (2016) Rose bengal–and riboflavin-mediated photodynamic therapy to inhibit
methicillin-resistant Staphylococcus aureus keratitis isolates. Am J Ophthalmol 166:194–202
Hassan N, Rafiq M, Hayat M, Shah AA, Hasan F (2016) Psychrophilic and psychrotrophic fungi: a
comprehensive review. Rev Environ Sci Biotechnol 15(2):147–172
Hassan N, Rafiq M, Rehman M, Sajjad W, Hasan F, Abdullah S (2019) Fungi in acidic fire: a
potential source of industrially important enzymes. Fungal Biol Rev 33(1):58–71
Hauser JT (1986) Techniques for studying bacteria and fungi. Carolina Biological Supply Company
Hearle HJ (2018) Physiological effect of saltwater on the “whisky fungus”,baudoinia sp “research
project in the school of science at the university of the west of scotland, session 2017–2018”.
Environ Health 40
Hesami A-A, Zakery-Asl M-A, Gardonpar H (2014) The effect of three amino acids (asparagine,
glutamine and gly-cine) on some quantity and quality characteristics of white button mushroom
(Agaricus bisporus). Intl J Farm Alli Sci 3(2):187–191
Homolka L (2013) Methods of cryopreservation in fungi. In: Laboratory protocols in fungal
biology. Springer, pp 9–16
Horikoshi K (2016) Alkaliphiles. In: Extremophiles. Springer, pp 53–78
Hu X, Webster G, Xie L, Yu C, Li Y, Liao X (2014) A new method for the preservation of axenic
fungal cultures. J Microbiol Methods 99:81–83
Huarte-Bonnet C, Paixão FR, Mascarin GM, Santana M, Fernandes ÉK, Pedrini N (2019) The
entomopathogenic fungus beauveria bassiana produces microsclerotia-like pellets mediated by
oxidative stress and peroxisome biogenesis. Environ Microbiol Rep 11(4):518–524
HujslováM, Gryndler M (2019) Fungi in biofilms of highly acidic soils. In: Fungi in extreme
environments: ecological role and biotechnological significance. Springer, pp 185–203
HujslováM, Bystrianský L, Benada O, Gryndler M (2019) Fungi, a neglected component of
acidophilic biofilms: do they have a potential for biotechnology? Extremophiles 23(3):267–275
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 27

Humber RA (1997) Fungi: preservation of cultures. In: Manual of techniques in insect pathology.
Elsevier, pp 269–279
Humber RA (2012) Preservation of entomopathogenic fungal cultures. Man Tech Inverte Pathol 2:
317–327
Ilyas M, Soeka YS (2019) Accelerated rate storage and viability test of Basidiomycetous fungal
strains were cryopreserved at –80 C. In IOP conference series: earth and environmental
science, 308; 1 (012069). IOP Publishing
Imathiu S, Edwards S, Ray R, Back M (2014) Artificial inoculum and inoculation techniques
commonly used in the investigation of Fusarium head blight in cereals. Acta Phytopathol
Entomol Hung 49(2):129–139
Iqbal S, Ashfaq M, Malik A, Khan K, Mathew P (2017) Isolation, preservation and revival of
Trichoderma viride in culture media. J Entomol Zool Stud 5(3):1640–1646
Jing L, Zhang X, Ali I, Chen X, Wang L, Chen H, Han M, Shang R, Wu Y (2020) Usage of
microbial combination degradation technology for the remediation of uranium contaminated
ryegrass. Environ Int 144:106051
Jong S-C, Birmingham JM (2001) Cultivation and preservation of fungi in culture. In: Systematics
and evolution. Springer, pp 193–202
Jong S, Dugan F, Edwards MJ (1996) Atcc filamentous fungi. American Type Culture Collection
Joshi S, Chettri U (2019) Fungi in hypogean environment: bioprospection perspective. In: Advanc-
ing frontiers in mycology and mycotechnology. Springer, pp 539–561
Kahraman SS, Gurdal IH (2002) Effect of synthetic and natural culture media on laccase production
by white rot fungi. Bioresour Technol 82(3):215–217
Kanazawa S, Kunito T (1996) Preparation of ph 3.0 agar plate, enumeration of acid-tolerant, and
al-resistant microorganisms in acid soils. Soil Sci Plant Nutr 42(1):165–173
Kannojia P, Kashyap AS, Manzar N, Srivastava D, Singh UB, Sharma SK, Sharma PK (2020)
Preservation of fungal culture with special reference to mineral oil preservation. In:
Microbiological advancements for higher altitude agro-ecosystems & sustainability. Springer,
pp 433–445
Karabıçak N, Karatuna O, Akyar I (2016) Evaluation of the viabilities and stabilities of pathogenic
mold and yeast species using three different preservation methods over a 12-year period along
with a review of published reports. Mycopathologia 181(5–6):415–424
Karaduman AB, Atli B, Yamac M (2012) An example for comparison of storage methods of
macrofungus cultures: Schizophyllum commune. Turk J Bot 36(2):205–212
Kent CR, Ortiz-Bermúdez P, Giles SS, Hull CM (2008) Formulation of a defined v8 medium for
induction of sexual development of Cryptococcus neoformans. Appl Environ Microbiol 74(20):
6248–6253
Khan MN, Lin H, Li M, Wang J, Mirani ZA, Khan SI, Buzdar MA, Ali I, Jamil K (2017)
Identification and growth optimization of a marine bacillus dk1-sa11 having potential of
producing broad spectrum antimicrobial compounds. Pak J Pharm Sci 30(3):839–853
Khan I, Gul S, Ahmad S, Rehman GB, Yunus AW, Gul I, Ali I, Akbar A, Islam M (2020a) Litter
decay in rangeland sites with varying history of human disturbance: a study with Hazargangi
Chiltan Mountain. J Mt Sci 17:898–906
Khan SA, Akbar A, Permpornsakul P, Yanwisetpakdee B, Chen X, Anwar M, Ali I (2020b)
Molecular diversity of halophilic fungi isolated from mangroves ecosystem of Miani Hor,
Balochistan, Pakistan. Pak J Bot 52(5):1823–1829
Kovács S, Pusztahelyi T (2018) Investigation of polyphenol resistance of aspergillus flavus on
cornmeal media. Acta Phytopathol Entomol Hung 53(2):163–170
Kumari S, Naraian R (2016) Decolorization of synthetic brilliant green carpet industry dye through
fungal co-culture technology. J Environ Manag 180:172–179
Kunert AT, Pöhlker ML, Krevert CS, Wieder C, Speth KR, Hanson LE, Morris CE, Schmale DG
III, Pöschl U, Fröhlich-Nowoisky J (2019) Highly active and stable fungal ice nuclei are
widespread among Fusarium species. Biogeosciences 16(23):4647–4659
28 K. M. Talib et al.

Leonian LH (1924) A study of factors promoting pycnidium-formation in some sphaeropsidales.
Am J Bot:19–50
Liu Q, Zhao Z, Dong H, Dong C (2018) Reactive oxygen species induce sclerotial formation in
Morchella importuna. Appl Microbiol Biotechnol 102(18):7997–8009
Lohnoo T, Yingyong W, Jongruja N, Krajaejun T (2018) Comparison of storage methods to
preserve the pathogenic oomycete Pythium insidiosum. SE Asian J Trop Med 49(3):421–427
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol Rev 64(3):461–488
Makhathini-Zincume AP (2017) Effects of antifungul treatments on some recalcitrant seeds.
University of KwaZulu-Natal, Westville
MarvanováL (2020) Maintenance of aquatic hyphomycete cultures. In: Methods to study litter
decomposition. Springer, pp 211–222
Mehta R, Anupama SW (2016) Evaluation of hicrome candida differential agar for species
identification of candida isolates from various clinical samples. Int J Contemp Med Res 3(4):
1219–1222
Merino N, Aronson HS, Bojanova DP, Feyhl-Buska J, Wong ML, Zhang S, Giovannelli D (2019)
Living at the extremes: extremophiles and the limits of life in a planetary context. Front
Microbiol 10:780
Nagano Y, Nagahama T (2012) Fungal diversity in deep-sea extreme environments. Fungal Ecol
5(4):463–471
Nakasone KK, Peterson SW, Jong S-C (2004) Preservation and distribution of fungal cultures.
Biodiversity of fungi: inventory and monitoring methods. Elsevier Academic Press,
Amsterdam, pp 37–47
Nannou TK, Jouhara H, Trembley J, Herrmann J (2016) Cryopreservation: methods, equipment and
critical concerns. Refrigeration Science and Technology, pp 247–258
Nireesha G, Divya L, Sowmya C, Venkateshan N, Lavakumar V (2013) Lyophilization/freeze
drying-an review. Int J Novel Trends Pharm Sci 3(4):87–98
Nonaka K, Todaka N, Ōmura S, Masuma R (2014) Combination cellulose plate (non-agar solid
support) and agar plate method improves isolation of fungi from soil. J Antibiot 67(11):755–761
Nugent LK, Sangvichen E, Sihanonth P, Ruchikachorn N, Whalley AJ (2006) A revised method for
the observation of conidiogenous structures in fungi. Mycologist 20(3):111–114
O’Donnell K, Peterson SW (2013) Isolation, preservation, and taxonomy. In: Biotechnology of
filamentous fungi: technology and products, p 7
Ohmasa M, Tsunoda M, Babasaki K, Hiraide M, Harigae H (1996) Fruit-body production of test
cultures of Flammulina velutipes preserved for seven years by freezing at three different
temperatures. Mycoscience 37(4):449–454
Okolo MO, Kim E, Tiri HS, Umar GZ, Envuladu EA, Tiri LY, Nwodo E, Onyedibe KI, Banwat EB,
Egah DZ (2017) Stability of candida albicans over long and short term storage in a resource-
limited setting. Niger J Med 26(2):163–166
Onions AH (1971) Chapter iv preservation of fungi. In: Methods in microbiology, vol 4. Elsevier,
pp 113–151
Ottow J, Glathe H (1968) Rose bengal-malt extract-agar, a simple medium for the simultaneous
isolation and enumeration of fungi and actinomycetes from soil. Appl Microbiol 16(1):170
Paola D, Patricio B, Milko J, Sharon V (2019) Occurrence of soil fungi in Antarctic pristine
environments. Front Bioeng Biotechnol 7:28
Paul JS, Tiwari K, Jadhav S (2015) Long term preservation of commercial important fungi in
glycerol at 4 c. Int J Biol Chem 9(2):79–85
Pegg DE (2015) Principles of cryopreservation. In: Cryopreservation and freeze-drying protocols.
Springer, pp 3–19
Perkins DD (1962) Preservation of neurospora stock cultures with anhydrous silica gel. Can J
Microbiol 8(4):591–594
Perkins D (1977) Details for preparing silica gel stocks. Fungal Genet Rep 24(1):23
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 29

Pisano R (2019) Alternative methods of controlling nucleation in freeze drying. In: Lyophilization
of pharmaceuticals and biologicals. Springer, pp 79–111
PlemenitašA, Lenassi M, Konte T, Kejžar A, Zajc J, Gostinčar C, Gunde-Cimerman N (2014)
Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective.
Front Microbiol 5:199
Polyak Y, Bakina L, Mayachkina N, Polyak M (2020) The possible role of toxigenic fungi in
ecotoxicity of two contrasting oil-contaminated soils–afield study. Ecotoxicol Environ Saf 202:
110959
Pommerville JC, Alcamo IE (2012) Alcamo’s fundamentals of microbiology: body systems edition.
Jones & Bartlett Publishers
Prakash O, Nimonkar Y, Shouche YS (2013) Practice and prospects of microbial preservation.
FEMS Microbiol Lett 339(1):1–9
Prasongsuk S, Lotrakul P, Ali I, Bankeeree W, Punnapayak H (2018) The current status of
Aureobasidium pullulans in biotechnology. Folia Microbiol 63(2):129–140
Raper KB (2014) The dictyostelids. Princeton University Press
Reddy GVS, Vijaya C (2020) A method of preservation of marine fungi in sterile marine water.
Asian J Biol Sci 9(1):99
Rey L (2016) Freeze-drying/lyophilization of pharmaceutical and biological products. CRC Press
Rezali NI, Sidik NJ, Saleh A, Osman NI, Adam NAM (2017) The effects of different strength of ms
media in solid and liquid media on in vitro growth of Typhonium flagelliforme. Asian Pac J Trop
Biomed 7(2):151–156
Richter DL, Dixon TG, Smith JK (2016) Revival of saprotrophic and mycorrhizal basidiomycete
cultures after 30 years in cold storage in sterile water. Can J Microbiol 62(11):932–937
Robinson CH (2001) Cold adaptation in Arctic and Antarctic fungi. New Phytol 151(2):341–353
Ryan M, Smith D, Jeffries P (2000) A decision-based key to determine the most appropriate
protocol for the preservation of fungi. World J Microbiol Biotechnol 16(2):183–186
Saeed N, Sattar M, Shakoor S, Farooqi J, Mehboob R, Zafar A, Jabeen K (2018) Identification of
candida species by direct inoculation of cornmeal tween™80 agar from blood culture bottles:
rapid and cost-effective approach. Biomed Biotechnol Res J (BBRJ) 2(2):122
Sakr N (2018) Evaluation of two storage methods for fungal isolates of Fusarium sp. and
Cochliobolus sativus. Acta Phytopathol Entomol Hung 53(1):11–18
Samiullah A, Tareen R, Khan N, Akber A, Ali I, Khan A, Alam F, Aslam S, Sajida A, Buzdar M
(2017) Determination of chemical composition, total phenolic content and antioxidant activity
of Xylanthemum macropodum. J Chem Soc Pak 39(1):83–91
Saroj P, Manasa P, Narasimhulu K (2020) Assessment and evaluation of cellulase production using
ragi (Eleusine coracana) husk as a substrate from thermo-acidophilic Aspergillus fumigatus
JCM 10253. Bioprocess Biosyst Eng 44:113–126
Sayed AM, Hassan MHA, Alhadrami HA, Hassan HM, Goodfellow M, Rateb ME (2020) Extreme
environments: microbiology leading to specialized metabolites. J Appl Microbiol 128(3):
630–657. https://doi.org/10.1111/jam.14386
Scott J, Malloch D, Gloer J (1993a) Brief article. Mycologia 85(3):503–508
Scott J, Malloch D, Gloer J (1993b) Polytolypa, an undescribed genus in the onygenales. Mycologia
85(3):503–508
Seirafinia M, Omidi A, Rasooli A, Mohebbi M (2019) Zearalenone production in sabouraud
dextrose broth and rice culture by various species of Fusarium fungi. J Biopest 12(2)
Sekova VY, Gessler NN, Isakova EP, Antipov AN, Dergacheva DI, Deryabina YI, Trubnikova EV
(2015) Redox status of extremophilic yeast Yarrowia lipolytica during adaptation to ph-stress.
Appl Biochem Microbiol 51(6):649–654. https://doi.org/10.1134/s0003683815060137
Shanley A (2017) Modernizing lyophilization. BioPharm Int 30(12):50–52
30 K. M. Talib et al.

Sharma SK, Kumar R, Vaishnav A, Sharma PK, Singh UB, Sharma AK (2017) Microbial cultures:
maintenance, preservation and registration. In: Modern tools and techniques to understand
microbes. Springer, pp 335–367
Sharma SK, Saini S, Verma A, Sharma PK, Lal R, Roy M, Singh UB, Saxena AK, Sharma AK
(2019) National agriculturally important microbial culture collection in the global context of
microbial culture collection centres. Proc Nat Acad Sci India Section B Biol Sci 89(2):405–418
Silber S (2018) Cryopreservation of sperm. In: Fundamentals of male infertility. Springer, pp
139–142
Simione F, Brown E, Buck C (1991) Atcc preservation methods. American Type Culture Collection
Singh SK (2017) Ex situ conservation of fungi: a review. In: Developments in fungal biology and
applied mycology. Springer, pp 543–562
Singh S, Puja G, Bhat D (2006) Psychrophilic fungi from Schirmacher Oasis, East Antarctica.
Curr Sci:1388–1392
Singh SK, Singh P, Gaikwad S, Maurya D (2018) Conservation of fungi: a review on conventional
approaches. In: Microbial resource conservation. Springer, pp 223–237
Singleton LL, Mihail JD, Rush CM (1992) Methods for research on soilborne phytopathogenic
fungi. vol 632, 4072 M4
Siwarungson N, Ali I, Damsud T (2013) Comparative analysis of antioxidant and
antimelanogenesis properties of three local guava (Psidium guajava l.) varieties of Thailand,
via different extraction solvents. J Food Meas Charact 7(4):207–214
Smith RS (1971) Maintenance of fungal cultures in presterilized disposable screw-cap plastic tubes.
Mycologia 63(6):1218–1221
Smith D, Onions AH (1994) The preservation and maintenance of living fungi, 2nd edn. CAB
International
Straub CT, Zeldes BM, Schut GJ, Adams MWW, Kelly RM (2017) Extremely thermophilic energy
metabolisms: biotechnological prospects. Curr Opin Biotechnol 45:104–112. https://doi.org/10.
1016/j.copbio.2017.02.016
Sun B, Xia Z, Yuan L, Wan C, Zhang L (2020) Streptomyces tailanensis sp. Nov., an actinomycete
isolated from riverside silt. Int J Syst Evol Microbiol 70(4):2760–2765
Szulc J, Ruman T, Gutarowska B (2017) Metabolome profiles of moulds on carton-gypsum board
and malt extract agar medium obtained using an aunpet saldi-tof-ms method. Int Biodeterior
Biodegradation 125:13–23
Tapia MS, Alzamora SM, Chirife J (2020) Effects of water activity (aw) on microbial stability as a
hurdle in food preservation. In: Water activity in foods: fundamentals and applications, pp
323–355
Tapia-Vázquez I, Sánchez-Cruz R, Arroyo-Domínguez M, Lira-Ruan V, Sánchez-Reyes A, del
Rayo Sánchez-Carbente M, Padilla-Chacón D, Batista-García RA, Folch-Mallol JL (2020)
Isolation and characterization of psychrophilic and psychrotolerant plant-growth promoting
microorganisms from a high-altitude volcano crater in Mexico. Microbiol Res 232:126394
Tariq A, Naz F, Rauf CA, Irshad G (2015) Long term and least expensive preservation methods for
various fungal cultures. Pakistan J Phytopathol 27(2):147–151
Thakur R, Sandhu SS (2010) Distribution, occurrence and natural invertebrate hosts of indigenous
entomopathogenic fungi of Central India. Indian J Microbiol 50(1):89–96
Tiquia-Arashiro SM, Grube M (2019) Fungi in extreme environments: ecological role and biotech-
nological significance. Springer
Tregoning GS, Kempher ML, Jung DO, Samarkin VA, Joye SB, Madigan MT (2015) A halophilic
bacterium inhabiting the warm, cacl2-rich brine of the perennially ice-covered lake Vanda,
Mcmurdo dry valleys, Antarctica. Appl Environ Microbiol 81(6):1988–1995
Trollope D (1975) The preservation of bacteria and fungi on anhydrous silica gel: an assessment of
survival over four years. J Appl Microbiol 38(2):115–120
Tsudome M, Deguchi S, Tsujii K, Ito S, Horikoshi K (2009) Versatile solidified nanofibrous
cellulose-containing media for growth of extremophiles. Appl Environ Microbiol 75(13):
4616–4619
1 Isolation, Culture, and Maintenance of Extremophilic Fungi 31

Valente T, Silva D, Gomes P, Fernandes M, Santos J, Sencadas V (2016) Effect of sterilization
methods on electrospun poly (lactic acid)(pla) fiber alignment for biomedical applications. ACS
Appl Mater Interfaces 8(5):3241–3249
Vanderwolf KJ, Malloch D, McAlpine DF (2019) No change detected in culturable fungal
assemblages on cave walls in Eastern Canada with the introduction of Pseudogymnoascus
destructans. Diversity 11(12):222
Verma S, Lindsay D, Grigg M, Dubey J (2017) Isolation, culture and cryopreservation of
Sarcocystis species. Curr Protoc Microbiol 45(1):20D.1.21–20D. 1.27
Waheeda K, Shyam K (2017) Formulation of novel surface sterilization method and culture media
for the isolation of endophytic actinomycetes from medicinal plants and its antibacterial activity.
J Plant Pathol Microbiol 8(399):2
Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J Pharm
203(1–2):1–60
Wang X-M, Zhang J, Wu L-H, Zhao Y-L, Li T, Li J-Q, Wang Y-Z, Liu H-G (2014) A mini-review
of chemical composition and nutritional value of edible wild-grown mushroom from China.
Food Chem 151:279–285
Wang M, Tian J, Xiang M, Liu X (2017) Living strategy of cold-adapted fungi with the reference to
several representative species. Mycology 8(3):178–188. https://doi.org/10.1080/21501203.
2017.1370429
Wang Z, Zhuang H, Wang M, Pierce NE (2019) Thitarodes shambalaensis sp. Nov. (Lepidoptera,
hepialidae): a new host of the caterpillar fungus Ophiocordyceps sinensis supported by genome-
wide SNP data. Zoo Keys 885:89
Wei W, Mo C, Guohua C (2012) Issues in freeze drying of aqueous solutions. Chin J Chem Eng
20(3):551–559
Weitzman I, Silva-Hutner M (1967) Non-keratinous agar media as substrates for the ascigerous
state in certain members of the gymnoascaceae pathogenic for man and animals. Sabouraudia
5(4):335–340
Widyana ED, Tarsikah N, Naimah N (2019) The effectiveness of rose flower (Rosa chinensis jacq)
on Candida albicans colonies in jelly (sabouraud dextrose agar) media. Public Health of
Indonesia 5(1):8–13
Windels CE, Burnes PM, Kommedahl T (1988) Five-year preservation of Fusarium species on
silica gel and soil. Phytopathology 78(1):107–109
Yanwisetpakdee B, Akbar A, Lotrakul P, Prasongsuk S, Punnapayak H, Ali I (2014) Purification,
characterization, and potential of saline waste water remediation of a polyextremophilic alpha-
amylase from an obligate halophilic Aspergillus gracilis. Biomed Res Int 2014:106937
Yegin S (2017) Single-step purification and characterization of an extreme halophilic, ethanol
tolerant and acidophilic xylanase from Aureobasidium pullulans nrrl y-2311-1 with application
potential in the food industry. Food Chem 221:67–75
Yu R, Shi L, Gu G, Zhou D, You L, Chen M, Qiu G, Zeng W (2014) The shift of microbial
community under the adjustment of initial and processing pH during bioleaching of chalcopyrite
concentrate by moderate thermophiles. Bioresour Technol 162:300–307
Zucconi L, Selbmann L, Buzzini P, Turchetti B, Guglielmin M, Frisvad J, Onofri S (2012)
Searching for eukaryotic life preserved in Antarctic permafrost. Polar Biol 35(5):749–757
32 K. M. Talib et al.

Modern Tools for the Identification
of Fungi, Including Yeasts 2
Ayesha Tahir, Irfana Iqbal, Kalhoro Muhammad Talib, Jing Luhuai,
Xiaoming Chen, Ali Akbar, Anam Asghar, and Imran Ali
Abstract
Fungi is a group of eukaryotic and multicellular heterotrophs with a wide range of
diversity of phenotypic characters. Therefore, the traditional practice of
identifying fungi solely based on morphological characters is incomplete and
outdated. With the advancement of molecular biology and bioinformatics, molec-
ular data surge, i.e., DNA and proteins based on which fungal species can be
identified. This chapter discusses DNA barcoding techniques using ITS, nuclear
ribosomal subunits, protein-coding genes, secondary DNA markers, and DNA
taxonomy to identify fungal species and their placement across different taxo-
nomic levels. In addition to the list of curated molecular databases, this chapter
provides a step-wise procedure for identifying extremophilic fungi.
Keywords
DNA barcoding · DNA taxonomy · Identification of extremophilic fungi
A. Tahir · I. Iqbal · A. Asghar
Department of Zoology, Lahore College for Women University, Lahore, Pakistan
K. M. Talib · J. Luhuai · X. Chen
School of Life Science and Engineering, Southwest University of Science and Technology,
Mianyang, Sichuan, China
A. Akbar
Faculty of Life Sciences, University of Balochistan, Quetta, Pakistan
I. Ali (*)
School of Life Science and Engineering, Southwest University of Science and Technology,
Mianyang, Sichuan, China
Faculty of Life Sciences, University of Balochistan, Quetta, Pakistan
Plant Biomass Utilization Research Unit, Botany Department, Chulalongkorn University, Bangkok,
Thailand
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_2
33

2.1 Introduction
Identification of fungi primarily depends on phenotypic characteristics such as mode
of reproduction (sexual/asexual) to form spore-producing structures, mycelial
growth pattern, and morphology of hyphae (Chethana et al. 2021). Using
morphology, fungal identification is crucial for understanding fungal evolution and
ontological studies at higher taxonomic levels, i.e., order and family levels (Aguilar-
Trigueros et al. 2015). Still, it provides insufficient knowledge at lower classifica-
tion, i.e., species level (Branco 2019). Morphological characterization of fungi is
problematic even for trained mycologists due to plasticity of characters (sporulation,
asexual structures, shape/size of spores) (Raja et al. 2017b), cryptic speciation
(Lücking et al. 2014; Sepúlveda et al. 2017), convergent evolution (Shang et al.
2016) and species hybridization (Brasier 2000). The International Code of Nomen-
clature does not accept this practice as trustworthy in fungal taxonomy (Hibbett and
Taylor 2013). As a result, there is much progress in using DNA sequence-based
methods, i.e., DNA barcoding, to identify the much-diversified group of fungi
(Hibbett et al. 2011; Insumran et al. 2021).
For sequence-based identification of fungus, two techniques are in use: DNA
barcoding and DNA taxonomies. DNA barcoding was developed to identify animals
at the species stage by using a short-standardized region of DNA (400–800 bp long)
(Kress and Erickson 2012). The concept of DNA barcoding states that variance
between species (interspecific differences) would be higher than variations among
the species (intraspecific variation). The difference between interspecific and intra-
specific variation is called the barcode gap, which is the actual measure for fungal
identification. DNA barcoding amplifies the ITS region of unknown fungi and
compares it to standard sequence databases to identify species based on similarity.
The European Nucleotide Sequencing Archive of the European Molecular Biology
Laboratory (EMBL) and DNA Data Bank of Japan (DDBJ); International Sequence
Database (INSD); GenBank at the National Center for Biotechnology Information
GenBank, NCBI, and UNITE (User-friendly Nordic ITS Ectomycorrhiza Database
are the main reference for sequences used to identify species (Batista et al. 2020).
DNA taxonomy studies the sequence alignments among two or more genes and
assesses the phylogenetic relationships by estimating the evolutionary relationship
between homologous sequences. However, the rapid evolution of DNA barcodes has
resulted in high divergence at lower taxonomic levels (species and genus); hence
could not be used in phylogenetic reconstruction at higher levels (familial and
ordinal) (Raja et al. 2017b).
2.2 Morphological Identification of Extremophilic Fungi
The isolated strains of extremophilic fungi are inoculated on medium plates, and the
colony culture characteristics are observed under the corresponding extreme envi-
ronment (Akbar et al. 2014). The slides can be cultured according to the method of
34 A. Tahir et al.

(Chung et al. 2019). The specific operation method is as follows: firstly, the colony
edge of the sterilized perforator is cut. The portion of the fungi can be placed on the
glass slide with a diameter of 50 mL on the glass slide. It is cultured in the
corresponding extreme environment for 4–7 days (Ahirwar et al. 2017). A sterile
absorbent cotton ball can be placed in the centrifuge tube to prevent the strain block
from losing water too fast. After the mycelium grows well, another clean slide can be
taken, dropping a drop of sterile water, taking off the cover glass of the mycelium,
and covering it. Then observing, photographing, and measuring the morphological
characteristics of the mycelium, conidia, and conidia under the microscope and
consulting the relevant data for identification. If one wants to make a permanent
slide, the emulsion phenol oil or the lactic acid phenol cotton blue dye can be used as
the floating carrier.
The morphology of yeast cells is observed after culture grown in YM and YMA
media and incubated at 30 C for 3 days (Kreger-van Rij 2013). Morphological
analysis cannot be ruled out from modern systematics, although fungal systematics
has stepped into an era of phylogenetic analysis. For preliminary identification of
pathogenic yeasts, their growth on CHROMagar medium is also examined. Prepared
CHROMagar Candida medium is used to prepare semisolid medium, and colonies
are patched on the medium. Various pathogenic yeasts showed the same
characteristics color of colonies after 5 days (Kurtzman et al. 2011; Nadeem et al.
2010).
2.3 Biochemical/Physiological Characterization
2.3.1 Physiological Characterization
According to Bridge (1985), growth and colonies morphology are investigated in the
influence of 0.032% sodium selenite, 0.005 or 0.001% crystal violet, or 0.05 or
0.001% copper sulfate. The tests were developed for sporulation in a liquid medium
containing glucose, citric acid, ethanol, ammonium oxalate, lactic acid, or ammo-
nium tartrate as the only source of carbon, as well as aesculin and gelatin hydrolysis.
After 30 days, growth at 4 C is assessed in malt extracted agar (MEA), and heat
tolerance of conidial aggregates is determined after a 5-min incubation at 75 C
(Bridge 1985).
Physiological characterization is essential for yeast identification. Yeasts are
mainly cultivated in a lower limit medium supplemented with a carbon or nitrogen
component as the only carbon or nitrogen resources to determine their capacity to
metabolize the spectrum of carbon or nitrogen substances. The growth of ascomy-
cetous yeasts in the influence of cycloheximide is also investigated (Hutzler et al.
2015; Kurtzman et al. 2011). Yeasts are also examined for their ability to use certain
carbon compounds anaerobically. This test is performed in an especially designed
durham tube and using the basic medium containing yeast extract 0.5% (w/v) and
test sugar—50 mM to the final concentration. Inoculated tubes are incubated at
25 C on a rotary shaker at 150 rpm for 10 days. The formation of an air column in
2 Modern Tools for the Identification of Fungi, Including Yeasts 35

the inner tube indicates the ability of yeast cells to utilize the carbon compound
anaerobically. For Basidiomycetous yeasts, positive response in starch formation,
urea hydrolysis, and DBB (Diazonium blue B) tests are considered important
diagnostic characters and are performed in small capped vials (Barnett et al. 1990;
Lloyd 2000).
2.3.1.1 Sporulation Test
All the six yeast isolates were tested for their ability to form spore on yeast malt
extract (YM) agar, PDA, 2% malt agar, 5% malt agar, corn meal agar, and
McClary’s acetate agar media, the incubation conditions being temperature 20 C
and 25 C and time 1 month. The inoculum used was cells from single (pure) or all
six (mixed) cultures. Since all the six isolates turned out to be the same based on
morphological and physiological data, one isolate, HG112, was finally selected for
further analysis.
2.4 Biochemical Characterization
Potato dextrose agar (PDA) is utilized to culture all single-spore isolates for 3 days at
25 C, mostly in darkness (Chung et al. 2019). Agar media blocks are taken from the
colonies’borders and in 250-mL Erlenmeyer flasks, employed as inocula of GYM
medium (Mugnai and Bridge 1989)at25C, without shaking, collected during
9 days, cleaned with sterile water, and centrifuged at 2000 rpm 3 g for 10 min at
4C. The resulting mycelial mass is clasped and grinded with a pestle before having
shaken for 5 min with 0.45-mm-diameter ballotini glass particles in a cold potter
containing 1.5 mL of sterile water. Light microscopy is performed to examine cell
disruption. The cell homogenates were moved to microcentrifuge tubes employing a
Pasteur pipette and centrifuged at 13,000 3 g for 2 min at 4 C. 0.45-mm-pore-size
The resulting precipitate has been filtered utilizing a Sterivex H-A filter (Millipore,
Bedford, Mass.) and saved in micro aliquots at 20 C awaiting isozyme investiga-
tion. Materials for electrophoresis were developed at a maximum concentration of
100 mg protein (Bradford 1976) via suspension 30 ml of mycelial isolate throughout
10 ml of 0.5 M Tris-HCl, pH 6.8, incorporating 1.2 mL of 0.5% bromophenol blue
as identifying dye in glycerol (1:4 dilution). Enzymes are isolated using the tech-
nique outlined. Procedures were applied to synthesize enzyme staining mixtures
(Paterson and Bridge 1994) and Shaw and Prasad (Shaw and Prasad 1970). Isoen-
zyme patterns are determined for alkaline phosphatase (ALP) (EC 3.1.3.1), catalase
(CAT) (EC 1.11.1.6), acid phosphatase (ACP) (EC 3.1.3.2), malate
dehydrogenase (MDH) (EC 1.1.1.37), alcohol esterase (EST) (EC 3.1.1.1) and
dehydrogenase (ADH) (EC 1.1.1.1) activities. Cellulase (1,4-(1,3:1,4)-b-D-glucan-
4-glucanohydrolase) (EC 3.2.1.4) is detectable via developing an overlying gel of
1% agarose in 0.05 M acetic acid-acetate buffer, pH 5.2, over a gel bond membrane,
afterward staining using Congo red according to Mateos et al. Isoenzymes are
implemented to determine well-resolved bands across all single-spore isolated for
yeast’s identification (Mateos et al. 1992).
36 A. Tahir et al.

2.5 Molecular Tools for Fungal Identification
2.5.1 Ribosomal Genes and ITS Region
Innis et al. (2012) described the fungal nuclear ribosomal operon primers two
decades ago after which the practice of using molecular data for the identification
of fungi flourished. These primers amplified three fungal DNA sequences; nuclear
ribosomal large subunit (nrLSU-26S or 28S), nuclear ribosomal smaller subunit
(nrSSU-18S), and the whole internal transcribed spacer region (ITS1, 5.8S, ITS2;
ca. 0.45–0.80 kb) that are being used in molecular phylogenetic sequence identifica-
tion of fungi (Bruns et al. 1991; Seifert et al. 1995). Various levels of evolution have
produced the slowest development in the SSU area and the quickest growth in the
ITS domain (Mitchell and Zuccaro 2006). As a result, the SSU region may be
transcribed and sequenced utilizing primers NS1 and NS4 for phylogenetic placing
at high taxonomic categories, such as family, order, class, and phylum (Innis et al.
2012). The LSU area may be amplified and sequencing employing the specific
primers such as LROR and LR6 for identifying and placing at intermediary taxo-
nomic stages, i.e., family and genus (Rehner and Samuels 1995; Vilgalys and Hester
1990). The hypervariable regions of LSU areas D1 and D2 individually (Liu et al.
2012) or combined with the ITS section are incredibly effective in fungal classifica-
tion (Porras-Alfaro et al. 2014; Raja et al. 2017a; Schoch et al. 2009; Seifert 2009).
Due to the fastest evolving portion of the rRNA cistron, the ITS region is suitable for
fungal identification at the species level. ITS region is the official barcode for fungi
due to its widespread use, easy amplification, and large barcode gap (Kõljalg et al.
2013). For species-level identification, the ITS region can also be used in combina-
tion with protein-coding genes.
2.6 Protein-Coding Genes in Conjunction with ITS Region
Because of the existence of intron sections, protein-coding genes are used to
determine species through bar codes, particularly, when compared to ITS, occasion-
ally progresses at a quicker pace (Tkacz and Lange 2004), and were utilized
phylogenetically because of to having an excellent resolution at higher taxonomic
rates compared to rRNA genes (Schoch et al. 2009). Additionally, these genes are
supposed to survive as a separate copy in fungi, are far less varying in lengths,
leading for easy identification of homology and convergence, as they accrue minimal
mutations within their exons, and are simple and easy to align than rRNA genes
since they contain little ambiguity related to codon regulations (Berbee and Taylor
2001; Einax and Voigt 2003). Protein-coding genes detected in fungal systematics
had also been widely utilized in the formation of molecular phylogenetic analysis to
identify a range and categorization of species, due mainly to the National Science
Foundation’s seminal research in systematic and taxonomy mycology, such as
Assembling the Fungal Tree of Life (Blackwell et al. 2006; Hibbett et al. 2007;
James et al. 2006; Lutzoni et al. 2004). Such attempts by fungal systematists
2 Modern Tools for the Identification of Fungi, Including Yeasts 37

contribute to our understanding of developmental interactions between fungi
(McLaughlin et al. 2009) and pave the way for a more reliable categorization of
such fungal kingdoms (Hibbett et al. 2007). Among the protein-coding markers (Liu
and Hall 2004; Matheny et al. 2002; Reeb et al. 2004; Stiller and Hall 1997), beta-
tubulin (tub2/BenA) (Glass and Donaldson 1995;O’Donnell and Cigelnik 1997) and
transforming growth factor 1-alpha (tef1), the highest (RPB1) and second-highest
(RPB2) RNA polymerase subunits would be most commonly implemented to
establish phylogenetic connections within fungi (James et al. 2006). Moreover, the
mini-chromosome management protein (MCM7) shows potential as a newly devel-
oped marker, implying higher and lower-level phylogenetic interactions (Gillot et al.
2015; Hustad and Miller 2015; Morgenstern et al. 2012; Raja et al. 2011; Schmitt
and Barker 2009). Compared to several extensively adopted protein-coding markers,
the MCM7 area provides a preferable gene for phylogenetic analysis (Aguileta et al.
2008). Furthermore, it functions effectively when combined alongside the large
subunit (LSU) gene, as displayed by Ascomycota members (Raja et al. 2011).
2.7 Secondary Barcode Marker
To obtain a more effective determination at the species stage (e.g., Aspergillus,
Penicillium, and Trichoderma), this could be required for the users to sequence one
or more single-copy protein-coding genes for specific fungal genera and/or lineages
as compared to utilizing the Interior transcriptional spacer (ITS) marker only for
identifying, which may not be sufficient in such fungal clades. Because of the
limitations of a single-marker barcoding technique in fungi, another community of
mycologists recently completed an experiment evaluating >1500 Dikarya
(Ascomycota and Basidiomycota) species (1931 strains or specimens) with various
ribosomal and single-copy protein-coding markers (Stielow et al. 2015). The study
concluded that an innovative, high-fidelity primer couple for tef-1 (EF1–1018F
GAYTTCATCAAGAACATGAT and EF1–1620R
GACGTTGAADCCRACRTTGTC) must have the most potent ability to facilitate
as a supplementary DNA barcode, providing superlative resolution to ITS (Stielow
et al. 2015), that has been already extensively employed as a phylogenetic marker in
mycology (Rehner and Buckley 2005).
The genetic sections tub2/BenA, RPB1, RPB2, and incomplete calmodulin
(CaM) are particularly favorable enabling species detection in specific lineages of
fungus such as Eurotiales, including Penicillium and Aspergillus, among of the
highly abundant genera of fungi for additional compounds, that contain several
therapeutic activities and industrial applications valuable species (Houbraken et al.
2011; Houbraken and Samson 2011; Samson et al. 2011,2014). Tub2/BenA has
been suggested as a supplementary barcoding marker for Penicillium sp. Barcoding
(Samson et al. 2014). For phylogenetic studies of Penicillium sp., RPB2 or CaM
genes are suggested. They are generally simpler to align than tub2/BenA, which
would be challenging to align primarily to the existence of many ambiguous areas on
Penicillium (Samson et al. 2014). The CaM gene is proposed for an additional
38 A. Tahir et al.

barcoding marker, whereas the tub2/BenA and RPB2 genes seem strongly suggested
for Aspergillus (Samson et al. 2014). The user is guided to Samson et al. for the
priming sequences with advance and reversal primers of genes utilized in segments
for Aspergillus and Penicillium for PCR procedures (Samson et al. 2014).
Although Trichoderma sp. having significant consequences in living creature and
wildlife health, industries, agriculture, and the ecosystem (Mukherjee et al. 2013;
Schuster and Schmoll 2010), recognizing Trichoderma is vital to natural products
investigation, particularly when applying for a position on peptaibols (Neumann
et al. 2015). Therefore, Trichoderma determination employing the ITS area could be
problematic because it lacks sufficient diversity to differentiate between species. The
ITS region can be used for BLAST search for this genus using the TrichoKeys
database, which is hosted on the ISHT webpage (Table 2.1) but provides information
only on species clusters. Hence, for species-level identification, the tef-1 region has
been widely used in the systematics and taxonomy of this genus (Degenkolb et al.
2008; Druzhinina et al. 2005; Lu et al. 2004; Montoya et al. 2016). However, using
tef-1 intron four in conjunction with intron five is extremely helpful for species
verification (Jaklitsch and Voglmayr 2015; Nagy et al. 2007). Furthermore, for
Trichoderma identification, a 1200 bp portion of RPB2 may be transcribed, and
sequencing via primer pairing RPB2–5f and RPB2–7cR (Liu et al. 1999)is
indicating advantages of applying phylogenetic techniques.
2.8 D1/D2 Domain and Identification of Yeasts
In the past, two gene regions have been used for the identification of
C. membranifaciens (Yamadazyma) phylogenetic clade species, namely the D1/D2
domains of the large subunit (LSU) and the nearly complete small subunit (SSU)
nrRNA gene (Sung-Oui et al. 2005). Recently Kurtzman et al. (2011) described the
genus Yamadazyma phylogenetically, employing the D1/D2 and almost entire SSU
sections and introducing an unexpected 11 CoQ-9-forming Candida species as a
component of this teleomorphic clade. Yamadazyma had also been classified as a
member of the Debaryomycetaceae family, according to Kurtzman et al. (2011). A
total of 5 samples extracted from plants in French Guiana and Thailand (four and one
strains, respectively) were described for this research based on physiological, mor-
phological, and phylogenetic characteristics and determined to categorize three
novel varieties of Yamadazyma. Including isolated, the ITS and D1/D2 domains
of the 26S nrDNA have indeed been identified. Because the ITS region was still only
publically available for a couple of the type strains of previously investigated
Yamadazyma species, additional strains were sequenced to evaluate the variance
across the ITS and the D1/D2 regions between the related species.
Except for CBS 8535T, the novel species’ITS and D1/D2 sequences do not
match any known yeast species in GenBank or the CBS yeast sequence database.
Their sequences matched those of an unidentified Candida species (CBS 9930).
S.H. identified a variant from the CBS yeast resource from Scheffleraoctophylla
leaves and placed it in the CBS stock cultures in 2004. Yang. The combination of
2 Modern Tools for the Identification of Fungi, Including Yeasts 39

Table 2.1 List of curated molecular databases
Name of Database URL Molecular markers Scope
Barcode of life database, BOLD http://www.boldsystems.org/
index.php/IDS_
OpenIdEngine
ITS
CBS-KNAW www.cbs.knaw.nl ITS Aspergillus and
PenicilliumDermatophytesFusariumindoor
fungimedical
fungiPhaeoacremoniumPseudallescheria/
ScedosporiumResupinateRussulalesRussulayeasts
FUSARIUM-ID http://isolate.fusariumdb.org ITS, tef1,RPB1,RPB2,
tub2
Fusarium
Fungal barcoding http://www.fungalbarcoding.
org
ITS
Fungal MLST database Q-Bank http://www.q-bank.eu/Fungi/ Partial actin, tub2,RPB1.
RPB2,tef1 among others
Quarantine organisms
ISHAM http://its.mycologylab.org ITS Human and animal pathogens
Naïve Bayesian Classifier http://rdp.cme.msu.edu/
classifier/classifier.jsp
28S, ITS
RefSeq Target Loci (RTL) http://www.ncbi.nlm.nih.
gov/refseq/targetedloci/
ITS, 18S, 28S Mainly sequences from type material,
re-annotated from INSDC
International Subcommision on Hypocrea
and Trichoderma (ISHT) TrichoKey and
TrichoBLAST (Trichoderma)
http://www.isth.info/tools/
blast/
ITS and tef1,RPB2
UNITE, user-friendly Nordic ITS
Ectomycorrhiza database
https://unite.ut.ee/ ITS Wide taxonomic range, re-annotated from INSDC
Adapted from the work of Robert et al. (2015) and Rebecca et al. (2016)
40 A. Tahir et al.

D1/D2 and ITS sequence alignments (placed in TreeBASE) that included 40 types
such as the group and out-group sequence used to have a length of 1120 characters,
of which these 130 were phylogenetically unspecific, 704 seemed stable, and
286 have been relevant (Groenewald et al. 2011).
Even though sequence analysis gave a more significant percentage of identifica-
tion than both phenotypic procedures for the D1/D2 or ITS2 regions, each of these
techniques had limitations. Sequence analysis of the ITS2 and D1/D2 sections
detected just 79 percent and 87 percent of the isolated species, respectively. The
identification rates for both the ITS and D1/D2 areas were lower than previously
reported for human diagnostic isolates (Hall et al. 2003; Leaw et al. 2006; Pryce et al.
2003; Putignani et al. 2008).
2.9 Curated Molecular Databases for Fungal Sequences
Various curated molecular databases for numerous fungal groupings had been
developed to identify ITS segments, ribosome subunits, and protein-coding
sequences (Paterson and Lima 2015). Table 2.1 provides a comprehensive overview
of selected molecular libraries, their URLs, molecular markers, and fungal sequenc-
ing reportage. It is based on the research of Robert et al. (2015).
2.10 DNA Taxonomic Studies for Phylogenetic Trees
Construction from DNA Sequences
DNA taxonomy, like DNA barcoding, is founded on the concept of categorizing taxa
by utilizing genetic diversity contained in sequences from different people. The
distinction between these two approaches is that an uncertain is identified depending
on a phylogenetic assumption (Tautz et al. 2003), promoting an evolutionary
viewpoint for detecting fungal species and employing a predicted strategy (Tkacz
and Lange 2004). Considering a specified fungus group, DNA classification could
depend on one or more protein-coding segments of rDNA. It may be obtained by
combining phylogenetic techniques with any gene region (Dissanayake et al. 2020).
Although DNA-based categorization relying on the phylogenetic concept can defi-
nitely assist via putting an alien species into a phylogenetic clade or group, thereby
giving a possible species identity, this could not consistently help identify a specific
genus of a fungus. Schmitt and Barker recently published a technique and software
description that helped do studies that inferred or used phylogenetic trees (Schmitt
and Barker 2009). Using sequences from known species with shared ancestors
(homologous sequences) might anticipate species characteristics like ecological
and metabolic (Tkacz and Lange 2004). Taylor and Fisher (2003) developed the
Genealogical Concordance Phylogenetic Species Recognition (GCPSR) concept to
define the boundaries of sexual species utilizing shared housekeeping genes based
2 Modern Tools for the Identification of Fungi, Including Yeasts 41

on the phylogenetic concordance of numerous unlinked genomic loci. Considering it
possesses more discriminating strength over other species ideas, like morphological
and biological species categories, the GCPSR concept is considerably quite helpful
in detecting fungus (Harrington and Rizzo 1999). A single gene marker can also be
exploited to enable broad species confirmation, while GCPSR needs two or even
more genetic loci to identify species (Tkacz and Lange 2004). Once the ribosomal
genes have supported a taxonomic category, including a genus or family, nuclear
protein-coding genes may be applied through phylogenetic experiments and/or
BLAST searches to determine further convincing species.
2.11 Protocol in Brief
2.11.1 DNA Extraction
DNA barcoding and DNA categorization are based on the concept of utilizing
intrinsic genetic diversity between distinct individual sequences to assess
classifications (Chung et al. 2019). A specific group of fungi’s DNA taxonomy
could be defined on protein-coding one or even other rDNA sections. It may be
produced via plant genetic techniques by adopting any gene section alone or
combination (Schoch et al. 2012). Whereas DNA-based categorization derived
from plant genetics hypothesis does not always facilitate accurately identify species
of fungus, it does help place undetectable species in plant genetics or groups,
allowing a specific species verification (Raja et al. 2017b). Mycelial powdered was
utilized, which is produced via crushing a tiny part of the fungal colony in liquid
nitrogen utilizing a pestle and mortar. The Doyle & Doyle technique may be used to
isolate DNA (Spadoni et al. 2019). Specific steps are: taking 10–50 mg of the
scraped fresh mycelium into a 1.5 mL centrifuge tube, adding sterilized quartz
sand and 0.01 g PVP, and first adding 100 μL Cetyltrimethylammonium Bromide
(CTAB) buffer solution of 65 C is grounded into homogenate with a sterilized
ground glass rod, and then 500 μL of the same CTAB buffer solution is added for
several times to make it evenly mixed (Worasilchai et al. 2018). The mixture is kept
in a 65 C water bath for 0.5–1 h after cooling; 600 μL saturated phenol: chloroform:
isoamyl alcohol (25:24:1) is added to the extraction solution: centrifugation at
12000 rpm for 10 min (Gonzalez-Franco et al. 2017). The supernatant is centrifuged
at 12000 rpm for 10 min again. The supernatant is added with an equal volume of
isopropanol and mixed evenly. The supernatant is placed at room temperature for
10–20 min, centrifuged (12,000 rpm, 15 min), and the supernatant is removed.
100 μL alcohol (75%) is washed two times, and then 75% ethanol is poured. After
drying, 100 μL TE buffer is added. The product is mixed with a 6Loading Buffer
with agarose gel of 0.8% for electrophoresis detection and stored at 20 C (Sandona
et al. 2019). Isolates can be identified with the services of National Center of
Biotechnology Information (NCBI) web by means of BLAST for the matches
amongst the sequences.
42 A. Tahir et al.

2.11.2 PCR Amplification
Metagenomic technologies are reliable tackles that can provide a more wide-ranging
assessment of fungal communities. The amplification of rDNA followed by next-
generation sequencing has given good results in studies aimed particularly at
extreme environments (Baeza et al. 2017). This technique is proved very useful
for easy and swift identification and classification of all the fungal species, rDNA
primers are effectively used for fungal detection and identification in samples
(Mohammad et al. 2017).
1. Primer synthesis: primers ITS1 and ITS4 for amplification and sequencing are
generally synthesized such as following (1–3) (Guo et al. 2020).
ITS1:50—TCCGTAGGTGAACCTGCGG —30
ITS4:50—TCCTCCGCTTATTGATATG —30
2. Amplification reaction system: 40 ul system, adding the following reagents
in turn:
dd H
2
0 30.4 μl
Buffer (10) 4.0 μl
ITSl (10 pmol/μl) 1.2 μl
ITS2 (10 pmol/μl) 1.2 μl
dNTP (2.5 mM) 2.0 μl
Taq (5 U/μl) 0.4 μl
DNA 0.8 μl
3. The amplification conditions are as follows (3–5) (Hashimoto and Kunieda
2017).
After 94 C for 4 min, Enter the following 30 consecutive cycles:
55 C 30 S
72 C 1.5 min
94 C 40 S
72 C 2 min, the last cycle is extended at 72 C for 10 min.
4. Purification of PCR products: 1% amplification of agarose gel is used to detect the
amplified products. The products show a single band with enough concentration
and are further purified.
5. Sequencing and Sequence Analysis
The purified PCR products are sequenced. The primer is used to determine the
positive direction of the fragment reverse primer retest. The reverse sequence is
reverse complementary by bio edit software and connected with the forward
sequence to form a complete sequence, and compared with the related sequence
in Gene Bank (Nicolaus et al. 2016).
2 Modern Tools for the Identification of Fungi, Including Yeasts 43

2.11.2.1 Phylogenetic Analyses
To evaluate protein-coding genes, the individual dataset is aligned employing
MAFFT’s electronic edition employing the E-INS-I option (Katoh et al. 2002).
Mesquite (version 2.74) is used to analyze aligned segments manually, and
adjustments are made (Morgenstern et al. 2012). The original alignment has been
modified to remove gap-rich regions with weak similarity within sequences,
locations represented by just one sequence, and the C- and N-terminal ends have
been trimmed. Protesters use the Akaike Information Criterion (AIC) to find the
most incredible amino acid replacement models independent of the number of
unchanging domains (Abascal et al. 2005). Most probable experiments are carried
out using RAxML 7.2.6, adopting both the quick bootstrapping and fast maximum
likelihood alternatives (Stamatakis 2006). It seems that every dataset’s branching
stability is calculated using 100 bootstrap repetitions. Sequences derived during the
research must be uploaded in GenBank and issued accession numbers.
The phylogeny is rebuilt by maximum likelihood by analyzing the ITS region
using MEGA 5.0 (Tamura et al. 2011). Along with Model Test, the Kimura
two-parameter nucleotide-substitution model is run with uniform rates and partial
deletion (95%) parameters (Posada and Crandall 1998).
2.12 Proteomics of Extremophilic Fungi
Proteomic studies of extremophilic fungal species had extended insignificance
hastily from when the biology of such creatures exposed their extraordinary con-
frontation to severe environmental and ecological situations (Tesei et al. 2019).
Proteomics techniques demonstrated the strength of an investigative attempt to
discover extremophilic fungi and deliver adequate information, predominantly
about quantitative dimensions of their cell biology. To identify the fungal proteome,
two primary proteomic techniques are used: lotion separating procedure combined
using mass spectrometry (MS) and shotgun (gel-free) methodology (Wasinger et al.
2013). A proteome comprehends the full information related to proteins that may be
produced, their profusion, disparities, alterations, interactions, and linkages.
Proteomic quantities are accomplished through a grouping of extraordinarily pro-
found equipment and the application of potent computational procedures to provide
interpretation of cellular pathways and procedures in a given cell (Pérez-Llano et al.
2020). On this principle, proteomics of extremophilic and extremotolerant fungi
allows recognizing them by the changes in structure and stability of proteins,
biologically dynamic during extreme conditions (Moreno et al. 2018).
Virtual proteomic describing approaches such as standard, Two-Dimensional
Difference Gel Electrophoresis (2D-E and 2D-DIGE) to label proteins and the
more latest shotgun proteomics methods are helpful to investigate response
mechanisms to various stress like temperatures, salinity, pH, dryness, Mars-like
environments (Blachowicz et al. 2019), and host–pathogen interaction
(Seyedmousavi et al. 2013). The combined use of diverse proteomics processes
and bioinformatics investigations provides advanced knowledge about protein
44 A. Tahir et al.

characteristics, functionalities, and cellular pathways during the survival of such
organisms (Gómez-Silva et al. 2019). The accessibility of whole-genome sequences
and annotation play a vital role in the exploration of mechanisms of adaptation.
Comparative genomics has demonstrated that extremophiles overall own a fantastic
set of genes and proteins that allow them with the natural ability to thrive in extreme
situations (Kumar et al. 2018).
Matrix-assisted laser desorption/ionization (MALDI) time of flight (MALDI-
TOF) is the latest proteomic technique used to identify microbes (Blättel et al.
2013; Moothoo-Padayachie et al. 2013). In this mass spectrometric technique, the
ion source is matrix-assisted laser desorption/ionization (MALDI), and the mass
analyzer is a time-of-flight (TOF) analyzer. The Sabouraud dextrose agar’inoculated
strains for 24 h (48 h if the colonies are not present or when the purity cannot be
verified after 24 h) at 30 C are transferred into 1.5- mL screws cap tubes and mixed
methodically in 0.3 mL of double-distilled ultrapure water. Pure ethanol (0.9 mL) is
added to the tubes, and they are centrifuged at 13,000 g for 2 min after vortexing.
The supernatant is castoff, and the pellet is mixed thoroughly with 50 μL of 70%
aqueous formic acid. The mixture is centrifuged at 13,000 g for 2 min after adding
50 μL of acetonitrile. A polished steel MALDI target plate (Bruker Daltonics) is used
to place one microliter of the microorganism extract supernatant in duplicate and
allowed it to dry at room temperature. Two microliters of a matrix solution are used
to overlay each sample consisting of saturated α-cyano-4-hydroxycinnamic acid in
50% acetonitrile–2.5% trifluoroacetic acid, and the plate is air-dried at room temper-
ature. The analysis is performed after the plate is loaded into MALDI-TOF mass
spectrometer. The spectra was automatically recorded in the linear positive ion mode
within a mass range from 4000 to 10,000 Da with delayed extraction at a laser
frequency of 20 Hz. Spectra are suitable for more analysis when the peaks have a
resolution better than 400 intensity in arbitrary units. Each run includes a bacterial
test standard with a characteristic protein and peptide profile, Bruker’s for calibra-
tion, a negative extraction control, and the reference QC strains. The same methods
were repeated using new colonies for failures (Yaman et al. 2012).
2.12.1 Identification of Extremophilic Fungi Using Molecular Tools
Extremophilic fungi show diversity in ecological, metabolism, morphological and
phylogenetic characteristics, especially their bioactive molecules that catch the
interests of scientists to discover novel chemicals for pharmaceutical, agricultural
and industrial applications (Zhang 2016). Despite the significance of extremophilic
fungi, their identification still vestiges an intimidating challenge for chemists,
particularly without coordination of mycologists (Chamekh et al. 2019). Currently,
there are numerous problems concerning identifying fungus just by morphology at
the species and strain levels; therefore, morphological and molecular data are
utilized for this objective. Three nuclear ribosomal genes are commonly utilized in
fungal identification. The potential benefits and drawbacks of the internal transcribed
spacer (ITS) region, the authorized DNA barcoding marker for species classification
2 Modern Tools for the Identification of Fungi, Including Yeasts 45

of fungus. The most current method is the use of NCBI-BLAST search for DNA
barcoding, with a cautionary note about its limitations; many systematized molecular
databases containing fungal sequences; several protein-coding genes used to
improve or replace ITS in species-level identification of specific fungus; and
technologies employed to build phylogenetic trees from DNA sequences to allow
identifying (Raja et al. 2017b).
References
Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit models of protein evolution.
Bioinformatics 21:2104–2105
Aguilar-Trigueros CA et al (2015) Branching out: towards a trait-based understanding of fungal
ecology. Fungal Biol Rev 29:34–41
Aguileta G et al (2008) Assessing the performance of single-copy genes for recovering robust
phylogenies. Syst Biol 57:613–627
Ahirwar S, Soni H, Prajapati BP, Kango N (2017) Isolation and screening of thermophilic and
thermotolerant fungi for production of hemicellulases from heated environments. Mycology 8:
125–134
Akbar A, Sitara U, Ali I, Muhammad N, Khan SA (2014) Isolation and characterization of
biotechnologically potent Micrococcus luteus strain from environment. Pak J Zool 46:967–973
Baeza M, Barahona S, Alcaíno J, Cifuentes V (2017) Amplicon-metagenomic analysis of fungi
from Antarctic terrestrial habitats. Front Microbiol 8:2235
Barnett JA, Payne RW, Yarrow D (1990) Yeasts: characteristics and identification, 2nd edn.
Cambridge University Press, Cambridge, p 1002
Batista TM et al (2020) Whole-genome sequencing of the endemic Antarctic fungus Antarctomyces
pellizariae reveals an ice-binding protein, a scarce set of secondary metabolites gene clusters and
provides insights on Thelebolales phylogeny. Genomics 112:2915–2921
Berbee ML, Taylor JW (2001) Fungal molecular evolution: gene trees and geologic time. In:
Systematics and evolution. Springer, pp 229–245
Blachowicz A et al (2019) Proteomic and metabolomic characteristics of extremophilic fungi under
simulated mars conditions. Front Microbiol 10:1013
Blackwell M, Hibbett DS, Taylor JW, Spatafora JW (2006) Research coordination networks: a
phylogeny for kingdom Fungi (Deep Hypha). Mycologia 98:829–837
Blättel V, Petri A, Rabenstein A, Kuever J, König H (2013) Differentiation of species of the genus
saccharomyces using biomolecular fingerprinting methods. Appl Microbiol Biotechnol 97:
4597–4606
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Branco S (2019) Fungal diversity from communities to genes. Fungal Biol Rev 33:225–237
Brasier C (2000) The rise of the hybrid fungi. Nature 405:134–135
Bridge PD (1985) An evaluation of some physiological and biochemical methods as an aid to the
characterization of species of Penicillium subsection Fasciculata. Microbiology 131:1887–1895
Bruns TD, White TJ, Taylor JW (1991) Fungal molecular systematics. Annu Rev Ecol Syst 22:525–
564
Chamekh R, Deniel F, Donot C, Jany J-L, Nodet P, Belabid L (2019) Isolation, identification and
enzymatic activity of halotolerant and halophilic fungi from the Great Sebkha of Oran in
Northwestern of Algeria. Mycobiology 47:230–241
Chethana K et al (2021) What are fungal species and how to delineate them? Fungal Divers 109:1–
25
Chung D, Kim H, Choi HS (2019) Fungi in salterns. J Microbiol 57:717–724
46 A. Tahir et al.

Degenkolb T et al (2008) The Trichoderma brevicompactum clade: a separate lineage with new
species, new peptaibiotics, and mycotoxins. Mycol Prog 7:177–219
Dissanayake A, Bhunjun C, Maharachchikumbura S, Liu J (2020) Applied aspects of methods to
infer phylogenetic relationships amongst fungi. Mycosphere 11:2652–2676
Druzhinina IS, Kopchinskiy AG, KomońM, Bissett J, Szakacs G, Kubicek CP (2005) An
oligonucleotide barcode for species identification in Trichoderma and Hypocrea. Fungal
Genet Biol 42:813–828
Einax E, Voigt K (2003) Oligonucleotide primers for the universal amplification of β-tubulin genes
facilitate phylogenetic analyses in the regnum. Fungi Organ Diver Evol 3:185–194
Gillot G et al (2015) Insights into Penicillium roqueforti morphological and genetic diversity. PLoS
One 10:e0129849
Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to
amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–
1330
Gómez-Silva B et al (2019) Metagenomics of atacama lithobiontic extremophile life unveils
highlights on fungal communities, biogeochemical cycles and carbohydrate-active enzymes.
Microorganisms 7:619
Gonzalez-Franco AC, Robles-Hernandez L, Strap JL (2017) Chitinase, chitosanase, and antifungal
activities from thermophilic streptomycetes isolated from compost Phyton. Int J Exp Bot 86:14–
27
Groenewald M, Robert V, Smith MT (2011) The value of the D1/D2 and internal transcribed
spacers (ITS) domains for the identification of yeast species belonging to the genus
Yamadazyma. Persoonia: Mol Phylog Evolut Fungi 26:40
Guo R, Zhao L, Zhang K, Gao D, Yang C (2020) Genome of extreme halophyte Puccinellia
tenuiflora. BMC Genomics 21:1–7
Hall L, Wohlfiel S, Roberts GD (2003) Experience with the MicroSeq D2 large-subunit ribosomal
DNA sequencing kit for identification of commonly encountered, clinically important yeast
species. J Clin Microbiol 41:5099–5102
Harrington TC, Rizzo DM (1999) Defining species in the fungi. In: Structure and dynamics of
fungal populations. Springer, pp 43–71
Hashimoto T, Kunieda T (2017) DNA protection protein, a novel mechanism of radiation tolerance:
lessons from tardigrades. Life 7:26
Hibbett DS, Taylor JW (2013) Fungal systematics: is a new age of enlightenment at hand? Nat Rev
Microbiol 11:129–133
Hibbett DS et al (2007) A higher-level phylogenetic classification of the fungi. Mycol Res 111:509–
547
Hibbett DS, Ohman A, Glotzer D, Nuhn M, Kirk P, Nilsson RH (2011) Progress in molecular and
morphological taxon discovery in fungi and options for formal classification of environmental
sequences. Fungal Biol Rev 25:38–47
Houbraken J, Samson R (2011) Phylogeny of Penicillium and the segregation of Trichocomaceae
into three families. Stud Mycol 70:1–51
Houbraken J, Frisvad JC, Samson R (2011) Taxonomy of penicillium section citrina. Stud Mycol
70:53–138
Hustad VP, Miller AN (2015) Studies in the genus Glutinoglossum. Mycologia 107:647–657
Hutzler M, Koob J, Riedl R, Schneiderbanger H, Mueller-Auffermann K, Jacob F (2015) Yeast
identification and characterization. In: Brewing microbiology. Elsevier, pp 65–104
Innis MA, Gelfand DH, Sninsky JJ, White TJ (2012) PCR protocols: a guide to methods and
applications. Academic press
Insumran Y, Jansawang N, Sriwongsa J, Reanruangrit P, Auyapho M (2021) Diversity and genetic
marker for species identification of edible mushrooms. Prawarun Agricult J 13:253–267
Jaklitsch W, Voglmayr H (2015) Biodiversity of Trichoderma (Hypocreaceae) in southern Europe
and Macaronesia. Stud Mycol 80:1–87
2 Modern Tools for the Identification of Fungi, Including Yeasts 47

James TY et al (2006) Reconstructing the early evolution of fungi using a six-gene phylogeny.
Nature 443:818–822
Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple
sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066
Kõljalg U et al (2013) Towards a unified paradigm for sequence-based identification of fungi. Wiley
Online Library
Kreger-van Rij NJW (2013) The yeasts: a taxonomic study. Elsevier
Kress WJ, Erickson DL (2012) DNA barcodes: methods and protocols. In: DNA barcodes.
Springer, pp 3–8
Kumar A et al (2018) Protein adaptations in extremophiles: an insight into extremophilic connec-
tion of mycobacterial proteome. In: Seminars in cell & developmental biology. Elsevier, pp
147–157
Kurtzman CP, Fell JW, Boekhout T (2011) The yeasts: a taxonomic study. Elsevier
Leaw SN, Chang HC, Sun HF, Barton R, Bouchara J-P, Chang TC (2006) Identification of
medically important yeast species by sequence analysis of the internal transcribed spacer
regions. J Clin Microbiol 44:693–699
Liu YJ, Hall BD (2004) Body plan evolution of ascomycetes, as inferred from an RNA polymerase
II phylogeny. Proc Natl Acad Sci 101:4507–4512
Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: evidence from
an RNA polymerse II subunit. Mol Biol Evol 16:1799–1808
Liu K-L, Porras-Alfaro A, Kuske CR, Eichorst SA, Xie G (2012) Accurate, rapid taxonomic
classification of fungal large-subunit rRNA genes. Appl Environ Microbiol 78:1523–1533
Lloyd D (2000) Yeasts characteristics and identification. J Eukaryot Microbiol 47:598–598
Lu B, Druzhinina IS, Fallah P, Chaverri P, Gradinger C, Kubicek CP, Samuels GJ (2004) Hypocrea/
Trichoderma species with pachybasium-like conidiophores: teleomorphs for T. minutisporum
and T. polysporum and their newly discovered relatives. Mycologia 96:310–342
Lücking R et al (2014) A single macrolichen constitutes hundreds of unrecognized species. Proc
Natl Acad Sci 111:11091–11096
Lutzoni F et al (2004) Assembling the fungal tree of life: progress, classification, and evolution of
subcellular traits. Am J Bot 91:1446–1480
Mateos P et al (1992) Cell-associated pectinolytic and cellulolytic enzymes in rhizobium
leguminosarum biovar trifolii. Appl Environ Microbiol 58:1816–1822
Matheny PB, Liu YJ, Ammirati JF, Hall BD (2002) Using RPB1 sequences to improve phyloge-
netic inference among mushrooms (Inocybe, Agaricales). Am J Bot 89:688–698
McLaughlin DJ, Hibbett DS, Lutzoni F, Spatafora JW, Vilgalys R (2009) The search for the fungal
tree of life. Trends Microbiol 17:488–497
Mitchell JI, Zuccaro A (2006) Sequences, the environment and fungi. Mycologist 20:62–74
Mohammad BT, Al Daghistani HI, Jaouani A, Abdel-Latif S, Kennes C (2017) Isolation and
characterization of thermophilic bacteria from Jordanian hot springs: bacillus licheniformis
and Thermomonas hydrothermalis isolates as potential producers of thermostable enzymes.
Int J Microbiol 2017:6943952
Montoya QV, Meirelles LA, Chaverri P, Rodrigues A (2016) Unraveling Trichoderma species in
the attine ant environment: description of three new taxa. Antonie Van Leeuwenhoek 109:633–
651
Moothoo-Padayachie A, Kandappa HR, Krishna SBN, Maier T, Govender P (2013) Biotyping
Saccharomyces cerevisiae strains using matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry (MALDI-TOF MS). Eur Food Res Technol 236:351–364
Moreno LF, Vicente VA, De Hoog S (2018) Black yeasts in the omics era: achievements and
challenges. Med Mycol 56:S32–S41
Morgenstern I et al (2012) A molecular phylogeny of thermophilic fungi. Fungal Biol 116:489–502
Mugnai L, Bridge PD, Evans HC (1989) A chemotaxonomic evaluation of the genus Beauveria.
Mycol Res 92:199–209
48 A. Tahir et al.

Mukherjee PK, Horwitz BA, Singh US, Mukherjee M, Schmoll M (2013) Trichoderma: biology
and applications. CABI
Nadeem SG, Hakim ST, Kazmi SU (2010) Use of CHROMagar Candida for the presumptive
identification of Candida species directly from clinical specimens in resource-limited settings.
Libyan J Med 5
Nagy V, Seidl V, Szakacs G, Komoń-Zelazowska M, Kubicek CP, Druzhinina IS (2007) Applica-
tion of DNA bar codes for screening of industrially important fungi: the haplotype of
Trichoderma harzianum sensu stricto indicates superior chitinase formation. Appl Environ
Microbiol 73:7048–7058
Neumann NK, Stoppacher N, Zeilinger S, Degenkolb T, Brückner H, Schuhmacher R (2015) The
peptaibiotics database–a comprehensive online resource. Chem Biodivers 12:743–751
Nicolaus B et al (2016) Pb2+ effects on growth, lipids, and protein and DNA profiles of the
thermophilic bacterium Thermus thermophilus. Microorganisms 4:45
O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a mono-
phyletic lineage of the fungusfusariumare nonorthologous. Mol Phylogenet Evol 7:103–116
Paterson R, Bridge P (1994) Biochemical techniques for filamentous fungi, vol 1. CaB International
Paterson RRM, Lima N (2015) Molecular biology of food and water borne mycotoxigenic and
mycotic fungi. CRC Press
Pérez-Llano Y, Soler HP, Olivano AR, Folch-Mallol JL, Cabana H, Batista-García RA (2020)
Laccases from extremophiles. In: Laccases in bioremediation and waste valorisation. Springer,
pp 213–238
Porras-Alfaro A, Liu K-L, Kuske CR, Xie G (2014) From genus to phylum: large-subunit and
internal transcribed spacer rRNA operon regions show similar classification accuracies
influenced by database composition. Appl Environ Microbiol 80:829–840
Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics
(Oxford, England) 14:817–818
Pryce T, Palladino S, Kay I, Coombs G (2003) Rapid identification of fungi by sequencing the ITS1
and ITS2 regions using an automated capillary electrophoresis system. Med Mycol 41:369–381
Putignani L, Paglia MG, Bordi E, Nebuloso E, Pucillo LP, Visca P (2008) Identification of
clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the
25–28S rRNA gene. Mycoses 51:209–227
Raja H, Schoch CL, Hustad V, Shearer C, Miller A (2011) Testing the phylogenetic utility of
MCM7 in the Ascomycota. MycoKeys 1:63
Raja HA, Baker TR, Little JG, Oberlies NH (2017a) DNA barcoding for identification of consumer-
relevant mushrooms: a partial solution for product certification? Food Chem 214:383–392
Raja HA, Miller AN, Pearce CJ, Oberlies NH (2017b) Fungal identification using molecular tools: a
primer for the natural products research community. J Nat Prod 80:756–770
Rebecca Y, Schoch CL, Dentinger BTM (2016) Scaling up discovery of hidden diversity in fungi:
impacts of barcoding approaches. Philos Trans R Soc B: Biol Sci 371(1702):20150336. https://
doi.org/10.1098/rstb.2015.0336
Reeb V, Lutzoni F, Roux C (2004) Contribution of RPB2 to multilocus phylogenetic studies of the
euascomycetes (Pezizomycotina, fungi) with special emphasis on the lichen-forming
Acarosporaceae and evolution of polyspory. Mol Phylogenet Evol 32:1036–1060
Rehner SA, Buckley E (2005) A Beauveria phylogeny inferred from nuclear ITS and EF1-α
sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia
97:84–98
Rehner SA, Samuels GJ (1995) Molecular systematics of the Hypocreales: a teleomorph gene
phylogeny and the status of their anamorphs. Can J Bot 73:816–823
Robert V, Cardinali G, Stielow B, Vu TD, Borges dos Santos F, Meyer W et al (2015) Fungal DNA
barcoding. In: Paterson RM, Lima N (eds) Metabolomics of food- and waterborne fungal
pathogens. Routledge & CRC Press
Samson R et al (2011) Phylogeny and nomenclature of the genus Talaromyces and taxa
accommodated in Penicillium subgenus Biverticillium. Stud Mycol 70:159–183
2 Modern Tools for the Identification of Fungi, Including Yeasts 49

Samson RA et al (2014) Phylogeny, identification and nomenclature of the genus Aspergillus. Stud
Mycol 78:141–173
Sandona K, Billingsley Tobias TL, Hutchinson MI, Natvig DO, Porras-Alfaro A (2019) Diversity
of thermophilic and thermotolerant fungi in corn grain. Mycologia 111:719–729
Schmitt I, Barker FK (2009) Phylogenetic methods in natural product research. Nat Prod Rep 26:
1585–1602
Schoch CL et al (2009) The Ascomycota tree of life: a phylum-wide phylogeny clarifies the origin
and evolution of fundamental reproductive and ecological traits. Syst Biol 58:224–239
Schoch CL et al (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal
DNA barcode marker for fungi. Proc Natl Acad Sci 109:6241–6246
Schuster A, Schmoll M (2010) Biology and biotechnology of Trichoderma. Appl Microbiol
Biotechnol 87:787–799
Seifert KA (2009) Progress towards DNA barcoding of fungi. Mol Ecol Resour 9:83–89
Seifert KA, Wingfield BD, Wingfield MJ (1995) A critique of DNA sequence analysis in the
taxonomy of filamentous ascomycetes and ascomycetous anamorphs. Can J Bot 73:760–767
Sepúlveda VE, Márquez R, Turissini DA, Goldman WE, Matute DR (2017) Genome sequences
reveal cryptic speciation in the human pathogen Histoplasma capsulatum. MBio 8:e01339–
e01317
Seyedmousavi S, Guillot J, De Hoog G (2013) Phaeohyphomycoses, emerging opportunistic
diseases in animals. Clin Microbiol Rev 26:19–35
Shang Y, Xiao G, Zheng P, Cen K, Zhan S, Wang C (2016) Divergent and convergent evolution of
fungal pathogenicity. Genome Biol Evol 8:1374–1387
Shaw CR, Prasad R (1970) Starch gel electrophoresis of enzymes—a compilation of recipes.
Biochem Genet 4:297–320
Spadoni A et al (2019) A simple and rapid method for genomic DNA extraction and microsatellite
analysis in tree plants journal of agricultural. Sci Technol 21:1215–1226
Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with
thousands of taxa and mixed models. Bioinformatics 22:2688–2690
Stielow JB et al (2015) One feungus, which genes? Development and assessment of universal
primers for potential secondary fungal DNA barcodes. Persoonia Mol Phyl Evolut Fungi 35:242
Stiller JW, Hall BD (1997) The origin of red algae: implications for plastid evolution. Proc Natl
Acad Sci 94:4520–4525
Sung-Oui S, Nguyen NH, Blackwell M (2005) Nine new Candida species near C. membranifaciens
isolated from insects. Mycol Res 109:1045–1056
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular
evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum
parsimony methods. Mol Biol Evol 28:2731–2739
Tautz D, Arctander P, Minelli A, Thomas RH, Vogler AP (2003) A plea for DNA taxonomy.
Trends Ecol Evol 18:70–74
Taylor JW, Fisher MC (2003) Fungal multilocus sequence typing—it’s not just for bacteria. Curr
Opin Microbiol 6:351–356
Tesei D, Sterflinger K, Marzban G (2019) Global proteomics of extremophilic fungi: mission
accomplished? In: Fungi in extreme environments: ecological role and biotechnological signifi-
cance. Springer, pp 205–249
Tkacz JS, Lange L (2004) Advances in fungal biotechnology for industry, agriculture, and
medicine. Springer
Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified
ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238–4246
50 A. Tahir et al.

Wasinger VC, Zeng M, Yau Y (2013) Current status and advances in quantitative proteomic mass
spectrometry. Int J Proteom 2013:180605
Worasilchai N, Chaumpluk P, Chakrabarti A, Chindamporn A (2018) Differential diagnosis for
pythiosis using thermophilic helicase DNA amplification and restriction fragment length poly-
morphism (tHDA-RFLP). Med Mycol 56:216–224
Yaman G, Akyar I, Can S (2012) Evaluation of the MALDI TOF-MS method for identification of
Candida strains isolated from blood cultures. Diagn Microbiol Infect Dis 73:65–67
Zhang S-H (2016) The genetic basis of abiotic stress resistance in extremophilic fungi: the genes
cloning and application. In: Fungal applications in sustainable environmental biotechnology.
Springer, pp 29–42
2 Modern Tools for the Identification of Fungi, Including Yeasts 51

Part II
Eco-physiology

Major Habitats and Diversity
of Thermophilic Fungi 3
Swapnil Chaturvedi and Indira P. Sarethy
Abstract
More than 80% of the earth surface has extreme environmental conditions, not
conducive for normal life, as assessed on anthropogenic parameters. Along with
the discovery of novel bacteria and archaea from various extreme environments,
it has been found that fungi also are capable of colonizing such areas. These fungi
have been shown to have diverse phylogenetic characteristics, belonging to many
genera. Conditions of extremes of pH, temperature, pressure, and radiation
necessitate special strategies for survival, involving genetic changes, which can
result in novel natural products. Thermophilic fungi occurrence is generally in
soil or in habitats where decomposition of plant material such as grains, compost,
husk, municipal refuse, and other organic material takes place, under humid and
aerobic environment conditions. In these habitats, thermophiles occur as either
resting propagules or as active mycelia depending on the nutrients and environ-
mental condition. The occurrence of thermophilic fungi is due to the dissemina-
tion of propagules from masses of organic material. Thermophilic fungi belong to
various genera such as Zygomycetes,Ascomycetes, and Deuteromycetes.
Metabolites from these organisms, not found in normal habitats, can be of good
value. Based on the biological activity and structure of compounds isolated from
such extremophilic fungi, more than 150 compounds have been documented from
thermophilic species of Penicillium,Aspergillus, and others. The current chapter
focuses on the major habitats of thermophilic fungi, their ecology, physiology,
and molecular biology, elicitation of specialized metabolites from them, and their
various documented activities.
S. Chaturvedi · I. P. Sarethy (*)
Department of Biotechnology, Jaypee Institute of Information Technology, Noida, Uttar Pradesh,
India
e-mail: indirap.sarethy@jiit.ac.in
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_3
55

Keywords
Thermophilic fungi · Extremophilic fungi · Metabolites · Metagenomics
3.1 Introduction
Thermophilic fungi are eukaryotic organisms, which generally grow at temperatures
of 60–62 C. According to Cooney and Emerson (1964), thermophiles are divided
into three categories: thermophiles grow at above 45 C, extreme thermophiles
66–79 C, and hyperthermophiles 80 C (Maria et al. 2016). Thermophilic fungi
grow at above 45 C and above, and the minimum temperature for growth is 20 C
(Oliveria et al. 2015). They occur in soil or in habitats where decomposing plant
materials such as grains, compost, husk, municipal refuse, and other organic material
are present. In these habitats, thermophiles occur as either resting propagules or as
active mycelia depend on the nutrients and the environmental conditions. These
fungi are spread by the dissemination of propagules from masses of organic material.
They belong to genera such as Zygomycetes,Ascomycetes, and Deuteromycetes.
They have a role in the production of thermostable enzymes and are used as
components of recombinant organisms for the production of proteases, pectinases,
and cellulases (Surrough et al. 2012). Thermophilic fungi can be cultivated in the
laboratory on different media such as yeast-starch agar (YpSs), oatmeal agar (OA),
and Czapek’s agar (C3) as expounded by Cooney and Emerson (1964). Themomyces
stellatus can grow at above 45 C (Morgenstern et al. 2012). These fungi contain
saturated fatty acids (Maheshwari et al. 2000) and can survive in stress conditions
such as high water pressure and desiccation (Mahajan et al. 1986). Thermophilic
fungi have an important role to play in environment as they can degrade organic
matter, act as biodeteriorants by producing intracellular and extracellular enzymes,
phenolic compounds, polysaccharides, antibiotics, and can also serve as single-cell
protein (SCP). Single-cell proteins can act as bioconversion agents, for instance such
as in preparation of mushroom compost. Chaetomium and Pulverulentum produce
SCP from lignocellulosic wastes. Chaetomium thermophile and Humicola
lanuginose compost are rich in different minerals such as sodium, potassium, and
phosphorus. Thermophilic activities of microbes are associated with the thermosta-
bility of enzymes and proteins. Advantages of using these thermostable enzymes and
protein are reduction in contamination risk of mesophilic microbes in industrial
processes and products; as the viscosity of culture medium decreases, solubility of
organic compounds increases and as the coefficient of diffusion of reactant and
product increases, the rate of reaction becomes higher.
56 S. Chaturvedi and I. P. Sarethy

3.2 Types
Thermophilic fungi grow at temperatures between 25 and 80 C, which is associated
with biological conditions. According to Cooney and Emerson (1964), the maxi-
mum temperature for their growth is above 50 C and minimum 25 C.
Based on various studies, some terms are given to thermophilic fungi to further
categorize them:
1. Thermophilic: Fungi that grow at or above 45 C (Maheshwari et al. 2000).
2. Thermotolerant: Those that grow best at a maximum of 50 C and minimum of
20 C (Mouchacca 2000).
3. Thermophilius fungi: They include both thermophilic and thermotolerant fungi
(Apinis 1963).
4. Thermoduric fungi: Fungi whose reproductive structures can resist temperature of
80 C or above, but normal growth is at 22–25 C (Apinis and Pugh 1967).
5. Transitional thermophile:Fungi that grow below 20 C but can survive at
temperature up to 40 C (Apinis and Pugh 1967).
6. Stenothermal: Those that grow at a narrow temperature range mostly found in a
habitat with a constant temperature range (Brock and Fred 1982).
7. Eurythermal: Fungi that grow at a wider temperature range mostly found in the
habitat where temperature fluctuates (Brock and Fred 1982).
3.3 History
Earlier studies on thermophilic fungi were more focused on their occurrence in
various habitats. The first thermophilic hyphomycetes, Thermomyces lanuginosus,
was reported from potato and described by Tsiklinskaya (1899). Four thermophilic
fungi, Thermoidium sulphureum,Mucor pusillus,Thermoascus aurantiacus, and
Thermomyces lanuginosus, are based on the growth of thermophilic fungi to the
thermogenesis of agriculture stock by Miehe (1907). Later he also studied the
thermophilic and mesophilic fungi to check the maximum temperature for their
growth. Led Kurt Noack (1920) isolated thermophilic fungi from different natural
substrates and observed that thermophilic fungi are present in habitats with varying
temperatures. Allen and Emerson (1949) isolated many thermophilic fungi, which
were able to grow at 60 C. They found that thermophilic microflora in retting
guayule decreased the amount of resin in the extract of crude rubber, which resulted
in improved physical properties. In 1950, La Touche discovered new cellulolytic
ascomycetes, which had industrial applications. Notable publications on thermo-
philic fungi during the sixties and seventies included ones by Apinis (1963), Eggins
and Malik (1969) for temperate climate soil, and Hedger (1974) and Gochenaur
(1975) for tropical regions. Morphology of thermophilic fungi is based on their
biological activities, which help in the discovery of industrial application. Approxi-
mately 75 types of thermophilic fungi species have been discovered till now,
3 Major Habitats and Diversity of Thermophilic Fungi 57

comprising around 0.1% of the total fungal species, from compost, soil, desert soil,
wood husk, organic material and coal, and many more.
3.4 Habitats
Several extreme conditions are found in the environment such as large or small
temperature ranges, acidity, radiation, and drought conditions; few organisms are
able to survive in these conditions, with a vast majority unable to adapt to such
variations. Thermophilic fungi prefer composts, snuff, municipal waste, wood husk,
grains, and other such organic material where the preferred humidity and aerobic
environmental conditions are present for their growth and development. Figure 3.1
shows various habitats from where thermophilic fungi have been isolated. Table 3.1
shows that the earliest studies of thermophilic fungi, which have shown these to be
Fig. 3.1 Habitats from where thermophilic fungi have been isolated
58 S. Chaturvedi and I. P. Sarethy

Table 3.1 Origin of thermophilic fungi and their habitats
Class Organism Habitat Reference
Ascomycetes Canariomyces
thermophile
Soil von Arx et al.
(1988)
Chaetomium
britannicum
Mushroom compost, soil Ames (1963),
Chen and Chen
(1996)
Chaetomidium
pingtungium
Sugarcane field
Decomposing leaves
Ames (1963)
Chaetomium
virginicum
Plant remains, seeds of Capsicum
annuum, soil
Ames (1963)
Chaetomium
senegalensis
Soil, decomposing wheat straw Ames (1963)
Corynascus
sepedonium
Soil, pasture soil, hay, coal spoil
tips, compost
von Arx (1975)
Coonemeria
crustacean
Coal spoil tips, bagasse, soil von Arx (1975)
Thielavia
australiensis
Nesting material of malleefowl Tansey and
Jack (1975)
Talaromyces
byssochlamydoides
Forest soil Guarro et al.
(1996)
Zygomycetes Rhizomucor
pusillus
Municipal wastes, horse dung,
composted wheat straw, guayule,
hay, seeds of cacao, barley, maize
and wheat, groundnuts
Schipper
(1978)
Rhizomucor miehei Soil, sand, coal mines, hay, stored
barley, compost
Cooney and
Emerson
(1964)
Eurotiomycetes Paecilomyces
themophila
Wheat straw Yang et al. (200
6)
Malbranchea
cinnamomea
Compost, soil Maijala et al.
(2012)
Sporotrichum
thermophile
Soil Sadaf and
Khare (2014)
Deuteromycetes Scytalidium
thermophilum
Compost, soil Narain et al.
(1983)
Scytalidium
thermophilum
Compost Robledo et al.
(2015)
Acremonium
thermophilum
Sugarcane bagasse Van Oorschot
(1977)
Chrysosporium
tropicum
Dung, soil, air Gams and
Lacey (1972),
Carmichael
(1962)
Acremonium
alabamense
Alluvial soil Malloch and
Cain (1973)
3 Major Habitats and Diversity of Thermophilic Fungi 59

widely present in decomposing organic matter or hot springs, are still the habitats
from where these continue to be isolated. In these habitats, thermophilic fungi may
occur as resting or active mycelia depending upon the favorable conditions of the
environment.
In 1939, Waksman and his team isolated thermophilic fungi from soil. As seen
from Table 3.1, thermophilic fungi have been isolated from a wide variety of
materials. Thermophilic fungi grow easily in temperate countries but not in tropical
countries. Their occurrence is due to self-heating masses of organic material
(Maheshwari et al. 1987). A thermophilic fungus was found in Australia in temper-
ate soil region (Ellis and Keane 1981). Thirty-two thermophilic and thermotolerant
fungi were found in coal soil with well-organized colonies (Evans 1971). Pine
hardwood pine scrub (Ward Jr and Cowley 1972) were found to be good sources
too. Approximately 75 types of thermophilic fungi species have been discovered till
now, comprising around 0.1% of the total fungal species, from compost, soil, desert
soil, wood husk, and organic material land coal.
3.4.1 Natural Habitats
In these habitats, where high temperature conditions prevail round the year, thermo-
philic fungi have been obtained using culture-dependent techniques. Some of the
characteristics of these habitats and the fungi isolated are discussed in this section.
3.4.1.1 Hot Springs
This is a type of thermal spring generated from geothermally heated water. Water
flows from hot spring and is heated either by geothermal process or when water
flows from the hot rock surface. On the other hand, in volcanic areas, water gets
heated on coming in contact with magma. Boiling water, which builds up steam
pressure, which comes out in the form of jet on the earth surface, is called a geyser.
Countries from which fungi have been documented from such hot springs are
Canada, New Zealand, Japan, Chile, Hungary, Israel, India, and Fiji. Hot spring
water contains many minerals, which provide the growth of microbiota. These hot
spring thermal areas are niches with some unique qualities, which can be explored
for biotechnological purposes. Isolation and characterization of thermophilic fungi
from hot spring thermal regions have been done by several researchers in the past
decades (Sharma et al. 2012). Thermophilic fungi are present in a larger population
in the hot spring region of Indonesia, but all of them are not able to grow under the
hot spring volcanic conditions and is restricted to some species. Five types of
thermophilic fungi were isolated from WU-Rai of northern Taiwan: Humicola
insolens,Rhizoctonia,Aspergillus fumigatus,Penicillium dupontii, and
Thermomyces lanuginosus, all of which could grow between 55 and 65 C (Chen
et al. 2000). A research of the mycoflora in Yangmingshan National Park, northern
Taiwan, from August 1999 to June 2000, of thermophilic and thermotolerant fungi
inhabiting sulfurous hot spring soils, identified 12 taxa (Chen et al. 2003). Further
four thermophilic fungi species were reported from Xiaoyoukeng sulfurous area:
60 S. Chaturvedi and I. P. Sarethy

Sporotrichum sp., Chrysosporium sp., Scytalidium thermophilum,andPapulaspora
thermophile. Pan et al. (2010) isolated thermophilic fungi from geothermal sites with
alkalescent hot spring in Tengchong Rehai National Park, China and utilized the ITS
region Internal transcribed spacer) sequencing system to classify fungi. More than a
hundred fungal strains were isolated such as Talaromyces byssochlamydoides,
Rhizomucor miehei,Thermoascus aurantiacus,Talaromyces thermophiles,
Thermomyces lanuginosus,Scytalidium thermophilum, and Coprinopsis species.
3.4.1.2 Soil
Soil is the upper land surface on earth, which is the mixture of all the organic
material present with minerals and other components, which provide support for the
growth of the organism. In soil, various microorganisms provide nutritious condition
to plant and animal species. Thermophilic fungi grow in soil; however, it depends on
the nature of the soil. According to researchers, such fungi are present in the upper
layer of the soil or the debris of the plant on the soil; Rajasekaran and Maheshwari
(1993) estimated the respiratory rates of thermophilic and mesophilic fungi. They
discovered that the respiratory rate of thermophilic organisms was especially recep-
tive to changes in temperature; however, that of mesophilic fungi was generally free
of such changes, suggesting that in thermally fluctuating conditions thermophilic
fungi might be at a physiological setback in contrast to mesophilic organisms. Their
work shows that thermophilic fungi are inactive components of soil microflora.
Different types of species were isolated from south-central Indiana soil, a
sun-heated soil: Myriococcum albomyces,Aspergillus fumigatus,Talaromyces
thermophilus,Humicola lanuginosa,Allescheria terrestris,Malbranchea pulchella
var. sulfurea,Thielavia heterothallica,Mucor pusillus,Chaetomium thermophile
var. dissitum,Thielavia minor,Thermoascus aurantiacus,Mucor miehei,Torula
thermophila,Humicola stellata,Acrophialophora nainana,Thielavia sepedonium,
Dactylomyces thermophilus,Talaromyces emersonii (Tansey and Jack 1976),
Malbranchea cinnamomea (Maijala et al. 2012), and Sporotrichum thermophile,
(Lu et al. 2013).
Ten species of thermophilic fungi was identified from different zones of Darjee-
ling in eastern Himalayan soil. It was observed that there was a decline in the
pervasiveness of thermophiles with expanding altitudes (Sandhu and Singh 1981).
Later, thermophilic and thermotolerant fungi were isolated from 77 regions of Iraq.
Out of these, six were true species of thermophilic fungi, while the rest were
thermotolerant. Aspergillus terreus,A. fumigatus,and A. niger were available with
frequencies of the event of 70%, 68%, and 60%, individually, and thus the investi-
gation revealed that thermophilic and thermotolerant fungi are widely present
mycoflora of Iraq soils (Abdullah and Al-Bader 1990).
3.4.1.3 Desert Soil
Desert soil has the amount of precipitation that falls based on temperature or due to
geographical location. Around one-third land surface of the earth is arid and semi-
arid. Researchers have isolated thermophilic fungi from the desert where the tem-
perature is up to 55 C or above. However, water is required for the growth, even
3 Major Habitats and Diversity of Thermophilic Fungi 61

when spores are present in dry soil. Abdel-Hafez (1982) studied the desert soil of
Saudi Arabia for thermophilic fungi and isolated 48 species of thermophilic fungi
from 24 genera on different laboratory media such as Czapek’s agar media and
containing cellulose or glucose. The organisms isolated were A. fumigatus,
Humicola grisea var,Aspergillus nidulans, and C. thermophile var. copropile.
Further, it was reported that 16 species were reported: Mucor pusillus,Talaromyces,
Thielavia,Myxococcus,Stilbella thermophile,A. fumigatus,C. thermophile var.
copropile,C. thermophile var. dissitum,Chaetomium virginicum,Torula thermo-
phile,Malbranchia pulchella var. sulfurea,Malbranchia pulchella var. sulfurea,
Sporotrichum pulverulentum,Talaromyces thermophilus,Myriococcum albomyces,
Allescheria terrestris,Papulaspora thermophile,Sporotrichum pulverulentum, and
Humicola lanuginose. In Middle East region (Egypt, Iraq, Syria, and Kuwait)
thermophilic and thermotolerant fungi from desert soil were isolated from which
Malbranchea cinnamomea,Scytalidium thermophilum,Myceliophthora thermo-
phile, and Thermomyces lanuginosus were obtained by Mouchacca (1995). Ther-
mophilic fungi from the Thar desert of India were isolated such as Aspergillus flavus,
A. niger,A. terreus,A.versicolor,Chaetomium,Emericella,Emericellanidulans,
Fusarium,Chlamydosporum,Penicillium,Scytalidium,Thanatephorus cucumeris,
Cunninghamella,Eurotium,Mucor,Chrysogenum,Talaromyces,Alternaria
alternata, and Rhizopus stolonifer (Sharma et al. 2010). Eight thermophilic fungi
were identified from desert area of Yard province: Ulocladium,Fusarium,Penicil-
lium,Aspergillus,Alternaria,Rhizopus,Stemphylium, and Paecilomyces (Rafiei and
Banihashemi 2019).
3.4.1.4 Coal Mine Soil
Thermophilic fungi were isolated from coal mine soil of Chandameta, Parasia, from
Chhindwara District of Madhya Pradesh, India. The normal temperature and precip-
itation rate of this region is 21 C and 64 cm. A total of 14 fungi species were
isolated: Achaetomium macrosporum,Emericella nidulans,Rhizopus
rhizopodiformis,Absidia corymbifera,Thermomyces lanuginosus,Thielavia
minor,Humicola grisea,Aspergillus fumigatus,Thermoascus aurantiacus,Torula
thermophile,Rhizopus microsporus,Penicillium sp., Sporotrichum sp., and Asper-
gillus fumigatus (Johri and Thakre 1975). Coal mine near Hazaribagh Jharkhand
exhibited the presence of thermophilic and thermotolerant fungi such as
Chrysosporium tropicum,Melanocarpus albomyces,Chaetomium piluliferum,
Chaetomium thermophile,Penicillium chrysogenum,Aspergillus fumigatus,
Curvularia lunata, and Cladosporium spp. (Tulsiyan et al. 2017).
3.4.1.5 Coal Soil Tips
Coal soil tip is waste accumulated together in one place. Thermophilic fungi can
grow on coal soil tip easily isolated thermophilic fungi from coal soil tip and
observed that thermophilic fungi grow in these habitats due to its high temperature,
organic material waste, and lack of soil crumbs. More than 30 thermophilic fungi
species from different genera were isolated: Aspergillus, Penicillium, Rhizopus,
Mucor, Chrysosporium, Acrophialophora, Aspergillus, Calcarisporium,
62 S. Chaturvedi and I. P. Sarethy

Chrysosporium, Geotrichum, Penicillium, Scolecobasidium, and Talaromyces
(Evans 1971).
3.4.2 Man-Made Habitats
Man-made habitats are largely those created and lived in by human beings. These
types of habitats having effluents, thermal insulation system, stored grains, and
compost piles provide a suitable environment for growth of thermophilic fungi.
Heating in these environments can be by natural means (such as the heat generated
during decomposition) or provided artificially by solar heating or other self-heating
systems in which temperature can rise to 70 C.
3.4.2.1 Manure
It is an organic material that is obtained from human, animal, and plant residues.
They contain nutrients in organic material form (Larney and Hao 2007). Agricultural
processes require a high demand for manure, but as manure has directly come in
contact with agricultural land, it may cause many adverse effects on soil, water, and
nutrient leaching. Manure compost provides better nourishment to the product as
they are rich in nutritious value. Holman et al. (2016) have isolated thermophilic
fungi such as Thermomyces lanuginosus and Remersonia thermophile.
3.4.2.2 Municipal Waste
Municipal waste is the waste material that has lignocellulosic material and some
inorganic waste material. According to a study by Kaiser et al. (1968), municipal
corporations generate more than 50% paper waste. Stutzenberger and their group
(1970) studied the microorganisms that could degrade lignocellulosic materials. In
municipal waste, the temperature can rise to 60 C, suitable for growth of thermo-
philic fungi. Kane and Mullins (1973) isolated Torula thermophile,Humicola
lanuginosa,Mucor pusillus,Aspergillus fumigatus,Chaetomium thermophilum,
and Thermoascus aurantiacus. Sen et al. (1979) studied thermophilic fungi such
as Mucor and Humicole, which were rich in nutrients such as nitrogen, potassium,
and phosphorus. From different parts of India, various representatives such as
Myceliophthora thermophile and Thermo mucor sp. have been isolated.
Thermotolerant fungi such as Cladosporium and Absidial spp. were isolated from
municipal waste in Iran (Ghazifard et al. 2001).
3.4.2.3 Wood Chip Piles
Wood chip piles are strong pieces of wood made by cutting or chipping parts of
freshly harvested wood and are commonly utilized for the production of wood mash
and as a crude material for specialized wood handling. Brown et al. (1994) found that
these wood chip pile consists more than 50% weight of water due to which bacteria
can grow easily by a fermentation process. Tansey (1971) studied that as the
temperature rises in these stored wood chip piles, an ignition process starts due to
which growth of thermophilic fungi takes place, and this causes deterioration in
3 Major Habitats and Diversity of Thermophilic Fungi 63

wood quality. He also isolated thermophilic fungi from wood chip piles obtained
from paper factory: Humicola lanuginose, Chaetomium thermophile var. dissitum,
Talaromyces emersonii, Chaetomium thermophile var. coprophile,Talaromyces
thermophiles, and Sporotrichum thermophile.
3.4.2.4 Hay
Hay is related to grass, vegetables, or different herbaceous plants that have been cut,
dried, and put away as bundles for later use as grain, especially for nibbling
creatures. The hay bundles are stacked one over another, and the moisture present
in these lead to the growth of microorganisms. Miehe (1907) isolated Thermoascus
aurantiacus,Rhizomucor pusillus, and Thermomyces lanuginosus from a self-
heating haystack.After inoculating hay and other organic material with a pure
culture of fungi, he observed that sterilized hay does not produce heat, whereas the
inoculated fungus does generate heat. Gregory and Lacey (1963) found that a
different type of microflora develops in haystack, which has some moisture content.
They demonstrated that hay having a 16% moisture content heated up moderately,
while that having 25% moisture heated up to 45 C, allowing the development of
thermotolerant molds. But the temperature in wet bales with 45% moisture soared to
60–65 C, and a range of thermophilic fungi, including Aspergillus fumigatus,
Mucor pusillus, and Humicola lanuginose, and some actinomycetes were also
recovered. Thermophilic fungal strains are capable of growing at 50–60 C, at pH
2.0. More than 70 fungal strains belonging to Ascomyota were isolated from
decaying plant matter and compost (Thanh et al. 2019).
3.4.2.5 Stored Grains
Stored grains have been used for centuries to fulfill the requirement for food
necessity. Tansey and Brock (1978) studied that thermophilic fungi, though present
on grains, do not damage grains prior to storage. Stored grains are a good substrate
for the colonization of thermophilic fungi. Clark (1967), Clarke (1969), and Lacey
(1971) discovered various thermophilic fungi from stored grains such as Aspergillus
candidus,Absidia corymbifera,Humicola lanuginosa, and Mucor pusillus. Mulinge
and Apinis (1969) isolated Eurotium amstelodami,Absidia spp., Monascus spp.,
Aspergillus fumigatus,Aspergillus terreus, and Dactylomyces crustaceus from
stored moist barley grains. Hansen and Welty (1970) isolated thermophilic fungi
from cocoa beans. Wareing (1997) isolated the thermophilic and thermotolerant
Aspergillus flavus,Paecilomyces variotii,Rhizomucor pusillus,Thermomyces
lanuginosus, and Thermoascus crustaceous from stored maize grain in
sub-Saharan Africa. Thermomyces,Fumigatus,Dupontii,Thermomyces
lanuginosces,Rhizomucor,and Thermoascus crustaceus were obtained from corn
grain at 52 C (Sandona et al. 2019).
64 S. Chaturvedi and I. P. Sarethy

3.5 Biodiversity of Thermophilic Fungi
3.5.1 Zygomycetes
1. Rhizomucor miehei form colonies with sporangia at 40–45 C. Initially colonies
are white and later turn into gray–brown (Schipper 1978). Spherical sporangia are
30–60 μm in diameter. The zygospore is subspherical and initially reddish-brown,
later turning a black color with a diameter of 30–50 μm. They are found on soil,
coal mines, hay, stored barley, and compost. They have been studied and
geographically found in the United States, India, Ghana, the United Kingdom,
and Saudi Arabia (Salar and Aneja 2007).
2. Rhizomucor nainitalensis produces colonies with sporangiospores and grows at
48 C and is very similar to R. miehei. Sporangiospores are ellipsoidal, dumb bell-
shaped and 3–6μm in diameter (Joshi 1982). They grow predominantly on
decomposed oak log and mainly have been reported in tropical places such as
India (Salar and Aneja 2007).
3. Rhizopus rhizopodiformis produces colonies and grows well at 45 C. Initially
colonies are white and later turn black (Thakre and Johri 1976). They have a
hyphae structure 3–7μm in diameter; sporangia are spherical with a smooth,
black surface and 76–175 μm in diameter and are found in coal mine soils, nesting
material of birds, bread, wooden slats, soil, seeds of Lycopersicon esculentum,
and oil palm effluents. They are found in India, the United Kingdom,
South Africa, China, Hong Kong, Indonesia, Malaysia, and Japan (Salar and
Aneja 2007).
3.5.2 Ascomycetes
1. Canariomyces thermophila colonies grow at 45 C under laboratory conditions
(Von arx et al. 1988). Ascospores are dark colored and irregularly shaped with
spore size 14.0–18.0 7.5–10.0 μm in diameter. They have been largely found in
Africa (Salar and Aneja 2007).
2. Chaetomidium pingtungium have colonies that grow at 45–50 C. They produce
dark globose cleistothecia with thick hairy walls and are 2–5μm wide and 350 μm
long. Asci are cylindrical in shape with a diameter of 40–52 7–9μm and have
an 8-spored structure (Chen and Chen 1996). Ascospores are uniseriate, dark
brown, and are thick-walled structures 8.5–10.0 6.5–8.5 μm diameter. They
have been reported from sugarcane fields of Taiwan (Salar and Aneja 2007).
3. Chaetomium britannicum produces colonies that grow at 45–50 C under labora-
tory conditions (Ames 1963). Ascomata are cylindrical in shape with a hairy
surface. Ascospores are brown in color and irregularly shaped
(19–24 11–14 μm diameter). They have been reported from mushroom com-
post and soil of the United Kingdom (Salar and Aneja 2007).
4. Chaetomium mesopotamicum has colonies that grow at 30–50 C. They are
different from other species (Abdullah and Zora 1993). It produces long and
3 Major Habitats and Diversity of Thermophilic Fungi 65

highly branched structures and has been isolated from date palm in Iraq (Salar and
Aneja 2007).
5. Coonemeria aegyptiaca produces colonies growing at 25–55 C under laboratory
conditions. Ascocarps are a crusty mass with red brown color during the initial
growth phase and have a coiled hyphae structure. Ascospores are single celled
and appear yellowish to pale reddish orange, are thick-walled and smooth.
Conidiophores are produced on aerial hyphae, with a smooth-walled surface,
and diameter of 50–300 5–7μm with apical parts irregularly branched.
Phialides are solitary or irregularly verticillate and cylindric structure, with
diameter size 12–30 3–6μm. They are usually found in soil and have been
reported from Egypt and Iraq (Ueda and Udagawa) Mouchacca 1997; Salar and
Aneja 2007).
6. Melanocarpus thermophilus has eight-spored asci (Guarro et al. 1996), with
ascospores being ovoid, dark brown, 7.5–9.0 6.0–7.5 μm, and each provided
with a single germ pore. This isolate has been reported from forest soil of Iraq
(Salar and Aneja 2007).
3.5.3 Deuteromycetes
1. Arthrinium pterospermum colonies have been shown to grow at 37 C. They have
hyphae like structure 2 μm in diameter (Apinis 1963). Conidiogenous cells are
colorless during the initial phase of growth and later become dark brown with a
diameter size of 7.6 5.3 μm. These have been described from hay and soil in the
USA (Salar and Aneja 2007).
2. Myceliophthora thermophile produces colonies at 45 C. Colonies have floccose
surface or granular (Van Oorschot 1977). Initially, they have white colored
colonies, which subsequently turn brown with a diameter of 2 μm. These have
been reported to grow in soil from various countries with diverse climatic
conditions such as Canada, India, Japan, and Australia (Salar and Aneja 2007),
3. Papulospora thermophila colonies have two types of mycelia: those with an
arrow diameter (1.5 μm) and those with a wider diameter (5–7μm) and with
highly branched structures (Fergus 1971). Bulbils are present on hyphae in the
form of globes to subglobose with diameter of 105 90 μm. These hyphae have
an irregular shape, and initially, they are yellow later orange in color. These have
been reported from mushroom compost and soil in India and Japan (Salar and
Aneja 2007).
4. Scytalidium indonesicum colonies grow at 45 C on laboratory media. Conidia
are brown with barrel-shaped to ellipsoid shape; they have a thick wall surface
with a diameter size 25–12 μm (Hedger et al. 1982). Once conidia are fully grown
they form irregular shaped brown structures with thick wall. These have been
reported from Indonesia (Salar and Aneja 2007).
66 S. Chaturvedi and I. P. Sarethy

3.6 Metagenomic Analyses of Thermophilic Fungi
Metagenomics-based analysis has considerable potential in understanding industrial
applications of thermophiles. The general strategy of metagenomics utilizes both
functional- and sequence-based techniques for a comprehensive understanding
(Rodriguez et al. 2018). Thermophiles surviving above 55 C have high potential
for biotechnological applications for production of biocatalysts, which can be
beneficial in reducing contaminations, especially in food industry. Thermoenzymes
are efficient under high pH, temperature, pressure, and high concentration of sub-
strate and hence are used in production of paper, textile, food, and pharmaceuticals
(DeCastro et al. 2016). Table 3.2 shows some cellulases obtained from thermophilic
fungi.
3.7 Metagenome-Based Classification
The classification of fungi in metagenomics studies is based on the Internal Tran-
scribed Spacer (ITS) sequence obtained from next-generation sequencing data
(Oliveria et al. 2015; Thanh et al. 2019). The so-obtained sequence data must be
deposited in a public repository such as the National Centre for Biotechnology
Information (NCBI). Table 3.3 shows the study of thermophilic fungi based on
taxonomy and diversity. Different repositories and tools (http://www.
indexfungorum.org/,http://www.mycobank.org/) are available where the descrip-
tion of each taxon is available based on their nomenclature and classification
database. Many of the sequences obtained from such studies have been identified/
tallied with appropriate MycoBank numbers, some of them mapped with fungi
which had been identified considerably earlier. An example is A. fumigatus,
identified in a study by Latge (1999), which has been mapped with an isolate
identified in 1863.
3.8 Phylogenetic Analysis: Case Study
A case study of Thermomyces lanuginosus (Fig. 3.2) shows that many isolates of this
species have been obtained, sequenced, and submitted in GenBank. A phylogenetic
analysis using Mega X (Kumar et al. 2018) shows that the isolates map close to each
other though many clades and subclades have formed. This indicates that consider-
able differences also exist among the isolates. This directly indicates possible genetic
diversity among produced compounds such as enzymes and other metabolites too.
3 Major Habitats and Diversity of Thermophilic Fungi 67

Table 3.2 Some cellulases obtained from thermophilic fungi
Enzyme
Optimum
temperature
(C)
Optimum
pH Source Reference
AtCel7A 60 5.0 Acremonium
thermophilum
Voutilainen et al.
(2008)
CBHI 55 3.0 Aspergillus aculeatus Takada et al.
(1998)
CtCel7A 65 4.0 Chaetomium
thermophilum
Voutilainen et al.
(2008)
CBH3 65 5.0 Chaetomium
thermophilum
Li et al. (2006)
CBH1 55 5.0 Humicola grisea var.
thermoidea
Takashima et al.
(1998)
TeCel7A 55 4.0 Talaromyces emersonii Voutilainen et al.
(2010)
Cel7A 65 3.0 Penicillium funiculosum Texier et al.
(2012)
TaCel7A 55 5.0 Thermoascus
aurantiacus
Voutilainen et al.
(2008)
CBHI 65 6.0 Trichoderma viride Song et al. (2010)
ThCBHI 50 5.0 Trichoderma harzianum Colussi et al.
(2011)
Bgl 60 5.0 Fusarium oxysporum Zhao et al. (2013)
Bgl4 55 6.0 Humicola grisea var.
thermoidea IFO9854
Takashima et al.
(1999)
Bgl1 55 5.5 Orpinomyces sp. PC-2 Li et al. (2004)
Bgl1G5 50 6.0 Phialophora sp. G5 Li et al. (2013)
Cel3A 50–60 5.0 Amnesia atro brunnea Colabardini et al.
(2016)
Cel3B 50–60 5.0 Amnesia atro brunnea Colabardini et al.
(2016)
BglB 52 5.5 Aspergillus nidulans Calza et al. (1985)
Bgl 50 5.0 Aspergillus oryzae Tang et al. (2014).
MoCel3A 50 5.5 Magnaporthe oryzae Takahashi et al.
(2011)
MoCel3B 50 5.5 Magnaporthe oryzae Takahashi et al.
(2011)
NfBGL1 80 5.0 Neosartorya fischeri Yang et al. (2014)
PtBglu3 75 6.0 Paecilomyces
thermophile
Yan et al. (2012)
RmBglu3B 50 5.0 Rhizomucor miehei Guo et al. (2015)
β-Glucosidase 75 4.5 Talaromyces aculeatus Lee et al. (2013)
CBH1 55 5.5 Fusarium lini Mishra et al.
(1983)
CBH11 55 5.5 Fusarium lini Mishra et al.
(1983)
(continued)
68 S. Chaturvedi and I. P. Sarethy

3.9 Metabolites
Thermophilic and thermotolerant fungi are considered as good sources of thermo-
stable enzymes, which can be useful in industrial processes that function at higher
temperatures. A variety of thermostable enzymes have been obtained from thermo-
philic fungi such as cellulases, lipases amylases, and proteases (Thanh et al. 2019).
Table 3.2 (continued)
Enzyme
Optimum
temperature
(C)
Optimum
pH Source Reference
S1 50 4.0 Irpex lactus Kanda et al.
(1980)
En1 50 5.0 Irpex lactus Kanda et al.
(1980)
E2A 60 5.0 Irpex lactus Kubo and
Nisizawa (1983)
E2B 60 5.0 Irpex lactus Kubo and
Nisizawa (1983)
GB1(E) 60 5.5 Pyricularia oryzae Hirayama et al.
(1978)
GB2(E) 45 5.0 Pyricularia oryzae Hiramaya and
Nagamaya (1979)
Table 3.3 Taxonomy of thermophilic fungi
Taxon name
MycoBank
number Reference
Aspergillus fumigatus MB#211776 Latge (1999)
Chaetomium thermophilum MB#807382 Amlacher et al.
(2011)
Myceliophthora fergusii MB#317954 van den Brink et al.
(2012)
Myceliophthora heterothallica MB#519877 Houbraken et al.
(2011)
Myceliophthora guttulata MB#802335 Zhang et al. (2014)
Rhizomucor miehei (¼Mucor miehei) MB#322483 Schipper (1978)
Rasamsonia byssochlamydoides MB#519877 Houbraken et al.
(2011)
Thermomyces dupontii MB#805186 Houbraken et al.
(2014)
Thermothelomyces heterothallica(¼Myceliophthora
heterothallica)
MB#809491 Stchigel et al. (2015)
Thermothelomyces thermophila (¼Myceliophthora
thermophila)
MB#809493 Stchigel et al. (2015)
Thermomyces lanuginosus (¼Humicola lanuginosa) MB#239786 Singh (2003)
3 Major Habitats and Diversity of Thermophilic Fungi 69

Fig. 3.2 Phylogenetic tree of 95 GenBank nucleotide sequences from isolates of T. lanuginosus.
Branch lengths are in the same units as those of the evolutionary distances, computed using the
Maximum Composite Likelihood method
70 S. Chaturvedi and I. P. Sarethy

Hence, the information provided in Sect. 3.8 can be relevant in further purifying and
better understanding of more potentially novel thermo-stable enzymes, considering
that many of the isolates of T. lanuginosus fall into distinct clades.
Many carbohydrate-active enzymes (CAZymes) have been found in thermophilic
fungi. For example, four polysaccharide lyases (PLs), 28 carbohydrate esterases
(CEs), and more than 50 enzymes with auxiliary activities (AAs) have been
described. The genome of Thielavia terrestris has been shown to encode nearly
500 CAZymes, including 212 glycoside hydrolases (GHs), 91 glycosyl transferases
(GTs), and 80 carbohydrate-binding modules (CBMs). Studies have shown that
common strategies for thermal adaptation include a reduced genome size and
increased frequency of the amino acids such as Ile, Val, Tyr, Trp, Arg, Glu, and
Leu (IVYWREL) in proteins (Thanh et al. 2019), contributing to thermostability.
T. lanuginosus has encouraging potential in production of xylanase too, as seen from
its genetic sequence (Oliveria et al. 2015).
Orfali and Perveen (2019) isolated two new compounds - 3-(furan 12-carboxylic
acid)-6-(methoxycarbonyl)-4-hydroxy-4-methyl-4 and 5-dihydro-2H-pyran 1 3-
α-methyl-7-hydroxy-5-carboxylic acid methyl ester-1-indanone - from a thermo-
philic Penicillium species that was initially isolated from Ghamiqa hot spring
sediments (Saudi Arabia). Austinol, emodin, and 2-methyl-penicinoline, already
documented, were also isolated. Emodin showed cytotoxicity against HTB-176 cell
line, while austinol exhibited antibacterial activity against Pseudomonas
aeruginosa. It is interesting that new bioactive molecules are elicited from thermo-
philic fungi and need further studies.
Metabolite profiling of T. lanuginosus and Scytalidium thermophilum (Yang et al.
2020) showed the presence of 23 metabolites from T. lanuginosus. Among these,
there was also a new metabolite, therlanubutanolide, and 15 known compounds. In
addition, seven known compounds were obtained from S. thermophilum. Also, a
polyketide synthase pathway-derived metabolite, three 3,4-dihydronaphthalen-1
(2H)-ones, was obtained from both fungi. This metabolite has been shown to possess
antimicrobial activity and is known to be a phytotoxin in plant pathogenic fungi.
Nematicidal compounds have been described from a thermophilic fungus,
belonging to a class of PKS-NRPS hybrid metabolites (Guo et al. 2012). These
compounds from Talaromyces thermophilus were found to have a 13-membered
lactam-bearing macrolactone, namely, thermolides A–F, as per NMR spectra. Two
compounds showed nematicidal activity against three destructive nematodes:
Meloidogyne incognita, Bursaphelenches siyophilus, and Panagrellus redivevus.
It is interesting to see that thermophilic fungi can not only synthesize bioactive
metabolites but also facilitate biotransformation. Sreelatha et al. (2018) have shown
that T. lanuginosus can convert spironolactone to the minor mammalian metabolites
7α-thiospironolactone (M1) canrenone (M2), 7α-thiomethylspironolactone (M3),
and 6β-OH-7α-thiomethylspironolactone (M4). Hence, possessing similar mamma-
lian enzyme systems, their potential in biotransformation, and production of impor-
tant metabolites warrants exploration.
These thermophilic fungi can also be utilized for metabolic engineering as
evidenced by the work of Li et al. (2020). They used the cellulolytic thermophilic
3 Major Habitats and Diversity of Thermophilic Fungi 71

filamentous fungus Myceliophthora thermophile in an attempt to produce ethanol
from glucose and cellobiose. They introduced the ScADH1 gene into the wild-type
strain and found that ethanol production was increased when glucose was used as
substrate. However, overexpression of a glucose transporter or cellodextrin transport
system from N. crassa resulted in increased ethanol production. Transcriptomic
analysis showed downregulation of genes involved in oxidation–reduction reactions
and stress response but upregulation of protein synthesis related genes.
3.10 Conclusion
It is obvious from this chapter that thermophilic fungi hold great potential for future
prospects in industry. While many such fungi have been isolated, identified, and
sequenced, whole-genome sequences and the mining of information from their genes
are yet to be comprehensively elucidated. Next-generation sequencing technologies
are set to play a vital role in better understanding this unique group of fungi.
Considering that metagenome analysis of thermophilic fungi, though very less, has
shown matches with fungi already documented, this can ease out the process of
information mining. It is expected that in the next few years, a wealth of information
repository would be developed focusing on thermophilic fungi.
Acknowledgement Authors acknowledge Jaypee Institute of Information Technology, Noida for
providing infrastructure.
Conflict of Interest Authors declare no conflict of interest.
References
Abdel-Hafez SII (1982) Thermophilic and thermotolerant fungi in the desert soils of Saudi Arabia.
Mycopathologia 80:15–20
Abdullah SK, Zora SE (1993) Chaetomium mesopotamicum a new thermophilic species from Iraqi
soil. Cryptogam Bot 3(4):387–389
Ames LM (1963) A monograph of the Chaetomiaceae. U.S. Army Research and Development,
Bibliotheca Mycologica Cramer, Lehrer No 2, pp 1–125
Apinis AE (1963) Occurrence of thermophilous microfungi in certain alluvial soils near
Nottingham. Nova Hedwigia 5:57–78
Calza RE, Irwin DC, Wilson DB (1985) Purification and characterization of two-
β-1,4-endoglucanases from Thermomonospora fusca. Biochemistry 24:7797–7804
Chen KY, Chen ZC (1996) A new species of Thermoascus with a Paecilomyces anamorph and
other thermophilic species from Taiwan. Mycotaxon 50:225–240
Colabardini AC, Valkonen M, Huuskonen A, Siika-aho M, Koivula A, Goldman GH, Saloheimo M
(2016) Expression of two novel β-glucosidases from Chaetomium atrobrunneum in
Trichoderma reesei and characterization of the heterologous protein products. Mol Biotechnol
58:821–831
Colussi F, Serpa V, Delabona PDS, Manzine LR, Voltatodio ML, Alves R, Mello BL, Pereira N,
Farinas CS, Golubev AM et al (2011) Purification, and biochemical and biophysical characteri-
zation of cellobiohydrolase I from Trichoderma harzianum. Microbiol Biotechnol 21:808–817
72 S. Chaturvedi and I. P. Sarethy

Cooney DG, Emerson R (1964) Thermophilic fungi: an account of their biology, activities, and
classification, vol 6. W.H. Freeman and Co, San Francisco, pp 1–137
DeCastro ME, Belmonte ER, Gonzalez-Siso MI (2016) Metagenomics of thermophiles with a focus
on discovery of novel thermozymes. Front Microbial 7:1521
Evans HC (1971) Thermophilous fungi of coal spoil tips. II. Occurrence, distribution and tempera-
ture relationships. Trans Br Mycol Soc 57:255–266
Fergus CL (1971) The temperature relationships and thermal resistance of a new thermophile
Papulaspora from mushroom compost. Mycologia 63:426–431
Guarro J, Abdullah SK, Al-Bader SM, Figueras MMJ, Gene J (1996) The genus Melanocarpus.
Mycol Res 100:75–78
Guo J-P, Zhu C-Y, Zhang C-P, Chu Y-S, Wang Y-L, Zhang J-X, Wu D-K, Zhang K-Q, Niu X-M
(2012) Thermolides, potent nematocidal PKS-NRPS hybrid metabolites from thermophilic
fungus Talaromyces thermophiles. J Am Chem Soc 134(50):20306–20309
Hiramaya T, Nagamaya HK (1979) Studies on cellulases of a phytopathogenic fungus. Pyricularia
oryzae Cavara III. Multiplicity of glucosidase and purification and properties of a second
component. J Biochem 85:591–599
Hirayama T, Horie S, Nagamaya H, Matusda K (1978) Studies on cellulases of a phytopathogenic
fungus. Pyricularia oryzae Cavara. II. Purification and properties of a (3-glucosidase). J
Biochem 84:3–27
Holman DB, Hao X, Topp E, Yang HE, Alexander TW (2016) Effect of co-composting cattle
manure with construction and demolition waste on the archaeal, bacterial and fungal microbiota
and on antimicrobial resistant determinants. PLoS One 11(6):E057539
Kanda T, Wakabayashi K, Nisizawa K (1980) Purification and properties of a loweer mlolecular
weight endocellulase from Irpex lacteus (polyporus tulipiferae). J Biochem 87:1625–1634
Kubo K, Nisizawa K (1983) Purification and properties of two endo-type cellulases from Irpex
lacteus (polyporus tulipiferae). J Ferment Technol 61:383–389
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics
analysis across computing platforms. Mol Biol Evol 35:1547–1549
Li X-L, Ljungdahl LG, Ximenes EA, Chen H, Felix CR, Cotta MA, Dien BS (2004) Properties of a
recombinant β-glucosidase from polycentric anaerobic fungus OrpinomycesPC-2 and its appli-
cation for cellulose hydrolysis. Appl Biochem Biotechnol 113:233–250
Li Y, Li D, Teng F (2006) Purification and characterization of a cellobiohydrolase from the
thermophilic fungus Chaetomium thermophilus CT2. Wei Sheng Wu Xue Bao 46:143–146
Li X, Zhao J, Shi P, Yang P, Wang Y, Luo H, Yao B (2013) Molecular cloning and expression of a
novel β-glucosidase gene from Phialophora sp. G5. Appl Biochem Biotechnol 169:941–949
Li J, Zhang Y, Li J, Sun T, Tian C (2020) Metabolic engineering of the cellulolytic thermophilic
fungus Myceliophthora thermophila to produce ethanol from cellobiose. Biotechnol Biofuels
13:23. https://doi.org/10.1186/s13068-020-1661-y
Mahajan MK, Johri BN, Gupta RK (1986) Influence of desiccation stress in a xerophilic thermo-
phile Humicola sp. Curr Sci 55:928–930
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol Rev 64:461–488
Maheshwari R, Kamalam PT, Balasubramanyam PV (1987) The biogeography of thermophilic
fungi. Curr Sci 56:151–155
Maijala P, Kango N, Szijarto N, Viikari L (2012) Characterization of hemicellulases from thermo-
philic fungi. Antonie Van Leeuwenhoek 101(4):905–917
Malloch D, Cain RF (1973) The genus Thielavia. Mycologia 65:1055–1077
Miehe H (1907) Die Selbsterhitzung des Heus. Eine. Biologische. Gustav Fischer, Jena, pp 1–127
Mishra C, Rao V, Despandey M (1983) Purification and properties of two exocellulaese from a
cellulolytic cultureof Fusarium lini. Enzym Microb Technol 51:430–434
Morgenstern I, Powlowski J, Ishmael N, Darmond C, Marqueteau S, Moisan M, Quenneville G,
Tsang A (2012) A molecular phylogeny of thermophilic fungi. Fungal Biol 116:489–502
3 Major Habitats and Diversity of Thermophilic Fungi 73

Oliveria TB, Gomes E, Raodrigues A (2015) Thermophilic fungi in the new age of fungal
taxonomy. Extremophiles 19:31–37
Orfali R, Perveen S (2019) New bioactive metabolites from the thermophilic fungus Penicillium
sp. isolated from Ghamiqa hot spring in Saudi Arabia. J Chem 2019:7162948
Pan WZ, Huang X, Biwei K, Zhang KQ (2010) Diversity of thermophilic fungi in Tengchong Rehai
National Park revealed by ITS nucleotide sequence analyses. J Microbiol 48(2):146–152
Rafiei V, Banihashemi Z (2019) Fungi in desert areas of Yazd province. Plant Pathol Sci 8(2):
110–121
Rodriguez JJE, DeCastro ME, Cerdan M, Belmont ER, Becerra M, Gonzalez-Siso MI (2018)
Cellulases from thermophiles found by metagenomics. Microoranism 6(3):66
Sadaf A, Khare S (2014) Production of Sporotrichum thermophile xylanase by solid state fermen-
tation utilizing deoiled Jatropha curcas seed cake and its application in xylooligosachharide
synthesis. Bioresour Technol 153C:126–113
Salar RK, Aneja KR (2007) Thermophilic fungi: taxonomy and biogeography. J Agricult Technol
3:77–107
Sandona K, Tobias TLB, Hutchinson MI, Natvig DO, Alfaro P (2019) Diversity of thermophilic
and thermotolerant fungi in corn grain. Mycologi 111(5):719–729
Schipper MAA (1978) On the genera Rhizomucor and Parasitella. Stud Mycol 17:53–71
Sharma N, Tanksale H, Kapley A, Purohit HJ (2012) Mining the metagenome of activated biomass
of an industrial wastewater treatment plant by a novel method. Indian J Microbiol 52:538–543
Song J, Liu B, Liu Z, Yang Q (2010) Cloning of two cellobiohydrolase genes from Trichoderma
viride and heterogenous expression in yeast Saccharomyces cerevisiae. Mol Biol Rep 37:2135–
2140
Sreelatha B, Shyam Prasad G, Koteshwar Rao V, Girisham S (2018) Microbial synthesis of
mammalian metabolites of spironolactone by thermophilic fungus Thermomyces lanuginosus.
Steroids 136:1–7
Takada G, Kawaguchi T, Sumitani J, Arai M (1998) Expression of Aspergillus aculeatus No. F-50
cellobiohydrolase I (cbhI) and beta-glucosidase 1 (bgl1) genes by Saccharomyces cerevisiae.
Biosci Biotechnol Biochem 62:1615–1618
Takahashi M, Konishi T, Takeda T (2011) Biochemical characterization of Magnaporthe oryzae
β-glucosidases for efficient β-glucan hydrolysis. Appl Microbiol Biotechnol 91:1073–1082
Takashima S, Iikura H, Nakamura A, Hidaka M, Masaki H, Uozumi T (1998) Isolation of the gene
and characterization of the enzymatic properties of a major exoglucanase of Humicola grisea
without a cellulose-binding domain. J Biochem 124:717–725
Takashima S, Nakamura A, Hidaka M, Masaki H, Uozumi T (1999) Molecular cloning and
expression of the novel fungal-glucosidase genes from Humicola grisea and Trichoderma
reesei. J Biochem 125:728–736
Tang Z, Liu S, Jing H, Sun R, Liu M, Chen H, Wu Q, Han X (2014) Cloning and expression of
A. oryzae β-glucosidase in Pichia pastoris. Mol Biol Rep 41:7567–7573
Tansey MR, Brock TD (1978) Microbial life at high temperature ecological aspects. In: Kushner D
(ed) Microbial life in extreme environments, pp 159–216
Tansey MR, Jack MA (1975) Thielavia australiensis sp. nov., a new thermophilic fungus from
incubator bird (mallee fowl) nesting material. Can J Bot 53:81–83
Texier H, Dumon C, Neugnot-Roux V, Maestracci M, O’Donohue MJ (2012) Redefining XynA
from Penicillium funiculosum IMI 378536 as a GH7 cellobiohydrolase. J Ind Microbiol
Biotechnol 39:1569–1576
Thakre RP, Johri BN (1976) Occurrence of thermophilic fungi in coal-mine soils of Madhya
Pradesh. Curr Sci 45:271–273
Thanh VN, Thuy NT, Huong HT, Hien DD, Hang DT, Anh DTK, Hüttner S, Larsbrink J, Olsson J
(2019) Surveying of acid-tolerant thermophilic lignocellulolytic fungi in Vietnam reveals
surprisingly high genetic diversity. Sci Rep 9(1):3674
Tulsiyan RK, Sinha NK, Kumar V (2017) Isolation and identification of fungi from coal mines near
hazaribagh and their diversity study. J Cell Sci Apopt 1:102
74 S. Chaturvedi and I. P. Sarethy

Van Oorschot CAN (1977) The genus Myceliophthora. Persoonia 9:404–408
Von Arx JA (1975) On thielavia and some similar genera of ascomycetes. Stud Mycol 8:1–32
von Arx JA, Figueras MJ, Guarro J (1988) Sordariaceous ascomycetes without ascospore ejacula-
tion. Beihefte zur nova hedwigia 94:1–104
Voutilainen SP, Puranen T, Siika-Aho M, Lappalainen A, Alapuranen M, Kallio J, Hooman S,
Viikari L, Vehmaanpera J, Koivula A (2008) Cloning, expression, and characterization of novel
thermostable family 7 cellobiohydrolases. Biotechnol Bioeng 101(3):515–528
Voutilainen SP, Murray PG, Tuohy MG, Koivula A (2010) Expression of Talaromyces emersonii
cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its
thermostability and activity. Protein Eng Des Sel 23:69–79
Ward JE Jr, Cowley GT (1972) Thermophilic fungi of some Central South Carolina forest soils.
Mycologia 64(1):200–205
Yang XY, Zhang JX, Ding QY, He ZC, Zhu CY, Zhang KQ, Niu XM (2020) Metabolites from two
dominant thermophilic fungal species Thermomyces lanuginosus and Scytalidium
thermophilum. Chem Biodivers 17(5):e2000137. https://doi.org/10.1002/cbdv.202000137
Zhao Z, Ramachandran P, Kim T-S, Chen Z, Jeya M, Lee J-K (2013) Characterization of an acid-
tolerant β-1,4-glucosidase from fusarium oxysporum and its potential as an animal feed additive.
Appl Microbiol Biotechnol 97:10003–10011
3 Major Habitats and Diversity of Thermophilic Fungi 75

Thermophilic Fungi: Habitats
and Morpho-Molecular Adaptations 4
Regina Sharmila Dass, Joy Elvin Dhinakar, Akriti Tirkey,
Mayukhmita Ghose, and Angeline Jessika Suresh
Abstract
The study of extremophilic fungi has received manifold global attention during
the past decade. Among the members belonging to the monophyletic fungal
kingdoms, very few species have the capacity to survive and proliferate between
the temperature range of 45–55 C. These temperatures are considered as high
temperatures while studying thermophilic and thermotolerant fungi. Earlier clas-
sification and studies were arbitrarily carried out based on their cardinal
temperatures. The temperature endured by the fungi are not as high as those
witnessed in bacteria and archaebacteria, adding to the very many reasons for not
receiving due publicity in the past. However, drastic improvements in the
methods employed for molecular fungal phylogeny and DNA-based studies has
eliminated such hassles and paved the way for the elucidation of thermophily as
an interesting phenomenon in fungi. Such fungal candidates have lent themselves
as tools and excellent laboratory material for classical, genetic, and applied
research. Their morphological, physiological, and molecular adaptations,
characteristics, diversity and their role in different habitats such as soils, compost
heaps, agricultural and forest debris, etc. have been reviewed and presented.
Keywords
Diversity · Fungi · Habitats · Molecular studies · Thermophilic · Thermotolerant
R. S. Dass (*) · A. Tirkey · M. Ghose · A. J. Suresh
Fungal Genetics and Mycotoxicology Laboratory, Department of Microbiology, School of Life
Sciences, Pondicherry University, Pondicherry, India
J. E. Dhinakar
Department of Studies in Biochemistry, University of Mysore, Mysore, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_4
77

4.1 Introduction
Fungi are widely distributed eukaryotes present in almost all the known habitats of
microorganisms. Their presence in diverse environments is owed to their structure
and ability to produce several exoenzymes to degrade and solubilize complex,
insoluble macromolecules. Fungi generally prefer dark and moist or humid
environments. Geared with an extremely diverse metabolic activity, fungi have
taken over several ecological habitats (Naranjo-Ortiz and Gabaldon 2019). Being
saprobes, fungi are abundantly present in soil rich in organic matter and play a major
role as decomposers of dead matter or recyclers of nutrients in the soil. They can
easily grow on and in between the soil aggregates. The presence of fungi in soil is
affected by several factors such as soil pH, moisture, temperature, salinity, structure,
and the presence of other organisms (Rouphael et al. 2015). Most of the fungi have
temperature optima between 20 and 25 C and prefer acidic conditions. Several
basidiomycetes have been reported to lose their tolerance above pH, 7 but many
other taxa of fungi have a wide pH tolerance range (Dangles and Chauvet 2003).
Fungi are found in varying types of soils ranging from grasslands, forest, and
agricultural soils to extreme environments like tundra regions and soils of the desert.
In grasslands, the fungi are important in terms of their nutrient recycling activity,
production of enzymes, and organic compounds due to interaction with plants,
further resulting in the inhibition of grassland biodiversity (Huhe et al. 2017).
Basidiomycetes are reported as the dominant taxon in grasslands, while ascomycetes
are the major decomposers of organic matter in agricultural soils (Deacon et al.
2006).
The ability to withstand and thrive in the extremes of temperature is found
throughout the domains of life. However, the extent of tolerance to high
temperatures varies, with Eukarya showing a more restricted nature of thermophily.
The taxonomically diverse group of thermophilic fungi is perhaps one of the most
enigmatic life forms on Earth, despite them occupying only a fraction of Kingdom
Fungi (Salar and Aneja 2007). There are numerous classification systems to define
thermophilic fungi based on minimum, optimum, and maximum temperatures of
growth starting from Cooney and Emerson (1964) classification (Oliveira et al.
2015). While on the other hand, Maheshwari et al. (2000)defines thermophilic
fungi with a more straightforward definition as those fungi whose optimal growth
temperature lays between 40 and 45 C or greater. Among multiple definitions for
thermophilic fungi, a recent one defines them as the fungi which demonstrate their
optimum growth between 40 and 50 C (Oliveira et al. 2015).
4.2 Terrestrial Thermophilic Fungi
The fungi are the most fascinating creatures of the eukaryotic microbial world. Of
the abundant fungal forms, a specific few species possesses the unique attribute to
survive, grow, and multiply between 45 and 55 C. These are referred to as
thermophilic or thermotolerant fungi, which are classified purely on their cardinal
78 R. S. Dass et al.

temperature requirements (Cooney and Emerson 1964; Crisan 1973). Unlike in
bacteria, thermophily in fungi does not necessarily exhibit growth at temperatures
close to 100 C as seen in archaebacteria or eubacteria isolated from solfatara
habitats, hydrothermal vents, and thermal springs (Brock 1995; Blochl et al.
1997). Ever since the discovery of thermophilic fungi, their identification had always
been a challenging and tedious task for mycologists due to the lack of availability of
sequences of reference strains in public/genetic databases. However, taxonomic
studies, advancement in sequencing technology leading to the discovery of rapid
DNA-based methodologies, chemo- and numerical taxonomic methods and, new
age sequencing (next-generation sequencing) have made their identification much
easier and faster (Oliveira et al. 2015; Raja et al. 2017).
4.3 Aquatic Thermophilic Fungi
Fungi are widespread in aquatic habitats. But most of them are still unknown to us,
since many locations across the globe are yet to be explored. This is due to the
challenges associated with aquatic sampling, especially the marine habitat. How-
ever, understanding the beneficiary role of aquatic fungi to the environment and in
turn to mankind has led scientists to undertake various fungal biodiversity studies in
aquatic habitats. Studies on such ecosystems report the prevalence of fungi of the
phyla Ascomycetes and Chytridiomycetes in aquatic habitats, while Basidiomycetes
have found be occurring the least. Moreover, species similarities were commonly
found between freshwater habitat and terrestrial habitats, while the marine habitat
shows uniqueness in its fungal biodiversity (Shearer et al. 2009) and therapeutic
potential.
Aquatic fungi are mostly found to be proliferating on submerged organic
substrates of either plant or animal origin. Some of these fungi are well adapted to
grow in freshwater habitats and hence are referred to as “resident”aquatic fungi,
while “transient”aquatic fungi are those which comprise spores from the adjoining
terrestrial habitat. Studying freshwater fungi is tedious because of their tight adher-
ence to their substrate through their elongated hyphae. Freshwater fungi mostly
thrive on dead coarse particulate organic matter, especially on plant litter, using them
as primary energy source and nutrient source. In turn, these fungi function in the
decomposition of plant litter in freshwater ecosystem, thereby transferring energy
and nutrients to higher trophic level, such as invertebrates (Gulis et al. 2009). The
importance of aquatic fungi in the ecosystem has been extensively studied (Krauss
et al. 2011). They can also serve as potential agents for pollution study, as climatic
change has a direct impact on them. Aquatic fungi play a major role in recycling
organic matter in aquatic environment and in maintaining the aquatic food web.
Brackish water is another aquatic habitat that has been investigated at a large
scale. Such water bodies have a higher salinity as compared with freshwater, thereby
favoring the growth of fungi of well-known marine taxa such of Halosphaeriaceae
and Lulworthiaceae family. Studies carried out in lakes (such as Lake Fuxian of
China, Lake of Italy and many more), containing brackish water, have reported
4 Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations 79

similar occurrences (Grasso and La Feria 1985; Cai et al. 2002) and were initially
referred to as the “saltwater fungi.”Besides, fungi show a great biodiversity in
brackish waters based on its climatic condition. As such, Ascomycetes supersede
mitosporic fungal community in temperate climate, while the tropical region reports
the contrary (Hyde and Goh 1998; Cai et al. 2002).
Marine habitat is the largest aquatic habitat in the world that offers an enormous
range of biodiversity. Of these, extremophiles and thermophilic fungi have been
widely exploited as a potential source of novel bioactive compounds in last 15 years.
In order to grow in such hypersaline environments alongside other stress-inducing
factors such as high pressure and extreme temperature, these extremophiles produce
special enzymes, which are of medicinal and industrial importance (Dalmaso et al.
2015).
4.4 Morpho-Molecular Adaptations
4.4.1 Morphological Adaptation
Records suggest that Lindt was the first scientist to isolate a thermophilic fungus
namely Mucor pusillusfrom bread. He was the pioneer for the world of thermophilic
fungi (Lindt 1886). Mucor pusillus is now rechristened as Rhizomucor pusillus.In
1899, another scientist named Tsiklinskaya studied yet another thermophilic fungus
growing on potatoes. The fungus was then identified as Thermomyces lanuginosus
(Tsiklinsky 1899). However, there was no information available at that time, regard-
ing nutritional requirements and natural habitats that favored growth of thermophilic
fungi. In the early twentieth century, Kurt Noack worked with thermophilic fungi
and isolated them from a vast range of natural substrates (Noack 1920). He used
respiration as a probe to determine if the metabolic rate of the thermophilic fungi had
any effect on their characteristic nature. Following him, another independent
research scientist Hugo Miehe intensively researched on thermophilic fungi and
accumulated substantial information regarding their primary habitat. He isolated four
species of thermophilic fungi, namely, Mucor pusillus,Thermomyces lanuginosus,
Thermoidium sulfureum, and Thermoascus aurantiacus (Miehe 1930), that set the
stage for future research. A compilation, such as this one, would rather be incom-
plete without acknowledging their pioneering work.
Thermophilic fungi are found across the fungal phyla. They are especially
abundant in the phylum Ascomycota followed by the “traditional”phylum
Zygomycota (Salar and Aneja 2007). These unique fungi are often found ubiqui-
tously in soils or in impermanent microhabitats consisting of degrading plant
biomass such as composting bins, lake sediments, and birds’nests (Oliveira et al.
2015). Much is yet to be discovered of the ecology of thermophilic fungi, but the
growth of these transient communities seems to be fostered by high temperatures and
precipitation (van Noort et al. 2013). Certain thermophiles can be pathogenic to
human beings, livestock, or plants while others are known to be saprophytic in
nature (Di Piazza et al. 2020).Taxonomic classification of thermophilic fungi has
80 R. S. Dass et al.

undergone major revision over the past few decades due to clashes in nomenclature,
misidentification of thermotolerant species as thermophilic species, and the recovery
of new fungal sequences from environmental samples (Oliveira et al. 2015). It is
suggested by many that the ancestors of the thermophilic fungi might have been
mesophilic, which eventually developed thermal tolerance. Some also believe that
this was a result of adaptation of fungi to seasonal changes and higher temperatures
during daytime and not specifically to occupy new thermophilic habitats (Powell
et al. 2012). Since then, several researchers have proposed cardinal temperature
points for the classification of thermophilic fungi based on their optimal growth
temperatures. This led to a divergence of these fungi into two groups: thermophilic
and thermotolerant. It was not before the monograph published by Cooney and
Emerson (1964) on thermophilic fungi that this group of fungi drew focused
attention of the mycologists across the world. Thermophilic fungi were seen to
play a key role in resin utilization in crude rubber, thereby making the extraction
of rubber from its shrub much feasible. Eventually Allen and Emerson’s work
provided evidence for the same. According to them, these thermophilic fungi
could thrive at temperature as high as 60 C (Allen and Emerson 1949). Later, the
term “thermophiles”was used by Apinis to describe fungi having optimal growth at
35–40 C (Apinis 1953), who’s descriptions recorded lower temperatures compared
with other researchers (Allen and Emerson 1949).
The fungal cells are encapsulated with a polysaccharide rich wall, which is a
pivotal entity for the preservation of cellular integrity, which in turn aid in their
protection from external invaders, namely, the environmental agents and host
infections (Geoghegan et al. 2017). The fungal cell wall is more than merely being
an external shield. It harnesses an exquisite architecture in form and its composition,
controlling the morphological structure and recognition of an extensive range of
external environmental stimuli. The cell wall of the fungi is thought to have a
stratified structure while under observation using an electron microscope;
the electrons are profound and densely present in the outer layer (Osumi 1998).
The composition in the inner layer of the fungal cell wall is majorly composed of the
chitin–glucan matrix that is cross-linked; mannosylated proteins are observed to be
rich in the outer layer. The substantial increase in the electrons observed in the inner
segment of the cell wall is because of the presence of melanin. Occasionally, the
fungal cell wall exhibits itself as a granular form if not as a distinct individual
electron rich layer (Latge 2007). In contrast to the genome reduction, duplication of
genes for hyphal melanization in thermophiles, which provides resistance against
desiccation, high temperature, and UV radiation, may provide newer insights into
their evolutionary process (van Noort et al. 2013). Due to climate change and global
warming, it is predicted that as the average global temperature rises, more
thermotolerant and thermophilic fungi will come into picture (Paterson and Lima
2017).
The fungal cell wall (FCW) composition by mass is composed of β-1, 3 glucans
being the most frequently occurring (approximately about 80 per cent) in the hyphal
cell wall of the rice blast fungus Magnaporthe oryzae (Samalova et al. 2016). Chitin
and of β-1, 6 glucan each comprise less than 10% of the entire polysaccharide,
4 Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations 81

whereas in a couple of species β-1, 6 glucan occurs as a single residue branch points
off β-1, 3 glucan chains rather than a polymer instead (Free 2013; Samalova et al.
2016; Geoghegan et al. 2017). Chitin turns out to be a straight polymer of β- 1,4 -N-
acetylglucosamine. The polymers with individual chains undergo hydrogen bonding
in the interchain region, giving rise to the genesis of microfibrils along with a
massively rigid structure. In comparison, the primary constituents of the fungal
isolates associated with the glucan portions of the cell wall are noncrystalline. The
β-1, 3 linked glucan chains form a branched structure, which is amorphous. Every
β-1,3 linked glucan chain is variegated with branch points, alongside β-1,6 linkages
(Borchani et al. 2016). Both β-1,3 and β-1,6 linked glucans can be interlinked to
chitin. The polysaccharide constituents present in the cell wall form an extremely
interwoven and interlinked matrix that imparts great strength. The protein
constituents present in the cell wall are usually a small cell wall component. They
incorporate the proteins possessing structural roles like sensor proteins, which are
membrane-bound and enzymes required for synthesis and rearrangement. The
proteins are mostly N-orO-glycosylated, with side chains comprising of
mannose-rich residues. The aforementioned proteins can be intensely attached to
the glucan network (Borchani et al. 2016). This in turn provides solid attachment to
the fungal cell wall matrix, with most of the cell wall proteins being soluble and
non-covalently bonded to the fungal wall with polysaccharide-binding modules
(Osumi 1998). The fungal cell wall is a balanced blend of mannoproteins, chitin,
alpha, and beta-glucans; they play a couple of roles, namely, rigidity, shape,
metabolism, ion exchange, and interactions with the host defense mechanisms
(Pan et al. 2010; Lord and Vyas 2019). There is a variation in the cell wall
compositions of fungi according to their species. Out of all, the major constituent
of most fungal cell wall is β-1,3 glucans. The fungal cell wall contains proteins that
play a role in tight adhesion or binding of the fungi to the substratum/substrate. This
is very useful for pathogenic fungi. The cell wall of fungi is essential for its viability,
considering the mechanical viewpoint; the cell wall facilitates tolerance of turgor
pressure, which aids in the prevention of cell lysis.
In order to survive, the thermophilic fungi must also find means to protect
themselves against oscillating temperatures experienced by their habitats by mount-
ing adequate responses. One such response is the heat shock response. A team of
researchers (Ianutsevich et al. 2016) probed into the molecular basis of heat shock
responses in thermophilic fungi, through studying membrane lipid composition and
the production of soluble carbohydrates. The group demonstrated an increase in
phosphatidic acids (PA) and sterols in thermophilic species Rhizomucor tauricus and
Myceliophthora thermophila while simultaneously observing a decrease in
phosphatidylcholines (PC) and phosphatidylethanolamines (PE). While a significant
alteration of membrane composition was reported, more insight about the specific
regulatory and functional roles of certain lipids in thermal adaptation is yet to be
elucidated. Interestingly, the group also reported that in the response to prolonged
heat shock, there was virtually no change in the levels of unsaturation, leading the
group to believe unsaturation does not play a role in heat shock responses of
thermophilic fungi. Apart from elevated temperatures, thermophilic fungi also
82 R. S. Dass et al.

need to withstand other environmental stresses such as cold shock (Gunde-
Cimerman et al. 2014), osmotic stress, and oxidative stress. In a study done by
Ianutsevich et al. (2020), thermophilic fungi Rhizomucor miehei was found to have
an increased degree of membrane polar lipid unsaturation and decreased ergosterol
concentration during cold shock. In the same report, osmotic stress was found to
trigger the production of compatible solutes such as glycerol and arabitol in the
thermophilic fungus. The role of disaccharide trehalose remains unclear in thermo-
philic eukaryotic organisms, although certain evidence suggests it may be required
for thermophilia (Zhou et al. 2014). However, the levels of trehalose were signifi-
cantly decreased under both heat shock and cold shock (Ianutsevich et al. 2016,
2020).
An extremely popular model that has been studied over the years to derive
maximum scientific information on the fungal cell wall is Saccharomyces cerevisiae.
This yeast from the baker’s armamentarium has been considered as the darling of
biochemical and genetic research, because of the manner in which it has lent itself
for cell biology studies too, apart from being credited as the first eukaryote, whose
genome was sequenced.
Individually the cells of fungi utilize a sequence of stress sensors, which cross-
ways with the plasma membrane of the cell to constantly monitor the cell wall
integrity. The structure of these sensors is quite indistinguishable with a short C
terminal cytoplasmic domain. As we venture to understand further, these sensors
have single transmembrane domains and periplasmic amino terminus, which are rich
in serine and threonine residues. These amino-terminal portions are necessary for the
process of endocytosis. The polarized dissemination of the wall stress component
Wsc1 is regulated endocytotically. This leads to polarized accumulation of the
protein in the fungal cell wall (Piao et al. 2006; Lord and Vyas 2019).
4.4.2 Molecular and Enzymatic Adaptations
Thermophilic fungi are the ones that live in high temperature conditions, while
thermotolerant fungi usually live at mesophilic temperatures but can also survive
at higher temperatures. One example of a thermotolerant fungus is Aspergillus
fumigatus, which also can grow at above 50 C (Mouchacca 2000a). Many fungi
are found in very dry ecosystems like dry and hot deserts or in regions where water is
present in the bound form, thus drastically reducing the availability of water.
Temperature greatly affects the structure and function of biological molecules,
thereby serving as one of the main regulators of growth and activity in thermophilic
fungi. These fungi do not seem to have a specific organelle or developmental pattern
different from that of mesophiles, but survival at elevated temperature requires
specific modifications of the already existing structures. In order to grow actively
at such high temperatures, these fungi have evolved themselves to adapt their DNA,
proteins, and cytoplasmic membrane to thermo-resistant mechanisms (Oliveira and
Rodrigues 2019).
4 Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations 83

The role of mycologists and taxonomists studying fungal phylogeny who have
followed painstaking, time-consuming, and elaborate procedures has been well-
documented ever since fungi were classified using the five-kingdom classification
(Whittaker 1959,1969) in a number of studies, up to the present-day classification
(Alexopoulos et al. 2010). Traditional classification systems have been based almost
entirely on morphological characteristics, for instance, nature of the asexual spore
formed as a result of mitosis, structure of spore-bearing structures, nature and
arrangement of sexual spores, which are a resultant of a meiotic process between
opposite mating types, etc. The nomenclature and taxonomic classification of fungi
with emphasis to thermophilic fungi is muddled, often leading to ambiguity and
confusion (Mouchacca 1997,2000b). Natural classification systems, which have
combined the use of molecular markers, have eliminated this chaos to a great extent.
The concept of fungal barcoding is extremely prevalent in the current times and has
been used as a resort by most mycologists during elaborate morphological identifi-
cation procedures, while following lengthy protocols. The evolution of fungal
classification using purely morphological methods to the present-day phylogenetic
approaches based on DNA-based and chemotaxonomic approaches is well outlined
in general (Raja et al. 2017) and particularly in thermophilic fungi (Pan et al. 2010).
The genomes of very few thermophilic fungi have been sequenced; several taxa and
their barcode sequences are unavailable in genetic databases. The dearth of such
genetic information certainly limits the identification and potential use of thermo-
philic fungi, thus impeding the way for the assessment of newer technologies for the
description of a thermophilic species. The paradigm shifts in fungal taxonomy were
witnessed, due to the advent and use of computers that led to numerical taxonomic
methods. While the role of conventional techniques routinely used in identification
of fungi using phenotypic characteristics is still important, chemotaxonomic
methods have played an enormous role, leading to the formation of three monophy-
letic kingdoms of fungi. When chemotaxonomic approaches are being used, cell
wall composition is a major parameter used in fungal phylogeny.
Of the six million fungal species that exist (as suggested by high-throughput
sequencing techniques), nearly over 1.5 lakh species of fungi that have been isolated
and identified till date, which constitute the most intriguing creatures of the micro-
bial world; both mycelial and unicellular forms are bound with the fungal cell wall.
This structure is not just vital to fungal growth and survival but also crucial in the
morphogenesis (e.g., cell compartmentalization and septal development) of a partic-
ular species. Adequate studies on the mutational analysis have evidenced the role of
the cell wall as a shielding barrier against a multitude of environmental parameters
such as high and low temperatures, desiccation, osmotic stress, and nutrition stress.
It is not surprising to note that, today, thermophily in kingdom fungi is believed to
have arisen as an adaptive mechanism to seasonal changes and high temperatures
prevailing and probably did not emerge due to occupying high-temperature ecologi-
cal niches. The recent modifications announced by the International Code of
Nomenclature for algae, fungi and plants could lead to simplifications in thermo-
philic fungal taxonomy, making the use of thermophilic fungi in biotechnological,
industrial, and research purposes more feasible. The survival of thermophilic fungi
84 R. S. Dass et al.

can mainly be attributed to the thermostability of its proteins, particularly its
enzymes such as lipases, esterases, amylases, peptidases, and cellulolytic enzymes.
Certain features of thermophilic adaptation have been observed across the domains
of life. For example, the substitution of lysine by arginine (known as amino acid
bias) has been observed on several occasions in thermophilic organisms. A research
team (van Noort et al. 2013) reported the increased levels of arginine and tryptophan
and decreased amounts of aspartic acid, lysine, and glycine in thermophilic species
Chaetomium thermophilum,Thielavia terrestris, and Thielavia heterothallica of the
order Sordariomycetes. The proteins of C. thermophilum were also found to contain
large amounts of cysteine, which, as hypothesized by the authors, increases the
thermostability of the proteins.
One of the major characteristics of thermophilic fungi is homeoviscous adapta-
tion (Sinensky 1974). The fungi have devised a cellular framework to overcome and
circumvent the challenges of experiencing varied temperatures by altering the fatty
acid composition of the phospholipids that are present in their cell membranes. This
is achieved by maintaining their fluidity of the membrane to keep it intact, for the
flawless functioning of the enzymes and transporters localized in the membrane. The
fungi recruit a high number of saturated fatty acids upon the exposure to high
temperatures; at lower temperatures, it produces adequate number of unsaturated
fatty acids. When there is a shift from low to high temperature, an enzyme called
fatty acid saturase comes into play. This enzyme converts fatty acid linoleate to
oleate in fungal cell membrane. In other words, oleic acid is the most dominant fatty
acid of fungal cell membrane at higher temperature (Kates and Baxter 1962; Mumma
et al. 1970; Sekura and Fergus 1971). Also, most microbes alter their fatty acid
composition of their cell membrane phospholipids, as one of their primary functions
in response to the temperatures prevailing in the growth environments. Conse-
quently, there is an increase in the saturated fatty acid content with the increase in
temperature. On the contrary, at lower temperatures, there is an increase in the
unsaturated fatty acid content. The adaptive phenomenon is referred to as
“homeoviscous adaptation.”Although, this is true with most microscopic creatures,
Wright et al. (1983) reported an exception with the fungal species Talaromyces
thermophilus, wherein a shift from a higher temperature (50 C) to a lower (30 C)
growth temperature failed to result in the increase in the degree of unsaturation. The
fatty acid composition remained virtually unaltered. There was, however, a differ-
ence when T.lanuginosus was studied and examined. When mycelia were grown in
50 C, the phospholipid unsaturated fatty acid composition was 0.88 in contrast to
mycelia grown at 30 C, which was equivalent to 1.00 (Rajasekaran and Maheshwari
1990). Chaetomium thermophile also showed a decrease in the degree of unsaturated
fatty acids, when a heat shock was applied (Oberson et al. 1999). These examples
point toward a certain inability to adjust membrane fluidity and could be the reason
for their high minimum growth temperature.
A comparative study on the lipid composition of psychrophilic, mesophilic,
thermophilic, and thermotolerant species of Mucor and Rhizopus by Sumner and
his colleagues gave enough evidence to support the aforementioned property of the
fungal cell wall (Evans 1969; Sumner and Morgan 1969). Their experiments
4 Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations 85

revealed that a high content of saturated fatty acids in thermophilic fungi is at high
and low growth temperature, while fatty acid composition of thermotolerant fungi
matched with that of psychrophiles and mesophiles at low temperature and to
thermophilic fungi at higher temperature. Additional and interesting facts that
emerged from their study showed that spores contain low amount of lipid but
predominating in saturated fatty acid as compared with fungal mycelia. They also
understood that molecular oxygen was crucial and played a pivotal role in fatty acid
desaturation (Sumner and Morgan 1969). In contrast, reduced oxygen tension at high
temperature activates saturases (Harris and James 1969). A study involving Saccha-
romyces cerevisiae subjected to both high (40 C) and low (26 C) temperatures,
revealed that a high amount of saturated fatty acids and negligible amount of
unsaturated fatty acids being present in their cell membrane at 40 C (Chang and
Matson 1972). Mumma and his coworkers elucidated the fact that thermophilic fungi
contain higher level of lipid content as compared with mesophilic fungal isolates of
same genera (Mumma et al. 1970; Sekura and Fergus 1971). Sterols are present in
significant amounts in cell membranes of thermophilic fungi in addition to saturated
fatty acids and propose a significant role in providing thermal stability. Thermophilic
fungi usually grow under aerobic conditions (Kane and Mullins 1973). However, the
thermophilic fungus, Humicolainsolens, is an exception and usually prefers anaero-
bic or microaerophilic environment for its growth.
Complete genome sequencing of thermophilic fungi has provided mycologists
with the inner workings of thermophilia in eukaryotic organisms. A research group
(van Noort et al. 2013) reported that thermophilic fungi tend to have reduced genome
sizes when compared with their mesophilic relatives. The team presented evidence
showing a large portion of genes found in the closely related mesophilic species
Chaetomium globosum and Neurospora crassa but not in thermophilic fungi.
C. thermophilum,T. terrestris, and T. heterothallica were related to transposable
elements. Based on their findings, the group suggested that the phenomenon of
transposition is not preferred at elevated temperatures. van Noort et al. (2013) also
noted that genes coding for certain enzymes (e.g., oxygenases) are present in fewer
copies in comparison with mesophilic species.
Over many years, fungi have evolved and acquired thermostability and thermo-
tolerance to overcome stressful and drastic temperatures. Some fungi have been
briefly exposed to sublethal temperatures and have adapted tolerance to be exposed
to lethal temperatures. Some of the mesophilic fungi synthesize a set of heat shock
proteins (HSPs) following a sudden exposure to higher temperatures. The synthesis
or the formation of heat shock proteins turns out to be an adaptive response to
encounter stressful conditions and thermotolerance (Trent et al. 1994). Based on the
research gone by, scientist groups have inferred that conidia of the fungal species
T. lanuginosus germinates at 50 C, while it is heat treated at 55 C for about 60 min
prior to the brief exposure of 58 C. These conidia priorly exposed to such heat
shock, exhibited prolonged survival compared with that of non-treated conidia
(Maheshwari et al. 2000). Therefore, based on this observation, the heat shock
proteins come into play in order to enhance the thermotolerance in fungi. If in case
the protein synthesis is blocked or inhibited during the heat shock period or heat
86 R. S. Dass et al.

shock interval using cycloheximide, it either deteriorated or eliminated the
thermotolerance in fungi. Pulse labeling of proteins during the heat shock interval
or the heat shock period led by the separation through sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), revealed an increase in the genesis
of the heat shock proteins. A transitory heat shock protein was also observed in
Chaetomium thermophile var. thermophile being specific a HSP60 (heat shock
protein) that was abundantly expressed in this species; this posed to be an important
member of thermophily (Oberson et al. 1999). The heat shock proteins (HSPs)
prompt fungal genera to withstand or overcome lethal temperatures. The enzymes
present in the fungi also enable them to survive in adverse conditions, thus making
them resilient to strive all odds in the environment. The fungi emerge out to become
more competent over a period of time after the exposure to all environmental fluxes.
As mentioned earlier, there are a set of enzymes that are present in thermophilic
fungi that aid in sustaining lethal and adverse conditions. These enzymes are called
the secretory enzymes and have found wide applications in industrial bioprocesses
(Maheshwari et al. 2000), like various food business organizations like fermentation
sectors, breweries, food and beverage industries, etc.
Just like prokaryotic extremophiles, several fungal species also make up the
eukaryotic extremophiles. AM (arbuscular mycorrhizal) fungi (Macek et al. 2011)
and soil yeast Occultifur mephitis sp. nov. (Sibanc et al. 2018) have been reported
from the mofette sites (natural CO
2
springs) of Slovenia, which are ambient temper-
ature areas, rich in gas vents of pure geological CO
2
rising to the surface from the
deep mantle. The elevated levels of CO
2
lead to reduction in O
2
concentration
making the soil hypoxic and high carbonic acid in the soil results in the lowering
of pH. Such fields are common in volcanically or tectonically active sites (Macek
et al. 2016).
Apart from thermophily, several psychrophilic fungi have been isolated in large
numbers from the sub-glacial ice of polythermal glaciers (Gunde-Cimerman et al.
2003). Populations of Penicillium crustosum have been reported from the glaciers of
Svalbard, Norway (Arctic), which have unique features like they are capable of
producing andrastin A, a secondary metabolite or are unable to use creatine as their
sole source of carbon (Sonjak et al. 2007,2009). Two novel endemic genotypes
Thelebolus globosus and T. ellipsoideus have been isolated from the extreme cold
environments of Antarctica (De Hoog et al. 2005). Halophilic fungal populations
have been abundantly and consistently reported from naturally hypersaline
ecosystems (Gunde-Cimerman et al. 2000), which have unique molecular
adaptations to survive at high ion concentrations and low water activity (Plemenitas
et al. 2008). Hortaea werneckii, a halotolerant fungus, is capable of growing in
almost saturated solutions of sodium chloride and also without it (Gunde-Cimerman
et al. 2000). Acidophilic and acid-tolerant fungi have been reported from acidic
environments like acid mine drainage, volcanic springs, and industrial wastewaters
(Gross and Robbins 2000). Aspergillus sp. and Geotrichum sp. have been isolated
from copper mine (Orandi et al. 2007). Eurotiomyces and Dothideomycetes are
found in highly acidic environments of acid mine drainage at Richmond Iron
Mountain where the pH was 0.8 (Baker et al. 2004,2009).
4 Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations 87

Ascomycetous, filamentous fungus Xeromyces bisporus is one of the most
xerophilic organisms, which can survive in extreme dry conditions and has a water
activity optimum at 0.85 (Grant 2004). Aspergillus penicillioides is a
polyextremophilic fungus as it demonstrates features of being a halophile (can
grow at high-salt concentrations), xerophile (has very low water activity), osmophile
(can withstand high sugar concentrations), and a psychrophile (can grow at near
0C) and can also grow anaerobically (Chin et al. 2010; Zhang et al. 2013; Nazareth
and Gonsalves 2014; Stevenson et al. 2015).
Thermophilic fungi are cosmopolitan. In both natural and man-made
environments, they have been observed to be present as propagules of active mycelia
(Oliveira and Rodrigues 2019). The dissemination of propagules or spores (sexual or
asexual) by air may be the cause behind their ubiquitous distribution (Le Goff et al.
2010). Forest soils are covered by plant debris and litter, which make a very
favorable environment for diverse groups of fungi. The terrestrial ecosystems,
which accumulate lignocellulosic biomass, are broken down by lignolytic or cellu-
lolytic fungi by the production of extracellular lignocellulases. Such enzymes also be
useful in a multitude of industrial and bioremediative processes. Thermophilic fungi
are also potential candidates for the production of thermostable enzymes. The use of
fungal isolates for the industrial production of such bioenzymes may greatly reduce
the costs involved in synthetic production (Banerjee et al. 2010). Their use in the
production of industrial enzymes has also increased economic feasibility of indus-
trial processes. Additionally, such utilization has minimized problems caused in
the environment as a result of its disposal (Reddy et al. 2003; Sanchez 2009). Among
the microbes cultivated using these biological wastes, filamentous fungi have topped
the list, especially by adopting solid state fermentation techniques (Leite et al. 2008;
Alves-Prado et al. 2010; Moretti et al. 2012). In a focused study by de Cassia Pereira
and team (2015) who aimed at obtaining new cellulases and xylanases from thermo-
philic fungi, they reported that Myceliophthora thermophila JCP1-4 was the best
producer of endoglucanase, β- glucosidase, and xylanase. The enzymes produced
remained active at 55–70 C and stable between 30 and 60 C. Many countries in the
world whose businesses run on agro-industries accumulate millions of tons of
lignocellulosic wastes. Such accumulated bio-wastes serve as culture medium for
thermophilic fungi or microbes in general. Maheshwari and his research colleagues
(2000) have documented such thermophilic mycelial forms and their role in indus-
trial processes, involving the production of enzymes, in large bio-reactors. These
enzymes are active and stable at higher temperatures, which serves as an important
feature in such industrial processes.
Self-heating environments like composting systems have high temperatures due
to the exothermic metabolic reactions of the microorganisms and therefore are one of
the most suitable habitats for thermophilic fungi. These fungi mostly occur as
propagules and are generally dispersed by the agency of wind (Rajasekaran and
Maheshwari 1993), especially from composting piles while turning the piles for
aeration and facilitate adequate degradative processes of biomass used. Thermo-
philic fungi produce and secrete a wide range of extracellular enzymes in their
environment with substrate specificity and in large quantities, which have shown
88 R. S. Dass et al.

to act in a synergistic way in the decomposition process (Johri and Rajani 1999;
Dashtban et al. 2009). When these enzymes were compared with the enzymes of
mesophilic fungal species, slight differences were found in their structure, sequence,
function, and thermodynamic features (Niehaus et al. 1999; Oliveira et al. 2018).
Thermophilic fungi possess a large potential for the commercial market (Singh
et al. 2016). They contain numerous genes coding for a secretion of various
industrially important enzymes such as proteases, amylases, xylanases, and other
lignocellulolytic enzymes (Maheshwari et al. 2000). These enzymes are often
thermostable and confer several advantages to an industrial process such as less
contamination by mesophilic organisms and reduced reaction times, therefore
minimizing overall energy costs (Oliveira et al. 2015). Thermomyces lanuginosus,
Myriococcum thermophilum,Thermoascus aurantiacus,Myriococcum
thermophilum, and Thermoascus aurantiacus are a few examples of thermophilic
fungi that have been investigated for industrial use. The biofuel sector especially
may greatly benefit from the use of thermophilic fungi, in which extracted thermo-
stable enzymes (e.g., glycoside hydrolases) are used for the bio-conversation of plant
biomass into fermentable sugars for the production of second-generation biofuels
such as bioethanol. Thermophilic fungi can also be useful for scientific purposes
(e.g., in structural studies or biochemical studies), the paper industry, and in food
biotechnology (van Noort et al. 2013; Zhou et al. 2014).
The rate of growth of thermophilic fungi is much slower than that of mesophilic
fungi (Brock 1967). Moreover, thermophilic fungi undergo slower metabolism than
their mesophilic counterparts (Prasad et al. 1979). In case of thermotolerant fungi,
certain nutritional supplements favors increase in their growth under conditions of
high temperature (Loginova et al. 1962). This is evident in the fungus, Coprinus
fimetarius (Fries 1953). In a particular research study by Morgenstern and
co-workers (2012), the growth performances of 30 fungal strains were studied
whose identity was established by previous studies as thermotolerant and thermo-
philic fungi (Maheshwari et al. 2000; Mouchacca 2000a). Mesophilic strains were
also used for comparative studies. Few to name are Calcarisporiella thermophila,
Chaetomium thermophilum,Paecilomyces,Remersonia thermophila,Rhizomucor
miehei,R. pusillus,Talaromyces thermophilus,andThermomyces ibadanensis.
Mesophilic fungal strains included Acremonium alcalophilum,Amorphotheca
resinae,Aureobasidium pullulans,Chaetomium globosum,Gloeophyllum trabeum,
Lentinula edodes,Pleurotus ostreatus, and Tramates versicolor. The classification
of thermophiles only with respect to their growth requirements has been a debatable
topic ever since the study of thermophilic fungi has been initiated. The upper and
lower limit temperatures set as per Cooney and Emerson (1964) requires further
investigations in the current age due to evolving nature of the fungi and classification
methodologies. Some species like Thielavia australiensis, which grows above 50 C
as they also do below 20 C, are not covered under traditional or conventional
definitions (Morgenstern et al. 2012) of thermophilic fungi.
A modification of the previous definitions was presented by a team of researchers
(Morgenstern et al. 2012) who redefined thermophilic species as those that could
exhibit faster growth at 45 C than at a mesophilic temperature of 34 C, in spite of
4 Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations 89

growth being observed at 20 C. This has led to a reclassification of Calcarisporiella
thermophila,Remersonia thermophila, and Thielavia australiensis as thermophiles,
though they were earlier classified as thermotolerants by different research groups
(Seifert et al. 1997; Mouchacca 2000b).
A reduction in the genome size of the thermophilic fungi has also been noticed
due to the loss of transposable elements, intergenic regions, protein-coding genes,
and shorter introns in comparison with their nearest mesophilic species (van Noort
et al. 2013). Upon comparison with phylogenetically related mesophilic fungi,
significant reduction in the number of peptidase coding genes in thermophilic
fungi has been reported (Oliveira et al. 2018).
An examination of the peptidases from thermophilic fungi revealed the presence
of Glu, Pro, Ala, Gly, Val, and Arg residues in larger proportion and Cys, Ile, His,
Met, Gln, Thr, Asn, Trp, and Ser in lesser proportion in comparison with mesophilic
peptidases. Also, the number of internal cavities in thermophilic peptidases was
reduced. According to the researcher, two possible scenarios for these modifications
are (1) loss of peptidases with large number of internal cavities and retention of only
compactly folded peptidases and (2) enzyme optimization to contain fewer cavities
(Oliveira et al. 2018).
4.5 Conclusions
The taxonomically diverse group of thermophilic fungi is perhaps one of the most
enigmatic life forms on Earth, despite them occupying only a fraction of kingdom
fungi. The genetic constitution of an organism is the key for it to be classified as
thermophilic, which is true even in case of thermophilic fungi. Thermophilic fungi
serve as a great source of heat stable enzymes, which are of industrial importance.
Once scientists became well aware of their remarkable potency in enzyme produc-
tion, this group of fungi will continue to draw much attention. Research in the last
five decades has undoubtedly increased our knowledge and understanding of the
biology and molecular mechanisms in thermophilic fungi, as they have educed
interests in the field of pharmaceuticals and enzymes. There will be more additions
to the list of thermophilic fungi, an understanding to their biology, lifestyle, etc., as
routine and advanced techniques to study them evolve. Both conventional and
non-conventional methodologies have been adopted in the current era, which have
contributed to the advancement of this area of research inquiries.
References
Alexopoulos CJ, Mims CW, Blackwell M (2010) Introductory mycology, 4th edn. Wiley
Allen PJ, Emerson R (1949) Guayule rubber, microbiological improvement by shrub retting. Ind
Eng Chem 41:346–365
Alves-Prado HF, Pavezzi FC, Leite RSR, Oliveira VM, Sette LD, DaSilva R (2010) Screening and
production study of microbial xylanase producers from Brazilian Cerrado. Appl Biochem
Biotechnol 161:333–346
90 R. S. Dass et al.

Apinis AE (1953) Distribution, classification and biology of certain soil inhabiting fungi. PhD
thesis, Nottingham University, United Kingdom
Baker BJ, Lutz MA, Dawson SC, Bond PL, Banfield JF (2004) Metabolically active eukaryotic
communities in extremely acidic mine drainage. Appl Environ Microbiol 70:6264–6271
Baker BJ, Tyson GW, Goosherst L, Banfield JF (2009) Insights into the diversity of eukaryotes in
acid mine drainage biofilm communities. Appl Environ Microbiol 75:2192–2199
Banerjee G, Scott-Craig JS, Walton JD (2010) Improving enzymes for biomass conversion: a basic
research perspective. Bioenergy Res 3:82–92
Blochl E, Rachel R, Burggraf S, Hafenbradl D, Jannasch HW, Stetter KO (1997) Pyrolobus fumarii,
gen. And sp. nov. represents a novel group of archaea, extending the upper temperature limit for
life to 113C. Extremophiles 1:14–21
Borchani C, Fonteyn F, Jamin G, Paquot M, Thonart P, Blecker C (2016) Physical, functional and
503 structural characterization of the cell wall fractions from baker’s yeast Saccharomyces
504 cerevisiae. Food Chem 194:1149–1155
Brock TD (1967) Life at high temperatures. Science 158:1012–1019
Brock TD (1995) The road to Yellowstone and beyond. Annu Rev Microbiol 49:1–28
Cai L, Tsui C, Zhang K, Hyde KD (2002) Aquatic fungi from Lake Fuxian, Yunnan, China. Fungal
Divers 9:57–70
Chang SB, Matson RS (1972) Membrane stability (thermal) and nature of fatty acids in yeast cells.
Biochem Biophys Res Commun 46:1529–1535
Chin JP, Megaw J, Magill CL, Nowotarski K, Williams JP, Bhaganna P (2010) Solutes determine
the temperature windows for microbial survival and growth. Proc Natl Acad Sci U S A A107:
7835–7840
Cooney DG, Emerson R (1964) Thermophilic fungi. An account of their biology, activities and
classification. W. H. Freeman & Co., San Francisco, CA
Crisan EV (1973) Current concepts of thermophilism and the thermophilic fungi. Mycologia 65:
1171–1198
Dalmaso GZ, Ferreira D, Vermelho AB (2015) Marine extremophiles: a source of hydrolases for
biotechnological applications. Mar Drugs 13(4):1925–1965
Dangles O, Chauvet E (2003) Effects of stream acidification on fungal biomass in decaying beech
leaves and leaf palatability. Water Res 37:533–538
Dashtban M, Schraft H, Qin W (2009) Fungal bioconversion of lignocellulosic residues;
opportunities and perspectives. Int J Biol Sci 5:578–595
De Cassia Pereira J, Marques P, Rodrigues A, Oliveira TB, Boscolo R, da Silva R, Gomes E,
Martins DAB (2015) Thermophilic fungi as new sources for the production of cellulases and
xylanases with potential use in sugarcane bagasse saccharification. J Appl Microbiol 118:928–
939
De Hoog GS, Göttlich E, Platas G, Genilloud O, Leotta G, van Brummelen J (2005) Evolution,
taxonomy and ecology of the genus Thelebolus in Antarctica. Stud Mycol 51:33–76
Deacon LJ, Pryce-Miller EJ, Frankland JC, Bainbridge BW, Moore PD, Robinson CH (2006)
Diversity and function of decomposer fungi from a grassland soil. Soil Biol Biochem 38:7–20
Di Piazza S, Houbraken J, Meijer M, Cecchi G, Kraak B, Rosa E, Zotti M (2020) Thermotolerant
and thermophilic mycobiota in different steps of compost maturation. Microorganisms 8(6):880
Evans HC (1969) The effect of growth temperature on the fatty acid composition of fungi in the
order Mucorales. Canad J Microbiol 15:515–520
Free SJ (2013) Fungal cell wall organization and biosynthesis. Adv Genet 81:33–82
Fries L (1953) Factors promoting growth of Coprinus fimetarius (L.) under high temperature
conditions. Physiol P1 (Copenhagen) 6:551–563
Geoghegan I, Steinberg G, Gurr SJ (2017) The role of the fungal cell wall in the infection of plants.
Review Trends in Microbiol 25(12):957–967
Grant WD (2004) Life at low water activity. Philos Trans R Soc Lond B 359:1249–1267
Grasso S, La Feria R (1985) Further investigations on the occurrence of lignicolous fungi in the
brackish lake of Faro (Messina, Italy). Mem Biol Mar Oceanogr 7:233–243
4 Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations 91

Gross S, Robbins EI (2000) Acidophilic and acid-tolerant fungi and yeasts. Hydrobiologia 433:91–
109
Gulis V, Kuehn KA, Suberkropp K (2009) Fungi. In: Likens G (ed) Encyclopedia of inland waters,
vol 3. Elsevier, Oxford, pp 233–243
Gunde-Cimerman N, Zalar P, De Hoog GS, Plemenitas A (2000) Hypersaline waters in salterns –
natural ecological niches for halophilic black yeasts. FEMS Microbiol Ecol 32:235–240
Gunde-Cimerman N, Sonjak S, Zalar P, Frisvad JC, Diderichsen B, Plemenitas A (2003)
Extremophilic fungi in Arctic ice: a relationship between adaptation to low temperature and
water activity. Phys Chem Earth 28:1273–1278
Gunde-Cimerman N, Plemenitas A, Buzzini P (2014) Change in lipids composition and fluidity of
yeast plasma membrane as response to cold. In: Buzzini P, Margesin R (eds) Cold-adapted
yeasts. Springer, Berlin, pp 225–242
Harris P, James AT (1969) The effect of low temperatures on fatty acid biosynthesis in plants.
Biochem J 112:325–330
Huhe YC, Chen X, Hou F, Wu Y, Cheng Y (2017) Bacterial and fungal community structures in
loess plateau grasslands with different grazing intensities. Front Microbiol 8:606
Hyde KD, Goh TK (1998) Fungi on submerged wood in Lake Barrine, North Queensland,
Australia. Mycol Res 102:739–749
Ianutsevich EA, Danilova OA, Groza NV, Kotlova ER, Tereshina VM (2016) Heat shock response
of thermophilic fungi: membrane lipids and soluble carbohydrates under elevated temperatures.
Microbiology 162(6):989–999
Ianutsevich EA, Danilova OA, Kurilov DV, Zavarzin IV, Tereshina VM (2020) Osmolytes and
membrane lipids in adaptive response of thermophilic fungus Rhizomucor miehei to cold,
osmotic and oxidative shocks. Extremophiles 24(3):391–401
Johri BN, Rajani (1999) Mushroom compost: microbiology and application. In: Bagyaraj BJ,
Varma A, Khanna K, Kheri HK (eds) Modern approaches and innovations in soil management.
Rastogi Publications, Meerut, pp 345–358
Kane BE, Mullins JT (1973) Thermophilic fungi in a municipal waste compost system. Mycologia
65:1087–1100
Kates M, Baxter RM (1962) Lipid composition of mesophilic and psychrophilic yeasts (Candida
species) as influenced by environmental temperature. Canad J Biochem Physiol 40:1213–1227
Krauss GJ, Solé M, Krauss G, Schlosser D, Wesenberg D, Bärlocher F (2011) Fungi in freshwaters:
ecology, physiology and biochemical potential. FEMS Microbiol Rev 35:620–651
Latge JP (2007) The cell wall: a carbohydrate Armour for the fungal cell. Mol Microbiol 66:279–
290
Le Goff O, Bru-Adan V, Bacheley H, Godon JJ, Wéry N (2010) The microbial signature of aerosols
produced during the thermophilic phase of composting. J Appl Microbiol 108:325–340
Leite RSR, Alves-Prado HF, Cabral H, Pagnocca FC, Gomes E, DaSilva R (2008) Production and
characteristics comparison of crude β-glucosidases produced by microorganisms Thermoascus
aurantiacus,Aureobasidium pullulans in agricultural wastes. Enzym Microb Technol 43:391–
395
Lindt W (1886) Mitteilungen über einige neue pathogene Shimmelpilze. Arch Exp Pathol
Pharmakol 21:269–298
Loginova LG, Gerasimova NF, Seregina LM (1962) Requirement of thermo-tolerant yeasts for
supplementary growth factors. Microbiology 31:21–25
Lord AK, Vyas JM (2019) Host defenses to fungal pathogens. In: Clinical Immunology. Elsevier,
pp 413–424.e1
Macek I, Dumbrell AJ, Nelson M, Fitter AH, Vodnik D, Helgason T (2011) Local adaptation to soil
hypoxia determines the structure of an arbuscular mycorrhizal fungal community in roots from
natural CO
2
springs. Appl Environ Microbiol 77:4770–4777
Macek I, Vodnik D, Pfanz H, Low-Décarie E, Dumbrell AJ (2016) Locally extreme environments
as natural long-term experiments in ecology. In: Dumbrell AJ, Kordas R, Woodward G (eds)
92 R. S. Dass et al.

Largescale ecology: model systems to global perspectives. Advances ecological research, vol
55. Elsevier, pp 283–323
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol Rev 64:461–488
Miehe H (1930) U¨ ber die Selbsterhitzung des Heues. Arb Dtsch Landwirtsch-Gesellsch Berlin
111:76–91
Naranjo-Ortiz MA, Gabaldon T (2019) Fungal evolution: major ecological adaptations and evolu-
tionary transitions. Biol Rev 94:1443–1476
Moretti MMS, Bocchini-Martins DA, Da-Silva R, Rodrigues A, Sette L, Gomes E (2012) Selection
of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under
solid-state fermentation. Braz J Microbiol 43(3):1062–1071
Morgenstern I, Powlowski J, Ishmael N, Darmond C, Marqueteau S, Moisan M, Quenneville G,
Tsang A (2012) A molecular phylogeny of thermophilic fungi. Fungal Biol 116:489–502
Mouchacca J (1997) Thermophilic fungi: biodiversity and taxonomic status. Crypt Mycol 18:19–69
Mouchacca J (2000a) Thermophilicfungi and applied research: a synopsis of name changes and
synonymies. World J Microbiol Biotechnol 16:881–888
Mouchacca J (2000b) Thermotolerant fungi erroneously reported in applied research work as
possessing thermophilic attributes. World J Microbiol Biotechnol 16:869–880
Mumma RO, Fergus CL, Sekura RD (1970) The lipids of thermophilic fungi: lipid composition
comparisons between thermophilic and mesophilic fungi. Lipids 5:100–103
Nazareth S, Gonsalves V (2014) Aspergillus penicillioides –a true halophile existing in hypersaline
and polyhaline econiches. Front Microbiol 5:412
Niehaus F, Bertoldo C, Kahler M, Antranikian G (1999) Extremophiles as a source of novel
enzymes for industrial applications. Appl Microbiol Biotechnol 51:711–729
Noack K (1920) Der Betriebstoffwechsel der thermophilen Pilze. Jahrb Wiss Bot 59:593–648
Oberson J, Rawyler A, Brandle R, Canevascini G (1999) Analysis of the heat-shock response
displayed by two Chaetomium species originating from different thermal environments. Fungal
Genet Biol 26:178–189
Oliveira TB, Rodrigues A (2019) Ecology of thermophilic fungi. In: Fungi in extreme
environments: ecological role and biotechnological significance, pp 39–57
Oliveira TB, Gomes E, Rodrigues A (2015) Thermophilic fungi in the new age of fungal taxonomy.
Extremophiles 19:31–37
Oliveira TB, Gostinčar C, Gunde-Cimerman N, Rodrigues A (2018) Genome mining for peptidases
in heat-tolerant and mesophilic fungi and putative adaptations for thermostability. BMC Geno-
mics 19:152
Orandi S, Yaghubpur A, Sahraei H (2007) Influence of AMD on aquatic life at Sar Cheshmeh
coppermine. In: Abstract Goldschmidt Conference, Cologne, August 2007
Osumi M (1998) The ultrastructure of yeast: cell wall structure and formation. Micron 29:207–233
Pan WZ, Huang XW, Wei KB, Zhang CM, Yang DM, Ding JM, Zhang KQ (2010) Diversity of
thermophilic fungi in Tengchong Rehai National Park revealed by ITS nucleotide sequence
analyses. J Microbiol 48:146–152
Paterson RRM, Lima N (2017) Thermophilic fungi to dominate aflatoxigenic/mycotoxigenic fungi
on food under global warming. Int J Environ Res Public Health 14:199
Piao S, Friedlingstein P, Ciais P, Zhou L, Chen A (2006) Effect of climate and CO2 changes on the
greening of the Northern hemisphere over the past two decades. Geophy Res Lett 33(L23402):
1–6
Plemenitas A, Vaupotic T, Lenassi M, Kogej T, Gunde-Cimerman N (2008) Adaptation of
extremely halotolerant black yeast Hortaea werneckii to increased osmolarity: a molecular
perspective at a glance. Stud Mycol 61:67–75
Powell AJ, Parchert KJ, Bustamante JM, Ricken JB, Hutchinson M, Natvig DO (2012) Thermo-
philic fungi in an aridland ecosystem. Mycologia 104:813–825
Prasad ARS, Kurup CKR, Maheshwari R (1979) Effect of temperature on respiration of a
mesophilic and thermophilic fungus. Plant Physiol 64:347–348
4 Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations 93

Raja HA, Miller AN, Pearce C, Oberlies NH (2017) Fungal identification using molecular tools: a
primer for the natural products research community. J Nat Products 80(3):1–17
Rajasekaran AK, Maheshwari R (1990) Effect of growth temperature on lipid composition of a
thermophilic fungus Thermomyces lanuginosus. Indian J Exp Biol 28:134–137
Rajasekaran AK, Maheshwari R (1993) Thermophilic fungi: an assessment of their potential for
growth in soil. J Biosci 18(3):345–354
Reddy GV, Ravindra Babu P, Komaraiah P, Roy KRRM, Kothari IL (2003) Utilization of banana
waste for the production of lignolytic and cellulolytic enzymes by solid substrate fermentation
using two Pleurotus species (P.ostreatus and P.sajor-caju). Process Biochem 38:1457–1462
Rouphael Y, Franken P, Schneider C, Schwarz D, Giovannetti M, Agnolucci M, De Pascale S,
Bonini P, Colla G (2015) Arbuscular mycorrhizal fungi act as bio-stimulants in horticultural
crops. Sci Hortic 196:91–108
Salar TK, Aneja KR (2007) Thermophilic fungi: taxonomy and biogeography. J Agric Technol
3(1):77–107
Samalova M, Melida H, Vilaplana F, Bulone V, Soanes DM, Talbot NJ, Gurr SJ (2016) The
β-1,3-glucanosyltransferases (gels) affect the structure of the rice blast fungal cell wall
duringappressorium-mediated plant infection. Cell Microbiol 19(e12659):1–14
Sanchez C (2009) Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnol
Adv 27:185–194
Seifert KA, Samson RA, Boekhaut T, Louis-Seize G (1997) Remersonia, a new genus for Stilbella
thermophila, a thermophilic mould from compost. Canad J Bot 75:1158–1165
Sekura RD, Fergus CL (1971) Thermophilic fungi: II, fatty acid composition of polar and neutral
lipids of thermophilic and mesophilic fungi. Lipids 6:584–588
Shearer CA, Raja HA, Miller AN, Nelson P, Tanaka K, Hirayama K, Marvanova L, Hyde KD,
Zhang Y (2009) The molecular phylogeny of freshwater Dothidiomycetes. Stud in Mycol 64:
145–153
Sibanc N, Zalar P, Schroers HJ, Zajc J, Pontes A, Sampaio JP, Maček I (2018) Occultifur mephitis
fa, sp. nov. and other yeast species from hypoxic and elevated CO
2
mofette environments. Intl
JSyst Evol Microbiol 68(7):2285–2298
Sinensky M (1974) Homeoviscous adaptation—a homeostatic process that regulates the viscosity
of membrane lipids in Escherichia coli. Proc Natl Acad Sci U S A 71:522–525
Singh B, Pocas-Fonseca MJ, Johri BN, Satyanarayana T (2016) Thermophilic molds: biology and
applications. Crit Rev Microbiol 42:1–22
Sonjak S, Frisvad JC, Gunde-Cimerman N (2007) Genetic variation among Penicillium crustosum
isolates from the arctic and other ecological niches. Microb Ecol 54:298–305
Sonjak S, Frisvad JC, Gunde-Cimerman N (2009) Fingerprinting using extrolite profiles and
physiological data shows sub-specific groupings of Penicillium crustosum strains. Mycol Res
113:836–841
Stevenson A, Burkhardt J, Cockell CS, Cray JA, Dijksterhuis J, Fox-Powell M (2015) Multiplica-
tion of microbes below 0.690 water activity: implications for terrestrial and extraterrestrial life.
Environ Microbiol 2:257–277
Sumner JL, Morgan ED (1969) The fatty acid composition of sporangiospores and vegetative
mycelium of temperature-adapted fungi in the order Mucorales. J Gen Microbiol 59:215–221
Trent JD, Gabrielsen M, Jensen B, Neuhard J, Olsen J (1994) Acquired Thermotolerance and heat
shock proteins in thermophiles from three phylogenetic domains. J Bacteriol 176(19):
6148–6152
Tsiklinsky P (1899) Sur les mucédinées thermophiles. Ann Inst Pasteur 13:500–505
Van Noort V, Bradatsch B, Arumugam M, Amlacher S, Bange G, Creevey C, Falk S, Mende DR,
Sinning I, Hurt E, Bork P (2013) Consistent mutational paths predict eukaryotic thermostability.
BMC Evol Biol 13:7
Whittaker RH (1959) On the broad classification of organisms. Q Rev Biol 34:210–226
94 R. S. Dass et al.

Whittaker RH (1969) New concepts of kingdoms of organisms. Science 163:150–160
Wright C, Kafkewitz D, Somberg EW (1983) Eucaryote thermophily: role of lipids in the growth of
Talaromyces thermophilus. J Bacteriol 156:493–497
Zhang XY, Zhang Y, Xu XY, Qi SH (2013) Diverse deep-sea fungi from the South China Sea and
their antimicrobial activity. Curr Microbiol 67:525–530
Zhou P, Zhang G, Chen S, Jiang Z, Tang Y, Henrissat B, Yan Q, Yang S, Chen CF, Zhang B, Du Z
(2014) Genome sequence and transcriptome analyses of the thermophilic zygomycete fungus
Rhizomucor miehei. BMC Genomics 15:294
4 Thermophilic Fungi: Habitats and Morpho-Molecular Adaptations 95

Modulation of Physiological and Molecular
Switches in Thermophilic Fungi: A Brief
Outlook
5
Tuyelee Das, Samapika Nandy, Abdel Rahman Al-Tawaha,
Potshangbam Nongdam, Ercan Bursal, Mahipal S. Shekhawat,
and Abhijit Dey
Abstract
Thermophiles are significantly important microorganisms utilize in biotechnolog-
ical research and industrial purposes. Thermophiles can grow at a temperature
ranges between 45 and 80 C. The survival of thermophilic fungi at such
conditions induced by genomic evolution and natural selection that in turn
gives thermophiles the thermotolerant abilities. Like other thermophilic
organisms, thermophilic fungi get inclusive attention due to their potential to
produce thermostable enzymes. Research studies mostly demonstrated physio-
logical aspects of thermophilic fungi based on their nutrition requirement, metab-
olism, growth, and their interaction with the environmental factors. Furthermore,
investigating molecular features such as genome size, protein, and nucleotides
screening is likewise significant. Moreover, the physiological and molecular
study gives us a concise idea about thermophilic fungi adaptation strategies and
parameter to sustain in an extreme environmental condition. Therefore, in this
chapter, we endeavor to convey thermophilic fungi physiological and molecular
highlights about adaptation to elevated temperature.
T. Das · S. Nandy · A. Dey (*)
Department of Life Science, Presidency University, Kolkata, West Bengal, India
e-mail: abhijit.dbs@presiuniv.ac.in
A. R. Al-Tawaha
Department of Biological Sciences, Al-Hussein Bin Talal University, Maan, Jordon
P. Nongdam
Department of Biotechnology, Manipur University, Imphal, Manipur, India
E. Bursal
Department of Biochemistry, Mus Alparslan University, Muş, Turkey
M. S. Shekhawat
Biotechnology Unit, Kanchi Mamunivar Government Institute for Postgraduate Studies and
Research, Puducherry, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_5
97

Keywords
Thermophilic fungi · Adaptations · Temperature · Molecular biology
5.1 Introduction
Thermophilic means heat-loving microorganisms that can be demarcated as hyper-
thermophile (>80 C), thermophilic (45–80 C), and mesophilic (<45 C). Most
hyperthermophiles are confined to the Archaea domain, whereas thermophilic
organisms belong to the Bacteria, Archaea, and Eukarya domain. A small number
of fungi can sporulate at 45–55 C. Thermophilic fungi cannot grow in as extreme as
bacteria and archaea can grow, excepts only a few species can grow at 113 C
(Blochl et al. 1997). Consequently, it gets important to see how thermophilic fungi
can grow in such extreme temperatures, while most living creature can’t endure.
Thermophily in fungi isn’t just about as outrageous as in eubacteria or archaea, a few
types of which can develop close or above 100 C in solfatara fields, hydrothermal
vents, or thermal springs (Brock 1995; Blochl et al. 1997). Definition for thermo-
philic organisms (bacteria and archaea) cannot embrace the same for thermophilic
fungi because temperature tolerance for them differs. According to Maheshwari et al.
(2000), fungi can grow optimally at or above 45 C called thermophilic fungi
(Maheshwari et al. 2000). Cooney and Emerson (1964) characterize fungi growing
between 50 Cand20 C temperature as considered thermophilic (Cooney and
Emerson 1964). Thermophilic fungi have an immense role in degrading organic
matter, production industrial production in extracellular and intracellular enzyme,
antibiotics, and organic acids. Maybe due to their moderate level of thermophily and
on the grounds that their habitats are not fascinating, thermophilic fungi have not
gotten a lot of exposure and attention. Additionally, research on this category of
extremophiles has been ignored. Moreover, uncertainty in taxonomic affiliation and
misidentification leading thermophilic fungi places them in a state of confusion
(Madigan and Orient 1999; Stetter et al. 1990).
For the industrial production of enzymes, one of the problems is the maintenance
of temperature during the entire farming procedure at an optimal level. Hence, the
utilization of thermophilic strains of fungi can be a real solution, so the study of their
thermostability related to genome or molecular level functioning is important. The
study of genetic engineering is an exceeding recent development. In different
environment conditions, thermophilic fungi act differently, but the research related
to this topic is scant. If we can understand the physiology of thermophilic fungi, we
undoubtedly use them for industrial production. Therefore, this chapter aims to
compile data from the past on whatever little information is available on the
physiology of thermophilic fungi that will assist with improving industrial produc-
tion. Figure 5.1 represents physiological and molecular adaptive features of thermo-
philic fungi. This chapter also focuses on the understanding of the genome structure,
size, and thermostable genes concerning thermostability.
98 T. Das et al.

5.2 Physiology of Thermophilic Fungi
5.2.1 Nutrition Requirements
Thermophilic fungi do not need any special nutrition for their growth. They can
grow in media composed of basic micronutrients and macronutrients. For the
growth, reproduction and maintenance of basic biosynthetic pathway, thermophilic
fungi need a constant supply of nutrients. Earlier, it was assumed that thermophilic
fungi require complex nutrition, but now, it is clear that they need simple media for
their growth contains carbon, nitrogen, and salts (Maheshwari et al. 2000). Addi-
tionally, it is known that they are autotrophic fungi.
Many thermophilic fungi observed to utilize polysaccharides (cellulose, hemicel-
lulose, starch, pectin, and lignin) as a carbon source to grow efficiently (Basu 1980;
Deploey 1976a,b; Satyanarayana and Johri 1984). Chauhan et al. (1985) revealed
Torula thermophila,Sporotrichum thermophile,Thermomucor indicae-seudaticae,
and Thermoascus aurantiacus can grow on methanol and formate (Chauhan et al.
1985). T. aurantiacus observed to grow densely on dulcitol, mannitol, oxalic acid,
and citric acid; however, Scytalidium thermophilum reported deprived growth on
oxalic and citric acids, with no growth on dulcitol (Subrahmanyam 1977). Thermo-
philic fungi Thermomyces lanuginosus and Penicillium duponti utilized a mixed
source (combination of glucose and sucrose) of nutrition at 50 C. The growth rate of
these two fungi is more in mixed culture rather than single carbon source culture,
Fig. 5.1 Physiological and molecular adaptative features of thermophilic fungi
5 Modulation of Physiological and Molecular Switches in Thermophilic Fungi:... 99

indicating that the two sugars proportionally affected their use in the combination.
The two sugars were likewise used simultaneously at 30 C but at almost indistin-
guishable rates. Combination of glucose and sucrose was picked on the grounds that
concentration of glucose and sucrose can easily determine in the medium utilizing
commercially available enzymes (Maheshwari and Balasubramanyam 1988). Ther-
mophilic fungi utilize amino acids as a nitrogen source was investigated.
Subrahmanyam (1980) reported poor growth and sporulation of T. aurantiacus in
the presence of magnesium nitrate, glycine, peptone, thiourea, L-serine,
DL-phenylalanine, DL-leucine, DL-alanine, and ammonium acetate
(Subrahmanyam 1980).
Satyanarayana and Johri (1984) revealed that nitrates of Na and K are a better
source of nitrogen than ammonium nitrate and sulfate (Satyanarayana and Johri
1984). Rosenberg in 1975 examined 21 species of thermophilic and thermotolerant
fungi growth in a glucose-containing mineral medium complemented with 0.01%
yeast extract. He reported that eight species required 0.01% yeast extract for growth
on a solid complex medium (Rosenberg 1975). Wali et al. (1978) reported organic
acid succinate needed in a concentration-dependent manner for the growth of
thermophilic fungi Humicola lanuginosa, P. duponti, S. thermophile, and Mucor
pusillus in addition to glucose media. The growth of these fungi stimulated because
of their buffering action. They also observed glucose utilization increase in the
presence of succinate (Wali et al. 1978). In contrast to Wali et al. (1978), Gupta
and Maheshwari (1985) reported that for the growth of T. lanuginosus, succinate is
not essential in addition to the need for organic acid that could be deleted if the pH of
the medium can properly maintain (Gupta and Maheshwari 1985). Oxygen with-
drawal from the growing medium of thermophilic fungus severely affected their
growth. Emerson 1968 reported that Humicola insolens grow better under anaerobic
conditions at elevated temperatures (Emerson 1968). Cooney and Emerson (1964)
detected that thermophilic Talaromyces duponti initiated conidial stage in aerobic
cultures, whereas the sexual stage was formed in agar culture only (Cooney and
Emerson 1964).
5.2.2 Growth and Metabolism
The growth of filamentous thermophilic fungi is complex and unpredictable. Aerial
hyphae from filamentous fungi get nutrition when mycelium interacts with the
medium. For that, nutrient transport needs more time to cover the distance from
mycelium to aerial hyphae. Therefore, continuous supply of nutrient source needs to
be maintained for the rapid growth of thermophilic fungi, mostly essential for
industrial purposes. T. aurantiacus grown at 45 C in minimal medium utilized
55% of sugars for the synthesis of growth and biomass production and 45% for
metabolism. Polysaccharide is utilized by thermophilic fungi so that they can
produce cellulose and hemicellulose. The fungi Chaetomium thermophilum and H.
insolens were recorded to grow better when they utilized xylan (Chang 1967).
However, later Bhat and Maheshwari (1987) informed that S. thermophile fungus
100 T. Das et al.

can grow on both cellulose and glucose identically (Bhat and Maheshwari 1987).
The growth rate of the fungus on cellulose was indistinguishable from that on
glucose uncovering the remarkable capacity of growth of this fungus to utilize
cellulose as efficiently as glucose (Gaikwad and Maheshwari 1994). Thermophilic
fungi growth rates increase in shake flasks than in static cultures (Prasad and
Maheshwari 1978). Similarly, Chaetomium cellulolyticum boomed in broth
containing 15% glucose with a growth rate of 0.14 per hour (Chahal and
Hawksworth 1976). Additionally, in 1978, Chahal and Wang displayed that
C. cellulolyticum can synthesized protein with 1% cellulose concentration in 0.09
per hour, where additional 1% cellulose in the medium help to synthesized protein
rapidly in just 0.3 per hour (Chahal and Wang 1978).
5.2.3 Lipids and Fatty Acids Composition
Thermophilic fungi grow at such temperatures at which most of the fungi cannot live
the required course of thermostability and permeability of the membranes. The lipid
components are mainly responsible for membrane stability at high temperature as
most of the cellular events are membrane linked. In low temperature, thermophiles
cannot produce unsaturated fatty acids so they are unable to grow. A low amount of
phosphatidic acid was reported in Malbranchea pulchella var. sulfurea and Absidia
ramose, while a very high level of phosphatide was observed in H. lanuginosa and
M. pulchella var. sulfurea. All the mentioned fungi contained fatty acids,
monoglycerides, diglycerides, and triglycerides, whereas sterols are detected in
A. ramose (Raju et al. 1976). A low amount of phosphatidic acid also is found in
Acremonium alabamensis and T. indicae-seudaticae. The lipid content of dry
mycelium of A. alabamensis ranged from 2.6% to 7.3% and of T. indicae-seudaticae
ranged from 8.5% to 13.0%. During growth, neutral lipid increased,
whereasphospholipids and polar lipids decreased (Satyanarayana et al. 1987).
Mumma et al. (1971a,b) compared neutral lipids and the fatty acid profile between
thermophilic and mesophilic fungi and reported thermophiles produced more
saturated neutral lipids than mesophiles (Mumma et al. 1971a). Mumma et al.
(1970) reported the same that the fatty acids of thermophilic fungi M. pusillus are
more saturated than mesophilic Mucor globosus. They also reported the presence of
palmitic, oleic, and linolenic in both species with considerable and no amount of
linolenic acid in mesophile and thermophile, respectively (Mumma et al. 1970). The
polar lipids of the thermophilic fungus Humicola grisea var. thermoidea consisted of
38.4–42.3%. Polar lipids consist of sterol glycosides and neutral lipids consist of
tri-glycerides, free fatty acids, sterols, sterol esters, and diglycerides (Mumma et al.
1971b). The unsaturated index in T. thermophila decreased as the temperature of
culture increased (Bruszewski et al. 1972). Wright et al. (1983) examined and
observed regulation of inability in membrane fluidity at high minimum temperature.
They reported shifting of growth temperature of Talaromyces thermophilus from
50 to 33 C was unable to increase the degree of unsaturation of fatty acids. Addition
of palmitic acids, oleic acids, stearic acids, and ergosterol induced fatty acid
5 Modulation of Physiological and Molecular Switches in Thermophilic Fungi:... 101

synthesis, which was before inhibited by the antibiotic cerulenin (Wright et al.
1983). However, Rajasekaran et al. noticed that growth of T. lanuginosus was
different in high (50 C) and low (30 C) temperature. Linoleic acid synthesis
increased at low temperature than at high temperature (Rajasekaran and Maheshwari
1990).
5.2.4 Ultrastructure
Thermophilic fungi reported to developed special dense vesicles for storage. A
unique papillate germ pore and pustulate surface ornamentation in C. thermophile
fungus are reported to be a notable ultrastructural feature compared with other
species of Chaetomium.Chaetomium semen-citrulli ascospores contained vacuoles,
many nuclei, mitochondria, and glycogen-like particles (Millner et al. 1977).
Another species Chaetomium brasiliense reported forming a typical ascus vesicle
close to the plasma membrane of asci (Rosing 1982).
In T. aurantiacus, subcellular events of ascosporogenesis take place. Events like
ascogenous hyphae formation, ascospores delimination, cell-wall formation between
ascospore-delimiting membranes, electron-transparent endospore, and an electron-
dense epispore were observed (Ellis 1981a). Perithecia are reported to be a remark-
able ultrastructural feature of Chaetomiun thermophile. It was revealed that
two-layered peridium formation may be a structural adaptation of Ascomycotina
fungi that defends the ascocarp centrum from desiccation (Ellis 1981c). Similarly,
two-layered peridium is also found in T. aurantiacus. Ascocarps of T. aurantiacus
was also reported to consist of well-defined cleistothecia (Ellis 1981b).
Furthermore, ultrastructural features of lipid bodies have been observed in
T. thermophilus (Bokhary et al. 1988). Goh and Hanlin (1998) reported in
Melanospora zamiae cell two morphologically distinct types of mitochondria such
as D-type and T-type that were present in different ascomal development stages. A
unique type of vesicles is found in M. zamiae cells at the distal end of the elongated
ascus (Goh and Hanlin 1998). Similarly, two different spore types such as type
1 conidia and type 2 chlamydospores reported in thermophilic H. insolens (Ellis
1982). Three species of hyphomycete T. lanuginosus,T. stellatus,and
T. ibadanensis reported developed thick-walled and terminal holoblastic conidia.
The presence of numerous dense body vacuoles and the formation of holoblastic
conidia are also reported. Vacuoles are observed only when T. lanuginosus,
T. stellatus,and T. ibadanensis grew in not only high temperature (52 C) but also
low temperature (40 C) (Ellis 1981d). Similarly, Thermomyces stellatus,
T. lanuginosa,Humicola insolans, and H. grisea var. thermoidea have been reported
to contain membrane-bound inclusion bodies in the cytoplasm when grown at high
temperatures (Garrison et al. 1975).
102 T. Das et al.

5.2.5 Pigments
The pigment formation gives the protection of several organisms under heat stress
condition. The pigmentation range differs depending upon temperature and age.
Some thermophilic fungi, T. duponti,T. aurantiacus,Malbranchea pulchella var.
sulfurea,T. lanuginosus, and H. grisea var. thermoidea, produce melanin pigments
in cell walls of ascocarp, ascospores, peridia, and conidia. By spectrometric studies,
it was reported that pigments were found to be similar to aphins (Somasundaram
et al. 1986). The pigment formation is completely sensitive to elevated temperature.
Mucor miehei sporulated at 35 C but produced a brown pigment after longer
incubation at 35 C (Lasure and Ingle 1976).
5.2.6 Abiotic Factors and Growth of Thermophilic Fungi
Thermophilic fungi, which grow at or above 50 C, are significant for the decompo-
sition of organic matter (Cooney and Emerson 1964) and mushroom compost
(Cooney and Emerson 1964; Fergus 1964; Renard and Cailleux 1972). Some
excellent sources are Talaromyces thermophilus,H. insolens,H. grisea,
S. thermophile,M. miehei, and Papulaspora for the investigation of the effects of
temperature (Chapman 1974). At 40 and 45 C, fungi grew fastest, whereas at
30, 35, 50, and 55 C, slower rates of fungi occurred. All of the species of fungi
formed spores with different temperature range (Chapman 1974). M. miehei pro-
duced zygospores after incubation for 51 h at 35 C, but not at 46 C in liquid
medium (Lasure and Ingle 1976). Chaetomium thermophile var. dissectum and one
strain of C. thermophile var. coprophile have shown a more restricted temperature
range for sexual and asexual reproduction; vegetative growth of thermophilic fungi
takes place in a wider temperature range (Tansey 1972). The mitochondria of
Talaromyces sp. contained higher amounts of oleic acid (39%) and palmitic acid
(30%) but considerably less amount of linoleic acid (10%) when grown at high
temperature (50 C). The higher amount of saturated fatty acids and lower unsatu-
rated fatty acids production is correlated with higher temperature. Mitochondrial
membranes of mesophilic and thermophilic fungi possess significant difference
(Asundi et al. 1974). The respiratory rates of thermophile T. lanuginosus reported
to be highly sensitive to temperature changes, but not in mesophile A. niger, where
respiration rates were insensitive to temperature changes (Prasad et al. 1979).
Complex nutrients contained factors cannot synthesize at the low temperatures.
Humicola,Thermoascus, and Aspergillus when incubated in liquid medium included
complex supplements at 20 C after ten days; they showed good growth, but they did
not grow below 30 C lacking any complex supplements (Maheshwari et al. 1987).
Wright et al. (1983) supported the observation of growth of T. thermophilus fungi at
33 C, which was helpful by supplemented culture medium with ergosterol (Wright
et al. 1983). Most of the thermophilic fungi survive exposure to 60 C for 1–48 h
observed by Satyanarayana and Johri (1984). Besides temperature, pH range is
another abiotic factor that influences the growth of thermophilic fungi. Rosenberg
5 Modulation of Physiological and Molecular Switches in Thermophilic Fungi:... 103

(1975) studied pH optima of 21 fungi and reported that the pH optima of
T. lanuginosus,Malbranchea cinnamomea, and T. thermophilus was found to be
near neutral (pH 7), whereas Talaromyces emersonii and Allescheria terrestris
optimally grew in an acidic environment (pH 3.4–6.0). Additionally, it observed
that between pH and temperature, no correlation was found (Rosenberg 1975). In
contrast, the temperature change is directly correlated with water availability. As
temperature increases, water availability also decreases. Humicola is a xerophilic
strain of thermophilic fungus found in the Thar Desert reported to produce higher
proline and sterol to overcome desiccation stress (Mahajan et al. 1986). For the
optimum growth of thermophilic fungi, sufficient humidity required. C. thermophile
var. coprophile ascospores survive longer in a dry state and stored at low relative
humidity with high temperatures than in water or stored dry at high relative humidity
(Celerin and Fergus 1971).
5.3 Acquired Thermotolerance
Acquired thermotolerance in fungi refers to the enhanced survival in lethal
temperatures subsequent exposure to sublethal high temperatures. Acquired
thermotolerance related with synthesis of heat-shock proteins (HSPs) under temper-
ature stress. Normal growth condition HSPs act as a molecular chaperone. All
organisms included fungi have grew in such a way that they developed a variety
of strategies for surviving in elevated temperature, and among these strategies, HSP
synthesis is one of them. Bacteria, Archaea, and Eucarya have been shown to
develop acquired thermotolerance by synthesizing HSPs after heat shock.
Trentet al. observed T. lanuginosus germinated at 50 C and heat shocked at
55 C for 60 min showed enhanced survival at 58 C. The synthesis of small
HSPs ranging from 31 to 33 kDa notably dominated in the heat-shock temperatures.
Thermotolerance in T. lanuginosus was removed by the effects of cycloheximide
when added during the heat shock period that subsequently inhibit protein synthesis
(Trent et al. 1994). Oberson et al. (1999) detected a transient HSP synthesis lasting
about 60 min in C. thermophile. Translation of HSPs stopped beyond 56 C
(Oberson et al. 1999).
5.4 Molecular Features
The molecular biology of thermophilic fungi has gained attention to molecular
structure and functioning of cell linked adaptation to extreme conditions. Molecular
genetic functioning has been effectively studied in thermophilic bacteria and
archaea, and little information is available on the thermophilic fungi.
104 T. Das et al.

5.4.1 Reduction in Genome Size
Thermophilic fungi genome size is small as compared with other fungi. Reduction of
genome size is one of the strategies to sustain in high temperature. The compact
genome is an interesting feature, as it helps in fast reproduction and reduces energy
consumption. Thermophiles living at a temperature of >60 C reported having
smaller genomes (4 Mb) than those species which grow at temperatures of <45 C
with larger genomes 6 Mb (Van Noort et al. 2013). Van Noort et al. (2013) revealed
some common strategies of thermophilic fungi Chaetomium thermophilum,
Thielavia terrestris, and Thielavia heterothallica. They found that a certain amino
acid residue for a specific protein contribute to thermostability. The steadiest pattern
showed the substitution of lysine by arginine (Van Noort et al. 2013). They also
compared the genome of those three thermophilic fungi to mesophilic fungi
Chaetomium globosum and Neurospora crassa and observed that the genome size
of mesophilic fungi is smaller than that of mentioned three thermophilic fungi.
Reduced genome size is due to fewer protein-coding genes and shorter introns and
intergenic regions (Van Noort et al. 2013). Besides those strategies, horizontal gene
transfer could be another evolutional strategy of thermophilic fungi to sustain in such
an extreme environment (Averhoff and Müller 2010).
5.4.2 GC Content
Thermophiles have a much more stable genomic structure than mesophiles. Higher
genome stability is correlated with higher guanine (G) and cytosine (C) content that
related to an adaptation to high temperature of the protein-coding genes of thermo-
phile. The genome sequence study of thermophilic M. thermophila and T. terrestris
when compared with the mesophile C. globosum and T. reesei showed higher GC
contents in thermophilic fungi coding regions in the genomes (Berkeley and Cancer
2011). Zygomycete fungus Rhizomucor miehei CAU432 exhibited a genome size of
27.6 Mb with a 43.8% GC content of fungal whole genome (Zhou et al. 2014). Other
thermophilic fungi T. lanuginosus, T. terrestris, and M. thermophila content are
52.14%, 54.7%, and 51.4% of GC, respectively, which reported to much higher than
other zygomycetes mesophilic fungi (Berkeley and Cancer 2011; Mchunu et al.
2013; Van Noort et al. 2013). The GC content of the whole genome of
T. lanuginosus Strain SSBP was 52.14% with higher GC content in the coding
region 55.6%. Thermal adaptation of T. lanuginosus fungus was associated with
DNA-related pathways. Activation of the ubiquitin degradation pathway in
T. lanuginosus is related to several stresses (Mchunu et al. 2013). On the other
hand, Zeldovich et al. (2007) revealed that the GC content in genomes does not
exhibit a significant correlation with optimum growth temperature. However, the
content of AG in the coding region of DNA is significantly correlated with optimum
growth temperature due to IVYWREL (Ile, Val, Tyr, Trp, Arg, Glu, and Leu) amino
acids (Zeldovich et al. 2007).
5 Modulation of Physiological and Molecular Switches in Thermophilic Fungi:... 105

5.4.3 Proteome
The amino acid arrangement of proteins of thermophilic organisms is related to their
adaptations to extreme environments. Proteome studies of thermophilic
C. thermophilum,T. terrestris, and T. heterothallica fungi were to provide new
paths to protein engineering related to stability. They reported that an enhanced level
of cysteines in C. thermophilum might be related to thermophily by forming
disulfide bridges and metal binding. A higher frequency of IVYWREL amino
acids was observed in C. thermophilum than in its mesophilic counterpart.
IVYWREL is observed to be related to optimum growth temperature and is also
related to the survival at higher temperatures (Van Noort et al. 2013). Lowering
glycine is correlated with acquiring thermostability. Thermophilic C. thermophilum
lowers glycine content, whereas mesophilic C. globosum has changed in the reverse
direction (Van Noort et al. 2013). A higher amount of IVYWREL, depletion of
glycine, and elevation of arginine and alanine likewise are observed in
T. lanuginosus and T. thermophilus when compared with the same subclass
mesophilic fungi Aspergillus fumigatus and Emericella nidulans (Van Noort et al.
2013). During translation, cysteine, lysine, and all the amino acids of IVYWREL are
involved with tRNA by class I aminoacyl tRNA synthetases and thus associated with
thermostable proteins synthesis (Eriani et al. 1990).
5.4.4 Genes Responsible for Thermostability
Until now, several numbers of genes have been isolated and screened from different
thermophilic fungi. β-Glucosidase is an important cellulose hydrolyzing enzyme that
has been widely utilized in several industrial fields. Zhao et al. (2015) expressed
mtbgl3b β-glucosidase of glycoside hydrolase (GH family 3) gene from
Myceliophthora thermophila in Pichia pastoris and reported properties of mtbgl3b
genes. Recombinant MtBgl3b was reported to show thermostability after incubated
at 60 and 65 C (Zhao et al. 2015). β-Glucosidase from T. aurantiacus fungus also
showed thermostability on semisolid fermentation medium (De Palma-Fernandez
et al. 2002). β-Glucosidase (cel3a) genes from Talalaromyces emersonii was
reported to show thermostability at 71.5 C after expressed in Trichoderma reesei
with a strong cbh1 promoter (Murray et al. 2004). Tachit1 and Ctchit1 chitinase
genes have been isolated from T. aurantiacus var. levisporus and C. thermophilum,
respectively, and expressed in P. pastoris. Single domain protein TaCHIT1 of
Tachit1 gene showed lower thermostability than CtCHIT1 of Ctchit1 gene, which
makes CtCHIT1 protein potentially suitable for chitin hydrolysis at high
temperatures (Li et al. 2010).
106 T. Das et al.

5.5 Conclusion
The biology of thermophiles has acquired consideration and anticipated the compre-
hension of adaptation at the molecular level. Thermophilic fungi grown at high
temperature have greater saturated lipids, palmitic, oleic and linolenic palmitic, oleic
and linolenic acid, and a low amount of phosphatidic acid. They can grow in basic
media containing amino acids, organic acids, etc. Sometimes, they grow in a
concentration-dependent manner. Ultrastructure study uncovered that thermophilic
fungi produced well-defined epispore, special dense vesicles, cleistothecia, and
membrane-bound inclusion bodies. The molecular biology of thermophiles has
recently gained attention both in terms of adaptation to temperature and the devel-
opment of manipulative genetic tools. Their genetic study discloses reduced high
G/C content, genomic size, shorter introns, enhanced level of cysteines, and
IVYWREL amino acids that exhibit adaptations. Future research on genetic engi-
neering and biotechnology related to thermophilic fungi is essential insight into
thermophilic fungi role in biology, ecology, and in economic importance, in addition
to understanding their ability to grow at high temperatures.
Acknowledgments Authors are extremely thankful to UGC, Government of India, for financial
assistance. Authors are highly grateful to Presidency University-FRPDF fund, Kolkata for
providing needed research facilities.
References
Asundi S, Mattoo AK, Modi VV (1974) Studies on a thermophilic Talaromyces Sp.: mitochondrial
fatty acids and requirements for a C4 compound. Indian J Microbiol 14:75–80
Averhoff B, Müller V (2010) Exploring research frontiers in microbiology: recent advances in
halophilic and thermophilic extremophiles. Res Microbiol 161:506–514. https://doi.org/10.
1016/j.resmic.2010.05.006
Basu PK (1980) Production of chlamydospores of Phytophthora megasperma and their possible
role in primary infection and survival in soil. Can J Plant Pathol 2:70–75
Berkeley L, Cancer B (2011) Comparative genomic analysis of the thermophilic biomass-degrading
fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29:922–927
Bhat KM, Maheshwari R (1987) Sprotrichum thermophile, growth, cellulose degrada- tion, and
cellulase activity. Appl Environ Microbiol 53:2175–2182
Blochl E, Rachel R, Burggraf S, Hafenbradl D, Jannasch HW, Stetter KO (1997) Pyrolobus fumarii,
gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for
life to 113C. Extremophiles 1:14–21
Bokhary HA, Abu-Zinada AH, Kunji ZA (1988) Biochemical and ultrastructural studies of
Penicillium chrysogenum, Talaromyces leycettanus and T. thermophilus. Folia Microbiol
(Praha) 33:29–33. https://doi.org/10.1007/BF02928010
Brock TD (1995) The road to Yellowstone—and beyond. Annu Rev Microbiol 49:1–28
Bruszewski TE, Fergus CL, Mumma RO (1972) Thermophilic fungi: IV. The lipid composition of
six species. Lipids 7:695–698. https://doi.org/10.1007/BF02533117
Celerin EM, Fergus CL (1971) Effects of nutrients, temperature and relative humidity on germina-
tion and longevity of the ascospores of Chaetomium thermophile Var. coprophile. Mycologia
63:1030–1045. https://doi.org/10.1080/00275514.1971.12019200
5 Modulation of Physiological and Molecular Switches in Thermophilic Fungi:... 107

Chahal DS, Hawksworth DL (1976) Chaetomium cellolyticum, a new thermotolerant and cellulo-
lytic chaetomium. Mycologia 68:600–610
Chahal DS, Wang WIC (1978) Chaetomium cellulolyticum growth behaviours on cellulose and
protein production. Mycologia 70:160–170
Chang Y (1967) The fungi of wheat straw compost II. Biochemical and physiological studies. Trans
Brit Mycol Soc 50:667–677
Chapman ES (1974) Effect of temperature on growth rate of seven thermophilic fungi. Mycologia
66:542–546. https://doi.org/10.1080/00275514.1974.12019640
Chauhan S, Prakash A, Satyanarayana T, Johri BN (1985) Utilization of C-1 compounds by
thermophilic fungi. Natl Acad Sci Lett B 8:167–169
Cooney DG, Emerson R (1964) Thermophilic fungi. An account of their biology, activities, and
classification. W. H. Freeman and Company, San Francisco, p 188
De Palma-Fernandez ER, Gomes E, Da Silva R (2002) Purification and characterization of two
β-glucosidases from the thermophilic fungus Thermoascus aurantiacus. Folia Microbiol (Praha)
47:685–690. https://doi.org/10.1007/BF02818672
Deploey JJ (1976a) Carbohydrate nutrition of Mucor miehei and M. pusillus. Mycologia 68:190–
194
Deploey JJ (1976b) Pectin utilization by thermophilic fungi. Proc Pennsylvania Acad Sci 50:179–
181
Ellis DH (1981a) Ultrastructure of thermophilic fungi. Trans Br Mycol Soc 76:467–478. https://doi.
org/10.1016/s0007-1536(81)80075-7
Ellis DH (1981b) Ultrastructure of thermophilic fungi: I. Ascocarp morphology of Thermoascus
aurantiacus. Trans Br Mycol Soc 76:457–466. https://doi.org/10.1016/s0007-1536(81)80074-5
Ellis DH (1981c) Ultrastructure of thermophilic fungi III. Ascocarp morphology of Chaetomium
thermophile and C. gracile. Trans Br Mycol Soc 77:165–177. https://doi.org/10.1016/s0007-
1536(81)80191-x
Ellis DH (1981d) Ultrastructure of thermophilic fungi IV. Conidial ontogeny in Thermomyces.
Trans Br Mycol Soc 77:229–241. https://doi.org/10.1016/s0007-1536(81)80025-3
Ellis DH (1982) Ultrastructure of thermophilic fungi. Trans Br Mycol Soc 78:129–139. https://doi.
org/10.1016/s0007-1536(82)80085-5
Emerson R (1968) Thermophiles. In: Ainsworth GC, Sussman AS (eds) The fungi, an advanced
treatise. Academic Press, Inc., New York, pp 105–128
Eriani G, Delarue M, Poch O, Gangloff J, Moras D (1990) Partition of tRNA synthetases into two
classes based on mutually exclusive sets of sequence motifs. Nature 347:203–206. https://doi.
org/10.1038/347203a0
Fergus CL (1964) Thermophilic and thermotolerant molds and actinomycetes of mushroom
compost during peak heating. Mycologia 56:267–284
Gaikwad JS, Maheshwari R (1994) Localization and release of β-glucosidase in the thermophilic
and cellulolytic fungus, Sporotrichum thermophile. Exp Mycol 18:300–310
Garrison RG, Boyd KS, Lane JW (1975) Ultrastructural studies on Thermomyces lanuginosa and
certain other closely related thermophilic fungi. Mycologia 67:961–971. https://doi.org/10.
1080/00275514.1975.12019829
Goh T-K, Hanlin RT (1998) Ultrastructural observations of ascomal development in Melanospora
zamiae. Mycologia 90:655–666. https://doi.org/10.1080/00275514.1998.12026954
Gupta SD, Maheshwari R (1985) Is organic acid required for nutrition of thermophilic fungi? Arch
Microbiol 141:164–169
Lasure LL, Ingle MB (1976) Some effects of temperature on zygospore formation in Mucor Miehei.
Mycologia 68:1145–1151. https://doi.org/10.1080/00275514.1976.12020003
Li AN, Yu K, Liu HQ, Zhang J, Li H, Li DC (2010) Two novel thermostable chitinase genes from
thermophilic fungi: cloning, expression and characterization. Bioresour Technol 101:5546–
5551. https://doi.org/10.1016/j.biortech.2010.02.058
Madigan MT, Orient A (1999) Thermophilic and halophilic extremophiles. Curr Opin Microbiol 2:
265–269
108 T. Das et al.

Mahajan MK, Johri BN, Gupta RK (1986) Influence of desiccation stress in a xerophilic thermo-
phile Humicola sp. Curr Sci 55:928–930
Maheshwari R, Balasubramanyam PV (1988) Simultaneous utilization of glucose and sucrose by
thermophilic fungi. J Bacteriol 170(7):3274–3280
Maheshwari R, Kamalam PT, Balasubramanyam PV (1987) The biogeography of thermophilic
fungi. Curr Sci 56:151–155
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol Rev 64:461–488. https://doi.org/10.1128/mmbr.64.3.461-488.2000
Mchunu N, Permaul K, Rahman AYA, Saito JA, Singh S, Alam M (2013) Xylanase superproducer:
genome sequence of a compost-loving thermophilic fungus, Thermomyces lanuginosus strain
SSBP. Genome Announc 1:4–5. https://doi.org/10.1128/genomeA.00388-13.Copyright
Millner PD, Motta JJ, Lentz PL (1977) Ascospores, germ pores, ultrastructure, and thermophilism
of Chaetomium. Mycologia 69:720–733. https://doi.org/10.1080/00275514.1977.12020116
Mumma RO, Fergus CL, Sekura RD (1970) The lipids of thermophilic fungi: lipid composition
comparisons between thermophilic and mesophilic fungi. Lipids 5:100–103. https://doi.org/10.
1007/BF02531102
Mumma RO, Sekura RD, Fergus CL (1971a) Thermophilic fungi: II. Fatty acid composition of
polar and neutral lipids of thermophilic and mesophilic fungi. Lipids 6:584–588. https://doi.org/
10.1007/BF02531140
Mumma RO, Sekura RD, Fergus CL (1971b) Thermophilic fungi: III. The lipids of Humicola
grisea var. thermoidea. Lipids 6:589–594. https://doi.org/10.1007/BF02531141
Murray P, Aro N, Collins C, Grassick A, Penttilä M, Saloheimo M, Tuohy M (2004) Expression in
Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the
moderately thermophilic fungus Talaromyces emersonii. Protein Expr Purif 38:248–257.
https://doi.org/10.1016/j.pep.2004.08.006
Oberson J, Rawyler A, Brändle R, Canevascini G (1999) Analysis of the heat-shock response
displayed by two Chaetomium species originating from different thermal environments. Fungal
Genet Biol 26:178–189
Prasad ARS, Maheshwari R (1978) Growth of and trehalase activity in the thermophilic fungus
Thermomyces lanuginosus. Proc Indian Acad Sci 87B:231–241
Prasad ARS, Kurup CKR, Maheshwari R (1979) Effect of temperature on respiration of a
mesophilic and a thermophilic fungus. Plant Physiol 64:347–348. https://doi.org/10.1104/pp.
64.2.347
Rajasekaran AK, Maheshwari R (1990) Effect of growth temperature on lipid-composition of a
thermophilic fungus Thermomyces-Langufinosus. Indian J Exp Biol 28:134–137
Raju KS, Maheshwari R, Sastry PS (1976) Lipids of some thermophilic fungi. Lipids 11:741–746
Renard Y, Cailleux R (1972) Contribution a !'etude des microorganismes du compost destine a la
culture du champignon de couche. Rev Mycol (Paris) 37:36–47
Rosenberg SL (1975) Temperature and pH optima for 21 species of thermophilic and
thermotolerant fungi. Can J Microbiol 21:1535–1540
Rosing WC (1982) Ultrastructure of ascus and ascospore development in Chaetomium brasiliense.
Mycologia 74:960–974. https://doi.org/10.1080/00275514.1982.12021616
Satyanarayana T, Johri BN (1984) Nutritional studies and temperature relationships of thermophilic
fungi of paddy straw compost. J Indian Bot Soc 63:165–170
Satyanarayana T, Sancholle M, Chavant L (1987) Lipid composition of thermophilic moulds
Acremonium alabamensis and Thermomucor indicae-seudaticae. Antonie Van Leeuwenhoek
53:85–91. https://doi.org/10.1007/BF00419504
Somasundaram T, Rao SRR, Maheswari R (1986) Pigments of thermophilic fungi. Curr Sci 55:
957–960
Stetter KO, Fiala G, Huber R, Segerer A (1990) Hyperthermophilic microorganisms. FEMS
Microbiol Rev 75:117–124
Subrahmanyam A (1977) Nutritional requirements of Torula thermophila Cooney & Emerson at
two different temperatures. Nova Hedwigia 19:85–89
5 Modulation of Physiological and Molecular Switches in Thermophilic Fungi:... 109

Subrahmanyam A (1980) A new thermophilic variety of Humicola grisea var. indica. Curr Sci 49:
30–31
Tansey MR (1972) Effect of temperature on growth rate and development of the thermophilic
fungus Chaetomium thermophile. Mycologia 64:1290–1299. https://doi.org/10.1080/
00275514.1972.12019380
Trent JD, Gabrielsen M, Jensen B, Neuhard J, Olsen J (1994) Acquired thermotolerance and heat
shock proteins in thermophiles from the three phylogenetic domains. J Bacteriol 176:6148–
6152
Van Noort V, Bradatsch B, Arumugam M, Amlacher S, Bange G, Creevey C, Falk S, Mende DR,
Sinning I, Hurt E, Bork P (2013) Consistent mutational paths predict eukaryotic thermostability.
BMC Evol Biol 13. https://doi.org/10.1186/1471-2148-13-7
Wali AS, Mattoo AK, Modi VV (1978) Stimulation of growth and glucose catabolite enzymes by
succinate in some thermophilic fungi. Arch Microbiol 118:49–53. https://doi.org/10.1007/
BF00406073
Wright CH, Kafkewitz DA, Somberg EW (1983) Eucaryote thermophily: role of lipids in the
growth of Talaromyces thermophilus. J Bacteriol 156:493–497
Zeldovich KB, Berezovsky IN, Shakhnovich EI (2007) Protein and DNA sequence determinants of
thermophilic adaptation. PLoS Comput Biol 3:0062–0072. https://doi.org/10.1371/journal.pcbi.
0030005
Zhao J, Guo C, Tian C, Ma Y (2015) Heterologous expression and characterization of a GH3
β-glucosidase from thermophilic fungi Myceliophthora thermophila in Pichia pastoris. Appl
Biochem Biotechnol 177:511–527. https://doi.org/10.1007/s12010-015-1759-z
Zhou P, Zhang G, Chen S, Jiang Z, Tang Y, Henrissat B, Yan Q, Yang S, Chen CF, Zhang B, Du Z
(2014) Genome sequence and transcriptome analyses of the thermophilic zygomycete fungus
Rhizomucor miehei. BMC Genomics 15:1–13. https://doi.org/10.1186/1471-2164-15-294
110 T. Das et al.

Psychrotrophic Microfungi: Major Habitats,
Diversity and Living Strategies 6
Kanak Choudhary, Najeeb Hussain Wani, Farooq Ahmad Ahanger,
Suhaib Mohamad Malik, Vinod Chourse, Abdul Majid Khan,
and Sanjay Sahay
Abstract
Ecology of extreme cold areas with subzero temperatures at least in some part of
the year is the subject of interest. Microfungi from these areas show special
morphological and physiological adaptations to avoid cold stresses. Some of
them are endemic, but majority are cosmopolitan in distribution. Except for a
few, mostly fungi from these areas show a wide range of growth temperatures.
Keywords
Cold-active microfungi · Psychrotrophic fungal diversity · Cold adaptations
6.1 Introduction
Cold climates are very low-temperature regions and known for their low water and
poor nutrient availability. Despite this, many fungal species are found here that can
grow and survive near-zero temperatures. Fungi can grow on various types of
substrate and can thrive or survive under many extreme environments. Hence their
abundance and diversity spread throughout the world. This category of
microorganisms was relatively neglected by the 1950s. These organisms possess
potential commercial value due to their cold-active enzymes. Many cold-active fungi
K. Choudhary · V. Chourse · A. M. Khan
Department of Biotechnology, Barkatullah University, Bhopal, Madhya Pradesh, India
N. H. Wani · F. A. Ahanger · S. M. Malik
Government Science and Commerce College, Bhopal, Madhya Pradesh, India
S. Sahay (*)
Sarojini Naidu Government Postgraduate Girl’s (Autonomous) College, Bhopal, Madhya Pradesh,
India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_6
111

are known as the active agent of spoilage of frozen foods, so it is important to study
these fungi as well (Margesin and Schinner 1994). These cold-active fungi are also
of interest to study due to their biogeographical and ecological importance. Cold
active organisms are those that are adapted to a climate characterized by very low
temperatures below 20 C. Around 85% of Earth including polar and alpine regions
and deep-sea exhibits this harsh condition. The cold-active organisms may be
psychrophilic or psychrotolerant, depending on their inability or ability to thrive
above a threshold lower temperature, respectively, and their threshold temperature
may be 15 or 20 C (Morita 1975; Robinson 2001; Cavicchioli et al. 2002).
It is well known that the rate of reaction decreases by half when the temperature
reduces by 10 C. This slow rate of reaction interferes with many biological
processes of fungi. Extremely low temperature, low water availability, and high
UV radiations are some general cold stress associated with the cold climate. High
UV radiation affects the cell by damaging the membrane lipoproteins, cellular
proteins, and DNA. Microorganisms protect themselves from the high UV radiation
by producing pigments such as melanin and mycosporines. Some fungi form micro-
niche in rocks or stones in cold ecosystems to protect them from high UV radiation.
Intracellular accumulation of osmoprotectants or compatible solutes protects the
cells from water loss. Low-temperature interferes with the membrane integrity and
enzyme activity of an organism. Fungi cope with this problem by altering the
membrane lipids and acquire flexibility in the membrane. Microfungi comprise a
group of filamentous fungi belonging to all major classes; the common characteristic
they exhibit is the inability to produce visible melanized fruiting bodies that can be
seen by naked eyes as found in most Ascomycetes and Basidiomycetes. The most
common microfungi are molds (bread molds, slime molds, and water molds),
powdery mildew, and rusts. This group includes both saprophytic and pathogenic
(for plants and animals) fungal species that comprise 4468 genera and 55,989
species. For a detailed classification of microfungi, readers are suggested to visit
the dedicated website (https://www.microfungi.org/).
Cold-active microfungi have been reported from almost all the cold areas exist on
the earth (Hassan et al. 2016). Their extraordinary capacity enables them to occupy
diverse types of niches such as the interior of living plants (endophytes), over the
surface of plants (epiphytes), on the decaying plant materials (saprobes), on or inside
the insects (entomopathogen), associated with roots (ectomycorrhiza), deep sea
animals (such as sponges) or sediments, soil, water, etc. in the cold environments.
They are expected to have faced diverse types of challenges enabling them to evolve
varied types of bio-molecules to respond to them. These bio-molecules are what we
are concerned with to feed our industries to get newer products or processes. The
most attractive features of all applications are the lower energy consumption either in
the bio-production (at low temperatures) of biomolecules or application of these
bio-molecules (in the process at low temperatures) or both and subsequent reduction
in CO
2
release. The chapter will unfold these aspects in detail.
112 K. Choudhary et al.

6.2 Habitats
Cold-active fungi exist in extensively diverse types of cold habitats (Hassan et al.
2016). Almost 85% of the Earth’s biosphere exhibits cold temperature (<5C) for a
longer period in the year. Specifically, this cold area includes deep ocean (about 90%
of the volume of the ocean shows temperature <5C), snow-covered land (35% of
land surface), permafrost (24% of land surface), sea ice (13% of the land surface),
glaciers (10% of land surface), cold water lakes, cold soils, and cold deserts. Major
cold land surfaces are present in the Arctic, Antarctic, and high-mountains
(Margesin and Miteva 2011; Hassan et al. 2016).
6.3 Terrestrial Cold Environments
6.3.1 Arctic Soil
Low nutrients availability, freeze-thaw cycles, low soil moisture, extreme cold, and
low annual precipitation are several limiting factors that reduce microbial activity in
arctic soils. Microbial communities in Finnish Lapland (Männistö and Häggblom
2006; Männistö et al. 2007) are dominated by a wide variety of these
microorganisms.
6.3.2 Alpine Soils
The term “alpine”implies a high altitude belt that lacks trees and is located above
continuous forests on mountains. Alpine soils are subjected to large temperature
fluctuations, a high number of frost and ice days, moisture fluctuations, regular
freeze-thaw events, and high precipitation. Microbial communities may vary sea-
sonally (Lipson 2007).
6.3.3 Antarctic Soils
Antarctic terrestrial ecosystems differ from those in the Arctic. Antarctic terrestrial
environment is colder (subzero temperatures down to –60 C), drier (moisture
content of 1–10%), lower availability of nutrients, and often alkaline. Due to the
extreme aridity, soils accumulate salts through precipitation and weathering. Micro-
bial diversity abundance in the terrestrial Antarctic region has been reviewed
recently. Cryptococcus and Mrakia are the dominating fungal species of Antarctic
soil (Aislabie et al. 2006; Smith et al. 2006; Babalola et al. 2009; Bej et al. 2009).
6.3.3.1 Historic Sites
Many historic sites present in the Antarctic environment have become a matter of
concern for their long preservation (Blanchette et al. 2002). Historic huts and pristine
6 Psychrotrophic Microfungi: Major Habitats, Diversity and Living Strategies 113

environment is a habitat of many diverse fungi that utilize wood as their nutrition
(source of carbon and nitrogen). They show temperature-dependent growth and
produce a significant amount of extracellular enzyme especially endo-
1,4-β-glucanase for wood degradation. Much cold-active fungal microbiota such
as Cadophora species (C. malorum, C. luteo-olivace, and C. cladosporioides) and
Geomyces sp. decompose the wood and organic matter of many artifacts and historic
huts located in the Antarctic pristine region (Held et al. 2003; Farrell et al. 2004).
These fungal species play an important role in nutrient cycling in a cold climate. Soft
rot are associated with the degradation of woody historic huts. The species of
Cadophora are known to be native saprophytes of the Antarctic region as they are
abundantly found in the environment with limited human interference. Cadophora
and Geomyces are the two most common fungal genera found in the historic hut
located on the Ross Island and are also found associated with many historic wooden
structures from various cold habitats and represent the fungal diversity in a cold
climate (Farrell et al. 2011).
6.3.3.2 Cryptoendolithic and Rock
Studies revealed that the total fungal diversity present in the Antarctic region is
composed of water molds (0.6%) and true fungi (99.4%). The cryptoendolithic
community especially lichen-dominated communities found in the deep zones of
porous sandstones of Antarctica. Filamentous fungi form cryptoendolithic lichen
association with chlorophycean algae. This fungal community is endemic as they
evolved with the harsh environments of Antarctica. Microscopic black melanized
fungi form microcolonies in rocks. Some filamentous hyphomycetes also found
associated with rocks. Melanized cell wall of these fungi enables them to survive
under cold stress (Ruisi et al. 2007).
6.4 Aquatic Cold Environments
6.4.1 Atmosphere and Clouds
The stratosphere and mesosphere layers are inhabited by many bacterial and fungal
cells as they associate with the aerosols present in the atmosphere and remain viable
in these layers. These microbes can stay here for a long time depending on the
capacity and environmental condition. Some of them can stay for only a week while
others can even stay for years. High UV radiation, desiccation, poor nutrients
availability, and oxidative stress in this ecosystem affect the survival of microbes
in this cold environment. However, the cloud is a more suitable environment for cold
fungi due to the presence of liquid water. Many bacterial and fungal communities
form a surface where water vapors condense and thus help in cloud formation and ice
nucleation. Thus atmosphere and clouds are considered as the ecosystem for diverse
microorganisms (Wainwright et al. 2004; Griffin2008; Pearce et al. 2009).
114 K. Choudhary et al.

6.4.2 Permafrost
Permafrost is the land surfaces such as soil, rock, or sediments usually at high
altitudes that are covered with ice for at least two consecutive years. Permafrost
regularly faces subfreezing temperatures. The mean temperature value for Arctic
permafrost is 10 C, while it ranges from 18.5 Cto27 C for Antarctic
permafrost. Bacteria and fungi of these permafrost exhibit growth and metabolic
activity up to a temperature of 39 C. Several viable Basidiomycetous yeasts were
found in the permafrost soil of Siberia. Genus Penicillium was found as dominating
fungus in Arctic permafrost (Margesin and Miteva 2011).
6.4.3 Snow
Snow is frozen atmospheric water and makes it a part of the cryosphere. Thirty-five
percent of the land surface of Earth is permanently or seasonally covered with snow.
Seasonal temperature variation, aerobic conditions, high light, and high UV radia-
tion are the characteristics of this ecosystem (Jones 1999; Cockell and Cordoba-
Jabonero 2004). Atmospheric microbes together with the dust particles reach the
snow and form a niche. Microorganisms inhabiting snow play important role in
nutrient cycling in the cold environment through exchanging of reactive oxygen
species. The microbial community present in snow also varies with changing latitude
and altitude (Hodson et al. 2008; Pearce 2009; Pearce et al. 2010). Many archaea,
bacteria, cyanobacteria, and fungi are isolated from this ecosystem. Many of them
are known psychrophilic and psychrotolerant species (Segawa et al. 2005; Amato
et al. 2007).
6.4.4 Polar and Alpine Lakes
Polar and high-altitude alpine lakes are the general cold lakes present on Earth that
possess significant limnological diversity. Due to different dominating temperatures,
these cold lakes show high diversity ranging from freshwater to hypersaline, from
highly oxygenated to anoxic, from highly acidic to alkaline, and permanently
ice-covered to ice-free (Vincent et al. 2008). The number of lakes in the Arctic
region (1432) exceeds the Antarctic region (174), and thus the two groups of lakes
exhibit different origin, geography, and environmental conditions (Ryanzhin et al.
2010).
6.4.5 Deep Sea and Sea Ice
Oceans cover around 71% of the Earth’s surface, and more than half of this area is
over 3000 m deep. The deep sea is defined as the lowest layer in the ocean at a depth
of more than 1000 fathoms (1 fathom ¼1.8288 m). The deep sea is characterized by
6 Psychrotrophic Microfungi: Major Habitats, Diversity and Living Strategies 115

poor light (dark), cold, high pressure, and low nutritional availability.
Microorganisms living in the deep sea adopt several unique features that allow
them to thrive in this extreme environment (Abe 2004; Deming 2009). Most isolated
strains from the deep sea are psychropiezophilic as they possess both psychrophilic
and piezophilic (optimal growth at pressures >0.1 MPa) properties and cannot be
cultured at temperatures higher than 20 C (Nogi 2008).
6.4.6 Glaciers
Glacier is a dynamic ecosystem as they possess high fluctuating conditions and
subfreezing temperatures. Glacial ice serves as a habitat for many fungal
communities. Penicillium species are abundantly found in the glacial ice of
Antarctica and source of various new bioactive secondary metabolites. The microbes
inhabit liquid veins or ice crystals in the glacial due to the presence of few amount of
liquid water. Glaciers are the important cold habitat for the genetic study of these
cold-active microbes as the ancient DNA of these microbes remains preserved in this
cold condition (Margesin and Miteva 2011).
6.4.7 Cryoconite Holes
Cryoconite holes are water-filled tiny cavities formed by atmospheric debris and dust
particles in the snow-free glaciers. These holes can be located at the upper surface or
subsurface in the glacier. The dust and other particles that form a cryoconite hole
absorb solar radiation at a high rate to melt the ice near the hole and thus forms a
micro-ecosystem for various microbes. Rhodotorula glacialis and Mrakiella
cryoconiti yeast are isolated from cryoconite holes (Margesin et al. 2007; Margesin
and Miteva 2011).
6.5 Artificial Ecosystem
Refrigerators, cold storage room, and refrigerated foods, where temperatures are
usually below 0 C, are man-made environment from many of the cold-adapted
fungi have also been isolated (Altunatmaz et al. 2012).
6.6 Fungal Investigations in Cold Environments
Fungi present in glaciers obtain its nutrient from archaea, bacteria, virus, and plants
and their metabolic products. However, embedded decaying vegetations and atmo-
spheric debris also provide nutrients to fungi in glaciers. This interactive ecology
enables the survival of various microbes in the glacial environment. Several fungi
116 K. Choudhary et al.

from high Arctic glaciers are known to produce acid phosphatase to solubilize the
inorganic phosphate and make it available for other species (Hassan et al. 2016).
6.7 Key Drivers of Fungal Abundance
During the fungal survey in Antarctica, the key drivers for fungal presence were
studied, and the percent composition of carbon and nitrogen in the soil was found to
be important in determining the fungal abundance (Arenz and Blanchette 2011).
Other factors such as soil moisture, pH, and salinity have hardly any correlation with
fungal abundance. The most frequently isolated fungi were the species of Geomyces
and Cadophora.
6.8 Diversity
The saying that “everything is everywhere but the environment selects”has served
as a motivational principle for surveying microbes from various ecosystems and
geographical regions (Beijerinck 1913; Baas-Becking 1934). Thus there is the
possibility of occurrence of similar phytotypes in similar habitats in different
geographical locations (Finlay and Clarke 1999). There are two more working
parameters, viz., environmental heterogeneity and spatial limitations that decide
the biodiversity of various geographical regions. Thus a geographically isolated
ecosystem may be dominated by endemic species (Papke et al. 2003; Souza et al.
2008; Whitaker et al. 2003). Advancement in molecular isolation techniques such as
“metagenomic one”has expedited the isolation of hitherto unknown species and
helped studying biogeographical distribution, dispersal, disseminating vectors, spe-
ciation, and the survival mechanisms and also verification of principles underlying
therein (Margesin and Miteva 2011).
Psychrophilic fungal communities have essential roles in that ecosystem includ-
ing nutrient cycle, water cycle, and energy flow and maintain an ecological balance
and ensuring the release of nutrient sources for other organisms (Treseder 2005;
Watling 2005).
Many psychrophilic fungi are cosmopolitan in distribution, e.g., Phoma
herbarum (Domsch et al. 1980; Singh et al. 2006). Again many of the psychrophilic
fungi exhibit broad growth temperature ranges, e.g., Epicoccum purpurascens can
grow in the temperature range of 3to4Cto45C with the optimum tempera-
ture being 23 to 28 C (Domsch et al. 1980; Hassan et al. 2016). Hardly a few
psychrophilic fungi exhibit a narrow growth temperature range (Table 6.1).
Fungi isolated from various cold areas are as follows:
6 Psychrotrophic Microfungi: Major Habitats, Diversity and Living Strategies 117

Table 6.1 Psychrotrophic fungi growing in narrow range of temperatures (Source: Wang et al.
2017)
Taxa
Growth
temperature Location/substrate/host Reference
Mucor strictus OGT below
10 C
Alpine soils Schipper (1967)
Coprinus
psychromorbidus
OGT between
8 and 3C
Plant pathogen infecting cereals
(winter wheat, oats), grass, and
conifers
Traquair and
Smith (1982)
Typhula
ishikariensis
OGT at 10 C Hoshino et al.
(1998)
Typhula incarnate OGT between
5 and 10 C
Nakajima and
Abe (1994)
Microdochium
nivale
OGT between
15 and 18 C
Sclerotinia borealis OGT between
4 and 10 C
Hoshino et al.
(2010)
Rhodotorula
himalayensis
–Soils of Himalayan mountain
ranges
Shivaji et al.
(2008)
Phoma herbarum OGT at 4 C Endophytic fungi of trees Moghaddam
and Soltani
(2014)
Humicola marvinii OGT below
15 C, MGT at
20 C
Soils in the maritime Antarctic Weinstein et al.
(1997)
Pseudogymnoascus
destructans
OGT at 4 C Hibernant bats Gargas et al.
(2009)
Pseudogymnoascus
pannorum
OGT at 5 C Soil and litter Flanagan and
Scarborough
(1974)
Thelebolus
microsporus
–Pangong Lake, Himalayan
region
Anupama et al.
(2011)
Mrakia robertii Antarctic and alpine soils
Mrakia blollopis MGT below
20 C
Thomas-Hall
et al. (2010)
Mrakiella
niccombsiis
Mrakiella
cryoconiti
MGT below
20 C
Northern Siberian glacier
sediment
Margesin and
Fell (2008)
Mrakia
psychrophila
OGT
10, MGT18
Antarctic soils Xin and Zhou
(2007)
Rhodotorula
psychrophila
OGT below
15 C
Alpine soils Margesin et al.
(2007)
Tetracladim
ellipsoideum
OGT below
15 C
Glacier soils in Qinghai–Tibet
plateau
Wang et al.
(2015)
Tetracladim
globosum
OGT below
15 C
Tetracladium
psychrophilum
OGT below
15 C, MGT
below 20 C
OGT optimum growth temperature, MGT maximum growth temperature
118 K. Choudhary et al.

6.8.1 Antarctica
6.8.1.1 Soil
Cadophora sp., C. fastigiata,C. luteo-olivacea,C. malorum,Chaetomium sp.,
Fusarium sp., Geomyces sp., Gliocladium sp., Hormonema sp., Hormonema
dematioides,Mortierella sp., Penicillium sp., P. echinulatum,P. expansum,
P. mali,P. roqueforti,Pestalotiopsis sp., Petriella sp., Pseudogymnoascus sp.,
Rhizopus sp., Talaromyces sp., Tetracladium sp., Thelebolus sp.
6.8.1.2 Lake
Acremonium sp., Cadophora luteo-olivacea,C. malorum,Davidiella tassiana,
Fontanospora sp., Geomyces pannorum,Mortierella sp., Penicillium commune,
P. dipodomyicola,Thelebolus globosus,Th. Ellipsoideus,Th. Microspores,
Trichoderma atroviride.
6.8.1.3 Endoliths
Cryomyces minteri,C. antarcticus,Friedmanniomyces simplex,F. endolithic,
Fusarium proliferatum,Geomyces sp., Penicillium sp., and some more from uniden-
tified members of Sporomiaceae.
6.8.2 Arctic
6.8.2.1 Soil
Aspergillus spp., A. aculeatus, A. nidulans, Cladophora finlandica, Cenococcum
geophilum, Geomyces spp., Mortierella spp., Myrothecium spp., Penicillium spp.,
Phialocephala fortinii, Phialophora spp.,Preussia spp.
6.8.2.2 Moss
Botrytis verrucosa, Corynespora sp., Fusarium oxysporum, Geomyces pannorum,
Microdochium sp., Mortierella sp., M. alpine,M. schmucker,M. simplex,Mucor sp.,
Mucor hiemalis,Penicillium sp., P. citrinum,P. frequentans,P. rugulosum,
Phialophora sp., Pithomyces chartarum.
6.8.2.3 Lichen
Acremonium sp., Alternaria sp., Antarctomyces sp., Arthrinium sp., Atradidymella
muscivora,Cadophora sp., C.thielavia,Cenococcum sp., Cosmospora sp.,
Elasticomyces elasticus,Fusarium sp., Herpotrichia sp., Lecanicillium sp. (insect
pathogen), Monodictys sp., Mortierella sp., Oidiodendron sp., Penicillium sp.,
Phaeosphaeria sp., Phialocephala sp., Polyblastia terrestris,Preussia sp.,
Rhizoscyphus ericae.
6 Psychrotrophic Microfungi: Major Habitats, Diversity and Living Strategies 119

6.8.3 Deep Sea
Acremonium sp., Alternaria sp., Aspergillus sp., A. terreus,A. ustus,A. versicolour,
Beauveria sp., Cadophora sp., Curvularia sp., Cylindrocarpon sp., Emericella sp.,
Emericellopsis sp., Eurotium sp., Fusarium sp., Galactomyces candidum,Geomyces
sp., Graphium sp., Gymnascella sp., Leptosphaeria sp., Mortierella sp.,
Paecilomyces sp. (insect pathogen), Penicillium sp., P.lagena,Pestalotiopsis sp.,
Petriella sp., Phoma sp., Pseudogymnoascus sp., Rhizoscyphus sp., Sagenomella
sp., Spicellum sp., Trichoderma sp., Xeromyces sp.
6.8.4 Alpine
Aspergillus sp., Beauveria sp., Cadophora sp., Curvularia sp., Fontanospora sp.,
Geomyces sp., Leotiomycetes sp., Mortierella sp., M.alpine,Mucor sp., Mucor
hiemalis,Penicillium sp., P. canesense,P. chrysogenum,Periconia sp., Phoma
sp., Phoma sclerotioides,Pseudogymnoascus pannorum,Ps. roseus,Pseudeurotium
sp., Scopulariopsis sp., Thelebolus microspores,Trichoderma sp., Truncatella
angustata,Umbelopsis isabellina.
6.8.5 Refrigerator
Acremonium sp., Aspergillus sp., Aspergillus ochraceus,Aspergillus flavus,Asper-
gillus niger,Aspergillus terreus Alternaria alternate,Botrytis sp., B. cinerea,Mucor
sp., M. racemosus, M. plumbeus,Penicillium sp., Penicillium italicum.
6.9 Living Strategies
Psychrotrophic fungi carry out ecological functions under extreme climatic
conditions through various forms such as symbionts, saprobes, parasites (plant and
animal), and pathogens and adopting various living strategies, some of which are
species-specific too.
6.9.1 Freeze Thaw and Desiccation
Freeze-thaw cycles and desiccation are two very hostile conditions impacting fungal
survival in a cold environment. The Arctic and Antarctic environments are
characterized by fluctuations in temperature near 0 C as an annual cycle. For
example in Arctic polar semidesert soil during winter, 30 freeze-thaw cycles have
been found at the depth of 3 cm (Coulson et al. 1995). Studies have shown that
filamentous fungi, e.g., spores of hyphomycete can tolerate lower temperatures (even
freezing ones), but they are not much tolerant to regular freeze-thaw cycles (Vishniac
120 K. Choudhary et al.

1996). In an experiments on an Antarctic yeast, Nadsoniella nigra var. Hesuelica,it
has been found that 33% and 10% of cells remain alive after one and ten cycles of
freeze (13 C) thaw cycle, respectively (Lyakh et al. 1984; Wynn-Williams 1990).
Desiccation is another very important factor impacting fungal survival in low
temperatures. Availability of free water around fungal cells at very low temperature
is only rarely or intermittently found in extremely cold areas (cold deserts), e.g.,
Antarctica (McRae and Seppelt 1999) that hampers fungal survival. Accumulation
of polyols makes the cell desiccation tolerant.
6.9.2 Molecular Strategies
A higher proportion of polyunsaturated and branched fatty acids maintain the fluid
condition of the membrane and ensure the transportability of nutrients under cold
conditions as in Rhodosporidium diobovatum (Turk et al. 2011) and Geomyces
pannorum (Weinstein et al. 2000). This is a very common strategy to avoid transport
constraints under freezing temperature. Accumulation of glycerol as cryoprotectant
as in Mrakia psychrophile (Su et al. 2016), compatible solutes such as polyols,
melanin, mycosporines, trehalose, and betaine (Ruisi et al. 2007) is another impor-
tant strategy. Reactive oxygen scavenging systems have also been observed in some
cold-active fungi such as Penicillium sp. p14 and Penicillium sp. m12 (Gocheva
et al. 2006) and Arctic fungi Keissleriella sp. YS 4108 and Aspergillus versicolor
(Sun et al. 2004).
6.10 Saprobes
Saprobes carry out a very important function of decomposition of dead plant/animal
or their detached parts with secreted hydrolases in the soil. The psychrotrophic
Cadophora and Geomyces spp. are found associated with the degradation of wooden
structures with secreted cellulases (historic huts and artifacts) in Antarctica (Arenz
et al. 2006; Arenz and Blanchette 2009; Farrell et al. 2011).
Cryptoendolithic fungi thrive in cold employing various morphological and
physiological adaptations. Important morphological adaptations include pigmenta-
tion and meristematic growth; the latter keeps the volume/surface ratio optimal,
minimizing the impact of external stressors. Important physiological adaptations
include an increased intracellular concentration of trehalose and polyol, secretion of
antifreeze proteins, and psychrophilic enzymes. Arthrobotrys ferox an Antarctica
fungus exhibits higher resistance to UV radiations because of the abundant pigments
its cell wall contains (Zucconi et al. 2002). Some fungi even produce extracellular
polymeric substances during biofilm formation that help them in tolerating stresses
arising out from desiccation, freeze-thaw cycles, etc. (De Los Ríos et al. 2002;
Selbmann et al. 2005).
6 Psychrotrophic Microfungi: Major Habitats, Diversity and Living Strategies 121

6.11 Plant Mutualists
Lichen and mycorrhiza comprise the common types of fungal mutualism in cold
areas. Mycorrhizae are fungi associated with the plant root, the association
benefitting both the partners. Fungi get carbohydrate from the plant while the plant
is helped in water and mineral absorption by fungi. The Arctic plants are deemed to
obtain 86% of N
2
with the help of mycorrhizae (Hobbie and Hobbie 2006;
Bjorbaekmo et al. 2010; Deslippe et al. 2011; Geml et al. 2012; Timling and Taylor
2012).
Lichens are another mutualistic association wherein algae can do photosynthesis
at temperature 20 C. They can absorb water vapor from the snow cover and
clouds and tolerate extreme desiccation conditions. All the three kinds of lichens,
viz., Crustose, Foliose (e.g., Xanthoria elegans), and Fruticose (e.g., Usnea
antarctica) exist in Antarctica. They can tolerate salinity and pigments such as
melanin, parietin, and usnic acid that are present in them and protect them from
UV radiation (Little 2009; Zhang et al. 2015).
6.12 Endophytes
Cold-active endophytic fungi studied in the plants belonging to Cupressaceae are
found to contain bioactive compounds with antibacterial, antifungal, and
antiproliferative activity conferring upon the plants’tolerance to abiotic and biotic
stress. The endophytic fungi Phoma and Dothideomycetes species show optimal
growth at 4 C (Moghaddam and Soltani 2014). Cold-active endophytic fungi
isolated from bryophytes, e.g., Barbilophozia hatchery, Chorisodontium
aciphyllum, and Sanionia uncinata in maritime Antarctica have been found to be
indispensable for the survival of the hosts (Zhang et al. 2013). Psychrotrophic
endophytic fungi from high altitude Baima Snow Mountain, Southwest China
belonging to genera Cephalosporium, Discula, Dothiorella, Phoma,
Seimatosporium, and Sirococcus exhibit optimal growth at 15 C (Li et al. 2012).
6.13 Parasites
Fungal pathogens in the cold area do develop survival strategies, for example, Snow
molds 0C pathogenizes several plants covered with snow in the polar regions and
thrive in that climate through spores and sclerotia. Some important diseases are
typhula blight caused by Typhula incarnate, Typhula idahoensis, and Typhula
ishikariensis; snow rots caused by Pythium iwayami and Pythium okanoganense;
snow scald caused by Myriosclerotinia borealis; and pink snow mold such as
Microdochium nivale associated diseases (Matsumoto 2009). Rust fungi
Melampsora causes mortality of willows and smuts (Ustilaginales) cause disease
in plants belongs to family Cyperaceae in the Arctic region (Parmelee 1989;
Scholler et al. 2003; Smith et al. 2004).
122 K. Choudhary et al.

References
Abe F (2004) Piezophysiology of yeast: occurrence and significance. Cell Mol Biol 50(4):437–445
Aislabie JM, Broady PA, Saul DJ (2006) Culturable aerobic heterotrophic bacteria from high
altitude, high latitude soil of La Gorce Mountains (8630’S, 147W), Antarctica. Antarct Sci
18(3):313–321
Altunatmaz SS, Issa G, Aydin A (2012) Detection of airborne psychrotrophic bacteria and fungi in
food storage refrigerators. Braz J Microbiol 43:1436–1443
Amato P, Hennebelle R, Magand O, Sancelme M, Delort AM, Barbante C, Boutron C, Ferrari C
(2007) Bacterial characterization of the snow cover at Spitzberg, Svalbard. FEMS Microbiol
Ecol 59(2):255–264
Anupama P, Praveen KD, Singh RK, Kumar S, Srivastava AK, Arora DK (2011) A psychrophilic
and halotolerant strain of thelebolus microsporus from Pangong Lake, Himalaya. Mycosphere
2:601–609
Arenz BE, Blanchette RA (2009) Investigations of fungal diversity in wooden structures and soils at
historic sites on the Antarctic Peninsula. Can J Microbiol 55(1):46–56
Arenz BE, Blanchette RA (2011) Distribution and abundance of soilfungi in Antarctica at sites on
the Peninsula, Ross Sea Region, and McMurdo dry valleys. Soil Biol Biochem 43(2):308–315
Arenz BE, Held BW, Jurgens JA, Farrell RL, Blanchette RA (2006) Fungal diversity in soils and
historic wood from the Ross Sea region of Antarctica. Soil Biol Biochem 38(10):3057–3064
Baas-Becking LGM (1934) Geobiologie; of inleiding tot de milieukunde. WP Van Stockum and
Zoon NV, The Hague, p 263
Babalola OO, Kirby BM, Le Roes-Hill M, Cook AE, Cary SC, Burton SG, Cowan DA (2009)
Phylogenetic analysis of actinobacterial populations associated with Antarctic Dry Valley
mineral soils. Environ Microbiol 11(3):566–576
Beijerinck MW (1913) De infusies en de ontdekking der bakterien. Jaarboek van de Koninklijke
Akademie van Wetenschappen:1–28
Bej AK, Aislabie J, Atlas RM (2009) Polar microbiology. The ecology, biodiversity and bioreme-
diation potential of microorganisms in extremely cold environments. CRC Press, Boca
Raton, FL
Bjorbaekmo MFM, Carlsen T, Brysting A, Vrålstad T, Høiland K, Ugland KI, Geml J,
Schumacher T, Kauserud H (2010) High diversity of root associated fungi in both alpine and
arctic Dryas octopetala. BMC Plant Biol 10(1):1–2
Blanchette RA, Held BW, Farrell RL (2002) Defibration of wood in the expedition huts of
Antarctica: an unusual deterioration process occurring in the polar environment. Polar Record
38:313–322
Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR (2002) Low-temperature extremophiles and
their applications. Curr Opin Biotechnol 13(3):253–261
Cockell CS, Cordoba-Jabonero C (2004) Coupling of climate change and biotic UV exposure
through changing snow-ice covers in terrestrial habitats. Photochem Photobiol 79(1):26–31
Coulson SJ, Hodkinson ID, Strathdee AT, Block W, Webb NR, Bale JS, Worland MR (1995)
Thermal environments of Arctic soil organisms during winter. Arct Alp Res 27(4):364–370
De Los Ríos A, Wierzchos J, Sancho LG, Grube M, Ascaso C (2002) Microbial endolithic biofilms:
a means of surviving the harsh conditions of the Antarctic. Eur Space Agency Publ Div 518:
219–222
Deming JW (2009) Extremophiles: cold environments. In: Schaechter M (ed) Encyclopedia of
microbiology. Elsevier, Oxford, pp 147–158
Deslippe JR, Hartmann M, Mohn WW, Simard SW (2011) Longterm experimental manipulation of
climate alters the ectomycorrhizal community of Betula nana in Arctic tundra. Glob Chang Biol
17(4):1625–1636
Domsch KH, Gams W, Anderson TH (1980) Compendium of soil fungi, vol I. Academic Press,
London
6 Psychrotrophic Microfungi: Major Habitats, Diversity and Living Strategies 123

Farrell RL, Blanchette RA, Auger M, Duncan SM, Held BW, Jurgens JE, Minasaki R (2004)
Scientific evaluation of deterioration in historic huts of Ross Island, Antarctica. In: Barr S,
Chaplin P (eds) Polar monuments and sites cultural heritage work in the Arctic and Antarctic
regions. ICOMOS Monuments and Sites No. VIII. Int Polar Heritage Committee, Oslo, pp
33–38
Farrell RL, Arenz BE, Duncan SM, Held BW, Jurgens JA, Blanchette RA (2011) Introduced and
indigenous fungi of the Ross Island historic huts and pristine areas of Antarctica. Polar Biol
34(11):1669–1677
Finlay BJ, Clarke KJ (1999) Ubiquitous dispersal of microbial species. Nature 400(6747):828
Flanagan PW, Scarborough AM (1974) Physiological groups of decomposer fungi on tundra plant
remains. In: Holding AJ, Heal OW, MacLean SF Jr, Flanagan PW (eds) Soil organisms and
decomposition in tundra. Tundra Biome Steering Committee, Stockholm, pp 159–181
Gargas A, Trest MT, Christensen M, Volk TJ, Blehert DS (2009) Geomyces destructans sp. nov.
associated with bat white-nose syndrome. Mycotaxon 108(1):147–154
Geml J, Kauff F, Brochmann C, Lutzoni F, Laursen GA, Redhead SA, Taylor DL (2012) Frequent
circumarctic and rare transequatorial dispersals in the lichenised agaric genus Lichenomphalia
(Hygrophoraceae, Basidiomycota). Fungal Biol 116(3):388–400
Gocheva Y, Krumova E, Slokoska L, Miteva J, Angelova M (2006) Cell response of Antarctic and
temperate strains of Penicillium spp. to different growth temperature. Mycol Res 110:1347–
1354
Griffin DW (2008) Non-spore forming eubacteria isolated at an altitude for 20,000 m in Earth’s
atmosphere: extended incubation periods needed for culture-based assays. Aerobiologia 24(1):
19–25
Hassan N, Rafiq M, Hayat M, Shah AA, Hasan F (2016) Psychrophilic and psychrotrophic fungi: a
comprehensive review. Rev Environ Sci Biotechnol 15(2):147–172
Held BW, Blanchette RA, Jurgens JA, Duncan S, Farrell RL (2003) Deterioration and conservation
issues associated with Antarctica’s historic huts. In: Koestler RJ, Koestler VR, Charloa AE,
Nieto-Fernandez FE (eds) Art, biology, and conservation: biodeterioration of works of art. The
metropolitan museum of art. New York and Yale University Press, New Haven, pp 370–388
Hobbie JE, Hobbie EA (2006) 15N in symbiotic fungi and plants estimates nitrogen and carbon flux
rates in Arctic tundra. Ecology 87(4):816–822
Hodson A, Anesio AM, Tranter M, Fountain A, Osborn M, Priscu J, Laybourn-Parry J, Sattler B
(2008) Glacial ecosystems. Ecol Monogr 78(1):41–67
Hoshino T, Tronsmo AM, Matsumoto N, Araki T, Georges F, Goda T, Ohgiya S, Ishizaki K (1998)
Freezing resistance among isolates of a psychrophilic fungus, Typhula ishikariensis, from
Norway. Proc NIPR Symp Polar Biol 11:112–118
Hoshino T, Terami F, Tkachenko OB, Tojo M, Matsumoto N (2010) Mycelial growth of the snow
mold fungus Sclerotinia borealis, improved at low water potentials: an adaptation to frozen
environment. Mycoscience 51(2):98–103
Jones HG (1999) The ecology of snow-covered systems: a brief overview of nutrient cycling and
life in the cold. Hydrol Process 13(14–15):2135–2147
Li HY, Shen M, Zhou ZP, Li T, Wei YL, Lin LB (2012) Diversity and cold adaptation of
endophytic fungi from five dominant plant species collected from the Baima Snow Mountain,
Southwest China. Fungal Divers 54(1):79–86
Lipson DA (2007) Relationships between temperature responses and bacterial community structure
along seasonal and altitudinal gradients. FEMS Microbiol Ecol 59(2):418–427
Little L (2009) Lichen Life in Antarctica: a review on growth and environmental adaptation of
Lichens. https://ir.canterbury.ac.nz
Lyakh SP, Kozlova TM, Salivonik SM (1984) Effect of periodic freezing and thawing of cells of the
Antarctic black yeast Nadsoniella nigra var. hesuelica. Microbiology 52:486–491
Männistö MK, Häggblom MM (2006) Characterization of psychrotolerant heterotrophic bacteria
from Finnsish Lapland. Sytemat Appl Microbiol 29(3):229–243
124 K. Choudhary et al.

Männistö MK, Tiirola M, Häggblom MM (2007) Bacterial communities in Arctic fields of Finnish
Lapland are stable but highly pH-dependent. FEMS Microbiol Ecol 59(2):452–465
Margesin R, Fell JW (2008) Mrakiella cryoconiti gen. nov., sp. nov., a psychrophilic, anamorphic,
basidiomycetous yeast from alpine and arctic habitats. Int J Syst Evol Microbiol 58(12):
2977–2982
Margesin R, Miteva V (2011) Diversity and ecology of psychrophilic microorganisms. Res
Microbiol 162(3):346–361
Margesin R, Schinner F (1994) Properties of cold-adapted microorganisms and their potential role
in biotechnology. J Biotechnol 33(1):1–14
Margesin R, Fonteyne PA, Schinner F, Sampaio JP (2007) Novel psychrophilic basidiomycetous
yeasts from Alpine environments: Rhodotorula psychrophila sp. nov. Rhodotorula
psychrophenolica sp. nov. and Rhodotorula glacialis sp. nov. Int J Syst Evol Microbiol 57:
2179–2184
Matsumoto N (2009) Snow molds: a group of fungi that prevail under snow. Microbes Environ
24(1):14–20
McRae CF, Seppelt RD (1999) Filamentous fungi of the Windmill Islands, continental Antarctica.
Effect of water content in moss turves on fungal diversity. Polar Biol 22(6):389–394
Moghaddam MSH, Soltani J (2014) Psychrophilic endophytic fungi with biological activity inhabit
Cupressaceae plant family. Symbiosis 63(2):79–86
Morita RY (1975) Psychrophilic bacteria. Bacteriol Rev 39(2):144–167
Nakajima T, Abe J (1994) Development of resistance to Microdochium nivale in winter wheat
during autumn and decline and the resistance under snow. Can J Bot 72(8):1211–1215
Nogi Y (2008) Bacteria in the deep sea: psychropiezophiles. In: Margesin R, Schinner F, Marx JC,
Gerday C (eds) Psychrophiles: from biodiversity to biotechnology. Springer, Berlin,
Heidelberg, pp 73–82
Papke RT, Ramsing NB, Bateson MM, Ward DM (2003) Geographical isolation in hot spring
cyanobacteria. Environ Microbiol 5(8):650–659
Parmelee JA (1989) The rusts (Uredinales) of arctic Canada. Can J Bot 67(11):3315–3365
Pearce DA (2009) Antarctic subglacial lake exploration: a new frontier in microbial ecology. ISME
J 3(8):877–880
Pearce DA, Bridge PD, Hughes KA, Sattler B, Psenner R, Russell NJ (2009) Microorganisms in the
atmosphere over Antarctica. FEMS Microbiol Ecol 69(2):143–157
Pearce DA, Hughes KA, Lachlan-Cope T, Harangozo SA, Jones AE (2010) Biodiversity of
air-borne microorganisms at Halley station, Antarctica. Extremophiles 14(2):145–159
Robinson CH (2001) Cold adaptation in Arctic and Antarctic fungi. New Phytol 151(2):341–353
Ruisi S, Barreca D, Selbmann L, Zucconi L, Onofri S (2007) Fungi in Antarctica. Rev Environ Sci
Biotechnol 6(1):127–141
Ryanzhin SV, Subetto DA, Kochkov NV, Akhmetova NS, Veinmeister NV (2010) Polar lakes of
the world: current data and status of investigations. Water Resour 37(4):427–436
Schipper MA (1967) Mucor strictus hagem, a psychrophilic fungus, and Mucor falcatus
sp. n. Antonie Van Leeuwenhoek 33(1):189–195
Scholler M, Schnittler M, Piepenbring M (2003) Species of Anthracoidea (Ustilaginales,
Basidiomycota) on Cyperaceae in Arctic Europe. Nova Hedwigia 76(3–4):415–428
Segawa T, Miyamoto K, Ushida K, Agata K, Okada N, Kohshima S (2005) Seasonal change in
bacterial flora and biomass in mountain snow from the Tateyama Mountains, Japan, analyzed by
16S rRNA gene sequencing and real-time PCR. Appl Environ Microbiol 71(1):123–130
Selbmann L, De Hoog GS, Mazzaglia A, Friedmann EI, Onofri S (2005) Fungi at the edge of life:
cryptoendolithic black fungi from Antarctic deserts. Stud Mycol 51(1):1–32
Shivaji S, Bhadra B, Rao RS, Pradhan S (2008) Rhodotorula himalayensis sp. nov., a novel
psychrophilic yeast isolated from Roopkund Lake of the Himalayan mountain ranges, India.
Extremophiles 12(3):375–381
Singh SM, Puja G, Bhat DJ (2006) Psychrophilic fungi from Schirmacher oasis, East Antarctica.
Curr Sci 90:1388–1392
6 Psychrotrophic Microfungi: Major Habitats, Diversity and Living Strategies 125

Smith JA, Blanchette RA, Newcombe G (2004) Molecular and morphological characterization of
the willow rust fungus, Melampsora epitea, from arctic and temperate hosts in North America.
Mycologia 96:1330–1338
Smith JJ, Tow LA, Stafford W, Cary C, Cowan DA (2006) Bacterial diversity in three different
Antarctic cold desert mineral soils. Microbal Ecol 51(4):413–421
Souza V, Eguiarte L, Siefert J, Elser J (2008) Microbial endemism: does phosphorus limitation
enhance speciation? Nat Rev Microbiol 6(7):559–564
Su Y, Jiang XZ, Wu WP, Wang MM, Hamid MI, Xiang MC, Liu XZ (2016) Provide insights into
the cold adaptation mechanism of the obligate psychrophilic fungus Mrakia psychrophila. G3:
genes, genomes. Genetics 6(11):3603–3613
Sun C, Wang JW, Fang L, Gao XD, Tan RX (2004) Free radical scavenging and antioxidant
activities of EPS2, an exopolysaccharide produced by a marine filamentous fungus Keissleriella
sp. YS 4108. Life Sci 75:1063–1073
Thomas-Hall SR, Turchetti B, Buzzini P, Branda E, Boekhout T, Theelen B, Watson K (2010)
Cold-adapted yeasts from Antarctica and the Italian Alps—description of three novel species:
Mrakia robertii sp. nov., Mrakia blollopis sp. nov. and Mrakiella niccombsii sp. nov.
Extremophiles 14:47–59
Timling I, Taylor DL (2012) Peeking through a frosty window: molecular insights into the ecology
of Arctic soil fungi. Fungal Ecol 5(4):419–429
Traquair JA, Smith DJ (1982) Sclerotial strains of Coprinus psychromorbidus, a snow mold
basidiomycete. Can J Plant Pathol 4(1):27–36
Treseder KK (2005) Nutrient acquisition strategies of fungiand their relation to elevated atmo-
spheric CO
2
. In: Dighton J, White JF, Oudemans P (eds) The fungal community: its organiza-
tion and role in the ecosystem. CRC Press, Boca Ration, FL, pp 713–731
Turk M, Plemenitas A, Gunde-Cimerman N (2011) Extremophilic yeasts: plasma-membrane
fluidity as determinant of stress tolerance. Fungal Biol 115:950–958
Vincent WF, Hobbie JE, Laybourn-Parry J (2008) Introduction to the limnology of high-latitude
lake and river ecosystems. In: Vincent WF, Laybourn-Parry J (eds) Polar lakes and rivers—
limnology of Arctic and Antarctic aquatic ecosystems, vol 11. Oxford University Press, pp 1–23
Vishniac HS (1996) Biodiversity of yeasts and filamentous microfungi in terrestrial Antarctic
ecosystems. Biodivers Conserv 5(11):1365–1378
Wainwright M, Wickramasinghe NC, Narlikar JV, Rajaratnam P (2004) Microorganisms cultured
from stratospheric air samples obtained at 41 km. FEMS Microbiol Lett 218(1):161–165
Wang M, Jiang X, Wu W, Hao Y, Su Y, Cai L, Xiang M, Liu X (2015) Psychrophilic fungi from the
world’s roof. Persoon Molecul Phylog Evolut Fungi 34:100
Wang M, Tian J, Xiang M, Liu X (2017) Living strategy of cold-adapted fungi with the reference to
several representative species. Mycology 8(3):178–188
Watling R (2005) Fungal conservation: some impressions –a personal view. In: Dighton J, White
JF, Oudemans P (eds) The fungal community: its organization and role in the ecosystem.
CRCPress, Boca Ration, FL, pp 881–896
Weinstein RN, Palm ME, Johnstone K, Wynn-Williams DD (1997) Ecological and physiological
characterization of Humicola marvinii, a new psychrophilic fungus from fellfield soils in the
maritime Antarctic. Mycologia 89(5):705–711
Weinstein RN, Montiel PO, Johnstone K (2000) Influence of growth temperature on lipid and
soluble carbohydrate synthesis by fungi isolated from fellfield soil in the maritime Antarctic.
Mycologia 92:222–229
Whitaker RJ, Grogan DW, Taylor JW (2003) Geographic barriers isolate endemic populations of
hyperthermophilic archaea. Science 301:976–978
Wynn-Williams DD (1990) Ecological aspects of Antarctic microbiology. Adv Microb Ecol 11:71–
146
126 K. Choudhary et al.

Xin MX, Zhou PJ (2007) Mrakia psychrophila sp. nov. a new species isolated from antarctic soil. J
Zhejiang Univ Sci B 8(4):260–265
Zhang T, Zhang Y-Q, Liu H-Y, Wei Y-Z, Li H-L, Su J, Zhao L-X, Yu L-Y (2013) Diversity and
cold adaptation of culturable endophytic fungi from bryophytes in the Fildes Region, King
George Island, maritime Antarctica. FEMS Microbiol Lett 341:52–61
Zhang T, Wei XL, Zhang YQ, Liu HY, Yu LY (2015) Diversity and distribution of lichen
associated fungi in the Ny-Ålesund Region (Svalbard, High Arctic) as revealed by
454 pyrosequencing. Sci Rep 5:1485
Zucconi L, Ripa C, Selbmann L, Onofri S (2002) Effects of UV on the spores of the fungal species
Arthrobotrys oligospora and A. ferox. Polar Biol 25:500–505
6 Psychrotrophic Microfungi: Major Habitats, Diversity and Living Strategies 127

Physiology and Molecular Biology
of Psychrotrophic Fungi: An Insight 7
Tuyelee Das, Samapika Nandy, Devendra Kumar Pandey,
Abdel Rahman Al-Tawaha, Potshangbam Nongdam, Ercan Bursal,
Mahipal S. Shekhawat, and Abhijit Dey
Abstract
Among other environmental parameters, the temperature is an important abiotic
factor in which the diversity of spices reduces at the extremes, and moreover, only
a few microorganisms become the main flora of that region. These extremophiles
represented bacteria, archaea, fungi, and algae that are found in both aquatic and
terrestrial cold and hot environments. Cold-tolerated microorganisms known as
psychrotrophic or psychrotolerant found in the snow, ice, and rocks. They have a
significant role in the ecology of that particular environment. Like other
extremophilic fungi, psychrotrophic fungi get broad attention due to their poten-
tial to produce cold-adapted enzymes, which have importance in pharmaceutical
and biotechnological fields. However, research data on psychrotrophic fungi are
lacking.Physiological studies of psychrotrophic fungi based on nutrition, growth
medium, pH requirement, and the interaction of fungi with environmental factors
are significantly important for fungus to grow optimally on a larger scale. The
T. Das · S. Nandy · A. Dey (*)
Department of Life Science, Presidency University, Kolkata, West Bengal, India
e-mail: abhijit.dbs@presiuniv.ac.in
D. K. Pandey
Department of Biotechnology, Lovely Professional University, Phagwara, Punjab, India
A. R. Al-Tawaha
Department of Biological Sciences, Al-Hussein Bin Talal University, Maan, Jordon
P. Nongdam
Department of Biotechnology, Manipur University, Imphal, Manipur, India
E. Bursal
Department of Biochemistry, Mus Alparslan University, Muş, Turkey
M. S. Shekhawat
Biotechnology Unit, Kanchi Mamunivar Government Institute for Postgraduate Studies and
Research, Puducherry, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_7
129

molecular basis of psychrotrophic or psychrotolerant fungi is reviewed in terms of
biochemical mechanisms in this chapter. Moreover, the physiological and molec-
ular study gives us a brief idea about psychrotroph adaptation strategies to sustain
in a low environmental condition. Therefore, in this chapter, we attempt to deliver
psychrotroph physiological and molecular features in relationship with adaptation
to low temperature.
Keywords
Psychrotolerant · Psychrotrophic fungi · Cold tolerant · Low temperature
7.1 Introduction
Psychrotrophs are organisms that have the ability to grow at low temperatures (0 C),
but their optimal growth occurs at 15 C, and maximal growth occurs above 20 C
(Ingram 1965; Morita 1975; Maheswari 2005). Psychrotrophs are also known as
psychrotolerant. The psychrotrophic term is more preferable by dairy
microbiologists, whereas the psychrotolerant term is utilized by environmental
microbiologists (Moyer et al. 2017). Cold places on earth range from deep-sea
ocean to cold desert, from frozen ground to terrestrial environment, and from high
mountain to glaciers. Cold environments considered as extreme environments when
rigid conditions like low water, low nutrient, and high pressure create hard
conditions. Microorganisms that flourish in the cold atmospheres have evolved
different adaptations strategies to live beneath such condition, which includes
production of cold-active enzymes that hydrolyze the compounds, which they can
utilize as nutrients (Buzzini and Margesin 2014; Alcaíno et al. 2015; Baeza et al.
2017). Microorganisms such as psychrophiles, mesophiles, and thermophiles have
been defined traditionally depends on basic temperature for growth of
psychrotrophic microorganisms that are found in cold environments in both terres-
trial aquatic regions. Psychrotrophic microorganisms can be isolated from meat,
milk, vegetables, cider, and fruit kept in the refrigerator. Psychrotrophs maintain the
ecology of cold regions, which cover a significant portion (80%) of the planet
(Hoshino and Matsumoto 2012). However, research related to psychrotrophs has
not been studied more intensively. In addition, psychrotrophs also could be used in
different biotechnological processes. Psychrotrophs grow in low temperature deter-
mined by membrane fluidity, nutrients transport, enzymes, and temperature-
dependent interaction with DNA, RNA, ribosome, and enzyme (Fukunaga and
Russell 1990; Russell 1990; Hamamoto 1994). The psychrotrophic microorganisms
possess the ability to grow at low temperature, but not at moderate temperature
owing to modification in their membrane proteins and lipids. Membrane protein
changes are genotypic, whereas lipids changes are genotypic or phenotypic, which
are significant for alternating membrane fluidity and permeability. The capacity to
acclimate solute uptake through membrane lipid alternation might differentiate
psychrotrophs from psychrophiles. If we can understand the physiology of
130 T. Das et al.

psychrotrophy, then we can easily utilize them for industrial cold-adapted enzyme
production. Figure 7.1 represents physiological and molecular adaptative features of
psychrotrophic fungi. Therefore, this chapter aims to compile data from the past on
whatever little information is available on the physiology of psychrotrophy that will
help to improve industrial production.
7.2 Nutritional Necessities
One cold-tolerant Himalaya strain of Penicillium pinophilum MCC 1049 is
supplemented with carbon, nitrogen sources, vitamins, and antibiotics and found
to produce lactase at 25∘C. Fructose, potassium nitrate, chloramphenicol, and folic
acid were found to be the top accompaniments for laccase production (Dhakar et al.
2014). Mortierella minutissima 01 fungi from arctic soils grew on agar plates with
limonene vapor in medium containing 0.8% substrate (Trytek and Fiedurek 2005).
Psychrotrophic micro-fungi (Aspergillus sp., Fusarium sp., Mucor sp., Rhizopus sp.,
and Penicillium sp.) grew best on different agar medium (CzapekDox Agar, Malt
extract agar Sabouraud dextrose agar, and Potato dextrose agar) (Maharana and Ray
2014). Cho et al. 2007 showed M. minutissima 01 improved perillyl alcohol produc-
tion to 0.3% by the addition of methanol at 15 C with 6.0 pH (Trytek and
Skowronek 2015). Penicillium chrysogenum 9 showed to produce the maximum
lipase at pH 6.0 but did not produce lipase at pH 3.0–5.0. (Cho et al. 2007).
P. chrysogenum 9 enzymes showed maximum activity at 30 C with pH 5.0 for
triacylglycerols substrate and with at pH 7.0 for natural substrates like oils (Cho et al.
2007).
Fig. 7.1 Physiological and molecular adaptative features of psychrotrophic fungi
7 Physiology and Molecular Biology of Psychrotrophic Fungi: An Insight 131

7.3 Pigments
The pigment formation gives the protection of fungal spores from stress. Thick
pigmented walls protected fungi from extreme cold conditions. A cold-tolerant
fungus Thelebolus microsporus produces carotenoid pigments detected by high-
performance liquid chromatography studies (Singh et al. 2014). Psychrotrophic
Antarctic sea fungus Arthrobacter agilis accumulates C-50 carotenoid
bacterioruberin and its glycosylated derivatives, regulating cellular membrane fluid-
ity (Fong et al. 2001). In cold weather, UV rays are present throughout the year.
Therefore, fungi from those places developed some strategies that protect against
UV light (Gorbushina 2003; Butler and Day 1998). Antarctic fungal taxa Alternaria
alternata,Stachybotrys chartarum and Ulocladium consortiale have melanized
strains that resist UV radiation (Hughes et al. 2003). Hughes et al. (2003) observed
that Phoma herbarum, isolated from Antarctica, was able to produce a brown
pigment, probably melanin, within 24 h of disclosure to high radiation of UV-B.
Psychrotolerant yeasts from the Antarctic region Cryptococcus gastricus,Dioszegia
sp., Leuconeurospora sp. (T27Cd2), Rhodotorula mucilaginosa, and Rhodotorula
laryngis showed the maximum UV-C radiation tolerance. Dioszegia sp.,
C. gastricus, and R. mucilaginosa showed production of the highest percentage of
carotenoids (torulene, 2-gamma carotene, gamma carotene, and lycopene)
(Villarreal et al. 2016). Penicillium cordubense produced yellow pigment at 25 C
optimal temperature (Pandey et al. 2016). The formation of green conidia has
importance in UV radiation tolerance (Braga et al. 2006). Greenish conidia produc-
tion observed in Trichoderma velutinum ACR-P1 fungus isolated from Shiwalik
hills, Jammu, and Kashmir (Sharma et al. 2016).
7.4 Antifreeze Proteins
Antifreeze proteins are structurally diverse polypeptides that attach to ice nucleators
and adsorb to ice surface that let down the freezing temperature without changing the
melting point (Urrutia et al. 1992). This phenomenon is known as thermal hysteresis,
in which the ranges vary from organism to organism. Antifreeze protein is a key
strategy of fungi in a low-temperature atmosphere (Duman and Olsen 1993). Anti-
freeze proteins alter the ice crystals shape led to inhibition of recrystallization
(Knight et al. 1984). Antifreeze protein from Antarctomycespsychrotrophicus was
identified as an extracellular protein that generated bipyramidal ice crystals and
showed thermal hysteresis phenomenon under alkaline conditions (Xiao et al.
2010). The psychrophilic or psychotropic snow molds are plant pathogen, which
also acts as a plant pathogen of grasses and cereals. Antifreeze proteins of Coprinus
psychromorbidus and Typhula ishikariensis present in extracellular space and repre-
sent 22 and 23 kDa molecular masses, respectively, which suppress the freezing of
the extracellular environment. Antifreeze proteins from T. ishikariensis do not form
proper hexagonal ice crystals instead bind with ice crystals to inhibit their growth
132 T. Das et al.

(Hoshino et al. 2003). Antifreeze protein binds to the various stratum of ice crystal
that suggested by observing ice crystal patterns linked with snow mold.
7.5 Compatible Solutes
Compatible solute accumulation results in a decrease in freezing point, which linked
to cytoplasmic macromolecules stability. The most extensive cryoprotective
substances are glycerol, mannitol, and trehalose that synthesized by induction of
low temperatures. Changes in polyol concentration led to typical physiological
adaptation for maintaining turgor pressure by fungi (Weinstein et al. 2000). Disac-
charide trehalose (a-D-glucopyranosyl(1–1)-aD-glucopyranoside) extensively found
in bacteria, yeasts, and fungi in different growing stages. Trehalose maintains fungi
survival in environmental stress conditions. Trehalose not only acts as a reserve
carbohydrate but also plays a role as an effective protectant that enhanced protection
against adverse conditions (Chi et al. 2003). Trehalose plays a significant role in
thermotolerance but the cold tolerance of the yeast. An Antarctic psychrotolerant
yeast Guehomyces pullulans 17-1 grows the best at 15 C. G. pullulans 17-1 cells
was grown at 10, 15, and 25 C and resulted in expression of trehalose-6-phosphate
synthase (Tps1) gene activity higher at 25 C than those grown at 10 and 15 C.
Trehalose synthesized by G. pullulans 17-1 linked with adaption to high temperature
(Zhang et al. 2013). These observations suggest that psychrotolerant G. pullulans
17-1 are more delicate to elevated temperature than to low temperature. Therefore,
the production of more trehalose needs to be synthesized in cell to protect the cells
grown at high temperature (Zhang et al. 2013). Glycogen and trehalose production is
detected in Penicillium olsonii p14 after 4–6 h of cold treatment. In this
psychrotolerant strain accumulation of glycogen and trehalose content increases up
to 2.0–2.5 times and 2.5-fold, respectively (Gocheva et al. 2009).
7.6 Lipid Composition
Membrane lipid composition changes in response to a different temperature.
Psychrophiles and psychrotrophs contain more short-chain branched unsaturated
and polyunsaturated fatty acids. In psychrophile, polyunsaturated fatty acid level
increased by decreasing the temperature. In low temperature, acyl chain length in the
phospholipids also decreased. Psychrophiles are capable of maintaining the higher
proportion of hexadecenoic (16:1) and octadecenoic (18:1) acids than are
mesophiles (Hamamoto et al. 1995). Geomyces pannorum in static liquid cultures
reported producing higher degrees of sterols, sterol esters, free fatty acids,
triacylglycerides, and mono/diacylglycerides. Unsaturated fatty acids and
triacylglycerides present more in Pseudogymnoascus destructans as compared
with G. pannorum (Pannkuk et al. 2014).The common fatty acids found in
G. pannorum and Geomyces vinaceus were tetradecanoic acid, 12-octadecadienoic
acid, exadecanoic acid, octadecanoic acid, cis 9-octadecenoic acid, cis
7 Physiology and Molecular Biology of Psychrotrophic Fungi: An Insight 133

9-exadecenoic acid, cis 9, and eicosanoic acid. G. vinaceus and C. merdarium fungi
showed a different unsaturation level at different temperatures (Finotti et al. 1993).
Two strains of G. pannorum, VKM FW-224 and VKM F-3808, showed an
improved level of linoleic acid (C 18:2) in a medium containing NaCl at 20 C
(Konova et al. 2009). Gram-negative psychrotolerant bacterial species were also
shown to contain branched-chain fatty acids (Fukunaga and Russell 1990). At low
temperatures, shorter chain unsaturated fatty acid production increases in both
bacteria and fungi that lead to membrane fluidity increase (Weete 1974; Tearle
and Richard 1987). T. microsporus produced myristic acid, palmitic acid, stearic
acid, heptadecanoic acid, linolenic acid, and linoleic acid, which detected by gas
chromatography. Presence of these fatty acids suggested that fungus growing at low
temperature modified membrane fluidity by altering membrane lipids (Singh et al.
2014).
7.7 DNA Methylation
Gene expression alternation in the response to cold stress has been studied in
prokaryotes and plants (Fan et al. 2013; Dai et al. 2015). Turchetti et al. (2020)
studied DNA methylation in psychrophilic (Naganishia antarctica) and
psychrotolerant (Naganishia albida) yeast species in different temperature. Both
the species showed a different level of DNA methylation. In N. antarctica total
methylated fragments did not change. In contrast, psychrotolerant N. albida showed
a nonsignificant increase of methylation when incubated at 4 C and also during the
regaining stage (Turchetti et al. 2020). DNA methylation is a significant epigenetic
modification. Numerous studies have associated with DNA methylation of gene;
however, those studies are quite controversial. DNA methylation studies in some
yeast displayed this modification, while some other confirmed the presence of DNA
methylation (Hattman et al. 1978; Bulkowska et al. 2007; Baum and Carbon 2011;
Tang et al. 2012; Capuano et al. 2014).
7.8 Enzymes
Psychrotrophic microorganisms utilized different enzymes for survival at a lower
temperature. Their survival strategies include reduced ionic pairs number, hydro-
phobic interactions, inter-subunit interactions, and increased solvent interaction,
nonpolar residues in the solvent, the decline in cofactor binding, and glycine
(D’Amico et al. 2006). Cold-tolerant microorganisms produce lipases that raise
their tolerance to survive in an extremely cold environment (Kour et al. 2019).
Various cold-adapted lipases have been studied from psychrotrophic and psychro-
philic microorganisms (Kavitha 2016). P. chrysogenum 9, Penicillium canesense,
and Pseudogymnoascus roseus were found to produced lipase (Bancerz et al. 2005;
Sahay and Chouhan 2018). The psychrotolerant Penicillium isolates (GBPI_P98,
GBPI_P228, and GBPI_P150) exhibited lipase production at 25 C. Mostly lipase
134 T. Das et al.

production increases at low temperature. All the isolates were shown to possess
tolerance from a temperature between 4 and 35 C (Pandey et al. 2016).
7.9 Cold Shock Proteins and Mycosporine
Cold shock (CS) proteins are multifunctional RNA/DNA binding proteins, enable
translation through RNA secondary structure destabilization. Fang and St. Leger
(2010) identified glycine-rich RNA binding proteins (GRPs) homologues in various
fungi. Nevertheless, Metarhizium anisopliae and Aspergillus clavatus possessed CS
domains. Crp1 and Crp2 displayed a high resemblance to CS proteins. CRP1 (cold
response protein 1) also protected M. anisopliae from oxidative stress and maintain
metabolism (Fang and St. Leger 2010). Psychrotolarent Leuco neurospora
creatinivora can produce mycosporines (Villarreal et al. 2016). The synthesis of
mycosporine mechanisms can reduce cell damage due to UV radiation in organisms
growing at high altitude (Margesin et al. 2007).
7.10 Temperature Effect
Psychrotrophic microorganisms are found in equally aquatic and terrestrial cold
environments. Psychrophilic and psychrotrophic microorganisms play a significant
role for maintain ecological balance, but, shockingly, research based on their
physiology has not yet been conducted intensely. Rhodotorula aurantiaca psychro-
philic Antarctic strain A19 cannot grow beyond 20 C, whereas Belgium strain
31,345 staring higher growth-limiting temperature was 32 C. These two yeast
produced maximum cell at 0 C and by increasing temperature cell production
decreased. Glucose uptake was increased at temperatures above l0 and 17 C for
the psychrophilic strain and psychrotrophic strain, respectively. The difference in
growth rate and substrate affinity was associated with the adaptation strategy of
R. aurantiaca strains to environmental conditions (Sabri et al. 2000). Snow mold
fungus Typhula incarnata formed small-sized sclerotia, which reduced by elevating
temperature but large-sized sclerotia formation increased by raising the temperature
(Hoshino et al. 2004). Gocheva et al. (2009) displayed that psychrotolerant strain
P. olsonii p14 from Antarctica can tolerate temperature downshift. Temperature
changes from 15 to 6 C directed cells to oxidative response, which linked to a drop
of biomass production and rise in the levels of superoxide dismutase (SOD) and
catalase (CAT) enzymes, storage carbohydrates, and oxidative damaged proteins
(Gocheva et al. 2009). Gray investigated Antarctic fungus growing in various
temperature was not found any psychrophilic species but two Antarctic
psychrotolerant nematophagous fungi Monacrosporium ellipsosporum and
Monacrosporium cionapagum, which grew between 4 and 15 C and adapted to
cold habitats (Gray 1982). Kerry (1990) investigated growth rates of fungi isolated
from the Antarctic Continent at temperatures ranging from 4 to 35 C. They specified
that those fungi that grew at 4 C were classified as psychrotrophs (Kerry 1990).
7 Physiology and Molecular Biology of Psychrotrophic Fungi: An Insight 135

Lipase production in different isolates of Penicillium spp. depends upon
temperatures. Maximum production of lipase was reported at 15 and 25 C
temperatures, while minimum production recorded at 5 and 35 C (Pandey et al.
2016). At temperature ranges from 4 to 50 C soil ascomycete fungus T. velutinum
ACR-P1 can grow and formed compact and hyphae with greenish conidia on solid
media (Sharma et al. 2016).
7.11 Conclusion
From the past few decades, the thermal features of psychrotrophs have been
established accurately; however, we are still understanding how psychrotrophs
have the ability to grow at such low temperatures. Psychrotrophs are naturally
exposed to very low temperature, high UV-B radiations, and low water and nutrient
available environment. Psychrotrophs survival is not possible without a proper
nutrient cycle in a functional ecological niche. The presence of Psychrotrophic
fungus in cold habitats like glaciers is quite interesting. The physiology of
psychrotrophs has gained attention and predicted the understanding of adaptation
at the molecular level. Psychrotrophs grown at low temperature have low acyl chain
length phospholipids, synthesis of melanin and mycosporine, production of cold-
active enzymes, and presence of compatible solutes. However, all the physiological
and molecular adaptive mechanisms still need more investigation. It is a fact that
psychrotrophic fungi show potential in biotechnological and pharmaceutical fields.
Future research on cold-active enzymes production, pharmaceutical metabolites, and
biotechnology related to psychotrophy is essential for understanding the role of
psychrotrophic or psychrotolerant fungi.
Acknowledgments Authors are extremely thankful to UGC, Government of India, for financial
assistance. Authors are highly grateful to Presidency University-FRPDF fund, Kolkata for
providing needed research facilities.
References
Alcaíno J, Cifuentes V, Baeza M (2015) Physiological adaptations of yeasts living in cold
environments and their potential applications. World J Microbiol Biotechnol 31:1467–1473
Baeza M, Alcaíno J, Cifuentes V, Turchetti B, Buzzini P (2017) Cold-active enzymes from cold-
adapted yeasts. In: Sibirny A (ed) Biotechnology of yeasts and filamentous fungi. Springer,
Cham, pp 297–232
Bancerz R, Ginalska G, Fiedurek J, Gromada A (2005) Cultivation conditions and properties of
extracellular crude lipase from the psychrotrophic fungus penicillium chrysogenum 90. J Ind
Microbiol Biotechnol 32:253–260. https://doi.org/10.1007/s10295-005-0235-0
Baum M, Carbon J (2011) DNA methylation regulates phenotype-dependent transcriptional activity
in Candida albicans. Proc Natl Acad Sci 108:11965–11970
Braga GUL, Rangel DEN, Flint SD et al (2006) Conidial pigmentation is important to tolerance
against solar-simulated radiation in the entomopathogenic fungus Metarhizium anisopliae.
Photochem Photobiol 82:418. https://doi.org/10.1562/2005-05-08-ra-52
136 T. Das et al.

Bulkowska U, Ishikawa T, Kurlandzka A, Trzcińska-Danielewicz J, Derlacz R, Fronk J (2007)
Expression of murine DNA methyltransferases Dnmt1 and Dnmt3a in the yeast Saccharomyces
cerevisiae. Yeast 24:871–882
Butler MJ, Day AW (1998) Fungal melanins. Can J Microbiol 44:1115–1136
Buzzini P, Margesin R (2014) Cold-adapted yeasts: a lesson from the cold and a challenge for the
XXI century. In: Buzzini P, Margesin R (eds) Cold-adapted yeasts. Springer, Berlin,
Heidelberg, pp 3–22
Capuano F, Mülleder M, Kok R, Blom HJ, Ralser M (2014) Cytosine DNA methylation is found in
Drosophila melanogaster but absent in Saccharomyces cerevisiae, Schizosaccharomyces
pombe, and other yeast species. Anal Chem 8:3697–3702
Chi ZM, Liu J, Ji J, Meng Z (2003) Enhanced conversion of soluble starch to trehalose by a mutant
of Saccharomycopsis fibuligera sdu. J Biotechnol 102:135–141
Cho HY, Bancez R, Ginalska G et al (2007) Culture conditions of psychrotrophic fungus, penicil-
lium chrysogenum and its lipase characteris. J Fac Agric Kyushu Univ 52:281–286. https://doi.
org/10.5109/9315
D’Amico S, Collins T, Marx JC et al (2006) Psychrophilic microorganisms: challenges for life.
EMBO Rep 7:385–389. https://doi.org/10.1038/sj.embor.7400662
Dai LF, Chen YL, Luo XD et al (2015) Level and pattern of DNA methylation changes in rice cold
tolerance introgression lines derived from Oryza rufipogon Griff. Euphytica 205:73–83. https://
doi.org/10.1007/s10681-015-1389-0
Dhakar K, Jain R, Tamta S, Pandey A (2014, 2014) Prolonged laccase production by a cold and pH
tolerant strain of Penicillium pinophilum (MCC 1049) isolated from a low temperature environ-
ment. Enzyme Res. https://doi.org/10.1155/2014/120708
Duman JG, Olsen MT (1993) Thermal hysteresis protein activity in bacteria, fungi, and
phylogenetically diverse plants. Cryobiology 30:322–328
Fan HH, Wei J, Li TC et al (2013) DNA methylation alterations of upland cotton (Gossypium
hirsutum) in response to cold stress. Acta Physiol Plant 35:2445–2453. https://doi.org/10.1007/
s11738-013-1278-x
Fang W, St. Leger RJ (2010) RNA binding proteins mediate the ability of a fungus to adapt to the
cold. Environ Microbiol 12:810–820. https://doi.org/10.1111/j.1462-2920.2009.02127.x
Fong N, Burgess M, Barrow K, Glenn D (2001) Carotenoid accumulation in the psychrotrophic
bacterium Arthrobacter agilis in response to thermal and salt stress. Appl Microbiol Biotechnol
56(5):750–756
Finotti E, Moretto D, Marsella R, Mercantini R (1993) Temperature effects and fatty acid patterns in
Geomyces species isolated from Antarctic soil. Polar Biol 13:127–130. https://doi.org/10.1007/
BF00238545
Fukunaga N, Russell NJ (1990) Membrane lipid composition and glucose uptake in two
psychrotolerant bacteria from Antarctica. J Gen Microbiol 136:1669–1673. https://doi.org/10.
1099/00221287-136-9-1669
Gocheva YG, Tosi S, Krumova ET et al (2009) Temperature downshift induces antioxidant
response in fungi isolated from Antarctica. Extremophiles 13:273–281. https://doi.org/10.
1007/s00792-008-0215-1
Gorbushina A (2003) Methodologies and techniques for detecting extraterrestrial (microbial)
life microcolonial fungi: survival potential of terrestrial vegetative structures. Astrobiology 3:
543–554
Gray NF (1982) Psychro-tolerant nematophagous fungi from the maritime Antarctic. Plant Soil 64:
431–435
Hamamoto T (1994) Effect of temperature and growth phase on fatty acid composition of the
psychrophilic Vibrio sp. strain no. 5710. FEMS Microbiol Lett 119:77–81. https://doi.org/10.
1016/0378-1097(94)90395-6
Hamamoto T, Takata N, Kudo T, Horikoshi K (1995) Characteristic presence of polyunsaturated
fatty acids in marine psychrophilic vibrios. FEMS Microbiol Lett 129(1):51–56
7 Physiology and Molecular Biology of Psychrotrophic Fungi: An Insight 137

Hattman S, Kenny C, Berger L, Pratt K (1978) Comparative study of DNA methylation in three
unicellular eucaryotes. J Bacteriol 135:1156–1157
Hoshino T, Matsumoto N (2012) Cryophilic fungi to denote fungi in the cryosphere. Fungal Biol
Rev 26:102–105
Hoshino T, Kiriaki M, Ohgiya S et al (2003) Antifreeze proteins from snow mold fungi. Can J Bot
81:1175–1181. https://doi.org/10.1139/b03-116
Hoshino T, Prończuk M, Kiriaki M, Yumoto I (2004) Effect of temperature on the production of
sclerotia by the psychrotrophic fungus Typhula incarnata in Poland. Czech Mycol 56:113–120.
https://doi.org/10.33585/cmy.56108
Hughes KA, Lawley B, Newsham KK (2003) Solar UV-B radiation inhibits the growth of Antarctic
terrestrial fungi. Appl Environ Microbiol 69:1488–1491
Ingram M (1965) Psychrophilic and psychrotrophic microorganism. Ann Inst Pasteur Paris 16:
111–118
Kavitha M (2016) Cold active lipases–an update. Front Life Sci 9:226–238. https://doi.org/10.1080/
21553769.2016.1209134
Kerry E (1990) Effects of temperature on growth rates of fungi from subantarctic Macquarie Island
and Casey, Antarctica. Polar Biol 10:293–299. https://doi.org/10.1007/BF00238428
Knight CA, Devries AL, Oolman LD (1984) Fish antifreeze protein and the freezing and recrystal-
lization of ice. Nature 308:295–229
Konova IV, Sergeeva YE, Galanina LA et al (2009) Lipid synthesis by Geomyces pannorum under
the impact of stress factors. Microbiology 78:42–47. https://doi.org/10.1134/
S0026261709010068
Kour D, Rana KL, Kumar A, Rastegari AA, Yadav N, Yadav AN et al (2019) Extremophiles for
hydrolytic enzymes productions: biodiversity and potential biotechnological applications. In:
Molina G, Gupta VK, Singh BN, Gathergood N (eds) Bioprocessing for biomolecules produc-
tion. Wiley, Hoboken, pp 321–372
Maharana AK, Ray P (2014) Screening of psychrotrophic micro-fungi for cold active extracellular
enzymes isolated from Jammu city, India. J Pure Appl Microbiol 8:2369–2375
Maheswari R (2005) Fungal biology in the 21st century. Curr Sci 88:1406–1418
Margesin R, Neuner G, Storey KB (2007) Cold-loving microbes, plants, and animals-fundamental
and applied aspects. Naturwissenschaften 94:77–99
Morita RY (1975) Psychrophilic bacteria. Bacteriol Rev 39:144–167
Moyer CL, Eric Collins R, Morita RY (2017) Psychrophiles and psychrotrophs. Ref Modul
Life Sci:1–6. https://doi.org/10.1016/b978-0-12-809633-8.02282-2
Pandey N, Dhaka K, Jain R, Pandey A (2016) Temperature dependent lipase production from cold
and pH tolerant species of Penicillium. Mycosphere 7:1533–1545. https://doi.org/10.5943/
mycosphere/si/3b/5
Pannkuk EL, Blair HB, Fischer AE et al (2014) Triacylglyceride composition and fatty acyl
saturation profile of a psychrophilic and psychrotolerant fungal species grown at different
temperatures. Fungal Biol 118:792–799. https://doi.org/10.1016/j.funbio.2014.06.005
Russell NJ (1990) Cold adaptation of microorganisms. Philos Trans R Soc Lond B Biol Sci 326:
595–611
Sabri A, Jacques P, Weekers F et al (2000) Effect of temperature on growth of psychrophilic and
psychrotrophic members of Rhodotorula aurantiaca. Appl Biochem Biotechnol Part A Enzym
Eng Biotechnol 84–86:391–399. https://doi.org/10.1007/978-1-4612-1392-5_30
Sahay S, Chouhan D (2018) Study on the potential of cold-active lipases from psychrotrophic fungi
for detergent formulation. J Genet Eng Biotechnol 16:319–325. https://doi.org/10.1016/j.jgeb.
2018.04.006
Sharma R, Singh VP, Singh D et al (2016) Optimization of nonribosomal peptides production by a
psychrotrophic fungus: Trichoderma velutinum ACR-P1. Appl Microbiol Biotechnol 100:
9091–9102. https://doi.org/10.1007/s00253-016-7622-5
Singh SM, Singh PN, Singh SK, Sharma PK (2014) Pigment, fatty acid and extracellular enzyme
analysis of a fungal strain Thelebolus microsporus from Larsemann Hills, Antarctica. Polar Rec
(Gr Brit) 50:31–36. https://doi.org/10.1017/S0032247412000563
138 T. Das et al.

Tang Y, Gao XD, Wang Y, Yuan BF, Feng YQ (2012) Widespread existence of cytosine
methylation in yeast DNA measured by gas chromatography/mass spectrometry. Anal Chem
84:7249–7255
Tearle PV, Richard KJ (1987) Ecophysiological grouping of Antarctic environmental bacteria by
API 20 NE and fatty acid finger- prints. J Appl Bacteriol 63:497–503
Trytek M, Fiedurek J (2005) A novel psychrotrophic fungus, Mortierella minutissima, for
D-limonene biotransformation. Biotechnol Lett 27:149–153. https://doi.org/10.1007/s10529-
004-7347-x
Trytek M, Skowronek M (2015) Biotransformation of (R)-(+)-Limonene by the psychrotrophic
fungus Mortierella minutissimainH
2
O
2
-Oxygenated culture. Food Technol Biotechnol 47:131
Turchetti B, Marconi G, Sannino C et al (2020) DNA methylation changes induced by cold in
psychrophilic and psychrotolerant naganishia yeast species. Microorganisms 8(2):296. https://
doi.org/10.3390/microorganisms8020296
Urrutia ME, Duman JG, Knight CA (1992) Plant thermal hysteresis proteins. Biochim Biophys
Acta 22:199–206
Villarreal P, Carrasco M, Barahona S et al (2016) Tolerance to ultraviolet radiation of
psychrotolerant yeasts and analysis of their carotenoid, Mycosporine, and ergosterol content.
Curr Microbiol 72:94–101. https://doi.org/10.1007/s00284-015-0928-1
Weete JD (1974) Fungal lipid biochemistry. Plenum Press, New York
Weinstein RN, Montiel PO, Johnstone K (2000) Influence of growth temperature on lipid and
soluble carbohydrate synthesis by fungi isolated from fellfield soil in the maritime Antarctic.
Mycologia 92:222–229
Xiao N, Suzuki K, Nishimiya Y et al (2010) Comparison of functional properties of two fungal
antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis. FEBS J
277:394–403. https://doi.org/10.1111/j.1742-4658.2009.07490.x
Zhang F, Wang ZP, Chi Z et al (2013) The changes in Tps1 activity, trehalose content and
expression of TPS1 gene in the psychrotolerant yeast Guehomyces pullulans 17-1 grown at
different temperatures. Extremophiles 17:241–249. https://doi.org/10.1007/s00792-013-0511-2
7 Physiology and Molecular Biology of Psychrotrophic Fungi: An Insight 139

Ecology, Physiology, and Diversity
of Piezophilic Fungi 8
Shyamji Shukla and Harshita Shukla
Abstract
Deep-sea hydrothermal ecosystems (>1000 m below sea level) are just like fertile
spot of life in oceans and symbolize one of the most extreme ocean environments.
Many indigenous species of Bacteria, Archaea, and other organisms, like annelids
and crabs, are reported since the late 1970s when the development of these
ecosystems took place. The deep-sea possesses great variety of microbial diver-
sity, in spite of the extreme environmental conditions viz., low temperatures, high
hydrostatic pressure, high salinity concentrations, absence of sunshine, etc. Con-
siderable information has been gathered about the variety of microorganisms in
deep-sea ecosystems, but the diversity of fungi has not been examined up to now.
These deep-sea fungi are called piezophilic fungi. Fungi are recognized as
important organisms in terrestrial ecosystems because of their environmental
functions and predominantly their capability to break down organic matter. The
paucity of information about fungal diversity in the deep-sea has generally
established a notion that they are terrestrial organisms. For many years, the
majority of the facts about deep-sea fungal diversity were revealed via culture-
dependent techniques. In recent times, with the advancements in efficient
sequencing techniques, there has been a speedy growth in the research of deep-
sea fungal flora. This chapter highlights the studies related to the variety, ecology,
and physiology of piezophilic fungi. The aim of this chapter is also to focus on the
evolution of the techniques to evaluate the diversity and ecological role of the
piezophilic fungi which could stimulate new prospecting for biomolecules from
these fungi.
S. Shukla (*)
Department of Biotechnology, Mata Gujri Mahila Mahavidyalaya (Autonomous), Jabalpur, India
H. Shukla
Department of Biotechnology, Sri Guru Tegh Bhadur Khalsa College, Jabalpur, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_8
141

Keywords
Deep sea · Ecosystem · Extremophile · Potential · Environment · Piezophilic fungi
8.1 Introduction
The deep-sea has great potential and is one of the most inexplicable and unknown
extreme environments. In spite of widespread studies on prokaryotes of deep-sea,
very little is known about the rich biodiversity of fungi which are ecologically very
important groups of eukaryotic microorganisms. Though, the occurrence of fungi in
these deep-sea ecosystems is now being acknowledged. Piezophiles dwell deep-sea
sediments (>3000 m depth) under high hydrostatic pressure and also inhabit the guts
of bottom-dwelling animals. Numerous fungi similar to terrestrial species have been
isolated by culture-dependent methods from various deep-sea environments. On the
other hand, these methods have also revealed the presence of various novel fungal
phylotypes viz., Cryptomycota, which do not possess the characteristic chitin-rich
fungal cell walls. Even though exact information about the diversity of fungi and its
function in deep-sea ecosystem remains not very clear, the purpose of this chapter is
to evaluate recent understanding of the ecology, physiology and variety of fungi in
these ecosystems recommending future path for investigating deep-sea fungal flora.
Fungi are a vital constituent of all ecosystems and are omnipresent in environ-
ment because of their extremely adaptable physiological character. Deep-sea fungi
inhabit sediments of deep-sea at a depth of quite 1000 m below the surface of the
ocean. The deep-sea is that part of the ocean which is around 1000 m deep, and
covers 60% of the Earth’s surface. It therefore makes the planet’s largest homoge-
neous ecological formation called biome. The geological, geochemical and physical
conditions of the seabed and water column define varied habitats, sheltering specific
biological communities. The bottom of the ocean consists of a variety of different
environments, viz., continental margins, abyssal plains, oceanic trenches, mid-ocean
ridges and seamounts. Around 90% of the deep-seabed contains fine sediments
composed of particles of different origin viz., biogenic (produced by living
organisms), terrigenous, volcanic and authigenic (originating from the rock where
it’s found). The seabed is however rockier on mid-ocean ridges and seamounts,
furthermore as in some isolated areas of continental slopes, into which submarine
canyons with abrupt cliffs is also cut. The marine environment is globally
characterized by an absence of sunlight, high, low, and comparatively constant
temperatures, low currents and oxygen content generally sufficient for animal life
to develop.
The deep-sea environment is among the most intense environments for living
organisms. Conditions like lack of sunlight, mostly lesser temperature (some places
like hydrothermal vents have a particularly warmth up to 400 C), excessive pH, and
high hydrostatic pressure (up to 110 MPa) create extreme environment in deep-sea
(Damare et al. 2006a; Burgaud et al. 2010). These environmental factors are known
to affect the abundance, diversity, activity, and distribution of fungi within the
142 S. Shukla and H. Shukla

natural habitats. Piezophilic fungi are able to survive in such extreme environmental
conditions and are found in large numbers in deep-sea environments (Gadanho and
Sampaio 2005; Wang et al. 2015). It is supposed that these fungi evolved from
terrestrial environments, and acquainted well to the deep-sea environmen. A current
estimation indicates that overall fungal species are approximately 2.2–3.8 million,
out of which only 120,000 species are explained by taxonomists, while some species
are identified from the deep-sea environments.
The biodiversity of deep-sea-derived fungal species and their applications in
biotechnology are not completely described, which raises the need of further
research to discover their potential. However some bioactive molecules having
antibacterial, antiviral, antidiabetics, anti-inflammatory, antitumor, and enzymatic
potential are isolated from deep-sea fungal communities (Wang et al. 2015). The aim
of this chapter is to provide an overview about Piezophilic fungal diversity, includ-
ing their ecology and physiology. This chapter also discusses the tools and
techniques employed in the collection, isolation, and identification of the Piezophilic
fungi.
8.2 Exploring Piezophilic Fungal Diversity
8.2.1 Collection of Sample
Collection of fungi from deep-sea waters can be performed using routine oceano-
graphic samplers such as Niskin or Nansen bottles or a Rossette or ZoBell sampler.
In-situ hydrostatic pressure is not maintained in these samplers. First water sampler,
having potential to sustain deep-sea pressure to collect water sample for isolation of
piezophilic bacteria was reported by Jannasch et al. (1973). Usually deep-sea fungi
procure their nutrients from organic matter present in the sediments and generate
hyphae, which extend and develop amid the sediment particles. It is more suitable to
search for deep-sea fungi in the sediments as they are not well adapted as free living
forms. Several types of sampling devices for collection of sediment samples have
been used successfully by mycologists.
A deep-sea workspace was created at JAMSTEC, Japan under the Deep-Star
program for collecting and culturing microorganisms from deep-sea conditions. This
system has a pressure-retaining sediment sampler, which is able to maintain hydro-
static pressure and low temperature of the sediment samples after collecting from the
bottom of deep-sea (Yanagibayashi et al. 1999). Whereas regular sampling of deep-
sea sediments for isolation of microbiological flora is done by techniques like
multiple corers, long gravity corers or box corers.
Box corers are more commonly used for sampling of flat oceanic floors. It is made
to penetrate the underside by lowering on a ship’s trawl wire. Since the corer is taken
out of the seabed, the top and bottom of the sample box are closed. The benefitofa
box corer is that it accumulates samples which are usually 20 cm long including
most of the deep-sea organisms. Research submarines can also be employed for
deep-sea sediments collection. An enquiry submarine is comparatively larger in size
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 143

and can carry a pilot and one or two scientists in a pressure sphere of about 2 m in
diameter. Around the sphere is a basket carrying equipments for support, propulsion,
ascent and descent, and those for scientific purposes like manipulator arms, cameras
and specialized payload in a very carrying basket.
Another method for sampling includes self propelled instrument packages viz.,
remotely operated vehicles. Some operate a two-way communications link at the top
of a cable that has power and hosts, while others are untethered, carrying their own
power, recording images and data. Instrument package consists of Propulsion unit,
sensors (particularly television), and manipulator arms. Their design enable them
either to fly or crawl, e.g., the Remote Underwater Manipulator over bed surface and
are further appropriate for sampling from deep-sea sediments.
8.2.2 Studying Microbial Communities
Microbial communities play essential role in diverse biogeochemical cycles of
different habitats. The major problem in correct estimation of microbes at a particu-
lar habitat is sampling and analyzing comparatively very small proportion of total
area. In addition, due to spatial heterogeneity of the species or the habitat, diversity
analysis of the microbes may be underestimated. Other factor for inability of correct
estimation of microbial communities at a particular habitat is their low culture
ability. It has been observed that approx. 99% of bacteria analyzed under a micro-
scope are not cultured by common laboratory techniques. The majority of the fungal
species also escape laboratory culturing techniques. Diversity of species includes
species richness, the overall number of species present, species constancy, and the
allocation of species. Some of the techniques used for the measurement of cultured
microbial diversity are as following:
8.2.3 Community Level Physiological Profiling
Applying this technique the culturable microorganisms may be recognized on the
basis of a range of carbon source utilization patterns. To perform this technique
designed Biolog plates containing all the well-known carbon sources are commer-
cially available. For examination, samples containing microbes are inoculated and
monitored over time for their ability to utilize substrates. Thereafter the speed at
which these substrates are utilized was also monitored. With the application of
various statistical techniques to the information, relative differences are assessed.
8.2.4 Fatty Acid Methyl Ester (FAME) Analysis
Fatty acids build up a comparatively invariable fraction of the cell biomass. Some
characteristic fatty acids exist that can distinguish chief taxonomic groups of a
community. Therefore, a modification in the fatty acid profile signifies a
144 S. Shukla and H. Shukla

transformation in the microbial population. Hence fatty acid profiles can be exploited
to analyze composition and population changes of a microbial community. Further
researches on bacteria have, revealed that fatty acid compositions affect the pheno-
typic characteristics of organisms based on DNA and rRNA homology.
Every bacterial species and strain depicts a particular fatty acid profile and
thereby can be identified on this basis. Yeast identification can also be performed
by using this method reported differential expression of fatty acids by different phyla
of fungi which can be in turn employed as a biomarker for their identification. For
example, fungi belonging to Oomycota express 20:5 fatty acids, Zygomycota
produce 18:3 fatty acids which are higher in number as compared to Dikaryotic
forms i.e., Ascomycota and Basidiomycota. On the other hand sterile forms of fungi
produce 22:0 fatty acid, which is not found in any other taxon.
8.2.5 Molecular Based Techniques
Polymerase Chain Reaction (PCR) targeting the 16S rDNA has been used compre-
hensively to examine the identification and diversity of prokaryotes. In case of
eukaryotes, particularly fungal species, 18S and internal transcribed spacer (ITS)
regions of SSU rDNA are increasingly used for study. Moreover, the D1/D2 domain
as well as different regions of Large Sub Unit (LSU) of 28S rDNA of eukaryotes can
also be employed for identification and analysis of diversity (Gadanho and Sampaio
2005; Burgaud et al. 2010). Besides this diversity analysis can also be done by
applying Restriction Fragment Length Polymorphism (RFLP) for the above
amplified products on the basis of DNA polymorphisms. RFLP is the dissimilarity
in banding patterns of DNA fragments obtained after electrophoresis of restriction
digests of DNA from different individuals of a species. The cause for the unlike
banding patterns is due to the occurrence of a restriction enzyme cleavage site at one
place in the genome in one individual and the lack of that specific site in another
individual. Therefore, strains can be identified and compared on the basis of these
different and specific banding patterns after cleavage with a particular restriction
enzyme obtained in electrophoresis of samples from different strains.
8.2.6 Culture-Dependent Approach
Recently some reports have described presence of fungi in deep-sea habitats using
culture-dependent approaches. This approach has been used for the isolation of
several fungi from various marine habitats. Besides it has also been known for the
isolation of few terrestrial fungi viz., Acremonium, Aspergillus, Penicillium, and
Trichoderma (Raghukumar et al. 1992). A wide range of culturable marine fungi has
been reported from deep-sea hydrothermal vents using this technique (Burgaud et al.
2009).
Their physiological characterization proved them to be more or less adapted to
deep-sea conditions. Burgaud et al. (2010), isolated yeast species belonging to
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 145

genera, Rhodotorula, Rhodosporidium, Candida, Debaryomyces, and Cryptococcus
from hydrothermal vent animals and identified them by analysis of 26 rRNA gene
sequences. Fungi were isolated in the earlier reports from the deep-sea sediments of
the Central Indian Basin (CIB) from different sites with geographical locations of
~10–16S and 73–77E (Raghukumar et al. 2004; Damare et al. 2006a). Earlier
studies have reported the identification of fungi only on the basis of classical
taxonomic method, which may give biased results about actual fungal diversity in
this region (Raghukumar et al. 2004; Damare et al. 2006a).
8.3 Ecological Distribution of Piezophilic Fungi
Around 65% area of the whole earth surface is covered with deep-sea. The main
characteristic features of deep-sea environment include intense conditions of tem-
perature, hydrostatic pressure and nutrient conditions. There is an increase in the
hydrostatic pressure with depth at a rate of 0.1 MPa/10 m (0.1 MPa is reminiscent of
1 bar hydrostatic pressure). Therefore, the hydrostatic pressure is ~50 MPa at 5000 m
depth. On the other hand, there is a decrease in temperature with increase in depth. It
is around ~2–3C at 5000 m depth. This environment is furthermore described by
low nutrient conditions and is intended to be physically stable.
Deep-sea fungi were primarily reported from the shells collected from deep-sea
waters of 4610 m depth (Höhnk 1969). Further reports suggests isolation of fungi
from subtropical ocean water samples having depth of 4500 m using sterile van Dorn
bags or Niskin samplers (Roth et al. 1964). Deep-sea fungi have also been isolated
by submerging wooden panels at a depth of 1615–5315 m directly (Kohlmeyer
1977). However, four fungi out of these were found growing on wooden panels
while one on chitin of a hydrozoan. Poulicek et al. 1986 depicted mycelial fungi
growing inside shells of mollusks at 4830 m depth within the Atlantic. Numerous
filamentous fungi were isolated using 1/5 diluted malt extract medium prepared with
seawater from surface sterilized calcareous fragments at a depth of 300–860 m
within the Bay of Bengal (Raghukumar et al. 1992). The high-pressure and
low-temperature tolerance assay can indicate whether the isolated fungal species
are native deep-sea forms or not (Kohlmeyer and Kohlmeyer 1979). In an another
study, it was demonstrated that conidia of Aspergillus ustus (Bainier) and Graphium
sp. isolated from the calcareous sediments germinated at 10 and 20 MPa pressure in
Czapek-Dox medium and on shells suspended in seawater (Raghukumar and
Raghukumar 1998). In a similar study culturable fungi, Penicillium lagena
(Delitsch) Stolk & Samson and Rhodotorula mucilaginosa (A. Jörgensen) F. C.
Harrison, were obtained from a depth of 10,500 m sediment samples from the
Mariana Trench within the ocean.
In marine ecosystem, fungi holds a significant position as they are present
everywhere and perform decomposition and mineralization of organic matter
(Kohlmeyer and Kohlmeyer 1979). The Deep-sea fungi were first observed in shells
collected from deep-sea waters at a depth of 4610 m. Presence of fungi inside
molluskan shells was reported by Poulicek et al. (1986). Isolation of cultures of
146 S. Shukla and H. Shukla

many marine yeasts viz., Debaryomyces sp., Rhodotorula sp., and Rhodosporidium
sp. over a range of temperature and hydrostatic pressure conditions have also been
recorded. Reports are also available on the presence of fungi in Mariana trench
within the Pacific at a depth of ~11,000 m.
A variety of filamentous fungi isolated from calcareous sediments show germi-
nation of spores at elevated hydrostatic pressure (Raghukumar and Raghukumar
1998). Fluorescent brightner calcofluor under epifluorescence microscope depicted
the presence of fungal filaments in calcareous fragments (Raghukumar and
Raghukumar 1998). The fungal filament isolated from the deep-sea sediments of
Chagos trench in the Indian Ocean is thought to be oldest in age (Raghukumar et al.
2004). More than 200 fungal isolates were procured in culture form from deep-sea
sediments of the CIB by the application of different techniques like dilution plating,
particle plating and pressure enrichment techniques (Damare et al. 2006a). Immuno-
fluorescence technique revealed that one of these isolates was a native of deep-sea
sediment.
8.4 Diversity of Piezophilic Fungi
Damare et al. (2006a) identified Aspergillus sp. by applying classical morphology
based taxonomy as the most dominant form from the CIB. Sixteen filamentous fungi
and 12 yeast species from CIB were reported by Singh et al. (2010) as culturable
fungi using ITS and 18S sequences of SSU rDNA. Occurrence of Sagenomella sp.,
Exophiala sp., Capronia coronata Samuels and Tilletiopsis sp. from deep-sea
sediments were reported for the first time by using this approach.It was observed
that the majority of the culturable filamentous fungi belonged to ascomycetes
whereas most of the yeast isolates belonged to basidiomycetes. Sixty-two filamen-
tous fungi associated with animals from a range of deep-sea hydrothermal vent sites
mostly belonging to ascomycetes were isolated by Burgaud et al. (2009). Inspite of
so many fungal species isolated from deep-sea, exploration of Piezophilic fungi
associated with deep-sea dwelling macrofauna and zooplankton should be
undertaken further to unravel their diversity and ecological role. A novel genus of
deep-sea ascomycetes, Alisea longicola including one new species was reported by
Dupont et al. (2009) via analyses of 18S and 28S rDNA sequences and morphologi-
cal characters. Its isolation was performed from sunken wood of Pacific Ocean at
Vanuatu Islands.
In a similar study Connell et al. (2009) isolated eight yeasts and yeast-like fungal
species from cold hydrothermal environment and basalt rock surface from an active
deep-sea volcano, Vailulu’u Seamount, Samoa. Recently, 32 isolates of yeasts
belonging to phyla Ascomycota and Basidiomycota were obtained by Burgaud
et al. (2010). These fungal species were associated with deep-sea fauna from
hydrothermal vents. On the other hand techniques like PCR amplification of SSU
rRNA genes and sequence analyses have revealed the occurrence and diversity of
microbial eukaryotes from a deep-sea in the aphotic zone at 250–3000 m depth in the
Antarctic polar front (Lopez-Garcia et al. 2001). With the advent of these reports
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 147

which showed fungi as one of the eukaryotic groups in these sediments, techniques
employing amplification of sediment DNA with fungal specific primers to study
culture-independent diversity attracted most of the scientific attention.
Investigation of culture-independent fungal diversity by examination of the
small-subunit rRNA gene sequences amplified by PCR using DNA extracts from
hydrothermal vent samples was performed by Le Calvez et al. (2009). Three fungal
phyla viz., Chytridiomycota, Ascomycota, and Basidiomycota were reported with
new species by them. On the other hand, particularly lesser variety of filamentous
fungi was reported by Bass et al. (2007). Only about 18 fungal 18S-types from
11 deep-sea sediment samples were obtained. It can therefore be concluded that
combination of culturing techniques with phylogenetic analysis gives much better
range of fungi within the deep-sea environment. Further, several researchers have
reported the diversity of fungi in deep-sea sediments isolated by culture-independent
methods and the challenges accompanied by this method (Lai et al. 2007; Takishita
et al. 2007; Le Calvez et al. 2009; Nagano et al. 2010; Biddle et al. 2008; Edgcomb
et al. 2010). Most of the fungi isolated so far from the deep-sea are like terrestrial
fungi. Though, Alker et al. (2001) and Zuccaro et al. (2004) reported some terrestrial
fungi from marine habitats, which have evolved into marine forms.
It is a matter of research that if these fungi are physiologically different from their
terrestrial counterparts in their nutritional requirements and enzyme activities. These
fungi are probably associated with deep-sea dwelling marine fauna. However some
of the novel fungi isolated from various deep-sea locations are true piezophiles as
described by several research groups Lopez-Garcia et al. 2001; Gadanho and
Sampaio 2005, Lai et al. 2007; Le Calvez et al. 2009; Nagano et al. 2010). But
still more techniques need to be developed for culturing fungi which are yet not
cultured. Moreover the host population and diversity in the deep-sea environment
are significantly affected by the association of many eukaryotic phylotypes with
parasitic organisms (Brown et al. 2009).
The Ocean Drilling Program has aided to a great extent in the examination of
microbial diversity from deep marine seafloor sediments in spite of the extreme
environment (Parkes et al. 2005). Ascomycetous fungi belonging to the genera
Cladosporium, Penicillium, and Acremonium spp. were recovered from sediment
core collected at 200 mbsf (meter below sea floor) of the Peru margin by direct
plating and enrichment culturing technique (Biddle et al. 2005). Identification of
these cultured fungi was performed by ITS sequencing. In a similar study Edgcomb
et al. (2010) obtained fungal sequences from the deep-sea sediment isolated fungal
trains. These fungi were isolated from the depth 37 m below the seafloor of the Peru
Margin and Peru Trench and were mostly found to be belonging to Basidiomycetous
phyla.
Besides this, fungal species belonging to different phyla has also been reported
from a few more extreme environments. Cryptococcus curvatus (Diddens & Lodder)
Golubev, a basidiomycetous yeast was found to be the main fungal phylotype in
oxygen-depleted sediments from deep-sea methane seeps (Takishita et al. 2006,
2007). These areas are particularly rich in methane and hydrogen sulfide
concentrations. On the other hand, gas-hydrate-bearing sediments containing
148 S. Shukla and H. Shukla

ice-like minerals that have crystallized under low temperature, elevated hydrostatic
pressure and methane concentrations are also known to possess rich fungal diversity
whose sequences have been recovered from there (Cao 2010). Gas hydrates are in
gas hydrates, where methane is the major hydrocarbon in the gas mixture held in
water molecules. Several fungal sequences in such methane hydrate-bearing deep-
sea sediments have been recovered that were not associated to any known fungi or
fungal sequences Lai et al. (2007). Besides, fungal clones showing similar
characteristics to Phoma, Cylindrocarpon, Hortaea, Cladosporium, Emericella,
Aspergillus, Malassezia, Cryptococcus, Lodderomyces, Candida, and Pichia were
also procured.
Several studies have confirmed the presence of diverse fungal forms from the
Indian Ocean (Raghukumar and Raghukumar 1998; Damare et al. 2006a). Identifi-
cation of these fungal species was carried out by classical taxonomic methods. But
the disadvantage of the classical identification method is that it cannot identify the
non-sporulating forms of fungi on the basis of morphological features. In recent
studies identification of fungal cultures is done by employing amplification and
sequencing of their 18S rDNA and ITS regions (Burgaud et al. 2009). With the aid of
these molecular techniques the identification of both sporulating as well as
non-sporulating forms of fungal cultures upto generic or species level is possible.
Culture-independent approach also has reported isolation of a large variety of
fungal species from different regions throughout the world using. In an another
study, Bass et al. (2007) revealed that yeast are among the most dominant fungal
forms found in the deep ocean. Fungal specific primers for SSU rDNA region were
used for this analysis. A similar study indicated the presence of fungal strains in
hydrothermal sediments of the mid-Atlantic ridge by analysis of 18S rDNA
sequences amplified with eukaryote-specific primers (Lopez-Garcia et al. 2003).
Likewise recovery of fungal sequences from different deep-sea habitats viz., meth-
ane hydrate bearing deep-sea sediments (Lai et al. 2007), hydrothermal vents, anoxic
sites etc. have been reported by several researchers.
Hardly any research work has confirmed the identification of fungi using molec-
ular methods from the CIB. In the previous researches classical morphological
methods have been used for the identification of fungal diversity however these
studies were not able to justify presence of unidentified non-sporulating forms with
classical methods. Consequently, in order to observe the exact diversity of fungi in
the deep-sea sediments of the CIB, both culture-dependent as well as culture-
independent approaches were applied for the study in addition to the classical
methods. Further culture identification and environmental gene libraries construction
was performed by targeting the 18S and ITS regions of SSU rDNA. Thereafter the
results obtained were compared with earlier reports on fungal diversity from the
same sampling area of the Indian Ocean.
Even though the deep-sea environment acts as a habitat for a huge number of
microbial species, but, the origin, diversity, and distribution of deep-sea fungal
community remain mostly unexplored. The first report on deep-sea fungi was
proposed by Höhnk (1969) when he isolated fungi from 4610 m depth below the
sea. Similarly, Roth et al. (1964) isolated fungi from the subtropical Atlantic Ocean
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 149

at a depth of 4500 m. However, these fungal isolates were not obtained in culture
form under laboratory conditions. In an another study numerous filamentous fungal
strains were primarily isolated from calcareous fragments of the Bay of Bengal at a
depth of 300–860 m using culture-dependent method Raghukumar et al. (1992).
Thereafter, several fungal species were isolated and identified from different deep-
sea environmental samples, such as from the Mariana Trench at a depth of 11,500 m,
the Chagos Trench 5500 m (Raghukumar et al. 2004), the CIB 5000 m (Singh et al.
2011), Gulf of Mexico sediment 2400 m, South China Sea 2400–4000 m, East India
Ocean 4000 m, Canterbury Basin (New Zealand) 4–1884 mbsf (below the seafloor),
Okinawa 1190–1589 m, and the pacific ocean off the Shimokita Peninsula, Japan,
1289–2466 mbsf (Liu et al. 2017).
Around 120 deep-sea fungal species have been isolated and identified by several
researchers using culture-dependent techniques (Gadanho and Sampaio 2005).
While some unknown fungi have also been isolated by the application of culture-
independent techniques. It has been reported that Ascomycota comprises about 78%
of the total species in deep-sea environment followed by Basidiomycota (17.3%),
Zygomycota (1.5%), and Chytridiomycota (0.8%). On the other hand unidentified
fungal isolates forms 2.4% of the total deep-sea microbial flora. Aspergillus sp.,
Penicillium sp., and Simplicillium obclavatum are the most diverse and common
fungal species. Whereas, Alternaria alternata,Aureobasidium pullulans,Crypto-
coccus liquefaciens,Exophiala dermatitidis,Epicoccum nigrum, and Neosetophoma
samarorum were reported to be the rarest fungal species found in the deep-sea
environments. Lately, 69 fungal isolates belonging to 27 species were isolated by
Liu et al. (2017) from deep coal-associated sediment samples collected at depths
ranging from 1289 to 2457 mbsf in the Pacific Ocean off the Shimokita Peninsula,
Japan. Around 88% of the isolated strains belonged to the phyla Ascomycota
dominated by Penicillium and Aspergillus, while only 12% of the strains belong to
Basidiomycota. Besides these a number of new fungal species were also reported.
In one such study, isolated six new phylotypes of genera Ajellomyces,
Podosordaria,Torula, and Xylaria from the sediment of the South China Sea at a
depth of 2400–4000 m. Similarly, one novel fungal phylotype, DSF-Group1, was
explored in deep-sea sediments at depths ranging from 1200 to 10,000 m by using
three fungal specific primer sets, targeting the ITS1–5.8S–ITS2-28S rRNA regions
(Nagano et al. 2010). In another study, environmental clade KML11, a group of the
parasitic genus Rozella in Cryptomycota, was also identified by using eukaryotic-
specific primers EK-82F and EK-1492R. Likewise, fungus-specific primers viz.,
nu-SSU- 081 and nu-SSU-1536-39 were used for the isolation and identification of a
novel fungal strain the BCGI clade. As majority of deep-sea fungal species exhibit
similar morphological characteristics with the terrestrial fungi, molecular phyloge-
netic analysis played vital role in the detection and identification of the unknown
fungi.
150 S. Shukla and H. Shukla

8.5 Role of in Deep-Sea Ecosystem
Deep-sea fungi play significant roles in the marine environments viz., parasitic,
symbiotic or pathogenic and is found in various associations. A marine basidiomy-
cete fungal species, Mycaureola dilseae, has been identified to parasitize the subtidal
alga, Dilsea carnosa. This marine macroalga is infected by two widespread, zoo-
sporic fungal pathogens viz., Eurychasma dicksonii and Chytridium polysiphoniae
(Sparrow 1960). Moreover some fungal species are known to have symbiotic
association with marine sponges which secrete useful antimicrobial compounds.
While some other fungi viz., Phoma sp. isolated from marine and estuarine environ-
ment are reported to be the parasites of seaweeds, sea-grasses, molluscs, and
sponges. Likewise few marine yeast species namely, Rhodotorula, and
Rhodosporidium are identified to be potential mycoparasites and phytopathogens.
These host-parasite interactions are facilitated by specific cellular structures referred
to as colacosomes present in the fungal species. Similarly, a black yeast isolated
from mussel and other animals residing in hydrothermal vent sites is reported to be
pathogenic (Moreira and López-García 2003). On the other hand a number of
potential pathogenic forms of Aspergillus sp. were isolated from deep-sea sediments
of the CIB (Damare et al. 2006a).
8.5.1 Aggregate Formation and Carbon Contribution
It is well-known fact that transformation of organic matter in terrestrial soil is one of
the major functions of bacteria and fungi. On the contrary, major studies on bacterial
flora of deep-sea sediments have revealed their lesser important role in organic
matter degradation (Turley and Dixon 2002). Since deep-sea fungi remain embedded
in water and cannot be often noticed, their role in the deep-sea sediments has
remained neglected (Damare and Raghukumar 2008). However, recent reports
have suggested the use of chelating agent, like EDTA which increases the visibility
of fungi in the deep-sea sediments by effectively solubilizing polysaccharides (Liu
et al. 2002). Recent studies also reveal the function of deep-sea fungi in humic
aggregate formation via mechanisms very similar to those in terrestrial sediments.
The humus formed is basically composed of some by-products of microbial metab-
olism viz., reducing sugars and amino acids, which form brown-colored products by
undergoing non-enzymatic polymerization (Tisdall and Oades 1982).
Microaggregates are formed by the combination of humic material with soil
particles. Fine particles of soil are trapped with the fungal hyphae further which
act as binding agents to form macro-aggregates (Kandeler et al. 1999). Therefore
fungi or bacteria stay sheltered and safe in some specific particle size classes
(Suberkropp and Meyers 1996). Cations such as Si
4+
,Fe
3+
,Al
3+
and Ca
2+
acts as
bridges between particles in terrestrial microaggregates (Bronick and Lal 2005).
Reports suggest more effective aggregate stabilization by deep-sea fungi as com-
pared to the other soil microflora. These aggregates are primarily formed through
hyphal entanglement of soil particles (Molope et al. 1987). Formations of micro
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 151

aggregates by fungi grown in sediment have been well described by Damare and
Raghukumar (2008) on extract medium under 20 MPa pressure. Similar to previous
reports on fungi from terrestrial environments, it is found that the microaggregates
formed by deep-sea fungi stain positively for humic substances indicating that the
fungal activities are responsible for the formation of humus possibly in deep-sea
sediments (Stevenson 1994). These findings therefore indicate that deep-sea fungi
play a significant role in nutrient cycle by acting upon from inside these
microaggregates. Aggregation is a key mechanism for accumulation and mainte-
nance of organic matter in soil thereby providing protection to the soil organic matter
(Beare et al. 1994). This is chiefly a useful characteristic in natural environment as it
inhibits the extracellular enzymes from diffusing away from the cells secreting it.
Therefore, the whole nutrient dynamics of the sediments is known to be largely
affected by the humic-enzyme complexes (Burns 1978). According to recent reports
fungi secrete a large quantity of exo-polysaccaharides (Selbmann et al. 2003), which
in turn leads to enrichment of the aggregates. Hyphal sheaths act as a connecting link
responsible for the attachment of fungal mycelium to surfaces and particle entrap-
ment (Hyde et al. 1986).
Fungal biomass forms a huge fraction of the probably mineralizable organic
matter present in the forest and grassland soils (Chiu et al. 2006). Damare and
Raghukumar (2008) evaluated fungal biomass in deep-sea sediments supported
fungal bio-volume. Conversion of this bio-volume into Carbon was reported at a
rate of 1 pg of C per μm
3
(Van Veen and Paul 1979). Similarly, the fungal organic
carbon contribution within the deep-sea sediments of CIB was reported to be 2.3 to
6.3 μgg
1
of dry sediment. Whereas the carbon contribution by bacterial species for
the same site was reported to be about1 to 4 mg g
1
of dry sediment (Raghukumar
et al. 2001).
Around 10–150 pg g
1
sediment of fungal carbon contribution within the coastal
waters off Goa has been reported (Jebraj and Raghukumar 2009). On the contrary,
about 453–3375 μgg
1
of dry sediment of fungal carbon contribution was reported
in soil from grassland and forests (Chiu et al. 2006). The results of fungal biomass
estimation obtained by hyphal length method are more accurate in comparison to
other biochemical methods viz., phospholipids (Balser et al. 2005), hexosamine
(Gessner and Newell 2001) or ergosterol (Mille-Lindblom et al. 2004) content.
Since the components of fungal cell wall viz., melanin and chitin, easily degradable,
as compared to phospholipids of bacterial cell walls thus carbon accumulation by
fungi is estimated to be more persistent in contrast to that stored by bacterial species
(Bailey et al. 2002). Therefore a comparative quantitative analysis using pure
cultures of deep-sea bacteria and fungi with
14
C-labeled sugars and amino acids
for growth, uptake kinetics and carbon sequestration under simulated deep-sea
conditions would help us in understanding their individual role to a particular extent.
152 S. Shukla and H. Shukla

8.5.2 Extracellular Enzymes
A number of enzymes are recognized to be involved in the cycling of nutrients in the
deep-sea and hence can be employed as possible indicators of nutrient cycling
processes. Among many such enzymes alkaline phosphatase activity (APA) in
aquatic ecosystems like that of the deep-sea, plays a significant role in the regenera-
tion of inorganic phosphate via catalysis of organic esters (Chróst 1991). Organic P
content as measured during one such study varied from 0.007 to 011% and alkaline
phosphatase activity was in the range of 0.06 to 7.8 U which is equivalent to μmol of
phosphate phosphorus released h
1
g
1
dry sediment (Raghukumar et al. 2006).
During laboratory microcosm experiments Damare (2007) tried to clear the
difference between the contribution of APA in deep-sea sediments by fungi and
bacteria under simulated deep-sea conditions.
APA was particularly very low in the plain sediment without any additional
nutrients. But as detritus was added the APA increased 20-fold, signifying sharp
rise in microbial activity. It was further observed that APA was elevated in the
presence of antibacterial agents, which is indicative of the fact that major share of
APA is coming from fungi. This indicates that fungi have a high degradative activity
toward detritic material might similar to that in the terrestrial environments (Newell
1996). In an another study the piezophilic fungal isolate A. terreus (#A4634),
showed a very high APA in the presence of antibacterial agents as compared to
APA exhibited by deep-sea bacteria in the presence of a fungicide under the same
experimental conditions. These results clearly depict the significant role of fungi in
the P cycle within the deep sea.
Besides their key role in the biogeochemical cycles, piezophilic microorganisms
growing under intense conditions are a potential source of some novel useful
enzymes with unusual properties (Synnes 2007; Dang et al. 2009). Out of various
extracellular enzymes being produced by the fungi, protease plays a significant role
in their nutritional requirements. Damare et al. (2006b) confirmed that entire fungal
flora isolated from CIB deep-sea sediments exhibited active protease production at
low temperatures. On the other hand these fungi when grown under elevated
pressure synthesized extracellular protease in good amounts. Enzyme production
and fungal growth are directly related therefore reduced growth at 5 C leads to low
levels of enzyme production. Whereas the enzyme production was increased in the
presence of several commercial detergents and 0.5 M NaCl, equivalent to 29 PSU
(Practical Salinity Unit) of seawater salinity. These factors are advantageous for
enzyme to be used as additive in detergents at industrial of enzymes level
(Raghukumar et al. 2009). Thus deep-sea fungal proteases are efficient detergent
enzymes.
Fungal polygalacturonases are yet another class of useful enzymes used for
clarification of fruit juices in the food industry. From the supernatant of deep-sea
yeast (strain N6) culture two novel endopolygalacturonases having activity at
0–10 C were purified. This deep-sea yeast (strain N6) was isolated from the
Japan Trench at a depth of 4500–6500 m (Miura et al. 2001; Abe et al. 2006). The
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 153

hydrolytic activity of these enzymes was found to be unaffected even at a hydrostatic
pressure of 100 MPa at 24 C (Abe et al. 2006).
According to another study strain of Cryptococcus was found to be tolerant to
CuSO
4
up to a concentration of 50 mM. Moreover this strain also exhibited high
activity of superoxide dismutase, an enzyme responsible for scavenging superoxide
radicals (Abe et al. 2001). It was also observed that high pressure shifted the required
temperature for any chemical reaction toward lower temperature (Daniel et al. 2006).
Therefore, in such reactions a mesophilic enzyme could be used in place of thermo-
philic enzyme.
8.6 A Novel Source of Extremozymes
Nearly three fourth of the Earth’s area is covered by ocean, having a depth of
3800 m, indicating that the majority of our planet consists of deep-sea environments.
However, the deep-sea is one of the most inexplicable and unexplored environments
on the earth. It inhabits a vast diversity of microbial communities that play vital
functions in biogeochemical cycles (Sogin et al. 2006). In addition the deep-sea is
acknowledged as an extreme environment, because it comprises certain intense
environmental conditions such as the lack of sunlight, the presence of mostly low
temperatures and high hydrostatic pressures. But sometimes environmental
conditions become even more harsh particularly in the habitats, like deep-sea
hydrothermal vents with their extremely high temperatures (approx. >400 C),
deep hyper saline anoxic basins with their extremely high salinities and abysses of
up to 11 km depth with their extremely high pressures.
Deep-sea extremophiles includes organisms that survive and grow in deep-sea
environments having extreme environmental conditions viz., pressure, temperature,
pH, salinity and redox potential etc. that are usually lethal to other organisms. The
majority of deep-sea extremophiles are prokaryotic microorganisms particularly
including the domains of Archaea and Bacteria (Horikoshi and Bull 2011; Harrison
et al. 2013). There is a huge diversity of extremophilic microorganisms inhabiting
the deep-sea. They are functionally diverse and cosmopolitan in taxonomy and are
classified into thermophiles (55–121 C), psychrophiles (2Cto20C), halophiles
(2–5 M NaCl or KCl), piezophiles (>500 atmospheres), alkalophiles (pH >8),
acidophiles (pH <4), and metalophiles (high concentrations of metals, e.g., copper,
zinc, cadmium and arsenic). Mostly deep-sea extremophiles tolerate more than one
extreme condition and therefore are called polyextremophiles (Cavicchioli et al.
2011). These extreme conditions are normally injurious to the majority of organisms,
but extremophilic microorganisms are capable to stay alive and flourish in them. It is
only because of their highly flexible metabolisms and therefore the unique structural
characteristics of their bio-macromolecules (Nath and Bharathi 2011; Dalmaso et al.
2015).
Since past few years, piezophilic microorganisms have attracted the attention of
researchers who are constantly searching for newer bioactive compounds like
enzymes that may be employed in the industries worldwide (Zhang and Kim
154 S. Shukla and H. Shukla

2010). The wide-ranging temperatures, salinities, pHs and pressures occurring
naturally in extreme deep-sea environments serve as promising sites to explore
enzymes having industrial potential (Samuel et al. 2012). Several studies have
demonstrated that the extremozymes secreted by deep-sea extremophilic
microorganisms have a number of industrial applications as a result of their high
activities and great stabilities under extreme conditions. Therefore the stability and
variety of activities of extremozymes make them important alternatives to ordinary
biotechnological processes. These enzymes possess considerable economic potential
within the agricultural, feed, food, beverage, pharmaceutical, detergent, leather,
textile, pulp, and biomining industries (Raddadi et al. 2015).
Even though till date many enzymes have been isolated and identified around the
world, the majority of which have been examined for industrial applications, the
commercial enzyme market remains insufficient in fulfilling industrial demands
(Van Den Burg 2003). The reason being that most of these enzymes are unable to
tolerate industrial conditions (Irwin and Baird 2004). The industrial process requires
biocatalysts that can tolerate a variety of intense conditions, viz., temperature, pH,
salinity, and pressure, while exhibiting high conversion rates and reproducibility’s
(Haki and Rakshit 2003). Besides this, ecological compatibility is also significant for
industrial enzymes used in technologies (Gao et al. 2019). Though only a few
extremozymes are currently being produced and used at the industrial level, the
improvement of novel industrial processes based on these enzymes is being pro-
moted by development in deep-sea extremophile and extremozyme research. The
rising demand for novel biocatalysts in industries, could be achieved by develop-
ment in deep-sea sampling techniques, rapid expansion of new molecular and omics
technologies, such as metagenomics, proteomics, protein engineering, gene-directed
evolution and synthetic biology (Ferrer et al. 2007). Thus, the priority in enzyme
research is the discovery of enzymes with unusual enzymatic activities and enhanced
stability (Raddadi et al. 2015).
8.7 Strategies to Explore Extremozymes
The conventional method employed for the discovery of novel extremozymes from
deep-sea microorganisms is that the cultivation of microorganisms followed by
screening for the particular enzymes. Although 99.9% of these deep-sea
microorganisms cannot be cultivated by using conventional laboratory techniques
but a number of extremozymes with great potential for industrial applications are
isolated from deep-sea environments using this method (Amann et al. 1990). To
overcome the need for isolation or cultivation of microorganisms metagenomic
technologies are developed as a tool for isolation and identification of novel genes
and enzymes directly from uncultured microorganisms (Madhavan et al. 2017).
Several reports suggest the application of metagenomes for exploring extremozymes
from deep-sea environments, thereby surpassing the disadvantages accompanied
with the conventional culturing methods of extremophiles (López-López et al.
2014).
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 155

Metagenomic analyses involves the direct isolation of genomic DNA from deep-
sea environmental samples by either sequence-based (isolation of putative enzymes
based on conserved sequences) or function-based methods (isolation of functional
enzymes on the basis of expressed features like a selected enzyme activity) (Popovic
et al. 2015). The sequence-based technique utilizes the colony hybridization
approach for screening metagenomic clones. It is performed by using an oligonucle-
otide primer or probes for the target gene, which is further amplified by polymerase
chain reaction (PCR) and consequently cloned into appropriate expression vectors.
Besides, this by application of metagenomics data followed by suitable bioinfor-
matic annotations particular gene sequences can also be isolated directly. This
sequence-based technique therefore leads to the discovery of novel sequences
which in turn increases the chances of identifying enzymes efficiently (Lee et al.
2010). Though, the potential of screening specific enzymes by employing this
method is based on classical bioinformatic analyses, several novel or unknown
activities may not be recognized.
8.8 Physiology of Piezophilic Fungi
Piezophilic fungi residing the deep-sea atmosphere possess some potential physio-
logical characteristics according to the prevailing extreme conditions. The deep-sea
environment is identified by certain characteristic conditions viz., high hydrostatic
pressure (1 bar/10 m), low temperatures (2–4C) or extremely high (>400 C)
(Nagano et al. 2010) high salinity concentrations (240 ppt) up to 500 ppt (Gladfelter
et al. 2019; Barone et al. 2019), and also the absence of sunlight (Nagano et al. 2010;
Wei et al. 2014). Out of all these conditions fungi has remarkable capacity to tackle
hydrostatic pressures. But most of the initial studies have not assessed the
barotolerance capacity of deep-sea fungi. In one such study it was observed that
Aspergillus ustus and Graphium sp., isolated from the Indian Ocean showed poten-
tial barotolerance, and also exhibited conidia germination at a pressure of 100 bar.
Other studies reported that Aspergillus sydowii can grow and sporulate at 500 bar
(50 MPa) (Damare et al. 2006a; Raghukumar et al. 2004). Recent research works
have depicted that Piezophilic fungi are actually facultative peizophile and are
therefore able to modify the composition of the deep-sea environment. These
fungi are also capable of changing the fluidity of their semipermeable membrane
in order to tolerate the pressure (Simonato et al. 2006).
The confirmation of these physiological adaptations has been made by the
recognition of elevated levels of transcripts involved in the biosynthesis of ergos-
terol, signifying that marine fungi undertake alterations in their membrane composi-
tion to bear high hydrostatic pressures (Pachiadaki et al. 2016). Up regulation of the
OLE1 gene involved in the synthesis of fatty acids was observed in case of
S. cerevisiae grown under a high hydrostatic pressure of 2000 bar (200 MPa)
(Simonato et al. 2006). In addition, another important feature of deep-sea fungi is
their ability to grow under high salt concentrations which in turn makes their survival
and growth easy in the deep-sea environments. Even though deep-sea fungi have
156 S. Shukla and H. Shukla

capacity to tolerate high salt concentrations, they’re not usually halophilic fungi
(Gladfelter et al. 2019). This ability of marine fungi to grow in presence of high salt
concentrations although salt is not one of the requirements for growth is referred to
as halotolerant or halophytic. On the contrary, halophilic fungi require a specific salt
concentration (from low to high concentrations) for their optimal growth (Burgaud
et al. 2010; Gunde-Cimerman et al. 2009). Some halotolerant species from the
genera Penicillium, Cladosporium, and Aspergillus growing at 5700 mbsl and are
able to sporulate in an extremely high salt concentration of 1.7 to 34 ppt, have been
reported.
Another halophilic species isolated from the genera Candida, dwelling in a deep-
sea hydrothermal vent, was able to grow only under a salinity concentration of 30 ppt
(Burgaud et al. 2010). In another research work it was demonstrated that deep-sea
hypersaline basins (salinity between 70 and 172 ppt) located in the Eastern Mediter-
ranean Sea harbored Ascomycota and Basidiomycota (Bernhard et al. 2014). During
a metatranscriptomic analysis of these fungal species it was observed that the
majority of the detected rRNA signatures were related to the genera Aspergillus
and Penicillium (Edgcomb et al. 2016).
The most commonly occurring genus in the deep-sea environments is Hortaea
(Lai et al. 2007; Singh et al. 2012; Singh et al. 2011; Burgaud et al. 2010; Li et al.
2019; Wei et al. 2019). Majority of its species particularly the H. werneckii species,
which is black yeast has been classified as halotolerant in Mediterranean deep-sea
waters (Romeo et al. 2020) and as halophilic in deep-sea hydrothermal vents
(Burgaud et al. 2010). Various potential bioactive secondary metabolites have
been isolated by marine-derived Penicillium and Aspergillus species (Lee et al.
2013; Liu et al. 2017). Penicillium and Aspergillus species isolated from the deep-
sea are reported to produce around 106 novel bioactive compounds (Zain Ul Arifeen
et al. 2019). Out of these some compounds were found to have potential cytotoxicity
activity on different cancer cell lines (Zain Ul Arifeen et al. 2019; Sun et al. 2012).
In a similar study few other deep-sea fungal species belonging to the
Simplicillium,Acaromyces, and Engyodontium genera have been recognized as an
attractive source of secondary metabolites with cytotoxicity (Deshmukh et al. 2018).
Zain Ul Arifeen et al. (2019) isolated six novel alkaloid-bioactive compounds from
Aspergillus species and demonstrated efficient antimicrobial activity against some
fungal and bacterial pathogens like Monilia albicans,Staphylococcus aureus, Pseu-
domonas aeruginosa, and Klebsiella pneumoniae. Therefore from different studies
it can be inferred that the vast diversity of fungal species within the deep-sea are an
efficient source of different bioactive compounds including anticancer, antibacterial,
antiviral, antifungal, antioxidant, and nontoxic antifouling compounds. Moreover,
the wide range of potential osmotic techniques can be applied to explore more novel
compounds from the deep-sea fungal flora (Barone et al. 2019; Zain Ul Arifeen et al.
2019; Wang et al. 2015; Zhang et al. 2014a,b).
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 157

8.8.1 Effect of Elevated Pressure and Low Temperature
Different pressures and temperatures magnitudes produce different effects on
organisms. Usually, high hydrostatic pressure equivalent to several dozen MPa,
have been proved to be nonlethal, but may exert adverse affects on organisms
inhabiting the environments with normal atmospheric pressures (Abe 2004). For
instance it has been demonstrated that the growth of mesophilic microorganisms gets
inhibited at a high pressure of 40–50 MPa. Along with inhibition of growth some
morphological modifications including formation of filaments in Escherichia coli
and cell chains or pseudomycelia in the marine yeast Rhodosporidium
sphaerocarpum have also been observed (Lorenz and Molitoris 1992). Application
of higher pressures, ranging from 100 MPa may also be used for sterilization
purposes. The growth inhibitory effects not only depends on the amount but also
on the duration of pressure applied in combination with temperature, pH, oxygen
supply and composition of culture media (Abe 2007). Initial steps of translation are
inhibited by both low temperature and elevated pressure. The cold-shock response
has been recommended to be an adaptive response to assist the gene expression
during initiation of translation.
Studies also suggest that conditions involving elevated pressures, results in the
controlled synthesis of proteins indicating decreased translation capacity. Lastly,
conditions involving low temperature and elevated pressure reduce membrane
fluidity, which affects a variety of membrane associated processes, including trans-
port of membrane ions, nutrient flux, and DNA replication.
E. coli,Bacillus subtilis, and the budding yeast S. cerevisiae have been selected as
model organisms to study the effect of increased hydrostatic pressure and low
temperature since their complete genomes have been sequenced, and are used as
powerful genetic tools by several workers. These microbes can be classified either as
piezophilic i.e., high-pressure loving or piezotolerant i.e., capable of tolerating
elevated pressure but showing better growth at atmospheric pressure. In 1979, the
first pure culture of a piezophilic bacterial isolate, strain CNPT-3 was reported by
Yayanos (1979). At an elevated pressure of about 50 MPa this spirillum-like
bacterium depicted efficient doubling rate, growth could be was not observed at
atmospheric pressure conditions even after incubation for several weeks. Isolation
and characterization of various piezophilic and piezotolerant bacteria have been
performed from deep-sea sediments of Japan at depths ranging from
2500–11,000 m. Among these isolates majority of the strains were observed to be
piezophilic as well as psychrophilic. Such strains exhibited optimum growth at low
temperatures and almost no growth at temperatures above 20 C. Additionally,
studies in E. coli confirmed high-pressure induction of heat-shock proteins (Grob
et al. 1994). Induction of heat-shock proteins has also been demonstrated in
piezophilic microorganisms upon decompression. In another research work
subsequent to shift to atmospheric pressure resulted in induction of stress protein
similar to heat- shock proteins in the deep-sea piezophilic hyperthermophilic bacte-
ria Thermococcus barophilus. As it is well known that high pressure and low
temperature produce similar effects on protein synthesis and membrane structure,
158 S. Shukla and H. Shukla

the initiation of both pressure-shock and cold-shock proteins may correspond to an
effort by a few bacterial isolates to improve the destructive effects of high pressure
on membrane structure, translation processes, and the macromolecules stability.
8.8.2 Response of Spores and Mycelia to Low Temperature
and Elevated Hydrostatic Pressure
Different environmental conditions of sea water viz., temperature, elevated hydro-
static pressure and low nutrient concentrations exert mycostatic effect on fungal
spore germination within the deep-sea (Kirk Jr. 1980). A characteristic feature of
spores is their capacity to develop into a complete individual without fusing with
other cell. The most important step during this process is spore germination,
followed by growth of germ tube producing a mycelium by elongation, septum
formation, and branching.
Comparative study on the effect of subsequent environmental conditions on the
germination of conidia of the deep-sea Aspergillus isolates, and their viability was
conducted by Damare et al. (2008). Germination of spores of three deep-sea and
terrestrial Aspergillus isolates and a Piezophilic Cladosporium isolate in sediment
extracts of various dilutions was found to be better at 20 MPa pressure and 30 C
temperature but no germination was obtained at 5 C. On the other hand, percentage
germination lowered steadily at high pressure. According to Damare (2007) the
spores did not germinate at a temperature of 5 C, even at 0.1 MPa pressure even
after 20 days.
Even though all fungal isolates were able to produce biomass at elevated hydro-
static pressure and became vasoconstrictive when began with mycelial inoculums,
spores did not germinate under these conditions. Therefore it seems that vasocon-
striction and not high hydrostatic pressure may be a limiting factor for spore
germination and further biomass production within the deep-sea. Dormancy of
spores in three Aspergillus species was broken by temperature shock of 15 min at
50 C resulting to their germination at 0.1 MPa at 5 C but not at 20 MPa/5 C. It can
be further inferred that fungal mycelial fragments which seem to be more active
metabolically are able to tolerate high hydrostatic pressure and temperature as
compared to the dormant fungal spores. Hence, mycelial fragments have greater
probability of proliferation under deep-sea conditions than the spores. As reported
by Ivanova and Marfenina (2001), the continued existence of fungal species in tense
extreme environments is largely influenced by the size of mycelial fragments.
8.8.3 Pressure Effects on Yeasts
The effect of air mass on growth, viability and cellular responses in living cells is
termed as piezophysiology (Abe 2004). It has been observed that growth and cellular
activity in S. cerevisiae is not affected at pressures below 20–30 MPa. Elevated
pressures exert several stress response via pressure inducible genes and proteins.
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 159

Piezotolerance is dependent on the period of air mass application. Yeast cells in
stationary phase are more resistant to pressure than actively growing cells (Abe
2004; Fernandes 2005).
Another study revealed various morphological changes in wild-type and treha-
lose-6-phosphate synthase (tsp1) mutant cells of S. cerevisiae caused by hydrostatic
pressure of 200 MPa for 30 min (Fernandes et al. 2001). These mutant cells are not
able to gather an eminent tissue layer protectant called trehalose, which is a disac-
charide, during unfavorable environmental conditions. When both wild and mutant
cells were subjected to preheat treatment at 40 C temperature for 60 min before
subjecting to the pressure treatment, both the types of cells gained resistance to the
pressure treatment. Similar induced pressure tolerance in S. cerevisiae via heat shock
was earlier reported by Iwahashi et al. (1991). Besides this many researchers have
studied the effect of lethal pressures on yeast cells (Iwahashi et al. 1991,2003;
Fernandes et al. 2004).
Iwahashi et al. (2003) examined the DNA microarrays of S. cerevisiae and
evaluated expression levels of approx. 6000 genes. The gene expression profiles
depicted that pressure of about 180 MPa at 4 C for 2 min resulted in damage to
cellular organelles just similar to that caused by detergents, oils, freezing and
thawing. Likewise Fernandes et al. (2004) applied whole genome microarray
hybridization and reported some characteristic modifications in retort to hydrostatic
pressure of 200 MPa for 30 min in S. cerevisiae.
It was confirmed by further researches that most of the upregulated genes played a
key role in stress defense and carbohydrate metabolism while, a variety of down-
regulated genes were involved in processes like cell cycle progression and protein
synthesis. It was also noticed that the effects of growth inhibiting pressures in
S. cerevisiae were different from those caused by lethal pressures (Abe 2004).
According to Iwahashi et al. (2005)S. cerevisiae grown under 30 MPa pressures
exhibited growth inhibiting genome-wide mRNA expression profile. They reported
upregulation of genes membrane metabolism which produced fundamentally diverse
response to pressures.
8.8.4 Pressure Effects on Filamentous Fungi
At high pressure microbial activity is inhibited as compared to at 0.1 MPa. Elevated
pressures affect features like growth, respiration, and specific biochemical processes
(Abe and Horikoshi 1995; Fernandes et al. 2004; Daniel et al. 2006; Abe 2007).
Marine bacteria possess pressure inducible genes, which aid in pressure adjustment
and withstand huge vertical changes within the water column (Bartlett 1991). On one
hand in the case of bacteria, the effects of pressure on organic phenomenon,
membranes, membrane proteins, DNA structure and organic process, are studied
thoroughly, on the other hand in the case of fungi the studies have been directed
within the deep-sea sediments toward growth under elevated pressure and produc-
tion of extracellular enzymes under elevated hydrostatic pressure (Raghukumar and
Damare 2008).
160 S. Shukla and H. Shukla

Prokaryotic and eukaryotic cells were found to secrete selected classes of proteins
under physiological stress conditions. This phenomenon is called as “stress
response”and also the newly formed transitory proteins are termed as “stress
proteins.”These proteins play a chief role in adaptation of cells and aid them in
defense against a number of stress conditions (Lindquist and Craig 1988). Since
specific kinds of proteins provide adaptation to different stresses or shock conditions
viz., heat-shock, cold-shock or antifreeze proteins. Peizophiles exhibit therefore
some kinds of proteins available for helping in adaptation toward different hydro-
static pressures.
Some fungal species isolated from deep-sea of CIB exhibited abnormal morphol-
ogy (Damare et al. 2006a). They possessed extremely long conidiophores with
vesicles being covered by long hyphae, rather than phialides of metulae or conidia,
similar to that of the Aspergillus species. Majority of these abnormal features
vanished after subculturing for 4–6 times. It was observed in case of many deep-
sea fungi that initially they remain non-sporulating, but ultimately begins producing
spores after repeated (~ 6–8 times) subculturing at 0.1 MPa pressure. Many such
fungi when grown under elevated hydrostatic pressure presented discrete swellings.
The morphology is believed to be greatly affected by the type of nutrients used for
culturing. In one such study the Piezophilic fungi produced fungal hyphae with
several swellings when grown in malt extract seawater broth, however when grown
in sediment extract medium the fungal hyphae produced were absolutely normal
(Damare and Raghukumar 2008). At a pressure of 10 MPa two deep-sea fungi
showed microcyclic conidiation during growth (Raghukumar and Raghukumar
1998). Immediate conidiation was observed after conidial germination without the
extension of vegetative mycelium. The studies suggest that under nutrient-limiting
conditions fungi produce microcyclic conidiation which in turn help them to com-
plete their life cycles in a very shorter time. Arrest of apical growth followed by
lateral differentiation of conidium producing cells might be the reason for this
occurrence.
8.8.5 Effect of Deep-Sea Conditions on Growth and Protein
Patterns
As compared to bacteria, very fewer studies have been made on the effect of high
hydrostatic pressure and low temperature conditions on fungal growth patterns.
Raghukumar et al. (2004), observed germination and growth of spores of deep-sea
fungus A. Sydowii, isolated from deep-sea sediments of Chagos Trench under a high
hydrostatic pressure and low temperature conditions. In another research work,
mycelial inocula of fungi isolated from deep-sea sediments of the CIB were found
to produce considerable growth at both 30 C and 5 C under elevated hydrostatic
pressure (Damare et al. 2006a). S. cerevisiae is a facultative anaerobe and therefore
is used in a variety of studies for evaluating the effects of elevated hydrostatic
pressure and low temperature. Because of its facultative anaerobic nature,
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 161

S. cerevisiae has been selected as most appropriate organism to survive under closed
pressure vessel condition in very low oxygen concentration (Abe 2007).
Approximately 6000 genes are encoded by the genome of this yeast out of which
more than 4800 are non essential ones. Recent studies have demonstrated the
presence of unexpected genes and metabolic pathways that are responsible for
environmental stresses tolerance. Moreover this yeast exhibited no growth at ele-
vated pressure and low temperature conditions when examined via genome-wide
gene expression profiles. However, high levels of upregulated genes involved in
energy metabolism transaction, protein and cell defense metabolism were recovered
from the stress conditions (Iwahashi et al. 2003). It was also observed that
S. cerevisiae survived more efficiently at elevated pressures after prior heat-shock
treatment (Iwahashi et al. 1991). The heat-shock treatments lead to increased
production of heat-shock proteins (Hsps) and trehalose metabolism in this yeast.
Out of all the heat-shock proteins (Hsp), Hsp104 plays a vital role in developing
tolerance by carrying out ATP- dependent unfolding of denatured intracellular
proteins. Additionally, synthesis rate of various proteins viz., ubiquitin, few glyco-
lytic enzymes, and a plasma membrane protein in S. cerevisiae, was found to be
enhanced greatly upon exposure to stress conditions. It was further observed that a
plasmid carrying the TAT2 gene, encoding a high affinity tryptophan permease,
supported S. cerevisiae growth at a pressure ranging between 15 and 25 MPa.
Moreover, cells producing high levels of the Tat2 protein became capable to grow
under low-temperature (10–15 C) as well as at high-pressure conditions. They also
reported that vacuoles in yeast cells were more acidified at hydrostatic pressures of
40–60 MPa and that the growth of yeast cells was highly affected by expression of
the tryptophan permease i.e., TAT2 gene.
Besides S. cerevisiae, very few other fungal isolates are utilized as model
organisms to study the effect of high hydrostatic pressure and cold stress conditions.
By application of advance techniques these stress conditions were applied for
determining their effects on filamentous and unicellular marine fungi. The marine
ecosystem is the world’s biggest ecosystem spreading over three-fourths of the
Earth’s surface. The average depth of the marine biome is 3800 m, and is subjected
to a pressure of 38 megapascals (MPa). Several factors affect the biodiversity
prevailing in oceans, but hydrostatic pressure is the most significant parameter out
of all.
Deep-sea microorganisms are categorized on the basis of their cardinal growth
pressures into different classes’viz., piezosensitive, piezotolerant, piezophiles, or
hyperpiezophiles. Recent studies suggest that Pyrococcus yayanosii, an obligate
piezophilic hyperthermophilic archaeon isolated from a deep-sea hydrothermal
vent, exhibited potential to grow even at high hydrostatic pressure ranging from
20 to 120 MPa. These results demonstrate the important role of hydrostatic pressure
in the life cycle of prokaryotic organisms within the deep-sea (Burgaud et al. 2015).
On the other hand numerous DNA-based studies have reported the presence of a
number of fungal communities in the deep-sea ecosystems along with prokaryotic
organisms (Abe 2007). These fungal communities live in deep sediments hydrother-
mal vents, sunken woods, cold seeps and even deep hypersaline anoxic basins
162 S. Shukla and H. Shukla

(Le Calvez et al. 2009). The sequencing techniques based on rRNA and mRNA
approaches have disclosed some important informations about these communities
viz., their metabolically functional components, their imperative ecological roles in
biogeochemical cycles or their interactions with prokaryotes and antibiotic defense
mechanisms (Takishita et al. 2007). The fungal communities thus recovered were
mainly belonging to a subkingdom Dikarya and two phyla Ascomycota and
Basidiomycota. In addition few species belonging to the basal phyla
Chytridiomycota and Cryptomycota were also obtained. Nevertheless culture-
based methods will always be preferred to isolate deep-sea fungal strains for
assessing their ability to tolerate hydrostatic pressure. This can develop our aware-
ness about the deep-sea fungal flora and their ecological roles in such stressful
environments.
Different effects of high pressure on biological processes include fluidity modifi-
cation of pressure-sensitive lipids, membrane permeability, multimeric associations
and stability of pressure-sensitive proteins, thereby inducing motility and organic
process and replication and transcription processes by pressure-stabilized DNA
hydrogen bonds. In a similar study the impact of high hydrostatic pressure on cell
physiology has been comprehensively demonstrated within the yeast cells. From
these studies it was indicated that the tryptophan auxotrophy and also the availability
of this organic compound affects the power of S. cerevisiae cells to grow at moderate
pressure, between 15 and 25 MPa. It was further observed that S. cerevisiae cells
were killed at pressures ranging from 100 to 200 MPa as a result of disruption of
microtubule ultrastructures, actin filaments or nuclear membranes.
A number of research works have especially paid attention on the development of
marine fungi growing under hydrostatic pressure. On the basis of these studies most
of the fungal species isolated were from the Aspergillus genus, while others were
marine yeasts namely Rhodotorula rubra,Debaryomyces hansenii, and
Rhodosporidium sphaerocarpum. However, almost all isolated fungal species
were considered as piezosensitive, since their growth was found to be much better
at gas pressure (0.1 MPa) in comparison to high hydrostatic pressures. Another study
highlighted that the three marine yeasts cultivated under high hydrostatic pressure
were able to grow at a pressure of 20 MPa, whereas only the basidiomycetous
species R. rubra and R. sphaerocarpum were observed to grow at 40 MPa, indicating
better piezotolerance of basidiomycetes compared to ascomycetes. Yeasts recovered
in these studies seems to be well adapted.
8.9 Conclusion
As a result of several research works it could be inferred that deep-sea sediments
emerge as one of the potential habitat for Piezophilic fungi. Therefore a technical
review of oceans of the world is generally suggested for determining the culture-
dependent and culture-independent fungi. Novel techniques and media compositions
to separate and culture them need to be designed. Moreover their function in macro
aggregate formation is very important in biogeochemical cycling of nutrients and
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 163

carbon fixation within the deep-sea. They are also an efficient source of bioactive
secondary metabolites and enzymes with new characteristics and applications (Pettit
2010). But still Piezophilic fungi are an unexplored part of the deep-sea environ-
ment. Inspite of their physiological adaptability to vasoconstrictive, and elevated
hydro pressure, and imperative roles within the ecosystem, the deep-sea fungal flora
have not been studied much. The diversity of deep-sea fungi currently known is just
like a drop from the ocean and a huge amount of the unidentified fungal community
is yet to be explored. Deep-sea fungi have shown great potential as a source of
interesting enzymes, exclusive metabolics, and novel secondary metabolites.
Numerous biotechnologically significant enzymes are produced by deep-sea fungal
community isolated from a range of marine habitats.
Piezophilic fungal diversity possesses genetic diversity and adaptive nature to
numerous extreme environmental conditions makes them an attractive candidate for
research works. For the investigation of deep-sea fungal diversity both cultural-
dependent and cultural-independent techniques are used. However, cultural-
independent techniques have been demonstrated to be more effective for the discov-
ery of unknown fungal species inhabiting the deep-sea, which are not able to
grow via standard culture-dependent techniques. These fungi are a promising source
of important products like extracellular bioactive compounds including
polysaccharides, enzymes, and other secondary metabolites. Enhanced coordinated
research work is necessary to expand our current knowledge about the diversity,
activity, and capability of deep-sea fungi to adapt to the extreme environmental
conditions of the marine ecosystem.
Future marine mycological research should focus particularly on the “unidenti-
fied”or “unclassified”sequences within the deep-sea fungal datasets, which not only
are of great biotechnological potential, but also represent key fungal species for
unravelling the origin of marine fungi. Besides more research works on marine
fungal biology are needed to disclose attractive biochemical and physiological
features of the deep-sea fungal flora. With the advent of recent scientific techniques
to assess the physiology and biochemistry of uncultured and unusual marine-derived
fungal flora would definitely pave the path for exploring potential of piezophilic
fungi.
References
Abe F (2004) Piezophysiology of yeast: occurrence and significance. Cell Mol Biol 50:437–445
Abe F (2007) Induction of DAN/TIR yeast cell wall mannoprotein genes in response to high
hydrostatic pressure and low temperature. FEBS Lett 581:4993–4998
Abe F, Horikoshi K (1995) Hydrostatic pressure promotes the acidification of vacuoles in Saccha-
romyces cerevisiae. FEMS Microbiol Lett 130:307–312
Abe F, Miura T, Nagahama T, Inoue A, Usami R, Horikoshi K (2001) Isolation of a highly copper-
tolerant yeast, Cryptococcus sp., from the Japan trench and the induction of superoxide
dismutase activity by Cu
2
+. Biotechnol Lett 23:2027–2034
164 S. Shukla and H. Shukla

Abe F, Minegishi H, Miura T, Nagahama T, Usami R, Horikoshi K (2006) Characterization of cold-
and high-pressure-active polygalacturonases from a deep-sea yeast, Cryptococcus liquefaciens
strain N6. Biosci Biotechnol Biochem 70:296–299
Alker AP, Smith GW, Kim K (2001) Characterization of Aspergillus sydowii (Thom & Church), a
fungal pathogen of Caribbean sea fan corals. Hydrobiologia 460:105–111
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S
rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial
populations. Appl Environ Microbiol 56:1919–1925
Bailey VL, Smith JL, Bolton H (2002) Fungal-to-bacterial ratios investigated for enhanced C
sequestration. Soil Biol Biochem 34:997–1007
Balser TC, Treseder KK, Ekenler M (2005) Using lipid analysis and hyphal length to quantify AM
and saprophytic fungal abundance along a soil chronosequence. Soil Biol Biochem 37:601–604
Barone G, Varrella S, Tangherlini M, Rastelli E, Dell’Anno A, Danovaro R, Corinaldesi C (2019)
Marine fungi: biotechnological perspectives from deep-hypersaline anoxic basins. Diversity 11:
113
Bartlett DH (1991) Pressure sensing in deep-sea bacteria. Res Microbiol 142:923–925
Bass D, Howe A, Brown N, Barton H, Demidova M, Michelle H, Li L, Sanders H, Watkinson SCC,
Willcock S, Richards TAA (2007) Yeast forms dominate fungal diversity in the deep oceans.
Proc R Soc B 274:3069–3077
Beare MH, Cabrera ML, Hendrix PF, Coleman DC (1994) Aggregate-protected and unprotected
pools of organic matter in conventional and no-tillage soils. Soil Sci Soc Am J 58:787–795
Bernhard JM, Kormas K, Pachiadaki MG, Rocke E, Beaudoin DJ, Morrison C, Visscher PT,
Cobban A, Starczak VR, Edgcomb VP (2014) Benthic protists and fungi of Mediterranean
deep hypsersaline anoxic basin redoxcline sediments. Front Microbiol 5:605
Biddle JF, House CH, Brenchley JE (2005) Microbial stratification in deeply buried marine
sediment reflects change in sulfate/methane profiles. Geobiology 3:287–295
Biddle JF, Fitz-Gibbon S, Schuster SC, Brenchley JE, House CH (2008) Metagenomic signatures of
the Peru margin subseafloor biosphere show a genetically distinct environment. Proc Natl Acad
Sci U S A 105:10583–10588
Bronick CJ, Lal R (2005) Soil structure and management: a review. Geoderma 124:3–22
Brown MV, Philip GK, Bunge JA, Smith MC, Bissett A, Lauro FM, Fuhrman JA, Donachi SP
(2009) Microbial community structure in the North Pacific Ocean, three-domain marine com-
munity composition analysis. ISME J 3:1374–1386
Burgaud G, Le Calvez T, Arzur D, Vandenkoornhuyse P, Barbier G (2009) Diversity of culturable
marine filamentous fungi from deep-sea hydrothermal vents. Environ Microbiol 11:1588–1600
Burgaud G, Arzur D, Durand L, Cambon-Bonavita M-A, Barbier G (2010) Marine culturable yeasts
in deep-sea hydrothermal vents: species richness and association with fauna. FEMS Microbiol
Ecol 73:121–133
Burgaud G, Hué NTM, Arzur D, Coton M, Perrier-Cornet J-M, Jebbar M, Barbier G (2015) Effects
of hydrostatic pressure on yeasts isolated from deep-sea hydrothermal vents. Res Microbiol
166(9):700–709
Burns RG (1978) Enzymes in soil: some theoretical and practical considerations. In: Burns RG
(ed) Soil enzymes. Academic Press, London, pp 295–339
Cao L (2010) Fungal communities of methane clathrate-bearing deep-sea sediments. In: Timmis
KN (ed) Handbook of hydrocarbon and lipid microbiology. Springer, Berlin, pp 2226–2230
Cavicchioli R, Amils R, Wagner D, McGenity T (2011) Life and applications of extremophiles.
Environ Microbiol 13:1903–1907
Chiu CY, Chen TH, Imberger K, Tian G (2006) Particle size fractionation of fungal and bacterial
biomass in subalpine grassland and forest soils. Geoderma 130:265–271
Chróst RJ (1991) Environmental control of the synthesis and activity of aquatic microbial
ectoenzymes. In: Chróst RJ (ed) Microbial enzymes in aquatic environment. Springer-Verlag,
New York, pp 29–59
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 165

Connell L, Barrett A, Templeton A, Staudigel H (2009) Fungal diversity associated with an active
deep-sea volcano: Vailulu’u Seamount, Samoa. Geomicrobiol J 26:597–605
Dalmaso G, Ferreira D, Vermelho A (2015) Marine extremophiles: a source of hydrolases for
biotechnological applications. Mar Drugs 13:1925–1965
Damare S (2007) Deep-sea fungi: occurrence and adaptations. PhD thesis, Goa University
Damare S, Raghukumar C (2008) Fungi and macroaggregation in deep-sea sediments. Microb Ecol
56:168–177
Damare S, Raghukumar C, Raghukumar S (2006a) Fungi in deep-sea sediments of the Central
Indian Basin. Deep-Sea Res I 53:14–27
Damare S, Raghukumar C, Muraleedharan UD, Raghukumar S (2006b) Deep-sea fungi as a source
of alkaline and cold-tolerant proteases. Enzym Microb Technol 39:172–181
Damare S, Nagarajan M, Raghukumar C (2008) Spore germination of fungi belonging to aspergil-
lus species under deep- sea conditions. Deep-Sea Res I 55:670–678
Dang H, Zhu H, Wang J, Li T (2009) Extracellular hydrolytic enzyme screening of culturable
heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough. World J
Microbiol Biotechnol 25:71–79
Daniel I, Oger P, Winter R (2006) Origins of life and biochemistry under high-pressure conditions.
Chem Soc Rev 35:858–875
Deshmukh SK, Prakash V, Ranjan N (2018) Marine fungi: a source of potential anticancer
compounds. Front Microbiol 8:2536
Dupont J, Magnin S, Rousseau F, Zbinden M, Frebourg G, Samadi S, de Forges BR, Jones EBG
(2009) Molecular and ultrastructural characterization of two ascomycetes found on sunken
wood off Vanuatu Islands in the deep Pacific Ocean. Mycol Res 113:1351–1364
Edgcomb VP, Beaudoin D, Gast R, Biddle JF, Teske A (2010) Marine subsurface eukaryotes: the
fungal majority. Environ Microbiol. https://doi.org/10.1111/j.1462-2920.2010.02318.x
Edgcomb VP, Pachiadaki MG, Mara P, Kormas KA, Leadbetter ER, Bernhard JM (2016) Gene
expression profiling of microbial activities and interactions in sediments under haloclines of
E. Mediterranean deep hypersaline anoxic basins. ISME J 10:2643–2657
Fernandes PMB (2005) How does yeast respond to pressure? Braz J Med Biol Res 38:1239–1245
Fernandes PMB, Farina M, Kurtenbach E (2001) Effect of hydrostatic pressure on the morphology
and ultrastructure of wild-type and trehalose synthase mutant cells of Saccharomyces cerevisiae.
Lett Appl Microbiol 32:42–46
Fernandes PMB, Domitrovic T, Cao CM, Kurtenbach E (2004) Genomic expression pattern in
Saccharomyces cerevisiae cells in response to high hydrostatic pressure. FEBS Lett 556:153–
160
Ferrer M, Golyshina O, Beloqui A, Golyshin PN (2007) Mining enzymes from extreme
environments. Curr Opin Microbiol 10:207–214
Gadanho M, Sampaio J (2005) Occurrence and diversity of yeasts in the Mid-Atlantic ridge
hydrothermal fields near the Azores Archipelago. Microb Ecol 50:408–417
Gao B, Li L, Wu H, Zhu D, Jin M, Qu W, Zeng R (2019) A novel strategy for e_cient agaro-
oligosaccharide production based on the enzymatic degradation of crude agarose in
Flammeovirga pacifica WPAGA1. Front Microbiol 10:1231
Gessner MO, Newell SY (2001) Biomass, growth rate and production of filamentous fungi in plant
litter. In: Hurst CJ, McInerney M, Stetzenbach L, Knudsen G, Walter M (eds) Manual of
environmental microbiology, 2nd edn. ASM Press, Washington DC
Gladfelter AS, James TY, Amend AS (2019) Marine fungi. Curr Biol 29:R191–R195
Grob M, Kosmoswosky IJ, Lorenz R, Molitoris HP, Jaenicke R (1994) Response of bacteria and
fungi to high-pressure stress as investigated by two-dimensional gel electrophoresis. Electro-
phoresis 15:1559–1565
Gunde-Cimerman N, Ramos J, Plemenitas A (2009) Halotolerant and halophilic fungi. Mycol Res
113:1231–1241
Haki G, Rakshit S (2003) Developments in industrially important thermostable enzymes: a review.
Bioresour Technol 89:17–34
166 S. Shukla and H. Shukla

Harrison JP, Gheeraert N, Tsigelnitskiy D, Cockell CS (2013) The limits for life under multiple
extremes. Trends Microbiol 21:204–212
Höhnk W (1969) Uber den pilzlichen Befall kalkiger Hartteile von Meerestieren. Ber Dtsch Wiss
Komm Meeresforsch 20:129–140
Horikoshi K, Bull AT (2011) Prologue: definition, categories, distribution, origin and evolution,
pioneering studies, and emerging fields of extremophiles. In: Extremophiles handbook.
Springer, Tokyo, pp 3–15
Hyde KD, Jones EBG, Moss ST (1986) Mycelial adhesion to surfaces. In: Moss ST (ed) The
biology of marine fungi. Cambridge University Press, Cambridge, pp 331–340
Irwin JA, Baird AW (2004) Extremophiles and their application to veterinary medicine. Irish Vet J
57:348
Ivanova AE, Marfenina OE (2001) The effect of ecological factors on spore germination and the
viability of the mycelial fragments of microscopic fungi. Microbiology 70:195–199
Iwahashi H, Kaul SC, Obuchi K, Komatsu Y (1991) Induction of barotolerance by heat shock
treatment in yeast. FEMS Microbiol Lett 80:325–328
Iwahashi H, Shimizu H, Odani M, Komatsu Y (2003) Piezophysiology of genome wide gene
expression levels in the yeast Saccharomyces cerevisiae. Extremophiles 7:291–298
Iwahashi H, Odani M, Ishidou E, Kitagawa E (2005) Adaptation of Saccharomyces cerevisiae to
high hydrostatic pressure causing growth inhibition. FEBS Lett 579:2847–2852
Jannasch HW, Wirsen CO, Winget CL (1973) A bacteriological pressure-retaining deep-sea
sampler and culture vessel. Deep-Sea Res 20:661–664
Jebraj CS, Raghukumar C (2009) Anaerobic denitrification in fungi from the coastal marine
sediments off Goa, India. Mycol Res 113:100–109
Kandeler E, Stemmer M, Klimanek EM (1999) Response of soil microbial biomass, urease and
xylanase within particle-size fractions to long-term soil management. Soil Biol Biochem 31:
261–273
Kirk PW Jr (1980) The mycostatic effect of seawater on spores of terrestrial and marine higher
fungi. Bot Mar 23:233–238
Kohlmeyer J (1977) New genera and species of higher fungi from the deep-sea (1615-5315m). Rev
Mycol 41:189–206
Kohlmeyer J, Kohlmeyer E (1979) Marine mycology: the higher fungi. Academic Press, New York,
690 p
Lai X, Cao L, Tan H, Fang S, Huang Y, Zhou S (2007) Fungal communities from methane hydrate-
bearing deep-sea marine sediments in South China Sea. ISME J 1:756–762
Le Calvez T, Burgaud G, Mahe S, Barbier G, Vandenkoornhuyse P (2009) Fungal diversity in deep-
sea hydrothermal ecosystems. Appl Environ Microbiol 75:6415–6421
Lee HS, Kwon KK, Kang SG, Cha S-S, Kim SJ, Lee JH (2010) Approaches for novel enzyme
discovery from marine environments. Curr Opin Biotechnol 21:353–357
Lee YM, Kim MJ, Li H, Zhang P, Bao B, Lee KJ, Jung JH (2013) Marine-derived Aspergillus
species as a source of bioactive secondary metabolites. Mar Biotechnol 15:499–519
Li W, Wang M, Burgaud G, Yu H, Cai L (2019) Fungal community composition and potential
depth-related driving factors impacting distribution pattern and trophic modes from epi- to
abyssopelagic zones of the western Pacific Ocean. Microb Ecol 78:820–831
Lindquist S, Craig EA (1988) The heat-shock proteins. Ann Rev Genet 22:631–677
Liu H, Herbert HP, Fang HP (2002) Extraction of extracellular polymeric substances (EPS) of
sludges. J Biotechnol 95:249–256
Liu S, Su M, Song SJ, Jung JH (2017) Marine-derived Penicillium species as producers of cytotoxic
metabolites. Mar Drugs 15(10):329
Lopez-Garcia P, Rodriguez-Valera F, Pedros-Allo C, Moreira D (2001) Unexpected diversity of
small eukaryotes in deep-sea Antarctic plankton. Nature 409:603–607
Lopez-Garcia P, Philip H, Gail F, Moreira D (2003) Autochthonous eukaryotic diversity in
hydrothermal sediment and experimental microcolonizers at the Mid-Atlantic Ridge. Proc
Natl Acad Sci U S A 100:697–702
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 167

López-López O, Cerdan ME, Gonzalez Siso MI (2014) New extremophilic lipases and esterases
from metagenomics. Curr Protein Pept Sci 15:445–455
Lorenz R, Molitoris HP (1992) High pressure cultivation of marine fungi: apparatus and method.
Colloq INSERM 224:537–539
Madhavan A, Sindhu R, Parameswaran B, Sukumaran RK, Pandey A (2017) Metagenome analysis:
Apowerful tool for enzyme bioprospecting. Appl Biochem Biotechnol 183:636–651
Mille-Lindblom C, Wachenfeldt E, Tranvik LJ (2004) Ergosterol as a measure of living fungal
biomass: persistence in environmental samples after fungal death. J Microbiol Methods 59:253–
262
Miura T, Abe F, Inoue A, Usami R, Horikoshi K (2001) Purification and characterization of novel
extracellular endopolygalacturonases from a deep-sea yeast, Cryptococcus sp. N6, isolated from
the Japan Trench. Biotechnol Lett 23:1735–1739
Molope MB, Grieve IC, Page ER (1987) Contributions of fungi and bacteria to aggregate stability
of cultivated soils. J Soil Sci 38:71–77
Moreira D, López-García P (2003) Are hydrothermal vents oases for parasitic protists? Trends
Parasitol 19:556–558
Nagano Y, Nagahama T, Hatada Y, Nunoura T, Takami H, Miyazaki J, Takai K, Horikoshi K
(2010) Fungal diversity in deep-sea sediments—the presence of novel fungal groups. Fungal
Ecol 3:316–325
Nath IA, Bharathi PL (2011) Diversity in transcripts and translational pattern of stress proteins in
marine extremophiles. Extremophiles 15:129–153
Newell SY (1996) Established and potential impacts of eukaryotic mycelial decomposers in marine/
terrestrial ecotones. J Exp Mar Biol Ecol 200:187–206
Pachiadaki MG, Redou V, Beaudoin DJ, Burgaud G, Edgcomb VP (2016) Fungal and prokaryotic
activities in the marine subsurface biosphere at Peru margin and Canterbury basin inferred from
RNA-based analyses and microscopy. Front Microbiol 7:1–16
Parkes RJ, Webster G, Cragg BA, Weightman AJ, Newberry CJ, Ferdelman TG, Kallmeyer J,
Jorgensen BB, Aiello IW, Fry JC (2005) Deep sub-seafloor prokaryotes stimulated at interfaces
over geological time. Nature 436:390–394
Pettit RK (2010) Culturability and secondary metabolite diversity of extreme microbes: expanding
contribution of deep-sea and deep-sea vent microbes to natural product discovery. Mar
Biotechnol. https://doi.org/10.1007/s10126-010-9294-y
Popovic A, Tchigvintsev A, Tran H, Chernikova TN, Golyshina OV, Yakimov MM, Golyshin PN,
Yakunin AF (2015) Metagenomics as a tool for enzyme discovery: hydrolytic enzymes from
marine-related metagenomes. In: Prokaryotic systems biology. Springer, Berlin/Heidelberg, pp
1–20
Poulicek M, Machiroux R, Toussaint C (1986) Chitin diagenesis in deep-water sediments. In:
Muzzarelli R, Jeunix C, Gooday GW (eds) Chitin in nature and technology. Plenum Press,
New York, pp 523–530
Raddadi N, Cherif A, Daffonchio D, Neifar M, Fava F (2015) Biotechnological applications of
extremophiles, extremozymes and extremolytes. Appl Microbiol Biot 99:7907–7913
Raghukumar C, Damare SR (2008) Deep-Sea fungi. In: Michiels C, Bartlett DH, Aertsen A (eds)
High-pressure microbiology. ASM Press, Washington, DC, pp 265–292
Raghukumar C, Raghukumar S (1998) Barotolerance of fungi isolated from deep-sea sediments of
the Indian Ocean. Aquat Microbiol Ecol 15:153–163
Raghukumar C, Raghukumar S, Sharma S, Chandramohan D (1992) Endolithic fungi from deep-
sea calcareous substrate: isolation and laboratory studies. In: Desai BN (ed) Oceanography of
the Indian ocean. Oxford & IBH, pp 3–9
Raghukumar C, Sheelu G, Loka Bharati PA, Nair S, Mohandass C (2001) Microbial biomass and
organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin. Mar Geores
Geotechnol 19:1–16
168 S. Shukla and H. Shukla

Raghukumar C, Raghukumar S, Sheelu G, Gupta SM, Nagender Nath B, Rao BR (2004) Buried
in time: culturable fungi in a deep-sea sediment core from the Chagos Trench, Indian Ocean.
Deep-Sea Res I 51:1759–1768
Raghukumar C, Nath NB, Sharma R, Loka Bharathi PA, Dalal SG (2006) Long-term changes in
microbial and biochemical parameters in the Central Indian Basin. Deep-Sea Res Part 1(53):
1695–1717
Raghukumar C, Damare SR, Muraleedharan UD (2009) A process for the production of low
temperature-active alkaline protease from a deep-sea fungus. EP1692296
Romeo O, Marchetta A, Giosa D, Giuffrè L, Urzì C, De Leo F (2020) Whole genome sequencing
and comparative genome analysis of the halotolerant deep-sea black yeast Hortaea werneckii.
Life 10:229
Roth FJ, Orpurt PA Jr, Ahearn DG (1964) Occurrence and distribution of fungi in a subtropical
marine environment. Can J Bot 42:375–383
Samuel P, Raja A, Prabakaran P (2012) Investigation and application of marine derived microbial
enzymes: status and prospects. Int J Ocean Mar Ecol Syst 1:1–10
Selbmann L, Stingele F, Petruccioli M (2003) Exopolysaccharide production by filamentous fungi:
the example of Botryosphaeria rhodina. Antonie Van Leeuwenhoek 84:135–145
Simonato F, Campanaro S, Lauro FM, Vezzi A, D’Angelo M, Vitulo N, Giorgio V, Bartlett DH
(2006) Piezophilic adaptation: a genomic point of view. J Biotechnol 126:11–25
Singh P, Raghukumar C, Verma P, Shouche Y (2010) Phylogenetic diversity of culturable fungi
from the deep-sea sediments of the Central Indian Basin and their growth characteristics. Fungal
Divers 40:89–102
Singh P, Raghukumar C, Verma P, Shouche Y (2011) Fungal community analysis in the deep-sea
sediments of the central Indian Basin by culture-independent approach. Microb Ecol 61:507–
517
Singh P, Raghukumar C, Meena RM, Verma P, Shouche Y (2012) Fungal diversity in deep-sea
sediments revealed by culture-dependent and culture-independent approaches. Fungal Ecol 5:
543–553
Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006)
Microbial diversity in the deep-sea and the underexplored “rare biosphere”. Proc Natl Acad Sci
U S A 103:12115–12120
Sparrow FK Jr (1960) Aquatic phycomycetes, 2nd edn. The University of Michigan Press, Ann
Arbor, pp 792–822
Stevenson FJ (1994) Humus chemistry, genesis, composition, reactions. Wiley, Hoboken, NJ, 497p
Suberkropp K, Meyers H (1996) Application of fungal and bacterial production methodologies to
decomposing leaves in streams. Appl Environ Microbiol 62:1610–1615
Sun Y, Takada K, Takemoto Y, Yoshida M, Nogi Y, Okada S, Matsunaga S (2012) Gliotoxin
analogues from a marine-derived fungus, Penicillium sp., and their cytotoxic and histone
methyltransferase inhibitory activities. J Nat Prod 75:111–114
Synnes M (2007) Bioprospecting of organisms from the deep sea: scientific and environmental
aspects. Clean Tech Environ Policy 9:53–59
Takishita K, Tsuchiya M, Reimer JD, Maruyama T (2006) Molecular evidence demonstrating the
basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sedi-
ment at the Kuroshima Knoll methane seep. Extremophiles 10:165–169
Takishita K, Yubuki N, Kakizoe N, Inagaki Y, Maruyama T (2007) Diversity of microbial
eukaryotes in sediment at a deep-sea methane cold seep: surveys of ribosomal DNA libraries
from raw sediment samples and two enrichment cultures. Extremophiles 11:563–576
Tisdall JM, Oades JM (1982) Organic matter and water-stable aggregates in soils. J Soil Sci 33:
141–163
Turley CM, Dixon JL (2002) Bacterial numbers and growth in surficial deep-sea sediments and
phytodetritus in the NE Atlantic: relationships with particulate organic carbon and total nitro-
gen. Deep-Sea Res I 49:815–826
8 Ecology, Physiology, and Diversity of Piezophilic Fungi 169

Van Den Burg B (2003) Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6:213–
218
Van Veen JA, Paul EA (1979) Conversion of biovolume measurements of soil organisms, grown
under various moisture tensions, to biomass and their nutrient content. Appl Environ Microbiol
37:686–692
Wang YT, Xue YR, Liu CH (2015) A brief review of bioactive metabolites derived from deep-sea
fungi. Mar Drugs 13:4594–4616
Wei X, Pang KL, Luo ZH (2014) High fungal diversity and abundance recovered in the deep-sea
sediments of the Pacific Ocean. Microb Ecol 68:688–698
Wei X, Gao Y-H, Gong L-F, Li M, Pang K-L, Luo Z-H (2019) Fungal diversity in the deep-sea
hadal sediments of the Yap Trench by cultivation and high throughput sequencing methods
based on ITS rRNA gene. Deep Sea Res Part I Oceanogr Res Pap 145:125–136
Yanagibayashi M, Nogi Y, Li L, Kato C (1999) Changes in the microbial community in Japan
trench sediment from a depth of 6292 m during cultivation without decompression. FEMS
Microbiol Lett 170:271–279
Yayanos AA (1979) Isolation of a deep-sea barophilic bacterium and some of its growth
characteristics. Science 205:808–810
Zain Ul Arifeen M, Ma YN, Xue YR, Liu CH (2019) Deep-Sea fungi could be the new arsenal for
bioactive molecules. Mar Drugs 18:9
Zhang C, Kim SK (2010) Research and application of marine microbial enzymes: status and
prospects. Mar Drugs 8:1920–1934
Zhang XY, Tang GL, Xu XY, Nong XH, Qi SH (2014a) Insights into deep-sea sediment fungal
communities from the East Indian Ocean using targeted environmental sequencing combined
with traditional cultivation. PLoS One 9:e109118
Zhang XY, Xu XY, Peng J, Ma CF, Nong XH, Bao J, Zhang GZ, Qi SH (2014b) Antifouling
potentials of eight deep-sea-derived fungi from the South China Sea. J Ind Microbiol Biotechnol
41:741–748
Zuccaro A, Summerbell RC, Gams W, Schroers H-F, Mitchell JI (2004) A new Acremonium
species associated with Fucus spp, and its affinity with a phylogenetically distinct marine
Emericellopsis clade. Stud Mycol 50:283–297
170 S. Shukla and H. Shukla

Halophilic, Acidophilic, Alkaliphilic,
Metallophilic, and Radioresistant Fungi:
Habitats and Their Living Strategies
9
Tuyelee Das, Abdel Rahman Al-Tawaha, Devendra Kumar Pandey,
Potshangbam Nongdam, Mahipal S. Shekhawat, Abhijit Dey,
Kanak Choudhary, and Sanjay Sahay
Abstract
The magnificent stress-resistant mechanism, capacity to transform extreme abi-
otic factors as triggers for genetic modulation and physiological evolution,
synced speciation in response to altered environment, and highly innovative
succession cum resource management skill have crowned the microorganisms
as the “specialist messenger of life”that thrive under extreme conditions. How-
ever, in recent decade, the ubiquitous fungi have gathered attention after archaea
and bacteria for their versatile ecological adaptation, morphological resilience,
and biochemical flexibility that allowed them to sustain and flourish under
nature’s deadliest environmental conditions. The inhospitable temperature, pres-
sure, radiation, desiccation, salinity, and pH (both acidic and basic)-induced
T. Das · A. Dey (*)
Department of Life Science, Presidency University, Kolkata, West Bengal, India
e-mail: abhijit.dbs@presiuniv.ac.in
A. R. Al-Tawaha
Department of Biological Sciences, Al-Hussein Bin Talal University, Maan, Jordan
D. K. Pandey
Department of Biotechnology, Lovely Professional University, Phagwara, Punjab, India
P. Nongdam
Department of Biotechnology, Manipur University, Imphal, Manipur, India
M. S. Shekhawat
Biotechnology Unit, Kanchi Mamunivar Government Institute for Postgraduate Studies and
Research, Puducherry, India
K. Choudhary
Department of Biotechnology, Barkarullah University, Bhopal, Madhya Pradesh, India
S. Sahay
Sarojini Naidu Government Postgraduate Girl’s (Autonomous) College, Bhopal, Madhya Pradesh,
India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_9
171

stress has capacitated a large number of extremophilic fungi with better
sustainability factors. The “extraterrestrial”type of existence has been reported
from hostile and lethal niches like frozen world of Antarctic and Arctic, deep sea
ice and hydrothermal vents, hot springs, areas of high salt concentration, barren
desert with extreme climate, toxic heavy metal and organic matter polluted
regions, ocean trenches with high pressure, radiation contaminated zones, etc.
The phylogenetic diversity of extremophilic fungi is highly complex exactly as
their multidimensional mechanism of primary and secondary resource manage-
ment, niche utilization, and physiological metabolism. From the bed of life-
enriched rainforests to barren worlds full of toxic materials and fluctuating
climate, this eukaryotic group has manifested great evolutionary plasticity and
molecular strategies that are the center of interdisciplinary research that connects
evolutionary biology, astrobiology, biochemistry, molecular biology, ecology,
and many related fields of science. The modification of genetic make-up and
introduction of specialized survival technique controlled via manipulation of
metabolic pathways are not only associated with successful colonization of
these fungal members but also important in terms of exploration of natural
products from unexplored sources.
Keywords
Halophiles · Acidophiles · Alkaliphiles · Living strategy
9.1 Introduction
Until microbiologist exposed that the extreme environment of earth is truly occupied
by a various range of microorganisms, humans assumed that in such extreme
parameters no organism can live. Nonetheless, lately, a diverse variety of
extremophiles has been discovered across a wide range of environment like hydro-
thermal vents, hot springs, polar regions, acid mine drainage sites, deserts, acidic
lake, saline–alkaline lakes, sodic lakes, etc. (Gunde-Cimerman et al. 2003; Gunde-
Cimerman and Zalar 2014; Plemenitašet al. 2014; Selbmann et al. 2013).
Extremophiles (eukaryotes, bacteria, and archaea) are microbes that have been
found at extremes of pressures of up to 110 MPa, pH (0–12.5), temperature
(122 C20 C), salinity (>1.0 M NaCl), and UV radiations. Archaea is the
most flourish group of extremophiles. Alternatively, fungi are the most adaptable,
ubiquitous, and effective ecological group having progressed gradually toward a
wide range of ecological niches. Accordingly, they need to utilize prime sources for
the establishment and production of essential enzymes. These fungi are additionally
impacted upon by main abiotic factors like pH, salinity, temperature, and water
availability and accessibility. Therefore, species of fungi occupy a respective niche
due to their unique kind of survival mechanism based on particular ecological abiotic
factors. Fungi apply diverse strategies to survive in different and extreme environ-
mental conditions. These strategies are mainly C-selected (combative), S-selected
(stress), and R-selected (ruderal) (Cooke and Whipps 1993). In this chapter, we are
172 T. Das et al.

specifically concerned with extremophilic fungi, which may use S-selected strategies
for growth and survival in a range of so-called extreme environments. Extremophilic
fungi are gaining ecological importance as well as biotechnical interest due to their
ability to produce different kinds of bioactive compounds, enzymes, and proteins
with prospective application in the industrial fields. Extremophilic fungi have some
unique feature that were evolved based on extreme environmental conditions. Types
of extremophilic fungi and its adaptative strategies to survive in extreme environ-
ment conditions are presented in Fig. 9.1. Many of the biomolecules, viz., enzymes
and proteins produced by these fungi, are attributed to some defense strategies for
their survival in the extreme environment. Apart from industrial benefits, these fungi
possess unique genes that promote the growth of plant when applied as biofertilizers
in sustainable agriculture (Yadav 2017). Thus, this chapter focuses on the strategies
adopted by the other extremophilic fungi (halophiles, acidophiles, and alkaliophiles)
to grow in harsh environments linked to some genes’expressions and the production
of natural products as a response, which lead to an ecological impact on the
environment.
9.2 Halophiles
Halophilic fungi require more than 0.2 M salt for their growth and are divided into
(1) slight halophiles (0.2–0.85 M; 2–5%), (2) moderate halophiles (0.85–3.4 M;
5–20%), and (3) extreme halophiles (3.4–5.1 M; 20–30%) (El Hidri et al. 2013;
Guesmi et al. 2013).
Fig. 9.1 Representative extremophilic fungi and their adaptive strategies to survive in extreme
environmental conditions
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 173

9.2.1 Habitats
Halophilic fungi have been reported from various habitats including the following.
9.2.1.1 Saline Soil
A saline soil is soil with high but variable sodium concentration.
9.2.1.2 Saline Water
Saline water is water with salinity 3% or above (De-Dekker 1983). It includes
brackish water, marine water, and water from salt lakes and salterns. The saline
water is broadly divided into two types, viz., NaCl-rich thalassohaline and MgCl
2
-
and CaCl
2
-rich athalassohaline. Of these, thalassohaline water is an important
habitat of halophilic life including fungi. Some typical thalassohaline habitats are
the Dead Sea, Grate Salt Lake of USA, and Natrun Valley of Egypt. The Dead Sea is
about 320 m in depth and a salt concentration of 78% NaCl. It has slightly acidic pH
and important ions such as Na+, Cl-, and Mg2+ (Javor 1989). The Great Salt Lake,
USA, has slightly alkaline pH and salinity of 33% NaCl (Javor 1989). The Solar
Lake, Egypt, may have salinity of 20% NaCl in the summer. Lakes at Natrun Valley
(Wadi El Natrun), Egypt, have salinity in the range of 3.1–8.6% NaCl,
9.2.1.3 Solar Salterns
These are manmade series shallow ponds for making salt. The ponds are fed by sea
water or other saline water bodies, the last in the series is crystallizer having salt
above 30% (Antón et al. 2000). Inland saltern of La Mala, Spain, has salinity of 18%
NaCl and other ions like Mg2+, Ca2+, and K+.
9.2.2 Halophilic and Halotolerant Fungi
The fungi isolated from various saline habitats are mostly halotolerant rather than
halophilic. They can grow in growth medium supplemented with or without salt.
They have been isolated from saline and nonsaline habitats (Plemenitašet al. 2008)
including from food as food contaminants. The orders Capnodiales, Eurotiales, and
Dothideales of Ascomycota and the genus Wallemia of Basidiomycota have been
reported to comprise halophilic or halotolerant species (Al-Abri 2011). They include
meristematic melanized yeast-like fungi, the so-called black yeasts such as Hortaea
werneckii (Zalar et al. 1999b), Phaeotheca triangularis (Zalar et al. 1999b,c),
Aureobasidium pullulans (Zalar et al. 1999b), and a new species Trimmatostroma
salinum (Zalar et al. 1999a), different related species of the genus Cladosporium
(Gunde-Cimerman et al. 2000; Zalar et al. 2007; Butinar et al. 2005a),
non-melanized yeasts Pichia guilliermondii,Debaryomyces hansenii,Yarrowia
lipolytica,Candida parapsilosis,Rhodosporidium sphaerocarpum,R. babjevae,
Rhodotorula laryngis,Trichosporon mucoides,Metschnikowia bicuspidata,Can-
dida atmosphaerica-like and Pichia philogaea-like (Butinar et al. 2005b), the
filamentous genera Wallemia,Scopulariopsis and Alternaria (Zalar et al. 2005;
174 T. Das et al.

Gunde-Cimerman et al. 2005), and different species of the anamorphic genera
Aspergillus and Penicillium, including some of their teleomorphic genera Eurotium
and Emericella (Butinar et al. 2005,2011). Of all, Wallemia ichthyophaga
(Basidiomycetes) is the most well-known and in true sense halophilic fungus that
requires a minimum of 10% NaCl for its growth (Zalar et al. 2005; Zajc et al. 2014).
9.2.3 Living Strategies
9.2.3.1 Lower Water Activity
Halotolerants are adapted to lower water activity (a
w
) and can thrive in the presence
of lower concentration of available water.
9.2.3.2 Compatible Solute
Fungi face in hypersaline environment two stresses, viz., osmotic and ionic ones.
Fungi adapted to life at a
w
do this by accumulating compatible solutes to counter the
impact of lowering turgor pressure in the presence of hypersaline environment. They
apply same strategy to counter salinity-related osmotic stress. The halophilic
W. ichthyophaga and halotolerants A. pullulans,H. werneckii, and other halotolerant
fungi accumulate primarily glycerol as compatible solute. In addition,
W. ichthyophaga does accumulate little amount of arabitol and traces of mannitol
to supplement glycerol (Zajc et al. 2013a,b). The black yeast H. werneckii, on the
other hand, at lower salinities produces mycosporine–glutaminol–glucoside (prime
function of mycosporine being involved in fungal sporulation and UV protection)
(Oren and Gunde-Cimerman 2007), and at higher salinities produces other polyols
(e.g., erythritol, arabitol, and mannitol) to supplement glycerol (Kogej et al. 2004,
2006). In case of salt-tolerant yeasts Debaryomyces hansenii,Candida versatilis,
Rhodotorula mucilaginosa,orPichia guilliermondii trehalose and other polyols
supplement glycerol (Andre et al. 1988; Prista et al. 1997; Almagro et al. 2000).
9.2.3.3 Ion Homeostasis
There are at least three physiological strategies halotolerant fungi apply to overcome
ion stress. The halotolerant H. werneckii is said to use the two salt-responsive P-type
(ENA-like) ATPases (Gorjan and Plemenitas 2006) to extrude Na + at higher
concentration of NaCl as supported by genomic data (Lenassi et al. 2013). The
halophilic Wallemia ichthyophaga, which lacks most cation transporters, seems to
use avoidance strategy by preventing entry of excess Na + with its extremely
thickened cell walls (Kralj Kuncic et al. 2010,2013; Zajc et al. 2013a,b).
.
9.2.3.4 Cell Wall Structure and Pigmentation
At the differential level of melanin on outer cell wall of H. werneckii in the presence
of different salt concentrations (e.g., thin layer of melanin when there is no NaCl, but
thick layer of melanin at optimal salt concentration) (Kogej et al. 2004,2006), the
melanin seemingly gives mechanical support to counter higher turgor pressure
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 175

(Kogej et al. 2004,2006). The meristematic growth of Wallemia ichthyophaga
forming bigger (fourfold) and compact multicellular clumps and thickened (three-
fold) at higher salinity (cf. growth phenotype at lower salt concentration) is consid-
ered as an important adaptation to tolerate extreme salinity (Kralj Kuncic et al. 2010,
2013).
9.2.3.5 Plasma Membrane Fluidity
It is generally seen that eukaryotic cells that accumulate glycerol as a compatible
solute, its back outflow has to be stopped by using active transport system (energeti-
cally costly) or by reducing fluidity of membrane through enhancing sterol content
(Oren 1999). In case of H. werneckii, it has been shown that membrane remains fluid
over a wide range of salinities (Turk et al. 2004,2007) and its sterol content remains
largely unchanged (Turk et al. 2004), suggesting that its hypermelanized cell wall
also helps maintain glycerol at higher concentrations in the cells even in the presence
of highly fluid membrane (Gostincar et al. 2009).
9.2.3.6 Molecular Basis
Halophilic and halotolerant fungi developed a novel molecular mechanism so that
they can maintain their growth in high salt condition. Halophilic fungi possess a few
features for osmotolerance via utilizing compatible solutes by activation of the HOG
pathway. The HOG pathway produces glycerol that reestablishes the osmotic bal-
ance in the cell (Gostinčar et al. 2011; Zajc et al. 2012; Hohmann 2009). Plemenitaš
et al. (2014) observed that halophilic W. ichthyophaga produced compatible solutes
(glycerol) by HOG pathway activation implicated to their survival in a high osmolar
environment. W. ichthyophaga also maintains high K
+
/Na
+
ratios since in a high
saline environment toxic Na
+
ions are over K
+
ions. Thus, halophilic fungi devel-
oped some mechanisms that can maintain high K
+
/Na
+
ratios (Plemenitašet al.
2014). Hydrophobin is a type of protein that contains a high number of acidic amino
acids. These acidic amino acids are exposed to the protein surface and bind with salt
and reduced salt-induced changes (Siglioccolo et al. 2011). Hydrophobins were
found to be present in both W. ichthyophaga and W. sebi (Zajc et al. 2013a,b).
Hydrophobins also induced microconidial chain formation in W. ichthyophaga,
which might involve the accumulation of cells for the formation of the cluster.
Production of haloadaptation is primarily attributed to the response against salt stress
(Fuchs et al. 2004; Gostincar et al. 2010). Hydrophobins can also maintain cell wall
rigidity so that halophilic fungi take advantage of osmolarity changes in stress
(Wosten 2001; Bayry et al. 2012). H. werneckii contains acidic proteins that are
involved in the accumulation of K
+
ions besides glycerol in response to hypersalinity
(Kogej et al. 2005).
176 T. Das et al.

9.3 Alkaliphiles
Biochemical processes can occur at different hydrogen ion concentrations. However,
biochemical events function better close to neutral pH. Very high or low pH harms
the activity of biochemical events mostly via damaging the protein structure.
Alkaliphiles have been defined as organisms that grow optimally at pH above
9. Alkaliphiles are further divided into obligate alkaliphiles (incapable of growing
at or below pH 7.0) and facultative alkaliphiles (capable of growing at pH 7.0)
(Padan et al. 2005; Slonczewski et al. 2009).
9.3.1 Habitats
Alkaline habitats have been classified into
1. High Ca
2+
environments (groundwaters bearing high CaOH). Various locations
of this type have been reported in California, Oman, the former Yugoslavia,
Cyprus, Jordan, and Turkey (Barnes et al. 1982; Jones et al. 1994).
2. Low Ca
2+
environments (e.g., soda lakes, soda soil, and deserts with major salt
being sodium carbonate) (Grant and Horikoshi 1989,1992). These are stable
environments with soda lakes being a productive system because of the presence
of favorable temperatures (30–45 C), high sunlight intensities, and abundance of
HCO3 for photosynthesis (Ulukanli and Diurak 2002). The soda lakes are
characterized by higher pH (11–12) and around of 5–30% salinity (NaCO
3
and
NaCl in almost equal proportion) conditions (Duckworth et al. 1996).
Alkaliphiles are also found in a few insect guts and littoral soils (Hicks et al.
2010).
9.3.2 Alkaliphilic Fungi
Alkaliphilic fungi are very rare and reported sporadically from soda soil, soda lake,
and limestone cave (Nagai et al. 1995,1998; Grum-Grzhimaylo et al. 2013a).
Alkalitolerant fungi Fusarium oxysporum,F. bullatum, and Penicillium variabile
capable of growing at pH have been isolated in 1923 (Johnson 1923). Okada et al.
(1993) isolated alkaliphilic fungus Acremonium alcalophilum growing optimally at
pH 9.0. Most of the fungi thus isolated were alkalitolerants that can grow at alkaline
pH of 10. For example, Acremonium alternatum,A. furcatum,Acremonium
sp. 6, Gliocladium cibotii (YBLF 575), Phialophora geniculata,Stachylidium
icolor, and Stilbella annulata isolated from soil Acremonium sp. 6 were said to be
alkalophile (Nagai et al. 1995). Likewise out of six Acremonium and Chrysosporium
species from limestone caves (stalactite caves) in Japan capable of growing at
alkaline pH, one species each of Acremonium sp. and Chrysosporium sp. were
alkalophiles (Nagai et al. 1998). Then eight species of alkaliphilic and alkalitolerant
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 177

soil fungi from Argentina have been reported belonging to families Bionectriaceae,
Trichocomaceae,Sporormiaceae,Ceratostomataceae, and Sordariaceae (Elíades
et al. 2006). Generally, the alkaliphilic fungi are anamorphic without forming any
sexual structure, for example, Acremonium or Verticillium species (Okada et al.
1993; Kladwang et al. 2003).An alkaliphilic fungus Sodiomyces alkalinus showing
optimal growth at alkaline pH, however, is able to form cleistothecium (Grum-
Grzhimaylo et al. 2013b). Another novel alkaliphilic fungus Emericellopsis alkalina
sp. nov. (grow at pH 4–11.2, but optimally at 10–10.2) besides several alkalitolerant
isolates of Acremonium has been reported (Grum-Grzhimaylo et al. 2013b).
9.3.3 Living Strategy
The fungi found in soda soil/water face at least three stresses, namely, high osmotic
pressures, low water potentials, and elevated ambient pH (>9) (Grum-Grzhimaylo
et al. 2013b).
Alkylophilic fungi regulate their internal pH near neutral through active and
passive regulation mechanisms. Passive regulation involves the low membrane
permeability and cytoplasmic pools of polyamines (PA). Active regulation mecha-
nism of homeostasis involves the sodium ion channels (Sharma et al. 2017). Cell
wall components are very different in alkaliphiles. Many acidic polymers are present
on the cell wall that reduces the pH. Altered membrane lipids and presence of
cytoprotectant molecules enable them to survive at alkaline pH (Masato et al.
2010). Na
+
/H
+
and K
+
/H
+
type of antiporters are used to produce acid to reduce
the internal pH and thus regulate the proton motive force (Charlesworth and Burns
2016). They employ different adaptation mechanisms against stress via accumula-
tion of cytoprotective compounds (carbohydrate osmolytes) and modification of the
composition of their membrane lipids. Sodiomyces alkalinus (Plectosphaerellaceae,
Sordariomycetes, Ascomycota) is an alkalophilic fungus that accumulates cytosol
carbohydrate trehalose, mannitol, phosphatidylcholines (PC), and PA in the myce-
lium of the fungus. Fruit bodies of this fungus were detected with high amounts of
trehalose, triacylglycerols (TAG), PC, and sterols (Kozlova et al. 2019a).
Bondarenko et al. (2018) observed trehalose, mannitol, and arabitol accumulation
in two obligate alkaliphilic fungi Sodiomyces magadii (Plectosphaerellaceae,
Sordariomycetes, Ascomycota) and S. alkaline (Plectosphaerellaceae,
Sordariomycetes, Ascomycota) with almost double proportion of PA and lower
proportions of PC and St (Bondarenko et al. 2018). Kozlova et al. (2019b)
demonstrated unique features of Ascomycete S. alkalinus, which in the early lysis
of cell walls of asci releases immature ascospores inside the fruit body whereas
pseudoparenchymal and peridium cells degradation occur long before the ascospores
maturation at extremely high pH of soda lakes. After maturity, these ascospores are
forcefully released due to higher turgor pressure by cracking the fruit body. It was
assumed that these features could develop to cope with the high pH (Kozlova et al.
2019b).
178 T. Das et al.

The fungi Fusarium oxysporum, was found to respond to hypersaline conditions
by the expression of gene ena1 encoding P-type Na
+
A-ATPase. This gene is also
upregulated when the pH of growth environment is increased (Caracuel et al. 2003).
This coincidence suggests commonality of alkalitolerance and halotolerance
mechanisms.
9.4 Acidophiles
Acidophiles are organisms that grow optimally at pH <4.0. Another criterion to
differentiate acidotolerant and acidophilious is the growth curve; the former exhibits
bimodal growth while the latter shows unimodal growth (Cavicchioli and Torsten
2000; Gimmler et al. 2001). Fungi are mostly found to be acidotolerants.
9.4.1 Habitats
The acidophilic fungi may be isolated from neutral or acidic habitats (pH <3) such
as acidic soil, lake, swamp, and peat bogs (Middelhoven et al. 1992). Some of the
highly studied sites are solfatara soil studied in the USA, Japan, Russia, Italy,
Iceland, New Zealand, acid rock drainage of São Domingos (Portugal) and Rio
Tinto (Spain), etc.
9.4.2 Acidophilic Fungi
Acidophilic fungi are rarely found; generally fungi growing at lower pH can also
grow at neutral to slightly alkaline pH and thus mostly they are acidotolerant. Fungal
biodiversity study in highly acidic Tinto river (Spain) revealed species of
Scytalidium,Bahusakala,Phoma,Heteroonium,Lecythophora,Acremonium, and
Mortierella (López-Archilla et al. 2004).
Three highly acidotolerant fungi Acidothrix acidophila (Amplistromataceae,
Sordariomycetes, Ascomycota), Acidea extrema, and Soosiella minima (Helotiales,
Leotiomycetes, Ascomycota) have been isolated from highly acidic soils in the
Czech Republic and a coastal site in the Antarctic Peninsula (Hujslováet al. 2014)
while another anamorphic brown mold fungus Scytalidium acidophilum was isolated
from acidic soil and acidic solutions in an industrial plant and a uranium mine that
show optimum growth at acidic pH (Sigler and Camichaeil 1974).
Acidophilous fungi have been explored from Iberian Pyrite Belt (IPB), and acid
rock drainage in two localities São Domingos (Portugal) and Rio Tinto (Spain). The
most acid-tolerant found was yeast Cryptococcus spp. 5 followed by Cryptococcus
spp. 3 and Lecytophora spp. Moderately tolerant species were Candida fluviatilis,
Rhodosporidium toruloides,Williopsis californica, and three unidentified yeasts
belonging to Rhodotorula and Cryptococcus (Gadanho et al. 2006).
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 179

A novel acidophilic fungus Teratosphaeria (Capnodiales, Dothideomycetes) was
reported from biofilms collected from an extremely acidic and hot spring. It is a
ascomycetous teleomorphic fungus belonging to ascomyetes; phylogenetically close
to Acidomyces acidophilus and Bispora spp., earlier reported acidophilic anamor-
phic fungi (Yamazaki et al. 2010).
From various studies, the domination of dematiaceous fungal species has been
found in various acidic habitats (Amaral Zettler et al. 2002,2003; Baker et al. 2004,
2009; Hujslováet al. 2010,2013; López-Archilla et al. 2004). Of these, the three
fungi Acidomyces acidophilus (Selbmann et al. 2008), Hortaea acidophila (Hölker
et al. 2004), and Acidomyces acidothermus (Yamazaki et al. 2010; Hujslováet al.
2013) have been considered as acidophilic ones. All these plus the acidotolerant
fungus Acidiella bohemica (Hujslováet al. 2013) belong to the family
Teratosphaeriaceae (Capnodiales, Dothideomycetes, Ascomycota). Moreover, the
three fungal species A. acidophilus,A. acidothermus, and H. acidophila along with
two unidentified fungal isolates Paecilomyces spp. and Penicillium sp. 4 can grow at
pH 1 (Gimmler et al. 2001; Hölker et al. 2004; Hujslováet al. 2010; Yamazaki et al.
2010).
9.4.3 Living Strategy
Fungi being eukaryotes face four main challenges: very high H
+
concentration,
higher concentration of toxic metals, oligotrophic conditions, and extreme
temperatures (Whitton 1970; Brock 1978; Brake and Hasiotis 2010). Extremely
low pH irreversibly destroys primary and secondary structures of proteins (Kapfer
1998; Nixdorf and Kapfer 1998).
The acidotolerants employ twin mechanisms to tolerate hyperacidic
environments; extrusion of protons out of the cell and maintaining low proton
membrane permeability (Nikolay et al. 2018). Fungi by virtue of these internal pH
regulation mechanisms exist commonly in acidic environments (Gross and Robbins
2000).
Acidophiles maintain the intracellular pH by preventing proton influx, buffering
of intracellular protons, and efflux of protons. Although a number of protein
transporter systems are located on the cell membrane to regulate the cytosolic pH
levels (Gupta et al. 2014; Sharma et al. 2017; Christel 2018).
Acidophiles have highly impermeable cell membrane or reduced size of mem-
brane pore to reduce entry of protons into the cytoplasm and maintain the pH
homeostasis (Mirete et al. 2017) or have efficient proton pumps, which maintain
the proton gradient across the cytoplasm and its pH at or near neutral pH (Mirete
et al. 2017). They cope with the heavy metals by rapid efflux of these metals,
inactivate them, or convert them into less toxic compounds (Charlesworth and
Burns 2016; Christel 2018) and manage their oxidative stress by regulating the
reacting oxygen species (ROS). They possess some antioxidants such as glutathione
to inactivate these ROS or possess some enzymatic machinery such as superoxidase
mutase or peroxidase to neutralize or inactivate the ROS (Christel 2018). They have
180 T. Das et al.

highly expressed chaperons that help them in rapid repair of the damaged proteins.
The protein protects the DNA and other proteins from damage caused by the low pH
(Mirete et al. 2017). An acid-tolerant strain of Penicillium funiculosum growing
actively at pH 1.0 possesses a major facilitator superfamily transporter (PfMFS)
involved in the acid resistance and intracellular pH homeostasis (Xu et al. 2014).
Acidophilic microorganisms are ecologically and economically important
extremophiles found in solfataric fields, hydrogen sulfide (H
2
S) emissions, active
or abandoned mines, acidic copper mine wastes, and geysers (Sharma et al. 2012).
Although a few acidophiles have been studied up to now, those data are not yet
sufficient to clearly understand the adaptive features of acidophilic fungi. Determi-
nation of endo-1,4-b-xylanase crystal structure from Scytalidium acidophilum
(Chaetomiaceae, Leotiomycetes, Ascomycota), XYL1 acidophilic fungi adds
understandings of low pH adaptation. This study revealed the changes in the
homologous enzyme to maintain stability in an acidic environment. Alterations
include modification in the surface charge, decreased number of salt bridges,
changes like the conserved residue of the active site, etc., at low pH (Michaux
et al. 2010). Bacteria control internal low pH through increasing ATPase pump
efficiency, which rapidly pumps out protons from the cells to raise the internal pH of
the cell. Bacterial adaptation in such an environment (low pH) includes alternation of
the cell membrane and controlling of flagella. This kind of observation is lacking in
fungi and needs to be elaborated to enable a better understanding of fungi present in
such ecological niches.
9.5 Metallophiles
Metallophiles are the organisms that thrive under metal-rich condition or environ-
ment with high metallic concentration. They are able to tolerate and detoxify high
concentration of heavy metals. Most of the metallophiles are acidophiles, thus
enhancing their survival 1000-fold than mesophiles and efficiently tolerate the
high level of heavy metals (Anahid et al. 2011; Gupta et al. 2014).
9.5.1 Habitat
Naturally metal-rich environment such as water bodies and land around mining areas
are the main habitats of metallophiles. Apart from these metal-contaminated areas
around industries are also habitats of such metallophiles.
9.5.2 Metallophilic Fungi
Penicillium verrucosum KNU3 is metallophilic as it shows increased growth in the
presence of Cr
3+
,Cu
2+
, and Pb
2+
at 1 mM concentration (Joo and Hussein 2012).
Similarly, Penicillium simplicissimum shows higher growth in the presence of heavy
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 181

metals at concentration up to 8000 ppm (Anahid et al. 2011). Other fungi Aspergillus
niger,Aspergillus foetidus and P. simplicissimum showing high tolerance to molyb-
denum and vanadium have been reported. Of these, P. simplicissimum and
A. foetidus are adapted to high concentration of heavy metals and show enhanced
growth in the presence of heavy metals up to concentration of 2000 ppm (Valix et al.
2001).
Fungi that are tolerant to various metals have also been reported. For example,
chromium- and nickel-resistant Aspergillus sp. tolerating chromium toxicity up to
10,000 mg/L chromium have been reported (Congeevaram et al. 2007).
Ectomycorrhizal fungi Hymenogaster sp., Scleroderma sp., and Pisolithus tinctorius
show higher tolerance against increased concentration of Al, Fe, Cu, and Zn (Tam
1995). Heavy metal biosorption analysis revealed that Aspergillus sp.1 accumulated
1.20 mg Cr and 2.72 mg Cd, Aspergillus sp. 2 accumulated 1.56 mg Cr and 2.91 mg
Cd while Rhizopus sp. accumulated 4.33 mg Cr and 2.72 mg Cd per gram of biomass
(Zafar et al. 2007). Saccharomyces cerevisiae and Rhizopus nigricans accumulate
zinc (Sprocati et al. 2006). Fusarium solani shows tolerance to Ag (I) up to 1100 mg/
L concentration (El Sayed and El-Sayed 2020). Another strain of fungus A. niger
tolerates high concentration of heavy metal (Acosta-Rodríguez et al. 2018).
Fomitopsis meliae,Trichoderma ghanense, and Rhizopus microsporus are some
other metalloresistant filamentous fungi isolated from gold and gemstone mine sites
that can tolerate various heavy metals such as Cu, Pb, and Fe (Oladipo et al. 2018).
9.5.3 Living Strategies
Presence of heavy metals such as Zn, Cd, Hg, Pb, Ag, Co, and Cr makes the
environment very toxic. Generally high metal concentration inhibits the growth
and functioning of microbes, but metallophiles develop the strategies to function
optimally under these conditions. Some metallophiles possess efficient efflux pumps
for the rapid removal of toxic metals while others associate these metals by binding
them with protein molecules (Gupta et al. 2014). Ascomycete fungi such as
S. cerevisiae,Schizosaccharomyces pombe, and Candida albicans have been studied
for their adaptations to cope with high concentration of heavy metals. Some fungi
chelate these heavy metals with thiolated peptides and make a complex that is either
accumulated in the vacuole or extruded out of the cell. Some produce an antioxidant
glutathione in high amount that prevents the oxidative stress. S. cerevisiae transports
the heavy metals into external environment through a plasma membrane transporter
Pca1 (Otohinoyi and Omodele 2015). They exhibit two general mechanisms: extra-
cellular and intracellular, to fight with the high concentration of heavy metals.
Extracellular mechanism involves the chelating and cell wall binding (biosorption)
of heavy metals to restrict the entry of heavy metals into the cell while intracellular
mechanism involves the binding of heavy metals to proteins to reduce the concen-
tration of heavy metals inside the cell and prevent itself from damage (Anahid et al.
2011).
182 T. Das et al.

9.6 Radioresistants
Radioresistants or radiophiles are the extremophiles that are highly resistant to high
level of ionizing and ultraviolet radiation. Radioresistant organisms tolerate extreme
radiations for longer period of time while radiotolerant organisms tolerate extreme
radiations for only a short period of time. Ionizing radiation such as gamma radiation
and nonionizing radiation such as ultraviolet radiation are the two major radiations
that cause lethal effect on an organism. Radiophiles are polyextremophiles as they
can tolerate extreme cold, dehydration, vacuum, and high acidic concentration
(Coker 2019).
9.6.1 Adaptations of Radiophiles
Gamma radiations causes double-stranded breaks in the DNA of an organism and
produce reacting oxygen species that interfere with the metabolic processes leading
to cell death. They also damage proteins and lipids and produce persistent oxidative
stress. UV radiations cause more destruction by DNA damage through formation of
thymine dimer and pyrimidine radio tolerant photoproducts. Radiophiles protect
them from gamma radiation by adapting efficient DNA repair mechanism that
rapidly repairs the damaged DNA, production of antioxidants, enzymatic defense
system (increased production of enzyme such as catalase to inactivate free radicals
and reactive oxygen species), and condensed nucleoid. UV-resistant radiophiles
protect them from radiation through multiple mechanisms. Their genome is com-
posed of very small number of bipyrimidine sequences. They possess gene duplica-
tion phenomenon causing polyploidy. Carotenoids, superoxide dismutase, and
hydroperoxidases reduce the stress developed by radiation (Coker 2019).
Radiophiles possess the capability to survive under starvation and high oxidative
stress condition. They can even survive in condition with high amount of DNA
damage. Ionizing radiations induce changes in upregulation of cell repair system and
genetic component of an organism. Some UV radiation-resistant radiophiles protect
their DNA from lethal radiation by the presence of UV-absorbing pigments such as
scytonemin in sheath around the cell while some radiophiles accumulate
UV-absorbing pigments such as mycosporine like amino acids in the cytoplasm of
the cell (Dighton et al. 2008; Kazak et al. 2010).
Fungi are resistant to chronic ionizing radiations evolved from various radiation
sources such as radioactive waste and nuclear disaster. The main strategy adopted by
the radiation-resistant fungi against high radiation stress is to scavenge reactive
oxygen species. They accumulate high amount of Mn
2+
metabolite antioxidant
complex for scavenging reactive oxygen species induced by the ionizing radiations
as Mn
2+
complexes with other compounds to inactivate the reactive oxygen species.
Low concentration of iron ions and high concentration of manganese ions protect the
cell from oxidative stress. Radiotolerant fungi possess high Mn
2+
/Fe
2+
ratio
(Dadachova and Casadevall 2008; Dighton et al. 2008; Matusiak 2016). Melanin
and some other pigments play an important role for the development of resistance to
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 183

radiations. A complex polymer melanin is important in energy transduction and
shielding as they possess the capability to absorb various kinds of electromagnetic
radiations. Radiation exposure causes fungal melanin pigment to alter the shape and
induce them to form a thick layer of melanin. Some fungi, especially melanized
fungi, harvest energy from the radiation with the help of melanin pigment and utilize
this energy for their growth and development (Dadachova and Casadevall 2008;
Dighton et al. 2008).
Ascomycota yeast possess resistance to chronic ionizing radiation is correlated
with Cr
+3
while resistance of Basidiomycete yeast to chronic ionizing radiation is
correlated with the highest temperature that allows the growth (Shuryak et al. 2019).
Biofilms of radioresistant fungi are adapted to high mutation rate and are more
resistant to ionizing radiation than other radioresistants (Ragon et al. 2011). Crypto-
coccus neoformans is a radioresistant fungi that generally can be found in high
radiation environment. Genome-wide radiation resistance analysis of this fungus
explains the upregulation of DNA repair machinery for reducing the radiation stress.
Rad53 protein kinase regulates the transcription factor Bdr1 and controls the tran-
scription (Jung et al. 2016).
9.7 Fungi in Exoplanet-like Environment
For the study of life outside of our planet, extremophilic organisms are considered
the best suitable model. As we already discussed, these organisms can survive in
extreme acidic, alkaline, heat, cold, salt, and pressure. The real challenges to grow
extremophilic fungi in exoplanet-like environment are space vacuum, solar, galactic
and ionizing radiation, and extreme cold and heat. The precondition for Mars would
be water availability. Fungi-producing melanin pigment are mostly colonized in the
Antarctic to the Arctic to high-altitude terrains. For growing in such regions,
extremophilic fungi have to deal with UV radiations, dry, and cold. So, melanized
fungi could be a suitable model for studies in Mars-like habitat. Microcolonial
fungus Cryomyces antarcticus (incertaesedis), Dothideomycetes, Ascomycota) can
live in Mars-like habitat in a good way. C. antarcticus in Mars-like habitat for 24 h
showed a decrease in protein number, but after 4- and 7-day treatment protein
number was increased again and protein patterns matched to normalcy. This result
indicated that C. antarcticus needs 1 week for recovery of its metabolic activity in a
Mars-like condition (Zakharova et al. 2014). Another melanin-forming fungi
Cryomyces minteri (incertaesedis, Dothideomycetes, Ascomycota) and known
C. antarcticus exposed in Mars-like habitat for 18 months resulted in 10% of the
sample being able to form colonies. Additionally, high stability in DNA is also
observed in the hostile conditions of space (Onofri et al. 2015). Onofri et al. (2018)
isolated C. antarcticus and C. minteri from cryptoendolithic microbial communities
in Antarctica. After the screening of their DNA, it was observed that C. antarcticus
displayed higher resistance than C. minteri. They concluded that the apparent
presence of thicker melanized cell wall of C. antarcticus could be a reason for
higher resistance (Onofri et al. 2018). Pacelli et al. (2019) experimented with black
184 T. Das et al.

fungus C. antarcticus with a simulated space vacuum or Mars-like condition and
found that this black fungus can tolerate such a condition with high integrity of DNA
even after the treatments (Pacelli et al. 2019) So the theory that in space biological
material can be preserved is somehow true as we cited that fungi DNA remains
undamaged in space. However, exact space condition cannot be created in the
laboratory.
9.7.1 Genes and/or Secondary Metabolites
The EhHOG gene has an important role in the osmoregulatory pathway. EhHOG
gene, isolated from Eurotium herbariorum from the dead sea, where salinity is the
utmost on earth, showed resistance against salt, water, and low- and high-
temperature stress. EhHOG genes encode mitogen-activated protein kinase
(MAPK), which is a homolog of the HOG gene from Aspergillus nidulans, Saccha-
romyces cerevisiae, Schizosaccharomyces pombe, and many other eukaryotes. In the
hog1 mutant gene of S. cerevisiae, when supplemented by the EhHOG gene, growth
of the fungi is restored in high salt stress condition. Additionally, glycerol content
also increased (Jin et al. 2005). Halophilic fungus Aspergillus glaucus contains
RPL44 (ribosomal protein L44), a conserved protein related to salt resistance (Liu
et al. 2014). Same kind of result was found in aquaglyceroporins (GlpFs), 60S
protease subunit, and AgRPS3aE, ribosomal subunit from A. glaucus.
Aquaglyceroporins transport glycerol and water, which are related to osmoregula-
tion (Liang et al. 2015; Liu et al. 2015). Altogether these genes are highly conserved;
they can support transgenic plants or cells surviving under high salt and heat stress
conditions. Analysis of these genes may further support genetic engineering tools
and crop improvement under high salt, water, and temperature stress. Extremophilic
fungi develop exclusive defenses to survive in extreme conditions like temperature,
salinity, pH, pressure, and desiccation, which leads to the production of diverse
secondary metabolites. Secondary metabolites have no direct role in the adaptation
process of extremophilic fungi. However, they have an indirect role by inhibiting the
different microorganisms (viruses, pathogenic fungi, and pathogenic bacteria) in a
competition to survive in an environment with limited nutrients (Table 9.1).
9.8 Conclusion
Extremophilic features are great parts of evolution, and scientists would get a better
understanding of the effect of different proteins, genes, or metabolites responsible
for survival in extreme environments. The presence of several harsh environmental
conditions can lead to weighty challenges for living, resulting in unique survival
strategies. Fungi are one of the most adaptable organisms for their splendid environ-
mental and structural flexibility. They are physiologically changed for vigorous
growth under extreme temperature, salt, pressure, pH, and minimal water availability
through employing biochemical pathways, which are responsible for synthesizing
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 185

Table 9.1 List of antimicrobial activity of metabolites from extremophilic fungi
Category Species Collected from Compound Antimicrobial activity against Reference
Acidophiles Penicilliumpurpurogenum
JS03–21
Red soil Purpurides B, purpurides C
berkedrimane B
Candida albicans, Enterobacter
aerogenes, Pseudomonas
aeruginosa
Wang
et al.
(2013)
Halophiles Aspergillus flavus, aspergillus
gracilis, aspergillus
penicillioides
Solar saltern Not mentioned Not mentioned Ali et al.
(2014)
Psychrophiles Penicillium chrysogenum Benthisenviornment Rugulosin, skyrin Staphylococcus aureus,
Escherichia coli, Candida albicans
Brunati
et al.
(2009)
Piezophiles Aspergillus sp. SCSIO
Ind09F01
Deep sea Liotoxin, 12,13-dihydroxy-
fumitremorgin C, helvolic
acid
Mycobacterium tuberculosis Luo et al.
(2017)
Aspergillus versicolor Deep sea Anthraquinone Methicillin-resistant
Staphylococcus aureus
Wang
et al.
(2018)
Neosartorya fennelliae KUFA
0811
Marine sponges Dihydrochromone dimer Staphylococcus aureus ATCC
29213, Enterococcus faecalis
ATCC 29212
Kumla
et al.
(2017)
Oidiodendron griseum
UBOCC-A-114129
Deep sea sediment Dihydrosecofuscin/
secofuscin
Enterococcus faecalis Navarri
et al.
(2017)
Thermophiles Elaphocordyceps
ophioglossoides
Soil Ophiosetin Not mentioned Putri et al.
(2010)
Xerophilies Aspergillus felis Atacama Desert Cytochalasins Paracoccidioides brasiliensis Pb18 Mendes
et al.
(2016)
186 T. Das et al.

compounds (organic compounds, glycerol, trehalose, mannitol, arabitol, erythritol,
etc.). In future, investigations on the extremophilic fungal genomes can be helpful to
reveal the alteration in their cellular response in response to the extreme environ-
ment. Extremophiles that can survive in a wide range of harsh environments can
further be used in a range of industrially important bioprocesses and in astrobiology
studies.
Acknowledgments The authors are extremely thankful to UGC, Government of India, for finan-
cial assistance. The authors are highly grateful to Presidency University-FRPDF fund, Kolkata, for
providing needed research facilities.
References
Acosta-Rodríguez I, Cárdenas-González JF, Rodríguez Pérez AS, Oviedo JT, Martínez-Juárez VM
(2018) Bioremoval of different heavy metals by the resistant fungal strain Aspergillus niger.
Bioinorg Chem Appl 2018:3457196
Al-Abri L (2011) Use of molecular approaches to study the occurrence of extremophiles and
extremodures in non-extreme environments. PhD thesis, University of Sheffield, UK
Ali I, Siwarungson N, Punnapayak H, Lotrakul P, Prasongsuk S, Bankeeree W, Rakshit SK (2014)
Screening of potential biotechnological applications from obligate halophilic fungi, isolated
from a man-made solar saltern located in Phetchaburi Province, Thailand. Pak J Bot 46:983–988
Almagro A, Prista C, Castro S, Quintas C, Madeira-Lopes A, Ramos J, Loureiro-Dias MC (2000)
Effects of salts on Debaryomyces hansenii and Saccharomyces cerevisiae under stress
conditions. Int J Food Microbiol 56:191–197
Amaral Zettler LA, Gomez F, Zettler E, Keenan BG, Amils R, Sogin ML (2002) Eukaryotic
diversity in Spain’s river of fire. Nature 417:137
Amaral Zettler LA, Messerli MA, Laatsch AD, Smith PJS, Sorgin ML (2003) From genes to
genomes: beyond biodiversity in Spain’s Rio Tinto. Biol Bull 204:205–209
Anahid S, Yaghmaei S, Ghobadinejad Z (2011) Heavy metal tolerance of fungi. Scientia Iranica
18(3):502–508
Andre L, Nillsson A, Adler L (1988) The role of glycerol in osmotolerance of the yeast
Debaromyces hansenii. J Gen Microbiol 134:669–677
Antón J, Rosselló-Mora R, Rodríguez-Valera F, Amann R (2000) Extremely halophilic Bacteria in
crystallizer ponds from solar salterns. Appl Environ Microbiol 66:3052–3057
Baker BJ, Lutz MA, Dawson SC, Bond PL, Banfield JF (2004) Metabolically active eukaryotic
communities in extremely acidic mine drainage. Appl Environ Microbiol 70(10):6264–6271
Baker BJ, Tyson GW, Goosherst L, Banfield JF (2009) Insights into the diversity of eukaryotes in
acid mine drainage biofilm communities. Appl Environ Microbiol 75(7):2192–2199
Barnes I, Presser TS, Saines M, Dickson P, Van Goos AFK (1982) Geochemistry of highly basis
calcium hydroxide groundwater in Jordan. Chem Geol 35:147–154
Bayry J, Aimanianda V, Guijarro JI, Sunde M, Latge JP (2012) Hydrophobins-unique fungal
proteins. PLoS Pathog 8:e1002700
Bondarenko SA, Ianutsevich EA, Sinitsyna NA, Georgieva ML, Bilanenko EN, Tereshina BM
(2018) Dynamics of the cytosol soluble carbohydrates and membrane lipids in response to
ambient pH in alkaliphilic and alkalitolerant fungi. Microbiology (Russian Fed) 87:21–32
Brake SS, Hasiotis ST (2010) Eukaryoteedominated biofilms and their significance in acidic
environments. Geomicrobiol J 27:534–558
Brock TD (1978) Thermophilic microeorganisms and life at high temperatures. Springer, New York
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 187

Brunati M, Rojas JL, Sponga F, Ciciliato I, Losi D, Göttlich E, de Hoog S, Genilloud O, Marinelli F
(2009) Diversity and pharmaceutical screening of fungi from benthic mats of Antarctic lakes.
Mar Genomics 2:43–50
Butinar L, Sonjak S, Zalar P, Plemenitas A, Gunde-Cimerman N (2005) Melanized halophilic fungi
are eukaryotic members of microbial communities in hypersaline waters of solar salterns. Bot
Mar 1:73–79
Butinar L, Zalar P, Frisvad JC, Gunde-Cimerman N (2005a) The genus Eurotium –members of
indigenous fungal community in hypersaline waters of salterns. FEMS Microbiol Ecol 51:155–
166
Butinar L, Santos S, Spencer-Martins I, Oren A, Gunde-Cimerman N (2005b) Yeast diversity in
hypersaline habitats. FEMS Microbiol Lett 244:229–234
Butinar L, Frisvad JC, Gunde-Cimerman N (2011) Hypersaline waters: a potential source of
foodborne toxigenic aspergilla and penicillia. FEMS Microbiol Ecol 77:186–199
Caracuel Z, Casanova C, Roncero MIG, Di Pietro A, Ramos J (2003) pH response transcription
factor PacC controls salt stress tolerance and expression of the P-type Na
+
-ATPase ena1 in
fusarium oxysporum. Eukaryot Cell 2:1246–1252
Cavicchioli R, Torsten T (2000) Extremophiles. In: Lederberg J (ed) Encyclopedia of microbiology,
2nd edn, vol 2. Academic Press Inc., San Diego, pp 317–337
Charlesworth J, Burns BP (2016) Extremophilic adaptations and biotechnological applications in
diverse environments. AIMS Microbiol 2(3):251–261
Christel S (2018) Function and Adaptation of Acidophiles in Natural and Applied Communities.
Doctoral dissertation, Linnaeus University Press
Coker JA (2019) Recent advances in understanding extremophiles. F1000Research 8:F10000
Faculty Rev-1917
Congeevaram S, Dhanarani S, Park J, Dexilin M, Thamaraiselvi K (2007) Biosorption of chromium
and nickel by heavy metal resistant fungal and bacterial isolates. J Hazard Mater 146(1–2):
270–277
Cooke RC, Whipps JM (1993) Ecophysiology of fungi. Blackwell, London
Dadachova E, Casadevall A (2008) Ionizing radiation: how fungi cope, adapt, and exploit with the
help of melanin. Curr Opin Microbiol 11(6):525–531
De-Dekker P (1983) Australian salt lakes: their history, chemistry, and biota-a review.
Hydrobiologia 105:231–244
Dighton J, Tugay T, Zhdanova N (2008) Fungi and ionizing radiation from radionuclides. FEMS
Microbiol Lett 281(2):109–120
Duckworth AW, Grant WD, Jones BE, Van Steenbergen R (1996) Phylogenetic diversity of soda
lake alkaliphiles. FEMS Microbiol Ecol 19:181–191
El Hidri D, Guesmi A, Najjari A, Cherif H, Ettoumi B, Hamdi C et al (2013) Cultivation-dependant
assessment, diversity, and ecology of haloalkaliphilic bacteria in arid saline systems of southern
Tunisia. Biomed Res Int 2013
El Sayed MT, El-Sayed AS (2020) Tolerance and mycoremediation of silver ions by fusarium
solani. Heliyon 6(5):e03866
Elíades LA, Cabello MN, Voget CE (2006) Contribution to the study of alkalophilic and alkali-
tolerant ascomycota from Argentina. Darwin 44:64–73
Fuchs U, Czymmek KJ, Sweigard JA (2004) Five hydrophobin genes in fusarium verticillioides
include two required for microconidial chain formation. Fungal Genet Biol 41:852–864
Gadanho M, Libkind D, Sampaio JP (2006) Yeast diversity in the extreme acidic environments of
the Iberian Pyrite Belt. Microb Ecol 52:552–563
Gimmler H, de Jesus J, Greiser A (2001) Heavy metal resistance of the extreme acidotolerant
filamentous fungus Bispora sp. Microb Ecol 42:87–98
Gorjan A, Plemenitas A (2006) Identification and characterization of ENA ATPasesHwENA1 and
HwENA2 from the halophilic black yeast Hortaea werneckii. FEMS Microbiol Lett 265:41–50
188 T. Das et al.

Gostincar C, Turk M, Plemenitas A, Gunde-Cimerman N (2009) The expressions of D9-,
D12-desaturases and an elongase by the extremely halotolerant Hortaea werneckii are salt
dependent. FEMS Yeast Res 9:247–256
Gostincar C, Grube M, de Hoog S, Zalar P, N. Gunde-Cimerman N (2010) Extremotolerance in
fungi: evolution on the edge. FEMS Microbiol Ecol 71:2–11
Gostinčar C, Lenassi M, Gunde-Cimerman N (2011) Fungal adaptation to extremely high salt
concentrations. Adv Appl Microbiol 77:71–96
Grant WD, Horikoshi K (1989) Alkaliphiles. In: Da Costa MS, Duarte JC, Williams RAD (eds)
Microbiology of extreme environments and its potential for biotechnology. Elsevier, London,
pp 346–366
Grant WD, Horikoshi K (1992) Alkaliphiles; ecology and biotechnological applications. In: Herbert
RA, Sharpe RJ (eds) Molecular biology and biotechnology of extremophiles. Blackie, Glasgow
and London, pp 143–162
Gross S, Robbins EI (2000) Acidophilic and acid-tolerant fungi and yeasts. Hydrobiologia 433(1):
91–109
Grum-Grzhimaylo AA, Debets AJ, van Diepeningen AD, Georgieva ML, Bilanenko EN (2013a)
Sodiomyces alkalinus, a new holomorphic alkaliphilic ascomycete within the
Plectosphaerellaceae. Persoonia. 31:147–158
Grum-Grzhimaylo AA, Georgieva ML, Debets AJM, Bilanenko EN (2013b) Are alkalitolerant
fungi of the Emericellopsis lineage (Bionectriaceae) of marine origin? IMA Fungus 4:213–228
Guesmi A, Ettoumi B, El Hidri D, Essanaa J, Cherif H, Mapelli F, Marasco R, Rolli E,
Boudabous A, Cherif A (2013) Uneven distribution of Halobacillus trueperi species in arid
natural saline systems of southern Tunisian Sahara. Microb Ecol 66(4):831–839
Gunde-Cimerman N, Zalar P (2014) Extremely halotolerant and halophilic fungi inhabit brine in
solar salterns around the globe. Food Technol Biotechnol 52:170–179
Gunde-Cimerman N, Zalar P, de Hoog GS, Plemenitas A (2000) Hypersaline waters in salterns –
natural ecological niches for halophilic black yeasts. FEMS Microbiol Ecol 32:235–240
Gunde-Cimerman N, Sonjak S, Zalar P, Frisvad JC, Diderichsen B, PlemenitašA (2003)
Extremophilic fungi in arctic ice: a relationship between adaptation to low temperature and
water activity. Phys Chem Earth 28:1273–1278
Gunde-Cimerman N, Butinar L, Sonjak S, Turk M, Ur V, Zalar P, Plemenitas A (2005) Halotolerant
and halophilic fungi from coastal environments in the arctics. In: Gunde-Cimerman N, Oren A,
Plemenitas A (eds) Adaptation to life at high salt concentrations in Archaea, bacteria, and
Eukarya, cellular origin. Life in extreme habitats and astrobiology, vol 9. Springer, Dordrecht,
pp 397–423
Gupta GN, Srivastava S, Khare SK, Prakash V (2014) Extremophiles: an overview of microorgan-
ism from extreme environment. Int J Agric Environ Biotechnol 7(2):371–380
Hicks DB, Liu J, Fujisawa M, Krulwich TA (2010) F1F0-ATP synthases of alkaliphilic bacteria:
lessons from their adaptations. Biochim Biophys Acta 1797:1362–1377
Hohmann S (2009) Control of high osmolarity signalling in the yeast Saccharomyces cerevisiae.
FEBS Lett 583:4025–4029
Hölker U, Bend J, Pracht R, Tetsch L, Müller T, Höfer M, de Hoog GS (2004) Hortaea acidophila, a
new acid-tolerant black yeast from lignite. Anton Leeuw 86:287–294
HujslováM, KubátováA, ChudíčkováM, KolaříkM (2010) Diversity of fungal communities in
saline and acidic soils in the Soos NationalNatural reserve, Czech Republic. Mycol Prog 9:1–15
HujslováM, KubátováA, Kostovčík M, Kolařík M (2013) Acidiella bohemica gen. Et sp. nov. and
Acidomyces spp. (Teratosphaeriaceae), the indigenous inhabitants of extremely acidic soils in
Europe. Fungal Divers 58:33–45
HujslováM, KubátováA, Kostovčík M, Blanchette RA, de Beer ZW, ChudíčkováM, Kolařík M
(2014) Three new genera of fungi from extremely acidic soils. Mycol Prog 13:819–831
Javor B (1989) Hypersaline environments: microbiology and biogeochemistry. Springer, Berlin
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 189

Jin Y, Weining S, Nevo E (2005) A MAPK gene from Dead Sea fungus confers stress tolerance to
lithium salt and freezing-thawing: prospects for saline agriculture. Proc Natl Acad Sci U S A
102:18992–18997
Johnson HW (1923) Relationships between hydrogen ion, hydroxyl ion and salt concentrations and
the growth of seven soil molds. Agricultural Experiment Station, Iowa State College of
Agriculture and the Mechanic Arts. Res Bull 76:307–344
Jones BE, Grant WD, Collins NC, Mwatha WE (1994) Alkaliphiles: diversity and identification. In:
Priest FG (ed) Bacterial diversity and systematics. Plenum Press, New York, pp 195–230
Joo JH, Hussein KA (2012) Heavy metal tolerance of fungi isolated from contaminated soil. Korean
J Soil Sci Fertil 45(4):565–571
Jung KW, Yang DH, Kim MK, Seo HS, Lim S, Bahn YS (2016) Unraveling fungal radiation
resistance regulatory networks through the genome-wide transcriptome and genetic analyses of
Cryptococcus neoformans. MBio 7(6):e01483–e01416
Kapfer M (1998) Assessment of the colonization and primary production of microphytobenthos in
the littoral of acidic mining lakes in Lusatia (Germany). Water Air Soil Pollut 108:331–340
Kazak H, Oner ET, Dekker RF (2010) Extremophiles as sources of exopolysaccharides. Develop-
ment, Properties and Applications, Carbohydrate Polymers, pp 605–619
Kladwang W, Bhumirattana A, Hywel-Jones N (2003) Alkaline-tolerant fungi from Thailand.
Fungal Div 13:69–83
Kogej T, Wheeler MH, Lanisnik Rizner T, Gunde-Cimerman N (2004) Evidence for
1,8-dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and
non-saline conditions. FEMS Microbiol Lett 232:203–209
Kogej T, Ramos J, PlemenitašA, Gunde-Cimerman N (2005) The halophilic fungus Hortaea
werneckii and the halotolerant fungus Aureobasidium pullulans maintain low intracellular
cation concentrations in hypersaline environments. Appl Environ Microbiol 71:6600–6605
Kogej T, Gostincar C, Volkmann M, Gorbushina AA, Gunde-Cimerman N (2006) Mycosporines in
extremophilic fungi –novel complementary osmolytes? Environ Chem 3:105–110
Kozlova MV, Ianutsevich EA, Danilova OA, Kamzolkina OV, Tereshina VM (2019a) Lipids and
soluble carbohydrates in the mycelium and ascomata of alkaliphilic fungus Sodiomyces
alkalinus. Extremophiles 23:487–494
Kozlova MV, Bilanenko EN, Grum-Grzhimaylo AA, Kamzolkina OV (2019b) An unusual sexual
stage in the alkalophilic ascomycete Sodiomyces alkalinus. Fungal Biol 123:140–150
Kralj Kuncic M, Kogej T, Drobne D, Gunde-Cimerman N (2010) Morphological response of the
halophilic fungal genus Wallemia to high salinity. Appl Environ Microbiol 76:329–337
Kralj Kuncic M, Zajc J, Drobne D, Tkalec PZ, Gunde-Cimerman N (2013) Morphological
responses to high sugar concentrations differ from adaptations to high salt concentrations in
xerophilic fungi Wallemia spp. Fung Biol 117:466–478
Kumla D, Aung TS, Buttachon S, Dethoup T, Gales L, Pereira JA, Inácio Â, Costa PM, Lee M,
Sekeroglu N, Silva AMS, Pinto MMM, Kijjoa A (2017) A new dihydrochromone dimer and
other secondary metabolites from cultures of the marine sponge-associated fungi Neosartorya
fennelliae KUFA 0811 and Neosartorya tsunodae KUFC 9213. Mar Drugs 15
Lenassi M, Gostincar C, Jackman S, Turk M, Sadowski I, Nislow C et al (2013) Whole genome
duplication and enrichment of metal cation transporters revealed by de novo genome sequencing
of extremely halotolerant black yeast Hortaea werneckii. PLoS One 8:e71328
Liang X, Liu Y, Xie L, Liu X, Wei Y, Zhou X, Zhang S (2015) A ribosomal protein agRPS3aE from
halophilic aspergillus glaucus confers salt tolerance in heterologous organisms. Int J Mol Sci
16:3058–3070
Liu XD, Xie L, Wei Y, Zhou X, Jia B, Liu J, Zhang S (2014) Abiotic stress resistance, a novel
moonlighting function of ribosomal protein RPL44 in the halophilic fungus Aspergillus
glaucus. Appl Environ Microbiol 80:4294–4300
Liu XD, Wei Y, Zhou XY, Pei X, Zhang SH (2015) Aspergillus glaucus aquaglyceroporin gene
glpF confers high osmosis tolerance in heterologous organisms. Appl Environ Microbiol 81:
6926–6937
190 T. Das et al.

López-Archilla AI, González AE, Terrón MC, Amils R (2004) Ecological study of the fungal
populations of the acidic Tinto River in southwestern Spain. Can J Microbiol 50:923–934
Luo X, Zhou X, Lin X, Qin X, Zhang T, Wang J, Tu Z, Yang B, Liao S, Tian Y, Pang X,
Kaliyaperumal K, Li JL, Tao H, Liu Y (2017) Antituberculosis compounds from a deep-sea-
derived fungus aspergillus sp. SCSIO Ind09F01. Nat Prod Res 31:1958–1962
Masato TAKJL, Fujisawa MM, Hicks MIDB (2010) 2.6 adaptive mechanisms of extreme
alkaliphiles. In: Horikoshi K (ed) Extremophiles handbook. Springer, Tokyo
Matusiak DM (2016) Radiotolerant microorganisms-characterization of selected species and their
potential usage. Postepy Mikrobiologii 55(2):182–194
Mendes G, Gonçalves VN, Souza-Fagundes EM, Kohlhoff M, Rosa CA, Zani CL, Cota BB, Rosa
LH, Johann S (2016) Antifungal activity of extracts from Atacama Desert fungi against
Paracoccidioides brasiliensis and identification of aspergillus felis as a promising source of
natural bioactive compounds. Mem Inst Oswaldo Cruz 111:209–217
Michaux C, Pouyez J, Mayard A, Vandurm P, Housen I, Wouters J (2010) Structural insights into
the acidophilic pH adaptation of a novel endo-1,4-β-xylanase from Scytalidium acidophilum.
Biochimie 92:1407–1415
Middelhoven WJ, Koorevaar M, Scchuur W (1992) Degradation of benzene compounds by yeasts
in acidic soils. Plant Soil 145:37–43
Mirete S, Morgante V, González-Pastor JE (2017) Acidophiles: diversity and mechanisms of
adaptation to acidic environments. AdaptiMicrobiLife Environ Extr:227–251
Nagai K, Sakai T, Rantiatmodjo R, Suzuki K, Gams W, Okada G (1995) Studies on the distribution
of alkalophilic and alkali-tolerant soil fungi. Mycoscience 36:247–256
Nagai K, Suzuki K, Okada G (1998) Studies on the distribution of alkalophilic and alkali-tolerant
soil fungi II: fungal flora in two limestone caves in Japan. Mycoscience 39:293–298
Navarri M, Jégou C, Bondon A, Pottier S, Bach S, Baratte B, Ruchaud S, Barbier G, Burgaud G,
Fleury Y (2017) Bioactive metabolites from the deep subseafloor fungus Oidiodendron griseum
UBOCC-A-114129. Mar Drugs 15:1–10
Nikolay A, Léon A, Schwamborn K, Genzel Y, Reichl U (2018) Process intensification of EB66®
cell cultivations leads to high-yield yellow fever and Zika virus production. Appl Microbiol
Biotechnol 102(20):8725–8737
Nixdorf B, Kapfer M (1998) Stimulation of phototrophic pelagic and benthic metabolism close to
sediments in acidic mining lakes. Water Air Soil Pollut 108:317–330
Okada G, Niimura Y, Sakata T, Uchimura T, Ohara N et al (1993) Acremonium alcalophilum,a
new alkalophilic cellulolytic hyphomycete. Trans Mycol Soc Japan 34:171–185
Oladipo OG, Awotoye OO, Olayinka A, Bezuidenhout CC, Maboeta MS (2018) Heavy metal
tolerance traits of filamentous fungi isolated from gold and gemstone mining sites. Brazilian J
Microbiol 49(1):29–37
Onofri S, De Vera JP, Zucconi L, Selbmann L, Scalzi G, Venkateswaran KJ, Rabbow E, De La
Torre R, Horneck G (2015) Survival of Antarctic Cryptoendolithic fungi in simulated Martian
conditions on board the international Space Station. Astrobiology 15:1052–1059
Onofri S, Selbmann L, Pacelli C, de Vera JP, Horneck G, Hallsworth JE, Zucconi L (2018) Integrity
of the DNA and cellular ultrastructure of cryptoendolithic fungi in space or mars conditions: a
1.5-year study at the international space station. Life 8:1–16
Oren A (1999) Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev 63:334–348
Oren A, Gunde-Cimerman N (2007) Mycosporines and mycosporine-like amino acids: UV
protectants or multipurposesecondary metabolites? FEMS Microbiol Lett 269:1–10
Otohinoyi DA, Omodele I (2015) Prospecting microbial extremophiles as valuable resources of
biomolecules for biotechnological applications. Int J Sci Rese 4(1):1042–1059
Pacelli C, Selbmann L, Zucconi L, Coleine C, De Vera JP, Rabbow E, Böttger U, Dadachova E,
Onofri S (2019) Responses of the black fungus Cryomyces antarcticus to simulated mars and
space conditions on rock analogs. Astrobiology 19:209–220
Padan E, Bibi E, Ito M, Krulwich TA (2005) Alkaline pH homeostasis in bacteria: new insights.
Biochim Biophys Acta 1717:67–88
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 191

PlemenitašA, VaupotičT, Lenassi M, Kogej T, Gunde-Cimerman N (2008) Adaptation of
extremely halotolerant black yeast Hortaea werneckii to increased osmolarity: a molecular
perspective at a glance. Stud Mycol 61:67–75
PlemenitašA, Lenassi M, Konte T, Kejžar A, Zajc J, Gostinčar C, Gunde-Cimerman N (2014)
Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective.
Front Microbiol 5:1–13
Prista C, Almagro A, Loureiro-Dias MC, Ramos J (1997) Physiological basis for the high salt
tolerance of Debaryomyces hansenii. Appl Environ Microbiol 6:4005–4009
Putri SP, Kinoshita H, Ihara F, Igarashi Y, Nihira T (2010) Ophiosetin, a new tetramic acid
derivative from the mycopathogenic fungus Elaphocordyceps ophioglossoides. J Antibiot
(Tokyo) 63:195–198
Ragon M, Restoux G, Moreira D, Møller AP, López-García P (2011) Sunlight-exposed biofilm
microbial communities are naturally resistant to Chernobyl ionizing-radiation levels. PLoS One
6(7):e21764
Selbmann L, de Hoog GS, Zucconi L, Isola D, Ruisi S, Gerrits van den Ende AHG, Ruibal C, De
Leo F, Urzì C, Onofri S (2008) Drought meets acid: three new genera in a dothidealean clade of
extremotolerant fungi. Stud Mycol 61:1–20
Selbmann L, Egidi E, Isola D, Onofri S, Zucconi L, de Hoog GS, Chinaglia S, Testa L, Tosi S,
Balestrazzi A, Lantieri A, Compagno R, Tigini V, Varese GC (2013) Biodiversity, evolution
and adaptation of fungi in extreme environments. Plant Biosyst 147:237–246
Sharma A, Kawarabayasi Y, Satyanarayana T (2012) Acidophilic bacteria and archaea: acid stable
biocatalysts and their potential applications. Extremophiles 16:1–19
Sharma A, Sharma R, Devi T (2017) Life at extreme conditions: extremophiles and their biocata-
lytic potential. Int J Adv Res Sci Eng 6:1413–1420
Shuryak I, Matrosova VY, Volpe RP, Grichenko O, Klimenkova P, Conze IH, Balygina IA,
Gaidamakova EK, Daly MJ (2019) Chronic gamma radiation resistance in fungi correlates
with resistance to chromium and elevated temperatures, but not with resistance to acute
irradiation. Sci Rep 9(1):1–1
Sigler L, Camichaeil JW (1974) A new acidophilic Scytalidiurn. Can J Microbiol 20:267–268
Siglioccolo A, Paiardini A, Piscitelli M, Pascarella S (2011) Structural adaptation of extreme
halophilic proteins through decrease of conserved hydrophobic contact surface. BMC Struct
Biol 11:50
Slonczewski JL, Fujisawa M, Dopson M, Krulwich TA (2009) Cytoplasmic pH measure- ment and
homeostasis in bacteria and archaea. Adv Microbiol Physiol 55:1–317
Sprocati AR, Alisi C, Segre L, Tasso F, Galletti M, Cremisini C (2006) Investigating heavy metal
resistance, bioaccumulation and metabolic profile of a metallophile microbial consortium native
to an abandoned mine. Sci Total Environ 366(2–3):649–658
Tam PC (1995) Heavy metal tolerance by ectomycorrhizal fungi and metal amelioration by
Pisolithus tinctorius. Mycorrhiza 5(3):181–187
Turk M, Méjanelle L, Sentjurc M, Grimalt JO, Gunde-Cimerman N, Plemenitas A (2004) Salt-
induced changes in lipid composition and membrane fluidity of halophilic yeast-like melanized
fungi. Extremophiles 8:53–61
Turk M, Abramovic Z, Plemenitac A, Gunde-Cimerman N (2007) Salt stress and plasma-membrane
fluidity in selected extremophilic yeasts and yeast-like fungi. FEMS Yeast Res 7:550–557
Ulukanli Z, Diurak M (2002) Alkaliphilic micro-organisms and habitats. Turk J Biol 26:181–191
Valix M, Tang JY, Malik R (2001) Heavy metal tolerance of fungi. Miner Eng 14(5):499–505
Wang H, Wang Y, Liu P, Wang W, Fan Y, Zhu W (2013) Purpurides B and C, two new
sesquiterpene esters from the aciduric fungus Penicillium purpurogenum JS03-21. Chem
Biodivers 10:1185–1192
Wang W, Chen R, Luo Z, Wang W, Chen J (2018) Antimicrobial activity and molecular docking
studies of a novel anthraquinone from a marine-derived fungus Aspergillus versicolor. Nat Prod
Res 32:558–563
192 T. Das et al.

Whitton BA (1970) Toxicity of heavy metals to fresh water algae: a review. Phykos 9:116–125
Wosten HA (2001) Hydrophobins: multipurpose proteins. Annu Rev Microbiol 55:625–646
Xu X, Chen J, Xu H, Li D (2014) Role of a major facilitator superfamily transporter in adaptation
capacity of Penicillium funiculosum under extreme acidic stress. Fungal Genet Biol 69:75–83
Yadav AN (2017) Beneficial role of extremophilic microbes for plant health and soil fertility. J
Agric Sci Bot 01
Yamazaki A, Toyama K, Nakagiri A (2010) A new acidophilic fungus Teratosphaeria acidotherma
(Capnodiales, Ascomycota) from a hot spring. Mycoscience 51:443–455
Zafar S, Aqil F, Ahmad I (2007) Metal tolerance and biosorption potential of filamentous fungi
isolated from metal contaminated agricultural soil. Bioresour Technol 98(13):2557–2561
Zajc J, Zalar P, PlemenitašA, Gunde-Cimerman N (2012) The mycobiota of the salterns. In:
Progress in molecular and subcellular biology, pp 133–158. https://doi.org/10.1007/978-3-642-
23342-5_10
Zajc J, Kogej T, Galinski EA, Ramos J, Gunde-Cimerman N (2013a) The osmoadaptation strategy
of the most halophilic fungus Wallemia ichthyophaga, growing optimally at salinities above
15% NaCl. Appl Environ Microbiol 80:247–256
Zajc J, Liu YF, Dai WK, Yang ZY, Hu JZ, Gostincar C et al (2013b) Genome and transcriptome
sequencing of the halophilic fungus Wallemia ichthyophaga: haloadaptations present and
absent. BMC Genomics 14:617. https://doi.org/10.1186/1471-2164-14-61
Zajc J, Kogej T, Galinski EA, Ramos J, Gunde-Cimermana N (2014) Osmoadaptation strategy of
the most halophilic fungus, Wallemia ichthyophaga, growing optimally at salinities above 15%
NaCl. Appl Environ Microbiol 80:247–256. https://doi.org/10.1128/AEM.02702-13
Zakharova K, Marzban G, De Vera JP, Lorek A, Sterflinger K (2014) Protein patterns of black fungi
under simulated Mars-like conditions. Sci Rep 4:1–7. https://doi.org/10.1038/srep05114
Zalar P, de Hoog GS, Gunde-Cimerman N (1999a) Trimmatostroma salinum, a new species from
hypersaline water. Stud Mycol 43:57–62
Zalar P, de Hoog GS, Gunde-Cimerman N (1999b) Ecology of halotolerant dothideaceous black
yeasts. Stud Mycol 43:38–48
Zalar P, de Hoog GS, Gunde-Cimerman N (1999c) Taxonomy of the endoconidial genera
Phaeotheca and Hyphospora. Stud Mycol 43:49–56
Zalar P, de Hoog GS, Schroers HJ, Frank JM, Gunde-Cimerman N (2005) Taxonomy and
phylogeny of the xerophilic genus Wallemia (Wallemiomycetes and Wallemiales, cl. Et Ord.
Nov.). Antonie Van Leeuwenhoek 87:311–328
Zalar P, de Hoog GS, Schroers HJ, Crous PW, Groenewald JZ, Gunde-Cimerman N (2007)
Phylogeny and ecology of the ubiquitous saprobe Cladosporium sphaerospermum, with
descriptions of seven new species from hypersaline environments. Stud Mycol 58:157–183
9 Halophilic, Acidophilic, Alkaliphilic, Metallophilic, and Radioresistant... 193

Ecology and Diversity of Microaerophilic
Fungi Including Endophytes 10
Deeksha Patil, Vishal Dawkar, and Umesh Jadhav
Abstract
This chapter explores the extremophilic microaerophilic fungi along with
endophytes. The relation between microaerophilic fungi and endophytes with
the environment which decide the diversity is discussed here. From the overall
population of one to five million fungal species, 1.5–1.6 million species are
predominating. These are widely spread in extreme conditions like deep sea
sediments, ionizing radiations along with areas where high salt concentrations
prevail. Another perspective is of endophytic fungi. They are classified majorly as
The Clavicipitaceae Fungal Endophyte (1) and the Non-Clavicipitaceae Fungal
Endophyte (2). The microaerophilic fungi in extreme conditions are not explored
much. There is a huge scope for the study of these microaerophilic fungi and
endophytes that can thrive in adverse conditions. These extreme environments
pose several stresses on fungi. The fungi use various mechanisms to adapt to
these adverse conditions.
Keywords
Microaerophilic fungi · Extremophiles · Fungal endophytes · Biotic stress ·
Abiotic stress · Adaptations
D. Patil · U. Jadhav (*)
Department of Microbiology, Savitribai Phule Pune University, Pune, Maharashtra, India
e-mail: ujadhav@unipune.ac.in
V. Dawkar
MITCON Biotechnology and Pharmaceutical Technology Business Incubator Center, Pune,
Maharashtra, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_10
195

10.1 Introduction
Human beings have a general tendency to look around the world as per their
perspective which tends to be normal for the Earth. But that is not the case; there
are things that thrive in extreme conditions that don’t seem to be normal for human
beings. MacElroy first introduced the term extremophiles (Macelroy 1974).
Extremophiles can live and thrive under harsh physical conditions like pressure,
radiation, and temperature and also in geochemical extremes like desiccation,
oxygen levels, pH, and salinity. These harsh conditions threaten the general
functions of life (Shrestha et al. 2018). All the three domains of life can survive in
extreme conditions, but they are dominated by prokaryotes. The growth limiting
factors for some organisms are listed in Table 10.1.
Talking about Endophyte, it means “in the plant,”where endon- within and
phyton- plant (Schulz and Boyle 2005). It was coined by the German scientist
Heinrich Anton De Bary. Endophytes are organisms living inside the plant tissue
(Vega et al. 2008). It colonizes the internal tissue of the plant without harming the
host (Sikora et al. 2007; Backman and Sikora 2008; Hardoim et al. 2015; Puri et al.
2016). This definition holds true for cultivable fungi and it doesn’t consider the
unculturable fungi (Lugtenberg et al. 2016). Many variations are witnessed in the
endophytic partners along with their relationships with each other (Schulz and Boyle
2005). They are ubiquitous in nature. They are accompanied with various organisms
(Hartley and Gange 2009). They colonize various body parts of host plant
(Saikkonen et al. 2006). They protect pants from herbivores. The grasses are infected
with endophytic infection artificially and this infected grasses act as biocontrol agent
against insects’pests of grasses (Clay 1989; Carroll 1995; Breen 1994; Saikkonen
et al. 1998). Some of the fungal endophytes shield host plants from pests, nematodes
attack and contribute to enhanced plant growth (Elmi et al. 2000; Sikora et al. 2007;
Backman and Sikora 2008; Ownley et al. 2008; Reddy et al. 2009; Akello and Sikora
2012; Biswas et al. 2012; Jaber and Enkerli 2016; Jaber and Araj 2017). On the other
hand, “Oecologie”or Ecology was the term first coined by Ernst Haeckel from Latin
words Oikos (home) and logos (study of) (Haeckel 1866). Taylor defined ecology as,
a science of all relations of all organisms to their entire environment (Taylor 1936). It
can also be said in other words, that ecology deals with the organisms and
Table 10.1 Organisms and their known limits for growth (Coker, 2019)
Organism Growth limiting factors
Psychromoas ingrahamii Minus 12 C
Geogemma barossii 129 C
Picrophilus torridus pH less than 0
Plectonemanos tocorum and Hydrogenophaga sp. pH 13
Shewanella benthica 100 Mpa pressure
Haloferax volcanii High concentration of NaCl and KCl
Halobacterium sp. NRC-1 and Deinococcus
radiodurans
Ultraviolet (UV) and gamma
radiation
196 D. Patil et al.

environment and is associated with the study of factors that control organism’s
abundance and distribution and clearly describes the characteristics needed to thrive
in natural environment (Winkelmann 2007).
10.2 Types of Microaerophilic Fungi with Endophytes
The Latin word fungus originated from the Greek word sphongos that meant having
morphology of mushroom (Ainsworth 1976; Simpson 1979). The five-kingdom
classification of Whittaker replaced the traditional classification of fungi as plants
and gave rise to three different kingdoms that are- animal, fungi, and plantae
(Baldauf and Palmer 1993).
These three multicellular eukaryotic kingdoms of life are believed to be ramified
from each other some billion years ago (Bruns 2006). Fungi are considered as key
players in terrestrial environment, where they perform essential functions like-
decomposers, operating nutrient cycles, transforming elements other than carbon
and behave as parasites and symbionts (Fig. 10.1) (Wainwright 1981; Wainwright
1988; James et al. 2006; Richards et al. 2012). Like other organisms’fungi also
perform basic metabolic function for which they require energy supply along with
carbon supply for the biosynthesis of cellular constituents. Basically, when fungi
grow aerobically and as heterotroph, to supply energy they generally use available
Functions
of Fungi
Act as
decomposers
Operates
nutrient cycles
Transformation
of elements
Behave as
parasites and
symbionts
Fig. 10.1 Basic metabolic functions performed by fungi
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 197

organic substrate. Here, oxygen plays an important role as terminal electron acceptor
for oxidation of substrate to CO
2
(Wainwright 1988; Grahl et al. 2012).
But it was seen that some rumen microorganisms that were recognized as
zoospores of anaerobic phycomycetous fungi that are now considered as normal
microbiota (Teunissen et al. 1991; Durrant 1996). Some of the scientists studied that,
there are few filamentous fungi along with yeasts that can grow in low oxygen
condition or in microaerophilic conditions (Waid 1962; Tabak and Cooke 1968).
Basidiomycete specie, Trichocladium canadense, Geotrichum sp., and Fusarium sp.
are said to require low oxygen concentrations for their growth and enzyme
productions (Pavarina and Durrant 2002). Fungi like Blastocladiella prings/
zeimiana, Penicillium roquefortii, along with several yeasts are found to have
somatic growth at low oxygen levels (Durrant et al. 1995). Scientific classification
for some of them is given in Table 10.2. Fungal endophytes occur naturally in many
host plants (Saikkonen et al. 1998). They showed presence in plants like wheat,
bananas, tomatoes (Vega et al. 2008). Various species are present in solitary vegetal
part like root, stem, leaf (Rodriguez et al. 2009; Fürnkranz et al. 2012). Higher
vascular plants exhibit symbiotic relationship with plant and endophytic fungi,
where the endophytes provide benefits to host as indirect defense (Vega et al.
2008) in exchange of nutrition from plants (Kim et al. 2008; Quesada-Moraga
et al. 2009; Lugtenberg et al. 2016). Endosymbiont derived from non-vascular
plants, ferns, conifers, monocots and dicots are associated with foliage and
compromises of ascomycetous fungi (Arnold and Lewis 2005). Many naturally
occurring insect pathogenic fungi like Beauveria bassiana, Clonostachys rosea,
Isaria farinosa, and Acremonium sp. have been isolated from symptomless body
parts (Cherry et al. 1999; Pimentel et al. 2006; Vega et al. 2008; Orole and Adejumo
2009; Quesada-Moraga et al. 2009). The host plants were artificial inoculated with
Metarhizium anisopliae,B. bassiana,Fusarium oxysporum, Hypocrea lixii,
Gibberella moniliformis, and Trichoderma asperellum and they were re-isolated
(Bing and Lewis 1991; Akello and Sikora 2012; Akello and Sikora 2012; Akutse
et al. 2013). Colonization of endophytic entomopathogenic fungi using artificial
methods of inoculation was successful in Triticum aestivum (Gurulingappa et al.
2010), Phaseolus vulgaris (Akutse et al. 2013), Zea mays (Bing and Lewis 1991),
Lycopersicon esculentum (Ownley et al. 2008), Glycine max (Russo et al. 2015),
Coffea spp. (Posada et al. 2007), Papaver somniferum (Quesada-Moraga et al.
2009), Manihot esculenta (Greenfield et al. 2016), Sorghum bicolor (Tefera and
Vidal 2009), Gossypium hirsutum (Ownley et al. 2008). Petrini (1991) propound that
some endophytic fungi exist in latent or inactive form. They become active due to
changes in environmental/host/pathogen conditions (Petrini 1991).
10.2.1 Classification of Fungal Endophytes
Although sundry endophytic species are present, but characterization of only few of
them has been done till now (Hawksworth 1991,2001). Fossil study suggested that
the plants were associated with endophytes from ancient time (Krings et al. 2007).
198 D. Patil et al.

Table 10.2 Classification of microaerophilic fungi
Kingdom Fungi Fungi Fungi Fungi Fungi
Division Ascomycota Ascomycota Blastocladiomycota Ascomycota Ascomycota
Sub division Pezizomycotina
Class Sordariomycetes Eurotiomycetes Blastocladiomycetes Saccharomycetes Sordariomycetes
Order Sordariales Eurotiales Blastocladiales Saccharomycetales Hypocreales
Family Chaetomiaceae Trichocomaceae Blastocladiaceae Dipodascaceae Nectriaceae
Genus Trichocladium Penicillium Blastocladiella Geotrichum Fusarium
Species P. Roqueforti
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 199

Fungal endophytes are categorized in two groups (Fig. 10.2) the clavicipitaceous
endophytes—they infect some grasses; and the nonclavicipitaceous endophytes.
They can be recovered from symptomless tissues of various vegetations (Lugtenberg
et al. 2016).
10.2.1.1 Clavicipitaceous Endophytes (C-Endophytes)
They are known as Class 1 endophytes. These were first noted in late nineteenth
Century by a group of European investigators in seeds of Lolium temulentum,
Lolium arvense, Lolium linicolum, and Lolium remotum (Rodriguez et al. 2009). It
was found that clavicipitaceous grow along with host and both of them benefited
(Clay 1988; Bacon and White, 2000). However, they are small in number. They
require particular media for their growth i.e., fastidious in nature. They are limited to
seasonal grass family like poaceae and sometimes in cyperacae (Rodriguez et al.
2009). Class 1 types are host specific. They cause systemic infections in plant shoots.
The family is said to be derived within the Hypocreales (order of fungi within the
class Sordariomycetes) (Spatafora and Blackwell, 1993; Rehner and Samuels 1995;
Spatafora et al. 2007). They involve plant pathogens, saprotrophs, and endophytes.
Some of them are capable of production of bioactive compounds. They are said to be
derived from insects’pathogens (Rodriguez et al. 2009). Transmission takes place
vertically (Saikkonen et al. 2002). Three types of clavicipitaceous endophytes are
observed (Clay and Schardl, 2002). They are said to add up in plant biomass. They
help to tolerate water shortage. They protect host by producing compounds that are
harmful to herbivores (Cheplick and Clay 1988; Rodriguez et al. 2009). Epichloë
species of endophytes has stromata production that helps in sexual reproduction
(White and Bultman 1987). They are found in Northern hemisphere. Whereas,
C-endophytes that are found in Southern hemisphere do not produce stromata and
Fungal
Endophytes
Clavicipitaceous
Endophytes
Class 1
Endophytes
Non-
Clavicipitaceous
Endophytes
Class 2
Endophytes
Class 3
Endophytes
Class 4
Endophytes
Fig. 10.2 Sorting of fungal endophytes
200 D. Patil et al.

reproduces asexually (Clay and Schardl 2002). They are referred as Type III
endophytes and classified as Neotyphodium species (White 1988; Moon et al.
1999; Leuchtmann et al. 2000).
These types of endophytes confer several beneficial effects to the host plant. First
being herbivore deterrence. They retard insect attack using several mechanisms.
They cause reduction in insect developmental rate. The insect growth is inhibited
through feeding deterrence. Some class 1 endophytes are found to show anti-
nematode activity. Mammal herbivory deterrence is also witnessed in USA, where
Achnatherum robustum also known as sleepy grass is associated with endophyte.
The domestic animals avoid consumption of such grasses. In South America there
are grasses that are harmful to mammals. These grasses are also getting infected by
Neotyphodium tembladerae.Achnatherum inebrians in Asia infects Neotyphodium
gansuense and is resistant to mammal herbivory. C-endophytes are also involved in
disease resistance. Epichloë festucae endophyte that infects turf grasses produces
derivatives like a sesquiterpene, and a diacetamide are resistant over leaf spot
pathogens and red thread disease. Ecophysiology of host plants is also enhanced
by C-endophytes. Abiotic stress like drought, metal contamination is countered by
infection of endophytes (Rodriguez et al. 2008).
10.2.1.2 Nonclavicipitaceous Endophytes (NC Endophytes)-
NC endophytes are varied and represent ascomycetous molds primarily. NC
endophytes are recovered from all terrestrial ecosystems (Arnold and Lutzoni
2007). Several properties of these endophytes attracted attention of scientists. The
diversity, ability to play an ecological role, and their endophytic and free-living
lifestyles are some of these properties (Vasiliauskas et al. 2007; Selosse et al. 2008;
Rodriguez et al. 2009). NC endophytes have presence of three functional classes—
Class 2, Class 3, and Class 4.
Class 2 Endophytes
They are diverse as compared to class 1 endophytes and they comprise of species
such as pezizomycotina (ascomycota), agaricomycotina, and pucciniomycotina
(basidiomycota) (Rodriguez et al. 2009; Lugtenberg et al. 2016). Diversity in
individual host plant is limited (Rodriguez et al. 2008). Roots, stem, leaves or
sometimes an entire plant is colonized by them i.e., extensive colonization of host
is possible. Colonization takes place through appressoria or hyphae. Transmission
can be vertical or horizontal. They are seen to enhance the fitness of their host to
enable the host to sustain habitat-specific stresses (Rodriguez et al. 2008). They are
found in both underground and above-ground tissues. C-2 endophytes are essential
for normal growth of plants. They are also seen to help plants to sustain in adverse
conditions. Not much study is done on ecology of class 2 endophytes and hence
limited information is available for distribution and abundance of same in rhizo-
sphere. Species like Phoma sp. and Arthrobotrys spp. are present in high number in
soil, whereas Fusarium culmorum, Colletotrichum magna, and Curvularia
protuberata are in low number (Rodriguez et al. 2008).
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 201

As stated above, endophytes increase root and shoot biomass. The possible
reason can be increased production of plant hormones by the host or their biosyn-
thesis by fungi (Tudzynski and Sharon 2002). Some endophytes are witnessed to
avoid stress by practicing plant symbiosis. Fusarium culmorum grow along all
non-embryonic tissues of coastal dunegrass. When they are grown individually,
they do not tolerate high salt concentration and growth is retarded. But if grown
symbiotically can tolerate salt concentration of 300–500 mM NaCl (Rodriguez et al.
2008). Class 2 endophytes provide protects plants in various ways against fungal
pathogens. It includes production of secondary metabolites, fungal parasitism, and
induction of systemic resistance. Resistance against virulent pathogen is seen in
Barley, as endophytic isolate Fusarium oxysporum had increased amount of pheno-
lic metabolite. Plants like Colletotrichum spp. that grows in agricultural fields,
Fusarium culmorum growing in coastal beaches or Curvularia protuberata that
grows in geothermal soils has the ability to infect plants asymptomatically (Redman
et al. 2001,2002).
Class 3 Endophytes
Class 3 endophytes are members of the Dikaryomycota (Ascomycota or
Basidiomycota), Pezizomycotina, Saccharomycotina. They grow in the tissues that
are above ground in localized manner. Their diversity is very high in individual plant
or tissue. Around>20 species may be present on single tropical leaf (Rodriguez et al.
2009). Variety of species associated with above-ground plant tissues are seen in
Class 3 endophytes. They are observed in photosynthetic and herbaceous tissues;
flowers, fruits, asymptomatic wood and inner bark. In Central Panama tropical
forest, it is seen that endophytes are diverse and individual leaves harbor around
one isolate/2mm
2
of leaf tissue. Class 3 endophytes exists in temperate and boreal
communities as well. These communities were recovered from Arctic endophytes.
Reproduction takes place by sexual or asexual way.
Ecological functions of class 3 endophytes are not explored yet. The infection of
plants with class 3 endophytes has insignificant effect on growth rate. Bark
endophytes protect trees against Dutch elm disease. Even if they are parasitic or
pathogenic, they may become mutualistic. It is also witnessed that class 3 endophytes
have negative effect on host.
Class 4 Endophytes
They are mostly observed in above-ground root and colonization is same as class
2 (Rodriguez et al. 2009). Transmission is horizontal. They are characterized based
on presence of darkly melanized septa. They are ascomycetous fungi, conidial or
sterile and referred as “dark septa endophytes”(DSE). Host or habitat specificity is
very less. They are associated with non-mycorrhizal plants from all types of
ecosystems. They are also found in boreal and temperate forests associated with
fine roots of trees and shrubs. It is witnessed that they are prevalent in highly adverse
conditions, ubiquitous and abundant in different ecosystems. DSE when studied
across North and South Pole was seen to be associated with 320 genera, 587 plant
species. Root colonization can be intercellular or intracellular.
202 D. Patil et al.

Like other endophytes, ecological role of DSE is not well defined. But it is seen
that DSE have symbiotic association like mycorhizza. They perform many roles. It is
also proposed that DSE acquisition can also play role in deterring pathogens. This
can be achieved by controlling carbon availability in rhizosphere. Secondary
metabolites production can be toxic to herbivores. Figure 10.3 shows interaction
of endophytes with host plant.
10.3 Major Habitats
Biodiversity means the biological variation on Earth (Hawksworth, 1991). It is
estimated to be around one to five million for fungi, where 1.5–1.6 million species
are predominating (Hawksworth 1991,2001; Richards et al. 2012; Hawksworth and
Lucking 2016). They are distributed worldwide and in extreme conditions like deep
sea sediments (Raghukumar and Raghukumar 1998), ionizing radiations
(Dadachova et al. 2007) along with areas where high salt concentrations prevail
(Vaupotic et al. 2008). The distribution studies of endophytic fungi showed their
presence in various ecosystems like terrestrial, semiaquatic, freshwater and marine
habitats.
10.3.1 Terrestrial Ecosystem
Relationship of growths with earthbound plant species has gotten significant consid-
eration contrasted with sea-going territories (Sandberg et al. 2014). Various geo-
graphic areas were assessed to study the occurrence of endophytic fungi like tropical,
subtropical, and temperate regions of forest trees (Sun and Guo 2012).
Fig. 10.3 Summary of impact of host-plant association with fungal endophytes
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 203

10.3.1.1 Trees and Shrubs
In this part we will discuss about the interrelation of endophytic fungi with trees and
shrubs. Diverse endophytic fungi are found on tropical trees and their leaves (Arnold
and Lutzoni 2007). Some endophytic fungi like Colletotrichum, Pestalotiopsis,
Phomopsis, and Xylaria are dominant irrespective of habitat. In India, Tectona
grandis host of Phomopsis sp. and Plumeria rubra in Colletotrichum sp.,
Phyllosticta sp. are dominant taxa in tropical trees, where as in Brazil, Tairira
guianensis host in Phomopsis sp., Euterpe oleraceae in Xylaria cubensis, Spondias
mombin in Phyllosticta sp. and Phomopsis sp., Malus domestica in Colletotrichum
spp. are dominant (Suryanarayanan 2011). Phoma spp. that infects Calluna vulgaris
was analyzed of Mediterranean plants stated that they are root endophytes and
confers fitness benefits to plants (Rayner 1915). Sun and Guo (2012) measured
endophytic fungi in Betula platyphylla, Quercus liaotungensis, and Ulmus
macrocarpa tree species and tissues from China’s woodland habitats. Overall,
48.5 to 65.6% of twigs were colonized. The leaves showed slightly more coloniza-
tion compared to twigs. In case of, Tectona grandis seasonal and geographical
variation of endophytes was observed (Singh et al. 2017). About 5089 isolates that
were recovered and assigned to 45 distinct morphotypes. Out of this, 43 morphotypes
were assigned to ascomycotina and 02 to basidiomycotina. In case of DSE, Zubek
et al. (2011) reported the co-occurrence of DSE with Arbuscular mycorrhizal
(AM) in the ten plant species of Pamir-Alay Mountains of Central Asia.
Bagyalakshmi et al. (2010) studied mycorrhizal types and DSE fungal associations
in Western Ghats region.
10.3.1.2 Medicinal Plants
The fungal endophytes are known for the production of a broad variety of secondary
metabolites (Kusari et al. 2014). They can be used for discovery of new medicinal
agents in health care (Fig.10.4) (Kharwar et al. 2011).
Xanthomonas oryzae bacterial disease of rice was targeted using several second-
ary metabolites produced by endophytic fungus Phomopsis longicolla. Antitumor
medicines like paclitaxel (Taxol), camptothecin, podophyllotoxin, vinblastine, and
vincristine are identified from fungal endophytes (Kharwar et al. 2011). Taxol is
produced by various endophytes associated with Japanese yew tree (Kumaran et al.
2010). Several endophytic fungi were isolated from liverwort Scapania verrucosa
that belonged to the Xylariaceae family. They were classified into seven genera and
showed antioxidant activities for DPPH radicals and hydroxyl radicals and proved to
be novel antioxidant compounds (Zeng et al. 2011). From these around fifty
endophtytic fungi were isolated from medicinal plants Alpinia calcarata, Bixa
orellana, Calophyllum inophyllum, and Catharanthus roseus. They were screened
for different enzymatic activities (Sunitha et al. 2013). Another study reported
isolation of several species of endophytic fungi from various plant parts of five
medicinal plant species present in the Western Ghats of India (Raviraja 2005).
204 D. Patil et al.

10.3.1.3 Ferns
In the tropical and subtropical ecosystems several thousand species of epiphytic
ferns are reported that are associated with pteridophyte (Jones 1947). Raviraja et al.
(1996) studied the roots of four riparian ferns. They observed colonization of
pteridophytes by aquatic hyphomycetes. Kumaresan et al. (2013) screened five
species of Pteridophytes, Adiantum sp., Gleichenia linearis, Lygodium flexuosum,
Pteris sp., and Selaginella sp. Out of the 40 species of fungal endophytes recorded,
Colletotrichum sp. 1 occurred in all the Pteridophyte species.
10.3.1.4 Orchids
The presence of endophytic and Rhizoctonia-like fungi in Aegean and Mediterra-
nean regions was studied. Fusarium, Papulaspora, and Rhizoctonia were isolated
(Gezgin and Eltem 2009). Ascomycetes—Helotiales, Hypocreales, and Xylariales
were found associated with five orchids from the tropical jungle of southern Ecuador
(Herrera et al. 2010). Ten orchid species have a place with the genera Dendrobium in
China which yielded high variety of endophytic organisms. The endophytes showed
varied degree of host specificity (Chen et al. 2011). Orchid Anoectochilus setaceus
which is found in Sri Lanka is associated with endophytic fungi Xylaria sp.
(Ratnaweera et al. 2014). Several isolates were obtained the roots of two orchids
(Yu et al. 2015).
Role of
Endophytic
Fungi
Antibacterial
and
Antifungal
Anticancer
Antioxidants
Biofuels
Industrial enzymes
Fig. 10.4 Role of endophytic fungi in different medicinal fields
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 205

10.3.2 Aquatic Ecosystem
10.3.2.1 Freshwater Habitats
The freshwater environments are less explored for the presence of endophytic fungi.
In such environments macrophytes act as host (Bärlocher 1992; Rajagopal et al.
2018). Endophytes from macrophytes uncovered low disengagement recurrence yet
contrasted in species extravagance, variety, and local area structure with closeness
with general earthbound networks (Sandberg et al. 2014). Aquatic medicinal fern
was found associated with hyphomycetes, Coelomycetes, and endophytic fungus
(Udayaprakash et al. 2018). Endophytic parasites were observed in riparian tree
species in two altitudinal reaches of the Western Ghats (Ghate and Sridhar 2017).
The variety of amphibian hyphomycetes was higher in mid elevation than high-
height (Raviraja et al. 1998). Several endophytic growths were recuperated with
predominance of Cladosporium, Fusarium, and Geotrichum from the three oceanic
and two riparian plant species (Li et al. 2010).
10.3.2.2 Marine Habitats
Mangroves
The plant species occurring in river mouths give possible living spaces for the
colonization of endophytic fungi. Suryanarayanan and Kumaresan (2000) isolated
four halophytes Acanthus ilicifolius (Acanthaceae), Arthrocnemum indicum,Suaeda
maritima (Chenopodiaceae), and Sesuvium portulacastrum (Aizoaceae) from the
mangrove plants. All of them harbored fungal endophytes in which Acremonium,
Phomopsis, Phyllosticta, and Sporormiella minima were common foliar endophytes.
Cheng et al. (2008) isolated endophytic fungus from mangrove Kandelia candel.
Using molecular and morphological evidence they identified the endophyte as
Diaporthe phaseolorum var. sojae. A wild legume Sesbania bispinosa was
evaluated for presence of endophytes. Several endophytic fungi with six dominant
taxa were identified (Shreelalitha and Sridhar 2015). In some cases, there is single
species dominance in endophytic fungi (Suryanarayanan and Kumaresan 2000;
Suryanarayanan et al. 1998; Kumaresan and Suryanarayanan 2001). The multispe-
cies domination was also witnessed (Ananda and Sridhar 2002; Kumaresan and
Suryanarayanan 2001).
Coastal Sand Dunes
The roots of three plant species—Ipomoea pes-caprae, Launaea sarmentosa, and
Polycarpaea corymbosa were studied for the presence of fugal endophytes. About
31 species were isolated—19 Deuteromycetes, 6 Ascomycetes, and 6 sterile fungi
(Beena et al. 2000). Chaetomium globosum was the most prevailing organism. It
plays an important role in plant protection. Just a single marine parasite was
endophytic. Wild vegetable of seaside sand and mangroves showed presence of
six endophytes (Shreelalitha and Sridhar 2015).
206 D. Patil et al.

Seaweeds
Ocean growth of wide geographic areas has endophytic parasites (Raghukumar
2008; Schulz et al. 2008; Ariffin et al. 2011; Flewelling et al. 2013; Singh et al.
2018). The endophyte Mycophycias ascophylli is related to the existence of certain
seaweeds—Ascophyllum nodosum and Pelvetia canaliculate. Macroalgae showed
presence of different types of endophytes than terrestrial plant species. The assess-
ment of seaweeds in coastal region showed presence of several endophytes (Sridhar
2019). In the seaweeds of Atlantic coast of Canada 79 endophytic fungi were
associated with red, brown, and green algae (Flewelling et al. 2013).
Seagrass
Seagrass has been studied as a possible niche for endophytic fungi colonization. The
east coast of India’s Halophila ovalis leaf blade, petiole, and rhizome showed lower
presence of endophytic fungi (Devarajan and Suryanarayanan 2002). Also, a culture-
based analysis, showed lower presence of endophytic fungi in coastal seagrass
species compared to terrestrial plant species (Venkatachalam et al. 2015). Around
26 endophytes are known from 3 seagrass (Sridhar 2019). Three endophytes—
Fusarium, Penicillium, and Nigrospora) were predominant. Strangely, Nigrospora
sp. showed antifungal property against the dermatophyte. An intensive study on the
endophytic growths in southern Thailand resulted in identification of four seagrass
species—Cymodocea serrulata, Enhalus acoroides, Halophila ovalis, and Thalassia
hemprichii (Supaphon et al. 2017).
10.4 Adaptations to Biotic and Abiotic Stress
Diverse environmental conditions are present on earth (Fig. 10.5). Drought, high and
low temperatures, scarcity of water, and salinity are few extreme environmental
conditions.
These conditions may influence the structure of types of plants present in a
particular region and thereby the types of fungal endophytes present. To survive
such extreme conditions the fungal endophytes adapt themselves in a variety of
ways. A study explored the endophytic fungal biota associated with plants adapted to
arid habitats. They reported diverse number of fungal endophytes. The dominant
genera among these were Aspergillus, Alternaria, Penicillium, Nigrospora,
Chaetomium and Curvularia (Sangamesh et al. 2018). Another study reported the
presence of endophytes in tree species of the desert area (Gehlot et al. 2008). The
pigmented fungi were also isolated from several cactus species (Suryanarayanan
et al. 2005; Gehlot et al. 2008; Massimo et al. 2015). These studies showed that the
isolates contained melanin and carotenoids such as lycopene. The increase in
melanin content may help to overcome oxidative stress (Singaravelan et al. 2008).
Such an adaptation in these endophytes (in the form of pigmentation) could provide
protection against UV light, pathogenicity, solar radiation, high temperature, chemi-
cal and radioactive pollution, drought, and environmental stresses (Zhdanova and
Vasil’evskaya 1988; Butler and Day 1998; Gupta et al. 2015). Ali et al. (2019)
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 207

showed that Thermomyces lanuginosus isolated from desert-adapted plant has the
ability to sustain high temperatures. The thermal adaptation of T. lanuginosus
originated from several DNA related pathways. Under the heat stress conditions
the cell signaling and DNA repair is governed by ubiquitin degradation,
histoneacetylation/deacetylation, and poly adenosine diphosphate ribosylation
(Mchunu et al. 2013; Ali et al. 2019).
The drought conditions cause disruption of water potential gradients, limitations
to gas diffusion, denaturation of proteins and enhanced production of Reactive
Oxygen Species (ROS) (Rao and Chaitanya 2016). The decrease in intracellular
CO
2
concentration results in production of ROS. It transfers electrons to photosys-
tem I (Reddy et al. 2004). This results in oxidative damage due to lipid peroxidation
(Rizhsky et al. 2004). It has been shown that P. indica an endophyte could counter-
act the ROS induced stress. It prevents the excess ROS formation under stress
conditions and avoid lipid degradation (Sun et al. 2010).
A wide range of compounds are secreted by fungal endophytes. Various fungal
endophytes are known for the production of plant hormones (Khan et al. 2011; Asaf
et al. 2018). These hormones help to tolerate the salinity stress. During the salinity
stress the presence of sodium chloride (NaCl) causes oxidative damage through
production of ROS. Several studies reported that the endophytic fungi produce
Fig. 10.5 Diverse environmental conditions on earth
208 D. Patil et al.

antioxidant enzymes under salt stress and eliminate the free radicals to overcome the
oxidative damage (Abogadallah 2011; Hashem et al. 2014; Ahmad et al. 2015;
Yasmeen and Siddiqui 2017).
Cold and freezing is another important stress needs to be studied with respect to
fungal endophytes. The fungal endophytes facing cold and freezing conditions may
rely on possible physiological and morphological adaptations. The biosynthesis of
unsaturated fatty acid and glycerol helps to maintain membrane fluidity and to
accumulate glycerol as a cryoprotectant. Also, the plants inhabited by the fungal
endophytes may help to survive the cold stress (Robinson 2001; Zhang et al. 2013).
Along with abiotic stresses the fungal endophytes have to face several biotic
stresses as well.
One of biotic stresses faced by fungal endophytes is plant defense compounds
(Bailey et al. 2005; Saunders and Kohn 2009). Over 10,000 secondary metabolites
are produced by plants which have antifungal properties (Dixon 2001). Along with
host plant defense compounds the fungal endophytes have to face the competitive
interactions exerted by other fungal species. These competitive fungal species also
produce biologically active compounds having antifungal activity (Schulz et al.
2002). The fungal endophytes adapt themselves to detoxify the toxic compound
and overcome these barriers (Cooney et al. 2001).
There are three primary mechanisms using which fungal endophytes associate
with plants and compete with fungal species. It includes tolerance: detoxification,
structural alteration of the toxin target, and activation of membrane transporters
(Carter et al. 1999; VanEtten et al. 2001; Saunders and Kohn 2009). The abiotic
stresses such as salinity, low temperature, and heavy metal toxicity hamper the
growth of plants. The adaptation of fungal endophytes to several environmental
stresses not only benefits their own survival but also toward the fitness of host plant.
Endophytic fungi facilitate plant growth through improved nutrient uptake, efficient
water use and curtailing of environmental stresses (Fig. 10.6). Endophytes confer
abiotic stress lenience in plants by triggering host stress response systems and by
synthesizing antistress biochemicals (Sun et al. 2010; Husaini et al. 2012; Ansari
et al. 2013; Gill et al. 2016; Lata et al. 2018). The fungal endophytes activate defense
related genes, abiotic stress responsive genes of the host to mitigate the stress
induced by high salinity and drought. They also enhance secretion of
osmoprotectants (proline, glycine betaine) (Waller et al. 2005; Zarea et al. 2012;
Ansari et al. 2013). This phenomenon is reported in Triticum aestivum (Zarea et al.
2012), Chinese cabbage (Sun et al. 2010) and strawberry (Husaini et al. 2012). The
overproduction of ROS occurs in plants due to abiotic stress and fungal and viral
infections. The excess ROS are removed from plant system with the help of fungal
endophytes. The fungal endophytes modulate the expression of antioxidant defense
enzymes other components of ROS-scavenging system of host plants (White and
Torres 2010; Foyer and Shigeoka 2011; Hamilton et al. 2012; Waller et al. 2005; Sun
et al. 2010). When the plants are exposed to pathogenic fungi, bacteria and virus the
fungal endophyes upregulate various defense related genes of host plant (Waller
et al. 2005; Serfling et al. 2007; Camehl and Oelmüller 2010; Molitor et al. 2011;
Johnson et al. 2013; Gill et al. 2016). The fungal endophytes produce compounds
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 209

having antagonistic activities to increase host defense mechanism (Waller et al.
2005; Gill et al. 2016).
10.5 Conclusions
Almost all the plant species harbor a diverse range of microaerophilic fungi includ-
ing endophytes. The research presented here showed various types of
microaerophilic fungi, their classification and the habitats. Research documented
here will help to understand endophyte ecology. Several factors influence the
diversity of microaerophilic fungi including endophytes. These include type of
host plant, competing species, and stress. The microaerophilic fungi including
endophytes use various mechanisms to sustain various stresses and overcome the
growth barriers. They use detoxification mechanisms. They produce several
osmoprotectants. They are also capable of secreting the defense enzymes or antioxi-
dant enzyme. These fungi not only protect themselves from several stresses but also
confer these properties to the host plant and help it to sustain adverse conditions.
Acknowledgments Ms. Deeksha Patil acknowledges Department of Biotechnology, India for the
research fellowship.
Fig. 10.6 Various mechanisms of stress tolerance in plants conferred by endophyte (Image
courtesy Gill et al., 2016)
210 D. Patil et al.

References
Abogadallah G (2011) Differential regulation of photorespiratory gene expression by moderate and
severe salt and drought stress in relation to oxidative stress. Plant Sci 180:540–547
Ahmad P, Hashem A, Abd-Allah E, Alqarawi A, John R, Egamberdieva D, Gucel S (2015) Role of
Trichoderma harzianum in mitigating NaCl stress in Indian mustard (Brassica juncea L)
through antioxidative defense system. Front Plant Sci 6:1–15
Ainsworth G (1976) Introduction to the history of mycology. Cambridge University Press,
Cambridge. ISBN 978-0-521-11295-6
Akello J, Sikora R (2012) Systemic acropedal influence of endophyte seed treatment on
Acyrthosiphonpisum and Aphis fabae offspring development and reproductive fitness. Biol
Control 61:215–221
Akutse K, Maniania N, Fiaboe K, Berg V, Ekesi S (2013) Endophytic colonization of Vicia faba
and Phaseolus vulgaris (Fabaceae) by fungal pathogens and their effects on the life-history
parameters of Liriomyzahuidobrensis (Diptera: Agromyzidae). Fungal Ecol 6:293–301
Ali A, Radwan U, El-Zayat S, El-Sayed M (2019) The role of the endophytic fungus, Thermomyces
lanuginosus, on mitigation of heat stress to its host desert plant Cullen plicata. Biol Fut 70:1–7
Ananda K, Sridhar K (2002) Diversity of endophytic fungi in the roots of mangrove species on the
west coast of India. Canadian J Microbiol 48:871–878
Ansari M, Bains G, Shukla A, Pant R, Tuteja N (2013) Low temperature stress ethylene and not
fusarium might be responsible for mango malformation. Plant Physiol Biochem 69:34–38
Ariffin S, Davis P, Ramasamy K (2011) Cytotoxic and antimicrobial activities of Malaysian marine
endophytic fungi. Bot Mar 54:95–100
Arnold A, Lutzoni F (2007) Diversity and host range of foliar fungal endophytes: are tropical leaves
biodiversity hotspots? Ecology 88:541–549
Asaf L, Hamayun M, Khan A, Waqas M, Khan M, Jan R, Lee I, Hussain A (2018) Salt tolerance of
Glycine max. L induced by endophytic fungus aspergillus flavus CSH1, via regulating its
endogenous hormones and antioxidative system. Plant Physiol Biochem 128:13–23
Backman P, Sikora R (2008) Endophytes: an emerging tool for biological control. Biol Control 46:
1–3
Bacon C, White J (2000) Physiological adaptations in the evolution of endophytism in the
Clavicipitaceae. In: Bacon CW, White JFJ (eds) Microbial endophytes. Marcel Dekker Inc.,
New York, pp 237–263
Bagyalakshmi G, Muthukumar T, Sathiyadash K, Muniappan V (2010) Mycorrhizal and dark
septate fungal associations in shola species of Western Ghats, southern India. Mycosci 51:44–52
Bailey J, Deckert R, Schweitzer J, Rehill B, Lindroth R, Gehring C, Whitham T (2005) Host plant
genetics affect hidden ecological players: links among Populus, condensed tannins, and fungal
endophyte infection. Canadian J Bot 83:356–361
Baldauf S, Palmer J (1993) Animals and fungi are each other’s closest relatives: congruent evidence
from multiple proteins. Proc Natl Acad Sci U S A 90:11558–11562
Bärlocher F (1992) Research on aquatic hyphomycetes: historical background and overview. In:
The ecology of aquatic hyphomycetes. Springer, Berlin, pp 1–15
Beena K, Ananda K, Sridhar K (2000) Fungal endophytes of three sand dune plant species of west
coast of India. Sydowia-Horn 52:1–9
Bing L, Lewis L (1991) Suppression of Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) by
endophytic Beauveria bassiana (Balsamo) Vuillemin. Environ Entomol 20:1207–1211
Biswas C, Dey P, Satpathy S, Satya P, Mahapatra B (2012) Endophytic colonization of white jute
(Corchorus capsularis) plants by different Beauveria bassiana strains for managing stem weevil
(Apion corchori). Phytoparasitica 41:17–21
Breen J (1994) Acremonium endophyte interactions with enhanced plant resistance to insects. Annu
Rev of Entomol 39:401–423
Bruns T (2006) A kingdom revised. Nature 443:758–761
Butler M, Day A (1998) Fungal melanins: a review. Can J Microbiol 44:1115–1136
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 211

Camehl I, Oelmüller R (2010) Do ethylene response factors-9 and- 14 repress PR gene expression
in the interaction between Piriformospora indica and Arabidopsis? Plant Signal Behav 5:932–
936
Carroll G (1995) Forest endophytes: pattern and process. Can J Bot 73:1316–1324
Carter J, Spink J, Cannon P, Daniels M, Osbourn A (1999) Isolation, characterization, and avenacin
sensitivity of a diverse collection of cereal-root-colonizing fungi. Appl Environ Microbiol 65:
3364–3372
Chen J, Hu K, Hou X, Guo S (2011) Endophytic fungi assemblages from 10 dendrobium medicinal
plants (Orchidaceae). World J Microbiol Biotechnol 27:1009–1016
Cheng Z, Tang W, Xu S, Sun S, Huang B, Yan X, Lin Y (2008) First report of an endophyte
(Diaporthepha seolorum var. sojae) from Kandeliacandel. J Forestry Res 19:277–282
Cheplick G, Clay K (1988) Acquired chemical defences in grasses: the role of fungal endophytes.
Oikos 52:309
Cherry A, Lomer C, Djegui D, Schulthess F (1999) Pathogen incidence and their potential as
microbial control agents in IPM of maize stem borers in West Africa. BioControl 44:301–327
Clay K (1988) Fungal endophytes of grasses: a defensive mutualism between plants and fungi.
Ecology 69:10–16
Clay K (1989) Clavicipitaceous endophytes of grasses: their potential as biocontrol agents. Mycol
Res 92:1–12
Clay K, Schardl C (2002) Evolutionary origins and ecological consequences of endophyte symbio-
sis with grasses. Am Nat 160:99–127
Coker J (2019) Recent advances in understanding extremophiles. Food Res 1917:1–8
Cooney J, Lauren D, di Menna M (2001) Impact of competitive fungi on trichothecene production
by fusarium graminearum. J Agric Food Chem 49:522–526
Dadachova E, Huang X, Schweitzer A, Aisen P, Nosanchuk J, Casadevall A (2007) Ionizing
radiation changes the electronic properties of melanin and enhances the growth of melanized
fungi. PLoS One 2:457
Devarajan P, Suryanarayanan T (2002) Endophytic fungi associated with the tropical seagrass
Halophila ovalis (Hydrocharitaceae). Ind J Mar Sci 31:73–74
Dixon R (2001) Natural products and plant disease resistance. Nature 411:843–847
Durrant L (1996) Biodegradation of lignocellulosic materials by soil fungi isolated under anaerobic
conditions. Int Biodeterior Biodegradation 37:189–195
Durrant L, Canale-Parola E, Leschine S (1995) Facultatively anaerobic cellulolytic fungi from soil.
Signific Regul Soil Biod 63:161–167
Elmi A, West C, Robbins R, Kirkpatrick T (2000) Endophyte effects on reproduction of a root-knot
nematode (Meloidogyne marylandi) and osmotic adjustment in tall fescue. Grass Forage Sci 55:
166–172
Flewelling A, Johnson J, Gray C (2013) Isolation and bioassay screening of fungal endophytes from
North Atlantic marine macroalgae. Bot Mar 56:287–297
Foyer C, Shigeoka S (2011) Understanding oxidative stress and antioxidant functions to enhance
photosynthesis. Plant Physiol 155:93–100
Fürnkranz M, Lukesch B, Muller H, Huss H, Grube M, Berg G (2012) Microbial diversity inside
pumpkins: microhabitat-specific communities display a high antagonistic potential against
phytopathogens. Microb Ecol 63:418–428
Gehlot P, Bohra N, Purohit D (2008) Endophytic mycoflora of inner bark of Prosopis cineraria-a
key stone tree species of Indian desert. Am Eur J Bot 1:01–04
Gezgin Y, Eltem R (2009) Diversity of endophytic fungi from various Aegean and Mediterranean
orchids (saleps). Turkish J Bot 33:439–445
Ghate S, Sridhar K (2017) Endophytic aquatic hyphomycetes in roots of riparian tree species of two
Western Ghat streams. Symbiosis 71:233–240
Gill S, Gill R, Trivedi D, Anjum N, Sharma K, Ansari M, Ansari A, Johri A, Prasad R, Pereira E,
Varma A, Tuteja N (2016) Piriformospora indica: potential and significance in plant stress
tolerance. Front Microbiol 7:332
212 D. Patil et al.

Grahl N, Shepardson K, Chung D, Cramer R (2012) Hypoxia and fungal pathogenesis: to air or not
to air? Eukaryot Cell 11:560–570
Greenfield M, Gomez-Jimenez M, Ortiz V, Vega F, Kramer M, Parsa S (2016) Beauveria bassiana
and Metarhizium anisopliae endophytically colonize cassava roots following soil drench
inoculation. Biol Control 95:40–48
Gupta V, Sreenivasaprasad S, Mach R (2015) Fungal bio-molecules: sources, applications and
recent developments, 1st edn. Wiley-Blackwell, Delhi, pp 117–136
Gurulingappa P, Sword G, Murdoch G, McGee P (2010) Colonization of crop plants by fungal
entomopathogens and their effects on two insect pests when in planta. Biol Control 55:34–41
Haeckel E (1866) Generelle Morphologie der Organismen. Georg Andreas Reimer Verlag, Berlin
Hamilton C, Gundel P, Helander M, Saikkonen K (2012) Endophytic mediation of reactive oxygen
species and antioxidant activity in plants: a review. Fungal Divers 54:1–10
Hardoim P, Overbeek L, Berg G, Pirttilä A, Compant S, Campisano A, Döring M, Sessitsch A
(2015) The hidden world within plants: ecological and evolutionary considerations for defining
functioning of microbial endophytes. Microbiol Mol Biol Rev 79:293–320
Hartley S, Gange A (2009) Impacts of plant symbiotic fungi on insect herbivores: mutualism in a
multitrophic context. Annu Rev Entomol 54:323–342
Hashem A, Abd Allah E, Alqarawi A, Al Huqail A, Egamberdieva D (2014) Alleviation of abiotic
salt stress in Ochradenus baccatus (Del.) by Trichoderma hamatum (bonord.) bainier. J Plant
Interact 9:857–868
Hawksworth D (1991) The fungal dimension of biodiversity: magnitude, significance and conser-
vation. Mycol Res 95:641–655
Hawksworth D (2001) The magnitude of fungal diversity: the 1.5 million species estimate revisited.
Mycol Res 105:1422–1432
Hawksworth D, Lucking R (2016) Fungal diversity revisited: 2.2 to 3.8 million species. Microbiol
Spectr 5:0052
Herrera P, Suárez J, Kottke I (2010) Orchids keep the ascomycetes outside: a highly diverse group
of ascomycetes colonizing the velamen of epiphytic orchids from a tropical mountain rainforest
in southern Ecuador. Mycology 1:262–268
Husaini A, Abdin M, Khan S, Xu Y, Aquil S, Anis M (2012) Modifying strawberry for better
adaptability to adverse impact of climate change. Curr Sci 102:1660–1673
Jaber L, Araj S (2017) Interactions among endophytic fungal entomopathogens (Ascomycota:
Hypocreales), the green peach aphid Myzuspersicae Sulzer (Homoptera: Aphididae), and the
aphid endoparasitoid Aphidius colemani Viereck (hymenoptera: Braconidae). Biol Control 116:
53–61
Jaber L, Enkerli J (2016) Effect of seed treatment duration on growth and colonization of Vicia faba
by endophytic Beauveria bassiana and Metarhizium brunneum. Biol Control 103:187–195
James T, Kauff F, Vilgalys R (2006) Reconstructing the early evolution of fungi using a six-gene
phylogeny. Nature 443:818–822
Johnson J, Sherameti I, Nongbri P, Oelmüller R (2013) Standardized conditions to study beneficial
and non beneficial traits in the Piriformospora indica/Arabidopsis thaliana interaction. In:
Varma A, Kost G, Oelmuller R (eds) Sebacinales-forms, functions and biotechnological
applications, soil biology series no.33. Springer-Verlag, Berlin, pp 325–343
Jones G (1947) An enumeration of Illinois Pteridophyta. Am Midland Nat 38:76–126
Khan A, Hamayun M, Kim Y, Kang S, Lee I (2011) Ameliorative symbiosis of endophyte
(Penicillium funiculosum LHL06) under salt stress elevated plant growth of Glycine max
L. Plant Physiol Biochem 49:852–861
Kharwar R, Mishra A, Gond S, Stierle A, Stierle D (2011) Anticancer compounds derived from
fungal endophytes: their importance and future challenges. Nat Prod Rep 28:1208
Kim J, Goettel M, Gillespie D (2008) Evaluation of Lecanicilliumlongisporum, Vertalec®for
simultaneous suppression of cotton aphid, Aphis gossypii, and cucumber powdery mildew,
Sphaerotheca fuliginea, on potted cucumbers. Biol Control 45:404–409
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 213

Krings M, Taylor T, Hass H, Kerp H, Dotzler N, Hermsen E (2007) Fungal endophytes in a
400-million-yr-old land plant: infection pathways, spatial distribution, and host responses. New
Phytol 174:648–657
Kumaran R, Kim H, Hur B (2010) Taxol promising fungal endophyte, Pestalotiopsis species
isolated from Taxus cuspidata. J Biosci Bioeng 110:541–546
Kumaresan V, Suryanarayanan T (2001) Occurrence and distribution of endophytic fungi in a
mangrove community. Mycol Res 105:1388–1391
Kumaresan V, Veeramohan R, Bhat M, Sruthi K, Ravindran C (2013) Fungal endophyte
assemblages of some Pteridophytes from Mahe, India. World J Sci Technol 3:7–10
Kusari S, Singh S, Jayabaskaran C (2014) Biotechnological potential of plant-associated endo-
phytic fungi: hope versus hype. Trends Biotechnol 32:297–303
Lata R, Chowdhury S, Gond S, White J Jr (2018) Induction of abiotic stress tolerance in plants by
endophytic microbes. Lett Appl Microbiol 66:268–276
Leuchtmann A, Schmidt D, Bush L (2000) Different levels of protective alkaloids in grasses with
stroma-forming and seed-transmitted epichloë/neotyphodium endophytes. J Chem Ecol 26:
1025–1036
Li H, Zhao C, Liu C, Xu X (2010) Endophytic fungi diversity of aquatic/riparian plants and their
antifungal activity in vitro. J Microbiol 48:1–6
Lugtenberg B, Caradus J, Johnson L (2016) Fungal endophytes for sustainable crop production.
FEMS Microbiol Ecol 92:194
Macelroy R (1974) Some comments on the evolution of extremophiles. BioSyst 6:74–75
Massimo N, Nandi Devan M, Arendt K, Wilch M, Riddle J, Furr S, Steen C, U’Ren J, Sandberg D,
Arnold A (2015) Fungal endophytes in aboveground tissues of desert plants: infrequent in
culture, but highly diverse and distinctive symbionts. Microb Ecol 70:61–76
Mchunu N, Permaul K, Abdul Rahman A, Saito J, Singh S, Alam M (2013) Xylanase super
producer: genome sequence of a compost-loving thermophilic fungus, Thermomyces
lanuginosus strain SSBP. Gen Announ 1:e00388–e00313
Molitor A, Zajic D, Voll L, Pons-Kuehnemann J, Samans B, Kogel K et al (2011) Barley leaf
transcriptome and metabolite analysis reveals new aspects of compatibility and Piriformospora
indica-mediated systemic induced resistance to powdery mildew. Mol Plant-Microbe Interact
24:1427–1439
Moon C, Tapper B, Scott B (1999) Identification of epichloë endophytes in planta by a
microsatellite-based pcr fingerprinting assay with automated analysis. Appl Environ Microbiol
65:1268–1279
Orole O, Adejumo T (2009) Activity of fungal endophytes against four maize wilt pathogens. Afr J
Microbiol Res 3:969–973
Ownley B, Griffin M, Klingeman W, Gwinn K, Moulton J, Pereira R (2008) Beauveria bassiana:
endophytic colonization and plant disease control. J Invertebr Pathol 98:267–270
Pavarina E, Durrant L (2002) Growth of lignocellulosic-fermenting fungi on different substrates
under low oxygenation conditions. Appl Biochem Biotechnol 98-100:663–678
Petrini O (1991) Fungalendophytes of tree leaves. In: Microbial ecology of leaves. Springer,
New York, pp 179–197
Posada F, Aime M, Peterson S, Rehner S, Vega F (2007) Inoculation of coffee plants with the
fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). Mycol Res 111:748–
757
Puri A, Padda K, Chanway P (2016) Evidence of nitrogen fixation and growth promotion in canola
(Brassica napus L.) by an endophytic diazotroph Paenibacilluspolymyxa P2b-2R. Biol Fertil
Soils 52:119–125
Quesada-Moraga E, Muñoz-Ledesma F, Santiago-Alvarez C (2009) Systemic protection of Papaver
somniferum L. against Iraella luteipes (hymenoptera: Cynipidae) by an endophytic strain of
Beauveria bassiana (Ascomycota: Hypocreales). Environ Entomol 38:723–730
Raghukumar C (2008) Marine fungal biotechnology: an ecological perspective. Fungal Diver 31:
19–35
214 D. Patil et al.

Raghukumar C, Raghukumar S (1998) Barotolerance of fungi isolated from deep-sea sediments of
the Indian Ocean. Aquat Microb Ecol 15:153–163
Rajagopal K, Meenashree B, Binika D, Joshila D, Tulsi P, Arulmathi R, Tuwar A (2018)
Mycodiversity and biotechnological potential of endophytic fungi isolated from hydrophytes.
Cur Res Environ Appl Mycol 8:172–182
Rao D, Chaitanya K (2016) Photosynthesis and antioxidative defense mechanisms in deciphering
drought stress tolerance of crop plants. Biol Plant 60:201–218
Ratnaweera P, Williams D, de Silva E, Wijesundera R, Dalisay D, Andersen R (2014) Helvolic
acid, an antibacterial nortriterpenoid from a fungal endophyte, Xylaria sp. of orchid
Anoectochilus setaceus endemic to Sri Lanka. Mycology 5:23–28
Raviraja N (2005) Fungal endophytes in five medicinal plant species from Kudremukh range,
Western Ghats of India. J Basic Microbiol 45:230–235
Raviraja N, Sridhar K, Barlocher F (1996) Endophytic aquatic hyphomycetes of roots of plantation
crops and ferns from India. Sydowia-Horn 48:152–160
Raviraja N, Sridhar K, Bärlocher F (1998) Fungal species richness in Western Ghat streams
(southern India): is it related to pH, temperature or altitude. Fungal Divers 1:179–191
Rayner M (1915) Obligate symbiosis in Calluna vulgaris. Ann Bot 29:97–133
Reddy A, Chaitanya K, Vivekanandan M (2004) Drought-induced responses of photosynthesis and
antioxidant metabolism in higher plants. J Plant Physiol 161:1189–1202
Reddy N, Khan A, Devi U, Sharma H, Reineke A (2009) Treatment of millet crop plant (Sorghum
bicolor) with the entomopathogenic fungus (Beauveria bassiana) to combat infestation by the
stem borer, Chilopartellus Swinhoe (Lepidoptera: Pyralidae). J Asia Pac Entomol 12:221–226
Redman R, Dunigan D, Rodriguez R (2001) Fungal symbiosis: from mutualism to parasitism, who
controls the outcome, host or invader? New Phytol 151:705–716
Redman R, Sheehan K, Stout R, Rodriguez R, Henson J (2002) Thermotolerance generated by
plant/fungal Symbiosis. Science 298:1581–1581
Rehner S, Samuels G (1995) Molecular systematics of the Hypocreales:a teleomorph gene
phylogeny and the status of their anamorphs. Can J Bot 73:816–823
Richards T, Jones M, Leonard G, Bass D (2012) Marine fungi: their ecology and molecular
diversity. Annu Rev Mar Sci 4:495–522
Rizhsky L, Davletova S, Liang H, Mittler R (2004) The zinc finger protein Zat12 is required for
cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J Biol Chem
279:11736–11743
Robinson C (2001) Cold adaptation in arctic and antarctic fungi. New Phytol 151:341–353
Rodriguez R, Henson J, Van Volkenburgh E, Hoy M, Wright L, Beckwith F, Kim Y, Redman R
(2008) Stress tolerance in plants via habitat-adapted symbiosis. ISME J 2:404–416
Rodriguez R, JrJ W, Arnold A, Redman R (2009) Fungal endophytes: diversity and functional
roles. New Phytol 182:314–330
Russo M, Pelizza S, Marta C, Stenglein S, Scorsetti A (2015) Endophytic colonisation of tobacco,
corn, wheat and soybeans by the fungal entomopathogen Beauveria bassiana (Ascomycota,
Hypocreales). Biocontrol Sci Tech 25:475–480
Saikkonen K, Faeth S, Helander M, Sullivan T (1998) Fungal endophytes: a continuum of
interactions with host plants. Annu Rev Ecol Syst 29:319–343
Saikkonen K, Ion D, Gyllenberg M (2002) The persistence of vertically transmitted fungi in grass
metapopulations. Proc R Soc Bio Sci 269:1397–1403
Saikkonen K, Lehtonen P, Helander M, Koricheva J, Faeth S (2006) Model systems in ecology:
dissecting the endophyte–grass literature. Trends Plant Sci 11:428–433
Sandberg D, Battista L, Arnold A (2014) Fungal endophytes of aquatic macrophytes: diverse host-
generalists characterized by tissue preferences and geographic structure. Microb Ecol 67:735–
747
Sangamesh M, Jambagi S, Vasanthakumari M, Shetty N, Kolte H, Ravikanth G, Nataraja K,
Shaanker R (2018) Thermotolerance of fungal endophytes isolated from plants adapted to the
Thar Desert, India. Symbiosis 75:135–147
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 215

Saunders M, Kohn L (2009) Evidence for alteration of fungal endophyte community assembly by
host defense compounds. New Phytol 182:229–238
Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686
Schulz B, Boyle C, Draeger S, Rommert A, Krohn K (2002) Endophytic fungi: a source of
biologically active secondary metabolites. Mycol Res 106:996–1004
Schulz B, Draeger S, dela Cruz T, Rheinheimer J, Siems K, Loesgen S, Krohn K (2008) Screening
strategies for obtaining novel, biologically active, fungal secondary metabolites from marine
habitats. Bot Mar 51:219–234
Selosse M, Vohnik M, Chauvet E (2008) Out of the rivers: are some aquatic hyphomycetes plant
endophytes? New Phytol 178:3–7
Serfling A, Wirsel S, Lind V, Deising H (2007) Performance of the biocontrol fungus
Piriformospora indica on wheat under green house and field conditions. Phytopathology 97:
523–531
Shreelalitha S, Sridhar K (2015) Endophytic fungi of wild legume Sesbania bispinosa in coastal
sand dunes and mangroves of the southwest coast of India. J Forestry Res 26:1003–1011
Shrestha N, Chilkoor G, Vemuri B, Rathinam N, Sani R, Gadhamshetty (2018) Extremophiles for
microbial-electrochemistry applications: a critical review. Bioresour Technol 255:318–330
Sikora R, Schafer K, Dababat A (2007) Modes of action associated with microbially induced in
planta suppression of plant-parasitic nematodes. Australas Plant Pathol 36:124–134
Simpson D (1979) Cassell’s Latin dictionary, 5th edn. Cassell Ltd., London, p 883. ISBN 978-0-
304-52257-6
Singaravelan N, Grishkan I, Beharav A, Wakamatsu K, Shosuke I (2008) Adaptive melanin
response of the soil fungus aspergillus Niger to UV radiation stress at Bevolution canyon,
Mount Carmel Israel. PLoS One 3:1–5
Singh D, Sharma V, Kumar J, Mishra A, Verma S, Sieber T, Kharwar R (2017) Diversity of
endophytic mycobiota of tropical tree Tectonagrandis Linn.F.: spatiotemporal and tissue type
effects. Sci Rep 7. https://doi.org/10.1038/s41598-017-03933-0
Singh V, Dwivedy A, Singh A, Asawa S, Dwivedi A, Dubey N (2018) Fungal endophytes from
seaweeds: an overview. In: Microbial biotechnology. Springer, Singapore, pp 483–498
Spatafora J, Blackwell M (1993) Molecular systematics of unitunicate Perithecial ascomycetes:the
Clavicipitales-Hypocreales connection. Mycologia 85:912–922
Spatafora J, Sung G, Sung J, Hywel-Jones N, White J Jr (2007) Phylogenetic evidence for an animal
pathogen origin of ergot and the grass endophytes. Mol Ecol 16:1701–1711
Sridhar K (2019) Diversity, ecology, and significance of fungal endophytes. In: Jha S
(ed) Endophytes and secondary metabolites. Reference series in Phytochemistry. Springer,
Cham. https://doi.org/10.1007/978-3-319-90484-9_5
Sun C, Johnson J, Cai D, Sherameti I, Oelmüller R, Lou B (2010) Piriformospora indica confers
drought tolerance in Chinese cabbage leaves by stimulating antioxidant enzymes, the expression
of drought-related genes and the plastid-localized CAS protein. J Plant Physiol 167:1009–1017
Sun X, Guo L (2012) Endophytic fungal diversity: review of traditional and molecular techniques.
Mycology 3:65–76
Sunitha V, Devi D, Srinivas C (2013) Extracellular enzymatic activity of endophytic strains isolated
from medicinal plants. World J Agric Sci 9:1–9
Suryanarayanan T (2011) Diversity of fungal endophytes in tropical trees. In: Endophytes of forest
trees. Springer, Dordrecht, pp 67–80
Suryanarayanan T, Kumaresan V (2000) Endophytic fungi of some halophytes from an estuarine
mangrove forest. Mycol Res 104:1465–1467
Suryanarayanan T, Kumaresan V, Johnson J (1998) Foliar fungal endophytes from two species of
the mangrove Rhizophora. Canadian J Microbiol 44:1003–1006
Suryanarayanan T, Wittlinger S, Faeth S (2005) Endophytic fungi associated with cacti in Arizona.
Mycol Res 109:635–639
Tabak H, Cooke W (1968) Growth and metabolism of fungi in an atmosphere of nitrogen.
Mycologia 60:115
216 D. Patil et al.

Taylor W (1936) What is ecology and what good is it? Ecology 17:333–346
Tefera T, Vidal S (2009) Effect of inoculation method and plant growth medium on endophytic
colonization of sorghum by the entomopathogenic fungus Beauveria bassiana. BioControl 54:
663–669
Teunissen M, den Camp H, Orpin C, Veld J, Vogels G (1991) Comparison of growth characteristics
of anaerobic fungi isolated from ruminant and non-ruminant herbivores during cultivation in a
defined medium. J Gen Microbiol 137:1401–1408
Tudzynski B, Sharon A (2002) Biosynthesis, biological role and application of fungal phyto-
hormones. In: Osiewacz HD (ed) The mycota X. industrial applications. Springer-Verlag,
Berlin, pp 183–212
Udayaprakash N, Ashwinkarthick N, Poomagal D, Susithra M, Chandran M, Bhuvaneswari S
(2018) Fungal endophytes of an aquatic weed Marsileaminuta Linn. Curr Res Environ Appl
Mycol 8:86–95
VanEtten H, Temporini E, Wasmann C (2001) Phytoalexin (and phytoanticipin) tolerance as a
virulence trait: why is it not required by all pathogens? Physiol Molecul Plant Pathol 59:83–93
Vasiliauskas R, Menkis A, Finlay R, Stenlid J (2007) Wood-decay fungi in fine living roots of
conifer seedlings. New Phytol 174:441–446
Vaupotic T, Jenoe P, Plemenitas A (2008) Mitochondrial mediation of environmental osmolytes
discrimination during osmoadaptation in the extremely halotolerant black yeast
Hortaeawerneckii. Fungal Genet Biol 45:994–1007
Vega F, Posada F, Aime MC, Pava-Ripoll M, Infante F, Rehner S (2008) Entomopathogenic fungal
endophytes. Biol Control 46:72–82
Venkatachalam A, Thirunavukkarasu N, Suryanarayanan T (2015) Distribution and diversity of
endophytes in seagrasses. Fungal Ecol 13:60–65
Waid J (1962) Influence of oxygen upon growth and respiratory behaviour of fungi from
decomposing rye-grass roots. Trans Br Mycol Soc 45:479–487
Wainwright M (1981) Mineral transformations by fungi in culture and in soils. Zeitschriftfür
Pflanzenernährung und Bodenkunde 144:41–63
Wainwright M (1988) Metabolic diversity of fungi in relation to growth and mineral cycling in soil -
a review. Trans Br Mycol Soc 90:159–170
Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M et al (2005) The endophytic
fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and
higher yield. Proc Natl Acad Sci USA 102:13386–13391
White J (1988) Endophyte-host associations in forage grasses. xi. A proposal concerning origin and
evolution. Mycologia 80:442–446
White J, Bultman T (1987) Endophyte-host associations in forage grasses. Viii. Heterothallism in
epichloëtyphina. Am J Bot 74:1716–1721
White J, Torres M (2010) Is plant endophyte-mediated defensive mutualism the result of oxidative
stress protection? Physiol Plant 138:440–446
Winkelmann G (2007) Ecology of siderophores with special reference to the fungi. Biometals 20:
379–392
Yasmeen R, Siddiqui Z (2017) Physiological responses of crop plants against Trichoderma
Harzianum in saline environment. Acta Bot Croat 76:154–162
Yu Y, Cui Y, Hsiang T, Zeng Z, Yu Z (2015) Isolation and identification of endophytes from roots
of cymbidium goeringii and Cymbidium faberi (Orchidaceae). Nova Hedwigia 101:57–64
Zarea M, Hajinia S, Karimi N, Goltapeh E, Rejali F, Varma A (2012) Effect of Piriformospora
indica and Azospirillum strains from saline or non-saline soil on mitigation of the effects of
NaCl. Soil Biol Biochem 45:139–146
10 Ecology and Diversity of Microaerophilic Fungi Including Endophytes 217

Zeng P, Wu J, Liao L, Chen T, Wu J, Wong K (2011) In vitro antioxidant activities of endophytic
fungi isolated from the liverwort Scapania verrucosa. Genet Mol Res 10:3169–3179
Zhang T, Zhang Y, Liu H, Wei Y, Li H, Su J, Zhao L, Yu L (2013) Diversity and cold adaptation of
culturable endophytic fungi from bryophytes in the Fildes region, King George Island, maritime
Antarctica. FEMS Microbiol Lett 341:52–61
Zhdanova N, Vasil’evskaya A (1988) Melanin-containing fungi in extreme conditions. Naukova
Dumka, Kiev, p 286
Zubek S, Blaszkowski J, Mleczko P (2011) Arbuscular mycorrhizal and dark septate endophyte
associations of medicinal plants. Acta Soc Bot Pol 80:285–292
218 D. Patil et al.

Fungi in Hypoxic Soils and Aquatic
Sediments 11
Irena Maček
Abstract
Under certain environmental conditions, O
2
availability in soils and aquatic
sediments can become a limiting factor for the survival of aerobic organisms,
including the majority of fungi. Here, hypoxia is presented in light of various
aspects of fungal ecology. Fungi are diverse in many extreme environments, but
many of these ecosystems have been poorly exploited to study general principles
of fungal biology and ecology, and these include hypoxic environments such as
submerged ecosystems and mofettes (natural CO
2
springs). Furthermore, with
global change accelerating the frequency of extreme events, hypoxic
environments are also becoming more common, with either permanent or tempo-
rary soil or sediment hypoxia caused by flooding or higher temperatures. Here, in
addition to a range of aquatic hypoxic environments, we present some new
insights and experiences on the response of fungi to hypoxia, derived from
research on a specific extreme mofette ecosystem. Findings on the response of
different groups of fungi in soil and sediments (e.g. yeasts, mycorrhizal fungi)
and in particular on their patterns of community formation are presented. Finally,
we report on the frontiers of fungal research in hypoxic environments, which
include some little-studied topics such as the bioprospecting of new extremophile
taxa, the study of fungal pathogens in humans, and extreme environments as
natural models for long-term experiments in ecology and evolution.
I. Maček (*)
Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Faculty of Mathematics, Natural Sciences and Information Technologies (FAMNIT), University of
Primorska, Koper, Slovenia
e-mail: irena.macek@bf.uni-lj.si
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_11
219

Keywords
Aquatic sediment · Arbuscular mycorrhiza · Arbuscular mycorrhizal fungi · Dark
septate endophyte · Elevated CO
2
· Extremophiles · Fungal pathogens ·
Filamentous fungi · Hypoxia · Mofette · Occultifur mephitis · Soil fungi · Soil
biodiversity · Yeasts
11.1 Introduction
Normally, soil air contains concentrations of O
2
similar to those in the atmosphere
(just below 21%). However, under certain environmental conditions, O
2
availability
in soil and also in aquatic sediments can become a limiting factor for the survival of
aerobic organisms, including most plants, animals, and fungi. There can be several
causes for hypoxia (low O
2
concentration compared to atmosphere) or even anoxia
(no O
2
). It can occur locally on a microscale or in a larger volume of soil or sediment.
In the case of local (small-scale) hypoxia, larger animals, plant roots, and possibly
some filamentous fungi may avoid it by moving or growing in another area where O
2
is more abundant. However, if hypoxia is present in a larger volume of soil or
sediment, and long-term at that, avoidance is not possible and other mechanisms of
O
2
supply must take place to support aerobic metabolism of aerophylls if they are
still present in such a system (e.g. in permanently submerged environments, in
terrestrial ecosystems during prolonged periods of flooding, or in some extreme
hypoxic ecosystems such as natural CO
2
springs or mofettes, see the description in
Sect. 3.1, Fig. 11.1).
In some cases (e.g. ecosystems with permanent soil hypoxia—natural CO
2
springs), specific terminology has been developed for plants based on their tolerance
to hypoxia, but not yet in general for fungi or other microorganisms (Maček et al.
2016b, Fig. 11.2). Plants in mofettes form three categories: (1) mofettophobic, which
strictly avoid geological CO
2
at concentrations above 2–3%; (2) mofettophilic,
which grow above high CO
2
concentrations (which can be over 80% in soil); and
(3) mofettovagous plants, which grow in both degassing and reference/control areas
(Pfanz 2008, Pfanz et al. 2019, Fig. 11.1). A similar categorisation could be applied
to other groups of organisms (animals and microbes) (Maček et al. 2016b) and has
recently been used for some Collembolae found in mofettovage areas (Russell et al.
2011; Hohberg et al. 2015).
In this chapter, a number of hypoxic environments are presented with their
specificities, in addition to insights into the response of different groups of fungi
and, in particular, the patterns of their community structure. With the new molecular
breakthroughs of the last decades, life in soils has become a new frontier of
biological research in a number of different ecosystems and its response to hypoxia
is an important topic to study. Hypoxia and its effects on various aspects of fungal
ecology and physiology were chosen for presentation for several reasons:
220 I. Maček

1. it is a common but little-studied stress in terms of its effects on the biology of
various fungal groups (Maček et al. 2011,2016b),
2. it is present in many natural ecosystems worldwide (Perata et al. 2011), both
terrestrial and aquatic, with climate change models predicting that it will become
even more common and severe in the future, due to larger floodplains and direct
and indirect effects of higher temperatures (Hirabayashi et al. 2013), and.
3. the new knowledge and experience on this phenomenon coming from our
research on a specific extreme hypoxic ecosystem—mofettes or natural CO
2
springs (e.g. Maček et al. 2011;Šibanc et al. 2014;Maček et al. 2016b;Šibanc
et al. 2018, Fig. 11.1, Sect. 3.1).
In the first part, the definition of an extreme environment and extremophiles is
presented. Then, different hypoxic ecosystems and their fungal inhabitants are
Fig. 11.1 Terrestrial grassland (above) and aquatic forest mofette (below) sites near the village of
Stavešinci, NE Slovenia. Reduced plant growth and altered plant community structure are seen at
both mofette sites. Subsurface geogenic CO
2
release leading to continuous bubbling of water is seen
in the forest-water aquatic mofette called Slepica (below). Dead animals that have suffocated in CO
2
traps near forest mofettes are often found at mofette sites (below). See also Maček et al. (2016b) for
more details on mofettes, their flora, fauna and microbes. Photo: I. Maček
11 Fungi in Hypoxic Soils and Aquatic Sediments 221

presented, starting with a specific extreme ecosystem where soil hypoxia is induced
by geogenic CO
2
exhalations in natural CO
2
springs or mofettes. These can be wet or
dry (Fig. 11.1). Submerged ecosystems are then addressed (hypoxia in aquatic
sediments), followed by less extreme, temporarily flooded and compacted soils
with induced soil hypoxia. The chapter ends with a section addressing the new
areas of studying fungi in hypoxic environments. This includes some little-studied
topics such as bioprospecting for new taxa, exploring fungal pathogens in hypoxic
environments, and a case study of mofettes that serve as natural long-term
experiments in the fields of evolution and ecology.
PLANTS
biomass,
photosynthesis
FUNGI
(soil & plant
endophytic), biomass
soil MICROALGAE
biomass, diversity
FAUN A
biomass, diversity
mobility, body size
ARCHAEA
methanogenes
BACTERIA
acidophiles,
anaerobes
MOFETTOPHILIC
MOFETTOPHOBIC MOFETTOPHOBIC
MOFETTOPHILICMOFETTOPHOBIC MOFETTOPHOBIC
MOFETTOPHILIC
CH4
MOFETTOPHOBIC MOFETTOPHOBIC
MOFETTOPHILICMOFETTOPHOBIC MOFETTOPHOBIC
CO2
CO2CO2
Fig. 11.2 Schematic representation of the effect of CO
2
venting at mofettes on biotic factors. The
gradients of the different factors are indicated by the thickness of the lenticular shapes. A biconvex
lens shape indicates a higher value of the specific parameter closer to the mofette centre with higher
CO
2
concentration, while a biconcave shape indicates the opposite. Mofette specific terminology is
presented (mofettophobic for organisms that strictly avoid geogenic CO
2
at concentrations above
2–3%, and mofettophilic based on their tolerance to hypoxia), see also Maček et al. (2016b) for
details
222 I. Maček

11.2 Extreme Environments and Extremophiles
Extreme environments have been defined as environments that have one or more
environmental parameters whose values are persistently close to the lower or upper
limits known to support life (Torossian et al. 2016) and have long been studied as
ecosystems of special interest. However, defining extreme environments remains
difficult. Microorganisms are able to use a wider range of different metabolic
pathways to survive in these environments. Therefore, it is clear that an extreme
for humans or higher (multicellular) organisms may not be an extreme for microbes.
In an analogous context, our current oxygen-rich atmosphere can be considered
extreme for anaerobic organisms that dominated over a large period of life’s
existence.
Extreme environments are also hotspots of biological discovery that have served
as rich sources of biotechnological compounds. Many initial studies have aimed to
describe extreme systems from many different aspects, including diversity of abiotic
factors and biological communities. By definition, an extremophile is an organism
that thrives in extreme environments. This can include hypoxic environments. The
ecology of extremophiles has long been a rich source of knowledge about the
evolution and functions relevant to stress adaptation of microbes from different
phylogenetic groups (e.g. Gostinčar et al. 2010). Ecological study, including biodi-
versity loss and the response of extreme ecosystems to global change, is becoming
increasingly important. Fungi are diverse in many extreme environments, but many
of these ecosystems have not been widely used to study general principles in fungal
biology and ecology, and these include hypoxic environments such as submerged
ecosystems and mofettes (Maček et al. 2016b;Maček 2017a,b). Extreme
environments characterised by specific gas composition are increasingly used as
natural experiments (e.g. Rothschild and Mancinelli 2001;Maček et al. 2005,2011,
2016b). Environments that are locally extreme relative to average regional
conditions (e.g. natural CO
2
springs or mofettes, see Sections 3.1 and 4) may be
less promising in terms of biological discovery, yet they provide some of the most
powerful natural experiments for studying ecology and evolution (Maček et al.
2016b, see Sect. 4.3).
11.3 Fungi in Hypoxic Soils and Aquatic Sediments
Fungi are a widespread, diverse and large group of eukaryotes with complex cell
structures consisting of various forms ranging from filamentous fungi, such as
mycorrhizal fungi, moulds and mushrooms to unicellular yeasts. Some fungi have
the ability to form tissues and organs typical of higher organisms. Like other
multicellular organisms, fungi are considered aerobes; however, their habitats can
often be hypoxic or even anoxic due to, for example, infiltration of soil with water,
metabolic activity in their environment, or during colonisation or infection of other
organisms (Maček et al. 2011; Grahl et al. 2012;Maček et al. 2016b; Grossart et al.
2019;Maček 2019). Fungi play a crucial role in carbon mineralisation and cycling in
11 Fungi in Hypoxic Soils and Aquatic Sediments 223

terrestrial environments, which are the habitat of the majority of known fungal taxa.
Soil organisms, including fungi, are subject to occasional hypoxia, although the
hypoxic response also begins in fungi (similar to multicellular eukaryotes) at oxygen
levels of about 6% (Simon and Keith 2008), but they have evolved to tolerate low or
rapidly changing oxygen levels (e.g. Maček et al. 2011). Yeasts are an exception in
the fungal kingdom, as many are known to have the ability to ferment (e.g. Šibanc
et al. 2018).
When hypoxia occurs in soils or aquatic sediments, respiration of roots and
rhizosphere and root-associated endophytic fungi is under stress. Especially in
severe hypoxia, root respiration is also impaired (Maček et al. 2005). Root respira-
tion decreases significantly only when plant roots are exposed to CO
2
concentrations
or water saturation in soil or sediment high enough to limit O
2
availability (above
50% CO
2
causes a decrease in O
2
saturation in liquids below 10%, Maček et al.
2005,2016b). Active nutrient uptake is an energetically costly process that requires
metabolic activity in all aerobic organisms (e.g. plants and fungi). Soil hypoxia can
inhibit ion uptake by roots or fungal hyphae due to limited aerobic metabolism and
higher dependence on less efficient anaerobic metabolism. In non-adapted plants,
fermentation occurs in root cells due to the lack of O
2
, leading to the production of
lactic acid and ethanol.
Plants that have adaptive traits such as the ability to form aerenchyma or other
systems for O
2
transfer to the roots and rhizosphere are more likely to survive in
these environments and are also more likely to support and form ecological
interactions with other aerobic organisms in the rhizosphere (Fig. 11.5). Aeren-
chyma are typical adaptive traits of plant species in wetlands, but can also form in
response to flooding or mineral deficiency (Videmšek et al. 2006; Vodnik et al.
2009; Marschner 2012). In plants, the formation of aerenchyma can enable a
relatively high O
2
content in the rhizosphere and maintain aerobic respiration of
roots even in the context of very high soil CO
2
concentrations or flooded soils. In
addition, plant-mediated changes in the rhizosphere (e.g. O
2
axial transport and
escape from roots) can affect other organisms that live in hypoxic systems (e.g. soil
and endophytic microbes and fauna) (Maček et al. 2016b, Fig. 11.5).
In some cases, hypoxic environments may be present locally, often aggregated
within a small geographic area, with relatively little variance between sites in
geophysical parameters. One such case of extreme environments with permanent
soil hypoxia is mofettes or natural sources of CO
2
, which may be terrestrial or
aquatic, with geologic CO
2
outgassing locally and inducing soil, sediment, and water
hypoxia. Apart from natural CO
2
springs (mofettes), hypoxia affects also a number
of other ecosystems, e.g. flooded, submerged or compacted soils and sediments
(Perata et al. 2011;Maček 2017a), which are specifically presented later in this book
chapter, together with their fungal inhabitants.
224 I. Maček

11.3.1 Fungi in Mofettes
Terrestrial and aquatic mofettes (Fig. 11.1) are extreme ecosystems that occur in
tectonically or volcanically active areas globally (Pfanz et al. 2004;Maček et al.
2016b). Mofettes are sites characterised by diffuse degassing of CO
2
of deep mantle
origin (above 99%) at ambient temperature and traces of other gases, including
methane (CH
4
), nitrogen (N
2
), hydrogen sulphide (H
2
S), or noble gases. Geogenic
CO
2
displaces O
2
from soil pores, so soil hypoxia or even anoxia is a common soil
feature at mofettes. Being heavier than air, mofette CO
2
can accumulate in local
landscape depressions, which, if large enough, can even form lakes of gases with
concentrations ranging from 5% to almost 100% (Kies et al. 2015;Maček et al.
2016b, Fig. 11.1). The main characteristics of practically all natural CO
2
spring sites
are high CO
2
concentrations and soil hypoxia (low O
2
concentration), while above-
ground CO
2
concentrations are more variable and lower. Aquatic sites with mofettes
usually exhibit subsurface CO
2
release, resulting in continuous effervescence of
water (e.g. Šibanc et al. 2018; Fazi et al. 2019, Fig. 11.1). The environment of
mofettes can therefore have both extremely high CO
2
levels and extremely low O
2
concentrations compared to the wider environment, in addition to low soil and water
pH due to dissolved CO
2
and the formation of carbonic acid, which dramatically
affects and limits the local ecology of these sites (e.g. Maček et al. 2005;Šibanc et al.
2014;Maček et al. 2011,2016b; Beulig et al. 2016;Šibanc et al. 2018).
Mofettes can be found in several places, in Europe in Czechia (Cheb Basin),
Germany (Eifel, Rhoen, Teutoburger Forest, NW Franconia), France (Massif Cen-
tral), Hungary, Iceland (regions with volcanoes), Italy (Tuscany), Romania
(Hargitha Mountains) and Slovenia (Radenci, Stavešinci area) (Maček et al.
2016b). Globally, they are found, for example, within the Yellowstone volcano
caldera or in the Inyo crater range, in the Cascades range (United States), in
geothermal fields of New Zealand (Bloomberg et al. 2012), Indonesia (Djeng
Plateau), Japan and Kamchatka.
Mofettes have been used to study communities of soil microbes, with the majority
of existing studies focusing on bacteria and archaea, but much less work published
on soil fungi (Maček et al. 2011,2016b;Šibanc et al. 2018). Soil gas composition in
natural CO
2
springs, e.g. soil hypoxia, has a significant impact on communities of
eukaryotic organisms that are predominantly obligate aerobes, such as soil fauna
(e.g. Hohberg et al. 2015), plants (e.g. Maček et al. 2016b), and fungi
(e.g. communities of arbuscular mycorrhizal fungi presented in Maček et al. 2011,
2016b; soil and aquatic yeasts as presented in Šibanc et al. 2018). Soil O
2
concen-
tration was also the strongest abiotic predictor of archaeal and bacterial soil commu-
nity composition in mofettes in Slovenia, while other soil factors were secondary,
including concentrations of CO
2
, pH, and availability of plant nutrients (Šibanc et al.
2014). Although most of the existing studies on natural CO
2
springs investigating
community composition of different organisms represent single (snapshot) studies or
at best a few time-points, this suggests a general and relatively stable pattern in the
evolution of archaeal and bacterial communities in mofette soils. Moreover, this
appears to extend to different groups of organisms, like fungi (Maček et al. 2011,
11 Fungi in Hypoxic Soils and Aquatic Sediments 225

2016b), with the recently described new species of yeast from soil, Occultifur
mephitis sp. nov. (Šibanc et al. 2018) (see Sect. 3.1.1, Fig. 11.3), and invertebrates
(Collembolae) (Schulz and Potapov 2010).
In contrast to more available published data on soil prokaryotes (e.g. Krüger et al.
2009; Oppermann et al. 2010; Krüger et al. 2011; Frerichs et al. 2013;Šibanc et al.
2014;Fazietal.2019), the diversity of fungi on mofettes is still largely unexplored
(Maček et al. 2016b;Maček 2019), and some publications have reported that the
majority of soil fungi are potentially excluded from mofette soil food webs due to
their sensitivity to soil hypoxia (Beulig et al. 2016). Indeed, most fungi are consid-
ered aerobes, although their habitats can often be hypoxic (low in O
2
) or even anoxic
(no O
2
), e.g. due to infiltration of soil with water or metabolic activity during
infection of other organisms, in the biosphere (Simon and Keith 2008; Drake et al.
2017). However, existing studies show that diverse fungal communities also inhabit
hypoxic mofette areas (Maček et al. 2011,2016b;Šibanc et al. 2018), with fungal
gene copy numbers decreasing only slightly at high CO
2
fluxes and sharply only at
the most extreme sites, as reported for Spanish mofettes (Fernández-Montiel et al.
2016). There is limited published work on diversity of fungi in mofettes (e.g. Maček
et al. 2011;Šibanc et al. 2018), and further research is urgently needed to understand
the complex processes and ecological interactions of the soil biota in this extreme
ecosystem, which include the fungal component.
In the study of soil fungi, there has been a particular focus on the ancient,
ubiquitous symbiotic interaction between arbuscular mycorrhizal fungi and plants
exposed to hypoxia in soil, while other filamentous groups of fungi from sites of
natural CO
2
springs have not yet been investigated. There is also a single report on
the diversity of yeasts in aquatic and terrestrial mofettes in Slovenia (Šibanc et al.
2018). Nevertheless, all existing studies confirm that natural CO
2
springs can be a
Fig. 11.3 Occultifur mephitis f.a., sp. nov. colonies in a Petri dish (9 cm diameter–left) and mofette
soil with reductive processes (right). Occultifur mephitis is a newly described yeast species isolated
from the Slovenian mofette soil in Stavešinci (NE Slovenia). The yeast was named after the Roman
Mephitis (me.phi¢tis. L. fem. gen. n. mephitis), a goddess of gasses emitting from the soil. The
photo of the yeast was taken from EXF-6436 (holotype) by N. Šibanc, the soil photo by I. Maček;
see Šibanc et al. (2018) for details
226 I. Maček

rich source of information on how organisms, populations and communities cope
with long-term environmental stresses in their environment. There is extensive
evidence that organisms in mofette fields are exposed to intense abiotic selection
pressures, such as soil hypoxia (Maček et al. 2011,2016b;Šibanc et al. 2014,2018).
11.3.1.1 Yeasts
Recently, a report on yeast diversity from sites of natural CO
2
springs in northeastern
Slovenia was published (Šibanc et al. 2018). Yeasts are known to be a group of fungi
that are widely distributed and colonise both terrestrial as well as aquatic systems. In
particular, yeasts are essential for ecosystem functioning in soils, as they are
involved in the mineralisation of organic matter and assimilation of plant
carbohydrates, as well as cycling of nutrients (Botha 2006,2011).
The inventory of cultivable yeasts from soils (terrestrial) and water bodies
(aquatic mofettes) (Šibanc et al. 2018, Fig. 11.1) includes a total of 142 isolated
and identified strains from highly geologically CO
2
-exposed soils and groundwater
in a meadow, a forest pond and stream water. They were assigned to six yeast genera
of the Basisiomicetes (6 species) and 11 genera of the Ascomycetes (18 species)
(Šibanc et al. 2018). Using high dilution plating of a soil sample, 4 strains of an
unknown basidiomycete species were isolated and described for the first time as
Occultifur mephitis f.a., sp. nov. (Fig. 11.3) (based on phylogeny and phenotype
criteria) (Šibanc et al. 2018). Occultifur mephitis did not show fermentative
capabilities and was unable to grow under 100% CO
2
in anaerobic chambers
(Šibanc et al. 2018). Nevertheless, it grew in an anaerobic vessel with a hypoxic
atmosphere of 100% N
2
(Šibanc et al. 2018). This suggests that hypoxic conditions
in soils of natural CO
2
springs likely include microenvironments with minimal O
2
supply, which are required for the survival of this species and likely others.
In Slovenia, the highest mofette yeast species richness (15 sp.) of ascomycetes
was found in forest mofette water (pond with visibly bubbling CO
2
) (Fig. 11.1),
which contained yeast species found only in forest water: Candida boleticola,
Debaryomyces hansenii, Kazachstania exigua, Kluyveromyces dobzhanskii, a repre-
sentative of the Metschnikowia pulcherrima species complex (Lachance 2016),
Metschnikowia pulcherrima, Pichia kudriavzevii, Suhomyces species and
Torulaspora delbrueckii (Šibanc et al. 2018). Isolates identified as Metschnikowia
pulcherrima species complex may represent another undescribed species as they
differ in 11/462 nucleotide positions of D1/D2 (98% identity) from the sequence of
the type strain of Metschnikowia fructicola. Of the taxa isolated, all ascomycetous
yeasts, with the exception of Debaryomyces hansenii, were able to grow and ferment
glucose under elevated CO
2
.Candida sophiae-reginae, Pichia fermentans and
Candida vartiovaarae were the dominant species in meadow and forest water with
elevated CO
2
exposure. Meyerozyma guilliermondii and Wickerhamomyces
anomalus dominated in highly CO
2
-exposed soils (Šibanc et al. 2018, Fig. 11.3).
The frequent occurrence of Meyerozyma guilliermondii and Wickerhamomyces
anomalus and their in vitro ability to grow in high CO
2
and N
2
atmospheres, as
well as their fermentative capacity, suggest that they might be well adapted to
ecological niches characterised by elevated CO
2
and consequently decreased O
2
.
11 Fungi in Hypoxic Soils and Aquatic Sediments 227

The same could be true for the majority of other yeast species from the ascomycete
group isolated from mofettes (Šibanc et al. 2018). Thereby, the most abundant yeasts
found in the natural CO
2
spring soils of the meadow are also described in the
literature as fermentative taxa (Kurtzman et al. 2011). Among the isolated yeast
species, all ascomycetous taxa except Debaryomyces hansenii, which were able to
ferment glucose, were also able to grow under elevated CO
2
(incubation under 100%
CO
2
- initially). Strains that were tested and representing these species were also able
to grow under 100% N
2
atmosphere (Šibanc et al. 2018). The low pH found in
mofette environments with high CO
2
may also favour yeasts known to survive in
environments with at least moderately reduced pH. This proves that mofette habitats
provide new insights into microbial responses and adaptations to long-term changes
in the abiotic soil environment and are a valuable source for the discovery of new
taxa (Fig. 11.3, see also Sect. 4).
11.3.1.2 Arbuscular Mycorrhizal Fungi
A second group of fungi that has been studied intensively in mofette areas are
arbuscular mycorrhizal fungi (Fig. 11.4). They were the first fungal group whose
diversity and community were characterised from a CO
2
springs area, the Stavešinci
mofette (Slovenia) (Maček et al. 2011). Arbuscular mycorrhizal fungi represent a
ubiquitous soil group with high functional importance and form diverse
communities even in hypoxic environments such as mofettes (Maček et al. 2011,
2016b). The widely distributed arbuscular mycorrhizal fungi are obligate biotrophic
plant root endosymbionts. They are present in all terrestrial ecosystems and are
estimated to colonise the roots of about two-thirds of plant species (Fitter and
Moyersoen 1996; Brundrett and Tedersoo 2018). In addition to natural
environments, they are also the most abundant fungal group in crops, which may
be important for promoting sustainable agricultural practises (Helgason et al.
1998; Smith and Read 2008; Säle et al. 2015; Johnson et al. 2017). For their host
plants, they are the main conduit for phosphorus uptake, and they can affect them in
several other ways, including pathogen defence, improving water relations and soil
structure, and micronutrient and nitrogen uptake (Smith and Read 2008; Johnson
et al. 2017).
Roots of plants of several species growing in the most extreme sites in the
Stavešinci mofette area (NE Slovenia) have consistently shown high colonisation
by arbuscular mycorrhizal fungi, despite the persistent stress and carbon cost of
colonisation (Maček et al. 2011,2012;Maček 2013). It is not yet clear how these
fungi cope, but soil mycelial growth is likely to be severely restricted in highly
hypoxic soils, and it is unknown whether plants benefit from mycorrhiza in this
environment (Maček et al. 2011;Maček 2017a). However, mofette fungal species
are probably not subsidised by mycelium in the surrounding soil, which could
explain how these fungal aerobes survive. Therefore, they must be adapted to
hypoxic conditions or at least competitive, presumably either tolerating low O
2
or
acquiring sufficient O
2
from roots; both explanations have profound implications for
their biology (Maček et al. 2011).
228 I. Maček

Acquisition of sufficient O
2
from roots (Maček et al. 2011) is an unexplored
concept of facilitation in the ecology of arbuscular mycorrhizal fungi (Maček et al.
2016b;Maček 2017a) that is relevant not only to mofette fungi (Maček et al. 2011)
but also to microbes colonising plants in aquatic environments (see Sections 3.2 and
3.3, Fig. 11.5). Starting from physically extreme environments, plant-fungal
interactions could therefore be extended beyond (trophic) nutrient interactions to
include the additional benefit of a positive effect of one species on another, by
reducing stresses in existing habitats and creating new habitats for arbuscular
mycorrhizal fungi (Maček et al. 2016b;Maček 2017a).
With respect to the ecology of arbuscular mycorrhizal fungal communities,
several studies suggest that under extreme environmental stress in soil, there are a
small number of arbuscular mycorrhizal fungal lineages that are better able to
tolerate these conditions, resulting in unique, adapted populations (Helgason and
Fig. 11.4 Arbuscular mycorrhizal fungi colonising the roots of maize (Zea mays L.), with visible
arbuscules (tree-like structures) important for nutrient exchange between the plant host and the
fungi. Photo: I. Maček
11 Fungi in Hypoxic Soils and Aquatic Sediments 229

Fitter 2009; Dumbrell et al. 2010;2011;Maček et al. 2011). Arbuscular mycorrhizal
fungi form an extensive hyphal network in the soil and will therefore be subject to
strong selection pressure from soil abiotic factors (e.g. Dumbrell et al. 2010;
2011;Maček et al. 2011). However, reports on community analyses and diversity
studies of arbuscular mycorrhizal fungi in extreme ecosystems based on molecular
studies remain scarce (e.g. Appoloni et al. 2008;Maček et al. 2011,2016a,b;Maček
2017b). Maček et al. (2011) reported significant levels of arbuscular mycorrhizal
Fig. 11.5 Water lily (Nymphaea sp.) and common reed (Phragmites australis) growing in a pond
in Ljubljana, Slovenia. Some plants can grow in open water with only the floating leaves and
flowers exposed to air, while rhizomes and roots with potential endophytic fungi are anchored deep
in rocky sand or mud and remain constantly submerged. Plant tolerance to soil and sediment
hypoxia may be directly related to aeration efficiency, which is achieved by the development of air
spaces in the tissue (Justin and Armstrong 1987; Evans 2003). Water lilies, for example, grow
rooted in water and mud and have a very well-developed aerenchyma of the same general type as in
some Rumex species, termed “honeycomb”by Justin and Armstrong (1987) and classified as
schizogenous (aerenchyma formed when intercellular gas spaces form during tissue development
without cell death). Mature aerenchyma contains broad lacunae interrupted by diaphragms with
small air spaces. Some of the diaphragms appear to be pierced by astrosclereids which presumably
support longitudinal airflow (see Seago Jr et al. (2000) for more details on aerenchyma formation in
wetland plants). On the other hand, in common reed (Phragmites australis), which can be seen in
the background of the photo, the rate of gas flow from the atmosphere may be increased by wind-
driven Venturi convection, which draws air into the subterranean system via dead culms cut above
ground level (Videmšek et al. 2006). Photo: I. Maček
230 I. Maček

fungal community turnover among soil types and numerical dominance of certain
arbuscular mycorrhizal fungal species in hypoxic mofette soils. This work shows
that direct environmental selection acting on arbuscular mycorrhizal fungi is a
significant factor in regulating fungal communities and their phylogeographic
patterns. Consequently, some arbuscular mycorrhizal fungi are more strongly
associated with local variation in the soil environment than with the distribution of
their host plants (Dumbrell et al. 2010;Maček et al. 2011). The higher temporal
predictability (stability) also emerges from preliminary results on arbuscular mycor-
rhizal fungal communities, which suggest that under permanent (long-term) selec-
tion pressure, community composition is more constant compared to control sites
(Maček et al. 2011,2016b). The case of arbuscular mycorrhizal fungal community
composition demonstrates the potential of mofettes to serve as model ecosystems to
study some of the important unresolved questions in microbial community ecology
(Maček et al. 2011,2016b; Fig. 11.6). Importantly, the observed community stability
at extreme sites may be more ubiquitous than currently recognised in many other
environments with long-term disturbances or specific selection pressures (Maček
et al. 2016b;Maček 2017b). The strong environmental gradients of mofette sites,
which include extreme and lethal conditions, thus make them ideal models for
further investigation of the rules of community establishment, temporal dynamics
(e.g. interannual variability), facilitation (plant O
2
supply), and symbiosis in
arbuscular mycorrhizal fungi, and provide insight into different pathways of plant
mineral assimilation and the role of the fungal partner in this process in hypoxic
environments (Maček et al. 2016b).
SOIL MICROBIAL
ECOLOGY &
EVOLUTION
TAXONO MY &
PHYLOGENY
(NEW TAXA)
PLANT
ENDOPHYTES
AND MYCORRHIZA
MOFETTE
RESEARCH
Fig. 11.6 Mofettes and
research fields of applied
microbial ecology for
bioprospecting potential
extremophilic taxa
11 Fungi in Hypoxic Soils and Aquatic Sediments 231

11.3.1.3 Other Filamentous Fungi
Apart from arbuscular mycorrhizal fungi and yeasts from the Slovenian mofette site
in Stavešinci, there is only one published study reporting fungal abundance from
mofette sites (Fernández-Montiel et al. 2016). Microbial communities were studied
in a series of CO
2
fluxes from a natural volcanic vent in Campo de Calatrava (Spain).
To assess changes in the diversity, abundance and functionality of the main groups
of the soil microbiota (fungi, archaea and bacteria), the researchers used a number of
different techniques, including quantitative polymerase chain reaction (qPCR),
denaturing gradient gel electrophoresis (DGGE) and Biolog EcoPlatesTM. A gen-
eral decrease for all variables studied was observed from control areas to high CO
2
areas. In contrast, at extreme CO
2
fluxes, bacterial and archaeal communities
increased in abundance and activity but remained less diverse. The fungal commu-
nity, on the other hand, showed a decrease in the number of fungal gene copies when
CO
2
fluxes increased. In the case of fungi, extreme CO
2
fluxes decreased fungal gene
copy number, with a significant decrease of three orders of magnitude in gene copy
number in the sampling site with high CO
2
flux (Fernández-Montiel et al. 2016). No
detailed data on general fungal community composition and taxa identity are
available for this site.
11.3.2 Fungi in Aquatic Sediments—Submerged Environments
Sediment is an environmental substance consisting of any particulate material that
can be transported by fluid flow and eventually deposited as a layer of solid particles
on the bottom of a body of water or other fluid. Based on recently developed high-
throughput molecular methods, datasets of fungal sequences come from both marine
and freshwater samples (Panzer et al. 2015), as well as those associated with aquatic
vegetation (e.g. rhizosphere of aquatic macrophytes, isoetid vegetation, mangroves)
that may also represent hypoxic environments.
Molecular tools have helped to increase our understanding of fungal diversity in
different marine and freshwater habitats (Pawlowski et al. 2021). They have shown
that aquatic fungi are highly diverse and that they play fundamental ecological roles
in aquatic systems (Panzer et al. 2015). More recently, aquatic fungal communities,
including those in sediment (Orsi et al. 2013), have been shown to contribute to
element cycling and mineralisation processes (Raghukumar 2012). Fungi can
account for a large proportion of sediment DNA in aquatic ecosystems, but reports
on their diversity in aquatic sediments, both marine and freshwater ecosystems, are
still relatively scarce (e.g. Manohar and Raghukumar 2013; Panzer et al. 2015).
Environmental studies using molecular tools have revealed the presence of fungi
from a wide range of marine habitats (Manohar and Raghukumar 2013; Panzer et al.
2015), such as deep-sea environments (Bass et al. 2007), hydrothermal vent
ecosystems (Le Calvez et al. 2009), coastal regions (Gao et al. 2010), anoxic regions
(Jebaraj et al. 2010), lakes (Monchy et al. 2011; Lefevre et al. 2012), rivers (Duarte
et al. 2015), or in association with aquatic animals (Chukanhom and Hatai 2004;
Amend et al. 2012), plants (Magnes and Hafellner 1991; Sakayaroj et al. 2010; Baar
232 I. Maček

et al. 2011; Sudováet al. 2015, Fig. 11.5) or algae (Zuccaro et al. 2003). However, it
is not clear how many of the identified groups are true marine or aquatic fungi, as
most of the clusters contain representatives from terrestrial regions (Manohar and
Raghukumar 2013), which could be accidentally transferred to aquatic environments
due to ecosystem connectivity.
With the intention of serving as a reference dataset for aquatic fungi, Panzer et al.
(2015) used all publicly available fungal 18S ribosomal RNA (rRNA) gene
sequences with the addition of new sequence data from a marine fungal culture
collection and further enrichment of the dataset by adding validated contextual data
(e.g. habitat type of samples assigning fungal taxa to ten different habitat categories).
The combined data allowed the authors of the paper to infer fungal community
patterns in aquatic systems (Panzer et al. 2015). Pairwise habitat comparisons
revealed significant phylogenetic differences, suggesting that habitat strongly
influences fungal community, with freshwater fungal community structure differing
most from all other habitat types and dominated by basal fungal lineages (Panzer
et al. 2015). For most communities, phylogenetic signals indicated clustering of
sequences, suggesting that environmental factors are the main drivers of fungal
community structure rather than species competition (Panzer et al. 2015). This
includes potential hypoxia, which is an important abiotic driver of fungal
communities in sediments. Several groups of Ascomycota, Basidiomycota,
Chytridiomycota and other basal fungal lineages were reported in the sediments
(Panzer et al. 2015). None of these formed a dominant group at higher taxonomic
ranks. Fungi must adapt to conditions inherent to the sediment (Orsi et al. 2013),
such as a strong oxygen gradient (potential hypoxia), a particular uptake capacity for
nutrients and organic matter, or selective entrainment transport (Hedges et al. 1999;
Avramidis et al. 2013). Therefore, fungal assemblages of sediments have been
reported to differ significantly from assemblages of almost all other habitat
categories (Panzer et al. 2015).
For the fungal groups associated with aquatic vegetation, the majority of studies
on sediment fungi involve symbiotic arbuscular mycorrhizal fungi, but there are few
reports on arbuscular mycorrhizal fungi in submerged environments associated with
permanent inundation of plant root and aerial systems in continental areas (e.g. Baar
et al. 2011; Kohout et al. 2012; Sudováet al. 2015) and marine environments—
mangrove forests (Wang et al. 2011; Wang et al. 2015; Wang et al. 2016). Most
studies on arbuscular mycorrhizal fungal diversity in submerged ecosystems (in the
context of permanent inundation of plant root and aerial systems) come from aquatic
macrophyte vegetation from the oligotrophic and ultraoligotrophic lakes of northern
Europe (e.g. Baar et al. 2011; Sudováet al. 2015). Only now, and strengthened by
newly developed molecular tools, have researchers investigated in more detail the
composition of the arbuscular mycorrhizal fungal community in these specific
ecosystems. Diverse arbuscular mycorrhizal fungal communities were found in the
roots of the aquatic macrophyte Littorella uniflora, with several arbuscular mycor-
rhizal fungal taxa present, including taxa from the genera Acaulospora,
Archaeospora and Glomus (Baar et al. 2011; Kohout et al. 2012). In addition, a
new arbuscular mycorrhizal fungal species, Rhizoglomus melanum, was described
11 Fungi in Hypoxic Soils and Aquatic Sediments 233

and isolated from the rhizosphere of aquatic macrophytes from the freshwater lake
Avsjøen in Norway (Sudováet al. 2015). These plants have characteristic aeration
systems (see also Fig. 11.5) that allow rapid O
2
diffusion from shoots to roots and are
known to have high radial O
2
losses from their roots into the sediment (Smolders
et al. 2002). Increasing plant nutrient uptake through arbuscular mycorrhizal
symbioses seems to be particularly relevant for some submerged plants when
nutrient availability in their environment is poor, e.g. for submerged vegetation of
oligotrophic lakes in Norway (Møller et al. 2013). In the latter study, extensive extra-
radical hyphal networks were found in the sediments of submerged isoetid plants
(Lobelia dortmanna and Littorella uniflora) with a high mean hyphal density (6 and
15 m cm
3
for each plant species, respectively). This is comparable to the density
typically found in terrestrial soils (Møller et al. 2013). The hyphal surface area
exceeded the root surface area by 1.7–3.2 times with the highest density in the main
root zone (Møller et al. 2013).
Arbuscular mycorrhizal fungi appear to be dependent on the high O
2
concentrations in the roots and surrounding root zones of aquatic plants (Wigand
et al. 1998, Fig. 11.5), and this appears to be a consistent and important component
of arbuscular mycorrhizal fungal habitats in hypoxic soils. Interestingly, in the
context of an extreme environment, this means that some plant species modify
conditions sufficiently to make life more hospitable to others that would otherwise
be unable to survive in that environment. The concept is known in the plant literature
as facilitation and is used to refer to beneficial (non-trophic) interactions that occur
between physiologically independent plants and are mediated by changes in the
abiotic environment (Brooker and Callaway 2009). The cross-trophic and cross-
system interactions in this concept of facilitation are still challenging a current
working definition of facilitation that is limited to plant-plant interactions, mostly
in terrestrial environments (Brooker and Callaway 2009;Maček et al. 2016b).
Arbuscular mycorrhizal fungi are also common in mangrove forests (Radhika and
Rodrigues 2007;D’Souza and Rodrigues 2013). Molecular analyses of arbuscular
mycorrhizal fungal communities in the roots of mangrove trees were not conducted
in a study of 16 mangrove species on riverine and fringing habitats in Goa, West
India, (D’Souza and Rodrigues 2013). However, in a second study conducted in four
semi-mangrove plant communities from the Qi’Ao Mangrove Forest Reserve in
southern China, arbuscular mycorrhizal fungal taxa were determined by molecular
approaches (e.g. Wang et al. 2011; Wang et al. 2015). The authors report six
operational taxonomic units (OTUs) from the family Glomeraceae that could not
be identified to the genus level and may represent new taxa, which is a common
feature in hypoxic environments (see Sudováet al. 2015). A second group of fungi
associated with marine aquatic vegetation belongs to the dark septate endophytes
(DSE), culturable root mycobionts of the seagrass Posidonia oceanica.In
Posidonia, roots have been reported to be colonised with new, undescribed taxa of
DSE (Vohník et al. 2016). Therefore, the observations from seagrass, mangrove
forests together with the new arbuscular mycorrhizal fungal species described from
oligotrophic lakes suggest that submerged environments are still a rich potential
source of new fungal taxa yet to be discovered.
234 I. Maček

11.3.3 Flooded Soils
As predicted by climate change models, the frequency and severity of flood events
will increase dramatically in the future, with the greatest increase predicted by
climate change models for the tropics and Western Europe (Hirabayashi et al.
2013). Therefore, to promote sustainable agriculture in the future, understanding
the response of different organisms to soil hypoxia, including the interaction of crops
with soil organisms, including symbiotic arbuscular mycorrhizal fungi, is becoming
increasingly important (Maček 2017a). Despite the fact that temporarily flooded
soils are not considered an extreme ecosystem but rather an environmental stress
event, studies on arbuscular mycorrhizal fungal communities conducted in flooded
soils show similar results as for submerged environments (see Sect. 3.2). For
example, flooded rice fields and rice plants in general are one such system where
considerable progress has been made in the interaction between plants and
arbuscular mycorrhizal fungi and their joint response to flooding (e.g. Vallino
et al. 2014). The latter includes adaptive traits such as the ability to form aerenchyma
or other systems for O
2
transfer to roots. Aerenchyma are typical adaptive traits of
wetland plant species (Fig. 11.5), but can also develop in response to flooding or
mineral deficiency (Marschner 2012). The results of the Vallino et al. (2014) study
on rice show that under flooding conditions arbuscular mycorrhizal fungal nutrient
transporters are regularly expressed, but the functional markers of arbuscular mycor-
rhizal symbiosis show a significant decrease in the expression of plant and fungal
nutrient transporters as flooding progresses (Vallino et al. 2014).
11.3.4 Compacted Soils
Apart from flooded and inundated substrates, hypoxic or even anoxic environments
can also be found in other habitats, such as microenvironments in soil aggregates,
subterranean borrows, and compacted soils. Here, hypoxia may be permanent or,
more commonly, intermittent (Hourdez 2012). Due to the use of heavy field equip-
ment and field traffic, soil compaction is common in agricultural fields, and soils are
particularly susceptible to soil compaction in wet conditions. In addition to directly
affecting soil structure, soil compaction can also affect roots (growth and activity)
and soil microbial functions. Soil porosity is an important factor affecting soil
aeration. Soil compaction reduces gas exchange in the soil through changes in
pore space size and distribution and soil strength. When soil particles are com-
pressed, the pore space between them is reduced, therefore compacted soils lack
large pores (DeJong-Hughes et al. 2001), and because the pore space in the soil is
reduced, bulk density increases. This can lead to local hypoxia or anoxia in the soil
and affect soil life, including soil fungi.
11 Fungi in Hypoxic Soils and Aquatic Sediments 235

11.4 Frontiers in Research of Fungi in Hypoxic Environments
In particular, long-term hypoxic soils (e.g. mofette soils) and submerged (aquatic)
environments (e.g. hypoxic sediments) show much potential for further research on
fungal biology in hypoxic environments in various fields, from soil ecology and
biodiversity research to bioprospecting for new extremophile taxa (Fig. 11.6). The
latter may have great potential for biotechnological applications, the study of
hypoxia-tolerant human pathogens, and others. In the following, recent and future
advances in the study of hypoxic soil and water environments and potential
applications are described, with a view to further developments in this field in the
future.
11.4.1 Bioprospecting for New Taxa
Extreme environments can serve as novel study systems to investigate how long-
term abiotic selection pressures drive natural communities and their evolution,
potentially leading to new specialised taxa (e.g. new yeast species Occultifur
mephitis isolated from Slovenian mofettes, Šibanc et al. 2018). Different fungal
groups have been studied at different depths in hypoxic environments. For example,
reports on any aspect of the important and ubiquitous symbiotic arbuscular mycor-
rhizal fungal biology from extreme habitats or hypoxic environments are relatively
scarce (Maček et al. 2011;Maček 2017a,b; Drake et al. 2017), whereas other fungal
groups (e.g. yeasts) have been studied more extensively in extreme environments
(see e.g. Cantrell et al. 2011; Rangel et al. 2018). Isolation of hypoxia- or stress-
tolerant microbes and microbial communities can have great potential in biotechnol-
ogy (e.g. new drug discovery). Since many biotechnological applications, such as
industrial fermentation, require the ability to grow in high CO
2
, low O
2
environments, mofettes (natural CO
2
sources), for example, along with other hyp-
oxic soils and substrates, are likely ideal sites for bioprospecting for industrially
important microbes (Figs. 11.1,11.2,11.3, and 11.6). To date, apart from initial
studies on arbuscular mycorrhizal fungi and yeasts (Maček et al. 2011,2016b;
Šibanc et al. 2018), there are no reports on the diversity, ecology or function of
fungi from mofette sites, while the biotechnological and medicinal potential of
mofette sites and their biota remains largely unknown and unexploited, and similar
is the case for submerged soils and aquatic sedimentary fungi.
In relatively recent reports, some endophytic fungi have also been isolated and
identified from hydrophites. For example, more than 200 isolates of endophytic
fungi of Nymphaea spp. (Fig. 11.5) have been reported growing at various sites in
India and Thailand (e.g. Rajagopal et al. 2018; Supaphon et al. 2018), showing some
antimicrobial activities, but there are no reports on the rhizosphere and root
endophytes of water lilies.
236 I. Maček

11.4.2 Exploration of Human Fungal Pathogens in Hypoxic
Environments
Since most eukaryotic human fungal pathogens are generally considered obligate
aerobes, O
2
availability during fungal pathogenesis may play a critical role in the
outcome of infection from the perspective of both the human host and the fungus
(Grahl et al. 2012). Among the most resistant human fungal pathogens are hypoxia-
and anoxia-tolerant microbes. In healthy tissues in the human body, O
2
levels of
2.5% to 9% are considered normal, whereas oxygen levels of 1%, as described in
wounds and tumours, are considered hypoxic (Arnold et al. 1987; Simmen et al.
1994; Dewhirst 1998; Nizet and Johnson 2009). In the context of microbial patho-
genesis, hypoxia is generally thought to occur at sites of infection and represents
significant environmental stress for most host and microbial pathogen cells
(e.g. Cramer et al. 2003; Peyssonaux and Johnson 2004; Nizet and Johnson 2009).
The most common fungal pathogens that may be exposed to an oxygen-limited or
even hypoxic microenvironment during fungal pathogenesis include Candida
albicans, which is commonly found in the gastrointestinal tract, which contains
significant regions of hypoxia (He et al. 1999; Karhausen et al. 2004), and Crypto-
coccus neoformans, which causes cryptococcal meningitis and is also exposed to
reduced O
2
levels during infection in the human brain (Erińska and Silver 2001;
Sharp and Bernaudin 2004). Human disease in immunodeficient individuals can be
caused by various moulds typically found in soil and decaying organic material, such
as Aspergillus fumigatus and Fusarium oxysporum, when O
2
levels in the tissues of
their hosts are very low (Grahl et al. 2012). Although most moulds are traditionally
considered obligate aerobes, Aspergillus fumigatus has been observed to tolerate O
2
levels as low as 0.1%, and some studies even suggest that Aspergillus fumigatus can
survive and grow anaerobically (Tabak and Cooke 1968; Hall and Denning 1994).
Moreover, Fusarium species appear to be particularly adept at tolerating hypoxic
and even anoxic conditions, consistent with their resident ecological niche in the soil
(Gunner and Alexander 1964; Hollis 1948). Therefore, soil environments where O
2
levels are low (e.g. submerged, flooded, compacted soils and mofettes) or can
change rapidly with microbial metabolic activity (e.g. in compost piles) may be a
source of human pathogenic fungi adapted to hypoxia (Grahl et al. 2012). Therefore,
research on hypoxic environments can identify and further investigate potential risks
for hypoxic habitats in nature that serve as reservoirs for pathogens.
11.4.3 Natural Long-Term Experiments in Ecology
Many natural phenomena and ecological processes occur very slowly, so that long-
term observations and experiments are necessary to study them (Franklin 1989).
However, the latter are still largely lacking for many microbial groups. Soil microbes
are a key driver of many vital biogeochemical cycles, with soil being one of the most
biodiverse habitats on the planet (Fitter 2005). Changes in the population density of
11 Fungi in Hypoxic Soils and Aquatic Sediments 237

soil microbes in response to long-term environmental factors have the potential to
influence plant community productivity and human health.
A natural study site with the potential to provide better predictions of ecosystem
impacts due to induced long-term environmental change is mofettes, which can
serve as natural long-term experiments in evolution and ecology (Maček et al.
2016b). Supported by advances in sequencing technologies in recent years
(e.g. Dumbrell et al. 2016), more and more studies are also focusing on the
subsurface. Not only the composition of soil communities, but also the various
interactions (networks) that occur between taxa help monitor the response of taxa
interactions to human changes in the environment, which is critical for ecosystem
conservation (Vacher et al. 2016). Ecological networks are now becoming a standard
method for representing and simultaneously analysing interactions among taxa
(e.g. Coyte et al. 2015; Vacher et al. 2016; Tylianakis and Morris 2017). However,
ecological networks in soils remain largely unknown. For example, the reduction of
the soil community to the microbial component that tolerates permanent soil hypoxia
induced by geogenic CO
2
makes mofettes a valuable model environment for study-
ing diversity effects on specific soil functions. Indeed, mofettes are characterised by
permanent exclusion of higher trophic levels and associated physical and ecological
features from local food webs, as most eukaryotes require an aerobic environment
(Maček et al. 2016b). Because a significant portion of the specific ecology of these
systems is microbial, mofettes provide an ideal opportunity to explore network-
based approaches to incorporate next generation sequencing-based data into the
ecology of food webs in hypoxic environments (e.g. Maček et al. 2016b; Vacher
et al. 2016).
Importantly, questions about long-term changes in soil microbial communities
are relevant not only to the study of hypoxia as a stressor, but also to many other
long-term anthropogenic drivers, including nutrient inputs, soil pollution, land-use
change, and more (e.g. Maček et al. 2016a,b;Maček 2017b). The study of mofettes
may be one of the ways to increase the knowledge of microbial and fungal ecology
under long-term environmental changes, in this case long-term hypoxia.
Acknowledgement The work was supported by the Slovenian Research Agency (ARRS) through
projects J4-5526, J4-7052 and the research programme P4-0085. We gratefully acknowledge the
support provided.
References
Amend AS, Barshis DJ, Oliver TA (2012) Coral-associated marine fungi form novel lineages and
heterogeneous assemblages. ISME J 6:1291–1301
Appoloni S, Lekberg Y, Tercek MT, Zabinski CA, Redecker D (2008) Molecular community
analysis of arbuscular mycorrhizal fungi in roots of geothermal soils in Yellowstone National
Park (USA). Microb Ecol 56:649–659
Arnold F, West D, Kumar S (1987) Wound healing: the effect of macrophage and tumour derived
angiogenesis factors on skin graft vascularization. Br J Exp Pathol 68:569–574
238 I. Maček

Avramidis P, Bekiari V, Kontopoulos N, Kokidis N (2013) Shallow coastal lagoon sediment
characteristics and water physicochemical parameters—Myrtari lagoon, Mediterranean Sea,
Western Greece. Fresenius Environ Bull 22:1628–1635
Baar J, Paradi I, Lucassen ECHET, Hudson-Edwards KA, Redecker D, Roelofs JGM, Smolders
AJP (2011) Molecular analysis of AMF diversity in aquatic macrophytes: a comparison of
oligotrophic and utra-oligotrophic lakes. Aquat Bot 94:53–61
Bass D, Howe A, Brown N, Barton H, Demidova M, Michelle H, Li L, Sanders H, Watkinson SC,
Willcock S, Richards TA (2007) Yeast forms dominate fungal diversity in the deep oceans. Proc
Royal Soc B-Biol Sci 274:3069–3077
Beulig F, Urich T, Nowak M, Trumbore SE, Gleixner G, Gilfillan GD, Fjelland KE, Küsel K (2016)
Altered carbon turnover processes and microbiomes in soils under long-term extremely high
CO
2
exposure. Nat Microbiol 1:1–9
Bloomberg S, Rissmann C, Mazot A, Oze C, Horton T, Kennedy B, Werner C, Christenson B,
Pawson J (2012) Soil gas flux exploration at the Rotokawa geothermal field on White Island,
New Zealand. Thirty-sixth Workshop on Geothermal Reservoir Engineering, pp 1–11
Botha A (2006) Yeast in soil. In: Rosa CA, Péter G (eds) The yeast handbook biodiversity and
ecophysiology of yeasts. Springer-Verlag, Berlin, pp 221–240
Botha A (2011) The importance and ecology of yeasts in soil. Soil Biol Biochem 43:1–8
Brooker RW, Callaway RM (2009) Facilitation in the conceptual melting pot. J Ecol 97:1117–1120
Brundrett MC, Tedersoo L (2018) Evolutionary history of mycorrhizal symbioses and global host
plant diversity. New Phytol 220:1108–1115
Cantrell SA, Dianese JC, Fell J, Gunde-Cimerman N, Zalar P (2011) Unusual fungal niches.
Mycologia 103:1161–1174
Chukanhom K, Hatai K (2004) Freshwater fungi isolated from eggs of the common carp (Cyprinus
carpio) in Thailand. Mycoscience 45:42–48
Coyte KZ, Schluter J, Foster KR (2015) The ecology of the microbiome: networks, competition,
and stability. Science 350:663–666
Cramer T, Yamanishi Y, Clausen BE, Förster I, Pawlinski R, Mackman N, Haase VH, Jaenisch R,
Corr M, Nizet V, Firestein GS, Gerber HP, Ferrara N, Johnson RS (2003) HIF-1alpha is
essential for myeloid cell-mediated inflammation. Cell 112:645–657
DeJong-Hughes J, Moncrief JF, Voorhees WB, et al (2001) Soil compaction: causes, effects and
control. On line paper: http://www.extension.umn.edu/agriculture/tillage/soil-compaction/
Dewhirst MW (1998) Concepts of oxygen transport at the microcirculatory level. Semin Radiat
Oncol 8:143–150
Drake H, Ivarsson M, Bengtson S, Heim C, Siljeström S, Whitehouse MJ, Broman C,
Belivanova V, Åström ME (2017) Anaerobic consortia of fungi and sulfate reducing bacteria
in deep granite fractures. Nat Commun 8:55
D’Souza J, Rodrigues BF (2013) Biodiversity of arbuscular mycorrhizal (AM) fungi in mangroves
of Goa in West India. J For Res 24(3):515–523
Duarte S, Barlocher F, Trabulo J, Cassio F, Pascoal C (2015) Stream-dwelling fungal decomposer
communities along a gradient of eutrophication unravelled by 454 pyrosequencing. Fungal
Divers 70:127–148
Dumbrell AJ, Ashton PD, Aziz N, Feng G, Nelson M, Dytham C, Fitter AH, Helgason T (2011)
Distinct seasonal assemblages of arbuscular mycorrhizal fungi revealed by massively parallel
pyrosequencing. New Phytol 190:794–804
Dumbrell AJ, Nelson M, Helgason T, Dytham C, Fitter AH (2010) Relative roles of niche and
neutral processes in structuring a soil microbial community. ISME J 4:337–345
Dumbrell AJ, Ferguson RMW, Clark DR (2016) Microbial community analysis by single-amplicon
high-throughput next generation sequencing: data analysis –from raw output to ecology. In:
McGenity TJ, Timmis KN, Nogales B (eds) Hydrocarbon and lipid microbiology protocols,
springer protocols handbooks. Springer, Heidelberg
Erińska M, Silver IA (2001) Tissue oxygen tension and brain sensitivity to hypoxia. Respir Physiol
128:263–276
11 Fungi in Hypoxic Soils and Aquatic Sediments 239

Evans DE (2003) Aerenchyma formation. New Phytol 161:35–49
Fazi S, Ungaro F, Venturi S, Vimercati L, Viggi CC, Baronti S, Ugolini F, Calzolari C, Tassi F,
Vaselli O, Raschi A, Aulenta F (2019) Microbiomes in soils exposed to naturally high
concentrations of CO
2
(Bossoleto mofette Tuscany, Italy). Front Microbiol 10:2238
Fernández-Montiel I, Pedescoll A, Bécares E (2016) Microbial communities in a range of carbon
dioxide fluxes from a natural volcanic vent in campo de Calatrava, Spain. Int J Greenhouse Gas
Cont 50:70–79
Fitter AH (2005) Darkness visible: reflections on underground ecology. J Ecol 93:231–243
Fitter AH, Moyersoen B (1996) Evolutionary trends in root-microbe symbioses. Philos Trans Royal
Soc B Biol Sci 351:1367–1375
Franklin JF (1989) Importance and justification of long-term studies in ecology. In: Likens GE
(ed) Long-term studies in ecology: approaches and alternatives. Springer, New York, pp 3–19
Frerichs J, Oppermann BI, Gwosdz S, Möller I, Herrmann M, Krüger M (2013) Microbial
community changes at a terrestrial volcanic CO
2
vent induced by soil acidification and anaero-
bic microhabitats within the soil column. FEMS Microbiol Ecol 84:60–74
Gao Z, Johnson ZI, Wang GY (2010) Molecular characterization of the spatial diversity and novel
lineages of mycoplankton in Hawaiian coastal waters. ISME J 4:111–120
Gostinčar C, Grube M, De Hoog S, Zalar P, Gunde-Cimerman N (2010) Extremotolerance in fungi:
evolution on the edge. FEMS Microbiol Ecol 71:2–11
Grahl N, Shepardson KM, Chung D, Cramer RA (2012) Hypoxia and fungal pathogenesis: to air or
not to air? Eukaryot Cell 11:560–570
Grossart H, Van den Wyngaert S, Kagami M, Wurzbacher C, Cunliffe M, Rojas-Jimenez K (2019)
Fungi in aquatic ecosystems. Nat Rev Microbiol 17:339–354
Gunner HB, Alexander M (1964) Anaerobic growth of fusarium oxysporum. J Bacteriol 87:1309–
1316
Hall LA, Denning DW (1994) Oxygen requirements of aspergillus species. J Med Microbiol 41:
311–315
Hedges JI, Hu FS, Devol AH, Hartnett HE, Tsamakis E, Keil RG (1999) Sedimentary organic
matter preservation: a test for selective degradation under oxic conditions. Am J Sci 299:529–
555
He G, Shankar RA, Chzhan M, Samouilov A, Kuppusamy P, Zweier JL (1999) Noninvasive
measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract
of living mice with spatial and spectral EPR imaging. Proc Natl Acad Sci U S A 96:4586–4591
Helgason T, Fitter AH (2009) Natural selection and the evolutionary ecology of the arbuscular
mycorrhizal fungi (phylum Glomeromycota). J Exp Bot 60:2465–2480
Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW (1998) Ploughing up the wood-
wide web? Nature 394:431
Hirabayashi Y, Mahendran R, Koirala S, Konoshima L, Yamazaki D, Watanabe S, Kim H, Kanae S
(2013) Global flood risk under climate change. Nat Clim Chang 3:816–821
Hohberg K, Schulz H-J, Balkenhol B, Pilz M, Thomalla A, Russell DJ, Pfanz H (2015) Soil faunal
communities from mofette fields: effects of high geogenic carbon dioxide concentration. Soil
Biol Biochem 88:420–429
Hollis JP (1948) Oxygen and carbon dioxide relations of fusarium oxysporum Schlecht and
fusarium eumartii carp. Phytopathology 38:761–775
Hourdez S (2012) Hypoxic environments. In: Bell EM (ed) Life at extremes: environments,
organisms and strategies for survival. Gutenberg Press, Malta, pp 438–453
Jebaraj CS, Raghukumar C, Behnke A, Stoeck T (2010) Fungal diversity in oxygen-depleted
regions of the Arabian Sea revealed by targeted environmental sequencing combined with
cultivation. FEMS Microbiol Ecol 71:399–412
Johnson NC, Gehrig C, Jansa J (eds) (2017) Mycorrhizal mediation of soil. Fertility, structure and
carbon storage. Elsevier, Amsterdam, p 509
Justin SHFW, Armstrong W (1987) The anatomical characteristics of roots and plant response to
soil flooding. New Phytol 106:465–495
240 I. Maček

Karhausen J, Furuta GT, Tomaszewski JE, Johnson RS, Colgan SP, Haase VH (2004) Epithelial
hypoxia-inducible factor-1 is protective in murine experimental colitis. J Clin Invest 114:1098–
1106
Kies A, Hengesch O, Tosheva Z, Raschi A, Pfanz H (2015) Diurnal CO
2
-cycles and temperature
regimes in a natural CO
2
gas lake. Int J Greenhouse Gas Cont 37:142–145
Kohout P, SýkorováZ, CtvrtlíkováM, RydlováJ, Suda J, Vohník M, SudováR (2012) Surprising
spectra of root-associated fungi in submerged aquatic plants. FEMS Microbiol Ecol 80:216–235
Krüger M, West J, Frerichs J, Oppermann B, Dictor M-C, Jouliand C, Jones D, Coombs P, Green K,
Pearce J, May F, Möller I (2009) Ecosystem effects of elevated CO
2
concentrations on microbial
populations at a terrestrial CO
2
vent at Laacher see, Germany. Energy Procedia 1:1933–1939
Krüger M, Jones D, Frerichs J, Oppermann BI, West J, Coombs P, Green K, Barlow T, Lister R,
Shaw R, Strutt M, Möller I (2011) Effects of elevated CO
2
concentrations on the vegetation and
microbial populations at a terrestrial CO
2
vent at Laacher see, Germany. Int J Greenhouse Gas
Cont 5:1093–1098
Kurtzman CP, Fell JW, Boekhout T (2011) The yeasts: a taxonomic study, 5th edn. Elsevier,
Amsterdam
Lachance MA (2016) Metschnikowia: half tetrads, a regicide and the fountain of youth. Yeast 33:
563–574
Le Calvez T, Burgaud G, Mahe S, Barbier G, Vandenkoornhuyse P (2009) Fungal diversity in
Deep-Sea hydrothermal ecosystems. Appl Environ Microbiol 75:6415–6421
Lefevre E, Letcher PM, Powell MJ (2012) Temporal variation of the small eukaryotic community in
two freshwater lakes: emphasis on zoosporic fungi. Aquat Microb Ecol 67:91–105
Maček I (2013) A decade of research in mofette areas has given us new insights into adaptation of
soil microorganisms to abiotic stress. Acta Agriculturae Slovenica 101:209–217
Maček I (2017a) Arbuscular mycorrhizal fungi in hypoxic environments. In: Varma A, Prasad R,
Tuteja N (eds) Mycorrhiza –function, diversity, state of the art. Springer, Cham, pp 329–348
Maček I (2017b) Arbuscular mycorrhizal fungal communities pushed over the edge –lessons from
extreme ecosystems. In: Lukac M, Grenni P, Gamboni M (eds) Soil biological communities and
ecosystem resilience, Book series: sustainability in plant and crop protection. Springer, Cham,
pp 157–172
Maček I (2019) Diversity and ecology of fungi in mofettes. In: Tiquia-Arashiro SM, Grube M (eds)
Fungi in extreme environments: ecological role and biotechnological significance. Springer,
Cham, pp 3–19
Maček I, Dumbrell AJ, Nelson M, Fitter AH, Vodnik D, Helgason T (2011) Local adaptation to soil
hypoxia determines the structure of an arbuscular mycorrhizal fungal community in roots from
natural CO
2
springs. Appl Environ Microbiol 77:4770–4777
Maček I, Kastelec D, Vodnik D (2012) Root colonization with arbuscular mycorrhizal fungi and
glomalin-related soil protein (GRSP) concentration in hypoxic soils from natural CO
2
springs.
Agric Food Sci 21:62–71
Maček I, Pfanz H, FrancetičV, BatičF, Vodnik D (2005) Root respiration response to high CO
2
concentrations in plants from natural CO
2
springs. Environ Exp Bot 54:90–99
Maček I, Šibanc N, Kavšček M, Lestan D (2016a) Diversity of arbuscular mycorrhizal fungi in
metal polluted and EDTA washed garden soils before and after soil revitalization with commer-
cial and indigenous fungal inoculum. Ecol Eng 95:330–339
Maček I, Vodnik D, Pfanz H, Low-Décarie E, Dumbrell AJ (2016b) Locally extreme environments
as natural long-term experiments in ecology. In: Dumbrell AJ, Kordas R, Woodward G (eds)
Large Scale Ecology: Model Systems to Global Perspectives, Advances in Ecological Research,
vol 55, pp 283–323
Magnes M, Hafellner J (1991) Ascomyceten auf Gefäßpflanzen an Ufern von Gebirgsseen in den
Ostalpen. Biblioth Mycol 139:1–182
Manohar, Raghukumar (2013) Fungal diversity from various marine habitats deduced through
culture-independent studies. FEMS Microbiol Letters 341:69–78
11 Fungi in Hypoxic Soils and Aquatic Sediments 241

Marschner P (2012) Marschner’s mineral nutrition of higher plants, 3rd edn. Academic Press, San
Diego, p 672
Monchy S, Sanciu G, Jobard M, Rasconi S, Gerphagnon M, Chabé M, Cian A, Meloni D, Niquil N,
Christaki U, Viscogliosi E, Sime-Ngando T (2011) Exploring and quantifying fungal diversity
in freshwater lake ecosystems using rDNA cloning/sequencing and SSU tag pyrosequencing.
Environ Microbiol 13:1433–1453
Møller CL, Kjøller R, Sand-Jensen K (2013) Organic enrichment of sediments reduces arbuscular
mycorrhizal fungi in oligotrophic lake plants. Freshw Biol 58:769–779
Nizet V, Johnson RS (2009) Interdependence of hypoxic and innate immune responses. Nat Rev
Immunol 9:609–617
Oppermann BI, Michaelis W, Blumenberg M, Frerichs J, Schulz HM, Schippers A, Beaubien SE,
Krüger M (2010) Soil microbial community changes as a result of long-term exposure to a
natural CO
2
vent. Geochim Cosmochim Acta 74:2697–2716
Orsi W, Biddle JF, Edgcomb V (2013) Deep sequencing of subseafloor eukaryotic rRNA reveals
active fungi across marine subsurface provinces. PLoS One 8:e56335
Panzer K, Yilmaz P, Weiß M, Reich L, Richter M, Wiese J, Schmaljohann R, Labes A, Imhoff JF,
Glöckner FO (2015) Identification of habitat-specific biomes of aquatic fungal communities
using a comprehensive nearly full-length 18S rRNA dataset enriched with contextual data. PLoS
One. https://doi.org/10.1371/journal.pone.0134377
Perata P, Armstrong W, Voesenek LACJ (2011) Plants and flooding stress. New Phytol 190:269–
273
Pawlowski et al (2021). https://doi.org/10.1016/j.scitotenv.2021.151783.https://www.
sciencedirect.com/science/article/pii/S0048969721068595
Peyssonaux C, Johnson RS (2004) An unexpected role for hypoxic response: oxygenation and
inflammation. Cell Cycle 3:168–171
Pfanz H, Vodnik D, Wittmann C, Aschan G, Raschi A (2004) Plants and geothermal CO
2
exhalations—survival in and adaption to high CO
2
environment. Progress in Botany 65:499–
538
Pfanz H (2008) Mofetten –kalter Atem schlafender Vulkane. RVDL-Verlag, Köln, p 85p
Pfanz H, Sassmannshausen F, Wittmann C, Pfanz B, Thomalla A (2019) Mofette vegetation as an
indicator for geogenic CO
2
emission: a case study on the banks of the Laacher see volcano,
Vulkaneifel, Germany. Geofluids UNSP 2019:9589306. https://doi.org/10.1155/2019/9589306
Radhika KP, Rodrigues BF (2007) Arbuscular mycorrhizae in association with aquatic and marshy
plant species in Goa, India. Aquat Bo 86:291–294
Raghukumar C (2012) Biology of marine fungi. Springer, Heidelberg
Rajagopal K, Meenashree B, Binika D, Joshila D, Tulsi PS, Arulmathi R, Kathiravan G, Tuwar A
(2018) Mycodiversity and biotechnological potential of endophytic fungi isolated from
hydrophytes. Curr Res Environ Appl Mycol 8(2):172–182
Rangel DEN, Finlay RD, Hallsworth JE, Dadachova E, Gadd GM (2018) Fungal strategies for
dealing with environment- and agriculture-induced stresses. Fungal Biol 122:602–612
Rothschild LJ, Mancinelli RL (2001) Life in extreme environments. Nature 409:1092–1101
Russell DJ, Schulz H-J, Hohberg K, Pfanz H (2011) Occurrence of collembolan fauna in mofette
fields (natural carbon-dioxide springs) of the Czech Republic. Soil Organisms 83:489–505
Seago JL Jr, Peterson CA, Kinsley LJ, Broderick J (2000) Development and structure of the root
cortex in Caltha palustris L. and Nymphaea odorata Ait. Ann Bot 86:631–640
Sakayaroj J, Preedanon S, Supaphon O, Jones EBG, Phongpaichit S (2010) Phylogenetic diversity
of endophyte assemblages associated with the tropical seagrass Enhalus acoroides in Thailand.
Fungal Divers 42:27–45
Säle V, Aguilera P, Laczko E (2015) Impact of conservation tillage and organic farming on the
diversity of arbuscular mycorrhizal fungi. Soil Biol Biochem 84:38–52
Schulz H-J, Potapov MB (2010) A new species of Folsomia from mofette fields of the northwest
Czechia (Collembola, Isotomidae). Zootaxa 2553:60–64
Sharp FR, Bernaudin M (2004) HIF1 and oxygen sensing in the brain. Nat Rev Neurosci 5:437–448
242 I. Maček

Simmen HP, Battaglia H, Giovanoli P, Blaser J (1994) Analysis of pH, pO
2
and pCO
2
in drainage
fluid allows for rapid detection of infectious complications during the follow-up period after
abdominal surgery. Infection 22:386–389
Simon MC, Keith B (2008) The role of oxygen availability in embryonic development and stem cell
function. Nat Rev Mol Cell Biol 4:285–296
Smith SE, Read DJ (2008) Mycorrhizal Symbiosis, 3rd edn. Academic Press, London, pp 11–145
Smolders AJP, Lucassen E, Roelofs JGM (2002) The isoetid environment: biogeochemistry and
threats. Aquat Bot 73:325–350
SudováR, SýkorováZ, RydlováJ, ČtvrtlíkováM, Oehl F (2015) Rhizoglomus melanum, a new
arbuscular mycorrhizal fungal species associated with submerged plants in freshwater lake
Avsjøen in Norway. Mycol Prog 14:1–9
Supaphon P, Keawpiboon C, Preedanon S, Phongpaichit S, Rukachaisirikul V (2018) Isolation and
antimicrobial activities of fungi derived from Nymphaea lotus and nymphaea stellate.
Mycoscience 59:415–423
Šibanc N, Dumbrell AJ, Mandić-Mulec I, Maček I (2014) Impacts of naturally elevated soil CO
2
concentrations on communities of soil archaea and bacteria. Soil Biol Biochem 68:348–356
Šibanc N, Zalar P, Schroers H, Zajc J, Pontes A, Sampaio JP, Maček I (2018) Occultifur mephitis f.
a., sp. nov. and other yeast species from hypoxic and elevated CO
2
mofette environments. Int J
Syst Evolut Microbiol 68:2285–2298
Tabak HH, Cooke WB (1968) Growth and metabolism of fungi in an atmosphere of nitrogen.
Mycologia 60:115–140
Torossian JL, Kordas RL, Helmuth B (2016) Cross-scale approaches to forecasting biogeographic
responses to climate change. Adv Ecol Res 55:371–433
Tylianakis JM, Morris RJ (2017) Ecological networks across environmental gradients. In: Futuyma
DJ (ed) Annual reviews of ecology, Evolution and Systematics, vol 48, pp 25–48
Vacher C, Tamaddoni-Nezhad A, Kamenova S, Peyrard N, Moalic Y, Sabbadin R, Schwaller L,
Chiquet J, Smith MA, Vallance J, Fievet V, Jakuschkin B, Bohan DA (2016) Learning
ecological networks from next-generation sequencing data. In: Woodward G, Bohan DA
(eds) Ecosystem Services: From Biodiversity to Society, PT2 Advances in Ecological Research,
vol 54. Elsevier, Amsterdam, pp 1–39
Vallino M, Fiorilli V, Bonfante P (2014) Rice flooding negatively impacts root branching and
arbuscular mycorrhizal colonization, but not fungal viability. Plant Cell Environ 37:557–572
Videmšek U, Turk B, Vodnik D (2006) Root aerenchyma –formation and function. Acta
Agriculturae Slovenica 87:445–453
Vodnik D, Strajnar P, Jemc S, Maček I (2009) Respiratory potential of maize (Zea mays L.) roots
exposed to hypoxia. Environ Exp Bot 65:107–110
Vohník M, Borovec O, Kolařík M (2016) Communities of cultivable root mycobionts of the
seagrass Posidonia oceanica in the Northwest Mediterranean Sea are dominated by a hitherto
undescribed pleosporalean dark septate endophyte. Microb Ecol 71:442–451
Wang Y, Huang Y, Qiu Q, Xin G, Yang Z, Shi S (2011) Flooding greatly affects the diversity of
arbuscular mycorrhizal fungi communities in the roots of wetland plants. PLoS One 6(9):e24512
Wang Y, Li T, Li Y, Qiu Q, Li S, Xin G (2015) Distribution of arbuscular mycorrhizal fungi in four
semi-mangrove plant communities. Ann Microbiol 65:603–610
Wang Y, Li Y, Bao X (2016) Response differences of arbuscular mycorrhizal fungi communities in
the roots of an aquatic and a semiaquatic species to various flooding regimes. Plant Soil 403:
361–373
Wigand C, Andersen FO, Christensen KK, Holmer M, Jensen HS (1998) Endomycorrhizae of
isoetids along a biogeochemical gradient. Limnol Oceanogr 43:508–515
Zuccaro A, Schulz B, Mitchell JI (2003) Molecular detection of ascomycetes associated with Fucus
serratus. Mycol Res 107:1451–1466
11 Fungi in Hypoxic Soils and Aquatic Sediments 243

Chaotolerant Fungi: An Unexplored Group
of Extremophile 12
Sanjay Sahay
Abstract
Chaophilic fungi constitute a small group of very special fungi that can complete
their life cycle in the presence of macromolecule-destabilizing chemicals known
as chaotropes, for example, MgCl
2
, CaCl
2
, etc. They, as concluded from the study
of a limited number of isolated fungi of this group, exhibit halotolerance/halo-
philic and xerotolerance characters simultaneously. There is hardly any informa-
tion about the unique physiological mechanism of chaotropicity that differentiates
them from halotolerants. But the harsh conditions they are exposed to seem to be
favorable for the evolution of exotic biomolecules in them. Thus, they may be
potential sources of novel molecules of human uses.
Keywords
Chaotropicity · Water activity · Halotolerance · Kosmotropes ·
Osmotic adaptation
12.1 Introduction
Fungi inhabiting extreme environments such as extreme temperatures, pH, metal
concentration, pressure, desiccation, and salinity are extremophilic fungi (Macelroy
1974). Of these, halophiles requiring >3% NaCl for growth (Wilson and Brimble
2009) and halotolerant are ecologically very important as they occupy and perform
ecological functions in an osmotically very hostile environment such as sea and
other saline waterbodies.
S. Sahay (*)
Sarojini Naidu Government Postgraduate Girl’s (Autonomous) College, Bhopal, Madhya Pradesh,
India
e-mail: ss000@redffmail.com
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_12
245

The saline water is broadly divided into two types, viz., NaCl-rich thalassohaline
and MgCl
2
- and CaCl
2
-rich athalassohaline. Fungi have been reported in large
numbers from thalassohaline water, but very limited efforts have been made to
isolate them from athalassohaline environment (Sonjak et al. 2010; Oren and
Gunde-Cimerman 2012; Zajc et al. 2014). The fungi isolated from bittern brines of
solar saltern (Sonjak et al. 2010) contrasted earlier belief that chaotrope-rich saline
water is devoid of life (Javor 1989). Chaotolerant fungi have also been reported from
littoral Anchialine caves golubinka and medova buža (Croatia) (Chlebicki and Jakus
2019) and indoor air (Chlebicki et al. 2018).
The chaotolerant fungi from highly life-limiting environment are bioresources
important from both ecological and biotechnological points of view.
12.2 Chaotropicity
The chaotropes (Greek chaos ¼disorder, tropes ¼behavior) consist of compounds
that destabilize macromolecules. Kosmotropes, on the other hand, are compounds
that provide stability to the macromolecules. As to its mechanism of action in living
systems, there are two possible theories available. One theory believes it acts via
breaking water (solvent) structure breaker vis-a-vis kosmotrope (water structure
maker) (Collins 1997). Another theory relies on direct action of chaotropes on
macromolecules via structural rearrangement of bulk liquid (Ball and Hallsworth
2015). They may extract water or take away hydrogen bonds and destabilize folding
patterns, thus destroying the two-dimensional structure of biomolecules, or they may
also enter hydrophobic parts, leading to their swelling or solubilization (Ball and
Hallsworth 2015). Chaotropes such as ethanol may cause oxidative (Russo et al.
2001; Bhaganna et al. 2016) and osmotic stress (Cray et al. 2015). The results of
direct action may be degradation of cell organelles such as membrane and ribosome,
which is fatal to the organisms (Hallsworth et al. 2003; Duda et al. 2004; Lo Nostro
et al. 2005; Bhaganna et al. 2016).
12.3 Chaotropicity Versus Water Activity
MgCl
2
is a highly water-soluble salt that exhibits chaotropicity as well as water
activities. It has been used to test whether water activity or chaotropicity is more
important a limiting factor to impact survival. Applying metagenomic technique,
presence of mRNA (an indicator of life) has been tested along the MgCl
2
gradient of
sea water (Hallsworth et al. 2007). It was found that life was available below 2.3 M
MgCl
2
in the absence of kosmotroph. In pure culture, the highest MgCl
2
concentra-
tion that can allow growth was found 1.26 M at which corresponding water activity
and chaotropicity values are 0.916 aw 26.1 kJ g
1
, respectively. Since the lowest
value of water activity still allowing growth is 0.61 (equivalent to 3.7 M MgCl
2
),
explicitly indicates that it is the chaotropicity of MgCl
2
that limits life and not the
water activity (Hallsworth et al. 2007). This finding was further verified using
246 S. Sahay

environmental xerophytic fungi and chaotropic solutes glycerol and fructose
(Williams and Hallsworth 2009). It was found that the xerophiles could not grow
on fructose added medium (0.760 a
w
), despite the water activity value (a
w
) being
above the minimum growth supporting one, viz., 0.710 a
w
(Pitt and Christian
1968). Chaotolerant organisms for this reason are adapted to even more life-limiting
conditions than halophiles and xerophiles.
12.4 Chaophilic Versus Chaotolerant
A very limited number of halophilic bacteria, archaea, and fungi tolerant to a higher
concentration of kosmotropic (NaCl, KCl, and MgSO
4
) and chaotropic (NaBr,
MgCl
2
, CaCl
2
) solutes have been reported. There is also overlapping between
halophiles and chaophiles; indeed, all chaophiles at the same time are halophiles
(Zajc et al. 2014). There are halophilic species and strains that require NaCl as
growth requirement (Gunde-Cimerman et al. 2009), but there has been no obligate
chaophile reported so far. The most chaotolerant fungal taxa Wallemia sp. (e.g.,
Wallemia mellicola), Talaromyces diversus, and Hortaea werneckii can well grow
on salt non-supplemented medium (Chlebicki et al. 2018), though W. ichthyophaga
was reported to need 10% NaCl as a growth requirement (Zalar et al. 2005; Zajc et al.
2014). The reason may be a general tendency among microbiologists to use NaCl
supplemented medium to isolate microbes from the saline environment. Hardly any
dedicated search for chaotropic microbes has been made (Zajc et al. 2014), thus all
the fungi so far reported as chaophiles are more appropriately the chaotolerant.
12.5 Chaotolerant Fungi
As chaotolerant microbes, fungi exhibit an edge over archaea since in the presence of
the highest concentration of MgCl
2
(without compensating kosmotropes) the latter
after 18 months of cultivation could continue to grow was 1.26 M (Hallsworth et al.
2007), which is lower than 1.5 M that the fungi could tolerate without compensating
NaCl (Hallsworth et al. 2007). The majority of fungi show a preference for a
relatively high concentration of kosmotropic salts, only a few of them having the
ability to tolerate higher concentrations of chaotropic salts.
The first fungus Xeromyces bisporus capable of growing at the lowest water
activity (Pitt and Hocking 2009) was described as exhibiting preference to
chaotropic conditions/solutes and growth in the presence of high concentration
(7.6 M) chaotropic glycerol (Williams and Hallsworth 2009). The fungus has also
been tolerant to a very high concentration of kosmotropic salts (Pitt and Hocking
2009).
In one study, isolation of chaophilic fungi from bittern brines (Sonjak et al. 2010)
of the Secovlje salterns (Slovenia) led to the identification of filamentous fungi,
Cladosporium sp., black and other yeasts that were able to grow at higher
12 Chaotolerant Fungi: An Unexplored Group of Extremophile 247

concentrations of MgCl
2
(1.5 M) (Sonjak et al. 2010), higher than that (1.26 M
MgCl
2
) reported earlier (Hallsworth et al. 2007).
In another survey, halotolerant rock-inhabiting fungi were isolated from
Golubinka and Medova Buža littoral anchialine caves in Croatia (Chlebicki and
Jakus 2019). There were six halotolerant isolates, viz., Cladosporium
psychrotolerans,C. delicatulum,Mucor circinelloides,Rhizopus stolonifer,
Aureobasidium pullulans var. pullulans, and Talaromyces diversus isolated of
which T. diversus was found to be chaophilic as well (Chlebicki and Jakus 2019).
T. diversus could thrive at 24% NaCl and 16% MgCl
2
, the tolerance being more than
that exhibited by Wallemia mellicola.
Another dedicated study on the tolerance of several halotolerant and/or
xerotolerant fungal strains to chaotropic solutes resulted in identification of 37 strains
of fungi 11 belonging to Hortaea werneckii and Wallemia ichthyophaga that can
thrive in 1.8 M MgCl
2
concentrations (Zajc et al. 2014). Though both could tolerate
the highest concentration of MgCl
2
,H. werneckii could but W. ichthyophaga could
not tolerate the highest concentration of CaCl
2
. The study revealed chaotropic
preferences of several known halotolerants such as H. werneckii,Aureobasidium
sp., Cladosporium sp., Wallemia sp., Eurotium, Emericella,Aspergillus, and Peni-
cillium. All of them were tolerant to higher concentration of kosmotropic salts and
could grow in the presence 1.5 M MgCl
2
and 1.2 M CaCl
2
(Table 12.1). Some
yeasts, provisionally named Candida atmosphaerica-like and Pichia philogaea-like,
have also been isolated from the MgCl
2
- rich athalassohaline habitat (bitterns)
(Butinar et al. 2005).
12.6 Ecology
There is not much information about the various habitats that the chaophilic fungi
occupy. However, they have been reported from solar saltern (Sonjak et al. 2010)
and saline waterbodies containing a high concentration of chaotropes especially
Table 12.1 Chaotolerant fungi and their tolerance to highest salt concentration (M) (Zajc et al.
2014)
Fungal species/strains
Kosmotropic salts Chaotropic salts
NaCl KCl MgSO
4
MgCl
2
CaCl
2
NaBr
H. werneckii 5.0 4.5 3.0 2.1 1.7 4.0
Aureobasidium spp. 4.0 3.0 <1.5 1.2
Cladosporium spp. 2.5–4.0 2.5–4.5 2.0–3.0
C. tenuissimum EXF-1943
C. Cladosporoides EXF-1824 2.0–2.1 1.7
Wallemia ichthyophaga >4.0 Sat Sat ––4.0
W. Ichthyophaga EXF-994 2.1 <1
Eurotium,Emericella
Aspergillus and Penicillium >3.0 3.5 2.0 >1.5 1.2 2.5
248 S. Sahay

MgCl
2
such as Dead Sea (Oren and Gunde-Cimerman 2012), etc. The ecological
role the chaophilic fungi play in such areas with very limited life forms has been only
scarcely explored. The chaotoleranr fungi, for example, Talaromyces diversus, have
been found to produce mitorubrinic acid (Yilmaz et al. 2014) that can dissolve
carbonate for, for example, limestone and thus seem to have an ecological role in
lithosere (Chlebicki and Jakus 2019).
12.7 Physiology
There is hardly any dedicated study on the physiology of chaotolerant fungus. Since
all chaotolerant fungi are at the same time halophilic, they should share many
physiological adaptations (osmotolerance) of halophiles. From a comparative
study on the salt tolerance mechanism in H. werneckii, a halotolerant fungus, and
W. ichthyophaga, a halophilic fungus, for example, it has been reported that both
accumulate small organic molecules (compatible solutes, e.g., glycerol) to balance
the osmotic pressure of surroundings and avoid high concentration of intracellular
toxic salts (NaCl, etc.) (Plemenitašet al. 2014). Cells do have the option to use efflux
and influx systems to discard surplus ions. The alkali-metal cation transporters in this
wake become important for osmotic adaptations to highly saline conditions. For
example in yeast, the Na
+
-exporting ATPase (EnaA) is a major mechanism to
tolerate salt (Ariño et al. 2010). Thus, the absence of EnaA in X. bisporus genome
may be responsible for its poor growth in the presence of salt (Leong et al. 2014), and
the presence of the same in multiple copies in W. werneckii (Lenassi et al. 2013), and
differentially expressed in W. ichtyophaga (Zajc et al. 2013) impart them higher
tolerance to salinity. In W. ichthyophaga cation-transporter genes are present in low
number and passive barriers play a crucial role in providing it tolerance against high
salinity conditions. Some of such barriers are unusually thick wall, clumping of cells,
and the cell wall proteins, hydrophobins, which is expressed under saline condition
(Zajc et al. 2013). Fungi may also apply such passive strategies as clumping of cells
(Kralj Kunčičet al. 2010), painting extracellular polysaccharides over cell wall or
thickening of cell wall (Kralj Kunčičet al. 2010), and pigmenting/melanizing the cell
wall (Kogej et al. 2006).
Halotolerant or halophilic fungi employ high osmolarity glycerol (HOG) signal
pathway to sense and respond to salt/osmolyte-led stress (Gostiňcar et al. 2011). The
activation of this pathway leads to the production of glycerol, the latter then plays an
active role in restoring the cell’s osmotic balance (Hohmann 2009). The channels
available in the cells are utilized to eject or take in glycerol as per requirement
(Ferreira et al. 2005). This strategy thus enables fungi to enjoy considerable flexibil-
ity in adapting to salinity.
The comparative study on the HOG pathways present in H. werneckii and
W. ichthyophaga by which both of these fungi synthesize glycerol and few other
compatible solutes after exposure to high salinity environment has revealed that the
key proteins of HOG pathway are conserved in both, but there is a difference in
regulation (Plemenitašet al. 2014). While the Hog1 kinase of H. werneckii
12 Chaotolerant Fungi: An Unexplored Group of Extremophile 249

phosphorylates when the extracellular salinity is 3 M NaCl (Turk and Plemenitaš
2002), that of W. ichthyophaga remains phosphorylated constitutively under normal
osmotic conditions and gets dephosphorylated under hyperosmotic or hypo-osmotic
conditions (Konte and Plemenitaš2013). Thus, Hog1 in H. werneckii may exercise
differential activation or repression of osmoresponsive genes or regulation of chro-
matin and RNA polymerase II via physical interaction with them under salinity stress
(Vaupotǐc and Plemenitaš2007). The kind of the Hog1 regulation in H. werneckii
seems to be compatible with its extreme halotolerance, the constitutive expression
(phosphorylation) of the Hog1 in W. ichthyophaga, on the other hand, might be
responsible for its obligate halophilicity. Furthermore, while all the HOG pathway
components in H. werneckii are present in at least two copies, in W. ichthyophaga
only Hog1 is present in two isoforms (Plemenitašet al. 2014).
12.8 Biotechnological Potential
Over the past many decades, injudicious agricultural practices and global climate
change have led to much deterioration of soil making it stressful for plants and
microbes (Thomas et al. 2005; Seager et al. 2007). Chaotolerant fungi may be
augmented to chaotrope (e.g., urea, NH
4
NO
3
, phenol, MgCl
2
, CaCl
2
)-polluted
agriculture soil to restore microbial activity, leading to structure-fertility enhance-
ment (Williams and Hallsworth 2009).
Pollution in the environment (land and water) especially with chemicals with
chaotropic activities of xenobiotics is a challenge to microbes (Hallsworth et al.
2003). Fungi with chaotolerance and bioremediation potential will be boon to
remediate such environments. The process can be more efficiently carried out at
low temperature as low temperature prevents disordering impact of chaotropes on
cellular structures (Hallsworth et al. 2003).
Chaotolerant fungi do show xerophytic feature, that is,. they have better survival
ability under low water activity conditions. Food protection from food spoiler
microbes is another area that may benefit from the work on chaotolerant fungi. A
deep insight into the survival mechanisms under these conditions (packaged food
products contain chaotropic stressors like sodium benzoate and are maintained under
low water activity) will be useful in eliminating food spoiling microbes/fungi.
The human pathogen Wallemia mellicola (Chlebicki et al. 2018) is a chaotolerant
fungus. Does chaotolerance provide this fungus with any pathogenic advantage?
Such a conclusion needs a dedicated study. Although literatures are available in
abundance on the biotechnological importance of whole cells and their products
from halophilic fungi, there is hardly any data about the application of chaotolerant
fungi per se.
250 S. Sahay

References
Ariño J, Ramos J, Sychrova H (2010) Alkali metal cation transport and homeostasis in yeasts.
Microbiol Mol Biol Rev 74:95–120
Ball P, Hallsworth JE (2015) Water structure and chaotropicity: their uses, abuses and biological
implications. Phys Chem Chem Phys 17:8297–8305
Bhaganna P, Bielecka A, Molinari G, Hallsworth JE (2016) Protective role of glycerol against
benzene stress: insights from the pseudomonas putida proteome. Curr Genet 62:419–429
Butinar L, Santos S, Spencer-Martins I, Oren A, Gunde-Cimerman N (2005) Yeast diversity in
hypersaline habitats. FEMSMicrobiol Lett 244:229–234
Chlebicki A, Spisak W, Saługa M (2018) Halotolerance (ionic NaCl) and chaotolerance (ionic
MgCl
2
) of the human pathogen Wallemia mellicola isolated, for the first time, from indoor air in
Poland. Acta Mycol 53:1104
Chlebicki A, Jakus N (2019) Halotolerant and chaotolerant microfungi from littoral anchialine
caves Golubinka and Medova Buža (Croatia). J Cave Karst Stud 81:153–161
Collins KD (1997) Charge density-dependent strength of hydration and biological structure.
Biophys J 72:65–76
Cray JA, Stevenson A, Ball P, Bankar SB, Eleutherio ECA, Ezeji TC, Singhal RS, Thevelein JM,
Timson DJ, Hallsworth JE (2015) Chaotropicity: a key factor in product tolerance of biofuel-
producing microorganisms. Curr Opinion Biotechnol 33:228–259
Duda VI, Danilevich VN, Suzina NE, Shorokhova AP, Dmitriev VV, Mokhova ON, Akimov VN
(2004) Changes in the fine structure of microbial cells induced by chaotropic salts. Microbiol-
ogy 73:341–349
Ferreira C, Van Voorst F, Martins A, Neves L, Oliveira R, Kielland-Brandt MC et al (2005) A
member of the sugar transporter family, Stl1p is the glycerol/H+ symporter in Saccharomyces
cerevisiae. Mol Biol Cell 16:2068–2076
Gostiňcar C, Lenassi M, Gunde-Cimermna N, PlemenitašA (2011) Fungal adaptation to extremely
high salt concentrations. Adv Appl Microbiol 77:71–96
Gunde-Cimerman N, Ramos J, PlemenitašA (2009) Halotolerant and halophilic fungi. Mycol Res
113:1231–1241
Hallsworth JE, Heim S, Timmis KN (2003) Chaotropic solutes cause water stress in Pseudomonas
putida. Environ Microbiol 5:1270–1280
Hallsworth JE, Yakimov MM, Golyshin PN, Gillion JLM, D’Auria G, de Lima AF, La Cono V,
Genovese M, McKew BA, Hayes SL, Harris G, Giuliano L, Timmis KN, McGenity TJ (2007)
Limits of life in MgCl2-containing environments: chaotropicity defines the window. Environ
Microbiol 9:801–813
Hohmann S (2009) Control of high osmolarity signaling in the yeast Saccharomyces cerevisiae.
FEBS Lett 583:4025–4029
Javor BJ (1989) Hypersaline environments. In: Schiewer U (ed) Microbiology and biogeochem-
istry. Springer-Verlag/Heidelberg GmbH & Co, Berlin, p 287
Kogej UT, Gorbushina AA, Gunde-Cimerman N (2006) Hypersaline conditions induce changes in
cell-wall melanization and colony structure in a halophilic and a xerophilic black yeast species
of the genus Trimmatostroma. Mycol Res 110:713–724
Konte T, PlemenitašA (2013) The HOG signal transduction pathway in the halophilic fungus
Wallemia ichthyophaga: identification and characterisation of MAP kinases WiHog1A and
WiHog1B. Extremophiles 17:623–636
Kralj KunčičM, Kogej T, Drobne D, Gunde-Cimerman N (2010) Morphological response of the
halophilic fungal genus Wallemia to high salinity. Appl Environ Microbiol 76:329–337
Lenassi M, Gostinèar C, Jackman S, Turk M, Sadowski I, Nislow C et al (2013) Whole genome
duplication and enrichment of metal cation transporters revealed by de novo genome sequencing
of extremely halotolerant black yeast Hortaea werneckii. PLoS One 8:e71328
12 Chaotolerant Fungi: An Unexplored Group of Extremophile 251

Leong S-LL, Lantz H, Pettersson OV, Frisvad JC, Thrane U, Heipieper HJ et al (2014) Genome and
physiology of the ascomycete filamentous fungus Xeromyces bisporus, the most xerophilic
organism isolated to date. Environ Microbiol. https://doi.org/10.1111/1462-2920.12596
Lo Nostro P, Ninham BW, Lo Nostro A, Pesavento G, Fratoni L, Baglioni P (2005) Specific ion
effects on the growth rates of Staphylococcus aureus and Pseudomonas aeruginosa. Phys Biol
2:1–7
Macelroy RD (1974) Some comments on the evolution of extremophiles. BioSystem 6:74–75
Pitt JI, Hocking AD (2009) Fungi and food spoilage. Springer, Dordrecht
Oren A, Gunde-Cimerman N (2012) Fungal life in the Dead Sea. In: Raghukumar C (ed) Biology of
marine fungi. Springer-Verlag, Berlin, pp 115–132
Pitt JI, Christian JHB (1968) Water relations of xerophilic fungi isolated from prunes. Appl
Microbiol 16:1853–1858
PlemenitašA, Lenassi M, Konte T, Kejžar A, Zajc J, Gostinčar C, Gunde-Cimerman N (2014)
Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective.
Front Microbiol 5:199
Russo A, Palumbo M, Scifo C, Cardile V, Barcellona ML, Renis M (2001) Ethanolinduced
oxidative stress in rat astrocytes: role of HSP70. Cell Biol Toxicol 17:153–168
Seager R, Ting MF, Held I, Kushnir Y, Lu J, Vecchi G et al (2007) Model projections of an
imminent transition to a more arid climate in southwestern North America. Science 316:1181–
1184
Sonjak S, Gürsu B, Gunde-Cimerman N (2010) MgCl
2
tolerant fungi from the bitterns. In: 8th
International Congress on Extremophiles AZORES 2010. Secretaria Regional da Ciência,
Tecnologia e Equipamentos, Ponta Delgada, p 109
Thomas DSG, Knight M, Wiggs GFS (2005) Remobilization of southern African desert dune
systems by twenty-first century global warming. Nature 435:1218–1221
Turk M, PlemenitašA (2002) The HOG pathway in the halophilic black yeast Hortaea werneckii:
isolation of the HOG1 homolog gene and activation of HwHog1p. FEMS Microbiol Lett 216:
193–199
Vaupotǐc T, PlemenitašA (2007) Differential gene expression and Hog1 interaction with
osmoresponsive genes in the extremely halotolerant black yeast Hortaea werneckii. BMC
Genomics 8:280
Wilson ZE, Brimble MA (2009) Molecules derived from the extremes of life. Nat Prod Rep 26:44–
71
Williams JP, Hallsworth JE (2009) Limits of life in hostile environments: no barriers to biosphere
function? Environ Microbiol 11:3292–3308
Yilmaz N, Visagie CM, Houbraken J, Frisvad JC, Samson RA (2014) Polyphasic taxonomy of the
genus Talaromyces. Stud Mycol 78:175–341
Zajc J, Liu YF, Dai WK, Yang ZY, Hu JZ, Gostinˇcar C et al (2013) Genome and transcriptome
sequencing of the halophilic fungus Wallemia ichthyophaga: haloadaptations present and
absent. BMC Genomics 14:617
Zajc J, Džeroski S, Kocev D, Oren A, Sonjak S, Tkavc R, Gunde-Cimerman N (2014) Chaophilic or
chaotolerant fungi: a new category of extremophiles? Front Microbiol 5:1–15
Zalar P, de Hoog GS, Schroers HJ, Frank JM, Gunde-Cimerman N (2005) Taxonomy and
phylogeny of the xerophilic genus Wallemia (Wallemiomycetes and Wallemiales, cl. Et Ord.
Nov.). Antonie Van Leeuwenhoek 87:311–328
252 S. Sahay

Xerophilic Fungi: Physiology, Genetics
and Biotechnology 13
Sanhita Sarkar, Namita Ashish Singh, and Nitish Rai
Abstract
Xerophilic fungi are the distinctive organism which can grow under conditions of
reduced water activity. The present work highlights the physiological adaptations
of xerophilic fungi which include osmoregulation through membrane
modifications, osmosensors-mediated sensing of low water activity (aw) and
utilisation of alternate substrates, namely, salt and sugar. We have also covered
the three unique strategies, namely combative, stress and ruderal, which is helpful
for their survival in unfavourable conditions. In this chapter, we have tried to
cover the molecular mechanism along with the genes expression responsible for
the adaptation of xerophilic fungi under water stress conditions. Further, this
chapter covers the various bioactive compounds produced by xerophilic fungi
along with their potential bioactivity. In the last section, we have discussed the
various aspects of xerophilic fungi such as enzyme and pigment production, air
biofiltration, biodeterioration in museums and libraries, etc. We have also covered
the health risks associated with the xerophilic fungi, namely fungal infections,
food spoilage and mycotoxin production.
Keywords
Xerophilic · Physiology · Adaptation · Biodeterioration · Food spoilage
S. Sarkar · N. Rai (*)
Department of Biotechnology, Mohanlal Sukhadia University, Udaipur, Rajasthan, India
e-mail: nitish.rai@mlsu.ac.in
N. A. Singh
Department of Microbiology, Mohanlal Sukhadia University, Udaipur, Rajasthan, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_13
253

13.1 Introduction
Several microorganisms have the abilities to survive under low availability of water,
but relatively few have become skilled at it through physiological adaptations that
enable them to thrive under such an environment. One way in which the water
availability could be expressed is water activity (aw), which is the ratio of the vapour
pressure of water in a sample to that of pure water in the sample (aw ¼1). Therefore,
aw of pure water is 1.00 and as the free-water content of substrate drops so does its
aw. Alternatively, water potential or Ψ, the sum of osmotic, matrix and turgor
potentials, is also used as a measure whose units are in Pascal (Pa); and it is related
to aw by formula Water potential(Ψ)¼RT/V log
n
aw(+P); where Ris the ideal gas
constant, Tthe absolute temperature, Pthe atmospheric pressure and Vthe volume of
1 mole of water.
The fungi which can grow and divide in a condition of reduced water activity, that
is, below 0.85 aw, is known as Xerophilic fungi. These fungi grow on a dried and
concentrated substrate where there is low water available in the matrix due to the
presence of high concentrations of soluble solids like salts and sugar (Su-lin et al.
2011; Rico-Munoz et al.2019). Usually, these fungi produce solutes like glycerol to
create an optimum osmotic pressure for reproduction and growth. The physiological
adaptations in these fungi involve osmoregulation via membrane modifications,
osmosensors-mediated sensing of low aw and utilising alternate salt and sugar
substrates that unfortunately causes spoilage in a variety of processed food. The
genera that come under xerophiles include Eurotium, Penicillium and Aspergillus
species.
In this chapter, xerophilic fungi with their different aspect relating to physiology,
genetics, adaptation, important bioactive secondary metabolites and biotechnologi-
cal importance are discussed. The chapter attempts to discuss the molecular players
which help the xerophilic fungi to adapt to the water stress by modulating the
expression of various genes. Further, the chapter discusses the role of xerophilic
fungi in various areas such as enzyme production, air biofiltration, food industry,
museum, environment and health.
13.2 Overview of Xerophilic Fungi
In general, depending on the physiological characteristics, the fungi are classified
into various categories such as thermophiles/psychrophiles (the ability to grow in
high/low temperature), alkaliphiles/acidophiles (grow in high/low pH), halophiles
(grow in high salt concentrations), oligotrophic (grow in low availability of
nutrients), piezophiles (grow in high pressure) and radiophiles (grow in high radia-
tion) and xerophiles (grow in low aw) (Abe and Horikoshi 2001; Rothschild and
Mancinelli 2001; Van den Burg 2003). In nature, some environments are associated
with one or more extreme condition and microorganisms inhabiting them are known
as polyextremophiles (Rothschild and Mancinelli 2001).
254 S. Sarkar et al.

The term “Xerophilic”is derived from Greek, meaning dry loving. Xerophilic
fungi may be classified into two groups (Pitt and Hocking 2009), namely, moderate
xerophile and extreme xerophile. Moderate xerophile, like Paecilomyces variotii,
Aspergillus pseudoglaucus, A. chevalieri, A. glaucus, A. montevidensis, A. ruber,
and Penicillium spp., are those that can grow at or below 0.85 aw values, and the
extreme xerophiles are those filamentous fungi that either get completely inhibited
under high aw or grow very slowly. Due to such physiology, moderate xerophiles
are notably responsible for food spoilage (Pitt and Hocking 2009; Rico-Munoz et al.
2019). Some studies have also shown that xerophilic fungi may have a dominant
existence in their habitat by producing toxic metabolite induced by a stress environ-
ment (Cray et al. 2013).
There are diverse groups of microorganisms present in the milieu of reduced aw
including archaea, eubacteria, algae, cyanobacteria, yeasts and filamentous fungi.
Most of these groups have species thriving in a highly saline environment, but only
the yeasts and filamentous fungi grow effectively under a high sugar environment.
Also, a far greater variety of fungi has been isolated from habitats with water
activities below 0.85 as compared to other groups combined.
13.3 Physiology and Adaptation
A constant supply of water is inevitable to maintain normal cellular functioning for
all life forms; however, often life is also found to thrive in minimal water conditions.
Since all life processes take place in solution form, therefore the physiological
adaptations used by the xerophilic fungus to adjust to osmotic alterations and aw
below 0.85 are quite fascinating. In fact, in terrestrial ecosystems both abiotic and
biotic factors spatiotemporally influence fungal activity, making the structure of
fungal community and dominance possible.
Although certain disturbances are transient, some are permanent features of a
habitat and communities inhabiting them employ three primal strategies and their
combinations as secondary strategies to survive and even flourish in these
unfavourable conditions. These strategies are known as combative, stress and
ruderal strategies. While combative or C-selected strategies ensure maximum
exploitation of resources in relatively ordinary conditions, stress or S-selected
strategies involve the development of adaptability, resilience and endurance required
for survival in continuous stress. Ruderal or R-selected strategies encourage a short
life span but high reproductive potential enabling success in severely chaotic and
poorly distributed nutrient conditions. Intercalation of these primary strategies in
different combinations gives rise to secondary survival strategies that in continuum
maximise survival potential of chaophilic organisms (Fig. 13.1).
Life exposed to low aw circumstances needs to check water loss and prevent
desiccation by osmosis. A certain level of turgor pressure needs to sustain in order to
support life. Two basic strategies followed are (1) achieving osmotic stability by
using counter-balancing levels of inorganic ions (usually KCl) and (2) synthesis and
accumulation of compatible solutes (a low molecular weight organic, polar,
13 Xerophilic Fungi: Physiology, Genetics and Biotechnology 255

uncharged osmolyte). These compatible solutes can easily pass through
biomembranes and become zwitterionic in intra-cellular environments, which can
make up for osmotic potential. The characteristic feature of compatible solutes
includes some common structural motifs which may be poly hydroxyl groups,
sugars, amino acid and their derivatives. Some suitable compatible solutes are
polyols such as glycerol, arabitol, mannitol; sugars like trehalose and sucrose; and
sugar derivatives like sulpho-trehalose and glucosyl-glycerol; betaines
(trimethylammonium compounds) and thetines (dimethyl sulphonium compounds);
amino acids, including proline, glutamate and glutamine; N-acetylated amino acids
such as N-acetylornithine; glutamine amide derivatives such as N-
carbamoylgutamine amide; ectoines, notably ectoine and β–hydroxyectoine.Fungi
and yeasts have been found to make use of these compatible solutes for survival in
low aw (Brewer 1999). Glycerol, being the smallest of sugar alcohols, plays a pivotal
role in osmoregulation and combats the greatest reduction in aw on a molar basis
compared with the other sugar alcohols accumulated by fungi during osmotic stress.
Growth at low aw requires continuous maintenance of cell turgor potential.
However, what is noteworthy is that optimum growth is influenced not only by aw
or turgor potential but also by interactions of other environmental factors like
temperature, pH and gas composition (Magan et al. 2004). A shift towards high
water potential affects all vital cellular processes such as nutrient uptake, protein
biosynthesis and enzyme activities. To enable normal cell functionality, compatible
solutes that can protect hydrated biopolymers and allow structural integrity under
low-water potential conditions are used. Multiple physiological adaptations have
been reported in different xerophilic fungi to combat physiological stresses like low
aw,disturbed turgor pressure and high osmotic potential. One such physiological
adaptation is modulation in the expression of enzymes that are crucial to glycerol
metabolism. A study using media containing 10% salt or 7.0 MPa water potential
Fig. 13.1 Characteristic comparison of fungi in relation to the three main ecological strategies
256 S. Sarkar et al.

showed an almost 40-fold increase in the enzyme glycerol-3-phosphate dehydroge-
nase production (Leong et al. 2015).
Xeromyces bisporus is a model candidate for studying the molecular mechanisms
utilised by typical xerophilic fungi, and its adaptation has been discussed in detail
below. Various experiments have demonstrated that X. bisporus notably use stress-
tolerant and ruderal strategies in response to competition whereas, combative
strategies are shut down to minimise the production of secondary metabolites (Zak
and Wildman 2004). It has been observed that X. bisporus at the genomic level is a
true S-strategist. As it shows apparent loss of all gene clusters that produce second-
ary metabolites which are the key molecules for competition and interaction with
other organisms, that is, abandonment of combative strategies. For example,
analyses of non-transcriptionally regulated events that are under allosteric control
show an increase in membrane fatty acid saturation and study of differentially
expressed genes during osmotic stress reinforce events like phospholipid and cho-
lesterol biosynthesis to achieve the best out of chaotropic situation.
They mainly apply two principal strategies in order to survive in extreme
conditions. Firstly, X. bisporus constitutively expresses genes to produce surplus
amounts of glycerol and increase their expression in hyper- or hypo-osmotic stress.
Overproduction of these solutes leads to leaked or secreted solutes and their associ-
ation with water comprise the bulk of the mycelial wet weight. This strategy
employed by X. bisporus helps to modulate the micro-environment around its
mycelium. Secondly, the low unsaturation index of membrane fatty acids, coupled
with high glycerol content, and modifications of phospholipids, sterols and cell wall
components, together lead to a unique ‘Xerophilic’stress response. Very innovative
self-conditioning physiological adaptations behind the success of X. bisporus
include not only sensing low aw through osmosensors and accumulating, utilising
and retaining compatible solutes for osmoregulation by membrane modifications,
but also preferences for alternate salt and sugar substrates that makes a huge variety
of processed food and stored commodities like jams and salterns susceptible to, what
we will know from now on as fungal Xerophiles (Pitt and Hocking 2009).
X. bisporus is an obligate ascomycete filamentous fungus, arguably the most ‘dry-
loving’organism known to mankind that grows mostly on sugary substrates where
aw is as low as 0.61 (Pitt and Christian 1968; Leong et al. 2011). It has a genome size
of 22 Mb and shows optimum growth in glycerol-supplemented growth media, at
30 C and 0.653 aw, and this demarcates X. bisporus as a classified chaophile
pointing to remodelling of cellular and subcellular structures. X. bisporus is known
to manage the most successful living in such chaotropic conditions amongst all other
xerophilic microorganisms.
The mechanisms deployed by xerophiles to achieve positive turgor potential for
active hyphal growth even at aw as low as 0.61 against strong internal and external
solute concentration gradients is pretty much alien and equally fascinating to the
scientific community. Although few hypotheses have been proposed to address the
above query, the underlying basic principles are apparently similar and we have tried
to comprehensively explain the physiological adaptations in this section.
13 Xerophilic Fungi: Physiology, Genetics and Biotechnology 257

Decoding the transcriptomes of gene clusters of X. bisporus revealed a great deal
of information about the strategies relinquished and employed by it to thrive in hypo
and hyper-osmotic stress. Usually in fungus genes that code for secondary
metabolites are present in clusters (Yu and Keller 2005). These clusters consist of
a backbone gene (Khaldi et al. 2010) as well as some regulatory elements responsi-
ble for transcription of these backbone genes (Fox and Howlett 2008) and some
enzyme coding genes required for modification of products of backbone genes
(Andersen et al. 2013). In Aspergillus nidulans (a xerotolerant fungal species),
66 gene clusters have been annotated as genes for secondary metabolites; out of
these, 63 clusters are associated to backbone enzymes (Inglis et al. 2013). However,
in X. bisporus, only six out of A. nidulans gene clusters are conserved and five extra
orthologues of backbone enzymes are found in isolated positions and not in
orthologous loci. For example, sidC, a gene involved in peroxisome metabolism,
is found to be conserved both in A, nidulans and X. bisporus (Gründlinger et al.
2013).
Xerophilic fungi that accumulate or synthesise compatible solutes have a rapid
response system that reacts to a sudden erosion of the environment. Some compati-
ble solutes are rapidly catabolised or polymerised to an osmotically inert state
(Trüper and Galinski 1990). Normally, certain uncharged molecules like simple
polyols may pass through bio-membranes pretty easily. The accumulation of
polyhydric alcohols (produced from sugar fermentation) that enables crucial physi-
ological processes alongside maintaining the osmotic balance between intracellular
and extracellular environment is the primary feature of xerophilic fungi. This
indicates either intrinsic differences in membrane structure of xerophiles or induced
alteration of membrane permeability upon sensing of low aw. In fact, glycerol is
quite efficient in decreasing mycelial water potential and a great choice for the
compatible solute to combat stress generated by extremely low water availability.
As a response to osmotic stress, some fungi increase glycerol production, while
others manipulate membrane permeability and its transportation across
biomembranes. Often, to prevent leakage of glycerol and counter-balance osmotic
stress energy-dependent active transportation strategies are assigned. For example,
to ensure optimum enzyme activities low intracellular Na+ is maintained and K+ and
H+ fluxes initialise osmoregulatory signals to which the cells respond with glycerol
production and accumulation. In fact, glycerol biosynthesis is triggered by rapid and
transient lowering in osmotic potential, consequently followed by K+ depletion.
Some species experience changes in membrane potential caused by Na+ efflux and
K+ retention which leads to high internal K+: Na+ ratio as a response to disturbance
in ionic gradient sensed by plasma membrane (Mager and Varela 1993). This loss in
turgor pressure triggers a complex series of molecular events involving protein
kinases, enzyme activation and gene expressions, for example, synthesis of trehalose
and certain heat shock proteins (HSPs) necessary for cell recovery.
Genomic arrays have confirmed the impact of solute on physiological stress by
making possible comparison of up- and downregulated genes under matric stress.
This opened up possibilities for a better understanding of biosynthetic pathways
responsible for active growth and survival in fluctuating ecosystems. In most fungi,
258 S. Sarkar et al.

response to osmotic stress relies on a well-conserved mitogen-activated protein
kinase or MAPK pathway. Differential transcriptome analysis of the filamentous
fungi in steady state under optimal and minimal water conditions has revealed the
following data. A few stress response elements were found to be upregulated, while
baseline expression levels of signal pathways mediated by kinase activities and
direct protein interactions remained rather unaltered. Seven transcriptional activators
and eight downstream genes have been postulated to be involved in stress response
event, including MsnA (Msn2p, Msx4p) as the key regulators (Causton et al. 2001).
Several downstream genes are found to be upregulated like Transmembrane sensor
Msb2p; ROS neutralizing enzyme, SOD,CatA and CatB;GfdA, a NAD + -dependent
glycerol-2-phosphate dehydrogenase necessary for glycerol metabolism (Påhlman
et al. 2001); Pmp3, a cation transporter (Navarre and Goffeau 2000); five genes
putatively involved with DNA repair (orthologues of RSC1,YAF9,RAD52/radC and
bimD); and the Stl1 H+ symport/glycerol transporter (Ferreira et al. 2005).
Genes linked to glycerol metabolism were upregulated sevenfold at low aw, for
example, glycerone kinase (orthologue of dak1) and NAD+ dependent glycerol-3-
phosphate dehydrogenase, GfdA. Both these gene products use dihydroxyacetone
phosphate (DHAP) as a substrate, which is the transition molecule from where the
intermediates of glycolysis are directed to glycerol production either via dihydroxy-
acetone/glycerone or glycerol-3-phosphate pathways. An alternative
FAD-dependent glycerol-3-phosphate dehydrogenase which is also orthologue of
gut2 was also found to be transcriptionally upregulated. In fact, an eightfold increase
in expression of glyceraldehyde-3-phosphate dehydrogenase (gpdA) and phospho-
glycerate kinase (pgkA) had been accounted for. However, X. bisporus possesses
only one copy of the genes responsible for glycerol synthesis. All of these evidence
point towards the hypothesis that, although glycerol production is modulated post-
transcriptionally by direct interactions of cytosolic signal transduction and enzy-
matic activations, reactions leading to DHAP and from DHAP play a major role in
glycerol flux modulation (Bouwman et al. 2011). HogA along with other sensory
and regulatory elements like transmembrane sensor ShoA and transmembrane mucin
Msb2p that act in coordination with actin cytoskeleton also are some widely studied
downstream response elements found in X. bisporus (Tatebayashi et al. 2007;
Tanaka et al. 2014). NikA, a cytoplasmic putative sensor from the sln1 signalling
pathway, is also upregulated in X. bisporus (Hagiwara et al. 2009).
Glycerol synthesis is regulated by a differentially expressing cluster of genes
known as hog genes or high osmolarity glycerol genes via HOG-MAPK stress
response pathway. Membrane osmosensor ShoA senses osmotic stress and activates
a MAPK kinase, Pbs2 which, in turn, phosphorylates HogA, a stress-activated
MAPK. Accumulation of phosphorylated HogA results in a cascade of reactions,
leading to glycerol synthesis and accumulation in the presence of osmotic stress.
However, upon return of favourable water conditions and with restricted energy
source rapid depletion of glycerol by leakage has been accounted alongside conidia
formation, indicating its utilisation as a metabolite (Hocking 1986).
Secretion of exopolysaccharide is another strategy deployed by extremophiles to
adapt to their respective environments (Nicolaus et al. 2010). Secretion of rinsable
13 Xerophilic Fungi: Physiology, Genetics and Biotechnology 259

components by the filamentous fungus is one such strategic move. Experiments have
demonstrated that the majority of the glycerol produced which is approximately
twice the amount of glycerol per colony area leaches out into media when not
required and is re-absorbed by ageing colonies (Hocking 1986), leading to an
increase in mycelial density. In fact, no tight regulation of glycerol synthesis
suggests its constitutive expression which is under allosteric control (Warringer
et al. 2010). Examination of X. bisporus mycelium by scanning electron microscopy
(SEM) has confirmed the presence of this viscous coating (Pettersson et al. 2011).
Additionally, rinsable components (glycerol) secreted around these mycelia lead to
16% gain in a wet weight of the organism, justifying the ‘mycelium density
hypothesis’which is a sure shot strategy for extreme xerophilicity.
Just as important as production, accumulation and retention of compatible
solutes, a certain transition of the membrane lipids and unsaturation index is also
necessary to inhibit massive loss of water, ions and compatible solutes (Gostinčar
et al. 2009). Glycerol retention is altogether a different story as it is dictated by cell
membrane composition and hence permeability. The sterol to phospholipid ratio and
saturation of fatty acids in the lipid bilayer vary greatly to modulate membrane
fluidity in xerophilic fungi. In fact, lower sterol to phospholipid ratio and increased
desaturation favours xerophilicity, as observed in the X. bisporus. This demonstrates
a tendency to increase saturation as an adaptive response. A decrease in the amount
of double-unsaturated linoleic acid and a corresponding increase in monounsaturated
oleic acid helps it achieve a more rigid membrane. Only single copies of the genes
that encode the key enzymes for fatty acid synthesis and modifications are found in
X. bisporus genome, and they are actively transcribed. These are namely fasA and
fasB that code for α-subunit and β-subunit of fatty acid synthase, accA acetyl-CoA
carboxylase, two elongases similar to FEN1 and ELO1, sdeAΔ9-stearic acid
desaturase and odeAΔ12-oleic acid desaturase. All the above-mentioned enzymes
are under post-translational or allosteric control (Cossins et al. 2002;O’Quin et al.
2010).
However, altering the unsaturation index is just one way to regulate membrane
fluidity in response to osmotic stress. Altering components like the lipid head group
or modifying sterol-phospholipid ratio are some other strategies employed by fungi
to achieve membrane rigidity. Differential expression of several genes have been
accounted in X. bisporus, which have putative roles in biosynthesis and metabolism
of phospholipid, sterols and sphingolipids. Ergosterol biosynthesis is modulated by
downregulation of IDI1, an isopentenyl-diphosphate Δ-isomerase, and ERG26, aC3
sterol dehydrogenase/C4 decarboxylase and side-by-side upregulation of ERG3
Δ-desaturase. A flotillin orthologue floA that putatively maintains sterol-rich plasma
domains, and two ceramide metabolism genes are also found to be upregulated. In
fact, differential expressions of genes engaged in biosynthesis and modulation of cell
wall components like four mannosyl transferases contributing to further rigidity, O-
glycosylation and anchorage of membrane proteins via glycosyl-
phosphatidylinositol (Orlean 1990; Bourdineaud et al. 1998; Sutterlin et al. 1998)
and further transcriptional regulation of β-glucanasesSCW11 and DSE4, respec-
tively, again suggest fine control of cell wall remodelling genes (Fig. 13.2).
260 S. Sarkar et al.

Fig. 13.2 Strategic physiological adaptations to water stress in Xerophilus bisporus. Transcriptionally regulated and allosterically controlled expression of
genes and their respective physiological roles leading to osmo-adaptation. The symbol ‘’indicates upregulated genes and ‘ ’ indicates downregulated genes
13 Xerophilic Fungi: Physiology, Genetics and Biotechnology 261

13.4 Bioactive Molecules from Xerophilic Fungi
The fungus Aspergillus felis isolated from rocks of the Atacama Desert were tested
in a microdilution assay against paracoccidioidomycosis causing fungi
Paracoccidioides brasiliensis Pb18. The fungi grown in Potato dextrose agar culture
and extracted with dichloromethane showed a MIC of 1.9 μg/mL against
P. brasiliensis and showed no cytotoxicity against normal mammalian cell line
(Mendes et al. 2016). The xerophilic fungus Chaetomium globosum, thriving as an
endophyte on Ephedra fasciculata (Mormon tea) in Sonoran Desert, is found to
possess three novel esters of orsellinic acid, Globosumones A, B and C. The
compounds Globosumones A and C were found to exhibit a moderate level of
cytotoxic activities against four cancer cell lines, namely MIA Pa Ca-2,
NCI-H460, SF-268 and MCF-7 with IC
50
values ranging from 6.5 to 30.2 μM
against doxorubicin as a positive control (IC
50
0.01–0.07 μM) (Bashyal et al. 2005).
Two new dioxopiperazine derivatives, Arestrictins A and B, are isolated from the
xerophilic fungus Aspergillus restrictus A-17 isolated from house dust; however,
their biological activity is not tested (Itabashi et al. 2006). The fungus Penicillium
citrinum HGY1-5 isolated from the ash of extinct volcano Huguangyan in
Guangdong, China, possessed 11 novel unique C25 steroid isomers with 20-O-
methyl-24-epicyclocitrinol, bicyclo[4.4.1]A/B rings named 24-epi-cyclocitrinol,
12R-hydroxycyclocitrinol, 20-O-methylcyclocitrinol, erythro-23-O-
methylneocyclocitrinol, 24-oxocyclocitrinol, isocyclocitrinol B, threo-23-O-
methylneocyclocitrinol, precyclocitrinol B and neocyclocitrinols B and D. Among
them, the compounds bicyclo[4.4.1]A/B rings named 24-epi-cyclocitrinol,
neocyclocitrinols B and threo-23-O-methylneocyclocitrinol induced the production
of cyclic AMP in GPR12-transfected CHO cells at a concentration of 10 μM (Du et al.
2008).
13.5 Biotechnological Importance of Xerophilic Fungi
Xerophilic fungi have applications in diverse areas related to the industries, environ-
ment, biodeterioration, food spoilage, agriculture, etc.. in the biotechnology field.
The xerophilic fungi have beneficial as well as adverse effects on the environment,
agriculture, human beings, etc., as depicted in Fig. 13.3 and discussed in detail as
follows.
13.5.1 Industrial Aspects
13.5.1.1 Enzyme and Pigment Production
A xerophilic fungus Aspergillus niger GH1 isolated from the Mexican semi-desert is
used for the production of invertase enzyme using substrates molasses and sugarcane
bagasse by solid state fermentation (Veana et al. 2014). Invertase is an important
enzyme which is used as a catalytic agent in the food industry for the development
262 S. Sarkar et al.

artificial sweetener (Ashokkumar et al. 2001). Méndez et al. (2011) reported the red
pigment production by xerophilic fungi Penicillium purpurogenum GH2 which can
be used in the food and textile industry.
13.5.1.2 Air Biofiltration
Janda et al. (2009) have studied the extracellular enzyme activities of some rapid
growing xerophilic fungi, that is, Aspergillus flavus, A. fumigatus, A. melleus,
A. nidulans, A. niger, A. parasiticus and Trichothecium roseum isolated from the
dried medicinal plants procured by herbal shops in Szczecin, Poland. They found
that A. melleus had the utmost hydrolytic activity on milk, tributyrin, gelatin, starch,
rapeseed oil and biodiesel oil agars and A. nidulans showed the highest hydrolase
activity. Among the hydrolases, β-glucosidase activity was the highest, followed by
others such as acid phosphatase, N-acetyl-β-glucosaminidase and naphthol-AS-BI-
phosphohydrolase activities. These xerophilic fungi can utilise various substrates
and therefore have high biodeterioration potential for biotechnological purposes, for
example, in air biofiltration and waste or soil bioremediation. Prenafeta-Boldú et al.
(2018) analysed the potential of xerophilic fungi Cladosporium cladosporioides for
the biofiltration of indoor air. During their study, they found that C. cladosporioides
can remove the total volatile organic compounds (VOCs) content by 96.1%.
Fig. 13.3 Biotechnological importance of xerophilic fungi in diverse fields
13 Xerophilic Fungi: Physiology, Genetics and Biotechnology 263

13.5.2 Environmental Aspects
13.5.2.1 Biodeterioration in Museums and Libraries
Biodeterioration is the undesirable change in the properties of materials due to the
biological agents. In museums as well as in libraries, xerophilic fungi play the most
important role in biodeterioration. Fungal contamination occurs due to exposure of
fungal spores in the air, by contact with contaminated materials or distribution by
vector organisms (e.g. arthropods) (Trovao et al. 2013). The major components of
paper used in books are fibre/fibrous material made up of hemp, cotton, linen,
bagasse, rice straw and wood with functional additives, namely sizing, optical
brighteners and consolidating agents such as gelatin, cellulose acetate, etc. Distinc-
tive fungal infections found in libraries that colonizing documents are the species of
slow-growing Ascomycetes as well as mitosporic xerophilic fungi of the genera
Aspergillus, Paecilomyces, Chrysosporium, Penicillium and Cladosporium (Pinzari
and Montanari 2011). Fungal spores may be viable for many years and will germi-
nate when the environmental conditions favour (Gallo et al. 2003). A xerophilic
fungus Eurotium halophilicum (anamorph Aspergillus halophilicus) has been
identified from the Library of Humanities (BAUM), at Ca0Foscari University,
Venice (Italy). This fungus produces white mycelia growth forming scattered
spots, mostly on books with leather or fabric bindings (Micheluz et al. 2015).
13.5.2.2 Xerophilic Fungi as Sensor/Detector
A fungal index which evaluates climates in the home environment was established
using moderately xerophilic Eurotium herbariorum and extremely xerophilic Asper-
gillus penicillioides. The sensor fungus which exhibited the greatest response in a
fungal detector (a device encapsulating spores of sensor fungi) provides a quantita-
tive and qualitative indicator of the environment tested, representing the type of
fungi that would contaminate the site. A fungal index would also be useful for
detecting wetness which induces fungal contamination, which has side effects on
human health (Abe 2012).
13.5.2.3 Fungal Infections Related to Home Dust
Fungal infections are mostly airborne with significant seasonal variations and high
numbers of spores can gather in the layers of home dust, namely, Penicillium/
Aspergillus/Paecilomyces variotii group, and Aspergillus penicillioides,
Aureobasidium pullulans, Cladosporium cladosporioides and Cladosporium
herbarum (Kaarakainen et al. 2009).
Xerophilic fungi have been isolated from Canadian and Hawaiian house dust
which include 1039 strains out of which 296 strains belong to Aspergillus,
representing 37 species. Aspergillus sect. Aspergillus was one of the most predomi-
nant groups which were isolated. Among all strains, two new species were found,
namely, A. mallochii and A. megaspores (Visagie et al. 2017). The disease occurring
due to fungal allergy is allergic bronchopulmonary aspergillosis usually caused by
A. fumigates which is an omnipresent indoor and outdoor fungus. This is an
inflammatory disease that mainly occurs in patients with asthma (up to 13% of
264 S. Sarkar et al.

asthmatic patients) or cystic fibrosis due to the poor clearance of secretions from the
airways (Agarwal and Chakrabarti 2013).
13.5.3 Health Aspects
13.5.3.1 Food and Food Products Spoilage
Eurotium species (previously known as ‘Aspergillus glaucus group’) are the most
universal foodborne genera with Aspergillus anamorphs (imperfect states). All the
species of Aspergillus glaucus are xerophilic and cause spoilage in low aw foods as
well as stored products (Hocking 2006). Wallemia spp. is now known as important
spoilage organisms for foods with low aw (Pitt and Hocking 2009). Moderate
xerophiles of the genera Aspergillus, Eurotium and Penicillium are the main spoilage
organisms. Some xerophiles are isolated from both high-salt and high-sugar
environments; others have a preference for either salt or sugar. Xerophiles can
cause spoilage of high sugary products, high salted foods, as well as stored food
and feed (Pettersson et al. 2011).
Ismail et al. (2012) analysed 55 samples from five baby food products chiefly
made of cereal flour(s) and found that the incidence of xerophilic fungi was 88% of
food samples. The highest contamination level by xerophiles was found in Mwebaza
rice porridge (a component of rice flour) and the lowest in Mukuza (a product of
maize, soyabean and sorghum flours). Eleven xerophilic species were found of
which Aspergillus and Eurotium (four species each) were the predominant, resulting
in 9.1% and 8.9% of the total CFU. Contamination of such foods is a matter of great
concern as these foods are used for babies.
Wallemia sebi is a common xerophile in cereals and spices, and Aspergillus
penicillioides as well as Aspergillus restrictus are early colonizers of stored
products. Nuts are more vulnerable to attack by Aspergillus spp.,especially Asper-
gillus flavus, Aspergillus niger, Aspergillus candidus, Aspergillus ochraceus and the
xerophiles, namely, Wallemia sebi and Aspergillus penicillioides (Hocking 2014).
The xerotolerant, xerophilic and halophilic Wallemia spp. are found in various
osmotically challenging conditions, such as dry, salted or highly sugared foods,
dry feed, salt crystals, indoor as well as outdoor air and agriculture aerosols globally.
Recently, eight species were recognised for the genus Wallemia, among which four
are commonly associated with foods: W. sebi, W. mellicola, W. muriae and
W. ichthyophaga (Zajc and Gunde-Cimerman 2018).
13.5.3.2 Mycotoxin Production
Mycotoxins are mainly present in the spores of fungal growth and in airborne dust
which affect the human’s respiratory system. These mycotoxins can cause the
allergological problem known as ‘farmer’s lung disease’and can also promote
infections in immunocompetent humans. Farmer’s lung disease may be combined
with pulmonary fibrosis which includes symptoms, that is, bronchial asthma, cough,
tiredness, headaches and fever/night sweats which depend on the severity of the
disease (Guarro et al. 2008; Gbaguidi-Haore et al. 2009). Xerophilic fungal species
13 Xerophilic Fungi: Physiology, Genetics and Biotechnology 265

are the prevailing species in crops under storage conditions when aw drops. After
harvest xerophilic fungi, namely Aspergillus spp. and Penicillium spp., germinate
and produce mycotoxins at a relative humidity of 80–90% and less (Manna and Kim
2017).
Al-Sohaibani et al. (2011) investigated black tea procured from the local markets
of Tamilnadu, India, for fungal contamination and reported the presence of two
xerophilic aflatoxigenic fungi, namely, Aspergillus niger ML01 and A. flavus ML02.
These xerophilic fungi are highly dangerous contaminants of tea because they are
related to tea quality deterioration which ultimately leads to serious health risk.
Ochratoxins are a group of mycotoxins produced by Penicillium verrucosum and
various species of Aspergillus moulds, namely, A. alliaceus, A. auricomus,
A. carbonarius, A. glaucus, A. melleus and A. niger which contaminate the crops
in the field and during storage (Peraica 2016). Jančičet al. (2016) investigated the
production of secondary metabolites produced by seven species of the genus
Wallemia, namely, W. sebi, W. muriae, W. mellicola, W. tropicali, W. canadensis,
W. hederae and W. ichtyhophaga at hypersaline conditions. They found that the
Wallemia spp. produce toxic metabolites, namely, walleminol, walleminone and
wallimidione, and their production increases proportionally to increased
concentrations of NaCl. Therefore, Wallemia spp. can be considered as a serious
health risk associated with food having high salt concentration.
Two xerophilic fungus Aspergillus chevalieri and Aspergillus amstelodami have
been isolated from peanuts which probably reduce their shelf life as well as the
quality of the kernels. The incidence of these two fungi on peanuts provides
favourable conditions for less xerophilic Aspergillus and other spoilage-related
fungal genera, particularly mycotoxin-producing species which lead to contamina-
tion of mycotoxin (Kamarudin and Zakaria 2018).
13.6 Conclusion
Most of the environments on Earth are populated by fungi due to their broad
adaptational amplitude. Xerophilic fungi are unique organism with the ability to
grow under conditions of aw. Their life cycles are completed on substrates having
high levels of soluble solids such as salts or sugars and as a result became dried or
concentrated. Some xerophilic fungi like Z. rouxii, H. werneckii and Wa. sebi are so
exceptionally adaptable that they can withstand water stress imposed by both salt
and sugar, and also diverse ranges of water activities. Other species are more
environment dependent and display a much narrower adaptation amplitude, with a
strong preference for a particular substrate.
Despite their huge importance in various areas of biotechnology, various related
aspects of xerophilic fungi are underexplored. It is also noteworthy that filamentous
fungi are hugely ignored as compared to others in the same group like yeast and their
adaptation to water stress. The area of xerophilic fungus and its importance demands
more studies exploring the utility in industries like food and medicine. Also, it has a
vital impact on the enterprises having recreational importance like museums and
266 S. Sarkar et al.

music instruments. The chapter has discussed, in detail, the xerophilic fungi and their
physiology, genetics, adaptation and industrially important bioactive metabolites.
The biotechnological importance of xerophilic fungi is also discussed in the context
of industry, environment and health.
Acknowledgement The authors are grateful to the authorities of Mohanlal Sukhadia University,
Udaipur, for supporting this work.
Conflict of Interest The authors declare no conflict of interest.
References
Abe K (2012) Assessment of home environments with a fungal index using hydrophilic and
xerophilic fungi as biologic sensors. Indoor Air 22:173–185
Abe F, Horikoshi K (2001) The biotechnological potential of piezophiles. Trends Biotechnol 19:
102–108
Agarwal R, Chakrabarti A (2013) Allergic bronchopulmonary aspergillosis in asthma: epidemio-
logical, clinical and therapeutic issues. Future Microbiol 8:1463–1474
Al-Sohaibani S, Murugan K, Lakshimi G, Anandraj K (2011) Xerophilic aflatoxigenic black tea
fungi and their inhibition by Elettaria cardamomum and Syzygiumaromaticum extracts. Saudi J
Biol Sci 18:387–394
Andersen MR, Nielsen JB, Klitgaard A, Petersen LM, Zachariasen M, Hansen TJ, Blicher LH,
Gotfredsen CH, Larsen TO, Nielsen KF, Mortensen UH (2013) Accurate prediction of second-
ary metabolite gene clusters in filamentous fungi. Proc Natl Acad Sci U S A 110(1):E99–E107.
https://doi.org/10.1073/pnas.1205532110. Epub 2012 Dec 17. PMID: 23248299; PMCID:
PMC3538241
Ashokkumar B, Nagarajan K, Paramasamy G (2001) Optimization of media for
β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation.
Process Biochem 37:331–338
Bashyal BP, Wijeratne EMK, Faeth SH, Gunatilaka AAL (2005) Globosumones ac, cytotoxic
orsellinic acid esters from the sonoran desert endophytic fungus Chaetomium globosum. J Nat
Prod 68:724–728
Bourdineaud JP, Der Vaart V, Marcel J, Donzeau M, De Sampaïo G, Verrips CT (1998) Pmt1
mannosyl transferase is involved in cell wall incorporation of several proteins in Saccharomyces
cerevisiae. Mol Microbiol 27:85–98
Bouwman J, Kiewiet J, Lindenbergh A, Van Eunen K, Siderius M, Bakker BM (2011) Metabolic
regulation rather than de novo enzyme synthesis dominates the osmo-adaptation of yeast. Yeast
28:43–53
Brewer MS (1999) Traditional preservatives—sodium chloride. Encyclopaedia of food microbiol-
ogy, vol 3. Academic, London
Causton HC, Ren B, Koh SS, Harbison CT, Kanin E, Jennings EG (2001) Remodeling of yeast
genome expression in response to environmental changes. Mol Biol Cell 12:323–337
Cossins AR, Murray PA, Gracey AY, Logue J, Polley S, Caddick M (2002) The role of desaturases
in cold-induced lipid restructuring. Biochem Soc Trans 30:1082–1086
Cray JA, Russell JT, Timson D, Singhal RS, Hallsworth JE (2013) A universal measure of
chaotropicity and kosmotropicity. Environ Microbiol 15:287–296
Du L, Zhu T, Fang Y, Gu Q, Zhu W (2008) Unusual C25 steroid isomers with bicycle [4.4.1]a/b
rings from a volcano ash-derived fungus Penicillium citrinum. J Nat Prod 71:1343–1351
13 Xerophilic Fungi: Physiology, Genetics and Biotechnology 267

Ferreira C, van Voorst F, Martins A, Neves L, Oliveira R, Kielland-Brandt MC (2005) A member of
the sugar transporter family, Stl1p is the glycerol/H + symporter in Saccharomyces cerevisiae.
Mol Biol Cell 16:2068–2076
Fox EM, Howlett BJ (2008) Secondary metabolism: regulation and role in fungal biology. Curr
Opin Microbiol 11:481–487
Gallo F, Pasquariello G, Valenti P (2003) Libraries and archives. In: Cultural heritage and
aerobiology. Springer, Dordrecht, pp 175–193
Gbaguidi-Haore H, Roussel S, Reboux G, Dalphin JC, Piarroux R (2009) Multilevel analysis of the
impact of environmental factors and agricultural practices on the concentration in hay of
microorganisms responsible for farmer's lung disease. Ann Agric Environ Med 16:219–225
Gostinčar C, Turk M, Gunde-Cimerman N (2009) Environmental impacts on fatty acid composition
of fungal membranes. In: Fungi from different environments, pp 278–325
Gründlinger M, Yasmin S, Lechner BE, Geley S, Schrettl M, Hynes M, Haas H (2013) Fungal
siderophore biosynthesis is partially localized in peroxisomes. Mol Microbiol 88:862–875
Guarro J, Gugnani HC, Sood N, Batra R, Mayayo E, Gene J, Kakkar S (2008) Subcutaneous
phaeohyphomycosis caused by Wallemiasebi in an immunocompetent host. J Clin Microbiol
46:1129–1131
Hagiwara D, Mizuno T, Abe K (2009) Characterization of NikA histidine kinase and two response
regulators with special reference to osmotic adaptation and asexual development in Aspergillus
nidulans. Biosci Biotechnol Biochem 73:1566–1571
Hocking AD (1986) Effect of water activity and culture ageon the glycerol accumulation patterns in
five fungi. J Gen Microbiol 132:269–275
Hocking AD (2006) Aspergillus and related teleomorphs. In: Food spoilage microorganisms, pp
451–487
Hocking AD (2014) Spoilage problems: problems caused by fungi. In: Batt CA, Tortorello ML
(eds) Encyclopedia of food microbiology. Academic Press, Oxford, pp 471–481
Inglis DO, Binkley J, Skrzypek MS, Arnaud MB, Cerqueira GC, Shah P, Sherlock G (2013)
Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of
Aspergillus nidulans, A. fumigatus, A. nigerand A. oryzae. BMC Microbiol 13:1–23
Ismail MA, Taligoola HK, Nakamya R (2012) Xerophiles and other fungi associated with cereal
baby foods locally produced in Uganda. Acta Mycology 47:75–89
Itabashi T, Matsuishi N, Hosoe T, Toyazaki N, Udagawa S, Imai T, Adachi M, Kawai K (2006)
Two new dioxopiperazine derivatives, arestrictins a and b, isolated from Aspergillus restrictus
and Aspergillus penicilloides. Chem Pharm Bull 54:1639
JančičS, Frisvad JC, Kocev D, Gostinčar C, Džeroski S, Gunde-Cimerman N (2016) Production of
secondary metabolites in extreme environments: food-and airborne Wallemia spp. produce toxic
metabolites at hypersaline conditions. PLoS One 11:12
Janda K, Ulfig K, Markowska-Szczupak A (2009) Further studies of extracellular enzyme profiles
of xerophilic fungi isolates from dried medicinal plants. Polish J Environ Stud 18:627–633
Kaarakainen P, Rintala H, Vepsäläinen A, Hyvärinen A, Nevalainen A, Meklin T (2009) Microbial
content of house dust samples determined with qPCR. Sci Total Environ 407:4673–4680
Kamarudin NA, Zakaria L (2018) Characterization of two xerophilic Aspergillus spp. from peanuts
(Arachis hypogaea). Malaysian J Microbiol 14:41–48
Khaldi N, Seifuddin FT, Turner G, Haft D, Nierman WC, Wolfe KH, Federova ND (2010)
SMURF: genomic mapping of fungal secondary metabolite clusters. Fungal Genet Biol 47:
736–741
Leong SL, Pettersson OV, Rice T, Hocking AD, Schnürer J (2011) The extreme xerophilic mould
Xeromycesbisporus–growth and competition at various water activities. Int J Food Microbiol
145:57–63
Leong SLL, Lantz H, Pettersson OV, Frisvad JC, Thrane U, Heipieper HJ, Schnürer J (2015)
Genome and physiology of the ascomycete filamentous fungus Xeromycesbisporus, the most
xerophilic organism isolated to date. Environ Microbiol 17:496–513
268 S. Sarkar et al.

Magan N, Aldred D, Sanchis V (2004) Role of spoilage fungi in seed deterioration. In: Fungal
biotechnology in agricultural, food and environmental applications, pp 311–123
Mager WH, Varela JCS (1993) Osmo-stress response of the yeast saccharomyces. Mol Microbiol
10:253–258
Manna M, Kim KD (2017) Control strategies for deleterious grain fungi and mycotoxin production
from pre-harvest to post-harvest stages of cereal crops: a review. Life Sci Nat Resour Res 25:13–
27
Mendes G, Gonçalves VN, Souza-Fagundes EM, Kohlhoff M, Rosa CA, Zani CL, Cota BB, Rosa
LH, Johann S (2016) Antifungal activity of extracts from Atacama Desert fungi against
Paracoccidioides brasiliensis and identification of Aspergillus felis as a promising source of
natural bioactive compounds. Mem Inst Oswaldo Cruz 111:209–217
Méndez A, Pérez C, Montañéz JC, Martínez G, Aguilar CN (2011) Red pigment production by
Penicillium purpurogenum GH2 is influenced by pH and temperature. J Zhejiang Univ Sci B 12:
961–968
Micheluz A, Manente S, Tigini V, Prigione V, Pinzari F, Ravagnan G, Varese GC (2015) The
extreme environment of a library: Xerophilic fungi inhabiting indoor niches. Interna Biodete
Biodegra 99:1–7
Navarre C, Goffeau A (2000) Membrane hyperpolarization and salt sensitivity induced by deletion
of PMP3, a highly conserved small protein of yeast plasma membrane. EMBO J 19:2515–2524
Nicolaus B, Kambourova M, Oner ET (2010) Exopolysaccharides from extremophiles: from
fundamentals to biotechnology. Environ Technol 31:1145–1158
O’Quin JB, Bourassa L, Zhang D, Shockey JM, Gidda SK, Fosnot S (2010) Temperature-sensitive
post-translational regulation of plant omega-3 fatty-acid desaturases is mediated by the endo-
plasmic reticulum associated degradation pathway. J Biol Chem 285:21781–21796
Orlean P (1990) Dolichol phosphate mannose synthase is required in vivo for glycosyl
phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein
in Saccharomyces cerevisiae. Mol Cell Biol 10:5796–5805
Påhlman AK, Granath K, Ansell R, Hohmann S, Adler L (2001) The yeast glycerol 3-phosphatases
Gpp1p and Gpp2p are required for glycerol biosynthesis and differentially involved in the
cellular responses to osmotic, anaerobic, and oxidative stress. J Biol Chem 276:3555–3563
Peraica M (2016) Mycotoxicoses. In: Environmental mycology in public health. Academic Press,
pp 45–49
Pettersson OV, Leong SL, Lantz H, Rice T, Dijksterhuis J, Houbraken J (2011) Phylogeny and
intraspecific variation of the extreme xerophile, Xeromycesbisporus. Fungal Biol 115:1100–
1111
Pinzari F, Montanari M (2011) Mould growth on library materials stored in compactus-type
shelving units. In: Sick building syndrome. Springer, Berlin, Heidelberg, pp 193–206
Pitt JI, Christian JHB (1968) Water relations of xerophilic fungi isolated from prunes. Appl
Microbiol 16:853–1858
Pitt JI, Hocking AD (2009) Fungi and food spoilage. Springer, New York
Prenafeta-Boldú FX, De Hoog GS, Summerbell RC (2018) Fungal communities in hydrocarbon
degradation. In: Microbial communities utilizing hydrocarbons and lipids: members,
metagenomics and ecophysiology, handbook of hydrocarbon and lipid microbiology. Springer,
Cham, pp 1–36
Rico-Munoz E, Samson RA, Houbraken J (2019) Mould spoilage of foods and beverages: using the
right methodology. Food Microbiol 81:51–62
Rothschild L, Mancinelli R (2001) Life in extreme environments. Nature 409:1092–1101
Su-lin LL, Pettersson OV, Rice T, Hocking AD, Schnürer J (2011) The extreme xerophilic mould
Xeromycesbisporus—growth and competition at various water activities. Int J Food Microbiol
145:57–63
Sutterlin C, Escribano M, Gerold P, Maeda Y, Mazon M, Kinoshita T (1998) Saccharomyces
cerevisiae GPI10, the functional homologue of human PIG-B, is required for
glycosylphosphatidylinositol-anchor synthesis. Biochem J 332:153–159
13 Xerophilic Fungi: Physiology, Genetics and Biotechnology 269

Tanaka K, Tatebayashi K, Nishimura A, Yamamoto K, Yang HY, Saito H (2014) Yeast
osmosensors hkr1 and msb2 activate the hog1 MAPK cascade by different mechanisms. Sci
Signal 7:21
Tatebayashi K, Tanaka K, Yang HY, Yamamoto K, Matsushita Y, Tomida T (2007) Transmem-
brane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG
pathway. EMBO J 26:3521–3533
Trovao J, Mesquita N, Paiva DS, Paiva de Carvalho H, Avelar L, Portugal A (2013) Can arthropods
act as vectors of fungal dispersion in heritage collections? A case study on the archive of the
University of Coimbra, Portugal. Int Biodeterior Biodegradation 79:49–55
Trüper HG, Galinski EA (1990) Biosynthesis and fate of compatible solutes in extremely halophilic
phototrophic bacteria. FEMS Microbiol Rev 75:181–186
Van den Burg B (2003) Xtremophiles as a source for novel enzymes. Curr Opin Microbiol 6:213–
218
Veana F, Martínez-Hernández JL, Aguilar CN, Rodríguez-Herrera R, Michelena G (2014) Utiliza-
tion of molasses and sugar cane bagasse for production of fungal invertase in solid state
fermentation using aspergillus Niger GH1. Braz J Microbiol 45:373–377
Visagie CM, Yilmaz N, Renaud JB, Sumarah MW, Hubka V, Frisvad JC, Chen AJ, Meijer M,
Seifert KA (2017) A survey of xerophilic Aspergillus from indoor environment, including
descriptions of two new section Aspergillus species producing eurotium-like sexual states.
Myco Keys 19:1–30
Warringer J, Hult M, Regot S, Posas F, Sunnerhagen P (2010) The HOG pathway dictates the short-
term translational response after hyperosmotic shock. Mol Biol Cell 21:3080–3092
Yu Y, Keller NP (2005) Regulation of secondary metabolism in fungi. Annu Rev Phytopathol 43:
437–458
Zajc J, Gunde-Cimerman N (2018) The genus Wallemia—from contamination of food to health
threat. Microorganisms 6:46
Zak JC, Wildman HG (2004) Fungi in stressful environments. In: Biodiversity of fungi: inventory
and monitoring methods. Academic Press, Burlington, pp 303–315
270 S. Sarkar et al.

Part III
Applications

Extremophilic Enzymes: Catalytic Features
and Industrial Applications 14
Kanak Choudhary, Mangesh Kumar Mankar, and Sanjay Sahay
Abstract
Extremophilic microbes are those that are adapted to very harsh environmental
conditions. Fungi constitute one of the important groups that by virtue of various
adaptation strategies learn to live in extreme environment and serve important
ecological functions. Their presence and activity are also essential for a large
variety of flora and fauna of these harsh environments. Although microbes have
developed a variety of strategies to successfully lead life in these extreme
conditions, the enzymes with unique combination of catalytic features they
have developed have especially enabled them to drive all metabolic and ecologi-
cal under extreme conditions. The chapter highlights some important catalytic
features found across a large number of important enzymes.
Keywords
Psychrophilic enzymes · Thermophilic enzymes · Alkaliphilic enzymes ·
Acidophilic enzymes · Halophilic enzymes · Radioresistant enzymes
14.1 Introduction
An organism needs physiological conditions that are essential for their survival.
Physiological conditions such as moderate temperature (10–37 C), near neutral pH
(pH between 6 and 8), pressure (1 atm), salinity (0.15–0.5 M NaCl), and availability
K. Choudhary · M. K. Mankar
Department of Biotechnology, Barkatullah University, Bhopal, MP, India
S. Sahay (*)
Sarojini Naidu Government Postgraduate Girl’s (Autonomous) College, Bhopal, Madhya Pradesh,
India
e-mail: ss000@redffmail.com
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_14
273

of water are required. Organisms that grow and survive under these moderate
environmental conditions are known as mesophiles or neutrophiles. However,
many bacteria, archaea, and eukaryotic organisms are found to survive and thrive
in toxic-heavy metal waste, refrigerated food, deep sea, solar salterns, hot and cold
deserts, and soda lakes. These environmental conditions are known as extreme
conditions, which are further categorized into physical and geochemical extremes.
Physical extreme involves extreme temperatures (very cold or very hot), high
pressure, and high level of radiations while geochemical extreme involves environ-
ment with high salinity and extreme pH (very low or very high). Extremophiles are
the organisms that can thrive under these physically and geochemically extreme
environments. The extremophiles can tolerate these nonphysiological conditions that
are inhospitable or lethal for the survival of mesophiles. Extremophiles need these
extreme conditions to grow while extremotolerants grow optimally at moderate
environment but can tolerate extreme conditions. Extremophiles are distributed
among all three domains of life that is bacteria, archaea, and eukarya. Most of the
eukaryotic extremophiles belong to fungi and algae (Kumar et al. 2011; Rampelotto
2013; Orellana et al. 2018).
They have evolved or adapted to survive under these extreme conditions. They
possess wide and versatile metabolic diversity. They are model organisms for the
study of the existence of extraterrestrial life. Most extremophiles can thrive and
survive under more than one type of extreme conditions, which are known as
polyextremophiles such as thermoacidophiles that thrive under high temperature
along with very low pH environment and haloalkaliphiles that thrive under high salt
concentration along with high pH environment. Extremophiles are important for the
study of mechanism of adaptation in extreme environment, metabolic pathways, and
novel products or metabolites. Extremophiles are the source of extreme enzymes
known as extremozymes and have ability to remain stable and active under these
conditions. Extremozymes adapted themselves to function or catalyze a reaction
optimally in extreme environment. When mesophilic enzymes are applied to
non-natural or nonphysiological conditions, their stability and catalysis is reduced.
In this case, extremozymes that have extreme stability become a key to overcome
this problem. Extremophiles are able to maintain neutral pH in their internal cellular
environment; thus, the intracellular enzymes do not possess any unique structural
adaptations. Therefore, extracellular extremozymes with unique structural properties
are used in many biotechnological industries. However, they do not possess any
major structural variations as the residues that build the active site are conserved.
Extremozymes make the biocatalysis specific, faster, and environmental friendly.
One another advantage to study the properties of extremozymes is to design novel
enzymes that possess similar properties with extremozyme (Gupta et al. 2014).
274 K. Choudhary et al.

14.2 Types of Extremophiles
Extremophiles are classified according to the extreme conditions. Thermophiles are
the organisms that can thrive under high-temperature environment with temperature
range between 45 and 80 C while hyperthermophiles can thrive under very high
temperature, that is, >80 C. To maintain the stability under high temperature, they
possess high GC content and more hydrophobic core in protein. They protect the cell
by synthesizing heat shock proteins. Psychrophiles are the organisms that can thrive
under very low-temperature condition <10

C. They grow at temperature between
-20 C and 20 C and possess optimal growth temperature <15 C. They maintain
the membrane fluidity by increasing the amount of unsaturated fatty acids in their
membrane. Accumulation and synthesis of cryoprotectants, anti-freeze proteins,
cold-shock proteins, and chaperons protect their RNA and proteins. High catalytic
activity, low thermostability at moderate temperature, and increased flexibility of
psychrophilic protein make them feasible for industrial application at lower temper-
ature. Psychrophilic enzymes are very economic as they decrease the energy con-
sumption. Some extremophiles can thrive in extreme pH. Those that thrive under
very high pH or alkaline environment are known as alkaliphiles while those that
thrive under very low pH or acidic environment are known as acidophiles.
Alkaliphiles acidify the cytosol as to neutralize the effect of high pH and protect
the cell by preventing the entry of hydroxide ions. Acidophiles protect themselves
from high proton by efficient proton pumping that rapidly remove the protons from
the cytoplasm. Halophiles are the extremophiles that thrive under high salt environ-
ment or environment with high ionic strength (higher than of seawater). They
prevent desiccation by increasing the osmotic concentration of the cell by
accumulating osmoprotectants or compatible solutes and selective influx of ions in
the cytoplasm. Some extremophiles thrive in environment rich in metals or heavy
metals, and high level of radiations are known as metallophiles and radioresistants,
respectively. Presence of heavy metals such as Zn, Cd, Hg, Pb, Ag, Co, and Cr make
the environment very toxic. Metallophiles, natural habitants of metal-rich environ-
ment, utilize these toxic-heavy metals and remove them from various heavy metal
contaminant areas. Generally high metal concentration inhibits the growth and
functioning of microbes, but metallophiles adapt the strategies to function optimally
at these conditions. Some metallophiles possess efficient efflux pumps for the rapid
removal of toxic metals while others associate these metals by binding them with
protein molecules. Metallophiles are used for ore-bioleaching, bioremediation, bio-
mineralization, and biomining of expensive metals from industrial effluents. High
level of radiations, mainly ultraviolet and gamma radiation, produces oxidative
stress that causes protein denaturation and mutations through DNA damage. UV
radiations are more lethal as they damage the nucleic acid of an organism.
Radiophiles or radioresistant organisms protect themselves from this radiation by
efficient DNA repair machinery, protecting substances such as chaperons or
carotenoids and active defense system for radiation stress. Radioresistant organisms
have the potential for bioremediation of radionuclide-contaminated sites and degra-
dation of organopollutants in radioactive waste. Organisms that can thrive under
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 275

high hydrostatic pressure are known as piezophiles or barophiles. They can opti-
mally grow under high pressure of 400 atm or more. Extreme barophiles grow
optimally at hydrostatic pressure 700 atm or more. They survive by developing
pressure gradient inside and outside of the cell and regulating membrane phospho-
lipid fluidity. Piezophilic proteins are adapted to specific stabilization in the presence
of higher pressure. This property of proteins is used by food industries for processing
and sterilization of food materials at high pressure (Oarga 2009; Rampelotto 2013;
Gupta et al. 2014; Parihar and Bagaria 2019).
14.3 Enzymes as Key to Metabolism
Metabolism is a series of biochemical reactions involved in the process of
synthesizing and breaking down compounds enabling cells to survive and reproduce.
Biochemical reactions occurring in cells are not catalyzed spontaneously. All cellu-
lar processes are carried out through biochemical reactions; the latter needs a specific
protein or a catalyst to precede that is known as enzyme. These proteins speed up the
rate of reaction by lowering the activation energy of the reaction without being
changed during the whole process. They are very specific to a reaction they
catalyzed. They do not shift the equilibrium of a reaction instead they help to attain
the equilibrium quickly. Various enzymes work together to form a metabolic
pathway that comprises a long chain of enzyme-catalyzed reactions. Metabolism
involves two types of reactions: anabolic reactions and catabolic reactions. Anabolic
reactions are usually endothermic that requires an input of energy for synthesizing
compounds while catabolic reactions are exothermic releasing energy after the
breakdown of complex organic molecules. Metabolism keeps the organism
maintained. Enzymes are very essential in metabolism as they perform two impor-
tant functions. They synthesize a large complex molecule from multiple substrates
and synthesize multiple products from a large substrate. The rate of anabolic and
catabolic reactions is dependent on enzyme and substrate concentration so that by
altering their concentration the metabolic rate can be controlled. The metabolic rate
can be regulated by feedback inhibition in which high product concentration signals
to slow down the process (Molnar and Gair 2012).
14.4 Catalytic Features of Enzyme
14.4.1 Effect of Temperature
Enzymes are proteinous in nature, and proteins are very sensitive to thermal changes.
They catalyze a reaction at optimum temperature, which is specific for each catalyst.
The optimal temperature is a particular temperature at which an enzyme shows
maximum activity. Initially, increase in temperature enhances the rate of reaction
as the kinetic energy of enzyme molecule increases with slight increase in tempera-
ture. Elevated temperature increases the vibrational energy of enzyme molecules that
276 K. Choudhary et al.

exert a pressure on hydrogen and ionic bonds present in enzyme. This pressure
breaks the bonds that causes change in active site of an enzyme and increased kinetic
energy denatures the protein’s three-dimensional structure also. Substrate molecules
are not able to bind with this altered active site. Thus at high temperature, the enzyme
loses its catalytic properties and the structure gets denatured. While at lower
temperature the enzyme becomes inactivated. However, rise in temperature allows
enzyme to reactivate (Robinson 2015).
14.4.2 Effect of pH
Enzymes work at specific pH and show maximum activity at optimum pH. Enzymes
are mostly active at or near neutral pH. Fluctuation in pH rapidly decreases the rate
of reaction as the concentration of H
+
and OH

ions interferes with the hydrogen and
ionic bond leads to the ionization of the functional group present on active site that
causes change in the shape of enzyme active site. The altered active site is no more
complementary with substrate, hence rate of reaction decreases. Small changes do
not cause permanent change as the broken bonds are reformed at original pH but
major changes in pH cause enzyme denaturation and enzyme permanently lose its
function (Robinson 2015).
14.4.3 Cofactors and Coenzymes
Most of the enzyme-catalyzed reactions cannot proceed without binding to a non-
protein molecule. This nonprotein molecule is known as cofactor while enzymes in
this kind of reaction are known as apozyme. Both apozyme and nonprotein part
make a complete catalytic part known as holozyme. Apozyme alone cannot catalyze
a reaction as their activity is dependent on nonprotein part. Cofactors can be organic
or inorganic and help enzyme to perform a reaction. Cofactors are categorized into
two parts: coenzymes that is organic molecule loosely binds to the active site of an
enzyme and metalloorganic compounds in which metal ions bind with the enzymes
and accelerate the enzyme-catalyzed reactions such as iron for catalase, zinc for
DNA polymerase, and magnesium for hexokinase. They are recycled and participate
in multiple enzymatic reactions. Some coenzymes bind reversibly with the active site
such as thiamine pyrophosphate while some bind to the enzyme other than active site
such as nicotinamide adenine dinucleotide. Coenzyme binds mostly by noncovalent
linkages with the enzyme. However, those that tightly bind with the enzyme by
covalent linkages are known as prosthetic groups such as heme group in hemoglobin
(Broderick 2001). In brief, cofactors and coenzymes are small nonprotein molecules
that enhance the enzyme’s function.
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 277

14.4.4 Inhibitions
Some molecules bind to the catalyst and directly or indirectly interfere with the
catalytic property of enzyme, thus reducing the activity of enzyme-catalyzed reac-
tion. These molecules are known as inhibitors, and the process is known as inhibi-
tion. Although high concentration of substrate or products can also inhibit the
enzyme activity. Competitive inhibition is a process of binding of enzyme substrate
analog to the active site of enzyme. In noncompetitive inhibition, inhibitor binds
with the enzyme at site other than active site. These inhibitors reduce the active
enzyme concentration and Vmax value but Km value is not altered. Uncompetitive
inhibition involves the binding of an inhibitor to the enzyme–substrate complex.
This reduces the Vmax and Km value that interferes with the equilibrium. In
irreversible inhibition, inhibitors permanently bind with the enzyme. This kind of
inhibition acts as a potent toxin for enzymes (Robinson 2015).
14.4.5 Co-Cooperativity
For enzymes that contain more than one active site, the phenomenon of binding of a
substrate to first active site influencing the binding of substrate of other site is known
as cooperativity. This simply means that binding of one substrate changes the
affinity for other substrate or fluctuations in affinity for binding of the other sites is
affected by the original binding site. The influence may be positive or negative. In
positive cooperativity, binding of first substrate increases the affinity for subsequent
substrate. An example of positive cooperativity is found in globin chain of hemo-
globin that facilitates the successive binding of oxygen molecule to alpha and beta
globin chains. In negative cooperativity, binding of first substrate decreases the
affinity for subsequent substrate (Bush et al. 2012).
14.5 Thermophilic Enzymes
14.5.1 Thermophiles
Thermophiles are the microorganisms that thrive under extreme temperatures. Mod-
erate thermophiles optimally grow at 50–60 C, extreme thermophiles optimally
grow at 60–80 C, and hyperthermophiles optimally grow at 80–110 C. They are
commonly found in thermal vents, hot springs, volcanic lakes, and boiling steam
vents (Kumar et al. 2011).
14.5.1.1 Catalytic Features of Thermophilic Enzymes/Proteins
Thermophilic enzymes can tolerate many extreme conditions. They are active and
stable even in high ionic strength medium, presence of denaturing agents, and
organic solvents. Thermophilic proteins possess high content of helices. Arginine
(propensity value 1.33) is a helix-favoring residue present in large amount in
278 K. Choudhary et al.

thermophilic proteins while cysteine (propensity value 0.87) and histidine (propen-
sity value 0.76) residues are present in negligible amount as they are unfavorable for
helix formation. Proline frequency in thermophilic α-helices is 0.7% as compared to
mesophilic protein for which the frequency of proline residue is 1.3%. This low
frequency of proline residues protects the helix from kinks. Thermolabile amino
acids asparagine, glutamine, methionine, and cysteine are present in very less
amount as they possess the ability to undergo oxidation or deamidation at elevated
temperature. Arginine and tyrosine are responsible for binding and folding of
proteins at high temperature that provide the stability to thermophilic proteins
(Kumar et al. 2000). Elevated temperature causes covalent modification in unstable
amino acids leading to denaturation of proteins. Some amino acid replacements
provide flexibility to these proteins due to their additional noncovalent interaction.
The amino acids alanine, threonine, and arginine are more common in thermophilic
proteins. Increased hydrophobicity is a feature of thermophilic protein. Hydrophobic
core provides tight packing of protein that possesses smaller cavities, resulting in
compact structure. Disulfide bonds form in between the cysteine residues. They are
important in oligomerization of protein. They provide rigidity to overall structure
and resist protein to unfold at high temperature. Increased number of disulfide bonds
prevents the alteration in quaternary structure of protein. Salt bridging allows the
interaction of charge–charge residues that increases the thermal stability of protein.
An increased surface charge provides stabilization and prevents aggregation of
protein (Reed et al. 2013). To prevent enzyme from nonspecific interactions, deep
sea thermophilic enzymes have shorter loops. Comparative study of mesophilic and
thermophilic proteins shows high alanine and leucine residues in thermophiles.
Many metal-dependent structures are bound with the membrane that provides
stability at high temperature (Li et al. 2019). Presence of tightly packed internal
core, tandem repeats, thermo stabilizing domains, and disulfide bridges at the ends or
α-helix region increases the activity of thermophilic enzymes (Collins et al. 2005).
As the rigidity in the thermophilic enzymes is high, rate of exchange of amide
protons becomes low. This will increase the resistance for proteolysis and denatur-
ation. Thermophilic enzymes are only marginally temperature dependent. Active site
amino acid residues are found to be conserved. Improved packing density and
specific local interactions are responsible for the thermostability (Jaenicke 1991;
Jin et al. 2019). Protein dynamics is inversely related with the protein stability. More
dynamic structure of a protein leads to the lower stability of that protein (Cipolla
et al. 2012). A study on thermophilic acylphosphatase suggests that the salt bridge
present in the active site is responsible for increased enzymatic activity and shows
stronger temperature dependency of enzymatic reaction (Lam et al. 2011). This is to
be noted that a particular factor is not responsible for the stability of an enzyme
because multiple factors contribute to the stability of a thermophilic enzymes.
14.5.1.2 Thermophilic Cellulases
Cellulases from thermophilic fungi have an approximately 30–250 kDa molecular
weight protein that shows maximum activity at pH 4.0–7.0 and 50–80 C tempera-
ture. Optimal thermal stability of this enzyme is at 60 C temperature. In comparison
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 279

with mesophilic fungal cellulases, thermophilic cellulases have longer half-lives at
70, 80, and 90 C. Catalytic efficiency of thermophilic cellulases is improved by the
structure-based rational site-directed mutagenesis and random mutagenesis through
directed evolution so that their stability at nonphysiological pH and elevated tem-
perature can be increased. Three-dimensional structure analyzes the characterization
of the different families of cellulases. The family 5 thermophilic cellulases were
studied in thermophilic fungi Trichoderma aurantiacus. This family consists of
endoglucanase type with β/α-barrel folding. Substrate binding cleft is present on
the C-terminus of the barrel that possesses seven glucose residues binding site and
has some extra barrel features. The family 6 cellulases studied in Humicola insolens.
They consist of endoglucanases and cellobiohydrolases type of thermophilic
cellulases that have distorted β/α-barrel structure where only seven parallel
β-strands are present in central barrel. Strands I and VII form the substrate binding
cleft that have six substrate binding sites. Around the active site, two extended
surface loops are present in endoglucanase, which forms an open cleft enabling them
to hydrolyze the bond internally in cellulose. The family 7 consists of
endoglucanases and cellobiohydrolases type and was studied in Trichoderma
emersonii fungi. The structure consists of two antiparallel β-sheets with six
β-strains in each sheet. A substrate binding tunnel (approximately 50 Ǻlong) is
formed by the loop that connects both concave and convex faces of strands that have
nine substrate binding sites. The family 12 of thermophilic cellulases was studied in
Humicola grisea. The structure consists of 15 β-strands folded into two antiparallel
β-sheets. Six antiparallel strands form the convex face while nine antiparallel strands
form the concave face. This concave site forms a long substrate binding cleft that
possess six substrate binding sites. The family 45 possesses endoglucanase type
isolated from Humicola grisea and Melanocarpus albomyces. They are flattened
sphere shape with dimensions about 32Ǻ32Ǻ22Ǻ. The structure is composed
of six stranded β-barrels with interconnected loops. These β-barrels and loop
structures form the approximately 40 Ǻlong, 10 Ǻdeep, and 12 Ǻwide substrate
binding cleft in between them (Li et al. 2019). Various thermophilic fungal cellulases
from different thermophilic fungi were expressed in their heterologous hosts and
their properties were evaluated (Table 14.1). Thermophilic fungi Chaetomium
thermophilum secreted three forms of cellulases. These three forms of
cellobiohydrolase are CBH1, CBH2, and CBH3. CBH1 and CBH2 consist of all
four domains such as signal peptide domain, cellulose binding domain, hinge region,
and catalytic domain while CBH3 consists of only signal peptide domain and
catalytic domain. Deletion of cellulose binding domain causes reduction in enzy-
matic activity (Li et al. 2019). Sporotrichum thermophile produces hemoflavoprotein
type of cellobiose dehydrogenase of 92–95 kDa molecular weights. This enzyme
possesses N-terminal flavin domain, heme domain, and C-terminal cellulose binding
domain. N-terminal flavin domain contains the active site. This enzyme possesses
maximum activity at 60–65

C temperature and pH 4 (Maheshwari et al. 2000).
Thermophilic fungal species Myceliophthora sp. viz., fungal species M.
thermophila,M.heterothallica,M.hinnulea, and M.fergusii optimally grow at
45

C secreted industrially valuable enzymes that have stability up to 70 C. They
280 K. Choudhary et al.

Table 14.1 Recombinant thermophilic fungal cellulases with their expression in heterologous hosts and their properties
Fungus Gene Family Host
Optimal
pH pI
Optimal
temperature (C) Thermal stability
Molecular
mass (kDa)
Acremonium
thermophilum
cel7a 7Trichoderma
reesei
5.5 4.67 60 NR 53.7
Chaetomium
thermophilum
cel7a 7Trichoderma
reesei
4 5.05 65 NR 54.6
Chaetomium
thermophilum
cbh3 7Pichia pastoris 4 5.15 60 T
1/2
: 45 min at 70 C 50.0
Humicola grisea egl2 5Aspergillus
oryzae
5 6.92 75 80% residual activity for
10 min at 75 C
42.6
Humicola grisea egl3 45 Aspergillus
oryzae
5 5.78 60 75% residual activity for
10 min at 80 C
32.2
Humicola grisea egl4 45 Aspergillus
oryzae
6 6.44 75 75% residual activity for
10 min at 80 C
24.2
Humicola grisea var
thermoidea
eg1 7Aspergillus
oryzae
5 6.43 55–60 Stable for 10 min at 60 C 47.9
Humicola grisea var
thermoidea
cbh1 7Aspergillus
oryzae
5 4.73 60 Stable for 10 min at 55 C 55.7
Humicola insolens avi2 6Humicola
insolens
NR 5.65 NR NR 51.3
Humicola insolens cbhII 6Saccharomyces
cerevisiae
9NR57 T
1/2
: 95 min at 63 CNR
Melanocarpus
albomyces
cel7b 7Trichoderma
reesei
6–8 4.23 NR NR 50.0
Melanocarpus
albomyces
cel7a 7Trichoderma
reesei
6–8 4.15 NR NR 44.8
Melanocarpus
albomyces
cel45a 45 Trichoderma
reesei
6–8 5.22 NR NR 25.0
(continued)
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 281

Table 14.1 (continued)
Fungus Gene Family Host
Optimal
pH pI
Optimal
temperature (C) Thermal stability
Molecular
mass (kDa)
Talaromyces
emersonii
cel3a 3Trichoderma
reesei
4.02 3.6 71.5 T
1/2
: 62 min at 65 C 90.6
Talaromyces
emersonii
cel7 7E. coli 5 4.0 68 T
1/2
: 68 min at 80 C 48.7
Talaromyces
emersonii
cel7A 7Saccharomyces
cerevisiae
4–565 T
1/2
: 30 min at 70 C 46.8
Thermoascus
aurantiacus
cbh1 7Saccharomyces
cerevisiae
6 4.37 65 80% residual activity for
60 min at 65 C
48.7
Thermoascus
aurantiacus
eg1 5Saccharomyces
cerevisiae
6 4.36 70 Stable for 60 min at 70 C 37.0
Thermoascus
aurantiacus
bgl1 3Pichia pastoris 5 4.61 70 70% residual activity for
60 min at 60 C
93.5
Thermoascus
aurantiacus
cel7a 7Trichoderma
reesei
5 4.44 65 NR 46.9
Source: Li et al. (2019) Fungi in Extreme Environments: Ecological Role and Biotechnological Significance.2019:395–417
282 K. Choudhary et al.

can grow and hydrolyze plant substrate efficiently, thus used in plant biomass
degradation process or biological conversion of pretreated plant biomass to ferment-
able sugars. Higher thermostability of these enzymes allows the saccharification of
plant biomass at increased temperatures resulting in shorter reaction time, higher
mass transfer, and reduced substrate viscosity (van den Brink et al. 2013).
M. thermophila produces thermophilic lignocellulolytic enzymes endoglucanase,
β-glucosidase, xylanase, and avicelase that show maximum activity at 55–70 C
and maximum stability at 30–60 C. Thermophilic avicelase efficiently hydrolyzes
the crystalline cellulose and thermostable β-glucosidase is resistant to glucose
inhibition making them specific to use in industries (de Cassia et al. 2015).
14.5.1.3 Themophilic Proteases
Thermophilic fungi Chaetomium thermophilum produce two thermostable proteases
that have molecular weight 33 kDa and 63 kDa. Both proteases possess high thermal
activity and optimal temperature 65 C. The 33 kDa protease shows maximum
activity at pH 10 along with Km value 6.6 mM and Vmax value 10.31 μmol/l/
min. The 63 kDa protease shows maximum activity at pH 5 along with Km value
17.6 mM and Vmax value 9.08 μmol/l/min. The activity of proteases is stimulated by
Ca
2+
and inhibited by phenylmethanesulfonyl fluoride (Li et al. 2007). Proteases
produced by thermophilic Myceliophthora species in solid state fermentation are
alkaline and thermostable. Optimal temperature and pH for the activity of proteases
are 50 C and 9, respectively, in solid-state fermentation. These thermostable
alkaline proteases have applications in leather processing, laundry detergents,
brewing, food, and pharmaceutical industries. Thermal stability of proteases
enhances when immobilized in alginate (Zanphorlin et al. 2010). Thermophilic
fungus Penicillium duponti K1014 produces thermostable acid proteases that are
stable at 50–60

C. Mucor pusillus aspartic protease possesses aspartic acid at their
active site. This enzyme shows maximum activity at 55 C temperature and 3–6
pH. Thermomyces lanuginosus produces alkaline protease, which is basically a
serine protease containing partially buried cysteine residue. This protease cleaves
the substrate C-terminus end of hydrophobic amino acid residues (Maheshwari et al.
2000).
14.5.1.4 Thermophilic Lipases
Extracellular lipases from thermophilic fungi Rhizopus oryzae and Rhizopus
rhizopodiformis isolated from palm oil mill effluent are active and stable at optimal
pH 6.0 and optimal temperature 45 C. Lipase from R. oryzae shows more activity at
acidic pH and broader substrate specificity as compared to lipases from
R. rhizopodiformis (Razak et al. 1997). Thermophilic fungus Humicola lanuginosa
produce lipases that are heat stable. Its optimal temperature is 60 C and optimal pH
is 8.0. However, it is stable at pH range 7.0–10.0. Metal ions such as Mn
2+
,Ca
2+
,
Ba
2+
,Mg
2+
, and Co
2+
show stimulatory effect while Cu
2+
and Hg
2+
show inhibitory
effect on the activity of lipases (Liu et al. 1973a,b). Humicola lipases possess high
number of hydrophobic amino acids than hydrophilic amino acid that provides the
compact structure to protect them from denaturation. Addition of sulfhydryl-
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 283

reducing agent and calcium ions does not affect the lipase activity. Addition of 8 M
urea accelerated the heat inactivation of lipases. They possess six half cysteine
residues per enzyme molecule (Liu et al. 1973a,b). Rhizomucor miehei produces
heat-stable lipases and contains 269 amino acid residues. Serine, histidine, and
aspartic acid present in the catalytic center are known as catalytic triad. Catalytic
site is covered with a lid composed of short α-helical loop that regulates the entry of
substrate and protects the site (Maheshwari et al. 2000).
14.5.1.5 Thermophilic Glucoamylases
Extracellular glucoamylases from thermophilic fungus Ther. lanuginosus are opti-
mally stable in the pH range 6.5–8.0 and 50 C temperature for 6 h. It shows optimal
enzyme activity at pH 5.0, substrate concentration 0.2%, soluble starch or 0.4%
maltose, and temperature 50 C. This 57 kDa molecular weight protein is highly
specific for α-D-1,4-glucosidic linkages and shows molecular activity of
4.76 10
3
min
1
. The rate of hydrolysis is significantly increased with increase
in the chain length of substrates. The unique semi-random or random coiled well-
hydrated structure provides thermostability to this enzyme (Basaveswara Rao et al.
1981).
Thermostable extracellular amylolytic enzyme glucoamylases produced by ther-
mophilic fungus C. thermophilum is a 64 kDa molecular weight protein. The
glucoamylase structure is composed of single polypeptide determined by Native
PAGE. Optimal pH and temperature for this enzyme are 4.0 and 65 C, respectively.
The enzyme is stable in pH range of 3.5–8.0. Metal ions Hg
2+
,Ag
+
, and Fe
2+
show
inhibitory effect while Ca
2+
,Na
+
, and K
+
show stimulatory effect on the enzyme
activity. N-terminus amino acid sequence of this enzyme shows high similarity with
glucoamylases from Neurospora crassa,H. grisea, and Thielavia terrestris. The
half-life of glucoamylase is 20 min at 70 C (Chen et al. 2005).
14.5.1.6 Thermophilic Xylanases
Thermophilic fungus Talaromyces byssochlamydoides strain YH-50 produces ther-
mostable xylanase that shows stability at optimal temperature 70 C and optimal
pH 5.5. This native xylanase hydrolyze xylan to the extent of 90% and produces
xylose in large amount along with arabinose, glucose, and galactose in small
amount. This thermostable xylanase is used to degrade hemicelluloses to produce
different monosaccharides (Yoshioka et al. 1981). Xylanase from Thermoascus
aurenticus are highly homologous to mesophilic xylanase. This enzyme has
301 amino acid residues. Substrate binding site possesses glutamate and histidine
residues that are linked with the negative charges. Proline residues present at
N-terminus decrease the conformational freedom of enzyme that makes them stable
under high temperature. Xylanase from Ther. lanuginosus possess a long cleft in
which active site is present. The active site adjusted the heptaxylan molecules
(Maheshwari et al. 2000). Comparative study of family 10 xylanase of
T. auranticus with its mesophilic homolog shows increased hydrophobic packing
and increased proline residues at the N-terminus in thermophilic xylanase. Although
comparative study of family 11 xylanase of thermophile and mesophile shows high
284 K. Choudhary et al.

threonine to serine ratio and an additional β-strand B1 at N-terminal in thermophilic
xylanase (Collins et al. 2005). A thermophilic filamentous fungi C. thermophilum
produce family 11 xylanase. Structure analysis shows that α-helix and highly twisted
two β-sheets contribute the structure. Glycerol molecule is incorporated into the
active site. Four sulfate ions and a calcium ion are present in this structure. Thermo-
philic xylanases contain more stable α-helix and an additional β-strand B1 at
N-terminal. Ser/Thr surface incorporated with arginine residues increases the opti-
mum temperature and thermotolerance of xylanase. Longer N-terminal as compared
to mesophile is involved in the high activity (Hakulinen et al. 2003).
14.5.1.7 Thermophilic Laccases
Extracellular phenol oxidase produced by thermophilic ascomycete T. aurantiacus
shows optimal oxidation temperature between 70 and 80 C. Maximum enzyme
activity is found at pH 6–8. Sodium azide and thioglycollic acid show highly
inhibitory effect on enzyme activity (Machuca et al. 1998). Thermophilic
actinomycetes Thermobifida fusca BCRC19214 produces 73.3 kDa laccase that
possess trimeric structure and shows 4.96 U/ml laccase activity. pH stability ranges
from 5.0 to 10.0. Optimal pH and temperature for laccase activity are 8.0 and 60 C,
respectively. Hg
2+
(1 mM), SDS, and sodium azide partially reduce the enzyme
activity while β-mercaptoethanol and EDTA strongly inhibit the laccase activity.
Acetonitrile and dimethylformamide stimulate the activity of enzyme. Internal
amino acid sequence analysis shows homology with laccase isolated from
Thermobifida fusca YX (Chen et al. 2013). Laccase from Myceliophthora
thermophila has heavy glycosylated structure. In total, 559 amino acid residues
form a mature enzyme. It contains three tightly packed cupredoxin like domain. A
and C domains form the trinuclear copper site while C domain contains T1 Cu site
that coordinates two histidine His 431 and His 508 and one cysteine Cys 503 residue.
T1 pocket shows high variability. A calcium ion involved in the crystal packing. Leu
363 carbonyl carbon and five water molecules coordinate with CA606 to form a
stabilized loop structure (Ernst et al. 2018).
14.5.1.8 Thermophilic b-Glucosidase
A thermophilic fungi C. thermophilum produces thermostable cellulolytic enzyme
β-glucosidase belonging to glycoside hydrolases family GH3. The crystal structure
(Fig. 14.1) shows that it consists of three domains: catalytic triose phosphate
isomerase barrel like domain, α/βsandwich domain, and fibronectin type III domain.
Modifications are found in loops and linker regions. Linker region that connects
domains 1 and 2 is found to be larger than other GH3 β-glucosidase except one from
N. crassa. Sequence alignment study revealed that this β-glucosidase shows 72%
similarity with N. crassa β-glucosidase. Glycosylated Asn 72 stabilizes the loop by
making bond with N-glycans. Asn 504 was found as new glycosylation site. Active
site is present near the first and second domain interface. Catalytic residue Asp 287 is
situated at the N-terminal of first domain and functions as nucleophile while Glu
517 is situated in α/βsandwich domain and functions as an acid-base catalyst. Both
catalytic residues are conserved. Second barrel β-strand are found shorter and
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 285

antiparallel near the active site that makes active site wider and more accessible.
Ramachandran plot shows 92.8% residues found in the most favorable region while
1% residues are found in the disallowed region. This enzyme has an optimum
activity at 50 C temperature. This enzyme retains half of the enzymatic activity
after incubation for 55 minutes at 65 C. Two thermostability parameters are
responsible for its stability. One of them is glycosylation and the other is the charged
residues that form electrostatic interactions. Thermophilic cellulases are preferred in
cellulose degradation process as high temperatures make the process easy due to
swelling of cellulose molecule (Mohsin et al. 2019).
Fig. 14.1 The three domains of Chaetomium thermophilum β-glucosidase shown in its crystal
structure. Different domains are indicated by blue, green, and yellow colors. Linker and loops are
also shown by different colors. N-glycans and glucose in the active site are shown in the form of
gray sticks. (Source: Mohsin et al. International journal of molecular sciences (2019) 20(23):5962)
286 K. Choudhary et al.

14.5.1.9 Thermophilic a-Amylases
The α-amylase isolated from thermophilic fungus of genus Aspergillum STG3 and
STG6 is thermostable that shows maximum enzyme activity at 55 C when
incubated for 10 min. Enzyme activity of STG3 and STG6 is 1.202 U/mg and
1.052 U/mg, respectively. pH optima for this enzyme is 6.0 for both strains. Ca
2+
,
Mg
2+
,andZn
2+
metal ions strongly inhibit the amylase activity (Teklebrhan Welday
et al. 2014). With increasing temperature, stability also increases. Increased enzyme
stability leads to decrease in entropy of enzymatic reaction and low flexibility.
Catalytic center of thermostable enzyme is compact and rigid due to which they
are not flexible. Decreased conformational flexibility increases the stability of
thermophilic enzyme. Thermophilic enzyme’s catalytic domains contain numerous
interactions that make them high temperature dependent, thus low temperature
decreases the enzymatic activity of thermophilic enzymes (D'Amico et al. 2003).
α-amylase from thermophilic actinomycete Thermobifida fusca is chloride depen-
dent. Removal of chloride ligand causes low affinity of enzyme. Kcat value is found
higher than its mesophilic and psychrophilic homolog. It possesses compact confor-
mation due to its higher affinity (Cipolla et al. 2012).
14.5.1.10 Thermophilic Phytase
Phytase is the enzyme that catalyzes the breakdown of phytic acid. One such phytase
is isolated from thermophilic fungus Ther. lanuginosus that is cloned and heterolo-
gously expressed to form a 60 kDa recombinant phytase. This phytase shows
superior catalytic efficiency. Maximum enzyme activity shows at optimum temper-
ature 65 C. pH stability of this enzyme is 3.0–7.5 and active at near neutral
pH. Specific activity of this recombinant phytase is 110 μmol/min/ mg of protein.
This enzyme is used in feed industries to reduce the high phosphate waste and also
reduces the phosphate content in manure (Berka et al. 1998).
14.6 Psychrophilic Enzymes
14.6.1 Psychrophiles
Psychrophiles are the organisms that can thrive under very low-temperature condi-
tion <10 C. They grow at temperature between -20 C and 20 C and possess
optimal growth temperature <15 C. Common habitats of psychrophiles are Arctic
and Antarctic regions, icebergs, glaciers, snow fields, refrigerated food, etc.
(Rampelotto 2013).
14.6.2 General Adaptations
Psychrophiles protect themselves from cold by increasing the amount of unsaturated
and short chain fatty acids to regulate the membrane fluidity. They synthesize cold
shock proteins and chaperons to protect the nucleic acid and proteins. Some
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 287

antifreeze proteins are found that has essential role in protecting the cell from cold.
Accumulation of cryoprotectants or compatible solutes enables them to survive and
tolerate extreme cold (Rampelotto 2013). But the highly studied adaptive strategy to
survive in extreme cold is their cold-adapted enzymes.
14.6.3 Catalytic Features of Psychrophilic Enzymes
Biochemical reaction in psychrophilic environment or very low temperature is
possible in the presence of psychrophilic enzymes, which is cold active and heat
labile. Psychrophilic enzymes enhance the mobility and flexibility of the active site
by decreasing the temperature dependence of the reaction. Catalytic center of
psychrophilic enzymes is identical to mesophilic enzyme, but local interactions are
different that provides high activity and improved catalysis at low temperature.
Destabilization of active site structure and reduced number of various weak
interactions enhances the dynamics of active site residues (Feller 2013). These
enzymes have very flexible structure. As temperature decreases rate of reactions, it
slowly decreases due to reduction in activation enthalpy, low kinetic energy, and
more negative activation entropy. Lower arginine to lysine ratio, more glycine
residues, reduced proline residues in loops, and increased proline residues in
α-helices are some amino acid replacements present in psychrophilic enzymes.
Reduced core hydrophobicity, decreased disulfide bridges, reduced oligomerization,
and less electrostatic interactions make them very flexible. When compared to its
mesophilic and thermophilic homologue; psychrophilic enzymes are more produc-
tive due to high catalytic efficiency. The active sites of these enzymes are the most
heat-labile structure that enables them to function at low temperatures (Cavicchioli
et al. 2011). Low proline content, less disulfide bond, and higher glycine clusters
increase the activity of cold-active enzymes at low temperature (D'Amico et al.
2003). A figure represents some modifications in structure (insertions and
extensions) and interactions of psychrophilic alkaline phosphate and psychrophilic
α-amylase respectively to understand the adaptations of psychrophilic enzymes to
cold environment (Fig. 14.2) (Santiago et al. 2016). From mutational studies, it is
revealed that mutation in the site of enzyme away from the active site increases the
enzyme activity. This also suggests that the local unfolding in psychrophilic
enzymes increases the entropy that causes high enzymatic activity (Deniz 2018).
For any biochemical reaction, activity constant is directly dependent on temperature.
Thus, lower temperature causes gradual decrease in enzyme activity of
mesophiles. Cold active enzymes have tenfold higher specific activity to cope with
the low temperature. Weak stability of psychrophilic enzymes and unfolding or
inactivation at moderate temperature make them responsible for its maximal activity.
Active site of cold-active enzymes is more heat labile as the enzyme inactivation
starts before protein unfolding. This means that active site is more flexible and active
structure. Noncatalytic part is stable as it is nonflexible. Large active site of cold-
active enzymes mitigates the effect of rate-limiting step (Feller 2013). Increased
conformational flexibility and solvent interactions are the most observed features of
288 K. Choudhary et al.

psychrophilic enzymes to cold environment (Karan et al. 2012). Deep sea cold-
active enzymes possess more α-helix as compared to mesophilic enzymes, which is
important for maintaining the flexibility of these enzymes. Their capacity of binding
to solvent molecule is very high and strong that is responsible for its high catalytic
rate at low temperature (Jin et al. 2019). Psychrophilic enzymes have high turnover
rate due to high value of catalytic constant Kcat. However, they also have high value
of K
M
as psychrophilic enzymes have weak substrate binding affinity due to their
flexible active sites. Although the catalytic efficiency of psychrophilic enzymes is
higher because Kcat value is relatively higher than K
M
value. Flexibility in psychro-
philic enzymes is more important than stability (Ichiye 2018). At low temperature,
kinetic energy of reacting molecules is low due to less number of weak interactions,
thus the rate of reaction is slow. Viscosity of the solvent increases at low tempera-
ture; higher viscosity weakens the affinity of the enzymes for their substrate. Hence,
psychrophilic enzymes have high specific activity, low thermostability, and
increased interactions with the solvent molecules. Lower activation enthalpy in
psychrophilic enzymes reduces the stability but enhances their flexibility that
reduces the additional energy cost and enhances the activity at low temperature
(Collins et al. 2007). The flexibility of protein is understood by its B-value, which is
a value that measures dynamic motions of atoms in a protein crystal. Psychrophilic
Fig. 14.2 Some important structural adaptations in psychrophilic enzymes vis-a-vis their
mesophilic counterparts (Source: Santiago et al. 2016)
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 289

enzymes possess high B-value in β-sheet along with turns. They possess signifi-
cantly high amount of buried water, which suggests that psychrophilic enzyme core
are more solvated than mesophilic counterpart. Their cavity is surrounded by high
number of acidic amino acid that contains more water molecules than mesophilic
homolog. It can be considered that psychrophilic enzymes either possess fewer
cavities with large cavity volumes or more cavities with equivalent cavity volume
as in mesophilic enzymes (Paredes et al. 2011).
14.6.4 Industrial Application
High catalytic efficiency of these enzymes enables them to use in biotechnological
industries. Solvent tolerance of these enzymes is useful for cleaning purposes and
removal of ester components in biofilms. These cold-active enzymes are used in food
and feed industry to prevent the changes in flavor, nutritional value, and food
spoilage. Transformation of heat-labile product by psychrophilic enzyme is benefi-
cial. Cold-adapted lipases and esterases are used in chiral drug production due to
high stereospecificity (Cavicchioli et al. 2011). They are used to stimulate ripening
process of slow ripened cheese. They are used in the removal of macromolecular
stains from fabrics and protect color of cloths during washing process at low
temperature. Peeling process of leather consumes much energy. Use of cold-active
proteases saves the energy as they work at the temperature of tap water. Cold-active
enzyme reduces the energy consumption of a process that needs low temperature.
Pulp and paper industries also need cold-active enzymes for the degradation of
polymers (Joshi and Satyanarayana 2013).
14.6.4.1 Psychrophilic Lipases
Psychotropic fungus Penicillium canesense BPF4 and Pseudogynmoascus roseus
BPF6 produce alkaline metallolipases BPF4 and BPF6, respectively, that is stable at
4C, 20 C, and 40 C as they retained its 80% and 90% residual activity after 2 h
and 1 h, respectively. pH optima for BPF4 and BPF6 are 9 and 11, respectively. Cu
2+
and Fe
3+
ions stimulate the activity of BPF4 and BPF6 lipases while the activity of
both lipases is inhibited by tween-20, HClO
3,
and H
2
O
2
. EDTA and Sn
2+
do not
affect BPF4 activity but enhance BPF6 activity. SDS reduces the activity of BPF6
while slightly enhances the activity of BPF4 lipase. These properties make them
potential to use in detergent formulation (Sahay and Chouhan 2018). Moesziomyces
antarcticus produces two types of cold-active lipases. Antarctic fungus Geomyces
species P7 lipases active and stable at optimum temperature 8–35 C, and at 0 C this
lipase shows 15% of its maximum activity. These cold-active lipases are used in
biodiesel production, wastewater treatment at low temperature, and cleaning
products (Duarte et al. 2018).
14.6.4.2 Psychrophilic Protease
Penicillium chrysogenum FS010 produces cold-active lipase that has 35 C optimum
production temperature. It shows high activity between 15 and 35 C and retained
290 K. Choudhary et al.

41% residual proteolytic activity at 0 C temperature. The activity of cold-active
proteases is increased by addition of Ca
2+
,Na
+
,Mg
2+
,K
+
, and NH
4+
while it reduces
by the addition of Cu
2+
and Co
2+
.Fe
3+
and EDTA strongly inhibit the activity (Joshi
and Satyanarayana 2013). Rhodotrula mucilaginosa L7 produces extracellular acid
proteases that are active at temperature between 15 and 60 C. Proteases from
Vanrija humicola show activity at temperature 0–45 C. Proteases from Antarctic
fungus Glaciozyma antarctica 107 are very cold active as they exhibit activity from
-10 Cto25C. They are used in food industries to preserve the quality of heat-
sensitive nutrients, softening of refrigerated meat products, cleaning of contact
lenses, and cheese ripening process. They are used in the textile industry for cold
washing process (Duarte et al. 2018).
14.6.4.3 Psychrophilic Amylases and Glucoamylases
Structural analysis of cold-active α-amylases from G. antarctica Pl12 shows that
they possess binding sites for conserved and nonconserved calcium ion and sodium
ion. Moe. antarcticus CBS6678 α-amylase and glucoamylase utilize high molecular
mass substrates. They are applicable in wine production in brewing industry and
juice clarification process at low temperature (Duarte et al. 2018). Psychrophilic
α-amylase possess strictly conserved catalytic cleft made up of 24 residues. The
catalytic cleft has large opening as compared to mesophilic homolog (Feller 2013).
Cold-active α-amylase from G, antarctica PI12 possesses binding sites for
conserved and nonconserved calcium ion and sodium ion (Duarte et al. 2018).
Low activation enthalpy of cold-active enzymes reduces the temperature depen-
dency of enzymatic reactions. Lesser extent of enthalpy-driven interaction makes
them heat labile. Low stability of active site of cold-active enzymes leads to the high
activity at low temperature. The active site of cols active enzymes is found to be
more heat labile than overall structure. Free and active state of active site is more
flexible due to which they possess more fluctuating structure. Activity of cold-active
enzyme increases by reducing the temperature dependency by destabilizing the
active site domain (D'Amico et al. 2003).
14.6.4.4 Psychrophilic Chitinases
Chitinolytic enzyme from fungus Lecanicillium muscarium CCFEE-5003 is a com-
plex structure made up of five proteins of molecular weight 20–75 kDa with
isoelectric point between 4.5 and 5.0 pH. Flexibility in this enzyme is due to
replacements of some amino acids in surface and loop regions identified in
G. antarctica PI12 chitinase. They are useful in chitin-rich waste treatment at low
temperature, preventing contamination in refrigerated foods and biocontrolling of
microbial spoilage (Duarte et al. 2018).
14.6.4.5 Psychrophilic b-Galactosidases
Marine Antarctic yeast Tausonia pollulans produces high amount of extracellular
β-galactosidase that shows high activity in cold environment. They are used to
reduce sugars in food and drugs, and reduce industrial effluents during
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 291

bioconversion of milk to whey and production of animal and human diet additives
(Duarte et al. 2018).
14.6.4.6 Psychrophilic b-Glucanase
β-Glucanase from marine psychrophilic yeast G. antarctica PI12 contains a
conserved motif G-E-x-D-x-x-E. Glu 214 and Glu 219 are highly conserved catalytic
residues of glucoside hydrolases family 16 performs acid–base catalysis and nucle-
ophile reactions. It shows optimum temperature 20 C and maximum activity at
pH 7. It comprises 28.6% α-helices and 42.8% β-strands. It possesses longer external
loops. Long broken β-sheets are connected by a loop. Salt bridges and hydrogen
bonds are lower in this enzyme as compared to mesophilic enzymes. The enzyme
activity is higher for lichenan. Recombinant β-glucanase shows optimum activity at
15 C and pH 7; however, it retains its activity at lower temperature and alkaline pH
(Mohammadi et al. 2021).
14.6.4.7 Psychrophilic Xylanases
Cold-active xylanases are used in biofuel production and chemical production from
lignocelluloses. Psychrophilic xylanase from Antarctic fungus Naganishia
adeliensis shows lower activation energy and higher catalytic efficiency at
0–20 C temperature. Structure analysis shows the presence of less compact and
more flexible molecular structure (Duarte et al. 2018). Psychrophilic xylanase
possess threefold higher activities than mesophilic xylanase at 5 C temperature.
They have short half-life and low denaturation temperature. Decreased stability of
cold-active xylanase increases its flexibility that is responsible for the catalysis at
low temperature (Collins et al. 2005).
14.6.4.8 Psychrophilic Laccases
An Antarctic soil fungi Penicillium commune AL2, Aspergillus fumigates AL3, and
Penicillium rugulosum AL7 produce laccases by utilizing phenol that is active at
5C temperature. These fungi degraded polycyclic aromatic hydrocarbons. Laccase
from Antarctic yeast is active at 15 C temperature. These enzymes are used in
textile industry and lignin degradation (Duarte et al. 2018).
14.6.4.9 Psychrophilic Cellulases
Many Antarctic filamentous fungi and yeast are found to produce cold-adapted
cellulases. Penicillium roqueforti, Cadophora malorum, Geomyces species, and
Mrakia blallopsis produce cellulase at 4-22 C temperature. These cold-adapted
enzymes are tolerant to organic solvents. Psychrophilic cellulases have the potential
to be used in the production of volatile and heat-sensitive compounds (Duarte et al.
2018).
14.6.4.10 Psychrophilic Catalases and Superoxide Dismutase
Antarctic fungus of genus Aspergillus, Cladosporium, and Penicillium produces
cold-active catalases. An Antarctic fungus Aspergillus glaucus 363 produces high
292 K. Choudhary et al.

amount of superoxide dismutase at 10 C. This cold-adapted enzyme is useful in
cosmetic formulation to prevent skin injury (Duarte et al. 2018).
14.6.4.11 Psychrophilic Tannases
These cold-active tannases are useful in the production of instant tea, garlic acid, and
fruit juice. Verticillium species P9 produces two types of extracellular tannase,
TAH1 and TAH2, which has temperature optima 25 and 20 C (Duarte et al. 2018).
14.7 Halophilic Enzymes
14.7.1 Halophiles
Organisms that live in high salt environment are known as halophiles. They grow
and survive at high salt concentration. Optimum growth of these organisms lies
between the salt concentration ranges 0.2–0.3 M NaCl. High concentration of ions,
toxic metals, and low water activity is the characteristic feature of hypersaline
environment (Edbeib et al. 2016).
14.7.2 Protein Adaptation
At high salt concentration, water activity is very low, which means water is less
available to the proteins. The low water activity causes hydrophobic amino acids in a
protein to lose hydration and aggregate. Non-halophilic proteins aggregate, precipi-
tate, or denature at high salt environment, but halophilic proteins remain functionally
active in that environment because of their haloadaptation. Halophiles adapted to
high salt remodel the protein at different levels. At cell physiology level, they have
large number of salt bridges allowing to stable at high salt. Another mechanism
includes large number of acidic or polar chain amino groups on their protein surfaces
and less bulky hydrophobic residues. At proteomics level, some enzymes like
cysteinyl-tRNA synthatases and P45 proteins provide additional stability to halo-
proteins. At genomic level, high DNA––protein interaction protects the protein from
denaturation at high salt concentration (Gomes and Steiner 2004; Kumar et al. 2018).
Stability of these proteins is also ensured due to the negative charges of acidic
amino acids on the surface of protein, hydrophobic groups on protein core, and their
hydration due to carboxylic group. High negative charges maintain the confirmation
of protein and prevent aggregation at high salt concentration through binding with
the hydrated cation and reduce the surface hydrophobicity of protein. Lysine amino
acid percentage is fewer in halophilic proteins (Kumar et al. 2018).
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 293

14.7.3 Catalytic Features
Haloenzymes are the enzymes produced by the halophiles. They have similar
characteristics to nonhalophilic homologues but have different structural properties.
Halophilic enzymes are the protein of halophilic origin (secreted by halophilic
microorganism). These enzymes are structurally adapted to function under high
saline environment. Nonhalophilic enzymes under this extreme condition lose
their structure and ability to perform catalysis efficiently. Halophilic enzymes
function in high salt because they have some unique features in their structure
(Charlesworth and Burns 2016). Low water activity and limited salvation in saline
environment disrupt the structure of water molecule near proteins, resulting in high
hydrophobicity. The scarcity of water molecule affects the solubility and stability of
protein. Thus, high salinity causes aggregation and precipitation of nonhalophilic
proteins (Reed et al. 2013).
Halophilic enzymes are more soluble in the presence of salt. They cannot perform
catalysis in low saline or nonsaline environment as they lose their enzymatic activity
due to the absence of salts. The role of salt is to maintain the native structure of
enzyme. Salt maintains the rigidity of secondary and tertiary structure of halophilic
enzymes against denaturants (Siglioccolo et al. 2011). Halophilic enzymes prevent
their unfolding with the help of Ca
+2
and regulate their catalytic activity with the
help of Na
+
. Thus, both the cations are essential for halophilic enzyme activity
(Sinha and Khare 2014). Halophilic enzymes are not limited to function under high
salt concentration but also they can be able to function under high temperature and
organic solvents. They can overcome the problems encountered during the industrial
processes such as high salt, high temperature, and high pH (Reed et al. 2013). At
high salt concentration, water is present in hydrated ionic form, which makes them
less available to the proteins. It is necessary to make protein molecule hydrated so
that halophilic enzymes contain large and multilayered hydration shell. There is
prevalence of glutamate residues in halophilic enzymes as they possess high water
binding capacity. Lysine residues are present in very negligible amount in protein
surface. Reduced surface hydrophobicity along with increased surface hydration
enables halophilic enzymes to function in low water activity environment (Karan
et al. 2012). The main problem in front of halophilic enzymes is to deal with the high
concentration of internal and external electrolytes. So they prefer highly hydrated
amino acid residues, especially glutamic acid and arginine, in their structure. They
possess low ratio of nonpolar to polar residues. Halophilic enzymes maintain its
flexibility at high salt concentration by reducing the hydrophobicity; thus, they
prefer amino acid of low hydrophobic nature in their structure (Jaenicke 1991).
Carboxylic groups of aspartic and glutamic acid make the protein surface hydrated.
The activity of halophilic enzymes is dependent on salt concentration so that low salt
concentration causes reduction in enzymatic activity and makes them less useful in
industrial process. This can be overcome by using these enzymes in nonaqueous
media as high salt concentration that leads to low water activity. Thus, they are
useful for pharmaceutical industry (Kumar et al. 2011; Jin et al. 2019).
294 K. Choudhary et al.

14.7.3.1 High Acidic Residues on their Surface
The halophilic enzymes contain high proportion of acidic amino acids such as
glutamic and aspartic acid residues on its surface. These acidic amino acid residues
help in the solvation (Gomes and Steiner 2004). An increased acidic residue
provides negative charges to the surface. These negative charges repel each other
and increase the flexibility of enzymes. Acidic surface is also essential for the
solvation of the biocatalyst (Kumar et al. 2018).
14.7.3.2 Low Hydrophobicity at the Core
These enzymes contain small proportion of aromatic hydrophobic amino acid in the
core. A lower bulky hydrophobic residue makes the hydrophobic interaction weaker
in these proteins. Thus, decreased hydrophobicity in halophilic proteins provides
them flexibility at high salt concentration (Siglioccolo et al. 2011; Reed et al. 2013;
Kumar et al. 2018).
14.7.3.3 Increased Salt Bridge Formation
Salt bridges are the electrostatic attraction between oppositely charged residues. The
presence of greater number of salt bridges in halophilic enzymes provides stability to
them (Sinha and Khare 2014; Kumar et al. 2018).
14.7.3.4 Salt-Dependent Folding
Salt-dependent folding is another catalytic feature of halophilic enzymes. Halophiles
utilize the salt to fold the protein and enable them to function under saline environ-
ment. Thus, they have salt-dependent catalytic activity (Reed et al. 2013; Sinha and
Khare 2014). In salt-free medium, proteins remain unfolded. This will reduce the
enzyme activity. However, addition of salt into medium induces refolding (Sinha
and Khare 2014) (Fig. 14.3).
In a study on the effect of salt on the catalytic activity of bacterial (Haloferax
volcanii) Ligase N, an NAD
+
-dependent DNA ligation enzyme, it was found that
high salt (KCl) concentration maintains its folded (active) structure and activates
it. The enzyme, however, remained inactive (unfolding of enzyme) in the presence of
NaCl. The enzyme has three domains, viz., DNA binding domain, an adenylation
domain with two sub-domains (Fig. 14.3b), and an OB fold domain. During cataly-
sis, adenylation of enzyme (transfer of an AMP molecule from NAD
+
to the protein
or adenylation of enzyme) occurs that close the subdomains.
14.7.3.5 Peptide Insertions
A peptide insertion consists of high number of acidic amino acid. This negatively
charged peptide insertion in halophilic enzyme is responsible for its flexibility (Reed
et al. 2013).
14.7.3.6 Low Lysine Content
Halophilic enzymes possess low frequency of lysine amino acid for stabilization
(DasSarma and DasSarma 2015).
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 295

14.7.3.7 Thermal Stability
Halophilic microorganisms have optimal growth at temperature range between
45 Cto60C. Hence, halophilic enzymes are soluble and thermostable at high
salt environment. Presence of ion–pair network in its structure is responsible for its
thermal stability. The decreased hydrophobicity and hydrogen bonding are impor-
tant parameters to maintain the stability of these enzymes at high temperature
(DasSarma and DasSarma 2015).
14.7.3.8 Stability and Activity in Organic Solvents
As the halophilic enzymes are adapted to function under low water activity environ-
ment, they are able to maintain their property in organic solvent too. Presence of
acidic residues on the surface and disulphide bonds in these enzymes enables them to
stable and active in organic solvents (DasSarma and DasSarma 2015). The enzy-
matic activity of halophilic enzymes increases in the presence of some salt such as
NaCl or KCl. Halophilic enzymes bind the water tightly due to which its solvation
and solubility is regulated under extreme salinity (Kumar et al. 2018).
Fig. 14.3 (a) Impact of salt (KCl) on ligase N structure and activity. (b) Note the adenylation
domain and its two subdomains in orange light blue: enzyme structure; active (folded) and inactive
(open) are related with adenylation and de-adenylated, respectively. (c) Relative enzyme activity
depends on KCl (not NaCl) concentration. (d) Representative examples of the chemical denatur-
ation profiles monitored by CD, for Hv dea-LigN and Hv ade-LigN. (Source: Ortega et al. 2011)
296 K. Choudhary et al.

14.7.4 Industrial Applications
The unique catalytic features and multi-resistant properties of these halophilic
enzymes make them stable and active under a wide range of salt concentration.
Halophilic hydrolases such as amylase, protease, lipase, cellulase, xylanase, and
laccase are the largest group of industrially important enzymes (Kumar et al. 2011).
14.7.4.1 Halophilic Protease
Proteases are the enzymes that catalyze hydrolysis of peptide bonds present in
protein. They have wide application in industries. They are used in leather industries
for skin dehairing. They have an important role in bioremediation process in the
treatment of highly saline wastewater. They enhance the protein stain removal action
of detergent, thus used in detergent industries. Salt stable proteases produced by
Nocardiopsis prasina are active at pH range 7–10 and temperature range 20–42 C
(Solanki and Kothari 2011).
14.7.4.2 Halophilic Xylanases
Xylanases are the xylan degrading enzymes that cleave the β-1,4 linkage of this
polysaccharide. In pulp and paper industries, cellulase-free xylanases are used for
pulp biobleaching. Xylanases produce by the Phoma species of mangrove fungi was
cloned and expressed in Pichia pastoris. This recombinant xylanase was found to be
active at pH 5 and 45 C temperature. This enzyme shows high degree of salt
resistance up to 4 M NaCl (Wu et al. 2018).
14.7.4.3 Halophilic Cellulases
Cellulases from halophilic microorganisms have higher catalytic activities as they
are stable at high salt and high temperature. These enzymes are used in the produc-
tion of bioethanol. Cellulases from a moderate halophile Aspergillus caesiellus are
active in salinity up to 2 M NaCl concentration. This cellulases has two isoforms of
approximately 50 and 35 kDa molecular mass. Optimal temperature and pH range
for enzyme activity are 60 C and 5–6 pH, respectively. These thermostable and
halostable cellulases are used in biorefinery process for the degradation of lignocel-
lulosic materials (Batista-García et al. 2014). Cellulases from Emericella nidulans
and Cladosporum cladosporioides are highly active at pH 4 and 10% NaCl and
pH 10 and 10% NaCl, respectively (Moubasher et al. 2016).
14.7.4.4 Halophilic Lipases
These enzymes catalyze the hydrolysis of triglycerides and produce glycerol and
fatty acids. Halophilic lipases are very useful in biodiesel production, bioremedia-
tion, and detergent formulation. Halophilic extracellular lipase produced by halo-
philic Fusarium solani using palm oil mill effluent is a very efficient approach for
low-cost production of halophilic lipase (Geoffry and Achur 2018).
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 297

14.7.4.5 Halophilic Amylase
Halophilic fungi Engyodontium album produce polyextremophilic α-amylases,
which have enzymatic activity at pH range between 5 and 9 and temperature range
between 40 and 60 C. They have optimal enzyme activity at pH 9 and temperature
60 C. However, they require 6–30% NaCl for their activity, optimal enzyme
activity being at 30% NaCl concentration. The catalytic activity is stimulated by
BaCl
2
, CaCl
2
, and HgCl
2
and inhibited by β-mercaptoethanol, EDTA, FeCl
2
, and
ZnCl
2
(Ali et al. 2014a,b). An obligate halophile Aspergillus gracilis produces
extracellular α-amylases, which are approximately 35 kDa molecular mass protein
and are of polyextremophilic nature. This enzyme has specific enzymatic activity
131.02 U/mg and optimal enzymatic activity is found at pH 5, temperature 60 C,
and salt concentration 30% NaCl. Thus, these salt-adapted enzymes are used in
bioremediation process for the treatment of saline and low water activity wastewater
(Ali et al. 2014a,b). An obligate halophile Aspergillus penicillioides TISTR3639
strain produces extracellular α-amylase that has approximately 42 kDa molecular
mass and utilizes soluble starch as a substrate. Specific activity, Vmax value, and
Km value of catalyst are 118.42 U/mg, 1.05 μmol/min, and 5.41 mg/mL, respec-
tively. This amylase is optically active at pH 9, 80 C temperature, and 300 g/L
NaCl. Enzyme activity increases with the addition of CaCl
2
at 2 mM concentration.
Enzyme activity of amylase is found strongly inhibited by ZnCl
2
and FeCl
2
while
EDTA at 2 mM concentration moderately inhibits the activity of amylase (Ali et al.
2015).
14.7.4.6 Halophilic Laccases
Laccases constitute a group of enzymes consisting of lignin peroxidase, phenol
peroxidase, phenol oxidase, manganese peroxidase, and tryrosine. They degrade
the lignocellulosic materials efficiently in the presence of salt. Halotolerant fungus
Pestalotiopsis species degrade lignin and toxic waste in elevated temperature and
salt. They are used in the treatment of 29 henolics wastewater and pulp-making
processes (Arfiet al. 2013). Halotolerant laccase from Chaetomium species,
Xylogone sphaerospora, and Coprinopsis species are chloride tolerant and used in
the transformation of aromatic pollutants like polycyclic aromatic hydrocarbon
(Qasemian et al. 2012).
14.7.4.7 Halophilic Chitinase
A thermohalophilic fungus Aspergillus flavus produces 30 kDa molecular mass
chitinase. This enzyme shows maximum catalytic activity at substrate concentration
0.9 g/l, pH 7.5, temperature 60 C, and NaCl concentration 0.8 M. The enzymatic
activity is stimulated by MnCl
2
and FeSO
4
. The kinetic parameter of this enzyme
such as Km value and Vmax value was found to be 0.18 g chitin/ml and 274.31 U/l,
respectively (Beltagy et al. 2018).
298 K. Choudhary et al.

14.8 Acidophilic Enzymes
14.8.1 Acidophiles
Organisms that thrive at extremely low pH and exhibit optimal growth below pH 3
are known as acidophiles. They maintain their cellular pH near neutral by regulating
the pH gradient.
14.8.2 Habitat of Acidophiles
Acidophiles are found in acid mine drainage, acidothermal hot springs, coal spoils,
solphataric fields, and bioreactor (Sharma et al. 2016).
14.8.3 Characteristics of Acidic Environment
Acidic environment is characterized by low pH value, high concentration of protons,
and low salinity. Some toxic-heavy metals and aggressive oxidative agents are also
present in acidic environment (Christel 2018).
14.8.4 Catalytic Features of Acidophilic Enzymes
The intracellular enzymes of acidophiles do not need to develop any strategy to
counter low pH as the organism maintains its internal pH near neutral. However, the
extracellular enzymes show adaptations against low pH. The acidophilic enzymes
have increased number of negatively charged amino acid residues on the surfaces
(negatively charged surface). They have glutamic acid and aspartic acid in excess
amount so that they can form a highly negative surface, which provides them
stability in acidic environment (Hassan et al. 2019). Reduced solvent access and
binding of metal co-factors enable them to catalyze at high acid environment
(Hassan et al. 2019). Enriched acidic amino acid residues along with low solvent
exposure makes them acid stable. They reduce the electrostatic repulsion by altering
charged amino acid with neutral polar amino acid. Isoleucine residues are also
present in high amount in acidophilic enzyme as compared to mesophilic enzymes
(Jaenicke 1991).
14.8.5 Industrial Applications
Acid-stable enzymes are used in many industries such as paper, leather, and food and
feed industry. Biobleaching, food industry, starch and baking industry, fruit juice
industry, wine industry, and animal feed are some industries that need acidophilic
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 299

enzymes (Charlesworth and Burn 2016; Sharma et al. 2016; Dumorné et al. 2017;
Christel 2018; Narayanasamy et al. 2018).
14.8.5.1 Acidophilic Laccase
Laccase from marine fungi Trematosphaeria mangrovei have maximum activity at
65 C temperature and pH 4.0. Presence of FeSO
4
and NaN
3
inhibits the activity of
enzyme (Atalla et al. 2013). A white rot fungus Ganoderma lucidium produces
laccase that have optimal activity at pH 3.5 and temperature 20 C (Ko et al. 2001).
Laccase from Aspergillus species HB-RZ4 has molecular weight approximately
62 kDa and optimal enzyme activity is 8.125 U/mL. This enzyme is stable and
active under 4.6–6.0 pH range and 20–60 C temperature; however, optimal pH is
4.5 and optimal temperature is 34 C. Glucose in the media highly increases the
productivity and glycerol in the media has very low productivity. Sodium azide,
halides, and fluorides inhibit the laccase activity. Cu
2+
,Mo
2+
,Mn
2+
, and Zn
2+
stimulated the activity of laccase. Kinetic parameters, Km and Vmax values, are
26.8 mM and 7132.6 mM/min, respectively. The attractive property is that during the
bleaching process it decolorizes the dye in the absence of a laccase mediator system,
which ultimately reduces the production cost (Sayyed et al. 2020). Fomitella
fraxinea acidophilic laccase has optimal activity at pH 3 and temperature 70 C
(Hassan et al. 2019).
14.8.5.2 Acidophilic Manganese Peroxidase
Manganese peroxidase from Phanerochaete chrysosporium is made up of two
domains. It possesses an extra disulfide bond between cys341 and cys 348 that is
essential for the formation of Mn
2+
binding site. Ten major and one minor helix
contribute the structure. Proximal His 173 of active site and Asp 242 make a
hydrogen bond responsible for iron low negative potential. His 46 and Arg
42 form H
2
O
2
binding pocket. Cd
2+
is found as a reversible competitive inhibitor
of Mn
2+
. Their activity in low pH makes them industrially important (Chandra et al.
2017).
14.8.5.3 Acidophilic Chitinase
A fungal species of genus Microbispora produces chitinases, which has acidophilic
and thermophilic property. The isolated enzymes have around 35 kDa molecular
mass. They have optimum enzymatic activity at pH 3.0 and temperature 60 C.
Although they retain their activity at pH range 3–11. The maximum activity is found
in the presence of p-nitro-β-D-N,N0-diacetylchitobiose (Nawani et al. 2002).
14.8.5.4 Acidophilic Xylanase
Acidophilic xylanase from acidophilic fungus Bispora are very stable under acidic
condition. The enzyme activity graphs of this acidophilic xylanase show two activity
peaks at pH 3 and 4.5. They are acid stable and resistant to Co
2+
,Mn
2+
,Cr
2+
, and
Ag
2+
(Wang et al. 2010). Acid stable xylanases are used in pulp and paper industries
for biobleaching process. As they have optimal activity at low pH, the metal ions in
the pulp are removed out easily. So that there is no requirement of the additional
300 K. Choudhary et al.

processes for metal ion removals such as application of chelating agents like EDTA
which lowers the production cost. In food and feed industry, these enzymes are used
for the release of nutrients from the diet, resulting in better digestibility. In biofuel
production process, the pretreatment requires the addition of dilute sulphuric acid for
making the catalysis easy. Thus, use of these acidophilic xylanases reduces the cost
of biofuel production as there is no need of pH adjustment (Rao and Li 2017; Sharma
et al. 2017; Dey and Roy 2018). An extracellular xylanase is isolated from acido-
philic Aspergillus flavus MTCC9390 is active and stable at pH 6 along with 60 C
temperature. Addition of metal salts or additives inhibits the activity of xylanase
while polyethylene glycol inhibits and interferes with the enzyme activity in a
concentration-dependent manner (Bharat et al. 2014). Trichoderma reesei produces
xylanase made up of 178 amino acid residues. Glu 75 and Glu 164 are catalytic
residues. Glu 75 is highly conserved and functions as nucleophile while Glu 164 is
less conserved and functions as acid base catalyst. It contains two β-sheets and one
α-helix. Three subsites are present at active site: one subsite at reducing end while
remaining two subsites at the nonreducing end near ligand binding. Xylanase C of
Aspergillus kawachii possess nucleophilic residue Glu79 and acid–base catalyst
residue Glu170. More acidic amino acid residues at the edge of catalytic cleft and
Asp 37 are responsible for low pH optima. Xylanase 1 from Scytalidium
acidophilum possess Glu 104 and Glu 192 catalytic residue and Asp 60 residue
near the acid base catalyst forms hydrogen bond with an angle of 2.45 Ǻ. Xylanase I
of Aspergillus niger possess same catalytic and aspartic acid residues as in the
xylanase of A. kawachii. This enzyme functions at pH 3. Asp 37 makes hydrogen
bond with Glu 170 with a bond angle 2.8 Ǻthat is responsible for low pH activity
(Dey and Roy 2018). Family 11 acidophilic xylanase contain aspartic acid residue
makes hydrogen bond with acid–base catalyst. They possess high number of acidic
residues and low number of salt bridges as compared to alkaliphilic xylanase
(Collins et al. 2005).
14.8.5.5 Acidophilic Polygalactouronse
Acidic polygalactouronase are used in food industries for clarification of vegetables
and fruits juices by the degradation of pectin and lowering the viscosity. They are
used in combination with the other hydrolases to produce the animal feed-in-feed
industries. The acid-stable hydrolases of A. kawachii is used in the fermentation
process (where acidic medium is required) as they are highly active under low pH
range 2.0–3.0 (Hassan et al. 2019).
14.8.5.6 Acidophilic b-Mannanase
β-Mannanases are enzymes that break β-mannosidic bonds in mannan. It has optimal
pH range of 2.4–6.0 that makes it very suitable for feed industries. A novel acidic
β-mannanase produced by Penicillium pinophilum C1 isolated from tin mine has
maximum activity at pH 3 and temperature 28 C (Hassan et al. 2019). Mannnanase
from acidophilic Bispora MEY-1 has high activity and stability over the pH range
1.0–6.0 and optimal temperature 65 C. They have strong resistance for proteases.
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 301

14.8.5.7 Acidophilic a-Amylases
Acidophilic α-amylases from acidophiles are used for the saccharification of starch
and hydrolysis of polysaccharides for the production of bioethanol. Acid-stable
α-amylases reduce the cost and time of the process that makes them economic and
time saving. These enzymes possess high number of basic residues that makes the
enzyme surface positive, which is responsible for the acid stability (Parashar and
Satyanarayana 2018).
14.9 Alkaliphilic Enzymes
14.9.1 Alkaliphiles
Extremophiles that grow optimally at pH value above 10 classified as alkaliphiles
(Dumorné et al. 2017).
14.9.2 Classification of Alkaliphiles
Organisms that grow at pH above 9 are alkaliphiles while those grow at pH above
9 along with the high salt concentration are referred to as haloalkaliphiles.
Alkaliphiles that grow only at alkaline pH above 9 but cannot grow at neutral pH
are obligate alkaliphiles while those grow at alkaline pH but can also grow at neutral
pH are known as facultative alkaliphiles. Microorganisms that can actively grow at
pH range 7–9 but can also survive in alkaline environment with pH value above 9 are
known as alkalitolerant (Gupta et al. 2014).
14.9.3 Habitat
Hypersaline water, high CaOH ground water, soda lake, soda desert, alkaline
drainage water, and alkaline carbonate lakes are native locations of alkaliphilic
microorganisms (Ulukanli and DIĞRAK 2002).
14.9.4 Challenges in Alkaline Environment
At high pH, the cytoplasm of a mesophile becomes alkaline as a result of influx of
hydroxyl ions in the cell or efflux of hydrogen ions from the cell. Proton motive force
is disrupted at high pH, which interferes with the substrate uptake, resulting in low
ATP synthesis. At alkaline condition, cell membrane structure becomes disturbed.
This decreases the specific permeability of cell membrane. Thus, maintenance of
internal pH is very essential for survival in alkaline environment (Gomes and Steiner
2004).
302 K. Choudhary et al.

14.9.5 Catalytic Features of Alkaliphilic Enzymes
The intracellular enzymes of alkaliphiles do not use any strategy to counter high pH
as the organism maintains its internal pH near neutral. However, the extracellular
enzyme shows adaptations against high pH.
14.9.5.1 Negatively Charged Amino Acid Residues
The alkaliphilic enzymes have increased number of negatively charged amino acid
residues on its surface (Kumar et al. 2018).
14.9.5.2 Increased Hydrogen Bonds
Higher number of hydrogen bonds and hydrophobic interactions in alkaliphilic
enzymes makes them alkaline stable (Kumar et al. 2018).
14.9.5.3 Fewer Hydrophobic Residues
The number of hydrophobic residues exposed to the solvent is very low, essential for
its activity in alkaline environment (Krulwich et al. 2010).
14.9.6 Industrial Applications
Alkaliphilic enzymes are active and stable at high pH. This property of enzyme is
very useful in biotechnological industries. Alkaliphilic proteases are used in hide
deharing processes and removing clogs in drain pipes. Alkaline cellulases are used in
saline wastewater treatment. Alkaline xylanases and pectinases are used in paper
manufacturing and wastewater treatment. Alkaline mannanase are used in food
processing. Use of alkaliphilic enzymes in detergent industries makes the product
stable and reduces the cost of production (Ulukanli and DIĞRAK 2002).
14.9.6.1 Alkaliphilic Laccase
Alkaliphilic laccases from Alkalitolerant fungi Myrothecium verrucaria have opti-
mal activity at pH 9 and temperature 70 C. Substrate specificity of this enzyme is
toward phenolics and aniline compounds (Sulistyaningdyah et al. 2004). Laccase
from basidiomyceteous fungus Coprinopsis cinerea is engineered and expressed in
Pichia pastoris. This mutated laccase is an alkali stabile and shows optimal pH 7–7.5
for dye decolorization process. They are active and stable at alkaline pH, thus highly
desirable in textile industries (Yin et al. 2019).
14.9.6.2 Alkaliphilic Xylanase
The alkaliphilic xylanases readily break down xylan that is soluble in alkaline
solution. Alkaliphilic extracellular xylanases isolated from Aspergillus nidulans
show optimal activity at pH 8.0 and temperature 55 C. However, they can be active
and stable over a wide range of pH (4.0–9.5) and temperature (40–55 C). The
enzyme activity is stimulated by the presence of Na
2+
and Fe
3+
while its activity is
inhibited by the presence of acetic anhydride, SDS, Hg
2+
, and Pb
2+
. Substrate
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 303

binding affinities of xylanases are higher for the xylans from hardwood. Studies on
inhibition show that the cysteine residues are present on the catalytic site. Positively
charged amino groups are essential for the catalytic activity of this enzyme (Taneja
et al. 2002). Penicillium citrinum extracellular xylanases have optimal enzymatic
activity at pH 8.5, temperature 50 C, and PI 3.6. Molecular mass were found to be
25 kDa. Their activity is stimulated by β-mercaptoethanol, dithiotheritol, cysteine,
NaCl, urea, and SDS. Hg
2+
and Mn
2+
inhibit the activity of enzymes. There is no
metal ion present in the active site (Dutta et al. 2007). Xylanase from an
alkalitolerant fungus Aspergillus niveus RS2 has approximately 22.5 kDa molecular
mass and shows maximum activity of 18.2 U/ml at pH 8. Enzymatic activity is
optimal at pH 7 and 50 C temperature. Km and Vmax values of enzyme are 2.5 mg/
ml and 26 μmol/mg/min, respectively. Hg
2+
strongly inhibits the enzyme activity
while it is moderately stimulated with the presence of Mn
2+
(Sudan and Bajaj 2007).
Alkaliphilic xylanase of family 11 type contains high number of salt bridges and less
number of acidic residues as compared to acidophilic xylanase. In alkaliphilic
xylanase, asparagine makes hydrogen bond with acid–base catalyst (Collins et al.
2005).
14.9.6.3 Alkaliphilic Proteases
Alkaliphilic protease increases the smoothness and dye affinity of wool (Kumar et al.
2011). Alkaliphilic protease from A. niger shows maximum activity at 45 C and
pH 8.5. It is thermostable and has a molecular mass of 38 kDa. The combination of
50 C temperature and pH 10 is mandatory for optimal enzymatic activity. Presence
of detergents stimulates the enzyme activity while Cu
2+
,Hg
2+
,Zn
2+
, EDTA, and
sodium azide inhibit the activity of protease (Devi et al. 2008). An alkaliphilic strain
of Streptomyces albidoflavus produces extracellular proteases, which are capable of
hydrolyzing keratin. This protease has optimal enzymatic activity at pH 9 and
temperature 60–70 C. Alkaline proteases have optimal pH range 9.0–11.0 and
optimal temperature range 50–70 C. They have molecular mass between 15 and
30 kDa. To maintain their active confirmation, they require metal ions or divalent
cations. Combination of metal ions or cations maximizes the enzyme activity. Hg
2+
,
phenylmethylsulphonylfluoride, and diisopropyl fluorophosphates are some
inhibitors of protease activity. They are more specific against aromatic or hydropho-
bic amino acid residues (El-Shafei et al. 2010).
Alkalitolerant P. citrinum produces 32.27 kDa halotolerant alkaline serine
proteases that are stable in acidic condition along with high salinity. They have
optimal activity at pH 8.0, temperature 40 C, and on substrate casein. They are
stable at broad range of pH 6.0–8.0 and temperature 4–30 C. The Km and Vmax
values of protease for casein were found as 1.93 mg/mL and 56.81 μg/min/mL,
respectively. They require salinity for their activity but higher NaCl concentration
interferes with their activity. However, many ions such as K
+
,Ca
2+
,Zn
2+
,Mg
2+
,
Fe
2+
, and Fe
3+
inhibit the protease activity. Phenylmethyl sulfonyl fluoride strongly
inhibited the activity of protease while presence of Mn
2+
stimulated the activity of
this enzyme (Dutta et al. 2007). Aspergillus strain KH17 produces alkaline protease
304 K. Choudhary et al.

that has maximum enzyme activity (215 U/ml) on casein. Enzymatic activity is
optimal at pH 9 and 25–30 C temperature (Palanivel et al. 2013).
14.9.6.4 Alkaline Cellulases
They catalyze a reaction under alkaline condition. In detergent industry, alkaline
cellulases are used as a laundry detergent additive, which enhances the cleaning
effect of detergent. These cellulases are known for their thermostability as they have
excellent heat resistance. Some Aspergillus and Penicillium strains isolated from
rainforest of Peru produce alkaline cellulases that are active at pH 9.4 (Vega et al.
2012; Rao and Li 2017).
14.9.6.5 Alkaline Pectinases
A class of enzyme that catalysis the breakdown of pectin substrates. Pectinases from
Penicillium italicum is stable at pH 8 and temperature 50 C. Amycolata species
secreted highly alkaline pectinase having optimum activity at pH 10.25 and temper-
ature 70 C. Pectinases from Streptomyces species QG-11-3 is active at pH 3–9 and
stable at optimal temperature 60 C. Alkaline pectinases are used in textile
processing, bioscouring of cotton fibers, degumming of plant bast fibers, retting of
plant fibers, and pretreatment of pectic wastewaters (Hoondal et al. 2002).
14.10 Piezophilic Enzymes
14.10.1 Piezophiles
Piezophiles also known as barophiles are the organisms that thrive under high
hydrostatic pressure often exceeding up to 400 atm. Extreme barophiles optimally
grow at 700 atm or more. Piezophiles are found in deep sea sediments, marine
trenches, hydrothermal vents, ocean floors, and deep rocks. Piezophilic
microorganisms requires high pressure to grow optimally while piezotolerant
microorganisms grow optimally at atmospheric pressure or high pressure but do
not obligately require high pressure (Ichiye 2018; Jin et al. 2019).
14.10.2 Structural/Catalytic Features of Piezophilic Proteins
High pressure causes reduction in lipid fluidity and membrane permeability, dena-
turation of DNA and protein, compactness in protein structure, protein unfolding,
and reduction in enzyme activity. Proteins from piezophiles do not exhibit any
adaptation to counter pressure effect. Compression and unfolding are the two
changes observed in proteins due to high pressure. Studies on protein domains and
internal cavities of different proteins show compaction at high pressure. Elevated
pressure reduces the volume in proteins resulting in the modification of interactions
between the polypeptide and water molecule leads to unfolding of protein. Protein
denaturation occurs at or more than 400 MPa hydrostatic pressure. At genomic level,
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 305

these organisms primarily adapt strategies to protect the DNA and protein from
damage and secondary adaptation involves the high repair rate. Some organisms
possess pressure-regulated operons. Transfer of mobile genetic element is very
significant as they possess pressure-adaptive genes. Cellular adaptations include
reduction in cell division, production of compatible osmolyte (piezolytes) to restrict
the changes caused by high pressure, and presence of increased number of polyun-
saturated fatty acids in membranes to increase the membrane fluidity. Increase in
1 kbar pressure causes reduction in reaction rate by 15% that indicates the sensitivity
of proteins to pressure unfolding. At proteomic level, these piezophilic proteins
include the high amount of arginine amino acid and increased number of small
amino acids such as glycine, lysine, and valine. Proline residues are present in very
less amount. Low stability of piezophilic enzymes provides high flexibility. High-
pressure structural stability is due to small volume and high amount of hydrophobic
interaction in protein. Arginine is known to be a barophilic amino acid as it is more
commonly found in piezophilic enzyme. Smallest volume and high percentage of
hydrophobic interactions in piezophilic enzymes makes them stable in high-pressure
environment. Small cavities present in piezophilic proteins allow the penetration of
water molecule at increased pressure. They possess high compressibility, which
protects them from pressure-induced deformation. High absolute activity and high
relative activity at high pressure increase its catalytic efficiency and make them
stable. Kcat values are relatively higher in most of the deep sea piezophilic enzymes.
Piezophilic enzymes and their mesophilic homolog show no structural variations.
However, variations are found in the flexibility and hydration of protein. Yeast
S. cerevisiae cope with the deep sea pressure by altering its membrane composition.
This is to be noted that higher and positive the value of change in free energies of
unfolded and folded state, greater will be the protein ability to prevent itself from
denaturation (Kumar et al. 2011; Ichiye 2018; Castell et al. 2019; Jin et al. 2019).
14.10.3 Industrial Application
Piezophilic proteins are used in processing and sterilization of food products that
need high pressure. High pressure provides better flavor to food products. At high
pressure, food products decolorize but use of piezophilic protein preserves the color
of food products. The high-pressure stable enzymes are very useful in high-pressure
bioreactors for food processing and production of antibiotics and other drugs. They
are also used in microbial-assisted oil recovery process (Ichiye 2018; Jin et al. 2019).
Yeast found in deep sea of Japan trench produces two types of polygalacturonase
that is active at 24 C temperature and 100 MPa hydrostatic pressures (Arifeen and
Liu 2008). These pressure-tolerant enzymes are also thermostable and hence cata-
lyze a biological reaction more efficiently under high pressure and high temperature.
During the fermentation process, microorganism produces certain undesirable
by-products that are reduced with increased pressure as the microorganism modifies
the metabolic pathway and produces the desirable products. Piezophiles are used in
the biopurification process of antigens where high pressure applied that increases the
306 K. Choudhary et al.

volume and causes dissociation of antigen–antibody complex to purify the desired
antigen. High pressure induces the dissociation of complexes. Therefore, piezophiles
and their enzymes are used in protein recovery process to purify protein from the
aggregate of incomplete folded protein. High pressure reduces the allergenicity of
protein. Hence, piezophiles are used to produce food products with very low or no
allergenicity. High pressure enhances the hydrolysis of inaccessible site of protein
that increases the digestibility of food products. Therefore, food products obtained
by piezophiles or with the help of their enzymes possess greater shelf life and
increased digestibility. Due to its thermostability and high-pressure resistance,
piezophiles and their enzymes are very useful in various biotechnological and
pharmaceutical processes. They are useful in the formation of gels and starch
granules (Castell et al. 2019). Piezophilic α-amylases from deep sea piezophiles
are important because they produce trisaccharides from maltooligosaccharides at
high pressure instead of producing maltobiose and tetrasaccharide. This low-energy
consumption process is very useful for food processing industries (Jin et al. 2019).
14.11 Metallophiles and Metallophilic Enzymes
14.11.1 Metallophiles
Metallophiles are the organisms that thrive under metal-rich condition or environ-
ment with high metallic concentration. They are able to tolerate and detoxify high
concentration of heavy metals. Most of the metallophiles are acidophiles, thus
enhancing their survival 1000-fold than mesophiles and efficiently tolerate the
high level of heavy metals (Anahid et al. 2011; Gupta et al. 2014).
Fungal metalloenzymes have hardly been studied from catalytic mechanism point
of views. There are reports of their finding from the study on models (synthetic) of
metalloenzymes (Ohta et al. 2012; Sunghee et al. 2012).
14.12 Radioresistants and Their Enzymes
14.12.1 Radioresistants
Radioresistants or radiophiles are the extremophiles that are highly resistant to high
level of ionizing and ultraviolet radiation. Radioresistant organisms tolerate extreme
radiations for longer period of time while radiotolerant organisms tolerate extreme
radiations for only a short period of time. Ionizing radiation such as gamma radiation
and nonionizing radiation such as ultraviolet radiation are the two major radiations
that cause lethal effect on an organism. Radiophiles are polyextremophiles as they
can tolerate extreme cold, dehydration, vacuum, and high acidic concentration
(Coker 2019).
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 307

14.12.2 Radioresistant Enzymes
Catalytic features of radioresistant fungi have not been deeply studied. They are
mainly studied for their adaptation to radiation mutations. Biofilm forming
radioresistant fungi are especially adapted to high mutation rate and are more
resistant to ionizing radiation than other radioresistants (Ragon et al. 2011).
Genome-wide radiation resistance analysis of Cryptococcus neoformans,a
radioresistant yeast, explains the upregulation of DNA repair machinery for reducing
the radiation stress. Rad53 protein kinase regulates the transcription factor Bdr1 and
controls the transcription (Jung et al. 2016).
14.12.3 Industrial Application
Radioresistants possess high potential for the treatment of radioactive environmental
waste. Radiophiles are useful in ore-bioleaching and biomineralization process.
Extracellular polymeric substances produced by this extremophile are used as
adhesive, used in cosmetic, pharmaceutical products as antitumor, antiviral, or
anti-inflammatory agent and oil recovery processes (Kazak et al. 2010; Parihar and
Bagaria 2019). Melanized fungi show increased growth in the presence of ionizing
radiation due to the changes in the electronic properties of melanin. Thus, these
melanized fungi can be used in high radiation-affiliated processes (Dadachova et al.
2007). Fungal hyphae possess the ability to absorb or adsorb radionuclides, which
are very useful in the treatment of industrial effluent contaminated with heavy metals
and radionuclides. Some melanized fungi such as Cladosporium sphaerospermum
optimally grow in radiation-rich environment such as radioactive nuclear plant and
show the ability to prevent ionizing radiations. They perform radiosynthesis using
melanin as an analog to energy harvesting pigment such as chlorophyll to convert
gamma radiations into chemical energy. This unique property is useful for industries
for making a passive radiation shields and used in deep space exploration mission to
protect against radiations (Shunk et al. 2020).
Radiation-tolerant fungi can be used as the indicators of radioactive contamina-
tion. Occurrence of fungal species such as Chaetomium aureum and Penicillium
liliacinus shows the high radiation environment (3.7x10
6
–3.7x10
8
Bq/Kg)
(Matusiak 2016). An acid-tolerant and gamma radiation-resistant yeast Rhodotorula
taiwanensis MD1149 isolated from an acid mine drainage possess the ability to cope
with the environment of high radiation, low pH, and high level of heavy metals. This
strain grows under 66 Gy/h of radiation exposure at pH 2.3 and high concentration of
mercury and chromium, which makes them very suitable for bioremediation of
acidic radioactive waste (Tkavc et al. 2018).
A study on filamentous fungi irradiated with gamma radiation was investigated
for their lignolytic enzyme production. The fungi Aspergillus awamori,Aspergillus
terreus, and Penicillium species produce lignin peroxidase, Manganese peroxidase
and laccase in high amount at 500 Gy dose. Lignolytic enzyme production can be
increased in these fungi through gamma radiation exposure (de Queiroz Baptista
308 K. Choudhary et al.

et al. 2015). Studies suggest that many fungal hyphae grow toward the ionizing
radiation source when they are exposed to high radiation environment. The fungi
exposed to radiations show positive radiotropism and some of them involved in the
decomposition of ribonuclide containing organic debris (Zhdanova et al. 2004).
Radioresistant microorganisms are used for making photoprotective devices.
Microbes that have the ability to withstand high radiation environment are very
useful for pharmaceutical industries. Microbial metabolic reserves or extremolytes
of these organisms can be used in the production of anticancer drugs as extremolytes
are resistant to UV radiations and possess antioxidant property. Thus, it can prevent
the skin from damage caused by harmful radiations (Singh and Gabani 2011).
References
Ali I, Akbar A, Anwar M, Prasongsuk S, Lotrakul P, Punnapayak H (2015) Purification and
characterization of a polyextremophilic α-amylase from an obligate halophilic aspergillus
penicillioides isolate and its potential for souse with detergents. Biomed Res Int 2015:245649
Ali I, Akbar A, Anwar M, Yanwisetpakdee B, Prasongsuk S, Lotrakul P, Punnapayak H (2014a)
Purification and characterization of extracellular, polyextremophilic α-amylase obtained from
halophilic Engyodontium album. Iran J Biotechnol 12(4):35–40
Ali I, Akbar A, Yanwisetpakdee B, Prasongsuk S, Lotrakul P, Punnapayak H (2014b) Purification,
characterization, and potential of saline waste water remediation of a polyextremophilic
α-amylase from an obligate halophilic aspergillus gracilis. Biomed Res Int 2014:106937
Anahid S, Yaghmaei S, Ghobadinejad Z (2011) Heavy metal tolerance of fungi. Scientia Iranica 18
(3):502–508
ArfiY, Chevret D, Henrissat B, Berrin JG, Levasseur A, Record E (2013) Characterization of salt-
adapted secreted lignocellulolytic enzymes from the mangrove fungus Pestalotiopsis sp. Nat
Commun 4(1):1–9
Arifeen MZU, Liu C (2008) Novel enzymes isolated from marine-derived fungi and its potential
applications. J Biotechnol Bioeng 2(4):1–12
Atalla MM, Zeinab HK, Eman RH, Amani AY, Abeer AAEA (2013) Characterization and kinetic
properties of the purified Trematosphaeria mangrovei laccase enzyme. Saudi J Biol Sci 20(4):
373–381
Basaveswara Rao V, Sastri NVS, Subba Rao PV (1981) Purification and characterization of a
thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus. Biochem
J 193(2):379–387
Batista-García RA, Balcázar-López E, Miranda-Miranda E, Sánchez-Reyes A, Cuervo-Soto L,
Aceves-Zamudio D, Atriztán-Hernández K, Morales-Herrera C, Rodríguez-Hernández R,
Folch-Mallol J (2014) Characterization of lignocellulolytic activities from a moderate halophile
strain of aspergillus caesiellus isolated from a sugarcane bagasse fermentation. PLoS One 9(8):
e105893
Beltagy EA, Rawway M, Abdul-Raouf UM, Elshenawy MA, Kelany MS (2018) Purification and
characterization of theromohalophilic chitinase producing by halophilic aspergillus flavus
isolated from Suez gulf. Egyptian J Aquat Res 44(3):227–232
Berka RM, Rey MW, Brown KM, Byun T, Klotz AV (1998) Molecular characterization and
expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus. Appl
Environ Microbiol 64(11):4423–4427
Bharat B, Ajay P, Satish K (2014) Production and partial characterization of extracellular xylanase
from acidophilic aspergillus flavus MTCC 9390 grown in SSF mode. Res J Biotechnol 9:5
Broderick JB (2001) Coenzymes and cofactors. In: Encyclopedia of life sciences. Wiley, West
Sussex
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 309

Bush EC, Clark AE, DeBoever CM, Haynes LE, Hussain S, Ma S, McDermott MM, Novak AM,
Wentworth JS (2012) Modeling the role of negative cooperativity in metabolic regulation and
homeostasis. PLoS One 7(11):e48920
Castell MS, Oger P, Peters J (2019) High-pressure adaptation of extremophiles and biotechnologi-
cal applications. In: Physiological and biotechnological aspects of extremophiles. Academic
Press, London. In press Hal-02122331
Cavicchioli R, Charlton T, Ertan H, Omar SM, Siddiqui KS, Williams TJ (2011) Biotechnological
uses of enzymes from psychrophiles. Microb Biotechnol 4(4):449–460
Chandra R, Kumar V, Yadav S (2017) Extremophilic ligninolytic enzymes. In: Extremophilic
enzymatic processing of lignocellulosic feedstocks to bioenergy 2017. Springer, Cham, pp
115–154
Charlesworth J, Burns BP (2016) Extremophilic adaptations and biotechnological applications in
diverse environments. AIMS Microbiol 2(3):251–261
Chen CY, Huang YC, Wei CM, Meng M, Liu WH, Yang CH (2013) Properties of the newly
isolated extracellular thermo-alkali-stable laccase from thermophilic actinomycetes,
Thermobifida fusca and its application in dye intermediates oxidation. AMB Express 3(1):49
Chen J, Li DC, Zhang YQ, Zhou QX (2005) Purification and characterization of a thermostable
glucoamylase from Chaetomium thermophilum. J Gen Appl Microbiol 51(3):175–181
Christel S (2018) Function and adaptation of acidophiles in natural and applied communities.
Doctoral dissertation, Linnaeus University Press. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:
diva-77666
Cipolla A, Delbrassine F, Da Lage JL, Feller G (2012) Temperature adaptations in psychrophilic,
mesophilic and thermophilic chloride-dependent alpha-amylases. Biochimie 94(9):1943–1950
Coker JA (2019) Recent advances in understanding extremophiles. 13 (8) F1000 Faculty Rev-1917.
https://doi.org/10.12688/f1000research.20765.1
Collins T, D'Amico S, Marx JC, Feller G, Gerday C (2007) Cold-adapted enzymes. In: Gerday C,
Glansdorff N (eds) Physiology and biochemistry of extremophiles. ASM Press, Washington,
DC, pp 165–179
Collins T, Gerday C, Feller G (2005) Xylanases, xylanase families and extremophilic xylanases.
FEMS Microbiol Rev 29(1):3–23
Dadachova E, Bryan RA, Huang X, Moadel T, Schweitzer AD, Aisen P, Nosanchuk JD, Casadevall
A (2007) Ionizing radiation changes the electronic properties of melanin and enhances the
growth of melanized fungi. PLoS One 2(5):e457
D’Amico S, Marx JC, Gerday C, Feller G (2003) Activity-stability relationships in extremophilic
enzymes. J Biol Chem 278(10):7891–7896
DasSarma S, DasSarma P (2015) Halophiles and their enzymes: negativity put to good use. Curr
Opin Microbiol 25:120–126
de Cassia PJ, Paganini Marques N, Rodrigues A, Brito de Oliveira T, Boscolo M, da Silva R,
Gomes E, Bocchini Martins DA (2015) Thermophilic fungi as new sources for production of
cellulases and xylanases with potential use in sugarcane bagasse saccharification. J Appl
Microbiol 118(4):928–939
de Queiroz Baptista NM, Solidônio EG, de Arruda FV, de Melo EJ, de Azevedo Callou MJ, de
Miranda R, Colaço W, de Gusmão NB (2015) Effects of gamma radiation on enzymatic
production of lignolytic complex by filamentous fungi. Afr J Biotechnol 14(7):612–621
Deniz AA (2018) Enzymes can adapt to cold by wiggling regions far from their active site. Nature
558:195–196
Devi MK, Banu AR, Gnanaprabhal GR, Pradeep BV, Palaniswamy M (2008) Purification, charac-
terization of alkaline protease enzyme from native isolate aspergillus Niger and its compatibility
with commercial detergents. Indian J Sci Technol 1(7):1–6
Dey P, Roy A (2018) Molecular structure and catalytic mechanism of fungal family G acidophilic
xylanases. 3. Biotech 8(2):78
Duarte A, Dos Santos JA, Vianna MV, Vieira J, Mallagutti VH, Inforsato FJ, Wentzel L, Lario LD,
Rodrigues A, Pagnocca FC, Pessoa Junior A, Durães Sette L (2018) Cold-adapted enzymes
310 K. Choudhary et al.

produced by fungi from terrestrial and marine Antarctic environments. Crit Rev Biotechnol
38(4):600–619
Dumorné K, Córdova DC, Astorga-Eló M, Renganathan P (2017) Extremozymes: a potential
source for industrial applications. J Microbiol Biotechnol 27(4):649–659
Dutta T, Sengupta R, Sahoo R, Sinha Ray S, Bhattacharjee A, Ghosh S (2007) A novel cellulase
free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and
characterization. Lett Appl Microbiol 44(2):206–211
Edbeib MF, Wahab RA, Huyop F (2016) Halophiles: biology, adaptation, and their role in
decontamination of hypersaline environments. World J Microbiol Biotechnol 32(8):135
El-Shafei H, Abdel-Aziz MS, Ghaly M, Abdalla A (2010) Optimizing some factors affecting
alkaline protease production by a marine bacterium Streptomyces albidoflavus. Afr J Biotech
6:125–142
Ernst HA, Jørgensen LJ, Bukh C, Piontek K, Plattner DA, Østergaard LH, Larsen S, Bjerrum MJ
(2018) A comparative structural analysis of the surface properties of asco-laccases. PLoS One
13(11):e0206589
Feller G (2013) Psychrophilic enzymes: from folding to function and biotechnology. Scientifica
2013:512840
Geoffry K, Achur RN (2018) Optimization of novel halophilic lipase production by fusarium solani
strain NFCCL 4084 using palm oil mill effluent. J Genet Eng Biotechnol 16(2):327–334
Gomes J, Steiner W (2004) The biocatalytic potential of extremophiles and extremozymes. Food
Technol Biotechnol 42(4):223–225
Gupta GN, Srivastava S, Khare SK, Prakash V (2014) Extremophiles: an overview of microorgan-
ism from extreme environment. Int J Agric Environ Biotechnol 7(2):371–380
Hakulinen N, Turunen O, Jänis J, Leisola M, Rouvinen J (2003) Three-dimensional structures of
thermophilic β-1, 4-xylanases from Chaetomium thermophilum and Nonomuraea flexuosa:
comparison of twelve xylanases in relation to their thermal stability. Eur J Biochem 270(7):
1399–1412
Hassan N, Rafiq M, Rehman M, Sajjad W, Hasan F, Abdullah S (2019) Fungi in acidic fire: a
potential source of industrially important enzymes. Fungal Biol Rev 33(1):58–71
Hoondal G, Tiwari R, Tewari R, Dahiya NBQK, Beg Q (2002) Microbial alkaline pectinases and
their industrial applications: a review. Appl Microbiol Biotechnol 59(4–5):409–418
Ichiye T (2018) Enzymes from piezophiles. Semin Cell Dev Biol 84:138–146
Jaenicke R (1991) Protein stability and molecular adaptation to extreme conditions. EJB reviews
1991:291–304
Jin M, Gai Y, Guo X, Hou Y, Zeng R (2019) Properties and applications of extremozymes from
deep-sea extremophilic microorganisms: a mini review. Mar Drugs 17(12):656
Joshi S, Satyanarayana T (2013) Biotechnology of cold-active proteases. Biology 2(2):755–783
Jung KW, Yang DH, Kim MK, Seo HS, Lim S, Bahn YS (2016) Unraveling fungal radiation
resistance regulatory networks through the genome-wide transcriptome and genetic analyses of
Cryptococcus neoformans. MBio 7(6):e01483–e01416
Karan R, Capes MD, DasSarma S (2012) Function and biotechnology of extremophilic enzymes in
low water activity. Aquat Biosyst 8(1):4
Kazak H, Oner ET, Dekker RF (2010) Extremophiles as sources of exopolysaccharides. In: Ito R,
Matsuo Y (eds) Carbohydrate polymers: development, properties and applications. Nova
Science, Hauppauge, NY, pp 605–619
Ko EM, Leem YE, Choi H (2001) Purification and characterization of laccase isozymes from the
white-rot basidiomycete Ganoderma lucidum. Appl Microbiol Biotechnol 57(1–2):98–102
Krulwich TA, Liu J, Morino M, Fujisawa M, Ito M, Hicks DB (2010) Adaptive mechanisms of
extreme alkaliphiles. In: Horikoshi K (ed) Extremophiles handbook. Springer, Heidelberg, pp
120–139
Kumar A, Alam A, Tripathi D, Rani M, Khatoon H, Pandey S, Ehtesham NZ, Hasnain SE (2018)
Protein adaptations in extremophiles: an insight into extremophilic connection of mycobacterial
proteome. Semin Cell Dev Biol 84:147–157
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 311

Kumar L, Awasthi G, Singh B (2011) Extremophiles: a novel source of industrially important
enzymes. Biotechnology 10(2):121–135
Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng 13(3):
179–191
Lam SY, Yeung RC, Yu TH, Sze KH, Wong KB (2011) A rigidifying salt-bridge favors the activity
of thermophilic enzyme at high temperatures at the expense of low-temperature activity. PLoS
Biol 9(3):e1001027
Li AN, Ding AY, Chen J, Liu SA, Zhang M, Li DC (2007) Purification and characterization of two
thermostable proteases from the thermophilic fungus Chaetomium thermophilum. J Microbiol
Biotechnol 17(4):624–631
Li D-C, Li A-N, Papageorgiou AC (2019) Cellulases from thermophilic fungi: recent insights and
biotechnological potential. Fungi in Extreme Environments: Ecological Role and Biotechno-
logical Significance 2019:395–417 Enzyme Research, vol. 2011, Article ID 308730,
9 pages, 2011
Liu WH, Beppu T, Arima K (1973a) Physical and chemical properties of the lipase of thermophilic
fungus Humicola lanuginosa S-38. Agric Biol Chem 37(11):2493–2499
Liu WH, Beppu T, Arima K (1973b) Purification and general properties of the lipase of thermo-
philic fungus Humicola lanuginosa S-38. Agric Biol Chem 37(1):157–163
Machuca A, Aoyama H, Durán N (1998) Production and characterization of thermostable phenol
oxidases of the ascomycete Thermoascus aurantiacus. Biotechnol Appl Biochem 27(3):
217–223
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol Rev 64(3):461–488
Matusiak DM (2016) Radiotolerant microorganisms-characterization of selected species and their
potential usage. Postepy Mikrobiologii 55(2):182–194
Mohammadi S, Hashim NH, Mahadi NM, Murad AM (2021) The cold-active Endo-β-1, 3 (4)-
Glucanase from a marine psychrophilic yeast, Glaciozyma antarctica PI12: heterologous
expression, biochemical characterisation, and molecular Modeling. Int J Appl Biol Pharm
Technol 12:279–300
Mohsin I, Poudel N, Li DC, Papageorgiou AC (2019) Crystal structure of a GH3 β-glucosidase
from the thermophilic fungus Chaetomium thermophilum. Int J Mol Sci 20(23):5962
Molnar C, Gair J (2012) Energy and metabolism. https://opentextbc.ca/biology/chapter/4-1-energy-
and-metabolism/
Moubasher AAH, Ismail MA, Hussein NA, Gouda HA (2016) Enzyme producing capabilities of
some extremophilic fungal strains isolated from different habitats of Wadi El-Natrun, Egypt.
Part 2: Cellulase, xylanase and pectinase. Eur J Biol Res 6(2):103–111
Narayanasamy M, Dhanasekaran D, Vinothini G, Thajuddin N (2018) Extraction and recovery of
precious metals from electronic waste printed circuit boards by bioleaching acidophilic fungi.
Int J Environ Sci Technol 15(1):119–132
Nawani NN, Kapadnis BP, Das AD, Rao AS, Mahajan SK (2002) Purification and characterization
of a thermophilic and acidophilic chitinase from Microbispora sp. V2. J Appl Microbiol 93(6):
965–975
Oarga A (2009) Life in extreme environments. Revista de Biologia e ciencias da Terra 9(1):1–10
Ohta S, Ohki Y, Hashimoto T, Cramer RE, Tatsumi K (2012) A nitrogenase cluster model
(Fe8S6O) with an oxygen unsymmetrically bridging two proto-Fe4S3 cubes: relevance to the
substrate binding mode of the FeMo cofactor. Inorg Chem 51:11217–11219
Orellana R, Macaya C, Bravo G, Dorochesi F, Cumsille A, Valencia R, Rojas C, Seeger M (2018)
Living at the frontiers of life: extremophiles in Chile and their potential for bioremediation.
Front Microbiol 9:2309
Ortega G, Laín A, Tadeo X et al (2011) Halophilic enzyme activation induced by salts. Sci Rep 1:6
Palanivel P, Ashokkumar L, Balagurunathan R (2013) Production, purification and fibrinolytic
characterization of alkaline protease from extremophilic soil fungi. Int J Pharm Bio Sci 4(2):
101–110
312 K. Choudhary et al.

Parashar D, Satyanarayana T (2018) An insight into ameliorating production, catalytic efficiency,
thermostability and starch saccharification of acid-stable α-amylases from acidophiles. Front
Bioeng Biotechnol 6:125
Paredes DI, Watters K, Pitman DJ, Bystroff C, Dordick JS (2011) Comparative void-volume
analysis of psychrophilic and mesophilic enzymes: structural bioinformatics of psychrophilic
enzymes reveals sources of core flexibility. BMC Struct Biol 11(1):1–9
Parihar J, Bagaria A (2019) The extremes of life and Extremozymes: diversity and perspectives.
ACTA Sci Microbiol 3(1):107–119
Qasemian L, Billette C, Guiral D, Alazard E, Moinard M, Farnet AM (2012) Halotolerant laccases
from Chaetomium sp., Xylogone sphaerospora, and Coprinopsis sp. isolated from a Mediterra-
nean coastal area. Fungal Biol 116(10):1090–1098
Ragon M, Restoux G, Moreira D, Møller AP, López-García P (2011) Sunlight-exposed biofilm
microbial communities are naturally resistant to Chernobyl ionizing-radiation levels. PLoS One
6(7):e21764
Rampelotto PH (2013) Extremophiles and extreme environments. Life 2013(3):482–485
Rao MN, Li WJ (2017) Microbial Cellulase and xylanase: their sources and applications. Adv
Biochem Appl Med 1:20
Razak CNA, Salleh AB, Musani R, Samad MY, Basri M (1997) Some characteristics of lipases
from thermophilic fungi isolated from palm oil mill effluent. J Mol Catal B Enzym 3(1–4):
153–159
Reed CJ, Lewis H, Trejo E, Winston V, Evilia C (2013) Protein adaptations in archaeal
extremophiles. Archaea 2013:373275
Robinson PK (2015) Enzymes: principles and biotechnological applications. Essays Biochem 59:
1–41
Sahay S, Chouhan D (2018) Study on the potential of cold-active lipases from psychrotrophic fungi
for detergent formulation. J Genet Eng Biotechnol 16(2):319–325
Santiago M, Ramírez-Sarmiento CA, Zamora RA, Parra LP (2016) Discovery, molecular
mechanisms, and industrial applications of cold-active enzymes. Front Microbiol 7:1408
Sayyed RZ, Bhamare HM, Marraiki N, Elgorban AM, Syed A, El-Enshasy HA, Dailin DJ (2020)
Tree bark scrape fungus: a potential source of laccase for application in bioremediation of
non-textile dyes. PLoS One 15(6):e0229968
Sharma A, Parashar D, Satyanarayana T (2016) Acidophilic microbes: biology and applications. In:
Rampelotto P (ed) Biotechnology of extremophiles: grand challenges in biology and biotech-
nology, vol 1. Springer, Cham, pp 215–241
Sharma A, Sharma R, Devi T (2017) Life at extreme conditions: extremophiles and their biocata-
lytic potential. International Journal of Advance Research in Science and Engineering 6:1413–
1420
Shunk GK, Gomez XR, Averesch NJ (2020) A Self-Replicating Radiation-Shield for Human Deep-
Space Exploration: Radiotrophic Fungi can Attenuate Ionizing Radiation aboard the Interna-
tional Space Station. bioRxiv
Siglioccolo A, Paiardini A, Piscitelli M, Pascarella S (2011) Structural adaptation of extreme
halophilic proteins through decrease of conserved hydrophobic contact surface. BMC Struct
Biol 11(1):1–12
Singh OV, Gabani P (2011) Extremophiles: radiation resistance microbial reserves and therapeutic
implications. J Appl Microbiol 110(4):851–861
Sinha R, Khare SK (2014) Protective role of salt in catalysis and maintaining structure of halophilic
proteins against denaturation. Front Microbiol 5:165
Solanki P, Kothari V (2011) Halophilic actinomycetes: salt-loving filaments. Int J Life Sci Technol
4(2):7
Sudan R, Bajaj BK (2007) Production and biochemical characterization of xylanase from an
alkalitolerant novel species aspergillus niveus RS2. World J Microbiol Biotechnol 23(4):
491–500
14 Extremophilic Enzymes: Catalytic Features and Industrial Applications 313

Sulistyaningdyah WT, Ogawa J, Tanaka H, Maeda C, Shimizu S (2004) Characterization of
alkaliphilic laccase activity in the culture supernatant of Myrothecium verrucaria 24G-4 in
comparison with bilirubin oxidase. FEMS Microbiol Lett 230(2):209–214
Sunghee K, Saracini C, Siegler MA, Drichko N, Karlin KD (2012) Coordination chemistry and
reactivity of a cupric hydroperoxide species featuring a proximal H-bonding substituent. Inorg
Chem 51:12603–12605
Taneja K, Gupta S, Kuhad RC (2002) Properties and application of a partially purified alkaline
xylanase from an alkalophilic fungus aspergillus nidulans KK-99. Bioresour Technol 85(1):
39–42
Teklebrhan Welday GDH, Abera S, Baye K (2014) Isolation and characterization of thermo-stable
fungal alpha amylase from geothermal sites of Afar, Ethiopia. Int J Adv Pharm Biol Chem 3
(1):102–127
Tkavc R, Matrosova VY, Grichenko OE, Gostinčar C, Volpe RP, Klimenkova P, Gaidamakova EK,
Zhou CE, Stewart BJ, Lyman MG, Malfatti SA, Rubinfeld B, Courtot M, Singh J, Dalgard CL,
Hamilton T, Frey KG, Gunde-Cimerman N, Dugan L, Malfatti SA (2018) Prospects for fungal
bioremediation of acidic radioactive waste sites: characterization and genome sequence of
Rhodotorula taiwanensis MD1149. Front Microbiol 8(8):2528
Ulukanli Z, Diğrak M (2002) Alkaliphilic micro-organisms and habitats. Turk J Biol 26(3):181–191
van den Brink J, van Muiswinkel GC, Theelen B, Hinz SW, de Vries RP (2013) Efficient plant
biomass degradation by thermophilic fungus Myceliophthora heterothallica. Appl Environ
Microbiol 79(4):1316–1324
Vega K, Villena GK, Sarmiento VH, Ludena Y, Vera N, Gutiérrez-Correa M (2012) Production of
alkaline cellulase by fungi isolated from an undisturbed rain forest of Peru. Biotechnol Res Int
2012:934325
Wang H, Luo H, Li J, Bai Y, Huang H, Shi P, Fan Y, Yao B (2010) An α-galactosidase from an
acidophilic Bispora sp. MEY-1 strain acts synergistically with β-mannanase. Bioresour Technol
101(21):8376–8382
Wu J, Qiu C, Ren Y, Yan R, Ye X, Wang G (2018) Novel salt-tolerant xylanase from a mangrove-
isolated fungus Phoma sp. MF13 and its application in Chinese steamed bread. ACS omega
3(4):3708–3716
Yin Q, Zhou G, Peng C, Zhang Y, Kües U, Liu J, Xiao Y, Fang Z (2019) The first fungal laccase
with an alkaline pH optimum obtained by directed evolution and its application in indigo dye
decolorization. AMB Express 9(1):151
Yoshioka H, Chavanich S, Nilubol N, Hayashida S (1981) Production and characterization of
thermostable xylanase from Talaromyces byssochlamydoides YH-50. Agric Biol Chem 45(3):
579–586
Zanphorlin LM, Facchini FDA, Vasconcelos F, Bonugli-Santos RC, Rodrigues A, Sette LD,
Gomes E, Bonilla-Rodriguez GO (2010) Production, partial characterization, and immobiliza-
tion in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora
sp. J Microbiol 48(3):331–336
Zhdanova NN, Tugay T, Dighton J, Zheltonozhsky V, Mcdermott P (2004) Ionizing radiation
attracts soil fungi. Mycol Res 108(9):1089–1096
314 K. Choudhary et al.

Biotechnological Application
of Extremophilic Fungi 15
Aneesa Fasim, H. K. Manjushree, A. Prakruti, S. Rashmi, V. Sindhuja,
Veena S. More, K. S. Anantharaju, and Sunil S. More
Abstract
White biotechnology (BT), a sustainable and eco-friendly technology, has taken
precedence over chemical industries in the last few decades. It has revolutionized
the industrial BT sector by exploiting abundant natural resources for the produc-
tion of important commodities benefiting mankind. Industries employ
microorganisms or biomolecules extracted from them for production and
processing in various industrial areas such as food and feed, beverages, agricul-
ture, pharmaceutical, textile, leather, paper, detergent, polymers, cosmetics, waste
management, etc. Despite the advantages, the use of biomolecules is not substan-
tial because they cannot tolerate harsh industrial conditions, which in turn affects
the production process. In the last decade, the industrial research focus has shifted
toward extremophiles, organisms that can survive extreme conditions. These
organisms have evolved defense mechanisms to survive severe conditions such
as high or low temperature, salinity, pressure, pH, radiation, and desiccation.
Biomolecules extracted from these organisms have robust characteristics to retain
optimum activity even under unnatural conditions. A class of eukaryotes called
extremophilic fungi are at the crux of this research focus as they are a reservoir of
sturdy biomolecules with many industrial applications. Fungal extremozymes can
be easily cultured on agro-industrial waste and also easily purified. All these
factors make fungal extremozymes an attractive resource for large-scale, cost-
effective, and eco-friendly industrial processes. In addition to extremozymes,
extremophilic fungi are an abundant resource of potent cytotoxic, antimicrobial
A. Fasim · H. K. Manjushree · A. Prakruti · S. Rashmi · V. Sindhuja · S. S. More (*)
School of Basic and Applied Sciences, Dayananda Sagar University, Bengaluru, Karnataka, India
V. S. More
Department of Biotechnology, Sapthagiri College of Engineering, Bangalore, Karnataka, India
K. S. Anantharaju
Department of Chemistry, Dayananda Sagar College of Engineering, Bangalore, Karnataka, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_15
315

drugs. This chapter focuses on various extremophilic fungi used in the BT
industry. It also covers the different extremozymes, biomolecules, and secondary
metabolites secreted by them and their potential biotechnological applications.
Keywords
Extremophilic fungi · Extremozymes · Natural products · Bioactive compounds ·
Biotechnological applications
15.1 Introduction
A sustainable bio-based economy is a ray of hope in response to the present
environmental crisis such as population expansion, climatic changes, exhaustion of
nonrenewable resources, global warming, pollution, etc. The advent of bioprocess
technology, also known as white biotechnology, has revolutionized the industrial
sector by exploiting natural resources for the production and processing of value-
added products that positively impact the global economy and environment. This
contemporary technology employs enzymes or microorganisms such as yeast, bac-
teria, fungi, and plant extracts in numerous industrial applications. Fungal sources
have been the major contributors in this field as many enzymes, organic acids,
antibiotics, etc., are produced on a commercial scale (Meyer et al. 2016). The
discovery of penicillin, fungal antibiotics along with the commercial production of
citric acid by Aspergillus niger, marked a milestone in the era of fungal biotechnol-
ogy, and since then many more discoveries have steadily transformed it into a
powerful and proficient technology. Fungi play a vital and irreplaceable role in
energy recycling of the ecosystem by helping in the decomposition and recycling of
organic matter. This versatile class of eukaryotes are omnipresent and can be found
in soil, desserts, glaciers, sea, freshwater bodies, and various other environments
including the stratosphere (van der Giezen 2011). Fungi have proven to be a valuable
resource to humanity from being consumed as food to combating infectious diseases
and many biomolecules with important industrial applications. Besides, helping in
the fermentation processes of baking, brewing, etc., they aid in the production of
enzymes, antibiotics, organic acids, pigments, vitamins, lipids, and numerous other
products that are economically important (Adrio and Demain 2003). Their fast
growth rate, short life cycles, ease of culture, and purification are highly favorable
attributes that benefit the industrial production processes (Hooker et al. 2019).
Fungi are highly resilient organisms that can adapt to diverse habitats and due to
their ecological plasticity, they can survive harsh environments precluded to most
life forms. They dwell in virtually all types of extreme habitats ranging from
extremely dry and cold deserts in the Antarctic and other very cold areas worldwide
to highest mountain peaks (Selbmann et al. 2008) to deep permafrost soils
(Ozerskaya et al. 2009; Selbmann et al. 2015), geothermal and fumarole soils in
volcanic areas, acid mine drainages with sulfuric acid (Selbmann et al. 2008), or in
highly alkaline sites (Gunde-Cimerman et al. 2009; Selbmann et al. 2013). Under
316 A. Fasim et al.

severe conditions and high competition, fungi acquire peculiar skills to exploit
natural or xenobiotic resources and such fungi are termed as extremophilic fungi
(Zhang et al. 2018).
These fungi have evolved defense mechanisms in the form of regulation and
expression of specific genes or production of robust enzymes that help them to
survive conditions such as high or low temperature, salinity, pressure, pH, radiation,
and desiccation. Biomolecules extracted from these organisms have robust
characteristics and retain optimum activity even under harsh industrial conditions.
All these factors make fungal extremozymes an attractive resource for large-scale,
cost-effective, and eco-friendly industrial processes, and the scope to use
extremophilic fungi for biotechnological applications is increasing with time
(Sarmiento et al. 2015).
The term “extremophile”was first proposed by MacElroy in 1974 to describe a
broad group of organisms that can live optimally under extreme conditions. They
belong to all three domains of life —Eucarya, Bacteria, and Archaea. Extremophiles
are classified into seven categories based on the extreme habitats they inhabit
(Fig. 15.1). Piezophiles can survive high hydrostatic pressure and have been isolated
from deep sea sediments (>3000 m deep). Thermophiles or hyperthermophiles are
organisms that inhabit hot springs, deep sea hydrothermal vents, and can tolerate
very high temperatures varying from 50 to 80 C or over 80 C (Raddadi 2015).
Some halotolerant fungi can tolerate high salt concentration and abiotic stress
Fig. 15.1 Extreme environments of the earth
15 Biotechnological Application of Extremophilic Fungi 317

(Gunde-Cimerman et al. 2003). This is why many fungi inhabit marine
environments. Alkalophiles can tolerate a pH range between 9 and 12, whereas
acidophiles can survive extremely low pH of 1–2 (Jin and Kirk 2018). Psychrophiles
are the next class that can tolerate extreme cold conditions of the Antarctic zone
(Selbmann et al. 2008) and some yeasts can survive ultraviolet rays (UV-B) expo-
sure even at lethal doses (Selbmann et al. 2011). Due to their uncommon adaptabil-
ity, fungi may also easily colonize stressful and extreme environments created by
anthropogenic activities, such as those polluted with heavy metals, toxic chemicals,
sewage, etc. (Ceci et al. 2019). Therefore, polluted sites are a rich source to screen
for extremophilic fungi. Fungal strains isolated from these environments are strongly
adapted to high toxicity and extreme physical parameters (i.e., high salt concentra-
tion and high pH). These strains are potentially useful in biotechnological
applications such as the biodegradation of the pollutants (Gomes and Steiner
2004; Selbmann et al. 2013) or they can be considered as sources of important
bioactive compounds, specific enzymes, biosurfactants, and antioxidants, useful for
applications in medicine or food, cosmetics, and chemical industry (Adrio and
Demain 2003). They are also employed in biofuel and bioenergy industries since
solar cells of specialized pigments work only under extreme conditions like polar
caps.
15.2 Biotechnological Applications
Biotechnological industries are exploiting a variety of enzymes as solutions to
numerous industrial processes. Fungi from the extreme environment are considered
a vital source of commercial hydrolytic enzymes due to their exceptional properties
of high catalytic activity, stability, high enzyme yield, ease of culture, and retention
of activity even under high-stress conditions. Lipases, amylases, proteases,
cellulases, xylanases, etc., are highly used in industries that require efficient break-
down of lignocellulosic biomass in the processing and production of good quality
biobased products. Hence, fungal extremozymes help in large-scale, cost-effective,
and eco-friendly industrial processes that could significantly affect the growth of the
biotechnology sector (Shukla and Singh 2020). Some of the important fungal
extremozymes are listed in Table 15.1. The important fields that use these enzymes
include decolorization of dyes in the textile industry, detoxify pesticides, degrade
agricultural waste to valuable by-products, delignify biomass for biofuel production,
bleach the kraft pulp in the paper industry, processing and stabilization of juice,
wine, bakery products in the food industry, bioremediation, and many other pro-
cesses (Baldrian 2006; Brijwani et al. 2010). Along with extremozymes, secondary
metabolites and bioactive peptides are also products of extremophilic fungi. Their
potential role in preventive medicine as antimicrobials, antivirals, cytotoxic agents,
antitumorigenic, antidiabetic, anti-inflammatory, lipid-lowering activities is also
illustrated in this chapter (Fig. 15.2).
318 A. Fasim et al.

Table 15.1 Extremophilic enzymes sources and uses in industries
Enzymes Organisms
Applications in
industries References
Proteases Penicillium buponti,
Malbranchea pulchella
var. sulfurea, Humicola
lanuginose
Rhodotorula mucilaginosa
L7
Leucosporidium
antarcticum
Acremonium sp. L1–4B
Pseudogymnoascus
pannorum
Candida humicola
Food, detergents,
leather,
pharmaceutical,
agricultural
industries
(Maheshwari et al. 2000)
(Lario et al. 2015)
(Turkiewicz et al. 2003)
(Evaristo da Silva
Nascimento et al. 2015)
(Krishnan et al. 2011)
(Ray et al. 1992)
Laccases Chaetomium
thermophilium
Corynascus thermophiles
aspergillus oryzae
Aigialus grandis,
Cirrenalia pygmea,
Gliocladium sp.,
Hypoxylon oceanicum,
Halosarpheia
ratnagiriensis,
Gongronella sp.,
Sordaria flmicola,
Verruculina enalia and
Zalerion varium.
Cladosporium
halotolerans,
Cladosporium
sphaerospermum,
Penicillium canescens.
Cerrena unicolor (MTCC
5159) and Penicillium
pinophilum (MCC 1049)
Paper and pulp,
Textile industry,
agriculture,
Food and beverages
(Chefetz et al. 1998)
(Babot et al. 2011; Berka
et al. 1997; Bulter et al.
2003; Xu et al. 1996)
(Raghukumar et al. 1994)
(Jaouani et al. 2014)
(D’Souza-Ticlo et al. 2009)
Cellulases Trichoderma resei
Chaetomiumthermophile,
Sporotrichum thermophile,
Humicola grisea var
thermoidea,
Humicola insolens,
Myceliopthera
thermophila,
Thermoascus aurantiacus
and Talaromyces emrsonii
Cadophora,
Pseudeurotium,
Geomyces, Wardomyces,
Pseudogymnoascus,
Biofuel production,
paper and pulp,
Textile
(Mandels and Weber 1969)
(Maheshwari et al. 2000)
(Krishnan et al. 2011; Tsuji
et al. 2014; Vaz et al. 2011;
Wang et al. 2013)
(continued)
15 Biotechnological Application of Extremophilic Fungi 319

Table 15.1 (continued)
Enzymes Organisms
Applications in
industries References
Verticillium, Cryptococcus
and Mrakia
Xylanases Aureobasidium pullulans
varmelangium,
Pencillium occitanis PO16,
Aureobasidium pullulans
Pencillium oxalicum
Pencillium citrinum,
Aspergillus fumigatus
Humicola insolensY1,
Sporotrichum thermophile
Rhizomucor pusillus,
Aspergillus gracilis,
Aspergillus penicillioides
Naganishia adeliensis.
Paper and pulp,
Animal feed,
Textile,
Food and brewery
(Ohta et al. 2001)
(Driss et al. 2011)
(Yegin 2017)
(Muthezhilan et al. 2007)
(Dutta et al. 2007)
(Deshmukh et al. 2016)
(Du et al. 2013)
(Sadaf and Khare 2014)
(Robledo et al. 2016)
(Ali et al. 2012)
(Gomes et al. 2003)
Lipases Rhizomucor miehei
Kurtzmanomyces sp. I-11
Moesziomyces antarcticus
Leucosporidium scottii
L117
Mrakia blollopis SK-4
Geomyces sp. P7
Biofuel, detergent,
food, and beverages
(Maheshwari et al. 2000)
(Kakugawa et al. 2002;
Goto et al. 1969)
(Goto et al. 1969)
(Duarte et al. 2015)
(Tsuji et al. 2013)
(Tsuji et al. 2013)
Amylases Rhizomucor pusillus,
Humicola lanuginose,
Myriococcum
thermophilum,
Thermomyces ibadanensis,
Thermomyces lanuginosus
Candida antarctica
Geomyces pannorum
Starch processing,
food and beverage,
paper and pulp,
Textile, and
pharmaceutical
(Adams 1994; Arnesen
et al. 1998; Barnett and
Fergus 1971; Bunni et al.
1989; Fergus 1969;
Jayachandran and
Ramabadran 1970;
Sadhukhan et al. 1992)
(Mot and Verachtert 1987)
(Gao et al. 2016)
Pectinases Aspergillus Niger
Cryptococcus albidus var.
albidus,
Aspergillus Niger
MTCC478,
Saccharomyces cerevisiae,
Penicillium sp. CGMCC
1669
Rhizomucor pusilis
Thermomucor indicae-
seudaticae
Arthrobotrys,
Aureobasidium,
Cladosporium,
Leucosporidium
Tetracladium
Biofuel production,
oil extraction, paper
and pulp, food, and
beverage
(Lara-Márquez et al. 2011)
(Federici 1985)
(Anand et al. 2017)
(Gainvors et al. 2000)
(Yuan et al. 2011)
(Siddiqui et al. 2012)
(Martin et al. 2010)
(Fenice et al. 1997)
(Carrasco et al. 2016)
(continued)
320 A. Fasim et al.

15.2.1 Food and Beverage Industry
Use of enzymes instead of chemicals improves the quality of the processed food and
creates superior products with improved yields. In addition, enzymes also play key
role in enhancing the nutrition and appeal of the products. Enzymes are used in
baking, making sugar syrups, cheese and dairy making, extraction and clarification
of juices, oil, as sweeteners, for flavor development, meat tenderizing, etc., and in
many other processes. From making food products to storage of food and beverage
all require extreme conditions making extremozymes an essential ingredient to
achieve food quality at low costs in this industry.
Cold-active enzymes produced by psychrophiles are flexible, resulting in higher
catalytic activity at low temperatures (Arora and Panosyan 2019). These enzymes
can be used to soften frozen meat products, preserve the heat-sensitive nutrients,
accelerate cheese ripening, and they are also effective against wine and juice
clarification. Rhodotorula mucilaginosa L7 is a yeast strain from the Antarctic
region that produces acid protease with an activity range between 15 C and 60 C
and pH 5 (Lario et al. 2015). A similar discovery of a psychrophilic and halotolerant
serine protease from Antarctic region resulted in isolation of Leucosporidium
antarcticum fungal strain where the enzyme was found most active at 10–25 C
Table 15.1 (continued)
Enzymes Organisms
Applications in
industries References
Chitinases Trichoderma, Oenicillium,
Penicillium, Lecanicillium,
Neurospora, Mucor,
Beauveria, Lycoperdon,
aspergillus, Myrothecium,
Conidiobolus,
Metharhizium,
Stachybotrys, Agaricus
Talaromyces emersonii,
Thermomyces lanuginosus
Dioszegia, Glaciozyma,
Lecanicillium,
Leuconeurospora, Mrakia,
Metschnikowia Phoma,
Sporidiobolus, Verticillium
lecanii
Glaciozyma antarctica
PI12
Pharmaceutical and
agricultural industry
(Hamid et al. 2013; Karthik
et al. 2014)
(McCormack et al. 1991)
(Zhang et al. 2012)
(Barghini et al. 2013;
Carrasco et al. 2012; Fenice
et al. 2012,1998,1997;
Onofri et al. 2000)
(Ramli et al. 2011)
Phytases Aspergillus Niger
Myceliophthora
thermophila, Talaromyces
Papiliotrema laurentii
AL27
Rhodotorula mucilaginosa
strain JMUY1
Bread making and
animal feed
(Haros et al. 2001)
(Maheshwari et al. 2000)
(Pavlova et al. 2008)
(Yu et al. 2015)
15 Biotechnological Application of Extremophilic Fungi 321

and 3.5% marine salt (Turkiewicz et al. 2003). Additionally, Laccases have many
applications like processing and stabilization of juice, wine, bakery products in the
food industry, and many other processes (Baldrian 2006; Brijwani et al. 2010).
Amylases are another class of enzymes highly used in food industry; they are also
used in various other industries such as starch processing, textile, food and beverage,
paper, pharmaceutical, and many other industries (Pandey et al. 2000).
Extremophilic fungal α-amylases have achieved an important place in industrial
enzymes. Many thermophilic fungal species studied so far are capable of secreting
amylases. Rhizomucor pusillus, and Humicola lanuginose,Myriococcum
thermophilum, Thermomyces ibadanensis, and Thermomyces lanuginosus are a
few of the thermophilic fungi found to produce amylase enzyme (Sadhukhan
et al.1992; Jayachandran and Ramabadran 1970; Fergus 1969; Bunni et al. 1989;
Barnett and Fergus 1971; Arnesen et al. 1998; Adams 1981; Adams 1994).
Psychotolerant fungi are also a good source of amylases. Candida antarctica from
Antarctic region was observed to produce both αand γamylases. Both enzymes
were active on high molecular weight polysaccharides with α-amylase showing
activity even on cyclodextrins (Mot and Verachtert 1987). Extremophilic fungal
xylanases and pectinases also have many benefits such as pulping, juice and wine
clarification, oil extraction etc. (Soni et al. 2017). Trichoderma sp, Aspergillus sp,
Penicillium sp, and Acido bacterium spp. are the major extremophilic fungal genera
Fig. 15.2 Representation of extremophilic fungi biotechnological applications
322 A. Fasim et al.

that contribute to the production of xylanases. Similarly, many acidic fungal
pectinases like Aspergillus niger between pH 3 and 5.5 (Lara-Márquez et al.
2011). Cryptococcus albidus var. albidus, pH 3.75 (Federici 1985), Aspergillus
niger MTCC478, pH 4 (Anand et al. 2016), Penicillium sp. CGMCC 1669,
pH 3.5 (Yuan et al. 2011), and Saccharomyces cerevisiae pH 3–5.5 (Gainvors
et al. 2000) have been screened. Novoshape (novozymes), pectinase 62 L
(biocatalysts), and lallzyme (lallemand) are few commercially available food-
based companies that use pectinase enzyme (Dumorné et al. 2017; Sarmiento et al.
2015). Acidic pectinases are one such enzyme used in the clarification of fruit juices,
beer, and wine as well (Kashyap et al. 2001). Recent research has indicatedS
screening of bacterial strains known to produce alkaline and thermophilic pectinases.
Anand et al. 2016 purified and characterized an alkaline pectinase from Aspergillus
fumigatus MTCC 2584 having a pH optima of 10. In another study, thermophilic
pectinase was purified from Rhizomucor pusilis having temperature optima of 55 C
was isolated (Siddiqui et al. 2012). Martin et al. 2010 also isolated a thermophilic
pectinase producing fungal strain Thermomucor indicae-seudaticae that could grow
at 45 C. Recently, psychrophilic and pectinolytic fungi were isolated from Antarctic
region. The representative genera are Arthrobotrys, Aureobasidium, Cladosporium,
and Leucosporidium showed the pectinase activities even at 5 C (Fenice et al.
1997). A cold-adapted pectinase-producing fungi was also isolated from
Tetracladium sp. with highest activity at 15 C (Carrasco et al. 2019).
15.2.2 Detergents
Extermophilic fungal lipases are sought-after enzymes in detergent industries as they
possess robust properties. Particularly esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3)
are important as they catalyze the cleavage of esterbonds and also help in reverse
reactions in organic solvents (Fuciños et al. 2012). Lipases help in acidolysis,
alcoholysis, aminolysis, esterification hydrolysis, interesterification, etc. (Daiha
et al. 2015), making them versatile and having many applications in organic and
fine chemical synthesis, and cleaning products. A thermostable lipase from
Humicola lanuginosa strain Y-38 was isolated from compost in Japan. The enzyme
was thermophilic having temperature optima of 60 C and alkalophilic with pH
optima of 8.0. Rhizomucor miehei, formerly called Mucor miehei, also produced
active lipase (Maheshwari et al. 2000). Kakugawa et al. (2002) reported a thermo-
stable and acidophilic lipase-producing yeast strain Kurtzmanomyces sp. I-11 with
optimum activity at 75 C and pH 2–4. Another noteworthy example of thermostable
and alkalophilic lipase is produced by Thermomyces lanuginosus, known as TLL
showing maximum lipase activity between 60 and 85 C and pH 10 (Avila-Cisneros
et al. 2014). Lipolase, Lipoclean, and Lipex are few of the genetically improved
lipases from the fungus Thermomyces lanuginosus included in detergent
formulations by Novozymes (Jurado-Alameda et al. 2012). Cellulases are the next
class of enzymes that have found applications in the detergent industry to increase
brightness and dirt removal from cotton mixed garments (Kuhad et al. 2011). Many
15 Biotechnological Application of Extremophilic Fungi 323

commercially available detergents have been reported where enzyme such as lipase,
protease, amylases, cellulases, and mannanases are included in the formulations
(Sarmiento et al. 2015).
15.2.3 Paper and Pulp Industry
In the paper and pulp industry, the significant application of enzymes is in the
prebleaching of kraft pulp. Xylanases, hemicellulases, and cellulases are the com-
monly used enzymes for this purpose due to its displayed efficiency. Enzymes have
also been used to raise water retention, pulp fibrillation, and decrease the beating
time in virgin pulps. Enzymes are also involved in increasing the freeness and in the
deinking process (Dumorné et al. 2017; Bajpai 1999). Fungal laccases are involved
in lignin degradation due to displayed efficiency (Alcalde 2007; Thurston 1994).
Due to high enzyme yield and higher redox potential, fungal laccases are preferred
over the plant or bacterial enzymes in the biotechnology sector (Thurston 1994).
Corynascus thermophilus is a fungal strain secreting highly active thermostable
laccase that was used to delignify eucalypt pulp. This laccase was heterologously
expressed in Aspergillus oryzae, characterized, and commercialized (Xu et al. 1996;
Berka et al. 1997; Bulter et al. 2003; Babot et al. 2011). Cellulases are also heavily
used in this industry. Penicillium roqueforti, Cadophora malorum, Geomyces sp.,
and Mrakia blollopis are few of the cold-adapted cellulase-producing fungal strains
(Carrasco et al. 2016; Duncan et al. 2006; Duncan et al. 2008). Trichoderma sp,
Aspergillus sp, Penicillium sp, and Acidobacterium spp are the major extremophilic
fungal genera that contribute to the production of xylanases.
15.2.4 Agricultural Applications
Many cellulolytic and xylanolytic fungi are acknowledged to have applications in
the field of agriculture by boosting the seed germination, improved root system and
flowering, increased crop yields, and rapid plant growth (Ahmed and Bibi 2018).
Fungal xylanases such as Pencillium oxalicum (Muthezhilan et al. 2007), Pencillium
citrinum (Dutta et al. 2007), Aspergillus fumigatus (Deshmukh et al. 2016),and
Humicola insolensY1(Du et al. 2013) are isolated showing optimum activity
between pH 8–9 and 45–55 C with H.insolensY1 also being highly thermophilic
with a temperature optima of 70–80 C. Other thermophilic xylanase-producing
fungi include Chaetomium sp. CQ31, Sporotrichum thermophile isolated from
composting soil having activity at neutral pH and 60–70 C temperature (Jiang
et al. 2010; Sadaf and Khare 2014). Rhizomucor pusillus and Aspergillus fumigatus
screened from the maize silage showed optimum xylanase activity at 75 C and pH 6
(Robledo et al. 2016). Many thermophilic xylanase-producing fungi such as
Chaetomium thermophilum, Humicola insolens, Melanocarpus sp., Malbranchea
sp., and Thermoascus aurantiacus were reported by Ghatora et al. 2006.
Halotolerant fungal xylanase Phoma sp isolated from mangrove sediments having
324 A. Fasim et al.

enzyme activity at pH 5, 45 C, and a high salt concentration of 4 M NaCl (Wu et al.
2018). Aspergillus gracilis and Aspergillus penicillioides were screened from
man-made solar saltern (Ali et al. 2012) and psychrophilic fungal xylanases were
isolated from Antarctic soils, marine sponges, etc. Cladosporium sp. from marine
sponge showed high xylanase activity at low temperatures (Del Cid et al. 2014).
Naganishia adeliensis are isolated from Antarctica (Gomes et al. 2003). Phytases are
another class of enzymes involved in seed germination, but they are also considered
antinutrients because they act as strong chelators of divalent mineral ions such as
calcium, magnesium, iron, and zinc. Chitinases have many applications, especially
as antiphytopathogenic and antifungal agents. They are used to protect crops to
control pathogens. Cola-active extremozymes are used in agriculture to enhance the
water management by plants, which are under deficiency stress (Dumorné et al.
2017).
15.2.5 Animal Feed Industry
Cellulases and xylanases have advantage in the animal feed industry in the treatment
of agricultural silage, grains, and seeds to enhance nutritional value. Cold-adapted
phytases have advantages as they can be directly included in the feed of monogastric
animals and also in aquaculture.
15.2.6 Bioremediation and Biodegradation: Major Application
of Extremozymes
Bioremediation and biodegradation employ microbes in the elimination of
pollutants, contaminants, and toxins from water, soil, and other environments.
Waste from any kind of industry is hazardous. It is highly acidic or alkaline, and
contains all kinds of biomass and proteinaceous waste. It also has a high content of
metal ions and many other toxins, dyes, chemicals, radioactive material, etc., making
it very harmful to the flora and fauna around it.
Certain microbes can be used to recycle and degrade pollutants as they produce
hydrolytic enzymes that can degrade and help clean up the contaminated sites.
Fungal extremozymes are extremely useful in these processes as they can sustain
harsh conditions and still work on organic toxins. Thermophiles convert recalcitrant
materials in bioprocessing and favor the in situ bioremediation process (Castro et al.
2019). As the solubility of the pollutants increases, the metabolic activity of
thermophiles also increases (Zeldes et al. 2015). Thermophilic fungi such as
Pyrodictium, Clostridium, and Methanopyrus can metabolize naphthalene, anthra-
cene, and phenanthrene (Ghosal et al. 2016). White rot fungi are the chief
representatives of the biodegradation of lignin substances (Deshmukh et al. 2016).
21 PAH degrading fungi were isolated from PAH-contaminated soils that could
efficiently degrade PAH. Aspergillus niger,Diaporthe sp., Coriolopsis byrsina,
Pestalotiopsis sp., and Cerrena are known to treat and bioremediate textile mill
15 Biotechnological Application of Extremophilic Fungi 325

effluents (Rani et al. 2014). Stenotrophomonas maltophilia strain AJH1 has been
isolated from Arabia, which was able to degrade low and high molecular weight
PAHs such as anthracene, naphthalene, phenanthrene, pyrene, and benzo(k)-
f
2
uoranthene (Rajkumari et al. 2019). D. radiouridans is another important fungus
used in bioremediation of radioactively contaminated sites (Brim et al. 2006).
Sulfolobus sulfataricus secrete lactonase enzyme that acts against organophosphates
(Hawwa et al. 2009). Thermoascus aurantiacus, another fungal strain, can secrete
phenol oxidase and target phenolic hydrocarbons (Machuca et al. 1998).
Rajkumari et al. (2019) studied different approaches of degradation of hydrocar-
bon waste. Candida, Aspergillus, Chlorella, and Penicillium were found to be most
suitable in the elimination of these wastes. A marine fungal laccase-mediated
detoxification and bioremediation of anthraquinone dye called reactive blue was
reported (Verma et al. 2012). These laccases could work under very high salinity.
Similarly, laccase from Fusarium incarnatum was able to degrade bisphenol A,
which is a endocrine-disrupting chemical (Chhaya and Gupte 2013). Other studies
indicated heavy metal and chloropyriphos bioremediation can be achieved by using
Aspergillus sp, Curvularia, and Acrimonium sp. (Akhtar et al. 2013; Silambarasan
and Abraham 2013); likewise, polychlorinated biphenyl degradation can be
degraded by Phoma eupyrena, Doratomyces nanus, Myceliophthora thermophila,
and D. verrucisporus (Barghini et al. 2013). Lugowski et al. 1998 has reported that
Pseudomonas sp is used for degradation of aromatic hydrocarbons. Halomonas
sp. and Pseudomonas aeruginosa strain is used for cleaving of aliphatic
hydrocarbons.
15.2.7 Bioactive Peptides from Marine Fungi
Oceans are the biggest resource for novel therapeutic compounds. Thousands of
secondary metabolites such as polyketides, lactones, alkaloids, steroids, and peptides
having pharmacological significance are discovered from marine fungal strains (Jin
et al. 2016). Sessile marine microorganisms usually harbor the fungal strains in a
symbiotic relationship where the marine fungi protect the host against predators and
disease by releasing bioactive compounds (Schueffler and Anke 2014). The unique
structural and functional diversity of the marine bioactive compounds is attributed to
the extreme conditions of salinity, pressure, and temperature that also give immense
stability from all kinds of degradation to these peptides, making them promising
candidates for drug discovery. Thus, isolating and characterizing novel bioactive
peptides and metabolites from marine fungi with therapeutic properties is a
promising avenue to explore in the prevention of human diseases. To date, thousands
of compounds have been isolated from many marine fungi, but curating them all is
not feasible. So, the data from two latest reviews covering last 15 years of research
(Ibrar et al. 2020; Youssef et al. 2019) on the fungal bioactive peptides and
compounds is adapted and a comprehensive summary is presented in Table 15.1
with additions and modifications made according to the relevance and scope of this
chapter.
326 A. Fasim et al.

15.2.7.1 Peptides
In the last five decades, a significant number of marine bioactive peptides are
discovered that either fall in the class of synthetic, non-ribosomally produced
peptides such as bacitracins, polymixins, glycopeptides, or gramicidins, etc., or
natural, ribosomal peptide class. The synthetic peptides are mostly produced by
bacteria, but natural peptides are produced by many species including marine fungi
with potent activities (Saleem et al. 2007). Many fungi belonging to various genus
produce potent peptides showing antimicrobial, antiviral, cytotoxic,
antitumorigenic, antidiabetic, anti-inflammatory, lipid-lowering activities. These
peptides are structurally diverse from being cyclic to N-methylated. Some are
dipeptides, nonapeptides, depsipeptides, or pentadecapeptides having complex
backbones and many side chains. Genus Aspergillus is found to be a rich source
of bioactive peptides with Aspergellicins A–E, Cyclodipeptide, Sclerotide A–B,
Terrelumamide A–B, Psychriphillin E–G, Aspersymmatide A, Cotteslosin A,
Diketopiperazine dimer, cyclic tetrapeptide, Aspergellipeptide D–E, and
14-hydroxycyclopeptine being produced by them showing cytotoxic, anticancer,
and anti-inflammatory properties (Table 15.1). Cordyhehptapeptides and efrapeptins
are certain other bioactive peptides isolated from Acremonium sp with cytotoxic and
antibacterial activities. Lajollamide A from Asteromyces, Dictyonamide A from
Certodictyon, Clonostachysins from Clonostachys, and Ungusin A, Emercellamide
from Emericella sp, and Rostratins from Exserohilium are cytotoxic,
antidinoflagellate, and antimicrobial in nature. Similarly, peptides from
Microsporum, Penicillium, Scytalidium, Simplicillium, Stachylidium, Talaromyces,
and Zygosporium fungi also show various toxic effects on cancers and microbes.
The general procedure for isolating fungal peptides involves culturing of fungi
under appropriate conditions and extraction of peptides using solvents such as ethyl
acetate. The extracted sample is lyophilized and further purified using chro-
matographic techniques until pure forms of peptides are obtained. 1D and 2D
NMR techniques in combination with mass spectrometry are used to determine the
structure of the peptides and Marfey’s and Mosher’s reactions are used to elucidate
the absolute configuration, amino acid composition, and structural modifications
(Wang et al. 2017) Biological activity of the purified peptide is measured using IC
50
or MIC (minimum inhibitory concentrations) values against cancer cell lines, patho-
genic bacteria, and many other microbes.
15.2.7.2 Bioactive Compounds
Marine secondary metabolites have gained a lot of attention in the recent past due to
their potent pharmacological properties. The accidental discovery of cephalosporin
C antibiotic from the marine Cephalosporium sp. fungus in 1949 started a trend to
explore marine habitats for bioactive compounds. Many other marine fungi-derived
products are currently available in the market such as antibacterial terpenoid fusidic
acid, polyketide griseofulvin antibiotic, penicillins, cephalosporins, macrolides,
statins, many alkaloids, glycosides, isoprenoids, lipids, etc. (Chandra and Arora
2009; Hamilton-Miller 2008), that exhibit potent toxicity towards tumors, cell
proliferation, microtubule formation, pathogenic bacteria, viruses, nematodes, foul
15 Biotechnological Application of Extremophilic Fungi 327

smells, and also exhibit photo-protective activities (Rateb and Ebel 2011). Bioactive
compounds are produced by all kinds of extremophilic fungi from psychotolerant, to
thermophiles, piezophiles, acidophiles, halotolerant, and xerophiles. Table 15.2
recapitulates different secondary metabolites and their biological activities. Many
bioactive compound-secreting fungal strains are discovered by exploring extremely
toxic environments such as Berkeley acid lake, hot springs, salt salterns, fumaroles,
deep sea sediments and vents, mangroves, Antarctic permafrost, etc. These places
have become rich biodiversity for the exploration of such value-added compounds
(Ibrar et al. 2020).
Bioactive compounds are also extracted and purified in the same way as peptides,
although the characterization techniques will differ. A bioassay-guided fractionation
procedure is employed to obtain pure compound fractions, where the potential
activity of the fractions is assessed. Most marine compounds have different chemical
composition so different polar compounds have to be used for the fractionation
method so that the active compound can be separated from the inactive fractions
depending on the partition coefficients of the analytes. Polyketides alkaloids, sugars,
steroids, and saponins are generally found in aqueous fractions, whereas peptides
need mildly polar solvents, and terpenes, hydrocarbons, and fatty acids are found in
low-polar fractions. The bioactive fractions are next subjected to gel permeation
chromatography to further purify the molecules. The purified compounds are then
structurally and chemically characterized by sophisticated techniques such as mass
spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. High-
resolution 1D and 2D NMR spectroscopy are routinely used for the structural
characterization of the bioactive compounds.
15.3 Conclusion
Biotechnological industries are using a variety of extremophilic fungi as solutions to
diverse industrial processes. The survival strategies of extremophilic fungi are
unique and associated with the production of extremozymes and various secondary
metabolites with robust qualities, making them a rich and abundant resource. Despite
their potential, a very small percent of extremophilic fungi are discovered. Explora-
tion of extremophilic organisms will make a huge impact and open new avenues in
biotechnology research. With the advancement in various technologies like
metagenomics, genetic engineering, in silico analysis, and technology that can
access uninhabitable and inaccessible places on earth, it is now possible to identify,
isolate, and extract potent compounds that can cater to the needs of almost every
sector of the biotech industry to help form a sustainable and efficient biobased
economy.
328 A. Fasim et al.

Table 15.2 Bioactive peptides and secondary metabolites isolated from marine fungi, their structure, sources, and biological activities
Bioactive peptides Marine fungi Features Source Biological activity References
Peptides
Cordyheptapeptide C
Cordyheptapeptide C
Acremonium persicinum
SCSIO 115
Cyclic
heptapeptides
Marine fungus Cytotoxic and antitumor
activity
(Chen et al.
2012)
Efrapeptin EαAcremonium Pentadecapeptides Marine fungus Cytotoxic activity (Gupta et al.
1992)
Efrapeptin F
Efrapeptin G
RHMI
Tolypocladium niueum
Acremonium sp
Acremonium sp.
Polypeptides
Polypeptide
N-methylated
linear octapeptides
Fractionated extract of T.
niueum
Cultured from a marine sponge
Cytotoxic activity
Cytotoxic and
antibacterial activity
Antibacterial activity
(Boot et al.
2006)
(Boot et al.
2007)
Aspergillicins A-E Aspergillus carneus Depsipeptides Estuarine sediment in
Tasmania
Cytotoxic activity (Capon et al.
2003)
Cyclo-(L-Trp-L-Tyr) Aspergillus Niger EN-13 Cyclic dipeptide Isolated from the marine brown
alga Colpomenia sinuosa
Cytotoxic activity (Zhang et al.
2010)
Sclerotide A
Sclerotide B
Aspergillus sclerotiorum
PT06–1
Cyclic hexapeptide Putian Sea salt field, China Antifungal activity
Antifungal, antibacterial,
and cytotoxic activity
(Zheng et al.
2009)
Similanamide Aspergillus similanensis
KUFA 0013
Cyclohexapeptide Ethyl acetate extracts of marine
unknown sponge
Cytotoxic and antitumor
activity
(Prompanya
et al. 2015)
Terrelumamide A
Terrelumamide B
Aspergillus terreus Linear lumazine
peptides
Marine sediments Improved insulin
sensitivity
(You et al.
2015)
Psychrophilin E Aspergillus sp Cyclic tropeptide Isolated from marine brown
algae Sargassum
Cytotoxic activity (Ebada et al.
2014)
Psychrophilin G Aspergillus
Versicolor ZLN-60
Cyclic peptides
with anthranilic
acid
Marine-derived fungi Lipid-lowering activity (Peng et al.
2014)
Aspersymmetide A Aspergillus
Versicolor
Cyclic hexapeptide Isolated from a gorgonian coral
Carijoa sp.
Cytotoxic activity (Hou et al.
2017)
(continued)
15 Biotechnological Application of Extremophilic Fungi 329

Table 15.2 (continued)
Bioactive peptides Marine fungi Features Source Biological activity References
Cotteslosin A Aspergillus versicolor
(MST-MF495)
Cyclic
pentapeptides
Isolated from Australian beach
sand
Cytotoxic and antitumor
activity
(Fremlin et al.
2009)
Diketopiperazine
dimer
Cyclic tetrapeptide
Aspergillus
Violaceofuscus
Diketopiperazine
dimer
Cyclic tetrapeptide
Isolated from marine sponge
Reniochalina sp.
Anti-inflammatory
activity
(Liu et al.
2018)
Aspergillipeptid D
Aspergillipeptid E
Aspergillussp. SCSIO
41501
Cyclic
pentapeptide
Tripeptide
Isolated from marine
gorgonians
Antiviral activity (Ma et al.
2017)
14-Hydroxy-
cyclopeptine
Aspergillus
sp. SCSIOW2
Cyclic dipeptide Isolated from deep sea (1000 m
depth) fugus
NO inhibition activity (Zhou et al.
2016)
Clavatustides A
Clavatustides B
Aspergillus clavatus
C2WU
Cyclodepsipeptides Xenograpsus testudinatus
Sulphur-rich hydrothermal
vents in Taiwan
Cytotoxic and
Antitumor activity
(Jiang et al.
2010)
Lajollamide A Asteromyces
Cruciatus
Pentapeptide Isolated from the
Coast of La Jolla, USA
Antibacterial (Gulder et al.
2012)
Dictyonamide A Fungus KO63 Linear
dodecapeptides
Isolated from marine red alga
Ceratodictyon spongiosum
CDK-4 inhibition (Komatsu et al.
2001)
Clonostachysin A
Clonostachysin B
Clonostachys
rogersoniana strain
HJK9
N-methylated
cyclic nona
peptides
Isolated from a sponge,
Halicondria japonica
Antidinoflagellate
activity
(Adachi et al.
2005)
Unguisin A
Emericellamide B
Emericella CNL-878 Cyclic
depsipeptides
Isolated from co-culture with
marine Salinispora arenicola
Antibacterial activity (Oh et al. 2007)
Microsporin A
Microsporin B
Microsporum
cf. gypseum
Cyclic
tetrapeptides
Isolated bryozoan Bugula sp.,
Virgin Islands USA
Inhibition of histone
deacetylases, cytotoxic
and antitumor activity
(Gu et al. 2007)
330 A. Fasim et al.

Penicimutide Penicillium
purpurogenum G59
Cyclic dipeptide Isolated from a neomycin-
resistant mutant marine
Penicillium purpurogenum
G59
Cytotoxic activity (Wang et al.
2016)
Psychrophilin D Penicillium algidum Cyclic nitropeptide Soil under a Ribes sp. east of
Oksestien, Greenland
Antimicrobial, antiviral,
anticancer, and
antiplasmodial
(Dalsgaard
et al. 2005)
Cis-Cyclo (Leucyl-
Tyrosyl)
Penicillium F37 Dipeptide Isolated from marine sponge
Axinella corrugate
Antibiofilim activity (Scopel et al.
2013)
Halovir A
Halovirs B–E
Scytalidium CNL240
Scytalidium
Linear, lipophilic
Peptides,
Deep sea-derived fungi Antiviral activity (Rowley et al.
2003,2004)
Simplicilliumtide A
Simplicilliumtide D
Simplicilliumtide E,
G and H
Simplicilliumtide J
Simplicillin obclavatum
EIODSF 020
Linear peptides Deep sea-derived fungi Cytotoxic activity
Antifouling activity
Cytotoxic and antitumor
activity
Antifungal and antiviral
activity
(Liang et al.
2017,2016)
Endolide A
Endolide B
Stachylidium sps. N-methylated
peptides
Isolated from a
Marine sponge
Binding to vasopressin
receptor
Binding to seratonin
receptor
(Almeida et al.
2016)
Talaropeptide A
Talaropeptide B
Talaromyces sps, N-methylated
linear peptides
Isolated from a marine
Tunicate.
Antibacterial activity (Dewapriya
et al. 2018)
Zygosporamide Zygosporium masonii Cyclic
depsipeptide
Marine-derived fungus Cytotoxic and antitumor
activity
(Oh et al. 2007;
Torres-García
et al. 2014)
(continued)
15 Biotechnological Application of Extremophilic Fungi 331

Table 15.2 (continued)
Bioactive peptides Marine fungi Features Source Biological activity References
Secondary metabolites
Fuscin,
dihydrofuscin,
dihydrosecofuscin,
and secofuscin
Oidiodendron griseum
UBOCC-A-114129
Polyketide 765 m below the seafloor Antibacterial, inhibited
CLK1 kinase
(Navarri et al.
2017)
Cytochalasin D Endophytic fungi
Xylaria sp
Polyketide amino
acid hybrid
From marine seaweed
Bostrychia tenella
Antitumor and antibiotic (de Felício
et al. 2015)
Pentacyclic
cytochalasin
Diaporthaceae sp
PSU-SP2/4
Polyketide amino
acid hybrid
Isolated from the marine
sponge
Antibacterial (Khamthong
et al. 2014)
Sterigmantocystin Aspergillus sp Polyketide
derivative
Marine algae derived, Germany Cytotoxic (Ebada et al.
2014)
Rugulosin and skyrin Pencillium
Chrysogenum
Polyketides Marine benthic-derived
Antarctic lake
Antimicrobial (Brunati et al.
2009)
Malbranpyrroles A–FMalbranchea sulfurea Polyketides Fumerole soil Cytotoxic (Yang et al.
2009)
Myceliothermophins
A–E
Myceliophthora
thermophila
Polyketides
containing tetramic
acid
Fumarole soil Cytotoxic (Yang et al.
2007)
Xanthone and
chromone
Penicillium sp. SCSIO
Ind16F01
Xanthones and
quinolones
Deep sea sediments Cytotoxic and
antimicrobial
(Liu and
Kokare 2017)
Anthraquinone Penicillium
sp. OUCMDZ 4736
Polyketide Sediment roots of mangrove Antiviral (Jin and Kirk
2018)
Anthraquinone Aspergillus Versricolor Polyketide Deep sea Antimicrobial (Wang et al.
2017)
Azophilones Pleurostomophora sp Polyketides Acidic Berkeley lake Antimicrobial (Stierle et al.
2015)
332 A. Fasim et al.

Berkeley lactones Penicillium fuscum and
P. camembertii /
clavigerum
Cyclic macrolides Acidic Berkeley lake Antimicrobial (Stierle et al.
2017)
Purpurquinones A-C Penicillium
purpurogenum JS03–21
Polyketides Red soil from Yunnan, China Antiviral (Wang et al.
2017)
Pennicitrinone C Penicillium citrinumB-
57
Citrinin dimers Jilantai salt field Antioxidant (Lu et al. 2008)
Terraquinone Aspergillus sp Curvularian
derivative
Sonoran Desert Cytotoxic (He et al. 2004)
Paecilin E Neosartorya fennelliae
KUFA 0811
Dihydrochromone
dimer
Marine sponge-associated
fungus
Antimicrobial (Kumla et al.
2017)
Curvularin
derivatives
Penicillium sp. Sf-5859 Lactone polyketide Marine sponge-associated
fungus
Anti-inflammatory (Ha et al. 2017)
Graphostrin A Graphostroma
sp. MCCC 3A00421
Chlorinated
polyketide
Deep sea hydrothermal sulfide
deposits
Anti-allergic (Niu et al.
2018)
Berkeleydioneand
berkeleytrione
Penicillium sp Polyketide-
terpenoid hybrid
Acidic Berkeley lake Cytotoxic (Stierle et al.
2004)
Berkazaphilones A-C
and many other
polyketides
Penicillium rubrum Polyketide
metabolites
Acidic Berkeley lake Cytotoxic (Stierle et al.
2012)
Phomopsolides Penicillium clavigerum Polyketides Alga associated from acidic
Berkeley lake
Cytotoxic (Stierle et al.
2014)
Eremophilanetype
sesquiterpenes
Penicillium sp. PR19N-1 Sesquiterpenes Prydz Bay, Antarctica Cytotoxic (Lin et al.
2014)
(Wu et al.
2013)
Purpurides B and C Penicillium
purpurogenum JS03–21
Sesquiterpene
esters
Red soil from Yunnan, China Antiviral (Wang et al.
2017)
(continued)
15 Biotechnological Application of Extremophilic Fungi 333

Table 15.2 (continued)
Bioactive peptides Marine fungi Features Source Biological activity References
Indole-diterpinoids Penicillium camemberti
OUCMDZ-1492
Terpenes Rhizospora apiculata roots
from acid niche, China
Antiviral (Fan et al.
2013)
Penicilliumin B Penicillium sp. F00120 Methylcyclopent-
enedione
sesquiterpene
Deep sea sediment Antioxidant and
antiallergic
(Lin et al.
2014)
Spirograterpene A Penicillium granulatum
MCCC3A00475
Spiro tetracyclic
diterpene
Deep sea sediment Antiallergic (Niu et al.
2017)
Bisabalone
sesquiterpenes, and
coumarin
Penicillium sp. Terpenes Acidic Berkeley lake Cytotoxic (Stierle et al.
2004)
Berkeley acetals A–CPenicillium sp. Meroterpenes Acidic Berkeley lake Cytotoxic (Stierle et al.
2007)
Berkidrimanes A and
B
Penicillium solitum Drimane
sesquiterpenes
Acidic Berkeley lake Cytotoxic (Stierle et al.
2012)
Gliotoxin Aspergillus SCSIO
Ind09F01
Diketopiperazine
alkaloids
Deep sea Cytotoxic and
antibacterial
(Luo et al.
2017)
Aspochalasins I, J,
and K
Aspergillus flavipes Cytochalasans
alkaloids
Rihizospere of plant Sonoran
Desert
Cytotoxic (Zhou et al.
2004)
Globosumones
AC
Chaetomium.Globosum Orsellinic acid
esters alkaloids
Endophytic fungi from
Sonoran Desert
Cytotoxic (Bashyal et al.
2005)
Terremides A–BA terreus PTO6–2Terremides Putian salt field Antimicrobial/antiviral (Wang et al.
2011)
Indole 3 ethanamide Aspergillus.Sclerotiorum
sp. PT06–1
Alkaloid Putian salt field Cytotoxic (Wang et al.
2011)
Variecolorquinones
A–B
Aspergillus.variecolorB-
17
Quinone alkaloid Jilantai salt field Cytotoxic (Wang et al.
2007)
Asperentin B Aspergillus sydowii Alkaloid Deep sea Tuyosine phosphatase
inhibitor
(Wiese et al.
2017)
334 A. Fasim et al.

Tyrosine derivatives Pithomyces sp Aromatic alkaloids Acidic Berkeley lake Antihypertensive and
antimigraine
(Stierle et al.
2007)
Berkeley amides A–
D
Penicillium sp. Amide Acidic Berkeley lake Cytotoxic (Stierle et al.
2008)
Glionitrin A Aspergillus fumigatus
KMC-901and
A. sphinogomonas KMK
001
Diketopiperazine
disulfide
Acid mine drainage Cytotoxic and
antimicrobial
(Park et al.
2009)
Talathermophilins A
and B
Talaromyces.
Thermophilus YM1–3
Prenylated Indol
alkaloids
Hot spring Nematocidal (Chu et al.
2010)
Thermolides Talaromyces.
Thermophilus YM3–4
Macrocyclic
PKS-NRPS
hybrids
Hot spring Nematocidal (Guo et al.
2012)
Dichotomocejs A–DDichotomomyces.Cejpii
F31–1
NRPS hybrid
dichotomocej A
Marine-lobophytum crissum
derived
Cytotoxic (Chen et al.
2017)
Brevianamides/
Mycochromenic acid
B.brevicompactum
DFFSCS025
Alkaloids Deep sea sediment Cytotoxic/antifouling (Xu et al. 2017)
15 Biotechnological Application of Extremophilic Fungi 335

References
Adachi K, Kanoh K, Wisespongp P, Nishijima M, Shizuri Y (2005) Clonostachysins a and B, new
anti-dinoflagellate cyclic peptides from a marine-derived fungus. J Antibiot (Tokyo) 58:145–
150
Adams PR (1981) Amylase production by Mucor pusillus and Humicola lanuginosa as related to
mycelial growth. Mycopathologia 76:97–101
Adams PR (1994) Extracellular amylase activities of Rhizomucor pusillus and Humicola
lanuginosa at initial stages of growth. Mycopathologia 128:139–141
Adrio JL, Demain AL (2003) Fungal biotechnology. Int Microbiol 6:191–199
Ahmed A, Bibi A (2018) Fungal Cellulase; production and applications: Minireview. Life Int J
Health Life-Sci 4:19–36
Akhtar S, Mahmood-ul-Hassan M, Ahmad R, Suthar V, Yasin M (2013) Metal tolerance potential
of filamentous fungi isolated from soils irrigated with untreated municipal effluent. Soil Environ
32:55–62
Alcalde M (2007) Laccases: biological functions, molecular structure and industrial
applications. In: Polaina J, MacCabe AP (eds) Industrial enzymes: structure, function and
applications. Springer, Dordrecht, pp 461–476
Ali I, Kanhayuwa L, Rachdawong S, Rakshit S (2012) Identification, phylogenetic analysis and
characterization of obligate halophilic fungi isolated from a man-made solar saltern in
Phetchaburi province, Thailand. Ann Microbiol 63:887–895
Almeida C, El Maddah F, Kehraus S, Schnakenburg G, König GM (2016) Endolides a and B,
vasopressin and serotonin-receptor interacting N-methylated peptides from the sponge-derived
fungus Stachylidium sp. Org Lett 18:528–531
Anand G, Yadav S, Yadav D (2017) Production, purification and biochemical characterization of an
exo-polygalacturonase from aspergillus Niger MTCC 478 suitable for clarification of orange
juice. 3 Biotech 7:122
Arnesen S, Havn Eriksen S, Olsen Ørgen J, Jensen B (1998) Increased production of α-amylase
from Thermomyces lanuginosus by the addition of tween 80. Enzym Microb Technol 23:249–
252
Arora NK, Panosyan H (2019) Extremophiles: applications and roles in environmental
sustainability. Environ Sustain 2:217–218
Avila-Cisneros N, Velasco-Lozano S, Huerta-Ochoa S, Córdova-López J, Gimeno M, Favela-
Torres E (2014) Production of thermostable lipase by Thermomyces lanuginosus on solid-
state fermentation: selective hydrolysis of sardine oil. Appl Biochem Biotechnol 174:1859–
1872
Babot ED, Rico A, Rencoret J, Kalum L, Lund H, Romero J, del Río JC, Martínez ÁT, Gutiérrez A
(2011) Towards industrially-feasible delignification and pitch removal by treating paper pulp
with Myceliophthora thermophila laccase and a phenolic mediator. Bioresour Technol 102:
6717–6722
Bajpai P (1999) Application of enzymes in the pulp and paper industry. Biotechnol Prog 15:147–
157
Baldrian P (2006) Fungal laccases –occurrence and properties. FEMS Microbiol Rev 30:215–242
Barghini P, Moscatelli D, Garzillo AMV, Crognale S, Fenice M (2013) High production of cold-
tolerant chitinases on shrimp wastes in bench-top bioreactor by the Antarctic fungus
Lecanicillium muscarium CCFEE 5003: bioprocess optimization and characterization of two
main enzymes. Enzym Microb Technol 53:331–338
Barnett EA, Fergus CL (1971) The relation of extracellular amylase, mycelium, and time, in some
thermophilic and mesophilic Humicola species. Mycopathol Mycol Appl 44:131–141
Bashyal BP, Wijeratne EMK, Faeth SH, Gunatilaka AAL (2005) Globosumones aC, cytotoxic
Orsellinic acid esters from the Sonoran Desert endophytic fungus Chaetomium globosum. J Nat
Prod 68:724–728
336 A. Fasim et al.

Berka RM, Schneider P, Golightly EJ, Brown SH, Madden M, Brown KM, Halkier T, Mondorf K,
Xu F (1997) Characterization of the gene encoding an extracellular laccase of Myceliophthora
thermophila and analysis of the recombinant enzyme expressed in aspergillus oryzae. Appl
Environ Microbiol 63:3151–3157
Boot CM, Amagata T, Tenney K, Compton JE, Pietraszkiewicz H, Valeriote FA, Crews P (2007)
Four classes of structurally unusual peptides from two marine-derived fungi: structures and
bioactivities. Tetrahedron 63:9903–9914
Boot CM, Tenney K, Valeriote FA, Crews P (2006) Highly N-methylated linear peptides produced
by an atypical sponge-derived Acremonium sp. J Nat Prod 69:83–92
Brijwani K, Rigdon A, Vadlani PV (2010) Fungal laccases: production, function, and applications
in food processing. Enzyme Res 2010:149748
Brim H, Osborne JP, Kostandarithes HM, Fredrickson JK, Wackett LP, Daly MJ (2006)
Deinococcus radiodurans engineered for complete toluene degradation facilitates Cr
(VI) reduction. Microbiology (Reading) 152:2469–2477
Brunati M, Rojas JL, Sponga F, Ciciliato I, Losi D, Göttlich E, de Hoog S, Genilloud O, Marinelli F
(2009) Diversity and pharmaceutical screening of fungi from benthic mats of Antarctic lakes.
Mar Gen 1(2):43–50
Bulter T, Alcalde M, Sieber V, Meinhold P, Schlachtbauer C, Arnold FH (2003) Functional
expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl Environ
Microbiol 69:987–995
Bunni L, McHale L, McHale AP (1989) Production, isolation and partial characterization of an
amylase system produced by Talaromyces emersonii CBS 814.70. Enzym Microb Technol 11:
370–375
Capon RJ, Skene C, Stewart M, Ford J, O’Hair RAJ, Williams L, Lacey E, Gill JH, Heiland K,
Friedel T (2003) Aspergillicins A–E: five novel depsipeptides from the marine-derived fungus
Aspergillus carneus. Org Biomol Chem 1:1856–1862
Carrasco M, Rozas JM, Barahona S, Alcaíno J, Cifuentes V, Baeza M (2012) Diversity and
extracellular enzymatic activities of yeasts isolated from King George Island, the
sub-Antarctic region. BMC Microbiol 12:251
Carrasco M, Villarreal P, Barahona S, Alcaíno J, Cifuentes V, Baeza M (2016) Screening and
characterization of amylase and cellulase activities in psychrotolerant yeasts. BMC Microbiol
16:21
Carrasco M, Rozas JM, Alcaíno J, Cifuentes V, Baeza M (2019) Pectinase secreted by
psychrotolerant fungi: identification, molecular characterization and heterologous expression
of a cold-active polygalacturonase from Tetracladium sp. Microb Cell Fact 18:1–11
Castro C, Urbieta MS, Plaza Cazón J, Donati ER (2019) Metal biorecovery and bioremediation:
whether or not thermophilic are better than mesophilic microorganisms. Bioresour Technol 279:
317–326
Ceci A, Pinzari F, Russo F, Persiani AM, Gadd GM (2019) Roles of saprotrophic fungi in
biodegradation or transformation of organic and inorganic pollutants in co-contaminated sites.
Appl Microbiol Biotechnol 103:53–68
Chandra P, Arora DS (2009) Antioxidant activity of fungi isolated from soil of different areas of
Punjab, India. Journal of Applied and Natural Science 1(2):123–128
Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium
thermophilium and its role in humification. Appl Environ Microbiol 64:3175–3179
Chen Z, Song Y, Chen Y, Huang H, Zhang W, Ju J (2012) Cyclic heptapeptides,
cordyheptapeptides C-E, from the marine-derived fungus Acremonium persicinum SCSIO
115 and their cytotoxic activities. J Nat Prod 75:1215–1219
Chen YX, Xu MY, Li HJ, Zeng KJ, Ma WZ, Tian GB, Xu J, Yang DP, Lan WJ (2017) Diverse
secondary metabolites from the marine-derived fungus Dichotomomyces cejpii F31–1. Mar
Drugs 15:339
Chhaya U, Gupte A (2013) Possible role of laccase from fusarium incarnatum UC-14 in bioreme-
diation of bisphenol a using reverse micelles system. J Hazard Mater 254–255:149–156
15 Biotechnological Application of Extremophilic Fungi 337

Chu YS, Niu XM, Wang YL, Guo JP, Pan WZ, Huang XW, Zhang KQ (2010) Isolation of putative
biosynthetic intermediates of prenylated indole alkaloids from a thermophilic fungus
Talaromyces thermophilus. Org Lett 12(19):4356–4359
D’Souza-Ticlo D, Sharma D, Raghukumar C (2009) A thermostable metal-tolerant laccase with
bioremediation potential from a marine-derived fungus. Mar Biotechnol (NY) 11:725–737
Daiha KDG, Angeli R, de Oliveira SD, Almeida RV (2015) Are lipases still important biocatalysts?
A study of scientific publications and patents for technological forecasting. PLoS One 10:
e0131624
Dalsgaard PW, Larsen TO, Christophersen C (2005) Bioactive cyclic peptides from the
Psychrotolerant fungus Penicillium algidum. J Antibiot 58:141–144
Del Cid A, Ubilla P, Ravanal MC, Medina E, Vaca I, Levicán G, Eyzaguirre J, Chávez R (2014)
Cold-active xylanase produced by fungi associated with Antarctic marine sponges. Appl
Biochem Biotechnol 172:524–532
Deshmukh RA, Jagtap S, Mandal MK, Mandal SK (2016) Purification, biochemical characteriza-
tion and structural modelling of alkali-stable β-1,4-xylan xylanohydrolase from aspergillus
fumigatus R1 isolated from soil. BMC Biotechnol 16:11
Dewapriya P, Khalil ZG, Prasad P, Salim AA, Cruz-Morales P, Marcellin E, Capon RJ (2018)
Talaropeptides A-D: structure and biosynthesis of extensively N-methylated linear peptides
from an Australian marine tunicate-derived Talaromyces sp. Front Chem 6:394
Driss D, Bhiri F, Elleuch L, Bouly N, Stals I, Miled N, Blibech M, Ghorbel R, Chaabouni SE (2011)
Purification and properties of an extracellular acidophilic endo-1,4-β-xylanase, naturally deleted
in the “thumb”, from Penicillium occitanis Pol6. Process Biochem 46:1299–1306
Du Y, Shi P, Huang H, Zhang X, Luo H, Wang Y, Yao B (2013) Characterization of three novel
thermophilic xylanases from Humicola insolens Y1 with application potentials in the brewing
industry. Bioresour Technol 130:161–167
Duarte AWF, Lopes AM, Molino JVD, Pessoa A, Sette LD (2015) Liquid–liquid extraction of
lipase produced by psychrotrophic yeast Leucosporidium scottii L117 using aqueous two-phase
systems. Sep Purif Technol 156:215–225
Dumorné K, Córdova DC, Astorga-Eló M, Renganathan P (2017) Extremozymes: a potential
source for industrial applications. J Microbiol Biotechnol 27(4):649–659
Duncan SM, Farrell RL, Thwaites JM, Held BW, Arenz BE, Jurgens JA, Blanchette RA (2006)
Endoglucanase-producing fungi isolated from Cape Evans historic expedition hut on Ross
Island, Antarctica. Environ Microbiol 8:1212–1219
Duncan SM, Minasaki R, Farrell RL, Thwaites JM, Held BW, Arenz BE, Jurgens JA, Blanchette
RA (2008) Screening fungi isolated from historic discovery hut on Ross Island, Antarctica for
cellulose degradation. Antarctic Sci 20:463–470
Dutta T, Sengupta R, SahooR RS, Bhattacharjee A, Ghosh S (2007) A novel cellulase free
alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and
characterization. Lett Appl Microbiol 44:206–211
Ebada SS, Fischer T, Hamacher A, Du FY, Roth YO, Kassack MU, Wang BG, Roth EH (2014)
Psychrophilin E, a new cyclotripeptide, from co-fermentation of two marine alga-derived fungi
of the genus aspergillus. Nat Prod Res 28:776–781
Evaristo da Silva Nascimento TC, de Sena AR, Gomes JEG, dos Santos WL, Agamez Montalvo
GS, Tambourgi EB, de Medeiros EV, Sette LD, Pessoa Junior A, Moreira KA (2015) Extracel-
lular serine proteases by Acremonium sp. L1-4B isolated from Antarctica: overproduction using
cactus pear extract with response surface methodology. Biocatal Agric Biotechnol 4:737–744
Fan Y, Wang Y, Liu P, Fu P, Zhu T, Wang W, Zhu W (2013) Indole-diterpenoids with anti-H1N1
activity from the aciduric fungus Penicillium camemberti OUCMDZ-1492. J Nat Prod 76:1328–
1336
Federici F (1985) Production, purification and partial characterization of an endopolygalacturonase
fromCryptococcus albidus var.albidus. Antonie Van Leeuwenhoek 51:139–150
de Felício R, Pavão GB, de Oliveira ALL, Erbert C, Conti R, Pupo MT, Furtado NAJC, Ferreira
EG, Costa-Lotufo LV, Young MCM, Yokoya NS, Debonsi HM (2015) Antibacterial, antifungal
338 A. Fasim et al.

and cytotoxic activities exhibited by endophytic fungi from the Brazilian marine red alga
Bostrychia tenella (Ceramiales). Rev Bras 25:641–650
Fenice M, Barghini P, Selbmann L, Federici F (2012) Combined effects of agitation and aeration on
the chitinolytic enzymes production by the Antarctic fungus Lecanicillium muscarium CCFEE
5003. Microb Cell Factories 11:12
Fenice M, Selbmann L, Di Giambattista R, Federici F (1998) Chitinolytic activity at low tempera-
ture of an Antarctic strain (A3) of Verticillium lecanii. Res Microbiol 149:289–300
Fenice M, Selbmann L, Zucconi L, Onofri S (1997) Production of extracellular enzymes by
Antarctic fungal strains. Polar Biol 17:275–280
Fergus CL (1969) The production of amylase by some thermophilic fungi. Mycologia 61:1171–
1175
Fremlin LJ, Piggott AM, Lacey E, Capon RJ (2009) Cottoquinazoline a and cotteslosins a and B,
metabolites from an Australian marine-derived strain of aspergillus versicolor. J Nat Prod 72:
666–670
Fuciños P, González R, Atanes E, Sestelo ABF, Pérez-Guerra N, Pastrana L, Rúa ML (2012)
Lipases and esterases from extremophiles: overview and case example of the production and
purification of an esterase from Thermus thermophilus HB27. Methods Mol Biol 861:239–266
Gainvors A, Nedjaoum N, Gognies S, Muzart M, Nedjma M, Belarbi A (2000) Purification and
characterization of acidic endo-polygalacturonase encoded by the PGL1-1 gene from Saccha-
romyces cerevisiae. FEMS Microbiol Lett 183:131–135
Gao B, Mao Y, Zhang L, He L, Wei D (2016) A novel saccharifying α-amylase of Antarctic
psychrotolerant fungi Geomyces pannorum: gene cloning, functional expression, and character-
ization. Starch - Stärke 68:20–28
Ghatora SK, Chadha BS, Badhan AK, Saini HS, Bhat MK (2006) Identification and characteriza-
tion of diverse xylanases from thermophilic and thermotolrant fungi. BioResources 1:18–33
Ghosal D, Ghosh S, Dutta TK, Ahn Y (2016) Current state of knowledge in microbial degradation
of polycyclic aromatic hydrocarbons (PAHs): a review. Front Microbiol 7:1369
Gomes J, Steiner W (2004) The biocatalytic potential of extremophiles and Extremozymes. Food
Technol Biotechnol 42:223–225
Gomes I, Gomes J, Steiner W (2003) Highly thermostable amylase and pullulanase of the extreme
thermophilic eubacterium Rhodothermus marinus: production and partial characterization.
Bioresour Technol 90:207–214
Goto S, Sugiyama J, Iizuka H (1969) A taxonomic study of Antarctic yeasts. Mycologia 61:748–
774
Gupta S, Krasnoff SB, Roberts DW, Renwick JAA, Brinen LS, Clardy J (1992) Structure of
efrapeptins from the fungus Tolypocladium niveum: peptide inhibitors of mitochondrial
ATPase. J Org Chem 57:2306–2313
Gu W, Cueto M, Jensen PR, Fenical W, Silverman RB (2007) Microsporins A and B: new histone
deacetylase inhibitors from the marine-derived fungus Microsporum cf. gypseum and the solid-
phase synthesis of microsporin A. Tetrahedron 63:6535–6541
Gulder TAM, Hong H, Correa J, Egereva E, Wiese J, Imhoff JF, Gross H (2012) Isolation, structure
elucidation and Total synthesis of Lajollamide a from the marine fungus Asteromyces cruciatus.
Mar Drugs 10:2912–2935
Gunde-Cimerman N, Ramos J, PlemenitašA (2009) Halotolerant and halophilic fungi. Mycol Res
113:1231–1241
Gunde-Cimerman N, Sonjak S, Zalar P, Frisvad JC, Diderichsen B, PlemenitašA (2003)
Extremophilic fungi in arctic ice: a relationship between adaptation to low temperature and
water activity. Phys Chem Earth Parts A/B/C 28:1273–1278
Guo JP, Zhu CY, Zhang CP, Chu YS, Wang YL, Zhang JX, Wu DK, Zhang KQ, Niu XM (2012)
Thermolides, potent Nematocidal PKS-NRPS hybrid metabolites from thermophilic fungus
Talaromyces thermophilus. J Am Chem Soc 134:20306–20309
15 Biotechnological Application of Extremophilic Fungi 339

Ha TM, Ko W, Lee SJ, Kim YC, Son JY, Sohn JH, Yim JH, Oh H (2017) Anti-inflammatory effects
of Curvularin-type metabolites from a marine-derived fungal strain Penicillium sp. SF-5859 in
lipopolysaccharide-induced RAW264.7 macrophages. Mar Drugs 15:282
Hamid R, Khan MA, Ahmad M, Ahmad MM, Abdin MZ, Musarrat J, Javed S (2013) Chitinases: an
update. J Pharm Bioallied Sci 5:21
Hamilton-Miller JMT (2008) Development of the semi-synthetic penicillins and cephalosporins. Int
J Antimicrob Agents 31:189–192
Haros M, Rosell CM, Benedito C (2001) Use of fungal phytase to improve breadmaking perfor-
mance of whole wheat bread. J Agric Food Chem 49:5450–5454
Hawwa R, Aikens J, Turner R, Santarsiero B, Mesecar A (2009) Structural basis for thermostability
revealed through the identification and characterization of a highly thermostable
phosphotriesterase-like lactonase from Geobacillus stearothermophilus. Arch Biochem Biophys
488:109–120
He J, Wijeratne EMK, Bashyal BP, Zhan J, Seliga CJ, Liu MX, Pierson EE, Pierson LS, VanEtten
HD, Gunatilaka AAL (2004) Cytotoxic and other metabolites of aspergillus inhabiting the
rhizosphere of Sonoran desert plants. J Nat Prod 67:1985–1991
Hooker C, Lee KZ, Solomon K (2019) Leveraging anaerobic fungi for biotechnology. Curr Opin
Biotechnol 59:103–110
Hou XM, Zhang YH, Hai Y, Zheng JY, Gu YC, Wang CY, Shao CL (2017) Aspersymmetide A, a
new centrosymmetric cyclohexapeptide from the marine-derived fungus Aspergillus versicolor.
Mar Drugs 15:363
Ibrar M, Ullah MW, Manan S, Farooq U, Rafiq M, Hasan F (2020) Fungi from the extremes of life:
an untapped treasure for bioactive compounds. Appl Microbiol Biotechnol 104:2777–2801
Jaouani A, Neifar M, Prigione V, Ayari A, Sbissi I, Ben Amor S, Ben Tekaya S, Varese GC,
Cherif A, Gtari M (2014) Diversity and enzymatic profiling of halotolerant Micromycetes from
Sebkha El Melah, a Saharan salt flat in southern Tunisia [WWW document]. Biomed Res Int
2014:439197
Jayachandran S, Ramabadran R (1970) Production of amylase by Thermoascus aurantiacus Miehe.
Indian J Exp Biol 8:344
Jiang W, Ye P, Chen CTA, Wang K, Liu P, He S, Wu X, Gan L, Ye Y, Wu B (2010) Two novel
hepatocellular carcinoma cycle inhibitory cyclodepsipeptides from a hydrothermal vent crab-
associated fungus Aspergillus clavatus C2WU. Mar Drugs 11:4761–4772
Jin L, Quan C, Hou X, Fan S (2016) Potential pharmacological resources: natural bioactive
compounds from marine-derived fungi. Mar Drugs 14:76
Jin Q, Kirk F (2018) pH as a primary control in environmental microbiology: 1. Thermodynamic
perspective. Front Environ Sci 6:21
Jin Y, Qin S, Gao H, Zhu G, Wang W, Zhu W, Wang Y (2018) An anti-HBV anthraquinone from
aciduric fungus Penicillium sp. OUCMDZ-4736 under low pH stress. Extremophiles 22:39–45
Jurado-Alameda E, Román MG, Vaz DA, Jiménez Pérez JL (2012) Fatty soil cleaning with ozone
and lipases—a way to develop more environmentally friendly washing processes. Househ Pers
Care Today 7:49–52
Kakugawa K, Shobayashi M, Suzuki O, Miyakawa T (2002) Purification and characterization of a
lipase from the glycolipid-producing yeast Kurtzmanomyces sp. I-11. Biosci Biotechnol
Biochem 66:978–985
Karthik N, Akanksha K, Pandey A (2014) Production, purification and properties of fungal
chitinases--a review. Indian J Exp Biol 52(11):1025–1035
Kashyap D, Vohra P, Chopra S, Tewari R (2001) Applications of pectinases in the commercial
sector: a review. Bioresour Technol 77:215–227
Khamthong N, Rukachaisirikul V, Phongpaichit S, Preedanon S, Sakayaroj J (2014) An
antibacterial cytochalasin derivative from the marine-derived fungus Diaporthaceae
sp. PSU-SP2/4. Phytochem Lett 10:5–9
Komatsu K, Shigemori H, Kobayashi J (2001) Dictyonamides A and B, new peptides from marine-
derived fungus. J Org Chem 66(18):6189–6192
340 A. Fasim et al.

Krishnan A, Alias SA, Wong CMVL, Pang KL, Convey P (2011) Extracellular hydrolase enzyme
production by soil fungi from King George Island, Antarctica. Polar Biol 34:1535–1542
Kuhad RC, Gupta R, Singh A (2011) Microbial Cellulases and their industrial applications. Enzyme
Res 2011:280696
Kumla D, Shine Aung T, Buttachon S, DethoupT GL, Pereira JA, Inácio Â, Costa PM, Lee M,
Sekeroglu N, Silva AMS, Pinto MMM, Kijjoa A (2017) A new Dihydrochromone dimer and
other secondary metabolites from cultures of the marine sponge-associated fungi Neosartorya
fennelliae KUFA 0811 and Neosartorya tsunodae KUFC 9213. Mar Drugs 15:375
Lara-Márquez A, Zavala-Páramo MG, López-Romero E, Camacho HC (2011) Biotechnological
potential of pectinolytic complexes of fungi. Biotechnol Lett 33:859–868
Lario LD, Chaud L, das Graças Almeida M, Converti A, Durães Sette L, Pessoa A (2015)
Production, purification, and characterization of an extracellular acid protease from the marine
Antarctic yeast Rhodotorula mucilaginosa L7. Fungal Biol 119:1129–1136
Liang X, Nong XH, Huang ZH, Qi SH (2017) Antifungal and antiviral cyclic peptides from the
Deep-Sea-derived fungus Simplicillium obclavatum EIODSF 020. J Agric Food Chem 65:
5114–5121
Liang X, Zhang XY, Nong XH, Wang J, Huang ZH, Qi SH (2016) Eight linear peptides from the
deep-sea-derived fungus Simplicillium obclavatum EIODSF 020. Tetrahedron 72:3092–3097
Lin A, Wu G, Gu Q, Zhu T, Li D (2014) New eremophilane-type sesquiterpenes from an Antarctic
deepsea derived fungus, Penicillium sp. PR19 N-1. Arch Pharm Res 37:839–844
Liu X, Kokare C (2017) Chapter 11 - microbial enzymes of use in industry. In: Brahmachari G
(ed) Biotechnology of microbial enzymes. Academic Press, Boston, pp 267–298
Liu J, Gu B, Yang L, Yang F, Lin H (2018) New anti-inflammatory cyclopeptides from a sponge-
derived fungus Aspergillus violaceofuscus. Front Chem 6:226
Lu ZY, Lin ZJ, Wang WL, Du L, ZhuTJ FYC, Gu QQ, Zhu WM (2008) Citrinin dimers from the
halotolerant fungus Penicillium citrinum B-57. J Nat Prod 71:543–546
Lugowski AJ, Palmateer GA, Boose TR, Merriman JE (1998) Biodegradation process for
de-toxifying liquid streams. WO1998005597A1
Luo X, Zhou X, Lin X, Qin X, Zhang T, Wang J, Tu Z, Yang B, Liao S, Tian Y, Pang X,
Kaliyaperumal K, Li JL, Tao H, Liu Y (2017) Antituberculosis compounds from a deep-sea-
derived fungus aspergillus sp. SCSIO Ind09F01. Nat Prod Res 31:1958–1962
Ma X, Nong XH, Ren Z, Wang J, Liang X, Wang L, Qi SH (2017) Antiviral peptides from marine
gorgonianderived fungus Aspergillus sp. SCSIO 41501. Tetrahedron Lett 58:1151–1155
Machuca A, Aoyama H, Durán N (1998) Production and characterization of thermostable phenol
oxidases of the ascomycete Thermoascus aurantiacus. Biotechnol Appl Biochem 27:217–223
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol Rev 64:461–488
Mandels M, Weber J (1969) The production of Cellulases. In: Cellulases and their applications,
advances in chemistry. American Chemical Society, Washington, pp 391–414
Martin N, Guez MAU, Sette LD, Da Silva R, Gomes E (2010) Pectinase production by a Brazilian
thermophilic fungus Thermomucor indicae-seudaticae N31 in solid-state and submerged fer-
mentation. Microbiology 79:306–313
McCormack J, Hackett TJ, Tuohy MG, Coughlan MP (1991) Chitinase production byTalaromyces
emersonii. Biotechnol Lett 13:677–682
Meyer V, Andersen MR, Brakhage AA, Braus GH, Caddick MX, Cairns TC, de Vries RP,
Haarmann T, Hansen K, Hertz-Fowler C, Krappmann S, Mortensen UH, Peñalva MA, Ram
AFJ, Head RM (2016) Current challenges of research on filamentous fungi in relation to human
welfare and a sustainable bio-economy: a white paper. Fungal Biol Biotechnol 3:6
Mot RD, Verachtert H (1987) Purification and characterization of extracellular α-amylase and
glucoamylase from the yeast Candida antarctica CBS 6678. Eur J Biochem 164:643–654
Muthezhilan R, Ashok R, Jayalakshmi S (2007) Production and optimization of thermostable
alkaline xylanase by Penicillium oxalicum in solid state fermentation. Afr J Microbiol Res 9:
20–28
15 Biotechnological Application of Extremophilic Fungi 341

Navarri M, Jégou C, Bondon A, Pottier S, Bach S, Baratte B, Ruchaud S, Barbier G, Burgaud G,
Fleury Y (2017) Bioactive metabolites from the deep subseafloor fungus Oidiodendron griseum
UBOCC-A-114129. Mar Drugs 15:111
Niu S, Fan ZW, Xie CL, Liu Q, Luo ZH, Liu G, Yang XW (2017) Spirograterpene a, a tetracyclic
Spiro-Diterpene with a fused 5/5/5/5 ring system from the Deep-Sea-derived fungus Penicillium
granulatum MCCC 3A00475. J Nat Prod 80:2174–2177
Niu S, Liu Q, Xia JM, Xie CL, Luo ZH, Shao Z, Liu G, Yang XW (2018) Polyketides from the
Deep-Sea-derived fungus Graphostroma sp. MCCC 3A00421 showed potent Antifood allergic
activities. J Agric Food Chem 66:1369–1376
Oh DC, Kauffman CA, Jensen PR, Fenical W (2007) Induced production of emericellamides a and
B from the marine-derived fungus Emericella sp. in competing co-culture. J Nat Prod 70:515–
520
Ohta K, Moriyama S, Tanaka H, Shige T, Akimoto H (2001) Purification and characterization of an
acidophilic xylanase from Aureobasidium pullulans var. melanigenum and sequence analysis of
the encoding gene. J Biosci Bioeng 92:262–270
Onofri S, Fenice M, Cicalini AR, Tosi S, Magrino A, Pagano S, Selbmann L, Zucconi L, Ocampo-
Friedmann R, Friedmann E (2000) Ecology and biology of microfungi from Antarctic rocks and
soils. Italian J Zoology 67:163–167
Ozerskaya S, Kochkina G, Ivanushkina N, Gilichinsky DA (2009) Fungi in permafrost. In:
Permafrost soils. Springer, Cham, pp 85–95
Pandey A, Soccol CR, Nigam P, Soccol VT (2000) Biotechnological potential of agro-industrial
residues. I: sugarcane bagasse. Bioresour Technol 74:69–80
Park HB, Kwon HC, Lee CH, Yang HO (2009) Glionitrin a, an antibioticantitumor metabolite
derived from competitive interaction between abandoned mine microbes. J Nat Prod 72:248–
252
Pavlova K, Gargova S, Hristozova T, Tankova Z (2008) Phytase from antarctic yeast strain
Cryptococcus laurentii AL27. Folia Microbiol 53:29–34
Peng J, Gao H, Zhang X, Wang S, Wu C, Gu Q, Guo P, Zhu T, Li D (2014) Psychrophilins E-H and
Versicotide C, Cyclic Peptides from the Marine-Derived Fungus Aspergillus versicolor
ZLN-60. J Nat Prod 77, 2218–2223
Prompanya C, Fernandes C, Cravo S, Pinto MMM, Dethoup T, Silva AMS, Kijjoa A (2015) A new
cyclic Hexapeptide and a new Isocoumarin derivative from the marine sponge-associated
fungus aspergillus similanensis KUFA 0013. Mar Drugs 13:1432–1450
Raghukumar C, Raghukumar S, Chinnaraj A, Chandramohan D, D’Souza T, Reddy C (1994)
Laccase and other lignocellulose modifying enzymes of marine fungi isolated from the coast of
India. Botanica Marina - BOT MAR 37:515–524
Rajkumari J, Bhuyan B, Das N, Pandey P (2019) Environmental applications of microbial
extremophiles in the degradation of petroleum hydrocarbons in extreme environments. Environ
Sustain 2:311–328
Ramli AN, Mahadi NM, Rabu A, Murad AM, Bakar FD, Illias RM (2011) Molecular cloning,
expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from
Glaciozyma antarctica PI12. Microb Cell Factories 10:94
Rani B, Kumar V, Singh J, Bisht S, Teotia P, Sharma S, Kela R (2014) Bioremediation of dyes by
fungi isolated from contaminated dye effluent sites for bio-usability. Braz J Microbiol 45:1055–
1063
Rateb ME, Ebel R (2011) Secondary metabolites of fungi from marine habitats. Nat Prod Rep 28:
290–344
Ray MK, Devi KU, Kumar GS, Shivaji S (1992) Extracellular protease from the antarctic yeast
Candida humicola. Appl Environ Microbiol 58:1918–1923
Robledo A, Aguilar CN, Belmares-Cerda RE, Flores-Gallegos AC, Contreras-Esquivel JC,
Montañez JC, Mussatto SI (2016) Production of thermostable xylanase by thermophilic fungal
strains isolated from maize silage. CyTA J Food 14:302–308
342 A. Fasim et al.

Rowley DC, Kelly S, Kauffman CA, Jensen PR, Fenical W, Halovirs A-E (2003) New antiviral
agents from a marine-derived fungus of the genus Scytalidium. Bioorg Med Chem 11
(19):4263–4274
Rowley DC, Kelly S, Jensen P, Fenical W (2004) Synthesis and structure-activity relationships of
the halovirs, antiviral natural products from a marine derived fungus. Bioorg Med Chem 12
(18):4929–4936
Sadaf A, Khare SK (2014) Production of Sporotrichum thermophile xylanase by solid state
fermentation utilizing deoiled Jatropha curcas seed cake and its application in
xylooligosachharide synthesis. Bioresour Technol 153:126–130
Sadhukhan R, Roy SK, Raha SK, Manna S, Chakrabarty SL (1992) Induction and regulation of
alpha-amylase synthesis in a cellulolytic thermophilic fungus Myceliophthora thermophila D14
(ATCC 48104). Indian J Exp Biol 30:482–486
Saleem M, Ali MS, Hussain S, Jabbar A, Ashraf M, Lee YS (2007) Marine natural products of
fungal origin. Nat Prod Rep 24:1142
Sarmiento F, Peralta R, Blamey J (2015) Cold and hot Extremozymes: industrial relevance and
current trends. Front Bioeng Biotechnol 3:148
Schueffler A, Anke T (2014) Fungal natural products in research and development. Nat Prod Rep
31:1425–1448
Scopel M, Abraham WR, Henriques AT, Macedo AJ (2013) Dipeptide cis-cyclo(Leucyl-Tyrosyl)
produced by sponge associated Penicillium sp. F37 inhibits biofilm formation of the pathogenic
Staphylococcus epidermidis. Bioorg Med Chem Lett 23:624–626
Selbmann L, de Hoog GS, Zucconi L, Isola D, Ruisi S, van den Ende AHGG, Ruibal C, De Leo F,
Urzì C, Onofri S (2008) Drought meets acid: three new genera in a dothidealean clade of
extremotolerant fungi. Stud Mycol 61:1–20
Selbmann L, Egidi E, Isola D, Onofri S, Zucconi L, de Hoog GS, Chinaglia S, Testa L, Tosi S,
Balestrazzi A (2013) Biodiversity, evolution and adaptation of fungi in extreme environments.
Plant Biosyst 147:237–246
Selbmann L, Isola D, Zucconi L, Onofri S (2011) Resistance to UV-B induced DNA damage in
extreme-tolerant cryptoendolithic Antarctic fungi: detection by PCR assays. Fungal Biol 115:
937–944
Selbmann L, Zucconi L, Isola D, Onofri S (2015) Rock black fungi: excellence in the extremes,
from the Antarctic to space. Curr Genet 61:335–345
Shukla AK, Singh AK (2020) Exploitation of potential extremophiles for bioremediation of
xenobiotics compounds: a biotechnological approach. Curr Genom 21:161–167
Siddiqui D, Pande V, Arif M (2012) Production, purification, and characterization of
Polygalacturonase from Rhizomucor pusillus isolated from Decomposting Orange peels.
Enzyme Res 2012:138634
Silambarasan S, Abraham J (2013) Ecofriendly method for bioremediation of Chlorpyrifos from
agricultural soil by novel fungus aspergillus Terreus JAS1. Water Air and Soil Pollution 224:1–
11
Soni H, Rawat H, Kango N (2017) Extremophilic xylanases. In: Extremophilic enzymatic
processing of lignocellulosic feedstocks to bioenergy. Springer, Cham, pp 73–88
Stierle A, StierleD MG, Snyder S, Antczak C, Djaballah H (2014) Phomopsolides and related
compounds from the alga-associated fungus, Penicillium clavigerum. Nat Prod Commun 9:87–
90
Stierle AA, Stierle DB, Decato D, Priestley ND, Alverson JB, Hoody J, McGrath K, Klepacki D
(2017) The Berkeleylactones, antibiotic macrolides from fungal Coculture. J Nat Prod 80:1150–
1160
Stierle AA, Stierle DB, Girtsman T (2012) Caspase-1 inhibitors from an Extremophilic fungus that
target specific leukemia cell lines. J Nat Prod 75:344–350
Stierle AA, Stierle DB, Girtsman T, Mou TC, Antczak C, Djaballah H (2015) Azaphilones from an
acid mine extremophile strain of a Pleurostomophora sp. J Nat Prod 78:2917–2923
15 Biotechnological Application of Extremophilic Fungi 343

Stierle AA, Stierle DB, Patacini B (2008) The Berkeleyamides, amides from the acid Lake fungus
Penicillum rubrum. J Nat Prod 71:856–860
Stierle DB, Stierle AA, Hobbs JD, Stokken J, Clardy J (2004) Berkeleydione and Berkeleytrione,
new bioactive metabolites from an acid mine organism. Org Lett 6:1049–1052
Stierle DB, Stierle AA, Patacini B (2007) The Berkeleyacetals, three Meroterpenes from a deep
water acid mine waste Penicillium. J Nat Prod 70:1820–1823
Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26
Torres-García C, Pulido D, Albericio F, Royo M, Nicolás E (2014) Triazene as a powerful tool for
solid-phase derivatization of phenylalanine containing peptides: Zygosporamide analogues as a
proof of concept. J Org Chem 79:11409–11415
Tsuji M, Yokota Y, Kudoh S, Hoshino T (2014) Improvement of direct ethanol fermentation from
woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low
temperature condition. Cryobiology 68:303–305
Tsuji M, Yokota Y, Shimohara K, Kudoh S, Hoshino T (2013) An application of wastewater
treatment in a cold environment and stable lipase production of Antarctic Basidiomycetous yeast
Mrakia blollopis. PLoS One 8:e59376
Turkiewicz M, Pazgier M, Kalinowska H, Bielecki S (2003) A cold-adapted extracellular serine
proteinase of the yeast Leucosporidium antarcticum. Extremophiles 7:435–442
Van der Giezen M (2011) Mitochondria and the rise of eukaryotes. Bioscience 61:594–601
Vaz ABM, Rosa LH, Vieira MLA, de Garcia V, Brandão LR, Teixeira LCRS, Moliné M,
Libkind D, Van Broock M, Rosa CA (2011) The diversity, extracellular enzymatic activities
and photoprotective compounds of yeasts isolated in Antarctica. Braz J Microbiol 42:937–947
Verma AK, Raghukumar C, Parvatkar RR, Naik CG (2012) A rapid two-step bioremediation of the
Anthraquinone dye, reactive blue 4 by a marine-derived fungus. Water Air Soil Pollut 223:
3499–3509
Wang W, Tianjiao Z, Hongwen T, Lu Z, Fang Y, Gu Q, Zhu W (2007) Two new cytotoxic quinone
type compounds from the halotolerant fungus Aspergillus variecolor. J Antibiot 60(10):
603–607
Wang H, Zheng JK, Qu HJ, Liu PP, Wang Y, Zhu WM (2011) A new cytotoxic indole-3-
ethenamide from the halotolerant fungus aspergillus sclerotiorum PT06-1. J Antibiot 64:679–
681
Wang N, Zang J, Ming K, Liu Y, Wu Z, Ding H (2013) Production of cold-adapted cellulase by
Verticillium sp. isolated from Antarctic soils. Electron J Biotechnol 16:10–10
Wang N, Cui CB, Li CW (2016) A new cyclic dipeptide penicimutide: the activated production of
cyclic dipeptides by introduction of neomycin-resistance in the marine-derived fungus Penicil-
lium purpurogenum G59. Arch Pharm Res 39:762–770
Wang X, Yu H, Xing R, Li P (2017) Characterization, preparation, and purification of marine
bioactive peptides. Biomed Res Int 2017:9746720
Wiese J, Aldemir H, Schmaljohann R, Gulder TA, Imhoff JF (2017) Asperentin B, a new inhibitor
of the protein tyrosine phosphatase 1B. Mar Drugs 15:191
Wu G, Lin A, Gu Q, Zhu T, Li D (2013) Four new Chloro-Eremophilane Sesquiterpenes from an
Antarctic Deep-Sea derived fungus, Penicillium sp. PR19N-1. Mar Drugs 11:1399–1408
Wu HY, Yang FL, Li LH, Rao YK, Ju TC, Wong WT, Hsieh CY, Pivkin MV, Hua KF, Wu SH
(2018) Ergosterol peroxide from marine fungus Phoma sp. induces ROS-dependent apoptosis
and autophagy in human lung adenocarcinoma cells. Sci Rep 8:1–14
Xu F, Shin W, Brown SH, Wahleithner JA, Sundaram UM, Solomon EI (1996) A Study of a series
of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox
potential, substrate specificity, and stability. Biochim Biophys Acta 1292:303–311
Xu X, Zhang X, Nong X, Wang J, Qi S (2017) Brevianamides and mycophenolic acid derivatives
from the Deep-Sea-derived fungus Penicillium brevicompactum DFFSCS025. Mar Drugs 15:43
Yang YL, Liao WY, Liu WY, Liaw CC, Shen CN, Huang ZY, Wu SH (2009) Discovery of new
natural products by intact-cell mass spectrometry and LC-SPE-NMR: malbranpyrroles, novel
polyketides from thermophilic fungus Malbranchea sulfurea. Chemistry 15:11573–11580
344 A. Fasim et al.

Yang YL, Lu CP, Chen MY, Chen KY, Wu YC, Wu SH (2007) Cytotoxic polyketides containing
tetramic acid moieties isolated from the fungus Myceliophthora Thermophila: elucidation of the
relationship between cytotoxicity and stereoconfiguration. Chemistry 13:6985–6991
Yegin S (2017) Single-step purification and characterization of an extreme halophilic, ethanol
tolerant and acidophilic xylanase from Aureobasidium pullulans NRRL Y-2311-1 with appli-
cation potential in the food industry. Food Chem 221:67–75
You M, Liao L, Hong SH, Park W, Kwon DI, Lee J, Noh M, Oh DC, Oh K, Shin J (2015) Lumazine
peptides from the marine-derived fungus aspergillus terreus. Mar Drugs 13:1290–1303
Youssef FS, Ashour ML, Singab ANB, Wink M (2019) A comprehensive review of bioactive
peptides from marine fungi and their biological significance. Mar Drugs 17:559
Yu P, Wang XT, Liu JW (2015) Purification and characterization of a novel cold-adapted phytase
from Rhodotorula mucilaginosa strain JMUY14 isolated from Antarctic. J Basic Microbiol 55:
1029–1039
Yuan P, Meng K, Huang H, Shi P, Luo H, Yang P, Yao B (2011) A novel acidic and low-
temperature-active endo-polygalacturonase from Penicillium sp. CGMCC 1669 with potential
for application in apple juice clarification. Food Chem 129:1369–1375
Zeldes BM, Keller MW, Loder AJ, Straub CT, Adams MWW, Kelly RM (2015) Extremely
thermophilic microorganisms as metabolic engineering platforms for production of fuels and
industrial chemicals. Front Microbiol 6:1209
Zhang X, Li SJ, Li JJ, Liang ZZ, Zhao CQ (2018) Novel natural products from Extremophilic fungi.
Mar Drugs 16:194
Zhang Y, Li XM, Feng Y, Wang BG (2010) Phenethyl-α-pyrone derivatives and cyclodipeptides
from a marine algous endophytic fungus aspergillus Niger EN–13. Nat Prod Res 24:1036–1043
Zheng J, Zhu H, Hong K, Wang Y, Liu P, Wang X, Peng X, Zhu W (2009) Novel cyclic
Hexapeptides from marine-derived fungus, aspergillus sclerotiorum PT06-1. Org Lett 11:
5262–5265
Zhou GX, Wijeratne EM, Bigelow D, Pierson LS, VanEtten HD, Gunatilaka AA (2004)
Aspochalasins I, J, and K: three new cytotoxic cytochalasans of Aspergillus flavipes from the
rhizosphere of Ericameria laricifolia of the Sonoran Desert. J Nat Prod 67(3):328–332
Zhou X, Fang P, Tang J, Wu Z, Li X, Li S, Wang Y, Liu G, He Z, Gou D (2016) A novel cyclic
dipeptide from deep marine-derived fungus Aspergillus sp. SCSIOW2. Nat Prod Res 30:52–57
15 Biotechnological Application of Extremophilic Fungi 345

Extremophilic Fungal Cellulases: Screening,
Purification, Catalysis, and Applications 16
Sangita Chouhan, Rajkumar Ahirwar, Tejpal Singh Parmar,
Ashiq Magrey, and Sanjay Sahay
Abstract
Cellulases constitute a consortium of enzymes that by their coordinated activity
hydrolyze cellulose to glucose. Commercially they are very important with their
varied industrial applications. Currently, Trichoderma and Aspergillus are the main
sources of commercially available cellulases, but for certain applications more
suitable enzymes are required that can be provided by extremophilic microbes
including fungi. During the last few decades cellulases from extremophilic fungi
exhibiting desirable features for specific applications such as psychrophilic
enzymes for textile and detergent applications and highly sturdy polyextremophilic
cellulases for biofuel application have been reported that they have paved the way
for the exploration and finally applications of cellulases from extremophilic fungi.
Keywords
Extremophilic microfungi · Extremocellulases · Cellulases production ·
Cellulases activity · Extremocellulases employability
16.1 Introduction
Cellulases are a group of enzymes which by their concerted action hydrolyze
cellulose. At least three members of cellulases, viz., endo-1,4-glucanase (also
named carboxymethyl cellulase; EC 3.2.1.4), an exo-β-1-4, glucanase
S. Chouhan · R. Ahirwar · T. S. Parmar · A. Magrey
Department of Biotechnology, Barkatullah University, Bhopal, MP, India
S. Sahay (*)
Sarojini Naidu Government Postgraduate Girl’s (Autonomous) College, Bhopal, Madhya Pradesh,
India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_16
347

(EC 3.2.1.91), and a β-glucosidase (also called cellobiase; EC 3.2.1.21), are required
in cellulose hydrolysis. Cellulose itself is the most abundant glucan composed of D-
glucose-linked β-1,4 glycosidic bonds (Kotchoni et al. 2006). It is the most abundant
renewable bioresource and raw material for various products such as biofuel, textile,
animal feed, adhesive, bioplastic, biopolymer, bionanomaterials, and phenolics
(Sahay 2020) produced by the plants through photosynthesis. Cellulose is found
intricately complexed with lignin and hemicelluloses (called lignocelluloses) in the
plant biomass (Sahay 2020). Current technology to utilize lignocellulose envisages
its pretreatment followed by its enzymatic hydrolysis. Apart from this, cellulases
have multiple applications such as in textile, food processing, and detergent
formulation.
Cellulases are present in plant, animal, and microbes, but microbes have been
found to be a better source for commercial exploitation because they can be easily
cultured in large quantities in a relatively short time period in a cost-effective way
(Acharya and Chaudhary 2012). Microbes do produce at a higher rate and their
enzymes are more stable than those from plants or animals (Headon and Walsh
1994). Among microbes, fungi are more amenable to solid-state fermentation, a
relatively cheaper method of enzyme production. Moreover, fungal cellulases are
found mostly as extracellular and free molecules rather than as complex cellulosome
that the bacteria produce.
Enzymes in general work optimally under a specific set of physicochemical
conditions which are indeed not always available in the industrial setup. Enzymes
from extremophilic microbes however in many instances are able to work under
harsh conditions because of various adaptations they developed during living in
hostile conditions (Gomes and Steiner 2004; Sujatha et al. 2005; Acharya and
Chaudhary 2012). Cellulases being the third largest enzyme from an industrial
point of view have multiple applications with each application requiring specific
set of catalytic features. Cellulases from extremophilic fungi are thus attractive.
16.2 Cellulases
Cellulases comprise a group of enzymes that coordinately catalyze hydrolysis of
cellulose into glucose. Except a few fungi, e.g., Neocallimastix,Piromyces, and
Orpinomyces (Tatsumi et al., 2006; Watanabe and Tokuda, 2010), that produce
cellulases in a complex form called cellulosomes, all other fungi produce cellulases
in free forms. Fungal cellulases comprise three hydrolytic enzymes viz., endo-
(1,4)-β-D-glucanase (EC 3.2.1.4), exo-(1,4)-β-D-glucanase (EC 3.2.1.91), and
β-glucosidase (EC 3.2.1.21) (Goyal et al. 1991).
Commercially, cellulases are obtained from fungi like Aspergillus and
Trichoderma spp. Aspergillus strains are also known for their ability to produce β–
glucosidase or cellobiohydrolase with much higher yields than Trichoderma spp.
(Damisa et al. 2011). The Trichoderma reesei produces cellulases at a high rate, but
its cellulases contain β-glucosidase in inadequate quantity affecting the efficiency of
cellulose hydrolysis into glucose. This in turn results in the accumulation of
348 S. Chouhan et al.

cellobiose (dimer of glucose) which is a strong inhibitor of endo- and exoglucanases
and ultimately overall process of hydrolysis (Gilkes et al. 1991). The addition of
exogenous β-glucosidase is thus indispensable to ensure efficient cellulose hydroly-
sis with T. reesei cellulases (Ting et al. 2009). The activities of the three enzymes are
as below (Fig. 16.1):
(a) Endo-β-1-4, glucanase: Catalyzes random breaking of internal β-1-4, linkages.
(b) Exo-β-1-4, glucanase: Catalyzes the removal of glucose units from the
non-reducing end of the cellulose chain and successively. The enzyme is
inhibited by glucose.
(c) β-glucosidases or cellobiases: It acts on cellobiose and catalyzes its hydrolysis
into glucose. The enzyme is also inhibited by glucose.
16.3 Screening
16.3.1 Rapid Test
The rapid cellulase screening method (Smith 1977) uses autoclaved soft agar (0.75%
w/v) medium without any carbon source dispensed in a screw-capped bottle. This is
to be overlaid by cellulose-azure containing medium. As recommended originally,
the cellulose-azure containing medium is prepared at first in two parts, first
containing only recipe of medium but with two-thirds volume of water required
and second with only cellulose-azure and one-third water required. After steriliza-
tion, the two are mixed while they are still warm, and then a small volume of it is
pipetted onto the medium kept in a screw-capped bottle. Originally, the medium
Fig. 16.1 Three members of cellulases and their sites of activity on substrate
16 Extremophilic Fungal Cellulases: Screening, Purification, Catalysis, and... 349

used was the Patterson medium (Coutts and Smith 1976), but it can be replaced with
another suitable medium as per the requirement of a specific microbe. The finally
prepared medium is inoculated with spores or cork-borer cut disc of fresh mycelium.
After suitable incubation, cellulolytic microbe is identified by its ability to release
blue color in the medium.
16.3.2 Congo Red Test
The fungal isolates are screened for their ability to produce cellulases according to
the method given earlier (Teather and Wood 1982) with minor modification by
various workers. One of the methods applies Czapek–Dox medium containing (g/l):
sucrose –30, NaNO
3
–2, K
2
HPO
4
–1, MgSO
4
–0.05, KCl –0.5, FeSO
4
–0.01,
carboxymethyl cellulose –1%, agar agar –20; pH of the medium has to be adjusted
according to fungal requirement viz., 3–4 (for acidophilic), 5–6 (thermo/psychro-
philic), and 9–11 (alkaliphilic). Medium may be supplemented with 5–10% NaCl as
per the requirement of halophilic fungus. After autoclaving, the medium is to be
poured into Petri plates and allowed to solidify. Cavities of definite size (e.g., 6 mm)
are made in the solidified medium and inoculated with 0.1 mm size of the fungal
colony (fungi forming mat) or spore suspension (10
6
/ml) from log phase culture.
Generally, the plate is incubated at around 30–37C (mesophilic), 20 C
(psychrotrophic), and >40C (thermophilic) for 3–4 days, slow grower
extremophiles may need a longer incubation period. After incubation, 10 ml of 1%
Congo Red staining solution is added to the plates and shaken at 50 rpm for 15 min.
The Congo Red staining solution is then discarded, and 10 ml of 1 N NaOH is added
and shaken again at 50 rpm for 15 min. Finally NaOH solution is also discarded and
the staining of the plates is analyzed by noticing the formation of clear or yellowish
zones around the fungal inoculated wells.
16.4 Production of Enzymes
There are two approaches to be followed viz., solid-state fermentation (SSF) and
submerged fermentation (SF). In SSF the growth medium contains agro-residue such
as wheat or rice straw as a carbon source. This is supplemented with a mixture of
inorganic compounds to supply various growth requirements. In SF the carbon
source used in the growth medium is microcrystalline cellulose (Avicel), which is
used generally. This is supplemented with other inorganic compounds. The pH and
salinity condition of the medium is adjusted as per the requirements of the fungus
used. Also, the incubation temperature depends upon the requirement of the fungus
for optimal growth. The submerged fermentation is carried out on an incubator
shaker. After the completion of fermentation a test is made for the presence of
enzyme activity in the medium. If expected enzyme activity is detected, the SF
medium is directly filtered through a four-layered cheese cloth and the filtrate
obtained is centrifuged at 5000 rpm for 10 min at 4 C and supernatant is used as
350 S. Chouhan et al.

a source of enzyme. In SSF after the completion of fermentation the medium is
washed with sterile distilled water. The wash is used as the source of enzyme.
16.5 Purification of Cellulases
Cellulases can be obtained in reasonably pure form applying the following steps:
Ammonium sulfate fractionation.
Supernatant is progressively subjected to increasing saturation with ammonium
sulfate, i.e., 0–10%, 10–20%, 20–30%, 30–40%, 40–50%, 50–60%, 60–70%,
70–80%, 80–90% following overnight incubation at 4C the precipitates after each
saturation are spun down. Precipitates thus obtained for each saturation are assayed
for each of the cellulase subunits, i.e., CMCase, FPase, and glucosidase. Finally,
enzymatically active fractions are pooled, dissolved in a buffer usually 1 M phos-
phate buffer (pH 6.5), and kept at 4 C until further purification steps.
16.5.1 Dialysis
Dissolved precipitates of CMCase, FPase, and glucosidase are to be desalted by
dialyzing against the same buffer overnight at 4 C using a dialysis membrane of
cut-off 14,000 Da. Alternatively, G-50 column may be used to desalt the enzyme
preparation.
16.5.2 Chromatographic Purification
Further purification may be carried out applying ion exchange (DEAE) chromatog-
raphy (Garsoux et al. 2004) or gel exclusion column chromatography (Bakare et al.
2005)oraffinity chromatography applying swollen Avicel as affinity matrix (Ncube
2013).
Before packing in the suitable column, the chromatographic material (matrix) is
pretreated with a suitable buffer (e.g., phosphate buffer). The sephadex G-100 is
incubated in phosphate buffer overnight and warmed in boiling water bath for 4–5h
to swell it before packing. The packing is carefully done to avoid entrapment of any
air bubbles in the matrix. A suitable sample size of dialyzed samples, i.e., CMCase,
FPase, and glucosidase, is loaded onto the column, washed with the same buffer
followed by elution of bound protein (s) with suitable elution buffer usually the same
buffer containing a linear gradient of ammonium sulfate or NaCl (usually 0–0.5 M).
Fractions of a specified volume, e.g., 3 ml, are collected manually or applying
automatic collector with constant rate, e.g., ½ml per minute. The fractions tested
for the presence of protein directly by measuring absorbance at 280 nm. Positive
fractions are then assayed for the presence of enzyme activity. Finally positive
fractions are pooled and again dialyzed and lyophilized. Finally, the enzymes are
16 Extremophilic Fungal Cellulases: Screening, Purification, Catalysis, and... 351

characterized by applying sodium dodecyl sulfate polyacrylamide gel electrophore-
sis (SDS PAGE).
Enzyme purification is currently done using fast and automatic system such as
FPLC (e.g., FPLC Mono Q Sepharose column) (Dutta et al. 2008).
16.6 Enzyme Assays
16.6.1 Total Cellulase or Filter Paperase (FPase)
It is determined as described earlier (Ghose 1987). The assay mixture (total volume
of 2 ml) contains 50 mg of Whatman No.1 strip (1 6 cm) in 1 ml of 50 mM citrate
buffer (pH 4.8) and 0.5 ml of diluted crude enzyme and is incubated at a suitable
temperature (varies for thermophilic, mesophilic, and psychrophilic enzymes) for
30 min. The buffer may be replaced with suitable ones in case of alkaliphilic/
acidophilic enzymes. The reaction mixture may be supplemented with suitable
concentration of salt in case of halophilic enzymes. Standard curve was prepared
with glucose. Overall cellulase activity is expressed as filter paper unit (FPU) per ml
of supernatant.
16.6.2 CMCase
Carboxy methyl cellulose (CMCase) activity is determined as described by Mandels
et al. (1975). The assay mixture, in a total volume of 2 ml, contains 0.5 ml of 1 mM
of carboxyl methyl cellulose (CMC) in 50 mM citrate buffer (pH 4.8) and 0.5 ml of
diluted crude enzyme. The reaction mix may be prepared with suitable buffer and
supplementation of salt and is incubated at a suitable temperature for 30 min as in the
case of FPase.
16.6.3 Cellobiase (b-Glucosidase)
Cellobiase activity is assayed by incubating 0.5 ml of supernatant with 0.5 ml of 2%
cellobiose in 0.05 M sodium citrate buffer (pH 4.8) at a suitable temperature for
30 min. The reaction mix may be prepared with suitable buffer and supplementation
of salt and is incubated at a suitable temperature for 30 min as in the case of FPase.
After incubation in each case, DNS mixture is added, boiled for 5 min, and
transferred immediately to a cold water bath. Then 20 ml of distilled water is added
to the tubes, mixed, and the developed color is measured at 540 nm to estimate the
amount of reducing sugars released. The enzymatic activity of CMCase, total FPase,
and endoglucanase is defined in international units (IU). One unit of enzymatic
activity is defined as the amount of enzyme that released 1 μmol reducing sugars
(measured as glucose) per ml per min.
The activity is calculated by the following formula:
352 S. Chouhan et al.

CMCase=FPase=Cellobiase activity IU=mlðÞ
¼mg glucose producedðÞ5560 22 2=30
Enzyme concentration ¼1 volume of enzyme sample in dilution
Total volume of dilution
1=dilution ¼1=20 ¼0:05
CMCase and FPase IU=mlðÞ
¼0:37=Enzyme concentration that releases 2:0 mg glucose
Cellobiase ¼0:0926=enzyme concentration that releases 1:0 mg glucose
16.6.3.1 Chromogenic Method for b-Glucosidase
β-glucosidase activity can also be determined using chromogenic substrates such as
p-nitrophenyl-β-D-glucoside, p-nitrophenyl β-D-1,4-glucopyranoside,-
β-naphthyl-β-D-glucopyranoside, 6-bromo-2-naphthyl-β-D-glucopyranoside, and
4-methylumbelliferyl-β-D-glucopyranoside. The enzyme acts on these substrates
and releases colored or fluorescent products. The most common substrate among
these is p-nitrophenyl β-D-1,4-glucopyranoside (pNPG) which releases
p-nitrophenyl that can be detected at 430 nm. pNPG (5 mM) is prepared by adding
0.1576 g of pNPG in 100 ml acetate buffer. 1.0 ml of 5 mM pNPG is mixed with
1.8 ml of suitable buffer and 0.2 ml of appropriately diluted enzyme and is incubated
at a suitable temperature. After adding 4.00 ml of glycine buffer to stop the reaction,
absorbance is read against blank at 430 nm. Standard curve is made with
4-nitrophenol (Zhang et al. 2009).
High-throughput multicolored chromogenic test has been developed for
cellulases assay by Kračun et al. (2015).
16.6.3.2 Effect of Glucose and Ethanol on b-Glucosidase Activities
β-glucosidase activity was quantified with the addition of glucose (100–1000 mmol)
or ethanol (50–400%) to the reaction mixture, according to Leite et al. (2008). The
assays were carried out in 100 mmol sodium acetate buffer, pH 50, at 50 C.
16.6.4 Protein Assay
Protein is assayed applying Lowry et al. (1951) method.
16.7 Enzyme Production
The commercial dominance of cellulases from the fungus Trichoderma spp. is due to
its higher production rate which in turn has resulted from tremendous research
efforts to improve strain and optimize production rate (Seiboth et al. 2011), even
16 Extremophilic Fungal Cellulases: Screening, Purification, Catalysis, and... 353

then it suffers from very low β-glucosidase activity (Knapp 1985). It has been
reported that the thermophilic fungi Sporotrichum thermophile (Coutts and Smith
1976) and Talaromyces emersonii (Folan and Coughlan 1978) match cellulase
production level with Trichoderma reesei. Moreover, the enzymatic activity of
cellulases from thermophilic fungi Chaetomium thermophile,Sporotrichum thermo-
phile, and Thermoascus aurantiacus has been found to be two- to three-fold higher
as compared to those of Trichoderma viridae (Tansey 1971). Halophilic fungus
A. terreus UniMAP AA-6 gas been found to produce highly sturdy ionic liquid
tolerant CMCase. Production of this enzyme has been optimized (2.2-fold) applying
Plackett–Burman experimental design (Gunny et al. 2015) implying the potential of
exploration of suitable extremophilic cellulases for various applications and their
production optimization.
16.8 Applications
Commercially cellulases hold a prominent place among industrially important
enzymes. They have applications in various industries such as textile, detergent,
pulp and paper, food, animal feed, biofuel, and molecular biology. In a market
research report, cellulases hold 32.84% of the Global market share among enzymes
in 2016. The same report forecasted a 5.5% compound annual growth in demand for
cellulases during 2018–2025 reaching 2300 million USD.
Currently, mesophilic cellulases produced from Trichoderma spp. and Aspergil-
lus spp. are commercially sold. Trichoderma spp. produces cellulases very effi-
ciently, but the proportion of β-glucosidase in them is low so they cannot hydrolyze
cellulose completely and efficiently (Knapp 1985). The addition of Aspergillus
β-glucosidase is so required in the enzyme cocktail. Moreover, some applications
require some special characteristics of enzyme, for example, food industries require
processing at a lower temperature to preserve nutritional values and aroma which is
possible if cold-active enzyme is available. Extremophilic microbes, e.g., fungi
produce cellulases with special features valuable for various industries (Table 16.1).
16.8.1 Food Industries
The food industry is one of the biggest industries worldwide that depends on huge
supply of enzymes. The following activities are especially dependent on enzymes.
16.8.2 Juice Clarification
Fruit and vegetable juice in the beginning looks hazy because of floating fibrous
materials of mostly carbohydrate nature. This is unattractive for consumers and thus
has to go. This is dealt with a cocktail of enzymes containing pectinases, cellulases,
and hemicellulases. The treatment not only clarifies the juice but also reduces the
354 S. Chouhan et al.

Table 16.1 Representative extremophilic cellulases with essential features from fungi
Fungal species Isolation site Enzymes
Optimum
ReferencepH Temp(C) Salinity
(A) Halophilic enzyme
Aspergillus flavus Solar saltern CMCase 10 60 200 g l
1
Bano et al. (2019)
Aspergillus ZJUBE-1 Marine All three 4–565 –Liu et al. (2012)
A. niger Marine 8% Liang and Xue (2017)
Penicillium chrysogenum FU42 Marine CMCase 7.5 60 0.1 M Lee et al. (2015)
Stachybotrys microspora Sodic soil CMCase 8.0 70 5.0 M Hmad et al. (2017)
(B) Acidophilic
Pleurotus ostreatus –CMCase 4.0 55 –Okereke et al. (2017)
Rasamsonia emersonii Plant litter CMCase 3.0 ––Thanh et al. (2019)
A. Fumigates CMCase 2.0 65 ––Grigorevski-Lima et al. (2009)
A. niger CMCase 4.0 70 ––Li et al. (2012)
A. nidulans CMCase 4.0 50 –Tavares et al. (2013)
(C) Alkaliphilic/tolerant
Sodiomyces alkalinus Soda soil 6.0–10 ––Grum-Grzhimaylo et al. (2017)
P. citrinum Soil CMCase 5.5/8.0 ––Dutta et al. (2008)
(D) Thermophilic
Myceliophthora thermophila JCP 1–4 CMCase 5.5 65
Cellobiase 5.5 70
Thermoascus aurantiacus CMCase 4.5–5.0 60–75 –Tong et al. (1980)
Cellobiase Do 70
Penicillium funiculosum All three 4.9 52–58 de Castro et al. (2010)
Aspergillus wentii CMCase 4.2 60 Giorgi (2017)
A. versicolor 4.0 60 Do
Chaetomium thermophile 4.5 55 Do
Sporotrichum pulverulentum 4.5 65 Do
(continued)
16 Extremophilic Fungal Cellulases: Screening, Purification, Catalysis, and... 355

Table 16.1 (continued)
Fungal species Isolation site Enzymes
Optimum
ReferencepH Temp(C) Salinity
Acremonium thermophilum CMCase 5.5 60 Voutilainen et al. (2008)
Humicola grisea var thermoidea Cellulase 5.0 55–60 Takashima et al. (1996)
Humicola insolens CMCase 5.0 50 Hayashida and Yoshioka (1980)
Talaromyces emersonii Cellobiohydrolase 5.0 68 Grassick et al. (2004)
Thermoascus aurantiacus Cellobiohydrolase 6.0 65 Hong et al. (2003)
Humicola grisea Cellobiase 5.0 75 Takashima et al. (1999)
Humicola grisea Do 5.0 60 Do
E. Psychrophilic
Glaciozyma Antarctica β-1,3-glucanase 7.0 20 Mohammadi et al. (2020)
Cladosporium spp. 5.0 60 Abrha and Gashe (1992)
Tausonia pullulans β-Galactosidase 4.0 50 Song et al. 2010
Verticillium sp. AnsX1 FPase 5.3 38 Wang et al. (2013)
Mrakia blollopsi CMCase 5.4 4–22 Carrasco et al. (2016)
Cadophora Malorum Berg (1978)
Penicillium spp. All three 4.9 52–58 Jorgensen et al. (2002)
A. terreus AKM-F3 CMCase 4.0 35 Maharana and Ray (2015)
F. Endophytes
Fusarium oxysporum 5–6 28 Dar et al. (2013)
A. niger DR02 CMCase Robii et al. 2013
Trichoderma atroviride
DR17,DR19 Cellobiase
Alternaria sp. DR45
Annulohypoxylon stigyum DR47
Talaromyces wortmannii DR49.
Penicillium glabrum All three Cabezas et al. (2012)
Induratia spp. All three Pereira et al. (2020)
356 S. Chouhan et al.

viscosity. Juice especially of citrus fruits is valued for vitamins (Vit C) which is
degenerated at higher temperatures. Thus, cold-active enzymes that preferably work
at storage temperature (<4C) are highly desired as they enable storing juice
immediately after extraction with enzymes. This practice preserves not only the
nutritional quality but also the aromatic profile of the juice.
In wine industry, cellulases in combination of pectinases are used to enhance the
release of juice, tannins, and color from grapes (Sahay 2019). Enzymes working
optimally at lower pH and temperatures (cold-active) are highly desirable.
16.8.3 Pigment Extraction
Natural pigments such as carotenoid are highly in demand nowadays as they are used
as safe food colorants. The peels of colored fruits are the sources of these pigments
whose extraction is highly accelerated in the presence of the cocktail of the cell wall
acting enzymes including cellulases.
16.8.4 Olive Oil Extraction
During extraction olive oil is subjected to the mixing step to facilitate the aggrega-
tion of small oil droplets into bigger ones. The addition of cellulases with or without
pectinases accelerates the process and also enhances the release of phenolic and
antioxidant compounds in the oil (Jayasekara and Ratnayake 2019).
16.8.5 Bakery
The quality of bakery products such as the softness of crumb, loaf volume, flavor,
and shelf life is enhanced substantially if the enzyme cocktail including cellulases is
added to the flour (Illingworth and Cook 1998).
The cold-active enzymes are especially useful in the food industries as they can
enable processing at lower temperatures which in turn ensures heat labile
compounds of nutritional values (e.g., vitamins and antioxidant compounds) and
aroma to be preserved.
16.8.6 Textile Industry
Textile comprises the most important part of the world’s one of the hugest fashion
industries. Cloths of unique feel, texture, shine, and colors are in huge demand.
Many of these qualities in the cloth depend on enzymatic treatments. Cellulases form
the third largest group of enzymes used in textile industries to catalyze processes
involved in improving these qualities (Xia and Cen 1999).
16 Extremophilic Fungal Cellulases: Screening, Purification, Catalysis, and... 357

16.8.6.1 Biostone Washing
Cellulases are used to replace conventional three-stepped biostone washing. The
conventional process involves the removal of starch coating from fabric with
amylases (desizing), subjecting the cloth to rubbing with pumice stone in washing
machine, and treating the cloth with sodium hypochlorite or potassium permanga-
nate. After the process is over pumice stones are removed manually which is
tiresome and pumice stone itself is abrasive to machine. The application of cellulases
deals with these problems. Cellulase (CMCase or endoglucanase) removes small
exterior fibers containing most of the indigo stains thus giving a faded look to the
cloth. One more problem, i.e., back staining whereby removed stain again binds to
treated cloth that remains even after treatment with cellulase. One of the solutions is
to use the cocktail of amylase, cellulase, and laccase; the last enzyme decolorizes the
removed stain preventing back staining (Cortez et al. 2001). T. reesei endoglucanase
II is found useful for biostone washing (Maryan and Montazer 2013).
16.8.6.2 Biopolishing or Biofinishing
Cotton and linen cloths look fuzzy due to protruding microfibers. If they are left
untreated, they may turn into an even worse ball-like appearance. All these are
unattractive themselves and also reduce the shine and brightness of color. Cellulases
are used to remove these microfibers thereby giving a glossy look, improved hand
feel, and brightness to color. Additionally enzyme also removes dirt spots and stains
trapped within fibers (Sreenath et al. 1996; Anish et al. 2007).
16.8.6.3 Bioscouring
The softness of the cloth can also be enhanced by removing non-cellulosic materials
(cuticle) from the surface. This is done with cellulase alone or in combination with
pectinases (Ibrahim et al. 2011).
16.8.6.4 Biocarbonization and Wool Scouring
Removal of extra cellulosic and other vegetative materials from raw wool, cotton, or
cotton blend fabric (carbonization) is also required to improve the quality of the
fabric. Earlier sulfuric acid was used for this that made the process costly, corrosive,
and hazardous. Now the enzyme cellulases have replaced the acid making the
process safe.
16.8.6.5 Defibrillation of Lyocell
Lyocell is a generic name given to pulp fabric, obtained by treating wood pulp with
amine oxide followed by pushing the dissolved materials through spinnerets. The
fabric material is regarded as sustainable but requiring the removal of superficial
tangled fibers (fibrillation) before use. The fibers are digested with cellulases giving
the fabric a glossy look and a smooth hand feel.
Textile enzymatic treatment requires exo- and endoglucolytic activities. There-
fore, T. reesei cellulase is mostly used in the treatment. There are now reports for the
use of cold-active alkaliphilic cellulases for textile because of certain advantages.
Cold-active enzymes enable process to be carried at low temperature under mild
358 S. Chouhan et al.

condition that would reduce wear and tear during processing. Cold-active enzymes
are generally heat labile, thus it is possible to denature the enzyme after heat
treatment that would reduce water use during the washing step.
16.8.7 Paper and Pulp
16.8.7.1 Pulping
Paper and pulp industry is also one of the largest industrial sectors globally. The
industry applies the most basic steps of cutting wood into pieces and their mechani-
cal pulping which are energy intensive processes and generate stiff pulps laden with
fines and bulk (Kuhad et al. 2011). Biopulping applying cellulases has been found to
reduce energy investment by 20–40% (Statista 2018), reduce fiber coarseness and
viscosity, and enhance bleachability (Pere et al. 1995; Mansfield et al. 1996).
Moreover, pulp generated through biopulping gives smaller particles during the
subsequent refining process which exhibits a lesser drainage rate during paper
making (Kuhad et al. 2011). Thus the quality of pulp/paper is improved and energy
cost and environmental impact are also reduced (Sharma et al. 2016).
16.8.7.2 Deinking
Recycling and reuse of papers require deinking of the used papers. Traditional
deinking process involves expensive and hazardous chemicals (alkali) that generate
toxic by-products (Zhang et al. 2013). Replacing them with celluloses called
bio-deinking is environment-friendly process that does yield whiter recycled paper
as it is done at acidic pH avoiding alkaline yellowing (Kuhad et al. 2011).
Pulping is done under more or less severe conditions due to the presence of wood-
generated phenolics, salts, and metals. So, sturdy and polyextremophilic or
polyextremotolerant (metallophilic, halophilic, thermophilic, and acidophilic)
cellulases may be more suitable to be used.
16.8.7.3 Detergent Industry
Use of enzymes in detergent formulations is now a common practice. Because of
this, the detergent industry was found to be the largest user of enzymes by 2014
according to the market report (Zhang et al. 2013). The major enzymes used in the
detergent are protease, lipase, cellulase, and amylase and the purpose behind their
use is to remove stains of various chemical natures under mild condition to preserve
cloth quality and longevity.
Cold-active, alkaliphilic, and heat labile cellulase is the most desirable one as it
would enable cold washing saving energy especially in cold regions. Because the
heat labile enzyme loses activity at a higher temperature, cloth after drying would
hardly be expected to retain residual activity thus giving health benefits.
16.8.7.4 Animal Feed
Cellulase-treated cereal-based animal feed has been found to benefit an animal in
proper digestion of food and subsequent growth and productivity (e.g., milk yield) of
16 Extremophilic Fungal Cellulases: Screening, Purification, Catalysis, and... 359

animals (Baker and Wicker 1996; Boutte et al. 2009; Vasco-Correa et al. 2016).
Cellulases have also been implicated in promoting cecal fermentation processes via
enhancing propionic acid production, which inhibits colonization by pathogenic
bacteria (Pascual 2001; Fortun-Lamothe et al. 2001; Pazarlioglu et al. 2005).
Cellulosic materials’hydrolysis encounters phenolics, metals, and salt; thus,
sturdy and polyextremophilic or polyextremotolerant cellulases may be more suit-
able to be used. Apart from them, acid-tolerant cellulases may remain active in the
intestine and give positive impact there.
16.9 Agriculture
Generation of protoplast and somatic hybridization are experiments which are
extensively used globally in improving plant. This is enzyme-dependent process
applying cocktail of cellulases, hemicellulases, and pectinases.
16.9.1 Medical Applications
People with metabolic disorder or weak digestion are prescribed enzyme blends
including cellulase. Cellulases enable digestion of cellulose-rich fibers available in
most of the food articles of plant origin. Apart from this direct use, cellulases have
also been reported to be useful in combination with chitinase to partially digest chitin
to get chitosan. Chitosan and its derivatives are used in various medical purposes
such as production of artificial skin, bone rebuilding, hemostatic dressing, anticoag-
ulant, and production of biopharmaceutics (Garcia-Ubasart et al. 2013).
16.9.2 Bioremediation
Waste water from paper, detergent, and textile industries containing cellulosic
residues and high concentration of salt requires halophilic and alkaliphilic cellulases
for bioremediation.
16.9.3 Biofuel Application
To mitigate current pace of climate change and ensure energy security, there is a
global effort to use bioethanol as gasoline blend. Also to avoid food vs fuel conflict,
second generation (2G) bioethanol technology is expected to use agro-forest-munic-
ipal residues instead of crop as feedstock. Currently, the most potential 2G
bioethanol technology envisages three-step process viz., pretreatment
(by autohydrolysis, dilute acid, alkali, or ionic liquid) of biomass to yield relatively
pure cellulose, enzymatic (cellulases) hydrolysis of residual cellulase to generate
glucose, and fermentation of thus obtained glucose.
360 S. Chouhan et al.

Cellulose obtained from pretreatment step thus may be acidic, alkaline, or with
residual ionic liquid. It does have degradation by-products of sugars (e.g., furfural
and hydroxymethyl furfural), lignin (array of phenolic compounds), and organic
acids (acetic acid, etc.). To hydrolyze this cellulose, very sturdy cellulases tolerant to
many of these inhibitory conditions are required. There are several reports where the
nonspecific and irreversible adsorption of cellulase to lignin has been observed
(Bernardez et al. 1993; Yang and Wyman 2004) that reduces the enzyme activity
and recovery. Moreover, currently available commercial cellulases work optimally
near 60C which is not compatible with yeast fermentation temperature (25–30C)
and thus with these cellulases simultaneous saccharification and fermentation (SSF)
which is considered to be cost-effective technology is not possible.
Taking into consideration of all of the facts there is a great scope to explore
suitable extremophilic cellulases that could commercialize 2G bioethanol technol-
ogy and enable various Governments to achieve stipulated bioethanol blending with
fossil fuels. Highly sturdy in nature that works optimally at 25–30C are the most
desirable features of the cellulase to be used in 2G bioethanol technology. Addition-
ally, if the cellulosic material is to be obtained by ionic liquid pretreatment of
biomass, then cellulases must also be tolerant to ionic liquid (Xu et al. 2014).
Ionic liquid disrupts hydrogen bonds and hydrophobic interaction and affects
water hydration cell of the protein thereby inhibiting enzyme activity. Cellulolysis
from lime pretreated biomass requires halophilic cellulases (Woolard and Irvine
1995). Cellulases from obligate halophilic fungus A. flavus have been reported to be
sturdier than available commercial cellulases under alkaline and saline conditions
(Bano et al. 2019). The global demand for such cellulases would be unexpectedly
much higher surpassing all other industrial uses.
References
Abrha B, Gashe BA (1992) Cellulase production and activity in a species of Cladosporium. World J
Microbiol Biotechnol 8:164–166
Acharya S, Chaudhary A (2012) Bioprospecting thermophiles for cellulase production: a review.
Braz J Microbiol 43:844–856
Anish R, Rahman MS, Rao M (2007) Application of cellulases from an alkalothermophilic
Thermomonospora sp. in biopolishing of denims. Biotechnol Bioeng 96:48–56
Bakare MK, Adewale IO, Ajayi A, Shonukan OO (2005) Purification and characterization of
cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens. Afr J
Biotechnol 4:898–904
Baker RA, Wicker L (1996) Current and potential applications of enzyme infusion in the food
industry. Trends Food Sci Technol 7:279–284
Bano A, Chen X, Prasongsuk S et al (2019) Purification and characterization of Cellulase from
obligate halophilic aspergillus flavus (TISTR 3637) and its prospects for bioethanol production.
Appl Biochem Biotechnol 189:1327–1337
Berg B (1978) Cellulose degradation and cellulase formation by Phialophora malorum. Arch
Microbiol 118:61–65
Bernardez TD, Lyford K, Hogsett DA, Lynd LR (1993) Adsorption of clostridium thermocellum
cellulases onto pretreated mixed hardwood, Avicel, and lignin. Biotechnol Bioeng 42:899–907
16 Extremophilic Fungal Cellulases: Screening, Purification, Catalysis, and... 361

Boutte TT, Sargent KL, Feng G (2009) Enzymatic dough conditioner and flavor improver for
bakery products. US Patent. 20090297659
Cabezas L, Calderon C, Medina LM et al (2012) Characterization of cellulases of fungal endophytes
isolated from Espeletia spp. J Microbiol 50:1009–1013
Cortez JM, Ellis J, Bishop DP (2001) Cellulase finishing of woven, cotton fabrics in jet and winch
machines. J Biotechnol 89:239–245
Coutts AD, Smith RE (1976) Factors influencing the production of cellulases by Sporotrichum
thermophile. Appl Environ Microbiol 31:819–825
Carrasco M, Villarreal P, Barahona S et al (2016) Screening and characterization of amylase and
cellulase activities in psychrotolerant yeasts. BMC Microbiol 16:2
Damisa D, Ameh JB, Egbe NEL (2011) Cellulase production by native Aspergillus niger obtained
from soil environments. Ferment Technol Bioeng 1:62–70
Dar R, Saba I, Shahnawaz M, Sangale M, Ade A, Rather S, Qazi P (2013) Isolation, purification and
characterization of carboxymethyl cellulase (CMCase) from endophytic fusarium oxysporum
producing podophyllotoxin. Adv Enzyme Res 1:91–96
de Castro AM, de Carvalho DA, Leite ML, S.G.F. et al (2010) Cellulases from Penicillium
funiculosum: production, properties and application to cellulose hydrolysis. J Ind Microbiol
Biotechnol 37:151–158
Dutta T, Sahoo R, Sengupta R, Ray SS, Bhattacharjee A, Ghosh S (2008) Novel cellulases from an
extremophilic filamentous fungi Penicillium citrinum: production and characterization. J Ind
Microbiol Biotechnol 35:275–282
Folan MA, Coughlan MP (1978) The cellulase complex in the culture filtrate of the thermophyllic
fungus, Talaromyces emersonii. Int J Biochem 9:717–722
Fortun-Lamothe L, Gidenne T, Debray L, Chalaye F (2001) Intake regulation, performances and
health status according to feeding strategy around weaning. In: Proceedings of the second
meeting of COST 848 working group, vol 4, pp 40–41. God¨ oll ¨ o, Hungary
Garcia-Ubasart J, Torres AL, Vila C, Pastor FIJ, Vidal T (2013) Biomodification of cellulose flax
fibers by a new cellulase. Ind Crop Prod 44:71–76
Garsoux G, Lamotte J, Gerday C, Feller G (2004) Kinetic and structural optimization to catalysis at
low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas
haloplanktis. Biochem J 384:247–253
Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268
Gilkes NR, Henrissat B, Kilburn DG, Miller RC, Warren RAJ (1991) Domains in microbial
ß-1,4-glycanases: sequence conservation, function,and enzyme families. Microbiol Rev 55:
303–315
Giorgi K (2017) Cellulases from extremophiles. Curr Trends Biomed Eng Biosci 4:555640
Gomes J, Steiner W (2004) The biocatalytic potential of extremophiles and extremozymes. Food
Technol Biotechnol 42:223–235
Goyal A, Ghosh B, Eveleigh O (1991) Characteristics of fungal cellulases. Bioresour Technol 36:
37–50
Grassick A, Murray PG, Thompson R et al (2004) Three dimensional structure of a thermostable
native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the
filamentous fungus, Talaromyces emersonii. Eur J Biochem 271:4495–4506
Grigorevski-Lima AL, Da Vinha FN, Souza DT, Bispo AS, Bon EP, Coelho RR, Nascimento RP
(2009 May) Aspergillus fumigatus thermophilic and acidophilic endoglucanases. Appl Biochem
Biotechnol 155(1–3):321–329. https://doi.org/10.1007/s12010-008-8482-y
Grum-Grzhimaylo A, Falkoski DL, Heuvel JVD, Min B, Choi IG, Henrissat B, Franssen HGJM,
Bilanenko EN, De Vries RP, van Kan JAL, Grigoriev IV, Debets AJM (2017) Genome of the
obligately alkaliphilic fungus Sodiomyces alkalinus reveals its adaptations to high pH. In:
Abstract book 29th fungal genetics conference, genetic Society of America, p 152
Gunny AAN, Arbain D, Jamal P, Gumba RE (2015) Improvement of halophilic cellulase produc-
tion from locally isolated fungal strain. Saudi J Biol Sci 22:476–483
362 S. Chouhan et al.

Hayashida S, Yoshioka H (1980) Production and purification of thermostable cellulases from
Humicola insolens YH-8. Agric Biol Chem 44:1721–1728
Headon DR, Walsh G (1994) The industrial production of enzymes. Biotechnol Adv 12:635–646
Hmad IB, Boudabbous M, Belghith H, Gargouri A (2017) A novel ionic liquid-stable halophilic
endoglucanase from Stachybotrys microspora. Process Biochem 54:59–66
Hong J, Tamaki H, Yamamoto K, Kumagai H (2003) Cloning of a gene encoding thermostable
cellobiohydrolase from Thermoascus aurantiacus and its expression in yeast. Appl Microbiol
Biotechnol 63:42–50
Ibrahim NA, El-Badry KB, Eid M, Hassan TM (2011) A new approach for bio finishing of
cellulosecontaining fabrics using acid cellulases. Carbohydr Polym 83:116–121
Illingworth CD, Cook SD (1998) Acanthamoeba keratitis. Surv Ophthalmol 42:493–508
Jayasekara S, Ratnayake R (2019) Microbial Cellulases: an overview and applications, cellulose.
Alejandro Rodríguez Pascual and María E, Eugenio Martín, IntechOpen
Jorgensen H, Eriksson T, Borjesson J, Tjerneld F, Olsson L (2002) Purification and characterisation
of five cellulases and one xylanase from Penicillium brasilianum IBT 20888. Enzyme Microbiol
Technol 32:851–861
Knapp JS (1985) Biodegradation of cellulose and lignins. Comprehen Biotechnol 4:835
Kotchoni SO, Gachomo EW, Omafuvbe BO, Shonukan OO (2006) Purification and biochemical
characterization of carboxymethyl cellulase (CMCase) from a catabolite repression insensitive
mutant of Bacillus pumilus. Int J Agri Biol 8:286–292
Kračun SK, Schückel J, Westereng B et al (2015) A new generation of versatile chromogenic
substrates for high-throughput analysis of biomass-degrading enzymes. Biotechnol Biofuels 8:
70
Kuhad RC, Gupta R, Singh A (2011) Microbial Cellulases and their industrial applications. Enzyme
Res 2011:280696
Lee H, Lee YM, Heo YM, Lee H, Hong J-H, Jang S, Park MS, Lim YW, Kim J-J (2015) Halo-
tolerance of marine-derived fungi and their enzymatic properties. BioRes 10:8450–8460
Leite RSR, Alves-Prado HF, Cabral H, Pagnocca FC, Gomes E, DaSilva R (2008) Production and
characteristics comparison of crude ß-glucosidases produced by microorganisms Thermoascus
aurantiacus & Aureobasidium pullulans in agricultural wastes. Enzyme Microb Technol
43:391–395
Li C-H, Wang H-R, Yan T-R (2012) Cloning, purification, and characterization of a heat- and
alkaline-stable endoglucanase B from aspergillus niger BCRC31494. Molecules 17(8):
9774–9789
Liang L, Xue D (2017) Kinetics of cellulose hydrolysis by halostable cellulase from a marine
aspergillus Niger at different salinities. Process Biochem 63:163–168
Liu J, Xue D, He K, Yao S (2012) Cellulase production in solid-state fermentation by marine
aspergillus ZJUBE-1 and its enzymological properties. Adv Sci Lett 16:381–386
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin
phenol reagent. J Biol Chem 193:265–275
Maharana AK, Ray P (2015) Optimization and characterization of cold-active endoglucanase
produced by aspergillus terreus strain AKM-F3 grown on sugarcane bagasse. Turk J Biol 39:
175–185
Mandels M, Sternberg D, Andreotti RE (1975) Growth and cellulase production by Trichoderma.
In: Bailey MT, Enari TM, Linko M (eds) Symposium on enzymatic hydrolysis of cellulose.
Technical Research Centre, Finland, pp 81–110
Mansfield SD, Wong KKY, Jong ED, Saddler JN (1996) Modification of Douglas-fir mechanical
and Kraft pulps by enzyme treatment. TAPPI J 79:125–132
Maryan AS, Montazer M (2013) A cleaner production of denim garment using one step treatment
with amylase/cellulase/ laccase. J Clean Prod 57:320–326
Mohammadi S, Hashim NHF, Mahadi NM, Murad AMA (2020) The cold-active endo-ß-1,3(4)-
glucanase from a marine psyhrophilic yeast, Glaiozyma Antarctica PI12: heterologous
16 Extremophilic Fungal Cellulases: Screening, Purification, Catalysis, and... 363

expression, biochemical characterization, and molecular modeling. Int J Appl Biol Pharm
Technol 12:279–300
Ncube T. Development of a fungal cellulolytic enzyme combination for use in bioethanol produc-
tion using hyparrhenia spp as a source of fermentable sugars [doctoral dissertation]. University
of Limpopo (Turfloop Campus); 2013
Okereke OE, Akanya HO, Egwim EC (2017) Purification and characterization of an acidophilic
cellulase from Pleurotus ostreatus and its potential for agrowastes valorization. Biocat Agri
Biotechnol 12:253–259
Pascual JJ (2001) Recent advances on early weaning and nutrition around weaning. In: Proceedings
of the 2nd meeting of COST 848 working group, vol 4, pp 31–36. Godöllö, Hungary. 2
Pazarlioglu NK, Sariisik M, Telefoncu A (2005) Treating denim fabrics with immobilized com-
mercial cellulases. Process Biochem 40:767–771
Pere J, Siika-aho M, Buchert J, Viikari L (1995) Effects of purified T. reesei cellulases on the fiber
properties of Kraft pulp. TAPPI J 78:71–78
Pereira MMC, Gonçalves TD, Mateus NE, de Vieira QM, Liparini PO, Gomes CP (2020) Enzyme
production by Induratia spp. isolated from coffee plants in Brazil. Braz Arch Biol Technol 63:
e20180673
Rob D, Delabona PDS, Mergel CM et al (2013) The capability of endophytic fungi for production
of hemicellulases and related enzymes. BMC Biotechnol 13:94
Sahay S (2019) Wine enzymes: potential and practices. In: Kuddus M (ed) Enzymes in food
biotechnology: principle, practices and future prospect. Academic Press, New York, pp 73–92
Sahay S (2020) Impact of pretreatment technologies for biomass to biofuels production. In:
Srivastava M, Srivastava N, Mishra PK, Ramteke PW (eds) Substrate analysis for effective
biofuel production. Springer Nature, Cham, pp 173–216
Seiboth B, Ivanova C, Seidl-Seiboth V (2011) Trichoderma reesei: a fungal enzyme producer for
cellulosic biofuels. In: Aurelio M, Bernardes DS (eds) Biofuel production –recent
developments and prospects. www.intechopen.com
Sharma A, Tewari R, Rana SS, Soni R, Soni SK (2016) Cellulases: classification, methods of
determination and industrial applications. Appl Biochem Biotechnol 179:8
Smith RE (1977) Rapid tube test for detecting fungal cellulase production. Appl Environ Microbiol
33:980–981
Song C, Liu GL, Xu JL et al (2010) Purification and characterization of extracellular
b-galactosidase from the psychrotolerant yeast 17-1 isolated from sea sediment in Antarctica.
Process Biochem 45:954–960
Sreenath HK, Shah AB, Yang VW, Gharia MM, Jeffries TW (1996) Enzymatic polishing of jute/
cotton blended fabrics. J Ferment Bioeng 81:18–20
Statista (2018) Statista: The Statistics Portal. Market Statistics of Paper Industry. Available from:
https://www.statista.com/topics/1701/paper-industry
Sujatha P, Bapi R, Ramana T (2005) Studies on a new marine streptomycete BT-408 producing
polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus aureus.
Microbiol Res 160:119–126
Takashima S, Nakamura A, Hidaka M, Masaki H, Uozumi T (1996) Cloning, sequencing, and
expression of the cellulase genes of Humicola grisea var. thermoidea. J Biotechnol 50:137–147.
Takashima S, Nakamura a, Hidaka M, Masaki H, Uozumi T (1999) molecular cloning and
expression of the novel fungal β-glucosidase genes from Humicola grisea and Trichoderma
reesei. J Biochem 125:728–736
Takashima S, Nakamura A, Hidaka M, Masaki HT, Uozumi T (1999) Molecular cloning and
expression of the novel fungal β-glucosidase genes from Humicola grisea and Trichoderma
reesei. J Biochem 125:728–736
Tansey MR (1971) Agar diffusion assay of cellulolytic ability of thermophilic fungi. Archive fur
Mikrobiologie 77:1–11
Tavares EQDP, Rubini MR, Mello-de-Sousa TM, Duarte GC, de Faria FP, Filho EXF, Kyaw CM,
Silva-Pereira I, Pocas-Fonseca MJ (2013) An acidic thermostable recombinant aspergillus
364 S. Chouhan et al.

nidulas endoglucanase is active towards distinct agriculture residues. Enzyme Research 2013:
287343
Teather RM, Wood PJ (1982) Use of Congo red–polysaccharide interaction in enumeration and.
characterisation of cellulytic bacteria from the bovine rumen. Appl Environ Microbiol 43:777–
780
Thanh VN, Thuy NT, Huong HTT, Hien DD, Hang DTM, Anh DTK, Hüttner S, Larsbrink J,
Olsson L (2019) Surveying of acid-tolerant thermophilic lignocellulolytic fungi in Vietnam
reveals surprisingly high genetic diversity. Sci Rep 9:3674
Ting CL, Makarov DE, Wang ZG (2009) A kinetic model for the enzymatic action of cellulase.
Phys Chem B 113:4970–4977
Tong CC, Cole AL, Shepherd MG (1980) Purification and properties of the cellulases from the
thermophilic fungus Thermoascus aurantiacus. Biochem J 191:83–94
Vasco-Correa J, Ge Y, Li J (2016) Biological pretreatment of lignocellulosic biomass. In: Biomass
fractionation Technologies for a Lignocellulosic Feedstock Based Biorefinery. Elsevier Science,
Amsterdam, pp 561–585
Voutilainen SP, Puranen T, Siika-Aho M et al (2008) Cloning, expression, and characterization of
novel thermostable family 7 cellobiohydrolases. Biotechnol Bioeng 101:515–528
Wang N, Jiane Z, Kaili M et al (2013) Production of cold adapted cellulase by Verticillium
sp. isolated from Antarctic soils. Electron. J Biotechnol 3:1–10
Woolard CR, Irvine RL (1995) Treatment of hypersaline wastewater in the sequencing batch
reactor. Water Res 29:1159–1168
Xia L, Cen P (1999) Cellulase production by solid state fermentation on lignocellulosic waste from
the xylose industry. Process Biochem 34:909–912
Xu J, Bingfang He B, Wu B, Wang B, Wang C-H, Hu L (2014) An ionic liquid tolerant cellulase
derived from chemically polluted microhabitats and its application in in situ saccharification of
rice straw. Bioresour Technol 157:166–173
Yang B, Wyman CE (2004) Effect of xylan and lignin removal by batch and flowthrough
pretreatment on the enzymatic digestibility of corn Stover cellulose. Biotechnol Bioeng 86:
88–95
Zhang YHP, Hong J, Ye X (2009) Cellulase assays. Methods Mol Biol 581:213–231
Zhang ZJ, Chen YZ, Hu HR, Sang YZ (2013) The beatability-aiding effect of aspergillus Niger
crude cellulase on bleached simao pine Kraft pulp and its mechanism of action. Bioresources 8:
5861–5870
16 Extremophilic Fungal Cellulases: Screening, Purification, Catalysis, and... 365

Extremophilic Fungal Xylanases: Screening,
Purification, Assay, and Applications 17
Aneesa Fasim, A. Prakruti, H. K. Manjushree, S. Akshay, K. Poornima,
Veena S. More, and Sunil S. More
Abstract
Lignocellulosic biomass is an abundant natural resource that can be utilized for
the production of commercial products beneficial to mankind. Biomass degrada-
tion and processing is the first step to harness its potential and this is achieved by
hydrolase enzymes. Xylanases are one such enzyme that plays a vital role in the
degradation of xylan, a major component of lignocellulose. During the last
decade, demand for xylanases has markedly increased due to its wide applications
not only in the biofuel industry but also in various other industries such as baking,
beverage, degumming, paper and pulp, and animal feed. Xylanases are produced
by many microorganisms but fungal xylanases are preferred due to their high
enzyme production, ease of culturing on cheap agro-industrial substrates, and
easy purification. The latest advancement in xylanase research is the discovery of
extremophilic fungi (EF) xylanases that have robust characteristics and can retain
activity even under harsh industrial conditions such as high or low pH, salt
concentrations, pressure, and temperatures. These qualities can drastically benefit
the global economics of biofuel production, paper and food industries, and many
other industrial production processes.
Thus, a detailed review of several techniques of isolation, screening, and
characterization of EF xylanases is discussed here. A comprehensive summary
of purification and different assay techniques has also been listed to better
understand and optimize enzyme extraction. Improved understanding of the
biochemical properties of fungal xylanases allows the exploration of xylanases
for various inventive industrial and biotechnological uses.
A. Fasim · A. Prakruti · H. K. Manjushree · S. Akshay · K. Poornima · S. S. More (*)
School of Basic and Applied Sciences, Dayananda Sagar University, Bengaluru, Karnataka, India
e-mail: drsunil@dsu.edu.in
V. S. More
Department of Biotechnology, Sapthagiri College of Engineering, Bangalore, Karnataka, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_17
367

Keywords
Lignocellulosic biomass · Xylan · Biomass degradation · Agroindustrial
substrates · Industrial applications
17.1 Introduction
Biomass, an abundant natural resource, has the power to end the world energy crisis
and create a sustainable economy, provided we have the right tools to harness its
untapped potential and produce value-added, chemical-free, and eco-friendly
products. Trees, woody plants, grasses, crops, agro-wastes, wood wastes, etc. are
all forms of terrestrial lignocellulosic biomass that can be degraded and processed to
harness the potential. An evolving trend is to employ hydrolytic enzymes secreted by
microorganisms to process lignocellulose which is a major component of the plant
cell walls. The cell wall is a multifaceted, molecular system that provides structural
integrity, rigidity, and protection to the cell. It allows the diffusion of nutrients and
intercellular signals within the cell via the plasma membrane. Growing plant cells are
surrounded by a thin layer of primary cell wall, but once the cell reaches its
maximum growth lignin starts depositing and a thick secondary cell wall starts
developing (Buchanan et al. 2000). The secondary plant cell wall is a highly resolute
and active network composed of hemicellulose (30–35%), lignin (15–20%), and
cellulose (40–50%) (Singla et al. 2012) held with covalent and non-covalent
interactions (Sánchez 2009). Hemicellulose is further made up of xylans
(xyloglucans and arabinoxylans) and mannans (glucomannans) with xylan forming
the major component of the lignocellulosic plant cell wall. Xylan makes up approxi-
mately 30% dry weight of the terrestrial plants. It is impermeable to water and
hydrolytic enzymes, thus protecting the plant cell from enzymatic degradation.
Xylan is a heteropolysaccharide made up of β-xylopyranose units forming the
backbone. Additionally, D-galactose, L-arabinose, D-mannose, α-glucuronic acids,
and α-arabinofuranose are also present as side branches to the backbone. Organic
acids such as acetic acid, ferulic acid, and glucuronic acid are found interwoven
together with the help of glycosidic and ester bonds to the xylan main chain (Collins
et al. 2005; Ahmed et al. 2007; Motta et al. 2013; Sharma 2017).
The percent and composition of xylan differ in different plants as well as different
parts of the plant. It is observed that the content of xylan is 10–35% in hardwood and
10–15% in softwood. Also, the content of acidic xylan is higher in hardwood
compared to softwood due to the presence of acetyl groups. In hardwood xylan,
one acidic group is present per 5–6D-xylose units, while in softwood it is one acidic
group per 9–12 D-xylose units (Bastawde 1992). Reports also claim that 95% of the
side chains of xylan consist of 4-O-methyl-α-D-glucuronic acid residues. Owing to
the complexity of the xylan, complete hydrolysis can only be achieved by the
synergistic action of many xylanases that depolymerize xylan to a simple monosac-
charide xylose that can be further utilized as a carbon source (Walia et al. 2017).
368 A. Fasim et al.

17.2 Xylanases
Xylanases are a group of hydrolase enzymes that specifically target xylans. As
xylans are structurally diverse, a set of xylanases act on different parts of the xylan
substrate and break it down. The enzymes that belong to this group consist of
endo-1,4-β-D-xylanase (EC number 3.2.1.8), β-D-xylosidases (EC number
3.2.1.37), α-glucuronidase (EC number 3.2.1.139), acetyl xylan esterase
(EC number 3.1.1.72), and ferulic acid esterase (EC number 3.1.1.73). Endo-
1,4-β-D-xylanase and β-D-xylosidases cleave the xylan backbone to release xylose
monomers, and the removal of side chain acid groups is catalyzed by α-L-
arabinofuranosidases. Likewise, α-Dglucuronidases and acetylxylan esterases act
on the phenolic and acetyl side branches; thus, these enzymes assist in complete
xylan degradation (Walia et al. 2017). Figure 17.1 gives an overview of the xylan
degradation by the xylanase enzymes.
Previously, xylanases were classified based on the crystal structure and kinetics
(Jeffries 1996), biochemical properties (molecular weight, isoelectric point) (Wong
et al. 1988), or the substrate specificity and product profile (Motta et al. 2013). But
with the discovery of novel enzymes, a more preferred comprehensive classification
of glycoside hydrolases (GH) of carbohydrate hydrolyzing enzymes (Cazy database)
was developed, and under this classification, xylanases were grouped under GH 5, 7,
8, 9, 10, 11, 12, 16, 26, 30, 43, 44, 51, and 62 families. The xylanases belonging to
16, 51, and 62 are bifunctional with two catalytic domains, whereas 5, 7, 8, 10,
11, and 43 have differences in their catalytic domain (Collins et al. 2005). GH10 and
Fig. 17.1 Cleaving site of different xylanases. (Adapted from Selvarajan and Veena 2017)
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 369

GH11 are the most extensively studied GH groups with plant, fungal, and bacterial
xylanases categorized under GH10 family and fungal, and few bacterial enzymes
included in the GH11 family (Chakdar et al. 2016). GH11 members are considered
as “true xylanases”as the endo-β-1, 4-xylanases belonging to this group catalyze
preferential cleavage of xylan backbone to release D-xylose (Henrissat and Bairoch
1993). GH11 group enzymes exhibit interesting characteristics such as optimum
activity at a wide range of temperature and pH, low molecular weight (<30 kDa),
high substrate selectivity, and catalytic effectiveness making them suitable for many
industrial applications (Paes et al. 2012). Overall, the structure of the GH11 family
comprises two large β-pleated sheets and a single α-helix that forms a partially
closed right hand (Torronen and Rouvinen 1997). Paes group conducted an exhaus-
tive study on 25 GH11 family xylanases where they combined structure and
sequence data along with biochemical properties to gain insights into the relationship
between the structure and enzyme properties. The superimposition studies revealed a
conserved β-jelly-roll domain with most residues in catalytic cleft also being
conserved. Based on the findings, a cartoon representation of GH11 family xylanase
structure was deduced (Fig. 17.2a). In another study, X-crystallography studies of a
thermostable xylanase from Thermoascus aurantiacus provided an understanding of
its structure and the presence of one disulfide and more than 10 salt bridges
contributed toward its stability at elevated temperatures (Fig. 17.2b) (Natesh et al.
1999), but Paes et al. (2012) concluded that every enzyme has unique properties to
acquire thermostability and the presence of disulfide bonds did not essentially confer
thermostability.
β- 1,4 Endo Xylanase (1TUX)
Xylanase from Thermoascus
aurantiacus
Cartoon representation of a typical
GH-11 member
a) b)
Fig. 17.2 (a) Cartoon representation of a typical GH-11 member. (Adapted from Paes et al. 2012)
and (b)β-1,4 Endo Xylanase (1TUX) Xylanase from Thermoascus aurantiacus. (Adapted from Liu
and Kokare et al. 2017)
370 A. Fasim et al.

17.3 Types of Xylanases
17.3.1 Endo-b-1,4-Xylanase
Endo-β-1,4-xylanase hydrolyzes O- as well as S-glycosyl compounds.
Endo-β-1,4-xylanases cleave the glycosidic linkage of xylan residues releasing
xylooligosaccharides (XOS) and xylose. XOSs are two to five xylose molecules
(xylobiose, xylotriose, and xylotetrose) linked by α1–4 glycosidic bonds. They are
used as prebiotics with many health benefits (Linares-Pastén et al. 2018; Ma et al.
2017). XOS has a huge market in the food industry where they are used to fortify
many food products (Aachary and Prapulla 2011).
17.3.2 b-D-Xylosidases
β-D-Xylosidases are exo-glycosidases. This class of enzymes hydrolyzes short
xylooligomers into single xylose residues. During xylan hydrolysis, short β-D-
xylopyranosyl oligomers get accumulated leading to inhibition of the endoxylanase
activity. β-xylosidases degrade these oligomers thereby enhancing the degradation
of xylan by endoxylanases (Manju and Singh Chadha 2011).
17.3.3 a-Glucuronidases
α-Glucuronidases are a class of hydrolases capable of hydrolyzing 4-O-methyl-D-
glucuronic acid not only in non-reducing ends of xylopyranosyl units but also
cleaves the internal xylosyl residues of xylan molecules (Dimarogona and Topakas
2016). α-Glucuronidases possess two groups within GH family 67 well-defined
either by bacterial or fungal origin. The α-glucuronidases of bacterial or fungal
origins differ in their molecular weight and quaternary structures. However, the
enzyme has a highly conserved active site which makes them different from other
class of xylanases (Yeoman et al. 2010).
17.3.4 Acetylxylan Esterase
Acetylxylan esterase is another group of xylanase that cleaves the O-acetyl group on
second and third positions of the beta-D-xylopyranosyl residues of xylan molecule
(Bajpai 2014). When acetyl side groups are removed from the xylan chain, it
facilitates the endoxylanase for the efficient hydrolysis of xylan. Therefore, acetyl
xylan esterase removes the acetyl groups. Biely (1985)first reported the existence of
acetylxylan esterase in hemicellulolytic and cellulolytic fungi such as Aspergillus
niger,Trichoderma reesei, and Schizophyllum commune.
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 371

17.3.5 Ferulic Acid Esterases
Ferulic acid esterases are classified under EC number 3.1.1.73. It cleaves the ester
linkage between the fifth carbon(O) position of arabinose in hydroxycinnamate and
hemicellulose. This linkage is denoted as FAX. It primarily forms a covalent linkage
between lignin and hemicellulose. FAE is used for the investigation of plant biomass
for biofuels, transesterification reactions utilizing hydroxycinnamic esters, and as
animal feed (Hunt et al. 2017).
Fungal xylanases are secreted by many microorganisms such as bacteria, fungi,
yeast, protozoa, crustaceans, snails, and insects to degrade the cell wall xylan, either
to get entry into the plant cell or to utilize the plant biomass as a carbon source
(Murashima et al. 2003). Bacillus,Micrococcus,Paenibacillus,Microbacterium,
Rhodothermus,Arthrobacter,Staphylococcus, and Pseudoxanthomonas are a few of
the bacterial genera that are reported to yield xylanases (Chakdar et al. 2016).
Similarly, Aspergillus spp., Trichoderma spp. are some of the fungal xylanase
producers (Sakthiselvan et al. 2015). Comparative studies prove that fungi secrete
much higher levels of this enzyme than bacteria and yeasts (Topakas et al. 2013).
Higher enzyme production along with ease of culturing fungi on agro-industrial
waste has helped industries bring down the production and processing costs drasti-
cally. Also, the utilization of agro-waste indirectly helps in building an eco-friendly
and sustainable bioprocess technology.
Although the fungal xylanase production is high, they are found to be optimally
active at temperatures around 42–65 C and neutral or slightly acidic pH implying
that they cannot tolerate harsh conditions, which can directly impact the industrial
production process. These problems lead scientists to focus on a novel group of
organisms called extremophilic fungi (EF). These fungi can survive extreme
conditions such as high or low pH, salt concentrations, pressure, and temperature
as they have evolved survival strategies. Xylanases from such fungi have robust
characteristics that can sustain harsh conditions, and the most extensively studied EF
xylanases are thermophiles, halophiles, alkalophiles, and acidophiles. Trichoderma
sp., Aspergillus sp., Penicillium sp., and Acidobacterium spp. are the major EF
species that contribute to the production of EF xylanases. Table 17.1 summarizes
the biochemical properties, sources, and purification techniques of different EF
fungal xylanases isolated to date.
17.4 Isolation and Screening of Extremophilic Xylanase
Producing Fungi
Bioprospecting is the first step toward finding efficient biomolecules for the produc-
tion and processing of industrial bioproducts. In recent years, screening and isolation
of EF fungal xylanases have received much attention. Many different sources are
explored to screen and isolate these EF fungi.
372 A. Fasim et al.

Table 17.1 Extremophilic fungi: screening, purification methods, and application
Fungi
Optimum
temperature/
pH Sources
Purification
techniques References
Acidophilic fungi
Aureobasidium
pullulans var.
melangium
50 C,
pH 5.0
Soil sample from
the crude sugar
industry
Ultrafiltration,
DEAE-Cellulofine
A-500
Sephacryl S-200 HR
Ohta et al.
(2001)
Penicillium
occitanis PO16
45 C, pH 3 Purchased from
Cayla corporation,
France
Ammonium sulfate
Precipitation
Biogel P-100 gel
filtration
chromatography
Mono Q anion
exchange
chromatography
Driss et al.
(2011)
Aureobasidium
pullulans
50 C, pH 4,
0–20%
NaCl
Agriculture
research service,
USA
Ammonium sulfate
precipitation
Cation exchange
chromatography—
SP Sepharose fast
column
Yegin
(2017b)
Alkaliphilic fungi
Penicillium
oxalicum
45 C, pH 8 Pichavaram
mangroves region
soil, Tamil Nadu
–Muthezhilan
et al. (2007)
Penicillium
citrinum
50 C,
pH 8.5
Soil samples from
Dhapa situated in
East Kolkata
Ammonium sulfate
Precipitation
Phenyl Sepharose
Dutta et al.
(2007)
Aspergillus
fumigatus
55C, pH 8 Soil samples from
decayed paper and
wood material
Ammonium sulfate
precipitation
Biogel P-100 gel
filtration
chromatography
Deshmukh
et al. (2016)
Humicola
insolens Y1
70–80 C,
pH 9
Commercially
obtained
Ultrafiltration
Dialysis
FPLC
Gel permeation
chromatography
Du et al.
(2013)
Thermophilic fungi
Rhizomucor
pusillus
Aspergillus
fumigatus
75 C, pH 6 Maize silage,
Mexico
–Robledo
et al. (2016)
Fusarium sp.
Aureobasidium
sp.
50 C, pH 7 Leaf debris, across
the Luzon Island,
Philippines
–Torres and
Dela Cruz
(2013)
(continued)
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 373

17.4.1 Sources
Xylanases are one of the important enzymes secreted by saprophytic fungi to utilize
biomass as the source of their food. Extreme environments with high availability of
biomass form a rich source to explore for the xylanase producing EF fungi. Screen-
ing samples from such environments enable isolation of robust xylanase producers.
Table 17.1 also summarizes xylanases secreting EF fungi isolated from different
environments
Mangrove forests are a preferred and most widely explored source for isolation of
the extremophilic fungi. Mangroves are a complex and interesting ecosystem,
usually present around the intertidal areas where the terrestrial and marine aquatic
ecosystems merge. These ecosystems have fluctuations in their salinity, temperature,
and pH creating an ideal extreme environment (Kathiresan and Bingham 2001).
Carbon recycling in the mangrove region is due to the decomposition of leaves and
roots, and this feat is accomplished by the hydrolytic enzymes secreted from the
microfauna, especially fungi that are efficient biomass degraders (Duarte and
Table 17.1 (continued)
Fungi
Optimum
temperature/
pH Sources
Purification
techniques References
Chaetomium
sp. CQ31
60–70 C,
pH 7
Composting soil
from Weihai,
China
SDS electrophoresis Jiang et al.
(2010)
Sporotrichum
thermophile
60–75 C,
pH 7
Soil composts –Sadaf and
Khare (2014)
Chaetomium
thermophilum,
Humicola
insolens,
Melanocarpus
sp.,
Malbranchea
sp.and
Thermoascus
aurantiacus
55–65C,
pH 7
Composting soil
samples
–Ghatora et al.
(2006)
Halophilic fungi
Phoma sp. 45 C, pH 5,
0.5 M–4M
NaCl
Mangrove
sediments from
National Nature
reserve, China
Ultrafiltration
Affinity
1 chromatography—
Nickel NTA column
Wu et al.
(2018)
Aspergillus
gracilis
Aspergillus
penicillioides
–Soil samples
around a
man-made solar
saltern, Thailand
–Ali et al.
(2014)
Aspergillus
nidulan sp.
55 C,
pH 7.8
Industrial effluents
of paper industry,
Punjab
Ammonium sulfate
precipitation
Taneja et al.
(2002)
374 A. Fasim et al.

Cebrian 1996, Kathiresan et al. 2011). Torres group collected leaf debris from
44 different locations across the Luzon Island in the Philippines and screened for
xylan degrading fungi. They were able to isolate and identify many fungal species
such as Aspergillus sp.,Fusarium sp.,Aureobasidium sp.,Paecilomyces sp.,Peni-
cillium sp.,Colletotrichum sp., and Phomopsis sp. (Torres et al. 2016). In another
study, Shankou mangrove, National Nature reserve in China, was explored and
novel halotolerant xylanase secreting fungi was isolated from the sediments of
mangrove and identified as Phoma herbarum (Wu et al. 2018). Similarly, 69 fungal
strains were isolated from Pichavaram mangroves, Tamil Nadu, India, and identified
as Aspergillus followed by Rhizopus,Alternaria,Mucor, and Penicillium. However,
Penicillium oxalicum was the highest xylanase producer among all the strains
isolated (Muthezhilan et al. 2007)
Another excellent source of xylanase producing fungi is the soil and effluent
samples around the industrial region. Aspergillus nidulans, an alkalophilic xylanase
producing fungus, was isolated from the Shreyas Paper Industry, Ahmedgarh,
Punjab effluents (Taneja et al. 2002). Penicillium citrinum was another
extremophilic strain isolated from soil samples from Dhapa, situated in east Kolkata
(Dutta et al. 2007)
Aureobasidium pullulans var. melanigenum (ATCC 20524), an endo-beta-1,4-
xylanase producing fungus, was screened from soil samples collected from the crude
sugar industry (Ohta et al. 2001), whereas Aspergillus fumigatus was isolated from
the wood material and decayed paper soil samples collected near Puducherry, India
(Deshmukh et al. 2016). Screening soil samples around a man-made solar saltern
situated in Phetchaburi province of Thailand resulted in the isolation of two obligate
halophilic fungal strains Aspergillus gracilis and Aspergillus penicillioides (Ali et al.
2014). Likewise, Chaetonium sp. was one more fungal strain isolated from
composting soil from Weihai city of Shandong province, China (Jiang et al. 2010).
Silage is another important source of xylanase producers. Maize silage is a type of
fermented fodder made from corn cobs and maize plants with excess sugar content.
It was collected from a local farm in the province of Chihuahua, Mexico and
21 fungal strains were isolated. The best xylanase producers Rhizomucor pusillus
and Aspergillus fumigatus were chosen, identified, and further characterized
(Robledo et al. 2016).
17.4.2 Screening Techniques
Screening plays a vital role in the isolation of a producer strain. Although samples
are collected from rich biodiversities, without screening it is impossible to isolate
specific biomolecule producing microorganisms. Generally, screening involves the
incorporation of specific nutritional requirements such as carbon or nitrogen source
essential for growth and propagation of the specific producer strain. For example, if
the growth media is supplemented with xylan, only those fungal strains that can
secrete the xylanase enzyme will be capable to utilize xylan as a carbon source and
survive.
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 375

Oat spelt xylan is one of the extensively used substrates in the primary screening
of xylanase producers. While the strains grow and degrade xylan on solid agar
plates, it is very difficult to identify the colonies. So dyes like iodine or Congo red are
added to the agar plates to visualize the xylanase producers. The principle behind the
Congo red or iodine assays is that these stains specifically stain only
polysaccharides. Xylanase produced by the microorganism hydrolyzes xylan in its
vicinity to produce xylose and xylooligosaccharides that do not take up the stain,
thus giving a clear zone of hydrolysis around the enzyme-producing colonies
(Sakthiselvan et al. 2015). Hence, most reports have followed staining as the primary
screening protocol. Imran A group performed screening for xylanase enzyme by
inoculating A. gracilis and A. penicillioides on potato dextrose agar (PDA)
containing agar plates with 1% xylan substrate. Small wells were drilled in the
agar plate using cock borer, and 50 μL of cell-free supernatant extracted from
culturing the fungi was added to the wells and incubated for 24 h at 37 C. After
incubation, the plates were flooded with iodine to observe the zone of clearance that
indicated xylanase activity (Ali et al. 2013). Screening of Penicillium oxalicum was
performed by flooding Congo red stain on the oat spelt agar plates aiding in the
formation of orange colored halos around the enzyme-producing colonies
(Muthezhilan et al. 2007). Another fungal strain, Aspergillus fumigatus, was also
screened for xylanase production by using 0.1% Congo red stain on xylan agar plates
for 10–15 min followed by 10-min treatment with 1 M sodium chloride and 5%
acetic acid. The best enzyme producer was isolated based on the biggest diameter of
the hydrolysis zone (Deshmukh et al. 2016) (Fig. 17.3).
An alternative technique is to infuse the dye in the agar media plate itself so that
clear zones appear as the colonies grow and the enzyme is produced. Rhizomucor
and Aspergillus fungal isolates were identified using this technique. The fungal
species were inoculated on agar plates containing 0.5% birchwood xylan, 0.1%
yeast extract, 0.5% Congo red, and 1.5% agar and incubated for 5 days at 55 C. The
formation of clear zones indicated the xylanase production (Yoon et al. 2007)
(Fig. 17.3).
Fig. 17.3 The representative agar plates show different screening techniques: (a) Congo red
staining, (b) iodine staining, and (c) Congo red stain infused agar plates showing zone of clearance
with the growth of colonies
376 A. Fasim et al.

17.4.3 Growth Media
Depending on the fungal growth requirements, various types of growth media are
utilized for culturing. Potato dextrose agar (PDA) is a well-known media often used
to culture fungi. Robledo group used serially diluted silage samples on PDA plates
and isolated thermophilic fungi after incubation for 5 days at 55 C (Robledo et al.
2016). In another study, halophilic fungi were primarily screened using wheat bran
agar media made with 50% seawater, 1 g wheat bran, 1 g NaNO
3
, 1.5 g agar, 0.5 g
MgSO
4
.7H
2
O, and streptomycin antibiotic to maintain the salinity of the sample.
The culture was grown at 28–30 C for 3–4 days and once the isolates were
identified, they were preserved on PDA slants containing seawater (potato infusion –
200 g, agar 15 g, dextrose 20 g, and 50% seawater) (Muthezhilan et al. 2007).
Penicillium occitanis PO16 mutant, a xylanase producing fungi purchased from
Cayla Co., France, was cultured in a liquefied medium with oat spelt xylan as carbon
source. For the production of the enzyme, Mandel’s medium (g/L) (KH
2
PO
4
–2;
NaNO
3
–5; MgSO
4
.7H
2
O–0.3; CaCl
2
–0.3; yeast extract –1; trace elements
solutions –1 mL/L, and tween 80 –1 mL/L, pH 5.5) supplemented with 2% glucose
was used (Driss et al. 2011). One more frequently used media component is
birchwood xylan. Aspergillus fumigatus was grown on the minimal media
supplemented with birchwood xylan (KH
2
PO
4
–0.5; MgSO
4
–0.25; NH
4
Cl –1;
yeast extract –0.1, birchwood xylan –0.1).The media was inoculated with soil
samples and incubated for 5–7 days at 45 C in a shaker. Production of the enzyme
was carried out using rice bran, an agro-industrial waste used as a solid substrate
fermentation media (Deshmukh et al. 2016). Phoma herbarum, a halo tolerant
fungus, was also isolated and cultured using birchwood xylan medium for
5–7 days at 25 C (Wu et al. 2018). Yeast mold (YM) is another medium of choice.
It was used to grow an extremely acidophilic and halophilic yeast-like fungus
Aureobasidium pullulans. The YM media is made of (g/L) yeast extract –3.0 g,
peptone –5.0 g, malt extract –3.0 g, glucose –10 g, and agar –20 g, and cultures
were grown at 24C for 3 days. Growth of fungi and production of xylanase enzyme
were carried out at 28 C at 150 rpm in the media composed of (g/L) xylose –10 g,
asparagine –2g,KH
2
PO
4
–5, yeast nitrogen base –6.7 g, KH
2
PO
4
–5 g, and pH 5
(Yegin 2017b).
17.5 Purification Techniques
The next step in enzyme extraction is purification. The enzymes are either
completely/partially purified or used directly as crude cell-free supernatant for
various applications. Most purification techniques to extract extracellularly secreted
enzymes follow a similar routine. After the growth of the fungi on submerged or
solid substrates, cultures are centrifuged to separate supernatant from the cell mass
and macromolecular debris followed by filtration using filter membranes or ultrafil-
tration techniques. Once the cell-free supernatant is relatively pure, it is subjected to
lyophilization. The lyophilized samples are then dissolved in the right buffer and
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 377

subjected to complete purification using ion exchange followed by gel permeation
chromatography techniques. The fractions are collected based on the absorbance at
280 nm or 220 nm, and appropriate assays are conducted to identify the xylanases.
Some examples of purification and characterization of extremophilic xylanases
are described below.
17.5.1 Acidophilic Xylanases
Xylanase from Aureobasidium pullulans var. melanigenum (ATCC 20524) showed
optimum activity at pH 2.0 and 50C making it a highly acidophilic enzyme. It has a
molecular weight of 24 kDa and a pI of 6.7. Anion exchange chromatography using
DEAE-cellulofine column combined with gel permeation chromatography using
Sephacryl S-100 was used to purify the enzyme (Ohta et al. 2001). Five days of
incubated culture was centrifuged at 2000 gfor 30 min and then subjected to
ultrafiltration using 3 kDa cutoff membrane in a stirred cell to concentrate the
supernatant. The concentrate was loaded onto DEAE-cellulofine A-500 column
pre-equilibrated with 20 mM Tris–HCl buffer, pH 8.5 buffer. To elute the protein,
0–0.5 M linear gradient of NaCl was used. Fractions displaying enzyme activity
were pooled and subjected to Sephacryl S-200 gel permeation chromatography to
separate proteins based on mass; 512 U/mg of specific activity was observed in a 4.2-
fold purified enzyme with 62.2% yield recovery after the final purification step.
In a similar study, another acidophilic xylanase from Penicillium occitanis POl6
having optimal xylanase activity at pH 3 and 45 C was purified using anion
exchange and size exclusion chromatography. They used a five-step purification
process where the sample was subjected to Biogel P-100 size exclusion column
twice. First, the cultures were filtered using a 0.45 μm nitrocellulose membrane
followed by dialysis and lyophilized. The sample was dissolved in 20 mM citrate-
phosphate buffer pH 3 and loaded on to Biogel P-100 column pre-equilibrated with
20 mM Tris–NaCl, pH 8 buffer. Fractions that exhibited xylanase activity were
pooled and loaded on to Mono-Q Sepharose anion exchanger. Again the fractions
exhibiting xylanase activity were pooled and loaded onto Biogel P100 for final
separation. All the purification steps lead to a recovery of 0.135% enzyme with 5.2-
fold-purity and 358 U/mg specific activity (Driss et al. 2011).
Deshmukh RA group followed a slightly different protocol where partial purifi-
cation by ammonium sulfate precipitation was employed followed by gel permeation
chromatography using Biogel-P60 on acidic xylanase from Aspergillus fumigatus.
They achieved a very high specific activity of 38196.22 U/mg from a 3.4-fold
purified enzyme with 54.81% recovery after the final purification step. The cell-
free supernatant obtained after culturing the fungus was centrifuged and subjected to
30–55% fractional ammonium sulfate precipitation followed by centrifugation at
10,000 gfor 10 min at 4 C. After extensive dialysis to remove salt, the sample was
lyophilized followed by separation on Biogel-P60 column equilibrated with 50 mM
phosphate buffer, pH 7. Absorbance at 280 nm was used as a reference to collect and
measure the activity of the fractions. Although the optimum pH and temperature of
378 A. Fasim et al.

the purified xylanase were 5 and 50 C, respectively, the enzyme was also found to
be stable at an alkaline pH of 8 and 9 (Deshmukh et al. 2016).
17.5.2 Alkaline Xylanases
Extracellular alkaline xylanase was purified from the fungi Penicillium citrinum with
a pH and temperature optima of 8.5 and 50 C, respectively. The fungus was
cultured on wheat bran solid substrate for 5 days before extracting the enzyme.
Wheat bran beds were subjected to agitation in 50 mM acetate buffer for 1 h at
150 revs/min, and the supernatant was collected by centrifugation at 3000 gfor
30 min. Partial purification of the enzyme was achieved using 80% ammonium
sulfate (NH
4
)
2
SO
4
precipitation. The precipitate was suspended in 50 mM sodium
acetate buffer, pH 5.5, and dialyzed to remove the salt. The sample was loaded onto a
Phenyl Sepharose hydrophobic column pre-equilibrated with 50 mM acetate buffer,
pH 5.5. Active fractions were pooled and concentrated using Centricon-10 ultrafil-
tration cups. A 14-fold purification with a yield of 28% and a specific activity of
361 U/mg was achieved using this purification protocol (Dutta et al. 2007).
Next, alkalophilic xylanase was partially purified from Aspergillus nidulans
(Taneja et al. 2002). The cell-free supernatant was obtained by centrifugation of
the culture broth grown on wheat bran substrate. To partially purify the enzyme,
40% saturated ammonium sulfate precipitation was used. The precipitate was
dialyzed and used for further studies. The optimum pH and temperature of the
partially purified enzyme were found to be 8 and 55 C, respectively.
17.5.3 Halotolerant Xylanase
One of the simplest purification techniques to achieve complete purification is
affinity chromatography using histidine-tagged recombinant proteins. Halotolerant
recombinant xylanase from Phoma sp. MF13 was purified using this technique. The
gene xynMF13A was cloned into pPICZα-C vector and heterogeneously expressed
in Pichia pastoris. Xylanase activity was detected in the supernatant after induction
with methanol for 120 h. The culture supernatants were concentrated using a
PES5000 ultrafiltration membrane and then loaded onto Ni
2+
-NTA agarose gel
column. A gradient of 20–200 mM imidazole in a Tris–NaCl buffer pH 7.6 was
used to elute the protein. The optimum pH and temperature of the recombinant
xylanase were found to be 5 and 45 C, respectively. The enzyme was highly active
in 0.5 M NaCl, and it retained approx. 54% of the activity even in the presence of
4 M NaCl (Wu et al. 2018).
An extremely halophilic and acidophilic xylanase was purified from
Aureobasidium pullulans using a single step chromatographic assay. The cell-free
supernatant obtained after centrifugation of the 126 h culture was concentrated by
lyophilization and desalted using PD-10 columns as the conductivity of the crude
sample was very high. The sample was then resuspended in 20 mM citrate buffer,
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 379

pH 5, and loaded onto the SP Sepharose cation exchange column. Elution was
performed using a gradient of 1 M NaCl in the same buffer, and the enzyme eluted
at 28% of the NaCl gradient with an 80% activity yield. The fractions with xylanase
activity were pooled and further characterized. The molecular weight of the enzyme
was found to be 21.6 kDa with pH and temperature optima at 4 and 50 C,
respectively. Even 20% NaCl did not hinder the activity of the enzyme making it
extremely halophilic xylanase (Yegin 2017b).
17.5.4 Thermophilic Xylanase
Thermophilic xylanases are stable at very high temperatures. To purify these
enzymes, similar chromatographic and gel permeation procedures are utilized. In a
study conducted by Y Du group, direct purification of one xylanase and heterolo-
gous expression of three more xylanases were reported from Humicola insolens Y1
strain. For direct purification, 6-day-old H. insolens Y1 wheat bran cultures were
harvested by centrifugation at 12,000 g4C for 10 min. The supernatant was
concentrated using a 5 kDa vivaflow 200 ultrafiltration membrane, dialyzed and
loaded on to HiTrap Q-Sepharose XL FPLC column pre-equilibrated with 20 mM
Tris–HCl buffer at pH 7. A gradient of 0–1 M NaCl was used to elute the proteins at
a 4 ml/min flow rate. Fractions showing xylanase activity were pooled, concentrated,
and subjected to size exclusion by loading onto Superdex 75 column. Elution was
carried out using 50 mM McIlvaine buffer with pH 6.0 at 0.5 mL/min flow rate. For
heterologous expression, XynA, XynB, and XynC xylanase encoding genes were
amplified from H. insolens Y1 cDNA, cloned into the pPIC9 vector, and transformed
into Pichia pastoris GS115 cells for extracellular xylanase expression. The expres-
sion was induced by methanol for 72 h with 90% xylanase found in the supernatant.
Purification was carried out using the same protocol and HiTrap Q Sepharose XL
FPLC column of anion exchange column. The xylanases showed optimal activity
between 70 and 80 C (Du et al. 2013). The specific activities of directly purified
xylanase were 236.2 U/mg, and recombinant xylanases of XynA, XynB, and XynC
were 527.3 U/mg, 278.1 U/mg, and 244.9 U/mg, respectively.
17.6 Assay Methods
Many different assay methods are employed to determine the xylanase enzyme
activity. These assays either quantify the released reducing sugars due to the action
of the enzyme on the substrate or estimate the viscosity/turbidity of the enzyme-
substrate reaction mixture before and after the assay.
The dinitrosalicylic acid (DNS) and Nelson–Somogyi (SN) methods are routine
and simple assay methods used to determine the xylanase activity, whereas other
methods listed here are not so common but are rapid, efficient, and accurate when
compared to conventional methods.
380 A. Fasim et al.

17.6.1 DinitroSalicylic Acid (DNS) Method (DNS)
The DNS method is a simple colorimetric estimation of reducing sugars released due
to the action of the enzyme on the substrate. The free carbonyl group present on the
released sugars reduces the dinitrosalicylic acid reagent to 3-amino 5-nitrosalicylic
acid, a reddish-brown colored complex formed under alkaline conditions that can be
measured colorimetrically or spectrophotometrically at 540 nm absorbance. An
appropriate concentration of enzyme is incubated with the substrate under optimum
conditions of pH and temperature for a given amount of time. The reaction is halted
by the addition of DNS followed by boiling the samples for 10–15 min. Once the
mixture is cooled, absorbance is measured using an enzyme or substrate blank as
controls (Gusakov et al. 2011). The absorbance is then converted to enzyme activity
using a standard product curve where the color produced is directly proportional to
the concentration of released sugars. This method is frequently used to estimate the
xylanase activity using xylan, oat spelts, larchwood, or birchwood as substrates.
However, this assay has certain disadvantages such as lack of sensitivity and
linearity, and it cannot be used to estimate samples containing high sugar content,
e.g., bread enhancing mixtures and chicken feeds (Bailey 1988).
17.6.2 Nelson–Somogyi (NS) Method
The NS method also relies on the principle of redox reaction by the released reducing
sugar to give a blue-colored molybdenum compound that can be quantified by
measuring the absorbance at 610 nm. After the enzyme-substrate reaction,
Somogyi’s alkaline copper tartrate reagent is added to stop the reaction followed
by boiling. The carbonyl group on the sugar reduces the reagent to cuprous oxide
which in turn reacts with Nelson’s arsenomolybdate reagent giving a blue-colored
molybdenum compound. The enzyme activity is calculated with the help of controls
where the blue color is directly proportional to the sugar released due to the enzyme
action (Gusakov et al. 2011). NS method is considered to be sensitive and more
accurate than the DNS method. However, the drawback of this method is the use of
arsenomolybdate, a highly toxic reagent (Nelson 1944). Phosphomolybdate reagents
are other alternative chromophores, but they lack color stability and are also toxic to
the environment (Hatanaka and Kobara 1980). According to McCleary and
McGeough (2015), NS is the best method suitable to determine the release of
reducing sugars by the endoxylanases as color intensity directly correlates with the
glycosidic bond cleavage liberating the reducing sugar. Another improvement to the
NS method is Somogyi’s iodometric method (Somogyi 1952). Somogyi’s alkaline
copper tartrate is added to the reaction mixture followed by the addition of 5 N
sulfuric acid. This solution is then titrated using 0.0025 N Na
2
S
2
O
3
and measured at
510 nm (Ghose and Bisaria 1987). The accuracy is very high in the iodometric
titration such that the error of analysis chiefly depends on the composition of the
copper reagent and condition affecting its sensitiveness and reproducibility during
the oxidation of sugar (Shaffer and Somogyi 1933).
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 381

17.6.3 Bicinchoninic Acid (BCA) Method
This assay is based on the methods of Fox and Robyt (1991) and Meeuwsen et al.
(2000) with a few modifications such as the assay volume, incubation time, and
wavelength of the sample mixture. BCA reagent is added to the enzyme-substrate
reaction mixture followed by incubation at 80 C for 1 h. The sample is then cooled
to room temperature before measuring the absorbance at 562 nm. The concentration
is determined by plotting the absorbance against the known substrates standard, e.g.,
birchwood xylan (sigma-Aldrich), beechwood xylan (Sigma-Aldrich), wheat
arabinoxylan (Megazyme), and xyloglucan (Megazyme) (Sydenham et al. 2014).
BCA method is considered to be much more accurate and sensitive compared to the
DNS method. The sensitivity of BCA assay is about 1–20 nmol per assay compared
to DNS which is 500 nmol per assay (Anthon and Barrett 2002; Moretti and Thorson
2008). Another major advantage of this assay is that it can quantify reducing sugars
with different chain lengths, unlike DNS or SN method where only mono or
disaccharides can be quantified. Many different debranching xylanase enzymes are
present and these enzymes can cleave the substrate and release a mixture of long-
chain oligosaccharides that can be quantified easily (Utsumi et al. 2009). Besides
these advantages, BCA assay method produces stable colored products upon reac-
tion with chromogenic reagents making it a reliable and accurate assay method.
17.6.4 Viscosity Assay (VA)
As the enzyme degrades the polymer, viscosity also decreases. The enzyme-
substrate reaction is carried out in a C-type viscometer that is pre-equilibrated with
buffer. The substrate solution is pipetted in it followed by the addition of the enzyme
that is mixed well and incubated at appropriate conditions. After the incubation
period, the rate of decrease in viscosity is measured by taking five falling time
readings (in s) for approximately 30 min. Each reading is taken as the elapsed time
from combining the enzyme/substrate solutions to the mean of the falling time. The
viscosity of the reaction is directly proportional to the falling number. Murphy group
has successfully used this method to estimate the xylan activity purified from Pichia
pastoris and Schizosaccharomyces pombe using arabinoxylan as the substrate
(Murphy et al. 2009). Viscosity assay is accurate and reliable, but very tedious.
However, from the industrial use perspective, it can be used as a reference method
for other assay methods (Buckee and Baker 1988).
17.6.5 Xylazyme AX Test
This is another simple test used to quantify the enzyme activity using commercial
substrate xylazyme AX tablets. The tablet contains azurine, an active constituent that
is cross-linked to wheat arabinoxylan. Upon hydrolysis by xylanase, a water-soluble
dyed fragment is generated which can be measured at 590 nm absorbance. The rate
382 A. Fasim et al.

of release of these fragments (increased absorbance) is directly related to the enzyme
activity that degrades the xylazyme AX tablets. The xylanase enzymes extracted by
A. niger and T. longibrachiatum were accurately quantified using this method
(Megazyme). It was observed that this test was sensitive and precise compared to
viscometric assay methods (European Symposium on Enzymes in Grain Processing
2000).
17.7 HPAEC-PAD Technique
This is one of the recent methods employed to estimate enzyme activity using HPLC
coupled to anion exchange chromatography (Cürten et al. 2017). The enzyme
activity was carried out in a temperature controlled Dionex™AS-AP autosampler
using HPLC vials. After the reaction, a gradient elution facilitated the separation of
soluble short-chain xylooligomers such as xylose, xylotriose, and long-chain
xylooligomers that were liberated throughout enzymatic hydrolysis. The enzyme
activity of the xylanase extracted from Bacillus subtilis,Bacillus
stearothermophilus, and Aspergillus niger was estimated using this technique
(Rothenhöfer et al. 2015). This method was accurate and the separation of even
very short chain oligomers could be achieved. It also helped in the detection and
distribution of separated molecules based on their chain length (Ballance et al.
2005).
17.8 Applications
During the last decade, demand for xylanases has markedly increased due to its wide
applications not only in the biofuel industry but also in various other industries such
as baking, beverage, degumming, paper and pulp, animal feed, and waste manage-
ment (Beg et al. 2001; Kuhad and Singh 1993; Virupakshi et al. 2005).
Xylooligosaccharides extracted from xylanase degradation are further used as food
additives (Pellerin et al. 1991). EF fungal xylanases are the latest tools in the
repertoire of hydrolytic enzymes used to unleash this untapped potential (Fig. 17.4).
17.8.1 Biofuel Industry
Degradation and saccharification of lignocellulosic biomass is the first step in the
generation of biofuels. Simple sugars released after the degradation process are
allowed to ferment in the presence of suitable microorganisms to produce biofuel,
bioethanol, and other useful products. Hence, biomass is subjected to pre-treatment
with hydrolytic enzymes that either modify lignocellulose structure or help in the
removal of hemicellulose to get access to the polysaccharide substrate (Jorgensen
et al. 2007). At this stage, enzymes like xylanases help in the release of
carbohydrates from the cell wall polysaccharides, and these sugars are then subjected
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 383

to fermentation for the production of bioethanol. Halophilic fungi A. gracilis and
A. penicillioides are both successfully employed in the biofuel production process
(Ali et al. 2014). Aureobasidium pullulans 477 NRRL Y-2311-1 is another ethanol
tolerant fungus suitable for the bioethanol production process. This fungus was able
to grow in the presence of ethanol which made it possible to co-culture this fungus in
the fermentation step itself (Yegin 2017a).
17.8.2 Paper and Pulp Industry
Paper is made from the wood pulp, and the quality of the paper is determined by low
lignin content and whiteness. First, the wood is subjected to pulping where it is
treated with organic solvents at very high temperatures to break down the lignocel-
lulosic biomass. This process is followed by bleaching, which is the removal of
residual lignin by the use of corrosive chemicals like chlorine dioxide/hypochlorite
(Singh et al. 2019). However, these procedures apart from being hazardous have
other disadvantages like the generation of toxic by-products and less yield. Effluents
released from these industries enter water bodies and adversely affect the environ-
ment and marine life (Singh et al. 2019). To combat these problems, biobleaching
and bio-pulping are introduced. Bio-pulping involves pre-treatment of wood with
hydrolytic enzymes produced from fungi. Debarked and chipped wood is first
decontaminated by steaming and then subjected to lignin degradation by fungi
(Scott et al. 1998). After this initial treatment, the chips are pulverized by either
mechanical or chemical means (López et al. 2017). In the biobleaching process,
Fig. 17.4 Applications of xylanase. (Adapted from Bhardwaj et al. 2019)
384 A. Fasim et al.

xylanase enzyme is used to remove residual lignin that imparts brightness to the
paper (Viikari et al. 1994). Xylanases used in biobleaching process should be able to
tolerate high temperatures and pH. Therefore, enzymes from extremophilic fungi
give us a huge advantage to manufacture paper in an efficient, eco-friendly, and
economical manner.
Sridevi et al. reported that biobleaching of paper pulp at 50 C with stable alkaline
xylanase from Trichoderma asperellum improved the brightness by 4 points due to
the release of chromophores present on lignin and also reduced the kappa number
(signifies the residual lignin) by 4.2 points. One of the reasons for the increase in
brightness was the breakdown of the bond by alkalophilic xylanase that retained its
activity even under alkaline conditions (Sridevi et al. 2017). Similarly, xylanase
from T. longibrachiatum and P. corylophilum effectively reduced the kappa number
and release of chromophores when kraft pulp from Eucalyptus was subjected to
biobleaching (Medeiros et al. 2007). In another study, it was observed that xylanase
extracted from Aspergillus sydowii SBS 45 retained 90% of its activity even after 5 h
of incubation at 40 C and pH 8.2 (Nair et al. 2010). A comparative study, using
thermostable and alkalophilic xylanases from C. thermophilum,Melanocarpus sp.,
Humicola insolens,Thermoascus aurantiacus, and Malbranchea sp. in the
biobleaching process of decker pulp, showed maximum brightness was achieved
by Malbranchea sp.Brightness increased by 2.04 ISO units with maximum release
of chromophores (Ghatora et al. 2006). Xylanase from alkalophilic fungus Aspergil-
lus nidulans KK-99 bleached and released chromophore at 1.01 U/g of pulp, 55 C,
and 3 h of incubation time. The kappa number was reduced by 5% with 1.7-fold
increase in the concentration of reducing sugars (Taneja et al. 2002).
17.8.3 Textile Industry
Desizing, scouring, and bleaching are the important processes involved in fabrica-
tion development. Non-cellulosic impurity of pectic substances, wax, ashes, and
lignin containing protein are eliminated by a chemical intensive process called as
scouring. Conventional scouring solutions affect non-precisely on cellulosic mate-
rial of fiber which results in loss of strength in the fabric. Xylanase positively
performs over the hemi-cellulosic materials by efficiently removing them. Enzy-
matic treatment does not lead to any loss of strength in the fiber, rather the fiber will
be developed to be more soft and smooth after desizing. During the later stages of
bleaching and finishing, fungi like Trichoderma reesei,Trichoderma
longibrachiatum produce xylanase which offers partial hydrolysis of seed coat
fragments by increasing the accessibility of the chemicals (Dhiman et al. 2008).
Garg et al. (2010) reported the enzyme pre-treatment resulted in the liberation of
additional reducing of sugar and weight loss when compared with that of control at
54 C incubation conditions. 120 min of incubation period was adequate to intensify
the whiteness and brightness of fabric up to 3.93 and 10.19%, respectively, and also
diminished the yellowness by 5.57%.
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 385

17.8.4 Baking Industry
Although bread making is an ancient process carried out in the conventional method,
many modifications are made to improve the overall quality and shelf life of bread.
One such modification is the use of enzymes (Butt et al. 2008). Wheat flour, the basic
ingredient in bread, contains 2–3% of arabinoxylan that negatively impacts the
quality of the bread. When arabinoxylan is treated with xylanase enzyme, it helps
in the release of sugars that influence the growth of yeast and production of CO
2
and,
at the same time, it makes arabinoxylan more water extractable to form gluten
network which drastically influences the quality of bread (Courtin and Delcour
2001; Wang et al. 2003; Yegin et al. 2018). The xylooligosaccharides formed by
the breakdown of arabinoxylan act as a prebiotic and help in the growth of beneficial
bacteria in the gut (Chapla et al. 2012; Yegin et al. 2018). Yegin et al. used xylanase
from Aureobasidium pullulans NRRL Y-231 strain to study its effect on bread-
making process. An increase of 35–40% in water absorption by the dough was
observed along with an increase in growth time and stability, whereas the firmness of
the crumb reduced. An overall improvement in dough and bread quality was
observed when xylanase was used in the bread preparation (Yegin et al. 2018).
17.8.5 Animal Feed
More than 900 agricultural ingredients are added to an animal feed that includes
straw, husk, grains, cereals, legumes, and many other agro-waste constituents.
Excess gluten in wheat, oats, etc. as well as β-glucans and arabinoxylans in the
cell walls of cereal grains linked with glucans, celluloses, galactans, and mannans
make digestion difficult in domestic animals. Enzymatic treatment of the feed greatly
refines the quality, and it also results in better digestion and growth rates in animals.
The addition of xylanase to the feed results in improved availability of
polysaccharides and increased nutrient absorption by thinning out the gut contents.
Xylanases also facilitate the conversion of hemicellulose to sugars that assists in
gaining sufficient energy from lesser feed to the animals, particularly chickens (Garg
and Tripathi 2017).
17.8.6 Beverage and Brewing Industry
Juice yield from fruits or vegetables can be increased when treated with xylanases.
Xylanases can also reduce the viscosity of the fruit juices along with improved
filterability. Xylanases also help in the extraction of sugars from barley when added
to the beer malt during the fermentation process (Garg and Tripathi 2017). Yanlong
Du et al. used three heterologously expressed xylanases from Humicola insolens Y1
in the mashing process. He observed a positive effect on the mashing process as the
xylanases reduced the viscosity and specificfiltration rate of the mash. However, the
386 A. Fasim et al.

cocktail mixture of xylanases was far more effective than individual preparation
(Du et al. 2013).
17.8.7 Fruit Ripening and Seed Germination
Germinating seeds naturally yield xylanases that aid in providing the required
nutrition for the growth of the sapling. Softening of fruits like papaya is one of the
major applications of xylanases. As the fruit begins to soften by ripening, xylanases
exhibit a significant part by altering the XOS in cell wall matrix. Consequently,
xylanases display a commercial role in the ripening of fruits (Kalim et al. 2015).
17.8.8 Degumming
Traditional water retting for fiber separation in the textile industry is a laborious and
harmful process. It also leads to low-quality fiber impacting the quality and cost of
the product. Bio-degumming is a process of degradation of non-cellulosic
components using hydrolytic enzymes (Duan et al. 2016). Xylanases are extensively
used in this process, and it is found that the degumming performed by a combination
of mild chemical pre-treatment along with enzymatic treatment resulted in an
enhanced yield of fiber. It is also reported that xylanases play a significant role in
the elimination of non-cellulosic as well as gummy substances from the bamboo
parts (Fu et al. 2008).
17.8.9 Pharmaceutical Industry
Xylooligosaccharides are the non-digestible sugar units composed of xylose
monomers. XOS has numerous purposes in the pharmaceutical and biotechnology
industry. XOS intensifies the growth of vital good bifid bacteria in the colon and
thereby limits the growth and proliferation of other harmful bacteria. Thus, XOS acts
as a prebiotic since it is not hydrolyzed or absorbed. XOSs are also found to have
anticancerous, immunomodulatory antimicrobial, anti-inflammatory, antioxidant,
and antihyperlipidemic activities (Bhardwaj et al. 2019). Therefore, XOSs are used
in beverages like soy milk, tea, and coffee, dairy products, desserts like pastries,
cakes, biscuits, puddings, jellies, and jams, and also as an active component of
symbiotic provisions. The action of the xylanase enzyme on animal feedstock makes
it easier to digest releasing the entrapped nutrients and thereby improving the
digestion in domestic animals. XOS can also be subsequently converted into simple
sugars, i.e., glucose and xylose that are useful in bioethanol production (Pellerin
et al. 1991).
The discussed applications thus prove that the employment of highly stable
extremophilic fungal xylanases in various industrial productions leads to the
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 387

development of value-added, chemical-free, and environmentally friendly products
(Table 17.2).
17.9 Conclusion
Biodegradation of biomass involves the action of numerous enzymes, among which
xylanases play a significant role. It has numerous applications in various industries,
and it is used in the production of value-added, chemical-free, and eco-friendly
products. Out of innumerable microorganisms isolated from different sources, fungal
strains are found to exhibit high potential for the production of xylanase enzyme.
The majority of xylanases from the fungal origin show optimal activity at mesophilic
temperatures (around 40–60 C) and neutral to slightly acidic pH. So exploring
extremophilic environments for robust fungal xylanases that can sustain harsh
industrial conditions is the focus of the current research. This chapter gives a detailed
review of the extremophilic fungal xylanase isolation, screening, and purification
techniques along with the industrial applications. Its role in biofuel production, paper
and pulp industry, animal feed, baking, and beverage industry is discussed in detail
Table 17.2 Applications of extremophilic xylanases
Microorganism Extremophilicity Applications References
Aureobasidium pullulans
477 NRRL Y-2311-1
Acidophilic
Ethanol tolerant
Halophilic
Fruit juice production
Wine-making and brewing
Bioethanol production and
processing of seafoods
Yegin (2017a)
Aureobasidium pullulans
NRRL Y-2311-1
Extremophilic Bread making Yegin et al.
(2018)
Phomopsis sp. MACA-J
Fusarium sp. KAWIT-A
Aureobasidium sp.
2LIPA-M
Alkaliphilic
Thermophilic
Thermophilic
Enzymatic pre-treatment of
recycled paper and pulp
Torres and
Dela Cruz
(2013)
Sporotrichum
thermophile
Thermophilic Xylooligosaccharide
synthesis (food industries)
Sadaf and
Khare (2014)
Chaetomium sp. CQ31 Thermophilic Chinese steamed bread Jiang et al.
(2010)
Humicola insolens Y1 Thermophilic Brewing industry Du et al.
(2013)
A. gracilis and
A. penicillioides
Halophilic Biofuel production Ali et al.
(2014)
Chaetomium
thermophilum,
Humicola insolens,
Melanocarpus sp.,
Malbranchea sp.and
Thermoascus
aurantiacus
Thermophilic Pulp bleaching Ghatora et al.
(2006)
Aspergillus nidulans
KK-99
Alkalophilic Pulp bleaching Taneja et al.
(2002)
388 A. Fasim et al.

here. However, further studies need to be conducted to gain knowledge about this
particular class of enzymes. New approaches, standardized and optimized protocols
to increase production will assist in deciphering more of its applications in the future.
The use of new technology involving genetic engineering, sequencing programs, in
silico approach to study the extremophilic xylanase, and xylanase family of enzymes
will further improve our understanding of these enzymes so that they can be
employed for an efficient, economical, and eco-friendly bioprocess technology.
References
Aachary AA, Prapulla SG (2011) Xylooligosaccharides (XOS) as an emerging prebiotic: microbial
synthesis, utilization, structural characterization, bioactive properties, and applications. Compr
Rev Food Sci Food Saf 10:2–16
Ahmed S, Jabeen A, Jamil A (2007) Xylanase from Trichoderma harzianum: enzyme characteriza-
tion and gene isolation. J Chem Soc Pak 29:176–182
Ali I, Kanhayuwa L, Rachdawong S, Rakshit SK (2013) Identification, phylogenetic analysis and
characterization of obligate halophilic fungi isolated from a man-made solar saltern in
Phetchaburi province, Thailand. Ann Microbiol 63(3):887–895
Ali I, Siwarungson N, Punnapayak H, Lotrakul P, Prasongsuk S, Bankeeree W, Rakshit SK (2014)
Screening of potential biotechnological applications from obligate halophilic fungi, isolated
from a man-made solar saltern located in Phetchaburi province, Thailand. Pak J Bot 46:983–988
Anthon GE, Barrett DM (2002) Determination of reducing sugars with 3-methyl-2-
benzothiazolinonehydrazone. Anal Biochem 305(2):287–289
Bailey MJ (1988) A note on the use of dinitrosalicylic acid for determining the products of
enzymatic reactions. Appl Microbiol Biotechnol 29:494–496
Bajpai P (2014) Chapter 3. Microbial xylanolytic systems and their properties. In: Bajpai P
(ed) Xylanolytic enzymes. Academic Press, Amsterdam, pp 19–36
Ballance S, Holtan S, Aarstad OA, Sikorski P, Skjåk-Bræk G, Christensen BE (2005) Application
of high-performance anion-exchange chromatography with pulsed amperometric detection and
statistical analysis to study oligosaccharide distributions—a complementary method to investi-
gate the structure and some properties of alginates. J Chromatogr A 1093:59–68
Bastawde KB (1992) Xylan structure, microbial xylanases, and their mode of action. World J
Microbiol Biotechnol 8:353–368
Beg Q, Kapoor M, Mahajan L, Hoondal G (2001) Microbial xylanases and their industrial
applications: a review. Appl Microbiol Biotechnol 56:326–338
Bhardwaj N, Kumar B, Verma P (2019) A detailed overview of xylanases: an emerging biomole-
cule for current and future prospective. Bioresour Bioprocess 6:40
Biely P (1985) Microbial xylanolytic systems. Trends Biotechnol 3(11):286–290
Buchanan B, Gruissem W, Jones R (2000) The cell wall. In: Biochemistry and molecular biology of
plants. American Society of Plant Physiologists, pp 52–158
Buckee GK, Baker CD (1988) Collaborative trial on the determination of beta-glucanase in malt by
viscometric and dye-labelled methods. J Inst Brewing 94:387–390
Butt MS, Tahir-Nadeem M, Ahmad Z, Sultan MT (2008) Xylanases and their applications in baking
industry. Food Technol Biotechnol 46(1):22–31
Chakdar H, Kumar M, Pandiyan K, Singh A, Nanjappan K, Kashyap PL, Srivastava AK (2016)
Bacterial xylanases: biology to biotechnology. 3 Biotech 6:150
Chapla D, Pandit P, Shah A (2012) Production of xylooligosaccharides from corncob xylan by
fungal xylanase and their utilization by probiotics. Bioresour Technol 115:215–221
Collins T, Gerday C, Feller G (2005) Xylanases, xylanase families and extremophilic xylanases.
FEMS Microbiol Rev 29(1):3–23
Courtin CM, Delcour JA (2001) Relative activity of endoxylanases towards water-extractable and
water-unextractable arabinoxylan. J Cereal Sci 33(3):301–312
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 389

Cürten C, Anders N, Juchem N, Ihling N, Volkenborn K, Knapp A, Jaeger KE, Büchs J, Spiess AC
(2017) Fast automated online xylanase activity assay using HPAEC-PAD. Anal Bioanal Chem
410(1):57–69
Deshmukh RA, Jagtap S, Mandal MK, Mandal SK (2016) Purification, biochemical characteriza-
tion and structural modelling of alkali-stable β-1, 4-xylan xylanohydrolase from aspergillus
fumigatus R1 isolated from soil. BMC Biotechnol 16(1):11
Dhiman SS, Sharma J, Battan B (2008) Industrial applications and future prospects of microbial
xylanases: a review. Bioresources 3:1377–1402
Dimarogona M, Topakas E (2016) Chapter 12. Regulation and heterologous expression of ligno-
cellulosic enzymes in Aspergillus. In: Gupta VK (ed) New and future developments in microbial
biotechnology and bioengineering. Elsevier, Amsterdam, pp 171–190
Driss D, Bhiri F, Elleuch L, Bouly N, Stals I, Miled N, Chaabouni SE (2011) Purification and
properties of an extracellular acidophilic endo-1, 4-β-xylanase, naturally deleted in the “thumb”,
from Penicillium occitanis Pol6. Process Biochem 46(6):1299–1306
Du Y, Shi P, Huang H, Zhang X, Luo H, Wang Y, Yao B (2013) Characterization of three novel
thermophilic xylanases from Humicola insolens Y1 with application potentials in the brewing
industry. Bioresour Technol 130:161–167
Duan S, Feng X, Cheng L, Peng Y, Zheng K, Liu Z (2016) Bio-degumming technology of jute bast
by Pectobacterium sp. DCE-01. AMB Express 6(1):86
Duarte CM, Cebrian J (1996) The fate of marine autotrophic production. Limnol Oceanogr 41:
1758–1766
Dutta T, Sengupta R, Sahoo R, Sinha Ray S, Bhattacharjee A, Ghosh S (2007) A novel cellulase
free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and
characterization. Lett Appl Microbiol 44(2):206–211
European Symposium on Enzymes in Grain Processing, Simoinen T, Valtion Teknillinen
Tutkimuskeskus (ed) (2000) 2nd European Symposium on Enzymes in Grain Processing:
ESEGP-2; Helsinki, Finland, 8–10 December 1999. Technical Research Centre of Finland,
Espoo. 340 p. (VTT symposium)
Fox JD, Robyt JF (1991) Miniaturization of three carbohydrate analyses using a microsample plate
reader. Anal Biochem 195(1):93–96
Fu J, Yang X, Yu C (2008) Preliminary research on bamboo degumming with xylanase. Biocatal
Biotransformation 26(5):450–454
Garg N, Mahatman KK, Kumar A (2010) Xylanase: applications and biotechnological aspects.
Lambert Academic Publishing AG & Co. KG, Mannheim
Garg SK, Tripathi M (2017) Microbial strategies for discoloration and detoxification of azo dyes
from textile effluents. Res J Microbiol 12:1–19
Ghatora SK, Chadha BS, Badhan AK, Saini HS, Bhat MK (2006) Identification and characteriza-
tion of diverse xylanases from thermophilic and thermotolerant fungi. Bioresources 1(1):18–33
Ghose TK, Bisaria VS (1987) Measurement of hemicellulase activities. Part 1: xylanases. Pure Appl
Chem 59(12):1739–1751
Gusakov AV, Kondratyeva EG, Sinitsyn AP (2011) Comparison of two methods for assaying
reducing sugars in the determination of carbohydrase activities. Int J Anal Chem 2011:283658
Hatanaka C, Kobara Y (1980) Determination of glucose by a modification of Somogyi-Nelson
method. Agric Biol Chem 44(12):2943–2949
Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on
amino acid sequence similarities. Biochem J 293:781–788
Hunt CJ, Antonopoulou I, Tanksale A, Rova U, Christakopoulos P, Haritos VS (2017) Insights into
substrate binding of ferulic acid esterases by arabinose and methyl hydroxycinnamate esters and
molecular docking. Sci Rep 7:17315
Jeffries TW (1996) Biochemistry and genetics of microbial xylanases. Curr Opin Biotechnol 7(3):
337–342
Jiang Z, Cong Q, Yan Q, Kumar N, Du X (2010) Characterisation of a thermostable xylanase from
Chaetomium sp. and its application in Chinese steamed bread. Food Chem 120(2):457–462
390 A. Fasim et al.

Jorgensen H, Kristensen JB, Felby C (2007) Enzymatic conversion of lignocellulose into ferment-
able sugars: challenges and opportunities. Biofuels Bioprod Biorefin 1(2):119–134
Kalim B, Böhringer N, Ali N, Schäberle TF (2015) Xylanases–from microbial origin to industrial
application. Biotechnol J Int 7:1–20
Kathiresan K, Bingham BL (2001) Biology of mangroves and mangrove ecosystems. Adv Mar Biol
40:81–251
Kathiresan K, Saravanakumar K, Anburaj R, Gomathi V, Abirami G, Sahu SK, Anandhan S (2011)
Microbial enzyme activity in decomposing leaves of mangroves. Int J Adv Biotech Res 2:382–
389
Kuhad RC, Singh A (1993) Lignocellulosic biotechnology: current and future prospects. Crit Rev
Biotechnol 13:151–172
Linares-Pastén JA, Aronsson A, Karlsson EN (2018) Structural considerations on the use of Endo-
xylanases for the production of prebiotic Xylooligosaccharides from biomass. Curr Protein Pept
Sci 19:48–67
Liu X, Kokare C (2017) Microbial enzymes of use in industry. Biotechnol Microb Enzym:267–298
López AMQ, Silva ALDS, Santos ECLD (2017) The fungal ability for biobleaching/biopulping/
bioremediation of lignin-like compounds of agro-industrial raw material. Química Nova 40(8):
916–931
Ma R, Bai Y, Huang H, Luo H, Chen S, Fan Y, Cai L, Yao B (2017) Utility of thermostable
xylanases of Mycothermus thermophilus in generating prebiotic Xylooligosaccharides. J Agric
Food Chem 65:1139–1145
Manju S, Singh Chadha B (2011) Chapter 9. Production of hemicellulolytic enzymes for hydrolysis
of lignocellulosic biomass. In: Pandey A, Larroche C, Ricke SC, Dussap C-G, Gnansounou E
(eds) Biofuels. Academic Press, Amsterdam, pp 203–228
McCleary BV, McGeough P (2015) A comparison of polysaccharide substrates and reducing sugar
methods for the measurement of endo-1, 4-β-xylanase. Appl Biochem Biotechnol 177(5):
1152–1163
Medeiros RG, Silva FGD Jr, Báo SN, Hanada R, Ferreira Filho EX (2007) Application of xylanases
from Amazon Forest fungal species in bleaching of eucalyptus Kraft pulps. Braz Arch Biol
Technol 50(2):231–238
Meeuwsen PJ, Vincken JP, Beldman G, Voragen AG (2000) A universal assay for screening
expression libraries for carbohydrases. J Biosci Bioeng 89(1):107–109
Moretti R, Thorson JS (2008) A comparison of sugar indicators enables a universal high-throughput
sugar-1-phosphate nucleotidyltransferase assay. Anal Biochem 377:251–258
Motta FL, Andrade CCP, Santana MHA (2013) A review of xylanase production by the fermenta-
tion of xylan: classification, characterization and applications. In: Chandel A (ed) Sustainable
degradation of lignocellulosic biomass—techniques, applications and commercialization.
InTech, London
Murashima K, Kosugi A, Doi RH (2003) Synergistic effects of cellulosomal xylanase and cellulases
from clostridium cellulovorans on plant cell wall degradation. J Bacteriol 185:1518–1524
Murphy TC, McCracken JK, McCann MEE, George J, Bedford MR (2009) Broiler performance
and in vivo viscosity as influenced by a range of xylanases, varying in ability to effect wheat
in vitro viscosity. Br Poultry Sci 50:716–724
Muthezhilan R, Ashok R, Jayalakshmi S (2007) Production and optimization of thermostable
alkaline xylanase by Penicillium oxalicum in solid state fermentation. Afr J Microbiol Res
1(2):20–28
Nair SG, Sindhu R, Shashidhar S (2010) Enzymatic bleaching of Kraft pulp by xylanase from
Aspergillus sydowii SBS 45. Indian J Microbiol 50(3):332–338
Natesh R, Bhanumoorthy P, Vithayathil PJ, Sekar K, Ramakumar S, Viswamitra MA (1999)
Crystal structure at 1.8 a resolution and proposed amino acid sequence of a thermostable
xylanase from Thermoascus aurantiacus. J Mol Biol 288(5):999–1012
Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose.
J Biol Chem 153(2):375–380
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 391

Ohta K, Moriyama S, Tanaka H, Shige T, Akimoto H (2001) Purification and characterization of an
acidophilic xylanase from Aureobasidium pullulans var. melanigenum and sequence analysis of
the encoding gene. J Biosci Bioeng 92(3):262–270
Paes G, Berrin JG, Beaugrand J, Purkarthofer H, Sinner MS (2012) GH11 xylanase: structure/
function/properties relationships and applications. Biotechnol Adv 30:564–592
Pellerin P, Gosselin M, Lepoutre JP, Samain E, Debeire P (1991) Enzymatic production of
oligosaccharides from corncob xylan. Enzy Microb Technol 13:617–622
Robledo A, Aguilar CN, Belmares-Cerda RE, Flores-Gallegos AC, Contreras-Esquivel JC,
Montañez JC, Mussatto SI (2016) Production of thermostable xylanase by thermophilic fungal
strains isolated from maize silage. CyTA J Food 14(2):302–308
Rothenhöfer M, Grundmann M, Bernhardt G, Matysik FM, Buschauer A (2015) High performance
anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the
sensitive determination of hyaluronan oligosaccharides. J Chromatogr B 988:106–115
Sadaf A, Khare SK (2014) Production of Sporotrichum thermophile xylanase by solid state
fermentation utilizing deoiled Jatropa cucas seed cake and its application in xylooligosachharide
synthesis. Bioresour Technol 153:126–130
Sakthiselvan P, Naveena B, Partha N (2015) Molecular characterization of a xylanase-producing
fungus isolated from fouled soil. Braz J Microbiol 45:1293–1302
Sánchez C (2009) Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnol
Adv 27:185–194
Scott GM, Akhtar M, Lentz MJ, Kirk TK, Swaney R (1998) New technology for papermaking:
commercializing biopulping. TAPPI J 81(11):220–225
Selvarajan E, Veena R (2017) Recent advances and future perspectives of thermostable xylanase.
Biomed Pharmacol J 10:261–279
Shaffer PA, Somogyi M (1933) Copperiodometric reagents for sugar determination. J Biol Chem
100:695–713
Sharma PK (2017) Xylanases current and future perspectives: a review. J New Biol Rep 6(1):12–22
Singh P, Srivastava N, Jagadish R, Upadhyay A (2019) Effect of toxic pollutants from pulp and
paper mill on water and soil quality and its remediation. Int J Lakes Rivers 12:1–20
Singh G, Kaur S, Khatri M, Arya SK (2019) Biobleaching for pulp and paper industry in India:
emerging enzyme technology. Biocatal Agric Biotechnol 17:558–565
Singla A, Paroda S, Dhamija SS (2012) Bioethanol production from xylose: problems and
possibilities. J Biofuels 3:1–17
Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:19–23
Sridevi A, Ramanjaneyulu G, Devi PS (2017) Biobleaching of paper pulp with xylanase produced
by Trichoderma asperellum. 3 Biotech 7(4):266
Sydenham R, Zheng Y, Riemens A, Tsang A, Powlowski J, Storms R (2014) Cloning and
enzymatic characterization of four thermostable fungal endo-1,4-β-xylanases. Appl Microbiol
Biotechnol 98:3613–3628
Taneja K, Gupta S, Kuhad RC (2002) Properties and application of a partially purified alkaline
xylanase from an alkalophilic fungus aspergillus nidulans KK-99. Bioresour Technol 85(1):
39–42
Topakas EG, Panagiotou G, Christakopoulos P (2013) Xylanases: characteristics, sources, produc-
tion and applications. Wiley, Hoboken, NJ
Torres JMO, Dela Cruz TE (2013) Production of xylanases by mangrove fungi from the Philippines
and their application in enzymatic pretreatment of recycled paper pulps. World J Microbiol
Biotechnol 29(4):645–655
Torres MJ, Brandan CP, Petroselli G, Erra-Balsells R, Audisio MC (2016) Antagonistic effects of
Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina phaseolina:
SEM study of fungal changes and UV-MALDI-TOF MS analysis of their bioactive compounds.
Microbiol Res 182:31–39
Torronen A, Rouvinen J (1997) Structural and functional properties of low molecular weight endo-
1,4-b-xylanases. J Biotechnol 57:137–149
392 A. Fasim et al.

Utsumi Y, Yoshida M, Francisco PB Jr, Sawada T, Kitamura S, Nakamura Y (2009) Quantitative
assay method for starch branching enzyme with Bicinchoninic acid by measuring the reducing
terminals of glucans. J Appl Glycosci 56:215–222
Viikari L, Kantelinen A, Sundquist J, Linko M (1994) Xylanases in bleaching: from an idea to the
industry. FEMS Microbiol Rev 13(2–3):335–350
Virupakshi S, Gireesh BK, Satish RG, Naik GR (2005) Production of a xylanolytic enzyme by a
thermoalkaliphilic bacillus sp. JB-99 in solid-state fermentation. Process Biochem 40:431–435
Walia A, Guleria S, Mehta P, Chauhan A, Parkash J (2017) Microbial xylanases and their industrial
application in pulp and paper biobleaching: a review. 3 Biotech 7(1):11
Wang M, Hamer RJ, Van Vliet T, Gruppen H, Marseille H, Weegels PL (2003) Effect of water
unextractable solids on gluten formation and properties: mechanistic considerations. J Cereal
Sci 37(1):55–64
Wong KKY, Tan LUL, Saddler JN (1988) Multiplicity of β-1,4 xylanase in microorganisms:
function and applications. Microbial Rev 52:305–317
Wu J, Qiu C, Ren Y, Yan R, Ye X, Wang G (2018) Novel salt-tolerant xylanase from a mangrove-
isolated fungus Phoma sp. MF13 and its application in Chinese steamed bread. ACS Omega
3(4):3708–3716
Yegin S (2017a) Single-step purification and characterization of an extreme halophilic, ethanol
tolerant and acidophilic xylanase from Aureobasidium pullulans NRRL Y-2311-1 with appli-
cation potential in the food industry. Food Chem 221:67–75
Yegin S (2017b) Xylanase production by Aureobasidium pullulans on globe artichoke stem:
bioprocess optimization, enzyme characterization and application in saccharification of ligno-
cellulosic biomass. Prep Biochem Biotechnol 47(5):441–449
Yegin S, Altinel B, Tuluk K (2018) A novel extremophilic xylanase produced on wheat bran from
Aureobasidium pullulans NRRL Y-2311-1: effects on dough rheology and bread quality. Food
Hydrocoll 81:389–397
Yeoman CJ, Han Y, Dodd D, Schroeder CM, Mackie RI, Cann IKO (2010) Chapter 1. Thermosta-
ble enzymes as biocatalysts in the biofuel industry. In: Advances in applied microbiology,
advances in applied microbiology. Academic Press, Amsterdam, pp 1–55
Yoon JH, Park JF, Suh DY, Hong SB, Ko SJ, Kim SH (2007) Comparison of dyes for easy
detection of extracellular cellulases in fungi. Mycobiology 35(1):21–24
17 Extremophilic Fungal Xylanases: Screening, Purification, Assay, and Applications 393

Extremophilic Fungal Lipases: Screening,
Purification, Assay, and Applications 18
J. Angelin and M. Kavitha
Abstract
Extremophiles are microorganisms which require or tolerate extreme environ-
mental conditions like high and low temperature, extreme acidic or basic pH, high
exposure to radiations, high salinity, low and high pressure, growth in the
presence of toxic wastes, organic solvents, heavy metals, and other habitats for
their survival. Among different fungal enzymes produced by these extremophiles,
lipases are considered to be more valuable natural resource to replace chemical
agents for industrial applications. Recombination and immobilization have
greatly improved the biocatalytic properties than the native lipases of
extremophilic fungal origin. It is more important to screen the molds or yeasts
using appropriate methods to determine their lipase producing capability. The
most crucial process is the purification of fungal lipases which aid to characterize
and distinguish their potential for utilization in various fields of interest. Evalua-
tion of lipolytic activity using different methods provides information about their
hydrolyzing property. Extremophilic fungal lipases play indispensable roles in
biotechnological applications such as detergent, food and feed, pharmaceuticals,
bioremediation, oleochemistry, fine chemicals, textiles, leather, pulp, and paper
industries. In this chapter, diverse methods for screening of lipases from fungi
isolated from different extreme regions, purification scheme, different lipase
assays, and the industrial applications of extremophilic fungal lipases are
discussed in detail.
Keywords
Extremophiles · Fungal lipases · Screening · Purification · Lipase assay · Industrial
applications
J. Angelin · M. Kavitha (*)
School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu,
India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_18
395

18.1 Introduction
Extremophiles are microorganisms which have the ability to survive and flourish in
harsh environmental conditions while normal organisms are incapable to confront
such situations. An organism which prefers harsh environments for their growth and
survival are known to be extremophiles whereas the organisms that withstands these
extreme conditions are known as extremotolerants. Interestingly, an organism which
endures more than one extreme environmental condition is known as poly-
extremophiles (Jin et al. 2019; Salwan and Sharma 2020). Based on their affinity
toward diverse regions, they are categorized on the basis of pH (acidophile and
alkaliphile), pressure (piezophile), radiation (radioresistant), redox potential (xero-
phile), salinity (halophile), and temperature (psychrotolerant, psychrophile,
mesophile, thermophile, and hyper-thermophile) (Sahay et al. 2017). The
extremophiles comprise bacteria, archaea, fungi, yeast, and few protozoans. These
extremophiles are discovered in places such as hot springs, deep-sea hydrothermal
vents, decaying plant matter, Antarctic, Arctic, glaciers, ashes, and deserts. Some of
the marine fungal species are found in Mediterranean and Red Sea DHABs (Deep-
Hypersaline Anoxic Basins) (Zhang et al. 2018). Figure 18.1 represents the preva-
lence of extreme environments around the world. In order to withstand in these
extreme conditions, extremophiles undergo certain modifications at structural, bio-
chemical, and molecular levels. The enzymes secreted by these extremophiles are
called as extremozymes that include amylases, cellulases, esterases, keratinases,
lipases, pectinases, peroxidases, proteases, and xylanases (Varela et al. 2012).
Especially, halo-tolerant fungi harbors stable and valuable enzymes compared to
enzymes isolated from terrestrial ecosystem. These extremozymes are known for
their tremendous attributes like high catalytic efficiency and high stability under
varied temperature and pH conditions, salinity, low water activity, low oxygen, and
more shelf life than enzymes of other origin. Among these extremozymes, lipases are
appraised as the third largest enzyme group, after proteases and carbohydrases based
on their market value. They occupy up to 10% enzyme market among the other
hydrolases (Basheer et al. 2011; Sharma et al. 2016a).
Lipases (EC 3.1.1.3) are triacylglycerol acyl hydrolases with an important physi-
ological role toward the hydrolysis of triglycerides into mono- and diglycerides, fatty
acids, and glycerol at oil–water interface; they break down carboxylic ester bonds
through catalytic triad composed of serine, histidine, and aspartate/glutamate to
perform various important reactions such as hydrolysis, esterification, and trans-
esterification reactions (alcoholysis, acidolysis, aminolysis, and inter-esterification)
(Gopinath et al. 2013). Lipases of microbial origin have attracted the scientific
communities than plant- and animal-derived lipases with profitable benefits and
functional ability at extreme conditions, stability in organic solvent, chemo-
selectivity, enantioselectivity, and they do not require any co-factor (Hasan et al.
2010; Singh and Mukhopadhyay 2012). Lipases acquire significant features to
perform at the interface between an aqueous and a non-aqueous phase (Kour et al.
2020). Lipases have a characteristic folding pattern of α/β-hydrolase with mostly
parallel β-sheets, flanked on both sides by α-helixes in the structure. The two distinct
396 J. Angelin and M. Kavitha

characteristics of lipases are (a) a lid covering the active site and (b) interfacial
activation. The lid influences the activity, specificity, enantioselectivity, and stability
of the enzyme. Lipases share a consensus sequence of G (glycine)-X
1
(histidine)-S
(serine)-X
2
(glutamic or aspartic acid) –G (glycine), whereby X may be any amino
acid residue. Lipase exhibits interfacial activation whereby it acts only on emulsified
substrates. The active site of lipase is covered by a lid-like α-helical structure. The lid
moves away upon binding to a lipid interface, causing the active site of lipase fully
accessible, enhancing hydrophobic interaction between the enzyme and lipid sur-
face. This lid experiences conformational rearrangement at the lipid water interface
when the substrate interacts with the active site of the enzyme which is termed as
“interfacial activation”(Balan et al. 2012; Sarmah et al. 2018). Lipase producing
microorganisms are ever-present to be isolated from soil, marine environment,
wastewater from fish industry, agro-industrial waste, waste volatile substances, air,
palm-oil mill effluent, intestine of silkworm, and human skin (Ilesanmi et al. 2020).
Figure 18.2 depicts the frequent sites for the isolation of extremophilic fungal
lipases.
Fig. 18.1 Prevalence of extreme environments around the world
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 397

Lipases are produced by plants, animals, bacteria, fungi, and archaea. Fungal
extremozymes are secreted by fungi belonging to the groups ascomycota and
basidiomycota that include Mucor,Penicillium,Candida,Aspergillus, etc. Antarctic
fungi encompasses the genera Beauveria,Candida,Cryptococcus,Geomyces,
Leucopsoridium,Moesziomyces,Mrakia,Pseudozyma,Penicillium,Phoma,
Pseudogymnoascus,Verticillium, and Trichosporon and have been recognized as
lipase producers (Martorell et al. 2019; Duarte et al. 2018). On the other hand, lipase
producing yeasts include Candida rugosa,Candida antarctica,Yarrowia lipolytica,
Pichia sp.,Rhodotorula sp.,Trichosporon sp., etc. In particular, among microbial
sources, filamentous fungi are good lipase producers and the extraction, purification,
and processing steps are relatively simple. Filamentous fungi are employed for
industrial purposes because it produces extracellular lipases which is much easier
to be recovered from the production medium than bacterial lipases and reduces
production expenses (Contesini et al. 2017). Lipases play significant role in multi-
faceted industrial applications such as pharmaceuticals, food, biodiesel,
agrochemicals, dairy and textile, detergent, and surfactant productions (Dumorné
et al. 2017; Raveendran et al. 2018). Cold-adapted lipases (CLPs) have developed
specific structural features which provide thermal flexibility around the active site
and high specific activity at low temperatures (Kavitha 2016; Joseph et al. 2008). On
the other hand, lipase producing microorganisms isolated from thermal
environments produce thermo-stable lipases with other unique properties including
solvent tolerance and resistance to chemical inactivation (Salihu and Alam 2015;
Casas-Godoy et al. 2012). Therefore, lipases from psychrophilic microorganisms are
some of the most widely used classes of enzymes in biotechnology applications,
organic chemistry, detergent industry, for bioremediation purposes, and in the food
industry (Dalmaso et al. 2015). In 2012, world lipase demand was US$255 million,
and this demand is expected to increase 6.2% annually to US$460 million in 2022,
driven by the growth in industrial and specialty markets (Group 2009).
Fig. 18.2 Frequent sampling sites for extremophilic fungal lipases
398 J. Angelin and M. Kavitha

Some researchers have increased and improved the effectiveness of the fungal
extremozymes employing recombination through heterologous expression using
other potential microorganisms such as bacteria (Escherichia coli) and yeast (Pichia
pastoris) (Xing et al. 2020; Ichikawa et al. 2020). In addition to that effort, lipases
are immobilized to ameliorate the technical and economic advantages which lower
the cost and enhance stability especially of psychrophilic lipases. Immobilization
facilitates the separation of products, lipase properties such as thermal stability and
activity, and provides more flexibility with enzyme/substrate contact by using
various reactor configurations (Yadav et al. 2017). For example, enzymatic synthesis
of glyceryl monoundecylenate (GMU) was achieved through immobilized Candida
antarctica lipase B preparation by esterification and could be referred for industrial
synthesis of fatty acid esters of glycerol (Yadav et al. 2017). Metagenomic
technologies have been developed to bypass the requirement for the isolation or
cultivation of microorganisms, and they could prove to be a powerful tool for
discovering novel genes and enzymes directly from uncultured microorganisms
(Madhavan et al. 2017). In the sequence-based approach, the colony hybridization
technique is used for screening metagenomic clones using an oligonucleotide primer
or probes for the target gene, and the desired gene may also be amplified by PCR
using specific or degenerate primers and subsequently cloned into suitable expres-
sion vectors (Kim et al. 2010). This chapter encompasses traditional as well as novel
methods for screening lipases from fungi of varied extreme regions, purification
schemes, different lipase assays, and industrial applications.
18.2 Screening from Extreme Environments
The lipid solubilizing microorganisms are retrieved from different environmental
sources to isolate, identify, and screen for hydrolyzing property. It is difficult for the
researchers to recreate the same environment for fungi obtained from harsh environ-
ment. But various attempts are made to retain the characteristics of the potent
organisms to be cultivated at laboratory level (Navvabi et al. 2018). Lipid materials
could be dissolved by the lipases produced by bacteria, yeasts, filamentous fungi,
and few protozoans (Gopinath et al. 2013). The samples obtained from various
extreme sources are processed by serial dilution followed by spread plating on solid
agar medium with a lipid source. Varied strategies for screening have been proposed
for identifying fungi producing lipases which involve culturing of fungi on solid agar
medium, a direct screening plate assay containing lipase substrates, or in liquid
media containing substrates which act as inducers and later test the filtrates for the
lipase activity (Kotogán et al. 2014). Table 18.1 and Fig. 18.3 show the various
screening techniques of lipase producers from different extremophilic fungi.
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 399

Table 18.1 Screening strategies for fungal lipases from extreme sources
S. no Type Fungus Source Screening medium/substrate Reference
1. Alkalophile Aspergillus
fumigatus
Oil contaminated soil—
Himachal Pradesh
Tributyrin agar Mehta et al. (2018a)
2. Mesophilic Rhizopus JK-1 Oil contaminated soil samples Rhodamine–olive oil agar
plates
Kantak et al. (2011)
3. Mild acidophile Penicillium
aurantiogriseum
Soils and oil refineries Tributyrin agar Pandey et al. (2018)
4. Osmo-tolerant Debaryomyces
hansenii
Dry-salted olives Rhodamine–olive oil agar Papagora et al. (2013)
5. Psychrophile Aspergillus
awamori
Arabian Sea water of Kerala
coast
Rhodamine B–olive oil agar Basheer et al. (2011)
6. Organic solvent tolerant Trichosporon sp. Soil sample (oil contaminated
sites)
PDA plates with olive oil and
rhodamine B
Cao et al. (2020)
7. Halotolerant Engyodontium sp. Marine water (Machilipatnam
coastal region)
Phenol red olive oil agar Lanka and B (2018)
8. Thermo-stable–organic
solvent stable
Rhizopus sp. Palm kernel cake (west oil
mill)—Malaysia
Phenol red olive oil agar and
tween 80 agar
Riyadi et al. (2017b)
9. Thermophilic fungal
strain
Neosartorya
fischeri P1
Acid wastewater—China Olive oil–rhodamine B agar Sun et al. (2016)
10. Organic solvent tolerant Candida tropicalis
SD7
Oil contaminated soil—China Tributyrin supplemented
medium
Peng et al. (2016)
11. Cold active yeast Crytococcus sp.
Y32
Nella lake—Antartica Tributyrin agar base Kumar Maharana and
Mohan Singh (2018)
400 J. Angelin and M. Kavitha

18.2.1 Direct Screening Methods
The direct screening methods are qualitative or semi-quantitative in nature.
Researchers have identified many screening methods to determine lipolytic activity.
Most frequently used method is solid medium-based screening with clear zone or
turbid appearance around the fungal colonies after certain period of incubation. The
substrates used to identify lipase production by extremophilic fungal stains as well as
other fungal sources include vegetable and fish oils, animal fats, synthetic
triacylglycerides, and Tween 20, 40, 60, and 80. Chromogenic substrates that
include rhodamine B, phenol red, Victoria blue, etc. are also used in addition with
the major substrates to recognize lipase activity. The most commonly employed
direct screening methods are listed below.
18.2.1.1 Olive Oil–Rhodamine B Agar Method
Olive oil contains high amount of oleic acid, which is a suitable substrate for lipase
production for both bacteria and filamentous fungi. It plays a vital role in the
detection and induction of lipase production and also serves as a carbon source for
growth. Olive oil-agar plate method is the best method to screen true lipase produc-
ing fungi. Lipases can be screened using carbon sources, such as olive oil added with
rhodamine B (Eugenia et al. 2016; Wagner et al. 2018; Singh et al. 2012). When
exposed to UV light of wavelength 350–370 nm, the hydrolyzed compounds such as
fatty acids, monoglycerides, and diglycerides combine with rhodamine B and form
orange fluorescence around the individual colonies (Akbari et al. 2016; Adan
Gökbulut and Arslanoǧlu 2013; Selvam et al. 2011). Trichoderma harzianum was
identified to show best lipolytic activity on olive oil–rhodamine B plates which is the
preliminary method of screening fungal species (Canseco-Pérez et al. 2018; Li et al.
Fig. 18.3 Screening of lipase producing extremophilic fungi on solid agar media
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 401

2018). Aspergillus carbonarius obtained from rotten cassava tuber was observed for
lipase production on rhodamine B agar to form orange halo around the fungal
colonies under UV light at 350 nm (Ire and Ike 2014). The true lipase secretion
from Penicillium sp. DS-39 was confirmed through fluorescence zone formation on
olive oil–rhodamine B agar under UV irradiation (Dsm et al. 2011). Cold active
Wickerhamomyces psychrolipolyticus, a novel yeast species producing two kinds of
lipases with activity at different temperatures of 25 C and 4 C, utilizes olive oil and
rapeseed oil as sole carbon source and shows lipase activity on the rhodamine B
medium (Shimizu et al. 2020). The three strains of T. lanuginosus (GSLMBKU-10,
GSLMBKU-13, and GSLMBKU-14) exhibited high lipase activity on rhodamine B
agar medium as evidenced by maximum orange fluorescent halo (Sreelatha et al.
2017).
18.2.1.2 Tributyrin Agar Method
Tributyrin is a fatty substance that can be digested by lipases, thus rendering it the
preferred assay for testing lipase-producing microorganisms. Tributyrin is a surface
active substance which is most appropriate and labor-saving substrate because it is
well dispersed in water by shaking or stirring without the addition of any emulsifiers
(Griebeler et al. 2011). Tributyrin is also supplemented to the agar medium through
filter sterilization to screen the lipases production (Ülker and Karaoĝlu 2012). Lipase
activity using tributyrin added agar plates was identified by hydrolysis indicating
that extracellular enzyme production and diameter of the clear zone around the
colonies are measured (Cho et al. 2007; Gopinath et al. 2013; Maharana and Ray
2015). In some cases, the samples were transferred to the enrichment culture medium
supplemented with olive oil. After certain period of incubation, the cultures were
diluted and then added to the medium containing tributyrin indicating lipase hydro-
lysis around the colonies (Peng et al. 2016; Mehta et al. 2018a). Alterneria sp. and
Aspergillus flavus isolated from oil contaminated sites were reported as best extra-
cellular lipase producers on tributyrin agar plates than other fungi (Wadia and Jain
2020). Since tributyrin added medium shows positive results for both lipase and
esterases, it is reported as a non-specific method (Kumar et al. 2012a). Cold active
zygomycetal fungal strains such as Dissophora,Gamsiella,Gilbertella,Mortierella,
Mucor,Rhizomucor,Rhizopus, and Umbelopsis were also screened with respect to
their ability to hydrolyze tributyrin (Kotogán et al. 2014). The Cladosporium
langeronii isolated from southern Caspian Sea showed higher lipolytic activity on
tributyrin agar plates (Sadati et al. 2015). Candida boidinii KF156789 alkaline cold-
adapted lipase obtained from spent olive derived from olive fruits showed lipolytic
activity (20 mm) on the tributyrin agar plate (Insaf et al. 2014). 17 lipase positive
fungi were determined using qualitative tributyrin phenol red agar method with
various degrees depending on the intensity of the produced yellow color. Among
them, Curvularia sp. DHE 5 producing alkaline lipase was considered as the most
prominent strain (El-Ghonemy et al. 2017).
402 J. Angelin and M. Kavitha

18.2.1.3 Tween 80 Agar Method
Screening using Tween 80 agar plates were also used as an indication of extracellular
lipase activity, where it exhibited opaque zone of precipitation around the fungi. This
is due to the deposition of insoluble calcium crystals salt formed by the liberation of
the fatty acid as the fungi grow on the Tween 80 agar plates (Akmoussi-Toumi et al.
2018; Riyadi et al. 2017a). Halotolerant and halophilic fungi isolated from Great
Sebkha of Oran (Algeria) such as P. vinaceum,G. halophilus,Wallemia sp.and
Ustilago cynodontis showed better appearance of an opaque precipitation around the
thallus on Tween 80 supplemented medium (Chamekh et al. 2019). Bromocresol
green agar medium containing Tween 80 was used in the isolation of lipolytic
A. niger ATCC 1015 on the basis of its ability to hydrolyze lipid in the medium
which resulted in the color change of the surrounding medium from green to yellow,
thus becoming acidic due to the release of fatty acids and glycerol (Bamitale Osho
and Quadri Adio 2015). Phenol red medium containing olive oil and tween 80 as
carbon source was suitable to identify lipolytic activity of Aspergillus aculeatus
(Triyaswati and Ilmi 2020).
18.2.1.4 Olive Oil Phenol Red Method
Fungi are screened qualitatively on chromogenic medium such as phenol red olive
oil agar plates for lipase production. Phenol red agar plate is a pH-based detection
method to detect lipase in plates and gels. Phenol red appeared reddish at pH above
neutrality, with a reddish end-point at pH 7.3–7.4. A slight decrease in pH (7.0–7.1)
due to the hydrolysis of the lipid substrate, which liberated fatty acids, turned the
agar plate color to yellow, denoting lipolysis. This change in color of phenol red was
used as an indicator of the enzyme activity. Psychrotrophic fungi were screened
using olive oil agar and palm oil agar with phenol red showed halo zones indicating
lipase production (Sahay and Chouhan 2018). Aspergillus sp. isolates that were able
to hydrolyze olive oil using the phenol red agar medium were further analyzed
depending on their ability to hydrolyze tributyrin (Alabdalall et al. 2020).
18.2.2 Other Direct Screening Methods
Aspergillus tamarii JGIF06 isolated from rhizosphere soil was monitored for its
lipolytic activity by observing deep blue color around the fungal colony on Spirit
blue agar (Das et al. 2016). For screening cold-adapted lipases, Victoria blue agar
plates were also used to evaluate the lipolytic activity of added substrates and color
change in indicator dye after incubation (Salwoom et al. 2019).
18.2.3 Quantitative Screening Methods
Aspergillus aculeatus was quantitatively screened through submerged fermentation
using the medium containing 1% glucose and 1% olive oil at pH 7.0 and temperature
of 30 C. After 96 and 120 h of incubation the enzyme activity was estimated to be
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 403

5.13 0.30 U/mL and 5.22 0.59 U/mL, respectively (Triyaswati and Ilmi 2020).
In cold-tolerant zygomycetal fungal strains, addition of mineral salts and olive oil to
the solid fermentation medium resulted in at least 1.5-fold increment in the enzyme
activities of the crude extracts. Tween 80 proved to be a good inductor for lipase
production since most of the investigated fungi displayed high enzyme activity when
this substrate was applied (Kotogán et al. 2014). Aspergillus japonicas (MTCC
no. 1975) strain was able to grow in SSF of castor bean waste and demonstrate
substantial lipase production. The maximum lipase activity reached was 24.8 U/g
and 18.9 U/g, about 48% and 47% greater when compared to activities reached
before the optimization process (Jain and Naik 2018). Fusarium solani isolated from
Arabian Sea has been reported to produce halophilic lipase using palm oil mill
effluent as substrate reveals a significant 3.2-fold increase with enzyme activity
7.8 U/mL (Geoffry and Achur 2018). The production medium containing olive oil
has promoted Fusarium solani strain SKWF7 to express its extracellular lipolytic
production with maximum activity of 36.9 U/mL (Kanmani et al. 2013). The novel
Penicillium sp. LBM 088 was the best producer of lipase isolated from Paranaense
rainforest with highest activity (1224 U/mL) (Ortellado et al. 2020). Two fungal
strains Arthrographis curvata and Rhodosporidium babjevae were isolated and
found to produce higher levels of lipases showing optimal activity at 40 C and
pH 9.0 for A. curvata and at 40 C and pH 8.0 for R. babjevae (Aamri et al. 2020).
18.2.4 Purification of Extremophilic Fungal Lipases
In general, enzyme purification plays a principle role in the determination of primary
amino acid sequence and three-dimensional structure that leads to X-ray studies of
pure lipases to enable the examination of structure–functional relationships. Purifi-
cation also contributes to transparency in kinetic mechanisms of lipase action on
hydrolysis, synthesis, and group exchange of esters (Mehta et al. 2017). Purification
of the lipase depends on the microbial origin and intracellular or extracellular nature
of enzyme from the filamentous fungi. Intracellular enzymes require cell disruption
to release the enzyme in fluid phase. The common techniques of cell wall disinte-
gration are ultrasound disintegration, homogenization in bead mill, application of
chemicals of various types, and osmotic shock. The enzyme purification scheme
includes extraction or concentration followed by purification by a combination of
different chromatographic methods and extraction protocols. Table 18.2 and
Fig. 18.4 present the purification strategies of lipases extracted from extremophilic
fungi.
18.2.5 Extraction
Lipases are initially subjected to pre-purification steps before proceeding with main
purification procedures. The cell-free supernatant obtained from the production
medium after a certain period of incubation undergoes filtration or centrifugation
404 J. Angelin and M. Kavitha

Table 18.2 Purification scheme for extremophilic fungal lipases
S. no Fungus
Extraction
method Purification method
Specific
activity
Purification
fold
Molecular
weight Yield References
1. P. chrysogenum
SNP5
Ammonium
sulfate
precipitation
DEAE-cellulose
ion-exchange resin
40.7 U/mL 10.6 and
26.28
40 kDa 39.6% Kumar et al.
(2012b)
2. Aspergillus
awamori
Ammonium
precipitation
Sephadex G100 ion
exchange chromatography
1164.63 U/mg –90 kDa 33.7% Basheer et al.
(2011)
3. Recombinant
Trichosporon
coremiiforme V3
lipase
Ultrafiltration DEAE sepharose fast flow
column
2549 U/mg 2.75 70 kDa –Wang et al.
(2015)
4. Trichosporon
coremiiforme V3
Ammonium
sulfate
precipitation
DEAE sepharose anion
exchange chromatography
8.0 U/mL 3.96 32.6 kDa 36.64% Wang et al.
(2015)
5. Mortierella
alliacea YN-15
Acetone
precipitation
and filtration
DEAE sepharose column
Phenyl-sepharose fast flow
column
Superdex 200 column
179 U/mg –11 kDa 4.0% Jermsuntiea
et al. (2011)
6. Mucor hiemalis
f. corticola
Ammonium
sulfate
precipitation
and dialysis
Gel filtration column
chromatography and ion
exchange chromatography
–12.63 46 kDa 27.7% Ülker and
Karaoĝlu
(2012)
7. Neosartorya
fischeri P1
Ultrafiltration HiPrep 26/10 desalting
column
870 U/mg –∼70 kDa –Sun et al.
(2016)
8. Aspergillus tamari Ammonium
sulfate
precipitation
and dialysis
DEAE Sepharose ion
exchange and Sephadex
G200 column
chromatography
260,210.46 U/
mg
7.9 50 kDa 43.1% Das et al.
(2016)
(continued)
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 405

Table 18.2 (continued)
S. no Fungus
Extraction
method Purification method
Specific
activity
Purification
fold
Molecular
weight Yield References
9. Penicillium
sp. DS-39
Ammonium
sulfate
precipitation
Sephacryl
®
100-HR–IQ
Sepharose
®
HP
Sephacryl
®
100-HR–II
308.73 U/mg 129.72 43 kDa 8.82% Dsm et al.
(2011)
10. Aspergillus
japonicus LAB01
Ammonium
sulfate
precipitation
Superose 12HR gel filtration
chromatography
Fast protein liquid
chromatography (FPLC)
3.5 10
4
U/
mg
3.9 25 kDa 44.2% Souza et al.
(2014)
11. Aureobasidium
pullulans
Ultrafiltration DEAE sepharose fast flow
column
17.7 U/mg 18.2 39.5 kDa 7.2% Li et al.
(2019)
12. Aspergillus
fumigatus
Ammonium
sulfate
precipitation
Octyl sepharose column 14.34 U/mg 6.96 35 kDa 11.03% Mehta et al.
(2018b)
13. Recombinant
lipase LK1 (Pichia
pastoris)
–Ni
2+
–NTA column
chromatography
1.82 U/mg 11.6 35.5 kDa 31.75% Nurul
Furqan and
Akhmaloka
(2020)
406 J. Angelin and M. Kavitha

followed by concentration by means of ultrafiltration or precipitation using ammo-
nium sulfate, acetone, or ethanol or extraction with organic solvents (Gaur et al.
2017). Precipitation enables modification in the solubility of proteins and favors the
formation of protein aggregates. Ammonium sulfate is a neutral salt which is
economically feasible with high solubility and lack of denaturing properties toward
most proteins. It exhibits stabilizing effect on many proteins and protects the
biological properties of proteins (Brígida et al. 2014). The addition of different
polar organic solvents to a protein solution can also promote precipitation; as such
solvents lower the di-electric constant of the aqueous solution. The hydro-organic
solution enables an increase in the electrostatic attraction between bodies with
opposite charges, such as proteins, leading to their precipitation (Melani et al.
2019). The protein precipitation is precipitation by ammonium sulfate which is
used about 60% of the time, while 35% use ethanol, acetone, or hydrochloric acid.
In general, precipitation is followed by chromatography which yields less protein
when compared to precipitation (Yagmurov et al. 2017). The increase in lipase
activity depends on the concentration of the ammonium sulfate used during partial
purification (Palekar et al. 2000). Partial purification of extracellular alkaline lipases
from novel fungus Curvularia sp. DHE 5 upon ammonium sulfate precipitation and
dialysis resulted in 3.1-fold purification with 50.6% recovery and 8.1–24.7 U/mg
specific activity (El-Ghonemy et al. 2017). Ultrafiltration is also a concentration
method recruited as an alternative or combined with other extraction methods where
separation is determined by particle size (Melani et al. 2019). After precipitation, the
enzymes are subjected to dialysis to remove salts and metal ions. Sometimes,
phenylmethylsulfonyl fluoride was added to the crude enzyme extract in order to
Fig. 18.4 Purification strategies for extremophilic fungal lipases
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 407

prevent enzyme degradation during the purification process. The concentrated lipase
was then filtered in order to remove larger molecules that could potentially clog the
gel filtration column (Tišma et al. 2019).
18.2.6 Purification
Chromatographic methods are separation processes where chromatographic matrices
(columns) are usually packed with hydrophilic materials that demonstrate no
interactions with the biomolecules (Melani et al. 2019). The selection of chro-
matographic techniques are determined in terms of source of microorganisms, size
of the proteins, and protein interaction with chromatographic resins, such as liquid
ionic charge, molecular weight, hydrophobicity, and specificity to purify lipases
(Bharathi and Rajalakshmi 2019; De Carvalho et al. 2019). The common chro-
matographic techniques include hydrophobic interaction chromatography, gel filtra-
tion, ion exchange chromatography, aqueous two-phase systems, reversed micellar
extraction, immune purification, and affinity chromatography (Jares et al. 2010;
Mehta et al. 2017). In some cases, purification may reduce the final yield of lipase
but enhances the lipolytic activity. Industrial applications such as in medical and
pharmaceutical industries demand high degree purity of enzymes. Ion-exchange
chromatography is the most frequently employed method, and the main anion and
cation exchangers are the diethyl-aminoethyl cationic group and carboxymethyl
anionic group, respectively. As lipases are well known for its hydrophobic nature
that possesses hydrophobic surfaces around the active site, affinity chromatographic
techniques are preferred. The most predominantly used chromatographic technique
involves columns packed with QAE sephadex, CM cellulose, DEAE cellulose,
phenyl-sepharose, etc. (Joseph et al. 2008). Psychrotrophic fungal lipases were
purified by ammonium sulfate precipitation, dialysis, and DEAE cellulose column
chromatography and the purity tested on SDS-PAGE gel electrophoresis to deter-
mine molecular mass of the lipase (Sahay and Chouhan 2018). The partial purifica-
tion of Geotrichum candidum lipase has been reported to achieve higher recovery
factors upon ammonium sulfate precipitation and reduces concentration period of
lipase with ethanol precipitation (Resende et al. 2015). 39 kDa monomeric lipase
from the newly isolated fungus Talaromyces thermophilus was purified from the
culture supernatant using ammonium sulfate precipitation, gel filtration, and anion
exchange chromatography (Romdhane et al. 2010). The routine chromatographic
techniques used in fungal lipase purification are dealt below.
18.2.6.1 Ion Exchange Chromatography
Ion exchange chromatography exhibits the principle of attraction between oppositely
charged stationary phase, known as an ion exchanger, and analyte. It is frequently
chosen for the separation and purification of proteins, peptides, nucleic acids,
polynucleotides, and other charged molecules, mainly because of its high resolving
power and high capacity. There are two types of ion exchanger, namely cation and
anion exchangers. Cation exchangers possess negatively charged groups and these
408 J. Angelin and M. Kavitha

will attract positively charged cations. These exchangers are also called acidic ion
exchangers because their negative charges result from the ionization of acidic
groups. Anion exchangers have positively charged groups that will attract negatively
charged anions. The term basic ion exchanger is also used to describe these
exchangers, as positive charges generally result from the association of protons
with basic groups (Lim et al. 2020; Maldonado et al. 2016; Tan et al. 2015). Cold
active lipase obtained from mesophilic yeast Pichia lynferdii NRRL Y-7723 was
purified by DEAE anion exchange chromatography column and further purified with
Sephacryl S-200 size exclusion chromatography column representing 33.1 purifica-
tion fold and 0.31% recovery (Bae et al. 2014). Lipase from Antarctic krill, with a
molecular weight of 71.27 kDa, was purified with ammonium sulfate precipitation
and a series of chromatographic separations over ion exchange (DEAE) and gel
filtration columns (Sephacryl S-100), resulting in 5.2% recovery with a 22.4-fold
purification ratio (Chen et al. 2020).
18.2.6.2 Gel Permeation Chromatography
This chromatographic technique is used for the separation of molecules on the basis
of their molecular size and shape and exploits the molecular sieve properties of a
variety of porous materials. The terms exclusion or permeation chromatography or
gel filtration describe all molecular separation processes using molecular sieves. The
general principle of exclusion chromatography is quite simple. A column of
microparticulate cross-linked copolymers generally of either styrene or
divinylbenzene and with a narrow range of pore sizes is in equilibrium with a
suitable mobile phase for the analytes to be separated. Large analytes that are
completely excluded from the pores will pass through the interstitial spaces between
the particles and will appear first in the eluate. Smaller analytes will be distributed
between the mobile phase inside and outside the particles and will therefore pass
through the column at a slower rate, hence appearing last in the eluate (Lim et al.
2020; Tan et al. 2015). The fungus Cunninghamella verticillata lipases were
reported as a monomeric proteins with molecular masses of 49 and 42 kDa as
determined by SDS–PAGE and gel filtration chromatography (Gopinath et al.
2002). Yarrowia lipolytica LgX64.81 lipase was purified by gel filtration on
Sephacryl S-100 to receive overall yield of 72% and a 3.5-fold increase in the
specific lipase activity (Turki et al. 2010). The lipase from Mucor hiemalis
f. corticola was purified to 12.63-fold with a final yield of 27.7% through following
purification steps; ammonium sulfate precipitation, dialysis, ion exchange chroma-
tography, and gel filtration column chromatography respectively (Ülker and
Karaoĝlu 2012). The lipase produced by Aspergillus niger was purified through
ammonium sulfate precipitation followed by Sephadex G-100 gel filtration. Molec-
ular mass of the purified lipase was 57 kDa as evident on SDS-PAGE analysis
(El-Ghonemy et al. 2021).
18.2.6.3 Affinity Chromatography
Affinity chromatography has been exclusively developed for the purification and
separation of enzymes based on extremely specific biological interactions and
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 409

reduces the number of steps necessary for lipase purification with greater yields of
purified enzymes (Mehta et al. 2018b). Affinity column chromatography purifies
proteins according to their specificaffinity toward a ligand. Such chromatography is
also known as immobilization, which is normally called immobilized metal affinity
chromatography. When the analyte molecules in the crude enzymes interact with the
solid resin, which has a covalent linkage with a polydentate metal-chelating group
binding to a metal ion, e.g., nickel (Ni
2+
), surface-exposed amino acid residues of the
enzyme of interest will exchange with the water molecule in the metal coordination
site, thus the enzyme is immobilized (Lim et al. 2020). Immunoaffinity chromatog-
raphy or immunopurification is a type of affinity chromatography when a protein of
interest is purified by applying an antibody–antigen principle. Immunopurification
as of today is considered one of the strongest and most selective methods of protein
purification, with the purification factor ranging from 1000- to 10,000-fold in one
single step procedure (Yagmurov et al. 2017). The choice depends on the availability
of monoclonal antibody against the target protein and the composition of the crude
preparation, namely, the type and the concentration of the contaminants
(Venkatanagaraju and Divakar 2017). Recombinant lipase produced by
Trichosporon asahii MSR54 was purified by affinity chromatography and the
molecular mass was recorded as monomeric 27 kDa with 1.7 purification fold
(Kumari and Gupta 2015). A recombinant lipase obtained from Aspergillus niger
GZUF36 purified on Ni
2+
(Nickel resin)-NTA affinity chromatography column and
estimated relative molecular mass of 35 kDa was recorded (Xing et al. 2020). A
recombinant acidic lipase purified by Ni
2+
-NTA affinity chromatography column
observed to have molecular weight of 60 kDa as determined by SDS-PAGE analysis
(Zhang et al. 2019).
18.2.6.4 Hydrophobic Interaction Chromatography
Since lipases are known to be hydrophobic in nature, with large hydrophobic
surfaces around the active site, the purification of lipases could be significantly
achieved by opting for hydrophobic interaction chromatography. The hydrophobic
amino acid residues of lipases would be exposed by addition of salt ions, and then
allowed to interact with hydrophobic groups such as butyl, octyl, and phenyl
attached to a matrix facilitating protein–matrix interaction. Lipases produced by
Aspergillus fumigatus was purified by ammonium sulfate precipitation and octyl
sepharose column chromatography, which resulted in sevenfold purification, and the
molecular weight found to be 35 kDa indicating the enzyme was homo-dimer
(Mehta et al. 2018b). The specific activity of Aspergillus fumigatus lipase was
observed to be 14.34 U/mg with a fold purification of 6.96 through purification
using octyl sepharose column (Mehta et al. 2020). The dialyzed extracellular filtrate
from Penicillium sp. section Gracilenta CBMAI 1583 was subjected to octyl
sepharose chromatography using ammonium acetate buffer and 52.9 kDa protein
presented esterification activity on octyl oleate (Turati et al. 2019). The lipases from
C. rugosa and G. candidum were purified using a single step purification via
interfacial adsorption on strongly hydrophobic support with the yield of 9.7% and
10.9% after complete purification (De Morais et al. 2016). Recombinant lipases
410 J. Angelin and M. Kavitha

produced by Rhizopus chinensis were sequentially purified via Q Sepharose™Fast
Flow anion exchange column chromatography and Phenyl-Sepharose 4 fast flow
hydrophobic chromatography column chromatography (Jiang et al. 2020). Lipase
recovered from Aspergillus fumigatus was purified by octyl sepharose column
chromatography to 6.96 purification fold and purified to homogeneity confirmed
by SDS and Native PAGE (Kaur et al. 2019).
18.2.6.5 Aqueous Two-Phase Systems
Aqueous two-phase system has been viewed as a powerful purification technique
used in the separation and purification of biomolecules. Upon mixing of aqueous
solutions containing two different incompatible polymers, they will separate into
two distinct phases because of steric exclusion. If it is a mixture of a polymer and a
high ionic strength salt, the phase separation phenomenon also occurs since the salt
retains huge amount of water. This phenomenon is used to separate enzymes,
without compromising its activity. The most used pairs are polyethylene glycol
and dextran and PEG–potassium phosphate (Carvalho et al. 2017). Aqueous
two-phase system composed of a hydrophilic organic solvent and an inorganic salt
solution has many advantages, which include rapid phase-separation, high extraction
efficiency, low viscosity, high polarity differences between the phases, a gentle
aqueous environment, and may be formed by inexpensive chemicals easy to recycle.
These systems, formed by adding a salt solution to an aqueous solution of an organic
compound, have been recently proposed and used for the partitioning of different
biomolecules, such as proteins, amino acids, and other natural products (Souza et al.
2015). The mutant Trichosporon laibacchii lipase was partially purified using
aqueous two-phase systems with activity recovery of 80.4%, and purification factor
of 5.84 (Zhang and Liu 2010). The research conducted in 2017 has proved that
aqueous two-phase system is evaluated as promising method for purification of
extracellular lipase from Yarrowia lipolytica than traditional methods such as pre-
cipitation and ultrafiltration (Carvalho et al. 2017). Purification factor of Aspergillus
carbonarius lipase has been increased using PEG/potassium phosphate aqueous
two-phase system (Panajotova et al. 2017).
18.2.6.6 Reverse Micellar Extraction
A reverse micelle is a system that combines an aqueous dispersed phase and a
non-aqueous continuous phase with the assistance of amphiphilic molecules (Chen
et al. 2020). Reverse micellar extraction of the protein is an attractive liquid–liquid
extraction method for the downstream processing of the enzymes. Reverse micelles
are used as a reaction system for enzymatic catalysis, liquid–liquid extraction of
proteins, and protein refolding in the field of biotechnology (Basheer and Thenmozhi
2010). Selective extraction of the target biomolecule from mixture of enzymes/
proteins in these reverse micelles can be achieved by varying parameters both in
organic phase and in aqueous phase (Gaikaiwari et al. 2012). More importantly, the
formation of reverse micelles could activate lipase, allow lipase to molecularly
disperse so as to interact with substances efficiently, and provide a large interface
for the reaction. The water/sodium 1,4-bis-2-ethylhexylsulfosuccinate /isooctane
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 411

reverse micelle system was set up as a reaction medium for Candida rugosa lipase
AY30 to synthesize β-sitosterol laurate (Chen et al. 2020).
18.3 Assay Methods
Lipase activity is examined by the release of either free fatty acids or glycerol from
triacylglycerol. Since lipases act at the oil–water interface, change in properties of
interface is an important criterion for measuring lipolysis (Nagarajan 2012). The
assay methods are involved for measuring the hydrolytic activity as well as the
detection of lipase. The methods are classified as: (1) titrimetry, (2) spectroscopy
(photometry, fluorimetry, and infrared), (3) chromatography, (4) interfacial tensiom-
etry, (5) radio activity, (6) conductimetry, (7) turbidimetry, (8) immunochemistry,
and (9) microscopy (Singh and Mukhopadhyay 2012). The most commonly used
lipase assay protocol is the titrimetric assay using olive oil as a substrate because of
its accuracy, simplicity, and reproducibility. The titrimetric assays are performed in
emulsion containing synthetic triacylglycerols (triacetin, tributyrate, tricaprylate,
tripalmitate, or tristearin). Other assays reported for lipase activity include fluores-
cence, gas chromatography, HPLC-based assay, monomolecular film technique, oil
drop method, atomic force microscopy, and IR spectroscopy (Nagarajan 2012). Both
bacterial and fungal lipases are able to hydrolyze para-nitrophenyl (p-NP) esters
having C2–C16 (p-NP acetate to p-NP palmitate) in their fatty acid chain. Kinetics of
lipases for substrate hydrolysis depends on different esters. It is determined by
Michaelis constant (Km), that is substrate concentration at which the rate of reaction
is half of the maximum rate (Vmax). Vmax is the maximum rate when an enzyme is
fully saturated with substrate concentration (Shamim et al. 2018). Table 18.3 and
Fig. 18.5 provide the detailed information on different types of lipase assays. The
common lipase assays employed are discussed below in detail.
18.3.1 Titrimetry
In this method, the free fatty acids released as a result of lipase hydrolysis with
suitable substrate were titrated against alkali. Lipase activity of filamentous fungi
can be evaluated through titrimetric method using olive oil as substrate and phenol-
phthalein as a pH indicator (Griebeler et al. 2011; Resende et al. 2015; Gutarra et al.
2009). Triton X-l00 and gum arabic were used in the assay mixture, and both act as
emulsifiers (Ayinla et al. 2017; Kantak et al. 2011). Penicillium canesense and
Pseudogymnoascus roseus produced BPF4 and BPF6 lipases which were found to
be alkaline metallolipase, showing maximum activity at pH 11 and 9 respectively
and at the temperature 40 C were assessed by titrimetric method (Sahay and
Chouhan 2018). Talaromyces thermophilus lipase showed specific activity of
about 7300 122 and 9868 139 U/mg using Tributyrin and olive oil emulsion
as substrates, respectively, at pH 9.5 and 50 C (Romdhane et al. 2010).
Engyodontium sp. was found to produce maximum lipase of 7.2 U/mL using olive
412 J. Angelin and M. Kavitha

Table 18.3 Assays for quantification of extremophilic fungal lipases
S.
no Fungus Substrate Method pH Temp Km Vmax
Enzyme
activity Reference
1Aspergillus tamarii Olive oil Titrimetric
method
437
C 330.4 mg 53,690 U/
mL/min
23,666.66 U/
mL/min
Das et al. (2016)
2Aspergillus
fumigatus
p-NP
benzoate
Spectrometric
method
9.0 40 C 6.59 mM 7.29 mmol/
min/mL
–Mehta et al.
(2018a)
3Penicillium
chrysogenum
p-NP
palmitate
Spectrometric
method
–– 0.4 mM 47.61 U/mL –Kumar et al.
(2012b)
4Debaromyces
hansenii
Olive oil Titrimetric
method
–– – – 7.44 U/mL Papagora et al.
(2013)
5A. awamori p-NP
caprylate
Spectrometric
method
740
C–– 495.0 U/mL Basheer et al.
(2011)
6Candida rugosa p-NP
butyrate
Spectrometric
method
5.0
&6
30 C and
40 C
129.21 μM 0.034 μmol/
min
12.3 0.2 U/
mL
De Morais et al.
(2016)
Geotrichum
candidum
p-NP
butyrate
Spectrometric
method
465.44 μM 0.384 μmol/
min
11.9 0.2 U/
mL
7Sporisorium
reilianum SRZ2
p-NP
hexanoate
Spectrometric
method
8.0 65 C 0.14 mmol/
L
–– Shen et al.
(2020)
8Aspergillus japonicus
LAB01
p-NP
palmitate
Spectrometric
method
–– 0.13 mM 12.58 μmol/
min)
–Souza et al.
(2014)
9Candida antarctica
ZJB09193
p-NP
acetate
Spectrometric
method
8.0 52 C 0.34 mM 7.36 μmol /
min/mg
–Liu et al. (2012)
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 413

oil as substrate compared to other fungal members by titration method (Lanka and B
2018). An Antarctic basidiomycete yeast Guehomyces pullalans showed maximum
lipase activity using olive oil as inducer at pH 8.0 and 40 C (Demera et al. 2019).
Purified lipase from Aspergillus fumigatus showed utmost enzyme activity at opti-
mum temperature of 40 and pH 9.0 with kinetic parameters of V
max
and K
m
as
10.42 μmol/min/mg and 9.89 mM, respectively (Mehta et al. 2018b). A recombinant
lipase from Aspergillus niger GZUF36 utilized olive oil as substrate to exhibit
highest activity of 7.02 0.05 U/mL at 35 C and pH 4.0 using alkali titration
method (Xing et al. 2020). The extracellular lipase produced by Trichoderma
harzianum displayed maximal lipase activity of 1.58 IU/mL/min 0.11 with olive
oil as substrate (Rihani and Soumati 2019). The major advantage of the titrimetric
assay is the resistance to turbidity. The assay also has a disadvantage as the strong
buffers make it hard to hold a stable pH, thus it is tough for the assay to be performed
(Pohanka 2019). Aspergillus niger acidic lipase when fused with small
Fig. 18.5 Distinct assays for lipase activity
414 J. Angelin and M. Kavitha

ubiquitin-related modifier possessed a higher catalytic efficiency toward p-NP
caprylate and long-chain triglycerides (olive oil, triolein, and tripalmitin) than
Aspergillus niger acidic lipase (Zhang et al. 2019). Despite its disadvantages like
long analysis time (two determinations per hour), low sensitivity (1 μmol/mL),
tedious measurements, and errors due to incomplete titration, the titrimetric method
for lipases activity determination remains in use as a reference method (Stoytcheva
et al. 2012).
18.3.2 Spectrophotometry
The spectrophotometric methods for lipase activity determination make use of
synthetic lipase substrates transformed upon enzyme catalyzed hydrolysis into
products able to be detected spectrophotometrically. The predominant substrates
are p-nitrophenyl and naphthyl esters of the long chain fatty acids, and thioesters.
The lipolysis of the p-NP esters (laurates, palmitates, and oleates) gives rise to the
yellow colored p-nitrophenol, measured at 405–410 nm. In the assay, p-NP acetate,
p-NP butyrate, p-NP valerate, p-NP caproate, p-NP decanoate, p-NP dodecanoate,
p-NP myristate, and p-NP palmitate are used as substrates for lipase to determine the
substrate specificities (Pohanka 2019). The deficiency of this method is related to the
pH dependence of the p-NP absorption coefficient and the total absence of absorp-
tion at acidic pH values. In addition, p-NP esters could undergo a non-enzymatic
hydrolysis. The cleavage of the naphthyl esters (naphthylcaprylate, naphthylacetate,
and naphthylpropionate) yields naphthol, which is complexed with diazonium salts
to produce a red colored complex measured at 560 nm. p-NP palmitate hydrolysis
was maximal at 40 C and pH 7.0 for the Rhizomucor miehei lipase, and at 30 C and
pH 5.2 for the Rhizopus oryzae enzyme. The enzymes showed almost equal affinity
to p-NP palmitate, but the Vmax of the R. oryzae lipase was about 1.13 times higher
than that determined for R. miehei using the same substrate (Km 2017). Lipase from
R. miehei,R. stolonifer, and M. echinosphaera produced by solid-state fermentation
were able to catalyze trans-esterification reactions in organic media (Kotogán et al.
2014; Maharana and Ray 2014,2015). The purified Aspergillus niger-lipase
displayed the maximal activity at 20 C and pH 6.5 with the specific activity of
1293 U/mg determined by spectrophotometric method (Cong et al. 2019). Purified
cold active lipase recovered from Pichia lynferdii Y-7723 exhibited Km and Vmax
values of 1.68 mM and 8.32 μmol/min/mg respectively by spectrophotometric
method using p-NP butyrate as substrate (Bae et al. 2014).
18.3.3 Fluorimetry
Fluorimetry is a sensitive analytical technique allowing continuous monitoring of
enzyme activity. The fluorimetric methods for lipase activity are classified as
methods using chromogenic substrates and methods based on the quantification of
the fatty acids released after their conversion into chromogenic products. A variety
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 415

of fatty acid esters derived from parinaric acid, coumarin (umbelliferone), pyrenic
compounds, resorufin, fluorescein, etc. are used as chromogenic substrates
(Stoytcheva et al. 2012). Fluorescence spectroscopy analysis is used to monitor the
biomolecular interactions between surfactants and proteins. Lipase secreted by
Y. lipolytica (YlLip2) contains three tryptophan residues Trp233, Trp271, and
Trp285 at active site. The fluorescence spectrum of YlLip2 in HEPES buffer
shows one peak at 337 nm. The addition of lipo-peptides significantly decreased
the fluorescence intensity of YlLip2, but without significant blue shift (Janek et al.
2020).
18.3.4 Turbidimetry
Turbidimetry is a method for determining the concentration of a substance in a
solution by measuring the change of intensity of light in the direction of propagation
of the incident beam, with reference to a standard solution. Lipase activity quantifi-
cation is performed by monitoring the decrease with time in the absorbance of a
triacylglycerol emulsion, due to its de-emulsification with the release of free fatty
acids (Stoytcheva et al. 2012). The two main strategies employed are (1) the use of
labeled lipase substrates and quantification of the liberated products, and (2) the use
of unlabeled substrates and determination of free fatty acids labeled once released.
The radioactive substrates commonly used are oleoyl glycerols labeled with
14
Cor
3
H, as well as
131
I triglyceride analogues. Radioactive assays are specific and
sensitive analytical methods. However, they do not allow continuous monitoring
and are time-consuming, because of the implicated extraction steps to remove the
fatty acids in addition to the use of hazardous radioactive substances (Stoytcheva
et al. 2012).
18.3.5 Immunoassays
Immunoassays are well known for their high specificity and sensitivity for lipase
activity. These methods are of primary importance for clinical diagnostics where
they are applied for lipases quantification in serum and plasma, tissues and cell
culture lysates, and duodenum. A number of ELISA-based clinical test kits have
been developed and are useful in the range up to 500 ng/mL lipase. These techniques
are not suitable for the evaluation of the activity of lipases originated from other
sources, because they require the selection of a wide range of specific antibodies. In
addition, the enzyme could form aggregates, which limits the accuracy of the
immunological assays (Stoytcheva et al. 2012).
416 J. Angelin and M. Kavitha

18.3.6 Conductimetry
Conductimetric evaluation of lipase activity is based on the measurement of the
variation in the solution conductance due to electrical charge concentration change
as a result of the release of free fatty acids. Triacetin is a suitable substrate due to its
water solubility. The limiting equivalent conductivity of the liberated acetate anions
is higher in comparison with that of the long-chain fatty acids, increasing the
sensitivity of the determinations. The drawback of the technique is that triacetin is
not a specific lipase substrate and conductimetric measurements suffer from high
temperature dependence (Stoytcheva et al. 2012).
18.4 Industrial Applications
Lipases are produced by plants, animals, and microorganisms of extreme environ-
mental conditions. Specifically, hydrolytic lipases of filamentous fungi have grabbed
more attention due to high yield in production, many varieties of catalytic activities
that satisfy industrial procedures, and ease of genetic manipulation (Hasan et al.
2006). Lipases are exploited in waste water treatment (degreasing of lipid clogged
drains), pharmaceutical (resolution of racemic mixtures), dairy (hydrolysis of milk,
fat), leather (removal of lipids from hides and skin), detergent (removal of oil/fat
stains), and medical (diagnostic tool in blood triglyceride assay) industries (Verma
and Sharma 2014). Lipases are able to catalyze acidolysis (where an ester and
carboxylic acid are involved), alcoholysis (where an ester and an alcohol are
involved), aminolysis (where an ester is allowed to react with an amine), and
inter-esterification (where two acyl groups are exchanged between two esters)
(Sharma et al. 2016a). The industrial process demands biocatalysts that can resist a
range of harsh conditions, including temperature, pH, salinity, and pressure, while
exhibiting high conversion rates and reproducibility (Jin et al. 2019). The original
physicochemical properties of fungal lipases are altered through various attempts so
that the efficiency of the enzymes would satisfy the industrial strategies. It is more
important to be aware about the characteristics of a particular enzyme before
indulging in modification techniques. These issues include the type and size of the
protein, the structure and size of the modifying reagent, the chemical reactions
involved in the modification procedures, and the conditions of such modifications
(Dalmaso et al. 2015). Lipases which are able to combat during industrial processes
are recruited for the development of several products. A major requirement for
commercial lipases is thermal stability which would allow enzymatic reaction to
be performed at higher temperatures and would be helpful to increase conversion
rates and substrate solubility. Industrial and biotechnological processes also require
thermo-stable lipases for use in processes such as grease esterification, hydrolysis,
trans-esterification, inter-esterification, and organic biosynthesis (Jin et al. 2019).
Aspergillus fumigatus is a potential lipase producing candidate for industrial
applications such as bioremediation, detergent, leather, and pharmaceutical
industries (Mehta et al. 2018a). Rhizopus chinensis produced novel mutant lipases
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 417

Table 18.4 Industrial applications of extremophilic fungal lipases
S. no Fungus/Lipase Mechanism Properties Applications References
1. CALB-type
(Sporisorium
reilianum SRZ2)
SRL
Short-chain flavor ester synthesis High thermo-stability, pH stability,
and good organic solvent tolerance
Used as flavor and fragrance
compounds in food, beverage,
cosmetic, personal care, chemical,
and pharmaceutical industries
Shen et al.
(2020)
2. Mucor hiemalis
f. corticola
–Stable in organic solvents Useful in organic synthesis and
biodiesel production
Ülker and
Karaoĝlu
(2012)
3. Candida
sp. 99–125
Wax esters from oleic acid and
cetyl alcohol
Dynamic viscosity, flash point,
solidifying point, pour point, and
acid value of wax esters are similar
to those of jojoba oil
Widely used in lubricant,
pharmaceutical, cosmetic, and
plasticizer industries
Deng et al.
(2011)
4. Trichosporon
laibachii
Synthesis of ((R)-5-acetoxy-1,3-
oxathiolan-2-yl)ethyl benzoate
using reversible hemi-thioacetal
transformation
–Useful in pharmaceutical industries Zhang
et al.
(2020)
5. CALB Butyl oleate synthesis by
immobilization
Thermal stability and operational
stability
Biodiesel production Silva et al.
(2012)
6. Aureobasidium
pullulans
Hydrolysis of oils Resistance to some organic
solvents, surfactants, and ions
Detergent production, biodiesel
synthetization, and food
manufacturing
Li et al.
(2019)
7. Thermomyces
lanuginosus
Fish oil ethanolysis through
adsorption
Improved stability Useful in food industry, biodiesel,
and fine chemical production
Jorge and
Ninow
(2016)
8. Thermomyces
lanuginosus
Synthesis of isoamyl oleate
(biolubricant) by esterification
reaction
High catalytic activity and
reusability
Hydraulic and metal working
fluids, hydraulic of harvesters,
drilling oils, slab and gear oils, and
lubricants for power saw chains
Lage et al.
(2015)
418 J. Angelin and M. Kavitha

9. Rhodotorula
glutinis HL25
Utilization of waste frying oils –Biodiesel production and organic
synthesis reactions
Taskin
et al.
(2016)
10. Aspergillus
fumigatus
Esterification process for the
synthesis of ethyl acetate and
ethyl lactate
–Useful in food industry as artificial
flavor enhancers
Mehta
et al.
(2020)
11. Aspergillus niger Synthesis of flavor ester (ethyl
lactate, butyl butyrate, and ethyl
caprylate) in soybean-solvent
system
Cold, acid, and alkaline tolerance Useful in food processing, such as
cheese ripening, alcoholic
beverage production, and other
fermented foods production
Cong et al.
(2019)
12. Penicillium sp.
Section Gracilenta
CBMAI 1583
Esterification reaction Thermo-tolerant and acid pH
tolerance
Treatment of dairy and industry
effluents, resolution of esters in the
pharmaceutical industry or in the
food industry
Turati
et al.
(2019)
13. Rhizomucor
endophyticus
Trans-esterification Regioselectivity Biofuel industry Yan et al.
(2016)
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 419

(Lipr27RCL-K64N and Lipr27RCL-K68T) with improved thermo-stability through
a combination of B factor analysis and site-directed mutagenesis, and these resultant
enzymes represent attractive candidates for use in industrial applications (Jiang et al.
2020). Table 18.4 and Fig. 18.6 display the industrial application of fungal lipases.
The applications of fungal lipases in various industries are explored here in detail.
18.4.1 Detergent Industry
Laundry detergents are widely used because they are required for washing purposes
but chemical agents deposited in the environment leads to contamination of ground
water. These pollutants are responsible for health-related issues which should be
replaced by other harmless stain removal agents. The enzyme-based detergents have
disclosed superior cleaning properties over synthetic agents that are active at low
washing temperatures and environmentally friendly. Additionally, enzyme carrying
detergents upgrade the fabric quality and keep the color bright (Hasan et al. 2010).
Lipases extend profit-oriented services in laundry detergents where thermo-stability
and tolerance in the alkaline environment are highly recommended. An estimated
1000 tons of lipases are added to approximately 13 billion tons of detergents
produced each year (Hasan et al. 2010; Verma and Sharma 2014). During launder-
ing, lipases adsorb onto the fabric surface to form a stable fabric–lipase complex and
then hydrolyze oil stains present on the fabric. The complex is resistant to the harsh
wash conditions and is retained on the fabric during laundering (Rigoldi et al. 2018).
To be detergent compatible, lipases should convince the following attributes: stabil-
ity at alkaline pH, solubility in water, tolerance to detergent proteases and
Fig. 18.6 Principal industrial applications of extremophilic fungal lipases
420 J. Angelin and M. Kavitha

surfactants, and low substrate specificity (Sharma et al. 2018; Maharana and Ray
2015). Talaromyces thermophilus produced thermo-alkaline lipase which could be
used as commercial wash and bleach agent in detergent industry (Romdhane et al.
2010). Cold active lipase produced by Cryptococcus sp. Y-32 established low
temperature and high pH stability, which has the ability to degrade lipid wastes in
cold regions and useful in detergent formulations for cold temperature washing of
delicate clothes (Kumar Maharana and Mohan Singh 2018). Lipases from
psychrotolerant fungi Penicillium canesense and Pseudogymnoascus roseus were
characterized as detergent compatible alkaline lipases that facilitate cold-washing if
included in detergent formulations (Sahay and Chouhan 2018). Aspergillus tamarii
JGIF06 lipase exhibited oil-de-staining efficiency in hot water when tested on cotton
fabric pieces stained with peanut oil (Das et al. 2016).
18.4.2 Medical and Pharmaceutical Industries
The main application of fungal lipases is in the treatment of diseases such as
dyspepsia, gastrointestinal disturbances, cutaneous manifestations of digestive
allergies, and cancer. Lipases also serve as a diagnostic tool in medicine, thereby
justifying the growing demands (Yagmurov et al. 2017). Immobilized lipase B from
Candida antarctica (Novozyme 435) catalyzes direct esterification of n-butanol and
lactic acid and the lactate esters thus produced are key intermediates in chemical
preparations for medical and pharmaceutical purposes (Knez et al. 2012). The three
recombinant lipases produced by Trichosporon asahii found to be enantioselective
which was determined by esterification of racemic 1-phenylethanol and capric acid.
These enantioselective lipases showed nearly 40% enantiomeric excess that is
indispensable in the field of pharmaceuticals for the chiral resolution of drugs
(Singh and Gupta 2016). The synthesis of both enantiomers of four new
phenylthiazole-based amines by enantiomer-selective acylation of racemic amines
and by hydrolysis of the corresponding racemic amides using lipase B from Candida
antarctica (Novozyme 435) as chiral catalyst was performed with good yields and
excellent enantioselectivities (Radu et al. 2014). A novel alkali tolerant recombinant
lipase TALipA produced by Trichosporon asahii MSR54 was found to be
enantioselective, regioselective, and long chain fatty acid selective which is the
major requirement of pharmaceutical industries (Kumari and Gupta 2015). Candida
antarctica lipase Awas investigated for its potential in trans-esterification of the
phenolic OH group of capsaicin and several capsaicin analogues using Capsicum
oleoresin for pharmaceutical purposes (Diaz-vidal et al. 2020). The efficient green
enzymatic process for L-ascorbyl palmitate synthesis by stable indigenously
immobilized Candida antarctica lipase B (CALB) lipase using underivatized
substrates with high space time yield of 15 g/L/h, and no by-product formation
with recycling of substrates makes the overall process clean and environment-
friendly (Yadav et al. 2018). Medium-chain, oleic (18:1n-9), and medium-chain
fatty acid and structured lipids were produced by acidolysis reaction in solvent-free
medium with capric (10:0) and lauric (12:0) free fatty acids and triolein or olive oil,
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 421

using Yarrowia lipolytica lipase as biocatalyst. This lipase showed promising
properties as a potential biocatalyst that may be effectively used in the production
of bioactive structured lipids, which might be applied for the prevention of metabolic
and inflammatory disorders related to obesity (Akil et al. 2020).
18.4.3 Food Industry
The majority of the food industries search for enzymes that exhibit catalytic speci-
ficity, thermo-stability, high catalytic activity in a wide range of pH and temperature,
and structural properties which can be immobilized with high catalytic efficiency
(Sharma et al. 2016b; Contesini et al. 2017). The literature presents several studies
that address lipase application in food industry in dairy processing—baking, oil,
meat, fish, and beverages (Coelho and Orlandelli 2020). The chemically synthesized
products cannot be regarded as “natural,”which is a fatal blow to the increasing
demand for “natural”flavor esters. In contrast, the enzymatic synthesis of flavor
esters catalyzed by lipases is a more efficient, economically benign, and promising
alternative approach to traditional methods (Shen et al. 2020). Thermomyces
lanuginosus lipase immobilized on styrene–di-vinyl benzene beads was observed
to produce butyl butyrate which could be employed as an artificial fruit flavor ester in
food industry (Martins et al. 2013). Thermomyces lanuginosus lipase immobilized
on PEGylated polyurethane particles was further coated with polyethyleneimine to
increase the stability of lipase enzyme. Therefore, it was efficiently used in the
production of ethyl esters from fish oil compared to the free enzyme (Jorge and
Ninow 2016). Since the enzymatic technique is milder and greener, and can mini-
mize oxidation, polymerization, and cis–trans isomerization of oil containing poly-
unsaturated fatty acids, lipase catalysis has been a promising technique among the
enrichment methods. Trichosporon sp. lipase F1–2 showed many good
characteristics for industrial applications, including high catalytic activity and sta-
bility under alkaline conditions, high activity without metal ions as cofactors,
tolerance to various organic solvents and sn-1,3 regioselectivity. Since the lipase
preferred short- and medium-chain fatty acid esters and discriminated against long-
chain fatty acid esters, its potential application in enrichment of eicosapentaenoic
acid and docosahexaenoic acid in fish oil was investigated (Cao et al. 2020).
Aspergillus fumigatus lipases are involved in the synthesis of two esters (ethyl
acetate and ethyl lactate) which could be used in high value-added products due to
their low toxicity. They can also be used in artificial fruit essence and give artificial
flavors such as pineapple, bananas, and strawberry in confectionary, ice-cream,
cakes, etc. (Mehta et al. 2020). The sn-1,3 selectivity of extracellular lipase from
A. niger GZUF36 has great potential in the synthesis of functional oils (Xing et al.
2020). Immobilization of CALB onto hydrophobic anion exchange resin Purolite
MN102 provided esterification catalytic mechanism to yield two valuable esters such
as isoamyl acetate and L-ascorbyl oleate to be used as food ingredients (Milivojevic
2016). Soybean oil deodorizer distillate, a by-product of vegetable oil refining
industries, can be hydrolyzed and esterified using Amano-30 (a crude lipase from
422 J. Angelin and M. Kavitha

Candida rugosa) and NS-40013 (an immobilized lipase from Candida antarctica)to
prepare functional foods like sterols and tocopherols. (Bengal and Bengal 2019).
The appropriate combination of S. lactis and Lactobacillus and the addition of lipase
(Aspergillus oryzae)efficiently improved the quality of yogurt-flavored bases. In
addition, the addition of fungal lipase to fermentation conducted by mixed lactic acid
bacteria significantly enhanced the physicochemical properties, especially total
volatile organic acids (Huang et al. 2020).
18.4.4 Biodiesel Production
Biodiesel is composed of methyl-esterified fatty acids derived from trans-
esterification of triglycerides by enzymatic action, providing a number of advantages
such as the reduction in the operational process in the manufacture and separation of
glycerol by-products (Dalmaso et al. 2015). The enzymatic trans-esterification is
more promising as it offers advantages with an environment-friendly option com-
pared to the chemical processes, such as mild reaction condition, less energy
intensity, higher yield in esters, as well as better recovery of glycerol and the
trans-esterification glycerides with high free fatty acid contents (Yan et al. 2016).
Lipases are versatile biocatalyst for biodiesel synthesis that requires relatively simple
downstream processing steps for the purification of biodiesel and its by-product
glycerol. Immobilized lipases obtained from filamentous fungi and yeasts are more
advantageous than free lipases because they are reusable, cost effective and also pos-
sess improved catalytic property (Aguieiras et al. 2015). Extremophilic fungal
lipases produced by Yarrowia lipolytica,Candida antarctica,Candida rugosa,
Penicillium camembertii,Rhizomucor miehei, and Thermomyces lanuginosus are
immobilized on solid support for biodiesel production by the following methods
such as cross-linking, physical adsorption, covalent bonding, and entrapment (Zhao
et al. 2015). Among fatty esters of industrial interest, 1-butyl oleate is used as
biodiesel additive to decrease the viscosity of diesel in winter use, polyvinyl chloride
plasticizer, water-resisting agent, and in hydraulic fluid (Séverac et al. 2011).
Usually, the synthesis of butyl oleate is conducted in the presence of concentrated
sulfuric acid through a chemical route, which has many disadvantages, such as
formation of by-products, disposal of acid wastewater, and equipment erosion by
the concentrated acid (Wang et al. 2010). Therefore, the enzymatic synthesis
becomes more and more interesting, since enzymes have high selectivity, specificity,
and activity in mild reaction conditions. Furthermore, lipases have broad availabil-
ity, low cost, mild reaction conditions, no need for co-factors, and substrate speci-
ficity. Since halophilic lipases are highly desirable for biodiesel production,
halophilic lipase obtained from Fusarium solani has been suggested to be utilized
for industrial purposes (Geoffry and Achur 2018). Lipases from Candida antarctica,
Thermomyces lanuginosus, and Rhizomucor miehei were covalently immobilized on
epoxy-functionalized silica and used to produce biodiesel by trans-esterification of
canola oil with methanol (Babaki et al. 2015). A recombinant CALB immobilized on
core-shell polymeric supports enables production of esters using residual fatty acids
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 423

as substrates which could be used for biodiesel and cosmetic production (Cipolatti
et al. 2018). A recombinant cold-adapted lipase belonging to Rhizomucor
endophyticus that possess high yield and excellent properties was conferred for its
great potential for biodiesel production in bioenergy industry (Yan et al. 2016).
Aspergillus niger lipase immobilized on magnetic nanoparticle was reported to
produce high quality biodiesel at 85.3% (Jambulingam et al. 2019).
18.4.5 Bioremediation
Due to escalated human population ultimately our environment has been
contaminated with improper disposal of waste discharges from various industrial
sectors such as dairy, food industries, oil refinery, poultry house, pesticides, and
wool processing factories. Therefore, biocatalysis process came into existence which
is cost-effective and energy-efficient targeting lipid-containing waste material and
conversion into value-added products (Kumar et al. 2019). The recombinant lipase
constructed by cloning lipase gene from Trichosporon coremiiforme V3 and func-
tionally expressed in Pichia pastoris X33 exhibited several significant industrially
important properties such as high temperature, pH stability, wide organic solvent
tolerance, and broad hydrolysis range on vegetable oils, which could be successfully
used in bioremediation of oil polluted sites (Wang et al. 2015). Lipase producing
Alternaria sp. and Aspergillus flavus were suggested for bioremediation of oil
contaminated sites (Wadia and Jain 2020). The lipase produced by Penicillium
chrysogenum showed promising results in the remediation of used cooking oil
(Kumar et al. 2012b). Aspergillus awamori BTMFW032 isolated from seawater
has potential for use in industries for the production of extracellular lipase under
submerged fermentation, which could be used in bioremediation of oil laden effluent
(Basheer et al. 2011). Penicillium sp. DS-39 lipase exhibited a broad substrate range
with distinct specificity for oils and triacylglycerols of long unsaturated fatty acids
and was found to be significantly stable in the presence of non-polar hydrophobic
solvents. These features render Penicillium sp. DS-39 lipase suitable for potential
applications in non-aqueous biocatalysis such as biodiesel production and enzymatic
restructuring, by inter-esterification of different oils and fats and biodegradation of
oil spills in the environment (Dsm et al. 2011). An alkaline lipase produced by
Cladosporium langeronii has been suggested for potential application in degradation
of oil and cleaning up the environment (Sadati et al. 2015). The fungal strains of
Thermomyces lanuginosus were stimulated to produce lipase in the presence of
either triacetin or olive oil suggesting the adaptive nature and contribute to the
deterioration of oil seeds during storage (Sreelatha et al. 2017). Mrakia blollopis
CBS8921 (Antarctic yeast) lipase assumed to be promising bio-remediation agent
for cleaning up unwanted milk fat curdles from dairy milk wastewater under low
temperature conditions (Tsuji et al. 2013). Lipase produced by Fusarium incarnatum
KU377454 was immobilized on Fe
3
O
4
nanoparticles and was found to be efficient
for degradation of waste cooking oil which pollutes environment to a greater extent
(Joshi et al. 2019).
424 J. Angelin and M. Kavitha

18.4.6 Cosmetic Industry
Active lipases can mainly be found in cosmetics for surficial cleansing, anti-cellulite
treatment, or overall body slimming, where they are responsible for the mild
loosening and removal of dirt and/or small flakes of dead corneous skin (i.e.,
peeling) and/or assist in breaking down fat deposits, often in combination with
further enzymes such as proteases (Ansorge-Schumacher and Thum 2013). Flavor
and fragrance compounds are important chemical ingredients of cosmetic products,
with further applications in the food, feed, chemical, and pharmaceutical industries.
The lipase retrieved from Aspergillus fumigatus synthesized methyl butyrate which
is an essential flavor enhancer in perfumes. Methyl butyrate is the methyl ester of
butyric acid having a characteristic sweet and fruity odor similar to that of apples and
pineapples. It can also be used as emulsifier in the food and cosmetic industries
(Kaur et al. 2019). The fatty amides (9Z,12Z)-N-dodecyloctadeca-9,12-dienamide
(3) and N-dodecyl-2-hydroxybenzamide (5), respectively derived from linoleic acid
and salicylic acid were synthesized through aminolysis reactions catalyzed by
CALB. These amphiphilic compounds receive greater attention from cosmetic
industry due to a range of beneficial properties for skin (Mouad et al. 2016).
18.4.7 Leather Industry
In leather processing, lipases are used in the removal of natural fat present in animal
skin. Separate degreasing step is required for animal skins with high fat content.
Insufficient removal of natural fat during processing prevents the chemicals from
penetrating into the leather which leads to negative impacts on the quality of finished
leather such as hardness without sufficient internal softness, fatty spew formation,
stained appearance due to chrome soap formation, weak bonding of the finishing
layer, and bad odor. Traditionally excess fat was removed using solvents and
emulsifiers but they add to pollution. Alternatively, lipases of microbial origin can
be used in degreasing to reduce pollution. Research on the use of lipase for
degreasing dated a few decades ago. Use of acid lipase of fungal origin in degreasing
on pickled pelts was reported in 1978 (Yeshodha et al. 1978). Acid lipase from
Rhizopus nodosus along with commercial degreaser was used in degreasing in 1982
(Muthukumaran and Dhar 1982). Recently, with the availability of commercial
lipases, the effectiveness of acid and alkaline lipases of commercial origin in
degreasing at various stages of leather processing was also reported (Afsar and
Cetinkaya 2008). In leather industries, tannery fleshing is a waste generated after
de-hairing of animal hides which are rich in proteins and fats. These wastes create
pollution which should be recovered through lipases and utilized as poultry feed and
bio-fertilizer. Application of extracellular lipase using A. tamarii MTCC 5152 on
tannery fleshing shows 92% of fat solubility. The enzymatically recovered fat can be
used for biodiesel manufacture and the residual protein as a fish or poultry feed
(Dayanandan et al. 2013).
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 425

18.4.8 Biosensors
A biosensor can be defined as a device resulting from the association between a
sensitive biological element and a transducer, which converts the biological signal
into a measurable physical signal. Biosensors are strong candidates for pesticide
residue determination and are becoming more relevant in environmental and food
analysis (De Moura et al. 2019). In the last few decades, the development of
enzymatic biosensors has attracted increasing attention and offered a new approach
to organophosphorus quantification. Many enzymes have been used in various
studies to develop biosensors for the detection of organophosphorus compounds in
aquatic environments. Biosensor system was developed with lipase as an alternative
to detect organophosphorus compounds which are toxic substances in polluted water
leading to serious health issues such as digestive diseases and damage of liver and
kidney (Kartal et al. 2007; Zehani et al. 2014b). Two novel impedimetric biosensors
for highly sensitive and rapid quantitative detection of diazinon (pesticide) in
aqueous medium were developed using two types of lipase, from Candida rugosa
(microbial source) and from porcine pancreas (animal source) immobilized on
functionalized gold electrode. Lipase is characterized to specifically catalyze the
hydrolysis of ester functions leading to the transformation of diazinon into diethyl
phosphorothioic acid and 2-isopropyl-4-methyl-6-hydroxypyrimidine. These
biosensors are very promising analytical tool for the detection of organophosphate
pesticides. It also used in the monitoring of detoxification processes for the treatment
of wastewaters generated by the industrial production of organophosphate pesticides
(Zehani et al. 2014a). A novel and simple optical biosensor to detect triglycerides has
been successfully constructed by using pectin hydrogel membrane as the indicator
pH and chromoionophore ETH 5294, with lipase (Candida antarctica) as the
catalyst (Hasanah et al. 2019). Chemical modification of elastin-like recombinamers
is carried out via enzymatic catalysis with CALB in a mild and effective one-pot
reaction. The coupling reaction with p-phenylazoaniline allowed to obtain photo-
responsive and electromagnetic radiation-sensitive biomaterials that could find a use
as sensors in devices for controlled drug delivery, whereas coupling with
4-[(2-amino) carbamoyl]phenylboronic acid led a glucose-responsive elastin-like
recombinamers with potential for the design of glucose-sensitive biosensors and
actuators for use in drug-delivery systems, for instance as insulin-delivery systems
for patients with diabetes mellitus (Testera et al. 2019).
18.5 Immobilization of Lipases
In general, enzymes have some drawbacks in their free form such as difficulty in
recovery, insoluble in some media, and unstable in organic solvents in pH and
temperature ranges. Thus enzymes are immobilized to improve the stability of
enzymes to heat and extreme pH conditions and allow their recovery, thereby
reducing the cost of enzymatic processes. Immobilization is a powerful tool not
only for the reuse of expensive enzymes in industrial processes, but also to improve
426 J. Angelin and M. Kavitha

catalyst stability, reaction selectivity, catalytic activity, resistance to inhibitors,
product purity, among others. Lipases are immobilized by the methods including
adsorption and covalent attachment, cross-linking, adsorption followed by cross-
linking, and physical entrapment using commercial carriers. Immobilization of
lipases has been performed by adsorption on hydrophobic adsorbents, including
glass beads coated with hydrophobic materials, methylated silica, phenyl-sepharose,
poly-(ethylene glycol)-sepharose, polypropylene particles, polypropylene hollow-
fibers and nonwoven fabric, and nitrocellulose membranes. Immobilized enzymes
are used in many commercialized products for higher yields (Sharma and Kanwar
2014). 2-Ethylhexyl oleate was synthesized using Candida antarctica lipase
immobilized on magnetic poly (styrene-co-divinylbenzene) particles in a continuous
packed-bed bioreactor. 2-Ethylhexyl oleate is an example of a commercially impor-
tant ester with applications in the cosmetics, pharmaceutical, food, and chemical
industries. These emollient esters are widely used as skin softening and moisturizing
agents in creams, lotions, sunscreen products, makeup, and antiperspirants (da Silva
et al. 2020). Esterification of lauric acid with n-butanol was catalyzed by
immobilized Candida antarctica lipase in aqueous-organic biphasic solvent system
(Shankar et al. 2013). Optimized glyceryl monoundecylenate synthesis can be used
as a useful reference for industrial synthesis of fatty acid esters of glycerol by the
immobilized CALB (Yadav et al. 2017).
Immobilization of Yarrowia lipolytica lipase on macroporous resin improved
reusability of lipase and provided a chance to expand the application of marine
microbial lipase in organic system to catalyze hydrolysis and esterification in harsh
condition (Sun et al. 2015). Green synthesis of pentaerythritol monoricinoleate was
carried out using CALB immobilized on hydrophobic adsorbent via interfacial
activation (Yadav et al. 2019). Rhizomucor miehei lipase (RML) immobilized on
octyl agarose–polyethylenimine–dextran sulfate has a reduced release of enzyme to
the medium under drastic conditions and enables reusability (Virgen-ort and
Fernandez-lafuente 2016). Biocompatible hybrid blend of cellulosic copolymers
made of hydroxypropyl methylcellulose and chitosan is designed for immobilization
of RML, in order to construct the robust biocatalytic system to synthesize industri-
ally important dodecanoate compounds. Butyl dodecanoate is a fatty acid ester,
which is a colorless liquid oil having pleasant fruity smell and is widely used in
pharmaceuticals, cosmetics, perfumery, food, and beverages as a flavoring ingredi-
ent (Badgujar et al. 2017). RML was immobilized on montmorillonite K-10 by
adsorption and in polyvinyl alcohol by entrapment to obtain a more stable and active
lipase preparation. The thermal stability of the enzyme was significantly enhanced
after immobilization and it showed 23.5-fold higher catalytic efficiency than that of
the free enzyme. It also exhibited better reusability performance than that of polyvi-
nyl alcohol-based lipase (Ece et al. 2019).
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 427

18.6 Conclusion
Extremophilic fungal lipases retrieved from diverse extreme resources have
exhibited more remarkable performance than other microbial lipases in industrial
sectors. This book chapter explored the diverse screening approaches to discover
novel extremophilic fungi with true lipase producing ability. In order to exploit
extremophilic fungal lipases to their exact potential and sketch out their inherent
characteristics, purification to homogeneity is inevitable. Detailed purification
schema was designated. Various assays used in the quantification of extremophilic
fungal lipases were described comprehensively. The operational behavior of
extremophilic lipases was strengthened through immobilization which is absolutely
necessary for successful industrial applications. Recombination had ameliorated
lipase yield with high specific activity and renders favorable roles in different
biotechnological applications. Extremophilic fungal lipases are extensively used in
industries such as dairy, beverage, food, detergent, pharmaceuticals, textile,
cosmetics, fuel, fat and oil, and agrochemicals, and in pollution control and produc-
tion of personal care products. The expansive research conducted on fungal lipases
of extreme origins and their immense potential for diverse industrial applications
evidences that these lipases are dominant over other counterparts and holds a highly
prospective future in commercial, health, and environment sectors.
References
Aamri LE, Hafidi M, Scordino F, Krasowska A, Lebrihi A, Orlando MG, Barresi C, Criseo G,
Barreca D, Romeo O (2020) Arthrographis curvata and Rhodosporidium babjevae as new
potential fungal lipase producers for biotechnological applications. Braz Arch Biol Technol
63. https://doi.org/10.1590/1678-4324-2020180444
Adan Gökbulut A, Arslanoǧlu A (2013) Purification and biochemical characterization of an
extracellular lipase from psychrotolerant Pseudomonas fluorescens KE38. Turk J Biol 37:
538–546. https://doi.org/10.3906/biy-1211-10
Afsar A, Cetinkaya F (2008) A research on increasing the effectiveness of degreasing process by
using enzymes. Tekstil Ve Konfeksiyon 18:278–283
Aguieiras ECG, Cavalcanti-Oliveira ED, Freire DMG (2015) Current status and new developments
of biodiesel production using fungal lipases. Fuel 159:52–67. https://doi.org/10.1016/j.fuel.
2015.06.064
Akbari E, Beheshti-maal K, Nayeri H (2016) Production and optimization of alkaline lipase by a
novel Psychrotolerant and halotolerant strain Planomicrobium okeanokoites ABN-IAUF-
2 isolated from Persian Gulf. Int J Med Res Heal Sci 5:139–148
Akil E, Pereira AS, El-bacha T, Amaral FF, Torres AG (2020) Efficient production of bioactive
structured lipids by fast acidolysis catalyzed by Yarrowia lipolytica lipase, free and immobilized
in chitosan-alginate beads, in solvent-free medium. Int J Biol Macromol 163:910–918. https://
doi.org/10.1016/j.ijbiomac.2020.06.282
Akmoussi-Toumi S, Khemili-Talbi S, Ferioune I, Kebbouche-Gana S (2018) Purification and
characterization of an organic solvent-tolerant and detergent-stable lipase from Haloferax
mediterranei CNCMM 50101. Int J Biol Macromol 116:817–830. https://doi.org/10.1016/j.
ijbiomac.2018.05.087
428 J. Angelin and M. Kavitha

Alabdalall AH, Alanazi NA, Aldakeel SA, Abdul Azeez S, Borgio JF (2020) Molecular, physio-
logical, and biochemical characterization of extracellular lipase production by aspergillus Niger
using submerged fermentation. Peer J 8:e9425. https://doi.org/10.7717/peerj.9425/table-5
Ansorge-Schumacher MB, Thum O (2013) Immobilised lipases in the cosmetics industry. Chem
Soc Rev 42:6475–6490. https://doi.org/10.1039/c3cs35484a
Ayinla ZA, Ademakinwa AN, Agboola FK (2017) Studies on the optimization of lipase production
by Rhizopus sp. ZAC3 isolated from the contaminated soil of a palm oil processing shed. J Appl
Biol Biotechnol 5:30–37. https://doi.org/10.7324/jabb.2017.50205
Babaki M, YousefiM, Habibi Z, Brask J (2015) Preparation of highly reusable biocatalysts by
immobilization of lipases on epoxy-functionalized silica for production of biodiesel from canola
oil. Biochem Eng J 101:23–31. https://doi.org/10.1016/j.bej.2015.04.020
Badgujar VC, Badgujar KC, Yeole PM (2017) Immobilization of Rhizomucor miehei lipase on a
polymeric film for synthesis of important fatty acid esters: kinetics and application studies.
Bioprocess Biosyst Eng 40:1463–1478. https://doi.org/10.1007/s00449-017-1804-0
Bae J, Kwon M, Kim I, Hou CT, Kim H (2014) Purification and characterization of a cold-active
lipase from Pichia lynferdii Y-7723: pH-dependent activity deviation. Biotechnol Bioprocess
Eng 19:851–857. https://doi.org/10.1007/s12257-014-0300-5
Balan A, Ibrahim D, Abdul Rahim R, Ahmad Rashid FA (2012) Purification and characterization of
a thermostable lipase from Geobacillus thermodenitrificans IBRL-nra. Enzyme Res 2012:
987523. https://doi.org/10.1155/2012/987523
Bamitale Osho M, Quadri Adio O (2015) Screening, optimization and characterization of extracel-
lular lipase of aspergillus Niger atcc 1015. J Microbiol Biotechnol Food Sci 05:40–44. https://
doi.org/10.15414/jmbfs.2015.5.1.40-44
Basheer SA, Thenmozhi M (2010) Reverse micellar separation of lipases: a critical review. Int J
Chem Sci 8:57–67
Basheer SM, Chellappan S, Beena PS, Sukumaran RK, Elyas KK, Chandrasekaran M (2011)
Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and
application in oil effluent treatment. N Biotechnol 28:627–638. https://doi.org/10.1016/j.nbt.
2011.04.007
Bengal W, Bengal W (2019) Functional foods from soybean oil deodorizer distillate using Candida
Rugosa and Candida Antarctica lipases. Chem Sci Trans 8:268–272. https://doi.org/10.7598/
cst2019.1567
Bharathi D, Rajalakshmi G (2019) Microbial lipases: an overview of screening, production and
purification. Biocatal Agric Biotechnol 22:101368. https://doi.org/10.1016/j.bcab.2019.101368
Brígida AIS, Amaral PFF, Coelho MAZ, Gonçalves LRB (2014) Lipase from Yarrowia lipolytica:
production, characterization and application as an industrial biocatalyst. J Mol Catal B: Enzym
101:148–158. https://doi.org/10.1016/j.molcatb.2013.11.016
Canseco-Pérez MA, Castillo-Avila GM, Chi-Manzanero B, Islas-Flores I, Apolinar-Hernández
MM, Rivera-Muñoz G, Gamboa-Angulo M, Sanchez-Teyer F, Couoh-Uicab Y, Canto-Canché
B (2018) Fungal screening on olive oil for extracellular triacylglycerol lipases: selection of a
Trichoderma harzianum strain and genome wide search for the genes. Genes (Basel) 9:e62.
https://doi.org/10.3390/genes9020062
Cao X, Liao L, Feng F (2020) Purification and characterization of an extracellular lipase from
Trichosporon sp. and its application in enrichment of omega-3 polyunsaturated fatty acids. LWT
118:108692. https://doi.org/10.1016/j.lwt.2019.108692
Carvalho T, Finotelli PV, Bonomo RCF, Franco M, Amaral PFF (2017) Evaluating aqueous
two-phase systems for Yarrowia lipolytica extracellular lipase purification. Process Biochem
53:259–266. https://doi.org/10.1016/j.procbio.2016.11.019
Casas-Godoy L, Duquesne S, Bordes F (2012) Lipases and phospholipases. Methods Mol Biol 861:
3–30. https://doi.org/10.1007/978-1-61779-600-5
Chamekh R, Deniel F, Donot C, Jany JL, Nodet P, Belabid L (2019) Isolation, identification and
enzymatic activity of halotolerant and halophilic fungi from the great Sebkha of Oran in
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 429

northwestern of Algeria. Mycobiology 47:230–241. https://doi.org/10.1080/12298093.2019.
1623979
Chen S, Li J, Fu Z, Wei G, Li H, Zhang B, Zheng L, Deng Z (2020) Enzymatic synthesis of
β-Sitosterol laurate by Candida rugosa lipase AY30 in the water/AOT/isooctane reverse micelle.
Appl Biochem Biotechnol 192(2):392–414. https://doi.org/10.1007/s12010-020-03302-0
Cho HY, Bancerz R, Ginalska G, Leonowicz A, Cho NS, Ohga S (2007) Culture conditions of
psychrotrophic fungus, Penicillium chrysogenum and its lipase characteristics. J Fac Agric
Kyushu Univ 52:281–286
Cipolatti EP, Costa M, Pinto C, Robert JDM (2018) Pilot-scale development of core-shell polymer
supports for the immobilization of recombinant lipase B from Candida antarctica and their
application in the production of ethyl ester. J Appl Polym Sci 135:46727. https://doi.org/10.
1002/app.46727
Coelho ALS, Orlandelli RC (2020) Immobilized microbial lipases in the food industry: a systematic
literature review. Crit Rev Food Sci Nutr 61:1689–1703. https://doi.org/10.1080/10408398.
2020.1764489
Cong S, Tian K, Zhang X, Lu F, Singh S, Prior B, Wang ZX (2019) Synthesis of flavor esters by a
novel lipase from Aspergillus niger in a soybean-solvent system. 3 Biotech 9:1–7. https://doi.
org/10.1007/s13205-019-1778-5
Contesini FJ, Calzado F, Valdo J Jr, Rubio MV, Zubieta MP (2017) Fungal metabolites. Fungal
Metab:639–666. https://doi.org/10.1007/978-3-319-25001-4
da Silva MVC, Souza AB, de Castro HF, Aguiar LG, de Oliveira PC, de Freitas L (2020) Synthesis
of 2-ethylhexyl oleate catalyzed by Candida antarctica lipase immobilized on a magnetic
polymer support in continuous flow. Bioprocess Biosyst Eng 43:615–623. https://doi.org/10.
1007/s00449-019-02257-9
Dalmaso GZL, Ferreira D, Vermelho AB (2015) Marine extremophiles a source of hydrolases for
biotechnological applications. Mar Drugs 13:1925–1965. https://doi.org/10.3390/md13041925
Das A, Shivakumar S, Bhattacharya S, Shakya S, Swathi SS (2016) Purification and characteriza-
tion of a surfactant-compatible lipase from Aspergillus tamarii JGIF06 exhibiting energy-
efficient removal of oil stains from polycotton fabric. 3 Biotech 6:1–8. https://doi.org/10.
1007/s13205-016-0449-z
Dayanandan A, Rani SHV, Shanmugavel M, Gnanamani A, Rajakumar GS (2013) Enhanced
production of Aspergillus tamarii lipase for recovery of fat from tannery fleshings. Braz J
Microbiol 1095:1089–1095
De Carvalho CR, Ferreira MC, Amorim SS, Hellen R, Catarine J, De Assis S, Zani CL, Rosa LH
(2019) Recent advancement in white biotechnology through fungi (eBook). https://doi.org/10.
1007/978-3-030-14846-1
De Morais WG, Kamimura ES, Ribeiro EJ (2016) Optimization of the production and characteri-
zation of lipase from Candida rugosa and Geotrichum candidum in soybean molasses by
submerged fermentation. Protein Expr Purif 123:26–34. https://doi.org/10.1016/j.pep.2016.
04.001
De Moura BA, Brunca Da Silva A, Mendonça Da Silva E, Pietro De Souza W, Soares MA, Gomes
De Vasconcelos L, Terezo AJ, Castilho M (2019) A biosensor based on microbial lipase
immobilized on lamellar zinc hydroxide-decorated gold nanoparticles for carbendazim determi-
nation. Anal Methods 11:5388–5397. https://doi.org/10.1039/c9ay01600g
Demera LL, Barahona PP, Javier E, Barriga C (2019) Production, extraction and characterization of
lipases from the antarctic yeast Guehomyces pullulans. Am J Biochem Biotechnol 15:75–82.
https://doi.org/10.3844/ajbbsp.2019.75.82
Deng L, Wang X, Nie K, Wang F, Liu J, Wang P, Tan T (2011) Synthesis of wax esters by lipase-
catalyzed esterification with immobilized lipase from Candida sp. 99-125. Chin J Chem Eng 19:
978–982. https://doi.org/10.1016/S1004-9541(11)60080-3
Diaz-vidal T, Rosales-rivera LC, Mateos-díaz JC, Rodríguez JA (2020) Research Paper. A series of
novel esters of capsaicin analogues catalyzed by Candida antarctica lipases, vol 103, pp
94–103. https://doi.org/10.1007/s12257-019-0290-4
430 J. Angelin and M. Kavitha

Dsm PD, Dheeman DS, Antony-babu S, Frías JM, Henehan GTM (2011) Enzymatic purification
and characterization of an extracellular lipase from a novel strain. J Mol Catal B 72:256–262.
https://doi.org/10.1016/j.molcatb.2011.06.013
Duarte AWF, dos Santos JA, Vianna MV, Vieira JMF, Mallagutti VH, Inforsato FJ, Wentzel LCP,
Lario LD, Rodrigues A, Pagnocca FC, Pessoa A, Durães Sette L (2018) Cold-adapted enzymes
produced by fungi from terrestrial and marine Antarctic environments. Crit Rev Biotechnol 38:
600–619. https://doi.org/10.1080/07388551.2017.1379468
Dumorné K, Córdova DC, Astorga-eló M, Renganathan P (2017) Extremozymes: a potential source
for industrial applications. J Microbiol Biotechnol 27:649–659
Ece OB, Deniz Y, Aysegul P, Baris B (2019) Immobilization of Rhizomucor miehei lipase onto
montmorillonite K-10 and polyvinyl alcohol gel Immobilization of Rhizomucor miehei lipase
onto montmorillonite K-10 and polyvinyl alcohol gel Ece Ozdemir Babavatan, Deniz Yildirim,
Aysegul Peksel & Baris B. Biocatal Biotransformation 38:274–282. https://doi.org/10.1080/
10242422.2019.1701660
El-Ghonemy DH, El-Gamal M, Tantawy AE, Ali TH (2017) Extracellular alkaline lipase from a
novel fungus Curvularia sp. DHE 5: optimization of physicochemical parameters, partial
purification and characterization. Food Technol Biotechnol 55:206–217. https://doi.org/10.
17113/ftb.55.02.17.4958
El-Ghonemy DH, Ali TH, Hassanein NM, Abdellah EM, Fadel M, Awad GEA, Abdou DAM
(2021) Thermo-alkali-stable lipase from a novel Aspergillus niger: statistical optimization,
enzyme purification, immobilization and its application in biodiesel production. Prep Biochem
Biotechnol 51:225–240. https://doi.org/10.1080/10826068.2020.1805759
Eugenia GOL, Isela QZ, Katiushka A (2016) Detection of extracellular enzymatic activity in
microorganisms isolated from waste vegetable oil contaminated soil using plate methodologies.
Afr J Biotechnol 15:408–416. https://doi.org/10.5897/ajb2015.14991
Gaikaiwari RP, Wagh SA, Kulkarni BD (2012) Efficient lipase purification using reverse micellar
extraction. Bioresour Technol 108:224–230. https://doi.org/10.1016/j.biortech.2011.11.126
Gaur R, Hemamalini R, Khare SK (2017) Lipolytic enzymes. Curr Dev Biotechnol
Bioeng:175–198. https://doi.org/10.1016/b978-0-444-63662-1.00008-7
Geoffry K, Achur RN (2018) Optimization of novel halophilic lipase production by fusarium solani
strain NFCCL 4084 using palm oil mill effluent. J Genet Eng Biotechnol 16:327–334. https://
doi.org/10.1016/j.jgeb.2018.04.003
Gopinath SCB, Hilda A, Priya TL, Annadurai G (2002) Purification of lipase from Cunninghamella
verticillata and optimization of enzyme activity using response surface methodology. World J
Microbiol Biotechnol 18:449–458. https://doi.org/10.1023/A:1015579121800
Gopinath SCB, Anbu P, Lakshmipriya T, Hilda A (2013) Strategies to characterize fungal lipases
for applications in medicine and dairy industry. Biomed Res Int 2013:31–34. https://doi.org/10.
1155/2013/154549
Griebeler N, Polloni AE, Remonatto D, Arbter F, Vardanega R, Cechet JL, Di Luccio M, de
Oliveira D, Treichel H, Cansian RL, Rigo E, Ninow JL (2011) Isolation and screening of lipase-
producing fungi with hydrolytic activity. Food Bioproc Tech 4:578–586. https://doi.org/10.
1007/s11947-008-0176-5
Group TF (2009) World enzymes. ORDER A J Theory Ordered Sets Its Appl:329
Gutarra MLE, Godoy MG, Maugeri F, Rodrigues MI, Freire DMG, Castilho LR (2009) Production
of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by
solid-state fermentation. Bioresour Technol 100:5249–5254. https://doi.org/10.1016/j.biortech.
2008.08.050
Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzyme Microb
Technol 39:235–251. https://doi.org/10.1016/j.enzmictec.2005.10.016
Hasan F, Shah AA, Javed S, Hameed A (2010) Enzymes used in detergents: lipases. Afr J
Biotechnol 9:4836–4844
Hasanah U, Md Sani ND, Heng LY, Idroes R, Safitri E (2019) Construction of a hydrogel pectin-
based triglyceride optical biosensor with immobilized lipase enzymes. Biosensors 9. https://doi.
org/10.3390/bios9040135
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 431

Huang Y, Yu J, Zhou Q, Sun L, Liu D, Liang M (2020) Preparation of yogurt-flavored bases by
mixed lactic acid bacteria with the addition of lipase. LWT- Food Sci Technol 131:109577.
https://doi.org/10.1016/j.lwt.2020.109577
Ichikawa K, Yoshida A, Shiono Y, Koseki T (2020) Biochemical characterization of a lipolytic
enzyme from aspergillus oryzae that hydrolyzes triacylglycerol and sterol esters. Appl Biochem
Biotechnol 192(3):910–922. https://doi.org/10.1007/s12010-020-03360-4
Ilesanmi OI, Adekunle AE, Omolaiye JA, Olorode EM, Ogunkanmi AL (2020) Isolation, optimi-
zation and molecular characterization of lipase producing bacteria from contaminated soil. Sci
African 8:e00279. https://doi.org/10.1016/j.sciaf.2020.e00279
Insaf B, Noreddine K, Jacqueline D, Annick L, Philippe T (2014) Screening of Candida boidinii
from Chemlal spent olive characterized by higher alkaline-cold adapted lipase production. Afr J
Biotechnol 13:1287–1294. https://doi.org/10.5897/ajb2013.13586
Ire FS, Ike VC (2014) Screening and optimization of process parameters for the production of lipase
in submerged fermentation by Aspergillus carbonarius (Bainer) IMI 366159. Annu Res Rev
Biol 4:2587–2602
Jain R, Naik SN (2018) Adding value to the oil cake as a waste from oil processing industry:
production of lipase in solid state fermentation. Biocatal Agric Biotechnol:181–184. https://doi.
org/10.1016/j.bcab.2018.06.010
Jambulingam R, Shalma M, Shankar V (2019) Biodiesel production using lipase immobilised
functionalized magnetic nanocatalyst from oleaginous fungal lipid. J Clean Prod 215:245–
258. https://doi.org/10.1016/j.jclepro.2018.12.146
Janek T, Mirończuk AM, Rymowicz W, Dobrowolski A (2020) High-yield expression of extracel-
lular lipase from Yarrowia lipolytica and its interactions with lipopeptide biosurfactants: a
biophysical approach. Arch Biochem Biophys 689:108475. https://doi.org/10.1016/j.abb.
2020.108475
Jares F, Branta D, Alves G, Grac M (2010) Enzymatic Aspergillus sp. lipase: potential biocatalyst
for industrial use. J Mol Catal B 67:163–171. https://doi.org/10.1016/j.molcatb.2010.07.021
Jermsuntiea W, Aki T, Toyoura R, Iwashita K, Kawamoto S, Ono K (2011) Purification and
characterization of intracellular lipase from the polyunsaturated fatty acid-producing fungus
Mortierella alliacea. N Biotechnol 28:158–164. https://doi.org/10.1016/j.nbt.2010.09.007
Jiang Z, Zhang C, Tang M, Xu B, Wang L, Qian W (2020) Improving the thermostability of
Rhizopus chinensis lipase through site-directed mutagenesis based on B-factor analysis. Front
Microbiol 11:1–8. https://doi.org/10.3389/fmicb.2020.00346
Jin M, Gai Y, Guo X, Hou Y, Zeng R (2019) Properties and applications of extremozymes from
deep-sea extremophilic microorganisms: a mini review. Mar Drugs 17:65. https://doi.org/10.
3390/md17120656
Jorge L, Ninow D (2016) Stabilization of lipase from Thermomyces lanuginosus by crosslinking in
PEGylated polyurethane particles by polymerization: application on fish oil ethanolysis.
Biochem Eng J 112:54–60. https://doi.org/10.1016/j.bej.2016.04.006
Joseph B, Ramteke PW, Thomas G (2008) Cold active microbial lipases: some hot issues and recent
developments. Biotechnol Adv 26:457–470. https://doi.org/10.1016/j.biotechadv.2008.05.003
Joshi R, Sharma R, Kuila A (2019) Lipase production from fusarium incarnatum KU377454 and its
immobilization using Fe3O4 NPs for application in waste cooking oil degradation. Bioresour
Technol Rep 5:134–140. https://doi.org/10.1016/j.biteb.2019.01.005
Kanmani P, Karthik S, Aravind J, Kumaresan K (2013) The use of response surface methodology as
a statistical tool for media optimization in lipase production from the dairy effluent isolate
fusarium solani. ISRN Biotechnol 2013:1–8. https://doi.org/10.5402/2013/528708
Kantak JB, Bagade AV, Mahajan SA, Pawar SP, Shouche YS, Prabhune AA (2011) Isolation,
identification and optimization of a new extracellular lipase producing strain of Rhizopus
sp. Appl Biochem Biotechnol 164:969–978. https://doi.org/10.1007/s12010-011-9188-0
Kartal F, Kilinç A, Timur S (2007) Lipase biosensor for tributyrin and pesticide detection. Int J
Environ Anal Chem 87:715–722. https://doi.org/10.1080/03067310701327741
432 J. Angelin and M. Kavitha

Kaur M, Mehta A, Gupta R (2019) Synthesis of methyl butyrate catalyzed by lipase from aspergil-
lus fumigatus. J Oleo Sci 68:989–993. https://doi.org/10.5650/jos.ess19125
Kavitha M (2016) Cold active lipases—an update. Front Life Sci 9:226–238. https://doi.org/10.
1080/21553769.2016.1209134
Kim EH, Cho KH, Lee YM, Yim JH, Lee HK, Cho JC, Hong SG (2010) Diversity of cold-active
protease-producing bacteria from arctic terrestrial and marine environments revealed by enrich-
ment culture. J Microbiol 48:426–432. https://doi.org/10.1007/s12275-010-0015-z
Km S (2017) Purification and properties of extracellular lipases with transesterification activity and
1,3-Regioselectivity from Rhizomucor miehei and Rhizopus oryzae. J Microbiol Biotechnol
27(2):277–288. https://doi.org/10.4014/jmb.1608.08005
Knez Ž, KavčičS, Gubicza L, Bélafi-Bakó K, Németh G, PrimožičM, Habulin M (2012) Lipase-
catalyzed esterification of lactic acid in supercritical carbon dioxide. J Supercrit Fluids 66:192–
197. https://doi.org/10.1016/j.supflu.2011.11.006
Kotogán A, Németh B, Vágvölgyi C, Papp T, Takó M (2014) Screening for extracellular lipase
enzymes with transesterification capacity in Mucoromycotina strains. Food Technol Biotechnol
52:73–82
Kour D, Rana KL, Kaur T, Singh B, Singh V (2020) Extremophiles for hydrolytic enzymes
productions: biodiversity and potential biotechnological applications. Bioprocess
Biomol Prod:321–372. https://doi.org/10.1002/9781119434436.ch16
Kumar Maharana A, Mohan Singh S (2018) Cold active lipases produced by Cryptococcus sp. Y-32
and Rhodococcus erythropolis N149 isolated from Nella Lake, Antarctica. Int J Curr Microbiol
App Sci 7:1910–1926. https://doi.org/10.20546/ijcmas.2018.703.227
Kumar D, Kumar L, Nagar S, Raina C, Parshad R, Gupta VK (2012a) Screening, isolation and
production of lipase/esterase producing bacillus sp. strain DVL2 and its potential evaluation in
esterification and resolution reactions. Arch Appl Sci Res 4:1763–1770
Kumar S, Mathur A, Singh V, Nandy S, Kumar S, Negi S (2012b) Bioresource technology
bioremediation of waste cooking oil using a novel lipase produced by Penicillium chrysogenum
SNP5 grown in solid medium containing waste grease. Bioresour Technol 120:300–304. https://
doi.org/10.1016/j.biortech.2012.06.018
Kumar A, Gudiukaite R, Gricajeva A, Sadauskas M (2019) Microbial lipolytic enzymes—
promising energy-efficient biocatalysts in bioremediation. Energy 192:116674. https://doi.org/
10.1016/j.energy.2019.116674
Kumari A, Gupta R (2015) Functional characterisation of novel enantioselective lipase TALipA
from Trichosporon asahii MSR54: sequence comparison revealed new signature sequence
AXSXG among yeast lipases. Appl Biochem Biotechnol 175:360–371. https://doi.org/10.
1007/s12010-014-1268-5
Lage FAP, Bassi JJ, Corradini MCC, Todero LM, Luiz JHH, Mendes AA (2015) Preparation of a
biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic
support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system.
Enzyme Microb Technol 84:56–67. https://doi.org/10.1016/j.enzmictec.2015.12.007
Lanka S, B TT (2018) Screening and isolation of lipase producing fungi from marine water obtained
from Machilipatnam costal region. Int J Pharmacogn Phytochem Res 9:928–932. https://doi.
org/10.25258/phyto.v9i07.11157
Li J, Shen W, Fan G, Li X (2018) Screening, purification and characterization of lipase from
Burkholderia pyrrocinia B1213. 3 Biotech 8:1–12. https://doi.org/10.1007/s13205-018-1414-9
Li Y, Liu TJ, Zhao MJ, Zhang H, Feng FQ (2019) Screening, purification, and characterization of an
extracellular lipase from Aureobasidium pullulans isolated from stuffed buns steamers. J
Zhejiang Univ Sci B 20:332–342. https://doi.org/10.1631/jzus.B1800213
Lim SJ, Hazwani-Oslan SN, Oslan SN (2020) Purification and characterisation of thermostable
α-amylases from microbial sources. Bioresources 15:2005–2029. https://doi.org/10.15376/
biores.15.1.2005-2029
Liu ZQ, Zheng XB, Zhang SP, Zheng YG (2012) Cloning, expression and characterization of a
lipase gene from the Candida antarctica ZJB09193 and its application in biosynthesis of vitamin
a esters. Microbiol Res 167:452–460. https://doi.org/10.1016/j.micres.2011.12.004
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 433

Madhavan A, Sindhu R, Parameswaran B, Sukumaran RK, Pandey A (2017) Metagenome analysis:
a powerful tool for enzyme bioprospecting. Appl Biochem Biotechnol 183:636–651. https://doi.
org/10.1007/s12010-017-2568-3
Maharana AK, Ray P (2014) Application of Plackett-Burman design for improved cold temperature
production of lipase by psychrotolerant pseudomonas sp. AKM-L5. Int J Curr Microbiol App
Sci 3:269–282
Maharana A, Ray P (2015) A novel cold-active lipase from psychrotolerant pseudomonas
sp. AKM-L5 showed organic solvent resistant and suitable for detergent formulation. J Mol
Catal B: Enzym 120:173–178. https://doi.org/10.1016/j.molcatb.2015.07.005
Maldonado RR, Lopes DB, Aguiar-Oliveira E, Kamimura ES, Macedo GA (2016) A review on
Geotrichum lipases: production, purification, immobilization and applications. Chem Biochem
Eng Q 30:439–454. https://doi.org/10.15255/CABEQ.2016.907
Martins AB, Friedrich JLR, Cavalheiro JC, Garcia-galan C, Barbosa O, Ayub MAZ, Fernandez-
lafuente R, Rodrigues RC (2013) Improved production of butyl butyrate with lipase from
Thermomyces lanuginosus immobilized on styrene—divinyl benzene beads. Bioresour Technol
134:417–422. https://doi.org/10.1016/j.biortech.2013.02.052
Martorell MM, Ruberto LAM, Fernández PM, De Figueroa LIC, Mac Cormack WP (2019)
Biodiversity and enzymes bioprospection of Antarctic filamentous fungi. Antarct Sci 31:3–12.
https://doi.org/10.1017/S0954102018000421
Mehta A, Bodh U, Gupta R (2017) Fungal lipases: a review. J Biotech Res 8:58–77
Mehta A, Bodh U, Gupta R (2018a) Isolation of a novel lipase producing fungal isolate aspergillus
fumigatus and production optimization of enzyme. Biocatal Biotransformation 36:450–457.
https://doi.org/10.1080/10242422.2018.1447565
Mehta A, Grover C, Gupta R (2018b) Purification of lipase from aspergillus fumigatus using Octyl
Sepharose column chromatography and its characterization. J Basic Microbiol 58:857–866.
https://doi.org/10.1002/jobm.201800129
Mehta A, Grover C, Bhardwaj KK, Gupta R (2020) Application of lipase purified from aspergillus
fumigatus in the syntheses of ethyl acetate and ethyl lactate. J Oleo Sci 69:23–29. https://doi.
org/10.5650/jos.ess19202
Melani NB, Tambourgi EB, Silveira E, Melani NB, Tambourgi EB, Silveira E (2019) Lipases: from
production to applications lipases: from production to applications. Sep Purif Rev 49:143–158.
https://doi.org/10.1080/15422119.2018.1564328
Milivojevic A (2016) Immobilization of Candida antarctica lipase B onto Purolite MN102 and its
application in solvent-free and organic media esterification. Bioprocess Biosyst Eng 40:23–34.
https://doi.org/10.1007/s00449-016-1671-0
Muthukumaran N, Dhar SC (1982) Comparative studies on the degreasing of skins using acid lipase
and solvent with reference to the quality of finished leathers. Leather Sci 29:417–424
Mouad AM, Taupin D, Lehr L, Yvergnaux F, Porto ALM (2016) Aminolysis of linoleic and
salicylic acid derivatives with Candida antarctica lipase B: a solvent-free process to obtain
amphiphilic amides for cosmetic application. J Mol Catal B: Enzym 126:64–68. https://doi.org/
10.1016/j.molcatb.2016.01.002
Nagarajan S (2012) New tools for exploring old friends-microbial lipases. Appl Biochem
Biotechnol 168:1163–1196. https://doi.org/10.1007/s12010-012-9849-7
Navvabi A, Razzaghi M, Fernandes P, Karami L, Homaei A (2018) Novel lipases discovery
specifically from marine organisms for industrial production and practical applications. Process
Biochem 70:61–70. https://doi.org/10.1016/j.procbio.2018.04.018
Nurul Furqan BR, Akhmaloka (2020) Heterologous expression and characterization of thermosta-
ble lipase (Lk1) in Pichia pastoris GS115. Biocatal Agric Biotechnol 23:2–5. https://doi.org/10.
1016/j.bcab.2019.101448
Ortellado LE, Lisowiec LA, Quiroga-Zingaretti AE, Villalba LL, Zapata PD, Fonseca MI (2020)
Exploring novel Penicillium lipolytic activity from the Paranaense rainforest. Environ
Technol:1–21. https://doi.org/10.1080/09593330.2020.1759697
Palekar AA, Vasudevan PT, Yan S (2000) Purification of lipase: a review. Biocatal Biotransforma-
tion 18:177–200. https://doi.org/10.3109/10242420009015244
434 J. Angelin and M. Kavitha

Panajotova HN, Strinska HN, Gandova VD, Dobreva VT, Zhekova BJ, Dobrev GT (2017)
Purification of lipase from aspergillus carbonarius NRRL369 by ATPS PEG/potassium phos-
phate. Bulg Chem Commun 49:130–136
Pandey H, Kestwal A, Chauhan D, Kumari S, Dhalwal V, Singh GJ, Singh P, Mann P, Sharma A,
Saxena G, Giri B (2018) Isolation and screening of potential fungi and standardization of a
process for the production of extracellular lipase, pp 116–123
Papagora C, Roukas T, Kotzekidou P (2013) Optimization of extracellular lipase production by
Debaryomyces hansenii isolates from dry-salted olives using response surface methodology.
Food Bioprod Process 91:413–420. https://doi.org/10.1016/j.fbp.2013.02.008
Peng R, Hong B, Zhou P (2016) Screening and production optimization of a new organic solvent-
tolerance fungal lipase, pp 194–201. https://doi.org/10.2991/bbe-16.2016.33
Pohanka M (2019) Biosensors and bioassays based on lipases, principles and applications, a review.
Molecules 24:616. https://doi.org/10.3390/molecules24030616
Radu A, MoisǎME, Toşa MI, Dima N, Zaharia V, Irimie FD (2014) Candida antarctica lipases
acting as versatile catalysts for the synthesis of enantiopure (R)- and (S)-1-(2-phenylthiazol-4-
yl)ethanamines this paper is dedicated to the anniversary of 95th birthday of professor Valer
Fǎrcǎşanu. J Mol Catal B: Enzym 107:114–119. https://doi.org/10.1016/j.molcatb.2014.05.007
Raveendran S, Parameswaran B, Ummalyma SB, Abraham A, Mathew AK, Madhavan A,
Rebello S, Pandey A (2018) Applications of microbial enzymes in food industry. Food Technol
Biotechnol 56:16–30. https://doi.org/10.17113/ftb.56.01.18.5491
Resende R, Aguiar-oliveira E, Massiero F, Giolo G, Alves G, Isabel M (2015) Biocatalysis and
Agricultural Biotechnology Evaluation of partial purification and immobilization of lipase from
Geotrichum candidum. Biocatal Agric Biotechnol:1–6. https://doi.org/10.1016/j.bcab.2015.
05.005
Rigoldi F, Donini S, Redaelli A, Parisini E, Gautieri A (2018) Review: engineering of thermostable
enzymes for industrial applications. APL Bioeng 2:011501. https://doi.org/10.1063/1.4997367
Rihani A, Soumati B (2019) Extracellular lipase production by trichoderma harzianum isolated
from oil contaminated soil. Sci Study Res Chem Chem Eng Biotechnol Food Ind 20:11–19
Riyadi FA, Alam MZ, Salleh MN, Salleh HM (2017a) Optimization of thermostable organic
solvent-tolerant lipase production by thermotolerant Rhizopus sp. using solid-state fermentation
of palm kernel cake. 3 Biotech 7:1–11. https://doi.org/10.1007/s13205-017-0932-1
Riyadi FA, Alam MZ, Salleh MN, Salleh HM (2017b) Thermostable and organic solvent tolerant
lipase producing fungi in solid state bioconversion of palm kernel cake. Asia Pac J Mol Biol
Biotechnol 25:98–107
Romdhane IB, Fendri A, Gargouri Y, Gargouri A, Belghith H (2010) A novel thermoactive and
alkaline lipase from Talaromyces thermophilus fungus for use in laundry detergents. Biochem
Eng J 53:112–120. https://doi.org/10.1016/j.bej.2010.10.002
Sadati R, Barghi A, Larki RA (2015) Isolation and screening of lipolytic fungi from coastal waters
of the southern Caspian Sea (north of Iran). Jundishapur J Microbiol 8:e16426. https://doi.org/
10.5812/jjm.8(4)2015.16426
Sahay S, Chouhan D (2018) Study on the potential of cold-active lipases from psychrotrophic fungi
for detergent formulation. J Genet Eng Biotechnol 16:319–325. https://doi.org/10.1016/j.jgeb.
2018.04.006
Sahay H, Yadav AN, Singh AK, Singh S, Kaushik R, Saxena AK (2017) Hot springs of Indian
Himalayas: potential sources of microbial diversity and thermostable hydrolytic enzymes.
3 Biotech 7:1–11. https://doi.org/10.1007/s13205-017-0762-1
Salihu A, Alam Z (2015) Thermostable lipases: an overview of production, purification and
thermostable lipases: an overview of production, purification and characterization. Biosci
Biotechnol Res Asia 11(3):1095–1107. https://doi.org/10.13005/bbra/1494
Salwan R, Sharma V (2020) Overview of extremophiles. In: Physiological and biotechnological
aspects of extremophils, pp 3–11. https://doi.org/10.1016/b978-0-12-818322-9.00001-0
Salwoom L, Rahman RNZRA, Salleh AB, Shariff FM, Convey P, Pearce D, Ali MSM (2019)
Isolation, characterisation, and lipase production of a cold-adapted bacterial strain pseudomonas
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 435

sp. LSK25 isolated from Signy Island, Antarctica. Molecules 24:1–14. https://doi.org/10.3390/
molecules24040715
Sarmah N, Revathi D, Sheelu G, Yamuna Rani K, Sridhar S, Mehtab V, Sumana C (2018) Recent
advances on sources and industrial applications of lipases. Biotechnol Prog 34:5–28. https://doi.
org/10.1002/btpr.2581
Selvam K, Vishnupriya B, Subhash Chandra Bose V (2011) Screening and quantification of marine
Actinomycetes producing industrial enzymes amylase, cellulase and lipase from south coast of
India. Int J Pharm Biol Arch 2:1481–1487
Séverac E, Galy O, Turon F, Pantel CA, Condoret JS, Monsan P, Marty A (2011) Selection of CalB
immobilization method to be used in continuous oil transesterification: analysis of the economi-
cal impact. Enzyme Microb Technol 48:61–70. https://doi.org/10.1016/j.enzmictec.2010.
09.008
Shamim S, Liaqat U, Rehman A, Campus DR, Chowk B, Genetics M, Campus N (2018) Microbial
lipases and their application—a review. Abasyn J Life Sci 1:54–76
Shankar S, Agarwal M, Chaurasia SP (2013) Study of reaction parameters and kinetics of esterifi-
cation of lauric acid with butanol by immobilized Candida antarctica lipase. Indian J Biochem
Biophys 50:570–576
Sharma S, Kanwar SS (2014) Organic solvent tolerant lipases and applications. Sci World J 2014:
625258. https://doi.org/10.1155/2014/625258
Sharma AK, Sharma V, Saxena J (2016a) A review on applications of microbial lipases. Int J
Biotech Trends Technol 19:1–5. https://doi.org/10.14445/22490183/ijbtt-v19p601
Sharma AK, Sharma V, Saxena J (2016b) A review on properties of fungal lipases. Int J Curr
Microbiol App Sci 5:123–130. https://doi.org/10.20546/ijcmas.2016.512.014
Sharma N, Yadav N, Bhagwani H, Chahar D, Singh B (2018) Of waste resources screening of lactic
acid bacteria from effluent samples of Jaipur dairy. Int J Water Resour 8:8–11. https://doi.org/
10.4172/2252-5211.1000332
Shen JW, Cai X, Dou BJ, Qi FY, Zhang XJ, Liu ZQ, Zheng YG (2020) Expression and characteri-
zation of a CALB-type lipase from Sporisorium reilianum SRZ2 and its potential in short-chain
flavor ester synthesis. Front Chem Sci Eng 14:868–879. https://doi.org/10.1007/s11705-019-
1889-x
Shimizu Y, Konno Y, Tomita Y (2020) Wickerhamomyces psychrolipolyticus f. a., sp. nov., a
novel yeast species producing two kinds of lipases with activity at different temperatures. Int J
Syst Evol Microbiol 70:1158–1165. https://doi.org/10.1099/ijsem.0.003894
Silva JA, Macedo GP, Rodrigues DS, Giordano RLC, Gonçalves LRB (2012) Immobilization of
Candida antarctica lipase B by covalent attachment on chitosan-based hydrogels using different
support activation strategies. Biochem Eng J 60:16–24. https://doi.org/10.1016/j.bej.2011.
09.011
Singh Y, Gupta R (2016) Novel S-enantioselective lipase TALipB from Trichosporon asahii
MSR54: heterologous expression, characterization, conformational stability and homology
modeling. Enzyme Microb Technol 83:29–39. https://doi.org/10.1016/j.enzmictec.2015.11.003
Singh AK, Mukhopadhyay M (2012) Overview of fungal lipase: a review. Appl Biochem
Biotechnol 166:486–520. https://doi.org/10.1007/s12010-011-9444-3
Singh SM, Singh SK, Yadav LS, Singh PN, Ravindra R (2012) Filamentous soil fungi from
Ny-Ålesund, spitsbergen, and screening for extracellular enzymes. Arctic 65:45–55. https://
doi.org/10.14430/arctic4164
Souza LTA, Oliveira JS, Dos Santos VL, Regis WCB, Santoro MM, Resende RR (2014) Lipolytic
potential of aspergillus japonicus LAB01: production, partial purification, and characterisation
of an extracellular lipase. Biomed Res Int 2014:108913. https://doi.org/10.1155/2014/108913
Souza RL, Lima RA, Coutinho JAP, Soares CMF, Lima ÁS (2015) Novel aqueous two-phase
systems based on tetrahydrofuran and potassium phosphate buffer for purification of lipase.
Process Biochem 50:1459–1467. https://doi.org/10.1016/j.procbio.2015.05.015.1
436 J. Angelin and M. Kavitha

Sreelatha B, Koteswara Rao V, Ranjith Kumar R, Girisham S, Reddy SM (2017) Culture conditions
for the production of thermostable lipase by Thermomyces lanuginosus. Beni Suef Univ J Basic
Appl Sci 6:87–95. https://doi.org/10.1016/j.bjbas.2016.11.010
Stoytcheva M, Montero G, Zlatev R, Leon JA, Gochev V (2012) Analytical methods for lipases
activity determination: a review. Curr Anal Chem 8:400–407. https://doi.org/10.2174/
157341112801264879
Sun J, Chen Y, Sheng J, Sun M (2015) Immobilization of Yarrowia lipolytica lipase on
macroporous resin using different methods: characterization of the biocatalysts in hydrolysis
reaction. Biomed Res Int 2015:139179. https://doi.org/10.1155/2015/139179
Sun Q, Wang H, Zhang H, Luo H, Shi P, Bai Y, Lu F, Yao B, Huang H (2016) Heterologous
production of an acidic thermostable lipase with broad-range pH activity from thermophilic
fungus Neosartorya fischeri P1. J Biosci Bioeng 122:539–544. https://doi.org/10.1016/j.jbiosc.
2016.05.003
Tan CH, Show PL, Ooi CW, Ng EP, Lan JCW, Ling TC (2015) Novel lipase purification
methods—a review of the latest developments. Biotechnol J 10:31–44. https://doi.org/10.
1002/biot.201400301
Taskin M, Ucar MH, Kara AA, Ozdemir M (2016) Lipase production with free and immobilized
cells of cold-adapted yeast Rhodotorula glutinis HL25. Biocatal Agric Biotechnol 8:97–103.
https://doi.org/10.1016/j.bcab.2016.08.009
Testera AM, Santos M, Girotti A, Arias FJ, Báñez JM, Alonso M, Rodríguez-Cabello JC (2019) A
novel lipase-catalyzed method for preparing ELR-based bioconjugates. Int J Biol Macromol
121:752–759. https://doi.org/10.1016/j.ijbiomac.2018.10.028
Tišma M, TadićT, Budžaki S, OstojčićM, ŠalićA, ZelićB, Tran NN, Ngothai Y, Hessel V (2019)
Lipase production by solid-state cultivation of Thermomyces lanuginosus on by-products from
cold-pressing oil production. PRO 7:465
Triyaswati D, Ilmi M (2020) Lipase-producing filamentous fungi from non-dairy creamer industrial
waste. Microbiol Biotechnol Lett 48:167–178. https://doi.org/10.4014/mbl.1912.12018
Tsuji M, Yokota Y, Shimohara K, Kudoh S, Hoshino T (2013) An application of wastewater
treatment in a cold environment and stable lipase production of Antarctic basidiomycetous yeast
Mrakia blollopis. PLoS One 8:e59376. https://doi.org/10.1371/journal.pone.0059376
Turati DFM, Almeida AF, Terrone CC, Nascimento JMF, Terrasan CRF, Fernandez-Lorente G,
Pessela BC, Guisan JM, Carmona EC (2019) Thermotolerant lipase from Penicillium sp. section
Gracilenta CBMAI 1583: effect of carbon sources on enzyme production, biochemical
properties of crude and purified enzyme and substrate specificity. Biocatal Agric Biotechnol
17:15–24. https://doi.org/10.1016/j.bcab.2018.10.002
Turki S, Ayed A, Chalghoumi N (2010) An enhanced process for the production of a highly purified
extracellular lipase in the non-conventional yeast Yarrowia lipolytica. Appl Biochem
Biotechnol 160:1371–1385. https://doi.org/10.1007/s12010-009-8599-7
Ülker S, Karaoĝlu ŞA (2012) Purification and characterization of an extracellular lipase from Mucor
hiemalis f. corticola isolated from soil. J Biosci Bioeng 114:385–390. https://doi.org/10.1016/j.
jbiosc.2012.04.023
Varela H, Lupo S, Calvin A (2012) Extracellular enzymes produced by microorganisms isolated
from maritime Antarctica. World J Microbiol Biotechnol 28:2249–2256. https://doi.org/10.
1007/s11274-012-1032-
Venkatanagaraju E, Divakar G (2017) Purification strategies for microbial pectinases. Curr Trends
Biotechnol Pharm 11:144–159
Verma S, Sharma KP (2014) Isolation, identification and characterization of lipase producing
microorganisms from environment. Asian J Pharm Clin Res 7:219–221
Virgen-ort LJJ, Fernandez-lafuente COR (2016) Physical crosslinking of lipase from Rhizomucor
miehei immobilized on octyl agarose via coating with ionic polymers: avoiding enzyme release
from the support. Process Biochem 54:81–88. https://doi.org/10.1016/j.procbio.2016.12.018
Wadia T, Jain SK (2020) Isolation, screening and identification of lipase producing fungi from oil
contaminated soil of Shani Mandir Ujjain. https://doi.org/10.20546/ijcmas.2017.607.223
Wagner A, Duarte F, Aparecida J, Vianna V, Maíra J, Vieira F, Mallagutti VH, José F, Costa L,
Wentzel P, Lario LD, Rodrigues A, Pagnocca FC, Junior AP, Sette LD, Vianna V, Maíra J,
18 Extremophilic Fungal Lipases: Screening, Purification, Assay, and Applications 437

Vieira F, Mallagutti VH, Inforsato FJ, Costa L, Wentzel P, Lario LD, Rodrigues A, Pagnocca
FC, Ma J, Lario LD, Rodrigues A, Carlos F, Pessoa A, Dur L (2018) Critical reviews in
biotechnology cold-adapted enzymes produced by fungi from terrestrial and marine Antarctic
environments. Crit Rev Biotechnol 0:600–619. https://doi.org/10.1080/07388551.2017.
1379468
Wang SG, Zhang WD, Li Z, Ren ZQ, Liu HX (2010) Lipase immobilized on the hydrophobic
polytetrafluoroethene membrane with nonwoven fabric and its application in intensifying
synthesis of butyl oleate. Appl Biochem Biotechnol 162:2015–2026. https://doi.org/10.1007/
s12010-010-8977-1
Wang JR, Li YY, Liu D (2015) Gene cloning, high-level expression, and characterization of an
alkaline and thermostable lipase from Trichosporon coremiiforme V3. J Microbiol Biotechnol
25:845–855. https://doi.org/10.4014/jmb.1408.08039
Xing S, Zhu R, Li C, He L, Zeng X, Zhang Q (2020) Gene cloning, expression, purification and
characterization of a sn-1,3 extracellular lipase from aspergillus Niger GZUF36. J Food Sci
Technol 57:2669–2680. https://doi.org/10.1007/s13197-020-04303-x
Yadav MG, Kavadia MR, Vadgama RN, Odaneth AA, Lali AM (2017) Green enzymatic produc-
tion of glyceryl monoundecylenate using immobilized Candida antarctica lipase B. Prep
Biochem Biotechnol 47:1050–1058. https://doi.org/10.1080/10826068.2017.1381621
Yadav MG, Kavadia MR, Vadgama RN, Odaneth AA, Lali AM (2018) Production of 6-O-l-
Ascorbyl palmitate by immobilized Candida antarctica lipase B. Appl Biochem Biotechnol
184:1168–1186. https://doi.org/10.1007/s12010-017-2610-5
Yadav MG, Vadgama RN, Kavadia MR, Odaneth AA, Lali AM (2019) Production of
Pentaerythritol Monoricinoleate (PEMR) by immobilized Candida antarctica lipase
B. Biotechnol Rep 23:e00353. https://doi.org/10.1016/j.btre.2019.e00353
Yagmurov ER, Kozlov GV, Pushkarev MA (2017) Lipase purification: the review of conventional
and novel methods. J Hyg Eng Des 20:60–69
Yan Q, Duan X, Liu Y, Jiang Z, Yang S (2016) Biotechnology for biofuels expression and
characterization of a novel 1, 3—regioselective cold—adapted lipase from Rhizomucor
endophyticus suitable for biodiesel synthesis. Biotechnol Biofuels 9:86. https://doi.org/10.
1186/s13068-016-0501-6
Yeshodha K, Dhar SC, Santappa M (1978) Studies on degreasing of skins using a microbial lipase.
Leather Sci 25:77–86
Zehani N, Dzyadevych SV, Kherrat R, Jaffrezic-Renault NJ (2014a) Sensitive impedimetric
biosensor for direct detection of diazinon based on lipases. Front Chem 2:1–7. https://doi.org/
10.3389/fchem.2014.00044
Zehani N, Kherrat R, Jaffrezic-Renault N (2014b) Immobilization of Candida Rugosa lipase on
Aluminosilicate incorporated in a polymeric membrane for the elaboration of an impedimetric
biosensor. Sens Transducers 27:371–373
Zhang YY, Liu JH (2010) Purification and in situ immobilization of lipase from of a mutant of
Trichosporon laibacchii using aqueous two-phase systems. J Chromatogr B Analyt Technol
Biomed Life Sci 878:909–912. https://doi.org/10.1016/j.jchromb.2010.01.045
Zhang X, Li SJ, Li JJ, Liang ZZ, Zhao CQ (2018) Novel natural products from extremophilic fungi.
Mar Drugs 16:1–36. https://doi.org/10.3390/md16060194
Zhang XF, Ai YH, Xu Y, Yu XW (2019) High-level expression of aspergillus Niger lipase in Pichia
pastoris: characterization and gastric digestion in vitro. Food Chem 274:305–313. https://doi.
org/10.1016/j.foodchem.2018.09.020
Zhang Y, Sun Y, Tang H, Zhao Q, Ren W, Lv K, Yang F, Wang F, Liu J (2020) One-pot enzymatic
synthesis of Enantiopure 1,3-Oxathiolanes using Trichosporon laibachii lipase and the kinetic
model. Org Process Res Dev 24:579–587. https://doi.org/10.1021/acs.oprd.0c00010
Zhao X, Qi F, Yuan C, Du W, Liu D (2015) Lipase-catalyzed process for biodiesel production:
enzyme immobilization, process simulation and optimization. Renew Sustain Energy Rev 44:
182–197. https://doi.org/10.1016/j.rser.2014.12.021
438 J. Angelin and M. Kavitha

Extremophilic Fungal Proteases: Screening,
Purification, Assay, and Applications 19
Sourav Bhattacharya and Arijit Das
Abstract
The use of extremophilic fungal proteases in industries has been an integral event
for decades together and some of these fungal cultures are efficient producers
of many hydrolytic enzymes. The fact that such proteases exhibit specific range of
action and vast diversity in terms of being active over a very wide range of
experimental conditions has made them attractive models for industrial usage.
Besides being widely distributed in nature, extremophilic fungi are preferred over
their mesophilic counterparts owing to their ability to tolerate harsher growth
conditions and to overproduce hydrolytic enzyme with extraordinary properties.
The search for novel extremophilc fungi as producers of proteases is a thrust area
of research since such extremophilc proteases may find major applications in
modern day food processing, beverage production, animal nutrition, leather and
textiles processing, detergent manufacture, etc. This chapter addresses the basic
characteristics and advantages of using extremophilc fungal proteases in
industries. The major focus has been on discussing the potential fungal sources,
screening strategies, assay techniques, purification regime of these valued
macromolecules, and already documented roles of these extremophilic proteases
in food, pharmaceutical, and beverage manufacturing sector.
Keywords
Extremophilic · Fungal protease · Thermophilic · Psychrophilic · Cold adaptive
S. Bhattacharya (*) · A. Das
Department of Microbiology, School of Sciences, JAIN (Deemed-to-be University), Bangalore,
Karnataka, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_19
439

19.1 Introduction
Proteases (peptidases or proteolytic enzymes) constitute a variety of enzymes that
hydrolyse peptide bonds in other proteins leading to degradation of proteinaceous
substrates into their constituent amino acids, or it can be specific, resulting in
selective protein cleavage for post-translational modification and processing
(Yu et al. 2020).
Proteases hold central importance in industrial applications by involving selective
degradation of proteins. The present cost for the sale of industrial enzymes is about
$8 billion of which 65% is contributed by proteases alone (Omrane Benmrad et al.
2016; Barzkar et al. 2018). The specific hydrolytic behaviour of proteases imparts
applications in leather, food, pharmaceutical, detergent, silk-degumming, silver
recovery, waste management, and peptide synthesis (Razzaq et al. 2019).
19.2 Protease Types
Depending on their site of action, proteases are subdivided into exopeptidases and
endopeptidases. The exopeptidases act only near the ends of polypeptide chains.
Based on their site of action at the N or C terminus, they are classified as amino and
carboxypeptidases (de Souza et al. 2015) (Fig. 19.1).
Aminopeptidases (EC 3.4.14) act at a free N terminus of the polypeptide
chain and liberate a single amino acid residue, a dipeptide, or a tripeptide. The
carboxypeptidases act at C terminals of the polypeptide chain and liberate a single
amino acid or a dipeptide. On the basis of the nature of the amino acid residues at the
active site of the enzymes, carboxypeptidases involve three major groups: serine
peptidases (EC 2.4.16), metallopeptidases (EC 2.4.17), and cysteine peptidases
Fig. 19.1 Types of proteases
440 S. Bhattacharya and A. Das

(EC 2.4.18). Endopeptidases are characterized by their preferential action at the
peptide bonds in the inner regions of the polypeptide chain. Considering their
catalytic mechanism, the endopeptidases are of four subgroups, i.e. serine proteases
(EC 2.4.21), cysteine proteases (EC 2.4.22), aspartic proteases (EC 2.4.23),
metalloproteases (EC 2.4.24) (Mótyán et al. 2013).
Serine proteases are characterized by the presence of a serine group in their active
site. They are generally active at neutral and alkaline pH, with optima at pH 7–11,
low molecular mass (18–35 kDa) and have applications in a number of industries
(Sundus et al. 2016). Aspartic acid proteases, commonly known as acidic proteases,
are the endopeptidases that depend on aspartic acid residues for their catalytic
activity. The activity of all cysteine proteases depends on a catalytic dyad consisting
of cysteine and histidine. Generally, cysteine proteases are active only in the
presence of reducing agents such as HCN or cysteine. Papain is the best-known
cysteine protease. Metalloproteases are the most diverse of the catalytic types of
proteases. They are characterized by the requirement for a divalent metal ion for their
activity (Nageswara et al. 2019).
19.3 Natural Sources of Proteases
Proteases occur in animals, plants, and microorganism and have critical role in many
physiological and pathological processes such as protein catabolism, blood coagula-
tion, cell growth and migration, tissue arrangement, morphogenesis in development,
inflammation, tumour growth and metastasis, activation of zymogens, release of
hormones and pharmacologically active peptides from precursor proteins, and trans-
port of secretory proteins across membranes (Gaur and Bartariya 2020). Extracellu-
lar proteases catalyse the hydrolysis of proteins into smaller peptides and amino
acids for subsequent absorption into cells, constituting a very important step in
nitrogen metabolism (de Souza et al. 2015).
19.4 Microbial Proteases
Proteases play an important role in physiological processes like growth and differ-
entiation, metabolic processes, gene expression, and cell signalling (Banerjee and
Ray 2017). Although plants and animals produce proteases, microbes are considered
as promising candidates because of their diverse biochemical properties, limited
space requirement, and ease of genetic modifications (Saggu and Mishra 2017).
Among the proteases produced by plants, animals, and microbes, about 40% of
the total sale is contributed by microbes alone. The microbial proteases serve as
preferred driver of increasing economy in the area of white biotechnology because of
their desired characteristics needed in the industrial processes. Bacteria produce
majority of the proteases as neutral and alkaline in nature, but proteases from
fungi are neutral, acidic, or alkaline in nature, and ~60% of these have been
commercialized till 2009 (Inacio et al. 2015). Various neutral proteases such as
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 441

Umamizyme find their use in food industry due to their specificity in breaking
hydrophobic bonds at neutral pH (Bagnasco et al. 2013).
19.5 Fungal Proteases
Filamentous fungi are used in many industrial processes for the production of
enzymes and metabolites. Among the many advantages offered by the production
of enzymes by fungi are low material costs coupled with high productivity, faster
production, and the ease with which the enzymes can be modified. Further, enzymes,
being normally extracellular, are easily recoverable from the media (Meyer et al.
2016). Proteases production of fungal origin has an advantage over bacterial prote-
ase as mycelium can be easily removed by filtration. Besides, the use of fungi as
enzyme producer is safer than the use of bacteria, since the former are normally
recognized as generally regarded as safe (GRAS) (de Souza et al. 2015). Many
filamentous fungal species including Aspergillus,Chrysosporium,Fusarium,Peni-
cillium,Pleurotus,Rhizopus,Scedosporium, and Trichoderma are known to produce
proteases (Omrane Benmrad et al. 2019). Besides these, proteases from
basidiomycetes such as Agaricus bisporus,Armillaria mellea,Flammulina
velutipes,Grifola frondosa,Pleurotus ostreatus,Pleurotus eryngii,Phanerochaete
chrysosporium, and Schizophyllum commune have also been reported for protease
production (Gurung et al. 2013).
Fungal proteases have high industrial demand due to high stability and catalytic
activity, broad diversity, and substrate specificity required for various bioengineer-
ing and biotechnological applications. Moreover, the extracellular enzyme produc-
tion from fungal species leads to cost-effective production because of easy
downstream processing and recovery of enzymes which is a major obstacle in
industrial processes (Singh and Bajaj 2017).
19.6 Extremophilic Fungal Proteases
19.6.1 Thermophilic Proteases
19.6.1.1 Sources
Fewer studies exist that report extremophilic fungi expressing proteases. Aspergillus
niger,Chaetomium thermophilum,Fusarium oxysporum,Penicillium oxalicum,
Thermomonospora fusca YX, and Trichoderma harzianum are the significant
cultures with promising protease production ability to satisfy industrial demand
(Maťaťa et al. 2019).
The fungi with minimum and maximum temperatures for growth as 20 and 50 C,
respectively, are known as thermophilic fungi. Whereas the ones that can tolerate
temperature below 20–55 C are referred to as thermotolerant. Thermotolerant fungi
are easily found to be associated with stacks of plant biomass and agricultural
products with trapped humidity, oxygen, and plentiful supply of organic residues.
442 S. Bhattacharya and A. Das

Fungal tolerance for high temperature is a less frequent event when compared to
bacteria (some tolerating up to 100 C) as among the 50,000 known fungi, a mere
30 species can tolerate 40–45 C. Talaromyces thermophilus,Thermoascus
aurantiacus,Thermomyces ibadanensis, and T. lanuginosus grow optimally at
42–52 C but are reported to tolerate till 61 C (Maheshwari et al. 2000).
The fungal thermophiles’ability to produce extracellular proteases with less
microbial contamination during protease production demands attention towards
understanding temperature tolerance of these fungi which comparatively has been
less explored (Chen et al. 2004). Proteases of thermophilic Humicola lanuginose,
Malbranchea pulchella var. sulfurea,Mucor pusillus, and Penicillium duponti are
active within 45–55 C and pH 3–6. Thus for their high specific activity and thermal
stability, proteases from thermophilic fungi hold promise for industrial applications
(Li and Li 2009) (Table 19.1).
Thermophilic fungi produce protein hydrolases with higher thermostability,
hydrolysis rates, and appreciable activity at elevated temperatures. A few fungal
species like Thermoascus aurantiacus and Thermomyces lanuginosus are consid-
ered as a source of thermostable acid protease. The T. aurantiacus enzyme produc-
tion involved SSF system (with wheat bran, at 60 C) (Merheb et al. 2007).
Optimum protease activity of the thermophilic fungi Thermomyces lanuginosus is
frequently reported to be 70, 50, and 45 C, while for Thermomucor indicae-
seudaticae it is 70 C (Macchione et al. 2008; Merheb-Dini et al. 2010). Proteases
from Aspergillus oryzae and Penicillium sp. are reported to show optimum activities
at 45 C and 55 C, respectively (Germano et al. 2003; Vishwanatha et al. 2010).
19.6.1.2 Screening
Strains of T. lanuginosus were isolated from composting site, soil, and dung on
Emerson’s yeast extract soluble starch agar. All strains isolated were evaluated for
their protease profile by inoculation onto yeast extract agar plates (with 1% skim
milk). Following their growth on the screening medium (for 72 h), a proteolytic zone
was observed around the colonies of T. lanuginosus. Among the 500 isolates, P
134
strain produced the widest proteolytic zone and was hence considered for further
evaluation (Li et al. 1997).
While studying the protease production by thermophilic fungi (Chaetomium
thermophile var. dissitum,Humicola lanuginosa,Malbranchea pulchella var.
sulfurea, and Sporotrichum thermophile), the primary screening (preliminary
Table 19.1 Protease characteristics from prominent thermophilic fungi (Li et al. 2007)
Organism Nature Thermostability (C) pH
Malbranchea pulchella var. sulfurea Alkaline protease 50 8.5
Torula thermophila Alkaline protease 60 8.0
Penicillium duponti K-1014 Acid protease 60 2.5
Humicola lanuginosa Thiol protease 55 8.0
Chaetomium thermophilum PRO33 Serine protease 60 10.0
C. thermophilum PRO63 Serine protease 60 5.0
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 443

indication of proteolysis) involved growth on casein agar (containing 1% w/v
casein). As a part of the secondary screening procedure, spore suspensions (prepared
using a detergent solution) from the cultures (grown on yeast–glucose agar) were
used as inoculum for modified Czapek-Dox medium (composed of 4% w/v casein),
maintained at pH 7.4. The culture was grown at 45 C under shaking condition. By
periodic sampling of the medium the maximum production of extracellular protease
was determined by assaying for caseinolytic activity at three different pH values
(pH 3, 7, and 9). During the maximum extracellular protease activity, the cultures
were harvested and the cell-free extracts prepared and the evaluated for protease
activity. The best producers were found to be H. lanuginosa and M. pulchella var.
sulfurea (Ong and Gaucher 1973).
Evaluation of protease production under solid-state and submerged fermentation,
respectively, by thermophiles (Aspergillus flavus 1.2, Aspergillus sp. 13.33, Asper-
gillus sp. 13.34, Aspergillus sp. 13.35, Rhizomucor pusillus 13.36, Rhizomucor
sp. 13.37, T. aurantiacus Miehe, T. lanuginosus, and T. lanuginosus TO.03) reports
using milk powder, soybean flour, soybean milk, rice, and wheat bran. The most
satisfactory screening results were observed in solid-state fermentation involving
wheat bran. Under solid-state fermentation, T. lanuginosus,T. lanuginosus TO.03,
Aspergillus sp. 13.34, Aspergillus sp. 13.35, and Rhizomucor sp. 13.37 produced
protease at appreciable levels. While under submerged fermentation, the significant
proteolytic cultures were T. aurantiacus,T. lanuginosus TO.03 and 13.37, respec-
tively (Macchione et al. 2008).
19.6.1.3 Purification
A thermotolerant protease produced by a Fusarium oxysporum was purified to
homogeneity by Sephadex G-200 gel filtration column and α-casein agarose gel
affinity chromatography. The purified F. oxysporum protease was reported to have a
specific activity of 93.88 U/mg protein. The purification magnitude was 7.7 and the
total yield was 20%. Purified protease had an optimum pH of 5.0, while the optimum
temperature was 40 C (Ja’afaru et al. 2020).
The crude protease (later found to be thermostable) from A. flavus was
precipitated by ammonium sulphate saturation (70%) and dialyzed (24 h, 40 C)
against phosphate buffer (50 mM, pH 7.0). The filtrate was passed into a DEAE-
cellulose column (equilibrated with phosphate buffer, 50 mM, pH 7.0) and eluted
with a linear NaCl concentration gradient (0–0.4 M) in the same buffer and 3.0 ml
fractions were collected (flow rate 20 ml/h). Following the purification steps, a 284 U
of protease activity, 0.37 mg of total protein, 170 U/mg of specific activity, 5.8-fold
purification, and 3.2% recovery, respectively, were recorded (Muthulakshmi et al.
2011).
While purifying a novel thermotolerant fibrinolytic protease from Fusarium
sp. CPCC 480097, the culture supernatant is reported to be treated using a
two-step ammonium sulphate salting-out procedure (first at 40%, and then at
60%). The crude precipitate of the 60% saturation centrifuged (8000 gfor
30 min at 4 C) and dissolved in Tris–HCl buffer (20 mmol/l, pH 7.4) is then loaded
onto a G-25 column (16 25 mm). The protease-containing fraction was applied to
444 S. Bhattacharya and A. Das

a MonoQ column (10 100 mm). The active fraction was concentrated by
lyophilized after desalting. The concentrated sample was loaded onto a Superdex
75 column (16 600 mm). After the three purification steps, the enzyme was
purified 158.5-fold, with a protein content of 1.8 mg, total activity of 13,700 U,
specific activity of 76,111 U/mg and a final yield of 6.8%, respectively (Wu et al.
2009).
A novel thermostable protease (SPPS) from Pleurotus sajor-caju CTM10057 is
known to be purified to homogeneity by heat-treatment (80 C for 20 min), followed
by ammonium sulphate precipitation (35–55%)-dialysis, FPLC based ion-exchange
chromatography (UNO Q-6 column), and finally gel filtration chromatography
(HPLC-ZORBAX PSM 300 HPSEC). Following purification, enzyme activity,
79 10
4
; total protein, 10 mg; specific activity, 79,000 U/mg of protein; protein
recovery, 15%; nine-fold purification, respectively are reported (Omrane Benmrad
et al. 2019).
A serine protease with a pH optimum from 7 to 9 and activity over the range of
pH 3–10 is isolated and purified from culture filtrates of Penicillium charlesii after
16 days of inoculation. The enzyme can be purified by the following sequence of
procedures: (1) gel permeation chromatography through Sephacryl S-200,
(2) DEAE-Sepharose anion-exchange chromatography, and (3) fast protein liquid
chromatography over Superose 12 (Abbas et al. 1989).
19.6.1.4 Assay
The protease activity of the crude proteases from the thermophile T. lanuginosus was
estimated using the modified protocol of Rick (1974). The enzyme (0.5 ml) was
added to casein (2.5 ml, 0.4%) dissolved in Tris-HCl (0.2 M, pH 9.0) or 0.2 M
CH
3
COOH/CH
3
COONa (pH 5.0) buffer and incubated (50 C, 60 min).
Trichloroacetic acid (2 ml, 10%) was added and incubated (room temperature,
30 min). The solution was filtered and to the filtrate (1 ml), water (5 ml) was
added, and absorbance was determined spectrophotometrically at 280 nm (Rick
1974).
Crude cell extracts of thermophilic fungi (Chaetomium thermophile var. dissitum,
C. thermophile var. coprophile,Humicola lanuginosa,H. insolens,H. grisea var.
thermoidea,Penicillium duponti,Malbranchea pulchella var. sulfurea,
Sporotrichum thermophile, and Talaromyces thermophilus) were prepared by
subjecting the slurry of lyophilized cultures to ultrasonication for protease release.
Centrifugation (48,000 g, 30 min) of the sonicate yielded supernatants to be
considered as the crude cell-free extracts. Before protease assay, the supernatants
were diluted with the buffers (0.2 M glycine-HCI, pH 3.0; 0.2 M phosphate, pH 7.0;
or 0.2 M glycine-NaOH, pH 9.0). The enzyme (0.25 ml) was added to casein
(1.75 ml, 0.5% w/v) in an appropriate buffer and incubated (37 C, 1 h).
Trichloroacetic acid (3 ml, 5%) was then added, and after standing at room tempera-
ture for 30 min, the solution was filtered. To the filtrate (1 ml), NaOH (5 ml, 0.4 N)
and diluted phenol reagent (1 ml, 1:2 v/v) were added consecutively and incubated
(room temperature, 30 min) for determining the absorbance at 660 nm (Ong and
Gaucher 1973).
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 445

The extracellular thermostable acidic protease activity of Aspergillus terreus
NCFT4269.10 was determined according to the method of van den Hombergh
et al. (1995). Each protease sample (450 μl) was incubated with BSA (50 μl, 1%
w/v) in sodium acetate buffer (0.1 M, pH 4.0) at 37 C. After 30 min of incubation,
the reaction was terminated with trichloroacetic acid (500 μl, 10% w/v). After
incubation at 0 C for 30 min, the precipitated proteins were removed through
centrifugation (6000 rpm, 5 min) and the absorbance of the TCA-soluble fraction
was estimated as per Lowry et al. (1951).
19.6.2 Applications
Talaromyces emersonii thermozyme cocktails are in use as baking enzymes as they
impart a positive effect on the final product. Enzyme screening studies reveal the
presence of hemicellulases, amylolytic enzymes, and proteases in all cocktails
(in various relative amounts) is essential for the manipulation of dough. The results
show that small quantities of crude enzyme mixtures positively affect the antistaling
properties, volume and softness of bread, thereby improving baking. This makes
T. emersonii a novel source of efficient thermozymes for the baking industry (Waters
et al. 2010).
Thermostable proteases, acting in temperature range of 65–85 C successfully
convert proteins into amino acids and peptides, thus finding applications in baking,
brewing, detergents, and the leather industry (Haki and Rakshit 2003; Merheb et al.
2007) (Fig. 19.2).
19.6.3 Psychrophilic Proteases
19.6.3.1 Sources
Fungi can flourish under low temperature locations and inhabit permafrost, banks of
polar waters, glaciers, icebergs, and freshwater ice (Tojo and Newsham 2012). The
fungi that survive and are able to tolerate extreme low temperature conditions
(optimum growth temperature being 10 C but may grow at lower temperature)
are referred to as psychrophilic fungi. Further, fungal forms that grow maximally at
20 C are psychrotrophic fungi (Hassan et al. 2016).
Extracellular proteases are documented from psychrophilic fungi (Candida
humicola,Glaciozyma antarctica, and Rhodotorula mucilaginosa) and mesophilic
fungi (Aspergillus niger and Trichoderma harzianum) (Duarte et al. 2018). Species
pertaining to the genus Aspergillus ustus,Cryptococcus gilvescens,Humicola
marvinii,H.fuscoatra,Mrakia gelida, and Rhodotorula laryngis are significant
protease producers (Damare et al. 2006; Turchetti et al. 2008). Several deep-sea
fungi are capable of producing psychrophilic protease including Aspergillus terreus,
Beauveria brongniartii, and Acremonium butyric (Srilakshmi et al. 2014).
Protease producing fungi from Antarctic locations are from the genera
Acremonium,Candida,Cryptococcus,Chrysosporium,Embellisia,Exophiala,
446 S. Bhattacharya and A. Das

Fig. 19.2 Applications of various extremophilic proteases
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 447

Geomyces,Glaciozyma,Glomerella,Leuconeurospora,Leucosporidium,Mrakia,
Phoma,Pseudogymnoascus,Rhodotorula,Trichoderma,Vanrija, and
Wickerhamomyces (Fenice et al. 1997).
The protease of Candida humicola is reported to be active at 0–45 C and is
resistant to freeze-thaw cycles (Ray et al. 1992). The serine protease of
Leucosporidium antarcticum 107 is highly active at 25 C and even at 10 C
(Turkiewicz et al. 2003). Proteolytic enzyme of Geomyces pannorum exhibits the
greatest activity at 4 C (Krishnan et al. 2011).
19.6.3.2 Screening
For the determination of proteolytic activity from cold-tolerant Rhodosporidiobolus,
Cystofilobasidium, and Yamadazyma, the isolates were inoculated onto a basal
medium containing skimmed milk as a protein source: (g/l) skimmed milk 1.0;
yeast extract 1.0; agar-agar 18.0; KH
2
PO
4
1.0; MgSO
4
7H
2
O 0.1; CaCl
2
.H
2
O
0.05; NaCl 5.0; Na
2
CO
3
1.0. Proteolytic activity was determined by the observation
of clear halo around the colony (Yadav et al. 2016).
While studying the enzymatic activity of halotolerant and halophilic fungi from
the Great Sebkha of Oran in Northwestern Algeria, protease activity was detected on
milk agar (containing 30% skim milk and 2% agar). After incubation, the degrada-
tion of casein was reflected by a clear zone around the thallus (Sarath et al. 1989).
Aspergillus subramanianii,A. terreus,A. calidoustus,Penicillium egyptiacum, and
Cladosporium ramotenellum were the significant cold-tolerant fungal forms to show
proteolysis (Chamekh et al. 2019).
19.6.3.3 Purification
The cold adaptive protease of Candida humicola isolated from the soil of
Schirmacher Oasis can be concentrated by ultrafiltration followed by anion-
exchange chromatography purification (Ray et al. 1992). Glaciozyma antarctica
isolated from the sub-glacial waters (200 m deep) in Admiralty Bay when subjected
to concentration by acetone precipitation followed by purification in Sephadex G75
anion-exchange chromatography Sephacryl S-100 column is reported to result in
1560-fold purity and 22.7% yield (Turkiewicz et al. 2003). Rhodotorula
mucilaginosa L7 from the Antarctic marine macroalgae when concentrated by
ultrafiltration followed by purification in cation-exchange chromatography
Sephacryl S-100 column results in 15.6-fold purity and 29.7% yield (Lario et al.
2015).
19.6.3.4 Assay
While studying the molecular cloning of cold-adapted serine protease expression in
Antarctic yeast Glaciozyma antarctica PI12, the proteolytic activity involves esti-
mation by the modified protocol of Alias et al. (2014) using azocasein. Azocasein
(0.5%) was solubilized in Tris-HCl (0.1 M, pH 7) and enzyme action initiated by
adding enzyme sample (100 μl). Incubation conditions involved 20 C for 30 min.
Same volume of trichloroacetic acid (10% w/v) was added to stop the reaction and
absorbance read (450 nm).
448 S. Bhattacharya and A. Das

The alkaline, cold-tolerant protease activity of deep-sea isolate of Aspergillus
ustus NIOCC#20 was assayed using crude culture filtrate (150 μl) and azocasein
(250 μl, 2% azocasein prepared in 0.1 M boric acid-borax buffer, pH 9). The reaction
mixture was allowed to incubate (5 and 30 C, 30 min). The reaction was stopped by
addition of trichloroacetic acid (1.2 ml, 10% w/v). Any developed turbidity was
removed by centrifugation (8000 rpm, 10 min). To the supernatant, NaOH (1.4 ml,
1 N) was added and the absorbance read (440 nm). One azocasein digestion unit is
defined as the increase in absorbance (0.001/min) under the assay conditions
(Damare et al. 2006).
19.6.3.5 Applications
Aspartic protease is one of the most versatile enzymes in food processing. Owing to
the increasing demand in cheese products, microbial aspartic proteases serve as
beneficial complements for animal-derived milk coagulant. A novel aspartic prote-
ase gene (P10) from an Antarctic psychrophilic fungus Geomyces pannorum can be
successfully expressed in Aspergillus oryzae when cultured at 20 C. Its potential
application in cheese-making provides novel insights into application of psychro-
philic fungal protease in the food industry (Gao et al. 2018).
Cold-adapted fungal proteases find their applications in the food industry to
fasten the ripening of cheese, making frozen meat products tender, protecting the
status of thermosensitive nutrients, and as an effective treatment for haze-associated
proteins in wine and malted beverages. In laundries, cold-adapted fungal proteases
can be incorporated in detergents powder for ‘cold washing’to wash fabrics at room
temperature (Peterson et al. 2013) (Fig. 19.2).
19.6.4 Acid/Aspartic Proteases
19.6.4.1 Sources
While studying the acid protease production in fungal root endophytes, multiple
isolates of Phialocephala fortinii,Meliniomyces variabilis,Umbelopsis isabellina,
Hebeloma incarnatulum,Laccaria bicolor, and Irpex lacteus can be considered.
H. incarnatulum and L. bicolor secrete metalloproteases having pH optima above
4, while other fungi produce aspartic proteases with lower pH optima. M. variabilis
and P. fortinii exhibit intermediate levels of protein utilization and M. variabilis
exhibits a very low pH optimum (Mayerhofer et al. 2015).
Aspartic proteases are sourced from moulds and yeasts and rarely from bacteria.
Aspartic protease enzymes from fungal sources mainly fall into two groups: pepsin-
like enzymes produced by Aspergillus,Penicillium,Rhizopus, and Neurospora and
rennin-like enzymes produced by Mucor miehei,M. pusillus, and Endothia
parasitica (Mamo and Assefa 2018).
19.6.4.2 Screening
Primary screening for protease produced by Aspergillus Z1BL1 can be tested using
skim-milk agar medium for the production of the clear zone (Saran et al. 2007). The
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 449

detection medium is prepared using skim milk, agar–agar dissolved in 200 ml
distilled water, and 600 ml of phosphate buffer (0.2 M, pH 5.0). The plates can be
subsequently inoculated with previously purified fungal isolates and incubated
(30 C, 2 days). The plates are examined for the formation of the clearing zone by
flooding them with a solution of trichloroacetic acid or tannic acid (10% v/v). The
relative enzyme activity is calculated using the inhibition zone diameter and colony
diameter.
In order to detect the acid protease activity of sixty-one fungal endophytes
(Alternaria sp., Aureobasidium pullulans,Beltrania rhombica,Chaetomium
fibripilium,Colletotrichum acutatum,Fusarium sp., Curvularia sp., etc.) a qualita-
tive dot blot method is used. A gel is prepared by mixing a solution containing
acrylamide/bisacrylamide (1 ml, 30%); substrate (0.4 ml, 2% autoclaved gelatin);
Tris-HCl buffer (2.3 ml, 50 mM, pH 9.0); TEMED (0.003 ml, 100%); and ammo-
nium persulphate (0.003 ml, 40%) and poured in a gel cassette. After polymeriza-
tion, it is topped with Bis-Tris buffer (pH 7.0) or sodium acetate buffer (pH 5.0).
Thus, a composite gel made of gel strips of pH 5.0, pH 7.0, and pH 9.0 is obtained.
Lyophilized crude enzyme (10 mg) mixed with 1 ml of appropriate buffer
(as mentioned above) is centrifuged (14,000 rpm, 5 min, 20 C) and the enzyme
solution (5 μl) was spotted on the appropriate gel fields of the composite gel and
incubated (10 h), stained with Coomassie Brilliant Blue (R 250, 0.025%) for 3 h,
washed with distilled water, and observed for the presence of clear zones on the deep
blue gels (indicating enzyme activity). Protease action is clearly visible as a
colourless spot on the dark blue background of the gel. Twenty-five isolates out of
the 61 endophytes elaborated acidic protease (Thirunavukkarasu et al. 2017).
19.6.4.3 Purification
In order to collect the cell-free extracts from Aspergillus terreus and Aspergillus
niger (3-days old) fungal mats, the biomass were washed twice with 1.0 mM
dithiothreitol (DTT), KCl (50 mM), and phosphate buffer (150 mM, pH 7.5). The
resulting homogenates were cheesecloth filtered, centrifuged (1500 rpm, 5 min), and
the supernatant considered as crude protease. The crude protein was precipitated
with ammonium sulphate (70% w/v) and suspended in phosphate buffer (20 ml, pH
7.5) followed by treatment with calcium phosphate (4 C, for 24 h). The supplement
was centrifuged (5000 rpm, 15 min, 4 C) and supernatant added to Sephadex G-200
affinity chromatography column. Elution was done with phosphate (50 mM, pH
7.5). Results indicated that for A. terreus, following the purification steps, the total
protein, specific activity, and yield were 1.9 mg of protein, 294.7 U/mg protein, and
16%, respectively. While for A. niger, after purification the total protein, specific
activity, and yield were 7.8 mg of protein, 179.4 U/mg protein, and 16%, respec-
tively. A 81.9 and 28.9-fold increase in purification was recorded for A. terreus and
A. niger, respectively (El-Shora and Metwally 2008).
Penicillium duponti K1014, a thermophile under submerged fermentation pro-
duced an acid protease that was purified by the combination of alcohol precipitation,
DEAE-cellulose column chromatography, batchwise treatment with O-
carboxymethyl-cellulose, and Sephadex G-200 gel filtration chromatography.
450 S. Bhattacharya and A. Das

These procedures were carried out at 0–4C. After purification the total activity,
specific activity, and yield were 279,842 U, 193 U/mg protein, and 25.5%, respec-
tively (Hashimoto et al. 1973).
The Aspergillus hennebergii HX08 mat growth on wheat bran was extracted with
acetic acid–sodium acetate buffer (0.1 mol/l, ratio of solvent to bran, 10:1 w/v, pH
5.4) for 12 h. The mixture was centrifuged (12,000 rpm, 15 min), and the supernatant
collected as crude enzyme. After membrane filtration (0.22 μm), the crude enzyme
solution was further purified by precipitation, ion-exchange chromatography, and
Sephacryl S-300 gel chromatography. All of the purification steps were carried out at
4C. After purification the total activity, specific activity, and yield were 2257.07 U,
103.67 U/ml, and 16.09%, respectively. A 22.94-fold increase in purification was
recorded (Huang et al. 2017).
Purification of the acid protease from Aspergillus brasiliensis strain BCW2 was
based on a three-step procedure involving initial precipitation with ammonium
sulphate (80% concentration), the precipitated fraction was centrifuged
(10,000 rpm, 4 C, 10 min), and the pellet re-suspended in phosphate buffer
(0.1 M, pH 7). Finally, the solution was subjected to dialysis overnight (membrane
cut-off 12,000 Da) and the dialyzed sample subjected to a Sephadex G-200 gel
filtration column (equilibrated with 0.1 M phosphate buffer, pH 7.0) to give a 7.7-
fold protein purification and 29% recovery, respectively (Chimbekujwo et al. 2020).
While purifying the acid protease of A. niger, the purification steps involved
ammonium sulphate precipitation (60–90% saturation), suspension of precipitate in
sodium sulphate buffer (0.02 M, pH 6.8), and solution dialyzed overnight against the
same buffer and the solution was loaded on DEAE-cellulose column (2 30 cm)
previously equilibrated with sodium phosphate buffer (0.02 M, pH 7). The column
elusion involved the same buffer with increase in morality of NaCl (0.02–0.5 M)
(Ahmed 2018).
The milk clotting acid protease of Rhizopus oryzae was precipitated between
saturation of ammonium sulphate (20–60%). The precipitate obtained after centrifu-
gation (10,000 g, 30 min) was suspended in phosphate buffer (50 mM, pH 6.0)
and dialyzed overnight against the same buffer. The obtained enzyme preparation
was further purified on a pre-equilibrated (with 50 mM phosphate buffer, pH 6.0)
DEAE-cellulose column (25 2.6 cm) and size-exclusion chromatography involv-
ing Sephadex G-100 column (65 1.5 cm). After purification the total activity,
specific activity, and recovery were 8279 U, 759.5 U/mg protein, and 26%, respec-
tively. A 91-fold increase in purification was recorded (Kumar et al. 2005).
An aspartic protease (Peptidase R) sourced from Rhizopus oryzae was purified to
homogeneity by anion-exchange chromatography (HiLoad 26/10 Q Sepharose
High-Performance column, equilibrated with 50 mM sodium phosphate buffer, pH
7.0) followed by the hydrophobic interaction chromatography column (HiTrap
Phenyl Sepharose column, equilibrated with 50 mM sodium phosphate buffer, pH
7.0) that resulted in total protein content of 87 mg, total activity of 5 10
6
, specific
activity of 57.5 10
6
, and 3.4-fold increase in purification fold, respectively (Hsiao
et al. 2014).
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 451

19.6.4.4 Assay
Aspergillus Z1BL1 acid protease activity was assayed according to the method of
Kembhavi et al. (1993) using haemoglobin as substrate. Enzyme solution (0.5 ml),
suitably diluted, was mixed with haemoglobin (1 ml, 2% w/v, in 100 mM glycine-
HCl, pH 3.0) and the mixture was incubated (50 C, 10 min). The reaction was
terminated by adding trichloroacetic acid (2 ml, 5% w/v). The mixture was allowed
to incubate (room temperature, 15 min) and then centrifuged (10,000 g, 15 min) to
remove the precipitate. The absorbance of the soluble fraction was measured at
280 nm. A standard curve was generated using tyrosine solutions (0–50 mg/l). One
unit of protease activity is defined as the amount of enzyme required to liberate 1 μg
of tyrosine per min under the experimental conditions.
Rhizopus oligosporus acid protease activity was performed by method as
described by Arima et al. (1970) with little modifications. Casein (2.5 ml, 1% w/v)
in acetate buffer (0.02 M, pH 4.0) and crude enzyme extract (0.5 ml) were mixed and
incubated (37 C, 10 min) and the reaction was terminated by adding trichloroacetic
acid solution (2.5 ml, 0.44 M). The precipitate formed was removed by filtration
through Whatman No 1 filter paper. Folin Ciocalteau reagent (1 ml) and sodium
carbonate solution (2.5 ml, 0.55 M) were added to the clear filtrate (1 ml) and
incubated (20 min, 37 C) for colour development. The absorbance at 660 nm
expressed the activity of enzyme in terms of proteolytic units.
Acid protease activity of 14 days cultures of fungal endophytes (grown on BSA
media) was quantified by the fluorescently labelled casein assay. The experimenta-
tion involved mixing of culture or control media (50 ml) with fluorescently labelled
casein (50 ml) in citrate–citric acid buffer in black 96-well microplates. The casein
buffer was adjusted to a pH range (pH 2.0–6.0). The microplates were incubated
(in dark, 18 h) and fluorescence read (excitation at 590 nm, emission at 645 nm)
(Mayerhofer et al. 2015).
19.6.4.5 Applications
(a) Food industry
The appreciable actionf of acid proteases in coagulating milk proteins advocates
their high demand in food processing where they are majorly applied to curdle
milk proteins, from which cheese is manufactured following the removal of
whey (Neelakantan and Mohanty 1999). The prominent forms of milk-curdling
enzymes include animal rennets, microbial proteases, recombinant chymosin,
and plant rennet (Ward et al. 2009). Worldwide, the dairy industry suffered a
scarcity of animal rennet, due to the increase in cheese demand and the resis-
tance from animal rights protection bodies (Furia 1980). This ushered the use of
alternate milk coagulation enzymes, primarily of microbial origin that would
cleave the specific peptide bond (Phe105-Met106 in bovine casein) in casein to
liberate macromolecules and para-casein (Rani et al. 2012). In similar lines, the
recombinant rennin (calf chymosin) is expressed in A. niger var. awamori for
dairy industry needs. Industrially, the most important protease for cheese
making is sourced from Rhizomucor miehei (Ward et al. 2009).
452 S. Bhattacharya and A. Das

Fresh cheese produced using an ochratoxin-free extracellular acid protease
from Aspergillus niger FFB1 and reconstituted cow milk as a substrate show
similar basic characteristics (pH 4.5, acid taste, and colour) as cheeses produced
with calf rennet (Fazouane-Naimi et al. 2010). The concentration of free amino
acid (FAA) and physiochemical characteristics is similar in the Turkish, white,
brined cheese produced using calf rennet and microbial rennet from Rhizomucor
miehei (Çepoğlu and Güler-Akın2013).
Besides their widespread involvement in the dairy industry, fungal-sourced
acid proteases have also been extensively harnessed in food seasonings and the
improvising protein-rich cereal based foods. Gluten (an insoluble protein found
in wheat flour determines the dough properties) can be enzymatically treated to
facilitate the handling of dough and mixing time reduction. Furthermore,
increased loaf volume and the accumulation of a wider range of products result
from wheat gluten following the action of acid proteases from A. oryzae (Rao
et al. 1998).
(b) Medical and pharmaceutical industry
Acid (aspartic) proteases are commercially available for tackling certain lytic
enzyme deficiency syndromes and in the formulation of digestive syrups
(Chanalia et al. 2011). The major aspartic proteases secreted in vitro by
C. albicans,C. parapsilosis, and C. tropicalis are Sap2, Sapp1, and Sapt1,
respectively (Rao et al. 1998). Several candidal aspartic proteases contribute to
human infections and the search for suitable aspartic protease inhibitors is of
interest for treating these infections (Aoki et al. 2012) (Fig. 19.2).
(c) Beverage industry
Industrially processed fruit-based beverages receive clarification steps to pre-
vent turbidity from haze. Acid proteases from A. saitoi (aspergillopepsin I) are
used in the manufacture of certain fruit juices and alcoholic beverages, to
degrade the proteins contributing to turbidity (Sumantha et al. 2006). In the
fermentation of sake (Japanese alcoholic beverage), A. oryzae acid proteases
decide the final taste of the product where they bring protein hydrolysis from the
steamed rice to release amino acids and peptides (Shindo et al. 1998).
Haze formation in fresh and fermented fruit and cereal based beverages is due
to the aggregation and precipitation of proteins that usually is associated with
microbial spoilage (Falconer et al. 2010). Addition of A. niger aspartic proteases
to kiwi fruit juice reduces the visible turbidity and prevents cold storage
associated with haze formation (Dawes et al. 1994). Addition of a commercial
fungal acid protease from A. niger to cherry juice resulted in a significant
reduction in the immediate turbidity. Results also indicated that during the
cold storage of the same there was a minimal impact on clarification (Pinelo
et al. 2010). Similar observations were also associated with the black currant
juice production where commercially available amino acid protease A (a form of
acid protease) from A. niger and Mucor miehei acid protease was used (Landbo
et al. 2006).
In beer manufacture, haze development is an outcome of the proteins from
autolyzed yeasts, dead bacteria from malt, presence of glucan from modified
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 453

malt, oxalate from calcium deficient worts, residual starch and carbohydrates. In
the breweries, fungal acid proteases are investigated to degrade proteins (related
to haze formation) during storage. A. niger acid proteases effectively prevent
chill-haze formation in beer, due to the hydrolysis of proline-rich proteins,
thereby liberating a peptide fraction that fails to form a complex with the
polyphenols (Steiner et al. 2010).
In a recent investigation, an acid protease (aspergillopepsin I and II) from
A. niger var. macrosporus was used together with flash pasteurization to
degrade haze proteins in white wine. Results indicate that addition of the
aspergillopepsin directly to the fermentation wort results in a 20% reduction
protein (Marangon et al. 2012). At wine-making temperatures, an acid protease
(BcAP8) from Botrytis cinerea effectively reduces haze and retains its activity
after completion of fermentation (Van Sluyter et al. 2013) (Fig. 19.2).
19.6.5 Alkaline Proteases
19.6.5.1 Sources
Olivieri et al. (2002) identified and characterized a serine protease from Fusarium
solani f. sp. eumartii that is found to be associated with potato tuber infections. The
synthesis of alkaline proteases by Fusarium culmorum, F. poae, and F.
graminearum was reported in grains of barley (Kudryavtseva et al. 2013). When
soil sample was cultured from Charm-Shahr, Iran, researchers successfully isolated
Aspergillus oryzae CH93 that is documented to produce an alkaline protease active
at pH 8 and 50 C (Salihi et al. 2017). The production of alkaline protease by an
Aspergillus flavus strain using solid-substrate fermentation is considered as a depil-
atory agent (Malathi and Chakraborty 1991).
Aspergillus clavatus was isolated from the Atlantic forest soil, Peruíbe city, SP,
Brazil. This strain shows the highest extracellular proteolytic activity that is primar-
ily an alkaline protease (Tremacoldi and Carmona 2005). An alkaline
metalloprotease produced by Aspergillus niger C-15 was purified and the optimum
pH and temperature for the protease activity are pH 8.0 and 60 C, respectively (Kim
2004). When cultivated in the presence of collagen (200 μg/ml) as sole nitrogen and
carbon source, Aspergillus fumigatus secretes an inducible alkaline protease
(AlPase) with maximum activity at pH 9.0 against azocollagen (Monod et al.
1991) (Table 19.2).
19.6.5.2 Screening
Aspergillus nidulans,A. glaucus,A. terreus, and A. fumigatus isolated from mush-
room compost and cow manure were screened for their alkaline protease activity on
Czapek-Dox agar media containing gelatin. Following incubation (45 C, 48 h)
resulting in the growth of organism, the plate was flooded with mercuric chloride
solution. Presence of a clear zone around the colony was an indication of an alkaline
protease activity (Singhania et al. 2018).
454 S. Bhattacharya and A. Das

In order to screen Trichoderma longibrachiatum and A. niger for their ability to
produce alkaline protease, the cultures were spot inoculated at the centre of casein
agar, skimmed milk agar, and gelatin agar (supplemented with 1 mg/100 ml strepto-
mycin), respectively. pH of these media was maintained at pH 8.5. Following
incubation at room temperature for 3 days, results indicated that both the isolates
resulted in >20 mm zone of clearance in the three media (Suryawanshi and Pandya
2017).
Production of alkaline protease by A. flavus and A. niger was detected by using
the plate assay method of Sudarkodi et al. (2015), using gelatin (1% w/v) as a protein
supplement in the growth medium. The fungal isolates were spot inoculated on the
growth medium. Following inoculation, the petridishes were incubated (28 1C,
3 days) and gelatinolysis was observed (as a clearing zone upon flooding the plate
with aqueous saturated solution of mercuric chloride reagent) around fungal
colonies. The gelatinolysis zone was measured and enzyme activity was calculated
using the concept of zone index.
19.6.5.3 Assay
Protease activity in the crude enzyme extract was determined according to the
method of Sudarkodi et al. (2015) by using casein as substrate. Casein solution
(5 ml, 0.65%) was added as a substrate and the enzyme (1 ml)–substrate mixture was
incubated (37 C, 30 min). The reaction was terminated by addition of
trichloroacetic acid solution (5 ml) and the solution was filtered using Whatman
No 1 filter paper. The filtrate was mixed with sodium carbonate (5 ml) and followed
by addition of Folin Ciocalteus phenol reagent (1 ml of two fold diluted). The
resulting solution was incubated in dark (30 min, room temperature) and the
absorbance measured at 660 nm against a reagent blank using tyrosine standard.
The Aspergillus flavus alkaline protease activity was assayed by the method of
Lovrien et al. (1985). Reaction mixture (3 ml) containing casein (0.5% in 2.95 ml of
0.1 M Tris-HCl buffer, pH 8.0) and enzyme (0.1 ml) was incubated (40 C, 30 min).
The reaction was stopped by adding cold trichloroacetic acid (3 ml, 10%). After 1 h,
each of the culture filtrate was centrifuged (8000 rpm, 5 min) to remove the
precipitate and absorbance of the supernatants was read (540 nm). The amount of
Table 19.2 Fungal sources of alkaline protease and (Sharma et al. 2019)
Organism Source pH Temperature (C)
Myceliopthora sp. Poultry soil 9.0 40–45
Penicillium sp. Soil 9.0 45
Aspergillus niger C-15 Poultry soil 8.0 60
Aspergillus clavatus Forest soil 9.5 40
Aspergillus parasiticus Sputum sample 8.0 40
Aspergillus terreus gr. Agricultural soil 11.0 50
Aspergillus nidulans HA-10 Poultry soil 8.0 35
Pleurotus citrinopileatus Fruiting bodies 10.0 50
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 455

amino acids released was calculated from a standard curve plotted against a range of
known concentrations of tyrosine.
19.6.5.4 Purification
While purifying an alkaline proteinase of Fusarium culmorum, the concentrated
culture medium was centrifuged (1700 g, 5 min) and the supernatant was applied
to a P-30 size-exclusion column (2.5 70 cm) equilibrated with ammonium acetate
(20 mM, pH 5.0). The column was eluted with the same buffer and the fractions that
voided the size-exclusion column were combined and subjected to carboxymethyl
cellulose cation-exchange chromatography column (1 6 cm). Immediately prior to
using, the enzyme was subjected to a final HPLC-cation exchange purification step
with a Shodex IEC CM-825, 8 75 mm column. Following the purification steps,
the purified alkaline protease had a protein content of 0.06 mg, enzyme activity of
150 U, specific activity of 2500 U/mg, 11% yield, and 8.3-fold purification, respec-
tively (Pekkarinen et al. 2002).
With the objective to purify an alkaline protease (SAPTEX) of P. chrysogenium
X5, the broth culture was centrifuged (20 min, 10,000 g). The clear supernatant
was heat-treated (10 min, 80 C), and by centrifugation (9000 gfor 30 min) all
insoluble materials removed. Ammonium sulphate saturation (between 30 and 50%)
was used for supernatant precipitation. Recovery of the precipitate was done by
centrifugation (20 min, 9000 g) followed by its resuspension in Tris-HCl (50 mM,
pH 8.5) and dialyzed overnight. The obtained supernatant was loaded to a previously
equilibrated UNO Q-12 FPLC column (1253 mm). The proteins were eluted with
the same buffer containing an increasing concentration of NaCl (0–500 mM). The
pooled active fractions were concentrated in centrifugal micro-concentrators
(30-kDa cut-off membranes). The purification profile of SAPTEX is as follows:
enzyme activity, 157 10
4
; total protein, 20 mg; specific activity, 78,500 U/mg of
protein; protein recovery, 35%; 9.3-fold purification, respectively (Omrane Benmrad
et al. 2018).
Broth culture of Trametes cingulata CTM10101was centrifuged (30 min,
10,000 g); the supernatant was used as the crude serine alkaline protease prepara-
tion and submitted to the following purification steps. It was initially saturated up to
40% ammonium sulphate, centrifuged (10,000 g, 20 min), and the supernatant
re-saturated with 60% with ammonium sulphate, re-centrifuged, re-suspended in
NaCl buffer (10 mM), and dialyzed overnight. The sample thus obtained was loaded
on a fast protein liquid chromatography system (735 mm UNO S-1 column
previously equilibrated with 20 mM Histidine buffer supplemented with 2 mM
CaCl
2
, pH 5.5). The proteins were eluted with the same buffer containing an
increasing concentration of NaCl (0–500 mM). Pooled fractions containing protease
activity were concentrated in centrifugal micro-concentrators with a cut-off mem-
brane (10 kDa). The purification profile is as follows: total activity, 141 10
4
U;
total protein, 15 mg; specific activity, 94,000 U/mg; purification fold, 6.27 and
protein recovery, 20%, respectively (Omrane Benmrad et al. 2016).
A halotolerant alkaline serine protease from Penicillium citrinum YL-1 (isolated
from traditional Chinese fish sauce) when purified by ammonium sulphate
456 S. Bhattacharya and A. Das

precipitation, dialysis, and DEAE 52-Cellulose column results in a 4.66-fold
increase in specific activity (110.68 U/mg) (Xie et al. 2016).
An alkaline protease is reported to be purified from culture broth of Penicillium
expansum by fractioning with acetone and column chromatography on Sephadex
G-100 and DEAE-Sephadex A-50. The purification profile is as follows: total
activity, 744 PU; total protein, 12.08 mg; specific activity, 61.59 PU/mg; purification
fold, 96.23 and protein recovery, 48%, respectively (Umar 1994).
19.6.5.5 Applications
Fungal alkaline proteases are involved in a variety of applications, primarily in the
detergent and food industries. In view of the recent trend of developing eco-friendly
technologies, fungal alkaline proteases are envisaged to have extensive applications
in leather treatment and in several bioremediation processes. These enzymes are also
used to develop high value added products (Kumar and Takagi 1999) (Fig. 19.2).
(a) Detergent industry.
Proteases are one of the standard ingredients of all kinds of detergents ranging
from those used for household laundering (Nehra et al. 2002) to reagents used
for cleaning contact lenses (Anwar and Saleemuddin 2000) or dentures. The
ideal detergent protease should possess substrate specificity to facilitate the
removal of a large variety of stains due to food, blood, and other body
secretions. Activity and stability at high pH and temperature and compatibility
with other chelating and oxidizing agents added to the detergents are among the
major prerequisites for the use of proteases in detergents. A number of published
reports are available on the compatibility of the fungal alkaline proteases with
detergents (Pundir et al. 2012). The fungal alkaline protease Conidiobolus
coronatus was consistent with commercial detergents used in India while
maintaining 43% of its activity in the presence of calcium (25 mM) and glycine
(M). These data imply that protease obtained from C. coronatus has potential for
use in laundry detergents (Lourdes et al. 2014) (Fig. 19.2).
(b) Leather industry.
Alkaline proteases with elastolytic and keratinolytic activity have been used in
leather processing, especially for the dehairing and debating of skins and hides.
The enzymatic process is easy to control, less time consuming, and also helps in
waste management and is therefore eco-friendly. In addition enzymatic treat-
ment destroys undesirable pigments and increases the skin area, thereby pro-
ducing clear hide (Arora 2003). Few fungal alkaline proteases have been
reported to be suitable and find application in leather industry (Malathi and
Chakraborty 1991; Pal et al. 1996) (Fig. 19.2).
(c) Dairy industry.
The major application of proteases in the dairy industry is in the manufacture of
cheese, proteolysis is responsible for characteristic of most varieties and is
indispensable for good flavour and textural development (Fox 1982). Perea
et al. (1993) have used alkaline protease for the production of whey hydrolysate
from the cheese whey although rennet is generally the enzyme of choice for
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 457

cheese making. In the dairy industry, bovine rennet is still widely used in
making cheese; fungi such as Rhizomucor miechie,R. pusillus, and A. oryzae
are extensively used for the production of proteases for use as milk coagulants
(Neelakantan and Mohanty 1999). Another extract with the powder of
coagulating milk, already produced industrially, is derived from the fungus
A. niger var. awamori (Neves Souza and Silva 2005) (Fig. 19.2).
(d) Pharmaceutical industry.
The wide diversity and specificity of fungal protease are used to great advantage
in developing effective therapeutic agents. Oral administration of protease from
Aspergillus oryzae has been used as a digestive aid to correct certain lytic
enzyme deficiency syndromes (Rani et al. 2012). Alkaline proteases have also
been used for developing production of medical use, such as for the treatment of
burns and purulent wound. A. niger LCF 9 alkaline protease has a high
collagenolytic activity and is being used for therapeutic application (Kumar
and Takagi 1999). Proteases from the tested Aspergillus strains exhibited
promising hydrolytic activities towards fibrinogen, fibrin, and blood clot
(El-Shora and Metwally 2008). Aspergillus protease can be used as digestive
aids in gastrointestinal disorders such as dyspepsia (Fig. 19.2).
(e) Brewing industry.
In brewing, proteases have two major applications. They can be used during the
cereal mashing process to increase the yield of extract. Though papain, brome-
lain, and papsin are the traditionally used proteases in chill proofing process,
microbial proteases also have been reported to be useful. The rennet produced
by Mucor pusillus has been reported to be effective for beer clarification (Nelson
and Witt 1973) (Fig. 19.2).
(f) Waste management.
Waste from poultry and leather industry is keratin rich whose polypeptide is
densely packed and stabilized by several weak interactions in addition to
disulphide bonds. Fungal keratinases from Aspergillus oryzae,Chrysoporium
inducum,Trichophyton sp., A. terreus,Microsporum gypseum,Fusarium
oxysporum have also been studied towards the degradation of keratin (Kim
2003; Ali et al. 2011; Sharma and De 2011).
19.7 Conclusions
The use of extremophilic fungal proteases in industries has been an integral event for
decades together and some of these fungal cultures are efficient producers of many
hydrolytic enzymes. The fact that such proteases exhibit specific range of action and
vast diversity in terms of being active over a very wide range of experimental
conditions has made them attractive models for industrial usage. Besides being
widely distributed in nature, extremophilic fungi are preferred over their mesophilic
counterparts owing to their ability to tolerate harsher growth conditions and to
overproduce hydrolytic enzyme with extraordinary properties. The search for
novel extremophilc fungi as producers of proteases is a thrust area of research
458 S. Bhattacharya and A. Das

since such extremophilc proteases find major applications in modern day food
processing, beverage production, animal nutrition, leather and textiles processing,
detergent manufacture, etc. However, only 5% of the protease producing
extremophilc fungi have been studied in great detail and applied in large scale
processing facilities. Thus in future, there lies a great opportunity to engineer
proteases using recombinant DNA technology for heterologous expression, site-
directed mutagenesis, and directed evolution of proteases with improved properties
and catalytic efficiency to meet newer industrial demands.
Acknowledgements We extend our sincere gratitude to the management of JAIN (Deemed-to-be
University) for providing the research facilities.
References
Abbas CA, Groves S, Gander JE (1989) Isolation, purification, and properties of Penicillium
charlesii alkaline protease. J Bacteriol 171:5630–5637
Ahmed ME (2018) Extraction and purification of protease from Aspergillus niger isolation. Pharm
Pharmacol Int J 6:96–99
Ali TH, Ali NH, Mohammed LA (2011) Production purification and some properties of extracellu-
lar keratinase from feathers-degradation by Aspergillus oryzae NRRL-447. J App Sci Environ
Sanit 6:123–136
Alias N, Mazian MA, Salleh AB, Basri M, Rahman RNZRA (2014) Molecular cloning and
optimization for high level expression of cold-adapted serine protease from Antarctic yeast
Glaciozyma antarctica PI12. Enzyme Res 2014:1–20
Anwar A, Saleemuddin (2000) Alkaline protease from Spilosoma obliqua, potential application in
bio-formulations. Biotechnol App Biochem 3(2):85–89
Aoki W, Kitahara N, Miura N, Morisaka H, Yamamoto Y, Kuroda K, Ueda M (2012) Candida
albicans possesses Sap7 as a pepstatin A in sensitive secreted aspartic protease. PLoS One 7:
e32513
Arima K, Yu J, Iwasaki S (1970) Milk clotting enzyme from Mucor pusillus var Lindt. In:
Perlmann G, Lorald L (eds) Methods in enzymology. Academic Press, New York, pp 446–459
Arora DK (2003) Handbook of fungal biotechnology, 2nd edn. CRC Press, Taylor & Francis, Boca
Raton
Bagnasco L, Pappalardo VM, Meregaglia A, Kaewmanee T, Ubiali D, Speranza G, Cosulich ME
(2013) Use of food-grade proteases to recover umami protein-peptide mixtures from rice
middlings. Food Res Int 50:420–427
Banerjee G, Ray AK (2017) Impact of microbial proteases on biotechnological industries.
Biotechnol Genet Eng Rev 8725:1–25
Barzkar N, Homaei A, Hemmati R, Patel S (2018) Thermostable marine microbial proteases for
industrial applications: scopes and risks. Extremophiles 22:335–346
Çepoğlu F, Güler-Akın MB (2013) Effects of coagulating enzyme types (commercial calf rennet,
Aspergillus niger var. awamorias recombinant chymosin and Rhizomucor miehei as microbial
rennet) on the chemical and sensory characteristics of white pickled cheese. Afr J Biotechnol 12:
5588–5594
Chamekh R, Deniel F, Donot C, Jany JL, Nodet P, Belabid L (2019) Isolation, identification and
enzymatic activity of halotolerant and halophilic fungi from the Great Sebkha of Oran in
Northwestern of Algeria. Mycobiology 2019:1–12
Chanalia P, Gandhi D, Jodha D, Singh J (2011) Applications of microbial proteases in pharmaceu-
tical industry: an overview. Rev Med Microbiol 22:96–101
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 459

Chen XG, Stabnikova O, Tay JH, Wang JY, Tay STL (2004) Thermoactive extracellular proteases
of Geobacillus caldoproteolyticus, sp. nov., from sewage sludge. Extremophiles 8:489–498
Chimbekujwo KI, Ja’afaru MI, Adeyemo OM (2020) Purification, characterization and optimiza-
tion conditions of protease produced by Aspergillus brasiliensis strain BCW2. Sci Afr 8:e00398
Damare S, Raghukumar C, Muraleedharan UD, Raghukumar S (2006) Deep-sea fungi as a source
of alkaline and cold-tolerant proteases. Enzym Microb Technol 39:172–181
Dawes H, Boyes S, Keene J, Heatherbell D (1994) Protein instability of wines—influence of protein
isoelectric point. Am J Enol Vitic 45:319–326
Duarte AWF, dos Santos JA, Vianna MV, Vieira JMF, Mallagutti VH, Inforsato FJ, Wentzel LC,
Lario LD, Rodrigues A, Pagnocca FC, Pessoa A Jr, Sette LD (2018) Cold-adapted enzymes
produced by fungi from terrestrial and marine Antarctic environments. Crit Rev Biotechnol 38:
600–619
El-Shora HM, Metwally MAA (2008) Production, purification and characterisation of proteases
from whey by some fungi. Ann Microbiol 58:495–502
Falconer RJ, Marangon M, Van Sluyter SC, Neilson KA, Chan C, Waters EJ (2010) Thermal
stability of thaumatin-like protein, chitinase, and invertase isolated from Sauvignon blanc and
Semillon juice and their role in haze formation in wine. J Agric Food Chem 58:975–980
Fazouane-Naimi F, Mechakra A, Abdellaoui R, Nouani A, Daga SM, Alzouma AM, Gais S,
Penninckx MJ (2010) Characterization and cheese-making properties of rennet-like enzyme
produced by a local Algerian isolate of Aspergillus niger. Food Biotechnol 24:258–269
Fenice M, Selbmann L, Zucconi L, Onofri S (1997) Production of extracellular enzymes by
Antarctic fungal strains. Polar Biol 17:275–280
Fox PF (1982) Proteolysis in milk and dairy products. Biochem Soc Trans 10:282–284
Furia TE (1980) Handbook of food additives. CRC Press, Boca Raton
Gao B, He L, Wei D, Zhang L (2018) Identification and magnetic immobilization of a pyrophilous
aspartic protease from Antarctic psychrophilic fungus. J Food Biochem 2018:e12691
Gaur A, Bartariya G (2020) Protease production by Aspergillus flavus under solid state fermentation
using cotton seeds and sugarcane bagasse. Int J Chem Stud 8:2117–2120
Germano S, Pandey A, Osaku CA, Rocha SN, Soccol CR (2003) Characterization and stability of
proteases from Penicillium sp. produced by solid-state fermentation. Enzym Microb Technol
32:246–251
Gurung N, Ray S, Bose S, Rai V (2013) A broader view: microbial enzymes and their relevance in
industries, medicine, and beyond. Biomed Res Int 2013:1–18
Haki G, Rakshit SK (2003) Developments in industrially important thermostable enzymes: a
review. Bioresour Technol 89:17–34
Hashimoto H, Kaneko Y, Iwaasa T, Yokotsuka T (1973) Production and purification of acid
protease from the thermophilic fungus, Penicillium duponti K1014. Appl Microbiol 25:584–588
Hassan N, Rafiq M, Hayat M, Shah AA, Hasan F (2016) Psychrophilic and psychrotrophic fungi: a
comprehensive review. Rev Environ Sci Biotechnol 15:147–172
van den Hombergh JPTW, van de Vondevoort PJI, van der Heijden NCBA, VisserNew J (1995)
Protease mutants in Aspergillus niger result in strongly reduced in vitro degradation of target
proteins: genetic and biochemical characterization of seven complementation groups. Curr
Genet 1995:28299–28308
Hsiao NW, Yeh C, Kuan Y, LeeY LS, Chan H, Kao C (2014) Purification and characterization of an
aspartic protease from the Rhizopus oryzae protease extract, peptidase R. Electron J Biotechnol
17:89–94
Huang Y, Wang Y, Xu Y (2017) Purification and characterisation of an acid protease from the
Aspergillus hennebergii HX08 and its potential in traditional fermentation. J Inst Brew 123:
432–441
Inacio FD, Ferreira RO, Araujo CAV, De Brugnari T, Castoldi R, Peralta RM, de Souza CGM
(2015) Proteases of wood rot fungi with emphasis on the genus Pleurotus. Biomed Res Int 2015:
10
460 S. Bhattacharya and A. Das

Ja’afaru MI, Chimbekujwo KI, Ajunwa OM (2020) Purification, characterization and de-staining
potentials of a thermotolerant protease produced by Fusarium oxysporum. Period Polytech
Chem Eng 64:539–547
Kembhavi AA, Kulkarni A, Pant A (1993) Salt-tolerant and thermostable alkaline protease from
Bacillus subtilis NCIM. Appl Biochem Biotechnol 38:83–92
Kim JD (2003) Keratinolytic activity of five Aspergillus sp. isolated from poultry farming soil in
Korea. Mycobiology 31:157–161
Kim JD (2004) Purification and characterization of an extracellular alkaline protease from Asper-
gillus niger C-15. Mycobiology 32:74–78
Krishnan A, Alias AS, Wong CMVL, Pang K, Convey P (2011) Extracellular hydrolase enzyme
production by soil fungi from King George Island, Antarctica. Polar Biol 34:1535–1542
Kudryavtseva NN, Sofyin AV, Revina TA, Gvozdeva EL, Ievleva EV, Valueva TA (2013)
Secretion of proteolytic enzymes by three phytopathogenic microorganisms. Appl Biochem
Microbiol 49:514–520
Kumar CG, Takagi H (1999) Microbial alkaline proteases: from a bioindustrial viewpoint.
Biotechnol Adv 17:561–594
Kumar S, Sharma NS, Saharan MR, Singh R (2005) Extracellular acid protease from Rhizopus
oryzae: purification and characterization. Process Biochem 40:1701–1705
Landbo AK, Pinelo M, Vikbjerg A, Let M, Meyer AS (2006) Protease assisted clarification of black
currant juice: synergy with other clarifying agents and effects on the phenol. J Agric Food Chem
54:6554–6563
Lario LD, Chaud L, Almeida MG, Converti A (2015) Production, purification and characterization
of extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7.
Fungal Biol 119:1129–1136
Li AN, Li DC (2009) Cloning, expression and characterization of the serine protease gene from
Chaetomium thermophilum. J Appl Microbiol 106:369–380
Li DC, Yang YJ, Shen CY (1997) Protease production by the thermophilic fungus Thermomyces
lanuginosus. Mycol Res 101:18–22
Li NAN, Ding AY, Chen J, Liu SA, Hang MZ, Li DC (2007) Purification and characterization of
two thermostable proteases from the thermophilic fungus Chaetomium thermophilum.J
Microbiol Biotechnol 17:624–631
Lourdes M, Polizeli TM, Rai M (2014) Fungal enzymes. Taylor & Francis, Boca Raton, p 106
Lovrien RE, Gusek T, Hart B (1985) Cellulase and protease specific activities of commercially
available cellulase preparations. J Appl Biochem 7:258–272
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the folin
phenol reagent. J Biol Chem 193:265–275
Macchione MM, Merheb CW, Gomes E, Da-Silva R (2008) Protease production by different
thermophilic fungi. Appl Biochem Biotechnol 146:223–230
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol Rev 64:461–488
Malathi S, Chakraborty R (1991) Production of alkaline protease by a new Aspergillus flavus isolate
under solid-substrate fermentation conditions for use as a depilation agent. Appl Environ
Microbiol 57:712–716
Mamo J, Assefa F (2018) The role of microbial aspartic protease enzyme in food and beverage
industries. J Food Qual 2018:1–15
Marangon M, Van Sluyter SC, Robinson EM, Muhlack RA, Holt HE, Haynes PA, Godden PW,
Smith PA, Waters EJ (2012) Degradation of white wine haze proteins by Aspergillopepsin I and
II during juice flash pasteurization. Food Chem 135:1157–1165
Maťaťa M, GaládováH, Varecka L, ŠimkovičM (2019) The study of intracellular and secreted high-
molecular-weight protease(s) of Trichoderma spp., and their responses to conidiation stimuli.
Can J Microbiol 65:653–667
Mayerhofer MS, Fraser E, Kernaghan G (2015) Acid protease production in fungal root
endophytes. Mycologia 107:1–11
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 461

Merheb CW, Cabral H, Gomes E, Da-Silva R (2007) Partial characterization of protease from a
thermophilic fungus, Thermoascus aurantiacus, and its hydrolytic activity on bovine casein.
Food Chem 104:127–131
Merheb-Dini C, Gomes E, Boscolo M, Da-Silva R (2010) Production and characterization of a
milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor
indicae-seudaticae N31 (milk-clotting protease from the newly isolated Thermomucor indicae-
seudaticae N31). Food Chem 120:87–93
Meyer V, Andersen MR, Brakhage AA et al (2016) Current challenges of research on filamentous
fungi in relation to human welfare and a sustainable bio-economy: a white paper. Fungal Biol
Biotechnol 3:1–17
Monod M, Togni G, Rahalison L, Frenk E (1991) Isolation and characterisation of an extracellular
alkaline protease of Aspergillus fumigatus. J Med Microbiol 35:23–28
Mótyán JA, Tóth F, Tőzsér J (2013) Research applications of proteolytic enzymes in molecular
biology. Biomol Ther 3:923–942
Muthulakshmi C, Gomathi D, Kumar DG, Ravikumar G, Kalaiselvi M, Uma C (2011) Production,
purification and characterization of protease by Aspergillus flavus under solid state fermentation.
Jordan J Biol Sci 4:137–148
Nageswara S, Guntuku G, Yakkali B (2019) Purification, characterization, and structural elucida-
tion of serralysin-like alkaline metalloprotease from a novel source. J Genet Eng Biotechnol 17:
1–15
Neelakantan S, Mohanty AK (1999) Production and use of microbial enzymes for dairy processing.
Curr Sci 77:143–148
Nehra KS, Dhillon S, Chaudhary K, Singh R (2002) Production of alkaline protease by Aspergillus
sp. under submerged and solid state fermentation. Indian J Microbiol 42:43–47
Nelson JH, Witt PR (1973) Mucor pusillus Lindt enzyme for chill proofing beer. US Patent,
3740233
Neves Souza RD, Silva RSSF (2005) Study of cost and yield of minas like fresh cheese produced
with added fat free soybean hydrosoluble extract powder with curd formed by different
coagulants agent. Ciencia Technol Alime 25:170–174
Olivieri F, Zanneti ME, Oliva CR, Covarrubias A, Casa-longué CA (2002) Characterization of an
extracellular seine protease of Fusarium eumarttii and its action on pathogenesis related
proteins. Eur J Plant Pathol 108:63–172
Omrane Benmrad M, Moujehed E, Ben Elhoul M, Zaraî Jaouadi N, Mechri S, Rekik H, Kourdali S,
El Hattab M, Badis A, Sayadi S, Bejar S, Jaouadi B (2016) A novel organic solvent- and
detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101. Int J Biol
Macromol 91:961–972
Omrane Benmrad M, Moujehed E, Ben Elhoul M, Mechri S, Bejar S, Zouari R, Baffoun A, Jaouadi
B (2018) Production, purification, and biochemical characterization of serine alkaline protease
from Penicillium chrysogenium strain X5 used as excellent bio-additive for textile processing.
Int J Biol Macromol 119:1002–1016
Omrane Benmrad M, Mechri S, Zaraî Jaouadi N, Ben Elhoul M, Rekik H, Sayadi S, Bejar S,
Kechaou N, Jaouadi B (2019) Purification and biochemical characterization of a novel thermo-
stable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with indus-
trial interest. BMC Biotechnol 19:43
Ong PS, Gaucher GM (1973) Protease production by thermophilic fungi. Can J Microbiol 19:129–
133
Pal S, Banerjee R, Bhattacharya BC, Chakraborty R (1996) Application of a proteolytic enzyme in
tanneries as a depilating agent. J Am Leather Chem Assoc 3:59–63
Pekkarinen AI, Jones BL, Niku-Paavola ML (2002) Purification and properties of an alkaline
proteinase of Fusarium culmorum. Eur J Biochem 269:798–807
Perea A, Ugalde U, Rodriguez I, Serra JL (1993) Preparation and characterization of whey protein
hydrolysates: applications in industrial whey bioconversion processes. Enzyme Microb Technol
15:418–423
462 S. Bhattacharya and A. Das

Peterson R, Kautto L, Sluyter SV, Nevalainen H (2013) In search of clarity: do cold-active proteases
from Antarctic fungi provide alternatives to heat-stabilisation with bentonite? Wine Vitic J 28:
20–23
Pinelo M, Zeuner B, Meyer AS (2010) Juice clarification by protease and pectinase treatments
indicates new roles of pectin and protein in cherry juice turbidity. Food Bioprod Proc 88:259–
265
Pundir RK, Rana S, Tyagi H (2012) Studies on compatibility of fungal alkaline protease with
commercially available detergents. Int J Modern Biochem 1:41–56
Rani K, Rana R, Datt S (2012) Review on latest overview of proteases. Int J Curr Life Sci 2:12–18
Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological
aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635
Ray MK, Devi KU, Kumar GS, Shivaji S (1992) Extracellular protease from the Antarctic yeast
Candida humicola. Appl Environ Microbiol 58:1918–1923
Razzaq A, Shamsi S, Ali A, Ali Q, Sajjad M, Malik A, Ashraf M (2019) Microbial proteases
applications. Front Bioeng Biotechnol 7:110
Rick W (1974) Chymotrypsin. In: Bergmeyer HU (ed) Methods of enzymatic analysis. Academic
Press, Berlin, pp 1045–1051
Saggu SK, Mishra PC (2017) Characterization of thermostable alkaline proteases from Bacillus
infantis SKS1 isolated from garden soil. PLoS One 12:e0188724
Salihi A, Asoodeh A, Aliabadian M (2017) Production and biochemical characterization of an
alkaline protease from Aspergillus oryzae CH93. Int J Biol Macromol 94:827–835
Saran S, Isar J, Saxena RK (2007) A modified method for the detection of microbial proteases on
agar plates using tannic acid. J Biochem Biophys Methods 70:697–699
Sarath G, De La Motte RS, Wagner FW (1989) Proteaseassay methods. In: Beynon RJ, Bonde JS
(eds) Proteolytics enzymes: a practical approach. Oxford University Press, Oxford, pp 25–54
Sharma N, De K (2011) Production, purification and crystallization of an alkaline protease from
Aspergillus tamarii [EF661565.1]. Agric Biol J N Am 2:1135–1142
Sharma M, Gat Y, Arya S, Kumar V, Panghal A, Kumar A (2019) A review on microbial alkaline
protease: an essential tool for various industrial approaches. Ind Biotechnol 15:69–78
Shindo S, Kashiwagi Y, Shiinoki S (1998) Sake brewing from liquefied rice with immobilised
fungal mycelia and immobilised yeast cells. J Inst Brew 104:277–281
Singh S, Bajaj BK (2017) Potential application spectrum of microbial proteases for clean and green
industrial production. Energy Ecol Environ 2:370–386
Singhania S, Ansari R, Neekhra N, Saini A (2018) Isolation, identification and screening of alkaline
protease from thermophilic fungal species of Raipur. Int J Life Sci Sci Res 4:1627–1633
de Souza PM, Bittencourt de AML, Caprara CC, de Freitas M, de Almeida RPC, Silveira D,
Fonseca et al (2015) A biotechnology perspective of fungal proteases. Braz J Microbiol 46:337–
346
Srilakshmi J, Madhavi J, Lavanya S, Ammani K (2014) Commercial potential of fungal protease:
past, present and future prospects. J Pharm Chem Biol Sci 2:218–234
Steiner E, Becker T, Gastl M (2010) Turbidity and haze formation in beer- insights and overview. J
Inst Brew 116:360–368
Sudarkodi C, Sundar SK, Murugan M (2015) Production and optimization of protease by filamen-
tous fungus isolated from paddy soil in Thiruvarur district Tamil Nadu. J Appl Biol Biotechnol
3:66–69
Sumantha A, Larroche C, Pandey A (2006) Microbiology and industrial biotechnology of food-
grade proteases: a perspective. Food Technol Biotechnol 44:211–220
Sundus H, Mukhtar H, Nawaz A (2016) Industrial applications and production sources of serine
alkaline proteases: a review. J Bacteriol Mycol Open Acces 3:191–194
Suryawanshi H, Pandya N (2017) Screening, identification of alkaline proteases producing fungi
from soil of different habitats of Amalner Tahsil [Maharashtra] and their applications. Int J Appl
Sci Biotechnol 5:397–402
19 Extremophilic Fungal Proteases: Screening, Purification, Assay, and Applications 463

Thirunavukkarasu N, Suryanarayanan TS, Rajamani T, Paranetharan MS (2017) A rapid and simple
method for screening fungi for extracellular protease enzymes. Mycosphere 8:131–136
Tojo M, Newsham KK (2012) Snow moulds in polar environments. Fungal Ecol 5:379–480
Tremacoldi CR, Carmona EC (2005) Production of extracellular alkaline proteases by Aspergillus
clavatus. World J Microbiol Biotechnol 21:169–172
Turchetti B, Buzzini P, Goretti M, Branda E, Diolaiuti G, D’Agata C et al (2008) Psychrophilic
yeasts in glacial environments of Alpine glaciers. FEMS Microb Ecol 63:73–83
Turkiewicz M, Pazgier M, Kalinowska H, Bielecki S (2003) A cold-adapted extracellular serine
proteinase of the yeast Leucosporidium antarcticum. Extremophiles 7:435–442
Umar DM (1994) Purification and some properties of alkaline protease from Penicillium expansum.
Med J Islamic World Acad Sci 7:100–105
Van Sluyter SC, Warnock NI, Schmidt S, Anderson P, Van Kan JA, Bacic A, Waters EJ (2013) An
aspartic acid protease from Botrytis cinerea removes haze forming proteins during white
winemaking. J Agric Food Chem 61:9705–9711
Vishwanatha KS, Appu Rao AG, Singh SA (2010) Production and characterization of a milk-
clotting enzyme from Aspergillus oryzae MTCC 5341. Appl Microbiol Biotechnol 85:1849–
1859
Ward OP, Rao MB, Kulkarni A (2009) Proteases production. In: Schaechter M (ed) Encylopedia of
microbiology. Elsevier, New York, pp 495–511
Waters DM, Murray PG, Ryan LA, Arendt EK, Tuohy MG (2010) Talaromyces emersonii
thermostable enzyme systems and their applications in wheat baking systems. J Agric Food
Chem 58:7415–7422
Wu B, Licheng W, Chen D, Yang Z, Luo M (2009) Purification and characterization of a novel
fibrinolytic protease from Fusarium sp. CPCC 480097. J Ind Microbiol Biotechnol 36:451–459
Xie L, Xiao Y, Gao X (2016) Purification and characterization of a halotolerant alkaline serine
protease from Penicillium citrinum YL-1 isolated from traditional Chinese fish sauce. Food
Biotechnol 30:137–153
Yadav AN, Sachan SG, Verma P, Kaushik R, Saxena AK (2016) Cold active hydrolytic enzymes
production by psychrotrophic bacilli isolated from three sub-glacial lakes of NW Indian
Himalayas. J Basic Microbiol 56:294–307
Yu S, Bech Thoegersen J, Kragh KM (2020) Comparative study of protease hydrolysis reaction
demonstrating normalized peptide bond cleavage frequency and protease substrate broadness
index. PLoS One 15:e0239080
464 S. Bhattacharya and A. Das

Extremophilic Fungal Amylases: Screening,
Purification, Assay, and Applications 20
Ragini Bodade and Krutika Lonkar
Abstract
Extremophiles are the microorganisms that grow under extreme environments of
high and low pH, high and low temperatures, deficient nutrient conditions, high
atmospheric pressure, high radiations, and low water availability. Development
of adaptation mechanism including strategies for survival, protection,
perpetuations, and metabolic diversity makes them to survive under these
extreme conditions. Moreover, studies through advanced techniques viz. proteo-
mics, genomics, and metabolomics revealed the production of many
extremozymes and extremolytes and their role in survival strategies.
Microorganisms including bacteria and archea are explored for extremolytes/
extremozymes compounds for their commercial production; however, fungi
needs more attention being equally metabolic diverse and easy to cultivate.
Extremozymes are stable and more catalytically active at these extreme
conditions, and thus have immense biotechnological applications. Amylase, an
important enzyme responsible for catalysis of starch to monomeric sugars,
thereby plays an important role in many industrial applications. This chapter
describes the status of isolation, screening, purification, and industrial
applications of fungal amylases in the last few decades.
Keywords
Extremophilic fungi · Extremozymes · Amylases · Isolation · Purification · Assay ·
Applications
R. Bodade (*) · K. Lonkar
Department of Microbiology, Savitribai Phule Pune University, Pune, Maharashtra, India
e-mail: ragini.bodade@unipune.ac.in
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_20
465

20.1 Introduction
“Everything is everywhere: but, the environment selects,”a well-known formulated
quote was described for the distribution of extremophilic microorganisms around the
world by the microbiologist Lourens Baas Becking in his book Geobiologie of
Inleiding Tot De Milieukunde in 1934. This revealed the ubiquitous nature of
microorganisms. From an anthropocentric point of view, physicochemical
parameters that deviate from the supporting mammalian or terrestrial life forms
have considered as extreme viz. pH (above 9 and below 4), temperature (range of
2to20C and 60 to 300 C), salt concentration (3–30% NaCl), UV-gamma
radiations (above 5 gy), and other factors. In 1974, MacElroy proposed the term
“extremophile”to define a broad group of organisms that thrive optimally under
these above-mentioned extreme physiochemical and geochemical conditions, how-
ever, still has Cavitate and lies in the eye of beholders. Some organisms can tolerate
these hostile conditions to some extent and are labeled as extremotolerant. More-
over, an organism growing in more than one extreme condition is termed as
polyextremophiles. Various adaptive features togetherly at the cellular level make
the extremophiles sustain in one or more of these extreme conditions (Selbmann
et al. 2013; Ma et al. 2010; Zhang et al. 2018).
Amylases are widely distributed in microorganisms, plants, and animals. Being a
hydrolase enzyme, amylase is responsible for the conversion of starch into simple
sugars or oligosaccharides. Structurally starch is a branched polysaccharide of
glucose monomer and consists of two polymers amylopectin and amylose. Amylo-
pectin constitutes 70–80% of starch containing branched glucose chain at every
15–45 glucose units linked by α-1,6-glycosidic bonds, while amylose creates
20–25% of starch made by linear chain of glucose with α-1,4-glycosidic bonds.
There are two types of hydrolases: endo-hydrolase and exo-hydrolase. Endo-
hydrolases cleave the chain from the interior of the substrate molecule, whereas
exo-hydrolases cleave from the terminal non-reducing ends. Nearly about
300 amylases are known to date. Depending upon the site of action, structure, and
mode of starch hydrolysis, amylases are of three types: α-amylase, β-amylase, and
γ-amylase (Table 20.1). TakkamylaseA (TAA) from Aspergillus oryzae is studied
first time for its catalytic and gene regulatory function (Sundarram and Murthy
2014).
α-Amylases (α-1,4-glucan-4-glucanohydrolase; EC 3.2.1.1), an endo-enzyme
belonging to glycoside hydrolase family-13 (GH), catalyze the hydrolysis of internal
α-1,4-glucosidic linkage in glycogen, starch, and various malto-oligosaccharides to
produce glucose, maltose, and maltotriose units. Other amylases belong to GH
family no. 57 and 119. α-Amylase is a metalloenzyme and contains Ca
2+
that
maintains most of the amylase structure and activity while other amylase forms
require divalent ions such as Mn
2+
,Zn
2+
, and Fe
2+
. Fungal α-amylases are of great
significance for bioconversion processes such as liquefaction, saccharification, and
isomerization in food, fermentation, detergent, textile, and paper industries and thus
share 30% of the total market. It is also used for desizing processes, such as
bio-finishing, stone washing, and surface fibril stain removal along with cellulases
466 R. Bodade and K. Lonkar

(Han et al. 2013; Kato et al. 2007). β-Amylases (EC 3.2.1.2) are exo-hydrolase of
plant origin responsible for catalyzing the non-reducing end of the substrate to form
maltose and β-limit dextrins and are incapable to hydrolyze the branched linkages in
glycogen or amylopectin. γ-Amylases (EC 3.2.1.2) cleave α-1,6-glucosidic linkage
as well as the last α-(1–4) glycosidic linkages at the non-reducing end of amylose
and amylopectin to form glucose. A wide variety of microorganisms are isolated,
screened, and studied from different niche with novel characteristics (Sundarram and
Murthy 2014; El-Enshasy et al. 2013; Kour et al. 2019).
20.2 Isolation and Screening
Extremophiles are found almost in all three domains such as Archea, Eubacteria, and
Eukarya, while fungi revealed most versatile entity among all the eukaryotes (Berde
et al. 2019). A plethora of research on distribution and adaptation of extremophilic
fungi confirmed their existence in hypersaline, extreme cold, and hot environments
of the earth. They have been isolated from deep oceans, dry rock surfaces, snow and
ice, permafrost, salterns, and lakes and compost soil, heated soil, manure, nuclear
reactor effluents, mines, agricultural residues, and vegetation associated with these
extreme environments. Many investigators have isolated extremophilic fungi for
commercial production of extremozymes and extremolytes (Gostincar et al. 2009;
Patel and Rawat 2021).
Temperature is one of the extremely important environmental factors that plays a
pivotal role in the survival, growth, distribution, and diversity of microorganisms on
the surface of the earth. Those fungi that show growth up to 60 C and grow
optimum between 45 and 50 C temperatures are called thermophilic fungi. Also,
few fungi grow up to 50–60 C but optimally at or below 20 C, termed
thermotolerant. Historically scientist Hugo Miehe isolated thermophilic fungi from
self-heating damp haystacks during his study on stored agricultural products
followed by scientist Kurt Noak from other natural substrates. Diverse types of the
thermophilic fungi have been reported from different soil types, pond sediments,
Table 20.1 Types of amylases
Type of amylase
Reaction
catalyzed Product Source Reference
α-Amylase
(EC 3.2.1.1)
α-1,4-Glycosidic
linkages
Glucose and small
chain
oligosaccharides
Animals,
plants,
microbes
El-Enshasy
et al.
(2013)
β-Amylase
(EC 3.2.1.2)
α-1,4-Glycosidic
linkages
Maltose and β-limit
dextrins
Bacterial,
fungal,
plants
Glucoamylases or
γ-amylase
(EC 3.2.1.3)
α-1,4-Glycosidic
linkages
α-1,6-Glycosidic
linkages
Glucose, maltose,
isomaltose
Animals,
plants,
microbes
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 467

composting systems, power plant cooling system, heaped masses of plant materials,
and piles of agricultural and forestry products belonging to phylum Phycomycetes,
Zygomycota, Ascomycota, fungi imperfecta, and Basidiomycota. These habitats are
mainly characterized with required temperature, water activity, humidity, and aero-
bic environments that support the fungal development. Thermophilic fungi from
taxonomic genus Canariomyces,Chaetomium,Coonemeria,Corynascus,
Dactylomyces,Malbranchea,Melanocarpus,Myceliophthora,Myriococcum,
Paecilomyces,Rhizomucor,Remersonia,Scytalidium,Stilbella,Talaromyces,
Thermoascus,Thermomyces, and Thielavia are described from advanced sequencing
methods viz. 18S rDNA, metagenomics, and ITS sequencing. In laboratory
conditions the thermophilic fungi can be cultivated in media supplemented with
yeast extract, ammonium sulfate, glucose, and an inducer (L-asparagine and succinic
acid) at pH 5.5–7 under aerobic conditions. Adaptations in thermophilic fungi
exhibited at cytoplasmic, nuclear, and cell membrane levels. Thermophilic fungi
incorporate high content of saturated and long fatty acids in their phospholipid
monolayer with ether linkages. At cytoplasmic level formation of heat shock
proteins, thermostable enzymes, and low metabolite transportation across the mem-
brane revealed as prominent adaptation as compared to mesophilic fungi. Moreover,
adaptation for reduced genome size, high G:C content, small sized introns, loss of
transposable elements, upgradation of hyphal melanization gene, and protein coding
genes carried at nucleus level. A wide industrial application of extremophiles
described for the production of extremozymes. Extremozymes are biocatalysts that
work at high salt concentrations and low and high temperature conditions, thus
having commercial interest. Thermophilic fungi produces thermostable enzyme viz.
cellulases, xylanases, amylases, lipases, and proteases with great catalytic activity
and stability at high temperatures due to their ionic interaction, self-aggregation
capability, hydrophobicity, and compact conformations. The enzymes are therefore
applied for reducing the viscosity of media, for hydrolysis of complex plant biomass
at extreme culture conditions (salt and temperature), and to avoid the contaminations
(Maheshwari et al. 2000; de Oliveira et al. 2015; Patel and Rawat 2021).
In contrast, psychrophiles are a group of microorganisms that exhibit growth at or
below 0 C and optimum growth between 15 C and 20 C cold temperature.
However, psychrotolerant fungi grow close to 0 C and require an optimum temper-
ature at 15 C and a maximum temperature above 20 C for development.
Psychrophiles may be obligate or facultative in nature. Earth covers diverse cold
habitats comprising deep sea to high mountains and from Antarctica to Arctic region
with 5 C temperature seasonally or permanently. Out of 85% of these biosphere
most other habitats are covered by oceans (~71%), terrestrial environments covering
snow (~35%), frozen ground (~24%), sea ice (~13%), and glacier (~10%) with 1
to 4 C temperature. Moreover, other low temperature environments comprise
permafrost, cold soils, lakes, caves, cryoconite glacier holes, and cold deserts. A
wide variety of yeasts, water molds (Chromista group), and filamentous fungi
(Helotiales/Pleosporales/Articulospora and Varicosporium) were isolated from
cryoconite sediments and Antarctica. The isolated fungal/yeast genus are from
Thielavia,Geomyces,Apiosordaria,Cadophora,Penicillium,Aspergillus,
468 R. Bodade and K. Lonkar

Fusarium,Curvularia,Cladosporium Cylindrocarpon,Golovinomyces,Phoma,
Antarctomyces,Thelebolus,Mortierella,Pythium,Sordariomycetes,Mycopappus,
Mrakia,Tetracladium,Phaeosphaeria,Venturia,Eurotiomycetes,Leotiomycetes,
Tremellomycetes,Alternaria,Lecanoromycetes,Arthoniomycetes,Geomyces,
Phialophore,Acremonium,Rhizosphaera,Candida,Cyptococcus,Rhosospoidium,
Pichia,Rhodotorula,Sporobolomyces, and Trichosporon. In most of these marine
ecosystems and at cold environment, psychrophilic fungi/yeast contributes in
recycling of nutrients and organic matter mineralization (geochemical cycles).
Adaptation in psychrophiles under a cold environment involved the production of
antifreeze proteins, cold shock proteins, nucleation proteins, compatible solutes
(glycerol and mannitol), cryoprotectant (trehalose and polyols), photo-protective
pigments (melanin), exopolysaccharide, and unsaturated branched fatty acid chains
in the cell membrane. The main adaptation of cold adapted enzymes from
psychrophiles is increase in flexibility and high site complementarity for substrate
with great catalytic activity. The structural flexibility can be achieved by more
glycine residues in surface loops, increased α-helices secondary structure, increased
surface hydrophobicity, reduced number of core hydrophobic amino acid (lysine,
arginine, and aromatic amino acids), and reduced ionic and electrostatic interactions.
Various extracellular enzymes like DNase, protease, phosphatase, amylase glucose
oxidase, lipases, and polygalacturonase have been reported with promising catalytic
activity than mesophilic enzyme (Hassan et al. 2016; Duarte et al. 2018).
Halophiles are the extremophiles capable of surviving in salt rich environments.
The word halophile is derived from the Greek, meaning “salt loving.”Fungi
showing optimum growth in hypersaline habitats, i.e., at NaCl concentrations
above 1.7–3 M salt concentration are categorized as halophiles. They may be slight
halophiles (0.3–0.8 M NaCl), moderate halophiles (0.8–3.4 M NaCl), and extreme
halophiles (3.4–5.1 M NaCl) depending upon the salt requirement for optimum
growth. These microorganisms can sustain in high salt concentrations as well as
other ions, UV radiation, and extreme pH. Most of the halophile microorganisms are
isolated and characterized from saline water, saline soil, salt lakes, soda lakes, salted
foods, and salterns. Moreover, different foods are supplemented with salts to
enhance flavor and as a preservative, thereby supporting the growth of halophiles
and halotolerant microorganisms. Halophilic fungi can grow in a wide salinity range
from freshwater to saturated NaCl solutions (1.7–3 M). Fungal halophiles have been
mostly isolated and characterized from the above salty habitats from genus
Cladosporium,Aspergillus,Penicillium,Emeicella,Alternaria,Scopulariopsis,
Eurotium,Aureobasidium,Debaryomyces,Phaetotheca,Trimmatostroma, and
yeast like Wallemia and Hortaea. Adaptation in fungi including accumulation of
cytoplasmic compatible solutes (erythritol, ribitol, arabinitol, xylitol, sorbitol, man-
nitol and galacticol, glycerol, trahlose, glutamic acid, alanine, and mycosporin),
enhanced secondary transporters/ionic pumps (Na
+
/H
+
and Na
+
/K
+
) and low ste-
rol-to-phospholipid, short fatty acid chains, ration in membrane are few important
strategies. Halophilic fungi mostly maintain the intracellular osmotic balance using
glycerol, trahlose, and other organic compatible solutes in their cytoplasm. Proteins
with high hydrophobicity, increased acidic residues, and reduced helices make
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 469

proteins and enzymes more stable in high salt concentrations. Many of the halophilic
fungi are reported for the production of bioactive compounds, enzymes, in bioreme-
diation (Gunde-Cimerman et al. 2009; Ali et al. 2019).
pH is another important parameter for the growth of microorganisms. Most of the
microorganisms easily thrive at neutral pH conditions. However, pH condition is not
the same throughout the surface of earth. There are places where the pH ranges from
0 to 4 and 11 to 14, and still some microorganisms are seen thriving with ease.
Acidophiles, a class among extremophiles, are capable of growing under acidic
conditions, i.e., below pH 4. Acidic natural environments having pH range from
3 to 4 are relatively common and include acid sulfate soils, acidic lakes, hydrother-
mal systems, mining effluents, coal tips, drainage water and industrial waste water,
metal and coal mines, swamps, and peat bogs. Acid-tolerant yeasts like Rhodotorula
spp., Candida,Cyptococcus, and Leucosporidium, from acid mine drainage (AMD)
and from sandy soils are reported. Filamentous fungi Bispora sp., Purpureocillium
lilacinum,Schizoblastosporion,Trichosporoncerebriae,Fusarium,Aspergillus,
Penicillium,Verticillium,Bipolaris,Acontiumcylatium, and Cephalosporium sp.,
Teratosphaeria acidotherma. Under these acidic conditions the fungi maintain the
pH toward neutrality by pumping the protons out of the cell and having low proton
membrane permeability (Gross and Robbins 2000; Johnson 1998).
The microorganisms exhibiting growth in alkaline conditions where the pH is
between 8 and 11 and optimally at 9 or above but not at neutral pH are grouped under
alkaliphiles. Alkaliphiles are found mainly in hypersaline soda lakes; hallow hydro-
thermal systems, alkali thermal hot springs, soda soils, and sewage. Different
isolated fungi viz. Acremonium sp., Stilbella,Veticeillium sp., Paecilomyces,
Metarhizium sp., Fusarium sp., Phialophora sp., Scopulariopsis sp., Mucor sp.,
Gliomastric sp., sodiomyces sp., Acrostalagmus sp., Emericellopsis sp., Thielavia
sp., Alternaria sp., Chordomyces sp., Acrostalagmus sp., Scopulariopsis sp., and
Cladosporium sp. are reported from soda lakes and soda soils using cultured media
buffered at pH 5–11. Neutralizing the cytoplasm pH by Na
+
/H
+
and K
+
/H
+
anti-ports
with continuous H
+
influx through ATPase revealed prominent adaptation in
alkalophiles. Accumulation of compatible solutes mannitol and arabitol are also an
adaptation for survival of microbes (Kladwang et al. 2003; Grum-Grzhimaylo et al.
2016).
The adaptations in the extreme conditions of such organism are some extent due
to well-adapted cellular biomolecules and product of the metabolic pathways.
Interestingly, some of these compounds show polyextremophilicity, i.e., stable and
catalytically active in more than one extreme condition. The products are referred to
as extremolytes/extremozymes viz. form of polysaccharides, proteins, primary/sec-
ondary metabolites, and enzymes. Though more than 3000 different enzymes for
biotechnological and industrial applications have been identified till date, still the
present enzyme stock fail to meet all demands like increased catalytic activity and
stability at extreme conditions (pH, temperature, salinity, pressure, and organic
solvents), enhancing the solubility of substrates, high mass transfer rate, and having
low risk of pollution during the process. The efficiency of these enzymes is improved
by genetic engineering and chemical modifications as well as by immobilization
470 R. Bodade and K. Lonkar

methods. Still search of novel enzymes from different niches with desired
characteristics for various indusial and biotechnological process is required. There-
fore, characterization of extremophiles that are capable to withstand in extreme
environment has drawn great attention. Such extremophiles can be a valuable source
of novel extremozymes (Niehaus et al. 1999; Van Den Burg 2003; Singh et al. 2016;
Dumorne et al. 2017).
Many investigators are carrying out a plethora of research to find novel fungal
cultures for amylase producer from different niches. Fungal amylase producers are
preferred for use in food and pharmaceutical industries due to their acceptable
characteristics like saccharifying efficiency, optimal conditions, and wide substrate
specificity and product. Fungi can grow on enrichment media like potato dextrose
agar, starch-yeast extract agar, malt extract agar, Saboraud dextrose agar, agricul-
tural waste materials, starchy foodstuffs, and wastewater. Several methods and
technologies are reported for studying the dynamic of extremophiles, such as
quantitative polymerase chain reaction (PCR), RNA stable isotope probing, proteo-
mics, genomics and metagenomics approach, microarray techniques, genetic engi-
neering, and recombinant technology (Singh et al. 2019).
The isolates of filamentous fungi were collected from different locations and were
enriched in their respective conditions. The selected fungi were examined for
amylase activity in starch agar medium, potato dextrose agar, yeast nitrogen base
agar, malt extract agar, Saboraud dextrose agar, and Czapek dox agar containing
0.5–2% starch. Halophilic fungal culture isolation required 5–10% of NaCl salt.
After the incubation, the amylolytic activity was assessed by flooding the plates with
potassium iodide solution. The activity of α-amylase can be investigated based on
the principle that starch and iodine react to form a blue colored complex. The ratio of
clear zone around the growth to colony indicates amylase-producing isolates
(Niknejad et al. 2013; Mukherjee et al. 2019).
20.3 Assay of Amylase
Alpha-amylase cleaves internal 1,4-glycosidic linkages of starch to produce glu-
cose, maltose, or dextrins, while glucoamylase cuts 1,4- and 1,6-glycosidic
linkages to release glucose from the non-reducing ends of starch. Both the enzymes
are widely used in the industries for the conversion of starch into sugars. Amylases
and glucoamylases are characterized by paper chromatography, high-performance
liquid chromatography, and thin-layer chromatography. Amylase activity is detected
using broth supernatant or crude extract by qualitative and quantitative assays.
Qualitative detection of starch hydrolysis mediated by α-amylase or glucoamylase
was done by paper chromatography. Using Whatman no.1 filter paper at room
temperature. The chromatogram is spot inoculated with hydrolysate, developed by
solvent butanol:acetic acid:water (4:1:5 v/v), and visualized by silver nitrate dip
(Spencer-Martins and Van Uden 1979).
Quantitative enzyme assay measures the reducing sugar formation as an action of
amylase on starch (Dinitrosalicylic acid assay or the Nelson–Somogyi assay).
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 471

Reducing sugars are sugars that contain aldehyde or ketone group (glucose, galac-
tose, lactose, and maltose). Different substrates viz. glycogen, pullulan, amylopectin,
and amylose can also be used to check the hydrolysis by amylase and glucoamylases
(Xiao et al. 2006).
20.3.1 DNS Method
In the 3,5-dinitrosalicylic acid method (DNSA), the aliquots of the substrate (1–2%
starch) solution is incubated with amylase enzyme for 10–15 min at 30–50 C.
DNSA reagent (chromogen) is added after incubation in the reaction mixture and
placed in boiling water bath for 5–10 min. Under alkaline condition (pH 7.5–8.5) the
3,5-dinitrosalicylic acid is reduced to 3-amino-5-nitro salicylic acid .Conversion of
yellow to brick red color indicates enzyme catalytic activity. After cooling to room
temperature, 10 mL of water was added and the absorbance of the reaction mixture is
read at 540 nm. One unit (U) of enzyme activity was defined as the amount of
enzyme that produced 1 μmol of glucose per minute under standard assay condition
(Ali et al. 2015; Gusakov et al. 2011).
20.3.2 Nelson–Somogyi Method
The Nelson–Somogyi method is also used for the quantitative determination of
reducing sugars. The enzyme is mixed with aliquots of 0.5% starch solutions in
50 mM Tris–HCl buffer (pH 9) and whole reaction mixture is incubated at 30–50C
temperature for 10 min. After incubation, Somogyi reagent (alkaline copper tartrate)
is added and heated in boiling water bath for 20 min. The reaction is terminated by
addition Arsenomolybolic acid reagent. Formation of yellow green to blue green
color indicates positive enzyme activity or starch hydrolysis, which can be estimated
spectrophotometrically at 620 nm. When the reducing sugar is heated with alkaline
copper tartrate solution, it reduces copper to cupric to cuprous (cuprous oxide).
Cuprous oxide reacts with arsenomolybdic acid and forms blue–green colored
molybdenum (Kobayashi et al. 1992; Nelson 1944; Somogyi 1945).
20.3.3 Glucose Oxidase-Peroxidase Assay (GOPOD)
A reliable, simple, micro-volumetric, rapid, and high-throughput analytical method
was developed for the detection and quantification of amylase/glucoamylase activity
using the glucose oxidase-peroxidase assay (GOPOD). The reaction mixture
contains amylase enzyme, aliquots of buffered starch, or maltose (0.2%) and
incubated at 30 C for 20 min. The formed maltose/glucose from starch hydrolysis
can be detected by glucose oxidase-peroxidase method. The maltose/glucose was
catalyzed by glucose oxidase (GOD) to form gluconic acid and hydrogen peroxide.
The product hydrogen peroxide was estimated by 4-aminoantipyrine and phenol in
472 R. Bodade and K. Lonkar

the presence of peroxidase enzyme after converting it to a quinone. The formed
chromogenic compound quinone is estimated by absorbance at 520 nm. The test is
recommended for screening amylase inhibitors and for glucoamylase activity detec-
tion (Visvanathan et al. 2016; Spencer-Martins and Van Uden 1979).
20.3.4 Hugget and Nixon Method
In this method glucoamylase/amylase activity was determined using p-nitro-
phenyl-α-D-glucopyranoside, the chromogenic substrates. The substrate is reacted
with crude enzyme in buffered reaction mixture (0.05 M citrate phosphate buffer,
pH 5.2) for 30 min at 50C. The released glucose is measured by GOPOD method.
α-Glucosidase/amylase unit was defined as the amount of enzyme which liberates
1μM of glucose per minute (Gautam and Gupta 1992). Other substrate ethylidene-
pNP-G7 is cleaved by α-amylase results in the release of the chromophore (p-nitro
phenol), and that can then be measured at 405 nm (Carrasco et al. 2017). All these
different methods are based on estimation of formed glucose by amylase/
glucoamylase catalytic activity. Details of other methods used to estimate glucose
concentration directly from the different biogenic samples are Otoluidine reagent,
Folin Wu reagent, glucose oxidase (GOD), and hexokinase enzyme-based
methods. Following addition of O-toluidine reagent (6% v/v in glacial acetic acid)
in reaction mixture and heating at 100 C for 10 min formation of stable green color
is estimated by reading absorbance at 630 or 635 nm that gives information about the
glucose concentration (Passey et al. 1977).
20.3.5 Sandstedt, Kneen, and Blish (SKB) Method
It is used for detection of amylases in bakery industry. The reaction mixture contains
buffered starch solution (0.5%) and enzyme solution, and the mixture is incubated at
30 C for 5 min. After incubation, 1 mL of iodine reagent was added. Transmittance
(T) at 660 nm was measured. One SKB unit is the amount of enzyme that will
dextrinize starch under assay conditions. The method expresses diastatic strength of
the malt (Ambade et al. 1998; Attia and Ali 1977). A similar method for determining
amylase activity in cereals and cereal products is described by Indian pharmacopeia
(ISI). Different aliquots of buffered starch and fixed volume of enzyme are incubated
at 40 C for 1 h. Addition of iodine solution confirms the enzyme activity. The starch
aliquot without blue colorization indicates the enzyme activity in terms of grams of
starch digested (Gupta et al. 2003).
20.3.6 Fuwa Method
Another assay method is based on the decreased staining value of blue starch–iodine
complexes, i.e., Fuwa method. In the Fuwa method, the reaction mixture contains
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 473

2.5 mL of buffered-amylase and 2.5 mL amylose (2 g/L), which is incubated at 37 C
for 30 min. After incubation, 5 mL of 1 N acetic acid is added to terminate the
reaction. After cooling, the mixture is then transferred into a 250 mL flask and
diluted to nearly 200 mL with water, followed by the addition of 5 mL of iodine
reagent (0.2% iodine and 2% potassium iodide). The amount of color development is
later determined by measuring the absorbance at 700 nm. Dextrinizing activity is
then calculated using the formula D¼(D
0
D) D
0
100 10, where Dis the
absorbance of the enzyme sample and D
0
is the absorbance of the amylo-sugars
degraded by amylase action. Control is without the addition of enzyme (Fuwa 1954).
20.3.7 Spencer-Martins Method
According to Spencer-Martins and Van Uden’s reported method, 1 mL of the culture
filtrate is incubated with 3 mL of 1% buffered soluble starch solution (0.05 M citrate-
phosphate buffer, pH 5.5). The reaction mixture is heated to 40 C for 15 min, and
then 0.5 mL is pipetted into 5 mL of iodine solution (0.15 iodine in 0.5% potassium
iodide solution) and diluted with distilled water to 20 mL. The formed blue color
read at 550 nm. An α-amylase unit is defined as the quantity of enzyme, mediating
0.1 ΔE 1 cm/550 nm under standard assay conditions (Spencer-Martins and Van
Uden 1979; Smith and Roe 1949; Gautam and Gupta 1992).
20.3.8 Viscometric Assay Method
In viscometric assay method decrease in viscosity of starch solutions during enzy-
matic reaction is measured. At low concentrations, starch solutions have high
viscosity. In the assay, the viscosity is reduced as the molecules are degraded by
the action of amylase. This method requires rapid visco analyzer. Viscosity of
cassava starch resulting after enzyme addition was rapidly measured with respect
to time. The enzyme activity in terms of decreasing viscosity of starch suspension
determined by falling number (FN) method and amylograph/farinograph test. In the
falling number method, the enzyme substrate preparations are assayed at 100 C.
The amylograph test employs the principle of the relationship between peak viscos-
ity of starch slurry and the enzyme activity. The lesser the viscosity of the starch
slurry, the more the activity of the enzyme (McCleary et al. 1997; Gonzalez et al.
2002).
20.4 Purification of Amylases
The industrial production of α-amylase was carried out by submerged fermentation
(SmF) and solid-state fermentation (SSF). SmF has been the choice of production
because of the ease of control of different parameters such as pH, temperature,
aeration and oxygen transfer, and moisture. However, SSF systems provide a
474 R. Bodade and K. Lonkar

resemblance to the natural habitat for culture to grow. Purification of enzymes is a
crucial step and is application specific. Enzymes used for industrial purposes gener-
ally require less downstream processing, whereas enzymes used in the application of
pharmaceutical or clinical applications are highly purified. Purity is also requiring
for enzymatic study, i.e., for structure–function relationships and biochemical
properties. Purification methods frequently employed are salt or solvent precipitation
(acetone, ethanol, 2-propanol, and ammonium sulfate), ultrafiltration, chromatogra-
phy (ion exchange, gel filtration, hydrophobicity interactions, and reverse phase
chromatography), and liquid–liquid extraction. A combination of all the above
methods is followed in a series of steps to yield high grade of enzyme purity. In a
study, the 5–14 days grown fungal culture broth was subjected to centrifugation and
the supernatant collected. This was followed by ammonium sulfate precipitation
with 20–90% salt saturation. This resulted in the precipitation of α-amylase and then
followed by series of dialysis, i.e., salting out. The dialysate was then subjected to
ion exchange and gel filtration. Different fractions were collected and tested for
α-amylase activity and total protein content (Fig. 20.1).
Although certain steps require expensive equipment at each step, making them
laborious, time consuming, barely reproducible, and may result in increasing loss of
the desired product, however, this process is widely employed in the chemical
industry due to its simplicity, low costs, and ease of scale up (Souza 2010;
Sundarram and Murthy 2014; Ali et al. 2015; Far et al. 2020).
Fig. 20.1 Isolation, purification, and application of α-amylases
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 475

Aspergillus species produce a large variety of extracellular enzymes including
amylases. Aspergillus gracilis TISTR 3638a halophilic fungus was isolated from
hypersaline man-made saltern from Thailand for α-amylase production using 10%
starch and 10% NaCl. The enzyme revealed polyextremophilic nature with maxi-
mum specific activity (131.02 U/mg) at pH 5, temperature 60 C, and 30% NaCl
concentration. Purification of enzyme using ammonium sulfate fractional precipita-
tion (up to 90%) followed by dialysis and Sephadex G-100 columns elution with Tris
buffer (100 mM, pH 6) revealed 35 kDa MW protein using SDS-PAGE. Being
active at high salt and temperature conditions, it could have application in bioreme-
diation (Ali et al. 2014a). Ali et al. (2015) reported another halophilic Aspergillus
penicillioides TISTR3639 strain revealed for amylase production (42 kDa protein;
specific activity 118.42 U/mg) with maximum activity at pH 9 and temperature
80 C. The metal CaCl
2
was found as activator while ZnCl
2
and FeCl
2
as inhibitors.
As the enzyme is stable at high salt concentration (300 g/L NaCl) and extreme pH
and temperature, it has a great potential in detergent industry. The enzyme was
purified by Sephadex G100 gel column after eluting with 25 mM Tris–HCl buffer
(pH 8) (Ali et al. 2015). Amylase production by Aspergillus niger strain UO-1 was
carried out in media supplemented with brewery (BW) and meat (MPW)
wastewaters along with different starch concentrations. The highest amylase activity
was obtained (70.29 and 60.12 EU/mL) in the BW and MPW media supplemented
with 40 g of starch/L after 88 h of fermentation respectively. Media supplemented
with casamino acids, peptone, or yeast extract further enhances the amylase produc-
tion. The enzyme was stable 50 C and pH 4.95 (Hernandez et al. 2006). Unal (2015)
isolated thermophilic Aspergillus niger,Aspergillus oryzae, and Aspergillus terreus
from soil and water samples collected form Afyon and Eskisehir in Turke. A.niger
showing the highest activity at optimum temperature 65 C and pH was 8 (Unal
2015). Acidophilic Aspergillus niger RBP7 was isolated from municipal dumping
area of Midnapore town, West Bengal, India for the production of α-amylase.
Enzyme was produced by SSF using potato peel. The cell-free supernatant signifi-
cantly hydrolyzes raw starchy foodstuff (taro, yam, malanga, and sweet potato) to
form glucose and maltose. Response surface methodology (RSM) enhanced enzyme
titer up to 1112.25 U/gds with an initial medium pH 3.0. These characteristics of the
enzyme may be suitable for use to treat digestive dysfunction (Mukherjee et al.
2019). Thermophilic two fungal isolates of genus Aspergillum produced amylase
were isolated from hot spring water and soil samples near to afar Ethiopia. Both the
isolates produce amylase at 55 C after 7 days of incubation with enzyme activity
2.73 0.0 U and 2.51 0.0 U. The enzyme activity was revealed at optimum
temperature between 45 and 65 C and pH 4–10. Enzyme activity was strongly
inhibited by Ca
2+
,Mg
2+
, and Zn
2+
(Teklebrhan Welday et al. 2014).
Thermotolerant Aspergillus fumigatus isolated from soil samples of Eastern
Nigeria produced amylase after 96 h at 30 C in a mineral medium containing 1%
starch and 1.5% organic nitrogen. Sorghum starch as carbon source and inorganic
(NH
4
Cl)/organic (soybean meal) as nitrogen sources led to higher concentration of
amylase in culture fluid. Amylase was optimally active at pH 6.0 and 60 C
temperature. The amylase enzyme was repressed by Fe
2+
and Mn
2+
but not inhibited
476 R. Bodade and K. Lonkar

by Cu
2+
,CO
2+
, and Hg
2+
at a concentration of 2 mM. The highest hydrolytic activity
of the A.fumigatus amylase was recorded for yam, followed by potato and cassava
starches. Another isolate Fusarium sp. produced amylase using 1% soybean meal,
0.2% NH
4
Cl, and 2.5% corn starch and elicited the highest amylase yield. Optimum
pH for the enzyme was 6.5 and at 50 C. Mg
2+
,Ca
2+
, and Zn
2+
stimulated catalytic
function of the crude amylase at 2 mM concentrations. The enzyme also hydrolyzed
cassava, potato, and yam starches effectively (Nwagu and Okolo 2011a,b). Ali et al.
(2014b) reported Engyodontium album TISTR 3645 polyextremophiles fungi from
man-made salterns for amylase production. The considerable enzyme specific activ-
ity 132.17 U/mg was observed for this 50 kDa amylase. The enzyme was well
purified by ammonium sulfate precipitation (90%) and gel elution by Sephadex G
100 with 25 mM Tris–HCl buffer. The enzyme gives maximum activity at tempera-
ture 60 C and pH 9. The enzyme strongly inhibited by FeCl
2
, ZnCl
2
, and activated
by BaCl
2
, CaCl
2
, HgCl
2
, and MgCl
2
(Ali et al. 2014b). Rhizomucor pusillus isolated
from compost heap, Thailand, produces glucoamylase on solid wheat bran medium
at 45 C. The crude enzyme was purified by ammonium sulfate fractional precipita-
tion (20–60%), followed by Sephadex G-75 gel filtration and DEAE-Sephadex A-50
column eluted with 0.15–0.5 M NaCl solution and finally by Bio-Gel P-100gel
column (specific activity 57.7 U/mg). The obtained purified enzyme was 68 kDa
with highest activity at temperature 65 C and pH 4.6 (Kanlayakrit et al. 1987).
Thermotolerant Rhizopus microsporus isolated from the soil of the Brazilian cerrado
region produced high levels of amylase (specific activity 88.25 U/mg) by submerged
fermentation using CarvalhoPeixoto (CP) medium at 45 C and pH 6 after 72 h
incubation. Purified enzyme revealed maximum activity at optimum temperature
65 C and pH 5.0 (Peixoto et al. 2003). Chakraborty et al. (2009) reported amylase
from marine Streptomyces sp. D1. The enzyme revealed maximum activity (specific
activity 113.64 U/mg) at optimum temperature at 85 C and pH 9. The enzyme has
molecular weight of 66 kDa. The enzyme retained 70–35% of its activity up to
7–90 days in the presence of commercial detergent, and 80–100% in the presence of
oxidizing agents (sodium hypochlorite and H
2
O
2
), activated by Ca
2+
and inhibited
by Hg
2+
and Fe
3+
ions, and thus has application in detergent industry (Chakraborty
et al. 2009). Gaur et al. (1993) reported a comparative amylase production from
Humicola and Paecilomyces sp. Humicola sp. produces thermostable amylase
enzyme (activity 3.9 U/mL) at 50 C, while Paecilomyces sp. with enzyme activity
4.2 U/mL and optimum stability at 35 C (Gaur et al. 1993). Thermophilic fungus
Malbranchea cinnamomea S 168 was studied for novel α-amylase production using
3.5% glutinous rice flour by shake flask culture method after 3 days of incubation at
35 C temperature with a specific activity 514.6 U/mg. The enzyme was purified
with 60–70% ammonium sulfate followed by DEAE gel column elution with Tris–
HCl buffer (20 mM, pH 8). The enzyme has a molecular mass of 60.3 kDa,
exhibiting maximal activity at pH 6.5 and temperature 65 C. The enzyme
hydrolyses starch, amylopectin, amylose, pullulan, cyclodextrin, and
maltooligosaccharides. The enzyme was crystallized and revealed resolution of
2.25 Awith 10 α-helices and 14 β-strands (PDB code 3VM7). The enzyme was
activated by Mn
2+
and Co
2+
and inhibited by Cr
3+
and Hg
2+
(Han et al. 2013).
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 477

Another species Malbranchea sulfurea was isolated from manuring cow dung and
demonstrated for extracellular amylase activity. The enzyme was produced in starch
yeast extract medium after 7 days of incubation at 45 C temperature with crud
enzyme activity 21.279 U (Gautam and Gupta 1992). A thermophilic fungi
Scytalidium thermophilum produced α-amylase of molecular weight 36 kDa and
maximum activity at pH 6 and temperature 60 C by submerged fermentation
method. Enzyme purification was carried out by dialyzed crude extract using
DEAE-cellulose column, eluted by gradation of 0–500 mM sodium chloride
containing 10 mM sodium acetate buffer, pH 5.5, followed by CM-cellulose column
and a Sepharose CL-6B gel filtration column. The enzyme was inhibited by HgCl
2
/
CuCl
2
and did not show any increased activity by other metals including Ca
2+
. The
α-amylase preferentially hydrolyzed starch, followed by amylopectin, maltose,
amylose, and glycogen (Aquino et al. 2003). Another thermophilic fungus
Paecilomyces varioti produced α-amylase showed optimum enzyme activity at
temperature 60 C and pH 4.0. The enzyme was activated by Ca
2+
and CO
2+
with
a molecular mass of 75 kDa and pI value of 4.5. The enzyme acts on starch, amylose,
and amylopectin as substrate with specific activity 81.1 U/mg. Enzyme purification
was done using DEAE cellulose ion exchange column followed by Sephadex 100 G
gel filtration (Michelin et al. 2010). In another study, thermophilic fungi were
isolated from Apete and Bodija refuse dump sites at Ibadan, Nigeria by Olagoke
for amylase production and identified as Humicola sp., Absidia corymbifera,
Chaetomium elatum,Gilmaniella humicola,Talaromyces helicus,Chaetomium
sp., and Rhizomucor pusillus. The amylase enzyme activity was observed at 45 C
temperature and pH 6.9 by all the isolated cultures (Olagoke 2014). In a parallel
study from Ethiopia, 61 amylase producers at 45 C temperature have been isolated
from hyperthermal springs named Arbaminch, Awassa, Nazret, Shalla and Abijata,
Wendo Genet, and Yirgalem (Haki and Gezmu 2012). Petrova et al. (2000) studied
the comparative production of amylase production using a wild and mutant type of
thermophilic fungus Thermomyces lanuginosus ATCC 34626. The molecular
weight of the purified α-amylases was 58 kDa by SDS-PAGE and pI 3. The optimal
enzyme activity at pH revealed 5.0 and 4.5 for the wild and mutant respectively.
Only Cu
2+
and Fe
3+
act as inhibitor and Mn
2+
,Ca
2+
,Mg
2+
,Ba
2+
, and Cd
2+
as
activator for enzyme activity. Mutant culture revealed 349 mg/U specific enzyme
activity. During enzyme purification culture supernatant was precipitated with
ice-cold 2-propanol (1:3 v/v), followed by ion exchange chromatography using
Mono Q HR 5/5 for FPLC system and lastly by Superose 12 column (Petrova
et al. 2000). Mishra and Maheshwai, 1996 isolated similar culture from manure
heap. The culture Thermomyces lanuginosus IISC91 produces 40 U amylase at
50 C temperature and is a homodimer protein of MW 42 kDa. The enzyme is active
maximally at pH 5.6 and at 65 C. Enzyme purification was done by ultrafiltration
followed by separation using DEAE-Sephadex A-50, UltrogelAcA 54 column eluted
with gradation of 0–400 mM NaCI in 50 mM, pH 5 acetate buffer, and 50 mM
sodium phosphate pH 8 buffer system. The purity of the protein was enhanced by
separation by Bio-Gel P-30 column (Mishra and Maheshwari 1996). Similarly
Kunamneni et al. (2005) reported extracellular amylase production by same culture
478 R. Bodade and K. Lonkar

by solid-state fermentation using wheat brane, rice brane, maize meal, millet cereal,
barley bran, and crushed maize. Maximum enzyme activity was obtained using
wheat brane (534 U/mg) after 120 h at 50 C temperature and pH 6 (Kunamneni
et al. 2005). A thermotolerant endophytic fungal isolate, Fusicoccum sp. BCC4124,
showed α-amylase production (25.9 U/mg specific enzyme activities) using agro-
industry substrates (wheat-bran soybean, Cassava pulp-soybean, cassava pulp, and
mixed media). The crude enzyme filtrates after membrane filtration were purified by
gel chromatography using DEAE-Sepharose column with MOPS buffer (50 mM,
pH 6.8). The enzyme worked optimally at 70 C in a neutral pH in the presence of
Ca
2+
. The hydrolyzed product of substrate starch was identified as maltose,
matotriose, and maltotetraose as well as glucose, maltose, and maltotriose after
prolonged incubations (Champreda et al. 2007). Thermophilic yeast Cryptococcus
flavus exhibited the highest amylase production (specific activity 842.85 U/mg) in
yeast nitrogen base media supplemented with 2% starch. The enzyme purified by
Sephacryl S-100 column was equilibrated and eluted with 50 mM acetate buffer
(pH 5.5), containing 1 mM CaCl
2
and 100 mM NaCl. The enzyme revealed
75 kDa MW having optimum enzyme activity at pH 5.5 and 50 C temperature.
The catalytic activity on starch substrate gives major products amylose and amylo-
pectin; pullulan and glycogen were maltose and maltotriose. Enzyme activity was
inhibited by Cu
++
,Fe
++
, and Hg
++
(Wanderley et al. 2004). Fossi et al. (2005)
isolated amylolytic yeast strains from starchy soils (flour mills environment, flour
market, and cassava farms) by enrichment media using 1% starch (pH 3). Enzyme
was produced on different starchy media viz. wheat, corn, potato, rice, cassava, and
starch. Eight percent wheat starch gives maximum activity, i.e., 298.5 0.1 U/mL at
30C and pH 4.5. The enzyme activity revealed maximum at temperature 70 C and
pH 5.5 (Fossi et al. 2005). Cold-adapted yeast Tetracladium sp. was isolated and
biochemically characterized. The enzyme was produced in yeast extract–malt extract
medium (pH 7) supplemented with soluble starch (1%). The culture filtrate was used
for ammonium fractional precipitation (20–80%) followed by desalting using a
HiTrap desalting column. The enzyme optimal activity at pH 6.0 and 30 C confirms
its cold-adapted nature and has no dependency on Ca
2+
for its hydrolytic activity.
The enzyme can be used for biofuel production (Carrasco et al. 2017). Enzyme
α-amylase and a glucoamylase were also reported from Candida antarcticus
(Moesziomyces antarcticus) by De Mot and Verachtert (1987). The amylase enzyme
was purified by protamine sulfate treatment followed by ammonium sulfate precipi-
tation, gel chromatography using Sephadex G-75, and UltrogelAcA 54, DEAE
Sephacel chromatography, and hydroxyapatite chromatography. While, for
glucoamylase the final step was carried out with affinity chromatography on
acarbose–AH-Sepharose 4B. Both the enzymes (amylase and glucoamylase) were
found to be glycoproteins with molecular weight of 50 kDa and 48.5 kDa, optimum
temperatures of 62 ¯C and 57 ¯C, and pH of 4.2 and 4.7, respectively (De Mot and
Verachtert 1987).
An Antarctic psychrotolerant fungi Geomyces pannorum produces amylase
enzyme. The α-amylase gene was cloned and overexpressed in Pichia pastoris and
studied for its production and purification. Enzyme purification was carried out by
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 479

ammonium sulfate precipitation (20–70%) followed by DEAE-Sephacel and
Sephadex G-100 column chromatography, equilibrated with 0.05 M phosphate
buffer at pH 6.0 and eluted with a linear gradient of 0–0.5 M NaCl and Sephadex
G-100 column). The SDS-PAGE confirms 54 kDa MW of the separated protein. The
enzyme was catalytically optimum at pH 6.0 and 70 C, and the specific activity was
9.78 10
3
U/mg (Gao et al. 2016). Psychrophilic fungi Glaciozyma antarctica PI12
(Leucosporidium antarcticum) has also been confirmed for amylase production
(Ramli et al. 2013). Another cold adapted yeast Rhodotorula glacialis has reported
for higher amylase activities at 10–22 C (Carrasco et al. 2016). Halotolerant fungal
species Penicillium chrysogenum,Fusarium incarnatum, and Penicillium
polonicum were isolated from saltwater Lake Urmia, Iran for amylase activity
using starch agar plates. Among the three selected isolates, only P. chrysogenum
was the most tolerant isolate that grew up to 25% salinity, whereas P. polonicum
isolate U4 was the most potent producer of amylase with a yield of 260.9 U/L in
yeast nitrogen base media supplemented with 5% NaCl and 1% starch after 7 days of
incubation at 27 1C temperature (Niknejad et al. 2013).
Mohapatra et al. (1998) reported amylase activity from marine sponge
Spirastrella sp., associated Mucor sp. grown at 30 C. The optimum catalytic
activity was observed at pH 5.0 and temperature 60 C. The metals NaCl, Ca
2+
,
and Mg
2+
have no effect on enzyme activity but strongly inhibited by EDTA
(Mohapatra et al. 1998). A halotolerant fungus Penicillium sp. was studied for
amylase production by submerged fermentation using 10% NaCl and 1% starch.
The purified enzyme revealed maximum activity at pH 9 and 11. Highest enzyme
production using maize meal obtained at 30 C and pH 11 was obtained followed by
other materials (barley grains, wheat grains, wheat bran, crushed wheat grains, birds
feed grains, and wheat meal). The enzyme activity was stable up to 10% NaCl, pH 9
and 30 C temperature (Gouda and Elbahloul 2008).
20.5 Applications of Amylase
Presently the usage of extremophilic amylases has increased because of its suitable
characteristics over mesophilic enzymes in food industry such as sweeteners, syrups,
beverages production, cheese ripening, starch liquefaction, bakery production, and
animal feed supplement process. Moreover, it has wide scope in biofuel production,
detergents production, laundry and textile industry, and paper industry. Apart from
these conventional applications, it is reported for wastewater purification, hydrogel
formation, in controlled drug delivery system, digestive syrup preparation, and as a
biofilm inhibitor in pharmaceutical industry (Paul et al. 2021). The commercial
production of enzyme was begun by chemist Christian Hansen in 1874 who for
the first time extracted the rennet from dried calves’stomachs for industrial use.
Soon the microbial enzyme demand was significantly increased in the twenty-first
century and still rising as it has enormous potential in the feed, food, pharmaceutical,
beverages, detergent, paper/pulp, and leather industries. The current global market
480 R. Bodade and K. Lonkar

for the enzyme has reached $6.2 billion in 2020 and still rising (Niehaus et al. 1999;
Van Den Burg 2003; Singh et al. 2016; Dumorne et al. 2017).
20.5.1 Pharmaceutical Industry
α-Amylase has been used as an ingredient in the preparation of digestive syrups to
treat digestive disorders. Acidophilic amylases are suitable for digestive syrup
preparation that can withstand the stomach pH. Along with polymer dextran or
dextrin, it forms copolymers useful for enzymatically controlled drug delivery
system (ECDR) (Rahmouni et al. 2001).
20.5.2 Starch Conversion
Amylase has been widely used in starch industry for hydrolysis to form glucose and
fructose syrup. The enzymatic conversion of starch comprises gelatinization (i.e.,
dissolution of starch granules to form a viscous solution), liquefaction (i.e., partial
hydrolysis and reduction in viscosity), and saccharification (involves formation of
glucose, maltose, and fructose). Thermophilic fungal cultures have remarkable
thermostability and thus have interest in large scale biotechnological industry
(Souza 2010).
20.5.3 Bioethanol Production
Demand for the renewable sources is increasing rapidly in current situation due to
fuel escalation, and bioethanol production from the starch can be a good choice.
Starch solubilization and liquefaction using microbial amylase can be carried out in
industry at large extent, followed by fermentation of sugars to ethanol.
Microorganisms are screened for conversion of sugars to bioethanol formation.
Natural polymers like starch, cellulose, pullulan, and lignocellulose are good source
of glucose; however, they require pretreatment either chemically or enzymatically at
high temperature. Genetically modified microbial culture further assists to enhance
the fermentation process (Rana et al. 2013).
20.5.4 Detergent Industry
Enzyme application in detergent industry is useful to remove the tough stains, thus
making the detergent environmentally safe. It is used in laundry and dishwasher
detergents to convert residual starch to dextrins and smaller oligosaccharides.
Amylase is fortified with detergent to remove the starchy food stains (gravies,
potatoes, and chocolate) from the surface and to increase the brightness of the cloths.
Almost all the detergent contains amylase enzymes as important ingredients. A
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 481

characteristic amylase working at low temperature, alkaline condition, and oxida-
tively resistant is suitable for detergent industry (Rana et al. 2013).
20.5.5 Textile Industry
Starch is widely used in textile industry as sizing agent because it is cheap and easily
available. It is applied to the yarn for increasing its strength during weaving process
before fabric production. Amylase is used for removing this starch after fabric
production called as desizing to give a smooth texture (Souza 2010).
20.5.6 Paper Industry
In the pulp and paper industry, starch is used as sizing agent for improving the
writing quality and erasability of the paper. The natural starch has high viscosity and
need pretreatment using the amylases, to convert it into low viscous by partial
hydrolysis. Sometimes starch coats on the paper to make it sufficiently smooth and
strong at first followed by hydrolysis using the amylase (Far et al. 2020).
20.5.7 Baking Industry
α-Amylases have been widely used in the baking industry to produce flavored,
sweet, soft bakery products by enhancing the fermentation rate and reducing the
viscosity of dough. During dough making, the addition of amylase degrades the
starch into dextrins, which can be fermented easily by yeast to form sugars, thereby
enhancing the product texture quality, volume, and shelf-life. In food industry, it is
used for digestive syrup preparation that improves the metabolism of the patient and
in the preparation of fruit juices it degrades starch into sugars giving a natural sweet
taste and clear appearance. Pretreatment of animal feed with amylase improves the
digestibility of fiber. Thermostable and cold adapted amylase thus has great potential
in breaker and food industry respectively (Sundarram and Murthy 2014).
20.5.8 Wastewater Treatment
The effluents from various industries like textile, paper, and food and sewage water
plant contain many organic chemicals and starch that can cause water pollution.
Amylases from extremophilic fungi can be able to degrade these pollutants and
starch, thus alleviating the pollution. (Priya and Renu 2018).
482 R. Bodade and K. Lonkar

20.6 Conclusion
Extremozymes, the metabolites of extremophilic microbes, have a great potential in
many industrial processes, including agricultural, chemical, and pharmaceutical
industries as catalyst, therapeutic drug, analytic reagents, and diagnostic tools.
Researchers are keen to understand the physiology and metabolic pathway running
in extremophiles to apply them for industrial applications. Various studies proved
their stability at extreme conditions of temperature, acidic and alkaline pH, and high
salt concentration that are requisite for industrial processes. Isolation and screening
of potential extremophiles for novel extremozymes are continuous processes in
research. Moreover, genetically modified mesophilic natural strain by cloning
extremophiles gene is of great interest in industrial research and development sector.
Amylases from extremophilic fungi will have increased demand in coming years to
avoid chemical methods and non-extreme enzymes in food, textile, paper, food,
biofuel, pharma industries, and wastewater treatment. Being genetically and meta-
bolically more stable, a systematic investigation in isolation and enzyme production
by extremophilic fungi is pivotal to open the new applications in health and
environmental sector.
Acknowledgments The authors are thankful to the Rashtriya Uchchatar Shiksha Abhiyan
(Reference No: RUSA-CBS-TH-1.6) for the financial support. The authors are also thankful to
the Head of Department of Microbiology, Savitribai Phule Pune University, Pune, MS, India, for
providing the necessary facility to conduct the RUSA/CBS/TH 1.6 project.
References
Ali I, Akbar A, Yanwisetpakdee B, Prasongsuk S, Lotrakul P, Punnapayak H (2014a) Purification,
characterization, and potential of saline waste water remediation of a polyextremophilic
α-amylase from an obligate halophilic Aspergillus gracilis. Biomed Res Int 2014:106937
Ali I, Akbar A, Anwar M, Yanwisetpakdee B, Prasongsuk S, Lotrakul P, Punnapayak H (2014b)
Purification and characterization of extracellular, polyextremophilic α-amylase obtained from
halophilic Engyodontium album. Iran J Biotechnol 12(4):35–40
Ali I, Akbar A, Anwar M, Prasongsuk S, Lotrakul P, Punnapayak H (2015) Purification and
characterization of a polyextremophilic α-amylase from an obligate halophilic Aspergillus
penicillioides isolate and its potential for souse with detergents. Biomed Res Int 2015:245649
Ali I, Khaliq S, Sajid S, Akbar A (2019) Biotechnological applications of halophilic fungi: past,
present, and future. In: Fungi in extreme environments: ecological role and biotechnological
significance. Springer, Cham
Ambade VN, Sharma YV, Somani BL (1998) Methods for estimation of blood glucose: a compar-
ative evaluation. Med J Armed Forces India 54(2):131–133
Aquino ACMM, Jorge JA, Terenzi HF, Polizeli MLTM (2003) Studies on a thermostable
α-amylase from the thermophilic fungus Scytalidium thermophilum. Appl Microbiol Biotechnol
61(4):323–328
Attia RM, Ali SA (1977) Simple and rapid colorimetric method for the microdetermination of alpha
amylase. ZblBakt II AbtBd 132(3):193–195
Berde CP, Berde VB, Sheela GM, Veerabramhachari P (2019) Discovery of new extremophilic
enzymes from diverse fungal communities. In: Recent advancement in white biotechnology
through fungi. Springer, Cham
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 483

Carrasco M, Villarreal P, Barahona S, Alcaíno J, Cifuentes V, Baeza M (2016) Screening and
characterization of amylase and cellulase activities in psychrotolerant yeasts. BMC Microbiol
16(1):1–9
Carrasco M, Alcaíno J, Cifuentes V, Baeza M (2017) Purification and characterization of a novel
cold adapted fungal glucoamylase. Microb Cell Fact 16(1):1–10
Chakraborty S, Khopade A, Kokare C, Mahadik K, Chopade B (2009) Isolation and characteriza-
tion of novel α-amylase from marine Streptomyces sp. D1. J Mol Catal B Enzym 58(1–4):17–23
Champreda V, Kanokratana P, Sriprang R, Tanapongpipat S, Eurwilaichitr L (2007) Purification,
biochemical characterization, and gene cloning of a new extracellular thermotolerant and
glucose tolerant maltooligosaccharide-forming α-amylase from an endophytic ascomycete
Fusicoccum sp. BCC4124. Biosci Biotechnol Biochem 71:2010–2020
De Mot R, Verachtert H (1987) Purification and characterization of extracellular α-amylase and
glucoamylase from the yeast Candida antarctica CBS 6678. Eur J Biochem 164(3):643–654
De Oliveira TB, Gomes E, Rodrigues A (2015) Thermophilic fungi in the new age of fungal
taxonomy. Extremophiles 19(1):31–37
Duarte AWF, Dos Santos JA, Vianna MV, Vieira JM, Mallagutti VH, Inforsato FJ, Wentzel LCP,
Lario LD, Rodrigues A, Pagnocca FC, Pessoa Junior A (2018) Cold-adapted enzymes produced
by fungi from terrestrial and marine Antarctic environments. Crit Rev Biotechnol 38(4):
600–619
Dumorne K, Cordova DC, Astorga-Elo M, Renganathan P (2017) Extremozymes: a potential
source for industrial applications. J Microbiol Biotechnol 27(4):649–659
El-Enshasy HA, Abdel Fattah YR, Othman NZ (2013) Amylases: characteristics, sources, produc-
tion, and applications. In: Bioprocessing technologies in biorefinery for sustainable production
of fuels, chemicals and polymers. Wiley, Hoboken, NJ
Far BE, Ahmadi Y, Khosroshahi AY, Dilmaghani A (2020) Microbial alpha-amylase production:
progress, challenges and perspectives. Adv Pharm Bull 10(3):350
Fossi BT, Tavea F, Ndouenkeu R (2005) Production and partial characterization of a thermostable
amylase from ascomycetes yeast strain isolated from starchy soils. Afr J Biotechnol 4(1):14–18
Fuwa H (1954) A new method for micro-determination CF amylase activity by the use of amylose
as the substrate. J Biochem 41(5):583–603
Gao B, Mao Y, Zhang L, He L, Wei D (2016) A novel saccharifying α-amylase of Antarctic
psychrotolerant fungi Geomycespannorum: gene cloning, functional expression, and character-
ization. Starch 68(1–2):20–28
Gaur R, Garg SK, Singh SP, Verma J (1993) A comparative study of the production of amylase
from Humicola and Paecilomyces species. Bioresour Technol 46(3):213–216
Gautam SP, Gupta AK (1992) Extracellular and mycelial amylases of the thermophilic fungus
Malbranchea sulfurea. Mycopathologia 119(2):77–82
Gonzalez CF, Farina JI, Figueroa LIC (2002) A critical assessment of a viscometric assay for
measuring Saccharomycopsis fibuligera α-amylase activity on gelatinized cassava starch.
Enzyme Microb Technol 30(2):169–175
Gostincar C, Grube M, De Hoog S, Zalar P, Gunde-Cimerman N (2009) Extremotolerance in fungi:
evolution on the edge. FEMS Microbiol Ecol 71(1):2–11
Gouda M, Elbahloul Y (2008) Statistical optimization and partial characterization of amylases
produced by halotolerant Penicillium sp. World J Agric Sci 4:359–368
Gross S, Robbins EI (2000) Acidophilic and acid-tolerant fungi and yeasts. Hydrobiologia
433(1–3):91–109
Grum-Grzhimaylo AA, Georgieva ML, Bondarenko SA, Alfons J, Debets M, Bilanenko EN (2016)
On the diversity of fungi from soda soils. Fungal Divers 76(1):27
Gunde-Cimerman N, Ramos J, Plemenitas A (2009) Halotolerant and halophilic fungi. Mycol Res
113(11):1231–1241
Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B (2003) Microbial α-amylases: a
biotechnological perspective. Process Biochem 38(11):1599–1616
484 R. Bodade and K. Lonkar

Gusakov AV, Kondratyeva EG, Sinitsyn AP (2011) Comparison of two methods for assaying
reducing sugars in the determination of carbohydrase activities. Int J Anal Chem 2011:283658
Haki GD, Gezmu B (2012) Detection of thermostable amylases produced by thermophilic fungi
isolated from some Ethiopian hyper-thermal springs. Green J Biol Sci 2:035–039
Han P, Zhou P, Hu S, Yang S, Yan Q, Jiang Z (2013) A novel multifunctional α-amylase from the
thermophilic fungus Malbranchea cinnamomea: biochemical characterization and three-
dimensional structure. Appl Biochem Biotechnol 170(2):420–435
Hassan N, Rafiq M, Hayat M, Shah AA, Hasan F (2016) Psychrophilic and psychrotrophic fungi: a
comprehensive review. Rev Environ Sci BioTechnol 15(2):147–172
Hernandez MS, Rodriguez MR, Guerra NP, Roses RP (2006) Amylase production by Aspergillus
niger in submerged cultivation on two wastes from food industries. J Food Eng 73(1):93–100
Johnson DB (1998) Biodiversity and ecology of acidophilic microorganisms. FEMS Microbiol
Ecol 27(4):307–317
Kanlayakrit W, Ishimatsu K, Nakao M, Hayashida S (1987) Characteristics of raw-starch-digesting
glucoamylase from thermophilic Rhizomucor pusillus. J Ferment Technol 65(4):379–385
Kato S, Shimizu-Ibuka A, Mura K, Takeuchi A, Tokue C, Arai S (2007) Molecular cloning and
characterization of an α-amylase from Pichia burtonii 15-1. Biosci Biotechnol Biochem 71(12):
3007–3013
Kladwang W, Bhumirattana A, Hywel-Jones N (2003) Alkaline-tolerant fungi from Thailand.
Fungal Divers 13(1):69–83
Kobayashi T, Kanai H, Hayashi T, Akiba T, Akaboshi R, Horikoshi K (1992) Haloalkaliphilic
maltotriose-forming alpha-amylase from the archaebacterium Natronococcus sp. strain ah-36. J
Bacteriol 174(11):3439–3444
Kour D, Rana KL, Kaur T, Singh B, Chauhan VS, Kumar A, Rastegari AA, Yadav N, Yadav AN,
Gupta VK (2019) Extremophiles for hydrolytic enzymes productions: biodiversity and potential
biotechnological applications. In: Bioprocessing for biomolecules production, pp 321–372
Kunamneni A, Permaul K, Singh S (2005) Amylase production in solid state fermentation by the
thermophilic fungus Thermomyces lanuginosus. J Biosci Bioeng 100(2):168–171
Ma Y, Galinski EA, Grant WD, Oren A, Ventosa A (2010) Halophiles 2010: life in saline
environments. Appl Environ Microbiol 76(21):6971
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol Rev 64(3):461–488
Mccleary BV, Gibson TS, Mugford DC (1997) Measurement of total starch in cereal products by
amyloglucosidase-α-amylase method: collaborative study. J AOAC Int 80(3):571–579
Michelin M, Silva TM, Benassi VM, Peixoto-Nogueira SC, Moraes LAB, Leao JM, Jorge JA,
Terenzi HF, Maria de Lourdes TM (2010) Purification and characterization of a thermostable
α-amylase produced by the fungus Paecilomyces variotii. Carbohydr Res 345(16):2348–2353
Mishra RS, Maheshwari R (1996) Amylases of the thermophilic fungus Thermomyces lanuginosus:
their purification, properties, action on starch and response to heat. J Biosci 21(5):653–672
Mohapatra BR, Banerjee UC, Bapuji M (1998) Characterization of a fungal amylase from Mucor
sp. associated with the marine sponge Spirastrella sp. J Biotechnol 60(1–2):113–117
Mukherjee R, Paul T, Soren JP, Halder SK, Mondal KC, Pati BR, Mohapatra PKD (2019)
Acidophilic α-amylase production from Aspergillus niger RBP7 using potato peel as substrate:
a waste to value added approach. Waste Biomass Valor 10(4):851–863
Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose.
J Biol Chem 153:375–380
Niehaus F, Bertoldo C, Kähler M, Antranikian G (1999) Extremophiles as a source of novel
enzymes for industrial application. Appl Microbiol Biotechnol 51(6):711–729
Niknejad F, Moshfegh M, Najafzadeh MJ, Houbraken J, Rezaei S, Zarrini G, Faramarzi MA,
Nafissi-Varcheh N (2013) Halotolerant ability and α-amylase activity of some saltwater fungal
isolates. Iran J Pharm Res 12(Suppl):113
Nwagu TN, Okolo BN (2011a) Growth profile and amylolytic activity of a thermophilic fungus
Aspergillus fumigatus isolated from soil. Asian J Biotechnol 3(1):46–57
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 485

Nwagu TN, Okolo BN (2011b) Extracellular amylase production of a thermotolerant Fusarium sp:
isolated from eastern Nigerian soil. Braz Arch Biol Technol 54(4):649–658
Olagoke OA (2014) Amylase activities of some thermophilic fungi isolated from municipal solid
wastes and palm-kernel stack. Am J Microbiol Biotechnol 1:64–70
Passey RB, Gillum RL, Fuller JB, Urry FM, Giles ML (1977) Evaluation and comparison of
10 glucose methods and the reference method recommended in the proposed product class
standard (1974). Clin Chem 23(1):131–139
Patel H, Rawat S (2021) Thermophilic fungi: diversity, physiology, genetics, and applications. In:
New and future developments in microbial biotechnology and bioengineering. Elsevier,
Amsterdam
Paul JS, Gupta N, Beliya E, Tiwari S, Jadhav SK (2021) Aspects and recent trends in microbial
α-amylase: a review. Appl Biochem Biotechnol 14:1–50
Peixoto SC, Jorge JA, Terenzi HF, Maria de Lourdes TM (2003) Rhizopus microsporus var.
rhizopodiformis: a thermotolerant fungus with potential for production of thermostable
amylases. Int Microbiol 6(4):269–273
Petrova SD, Ilieva SZ, Bakalova NG, Atev AP, Bhat MK, Kolev DN (2000) Production and
characterization of extracellular α-amylases from the thermophilic fungus Thermomyces
lanuginosus (wild and mutant strains). Biotechnol Lett 22(20):1619–1624
Priya FS, Renu A (2018) Efficacy of amylase for wastewater treatment from Penicillium SP. SP2
isolated from stagnant water. J Environ Biol 39(2):189–195
Rahmouni M, Chouinard F, Nekka F, Lenaerts V, Leroux JC (2001) Enzymatic degradation of
cross-linked high amylose starch tablets and its effect on in vitro release of sodium diclofenac.
Eur J Pharm Biopharm 51(3):191–198
Ramli ANM, Azhar MA, Shamsir MS, Rabu A, Murad AMA, Mahadi NM, Illias RM (2013)
Sequence and structural investigation of a novel psychrophilic α-amylase from Glaciozyma
antarctica PI12 for cold-adaptation analysis. J Mol Model 19(8):3369–3383
Rana N, Walia A, Gaur A (2013) α-Amylases from microbial sources and its potential applications
in various industries. Natl Acad Sci Lett 36(1):9–17
Selbmann L, Egidi E, Isola D, Onofri S, Zucconi L, de Hoog GS, Chinaglia S, Testa L, Tosi S,
Balestrazzi A, Lantieri A, Compagno R, Tigini V, Varese C (2013) Biodiversity, evolution and
adaptation of fungi in extreme environments. Plant Biosyst 147(1):237–246
Singh R, Kumar M, Mittal A, Mehta PK (2016) Microbial enzymes: industrial progress in 21st
century. 3 Biotech 6(2):1–15
Singh P, Jain K, Desai C, Tiwari O, Madamwar D (2019) Microbial community dynamics of
extremophiles/extreme environment. In: Microbial diversity in the genomic era. Academic
Press, San Diego, CA
Smith BW, Roe JH (1949) A photometric method for the determination of α-amylase in blood and
urine, with use of the starch-iodine color. J Biol Chem 179(1):53–59
Somogyi M (1945) A new reagent for the determination of sugars. J Biol Chem 160:61–68
Souza PMD (2010) Application of microbial α-amylase in industry—a review. Braz J Microbiol
41(4):850–861
Spencer-Martins I, Van Uden N (1979) Extracellular amylolytic system of the yeast Lipomyces
kononenkoae. Eur J Appl Microbiol Biotechnol 6(3):241–250
Sundarram A, Murthy TPK (2014) α-Amylase production and applications: a review. J Appl
Environ Microbiol 2(4):166–175
Teklebrhan Welday G, Abera S, Baye K (2014) Isolation and characterization of thermo-stable
fungal alpha amylase from geothermal sites of Afar, Ethiopia
Unal A (2015) Production of α-amylase from some thermophilic Aspergillus species and optimiza-
tion of its culture medium and enzyme activity. Afr J Biotechnol 14(47):3179–3183
Van Den Burg B (2003) Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6(3):
213–218
486 R. Bodade and K. Lonkar

Visvanathan R, Jayathilake C, Liyanage R (2016) A simple microplate-based method for the
determination of α-amylase activity using the glucose assay kit (GOD method). Food Chem
211:853–859
Wanderley KJ, Torre FA, Moraes LM, Ulhoa CJ (2004) Biochemical characterization of α-amylase
from the yeast Cryptococcus flavus. FEMS Microbiol Lett 231(2):165–169
Xiao Z, Storms R, Tsang A (2006) A quantitative starch-iodine method for measuring alpha-
amylase and glucoamylase activities. Anal Biochem 351(1):146–148
Zhang X, Li SJ, Li JJ, Liang ZZ, Zhao CQ (2018) Novel natural products from extremophilic fungi.
Mar Drugs 16(6):194
20 Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications 487

Extremophilic Fungi as a Source
of Bioactive Molecules 21
Annada Das , Kaushik Satyaprakash , and Arun Kumar Das
Abstract
Extremophiles are the organisms that survive the harshest and extreme
environments on earth, notably deep-sea sediments, permafrost, deserts, hypersa-
line water, etc., of the extremophiles, various fungal species like Aspergillus spp.,
Emericella spp., Eutypella spp., Microsporum spp., Penicillium spp.,
Trichoderma spp., Wallemia spp., etc. produce a number of bioactive molecules
categorized into polypeptides, polyketides, terpenoids, alkaloids, sterols, etc.
These metabolites are proven to possess antibacterial, antiviral, antifungal, anti-
inflammatory, anticancer, and anti-diabetic activities. These compounds have
significant scope in biomedical research and can be explored as potential
candidates for new drug discovery. Apart from it, they can be substantially
applied in the fields of environmental, industrial, and food technology. There is
a need to isolate, propagate, and conserve these novel microorganisms and their
active metabolites. This chapter provides an overview of various bioactive
molecules obtained from different genera of extremophilic fungi isolated from
extreme habitats on this planet.
Keywords
Extremophiles · Extreme environments · Fungi · Bioactive molecules · Drug
discovery
A. Das (*)
Department of Livestock Products Technology, Faculty of Veterinary and Animal Sciences, West
Bengal University of Animal and Fishery Sciences, Kolkata, West Bengal, India
K. Satyaprakash
Department of Veterinary Public Health and Epidemiology, Faculty of Veterinary and Animal
Sciences, Banaras Hindu University, Mirzapur, Uttar Pradesh, India
A. K. Das
Eastern Regional Station, ICAR-Indian Veterinary Research Institute, Kolkata, West Bengal, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_21
489

21.1 Introduction
Emergence of antimicrobial resistance to conventional drugs has prompted scientists
and researchers to search for various alternatives in the unexplored sources, viz. the
extreme environments. A number of literatures published over time have revealed
about the microorganisms surviving and growing in extreme habitats by producing
specialized metabolites that have some inherent bioactive properties.
If we look at extremophiles, these are the organisms which can survive and/or
multiply in physically and chemically extreme environmental conditions which are
detrimental to most of the life forms on this planet. Most of the extremophiles are
microorganisms—fungi constitute one of the big classes among them.
Extremophiles can be classified according to the environmental condition in which
they survive and reproduce and these are thermophiles (60–80 C),
hyperthermophiles (>80 C), psychrophiles (<20 C), acidophiles (pH <3),
alkalophiles (pH >9), piezophiles (>500 atm. pressure), halophiles (>0.2 M
NaCl), oligotrophic (low nutrient availability), xerophiles (very dry climate),
metallophiles (high concentration of metals), and endolithic (within rock or within
pores of mineral grains). Some extremophiles are tolerant to a combination of
extreme physicochemical factors, hence called polyextremophiles, viz.
thermoacidophiles and haloalkalophiles (Gupta et al. 2014).
Extremophilic organisms have developed robust defense mechanisms to survive
under extremely harsh environments, leading to the synthesis of biomolecules with
diverse biological activities. The ability of extremophiles to adapt to adverse envi-
ronmental factors is probably due to the regulation and expression of specific genes
in their genome. The strategy for survival in extremes is due to the production of
enzymes and other secondary metabolites. Such compounds have many biotechno-
logical applications, viz. environmental (bioremediation, biodegradation, and bio-
control), industrial (biomining, biofuel, and food), and medical (antibiotics, antiviral,
antifungal, anti-tumor compounds) (Dhakar and Pandey 2016; Zhang et al. 2018). In
order to survive the extreme pressure, temperature, salinity, water availability, and
pH conditions, the biosynthesis of various novel natural products with diversified
biological activities takes place, which could lead to their industrial application
(Rogozhin et al. 2018). Biotechnological process frequently happens in inhospitable
conditions to microbes in the industrials setup like extreme temperature, salinity, and
pH which could be comparable to that of the natural habitats of extremophiles
(Elleuche et al. 2015). Many bioactive compounds are produced by extremophilic
fungi, yet their potential has not been fully evaluated. Keeping this in view, this
chapter focuses on the bioactive molecules produced by different extremophilic
fungi which can provide a plethora of novel biomolecules for the industry to
work with.
490 A. Das et al.

21.2 Bioactive Compounds from Extremophilic Fungi
The abundant presence of diverse genera of fungi in extreme habitats makes them
ideal sources for search of bioactive compounds which could be useful in designing
new drugs. Fungi living in extreme environments might have made adjustments at
genome level to sustain life by augmenting the chemical defense and communication
(Deshmukh et al. 2018). Cephalosporin C was the first compound obtained from
deep-sea fungus Cephalosporium spp. in 1949. Thereafter a number of important
biochemicals have been isolated from marine fungi.
Of the specialized compounds obtained from extremophilic microorganisms,
antibacterial activities are exhibited by 33.6% of compounds followed by anticancer
(25.6%), antifungal and anti-inflammatory (9.6% each), and antiviral (8.0%)
activities which possess the potential to be future candidates of lifesaving drugs
(Sayed et al. 2019).
21.2.1 Desert Fungi
Fungi are the most stress-resistant eukaryotes (Sterflinger et al. 2012). In the
challenging desert environments, they have exhibited a wide range of adaptive
mechanisms (Onofri et al. 2007; Stevenson et al. 2017; Santiago et al. 2018).
Early isolation studies from soil of Negev Desert in Southern Israel and Sonoran
Desert in North America revealed extensive fungal diversity (Taylor-George et al.
1983). The Atacama Desert in Chile has been the hunting ground for scientist in
search of microflora in desert habitats (Bull et al. 2016,2018; Bull and Goodfellow
2019) among the studies in all non-polar deserts (Kurapova et al. 2012; Tiwari et al.
2015; Ouchari et al. 2018) of the world. Although novel fungal species have been
discovered from desert ecosystem, yet their utility in bioprospecting campaigns is
limited. For instance, 13 distinct fungal genera, notably that of Alternaria spp. and
Ulocladium spp. were isolated from Atacama Desert soil (Conley et al. 2006);
135 novel species dominated by Ascomycetes from Makhtesh Ramon Desert soil
in Israel (Grishkan and Nevo 2010); 77 lichenoid fungal species including four novel
species (Amandinea efflorescens,Diploicia canescens,Myriospora smaragdula, and
Rhizocarpon simillimum) from two altitudinal transects of Alto Patache in Atacama
Desert (Castillo and Beck 2012); Cladosporium,Neucatenulostroma, and Penicil-
lium spp. from high altitude rocks of Atacama Desert (Gonçalves et al. 2016); three
novel fungal species (Diversispora omaniana,Septoglomus nakheelum, and
Rhizophagus arabicus) from an Omani Desert (Symanczik et al. 2014) and two
novel halophilic fungi (Aspergillus atacamensis and Aspergillus salisburgensis)
from a cave at the Atacama Desert (Martinelli et al. 2017) have been isolated. The
deserts of the Middle East are also home to some extensive diverse fungal species
(Murgia et al. 2018). The chemical structures of some bioactive compounds obtained
from desert fungi are given in Fig. 21.1.
21 Extremophilic Fungi as a Source of Bioactive Molecules 491

21.2.1.1 N-Containing Compounds
Recent studies on fungi from desert ecosystem underline their potential as a prolific
source of novel bioactive chemicals (Santiago et al. 2018). Wallemia sebi, isolated
from the Atacama Desert was found to produce wallimidione (Desroches et al.
2014), 15-azasterol, and 24, 28-dihydro-15-azasterol (Jančičet al. 2016). Molecules
UCA 1064-A and UCA 1064-B exhibited in vivo anti-tumor activity against mam-
mary tumor in mouse and in vitro anti-proliferative activity against HeLa cells,
respectively. Further, antifungal activity against Saccharomyces cerevisiae and
inhibition of G+ve bacteria were also exhibited by the above compounds (Jančič
et al. 2016). Cyclopentenopyridine (an alkaloid from a halophilic strain of W. sebi)
was found to inhibit Enterobacter aerogenes (Peng et al. 2011).
21.2.1.2 Terpenes
The unique terpenes, Walleminone and walleminol have been produced by W. sebi
(Jančičet al. 2016).
21.2.2 Permafrost Soil Fungi
The layer beneath the earth’s crust which remains at a sub-zero temperature for at
least 2 consecutive years is known as permafrost layer (Jansson and Tas 2014).
Water availability, temperature, high viscosity, and low thermal energy are the most
important abiotic factors for the microorganisms in the permafrost (Jansson and Tas
2014). Normal cell cycle is inhibited under such conditions and the transmembrane
and intracellular proteins can denature leading to loss of cell structure and cell
Fig. 21.1 Chemical structures of compounds obtained from desert fungi (Sayed et al. 2019)
492 A. Das et al.

membrane fluidity (Chattopadhyay 2006;D’Amico et al. 2006). These cryophilic
microorganisms have developed survival strategies, viz. initiating a state of dor-
mancy, generation of specialized metabolites and proteins, storing energy in the
form of polyphosphates, triglycerides, wax, esters, and glycogen, thereby reducing
the cellular metabolism (Unell et al. 2007; Bowman 2008; Bakermans et al. 2009).
In Arctic permafrost, the fungi of genera Aspergillus,Cladosporium, Geomyces,
and Penicillium (Ozerskaya et al. 2009) are predominant. Of nearly 400 taxonomi-
cally distinct fungal genera isolated from Antarctic soil (Bridge and Spooner 2012),
Cladospora,Geomyces, and Thielava from Cape Royds, Antarctica (Blanchette
et al. 2010) are the important ones. Fungal diversity in the Antarctic lichens of
King George Island includes Arthoniomycetes,Eurotiomycetes,Lecanoromycetes,
Leotiomycetes,Sordariomycetes of Ascomycota, and Cystobasidiomycetes and
Tremellomycetes of Basidiomycota (Park et al. 2015). The chemical structures of
some bioactive compounds obtained from permafrost fungi are given in Fig. 21.2.
21.2.2.1 N-Containing Compounds
Fungi of polar region have the ability to metabolize rapidly a variety of substrates.
Oidiodendron truncatum, a psychrophilic fungus found in Antarctica, was found to
produce two novel epipolythiodioxopiperazines (chetracins B and chetracin C) and
five novel diketopiperazines (oidioperazines A–D and chetracin D). The sulfide
bridge in chetracin B and chetracin C is responsible for their anticancer activity
against human cancer cell lines (Li et al. 2012). Cytochalasins Z
24
(moderate
cytotoxicity towards human breast cancer cells (MCF-7)), cytochalasins Z
25
, and
cytochalasins Z
26
were recovered from Eutypella sp. D1, isolated from Arctic soil on
London Island of Fongsfjorden (Liu et al. 2014). Antibacterial activity against G+ve
and Gve bacteria was observed for Libertellenone G (a N-containing diterpene),
obtained from Eutypella spp. Another N-containing diterpene, eutypenoid B
obtained from the same organism was found to be a potent immunosuppressant
(Lu et al. 2014; Zhang et al. 2016a,b). Similarly, Lindgomycin and ascosetin
obtained from Arctic sponge-derived fungal strain of family Lindgomycetaceae
have exhibited potent antimicrobial activity against methicillin-resistant Staphylo-
coccus aureus and Candida albicans (Ondeyka et al. 2014; Wu et al. 2015).
21.2.2.2 Polyketides
Penilactone A and penilactone B (highly oxygenated polyketides), isolated from an
Antarctic deep-sea-derived fungus Penicillium crustosum were found to exhibit
inhibitory action against nuclear factor-κB (Wu et al. 2012). Pseudogymnoascus
spp., isolated from an Antarctic marine sponge was found to produce
pseudogymnoascin A–C and 3-nirtoasterric acid, but they failed to exhibit antifungal
or antibacterial activity may be due to the presence of nitro-group (Figueroa et al.
2015). Again, ochraceopones A–E, isoasteltoxin, and asteltoxin B (highly
oxygenated polyketides) were obtained from Aspergillus ochraceopetaliformis,of
which anti-influenza activity against H1N1 and H3N2 viruses was observed for
ochraceopones A and isoasteltoxin (Wang et al. 2016).
21 Extremophilic Fungi as a Source of Bioactive Molecules 493

Fig. 21.2 Chemical structures of compounds obtained from permafrost soil fungi (Sayed et al.
2019)
494 A. Das et al.

21.2.2.3 Terpenes
Four new diterpenes, scoparasin B, libertellenone H, and eutypenoids A and C, were
isolated from Eutypella strain from Arctic soil on London Island (Liu et al. 2014).
Antarctic moss-derived fungus Penicillium funiculosum has been found to contain
the meroterpenoids, chrodrimanin I and chrodrimanin J along with five known
structurally related chrodrimanins, but the novel chrodrimanins revealed a weak
inhibitory activity against H1N1 virus (Zhou et al. 2015).
21.2.3 Deep-Sea Sediments Fungi
The ocean is home to taxonomically distinct microorganisms. Less attention has
been given to the deep-sea sediments especially in the Polar Regions and deep
trenches, may be due to the difficulty in accessing the samples. The deep-sea
environment (water depths below 1000 m) is a potentially difficult habitat for
sustenance of life with respect to variation in temperature, lack of dissolved oxygen,
limited or no penetration of sunlight, high hydrostatic pressure (up to 400 atm.), and
limited nutrient availability (Danovaro et al. 2017; Barone et al. 2019). Organisms
surviving and growing in these environments must have developed some sort of
mechanisms to tolerate the extremes of temperature, pH, salinity, osmolarity as well
Fig. 21.2 (continued)
21 Extremophilic Fungi as a Source of Bioactive Molecules 495

as production of certain bioactive molecules to protect themselves in these harsh
conditions.
Some of the important fungi isolated from deep-sea sediments include Graphium
spp. from Northern Antarctic Peninsula (Gonçalves et al. 2017), filamentous fungi
belonging to the phylum Ascomycota from the Central Indian Basin (Singh et al.
2010,2012) and Candida,Cryptococcus,Pichia, and Rhodotorula spp. from deep
Polar sea (Nagano et al. 2013). The chemical structures of some bioactive
compounds obtained from deep-sea sediment fungi are given in Fig. 21.3.
21.2.3.1 N-Containing Compounds
Eremophilane (a lactam-type metabolite) from Antarctic deep-sea fungus Penicil-
lium sp. PR19N-1 (Lin et al. 2014) and circumdatin K and circumdatin L (benzodi-
azepine alkaloids), 10-epi-sclerotiamide and 5-epi-sclerotiamide (indole alkaloids)
along with aspergilliamide B (a novel amide) from Aspergillus westerdijkiae
DFFSCS013 (Peng et al. 2013) have been isolated. Meleagrins B–E, roquefortines
F–I, and breviones F–H (all alkaloids) isolated from deep-sea sediment-derived
Penicillium spp. have displayed cytotoxic activity against MOLT4, HL60, A549,
BEL7402, and HeLa cell lines (Li et al. 2012).
21.2.3.2 Terpenes
A moderate cytotoxic effect against cancer cell lines HL-60 and A549 has been
exhibited by chlorinated eremophilane sesquiterpenes obtained from the Penicillium
spp. PR19N-1 isolated from Antarctic region (Wu et al. 2013). Further, five new
cytotoxic eremophilane-type sesquiterpenes have been identified from the above
strain (Lin et al. 2014).
21.2.3.3 Polyketides
Two novel polyketides, penilactone A and penilactone B, with unusual highly
oxygenated structures were identified from an Antarctic deep-sea fungus Penicillium
crustosum PRB-2 (Wu et al. 2012).
21.2.4 Marine Fungi
(a) Halophiles
Halophiles are organisms that can thrive in saline environments with
concentrations higher than 0.2 M NaCl (Gupta et al. 2014). Halophiles can be
found in hypersaline environments like tide pools, deep-sea, salt lakes, etc. In
order to cope with the osmotic stress, the halophilic eukaryotes have developed
a mechanism for intracellular accumulation of compatible solutes or
osmoprotectants or osmolytes like polyols, soluble sugars, amino acids, and
quaternary ammonium compounds in contrast to salts (Lentzen and Schwarz
2006). Debaryomyces hansenii,Hortaea werneckii, and Wallemia
ichthyophaga have been isolated from natural hypersaline environments (Prista
et al. 1997; Gunde-Cimerman et al. 2009).
496 A. Das et al.

Fig. 21.3 Chemical structure of compounds obtained from deep-sea sediment-derived fungi
(Sayed et al. 2019)
21 Extremophilic Fungi as a Source of Bioactive Molecules 497

Fig. 21.3 (continued)
498 A. Das et al.

H. werneckii and Trimmatostroma salinum have been found to produce
extracellular hydrolytic enzymes having xylanolytic, lignolytic, and cellulolytic
activities (Plemenitǎs et al. 2008; Zalar et al. 2005). Salt (NaCl) concentration
affects the production of some secondary metabolites (wallimidione,
walleminol, walleminone, UCA 1064-A, and UCA 1064-B) from W. sebi with
substantial biological activities (Botíc et al. 2012; Jančičet al. 2016). The
marine-derived fungus, Emericellopsis spp. produces an array of peptide
antibiotics, viz. emericellipsin A, zervamicins, bergofungins A, B, C, and D,
heptaibin, and emerimicines with antibacterial and antifungal activities with
emericellipsin A also exhibiting a significant cytotoxic effect against HepG2
and HeLa tumor cell lines (Rogozhin et al. 2018).
The compounds (22R,23S)-epoxy-3β,11α,14β,16β-tetrahydroxyergosta-5,7-
dien-12-one and 6-(1H-pyrrol-2-yl)-hexa-1,3,5-trienyl-4-methoxy-2H-pyran-2-
one (existed as a pair of epimers), along with nine other compounds were
isolated and identified from the fermentation broth of Aspergillus flocculosus
PT05-1, a halotolerant fungus obtained from sediments of Putian saltern of
Fujian Province of China which exhibited cytotoxic activity against HL-60
and BEL-7402 cells, as well as antimicrobial activity against Enterobacter
aerogenes,Pseudomonas aeruginosa, and Candida albicans (Zhang et al.
2013). Alternaroside A–C (cerebrosides) and alternarosin A (diketopiperazine
alkaloid) obtained from the halotolerant fungus, Alternaria raphani isolated
from Hongdao sea salt field, Qingdao, China were found to possess weak
antibacterial activity (Wang et al. 2009).
Fig. 21.3 (continued)
21 Extremophilic Fungi as a Source of Bioactive Molecules 499

(b) Piezophiles
The piezophilic fungus (previously known as barophilic fungus) is a class of
extremophiles that can thrive and grow in high-pressure habitats, especially the
oceans (Pettit 2011). Microorganisms in the deep-sea environment are affected
by elevated hydrostatic pressure, low temperature, and low nutrients. The high
pressure alters the fluidity and permeability of cell membranes, the stability of
H-bonds in DNA, thereby affecting the cellular integrity and cell division
(Burgaud et al. 2015).
The piezophilic fungus Phialocephala spp. FL30r was reported as a natural
producer of trisorbicillinone A (a sorbicillin trimer) and oxosorbiquinol and
dihydrooxosorbiquinol (bisorbicillinoids). All sorbicillin compounds showed
cytotoxicity against P388, A549, HL60, BEL-7402, and K562 cell lines
(Li et al. 2007a,b). A total of 16 steroids with bicycle[4.4.1] A/B rings were
obtained from Penicillium citrinum HGY1-5 from Huguangyan, China of which
24-epi-cyclocitrinol, cyclocitrinol, neocyclocitrinol C, and threo-23-O-
methylneocyclocitrinol were found to enhance cAMP production in GPR12-
transfected CHO cells (Du et al. 2008). Penicillium spp. isolated from Berkeley
Pit Lake of Bute (Montana) produced the bioactive compounds like
berkeleydione and berkeleytrione (having anticancer activity, inhibiting
MMP-3 and caspase-1), preaustinoid A and preaustinoid A1, berkeleyones A–
C (Stierle et al. 2011), while Pleurostomophora spp. isolated from the same lake
was found to produce the secondary metabolites berkchaetoazaphilones A–C,
the red pigment berkchaetorubramine, and 4-(hydroxymethyl) quinolone
(Stierle and Stierle 2014). Berkchaetoazaphilone B inhibited the production of
IL-1β, TNF-α, and IL-6 and showed cytotoxicity on human retinoblastoma cell
line Y79, leukemia cell lines CCRF-CEM and SR, and the melanoma cell line
LOX IMVI (Stierle and Stierle 2014). Dicitrinone B, obtained from P. citrinum
HGY1-5 from a dead volcano in Huguangyan, China was observed to induce
apoptosis in tumor cells. The fungal strain Leptosphaeria spp. isolated from
Tanabe Bay, Japan produced leptosins A-N1, of which leptosins I, J, M, M1, N,
and N1 exhibited cytotoxicity against a panel of 39 human cell lines (Takahashi
et al. 1994; Yamada et al. 2002).
(c) Psychrophiles
Psychrophiles are cold-adapted microorganisms with an optimum growth tem-
perature of 15 C or lower. The survivability of microorganisms in cold envi-
ronment has been very challenging. At least, the temperature of 90% of the
ocean volume is below 5 C (Anwar et al. 2020). The decrease in temperature
may damage the cell membrane and generate reactive oxygen species (ROS)
which can degrade the genetic materials, proteins, and lipids (Gocheva et al.
2009). Microorganisms residing in cold climates especially at the Antarctic
region must have made some structural and functional adaptations at the cellular
constituents, cell membrane, metabolic pathways, production of radical scav-
enging molecules, and mechanisms to inhibit the formation of intracellular ice
crystals. The production of different metabolites in response to these extreme
situations has a tremendous scope for the biotechnological and pharmaceutical
industries to search for potent bioactive molecules.
500 A. Das et al.

A variety of polyketides, steroid, and indole-derivatives, sesquiterpenoids,
alkaloids, aromatic compounds, fatty acids, pyrone analogs, sorbicillin,
breviane-derivatives, and compounds containing amino acid structures have
been found to exhibit anticancer activities, while some other compounds, viz.
prenylxanthones, depsidone-based analogs, triple benzene compounds, and
citromycetin analog were shown to possess antibacterial activity (Wang et al.
2015). A hybrid polyketide, cladosin C, produced by Cladosporium
sphaerospermum 2005-01-E3 showed antiviral activity against H1N1 influenza
virus (Wu et al. 2014). The deep-sea fungus Aspergillus versicolor CXCTD-06-
6 was described as a source of antifungal and antifouling agents, viz.
diketopiperazine, brevianamide W, diketopiperazine V, brevianamide Q, R, K,
and E (Kong et al. 2014). P-hydroxyphenopyrrozin and terphenyl isolated from
deep-sea fungi Chromocleista spp. (Park et al. 2006) and Aspergillus candidus
(Liu et al. 2013), respectively, have shown antifungal activity against Candida
albicans. The compound terphenyl also showed antibacterial activity against
Staphylococcus aureus,Bacillus subtilis, and Vibrio spp. (Liu et al. 2013). The
β-diversonolic ester obtained from deep-sea fungus Penicillium spp. SCSIO
06720 from the Indian Ocean showed moderate antibacterial activity against
S. aureus ATCC 29213 and methicillin-resistant S. aureus-shh-1 (Guo et al.
2020). An Antarctic strain of Geomyces pannorum has been associated with
non-enzymatic antioxidant response and production of phenolic compounds
(Maggi et al. 2013). Rugulosin and skyrin (bis-anthraquinones) from Penicil-
lium chrysogenum isolated from a saline lake in Vestfold Hills were to electively
inhibit S. aureus,Enterococcus faecium, and E. coli (Brunati et al. 2009).
Aspergillus sydowii,Penicillium allii-sativi,Penicillium brevicompactum,
P. chrysogenum, and Penicillium rubens were shown to produce antibacterial,
antifungal, anticancer, herbicidal, and antiprotozoal compounds. Extracts from
P. allii-sativi,P. brevicompactum, and P. chrysogenum exhibited a high
antiviral activity against dengue virus 2 and antiprotozoal activity against
Trypanosoma cruzi; strong antifungal activity against the phytopathogen
Colletotrichum gloeosporioides as well as herbicidal activity (Godinho et al.
2015). P. chrysogenum obtained from the endemic Antarctic macroalga
Palmaria decipiens revealed to possess selective antifungal and trypanocidal
activities (Godinho et al. 2015). Penicillium tardochrysogenum isolated from
McMurdo Dry Valley in Antarctica produces penicillins, secalonic acids D
and F, asperentins, and the uncharacterized extrolite met Ø (Houbraken et al.
2012). The extracts from the endophytic fungi belonging to the genera
Alternaria,Antactomyces,Cadophora,Davidiella,Hegardia,Herpotrichia,
Microdochium,Oculimacula,Phaeosphaerica isolated from plants
Deschampsia antarctica and Colobanthus quitensis collected in Antarctica
showed leishmanicidal activity and a specific cytotoxic activity was found for
Microdochium phragmitis extract on UACC-62 cell line (Santiago et al. 2012).
The chemical structures of different bioactive compounds obtained from
marine fungi are given in Figs. 21.4,21.5,21.6,21.7,21.8, and 21.9.
21 Extremophilic Fungi as a Source of Bioactive Molecules 501

Fig. 21.4 Chemical structures of N-containing compounds obtained from marine fungi (Arifeen
et al. 2020)
502 A. Das et al.

21.2.4.1 N-Containing Compounds
The majority of bioactive compounds from marine fungi have been isolated from
two fungal genera, i.e., Penicillium (41.2% of compounds) and Aspergillus (28.1%
of compounds). Some noteworthy novel alkaloids from deep-sea Penicillium spp.
include brevicompanines D–H and cyclopiamide B–J which exhibited cytotoxicity
against BV2 cells, brine shrimp, SMMC-7721, and BEL-7402 cell lines (Cardoso-
Martínez et al. 2015; Fredimoses et al. 2015; Hu et al. 2019). The novel alkaloids
from deep-sea Aspergillus spp. include brevianamide R, circumdatin G,
circumdatin F, oximoaspergillimide, neohydroxyaspergillic acid, and neoaspergillic
acid, of which the first one and the last two exhibited antibiotic activities against
BCG, B. subtilis,S. aureus,P. aeruginosa,B. cereus,Klebsiella pneumonia, and
E. coli. Varioxepine A and Neoechinulin A extracted from fungi other than Penicil-
lium or Aspergillus showed antimicrobial activity against E. coli,Aeromonas
hydrophila,Micrococcus luteus,S. aureus,Vibrio anguillarum,Vibrio harveyi,
Fig. 21.4 (continued)
21 Extremophilic Fungi as a Source of Bioactive Molecules 503

Fig. 21.5 Chemical structure of polyketide compounds obtained from marine fungi (Arifeen et al.
2020)
504 A. Das et al.

and Vibrio parahaemolyticus and cytotoxic activity against HeLa cells, respectively
(Li et al. 2011; Wijesekara et al. 2013; Zhang et al. 2014,2018; Xu et al. 2015a,b).
21.2.4.2 Polyketide Compounds
Some noteworthy polyketide compounds from deep-sea Penicillium spp. include
methylisoverrucosidinol, showing antimicrobial activity against B. subtilis and
penilactone A showing NF-κβ inhibition activity (Wu et al. 2012; Pan et al. 2016).
Aspiketolactonol, aspilactonol A–F, aspyronol, and epiaspinonediol obtained from
Aspergillus sp. 16-02-1 exhibited cytotoxicity against various cancer cell lines, viz.
K562, HL-60, HeLa, and BGC-823 (Chen et al. 2014a,b). Ascomycotin A and
diorcinol from Ascomycota sp. Ind19F07 and lindgomycin and ascocetin from
Lindgomycetaceae strains KF970 and LF327 were found to exhibit strong antibiotic
activity against B. subtilis,Acenetobacter baumanii,E. coli,S. aureus,Enterococcus
faecalis,Staphylococcus epidermidis, and Propionibacterium acnes, whereas
engyodontiumone A–J obtained from Engyodontium album DFFSC021 exhibited
strong cytotoxic activity against U937 cells (Yao et al. 2014; Tian et al. 2015;Wu
et al. 2015).
Fig. 21.5 (continued)
21 Extremophilic Fungi as a Source of Bioactive Molecules 505

Fig. 21.6 Chemical structures of terpenoid compounds obtained from marine fungi (Arifeen et al.
2020)
506 A. Das et al.

Fig. 21.7 Chemical structure of polypeptide compounds obtained from marine fungi (Arifeen et al.
2020)
21 Extremophilic Fungi as a Source of Bioactive Molecules 507

21.2.4.3 Terpenoids
Brevione F–I, obtained from sediment-derived fungus Penicillium spp. has shown
cytotoxic activity against HeLa, MCF-7, and A549 cells and growth inhibition of
HIV-1 against C8116 cells (Li et al. 2012; Zhang et al. 2018). Aspewentin A,
aspewentin D–H, asperether A–E, asperoloid D–E from Aspergillus wentii, and
6b,9a-dihydroxy-14-p-nitrobenzoylcinnamolide and insulicolide A from Aspergillus
ochraceus Jcma1F17 showed antimicrobial, anti-inflammatory, antiviral, and cyto-
toxic activities (Fang et al. 2014; Li et al. 2016a,b,c). Sesquiterpenoids (sydonol
and its derivatives) obtained from Aspergillus sydowii have shown anti-
inflammatory and anti-diabetic activities (Chung et al. 2013).
Fig. 21.7 (continued)
508 A. Das et al.

Fig. 21.8 Chemical structures of esters and phenolic compounds obtained from marine fungi
(Arifeen et al. 2020)
21 Extremophilic Fungi as a Source of Bioactive Molecules 509

Fig. 21.9 Chemical structure of piperazine and other compounds obtained from marine fungi
(Arifeen et al. 2020)
510 A. Das et al.

21.2.4.4 Polypeptides
Canescenin A and canescenin B obtained from Penicillium canescens SCSIO z053
revealed antibacterial activities against Bacillus amyloliquefaciens and
P. aeruginosa (Dasanayaka et al. 2020); clavatustide A and clavatustide B from
Aspergillus clavatus C2WU and aspergillamide C & aspergillamide D from Asper-
gillus terreus SCSIO 41008 showed cytotoxic activity against HepG2, SMMC-
7721, Bel-7402 and human glioma U87 cell lines (Jiang et al. 2013a,b; Luo et al.
2019); simplicilliumtides A–I from Simplicillium obclavatum EIODSF
020 displayed cytotoxicity against human leukemia HL-60 and K562 cell lines
(Liang et al. 2016) and asperelines A–F from Trichoderma asperellum were found
to exhibit antibacterial activity against B. amyloliquefaciens,S. aureus,
P. aeruginosa, and E. coli (Ren et al. 2009).
Fig. 21.9 (continued)
21 Extremophilic Fungi as a Source of Bioactive Molecules 511

21.2.4.5 Ester and Phenolic Compounds
The esters (7-chlorofolipastatin, folipostatin B, Unguinol, 2-chlorounginol,
2,7-dichlorounguinol, and nornidulin) extracted from Aspergillus ungui NKH-007
were found to be potent candidates for anti-atherosclerotic agents along with
showing some antibiotic and cytotoxic activities (Uchida et al. 2016). The phenolic
compounds (pestalotionol, aspergilol G–I and coccoquinone A) extracted from
Penicillium sp. Y-5-2 and Aspergillus versicolor SCSIO 41502 showed potent
antimicrobial activity against S. aureus and B. subtilis along with some anti-HSV-
1, antioxidant, and antifouling properties (Huang et al. 2017; Pan et al. 2017a).
21.2.4.6 Piperazine Derivatives
Piperazine derivatives, viz. fusaperazine F, brevianamides, and versicolorin B
obtained from deep-sea sediment-derived marine fungi of genera Penicillium,Asper-
gillus, and Dichotomomyces showed strong cytotoxicity against K562 and mouse
lymphoma cell lines and also exhibited antibacterial activity against S. aureus
(Hu et al. 2019; Liu et al. 2019).
21.2.4.7 Other Compounds
Some other secondary metabolites, viz. steroloic acid, dicitrinone B, penipacids A–
F, butanolide A obtained from deep-sea sediment-derived Penicillium spp. showed
cytotoxic activities against RKO, MCF-7, PTP1B, and A375 cancer cell lines
(Li et al. 2012,2013; Chen et al. 2014a,b; Zhou et al. 2018). Isocoumarins
(penicillisocoumarin A–D) from Penicillium and another isocoumarin,
aspergillumarin B have exhibited antibacterial activities (Pan et al. 2017b).
Wentilactones having anti-tumor activities were obtained from Aspergillus spp.
(Xu et al. 2015a,b; Pan et al. 2017a).
21.2.5 Highly Acidic Habitat Fungi
Acidophiles are the microorganisms that grow below the optimum pH of 3.0
(Johnson and Quatrini 2016).
21.2.5.1 Polyketides
Penicillium rubrum obtained from a lake of acid mine in Montana, USA has been
associated with production of various polyketides, viz. azaphilone-type polyketides,
berkazaphilones A, B, and C, berkedienoic acid, berkedienolactone, vermistatin,
dihydrovermistatin, penisimplicissin, and methylparaconic acid. Berkazaphilones B,
berkazaphilones C, and penisimplicissin were found to inhibit leukemia cancer cell
lines by inhibiting caspase-1 enzyme (Stierle et al. 2012a,b).
21.2.5.2 Terpenes
Two new drimane sequiterpene lactones, berkedrimane A and berkedrimane B and a
new carboxylic acid were obtained from Penicillium solitum, isolated from an acid
mine waste lake of Montana, USA. Berkedrimanes A and B exhibited in vitro
512 A. Das et al.

anti-inflammatory activity by inhibiting caspase-1 and caspase-3 enzymes (Stierle
et al. 2012a,b).
21.2.6 Saline and Hypersaline Habitat Fungi
A habitat having salt concentration similar to that of sea water (3.5% w/v of total
dissolved salts) can be considered as a saline habitat (Díaz-Cárdenas et al. 2017).
Hypersaline habitats are the environments having salt concentration >100 g/l
(Enache et al. 2017), viz. some lakes and biomes of Antarctic region where the
microorganisms have to sustain the osmotic pressure and low water activities (Oren
1999; Gunde-Cimerman and Zalar 2014; Waditee-Sirisattha et al. 2016).
Extremely halotolerant and halophilic fungi, viz. Aspergillus spp., Cladosporium
spp., Penicillium spp., Emericella spp., Eurotium spp., and Wallemia spp. have been
isolated from solar salterns across the world (Gunde-Cimerman and Zalar 2014).
21.2.7 High-Temperature Environments
Thermophilic (optimum growth temperature of 55 C) and hyperthermophilic (opti-
mum growth temperature of 80 C) microorganisms are often found in hot-springs
and deep-sea hydrothermal vents (Rastogi et al. 2010; Urbieta et al. 2015). The
polyamines present in hyperthermophiles are responsible for their survival at high
temperatures (Hidese et al. 2018).
21.2.7.1 Polyketides and N-Containing Compounds
Malbranpyrroles A–F, the photosensitive polyketides were discovered from the
thermophilic fungus Malbranchea sulfurea from Sihchong River Hot Spring Zone
of Taiwan. Two cyclodepsipeptides (clavatustides A and B) were obtained from the
fungus Aspergillus clavatus C2WU isolated from a crab, Xenograpsus testudinatus,
that was collected at the sulfur-rich hydrothermal vents in Taiwan. They exhibited
anticancer activity against hepatocellular carcinoma cell lines HepG2 (Jiang et al.
2013a,b). Nematicidal activity of two prenylated alkaloids (talathermophilins A and
B) obtained from the fungus Talaromyces thermophilus was observed against the
parasite Panagrellus redivevus (Chu et al. 2010).
The extracts from fungal strains isolated from the Atacama Desert, Chile have
been shown to be effective against Paracoccidioides brasiliensis Pb18, the causative
agent for South American blastomycosis. The antifungal agent cytochalasin was
obtained from organic extract of Aspergillus felis from the same area (Mendes et al.
2016).
21.2.7.2 Thermozymes
Thermozymes, the extracellular enzymes from thermophilic fungi have been
explored commercially in bioprocesses due to their intrinsic thermostability and
catalytic properties (Maheshwari et al. 2000; Fotouh et al. 2016). Different
21 Extremophilic Fungi as a Source of Bioactive Molecules 513

extracellular enzymes, viz. proteases from Achaetomium, Chaetomium, Penicillium,
Rhizopus, Torula spp. (Dhakar and Pandey 2016); a proteolytic enzyme with milk-
curdling activity at 50 C from Mucor pusillus (Nouani et al. 2009); lipase from
Humicola lanuginose strain Y-38 (Arima et al. 1972); α-amylase from Thermomyces
lanuginosus (Arnesen et al. 1998) and Myceliophthora thermophila D14 (ATCC
48104) (Sadhukhan et al. 1992); glucoamylase from T. lanuginosus (Taylor et al.
1978) and Humicola grisea var. thermoidea (Tosi et al. 1993); exocellulase,
endocellulase, and cellobiase from Thermomyces aurantiacus (Khandke et al.
1989); cellobiose dehydrogenases from Sporotrichum thermophile (Coudray et al.
1982; Schou et al. 1998) and Humicola insolens (Schou et al. 1998); xylanases from
Melanocarpus albomyces (Prabhu and Maheshwari 1999), Humicola grisea var.
thermoidea (Monti et al. 1991)andTalaromyces emersonii (Tuohy et al. 1993);
polygalacturonases (exo-TePG28a and endo-TePG28b) from Talaromyces
leycettanus JCM12802 (Li et al. 2017); laccases from Chaetomium thermophilum
(Chefetz et al. 1998); phytases (myo-inositol hexakisphosphate phosphohydrolases)
from Myceliophthora thermophila and Talaromyces thermophilus (Wyss et al. 1999)
have been isolated, purified, and characterized.
21.3 Conclusion
The growing concern over antimicrobial resistance in human and animal pathogens
has led to the search for alternative strategies to combat them. This has resulted in the
discovery of some novel bioactive substances produced by microbial species
isolated from extreme habitats. These compounds can be exploited to control
multidrug-resistant pathogens and treat chronic ailments like cancer, diabetes, epi-
lepsy, blood pressure, etc. As per the available literatures, taxonomically diverse
extremophilic and extremotolerant fungi (e.g., Penicillium spp. and Aspergillus spp.)
were the source of several novel specialized metabolites, of which the N-containing
compounds and polyketides were the most frequently isolated classes of metabolites.
It is observed that the wide range of bioactive compounds obtained from
extremophilic fungi showed several activities, but majority of them showed
antibacterial and anticancer activities. Fungal strains have been isolated from all
the extreme biomes like highly acidic habitats, saline and hypersaline habitats, high-
temperature environments, etc. Terpenoid derivatives from extremophilic fungi
showed stronger antibacterial and cytotoxic potentials and have the possibility of
being future candidates for anticancer drug development.
The major challenges in growth, production, and extraction of extremophilic
fungi in laboratory scale are the difficulty in achieving the optimum conditions for
growth and high cost involvement. There remains a huge difficulty in producing
enough biomass for large scale industrial production. The knowledge on
extremophiles is increasing but is still insufficient considering the hugeness in
diversity of compounds and variety of organisms present. More research in this
area can lead to identification of novel bioactive compounds of clinical importance.
514 A. Das et al.

In general, extremophilic fungi can be considered as “chemical factory.”Hence, it
can be presumed that the extremophilic fungi as source of bioactive compounds
could be the new arsenal for modern science.
References
Anwar UB, Zwar IP, de Souza AO (2020) Biomolecules produced by extremophiles
microorganisms and recent discoveries. In: Rodrigues AG (ed) New and future developments
in microbial biotechnology and bioengineering. Microbial biomolecules: properties, relevance,
and their translational applications. Elsevier, Amsterdam, pp 247–270
Arifeen MZ, Ma YN, Xue YR, Liu CH (2020) Deep-sea fungi could be the new arsenal for
bioactive molecules. Mar Drugs 18(9):1–15
Arima K, Liu W-H, Beppu T (1972) Studies on the lipase of thermophilic fungus Humicola
lanuginose. Agric Biol Chem 36:893–895
Arnesen S, Eriksen SH, Olsen J, Jensen B (1998) Increased production of α-amylase from
Thermomyces lanuginosus by the addition of Tween 80. Enzym Microb Technol 23:249–252
Bakermans C, Bergholz PW, Ayala-del-Río H, Tiedje J (2009) Genomic insights into cold adapta-
tion of permafrost bacteria. In: Margesin R (ed) Permafrost soils. Springer, Berlin, pp 159–168
Barone G, Varrella S, Tangherlini M, Rastelli E, Dell’Anno A, Danovaro R, Corinaldesi C (2019)
Marine fungi: biotechnological perspectives from deep-hypersaline anoxic basins. Diversity 11:
113
Blanchette RA, Held BW, Arenz BE, Jurgens JA, Baltes NJ, Duncan SM, Farrell RL (2010) An
Antarctic hot spot for fungi at Shackleton’s historic hut on Cape Royds. Microb Ecol 60:29–38
Botíc T, KunčičMK, Sepčíc K, Knez Z, Gunde-Cimerman N (2012) Salt induces biosynthesis of
hemolytically active compounds in the xerotolerant food-borne fungus Wallemia sebi. FEMS
Microbiol Lett 326:40–46
Bowman JP (2008) Genomic analysis of psychrophilic prokaryotes. In: Margesin R, Schinner F,
Marx J-C, Gerday C (eds) Psychrophiles: from biodiversity to biotechnology. Springer, Berlin,
pp 265–284
Bridge PD, Spooner BM (2012) Non-lichenized Antarctic fungi: transient visitors or members of a
cryptic ecosystem. Fungal Ecol 5:381–394
Brunati M, Rojas JL, Sponga F, Ciciliato I, Losi D, Göttlich E, de Hoog S, Genilloud O, Marinelli F
(2009) Diversity and pharmaceutical screening of fungi from benthic mats of Antarctic lakes.
Mar Genomics 2:43–50
Bull AT, Goodfellow M (2019) Dark, rare and inspirational microbial matter in the
extremobiosphere: 16000 meters of bioprospecting campaigns. Microbiology (Reading) 165:
1252–1264
Bull AT, Asenjo JA, Goodfellow M, Gomez-Silva B (2016) The Atacama Desert: technical
resources and the growing importance of novel microbial diversity. Ann Rev Microbiol 70:
215–234
Bull AT, Andrews BA, Dorador C, Goodfellow M (2018) Microbiology of the Atacama Desert.
Antonie Van Leeuwenhoek 111:1269–1491
Burgaud G, Hue’NT AD, Coton M, Perrier-Cornet JM, Jebbar M, Barbier G (2015) Effects of
hydrostatic pressure on yeasts isolated from deep-sea hydrothermal vents. Res Microbiol 166:
700–709
Cardoso-Martínez F, de la Rosa JM, Díaz-Marrero AR, Darias J, D’Croz L, Cerella C, Diederich M,
Cueto M (2015) Oximoaspergillimide, a fungal derivative from a marine isolate of Aspergillus
sp. Eur J Org Chem 2015:2256–2261
Castillo RV, Beck A (2012) Photobiont selectivity and specificity in Caloplaca species in a
fog-induced community in the Atacama Desert, northern Chile. Fungal Biol 116:665–676
21 Extremophilic Fungi as a Source of Bioactive Molecules 515

Chattopadhyay MK (2006) Mechanism of bacterial adaptation to low temperature. J Biosci 31:157–
165
Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium
thermophilum and its role in humification. Appl Environ Microbiol 64:3175–3179
Chen L, Gong M-W, Peng Z-F, Zhou T, Ying M-G, Zheng Q-H, Liu Q-Y, Zhang Q-Q (2014a) The
marine fungal metabolite, dicitrinone B, induces A375 cell apoptosis through the ROS-related
caspase pathway. Mar Drugs 12:1939–1958
Chen X-W, Li C-W, Cui C-B, Hua W, Zhu T-J, Gu Q-Q (2014b) Nine new and five known
polyketides derived from a deep sea-sourced Aspergillus sp. 16-02-1. Mar Drugs 12:3116–3137
Chu YS, Niu XM, Wang YL, Guo JP, Pan WZ, Huang XW, Zhang KQ (2010) Isolation of putative
biosynthetic intermediates of prenylated indole alkaloids from a thermophilic fungus
Talaromyces thermophilus. Org Lett 12:4356–4359
Chung Y-M, Wei C-K, Chuang D-W, El-Shazly M, Hsieh C-T, Asai T, Oshima Y, Hsieh T-J,
Hwang T-L, Wu Y-C (2013) An epigenetic modifier enhances the production of anti-diabetic
and anti-inflammatory sesquiterpenoids from Aspergillus sydowii. Bioorg Med Chem 21:3866–
3872
Conley CA, Ishkhanova G, Mckay CP, Cullings K (2006) A preliminary survey of non-lichenized
fungi cultured from the hyperarid Atacama Desert of Chile. Astrobiology 6:521–526
Coudray M-R, Canevascini G, Meier H (1982) Characterization of a cellobiose dehydrogenase in
the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile. Biochem J 203:277–284
D’Amico S, Collins T, Marx JC, Feller G, Gerday C (2006) Psychrophilic microorganisms:
challenges for life. EMBO Rep 7:385–389
Danovaro R, Corinaldesi C, Dell’Anno A, Snelgrove PV (2017) The deep-sea under global change.
Curr Biol 27:R461–R465
Dasanayaka S, Nong X-H, Liang X, Liang J-Q, Amin M, Qi S-H (2020) New dibenzodioxocinone
and pyran-3, 5-dione derivatives from the deep-sea-derived fungus Penicillium canescens
SCSIO z053. J Asian Nat Prod Res 22:338–345
Deshmukh SK, Prakash V, Ranjan N (2018) Marine fungi: a source of potential anticancer
compounds. Front Microbiol 8:25–36
Desroches TC, McMullin DR, Miller JD (2014) Extrolites of Wallemia sebi, a very common fungus
in the built environment. Indoor Air 24:533–542
Dhakar K, Pandey A (2016) Wide pH range tolerance in extremophiles: towards understanding an
important phenomenon for future biotechnology. Appl Microbiol Biotechnol 100:2499–2510
Díaz-Cárdenas C, Cantillo A, Rojas LY, Sandoval T, Fiorentino S, Robles J, Ramos FA, Zambrano
MM, Baena S (2017) Microbial diversity of saline environments: searching for cytotoxic
activities. AMB Express 7:223
Du L, Zhu T, Fang Y, Gu Q, Zhu W (2008) Unusual C25 steroid isomers with bicyclo [4.4.1]A/B
rings from a volcano ash-derived fungus Penicillium citrinum. J Nat Prod 71:1343–1351
Elleuche S, Schäfers C, Blank S, Schröder C, Antranikian G (2015) Exploration of extremophiles
for high temperature biotechnological processes. Curr Opin Microbiol 25:113–119
Enache M, Teodosiu G, Itoh T, Kamekura M, Stan-Lotter H (2017) Halophilic microorganisms
from manmade and natural hypersaline environments: physiology, ecology, and biotechnologi-
cal potential. In: Stan-Lotter H, Fendrihan S (eds) Adaption of microbial life to environmental
extremes. Springer, Berlin, pp 201–226
Fang W, Lin X, Zhou X, Wan J, Lu X, Yang B, Ai W, Lin J, Zhang T, Tu Z (2014) Cytotoxic and
antiviral nitrobenzoyl sesquiterpenoids from the marine-derived fungus Aspergillus ochraceus
Jcma1F17. Med Chem Comm 5:701–705
Figueroa L, Jiménez C, Rodríguez J, Areche C, Chávez R, Henríquez M, de laCruz M, Díaz C,
Segade Y, Vaca I (2015) 3-Nitroasterric acid derivatives from an Antarctic sponge derived
Pseudogymnoascus sp. fungus. J Nat Prod 78:919–923
Fotouh DMA, Bayoumi RA, Hassan MA (2016) Production of thermoalkaliphilic lipase from
Geobacillus thermoleovorans DA2 and application in leather industry. Enzyme Res 2016:
9034364
516 A. Das et al.

Fredimoses M, Zhou X, Ai W, Tian X, Yang B, Lin X, Xian J-Y, Liu Y (2015) Westerdijkin A, a
new hydroxyphenylacetic acid derivative from deep sea fungus Aspergillus westerdijkiae
SCSIO 05233. Nat Prod Res 29:158–162
Gocheva YG, Tosi S, Krumova ET (2009) Temperature downshift induces antioxidant response in
fungi isolated from Antarctica. Extremophiles 13:273–281
Godinho VM, Gonçalves VN, Santiago IF, Figueredo HM, Vitoreli GA, Schaefer CEGR, Barbosa
EC, Oliveira JG, Alves TMA, Zani CL, Junior PAS, Murta SMF, Romanha AJ, Kroon EG,
Cantrell CL, Wedge DE, Duke SO, Ali A, Rosa CA, Rosa LH (2015) Diversity and
bioprospection of fungal community present in oligotrophic soil of continental Antarctica.
Extremophiles 19:585–596
Gonçalves VN, Cantrell CL, Wedge DE, Ferreira MC, Soares MA, Jacob MR, Oliveira FS,
Galante D, Rodrigues F, Alves TMA, Zani CL, Junior PAS, Murta S, Romanha AJ, Barbosa
EC, Kroon EG, Oliveira JG, Gomez-Silva B, Galetovic A, Rosa CA, Rosa LH (2016) Fungi
associated with rocks of the Atacama Desert: taxonomy, distribution, diversity, ecology and
bioprospection for bioactive compounds. Environ Microbiol 18:232–245
Gonçalves VN, Vitoreli GA, de Menezes GC, Mendes CR, Secchi ER, Rosa CA, Rosa LH (2017)
Taxonomy, phylogeny and ecology of cultivable fungi present in seawater gradients across the
Northern Antarctica Peninsula. Extremophiles 21:1005–1015
Grishkan I, Nevo E (2010) Spatiotemporal distribution of soil microfungi in the Makhtesh Ramon
area, Central Negev desert, Israel. Fungal Ecol 3:326–337
Gunde-Cimerman N, Zalar P (2014) Extremely halotolerant and halophilic fungi inhabit brine in
solar salterns around the globe. Food Technol Biotechnol 52:170–179
Gunde-Cimerman N, Ramos J, Plemenitǎs A (2009) Halotolerant and halophilic fungi. Mycol Res
113:1231–1241
Guo C, Lin X-P, Liao S-R, Yang B, Zhou X-F, Yang X-W, Tian X-P, Wang J-F, Liu Y-H (2020)
Two new aromatic polyketides from a deep-sea fungus Penicillium sp. SCSIO 06720. Nat Prod
Res 8:1–9
Gupta GN, Srivastava S, Khare SK, Prakash V (2014) Extremophiles: an overview of microorgan-
ism from extreme environment. Int J Agric Environ Biotechnol 7:371–380
Hidese R, Fukuda W, Niitsu M, Fujiwara S (2018) Identification of branched-chain polyamines in
hyperthermophiles. In: Alcázar R, Tiburcio AF (eds) Polyamines. Humana Press, New York, pp
81–94
Houbraken J, Frisvad JC, Seifert KA, Overy DP, Tuthill DM, Valdez JG, Samson RA (2012) New
penicillin-producing Penicillium species, an overview of section Chrysogena. Persoonia 29:78–
100
Hu J, Li Z, Gao J, He H, Dai H, Xia X, Liu C, Zhang L, Song F (2019) New diketopiperazines from
a marine-derived fungus strain Aspergillus versicolor MF180151. Mar Drugs 17:262
Huang Z, Nong X, Ren Z, Wang J, Zhang X, Qi S (2017) Anti-HSV-1, antioxidant and antifouling
phenolic compounds from the deep-sea-derived fungus Aspergillus versicolor. SCSIO 41502.
Bioorg Med Chem Lett 27:787–791
JančičS, Frisvad JC, Kocev D, Gostiňcar C, Ďzeroski S, Gunde-Cimerman N (2016) Production of
secondary metabolites in extreme environments: food-airborne Wallemia spp. produce toxic
metabolites at hypersaline conditions. PLoS One 11:1–20
Jansson JK, Tas N (2014) The microbial ecology of permafrost. Nat Rev Microbiol 12:414–425
Jiang P, Du W, Mancuso A, Wellen KE, Yang X (2013a) Reciprocal regulation of p53, malic
enzymes modulates metabolism senescence. Nature 493:689–693
Jiang W, Ye P, Chen C-T, Wang K, Liu P, He S, Wu X, Gan L, Ye Y, Wu B (2013b) Two novel
hepatocellular carcinoma cycle inhibitory cyclodepsipeptides from a hydrothermal vent crab-
associated fungus Aspergillus clavatus C2WU. Mar Drugs 11:4761–4772
Johnson DB, Quatrini R (2016) Acidophile microbiology in space and time. In: Quatrini R, Johnson
B (eds) Acidophiles: life in extremely acidic environments. Caister Academic Press, Norfolk, pp
3–16
21 Extremophilic Fungi as a Source of Bioactive Molecules 517

Khandke KM, Vithayathil PJ, Murthy SK (1989) Purification, characterization of an α-D-glucuron-
idase from a thermophilic fungus, Thermoascus aurantiacus. Arch Biochem Biophys 274:511–
517
Kong X, Cai S, Zhu T-J, Luan Y (2014) Secondary metabolites of a deep sea derived fungus
Aspergillus versicolor CXCTD-06-6a and their bioactivity. J Ocean 13:691–695
Kurapova I, Zenova GM, Sudnitsyn II, Kizilova AK, Manucharova NA, Norovsuren ZH,
Zvyagintsev DG (2012) Thermotolerant and thermophilic actinomycetes from soils of Mongolia
Desert Steppe Zone. Microbiology 81:98–108
Lentzen G, Schwarz T (2006) Extremolytes: natural compounds from extremophiles for versatile
applications. Appl Microbiol Biotechnol 72:623–634
Li D, Wang F, Cai S, Zeng X, Xiao X, Gu Q, Zhu W (2007a) Two new bisorbicillinoids isolated
from a deep-sea fungus, Phialocephala sp, FL30r. J Antibiot 60:317–320
Li D, Wang F, Xiao X, Fang Y, Zhu T, Gu Q, Zhu W (2007b) Trisorbicillinone A, a novel
sorbicillin trimer from a deep sea fungus, Phialocephala sp. FL30r. Tetrahedron Lett 48:5235–
5238
Li C-S, An C-Y, Li X-M, Gao S-S, Cui C-M, Sun H-F, Wang B-G (2011) Triazole and
dihydroimidazole alkaloids from the marine sediment-derived fungus Penicillium paneum
SD-44. J Nat Prod 74:1331–1334
Li Y, Ye D, Shao Z, Cui C, Che Y (2012) A sterol and spiroditerpenoids from a Penicillium
sp. isolated from a deep sea sediment sample. Mar Drugs 10:497–508
Li C-S, Li X-M, Gao S-S, Lu Y-H, Wang B-G (2013) Cytotoxic anthranilic acid derivatives from
deep sea sediment-derived fungus Penicillium paneum SD-44. Mar Drugs 11:3068–3076
Li X, Li X-M, Li X-D, Xu G-M, Liu Y, Wang B-G (2016a) 20-Nor-isopimarane cycloethers from
the deep-sea sediment-derived fungus Aspergillus wentii SD-310. RSC Adv 6:75981–75987
Li X-D, Li X, Li X-M, Xu G-M, Zhang P, Meng L-H, Wang B-G (2016b) Tetranorlabdane
diterpenoids from the deep sea sediment-derived fungus Aspergillus wentii SD-310. Planta
Med 82:877–881
Li X-D, Li X-M, Li X, Xu G-M, Liu Y, Wang B-G (2016c) Aspewentins D-H, 20-nor-isopimarane
derivatives from the deep sea sediment-derived fungus Aspergillus wentii SD-310. J Nat Prod
79:1347–1353
Li Y, Wang Y, Tu T, Zhang D, Ma R, You S, Wang X, Yao B, Luo H, Xu B (2017) Two acidic,
thermophilic GH28 polygalacturonases from Talaromyces leycettanus JCM 12802 with appli-
cation potentials for grape juice clarification. Food Chem 237:997–1003
Liang X, Zhang X-Y, Nong X-H, Wang J, Huang Z-H, Qi S-H (2016) Eight linear peptides from the
deep-sea-derived fungus Simplicillium obclavatum EIODSF 020. Tetrahedron 72:3092–3097
Lin A, Wu G, Gu Q, Zhu T, Li D (2014) New eremophilane-type sesquiterpenes from an Antarctic
deepsea derived fungus Penicillium sp. PR19N-1. Arch Pharm Res 37:839–844
Liu F, Damm U, Cai L, Crous PW (2013) Species of the Colletotrichum gloeosporioides complex
associated with anthracnose diseases of Proteaceae. Fungal Divers 61:89–105
Liu JT, Hu B, Gao Y, Zhang JP, Jiao BH, Lu XL, Liu XY (2014) Bioactive tyrosine-derived
cytochalasins from fungus Eutypella sp. D-1. Chem Biodivers 11:800–806
Liu C-C, Zhang Z-Z, Feng Y-Y, Gu Q-Q, Li D-H, Zhu T-J (2019) Secondary metabolites from
Antarctic marine-derived fungus Penicillium crustosum HDN153086. Nat Prod Res 33:414–
419
Lu XL, Liu JT, Liu XY, Gao Y, Zhang J, Jiao B-H, Zheng H (2014) Pimarane diterpenes from the
Arctic fungus Eutypella sp. D-1. J Antibiot 67:171–174
Luo XW, Yun L, Liu YJ, Zhou XF, Liu YH (2019) Peptides and polyketides isolated from the
marine sponge-derived fungus Aspergillus terreus SCSIO 41008. Chin J Nat Med 17:149–154
Maggi O, Tosi S, Angelova M, Lagostina E, Fabbri AA, Pecoraro L, Altobelli E, Picco AM,
Savino E, Branda E, Turchetti B, Zotti M, Vizzini A, Buzzini P (2013) Adaptation of fungi,
including yeasts, to cold environments. Plant Biosyst 147:247–258
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol 64:461–488
518 A. Das et al.

Martinelli L, Zalar P, Gunde-Cimerman N, Azua-Bustos A, Sterflinger K, Piñar G (2017) Asper-
gillus atacamensis and A. salisburgensis: two new halophilic species from hypersaline/arid
habitats with a phialosimplex-like morphology. Extremophiles 21:755–773
Mendes G, Gonçalves VN, Souza-Fagundes EM, Kohlhoff M, Rosa CA, Zani CL, Cota BB, Rosa
LH, Johann S (2016) Antifungal activity of extracts from Atacama Desert fungi against
Paracoccidioides brasiliensis, identification of Aspergillus felis as a promising source of natural
bioactive compounds. Mem Inst Oswaldo Cruz 111:209–217
Monti R, Terenzi HF, Jorge JA (1991) Purification, properties of an extracellular xylanase from the
thermophilic fungus Humicola grisea var. thermoidea. Can J Microbiol 37:675–681
Murgia M, Fiamma M, Barac A, Deligios M, Mazzarello V, Paglietti B, Cappuccinelli P,
Al-Qahtani A, Squartini A, Rubino S, Al-Ahdal MN (2018) Biodiversity of fungi in hot desert
sands. Microbiol Open 8:e00595
Nagano Y, Nagahama T, Abe F (2013) Cold-adapted yeasts in deep-sea environments. In:
Buzzini P, Margesin R (eds) Cold-adapted yeasts. Springer, Berlin, pp 149–171
Nouani A, Belhamiche N, Slamani R, Belbraouet S, Fazouane F, Bellal MM (2009) Extracellular
protease from Mucor pusillus: purification and characterization. Int J Dairy Technol 62:112–117
Ondeyka JG, Smith SK, Zink DL, Vicente F, Basilio A, Bills GF, Polishook JD, Garlisi C,
Mcguinness D, Smith E, Qiu H, Gill CJ, Donald RG, Phillips JW, Goetz MA, Singh SB
(2014) Isolation, structure elucidation and antibacterial activity of a new tetramic acid,
ascosetin. J Antibiot 67:527–531
Onofri S, Selbmann L, De Hoog GS, Grube M, Barreca D, Ruisi S, Zucconi L (2007) Evolution and
adaptation of fungi at boundaries of life. Adv Space Res 40:1657–1664
Oren A (1999) Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev 63:334–348
Ouchari L, Boukeskasse A, Bouizgarne B, Ouhdouch Y (2018) Antimicrobial potential of
actinomycetes isolated from unexplored hot Merzouga desert and their taxonomic diversity.
Biol Open 8:bio035410
Ozerskaya S, Kochkina G, Ivanushkina N, Gilichinsky DA (2009) Fungi in permafrost. In:
Margesin R (ed) Permafrost soils, Soil biology, vol 16. Springer, Berlin, pp 85–95
Pan C, Shi Y, Auckloo B, Chen X, Chen C-T, Tao X, Wu B (2016) An unusual conformational
isomer of verrucosidin backbone from a hydrothermal vent fungus, Penicillium sp. Y-50-10.
Mar Drugs 14:156
Pan C, Shi Y, Chen X, Chen C-TA, Tao X, Wu B (2017a) New compounds from a hydrothermal
vent crab-associated fungus Aspergillus versicolor XZ-4. Org Biomol Chem 15:1155–1163
Pan C, Shi Y, Auckloo BN, ul Hassan SS, Akhter N, Wang K, Ye Y, Chen C-TA, Tao X, Wu B
(2017b) Isolation and antibiotic screening of fungi from a hydrothermal vent site and character-
ization of secondary metabolites from a Penicillium isolate. Mar Biotechnol 19:469–479
Park YC, Gunasekera SP, Lopez JV, McCarthy PJ, Wright AE (2006) Metabolites from the marine-
derived fungus Chromocleista sp. Isolated from a deep-water sediment sample collected in the
Gulf of Mexico. J Nat Prod 69:580–584
Park CH, Kim KM, Elvebakk A, Kim OS, Jeong G, Hong SG (2015) Algal and fungal diversity in
Antarctic lichens. J Eukaryot Microbiol 62:196–205
Peng XP, Wang Y, Liu PP, Hong K, Chen H, Yin X, Zhu W-M (2011) Aromatic compounds from
the halotolerant fungal strain of Wallemia sebi PXP-89 in a hypersaline medium. Arch Pharm
Res 34:907–912
Peng J, Zhang XY, Tu ZC, Xu XY, Qi SH (2013) Alkaloids from the deep-sea-derived fungus
Aspergillus westerdijkiae DFFSCS013. J Nat Prod 76:983–987
Pettit RK (2011) Culturability and secondary metabolite diversity of extreme microbes: expanding
contribution of deep sea, deep-sea vent microbes to natural product discovery. Mar Biotechnol
13:1–11
Plemenitǎs A, Vaupotiˇc T, Lenassi M, Kogej T, Gunde-Cimerman N (2008) Adaptation of
extremely halotolerant black yeast Hortaea werneckii to increased osmolarity: a molecular
perspective at a glance. Stud Mycol 61:67–75
21 Extremophilic Fungi as a Source of Bioactive Molecules 519

Prabhu KA, Maheshwari R (1999) Biochemical properties of xylanases from a thermophilic fungus,
Melanocarpus albomyces, their action on plant cell walls. J Biosci 2:461–470
Prista C, Almagro A, Loureiro-Dias MC, Ramos J (1997) Physiological basis for the high salt
tolerance of Debaryomyces hansenii. Appl Environ Microbiol 63:4005–4009
Rastogi G, Bhalla A, Adhikari A, Bischoff KM, Hughes SR, Christopher LP, Sani RK (2010)
Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains.
Bioresour Technol 101:8798–8806
Ren J, Xue C, Tian L, Xu M, Chen J, Deng Z, Proksch P, Lin W (2009) Asperelines A-F, peptaibols
from the marine-derived fungus Trichoderma asperellum. J Nat Prod 72:1036–1044
Rogozhin EA, Sadykova VS, Baranova AA, Vasilchenko AS, Lushpa VA, Mineev KS, Georgieva
ML, Kul’ko AB, Krasheninnikov ME, Lyundup AV, Vasilchenko AV, Andreev YA (2018) A
novel lipopeptaibol emericellipsin a with antimicrobial and antitumor activity produced by the
extremophilic fungus Emericellopsis alkaline. Molecules 23(11):2785
Sadhukhan R, Roy SK, Raha SK, Manna S, Chakrabarty SL (1992) Induction, regulation of
α-amylase synthesis in a cellulolytic thermophilic fungus Myceliophthora thermophila D14
(ATCC 48104). Indian J Exp Biol 30:482–486
Santiago IF, Alves TM, Rabello A, Sales-Júnior PA, Romanha AJ, Zani CL, Rosa AC, Rosa LH
(2012) Leishmanicidal and antitumoral activities of endophytic fungi associated with the
Antarctic angiosperms Deschampsia antarctica desv. and Colobanthus quitensis (kunth) bartl.
Extremophiles 16:95–103
Santiago IF, Gonçalves VN, Gomez-Silva B, Galetovic A, Rosa LH (2018) Fungal diversity in the
Atacama Desert. Antonie Van Leeuwenhoek 111:1345–1360
Sayed AM, Hassan MHA, Alhadrami HA, Hassan HM, Goodfellow M, Rateb ME (2019) Extreme
environments: microbiology leading to specialized metabolites. J Appl Microbiol 128:630–657
Schou C, Christensen MH, Schülein M (1998) Characterization of a cellobiose dehydrogenase from
Humicola insolens. Biochem J 330:565–571
Singh P, Raghukumar C, Verma P, Shouche Y (2010) Phylogenetic diversity of culturable fungi
from the deepsea sediments of the Central Indian Basin and their growth characteristics. Fungal
Diver 40:89–102
Singh P, Raghukumar C, Meena RM, Verma P, Shouche Y (2012) Fungal diversity in deep-sea
sediments revealed by culture-dependent and culture-independent approaches. Fungal Ecol 5:
543–553
Sterflinger K, Tesei D, Zakharova K (2012) Fungi in hot and cold deserts with particular reference
to microcolonial fungi. Fungal Ecol 5:453–462
Stevenson A, Hamill PG, O’kane CJ, Kminek G, Rummel JD, Voytek MA, Dijksterhuis J,
Hallsworth JE (2017) Aspergillus penicillioides differentiation and cell division at 0.585
water activity. Environ Microbiol 19:687–697
Stierle AA, Stierle DB (2014) Bioactive secondary metabolites from acid mine waste
extremophiles. Nat Prod 9:1037–1044
Stierle DB, Stierle AA, Patacini B, Mcintyre K, Girtsman T, Bolstad E (2011) Berkeleyones and
related meroterpenes from a deep water acid mine waste fungus that inhibit the production of
interleukin 1-βfrom induced inflammasomes. J Nat Prod 74:2273–2277
Stierle AA, Stierle DB, Girtsman T (2012a) Caspase-1 inhibitors from an extremophilic fungus that
target specific leukemia cell lines. J Nat Prod 75:344–350
Stierle DB, Stierle AA, Girtsman T, McIntyre K, Nichols J (2012b) Caspase-1 and-3 inhibiting
drimane sesquiterpenoids from the extremophilic fungus Penicillium solitum. J Nat Prod 75:
262–266
Symanczik S, Błaszkowski J, Chwat G, Boller T, Wiemken A, Al-Yahyaei N, Al-Yahya’ei M
(2014) Three new species of arbuscular mycorrhizal fungi discovered at one location in a desert
of Oman: Diversispora omaniana,Septoglomus nakheelum and Rhizophagus arabicus. Mycol-
ogy 106:243–259
Takahashi C, Numata A, Matsumura E, Minoura K, Eto H, Shingu T, Ito T, Hasegawa T (1994)
Leptosins I, J: cytotoxic substances produced by a Leptosphaeria sp. J Antibiot 47:1242–1249
520 A. Das et al.

Taylor PM, Napier EJ, Fleming ID (1978) Some properties of glucoamylase produced by the
thermophilic fungus Humicola lanuginose. Carbohydr Res 61:301–308
Taylor-George S, Palmer F, Staley JT, Borns DJ, Curtiss B, Adams JB (1983) Fungi and bacteria
involved in desert varnish formation. Microb Ecol 9:227–245
Tian Y-Q, Lin X-P, Liu J, Kaliyaperumal K, Ai W, Ju Z-R, Yang B, Wang J, Yang X-W, Liu Y
(2015) Ascomycotin A, a new citromycetin analogue produced by Ascomycota sp. Ind19F07
isolated from deep sea sediment. Nat Prod Res 29:820–826
Tiwari K, Upadhyay DJ, Mosker E, Sussmuth R, Gupta RK (2015) Culturable bioactive
actinomycetes from the Great Indian Thar Desert. Ann Microbiol 65:1901–1914
Tosi LRO, Terenzi HF, Jorge JA (1993) Purification, characterization of an extracellular
glucoamylase from the thermophilic fungus Humicola grisea var. thermoidea. Can J Microbiol
39:846–852
Tuohy MG, Puls J, Claeyssens M, VřsanskáM, Coughlan MP (1993) The xylan-degrading enzyme
system of Talaromyces emersonii: novel enzymes with activity against aryl β-D-xylosides and
unsubstituted xylans. Biochem J 290:515–523
Uchida R, Nakajyo K, Kobayashi K, Ohshiro T, Terahara T, Imada C, Tomoda H (2016)
7-Chlorofolipastatin, an inhibitor of sterol O-acyltransferase, produced by marine-derived
Aspergillus ungui NKH-007. J Antibiot 69:647–651
Unell M, Kabelitz N, Jansson JK, Heipieper HJ (2007) Adaptation of the psychrotroph
Arthrobacter chlorophenolicus A6 to growth temperature and the presence of phenols by
changes in the anteiso/iso ratio of branched fatty acids. FEMS Microbiol Lett 266:138–143
Urbieta MS, Donati ER, Chan KG, Shahar S, Sin LL, Goh KM (2015) Thermophiles in the
genomic era: biodiversity, science, and applications. Biotechnol Adv 33:633–647
Waditee-Sirisattha R, Kageyama H, Takabe T (2016) Halophilic microorganism resources and their
applications in industrial and environmental biotechnology. AIMS Microbiol 2:42–54
Wang W, Tao H, Wang Y, Liu P, Zhu W, Peng X (2009) Cerebrosides of the halotolerant fungus
Alternaria raphani isolated from a sea salt field. J Nat Prod 72:1695–1698
Wang YT, Xue YR, Liu CH (2015) A brief review of bioactive metabolites derived from deep-sea
fungi. Mar Drugs 13:4594–4616
Wang J, Wei X, Qin X, Tian X, Liao L, Li K, Zhou X, Yang X, Wang F, Zhang T, Tu Z, Chen B,
Liu Y (2016) Antiviral merosesquiterpenoids produced by the Antarctic fungus Aspergillus
ochraceopetaliformis SCSIO 05702. J Nat Prod 79:59–65
Wijesekara I, Li Y-X, Vo T-S, Van TQ, Ngo D-H, Kim S-K (2013) Induction of apoptosis in human
cervical carcinoma HeLa cells by neoechinulin A from marine-derived fungus Microsporum
sp. Process Biochem 48:68–72
Wu G, Ma H, Zhu T, Li J, Gu Q, Li D (2012) Penilactones A and B, two novel polyketides from
Antarctic deep-sea derived fungus Penicillium crustosum PRB-2. Tetrahedron 68:9745–9749
Wu G, Lin A, Gu Q, Zhu T, Li D (2013) Four new chloro-eremophilane sesquiterpenes from an
Antractic deep-sea derived fungus Penicillium sp. Pr19n-1. Mar Drugs 11:1399–1408
Wu G, Sun X, Yu G, Wang W, Zhu T, Gu Q, Li D (2014) Cladosins A-E, hybrid polyketide from a
deep-sea-derived fungus Cladosporium sphaerospermum. J Nat Prod 77:270–275
Wu B, Wiese J, Labes A, Kramer A, Schmaljohann R, Imhoff J (2015) Lindgomycin, an unusual
antibiotic polyketide from a marine fungus of the Lindgomycetaceae. Mar Drugs 13:4617–4632
Wyss M, Pasamontes L, Friedlein A, Rémy R, Tessier M, Kronenberger A, Middendorf A,
Lehmann M, Schnoebelen L, Röthlisberger U, Kusznir E, Wahl G, Müller F, Lahm H-W,
Vogel K, van Loon APGM (1999) Biophysical characterization of fungal phytases
(myo-insoitol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern,
engineering of proteolytic resistance. Appl Environ Microbiol 65:359–366
Xu R, Xu G-M, Li X-M, Li C-S, Wang B-G (2015a) Characterization of a newly isolated marine
fungus Aspergillus dimorphicus for optimized production of the anti-tumor agent wentilactones.
Mar Drugs 13:7040–7054
Xu X, Zhang X, Nong X, Wei X, Qi S (2015b) Oxindole alkaloids from the fungus Penicillium
commune DFFSCS026 isolated from deep-sea-derived sediments. Tetrahedron 71:610–615
21 Extremophilic Fungi as a Source of Bioactive Molecules 521

Yamada T, Iwamoto C, Yamagaki N, Yamanouchi T, Minoura K, Yamori T, Uehara Y, Andoh T,
Uemura K, Numata A (2002) Leptosins M-N1, cytotoxic metabolites from a Leptosphaeria
species separated from a marine alga. Structure determination and biological activities. Tetra-
hedron 58:479–487
Yao Q, Wang J, Zhang X, Nong X, Xu X, Qi S (2014) Cytotoxic polyketides from the deep-sea-
derived fungus Engyodontium album DFFSCS021. Mar Drugs 12:5902–5915
Zalar P, Kocuvan M, Plemenitǎs A, Gunde-Cimerman N (2005) Halophilic black yeast colonize
wood immersed in hypersaline water. Bot Mar 48:323–326
Zhang XY, Zhang Y, Xu XY, Qi SH (2013) Diverse deep-sea fungi from the South China Sea, their
antimicrobial activity. Curr Microbiol 67:525–530
Zhang P, Mandi A, Li X-M, Du F-Y, Wang J-N, Li X, Kurtan T, Wang B-G (2014) Varioxepine A,
a 3 H-oxepine-containing alkaloid with a new oxa-cage from the marine algal-derived endo-
phytic fungus Paecilomyces variotii. Org Lett 16:4834–4837
Zhang LQ, Chen XC, Chen ZQ, Wang GM, Zhu S-G, Yang Y-F, Chen K-X, Liu X-Y, Li YM
(2016a) Eutypenoids A-C: novel pimarane diterpenoids from the Arctic fungus Eutypella
sp. D-1. Mar Drugs 14(3):44
Zhang T, Wang NF, Liu HY, Zhang YQ, Yu LY (2016b) Soil pH is a key determinant of soil fungal
community composition in the Ny-Ålesund region, Svalbard (high Arctic). Front Microbiol 7:
227
Zhang X, Li SJ, Li JJ, Liang ZZ, Zhao CQ (2018) Novel natural products from extremophilic fungi.
Mar Drugs 16:1–36
Zhou H, Li L, Wang W, Che Q, Li D, Gu Q, Zhu T (2015) Chrodrimanins I and J from the Antarctic
moss-derived fungus Penicillium funiculosum GWT2-24. J Nat Prod 78:1442–1445
Zhou Y, Li Y-H, Yu H-B, Liu X-Y, Lu X-L, Jiao B-H (2018) Furanone derivative and sesquiter-
pene from Antarctic marine-derived fungus Penicillium sp. S-1-18. J Asian Nat Prod Res 20:
1108–1115
522 A. Das et al.

Piezophilic Fungi: Sources of Novel Natural
Products with Preclinical and Clinical
Significance
22
Tuyelee Das, Puja Ray, Samapika Nandy, Abdel Rahman Al-Tawaha,
Devendra Kumar Pandey, Vijay Kumar, and Abhijit Dey
Abstract
Extremophiles span through all taxonomic ranges starting from prokaryotes to
Eucarya as well as Archaea. Extremophiles are categorized into seven groups on
the basis of their various extreme habitats. Piezophiles withstand high hydrostatic
pressure and reside at deep-sea sediments or are isolated from the bottom-
dwelling animals’guts. Piezophilic fungi are known to produce plethora of
natural compounds with tremendous preclinical and clinical significance. Deep-
sea-derived fungi such as different strains of Phialocephala sp., Penicillium sp.,
Ascomycota sp., Aspergillus sp., and Emericella sp. have been known to produce
an array of pharmacologically active compounds such as diketopiperazine and
oxindole alkaloids, sorbicillin-type compounds, diterpenes like
brevianespiroditerpenoids, prenylxanthones, hydroxyphenyl acetic acid, etc.
exhibiting antimicrobial, antiviral, anticancer, α-glucosidase inhibitory, and anti-
oxidant properties in a number of preclinical investigations. The present review
comprehensively retrieves the reports on novel piezophilic fungi-derived natural
products mainly focusing on the recent literature emphasizing on their structures,
biological activities, and structure–activity analyses with a note on future clinical
implications of such piezophiles-derived drugs.
T. Das · P. Ray · S. Nandy · A. Dey (*)
Department of Life Sciences, Presidency University, Kolkata, West Bengal, India
e-mail: abhijit.dbs@presiuniv.ac.in
A. R. Al-Tawaha
Department of Biological Sciences, Al-Hussein Bin Talal University, Maan, Jordon
D. K. Pandey · V. Kumar
Department of Biotechnology, Lovely Faculty of Technology and Sciences, Lovely Professional
University, Phagwara, Punjab, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_22
523

Keywords
Extremophiles · Piezophilic · Natural Products · Structure-activity · Bioactivity
22.1 Introduction
Extremophiles are a wide group of organisms present in extreme conditions.
Extremophiles include bacteria, archaea, and eukarya (Woese et al. 1990). Based
on different extreme environmental conditions, extremophiles are categorized into
piezophiles (high hydrostatic pressure) (Skropeta 2008; Yayanos 1995; Horikoshi
1998), thermophiles (50–80 C or more than 80 C) (Wilson and Brimble 2009),
psychrophiles (present in extreme habitats like in the Antarctic, the Arctic, and
glacial regions) (Deming 2002), halophiles (requires >3% NaCl for growth) (Wilson
and Brimble 2009), xerophiles (ashes and deserts) (Evans and Johansen 1999),
acidophiles (present in an environment with pH values less than 4), and alkaliphiles
(pH values more than 9) (Wilson and Brimble 2009). Due to this extreme
ecosystem’s extremophiles possess some unique strategies for their reproduction
and survival, which needs the involvement of metabolic pathways and genetic
regulation. Metabolic pathways that involve in their survival produced some note-
worthy and precious compound, which can be used as natural products alternative to
chemically synthesised drugs for the treatment of some most important human
diseases (Stetter 1999; Madern et al. 2000; Rothschild and Mancinelli 2001;
Soldatou and Baker 2017). Natural products include bioactive secondary
metabolites, which are produced by all ranges of organism prokaryotes to
eukaryotes. These compounds exhibit several biological activities including antiox-
idant, antibacterial, antifungal, antiviral, cytotoxic, and anti-inflammatory. Most of
the species from extreme ecosystems are still unexplored. Fungi from such environ-
ment hold undiscovered metabolites.
Deep-sea is defined by the depths of 200–300 m beyond the euphotic zone (van
Dover et al. 2002). Some of the world’s remote ecosystems are present in the ocean
more than 1000 m deep, with every 10 m of depth 1 atm hydrostatic pressure
increases (Skropeta and Wei 2014; Takami et al. 1997). World’s 71% of marine
environment and polar region is cold. Piezophiles are present in the sea below the
thermocline layer where cold environment is constant throughout the year. Water
present below the thermocline layer is having extreme hydrostatic pressure (Moyer
et al. 2017). Piezophilic organisms can grow at 70–80 MPa, though unable to sustain
below 50 MPa (Kato et al. 1998). Oger and Jebbar 2010 described that piezophiles
grow and complete reproduction under high pressure 100 MPa (Oger and Jebbar
2010). These organisms habituated from deep-sea obtain their nutrients from the
deep-sea sediment or isolated waters. However, wide varieties of deep-sea fungi are
present but proper sampling methods of those fungi are the major limitations for the
discovery of new bioactive chemicals. ZoBell describes “barophile”(also known as
piezophile) term for the first time and Roth et al. (1964) studied and published the
first paper on deep-sea piezophilic fungi in 1964 (ZoBell and Johnson 1949; Roth
524 T. Das et al.

et al. 1964). The piezophilic fungi are a novel and sustainable source for the specific
and wide varieties of bioactive molecules that can treat cancer, bacterial, viral, and
fungal diseases.
Deep-sea fungi (piezophiles) can be isolated from the different sources such as
from the sediment, free suspension, any attached flocculated material, any surfaces,
or from association with alga, plant, or other living partners without changing the
pressure and temperature. After collection, this sample can be preserved in liquid
nitrogen for as long as decades. The extraction of fungi from the mycelium of broth
culture settled into different solvents such as acetone, hexane, n-butanol, chloroform,
ethyl acetate (EtOAc), methanol (MeOH), carbon tetrachloride, and DCM. Bioactive
compounds are generally categorized in six classes: alkaloids, polyketides, peptides,
shikimates, sugars, and terpenes. Because of the mixed and wide diversity of
bioactive compounds, separation is difficult. TLC, HPLC, and other types of column
chromatography are good choices for the separation of the extracted compounds
(Zhang et al. 2011; Parkes et al. 2009). Structures of all the bioactive compounds are
generally screened by spectroscopy analysis (NMR, MS, and FTIR) and X-ray
crystallographic studies. The present chapter focuses on piezophillic fungal bioac-
tive compounds, their source, structures, biological activities, and structure–activity
relationship analysis.
22.2 Diversity of Deep-Sea Fungi
The ecosystem of deep-sea rich in different microbial organisms not only contains
bacteria and Achaea but also contains a wide variety of fungi. The investigated deep-
sea environments include South Atlantic Ocean, South China Sea, Suruga-Bay,
Japan Prydz Bay, Antarctica Gulf of Mexico Hsinchu, Taiwan Bohai Sea, West
and South Pacific Ocean, Paracel Islands, and Indian Ocean. Deep-sea fungi
included in this chapter are Ascomycota,Acremonium,Aspergillus, Cladosporium,
Chromocleista,Dichotomomyces,Diaporthe,Emericella,Engyodontium,Penicil-
lium,Phialocephal,Phoma,Spiromastix, and Simplicillium. In this review, we
summarized new and important bioactive compounds obtained from deep-sea
fungi during the last 15 years (Table 22.1). Chemical structures of antibacterial
and anticancer fungi derived natural products and some other bioactivity of fungi
derived natural products are described in Figs. 22.1 and 22.2. Figure 22.3 presents
some other bioactivity of fungi derived natural products.
22.3 Biological Activities
22.3.1 a-Glucosidase Inhibitory Activity
α-Glucosidase inhibitors are broadly used for the treatment of type 2 diabetes.
Natural products could be also an important source for α-glucosidase inhibitors.
Deep-sea fungus Dichotomomyces cejpii FS110 (South China Sea, depth 3941 m)
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 525

Table 22.1 Piezophilic fungi bioactive compounds and their activities
Species Habitat
Location
of
collection Bioactive compounds
Structures were
elucidated by Activity
Active against
bacteria/fungus/
virus/larva or
inhibited cell line
Activity value (MIC/IC
50
/
EC
50
/LC
50
) References
Acaromyces ingoldii FS121 Deep-sea
sediment
South
China Sea
Acaromycin A, cryptosporin Spectroscopic
analysis, ECD
Cytotoxicity MCF-7,
NCI-H460,
SF-268, HepG-2
IC
50
:<10 μM Gao et al.
(2016)
Arthrinium sp.UJNMF0008 Deep-sea
sediment
South
China Sea
4-Hydroxy-2-pyridone alkaloids
arthpyrones D–K,
apiosporamide, arthpyrone B
Spectroscopic
analysis
Antibacterial M. smegmatis,
S. aureus
IC
50
: 1.66–42.8 μM Bao et al.
(2018)
Cytotoxicity U2OS, MG63 IC
50
: 19.3 μM (U2OS)
11.7 μM (MG63)
Ascomycota sp. CYSK-4 Mangrove
endophytic
Pluchea
indica
Guangxi
Province,
China
Desmethyldichlorodiaportintone Spectroscopic
analysis
Anti-
inflammatory
LPS-induced
RAW 264.7 cells
IC
50
: 15.8 μM Chen et al.
(2018)
Dichlorodiaportintone,
desmethyldichlorodiaportin,
dichlorodiaportin
Spectroscopic
analysis
Anti-
inflammatory
LPS-induced
RAW 264.7 cells
IC
50
: 41.5 μM
(dichlorodiaportintone),
33.6 μM
(desmethyldichlorodiaportin),
67.2 μM (dichlorodiaportin)
Antibacterial S. aureus,
B. subtilis,E. coli,
K. pneumoniae,
A. calcoaceticus
MIC: 25–50 μg/mL
Aspergillus sp. SCSIO
Ind09F01
Deep-sea
sediment
Indian
Ocean
Sydoxanthone C, acremolin B,
diorcinol, cordyol C,
3,7-dihydroxy-1,9-
dimethyldibenzofuran
1D, 2D-NMR,
HRESIMS
Cytotoxicity HeLa, DU145,
U937
IC
50
: 2.4 μM (diorcinol),
7.1 μM (cordyol C), 10.6 μM
(3,7-dihydroxy-1,9-
dimethyldibenzofuran)
Tian et al.
(2015)
Aspergillus fischeri FS452 Deep-sea
sludge
Indian
Ocean
Fiscpropionates A–D J-HMBC, ECD Inhibitory M. tuberculosis
protein tyrosine
phosphatase B
IC
50
: 5.1, 12, 4.0, 11 μM Liu et al.
(2019)
Aspergillus ochraceus
Jcma1F17
Marine alga
Coelarthrum
sp.
Paracel
Islands,
Nitrobenzoyl sesquiterpenoids NMR, MS, CD,
optical rotation
analysis
Cytotoxicity H1975, U937,
K562, BGC-823,
Molt-4, MCF-7,
IC
50
: 1.95–6.35 μM Fang et al.
(2014)
526 T. Das et al.

South
China Sea
A549, Hela,
HL60, Huh-7
Antiviral H3N2, EV71 IC
50
: 17.0 μM, 9.4 μM
Aspergillus sp. SCSIO
Ind09F01
Deep-sea
sediment
–Gliotoxin, 12,13-
dihydroxyfumitremorgin C,
helvolic acid
NMR, ESIMS Anti-
tuberculosis
M. tuberculosis MIC
50
:<0.03, 2.41 and
0.894 μM
Luo et al.
(2017)
Gliotoxin NMR, ESIMS Cytotoxicity K562, A549,
Huh-7
IC
50
: 0.191 μM, 0.015 μM,
95.4 μM
Aspergillus sydowii YH11–2 Deep-sea
sediment
Guam (2R)-2, 3-Dihydro-7-hydr oxy-6,
8-dimethyl-2-[(E)-propl-enyl]
chromen-4-one; 2, 4-dihydroxy-
3, 5, 6-trimethylbenzaldehyde
Spectroscopic
analysis
Cytotoxicity P388 IC
50
: 0.14, 0.59 μM Tian et al.
(2007)
Aspergillus sydowii
C1-S01-A7
Deep-sea
sediment
West
Pacific
Ocean
2-Hydroxy-6-formyl-
vertixanthone, 12-O-acetyl-
sydowinin A, aspergillus one A,
AGI-B4, 8- demethoxy-10-
methoxy-wentiquinone C,
emodin
Spectroscopic
analysis
Antibacterial MRSA MIC: 15–32 μg/mL Wang et al.
(2019)
AGI-B4 Spectroscopic
analysis
Cytotoxicity A549 IC
50
:<10 μM
Aspergillus terreus YPGA10 Deep-sea
water
Yap Trenc,
West
Pacific
Ocean
Aspernolide A, Aspernolide E NMR, MS Inhibitory α-Glucosidase IC50: 6.98 (aspernolide A),
8.06 μM (aspernolide E)
Cheng et al.
(2019)
Aspergillus versicolor
MF030
Deep-sea
sediment
Bohai Sea,
China
Brevianamide S Spectroscopic
analysis
Antibacterial Bacille Calmette-
Guѐrin
MIC: 6.25 μg/mL Song et al.
(2012)
Aspergillus versicolor
A-21-2-7
Deep-sea
sediment
Southern
China Sea
Aspergilols A–F, cordyol C,
methylgerfelin, violaceol II,
lecanoric acid
Spectroscopic
analysis
Antioxidant Activates Nrf2-
regulated gene
–Wu et al.
(2016)
Aspergillus versicolor
SD-330
Deep-sea
sediment
South
China Sea
Sesquiterpenoid,
aspergoterpenin C,
engyodontiumone I
NMR,
HRESIMS,
X-ray
crystallographic
analysis, ECD
Antimicrobial A. hydrophilia,
E. coli,E. tarda,
V. harveyi
MIC: 1.0–8.0 μg/mL Li et al.
(2019)
(continued)
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 527

Table 22.1 (continued)
Species Habitat
Location
of
collection Bioactive compounds
Structures were
elucidated by Activity
Active against
bacteria/fungus/
virus/larva or
inhibited cell line
Activity value (MIC/IC
50
/
EC
50
/LC
50
) References
Aspergillus versicolor
SCSIO 41502
Deep-sea
sediment
South
China Sea
4-Carbglyceryl-3,30-dihydroxy-
5,50-dimethyldiphenyl ether,
sydowiol B, sydowiol E,
sydowiol D, 2,4-dihydroxy-6-
(4S-hydroxy-2-oxopentyl)-3-
methylbenzaldehyde
Spectroscopic
analysis
Antifouling
activity
B. neritina EC
50
: 1.28 μg/mL, 2.61 μg/
mL, 5.48 μg/mL, 1.59 μg/mL,
3.40 μg/mL
Huang et al.
(2017)
Aspergillus versicolor
SCSIO 05879
Deep-sea-
derived
Indian
Ocean
Versicoloid A, versicoloid B,
versicone A, cottoquinazoline A
NMR, X-ray
single crystal
diffraction
Antifungal C. acutatum MIC: 1.6 μg/mL Wang et al.
(2016)
Aspergillus westerdijkiae
SCSIO 05233
Deep-sea
sediment
South
China Sea
Circumdatin G NMR, optical
rotation analysis
Cytotoxicity K56, HL-60 IC
50
: 25.8 μM (K56), 44.9 μM
(HL-60)
Fredimoses
et al. (2015)
Circumdatin F NMR, optical
rotation analysis
Antifouling Balanus
amphitrite
EC
50:
8.81 μg/mL
Aspergillus sydowii Deep-sea
sediment
Hsinchu,
Taiwan
Diorcinol, bisabolane-type
sesquiterpenoids, AGI-B4,
sydowinin B
NMR, HPLC,
MS-ESI
Anti-
inflammatory
Inhibits
superoxide anion
and elastase
release
–Chung et al.
(2013)
Chaetomium globosum Deep-sea
sediment
Indian
Ocean
Cytoglobosins H and I,
cytochalasan alkaloids
1D, 2D NMR,
MS
Cytotoxicity MDA-MB-231,
B16F10
IC
50
: 0.62 (MDA-MB-231),
2.78 μM (B16F10)
Zhang et al.
(2016c)
Chromocleista sp. Sediment Gulf of
Mexico
p-Hydroxyphenopyrrozin NMR, y X-ray
crystallography
Antifungal C. albicans MIC: 25 μg/mL Park et al.
(2006)
Cladosporium
cladosporioidesHDN14–342
Deep-sea Indian
Ocean
Clindanones A and B,
cladosporols F and G
MS, NMR,
TD-DFT, ECD
Cytotoxicity HeLa IC
50
: 3.9 μM Zhang et al.
(2016b)
Cladosporium
sphaerospermum2005–01-
E3
Deep-sea
sludge
Pacific
Ocean
Cladosin C NMR Antiviral Influenza A H1N1
virus
IC
50
: 276 μM Wu et al.
(2014)
528 T. Das et al.

Dichotomomyces cejpii
FS110
Deep-sea
sediment
South
China Sea
Dichotocejpins A HRESIMS,
NMR, X-ray
crystallography,
ECD
Inhibitory
activity
α-Glucosidase IC
50
: 138 μM Fan et al.
(2016)
6-Deoxy-5a,6-
didehydrogliotoxin, gliotoxin,
acetylgliotoxin
HRESIMS,
NMR, X-ray
crystallography,
ECD
Cytotoxicity MCF-7,
NCI-H460,
HepG-2, SF-268
IC
50
: 0.08–1.52 μM
Diaporthe phaseolorum
FS431
Deep-sea
sediment
Indian
Ocean
Phaseolorin A–E Spectroscopic
analysis, single-
crystal X-ray
diffraction
Cytotoxicity HepG-2, MCF-7,
SF-268
–Guo et al.
(2019)
Emericella sp.SCSIO 05240 Deep-sea
sediment
South
China Sea
Emerixanthones A, B NMR, HSQC,
HMBC, 1H-1H
COSY, MS, CD
Antibacterial E. coli,
K. pneumonia,
S. aureus,
E. faecalis,
A. baumannii,
A. hydrophila
–Fredimoses
et al. (2014)
Emerixanthone D NMR, HSQC,
HMBC, 1H-1H
COSY, MS, CD
Antifungal Fusarium sp.,
Penicillium sp.,
Aspergillus niger,
Rhizoctonia
solani,Fusarium
oxysporium
–
Engyodontium album
DFFSCS021
Deep-sea
sediment
South
China Sea
Engyodontiumone H Spectroscopic
analysis
Cytotoxicity U937 IC
50
: 4.9 μM, Yao et al.
(2014)
Antibacterial E. coli,B. subtilis MIC 64 μg/mL
Phialocephala sp. FL30r Deep-sea
sediment
East
Pacific site
W2003–03
Dihydrotrichodermolide,
dihydrodemethylsorbicillin,
phialofurone
Spectroscopic
analysis
Cytotoxicity P388 IC
50
:11.5 1.4 μM
(dihydrotrichodermolide),
0.1 0.1 μM
(dihydrodemethylsorbicillin),
0.2 0.01 μM
Li et al.
(2011a,b)
K562 IC
50
: 22.9 0.8 μM
(dihydrodemethylsorbicillin,
(continued)
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 529

Table 22.1 (continued)
Species Habitat
Location
of
collection Bioactive compounds
Structures were
elucidated by Activity
Active against
bacteria/fungus/
virus/larva or
inhibited cell line
Activity value (MIC/IC
50
/
EC
50
/LC
50
) References
phialofurone), 4.8 0.3 μM
(dihydrodemethylsorbicillin),
22.4 0.9 μM (phialofurone)
Phialocephala sp. FL30r Deep-sea
sediment
–Trisorbicillinone A Spectroscopic
analysis
Cytotoxicity P388 IC
50
: 9.10 μM Li et al.
(2007b)
HL60 IC
50
: 3.14 μM
Penicillium sp. SCSIO
06720
Deep-sea
sediment
Indian
Ocean
b-Diversonolic ester NMR, MS
HPLC, ECD
Antibacterial
activity
MRSA MIC: 10.4 3.7 μg/mL Guo et al.
(2020)
S. aureus-shh-1 MIC: 46.9 29.7 μg/mL
Penicillium sp. PR19N-1 Deep-sea
sediment
Prydz Bay,
Antarctica
Chloro-trinor eremophilane
sesquiterpene
IR, HRMS, 1D,
2D NMR
Cytotoxic
activity
HL-60, IC
50
: 11.8 0.2 μM, Wu et al.
(2013)
A549 IC
50:
12.2 0.1 μM
Penicillium sp. JMF034 Deep-sea
sediment
Fujikawa,
Suruga-
bay, Japan
Gliotoxin, gliotoxin G ESIMS,
1
H
NMR
Cytotoxic
activity
P388 0.024 μM (gliotoxin),
0.020 μM (gliotoxin G)
Sun et al.
(2012)
Penicillium brevicompactum
DFFSCS025
Deep-sea
sediment
South
China Sea,
Sansha
City,
Hainan
Province
Brevianamides, mycochromenic
acid
Spectroscopic
analysis
Antifouling Bugula neritina EC
50
: 13.7 μM Xu et al.
(2017)
Cytotoxicity HCT116 IC
50
: 15.6 μM
Penicillium chrysogenum
MCCC 3A00292
Deep-sea
sediment
South
Atlantic
Ocean
Peniciversiols A HRESIMS,
NMR, ECD
Cytotoxicity BIU-87 IC
50
:10.21 μM Niu et al.
(2019)
Decumbenone A,
decumbenone B,
3,30-dihydroxy-
5,50-dimethyldiphenyl ether,
violaceol-II,3,8-dihydroxy-4-
(2,3-dihydroxy-1-
hydroxymethylpropyl)-1-
HRESIMS,
NMR, ECD
Cytotoxicity ECA109, BIU-87,
BEL-7402
IC
50
: 7.70 to >20 μM
530 T. Das et al.

methoxyxanthone, asperdemin,
cyclopenol
Penicillium commune
SD-118
Deep-sea
sediment
South
China Sea
Xanthocillin X NMR Antimicrobial S. aureus MIC: 2 μg/mL Shang et al.
(2012)
E. coli MIC: 1 μg/mL
Antifungal A. brassicae MIC: 32 μg/mL
Cytotoxicity MCF-7 IC
50
: 12.0 μg/mL
HepG2 IC
50
: 7.0 μg/mL,
NCI-H460 IC
50
: 10.0 μg/mL, 10.0 μg/mL
HeLa,
MDA-MB231,
DU145,
IC
50
: 8.0 μg/mL
Penicillium
fellutanumHDN14–323
Deep-sea
sediment
Indian
Ocean
Peniphenylanes Spectroscopic
analysis
Cytotoxicity HeLa IC
50
: 9.3 μM Zhang et al.
(2016a)
Penicillium paneumSD-44 Deep-sea
sediment
South
China Sea
Anthranilic acid, penipacids
A-E
NMR, MS Cytotoxicity RKO IC
50
: 8.4 μM, 9.7 μM Li et al.
(2013)
Hela IC
50
: 6.6 μM
Phomopsistersa FS441 Deep-sea
sediment
Indian
Ocean
Tersone E, citridone A Spectroscopic
analysis, single-
crystal X-ray
diffraction
experiments,
ECD
Antibacterial S. aureus ATCC
29
MIC: 31.5 μg/mL Chen et al.
(2019)
Tersone E Cytotoxicity SF-268 IC
50:
32.0 μM
MCF-7 29.5 μM
HepG-2 39.5 μM
A549 33.2 μM
Simplicillium obclavatum
EIODSF 020
Deep-sea
sediment
East Indian
Ocean
Simplicilliumtide J, verlamelins
A and B
Spectroscopic
analysis
Antiviral HSV-1 IC
50:
14.0 μM
(simplicilliumtide J), 16.7 μM
(verlamelin A), 15.6 μM
(verlamelin B)
Liang et al.
(2017)
Antifungal A. versicolor,
C. australiensis
MIC: 14.0 μg/disc
(simplicilliumtide J), 16.7 μg/
disc (verlamelin A), 0.156
(verlamelin B) μg/disc
(continued)
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 531

Table 22.1 (continued)
Species Habitat
Location
of
collection Bioactive compounds
Structures were
elucidated by Activity
Active against
bacteria/fungus/
virus/larva or
inhibited cell line
Activity value (MIC/IC
50
/
EC
50
/LC
50
) References
Spiromastix sp.,
Simplicillium
Deep-sea
sediment
South
Atlantic
Ocean
Spiromastixones A–O NMR Antibacterial MARS, MRSE MIC: 0.1–8.0 μg/mL Niu et al.
(2014)
Spiromastix sp. MCCC
3A00308
Deep-sea
sediment
South
Atlantic
Ocean
Spiromastols A–K NMR, MS Antibacterial X. vesicatoria,
P. lachrymans,
A. tumefaciens,
R. solanacearum,
B. thuringensis,
S. aureus,
B. subtilis
MIC: 0.25–4μg/mL Niu et al.
(2018)
532 T. Das et al.

derived biologically significant dichotocejpin A compound showed inhibitory activ-
ity against α-glucosidase with IC
50
: 138 μM (Fan et al. 2016). One recent study by
Chen et al. (2019) reported that ethyl acetate extract of deep-sea water Aspergillus
terreus YPGA10 fungus produced butyrolactone I, butyrolactone VII,
aspernolide A, and aspernolide E which also inhibited α-glucosidase (Cheng et al.
2019). This study could be helpful for the development of new potential inhibitors of
α-glucosidase.
22.3.2 Antibacterial Activity
Antibacterial drugs are one of the most important drugs against human pathogenic
bacterial diseases. Deep-sea fungal strains and its bioactive compounds are new and
noteworthy potential alternative source, which by screening can be designed as
drugs against bacterial diseases.
New polyphenols spiromastols A–K were isolated from the fermentation broth of
the deep-sea-derived fungus Spiromastix sp. MCCC 3A00308 (South Atlantic
Ocean). All these polyphenols inhibited the growth of Staphylococcus aureus
ATCC 25923, Xanthomonas vesicatoria ATCC 11633, Ralstonia solanacearum
ATCC11696, Pseudomonas lachrymans ATCC11921, Bacillus thuringensis
Fig. 22.1 Chemical structure of antibacterial natural products from piezophilic fungi.
1. Spiromastol A, 2. Spiromastol B, 3. Spiromastol C, 4. Spiromastol D, 5. Spiromastol E,
6. Spiromastol F, 7. Spiromastol G, 8. Spiromasto H, 9. Spiromasto l, 10. Prenylxanthone, 11.
Xanthocillin X, 12. Spiromastixones A, 13. Spiromastixone B, 14. Spiromastixone C, 15.
Spiromastixone D, 16. Spiromastixone E, 17. Spiromastixone F, 18. Spiromastixone G, 19.
Spiromastixone H, 20. Spiromastixone I, 21. Spiromastixone J, 22. Spiromastixone k, 23.
Spiromastixone L, 24. Spiromastixone M, 25. Spiromastixone N, 26. Spiromastixone O
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 533

Fig. 22.2 Chemical structure of natural products from piezophilic fungi with cytotoxic activity.
27. Arthpyrone B, 28. Arthpyrone D, 29. Arthpyrone E, 30. Arthpyrone F, 31. Arthpyrone G, 32.
Arthpyrone H, 33. Arthpyrone I, 34. Arthpyrone J, 35. Arthpyrone K, 36. Apiosporamide, 37.
Cryptosporin, 38. Acremolin B, 39. Diorcinol, 40. Cordyol C, 41. 3,7-Dihydroxy-1,9-
dimethyldibenzofuran, 42. Gliotoxin, 43. Engyodontiumone H, 44. Trisorbicillinone A, 45.
Trisorbicillinone B, 46. Trisorbicillinone C, 47. Trisorbicillinone D, 48. Oxosorbiquinol, 49.
Dihydrooxosorbiquinol, 50. Racemates, 51. Tersone A, 52. Tersone B, 53. Tersone C, 54.
534 T. Das et al.

ATCC 10792, and Bacillus subtilis CMCC 63501 with MIC values between 0.25
and 4 μg/mL (Niu et al. 2015). Prenylxanthone obtained from EtOAc extract of
Emericella sp. SCSIO 05240 collected from depth 3258 m in the South China Sea
displayed week antibacterial activity against Escherichia coli,Klebsiella
pneumoniae,S. aureus,Enterococcus faecalis,Acinetobacter baumannii, and
Aeromonas hydrophila (Fredimoses et al. 2014).One isocyanide compound
xanthocillin X have been described as metabolite from Penicillium commune
SD-118 (South China Sea) fungus that showed antibacterial activity against
S. aureus and E. coli. The yield of xanthocillin X compound from P. commune
was 361.55 mg/L in the liquid fermentation method (Shang et al. 2012). Deep-sea-
derived fungi are also useful for the treatment of several strains of multidrug-
resistant bacteria. Penicillium sp. SCSIO 06720 reported for the production of
b-diversonolic ester, which displayed antibacterial activity against methicillin resis-
tant (MR) S. aureus-shh-1 and S. aureus with MIC (microbial inhibitory concentra-
tion) values 46.9 29.7 μg/mL and 10.4 3.7 μg/mL, respectively (Guo et al.
2020). Additionally, depsidone-based analogues spiromastixones A–O from
Spiromastix sp. displayed inhibitory effects against methicillin resistant strain of S.
aureus ATCC 29213 and Staphylococcus epidermidis with MIC: 0.1–8.0 μg/mL.
Spiromastixones J also inhibits VRE (vancomycin-resistant enterococci) strains of
E. faecalis and E. faecium (Niu et al. 2014). Pyridone alkaloids and isocoumarins
obtained from marine sediment and mangrove endophyte Arthrinium sp.
UJNMF0008 and Ascomycota sp. CYSK-4 are active against B. subtilis,E. coli,
K. pneumoniae,Acinetobacter calcoaceticus,Mycobacterium smegmatis, and
Staphylococcus aureus bacteria. MIC values ranges between 25 and 50 μg/mL for
isocoumarins (Chen et al. 2018). (+)-tersone E and ent-citridone A have been
isolated from the Phomopsis tersa, which was collected from the deep-sea sediment
in the depth of 3000 m. Results showed strong antibacterial activity against S. aureus
ATCC 29 with the MIC value 31.5 μg/mL (Chen et al. 2019).
22.3.3 Anticancer Activity
Worldwide, cancer becomes a significant health-related problem, with a high mor-
tality rate, with approximately 9.6 million deaths in 2018 (https://www.who.int/
cancer/PRGlobocanFinal.pdf). In 2020, approximately 1.8 million new cancer cases
and 600,000 cancer deaths were recorded in the USA. Cancer treatments available
are chemotherapy, immunotherapy, radiation therapy, and targeted therapy; how-
ever, chemo-resistance is still a problem, which we need to overcome. However,
several bioactive plant or microbes metabolites could be a good choice for treatment
⁄
Fig. 22.2 (continued) Tersone D, 55. Tersone E, 56. Tersone F, 57. Tersone G, 58. Penipacid A,
59. Penipacid B, 60. Penipacid C, 61. Penipacid D, 62. Penipacid E, 63. Penipanoid B, 64.
Penipanoid C, 65. Gliotoxin, 66. Gliotoxin G
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 535

of cancer. Below we discussed the possibilities of piezophillic fungi in cancer
treatment.
One new compound acaromycin A and one known compound (+)-cryptosporin
were obtained from the fermentation broth of Acaromyces ingoldii FS121, and
collected from the South China Sea from the depth of 3415 m that displayed
cytotoxic activities against MCF-7, NCI-H460, SF-268, and HepG-2 with IC
50
values less than 10 μM (Gao et al. 2016). Marine Arthrinium fungi are distributed
throughout the world and known for its cytotoxicity, antimicrobial, and AChE
inhibitory activities. Bao et al. (2018) studied UJNMF0008 strain of Arthrinium
sp. (South China Sea, depth 3858 m) fungi and reported the presence of new eight
4-hydroxy-2-pyridone alkaloids arthpyrones D–K compound with apiosporamide
and arthpyrone B analogs. These compounds displayed cytotoxicity against U2OS
and MG63 cell lines (Bao et al. 2018). Bioactive compounds of deep-sea-derived
strains of Aspergillus sp. revealed significant cytotoxicity activity. One xanthone
sydoxanthone C, one alkaloid acremolin B, diphenyl ether diorcinol, cordyol C,
3,7-dihydroxy-1,9-dimethyldibenzofuran, nitrobenzoyl sesquiterpenoids,
diketopiperazine, fumiquinazoline, 2-hydroxy-6-formyl-vertixanthone, and 12-O-
acetyl-sydowinin A are some bioactive compounds isolated from deep-sea-derived
Aspergillus sp. Tian et al. (2015) reported that diorcinol, cordyol C, and
3,7-dihydroxy-1,9-dimethyldibenzofuran displayed COX-2 inhibitory activity
against HeLa, DU145, and U937 cell lines. COX-2 produces enzymes, which
activates tumor-inducing promoters, cytokines, and mitogens. Gliotoxin induces
Aspergilols A–F, Cordyol, Fumalic acid, Lecanoric acid, Methylgerfelin,
Violaceol II
Simplicilliumtides, Verlamelins A and B, Versicoloids A and B, Versicone A,
Cottoquinazoline A, Emerixanthones D
Brevianamides, Circumdatin F, 4-carbglyceryl-3,3'-dihydroxy-5,5'-
dimethyldiphenyl ether, Sydowiol B, Sydowiol E, Sydowiol D,
Mycochromenic acid
Dichotocejpin A, Butyrolactone I, Butyrolactone VII,
aspernolide A, Aspernolide E
Desmethyldichlorodiaportintone, Diorcinol, AGI-B4,
Sydowinin B, Brevicompanines D–H
Fiscpropionates A−D, Gliotoxin, 12, 13-
dihydroxyfumitremorgin C, Helvolic acid
Insulicolide A, Simplicilliumtide I,
Simplicilliumtides J−M, Verlamelins A and B,
Cladosin C
Anti-oxidant
Anti-fungal
Anti-viral
Anti-tuberculosis
Anti-inflammatory
α-glucosidase
inhibitory
Anti-fouling
Fig. 22.3 Some other bioactivity of fungi derived natural products
536 T. Das et al.

apoptosis (McDougall 1969). Luo et al. (2017) reported the presence of cytotoxic
gliotoxin in deep-sea-derived Aspergillus sp. SCSIO Ind09F01 fungus showed
inhibitory activity against K562, A549, and Huh-7 cell lines with IC
50
values of
0.191, 0.015, and 95.4 μM, respectively. Engyodontiumone H was obtained from
Engyodontium album DFFSCS021 collected from deep-sea sediment of South China
Sea in 3739 m depth. Engyodontiumone H revealed cytotoxic activity against U937
cells with IC
50
value of 4.9 μM (Yao et al. 2014).
Phialocephala sp. FL30r obtained from the underwater sample (depth 5059 m,
the east Pacific) was a powerful producer of diverse fungal polyketides such as
sorbicillin-type compounds (cyclohexanone ring with a sorbyl side chain),
sorbicillinoids, benzofuranone derivatives (Li et al. 2011a,b), oxosor-biquinol,
dihydrooxosorbiquinol (Li et al. 2007a), and trisorbicillinone A, B, C, and D
(Li et al. 2010,2007b). Sorbicillinoids and phialofurone compounds revealed
cytotoxic effects against P388 and K562 cell lines (Li et al. 2011a,b).
Bisorbicillinoids (oxosor-biquinol and dihydrooxosorbiquinol) displayed cytotoxic
effects on P388, A-549, HL60, BEL7402, and K562 cell lines evaluated by the MTT
method. Oxosorbiquinol showed moderate cytotoxicity against the A549 cell line
(Li et al. 2007a). Trisorbicillinone A exhibited cytotoxicity against P388 and HL60
cells. IC
50
value for Trisorbicillinone A was 9.10 μM and 3.14 μM, against P388 and
HL60 cells, respectively (Li et al. 2007b). Trisorbicillinone B, C, and D also showed
cytotoxicity against P388 and K562 cell lines which were detected by the MTT
method. The IC
50
values for P388 cells were 77.1 μM, 78.3 μM, and 65.7 μM and for
K562 cells were 88.2 μM, 54.3 μM, and 51.2 μM respectively (Li et al. 2010).
Deep-sea-derived FS441 strains of Phomopsis tersa (depth 3000 m, Indian
Ocean) are known for the production of novel metabolites such as
phenylfuropyridone racemates, ()-tersones A, B, C, D, E, F, and G phenylpyridone
racemate, pyridine alkaloid, and phenylfuropyridone. All these compounds
exhibited in vitro cytotoxic activity against SF-268, MCF-7, HepG-2, and A549
cell lines (Chen et al. 2019). After the discovery of penicillin from Penicillium,it
became an attraction to scientists for the production of several metabolites. Deep-
sea-derived Penicillium (P. paneum,P. fellutanum,P. brevicompactum, and
P. chrysogenum) become attracted from the last decades for the presence of novel
metabolites which showed cytotoxic activity against RKO, BIU-87, ECA109,
BEL-7402, PANC-1, HCT116, Hela-S3, HeLa, HL-60, A549, and HCT-116 cell
lines. Triazole carboxylic acid (penipacids A–E), penipanoids B and C,
quinazolinone alkaloids, and quinazolinone derivative reported from deep-sea sedi-
ment fungi P. paneum SD-44 (South China Sea). Structures were screened and
elucidated by NMR and MS analysis. Penipacid A, penipacid E, and quinazolinone
derivative displayed cytotoxicity against the human colon cancer RKO cell line,
while compound 6 displayed cytotoxic activity against SMMC-7721 and RKO cell
line (Li et al. 2011a,b,2013). Penipacid A inhibits SMMC-7721 and RKO cell line
with an IC
50
value of 54.2 μM (Li et al. 2013) and 8.4 (Li et al. 2011a,b),
respectively, while penipacid E inhibits the RKO cell line with IC
50
value 9.7 μM
(Li et al. 2011a,b). Trimeric peniphenylanes A–B and dimeric peniphenylanes C–G
were produced by deep-sea sediment derived fungus P. fellutanum HDN14–323
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 537

(depth 5725 m, Indian Ocean). These new 6-methylsaligenin derivatives exhibited
cytotoxic activity against HeLa, HL-60, and HCT-116 cell lines (Zhang et al.
2016a). Chloro-trinoreremophilane sesquiterpene 1, chlorinated eremophilane
sesquiterpenes 2–4, and eremofortine C were produced by Penicillium sp. PR19N-
1 (Prydz Bay, Antarctica). Among them chloro-trinoreremophilane sesquiterpene
displayed moderate cytotoxicity against HL-60 and A549 cell lines with IC
50
values
of 11.8 μM and 12.2 μM, respectively (Wu et al. 2013). Additionally, five new
eremophilane-type sesquiterpenes were also isolated from Penicillium sp. PR19N-1
fungus showed cytotoxicity against similar cell lines but with a low IC
50
value5.2 μM
against the A549 cell line (Lin et al. 2014). Penicillium chrysogenum MCCC
3A00292 fungus from the South Atlantic Ocean, depth of 2076 m, contain
peniciversiols A, B, C, and penicilactones A and B, where speniciversiols A
inhibited BIU-87 cells with IC
50
value of 10.21 μM (Niu et al. 2019). Gliotoxin
and gliotoxin related compounds (bis(dethio)bis-(methylthio)-5a,6-
didehydrogliotoxin, 6-didehydrogliotoxin, and gliotoxin G) were isolated from
deep-sea sediments of the Penicillium sp.JMF034 fungus (Suruga-Bay, Japan).
Among them, gliotoxin and gliotoxin G reported the most potent one which
inhibited P388 cell with IC
50
0.024 and 0.020, respectively (Sun et al. 2012).
22.3.4 Antifungal Activity
Simplicilliumtides, verlamelins A and B isolated from the deep-sea sediment fungus
Simplicillium obclavatum EIODSF 020 (East Indian Ocean) and screened based on
spectroscopic analysis. The MICs of these compounds range between 0.156 and
16.7 μg/disc against Aspergillus versicolor and Curvularia australiensis (Liang et al.
2017). Versicoloids A and B, versicone A, and cottoquinazoline A have been
isolated from the deep-sea-derived A. versicolor SCSIO 05879 (Indian Ocean,
depth 3927 m) and characterized based on NMR, and structure elucidates by
X-ray single-crystal diffraction. These compounds showed antifungal activity
against Colletotrichum acutatum fungi. A phytopathogenic C. acutatum fungus is
known for global economic loss in agricultural crops. Wang et al. (2016) reported
that versicoloids A and B showed strong antifungal activity and could be a promising
candidate for the production of natural products (Wang et al. 2016). Emerixanthones
D are also active against agricultural pathogens Fusarium sp., Fusarium sporium
f. sp. cucumeris,Aspergillus niger,Fusarium oxysporium f. sp. niveum,Penicillium
sp., and Rhizoctonia solani with the diameter of zone of inhibition ranges between
3 and 4 mm. Emerixanthones D derived from deep-sea fungus Emericella sp. SCSIO
05240 (depth 3258 m, South China Sea) (Fredimoses et al. 2014). Candida albicans
is a harmful human pathogenic bacteria which can be inhibited by
p-hydroxyphenopyrrozin metabolite from deep-sea-derived Chromocleista sp. with
MIC 25 μg/mL (Park et al. 2006).
538 T. Das et al.

22.3.5 Antifouling Activity
Fredimoses et al. (2015) reported the presence of circumdatin F isolated from EtOAc
extract of deep-sea-derived Aspergillus westerdijkiae SCSIO 05233 (depth 4593 m,
South China Sea). Circumdatin F showed antifouling activity against Balanus
amphitrite with EC
50
value 8.81 μg/mL (Fredimoses et al. 2015). Strong antifouling
activity against Bugula neritina larva took place by 4-carbglyceryl-3,30-dihydroxy-
5,50-dimethyldiphenyl ether, sydowiol B, sydowiol E, sydowiol D, 2,4-dihydroxy-6-
(4S-hydroxy-2-oxopentyl)-3-methylbenzaldehyde metabolites isolated from Asper-
gillus versicolor SCSIO 41502 (South China Sea) fungus with EC
50
values of 1.28,
2.61, 5.48, 1.59, and 3.40 μg/mL, respectively (Huang et al. 2017). Two new
mycochromenic acid derivatives brevianamides and mycochromenic acid
metabolites from Penicillium brevicompactum DFFSCS025 (South China Sea,
Sansha City, 3928 m depth) also displayed antifouling activity against B. neritina
but with high EC
50
value than that Huang et al. (2017) reported (Xu et al. 2017).
22.3.6 Anti-Inflammatory Activity
Desmethyldichlorodiaportintone was obtained from Ascomycota sp. CYSK,
which was collected from mangrove endophytic Pluchea indica.
Desmethyldichlorodiaportintone showed LPS-induced anti-inflammatory
activity in RAW 264.7 cells with IC
50
value of 15.8 μM (Chen et al. 2018).
Diorcinol, bisabolane-type sesquiterpenoids, AGI-B4, sydowinin B isolated
from the deep-sea fungus Aspergillus sydowii and characterized on the basis of
NMR, HPLC, MS-ESI studies. All these compounds act as an anti-inflammatory
compound by inhibiting superoxide anion generation and elastase releases. This
study also reported that the addition of a 5-azacytidine, enhanced the production of
diorcinol, (7S)-(+)-7-O-methylsydonol, (S)-(+)-sydonol (4), (S)-(+)-sydonic acids.
5-azacytidine act as epigenetic modifiers (Chung et al. 2013). New diketopiperazine
alkaloids brevicompanines D–H obtained from deep-sea sediment fungus Penicillium
sp. F23–2showedanti-inflammatory activity on LPS-challenged BV2 cells with IC
50
values of 27 and 45 μg/mL, respectively (Du et al. 2010).
22.3.7 Anti-Oxidant Activity
Oxidative stress leads to a wide range of human diseases including cancers, aging,
Alzheimer’s disease, Parkinson’s disease, and inflammations. Antioxidants are
neutralizing free radicals. New phenolic bioactive compounds Aspergilols A–F
obtained from ethyl acetate extract of deep-sea fungus Aspergillus versicolor
(A-21-2-7) which, collected from the South China Sea from 3002 m depth, displayed
antioxidant activity that detected by the DPPH assay. Cordyol, violaceol II,
1-methylpyrogallol, and fumalic acid showed strong free-radical scavenging effects.
Aspergilol E, cordyol C, methylgerfelin, violaceol II, and lecanoric acid compounds
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 539

displayed significantly activated Nrf2, which is a potent therapeutic factor for
neurodegenerative diseases (Wu et al. 2016).
22.3.8 Anti-Tuberculosis Activity
Polypropionate derivatives (Fiscpropionates A–D) were obtained from the deep-sea
sludge of Aspergillus fischeri FS452 fungus (Indian Ocean, depth 3000 m) and
displayed anti-tuberculosis activity against MptpB protein of M. tuberculosis bacte-
ria with IC
50
ranges between 5.1 and 11 μM (Liu et al. 2019). Another strain of
Aspergillus sp. SCSIO Ind09F01 produced gliotoxin, 12,13-
dihydroxyfumitremorgin C, helvolic acid which also showed anti-tuberculosis activ-
ity with MIC
50
<0.03, 2.41, and 0.894 μM, respectively (Luo et al. 2017).
22.3.9 Antiviral Activity
Nitrobenzoyl sesquiterpenoid (6β,9α-dihydroxy-14-p-nitrobenzoylcinnamolide) and
its analogue insulicolide A obtained from marine alga Coelarthrum sp. derived
Aspergillus ochraceus Jcma1F17 (Paracel Islands, South China sea) showed
antiviral activity against H3N2 and EV71 virus with 50% inhibitory effects
17.0 μM and 9.4 μM, respectively (Fang et al. 2014). Another deep-sea-derived
fungus Simplicillium obclavatum EIODSF 020 (East Indian Ocean) produces natural
bioactive compound peptides like simplicilliumtide I, simplicilliumtides J–M, and
verlamelins A and B which displayed antiviral activity against HSV-1 virus (Liang
et al. 2017). Cladosin C is a polyketide which was reported by Wu and colleagues
from the Cladosporium sphaerospermum 2005–01-E3, a deep-sea fungus displayed
a decent inhibitory activity of H1N1 influenza A virus (Wu et al. 2014).
22.4 Structure–Activity Relationships
Structure–activity relationship is an influential technology that predicts the
biological activity from the molecular structure of compounds, which leads to the
development of new desirable drugs. Very few studies are present about SAR for
bioactive compounds of deep-sea piezophilic fungi. Comparing the structures and
bioactivities of simplicilliumtides J–M, verlamelins A and B showed that the
lactonized 5-hydroxytetradecanoic acid residue played an important role in the
antifungal and antiviral activities. When lactone linkage is open, bioactivities will
lose. Simplicilliumtides J and verlamelins A and B showed more antiviral and
antifungal activity because of the absence of C-13/C-14 of the
5-hydroxytetradecanoic acid residue, which is present in simplicilliumtides K, L,
and M (Liang et al. 2017). Niu et al. (2015) reported that antibacterial activities of
spiromastols were based on ring A and B substitution by the analysis of the SAR.
This study also reported that isocoumarin is more potent than dihydroisocoumarin
540 T. Das et al.

scaffold for bacterial inhibition. Ester bonds that connected A and B rings displayed
stronger effects than ether bond. Agrobacterium tumefaciens,B. thuringensis,
B. subtilis,Pseudomonas lachrymans,R. solanacearum,S. aureus, and
X. vesicatoria inhibited because of dichlorinated ring A, while carboxylic acid
analogues significantly decreased antibacterial activity at C-1 (Niu et al. 2015).
Meleagrin B is already known for its significant cytotoxic activity for cancer cell
lines. Acetate–mevalonate-derived C5 or C9 side chains addition on N-17
suppressed the meleagrin alkaloids activities. However, diterpene moiety
substitutions on imidazole ring enhance the cytotoxic activities of meleagrin
alkaloids (Du et al. 2010). Brevicompanine E and brevicompanine H showed
average activities that inhibited lipopolysaccharide (LPS)-induced nitric oxide
(NO) production in BV2 microglial cells with IC
50
values of 27 mg/mL and
45 mg/mL. Substitution at the N-6 position contributes to anti-inflammatory
activities by inhibition of NO production (Du et al. 2010).
22.5 Conclusion and Future Prospectives
Deep-sea fungi known as piezophilic fungi are an important and novel source of
bioactive secondary metabolites including mostly in alkaloids, lactones, polyketides,
terpenoids, peptides, and steroids categories. This chapter reviewed the chemical
structures and biological activities of deep-sea fungi genera including Ascomycota,
Acremonium,Aspergillus,Cladosporium,Chromocleista,Dichotomomyces,
Diaporthe,Emericella,Engyodontium,Penicillium,Phialocephala,Phoma,
Spiromastix, and Simplicillium, where Aspergillus,Penicillium, and Phialocephala
are extensively reviewed. Our chapter highlighted that fungi from the deep-sea
extreme ecosystems are the most diverse source for the production of natural
products. Research on deep-sea piezophillic fungi are limited due to challenges in
sample collection and fungal culture techniques. Meleagrin increases cytotoxicity by
the addition of acetate–mevalonate-derived C5 or C9 side chains, whereas LPS
induced NO inhibition capacity of brevicompanine E and brevicompanine H
increases by substitution at the N-6 position. Additionally, simplicilliumtides J and
verlamelins A and B antimicrobial activity depend on lactone linkage. All the new
isolated compounds screened and detected by 1D and 2D NMR, HRMS, HRESIMS,
TD-DFT, IR ESIMS, and CD. Most of the isolated compounds showed cytotoxic
and antimicrobial activity with few shows antioxidant, antifouling, anti-
inflammatory, and anti-tuberculosis activity. Synopsis of bioactive compounds
from deep-sea fungi, their sources, biological activities, and references will help
readers to better understand the bioactive compounds’prime potential as drug
candidates that further could provide solutions to new drug discovery.
Acknowledgments The authors acknowledge UGC and DST-INSPIRE for providing the funding
and the authors are thankful to the Presidency University-FRPDF fund.
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 541

References
Bao J, Zhai H, Zhu K, Yu JH, Zhang Y, Wang Y, Jiang CS, Zhang X, Zhang Y, Zhang H (2018)
Bioactive pyridone alkaloids from a deep-sea-derived fungus Arthrinium sp. UJNMF0008. Mar
Drugs 16(5):174
Chen Y, Liu Z, Liu H, Pan Y, Li J, Liu L, She Z (2018) Dichloroisocoumarins with potential anti-
inflammatory activity from the mangrove endophytic fungus Ascomycota sp. CYSK-4. Mar
Drugs 16(2):54
Chen SC, Liu ZM, Tan HB, Chen YC, Li SN, Li HH, Guo H, Zhu S, Liu HX, Zhang WM (2019)
Tersone A-G, new pyridone alkaloids from the deep-sea fungus Phomopsis tersa. Mar Drugs
17:1–14
Cheng Z, Li Y, Liu W, Liu L, Liu J, Yuan W, Luo Z, Xu W, Li Q (2019) Butenolide derivatives
with-glucosidase inhibitions from the deep-sea-derived fungus aspergillus terreus ypga10. Mar
Drugs 17(6):332
Chung YM, Wei CK, Chuang DW, El-Shazly M, Hsieh CT, Asai T, Oshima Y, Hsieh TJ, Hwang
TL, Wu YC, Chang FR (2013) An epigenetic modifier enhances the production of anti-diabetic
and anti-inflammatory sesquiterpenoids from Aspergillus sydowii. Bioorg Med Chem 21(13):
3866–3872
Deming JW (2002) Psychrophiles and polar regions. Curr Opin Microbiol 5:301–309
Du L, Feng T, Zhao B, Li D, Cai S, Zhu T, Wang F, Xiao X, Gu Q (2010) Alkaloids from a deep
ocean sediment-derived fungus Penicillium sp. and their antitumor activities. J Antibiot (Tokyo)
63(4):165–170
Evans RD, Johansen JR (1999) Microbiotic crusts and ecosystem processes. Crit Rev Plant Sci
18(2):183–225
Fan Z, Sun ZH, Liu Z, Chen YC, Liu HX, Li HH, Zhang WM (2016) Dichotocejpins A–C: new
diketopiperazines from a deep-sea-derived fungus Dichotomomyces cejpii FS110. Mar Drugs
14(9):164
Fang W, Lin X, Zhou X, Wan J, Lu X, Yang B, Ai W, Lin J, Zhang T, Tu Z, Liu Y (2014) Cytotoxic
and antiviral nitrobenzoyl sesquiterpenoids from the marine-derived fungus Aspergillus
ochraceus Jcma1F17. Med Chem Commun 5(6):701–705
Fredimoses M, Zhou X, Lin X, Tian X, Ai W, Wang J, Liao S, Liu J, Yang B, Yang X, Liu Y (2014)
New prenylxanthones from the deep-sea derived fungus Emericella sp. SCSIO 05240. Mar
Drugs 12(6):3190–3202
Fredimoses M, Zhou X, Ai W, Tian X, Yang B, Lin X, Xian JY, Liu Y, Westerdijkin A (2015)
Westerdijkin a, a new hydroxyphenylacetic acid derivative from deep-sea fungus Aspergillus
westerdijkiae SCSIO 05233. Nat Prod Res 29(2):158–162
Gao XW, Liu HX, Sun ZH, Chen YC, Tan YZ, Zhang WM (2016) Secondary metabolites from the
deep-sea derived fungus Acaromyces ingoldii FS121. Molecules 21:1–7
Guo H, Liu ZM, Chen YC, Tan HB, Li SN, Li HH, Gao XX, Liu HX, Zhang WM (2019)
Chromone-derived polyketides from the deep-sea fungus Diaporthe phaseolorum FS431. Mar
Drugs 17(3):182
Guo C, Lin XP, Liao SR, Yang B, Zhou XF, Yang XW, Tian XP, Wang JF, Liu YH (2020) Two
new aromatic polyketides from a deep-sea fungus Penicillium sp. SCSIO 06720. Nat Prod Res
34(9):1197–1205
Horikoshi K (1998) Barophiles: deep-sea microorganisms adapted to an extreme environment. Curr
Opin Microbiol 1(3):291–295
Huang Z, Nong X, Ren Z, Wang J, Zhang X, Qi S (2017) Anti-HSV-1, antioxidant and antifouling
phenolic compounds from the deep-sea-derived fungus Aspergillus versicolor SCSIO 41502.
Bioorg Med Chem Lett 27(4):787–791
Kato C, Li L, Nogi Y, Nakamura Y, Tamaoka J, Horikoshi K (1998) Extremely barophilic bacteria
isolated from the Mariana trench, challenger deep, at a depth of 11000 meters. Appl Environ
Microbiol 64(4):1510
542 T. Das et al.

Li D, Wang F, Cai S, Zeng X, Xiao X, Gu Q, Zhu WT (2007a) Two new Bisorbicillinoids isolated
from a deep-sea fungus, Phialocephala sp. FL30r. J Antibiot 60(5):317–320
Li D, Wang F, Xiao X, Fang Y, Zhu T, Gu Q, Zhu W (2007b) Trisorbicillinone a, a novel sorbicillin
trimer, from a deep sea fungus, Phialocephala sp. FL30r. Tetrahedron Lett 48(30):5235–5238
Li D, Cai S, Zhu T, Wang F, Xiao X, Gu Q (2010) Three new sorbicillin trimers,
trisorbicillinones B, C, and D, from a deep ocean sediment derived fungus, Phialocephczlcz
sp. FL3Or. Tetrahedron 66(27–28):5101–5106
Li CS, An CY, Li XM, Gao SS, Cui CM, Sun HF, Wang BG (2011a) Triazole and
dihydroimidazole alkaloids from the marine sediment-derived fungus Penicillium paneum
SD-44. J Nat Prod 74(5):1331–1334
Li DH, Cai SX, Zhu TJ, Wang FP, Xiao X, Gu QQ (2011b) New cytotoxic metabolites from a deep-
sea-derived fungus, Phialocephala sp., strain FL30r. Chem Biodivers 8(5):895–901
Li CS, Li XM, Gao SS, Lu YH, Wang BG (2013) Cytotoxic anthranilic acid derivatives from deep
sea sediment-derived fungus Penicillium paneum SD-44. Mar Drugs 11(8):3068–3076
Li XD, Li X, Li XM, Yin XL, Wang BG (2019) Antimicrobial bisabolane-type sesquiterpenoids
from the deep-sea sediment-derived fungus Aspergillus versicolor SD-330. Nat Prod Res:1–7
Liang X, Nong XH, Huang ZH, Qi SH (2017) Antifungal and antiviral cyclic peptides from the
deep-sea-derived fungus Simplicillium obclavatum EIODSF 020. J Agric Food Chem 65(25):
5114–5121
Lin A, Wu G, Gu Q, Zhu T, Li D (2014) New eremophilane-type sesquiterpenes from an Antarctic
deep-sea derived fungus, Penicillium sp. PR19 N-1. Arch Pharm Res 37(7):839–844
Liu Z, Wang Q, Li S, Cui H, Sun Z, Chen D, Lu Y, Liu H, Zhang W (2019) Polypropionate
derivatives with Mycobacterium tuberculosis protein tyrosine phosphatase B inhibitory
activities from the deep-sea-derived fungus Aspergillus fischeri FS452. J Nat Prod 82(12):
3440–3449
Luo X, Zhou X, Lin X, Qin X, Zhang T, Wang J, Tu Z, Yang B, Liao S, Tian Y, Pang X (2017)
Antituberculosis compounds from a deep-sea-derived fungus Aspergillus sp. SCSIO Ind09F01.
Nat Prod Res 31(16):1958–1962
Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzymes. Extremophiles 4(2):91–98
McDougall J (1969) Antiviral action of gliotoxin. Arch Virol 27(2–4):255–267
Moyer CL, Eric Collins R, Morita RY (2017) Psychrophiles and psychrotrophs. In: Reference
module in life sciences, pp 1–6
Niu S, Liu D, Hu X et al (2014) Spiromastixones A-O, antibacterial chlorodepsidones from a deep-
sea-derived Spiromastix sp. fungus. J Nat Prod 77:1021–1030
Niu S, Liu D, Proksch P, Shao Z, Lin W (2015) New polyphenols from a deep sea Spiromastix
sp. fungus, and their antibacterial activities. Mar Drugs 13(4):2526–2540
Niu S, Xia M, Chen M, Liu X, Li Z, Xie Y, Shao Z, Zhang G (2019) Cytotoxic polyketides isolated
from the deep-sea-derived fungus Penicillium chrysogenum MCCC 3A00292. Mar Drugs
17(12):686
Oger PM, Jebbar M (2010) The many ways of coping with pressure. Res Microbiol 161(10):
799–809
Park YC, Gunasekera SP, Lopez JV, McCarthy PJ, Wright AE (2006) Metabolites from the marine-
derived fungus Chromocleista sp. isolated from a deep-water sediment sample collected in the
Gulf of Mexico. J Nat Prod 69(4):580–584
Parkes RJ, Sellek G, Webster G, Martin D, Anders E, Weightman AJ, Sass H (2009) Culturable
prokaryotic diversity of deep, gas hydrate sediments: first use of a continuous high-pressure,
anaerobic, enrichment and isolation system for subseafloor sediments (DeepIsoBUG). Environ
Microbiol 11(12):3140–3153
Roth FJ Jr, Orpurt PA, Ahearn DG (1964) Occurrence and distribution of fungi in a subtropical
marine environment. Can J Bot 42(4):375–383
Rothschild LJ, Mancinelli RL (2001) Life in extreme environments. Nature 409(6823):1092–1101
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 543

Shang Z, Li X, Meng L, Li C, Gao S, Huang C, Wang B (2012) Chemical profile of the secondary
metabolites produced by a deep-sea sediment-derived fungus Penicillium commune SD-118.
Chinese J Oceanol Limnol 30(2):305–314
Skropeta D (2008) Deep-sea natural products. Nat Prod Rep 25(6):1131–1166
Skropeta D, Wei L (2014) Recent advances in deep-sea natural products. Nat Prod Rep 31(8):
999–1025
Soldatou S, Baker BJ (2017) Cold-water marine natural products, 2006 to 2016. Nat Prod Rep
34(6):585–626
Song F, Liu X, Guo H, Ren B, Chen C, Piggott AM, Yu K, Gao H, Wang Q, Liu M, Liu X (2012)
Brevianamides with antitubercular potential from a marine-derived isolate of Aspergillus
versicolor. Org Lett 14(18):4770–4773
Stetter KO (1999) Extremophiles and their adaptation to hot environments. FEBS Lett 452:22–25
Sun Y, Takada K, Takemoto Y, Yoshida M, Nogi Y, Okada S, Matsunaga S (2012) Gliotoxin
analogues from a marine-derived fungus, Penicillium sp., and their cytotoxic and histone
methyltransferase inhibitory activities. J Nat Prod 75(1):111–114
Takami H, Inoue A, Fuji F, Horikoshi K (1997) Microbial flora in the deepest sea mud of the
Mariana trench. FEMS Microbiol Lett 152(2):279–285
Tian L, Cai SX, Li DH, Lin ZJ, Zhu TJ, Fang YC, Liu PP, Gu QQ, Zhu WM (2007) Two new
metabolites with cytotoxicities from deep-sea fungus, Aspergillus sydowi YH11-2. Arch Pharm
Res 30(9):1051–1054
Tian Y, Qin X, Lin X, Kaliyaperumal K, Zhou X, Liu J, Ju Z, Tu Z, Liu Y (2015) Sydoxanthone C
and acremolin B produced by deep-sea-derived fungus Aspergillus sp. SCSIO Ind09F01. J
Antibiot (Tokyo) 68(11):703–706
Van Dover CL, German CR, Speer KG, Parson LM, Vrijenhoek RC (2002) Evolution and
biogeography of deep-sea vent and seep invertebrates. Science 295(5558):1253–1257
Wang J, He W, Huang X, Tian X, Liao S, Yang B, Wang F, Zhou X, Liu Y (2016) Antifungal new
oxepine-containing alkaloids and xanthones from the deep-sea-derived fungus Aspergillus
versicolor SCSIO 05879. J Agric Food Chem 64(14):2910–2916
Wang W, Gao M, Luo Z, Liao Y, Zhang B, Ke W, Shao Z, Li F, Chen J (2019) Secondary
metabolites isolated from the deep sea-derived fungus Aspergillus sydowii C1-S01-A7. Nat
Prod Res 33(21):3077–3082
Wilson ZE, Brimble MA (2009) Molecules derived from the extremes of life. Nat Prod Rep 26(1):
44–71
Woese CR, Kandler O, Wheelis ML (1990) Towards a natural system of organisms: proposal for the
domains archaea, bacteria, and eucarya. Proc Natl Acad Sci 87(12):4576–4579
Wu G, Lin A, Gu Q, Zhu T, Li D (2013) Four new chloro-eremophilane sesquiterpenes from an
Antarctic deep-sea derived fungus, Penicillium sp. PR19N-1. Mar Drugs 11(4):1399–1408
Wu G, Sun X, Yu G, Wang W, Zhu T, Gu Q, Li D (2014) Cladosins A-E, hybrid polyketides from a
deep-sea-derived fungus, Cladosporium sphaerospermum. J Nat Prod 77(2):270–275
Wu Z, Wang Y, Liu D, Proksch P, Yu S, Lin W (2016) Antioxidative phenolic compounds from a
marine-derived fungus Aspergillus versicolor. Tetrahedron 72(1):50–57
Xu X, Zhang X, Nong X, Wang J, Qi S (2017) Brevianamides and mycophenolic acid derivatives
from the deep-sea-derived fungus Penicillium brevicompactum DFFSCS025. Mar Drugs 15(2):
43
Yao Q, Wang J, Zhang X, Nong X, Xu X, Qi S (2014) Cytotoxic polyketides from the deep-sea-
derived fungus Engyodontium album DFFSCS021. Mar Drugs 12:5902–5915
Yayanos AA (1995) Microbiology to 10,500 meters in the deep sea. Annu Rev Microbiol 49(1):
777–805
Zhang Y, Arends JB, Van de Wiele T, Boon N (2011) Bioreactor technology in marine microbiol-
ogy: from design to future application. Biotechnol Adv 29(3):312–321
544 T. Das et al.

Zhang Z, Guo W, He X, Che Q, Zhu T, Gu Q, Li D (2016a) Peniphenylanes A–G from the deep-
sea-derived fungus Penicillium fellutanum HDN14-323. Planta Med 82(09/10):872–876
Zhang Z, He X, Liu C, Che Q, Zhu T, Gu Q, Li D (2016b) Clindanones A and B and Cladosporols F
and G, polyketides from the deep-sea derived fungus Cladosporium cladosporioides
HDN14-342. RSC Adv 6(80):76498–76504
Zhang Z, Min X, Huang J, Zhong Y, Wu Y, Li X, Deng Y, Jiang Z, Shao Z, Zhang L, He F (2016c)
Cytoglobosins H and I, new antiproliferative cytochalasans from deep-sea-derived fungus
Chaetomium globosum. Mar Drugs 14(12):233
ZoBell CE, Johnson FH (1949) The influence of hydrostatic pressure on the growth and viability of
terrestrial and marine bacteria. J Bacteriol 57(2):179
22 Piezophilic Fungi: Sources of Novel Natural Products with Preclinical and... 545

Biotechnological Applications
of Microaerophilic Species Including
Endophytic Fungi
23
Beenish Sarfaraz, Mehwish Iqtedar, Roheena Abdullah,
and Afshan Kaleem
Abstract
Fungi are very diverse in nature and can be found in any ecosystem. Until now
around 10,000 species have been characterized and many remain to be identified.
Fungi play an integral role in the stability of an ecosystem as they perform a
multitude of environmental tasks including but not limited to the decomposition
of organic matter, forming mutualistic mycorrhizal association with plants, and
cause of some diseases. They are capable of growing in different environmental
conditions, i.e., low and high oxygen environments, temperatures, and pH values.
Fungi can grow in a wide range of oxygen gradients. Their unique characteristics
combined with extensive metabolic activity under different oxygen conditions
make them valuable in many biotechnological applications. In this chapter,
different fungal species have been explored for their biotechnological potential
under microaerophilic conditions. Microaerophilic conditions are deemed most
suitable for ethanol and hydrocarbon production. Under microaerophilic
conditions fungi can effectively degrade industrial dyes and produce cheese,
carotenoid, and even nanoparticles. Different fungal species have been studied
in this regard and further research is required to explore their potential under these
conditions.
Keywords
Fungi · Extremophiles · Microaerophilic fungi · Fungal biodegradation · Fungal
enzymes
B. Sarfaraz
Department of Biology, Lahore Garrison University, Lahore, Pakistan
M. Iqtedar (*) · R. Abdullah · A. Kaleem
Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_23
547

23.1 Introduction
The biosphere comprises a multitude of ecosystems and biomes. Each ecosystem has
a characteristic unique environment that caters to its biotic community with appro-
priate living conditions, i.e., habitat, food, energy, biomass, soil, oxygen, etc.
(Haines and Potschin 2005). Oxygen is one of the key requirements of all living
organisms. Each habitat has different oxygen gradients which contribute to diverse
microbial communities. Bacterial and fungal communities thrive in diverse oxygen
concentrations. Some prefer high O
2
levels, while some are habituated to oxygen-
free environments. Fungi are considered chief decomposers in the biosphere along
with bacteria. They are present in different ecosystems thriving in deep marine
sediments of the Indian Ocean (Raghukumar and Raghukumar 1998), in air over
the Atlantic ocean (Pady and Kapica 1955), in volcanic landscape (Tadiosa and
Briones 2013), in rivers (Pascoal and Cássio 2004), in soil (Garrett 1963,1981), and
in plant roots as mycorrhizal fungi (Miller and Jastrow 1992,2000; Miransari 2011;
Zhang et al. 2020).
They are powerful contributors to global carbon cycling, biogeochemical cycles,
decomposition cycles, and in mobilizing macroelements (Gadd 2006). They form
mutualistic relationships with plant roots on one hand (Zhu and Miller 2003) and act
as notorious pathogens on other hand (Anaissie et al. 1989). Microaerophiles are
generally defined as organisms that require less than 21% of O
2
for their maximum
growth (Krieg and Hoffman 1986), but that term is open to interpretation for
microbiologists and considered too restrictive. Based on oxygen demand microbes
are divided into aerotolerant anaerobes, facultative anaerobes, obligate aerobes,
obligate anaerobes, and nanoaerobes (Baughn and Malamy 2004). The term
“microaerobe”has been recently used by Morris and Schmidt (2013) which is
defined as any microbe that can respire oxygen using high-affinity terminal oxidase
within microoxic environments.
Oxygen affects microaerophiles in two ways. It could be deleterious to them or
beneficial as it can be used for generating energy in the respiration process. Some
microaerophiles can grow anaerobically or using fermentative pathways, but their
preference of using O
2
as a terminal electron acceptor during low O
2
levels sets them
apart from other categories. Also, they can survive in low O
2
levels (Krieg and
Hoffman 1986).
Fungi thrive in various oxygen conditions, most of the fungal species flourish in
aerobic conditions and they have been long considered obligate aerobes requiring
oxygen for their growth and metabolism (Tabak and Cooke 1968). However, the
relationship of fungi with oxygen varies significantly from species to species and
some can grow in a wide range of oxygen conditions based on their desired function.
Some studies have demonstrated that Blastocladiomycota clades in aquatic
environments can survive in low oxygen environments (Naranjo-Ortiz and
Gabaldón 2019). Some studies have shown that filamentous fungi and yeast species
can grow in microaerophilic conditions as well (Brown 1922; Durbin 1955; Fellows
1929; Tabak and Cooke 1968). Various studies have proved that fungi were able to
grow at low oxygen conditions like genus Trichocladium which is a saprophytic
548 B. Sarfaraz et al.

fungus found in aquatic environments and soil. It can display excellent cellulolytic
activity under microaerophilic conditions and certain Trichocladium species can
convert up to 96% of cellulose to ethanol (Eichorst and Kuske 2012). Fungal
pathogens are considered obligate aerobes requiring oxygen for pathogenesis. Can-
dida albicans, one of the common human fungal pathogens, is located in the
gastrointestinal tract which has low oxygen tensions. Similarly, Cryptococcus
neoformans also survive in low oxygen conditions in the brain. Apart from fungal
pathogens, typical soil fungi are also present in poor oxygen environments and thrive
there and perform all essential metabolic activities, which proved that fungi could
survive in low O
2
conditions due to their adaptations to survive in rapidly changing
oxygen conditions. Aspergillus fumigatus can tolerate up to 0.1% oxygen and it can
even grow anaerobically (Grahl et al. 2012). Although most of the studies are
focused on the growth of fungi in aerobic or anaerobic conditions, different studies
have been done on fungal growth and metabolism in microaerophilic conditions. As
fungi can grow across a range of O
2
levels, studies are included on the basis of the
fact that maximum activity and growth were observed under microaerophilic
conditions.
23.2 Biotechnological Applications
The potential interactions between microorganisms and the O
2
in their environment
provide a useful basis for the physiological and ecological characterization of
microorganisms. Microaerophilic fungal species are ecologically diverse with
unique features. Below are some of the major biotechnological applications of
fungi that are grown under microaerophilic conditions. Tabak et al. (1968) isolated
the fungal species of Fusarium solani,Mucor hiemalis,Geotrichum candidum,
Fusarium oxysporum,Phoma herbarum,Rhodotorula mucilaginosa,Candida
parapsilosis,Aspergillus niger,A. fumigatus,Aureobasidium pullulans,
Phialophora jeanselmei,Penicillium oxalicum, and Penicillium brevi from the
enriched soils, polluted water, and sewage sludge samples. It was observed that
many fungal species needed vitamins for their growth in microaerophilic and
anaerobic conditions apart from Geotrichum candidum,Fusarium oxysporum, and
F. solani. Different fungi metabolized organic substrates and produced cell biomass
under microaerophilic and anaerobic conditions which could help to understand their
role in bioremediation, especially in sewage treatment systems.
23.2.1 Bioethanol Production
Lignocellulosic material degradation is a widely studied topic. Various organisms
have been explored for their ability to carry out enzymatic degradation of these
complex compounds. Fungi are mostly nominated for performing this task owing to
the presence of an extensive enzyme system capable of carrying out this degradation
process. Fusarium species, Basidiomycete sp., Geotrichum sp., and Trichocladium
23 Biotechnological Applications of Microaerophilic Species Including... 549

canadense produce enzymes to hydrolyze sawdust and sugarcane bagasse under
both aerobic and microaerophilic conditions efficiently. However, these microbes
preferred low oxygen environment (5–15% O
2
) for enzyme production and growth
whereas variable lignocellulosic activity. Fungal CMCase, avicelase, and
β-glucosidase activity have been reported highest in both combined and
microaerophilic conditions (Pavarina et al. 1999; Pavarina and Durrant 2002).
Lignocellulosic biomass is widely degraded in aerobic conditions but sometimes
low O
2
conditions also support the degradation process in the soil or mud. O
2
mostly
determines the fate of lignin degradation where higher concentration accelerates the
decay process, but tree logs and trunks inhabiting fungi also take part in the decay
process despite the presence of low O
2
conditions which means low oxygen concen-
tration can also support the process (Blanchette et al. 1989). White rot fungi degrade
lignin inside wooden substrates under different gas levels (Tuor et al. 1995). Some
fungi have the capability to grow on a wide range of substrates. Trichocladium
canadense and Basidiomycete species exhibited high levels of xylanase, avicelase,
lactase, β-glucosidase, and CMase activity that could carry out cellulose fermenta-
tion by successfully consuming lignin, xylan, cellobiose, pentose, and hexose under
microaerophilic and aerobic growth conditions. However, the efficiency of both
strains achieved faster cellulose utilization under microaerophilic conditions
(Durrant 1996a,b).
Some other fungi that are equipped with biosynthetic machinery to actively
degrade recalcitrant lignin compounds in both anaerobic and microaerophilic
conditions by producing lignin-degrading enzymes are Verticillum sp. that are
microaerophilic and can tolerate 10–15% O
2
with 10% CO
2
.Basidiomycete
sp. has demonstrated higher lignin-peroxidase, manganese-peroxidase, and laccase
activities under microaerophilic conditions. Lignosulfonic acid was degraded to
27.5% by Verticillum sp. in microaerophilic conditions, whereas Fusarium
oxysporum and Aspergillus sp. showed higher LiP, Lac, and MnP activities under
microaerophilic conditions (Silva et al. 2009).
Different fungal species such as Pichia stipitis Y7124, Pachysolen tannophilus
Y2460, Kluyveromyces marxianus Y2415, and Candida shehatae Y12878 exhibited
a different rate of ethanol production under different O
2
levels, whereas under
microaerophilic conditions, Pichia stipitis produced the highest amount of ethanol:
3.5 g of ethanol per gram dry cell per day (Delgenes et al. 1986). Pichia stipitis
NRRL Y-7124 was also able to produce high sugar and ethanol from hemicellulose
acid hydrolysate of Eichhornia crassipes under microaerophilic conditions (Nigam
2002). Pichia stipites has unique physiology; it not only produces a high amount of
ethanol (41 g ethanol per liter) but also helps in bioremediation process. It has a thick
cell wall, simple nutritional needs, and high resistance to contaminants. This species
grows ideally in microaerophilic conditions and has a low sugar consumption rate
(Agbogbo and Coward-Kelly 2008).
550 B. Sarfaraz et al.

23.2.2 Hydrocarbon Production
Endophytes are fungal or bacterial species capable of surviving inside plants. They
could form a highly beneficial mutualistic relationship or a pathogenic relationship
with plants. Endophytic fungi, which are estimated to be 1.5 million fungal species,
reside in plants forming a symbiotic relationship with their hosts. Fungal strains
could not only degrade complex carbon but also can produce hydrocarbons. One
study analyzed the cellulose and hydrocarbons degrading ability of endophytic fungi
belonging to the genus Gliocladium under the microaerophilic conditions which
could help to synthesize pretreatment-free microbial biofuels. Endophytic fungi
produce various hydrocarbons including benzene, heptane, tridecane,
3,4-dimethyl, m-xylene, hexane, hexadecane, heptane, and others by using cellulosic
biomass. Cocultures of Escherichia coli and Gliocladium sp. demonstrated
100 times higher hydrocarbon production ability than the single inoculum culture
of Gliocladium sp. owing to the stability of dry mycelial mass in stationary phase in a
coculture (Ahamed and Ahring 2011).
In a host plant, Eucryphia cordifolia volatile hydrocarbons were produced from
the growth of Gliocladium roseum (NRRL 50072). This fungus synthesized exten-
sive varieties of acetic acid esters of straight-chained alkanes including those of
pentyl, hexyl, heptyl, octyl, sec-octyl, and decyl alcohols. It also produced undecane,
2,6-dimethyl; decane, 3,3,5-trimethyl; cyclohexene, 4-methyl; decane, 3,3,6-
trimethyl; and undecane, 4,4-dimethyl, and VHCs including benzene, heptane, and
octane were also produced. Fungal endophyte was also producing lipids and fatty
acids. This elaborate hydrocarbon profile of G. roseum helped to obtain a new name
“myco-diesel”for its volatiles (Strobel et al. 2010). Increased production of VHCs
under microaerophilic conditions was due to similar conditions in host tissue as
endophytes frequently develop intracellularly in plant tissues. VHCs could possibly
be produced under microaerophilic conditions in the plant apoplast (Stadler and
Schulz 2009).
An endophyte Nodulisporium sp. (designated TI-13) produced volatile organic
compound (VOC) under microaerophilic conditions in a solid state reactor utilizing
agricultural waste beet pulp as a substrate. Ester compound production rate escalated
under microaerophilic conditions. These conditions are similar to the natural grow-
ing condition of fungi inside plants, and these VOCs are a kind of protective gear of
fungi against invading organisms (Schoen et al. 2017).
23.2.3 Dye Degradation
The industrial revolution has changed human life. Industrial products are produced
using toxic chemicals which end up being discharged into water bodies, affecting the
freshwater and marine flora and fauna. Microbes have been commonly studied for
their bioremediation potentials. One possible application of microbes would be the
natural degradation of these chemicals for reducing pollution hazards. In a similar
context, a study was conducted using a consortium of fungal and bacterial species
23 Biotechnological Applications of Microaerophilic Species Including... 551

(Aspergillus ochraceus NCIM-1146 and Pseudomonas sp. SUK1) to assess their
ability to biodegrade and decolorize azo dye Rubine GFL and textile effluent. This
developed consortium-AP helped to obtain the decolorization rate of 95% and 98%
in dye and effluent, respectively, in aerobically grown A. ochraceus NCIM-1146 and
Pseudomonas sp. grown in microaerophilic conditions possibly due to involvement
of oxygen-sensitive reductase. Individually both microbes showed reduced levels of
decolorization as compared to consortium-AP. Microbes grown in aerobic
conditions showed 46% and 34% decolorization of dye, whereas microbes grown
in microaerophilic conditions demonstrated 43% and 63% dye decolorization.
Biological oxygen demand (BOD; 82%), chemical oxygen demand (COD; 96%),
and total organic carbon (TOC; 48%) were also reduced in consortium-AP-treated
textile effluent in microaerophilic conditions (Lade et al. 2012).
Azo dyes are stable compounds with various applications in textile and other
industries. They are not easily degraded due to their recalcitrant xenobiotic nature,
but different microbes can perform this important task under different oxygen
conditions. A new consortium GG-BL comprising of fungus Galactomyces
geotrichum MTCC 1360 and bacterium Brevibacillus laterosporus NCIM 2298
cultures could produce reusable water by converting toxic form of Golden Yellow
HER (GYHER), a mono azo dye to fewer toxic forms demonstrating higher decol-
orization activity (98%) under microaerophilic conditions as compared to individual
microbial decolorization. Even 100% dye was degraded in consortium in the mixed
aeration condition (12 h aerobic followed by 12 h microaerophilic). COD and TOC
levels’reduction was also reported in addition to reduced phytotoxicity to dye
metabolites after decolorization in Phaseolus mungo and Sargassum vulgare plants
(Waghmode et al. 2011).
The ability of bacterial and fungal-based consortium to degrade textile effluents
under microaerophilic conditions using Aspergillus ochraceus NCIM 1146 and
Providencia rettgeri strain HSL1 was also observed by Lade et al. (2016). Consor-
tium successfully degraded 92% of ADMI textile effluents in 30 h under
microaerophilic conditions, whereas consortium only degraded 4% textile effluents
in aerobic conditions at 30 C. This study again proved the need for carrying out the
degradation of azo dyes under microaerophilic conditions. These low oxygen
conditions are present during effluent treatment plants and coculture accelerated
the degradation process due to efficient enzyme activity which also reduced
phytotoxicity of dyes (Lade et al. 2016).
Azo dye degradation under microaerophilic conditions was also exhibited from a
yeast species Issatchenkia occidentalis which is an ascomycete. More than 80% of
the dye degraded under microaerophilic conditions in 15 h. Yeast species degraded
dye during late log phase and in acidic pH. I. occidentalis managed to decolorize
dyes but in lower quantities. Reduction mostly depends on the stage of microbial
growth and enzyme activity where 1-amino-2-naphthol and N,N-dimethyl-p-
phenylenediamine, which are dye degradation products, were used as carbon and
nitrogen sources (Ramalho et al. 2004).
552 B. Sarfaraz et al.

23.2.4 Cheese Production
Cheese production is a billion dollar industry with more than 1000 varieties of
cheese around the world. Bacterial and fungal species form a rind on cheese surface
imparting unique properties to each cheese. More than 30 yeast species have been
found to be prevalent on the ripened cheese surface including Geotrichum candidum
and Debaryomyces hansenii. Yeast helps in the development of flavor and texture
due to lipolytic, proteolytic, and deacidifying activities. Geotrichum candidum is a
microaerophilic fungi with vast applications in cheese industry (Fröhlich-Wyder
et al. 2019). In the study of Livarot cheese, researchers found out that different yeast
species are involved in the fermentation process but Geotrichum candidum being the
most prevalent although it is a salt-sensitive species. It helped in curd deacidification
and aroma compound production (Larpin et al. 2006). Geotrichum candidum yeast is
able to carry out primary and secondary proteolysis while degrading casein at the
cheese surface. This action increased the concentration of free amino acids in cheese
from early weeks till late ripening (Boutrou et al. 2006).
23.2.5 Other Applications
Selenium is a trace element required in human diet for maintaining growth and
health. Elemental selenium is a nontoxic form which has great health benefits.
Selenium nanoparticles can be synthesized using different green chemistry methods
for use in the health sector. Saccharomyces cerevisiae was used in a study that
produced extracellular SeNPs ideally under microaerophilic conditions.Selenium/
protein NPs were fabricated in vivo and moved outside of the cell in vesicles in low
oxygen conditions. These NPs were approximately 100 nm in diameter and spherical
in shape. Yeast proteins act as a capping agent for NPs formation. FTIR analysis
confirmed the presence of proteins and other biomolecules in SeNPs. Microaerobic
environment supported the reduction of selenite to elemental selenium (Zhang et al.
2012).
Yeast species Phaffia rhodozyma can produce carotenoid pigments. The ability to
produce pigments depends on the culture conditions and astaxanthin was
synthesized under exponential growth phase of yeast. Xanthophyll pigment signifi-
cantly improved from 90 to 406 concentration μg (g yeast)
1
during fermentation.
Oxygen concentration dictated the optimum yields of yeasts and pigments. Under
microaerophilic conditions yeast accumulated β-carotene and the monoketone
echinenone (Johnson and Lewis 1979).
Microaerophiles are an integral part of an ecosystem and should be further
explored in order to find their applications. Due to their different nutritional require-
ment and metabolic processes, these fungi can be successfully applied to processes
where microaerophilic to anaerobic conditions prevail.
23 Biotechnological Applications of Microaerophilic Species Including... 553

References
Agbogbo FK, Coward-Kelly G (2008) Cellulosic ethanol production using the naturally occurring
xylose-fermenting yeast, Pichia stipitis. Biotechnol Lett 30:1515–1524. https://doi.org/10.1007/
s10529-008-9728-z
Ahamed A, Ahring BK (2011) Production of hydrocarbon compounds by endophytic fungi
Gliocladium species grown on cellulose. Bioresour Technol 102:9718–9722
Anaissie E, Bodey G, Rinaldi M (1989) Emerging fungal pathogens. Eur J Clin Microbiol Infect Dis
8:323–330. https://doi.org/10.1007/BF01963467
Baughn AD, Malamy MH (2004) The strict anaerobe Bacteroides fragilis grows in and benefits
from nanomolar concentrations of oxygen. Nature 427:441–444. https://doi.org/10.1038/
nature02285
Blanchette RA, Nilsson T, Daniel G, Abad A (1989) Biological degradation of wood. In:
Archaeological wood: properties, chemistry, and preservation, vol 225. ACS, Washington,
DC, pp 141–174. https://doi.org/10.1021/ba-1990-0225.ch006
Boutrou R, Kerriou L, Gassi JY (2006) Contribution of Geotrichum candidum to the proteolysis of
soft cheese. Int Dairy J 16:775–783. https://doi.org/10.1016/j.idairyj.2005.07.007
Brown W (1922) On the germination and growth of fungi at various temperatures and in various
concentrations of oxygen and of carbon dioxide. Ann Bot 36:257–283
Delgenes J, Moletta R, Navarro J (1986) The effect of aeration on D-xylose fermentation by
Pachysolen tannophilus, Pichia stipitis, Kluyveromyces marxianus and Candida shehatae.
Biotechnol Lett 8:897–900. https://doi.org/10.1007/BF01078656
Durbin RD (1955) Straight-line function of growth of microorganisms at toxic levels of carbon
dioxide. Science 121:734–735
Durrant L (1996a) Biodegradation of lignocellulosic materials by soil fungi isolated under anaero-
bic conditions. Int Biodeter Biodegr 37:189–195
Durrant LR (1996b) Ethanol production from cellulose by two lignocellulolytic soil fungi. In:
Seventeenth symposium on biotechnology for fuels and chemicals. Springer, Berlin. https://doi.
org/10.1007/978-1-4612-0223-3_36
Eichorst SA, Kuske CR (2012) Identification of cellulose-responsive bacterial and fungal
communities in geographically and edaphically different soils by using stable isotope probing.
Appl Environ Microbiol 78:2316–2327
Fellows H (1929) The influence of oxygen and carbon dioxide on the growth of ophiobolus
graminis in pure. J Agric Res 37:349–355
Fröhlich-Wyder MT, Arias-Roth E, Jakob E (2019) Cheese yeasts. Yeast 36:129–141. https://doi.
org/10.1002/yea.3368
Gadd GM (2006) Fungi in biogeochemical cycles. Cambridge University Press, Cambridge. https://
doi.org/10.1017/CBO9780511550522
Garrett SD (1963) Soil fungi and soil fertility. In: The commonwealth and international library of
science, technology, engineering and Liberal studies, vol 8., 165 seiten. 10 s. 6 d. net. Pergamon
Press, The Macmillan, London; New York. https://doi.org/10.1002/jpln.3591050110
Garrett SD (1981) Soil fungi and soil fertility: an introduction to soil mycology. Elsevier,
Amsterdam
Grahl N, Shepardson KM, Chung D, Cramer RA (2012) Hypoxia and fungal pathogenesis: to air or
not to air? Eukaryot Cell 11:560–570. https://doi.org/10.1128/EC.00031-12
Haines YR, Potschin M (2005) The links between biodiversity, ecosystem services and human well-
being. Millennium ecosystem assessment, p 18
Johnson EA, Lewis MJ (1979) Astaxanthin formation by the yeast Phaffia rhodozyma. Microbiol-
ogy 115:173–183. https://doi.org/10.1099/00221287-115-1-173
Krieg N, Hoffman P (1986) Microaerophily and oxygen toxicity. Annu Rev Microbiol 40:107–130.
https://doi.org/10.1146/annurev.mi.40.100186.000543
Lade HS, Waghmode TR, Kadam AA, Govindwar SP (2012) Enhanced biodegradation and
detoxification of disperse azo dye Rubine GFL and textile industry effluent by defined fungal-
554 B. Sarfaraz et al.

bacterial consortium. Int Biodeter Biodegr 72:94–107. https://doi.org/10.1016/j.ibiod.2012.
06.001
Lade H, Kadam A, Paul D, Govindwar S (2016) Exploring the potential of fungal-bacterial
consortium for low-cost biodegradation and detoxification of textile effluent. Arch Environ
Prot 42:12–21. https://doi.org/10.1515/aep-2016-0042
Larpin S, Mondoloni C, Goerges S, Vernoux J-P, Guéguen M, Desmasures N (2006) Geotrichum
candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese.
FEMS Yeast Res 6:1243–1253
Miller R, Jastrow J (1992) The role of mycorrhizal fungi in soil conservation. In: Mycorrhizae in
sustainable agriculture. ASA Special Publications, vol 54, pp 29–44. https://doi.org/10.2134/
asaspecpub54.c2
Miller R, Jastrow J (2000) Mycorrhizal fungi influence soil structure. In: Arbuscular mycorrhizas:
physiology and function. Springer, Berlin, pp 3–18. https://doi.org/10.1007/978-94-017-
0776-3_1
Miransari M (2011) Interactions between arbuscular mycorrhizal fungi and soil bacteria. Appl
Microbiol Biotechnol 89:917–930. https://doi.org/10.1007/s00253-010-3004-6
Morris RL, Schmidt TM (2013) Shallow breathing: bacterial life at low O
2
. Nat Rev Microbiol 11:
205–212. https://doi.org/10.1038/nrmicro2970
Naranjo-Ortiz MA, Gabaldón T (2019) Fungal evolution: diversity, taxonomy and phylogeny of the
fungi. Biol Rev 94:2101–2137
Nigam J (2002) Water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to motor fuel
ethanol by xylose-fermenting yeast. J Biotechnol 97:107–116. https://doi.org/10.1016/s0168-
1656(02)00013-5
Pady SM, Kapica L (1955) Fungi in air over the Atlantic Ocean. Mycologia 47:34–50. https://doi.
org/10.1080/00275514.1955.12024427
Pascoal C, Cássio F (2004) Contribution of fungi and bacteria to leaf litter decomposition in a
polluted river. Appl Environ Microbiol 70:5266–5273. https://doi.org/10.1128/AEM.70.9.
5266-5273.2004
Pavarina EC, Durrant LR (2002) Growth of lignocellulosic-fermenting fungi on different substrates
under low oxygenation conditions. Appl Biochem Biotechnol 98:663–677. https://doi.org/10.
1385/ABAB:98-100:1-9:663
Pavarina EC, Sette LD, Anazawa TA, Durrant LR (1999) Enzymes produced by soil fungi
following microaerobic growth on lignocellulosic materials. Appl Biochem Biotechnol 82:
153–163. https://doi.org/10.1385/ABAB:82:2:153
Raghukumar C, Raghukumar S (1998) Barotolerance of fungi isolated from deep-sea sediments of
the Indian Ocean. Aquat Microb Ecol 15:153–163. https://doi.org/10.3354/ame015153
Ramalho PA, Cardoso MH, Cavaco-Paulo A, Ramalho MT (2004) Characterization of azo reduc-
tion activity in a novel ascomycete yeast strain. Appl Environ Microbiol 70:2279–2288. https://
doi.org/10.1128/AEM.70.4.2279-2288.2004
Schoen HR, Knighton WB, Peyton BM (2017) Endophytic fungal production rates of volatile
organic compounds are highest under microaerophilic conditions. Microbiology 163:1767–
1777. https://doi.org/10.1099/mic.0.000555
Silva SI, Grossmanb M, Durranta RL (2009) Degradation of polycyclic aromatic hydrocarbons (2-7
rings) under microaerobic and very-low-oxygen conditions by soil fungi. Int Biodeter Biodegr
63(2):224–229. https://doi.org/10.1016/j.ibiod.2008.09.008
Stadler M, Schulz B (2009) High energy biofuel from endophytic fungi? Trends Plant Sci 14:353–
355. https://doi.org/10.1016/j.tplants.2009.05.001
Strobel GA, Knighton WB, Kluck K, Ren Y, Livinghouse T, Griffin M, Spakowicz D, Sears J
(2010) The production of myco-diesel hydrocarbons and their derivatives by the endophytic
fungus Gliocladium roseum (NRRL 50072). Microbiology 156:3830–3833
Tabak HH, Cooke WB (1968) Growth and metabolism of fungi in an atmosphere of nitrogen.
Mycologia 60:115–140
23 Biotechnological Applications of Microaerophilic Species Including... 555

Tadiosa ER, Briones RU (2013) Fungi of Taal volcano protected landscape, southern Luzon,
Philippines. Asian J Biodivers 4:46–64
Tuor U, Winterhalter K, Fiechter A (1995) Enzymes of white-rot fungi involved in lignin degrada-
tion and ecological determinants for wood decay. J Biotechnol 41:1–17. https://doi.org/10.1016/
0168-1656(95)00042-O
Waghmode TR, Kurade MB, Khandare RV, Govindwar SP (2011) A sequential aerobic/
microaerophilic decolorization of sulfonated mono azo dye Golden yellow HER by microbial
consortium GG-BL. Int Biodeter Biodegr 65:1024–1034. https://doi.org/10.1016/j.ibiod.2011.
08.002
Zhang L, Li D, Gao P (2012) Expulsion of selenium/protein nanoparticles through vesicle-like
structures by Saccharomyces cerevisiae under microaerophilic environment. World J Microbiol
Biotechnol 28:3381–3386. https://doi.org/10.1007/s11274-012-1150-y
Zhang HS, Zhou MX, Zai XM, Zhao FG, Qin P (2020) Spatio-temporal dynamics of arbuscular
mycorrhizal fungi and soil organic carbon in coastal saline soil of China. Sci Rep 10:1–13.
https://doi.org/10.1038/s41598-020-66976-w
Zhu Y-G, Miller RM (2003) Carbon cycling by arbuscular mycorrhizal fungi in soil–plant systems.
Trends Plant Sci 8:407–409. https://doi.org/10.1016/S1360-1385(03)00184-5
556 B. Sarfaraz et al.

Whole Cell Application Potential
of Extremophilic Fungi in Bioremediation 24
Sunil Bhapkar, Rushikesh Pol, Deeksha Patil, Anupama Pable,
and Umesh U. Jadhav
Abstract
Various microorganisms are known for their potential to metabolize complex
pollutants and this ability of microorganisms has been utilized in remediation
processes. The bioremediation process in which the fungi are used is referred to as
myco-remediation. When it comes to the whole cell application of
microorganisms at extreme contaminated sites, fungi are a good choice because
of their wide habitats and ability to digest complex pollutants. They either
catalyze the contaminants through extracellular secretion of active components
or may uptake them inside the cells. The source sites for such kind of
extremophiles are either natural sources like hot springs, cold deserts, soda
lakes, or man-made contaminated sites. These fungi are isolated from such sites
and enriched for their potential to metabolize the contaminants. Many of such
fungal organisms have been identified and applied for their potential to remove
the contaminants like heavy metals, radioactive elements, hydrocarbons, phenols
and phenolic derivatives, etc. from the polluted sites. This chapter reviews
various reports on extremophilic fungi and their whole cell application potential
in bioremediation.
Keywords
Fungi · Bioremediation · Extremophiles · Heavy metals · Radioactive elements ·
Hydrocarbons · Phenols
S. Bhapkar · R. Pol · D. Patil · A. Pable · U. U. Jadhav (*)
Department of Microbiology, Savitribai Phule Pune University, Pune, Maharashtra, India
e-mail: ujadhav@unipune.ac.in
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_24
557

24.1 Introduction
Increasing globalization and population explosion gave rise to increasing industrial-
ization which ultimately led to tremendous increase in pollution in developed as well
as developing countries. Various pollutants introduced into the environment may
include polyaromatic hydrocarbons (PAHs), phenol and phenol derivatives, heavy
metals, etc. Many of them are toxic as well as recalcitrant providing harm to
biosphere in different ways. There are various methods like physical, chemical,
and biological methods to remove or clean up these toxic compounds from the
contaminated sites including soil and water.
Each method has its own advantages and limitations. When biological agents are
used to serve the purpose, it is called as bioremediation (Fig. 24.1). Bioremediation
is defined as the use of living organisms to reduce or eliminate environmental
hazards resulting from accumulation of toxic chemicals and other hazardous
compounds (Gibson and Saylor 1992). In other words, it is a process in which the
waste and hazardous materials are transformed into non-hazardous or less-hazardous
Fig. 24.1 The concept of bioremediation
558 S. Bhapkar et al.

substances by microorganisms such as bacteria, fungi, and algae. Nowadays, they
are deliberately released into the polluted environment to clean up the pollutants.
24.2 Bioremediation
Different life forms like plants, bacteria, fungi, and algae have been shown potential
for bioremediation. Depending on the contaminated site (like soil, water, etc.) their
selection is favored. When the plants are used to serve the purpose, it is referred to as
phytoremediation while the use of fungi is called mycoremediation. Most of the
agents are isolated from contaminated sites or natural reserved sites. A study
reported that a number of microorganisms are widely distributed in nature which
can be used for pollutant degradation. These microorganisms can be enriched to
remove various contaminants from the contaminated sites (Zobell 1946). Figure 24.2
shows the various strategies used by microorganisms to remove the contaminants
and includes the following processes. (a) Biosorption is metabolically independent
process and is based on ionic interactions between the extracellular surface of
biomass and metal ions. (b) In bioaccumulation, microorganisms use proteins to
absorb metal ions inside their intracellular space, (c) while bioprecipitation is a
process where soluble metal ions are immobilized through redox reactions, enzymes,
and metabolites present on the extracellular surface of microorganisms.
(d) Bioreduction is a transformation process where toxic metals/metalloids are
altered to non-toxic elements through biological reduction and oxidation process.
(e) Lastly bioemulsification process makes use of proteins or metabolites to form
emulsions in two immiscible liquid phases (Jeong and Choi 2020). A number of
Fig. 24.2 Microbial bioremediation strategies for the removal of diverse toxic pollutants (Jeong
and Choi 2020)
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 559

microorganisms are capable of using toxic organic compounds as the sole source of
carbon and energy to drive their metabolism. This ability of transforming or
hydrolyzing toxic compounds into nontoxic or less toxic compounds is by the virtue
of their biochemical pathways. Since, the emergence of genetic engineering, various
tools and techniques are being used to enhance the natural remediation potential of
these agents. These organisms may perform their metabolism under aerobic, anaer-
obic, or facultative aerobic/anaerobic conditions. The applications of fungal
microorganisms in bioremediation have been well known as fungal bioremediation
or mycoremediation. Fungi are competent for metabolizing various environmental
chemicals and utilize its product for survival without any additional need for
nutrition. The fungi decompose the biomass residues and chemical pollutants by
producing several enzymes. These microorganisms are used for the biotransforma-
tion of organic waste materials and the removal of pollutants from the environment
(Balabanova et al. 2018). They are also adapted to survive in diverse conditions
continuously. Though a wide range of fungal species is used for the treatment of
various pollutants like heavy metals, various hydrocarbons and pesticides, radioac-
tive compounds, etc., this chapter focuses on the applications of only extremophilic
fungi used in bioremediation.
24.3 Extremophilic Fungi for Bioremediation
Many species belonging to fungi can able to survive and sustain themselves in
extreme habitats such as extreme temperature and pressure, high salinity, acidic and
alkaline substrate or soil, and limited nutrient conditions (Abdel-Hafez 1982). Along
with natural extreme habitat, fungal species have also been isolated from man-made
extreme habitats such as the acidic and alkaline industrial effluent plant of wastewa-
ter (Kumar and Dwivedi 2019), heavy metal contaminated soil, and hazardous
chemicals contaminated industrial (Bordjiba et al. 2001) and agricultural (Zafar
et al. 2007) areas. Considering the harsh environment of contaminated soil and
water, it is very essential for organisms to sustain within a harmful environment of
pollutants, toxins chemicals, and heavy metals, and to remove the hazardous effect
of contaminants. The ability to grow in the above-mentioned extreme conditions
thus makes extremophilic fungi a potential eco-friendly candidate for bioremedia-
tion. Various strategies are used by fungi for their survival in these extreme
conditions and those properties are essential for the selection of these fungi in the
bioremediation process. The enzymes produced by extremophiles are considered
superior than mesophilic enzymes as they are able to perform reaction under extreme
and adverse conditions in which conventional proteins are completely denatured.
The common extremophilic enzymes are the oxidative enzymes such as peroxidases,
oxygenases, laccases, etc., and these enzymes producing fungi have shown a role in
the bioremediation of contaminated water and soil. Peroxidases catalyze the degra-
dation of lignin and phenolic compounds. The role of fungal cytochrome P450
monooxygenase has been studied for detoxification of toxic compounds from soil
and water (Hernández-López et al. 2016). Laccases are oxidases, the enzymes
560 S. Bhapkar et al.

capable of oxidization of a large number of phenolic and non-phenolic compounds
(Arregui et al. 2019). Fungi are one of the organisms having ability to produce higher
amount of laccase and have been applied for bioremediation of phenolic compounds,
aromatic hydrocarbons (Tavares et al. 2009), and dyes (Iark et al. 2019; Yin et al.
2019). Filamentous fungi are suitable and low cost candidates for biosorption and
adsorption of heavy metals by using either living or inactivated biomass (Ting and
Choong 2009; Cai et al. 2016). Further, use of halophilic fungi increases the
efficiency of biosorption under the conditions found at heavy metal contaminated
sites (Bano et al. 2018; Ali et al. 2019). The application of filamentous fungi may be
advantageous for those situations where translocation of essential factors (nutrients,
water, the pollutant itself, and so on) is required for the transformation or detoxifica-
tion of the pollutant (Harms et al. 2011).
24.4 Need of Extremophilic Fungi in Bioremediation
Different methods applied for bioremediation include natural attenuation,
biostimulation, and bioaugmentation, or a combination thereof.
These methods are environmentally friendly as well as cost-effective and can be
managed easily. When the catalysis process can be controlled in a single step, only
single cells can be applied but when it comes to the catalysis of complex environ-
mental pollutants through multiple steps, the application of consortia of different
species is must. Together, they metabolize pollutants. The degradation of the
contaminants may occur intracellularly or extracellularly depending on the nature
of the contaminant and the type of biological agent (Fig. 24.3). Considering the
example of recalcitrant compound, Fig. 24.4 depicts the various mechanisms used by
fungi to remove/detoxify the recalcitrant compound from the affected site
(Deshmukh et al. 2016).
However when it comes to the recreation of contaminated sites having extreme
conditions (like pH, salinity, radiations, or temperature) of normal species cannot
perform their function efficiently. This raises the need for extremophiles which can
grow easily at the extreme sites and carry out bioremediation more efficiently.
Microorganisms from extreme natural or man-made environments provide robust
enzymatic and whole cell biocatalytic systems that are attractive under conditions
that limit the effectiveness of typical bioconversions. A variety of extremophilic
systems adapted to specific niche environments have been shown in Fig. 5.
These bioconversion operations attempt to augment naturally occurring remedia-
tion capacity and accelerate reactive processes by removing limitations on microbial
action. Many environmental sites are contaminated with multiple pollutants and
support multiple modes of extremophily that operate in concert to facilitate chemical
transformation. In order to apply the extreme microorganisms, scientists have
deepened their knowledge and understanding of molecular systems that regulate
extremozyme action, stability, and expression. Similarly, they are focusing on
insights into metabolic strategies for whole-cell catalysis under extreme conditions.
Technological advances in the recruitment of extremophilic systems for remediation
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 561

operations are made through evaluating selective conditions that dominate
contaminated environments, identifying extremophilic organisms that can grow
and adapt in contaminated environments, and enhancing the remediation capacity
with the help of genetic engineering into organisms that can survive under extreme
selective pressures (Peeples 2014). Various extremophilic organisms along with
their sources are mentioned in Fig. 24.5. These sources include deep-sea hydrother-
mal vents, hot springs, sulfataric fields, soda lakes, inland saline systems, solar
salterns, hot and cold deserts, environments highly contaminated with nuclear
waste or heavy metals as well as lithic or rock environments.
24.5 Heavy Metal Tolerant Fungi
24.5.1 Metal Tolerants from Coastline, Mangroves, and Salterns
Nazareth and Marbaniang (2008) from India isolated four different heavy metal
tolerant species based on morphological differences. Their isolates were collected
from a well-situated close to copper smelting plant near the coastline (WP), from the
mangroves (MP), and from salterns (SP) of Goa, India. Based on their penicillial
heads morphology, WP1 was biverticillate symmetric, MP2 observed as
Fig. 24.3 Schematics of intracellular and extracellular inactivation/degradation of contaminants
562 S. Bhapkar et al.

Fig. 24.4 Mechanisms adopted by fungi for bioremediation of toxic, recalcitrant compounds (Deshmukh et al. 2016)
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 563

Fig. 24.5 Various types of extremophiles applied in the process of bioremediation
564 S. Bhapkar et al.

biverticillate asymmetric, MP4 terverticillate, and SP10 monoverticillate. Further,
they were compared for heavy metal resistance against the presence of lead, copper,
and cadmium salts. The cultures were grown on Czapek-Dox agar supplemented
with concentrations of Pb(NO
3
)
2,
CuSO
4
.5H
2
O, and Cd(NO
3
)
2
.4H
2
O. The growth
was examined in terms of colony diameter. All four cultures were resistant to Pb
2+
at
a concentration of 7.5 mM, with no decrease in growth up to 5 mM; WP1 and MP2
were resistant to 2 mM Cu
2+
and very slightly to 3 mM Cu
2+
, and MP4 was resistant
to 5 mM Cu
2+
, while SP10 showed no resistance to Cu
2+
; WP1 could resist Cd
2+
as
Cd(NO
3
)
2
up to 1 mM but not as CdSO
4
, while MP2 and SP10 could resist only
CdSO
4
at 1 mM concentration; MP4 tolerated both nitrate and sulfate salts of
cadmium up to 5 mM and 4 mM, respectively. Thus, lead was the most easily
tolerated of the heavy metals tested and caused the least variations in cultural and
morphological characteristics, while cadmium was the most toxic, causing signifi-
cant cultural and morphological variations. More striking was the observation that
resistance to the heavy metals was highest by the terverticillate Penicillium, decreas-
ing in the biverticillate isolates, the monoverticillate isolate showing the least
resistance (Nazareth and Marbaniang 2008). Massaccesi et al. (2002) worked on
Cd removal using filamentous fungi isolated from heavily polluted streams near to
some Argentinian industrial area. The identified species included Aspergillus
terreus,Clodosporium clodosporioides,Fusarium oxysporum,Gliocladium roseum,
Penicillium spp.,Talaromyces helices, and Trichoderma koningii. The concentration
of Cd in the sediments from where these fungal species were isolated ranges between
0.25 and 0.50 mg/L. They were isolated in Cd basal medium. Penicillium spp. was
able to grow and remove a 100-fold higher Cd level after 13 days of incubation by an
absorption process. Most of the above-mentioned species were found highly efficient
in detoxifying Cd and helping in remediation of chronically contaminated sites
(Massaccesi et al. 2002).
24.5.2 Metal Biosorption by Obligate Halophilic Fungi
Bano et al. (2018) studied a halophilic group of fungal species having the ability to
perform biosorption of heavy metals from contaminated sites. The obligate halo-
philic fungal species comprised of Aspergillus flavus,Aspergillus gracilis,Aspergil-
lus penicillioides (sp. 1), Aspergillus penicillioides (sp. 2), Aspergillus restrictus,
and Sterigmatomyces halophilus. The heavy metals included cadmium, copper,
ferrous, manganese, lead, and zinc. During studies, the metals were supplemented
as salts in media for the growth of obligate halophilic fungi and incubated for
14 days. Amongst all the fungi, A. flavus (86%) and S. halophilus (83%) exhibited
best average adsorption of all the heavy metals. The rest of all the tested fungi
showed moderate to high adsorption of heavy metals. On an average, Fe and Zn were
best removed from the liquid media with an average of 85 and 84%, respectively.
The study involved the use of inexpensive media in stagnant conditions which
provides a cost-effective environmental solution for the removal of heavy metals
(Bano et al. 2018).
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 565

24.5.3 Bioremediation of Cadmium by Penicillium chrysogenum XJ-1
Xu et al. (2015) conducted interesting studies using the ability of cadmium bioreme-
diation by Penicillium chrysogenum XJ-1. Using physicochemical and biochemical
methods, they investigated the morphological and antioxidative response of the
fungus to different concentrations of Cd (1–10 mm). They found that with the
increase in Cd concentration, malondialdehyde level was increased by
14.82–94.67 times and Cd was mainly bound to the cell wall of the fungus. The
results also reported that the biosorption, cellular sequestration, and antioxidant
defense were involved in Cd detoxification. At 1 mM Cd concentration, superoxide
dismutase (SOD), glutathione reductase (GR), and glucose-6-phosphate dehydroge-
nase (G6PDH) levels were increased and at 5 mM concentration, catalase level was
peaked. The glutathione/oxidized glutathione ratio was also increased on exposure to
Cd. The group also studied in situ application of the XJ-1 fungus where they sowed
pak choi plant seeds in the Cd polluted soil (5–50 mg/kg) and found that plant yield
was increased through reduced Cd bioavailability (Xu et al. 2015). The in situ results
obtained signify the potential importance of the fungus to remediate Cd polluted
sites as a promising candidate (Table 24.1).
24.6 Radio Tolerant Species
24.6.1 Uranium Tolerant Saccharomyces
Strandberg et al. (1981) studied the uranium accumulation ability of Saccharomyces
cerevisiae and Pseudomonas aeruginosa.S. cerevisiae accumulates the element on
extracellular surface while Pseudomonas aeruginosa accumulates it intracellularly.
Additionally, it was found superior to S. cerevisiae in terms of accumulation rate.
Environmental parameters such as pH, temperature, and interference by certain
anions and cations affected the rate and accumulation ability of S. cerevisiae.
Pseudomonas aeruginosa observed not giving any response to environmental
parameters. Both the organisms work irrespective of metabolism for metal uptake.
Formaldehyde and HgCl
2
pretreatments increased rate of uranium uptake by
S. cerevisiae cells but it did not affect the other. The solution of the pH and
temperature both considerably affected the metal uptake by S. cerevisiae. The
accumulated uranium could be removed chemically from S. cerevisiae cells, and
the cells could then be reused as a biosorbent (Strandberg et al. 1981).
24.6.2 Uranium and Thorium Biosorption by Rhizopus arrhizus
Tsezos and Volesky (1981) from Canada studied biosorption of uranium and
thorium by various living beings and materials. They found that Rhizopus arrhizus
at pH 4 exhibited the highest biosorption capacity for both the elements in excess of
180 mg/g. It was also found that R.arrhizus removed approximately 2.5 and 3.3
566 S. Bhapkar et al.

times more uranium than the ion exchange resin and activated carbon, respectively
when uranium was placed at the equilibrium concentration of 30 mg/L. Talking
about thorium, under the same conditions, R. arrhizus removed 20 times more
thorium than the ion exchange resin and 2.3 times more than the activated carbon.
R. arrhizus showed higher uptake and a generally more favorable isotherm for both
uranium and thorium than all other biomass types examined in the studies. Other
biomass used included Aspergillus niger,Aspergillus terreus,Penicillium
chrysogenum,Pseudomonas fluorescens,Streptomyces niveus (Tsezos and Volesky
1981) (Table 24.2).
24.7 Hydrocarbon Degradation
24.7.1 n-Alkanes Degradation
Barnes et al. (2018) conducted a study to identify potential hydrocarbon degrading
fungal species from marine niches in India. In their study, they identified 10 potential
isolates using ITS rDNA sequencing. Out of ten, six are Aspergillus spp., two are
Fusarium, and one each of Penicillium and Acremonium spp. The sources included
mangrove sediments, Arabian Sea sediments, and tarballs. Penicillium citrinum
(#NIOSN-M126) showed the highest efficiency of 77% reduction in crude oil
content. For the identification, they used a mineral salt medium with 5% petrol as
a sole carbon source. The isolates that were able to grow in the medium and showing
increased biomass were selected. The 10 isolates were coded as IOSNM113,
NIOSN-M126, NIOSN-M142, NIOSN-M109, NIOSN-SK56S57, NIOSN-
Table 24.1 Metalophilic fungal species
S. no. Fungal sp.
Heavy
metal Reference
1Penicillium sp. Pb, Cu, Cd Nazareth and
Marbaniang
(2008)
2A. terreus,C. clodosporioides,F. oxysporum,
G. roseum,Penicillium spp.,T. helices,T. koningii
Cd Massaccesi et al.
(2002)
3A. flavus,A. gracilis,A. penicillioides,A. restrictus,
S. halophilus
Cd, Cu, Fe,
Mn, Pb, Zn
Bano et al.
(2018)
4P. simplicissimum Cd and Zn Fan et al. (2008)
5P. chrysogenum Cd Holan and
Volesky (1995)
6P. canescens Cd, Pb, As,
Hg
Say et al. (2003)
7P. purpurogenum Cr Say et al. (2004)
8P. cyclopium Cu Ianis et al.
(2006)
9P. italicum Mn, Fe, Ni,
Co
Mendil et al.
(2008)
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 567

SK56S32, NIOSN-SK56S22, NIOSN-SK56C42, NIOSN-T5, and NIOSN-T4. Four
of them obtained from mangrove environments (NIOSN-M113, NIOSN-M126,
NIOSN-M142, and NIOSN-M127), four from marine environments (NIOSN-
SK56S57, NIOSNSK56S32, NIOSN-SK56S22, and NIOSN-SK56C42), and two
from tarballs (NIOSN-T5 and NIOSN-T4). For qualitative analysis, gas chromatog-
raphy was performed to detect n-alkanes in the incubated medium. Penicillium
citrinum NIOSN-M126 showed the highest efficiency of degradation of the long-
chain n-alkanes (C29–C20), while Aspergillus sydowii NIOSN-SK56C42 was least
efficient (Barnes et al. 2018). Ferrari et al. (2011) from Australia recovered 91 fungal
species from contaminated and non-contaminated soils from sub-Antarctic
Macquarie Island and evaluated their potential for hydrocarbon degradation. In
their studies, they used both of traditional high nutrient and a low nutrient broth
approach to recover greater fungal species diversity from both pristine and SAB
(special Antarctic blend) diesel fuel contaminated soil. The species were identified
using standard molecular techniques targeting the highly variable ITS region. The
total of 91 recovered fungi includes 63 unidentified fungal species. Few of the
identified species include Trichophyton sp.,Antarctomyces psychrotrophicus,
Pseudeurotium bakeri,Aspergillus fumigatus,Penicillium sp., etc. Few of the
recovered species (Cladophialophora and Exophiala genera) have also been reported
for their potential to breakdown aromatic hydrocarbons (Ferrari et al. 2011).
Borowik et al. (2017) studied fungal diversity in diesel contaminated sites. In their
270-day study, it was found that the fungal diversity was altered than that of the
unpolluted site. In diesel oil polluted soil, the colony development index was
significantly increased but the fugal diversity index was decreased. The polluted
soil was characterized by higher activity of oxidoreductases and a higher number of
fungi as compared to unpolluted soil but the lasting effect of diesel oil led to a
decrease in functional diversity of fungal communities. The studied soil was not
subjected to remediation activities. After 270-day study, 64% of four-ringed, 28% of
five-ringed, 21% of 2–3 ringed, and 16% of six-ringed PAHs underwent degrada-
tion. The polluted soil included mainly Fusarium (37.9%), Candida (13.8%),
Microsporum (13.8%), Penicillium (13.8%) genera, and the unpolluted soil showed
the dominance of Penicillium (26.5%), Microsporum (21.1%), Fusarium (21.1%),
and Candida (15.8%). It is possible to reduce the hydrocarbons considerably using
identified fungal species (Borowik et al. 2017).
Table 24.2 Uranium and thorium tolerant fungi
S. no. Organism Radioactive element Reference
1S. cerevisiae,P. aeruginosa Uranium Strandberg et al. (1981)
2R. arrhizus Uranium and thorium Tsezos and Volesky (1981)
568 S. Bhapkar et al.

24.7.2 High Molecular Weight PAHs Degradation by Bacteria–Fungi
Coculture
An Australian research group investigated the biodegradation of high-molecular-
weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil. They
used bacterial Stenotrophomonas maltophilia (VUN 10,009 and 10,010) and a
fungus Penicillium janthinellum (VUO 10,201) which were isolated from separate
creosote and manufactured-gas plant-contaminated soils. The bacteria used pyrene
as its sole carbon and energy source in a basal salt medium (BSM) and successfully
mineralized significant amount of benzo[a]pyreneco metabolically when pyrene was
also present in BSM. P. janthinellum (VUO 10,201) alone failed to utilize any high
molecular weight PAH as the sole carbon and energy source. But when cultured in
nutrient broth, it partially degraded them. The axenic cultures do not grow signifi-
cantly though they support degradation of small amounts of chrysene, benz[a]
anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene in BSM containing a single
PAH. However, when either bacterial consortium VUN 10,009 or VUN 10,010
cocultivated with P. janthinellum VUO 10,201 (fungal–bacterial cocultures), signif-
icant degradation along with microbial growth was achieved. It was achieved on
pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene,
each as a single PAH in BSM. The cocultures mineralized about 25% of the benzo[a]
pyrene to CO
2
over 49 days, accompanied by transient accumulation and disappear-
ance of intermediates detected by high-pressure liquid chromatography. When these
fungal-bacterial cocultures were inoculated into PAH-contaminated soil, it signifi-
cantly improved degradation of high-molecular-weight PAHs, benzo[a]pyrene min-
eralization (53% of added [
14
C]benzo[a]pyrene was recovered as
14
CO
2
in
100 days), and reduction in the mutagenicity of organic soil extracts, compared
with the indigenous microorganisms and soil amended with only axenic inocula
(Boonchan et al. 2000).
24.7.3 Heavy Hydrocarbon Degradation by Bioaugmentation
of Native Fungi
Medaura et al. (2021) applied bioaugmentation of fungal species to cure aged
industrially polluted soil containing heavy hydrocarbons. They applied defined
consortium of six potentially hydrocarbonoclastic fungi belonging to the genera
Penicillium,Ulocladium,Aspergillus, and Fusarium in the microcosm assays for
bioaugmentation and biostimulation. All the fungi were previously isolated from
polluted sites. The bioaugmentation applied degradation performance was compared
with biostimulation (water and nutrient addition) as well as with untreated soil as a
control. The results obtained showed that fungal bioaugmentation degraded more
efficiently than biostimulation. In bioaugmentation microcosm, total petroleum
hydrocarbons (TPH) C14–C35 decreased by 39.90 1.99% while biostimulated
microcosms resulted in a 24.17 1.31% reduction in TPH (C14–C35). Similarly,
the effect on high molecular weight polycyclic aromatic hydrocarbons
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 569

(HMW-PAHs) was also studied. In bioaugmentation microcosm, it was found that
the five-ringed benzo(a)fluoranthene and benzo(a)pyrene were reduced by 36% and
46%, respectively, while the six-ringed benzoperylene decreased by 28%, after
120 days of treatment. On the other hand, biostimulation microcosm showed a
reduction of five- and six-ringed PAHs only 8% and 5% respectively. Further studies
revealed that bioaugmentation promoted the growth of autochthonous active
hydrocarbon-degrading bacteria (Medaura et al. 2021).
24.7.4 Phenanthrene Removal by Penicillium frequentans
The study conducted by Meléndez-Estrada et al. (2006) with Penicillium
frequentans showed that oxygen concentration has a significant effect on phenan-
threne removal. P. frequentans was grown on sugarcane bagasse pith mixed with
soil spiked supplemented with 200 mg/L of phenanthrene to obtain a final bagasse/
soil ratio of 1:16.The C/N ratio was adjusted to 60 and moisture content adjusted to
40%. Various oxygen concentrations were adjusted to 20%, 10%, 5%, and 2% and
almost 0% in the soil gas phage for each treatment. A high removal rate was obtained
for the higher oxygen concentrations, reaching 52% removal after 17 days with 20%
oxygen. An opposite phenanthrene removal trend was found under low oxygen
concentrations, reaching only 13% at close to 0% oxygen after 17 days of incuba-
tion. An explanation was presented based on the adsorption of soil components due
to the higher content in organic matter and clay (Meléndez-Estrada et al. 2006).
24.7.5 Fluorene Degradation by Absidia cylindrospora
Garon et al. (2002) studied fluorine degradation in soil slurry using various fungal
isolates and with reference to bioaugmentation. Totally, 47 fungal species were
isolated from a contaminated site and amongst them, Absidia cylindrospora was
found to be the most potent one. The strain was further used for bioaugmentation
process where more than 90% degradation rate was achieved. In the absence of
bioaugmentation process, it took 576 h to achieve the same rate and the
bioaugmentation process reduced the required time to 288 h. Malstosyl-cyclodextrin
improves the solubility of fluorine by forming inclusion complexes. This helps to
enhance the bioavailability of fluorine in the soil slurry. The study helps to utilize
Absidia cylindrospora as a potent tool for fluorine degradation in soil through
bioremediation process (Garon et al. 2004). In their prior studies, Garon et al.
(2002) also found that the addition of surfactant Tween 80 enhances the biodegra-
dation of fluorine by Doratomyces stemonitis and Penicillium chrysogenum. In the
case of Doratomyces stemonitis, Tween 80 concentration of 0.324 mM resulted
change in biodegradation from 46% to 62% in 2 days. Similarly, the change for
Penicillium chrysogenum with the same Tween 80 concentration within the same
time period is from 28% to 61%.
570 S. Bhapkar et al.

24.8 Degradation of Phenol and Its Derivatives
24.8.1 Biodegradation of Phenols at Low Temperatures
Phenol is toxic even at low concentrations to many living organisms. This retards the
growth of many biodegradative agents at the contaminated sites and affects the
process of bioremediation. The reporting of cold adapted microorganisms tolerant to
phenol is a hope for phenol degradation at low temperature sites. Margesin et al.
(2005) isolated two phenol degrading psychrophilic fungal species, both from
Rhodotorula species: Rhodotorula psychrophenolica sp. nov. (type strain AG21T
5CBS 10,438T 5DSM 18,767T) isolated from mud at the foot of a glacier (thawing
zone) and Rhodotorula glacialis sp. nov. (type strain A19T 5CBS 10,436T 5DSM
18,766T) isolated from a glacier cryoconite. These species utilized phenol concen-
tration up to 12.5 mM and 5 mM as a sole carbon source at 10 C respectively
(Margesin et al. 2007). Another fungal species Trichosporondulcitum and
Urediniomycetes isolated from hydrocarbon contaminated alpine soils showed the
potential of degrading phenol. The optimum temperature for Trichosporondulcitum
was found at 20 C while for Urediniomycetes strain it is 10 C. From the same soil
sample, two bacterial species of Rhodococcus spp. were isolated. The phenol
degrading activity of Urediniomycetes strain at 1 C was found faster than that of
Rhodococcus spp. at 10 C. Under fed-batch systems provided with mineral medium
and phenol as sole carbon source, both the bacteria and fungi were assessed for
phenol degradation at 10 C. The yeast strains degraded phenol concentration up to
15 mM while both bacteria degraded up to 12.5 mM of phenol under provided
environment (Margesin et al. 2005). Another group isolated a fungus Aureobasidium
pullulans FE13 from stainless steel effluents. It was examined for phenol degrada-
tion through immobilization and as free cells. The rate of degradation with free cells
was found to be 18.35 mg/L/h while alginate immobilized cells showed the degra-
dation rate of 20.45 mg/L/h. However, the significant information is that
immobilized cells showed longer viability compared to free cells and thus increasing
phenol degradation efficiency (dos Santos et al. 2009). Candida tropicalis HP
15 showed the phenol degradation up to 2.5 g/L through beta-ketoadipate pathway
(Krug et al. 1985).
24.8.2 Halophilic Fungi for Phenol Degradation
Jiang et al. (2016) isolated Debaryomyces sp. on the basis of phenol tolerance from
activated sludge of a pharmaceutical factory in Wuhan, China. The isolation medium
was supplied with 5% NaCl. The species showed close resemblance with
Debaryomyces hansenii and Debaryomyces subglobosus. To examine the phenol
degradation potential of the fungus, the growth medium was supplemented with
100–1300 mg/L of phenol. The growth temperature provided was 30 C at 160 rpm.
It was observed that increasing phenol concentration was associated with a decrease
in cellular biomass. In the results, it was found that phenol concentrations up to
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 571

500 mg/L degraded in a short time (around 32 h) as compared to the higher
concentrations. Concentrations above 900 mg/L required more time (96 h and
more) for complete degradation of phenol in the media. Other parameters checked
included salt and metal tolerance, optimum pH, and agitation speed. At NaCl
concentration of 1%, maximum fungal growth and phenol degradation were
achieved. The phenol degradation was affected by Co and Ni and not by Mn and
Zn. The fungus growth was maximum at neutral pH while the optimum pH value for
phenol degradation was 6.0. The agitation speed at 200 rpm helped to degrade
phenol with higher efficiency (Jiang et al. 2016).
24.8.3 Bioremediation by Specific Extremofungal Species
24.8.3.1 By Halotolerant Trichoderma
Divya et al. (2014) from Kerala studied the ability of halotolerant Trichoderma
viridae strain having the ability to remediate the phenol polluted sites. The group
found that the isolated halotolerant strain Trichoderma viridae Pers. NFCCI-2745 is
also phenol-tolerant. It produces laccase enzyme and the ability of laccase to oxidize
phenol is very well known. The source of the strain is a phenol contaminated saline
environment. Additionally, at a 5–10 ppm saline environment, enhanced laccase
production was found. The organism is resistant to even 30 ppm salinity but it results
in reduced biomass and reduced laccase production. Even, the enzyme secretion was
also found in response to phenol concentration. Different phenolic compounds were
used in this study and their optimum concentration ranges between 20 and 80 mg/L.
Beyond 200 mg/L concentration of phenolic compounds, the enzyme activity was
decreased and completely stopped at 800 mg/L. The ability of the strain can also be
utilized to remove the sites which are polluted by industrial effluents containing
phenolic compounds (Divya et al. 2014).
24.8.3.2 By Candida tropicalis
Chang et al. (1998) produced fusant of Candida tropicalis using protoplast fusion as
a selective technique. Then they studied the comparative of phenol degradation
abilities of Candida tropicalis and the produced fusant. The studies were carried
out under batch and high concentration conditions. They found that the oxygen
uptake activities of both yeast and fusant peak at pH 7.0 and 32 C. But surprisingly
the fusant was more active than the control strain. The fusant showed less suscepti-
bility to phenol inhibition than that of the control strain. The fusant also showed
better phenol degradation than the control. It was observed that when the phenol
concentration is 3300 mg/L, the ability of C. tropicalis was completely inhibited,
whereas for the C. tropicalis fusant complete inhibition is absent until the phenol
concentration is 4000 mg/L. However, they were virtually identical and remain
fairly constant at approximately 0.5 mg MLVSS/mg C6H5OH (MLVSS: mixed
liquor volatile suspended solids) for both the strains (Chang et al. 1998).
572 S. Bhapkar et al.

24.8.3.3 By Halotolerant Penicillium
A research group isolated a fungus from a salt mine in Portugal. The isolate was
enriched in the presence of phenol and high salt concentration. Further, it was
identified as a halotolerant strain of Penicillium chrysogenum and was able to
tolerate up to 5.8% NaCl in the medium.Further, it was found to utilize at least
300 mg/L of phenol as a sole carbon and energy source and did not accumulate any
intermediates. The fungus reduced the phenol toxicity under saline conditions. At
5.9% NaCl, P. chrysogenum CLONA2 was able to rapidly detoxify phenol. Degra-
dation of phenol in the presence of glucose suggested that it could proceed via
hydroquinone. Based on the results achieved, the strain can possibly be used in the
biological treatment of phenol-containing wastewaters like that of petrochemical
effluents (Leitão et al. 2007).
24.8.3.4 By Penicillium frequentans
Hofrichter et al. (1994) studied a soil isolated fungus Penicillium frequentans Bi 7/2
strain for its activity to degrade halogenated phenols. These halogenated phenols are
frequently released as major pollutants from chemical industries. The fungus was
isolated from a soil contaminated with polyaromatic hydrocarbons. The crude
extract made of the fungus was found capable of degrading various
monohalogenated phenols. The fungus also metabolized phenol derivatives like
3,4-dichlorophenol, 2,4-dichlorophenol in the presence of phenol as a cosubstrate.
The degradation process occurs through many reactions and mainly occurs because
of unspecific intracellular enzymes like phenol hydroxylase, catechol-1,2-
dioxygenase, and muconatecycloisomerase I. The authors have proposed a pathway
for the degradation process of both halogenated phenols and dichlorophenols
(Hofrichter et al. 1994).
24.9 Bioremediation at Low Temperature
Cold regions like Arctic, Alpine, and Antarctic environments are being studied for
various objectives involving human activities. They are being under focus for
petroleum exploration, production, transport, etc. Such kind of activities increases
the risk of accidental oil release in these areas (Buzzini and Margesin 2014). Various
hydrocarbon-degrading microorganisms have also been described in cold
environments. When some contaminant is introduced to their natural habitat, they
may adapt to the contamination in order to survive. This has been reported where the
introduction of pollutant induced a number of degradative microorganisms in partic-
ular environments (Aislabie et al. 2001; Margesin and Schinner 2001; Bej et al.
2010; Greer et al. 2010). Bioremediation in hydrocarbon contaminated cold
environments like aquatic and terrestrial sites has been reported (Brakstad 2008;
Filler et al. 2008; Bej et al. 2010; Greer et al. 2010). Along with this, successful
implementation of different remediation schemes like biopiles, land farming, and
engineered bioremediation have been achieved (Filler et al. 2008). Cold adapted
yeasts can be utilized for bioremediation of contaminated sites containing various
hydrocarbons like petroleum products and phenolic compounds. For soil
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 573

bioremediation, biostimulation and bioaugmentation approaches can be applied.
Growing in natural conditions imparts many limitations over the growth of
microorganisms like nutrient deprivation, pH adversity, temperature, and oxygen
conditions. These limiting conditions reduce the rate of bioremediation. When the
nutrients and optimum conditions are provided to the indigenous microorganisms, it
stimulates their growth and speeds up the bioremediation rate. This approach is
referred to as biostimulation. In bioaugmentation, external microorganisms are
added to the contaminated sites to increase the rate of the biodegradation process.
24.10 Salt Crystallization Cleanup by Halophilic Fungi
Mansour (2017) studied the cleanup of salt crystals formed on sandstones on the site
of Nile river in Egypt. Various halophilic fungi like Cladosporium
sphaerospermum,Wallemia sebi,Aureobasidium pullulans, and Aspergillus
nidulans were evaluated for their ability to clean up halites formed on the surface
of sandstones. Analytical studies like X-ray diffraction (XRD), scanning electron
microscopy (SEM), and total dilation salt (TDS) confirmed that the stones treated
with fungi showed lowered concentrations of salt as compared to control. Further,
W. sebi,A. pullulans, and A. nidulans were grown on NaCl concentration ranging
from 0 up to 25% to check growth stimulation. At 5% NaCl, maximum mycelial
growth was achieved. However, C. sphaerospermum showed best growth without
NaCl and W. sebi was able to grow even at 20% NaCl. In conclusion, it was found
that W. sebi consumed maximum halites while A. nidulans gave the worst results
showing the change in coloration of sandstone. Furthermore, W. sebi and
A. pullulans are suggested for desalination of archaeological stone monuments
(Mansour 2017).
24.11 Acidophiles in Bioremediation
Fungal acidophiles like Acidomyces acidophilus and A. richmondensis are described
for their bioremediation potential of contaminated sites where extreme conditions
like that of pH and toxic compounds are present. Especially Acidomyces acidophilus
has a great potential in bioremediation. It is a pigmented ascomycete capable of
growing in extremely acidic conditions in the pH range of 1–3 (Chan et al. 2019).
The ability of the fungus to survive in adverse environmental conditions such as
extreme pH, temperature, and toxins is by the virtue of its melanin-containing cell
wall (Hujslováet al. 2013). The intracellular enzymatic regulation mechanisms of
A. acidophilus at low pHs, reactive oxygen species, and extreme temperatures are
now widely considered as valuable resources for their exploitation in novel biotech-
nological applications (such as cleaning of contaminated soils or water) and in the
field of biocatalysis (Chan et al. 2019). The first report of Acidomyces acidophilus is
from a highly acidic, sulfate-containing industrial water. Subsequently, more
Acidomyces species were isolated and identified successfully from various extreme
574 S. Bhapkar et al.

environments (Chan et al. 2019). Another species isolated by Baker et al. (2004),
Acidomyces richmondens, is from sulfuric acid ore mine drainage in Richmond,
USA, even at pH 0.5 and 0.9 and thermophilic temperature in the range of 35–57 C
(Baker et al. 2004). Along with being acidophilic, A. acidophilus has developed
tolerance toward high concentration of toxic metals and metalloids stresses such as
Al, As, Cu, Fe, or U (Chan et al. 2018). Jambon et al. (2019) reported biodegradation
of chlorendic acid by fungi isolated from polluted sites in Belgium. Chlorendic acid
is a highly chlorinated and recalcitrant organic pollutant. The soil samples were
collected from around the roots of the plants common bent (Agrostis capillaris) and a
hybrid poplar [Populus deltoides (Populus trichocarpa P. deltoids)
cv. Grimminge]. Both plants are able to grow on chlorendic acid contaminated
sites in Belgium. Out of the 75 fungi isolated from the samples, 16 degraded
chlorendic acid between 10 and 20% in liquid media and 4 removed 20–50%.
Chlonostachys sp. 2C-2c degraded up to 71% in the liquid phase. The fungal isolates
were observed producing higher levels of hydroxyl radicals on exposure to
chlorendic acid indicating oxidative degradation of the pollutant through the pro-
duction of Fenton-mediated hydroxyl radicals. One of the ascomycetes isolate
Penicillium sp. 1D-2a was found to degrade 58% chlorendic acid in the soil after
28 days. This study gives a new perspective for in situ degradation of the recalcitrant
compound chlorendic acid (Jambon et al. 2019). Tkavc et al. (2018) have
characterized an acidophilic fungi Rhodotorula taiwanensis MD1149. The group
studied 27 fungal species to find a suitable candidate for bioremediation of acidic
radioactive environmental sites. They were having characteristics for resistance to
ionizing radiation (chronic and acute), heavy metals, pH minima, temperature
maxima and optima, and their ability to form biofilms. Remarkably, many yeasts
are extremely resistant to ionizing radiation and heavy metals. The fungal species
included 16 ascomycetous and 11 basidiomycetous yeasts isolated from diverse
environments including arctic ice, acid mine drainage, red wine, and apple juice,
as well as dry environments with elevated temperatures. However, the studies
identified only one potential fungus strain, i.e., Rhodotorula taiwanensis MD1149
to fulfill the expectations. The strain was isolated from a sediment sample from an
abandoned acid mine drainage in Maryland, USA. The fungus is a red pigmented,
unicellular, non-sporulating, ovoidal, obligately aerobic, budding yeast. MD 1149
showed resistance to gamma radiations. It was found capable of growing under
66 Gy/h at 2.3 pH value and in the presence of high concentrations of heavy metal
compounds of mercury and chromium. The strain was found forming biofilms under
high-level cgronic radiation and low pH. The strain has undergone whole genome
sequencing and presents a strong potential role in the bioremediation of acidic
radioactive sites (Tkavc et al. 2018).
24.12 Conclusions
Extremophiles of different kinds are being studied for their novel characteristic to
survive in extreme conditions of pH, temperature, salt concentration, hydrocarbon
contamination, etc. The approach finds applications in industrial and environmental
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 575

issues. An important source of biochemicals like enzymes and proteins will help in
industrial catalysis. Fungi have a wide range of metabolism. Especially,
extremophilic fungi can help in the degradation or inactivation of various environ-
mental contaminants at the sites where normal microorganisms cannot help. They
have been isolated from diverse sources like low and high temperatures, acidic
conditions and high concentration of heavy metals, etc. Many of the isolated species
show a greater potential in bioremediation of such contaminated sites. Further
studies will help to identify new species and help them to perform the activity
with enhanced efficiency.
Acknowledgments Mr. Sunil Bhapkar acknowledges Council of Scientific & Industrial Research
for the research fellowship. Mr. Rhushikesh Pol acknowledges Indian Council of Medical Research
for the research fellowship. Ms. Deeksha Patil acknowledges Department of Biotechnology, India
for the research fellowship.
References
Abdel-Hafez SII (1982) Osmophilic fungi of desert soils in Saudi Arabia. Mycopathologia 80:9–14.
https://doi.org/10.1007/BF00437172
Aislabie J, Fraser R, Duncan S, Farrell RL (2001) Effects of oil spills on microbial heterotrophs in
Antarctic soils. Polar Biol 24:308–313. https://doi.org/10.1007/s003000000210
Ali I, Khaliq S, Sajid S, Akbar A (2019) Biotechnological applications of halophilic fungi: past,
present, and future. In: Fungi in extreme environments: ecological role and biotechnological
significance. Springer International, Cham, pp 291–306
Arregui L, Ayala M, Gómez-Gil X et al (2019) Laccases: structure, function, and potential
application in water bioremediation. Microb Cell Fact 18:1–33
Baker BJ, Lutz MA, Dawson SC et al (2004) Metabolically active eukaryotic communities in
extremely acidic mine drainage. Appl Environ Microbiol 70:6264–6271. https://doi.org/10.
1128/AEM.70.10.6264-6271.2004
Balabanova L, Slepchenko L, Son O, Tekutyeva L (2018) Biotechnology potential of marine fungi
degrading plant and algae polymeric substrates. Front Microbiol 9:1–15. https://doi.org/10.
3389/fmicb.2018.01527
Bano A, Hussain J, Akbar A et al (2018) Biosorption of heavy metals by obligate halophilic fungi.
Chemosphere 199:218–222. https://doi.org/10.1016/j.chemosphere.2018.02.043
Barnes NM, Khodse VB, Lotlikar NP et al (2018) Bioremediation potential of hydrocarbon-
utilizing fungi from select marine niches of India. 3 Biotech 8:1–10. https://doi.org/10.1007/
s13205-017-1043-8
Bej A, Aislabie J, Atlas R (2010) Polar microbiology: the ecology, biodiversity and bioremediation
potential of microorganisms in extremely cold environments. Taylor & Francis, Boca Raton
Boonchan S, Britz ML, Stanley GA (2000) Degradation and mineralization of high-molecular-
weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures. Appl Environ
Microbiol 66:1007–1019. https://doi.org/10.1128/AEM.66.3.1007-1019.2000
Bordjiba O, Steiman R, Kadri M et al (2001) Removal of herbicides from liquid media by fungi
isolated from a contaminated soil. J Environ Qual 30:418–426. https://doi.org/10.2134/jeq2001.
302418x
Borowik A, Wyszkowska J, Oszust K (2017) Functional diversity of fungal communities in soil
contaminated with diesel oil. Front Microbiol 8:1–11. https://doi.org/10.3389/fmicb.2017.
01862
576 S. Bhapkar et al.

Brakstad OG (2008) Natural and stimulated biodegradation of petroleum in cold marine
environments. In: Psychrophiles: from biodiversity to biotechnology. Springer-Verlag, Berlin,
Heidelberg, pp 389–407
Buzzini P, Margesin R (2014) Cold-adapted yeasts: biodiversity, adaptation strategies and biotech-
nological significance. Springer, Berlin
Cai CX, Xu J, Deng NF et al (2016) A novel approach of utilization of the fungal conidia biomass to
remove heavy metals from the aqueous solution through immobilization. Sci Rep 6:36546.
https://doi.org/10.1038/srep36546
Chan WK, Wildeboer D, Garelick H, Purchase D (2018) Competition of as and other group
15 elements for surface binding sites of an extremophilic Acidomyces acidophilus isolated
from a historical tin mining site. Extremophiles 22:795–809. https://doi.org/10.1007/s00792-
018-1039-2
Chan WK, Wildeboer D, Garelick H, Purchase D (2019) Acidomyces acidophilus: ecology,
biochemical properties and application to bioremediation. In: Fungi in extreme environments:
ecological role and biotechnological significance. Springer Nature Switzerland AG, Cham, pp
505–515
Chang YH, Li CT, Chang MC, Shieh WK (1998) Batch phenol degradation by Candida tropicalis
and its Fusant. Biotechnol Bioeng 60:391–395
Deshmukh R, Khardenavis AA, Purohit HJ (2016) Diverse metabolic capacities of fungi for
bioremediation. Indian J Microbiol 56:247–264. https://doi.org/10.1007/s12088-016-0584-6
Divya LM, Prasanth GK, Sadasivan C (2014) Potential of the salt-tolerant laccase-producing strain
Trichoderma viride Pers. NFCCI-2745 from an estuary in the bioremediation of phenol-polluted
environments. J Basic Microbiol 54:542–547. https://doi.org/10.1002/jobm.201200394
dos Santos VL, de Monteiro A, Braga DT, Santoro MM (2009) Phenol degradation by
Aureobasidium pullulans FE13 isolated from industrial effluents. J Hazard Mater 161:1413–
1420. https://doi.org/10.1016/j.jhazmat.2008.04.112
Fan T, Liu Y, Feng B et al (2008) Biosorption of cadmium(II), zinc(II) and lead(II) by Penicillium
simplicissimum: isotherms, kinetics and thermodynamics. J Hazard Mater 160:655–661. https://
doi.org/10.1016/j.jhazmat.2008.03.038
Ferrari BC, Zhang C, van Dorst J (2011) Recovering greater fungal diversity from pristine and
diesel fuel contaminated sub-antarctic soil through cultivation using both a high and a low
nutrient media approach. Front Microbiol 2:1–14. https://doi.org/10.3389/fmicb.2011.00217
Filler D, Snape I, Barnes D (2008) Bioremediation of petroleum hydrocarbons in cold regions.
Cambridge University Press, Cambridge
Garon D, Krivobok S, Wouessidjewe D, Seigle-Murandi F (2002) Influence of surfactants on
solubilization and fungal degradation of fluorene. Chemosphere 47:303–309. https://doi.org/10.
1016/S0045-6535(01)00299-5
Garon D, Sage L, Seigle-murandi F (2004) Effects of fungal bioaugmentation and cyclodextrin
amendment on fluorene degradation in soil slurry. Biodegradation 15:1–8
Gibson DT, Saylor GS (1992) Scientific foundations of bioremediation: current status and future
needs. American Academy of Microbiology, Washington, DC
Greer CW, Whyte LG, Niederberger TD (2010) Microbial communities in hydrocarbon-
contaminated temperate, tropical, alpine, and polar soils. Springer-Nerla, Berlin, Heidelberg
Harms H, Schlosser D, Wick LY (2011) Untapped potential: exploiting fungi in bioremediation of
hazardous chemicals. Nat Rev Microbiol 9(3):177–192
Hernández-López EL, Perezgasga L, Huerta-Saquero A et al (2016) Biotransformation of petro-
leum asphaltenes and high molecular weight polycyclic aromatic hydrocarbons by Neosartorya
fischeri. Environ Sci Pollut Res 23:10773–10784. https://doi.org/10.1007/s11356-016-6277-1
Hofrichter M, Bublitz F, Fritsche W (1994) Unspecific degradation of halogenated phenols by the
soil fungus Penicillium frequentans bi 7/2. J Basic Microbiol 34:163–172. https://doi.org/10.
1002/jobm.3620340306
Holan ZR, Volesky B (1995) Accumulation of cadmium, lead, and nickel by fungal and wood
biosorbents. Appl Biochem Biotechnol 53:133–146. https://doi.org/10.1007/BF02788603
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 577

HujslováM, KubátováA, Kostovčík M, Kolařík M (2013) Acidiella bohemica gen. Et sp. nov. and
Acidomyces spp. (Teratosphaeriaceae), the indigenous inhabitants of extremely acidic soils in
Europe. Fungal Divers 58:33–45. https://doi.org/10.1007/s13225-012-0176-7
Ianis M, Tsekova K, Vasileva S (2006) Copper biosorption by penicillium cyclopium: equilibrium
and modelling study. Biotechnol Biotechnol Equip 20:195–201. https://doi.org/10.1080/
13102818.2006.10817332
Iark D, dos Buzzo AJ, Garcia JA et al (2019) Enzymatic degradation and detoxification of azo dye
Congo red by a new laccase from Oudemansiella canarii. Bioresour Technol 289:121655.
https://doi.org/10.1016/j.biortech.2019.121655
Jambon I, Thijs S, Torres-FarradáG et al (2019) Fenton-mediated biodegradation of chlorendic
acid—a highly chlorinated organic pollutant—by fungi isolated from a polluted site. Front
Microbiol 10:1–13. https://doi.org/10.3389/fmicb.2019.01892
Jeong SW, Choi YJ (2020) Extremophilic microorganisms for the treatment of toxic pollutants in
the environment. Molecules 25:13–15. https://doi.org/10.3390/molecules25214916
Jiang Y, Shang Y, Yang K, Wang H (2016) Phenol degradation by halophilic fungal isolate JS4 and
evaluation of its tolerance of heavy metals. Appl Microbiol Biotechnol 100:1883–1890. https://
doi.org/10.1007/s00253-015-7180-2
Krug M, Ziegler H, Straube G (1985) Degradation of phenolic compounds by the yeast Candida
tropicalis HP 15. J Basic Microbiol 25:103–110
Kumar V, Dwivedi SK (2019) Hexavalent chromium reduction ability and bioremediation potential
of aspergillus flavus CR500 isolated from electroplating wastewater. Chemosphere 237:124567.
https://doi.org/10.1016/j.chemosphere.2019.124567
Leitão AL, Duarte MP, Oliveira JS (2007) Degradation of phenol by a halotolerant strain of
Penicillium chrysogenum. Int Biodeter Biodegr 59:220–225. https://doi.org/10.1016/j.ibiod.
2006.09.009
Mansour MMA (2017) Effects of the halophilic fungi Cladosporium sphaerospermum, Wallemia
sebi, Aureobasidium pullulans and aspergillus nidulans on halite formed on sandstone surface.
Int Biodeter Biodegr 117:289–298. https://doi.org/10.1016/j.ibiod.2017.01.016
Margesin R, Schinner F (2001) Biodegradation and bioremediation of hydrocarbons in extreme
environments. Appl Microbiol Biotechnol 56:650–663. https://doi.org/10.1007/s002530100701
Margesin R, Fonteyne PA, Redl B (2005) Low-temperature biodegradation of high amounts of
phenol by Rhodococcus spp. and basidiomycetous yeasts. Res Microbiol 156:68–75. https://doi.
org/10.1016/j.resmic.2004.08.002
Margesin R, Fonteyne PA, Schinner F, Sampaio JP (2007) Rhodotorula psychrophila sp. nov.,
Rhodotorula psychrophenolica sp. nov. and Rhodotorula glacialis sp. nov., novel psychrophilic
basidiomycetous yeast species isolated from alpine environments. Int J Syst Evol Microbiol 57:
2179–2184. https://doi.org/10.1099/ijs.0.65111-0
Massaccesi G, Romero MC, Cazau MC, Bucsinszky AM (2002) Cadmium removal capacities of
filamentous soil fungi isolated from industrially polluted sediments, in La Plata (Argentina).
World J Microbiol Biotechnol 18:817–820. https://doi.org/10.1023/A:1021282718440
Medaura MC, Guivernau M, Prenafeta-boldú FX, Viñas M (2021) Bioaugmentation of native fungi,
an efficient strategy for the bioremediation of an aged industrially polluted soil with heavy
hydrocarbons. Front Microbiol 12:1–18. https://doi.org/10.3389/fmicb.2021.626436
Meléndez-Estrada J, Amezcua-Allieri MA, Alvarez PJJ, Rodríguez-Vázquez R (2006) Phenan-
threne removal by Penicillium frequentans grown on a solid-state culture: effect of oxygen
concentration. Environ Technol 27:1073–1080. https://doi.org/10.1080/09593332708618720
Mendil D, Tuzen M, Soylak M (2008) A biosorption system for metal ions on Penicillium
italicum—loaded on Sepabeads SP 70 prior to flame atomic absorption spectrometric
determinations. J Hazard Mater 152:1171–1178. https://doi.org/10.1016/j.jhazmat.2007.07.097
Nazareth S, Marbaniang T (2008) Effect of heavy metals on cultural and morphological growth
characteristics of halotolerant Penicillium morphotypes. J Basic Microbiol 48:363–369. https://
doi.org/10.1002/jobm.200800006
Peeples TL (2014) Bioremediation using extremophiles. Elsevier, Amsterdam
578 S. Bhapkar et al.

Say R, Yilmaz N, Denizli A (2003) Removal of heavy metal ions using the fungus penicillium
canescens. Adsorpt Sci Technol 21:643–650. https://doi.org/10.1260/026361703772776420
Say R, Yilmaz N, Denizli A (2004) Removal of chromium(VI) ions from synthetic solutions by the
fungus Penicillium purpurogenum. Eng Life Sci 4:276–280. https://doi.org/10.1002/elsc.
200420032
Strandberg GW, Shumate SE, Parrott JR (1981) Microbial cells as biosorbents for heavy metals:
accumulation of uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa. Appl
Environ Microbiol 41:237–245. https://doi.org/10.1128/aem.41.1.237-245.1981
Tavares APM, Cristóvão RO, Gamelas JAF et al (2009) Sequential decolourization of reactive
textile dyes by laccase mediator system. J Chem Technol Biotechnol 84:442–446. https://doi.
org/10.1002/jctb.2060
Ting ASY, Choong CC (2009) Bioaccumulation and biosorption efficacy of Trichoderma isolate
SP2F1 in removing copper (Cu(II)) from aqueous solutions. World J Microbiol Biotechnol 25:
1431–1437. https://doi.org/10.1007/s11274-009-0030-6
Tkavc R, Matrosova VY, Grichenko OE et al (2018) Prospects for fungal bioremediation of acidic
radioactive waste sites: characterization and genome sequence of Rhodotorula taiwanensis
MD1149. Front Microbiol 8:1–21. https://doi.org/10.3389/fmicb.2017.02528
Tsezos M, Volesky B (1981) Biosorption of uranium and thorium. Biotechnol Bioeng 23:583–604
Xu X, Xia L, Zhu W et al (2015) Role of Penicillium chrysogenum XJ-1 in the detoxification and
bioremediation of cadmium. Front Microbiol 6:1–10. https://doi.org/10.3389/fmicb.2015.
01422
Yin Q, Zhou G, Peng C et al (2019) The first fungal laccase with an alkaline pH optimum obtained
by directed evolution and its application in indigo dye decolorization. AMB Express 9:151.
https://doi.org/10.1186/s13568-019-0878-2
Zafar S, Aqil F, Ahmad I (2007) Metal tolerance and biosorption potential of filamentous fungi
isolated from metal contaminated agricultural soil. Bioresour Technol 98:2557–2561. https://
doi.org/10.1016/j.biortech.2006.09.051
Zobell CE (1946) Action of microorganisms on hydrocarbons. Bacteriol Rev 10:1–49
24 Whole Cell Application Potential of Extremophilic Fungi in Bioremediation 579

Extremophilic Fungi: Potential Applications
in Sustainable Agriculture 25
Sanjay Sahay
Abstract
Food is the most basic requirement of life which has been hard hit due to climate
change effect. Unfortunately, unsustainable agricultural practices pursued for
many decades to produce more have left behind a substantial proportion of
polluted and depleted water sources and highly affected soil concerning its
structure, and chemical and microbiological composition. To ensure continued
supply of food to billions of stomachs across the world, remediation of the
so-called problem soils and sustainable use of soil in general have now become
imperative. Fungi constitute an important part of soil microbiome which by their
efficient secretomes and plant growth-promoting activities play a key role in soil
structure and fertility maintenance. Extremophilic fungi with their capacity to
function under various stress conditions and to confer upon plants’tolerance to
various abiotic and biotic stresses hold a key to bioremediate problem soils,
restore soil fertility, and augment stress tolerance to the plants.
Keywords
Extreme-fungi in soil management · Extreme-fungi in crop improvement ·
Extreme-fungi and photosynthesis · Extreme-fungi as biocontrol · Extreme-fungi
and abiotic stress
S. Sahay (*)
Sarojini Naidu Government Postgraduate Girl’s (Autonomous) College, Bhopal, Madhya Pradesh,
India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_25
581

25.1 Introduction
Fungi have been serving humanity since very early in civilization by providing
fermented food and beverages. Advancement in the genetics and biochemistry of
fungi has opened up the possibility for their diverse applications in industrial and
environmental fields. Fungi as saprotrophs, phytopathogens, endophytes, and
Arbuscular mycorrhizal forms perform varied services to agriculture including
structural and fertility management of soil and conferring tolerance to various
stresses upon plants. As decomposer, the importance of fungi in maintaining the
physicochemical nature of the soil is well known.
Fungi as part of decomposers perform an important function in unlocking simpler
organic matters from dead animals and plants and their detached parts thus adding
organic matters to the soil (Aitken 1993). Fungi also produce organic substances
such as glomalin that improve soil structure (Spurgeon et al. 2013). Fungi also
improve the availability of plant nutrients, for example, by transforming complex
insoluble and unusable phosphate into a soluble and usable form, carrying immobile
metal nutrients close to root hair, and producing plant growth-promoting and
phytopathogen inhibitory compounds. In soil, fungi vis-à-vis bacteria like mild
acidic conditions, not many disturbed soils, perennial plant community, nutrients
supply from nearby plants and higher carbon to nitrogen (C:N) content (Lavelle and
Spain 2005). The mycelial structure of fungi enables them to grow deeper in the soil
and connect roots to more distant nutrient sources (Lowenfels and Lewis 2006).
Unfortunately unsustainable practices in agriculture such as the unjudicious applica-
tion of synthetic fertilizers and pesticides have apart from polluting soil and water
also altered the soil microbiome including soil fungi.
Fungi in the extreme climate have evolved novel ability to tolerate various abiotic
stresses such as tolerance to extreme climates (high and low temperatures), extreme
pH conditions (alkaline and acidic), extreme salt (sodic), and higher metal
concentrations. During the last few decades, surveys in such extremophilic climates
have revealed the existence of strains and species of extremophilic fungi that can not
only perform their normal functions under extreme conditions but also exhibit
various ameliorative activities. Further physiological and genetical studies on them
have led to the discovery of mechanisms of tolerance to specific extreme conditions.
All these developments have opened up vistas in applying extremophilic fungi and
their biomolecules to bioremediate problem soils, maintain decomposition activity
under extreme conditions, restore fertility in problem soils, manage soil
phytopathogens, and ensure beneficial activities of endophytes and AMF under
extreme conditions.
25.2 Agricultural Constraints and Fungal Intervention
An ideal agriculture system requires at least fertile soil, a proper supply of water and
nutrients, and suitable crop variety. However, global climate change and
non-judicious agricultural practices to produce more have led to such problems as
582 S. Sahay

depletion of water sources and highly degraded soil concerning its structure, and
chemical and microbiological compositions in many parts of the world. The current
focus to restrict the use of chemical fertilizers and pest controls has also enhanced
nutritional and biotic stress respectively for the crop. Agriculture under these
constraints put plants in mainly three abiotic stresses viz., water, temperature (low
or high), and salt stresses (Egamberdieva et al. 2008; Egamberdieva and Lugtenberg
2014). The problems may be tackled at the level of the soil (by amending soil) and
plant (by conferring stress tolerance). Plant growth-promoting and pathogen
inhibiting activities of microbes especially fungi may be useful. Mainly three
categories of fungi viz., open soil dweller, Arbuscular mycorrhizic fungi (AMF),
and endophytic fungi (EPF) have been studied concerning their role in promoting
plant growth and yield under normal and various stress conditions. The three
categories of extremophilic fungi on the other hand are more potential to provide
these benefits as they are acclimatized to carry out their functions under stressful
conditions with variously upgraded tools (e.g., enzymes).
25.2.1 Soil Management
25.2.1.1 Soil Structure Improvement
Many fungi produce coagulating substances, of which glomalin is a highly studied
one. These substances play a very effective role in aggregating the soil particles into
microaggregates (Wright and Upadhyaya 1998). Several studies have demonstrated
the direct relationship between glomalin-producing fungi and aggregate stability
(Spurgeon et al. 2013; Wright and Upadhyaya 1998). As in the case of natural
grassland and forests as well as agricultural fields, soil receives green litter either as
fallen plant parts due to senescence (Girisha et al. 2003) or plant residues left after
harvesting (Cookson et al. 2008). The litter in abundant quantity is also received
from thinned trees during the process of thinning where it is in practice (Tian et al.
2010). Thus litter in different amount and composition is incorporated in various
categories of forests which in turn determine the soil microbial community
according to their food preference/enzymatic capacity. Among microbes, fungi
play a crucial role in the decomposition of lignoellulosic mass and contribution of
thus formed organic matter to the soil. The organic matter in the soil helps in forming
favorable soil structure (microaggregate formation) and enhances the nutritional
status and water retention capacity of the soil (Jiménez-Morillo et al. 2016). Soil
organic matter does play an effective role in maintaining soil resistance and stability,
though a scientific explanation of this relationship is not available. Deforestation-
led-degraded soil that may exhibit poor fungal diversity and soil functional stability
partially explains fungal diversity and soil functional stability relationship (Chaer
et al. 2009). The extremophilic fungi have a potential to carry out this function under
extreme climatic or soil conditions.
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 583

25.2.1.2 Soil Amendments
There are the so-called problem soils such as saline and sodic, acidic, and acid sulfate
soils which constitute a considerable proportion of arable land globally. For exam-
ple, India has approximately 60 million hectares of such problem soils out of about
140 million hectares of net cultivable area (Goswami 1982). Of these problem soils,
saline and sodic soils are the most problematic ones. Accumulation of higher
concentration of salts in arid and semi-arid soil as a result of natural causes such
as rainfall and evapotranspiration dynamics (Zhu 2001; Zheng et al. 2009), and
man-made practices such as assisted irrigation (Allbed and Kumar 2013) are respon-
sible for the development of these types of soil. They have Na
+
and Cl

in the toxic
concentrations that cause, in plants, disruption of enzymes’structure and damages to
cell organelles, and negatively affect metabolic activities such as protein biosynthe-
sis. Soil exhibits waterlogging and poor aeration. Plants in saline soil are exposed to
osmotic, ionic, and oxidative stress, and thus exhibit highly reduced viability and
productivity (Saxena et al. 2017). The saline-sodic soils are also characterized by a
paucity of fungal diversity. The problem soils are recognized as the main constraints
in the way of food security (FAO 2009).
Various types of extremophilic fungi may be used to augment such soils to amend
them. For example, the haloalkaliphilic fungi can ameliorate such soils by secreting
organic acids, absorbing salt ions, and adding biomolecules such as cellulases
that have a positive impact on soil physical properties, fertility, and even health
(Wei and Zhang 2019). The potential alkaliphilic/alkalitolerant fungi have been
reported from various studies. In one study, taxa belonging to Emericellopsis lineage
(Hypocreales and Hypocreomycetidae) were found to dominate followed by the
members of families Plectosphaerellaceae (Hypocreomycetidae), Pleosporaceae
(Dothideomycetes), and Chaetomiaceae (Sordariomycetidae) in alkaline/saline
soil. The fungal species belonging to these families such as Sodiomyces species
(Plectosphaerellaceae), Acrostalagmus luteoalbus (Plectosphaerellaceae),
Emericellopsis alkaline (Hypocreales), Thielavia spp. (Chaetomiaceae), and
Alternaria sp. were especially found to dominate the scene. Members of
Scopulariopsis (Microascales) and species of Fusarium,Cladosporium, and
Acremonium-like fungus from Bionectriaceae were those found as moderate
alkaliphilic ones (Grum-Grzhimaylo et al. 2016). In another study, the species of
fungi reported in moderate saline soil were Trichoderma spp., Penicillium spp.,
Fusarium spp., Geocladium spp., and Paceliomyces and in hypersaline soil
Eurotium spp., Wallemia spp., and Phaeotheca spp. (Bronicka et al. 2007). Fungi
isolated from salterns are represented by black yeasts (Hortaea werneckii,
Phaeotheca triangularis,Aureobasidium pullulans, and Trimmatostroma salinum),
Cladosporium spp., Aspergillus spp., and Penicillium spp. (Chung et al. 2019).
Apart from these, chaotolerant fungi such as Hortaea werneckii and Wallemia
ichthyophaga that can thrive in 1.8 M MgCl
2
concentrations (Zajc et al. 2014) are
available to be used in the case of chaotrope (e.g., MgCl
2
, CaCl
2
, urea, NH
4
NO
3
,
phenol, and NaBr)-polluted soils (Hallsworth et al. 2003).
584 S. Sahay

25.2.1.3 Soil Bioremediation
Heavily contaminated soils with hazardous chemicals such as heavy metals (due to
atmospheric deposition), complex chemicals (via use of pesticides), or residual
organic compounds (via operations like wastewater and sludge treatment and com-
post landfilling) need to be remediated. The available main processes are bioslurry
reactor, biopile, and land farming which are compatible with the application of
fungal cultures or enzymes (Mougin et al. 2009). The ligninolytic white rot fungi
have been in the center for any of such applications because of their capability of
producing required enzymes in higher amounts (Aitken 1993; Barr and Aust 1994).
Fungal oxidases, laccases, and peroxidases are the main enzymes used in soil
bioremediation of soil. Among the three important remediation technologies,
bioslurry has been finding more attention as this ensures large-scale operation,
rapid enzyme/fungi-contaminant contact, and degradation of pollutant efficiently
(for details, see Mougin et al. 2009). Extremophilic fungi that thrive under harsh
conditions produce variously adapted enzymes such as metal tolerant, thermophilic,
and psychrophilic. These enzymes are expected to be of immense potential for
applying for soil bioremediation purposes. Extremophilic laccases (Sahay et al.
2019), peroxidases (Chandra et al. 2017), cellulases (Dutta et al. 2008), etc. have
been characterized from extremophilic fungi. Extremophilic fungi have also been
evaluated for bioremediation purposes (Baker et al. 2004; Bano et al. 2018; Bej et al.
2010).
25.2.1.4 Soil Fertility Restoration/Biofertilizer
Filamentous fungus, for example, Mortierella, exists in diverse types of
environments including extreme ones and serves as an efficient decomposer, and
plant growth-promoting fungus (PGPF) by improving the availability of P and Fe,
synthesizing phytohormones and 1-aminocyclopropane-1-carboxylate (ACC) deam-
inase, and antagonizing pathogenic fungi in soil. The strains of this fungus are found
in bulk soil, rhizosphere, and plant tissues under extreme conditions (Ozimek and
Hanaka 2021).
Arbuscular mycorrhizal fungi (AMF) constitute an important group of fungi that
help in maintaining soil fertility in agricultural, forest, and horticultural soils (Smith
and Read 1997). Several experiments proved the beneficial role of AMF in increas-
ing crop yield following AMF inoculation of crop plants (Bagyaraj and Ashwin
2017). The AMF symbiosis has been reported to exhibit a positive impact on soil
structure, nutrient cycling, plant root formation and establishment, plant tolerance
to stresses, plant community diversity, and plant’s ability to take up low mobility
ions (Azcón-Aguilar and Barea 1997). AMF in combination with plant growth-
promoting rhizobacteria (PGPR) act synergistically to improve soil structure and
fertility (Bagyaraj and Ashwin 2017). In forest soil, ectomycorrhizae on the roots
produce extractable dissolved organic carbon that stimulates the growth and prolif-
eration by other microbes (Högberg and Högberg 2002) and also control the water
retaining capacity of the soil (Jiménez-Morillo et al. 2016). AMF increase nutrient
uptake for the plants, particularly immobile nutrients such as phosphorus (P), copper
(Cu), and zinc (Zn) in soil which are not accessible to plant roots in normal condition
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 585

due to slow immobility (Marschner 2012; Ortas 2003). Additionally, AMF assist the
plants to tolerate various environmental stresses such as salinity, drought, heat,
and pollutants in the rhizosphere (Aranda et al. 2013; Bowles et al. 2016;
Chandrasekaran et al. 2016; Maya et al. 2014). The mycorrhizal application also
reduces the quantitative use of chemical fertilizer input especially phosphate fertil-
izer (Charron et al. 2001; Ortas 2012). The effect of nutrient- and water-deficient
conditions in the field can be reduced in a well-managed rhizosphere with increased
microbial community and nutrient availability which benefit plant-soil quality
(Barea et al. 2005).
In the Arctic ecosystem where low water and nutrient content are major
constraints, the role of mycorrhizal fungi becomes very important (Timling and
Taylor 2012). It has been reported that as much as 88% of nitrogen is provided by
mycorrhizae especially ectomycorrhizae (ECM) (Hobbie and Hobbie 2006). A large
number of ECM fungi have been reported from the Arctic ecosystems (Bjorbaekmo
et al. 2010; Deslippe et al. 2011; Geml et al. 2012) such as Thelephora,Sebacina,
and Clavulina. ECM isolated and reported from root tips and soil clones in the Arctic
showed higher species richness (Bjorbaekmo et al. 2010; Walker et al. 2011; Geml
et al. 2012). They are highly potential bioresurces to provide growth-promoting
factors to the plants in novel cold areas.
The psychrotrophic yeast strains isolated from Nothofagus spp. and Vaccinium
spp. in Northwest-Patagonia have been found to exhibit various plant growth-
promoting activities. The yeast Aureobasidium pullullans, for example, was
identified as a producer of auxin-like and siderophores compounds while yeasts
Holtermaniella takashimae and Candida maritime as phosphate solubilizers (Mestre
et al. 2016).
25.2.2 Crop’s Stress Management
25.2.2.1 Abiotic Stress
Plants in agriculture systems face mainly three abiotic stresses viz., water, tempera-
ture (low or high), and salt stresses (Egamberdieva et al. 2008; Egamberdieva and
Lugtenberg 2014) that can also be handled at plant level by conferring various stress
tolerance upon plants. There are at least three physiological targets whose improve-
ment has been considered to bring about overall tolerance to various abiotic stresses
by the fungi of various types. Apart from these, other mechanisms imparting
tolerance to various abiotic stresses have also been reported (Fig. 25.1) (Malhi
et al. 2021).
Water Relations
Water status is very important in plant growth and physiology. The contribution of
fungi to improve plants’water relations is mainly credited to AMF. AM plants
(plants with associated AMF) exhibit in many cases a better water status under water
stress conditions as compared to non-AM plants (Augé 2001). Rather improved
water-use efficiency for the plant has been reported by AMF application (Bowles
586 S. Sahay

et al. 2016). For instances, AM maize has been shown to exhibit higher water
holding capacity and relative water content under low- and high-temperature stress
(Zhu et al. 2010a,2011; Liu et al. 2014b) and AM bean (Phaseolus vulgaris) has
been found to have higher leaf water potential under low-temperature stress
(El Tohamy et al. 1999). In contrast to these reports, under low-temperature stress
conditions, AM plants have also been found to have the same water content as
non-AM plants (Aroca et al. 2007; Liu et al. 2014a). Uptake of water by roots
depends upon root hydraulic conductivity, stomatal movement, transpiration rate,
and the activity of aquaporin belonging to the intrinsic membrane protein family
(Luu and Maurel 2005). AM plants’better water status has been ascribed to the
increased water extraction by the external hyphae of AMF (Faber et al. 1991), higher
hydraulic conductivity of AM plant roots (Augé and Stodola 1990), higher stomatal
conductance, and transpiration rate of AM maize (Zhu et al. 2010a,2011).
Aquaporins that facilitate the passive water flow through membranes (Kruse et al.
2006) have been found to exhibit enhanced activity in AM plants (Liu et al. 2014b).
AMF can regulate in addition to their host plants aquaporin genes (PvPIP1;3) thus
improving the water status of the host (Liu et al. 2014b). AM plants exhibit under
low-temperature stress higher expression of PvPIP1;3 gene and higher abundance of
Fig. 25.1 Schematic representation of mechanisms imparting tolerance to various abiotic stresses
(Malhi et al. 2021)
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 587

PIP protein (Aroca et al. 2007). Upregulation of other membrane intrinsic protein
genes (PIP1;1,PIP1;3,PIP2;1,andPIP2;5) has also been reported in AM rice
(Oryza sativa) plants (Liu et al. 2014b).
Ectomycorrhizal fungi or ECM occupy the surface of lateral roots superficially
near root tips forming three distinct regions related to water transport viz., the
extraradical network of mycelia, mantle sheath, and Hartig net (Fig. 25.2). The
growing mycelial tips take up water and nutrient actively. A part of hyphae is
grouped together and differentiated into rope-like rhizomorphs (Agerer 2001).
Water and nutrients flowing along rhizomorphs may follow two paths at hyphae–
root interface—the hyphal mantle sheath occupying the surface of root tip mainly
meant for storage, and the intercellular hypha Hartig net in the plant epidermis and
outer cortex meant for water-nutrient exchange between hyphae and plant cells
(Smith and Read 1997).
Fungal aquaporin is believed to enhance hydration at the hyphal-root cell inter-
face and consequent apoplastic water transport. A direct relationship between
overexpression of aquaporin gene in mycorrhiza Laccaria bicolor and enhancement
of root hydraulic conductance of its host Picea glauca has proved fungal aquaporin’s
ability to regulate host plant water status (Xu 2015).
Photosynthesis
Photosynthesis is a crucial physiological process in plants; any alteration in its rate
has direct implications in stress tolerance ability including temperature-related stress
(Wahid et al. 2007). AM plants under temperature stress have been reported to
exhibit a higher net photosynthetic rate than their non-AM counterparts
(Ruotsalainen and Kytöviita 2004; Zhu et al. 2010a,2011,2015). Temperature
stress suppresses chlorophyll biosynthesis or promotes chlorophyll degradation
resulting in the reduction of chlorophyll concentration in the plants. AM plants
have been reported to exhibit higher chlorophyll concentration as compared to their
non-AM counterparts under temperatures stress (Paradis et al. 1995; Zhu et al.
2010a,2011). These findings imply that AMF inoculation alleviates temperature
stress-related reduction in chlorophyll synthesis or degradation of chloroplasts,
thereby maintaining the photosynthetic rate (Evelin et al. 2009). In another report
AM maize has been found to have higher carotenoid concentration compared to
non-AM maize (Zhu et al. 2011). Carotenoid is known to add light-harvesting during
photosynthesis and also helps photoprotection of chloroplast (Young 1991). AMF
inoculation in maize thus adds to stabilization of membrane lipid of the thylakoid
and photoprotection of chloroplast (Karim et al. 1999; Wahid et al. 2007). Another
target of temperature stress injury in chloroplast has been reported to be the oxygen-
evolving complex of photosystem II (Allakhverdiev et al. 2008). Two indicators
viz., the ratio of Fv/maximum fluorescence (Fm) (maximum quantum efficiency of
PSII primary photochemistry) and of Fv/primary fluorescence (Fo) (potential photo-
chemical efficiency of PSII) have been used to evaluate the damage caused by
environmental stresses (Krause and Weis 1991; Maxwell and Johnson 2000). AM
maize has been reported to exhibit higher Fv/Fm and Fv/Fo as compared to non-AM
maize plants under temperature stresses (Zhu et al. 2010a,2011). The finding
588 S. Sahay

suggests that AMF protects the PSII reaction center and the chloroplast from the
damaging impact of temperature stress. Temperature stress may thus affect the
reaction center of PSII, the number of open PSII units, PSII photochemical reaction,
and electron transport in chloroplast negatively (Camejo et al. 2005; Baker 2008).
Fig. 25.2 Schematic representation of water transport pathways and aquaporin participation in
ECM associated plant (Xu and Zwiazek 2020). (a) ECM–root interaction as originally given earlier
(Steudle and Peterson 1998; Lehto and Zwiazek 2011). (b) Fungal and plant aquaporins at the
hypha–root interface participating in water transport activity. (B1) Water transport pathway involv-
ing apoplast and cell–cell route in a non-ECM associated root; (B2) Water transport in ECM
associated root highlighting changes in normal water transport pathway and enhancement in rate of
hydration due to involvement of mycorrhizal hyphae and aquaporins; (B3) Enhancement of
hydration rate as a result of upregulation of fungal aquaporins; (B4) Upregulated fungal aquaporins
may alternatively lead to acquisition of more water by fungal hyphae. (Reproduced with permission
from author)
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 589

AM symbiosis can mitigate all these unfavorable effects of temperature stress on the
PSII reaction center which in turn improves the efficiency of PSII photochemistry
and photosynthetic ability of chloroplast. Contrasting reports, however, show that in
wheat (Triticum aestivum) AM symbiosis has no impact on electron transfer, the
quantum yield, and non-photochemical quenching under high-temperature condition
(Cabral et al. 2016). Similarly, AMF inoculation could not cause any impact on the
photosynthetic rate in the citrus plants (Wu and Zou 2010) under low-temperature
stress. Charest et al. (1993) also found that the chlorophyll concentration of AM
maize plants was lower than the non-AM plants.
Salt Stress Tolerance
Salt tolerance is carried out via modulation of several physiological activities such as
reduction in lipid peroxidation, enhancement of proline accumulation, NHX1
antiporter gene expression, and ecophysiological performance. AMF inoculation in
maize plants with three native AMF was reported to cause photosystem II enhance-
ment and stomatal conductance, reduce ROS production, the oxidative damage to
lipids, and the membrane electrolyte leakage (Estrada et al. 2013), and also reduce
malondialdehyde (MDA)/electrolyte leakage in leaves (Wang et al. 2020).
AMF and Abiotic Stress
In saline–sodic soil the density of AMF was generally found low and represented by
Glomus,Scutellospora,Acaulospora,Sclerocystis, and Gigaspora,Acaulospora and
Glomus in the rhizosphere of maize, Scutellospora and Glomus in the rhizosphere of
tulsi, onion, and rice, Glomus, and Sclerocystis in the rhizosphere of guava, and
Gigaspora and Glomus in the rhizosphere of bamboo (Srimathi Priya et al. 2014).
AMF were found to be low in diversity in saline–alkaline soil of Yellow River Delta,
species richness being even lower in winter (Wang and Liu 2001). Salinity has been
found to be inhibitory to AMF (Aliasgharzad et al. 2001). AMF have also been
found in very low density in the upper Arctic (Kytöviita 2005) and Antarctica soils
(Barbosa et al. 2017). Thus although AMF confer stress tolerance on symbiont
plants, they are less tolerant to various abiotic stresses.
AMF are known to impart tolerance to temperature stresses in plants (Duhamel
and Vandenkoornhuyse 2013). They help alter plant physiology toward
withstanding stress conditions (Miransari et al. 2008). Thus under high- and
low-temperature stress, AM plants perform better than non-AM plants showing
higher shoot and root dry weights (Zhu et al. 2010b; Latef and Chaoxing 2011;
Liu et al. 2014a). AMF have been reported to enhance leaf and root number, crown
diameter, and leaf area in strawberry (Fragaria ananassa) (Matsubara et al. 2004)
and root length and root diameter in Dichanthelium lanuginosum (Bunn et al. 2009).
Similarly, a 1.53 C increase in temperature enhances seed number and plant
biomass in Medicago truncatula colonized by Rhizophagus irregularis (Hu et al.
2015). The ability to perform better under temperature stresses has partially been
attributed to AMF’s contribution to enhancing hosts’photosynthesis and nutrient
uptake (especially phosphate).
590 S. Sahay

Contrasting reports are also available that show that AM symbiosis causes no or
negative growth enhancement in host plants under temperature stress. For example,
AM maize showed similar shoot and root dry weights to non-AM maize under
temperature stress (Zhu et al. 2015), while under high-temperature stress AM
cyclamen (Cyclamen persicum) showed lower root and tuber dry weights as com-
pared to non-AM plants (Maya and Matsubara 2013). The reasons ascribed to these
discrepancies are the possible underperformance of AMF in nutrition mobilization
and/or the high carbon cost–benefit ratio of AMF with the host plants (Martin and
Stutz 2004; Chen et al. 2014).
Endophytes and Abiotic Stress
Endophytic fungi occupy the inner tissue of plants deriving food and protection from
the host and in return bestow upon the host an enhanced ability to resist various
biotic and abiotic stresses (Saikkonen et al. 1998). They may spend the whole or part
of their lifecycle in the host without causing any infection (Tan and Zou 2001).
The endophytic fungus Curvularia crepinii isolated from the roots of Hedyotis
diffusa growing in the geothermal ecosystems of Southwest China has been reported
to improve thermotolerance of host plants significantly under laboratory conditions.
The endophyte-treated plant showed a lower death rate as compared to non-treated
plants under thermal stress conditions (Zhou et al. 2015). Thermomyces sp., a
thermophilic endophytic (CpE) fungus, isolated from Cullen plicata (a desert
plant) roots was found to assist cucumber plants in heat stress tolerance grown in
the summer season (Ali et al. 2018). The fungal inoculants could alleviate heat
stress-related adverse effects in this plant by avoiding alteration in the PSII quantum
efficiency, photosynthesis rate, water use efficiency, and root length vis-à-vis
noninoculated plants. The inoculated plant also showed a higher amount of total
sugars, flavonoids, saponins, soluble proteins, and antioxidant enzyme activities (Ali
et al. 2018).
The role of introduced salt-tolerant Antarctica microorganisms (endosymbiotic
fungus) has been tested in causing overall growth promotion of four plants under salt
stress viz., tomato, onion, cayenne, and lettuce (Acuña-Rodríguez et al. 2019). The
following four types of treatments were used: (a) uninoculated plants without saline
stress (control), (b) uninoculated plants with saline stress (200 mM NaCl) (control),
(c) plants inoculated with the microorganism consortium with no saline stress, and
(d) inoculated plants subjected to saline stress. The inoculated plants were found in
general to show improved survival in presence of salt stress as compared to uninoc-
ulated plants. Although saline stress caused a negative impact on all the selected
measurable traits (lipid peroxidation, proline accumulation, expression of NHX1
antiporter gene, and ecophysiological performance) of inoculated and uninoculated
plants, impact was significantly less in inoculated plants. In salt stress conditions, the
ecophysiological performance, proline accumulation, and NHX1 expression were
found to higher while lipid peroxidation to be lower in inoculated plants vis-à-vis
uninoculated ones. Overall, the biomass of inoculated plants under normal and salt
stress conditions was found to be the same indicating the salt stress alleviating effect
of inoculants (Acuña-Rodríguez et al. 2019). The two fungal endophytes from
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 591

Antarctica viz., Penicillium brevicompactum and P. chrysogenum have been
reported to improve sequestration of Na
+
in vacuoles in case of tomato and lettuce
plants by upregulating the expression of vacuolar NHX1 Na
+
/H
+
antiporters apart
from causing a positive impact on nutrients availability, net photosynthesis, water
use efficiency, yield, and survival under salt stress conditions (Molina-Montenegro
et al. 2020).
Endophytic fungi have also been reported to impart UV protection in the
Antarctica plant Colobanthus quitensis (Barrera et al. 2020). The fungal inoculant
reduced lipid peroxidation and improved photosynthesis efficiency in the presence
of high UV-B radiation conditions. Analysis at the molecular level revealed that the
inoculants reduced CqUVR8,CqHY5, and CqFLS transcript levels, and enhanced
(eightfold) the content of quercetin, a ROS-scavenger flavonoid, in leaves under
high UV-B after 48 h of treatment (Barrera et al. 2020).
Endophytic fungi from the plants of desert areas have also been found to help
symbiont tolerating abiotic stress, such as heat and drought. For example,
thermotolerant endophytic fungal species Thermomyces lanuginosus has been
reported to equip the desert plant Cullen plicata Delile with the ability to show a
higher growth rate and resistance to drought and heat stress (Ali et al. 2019). The
fungus Piriformospora indica, originally isolated from the spore of Glomus mosseae
in the rhizosphere xerophytic plants (Verma et al. 1998), can colonize root of broad
host plants belonging to monocotyledons and dicotyledons and confer tolerance to
abiotic (drought, low and high temperatures, salinity, and heavy metals) and biotic
(root and foliar pathogens) stresses (Johnson et al. 2014).
Some reports tell that plants in various extreme climates harbor fungal
endophytes capable of conferring tolerance to such climates related stresses
(Rodriguez and Redman 2008). Thus grass species Leymus mollis (dunegrass)
from coastal habitat is home to symbiotic fungal endophytes F. culmorum that can
confer salinity tolerance to the host plants (Rodriguez and Redman 2008). The
endophytic fungi from halotolerant Ipomeapes-caprae LR Br have been found to
impart salinity tolerance in Oryza sativa L. (Manasa et al. 2020). The grass,
Dichanthelium lanuginosum, from geothermal soils in Yellowstone National Park
harbors endophyte, Curvularia protuberate, which provides heat tolerance to the
host plant (Redman et al. 2002). In this case, it was later found that the heat tolerance
was indeed provided by the virus inhabited by the fungus (Marquez et al. 2007). The
habitat-specific endophytes were found to exhibit a broad host range and thus very
important for managing plants under stressful climates. The habitat-specific fungal
endophytes (F. culmorum and Cu. protuberata) have been used to inoculate two
commercial rice varieties to inculcate salt and drought tolerance in them (Redman
et al. 2011). The inoculated plants were found to develop salt and drought tolerance,
exhibit water consumption reduced by 20–30%, and growth and yield increased
significantly (Redman et al. 2011).
25.2.2.2 Biotic Stress
Fungi also regulate plant pathogens in the soil (Frąc et al. 2015). Mycoparasitic fungi
that can derive nutrition from other live fungi are considered to be important for such
592 S. Sahay

regulation. Mycoparasitic fungi may be contact necrotroph [when there is no pene-
tration of host mycelium but host mycelium may lyse], invasive nectrotroph [when
host mycelium is penetrated followed by degeneration of cytoplasm e.g., Chytrids
and members of the Oomycota, including Spizellomyces and Pythium-like fungi,
and, Fusarium spp., Gliomastix spp. (both Ascomycota) and Mortierella
ramanniana (Mortierellomycotina) which have been reported from dead and dying
spores of AMF], intracellular biotroph [when complete mycoparasite mycelium is
present in the host and host cytoplasm remains healthy], houstorial biotroph [when
only haustoria are sent inside host mycelium and host cytoplasm remains healthy
e.g., parasitic species of Piptocephalis (Zoopagomycotina) and Dimargaris
(Kickxellomycotina) growing on living saprotrophic hosts such as Pilobolus,Pilaira
and Phycomyces (Mucoromycotina)], and fusion biotroph [when mycoparasite and
host mycelium are closely appressed often former’s hyphae coil around latter’s one
and cytoplasmic contact is ensured via micropore(s) or short penetrative hyphal
branch] (Jeffries 1995). Nematode trapping fungi such as Arthrobotrys has been
found to attack not only nematodes but also fungus (Nordbring-Hertz 2004). The
species of Trichoderma have special significance in their role as competitive
saprotrophs, opportunistic mycoparasite, and possibly plants’symbionts (Harman
et al.2004). Trichoderma viride, for example, attacks Rhizoctonia solani (damping
off fungus) and Armillaria and Armillariella (trees’pathogen). There are strains of
Trichoderma and Gliocladium developed to serve as biocontrol for crop pathogens.
Mycoparasitic fungi such as Trichoderma sp. play a special role in controlling
pathogenic fungi in the soil (Dawidziuk et al. 2016). Soil health is crucial for
sustainable agriculture (Cardoso et al. 2013). The antagonistic fungi (Glomus spp.
and Trichoderma spp.) are recommended to be used to inhibit fungal pathogens
(Dawidziuk et al. 2016). The Trichoderma species (T. asperellum, T. atroviride,
T. harzianum, T. virens, and T. viride), for example, are recommended as biocontrol
in horticulture (López-Bucio et al. 2015). The species of Trichoderma have been
reported from various cold areas (Sahay 2021) and Mediterranean sponges
(Gal-Hemed et al. 2011). Apart from these psychrotolerant and halotolerant strains,
thermo cum halotolerant strains T. asperellum, TaDOR673 and T. asperellum,
TaDOR7316 have also been isolated (Poosapati et al. 2014) implying their applica-
bility as a biocontrol in extremophilic areas.
The role of endophytes and their volatile products in controlling plant pathogens
have especially been studied. The fungal endophyte, for example, Epicoccum
nigrum from sugarcane and Colletotrichum gloeosporioides and Clonostachys
rosea from cacao plant (Theobroma cacao) antagonize several pathogens (de Lima
Favaro et al. 2012; Mejia et al. 2008). The inoculation of Pi. indica has been
demonstrated to confer resistance to various root diseases in the case of barley
(Waller et al. 2005), maize (Kumar et al. 2009), tomato (Fakhro et al. 2010), and
wheat (Rabiey et al. 2015). The fungus has been reported to repress even viral
(Pepino Mosaic Virus) infection (Fakhro et al. 2010) (Table 25.1).
The secondary metabolites from sugarcane endophyte E. nigrum (Brown et al.
1987) such as epicorazines A–B (Baute et al. 1978) and flavipin (Bamford et al.
1961) have been associated with E. nigrum biocontrol activity (Brown et al. 1987;
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 593

Table 25.1 Antimicrobial compounds and their targets obtained from endophytic fungi
Endophyte Source plant
Bioactive
compound Target pathogen Host Reference
Piriformospora
indica
Glomus
mosseae
(spore)
–Fusarium culmorum Barley Waller et al. (2005)
Wheat Serfling et al. (2007)
F. graminearum Barley Deshmukh and Kogel
(2007)
F. verticillioides Maize Kumar et al. (2009)
F. oxysporum Tomato Qiang et al. (2012)
Verticillium dahliae Tomato Fakhro et al. (2010)
Rhizoctonia solani Barley Qiang et al. (2012)
Blumeria graminis f. sp. hordei Barley Waller et al. (2005)
B. graminis f. sp. tritici Wheat Serfling et al. (2007)
Pepino mosaic virus Tomato Fakhro et al. (2010)
Epicoccum nigrum Sugarcane Flavipin
Epicorazines A–B
Sclerotinia sclerotiorum Sunflower
Pythium Cotton
Phytoplasma Apple
Monilinia spp. Peaches de Lima Favaro et al.
(2012)
Nectarines Brown et al. (1987)
P. brevicompactum Barley Cochiolobus,fusarium Barley
Pyrenophora and Rhynchosporium Murphy et al. (2015)
Colletotrichum sp., Artemisia
annua
Colletonoic acid Bacteria and fungi Hussain et al. (2014) and
Bills et al. (2002)
Co.
gloeosporioides
Theobroma
cacao
–Phytophthora palmivora Cacao Mejia et al. (2008)
594 S. Sahay

Do A. mongolica Colletotric acid Bacteria and fungi
Helminthosporium sativum Zou et al. (2000)
Clonostachys
rosea
Do Moniliophthora roreri Do Do
Pestalatiopsis
jester
–Jesterone Anti-oomycete activity Li and Strobel (2001)
Pe. microspora Terminalia
morobensis
Anti-microbial activity Strobel et al. (2002) and
Strobel and Daisy (2003)
Pe. microspora Rain forest’s
tree
Ambuic acid Fusarium spp. and Pythium ultimum Li et al. (2001)
Cordyceps
dipterigena
Cordycepsidone
A
Gibberella fujikuroi Varughese et al. (2012)
Cryptosporiopsis
quercina,
hardwood
Cryptocandin Sclerotinia sclerotiorum, Botrytis cinerea Strobel et al. (1999)
Do Do Cryptocin Pyricularia oryzae
Fusarium oxysporum
Geotrichum candidum,
Rhizoctonia solarium
S. sclerotiorum,Py. ultimum,
Phytophthora cinnamon and Ph. citrophthora Li et al. (2000)
Phomopsis sp. Phomopsichalasin Bacillus subtilis,Salmonella enterica, and
Staphylococcus aureus
Horn et al. (1995)
Muscodor albus Cinnamomum
zeylanicum
28 volatile
compounds
Bacteria and fungi Worapong et al. (2001)
and Strobel et al. (2001)
Muscodor crispans Ananas
ananassoides
Mixed volatile
compounds
Bacteria and fungi
Pythium ultimum
(continued)
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 595

Table 25.1 (continued)
Endophyte Source plant
Bioactive
compound Target pathogen Host Reference
Alternaria helianthi,
Botrytis cinerea,
Fusarium culmorum,
F. oxysporum,
Phytophtora cinnamomi,
Ph. palmivora,
Rhizoctonia solani,
Sc. Sclerotiorum, and
Verticillium dahlia
Xanthomonas axonopodis pv. citri Mitchell et al. (2010)
Daldinia
concentrica
Olive tree 27 volatiles Aspergillus niger Peanuts
Penicillium digitatum
Botrytis cinerea, etc. 12 fungi belonging to asco,
Basiodio, and oomycetes and post-harvest fungi
of plums, apricot, and resin
Liarzi et al. (2016)
596 S. Sahay

Madrigal et al. 1991; Madrigal and Melgarejo 1995). The secondary metabolite
ambuic acid from the endophytic fungus Pestalotiopsis microspora shows inhibitory
activity against Fusarium and Pythium ultimum (Li et al. 2001) and Colletonoic acid
(Bills et al. 2002) from Colletotrichum sp., against many bacteria, fungi, and algae
activities (Hussain et al. 2014). Other important secondary metabolites from
endofungus Cryptosporiopsis quercina such as Cryptocandin inhibits many phyto-
pathogenic fungi, e.g., Sclerotinia sclerotiorum and Botrytis cinerea (Strobel et al.
1999) and Cryptocin (Li et al. 2000) antagonizes phytogens Pyricularia oryzae,
F. oxysporum,Geotrichum candidum,Rhizoctonia solarum,S. sclerotiorum,
Pythium ultimum,Phytophthora cinnamon, and Ph. citrophthora (Li et al. 2000)
(Table 25.1).
Furthermore, volatile compounds (chemically alcohols, esters, ketones, acids,
and lipids) from the endophytic fungus Muscodor albus isolated from cinnamon
(Cinnamomum zeylanicum) plant (Worapong et al. 2001), M. crispans isolated from
pineapple (Ananas ananassoides), and Daldinia concentric from olive tree are
inhibitory to many of the phytopathogenic fungi. The antimicrobial volatile
compounds from M. crispans have additional advantages, i.e., they are harmless
and can be used as safe antimicrobials in food and agriculture (Mitchell et al. 2010)
(Table 25.1).
Endophytes also generate compounds that are insecticidal or insect repellant
(Table 25.2). Apart from these, entomopathogenic fungal endophytes have also
been reported to have potential activity against insect pests of important crops
reducing their survival and/or reproduction (Table 25.3).
Losses during storage of fruits, vegetables, and grains as caused by fungal
pathogens such as Alternaria,Aspergillus,Botrytis,Fusarium,Geotrichum,
Table 25.2 Insecticidal compounds /insect repellants from endophytes and their target insects
Endophyte Source plant
Bioactive
compound Target insect Reference
Epichloë
endophytes
Ryegrass Peramine Argentine stem weevil Johnson et al.
(2013)
E. endophytes
R-37
Epoxy-
janthitrems
Broad pesticidal effect Finch et al.
(2012)
Beauveria
bassiana
Coffee berry borer Vega et al.
(2010)
Clonostachys
rosea
Do
Nodulisporium
sp.
Bontia
daphnoides
Nodulisporic
acids
Larvae of the blowfly Demain
(2000)
Muscodor
vitigenus
Lianas Naphthalene General insect repellent
Cephus cintus
Daisy et al.
(2002)
Be. bassiana
Clonostachys
rosea
Coffee berry borer Vega et al.
(2010)
Curvularia
lunata
Melia
azedaracht
Instar larvae of the cotton
leaf worm
Saad et al.
(2019)
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 597

Table 25.3 Entomopathogenic fungal endophytes as potential biocontrol against insect pests of
important crops (adapted from Mantzoukas and Eliopoulos 2020)
Endophytic
species Crop Target pest Activities Reference
Beauveria.
bassiana
Coffee Hypothenemus
hampei
Pathogenize Vega et al. (2008)
Maize Ostrinia
nubilalis
Reduce pest
population
Bing and Lewis (1991)
Sesamia
calamistis
Reduce
larval
tunneling
Cherry et al. (2004)
Banana Cosmopolites
sordidus
Reduce
larval
survival
Akello et al. (2008)
Tomato Helicoverpa
zea
Hardly affect
larval
mortality
Powell et al. (2009)
Helicoverpa
armigera
Reduce
infestation
Qayyum et al. (2015)
Sorghum Chilo partellus Reduce
larval
tunneling
Reddy et al. (2009)
Sesamia
nonagrioides
Reduce
infestation
Mantzoukas et al.
(2015) and Mantzoukas
and Grammatikopoulos
(2019)
Opium
poppy
Iraella luteipes Reduce
larval
survival
Quesada-Moraga et al.
(2009)
Cotton Aphis gossypii Reduce
reproduction
Castillo-Lopez et al.
(2014)
Chortoicetes
terminifera
Reduce
growth rate
Gurulingappa et al.
(2010)
Rachiplusia nu Reduce
larval
feeding
Russo et al. (2019)
Melon Aphis gossypii Reduce
reproduction
No effect on
natural
enemies
González-Mas et al.
(2019)
Fava bean Helicoverpa
armigera
Reduce
larval
survival
Vidal and Jaber (2015)
Liriomyza
huidobrensis
Reduce pest
population
Akutse et al. (2013)
Acyrthosiphon
pisum
Reduce pest
population
Akello and Sikora
(2012)
(continued)
598 S. Sahay

Table 25.3 (continued)
Endophytic
species Crop Target pest Activities Reference
Common
bean
Helicoverpa
armigera
Reduce
larval
survival
Vidal and Jaber (2015)
Liriomyza
huidobrensis
Reduce pest
population
Akutse et al. (2013)
White jute Apion corchori Reduce
infestation
Biswas et al. (2013)
Soybean Aphis glycines Hardly affect
pest
population
Clifton et al. (2018)
Helicoverpa
gelotopoeon
Decrease
larval
feeding
Russo et al. (2018)
Grapevine Planococcus
ficus
Reduce
infestation
Rondot and Reineke
(2018)
Empoasca vitis Reduce
infestation
Do
Pepper Myzus persicae Increase pest
mortality
Mantzoukas and
Lagogiannis (2019)
Reduce
development
and fecundity
Jaber and Araj (2018)
Strawberry Myzus persicae Reduce
feeding
Manoussopoulos et al.
(2019)
Cauliflower Bemisia tabaci Reduce pest
survival
Jaber et al. (2018)
Pecan Melanocallis
caryaefoliae
Reduce pest
population
Ramakuwela et al.
(2020)
Monellia
caryella
Do
Lemon Diaphorina
citri
Reduce
reproduction
and survival
Peña-Peña et al. (2015)
Lecanicillium
lecanii
Cotton Aphis gossypii Reduce
reproduction
Gurulingappa et al.
(2010)
Lecanicillium
muscarium
Cauliflower Plutella
xylostella
Increase
larval
mortality
Kuchár et al. (2019)
Aspergillus
parasiticus
Cotton Chortoicetes
terminifera
Reduce
growth rate
Gurulingappa et al.
(2010)
Metarhizium
anisopliae
Fava bean Acyrthosiphon
pisum
Hardly pest
population
Akello and Sikora
(2012)
Pepper Myzus persicae Increase pest
mortality
Mantzoukas and
Lagogiannis (2019)
(continued)
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 599

Gloeosporium,Mucor,Monilinia,Penicillium,Rhizopus, and other genera called
postharvest loss are significant. There is a worldwide concern to reduce it. Some of
the recommended practices to control postharvest losses include avoiding injury
during harvesting and handling, sanitation practices, and application of fungicides.
But all these are either insufficient or unsustainable (Pimenta et al. 2009). One of the
promising alternatives proposed is to use antagonistic microbes such as bacteria,
fungi, and yeasts (Schisler et al. 2011). Among these microbes yeasts have been
found more suitable for their inherent features such as ability to establish on
the wounds, fast scavenge sugary exudates making the wound unsuitable for forth-
coming pathogens, survive at a low temperature, acidic pH, and conditions of
osmotic stress, resist desiccation, and tolerate to chemicals. Moreover, the ease of
their cultivation, fast-growth, and low input requirements make them attractive for
biocontrol formulations (Schisler et al. 2011). Yeasts that could reduce postharvest
Table 25.3 (continued)
Endophytic
species Crop Target pest Activities Reference
Rapeseed Aphis fabae Reduce
larval
survival
Batta (2013)
Plutella
xylostella
Reduce
larval
survival
Batta (2013)
Strawberry Myzus persicae Reduce
feeding
Manoussopoulos et al.
(2019)
Metarhizium
brunneum
Soybean Aphis glycines Increase pest
population
Clifton et al. (2018)
Cauliflower Bemisia tabaci Reduce pest
survival
Jaber et al. (2018)
Melon Aphis gossypii Reduce
reproduction
& survival
González-Mas et al.
(2019)
Metarhizium
robertsii
Sorghum Sesamia
nonagrioides
Reduce
infestation
Mantzoukas and
Grammatikopoulos
(2019)
Clonostachys
rosea
Coffee Hypothenemus
hampei
Pathogenize Vega et al. (2008)
Purpureocillium
lilacinum
Cotton Aphis gossypii Reduce
reproduction
Castillo-Lopez et al.
(2014)
Isaria
fumosorosea
Sorghum Sesamia
nonagrioides
Reduce
infestation
Mantzoukas and
Grammatikopoulos
(2019)
Pepper Myzus persicae Increase pest
mortality
Mantzoukas and
Lagogiannis (2019)
Lemon Diaphorina
citri
Reduce
reproduction
and survival
Peña-Peña et al. (2015)
600 S. Sahay

pathogens of various crops have been reported as follows: Au. pullulans in cherry
fruit, Au. pullulans and Rhodotorula glutinis in apple fruit, Candida sake in straw-
berry fruit, Cryptococcus albidus in pear fruit, Cr. laurentii,Rh. glutinis in mango
fruit, etc. (Schisler et al. 2011).
The psychrotrophic yeast strains isolated from Nothofagus sp. and Vaccinium
sp. in Northwest-Patagonia have been found to exhibit an inhibitory effect on some
phytopathogen like Verticillium dahliae PPRI5569 and Pythium aphanidermatum
PPRI 9009, but could not affect the growth of F. oxysporum PPRI5457 (Mestre
et al. 2016). Marine yeasts (Debaryomyces hansenii and Rhodotorula minuta)in
combination with ClO
2
were found to control postharvest anthracnose (Co.
gloeosporioides) of mango fruits (Reyes-Perez et al. 2019).
Chitinase as Biocontrol
The enzyme chitinase has also been studied as a potential tool to manage pathogenic
fungi. Of the seven chitinases purified from the thermophilic fungus, Thermomyces
lanuginosus SSBP (Chit1), six have been found to inhibit Aspergillus niger,
A. flavus,A. alliaceus,A. ochraceus,F. verticillioides, and Mucor sp. (Okongo
et al. 2019). An exochitinase (MtChit) purified from thermophilic mold
Myceliopthora thermophila was found to inhibit the growth of plant pathogenic
fungi such as F. oxysporum and Cu. lunata. The enzyme is acid and organic acid
solvent tolerant and thermostable that could be produced in heterologous system
Pichia pastoris in high titers expressed from GAP promoter (Dua et al. 2017).
Cold active chitinases from Antarctica fungus Lecanicillium muscarium CCFEE
5003 have also been found to be very effective antimycotic molecules that could be
used to control pathogenic fungi including oomycetous one in a cold environment.
Production of this enzyme has been optimized at the bench-top bioreactor level
(Fenice 2016).
Antifungal Protein (AFuP) as Biocontrol
AFuPs are small, cysteine-rich, and cationic proteins that inhibit many of the species
of fungi (Lee et al. 1999). Genome mining has revealed that fungi harbor many
AFuP-like sequences falling in four major classes viz., A, B, C, and the newer ones
(Tóth et al. 2016; Garrigues et al. 2018). Various fungi produce more than one type
of any or many of these classes of AFuPs. The genome of fungus P. chrysogenum,
for example, codes for one AFuP of each of the three classes (classes A, B, and C)
while that of Penicillium digitatum only one (class B) (Garrigues et al. 2017) and
Neosartorya fischeri one each of two AFuPs (classes A and C).
The fungal species P. chrysogenum (Khan et al. 2020; Leitão et al. 2012),
P. brevicompactum (Zhang et al. 2018), and Monascus pilosus (Zheng et al. 2021)
which have extremophilic strains produce AFuPs. The representatives of class B
AFuP is produced by P. chrysogenum (Huber et al. 2018) and Mo. pilosus (Tu et al.
2016), and that of class C, i.e., the BP protein, is produced by P. brevicompactum
(Seibold et al. 2011) and the Pc-Arctin is produced by P. chrysogenum (Chen et al.
2013). AFuPs such as PAF and PAFB from P. chrysogenum were also found to be
active against human pathogenic yeasts and molds such as A. fumigates,A. terreus,
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 601

and Trichophyton rubrum (Huber et al. 2018). P. expansum codes for A, B, and C
AfuP of which A type one (PeAFPs) was found to have efficient activity against
fungal infections caused by Botrytis cinerea in tomato leaves and P. digitatum in
oranges (Garrigues et al. 2018).
The psychrotolerant and osmotolerant yeast Starmerella bacillaris (Nadai et al.
2018) isolated from overripe and botrytized grape berries have been reported to
exhibit antifungal activity against Botrytis cinerea, a wine degrader and P. expansum
causing postharvest rot in apples (Lemos et al. 2016).
25.2.3 Crop Improvement
25.2.3.1 AFP Coding Gene for Crop Improvement
Antifreeze protein (AFP) and glycoprotein (AFGP) are stress proteins that protect
the organisms from freezing injury. They do so by reducing the freezing point by the
adsorption–prevention process. They have been isolated from bacteria, fungi, polar
fishes, insects, and plants. AFPs bind the growing ice surface, give it a specific
shape, and discourage its further enlargement. Besides, AFPs stop the fusion of ice
crystals forming large aggregates that may be fatal to cells. AFPs lower the freezing
point but not the melting point, the process being called thermal hysteresis or
TH. TH exhibited by plants is mild TH (0.1–0.5 C), by fishes is moderate TH
(1–5C), and by insects is high (5 C). TH of yeast Glaciozyma sp. AY30 is ~0.42 C
falling in the mild range (Chakraborty and Jana 2019).
Both AFP and AFGP have been reported to be useful in imparting cold tolerance
to plants (e.g., tobacco, tomato, and potato) (Hightower et al. 1991; Fan et al. 2002;
Gupta and Deswal 2014). Thus obtained transgenic plants exhibit different
psychrotolerant phenotypes. For example, transgenic tobacco carrying carrot AFP
gene DcAFP exhibited retardation in ice recrystallization, enhancement of TH from
0.35 to 0.56 C, reduction in ion leakage, and faster recovery from cold stress (Fan
et al. 2002). Likewise, AFP transgene from Lolium perenne in transgenic tomato
showed three times more relative water content and 2.6 times less electrolyte leakage
(cf wild type tomato plant) (Balamurugan et al. 2018) and in transgenic Arabidopsis
and E. coli enhanced cold hardiness (Zhang et al. 2010). In another report,
Arabidopsis plants carrying transgene LpIRI-a/b exhibited a survival rate of
85–100% (cf 73% shown by wild type) when exposed to cold at 4C for 7 days
(Zhang et al. 2010). AFP transgenic plants have been found in general to exhibit
retardation in ice recrystallization, production of TH (Griffith and Yaish 2004), and
enhancement of general cold hardiness (Gupta and Deswal 2014). In one report
chimeric AFP gene (spa-afa-5) has been found to exhibit 10 times more effect than
the original AFP gene (afa-3) (Hightower et al. 1991) in a transgenic tomato plant,
afa-3 being the AFP III from winter flounder and spa being staphylococcal
protein-A.
Ice binding proteins (AFP) have been isolated and studied from psychrophilic
fungi Antarctomyces pellizariae and A. psychrotrophicus (Thelebolales) of
Ascomycetes (Batista et al. 2020) and Typhula ishikariensis (Kondo et al. 2012),
602 S. Sahay

yeasts Goffeauzyma gastric (Villarreal et al. 2018), Leucosporidium sp. (Lee et al.
2012), and Glaciozyma antarctica (Hashim et al. 2013). Although THs of plant and
yeast AFPs are almost parallel, plant AFPs have been used to test their ability to
confer cold-hardiness in mesophilic plants (Gupta and Deswal 2014). Yeast/fungal
AFP is yet to be tested and applied to impart cold tolerance to plants.
25.2.3.2 Chitinase Coding Genes and Crop Improvement
The transgenic potato and tobacco-containing endochitinase transgene from
T. harzianum introduced by Agrobacterium tumefacience mediated transformation
have been found to exhibit tolerance to complete resistance to many phytogenic
fungi such as Alternaria alternate,A. solani,B. cinerea, and R. solani (Lorito et al.
1998). Endochitinase gene from T. virens has also been found to impart resistance in
transgenic cotton and tobacco against soil-borne fungus Rhizoctonia solani and a
foliar pathogen, Alternaria alternate (Emani et al. 2003).
25.2.3.3 AFuP Coding Gene for Crop Improvement
Antifungal protein-coding genes from A. giganteus have been expressed from the
promoters of three maize pathogenesis-related (PR) genes ZmPR4, mpi, and, PRms
in transgenic rice, and their impact was assessed against the most devastating fungal
pathogen Magnaporthe grisea causing rice blast disease. The transgenic rice plants
showed resistance to M. grisea at various levels indicating the effectiveness of the
strategy (Moreno et al. 2005). Recently, the antifungal protein gene PeafpA from
P. expansum has been cloned and expressed using P. chrysogenum based expression
cassette pSK275 and its antifungal activities have been tested (Garrigues et al. 2018).
Both P. chrysogenum and P. expansum are found in Antarctica soil (Sahay 2021).
This result shows potential for its use to engineer phytopathogenic fungal-resistant
transgenic crops.
Although the PeAFPA has not shown hemolytic activity, it has to face other
biosafety and public acceptance tests.
References
Acuña-Rodríguez IS, Hansen H, Gallardo-Cerda J, Atala C, Molina-Montenegro MA (2019)
Antarctic extremophiles: biotechnological alternative to crop productivity in saline soils.
Front Bioeng Biotechnol 7:22
Agerer R (2001) Exploration types of ectomycorrhizae—a proposal to classify ectomycorrhizal
mycelial systems according to their patterns of differentiation and putative ecological impor-
tance. Mycorrhiza 11:107–114
Aitken MD (1993) Waste treatment applications of enzymes: opportunities and obstacles. Chem
Eng J 52:B49–B58
Akello J, Sikora R (2012) Systemic acropedal influence of endophyte seed treatment on
Acyrthosiphon pisum and Aphis fabae offspring development and reproductive fitness. Biol
Control 61:215–221
Akello J, Dubois T, Coyne D, Kyamanywa S (2008) Endophytic Beauveria bassiana in banana
(Musa spp.) reduces banana weevil (Cosmopolites sordidus) fitness and damage. Crop Prot 27:
1437–1441
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 603

Akutse KS, Maniania NK, Fiaboe KKM, Van den Berg J, Ekesi S (2013) Endophytic colonization
of Vicia faba and Phaseolus vulgaris (Fabaceae) by fungal pathogens and their effects on the
life-history parameters of Liriomyza huidobrensis (Diptera: Agromyzidae). Fungal Ecol 6:293–
301
Ali AH, Abdelrahman M, Radwan U, El-Zayat S, El-Sayed MA (2018) Effect of Thermomyces
fungal endophyte isolated from extreme hot desert-adapted plant on heat stress tolerance of
cucumber. Appl Soil Ecol 124:155–162
Ali AH, Radwan U, El-Zayat S et al (2019) The role of the endophytic fungus, Thermomyces
lanuginosus, on mitigation of heat stress to its host desert plant Cullen plicata. Biol Futura 70:1–
7
Aliasgharzad N, Rastin SN, Towfighi H, Alizadeh A (2001) Occurrence of arbuscular mycorrhizal
fungi in saline soils of the Tabriz plain of Iran in relation to some physical and chemical
properties of soil. Mycorrhiza 11(3):119–122
Allakhverdiev SI, Kreslavski VD, Klimov VV et al (2008) Heat stress: an overview of molecular
responses in photosynthesis. Photosynth Res 98:541–550
Allbed A, Kumar L (2013) Soil salinity mapping and monitoring in arid and semi-arid regions using
remote sensing technology: a review. Adv Remote Sens 2:373–385
Aranda E, Scervino JM, Godoy P et al (2013) Role of arbuscular mycorrhizal fungus Rhizophagus
custos in the dissipation of PAHs under root-organ culture conditions. Environ Pollut 181:182–
189
Aroca R, Porcel R, Ruiz-Lozano JM (2007) How does arbuscular mycorrhizal symbiosis regulate
root hydraulic properties and plasma membrane aquaporins in Phaseolus vulgaris under
drought, cold or salinity stresses? New Phytol 173:808–816
Augé RM (2001) Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. Mycor-
rhiza 11:3–42
Augé RM, Stodola JW (1990) An apparent increase in symplastic water contributes to greater turgor
in mycorrhizal roots of droughted Rosa plants. New Phytol 115:285–295
Azcón-Aguilar C, Barea JM (1997) Applying mycorrhiza biotechnology to horticulture: signifi-
cance and potentials. Sci Hortic 68:1–24
Bagyaraj DJ, Ashwin R (2017) Soil biodiversity: role in sustainable horticulture. Biodivers Hortic
Crops 5:1–18
Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu Rev Plant Biol
59:89–113
Baker BJ, Lutz MA, Dawson SC, Bond PL, Banfield JF (2004) Metabolically active eukaryotic
communities in extremely acidic mine drainage. Appl Environ Microbiol 70:6264–6271
Balamurugan S, Ann JS, Varghese IP, Murugan SP, Harish MC, Kumar SR, Sharma RK,
Sathishkumar R (2018) Heterologous expression of Lolium perenne antifreeze protein confers
chilling tolerance in tomato. J Integr Agric 17:1128–1113
Bamford PC, Norris GLF, Ward G (1961) Flavipin production by Epicoccum spp. T Brit Mycol Soc
44:354–356
Bano A, Hussain J, Akbar A, Mehmood K, Anwar M, Hasni MS, Ullah S, Sajid S, Ali I (2018)
Biosorption of heavy metals by obligate halophilic fungi. Chemosphere 199:218–222
Barbosa MV, Pereira EA, Cury JC, Carneiro MAC (2017) Occurrence of arbuscular mycorrhizal
fungi on King George Island, South Shetland Islands, Antarctica. An Acad Bras Cienc 89:1737–
1743
Barea JM, Pozo MJ, Azcon R et al (2005) Microbial co-operation in the rhizosphere. J Exp Bot 56:
1761–1778
Barr DP, Aust SD (1994) Mechanisms white-rot fungi use to degrade pollutants. Environ Sci
Technol 28:78A–86A
Barrera A, Hereme R, Ruiz-Lara S, Larrondo LF, Gundel PE, Pollmann S, Molina-Montenegro
MA, Ramos P (2020) Fungal endophytes enhance the photoprotective mechanisms and photo-
chemical efficiency in the Antarctic Colobanthus quitensis (Kunth) Bartl. exposed to uv-B
radiation. Front Ecol Evol 8(122)
604 S. Sahay

Batista TM, Hilario HO, de Brito GAM, Moreira RG, Furtado C, de Menezes GCA, Rosa CA, Rosa
LH, Franco GR (2020) Whole-genome sequencing of the endemic Antarctic fungus
Antarctomyces pellizariae reveals an ice-binding protein, a scarce set of secondary metabolites
gene clusters and provides insights on Thelebolales phylogeny. Genomics 112:2915–2921
Batta YA (2013) Efficacy of endophytic and applied Metarhizium anisopliae (Metch.) Sorokin
(Ascomycota:Hypocreales) against larvae of Plutella xylostella L. (Yponomeutidae: Lepidop-
tera) infesting Brassica napus plants. Crop Prot 44:128–134
Baute MA, Deffieux G, Baute R, Neveu A (1978) New antibiotics from the fungus Epicoccum
nigrum. I. Fermentation, isolation and antibacterial properties. J Antibiot 31:1099–1101
Bej A, Aislabie J, Atlas R (2010) Polar microbiology: the ecology, biodiversity and bioremediation
potential of microorganisms in extremely cold environments. Taylor & Francis, Boca Raton
Bills G, Dombrowski A, Peleaz F et al (2002) Recent and future discoveries of pharmacologically
active metabolites from tropical fungi. In: Watling R, Frankland JC, Ainsworth AM et al (eds)
Tropical mycology: micromycetes, vol 2. CABI, Wallingford, Oxon, pp 165–194
Bing LA, Lewis LC (1991) Suppression of Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) by
endophytic Beauveria bassiana (Balsamo) Vuillemin. Environ Entomol 20:1207–1211
Biswas C, Dey P, Satpathy S, Satya P, Mahapatra BS (2013) Endophytic colonization of white jute
(Corchorus capsularis) plants by different Beauveria bassiana strains for managing stem weevil
(Apion corchori). Phytoparasitica 41:17–21
Bjorbaekmo MFM, Carlsen T, Brysting A, Vrålstad T, Høiland K, Ugland KI, Geml J,
Schumacher T, Kauserud H (2010) High diversity of root associated fungi in both alpine and
arctic Dryas octopetala. BMC Plant Biol 10:244
Bowles TM, Barrios-Masias FH, Carlisle EA et al (2016) Effects of arbuscular mycorrhizae on
tomato yield, nutrient uptake, water relations, and soil carbon dynamics under deficit irrigation
in field conditions. Sci Total Environ 566:1223–1234
Bronicka M, Raman A, Hodgkins D, Nicol H (2007) Abundance and diversity of fungi in saline soil
in central-west New South Wales. Sydowia 59:7–24
Brown AE, Finlay R, Ward JS (1987) Antifungal compounds produced by Epicoccum
purpurascens against soil-borne plant pathogenic fungi. Soil Biol Biochem 19:657–664
Bunn R, Lekberg Y, Zabinski C (2009) Arbuscular mycorrhizal fungi ameliorate temperature stress
in thermophilic plants. Ecology 90:1378–1388
Cabral C, Ravnskov S, Tringovska I et al (2016) Arbuscular mycorrhizal fungi modify nutrient
allocation and composition in wheat (Triticum aestivum L.) subjected to heat-stress. Plant and
Soil 408(1):385–399
Camejo D, Rodríguez P, Morales MA et al (2005) High temperature effects on photosynthetic
activity of two tomato cultivars with different heat susceptibility. J Plant Physiol 162:281–289
Cardoso EJBN, Vasconcellos RLF, Bini D, Miyauchi MYH, dos Santos CA, Alves PRL, de Paula
AM, Nakatani AS, Pereira JD, Nogueira MA (2013) Soil health: looking for suitable indicators.
What should be considered to assess the effects of use and management on soil health? Sci Agric
70:274–289
Castillo-Lopez D, Zhu-Salzman K, Ek-Ramos MJ, Sword GA (2014) The entomopathogenic fungal
endophytes Purpureocillium lilacinum (formerly Paecilomyces lilacinus) and Beauveria
bassiana negatively affect cotton aphid reproduction under both greenhouse and field
conditions. PLoS One 9:e103891
Chaer G, Fernandes M, Myrold D, Bottomley P (2009) Comparative resistance and resilience of soil
microbial communities and enzyme activities in adjacent native forest and agricultural soils.
Microb Ecol 58:414–424
Chakraborty S, Jana B (2019) Antifreeze proteins: an unusual tale of structural evolution, hydration
and function. Proc Indian Natl Sci Acad 85:169–187
Chandra R, Kumar V, Yadav S (2017) Extremophilic ligninolytic enzymes. In: Sani R, Krishnaraj R
(eds) Extremophilic enzymatic processing of lignocellulosic feedstocks to bioenergy.
Springer, Cham
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 605

Chandrasekaran M, Kim K, Krishnamoorthy R et al (2016) Mycorrhizal symbiotic efficiency on
C-3 and C-4 plants under salinity stress—a meta-analysis. Front Microbiol 7:1246
Charest C, Dalpé Y, Brown A (1993) The effect of vesicular-arbuscular mycorrhizae and chilling on
two hybrids of Zea mays L. Mycorrhiza 4:89–92
Charron G, Furlan V, Bernier-Cardou M et al (2001) Response of onion plants to arbuscular
mycorrhizae. Mycorrhiza 11:187–197
Chen Z, Ao J, Yang W, Jiao L, Zheng T, Chen X (2013) Purification and characterization of a novel
antifungal protein secreted by Penicillium chrysogenum from an Arctic sediment. Appl
Microbiol Biotechnol 97:10381–10390
Chen XY, Song FB, Liu FL et al (2014) Effect of different arbuscular mycorrhizal fungi on growth
and physiology of maize at ambient and low temperature regimes. Sci World J 2014:956141
Cherry AJ, Banito A, Djegui D, Lomer C (2004) Suppression of the stem-borer Sesamia calamistis
(Lepidoptera; Noctuidae) in maize following seed dressing, topical application and stem
injection with African isolates of Beauveria bassiana. Int J Pest Manag 50:67–73
Chung D, Kim H, Choi HS (2019) Fungi in salterns. J Microbiol 57(9):717–724
Clifton EH, Jaronski ST, Coates BS, Hodgson EW, Gassmann AJ (2018) Effects of endophytic
entomopathogenic fungi on soybean aphid and identification of Metarhizium isolates from
agricultural fields. PLoS One 13:e0194815
Cookson WR, O’Donnell AJ, Grant CD, Grierson PF, Murphy DV (2008) Impact of ecosystem
management on microbial community level physiological profiles of postmining forest rehabili-
tation. Microb Ecol 55:321–332
Daisy BH, Strobel GA, Castillo U et al (2002) Naphthalene, an insect repellent, is produced by
Muscodor vitigenus, a novel endophytic fungus. Microbiology 148:3737–3741
Dawidziuk A, Popiel D, Kaczmarek J, Strakowska J, Jedryczka M (2016) Morphological and
molecular properties of Trichoderma species help to control stem canker of oilseed rape.
BioControl 61:755–768
de Lima Favaro LC, de Souza Sebastianes FL, Araujo WL (2012) Epicoccum nigrum P16, a
sugarcane endophyte, produces antifungal compounds and induces root growth. PLoS One 7:
e36826
Demain AL (2000) Microbial natural products: a past with a future. In: Wrigley SK, Hayes MA,
Thomas R et al (eds) Biodiversity: new leads for pharmaceutical and agrochemical industries.
The Royal Society of Chemistry, Cambridge, pp 3–16
Deshmukh S, Kogel KH (2007) Piriformospora indica protects barley from root rot disease caused
by Fusarium. J Plant Dis Prot 114:262–268
Deslippe JR, Hartmann M, Mohn WW, Simard SW (2011) Longterm experimental manipulation of
climate alters the ectomycorrhizal community of Betula nana in Arctic tundra. Glob Chang Biol
17:1625–1636
Dua A, Joshi S, Satyanarayana T (2017) Recombinant exochitinase of the thermophilic mould
Myceliopthora thermophila BJA: characteristics and utility in generating N-acetyl glucosamine
and in biocontrol of phytopathogenic fungi. Biotechnol Prog 33:70–80
Duhamel M, Vandenkoornhuyse P (2013) Sustainable agriculture: possible trajectories from
mutualistic symbiosis and plant neodomestication. Trends Plant Sci 18(5):97–600
Dutta T, Sahoo R, Sengupta R, Ray SS, Bhattacharjee A, Ghosh S (2008) Novel cellulases from an
extremophilic filamentous fungi Penicillium citrinum: production and characterization. J Ind
Microbiol Biotechnol 35:275–282
Egamberdieva D, Lugtenberg B (2014) Use of plant growth-promoting rhizobacteria to alleviate
salinity stress in plants. In: Miransari M (ed) Use of microbes for the alleviation of soil stresses,
vol 1. Springer, New York, pp 73–96
Egamberdieva D, Kamilova F, Validov S et al (2008) High incidence of plant growth-stimulating
bacteria associated with the rhizosphere of wheat grown on salinated soil in Uzbekistan. Environ
Microbiol 10:1–9
El Tohamy W, Schnitzler WH, El Behairy U et al (1999) Effect of VA mycorrhiza on improving
drought and chilling tolerance of bean plants (Phaseolus vulgaris). J Appl Bot 73:178–183
606 S. Sahay

Emani C, Garcia JM, Lopata-Finch E, Pozo MJ, Uribe P, Kim DJ, Sunilkumar G, Cook DR,
Kenerley CM, Rathore KS (2003) Enhanced fungal resistance in transgenic cotton expressing an
endochitinase gene from Trichoderma virens. Plant Biotechnol J 1:321–336
Estrada B, Aroca R, Barea JM, Ruiz-Lozano JM (2013) Native arbuscular mycorrhizal fungi
isolated from a saline habitat improved maize antioxidant systems and plant tolerance to salinity.
Plant Sci 201–202:42–51
Evelin H, Kapoor R, Giri B (2009) Arbuscular mycorrhizal fungi in alleviation of salt stress: a
review. Ann Bot 104:1263–1280
Faber BA, Zasoski RJ, Munns DN, Shackel K (1991) A method for measuring hyphal nutrient and
water uptake in mycorrhizal plants. Can J Bot 69:87–94
Fakhro A, Andrade-Linares DR, von Bargen S, Bandte M, Büttner C, Grosch R, Schwarz D,
Franken P (2010) Impact of Piriformospora indica on tomato growth and on interaction with
fungal and viral pathogens. Mycorrhiza 20:191–200
Fan Y, Liu B, Wang H, Wang S, Wang J (2002) Cloning of antifreeze protein gene from carrot and
its influence on coldtolerance in transgenic tobacco plants. Plant Cell Rep 21:296–301
FAO (2009) The state of food insecurity in the world
Fenice M (2016) The psychrotolerant Antarctic fungus Lecanicillium muscarium CCFEE 5003: a
powerful producer of cold-tolerant Chitinolytic enzymes. Molecules 21(4):447
Finch SC, Fletcher LR, Babu JV (2012) The evaluation of endophyte toxin residues in sheep fat. N
Z Vet J 60:56–60
Frąc M, Jezierska-Tys S, Takashi Y (2015) Occurrence, detection, and molecular and metabolic
characterization of heat-resistant fungi in soils and plants and their risk to human health. Adv
Agron 132:161–204
Gal-Hemed I, Atanasova L, Komon-Zelazowska M, Druzhinina SI, Viterbo A, Yarden O (2011)
Marine isolates of Trichoderma as potential halotolerant agents of biological control for arid-
zone agriculture. Appl Environ Microbiol 77(15):5100–5109
Garrigues S, Gandía M, Popa C, Borics A, Marx F, Coca M et al (2017) Efficient production and
characterization of the novel and highly active antifungal protein AfpB from Penicillium
digitatum. Sci Rep 7:14663
Garrigues S, Gandía M, Castillo L, Coca M, Marx F, Marcos JF, Manzanares P (2018) Three
antifungal proteins from Penicillium expansum: different patterns of production and antifungal
activity. Front Microbiol 9:2370
Geml J, Kauff F, Brochmann C et al (2012) Frequent circumarctic and rare transequatorial
dispersals in the lichenised agaric genus Lichenomphalia (Hygrophoraceae, Basidiomycota).
Fungal Biol 116:388–400
Girisha GK, Condron LM, Clinton PW, Davis MR (2003) Decomposition and nutrient dynamics of
green and freshly fallen radiata pine (Pinus radiata) needles. For Ecol Manage 179:169–181
González-Mas N, Sánchez-Ortiz A, Valverde-García P, Quesada-Moraga E (2019) Effects of
endophytic entomopathogenic ascomycetes on the life-history traits of Aphis gossypii glover
and its interactions with melon plants. Insects 10:165
Goswami NN (1982) Distribution and characteristics of problem soils in India and their manage-
ment for crop production, pp 25–40. https://www.jircas.go.jp›tars›tars15-25-39
Griffith M, Yaish MW (2004) Antifreeze proteins in overwintering plants: a tale of two activities.
Trends Plant Sci 9:399–405
Grum-Grzhimaylo AA, Georgieva ML, Bondarenko SA, Debets AJM, Bilanenko EN (2016) On
the diversity of fungi from soda soils. Fungal Divers 76:27–74
Gupta R, Deswal R (2014) Antifreeze proteins enable plants to survive in freezing conditions. J
Biosci 39:931–944
Gurulingappa P, Sword GA, Murdoch G, McGee PA (2010) Colonization of crop plants by fungal
entomopathogens and their effects on two insect pests when in planta. Biol Control 55:34–41
Hallsworth JE, Heim S, Timmis KN (2003) Chao tropic solutes cause water stress in pseudomonas
putida. Environ Microbiol 5:1270–1280
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 607

Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species—opportunistic,
avirulent plant symbionts. Nat Rev Microbiol 2:43–56
Hashim NH, Bharudin I, Nguong DL, Higa S, Bakar FD, Nathan S, Rabu A, Kawahara H, Illias
RM, Najimudin N, Mahadi NM, Murad AM (2013) Characterization of Afp1, an antifreeze
protein from the psychrophilic yeast Glaciozyma antarctica PI12. Extremophiles 17:63–73
Hightower R, Baden C, Penzes E, Lund P, Dunsmuir P (1991) Expression of antifreeze proteins in
transgenic plants. Plant Mol Biol 17:1013–1021
Hobbie JE, Hobbie EA (2006) 15N in symbiotic fungi and plants estimates nitrogen and carbon flux
rates in Arctic tundra. Ecology 87:816–822
Högberg MN, Högberg P (2002) Extramatrical ectomycorrhizal mycelium contributes one-third of
microbial biomass and produces, together with associated roots, half the dissolved organic
carbon in a forest soil. New Phytol 154:791–795
Horn WS, Simmonds MSJ, Schwartz RE et al (1995) Phomopsichalasin, a novel antimicrobial
agent from an endophytic Phomopsis spp. Tetrahedron 51:3969–3978
Hu Y, Wu S, Sun Y et al (2015) Arbuscular mycorrhizal symbiosis can mitigate the negative effects
of night warming on physiological traits of Medicago truncatula L. Mycorrhiza 25(2):131–142
Huber A, Hajdu D, Bratschun-Khan D, Gáspári Z, Varbanov M, Philippot S et al (2018) New
antimicrobial potential and structural properties of PAFB: a cationic, cysteine-rich protein from
Penicillium chrysogenum Q176. Sci Rep 8:1751
Hussain H, Root N, Jabeen F et al (2014) Seimatoric acid and colletonoic acid: two new compounds
from the endophytic fungi, Seimatosporium sp. and Colletotrichum sp. Chin Chem Lett 25:
1577–1579
Jaber LR, Araj SE (2018) Interactions among endophytic fungal entomopathogens (Ascomycota:
Hypocreales), the green peach aphid Myzus persicae Sulzer (Homoptera: Aphididae), and the
aphid endoparasitoid Aphidius colemani Viereck (hymenoptera: Braconidae). Biol Control 116:
53–61
Jaber LR, Araj SE, Qasem JR (2018) Compatibility of endophytic fungal entomopathogens with
plant extracts for the management of sweetpotato whitefly Bemesia tabaci Gennadius
(Homoptera: Aleyrodidae). Biol Control 117:164–171
Jeffries P (1995) Biology and ecology of mycoparasitism. Can J Bot 73:S1284–S1290
Jiménez-Morillo NT, González-Pérez JA, Jordán A, Zavala LM, de la Rosa JM, Jiménez-González
MA et al (2016) Organic matter fractions controlling soil water repellency in sandy soils from
the Doñana National Park (southwestern Spain). Land Degrad Dev 27:1413–1423
Johnson LJ, De Bonth ACM, Briggs LR et al (2013) The exploitation of epichloae endophytes for
agricultural benefit. Fungal Divers 60:171–188
Johnson JM, Alex T, Oelmuller R (2014) Piriformospora indica: the versatile and multifunctional
root endophytic fungus for enhanced yield and tolerance to biotic and abiotic stress in crop
plants. J Trop Agric 52:103–122
Karim MA, Fracheboud Y, Stamp P (1999) Photosynthetic activity of developing leaves of Zea
mays is less affected by heat stress than that of developed leaves. Physiol Plant 105:685–693
Khan I, Zhang H, Liu W, Zhang L, Peng F, Chen Y, Zhang Q, Zhang G, Zhang W, Zhang C (2020)
Identification and bioactivity evaluation of secondary metabolites from Antarctic-derived Peni-
cillium chrysogenum CCTCC M 2020019. RSC Adv 10:20738–20744
Kondo H, Hanada Y, Sugimoto H, Hoshino T, Garnham CP, Davies PL, Tsuda S (2012) Structure
of fungal antifreeze protein. Proc Natl Acad Sci 109(24):9360–9365
Krause G, Weis E (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu Rev Plant
Physiol Plant Mol Biol 42:313–349
Kruse E, Uehlein N, Kaldenhoff R (2006) The aquaporins. Genome Biol 7:206
Kuchár M, Glare TR, Hampton JG, Dickie IA, Christey MC (2019) Virulence of the plant-
associated endophytic fungus Lecanicillium muscarium to diamond backmoth larvae. N Z
Plant Prot 72:253–259
Kumar M, Yadav V, Tuteja N et al (2009) Antioxidant enzyme activities in maize plants colonized
with Piriformospora indica. Microbiology 155:780–790
608 S. Sahay

Kytöviita MM (2005) Asymmetric symbiont adaptation to Arctic conditions could explain why
high Arctic plants are non-mycorrhizal. FEMS Microbiol Ecol 53:27–32
Latef AHA, Chaoxing H (2011) Effect of arbuscular mycorrhizal fungi on growth, mineral
nutrition, antioxidant enzymes activity and fruit yield of tomato grown under salinity stress.
Sci Hortic 127:228–233
Lavelle P, Spain AV (2005) Soil organisms. Soil ecology. Springer, New Delhi
Lee DG, Shin SY, Maeng C-Y, Jin ZZ, Kim KL, Hahm K-S (1999) Isolation and characterization of
a novel antifungal peptide from Aspergillus niger. Biochem Biophys Res Commun 263:646–
651
Lee SG, Koh HY, Lee JH, Kang SH, Kim HJ (2012) Cryopreservative effects of the recombinant
ice-binding protein from the arctic yeast Leucosporidium sp. on red blood cells. Appl Biochem
Biotechnol 167:824–834
Lehto T, Zwiazek JJ (2011) Ectomycorrhizas and water relations of trees: a review. Mycorrhiza 21:
71–90
Leitão AL, García-Estrada C, Ullán RV, Guedes SF, Martín-Jiménez P, Mendes B, Martín JF
(2012) Penicillium chrysogenum var. halophenolicum, a new halotolerant strain with potential
in the remediation of aromatic compounds in high salt environments. Microbiol Res 167:79–89,
Lemos WJ Jr, Bovo B, Nadai C, Crosato G, Carlot M, Favaron F et al (2016) Biocontrol ability and
action mechanism of Starmerella bacillaris (synonym Candida zemplinina) isolated from wine
musts against gray mold disease agent Botrytis cinerea on grape and their effects on alcoholic
fermentation. Front Microbiol 7:1249
Li JY, Strobel GA (2001) Jesterone and hydroxy-jesterone antioomycete cyclohexenone epoxides
from the endophytic fungus Pestalotiopsis jesteri. Phytochemistry 57:261–265
Li JY, Strobel G, Harper J et al (2000) Cryptocin, a potent tetramic acid antimycotic from the
endophytic fungus Cryptosporiopsis cf. quercina. Org Lett 2:767–770
Li JY, Harper JK, Grant DM et al (2001) Ambuic acid, a highly functionalized cyclohexenone with
antifungal activity from Pestalotiopsis spp. and Monochaetia sp. Phytochemistry 56:463–468
Liarzi O, Bar E, Lewinsohn E, Ezra D (2016) Use of the endophytic fungus Daldinia cf. concentrica
and its volatiles as bio-control agents. PLoS One 11(12):e0168242
Liu A, Chen S, Chang R et al (2014a) Arbuscular mycorrhizae improve low temperature tolerance
in cucumber via alterations in H2O2 accumulation and ATPase activity. J Plant Res 127:775–
785
Liu ZL, Ma LN, He XY et al (2014b) Water strategy of mycorrhizal rice at low temperature through
the regulation of PIP aquaporins with the involvement of trehalose. Appl Soil Ecol 84:185–191
López-Bucio J, Pelagio-Flores R, Herrera-Estrell A (2015) Trichoderma as biostimulant: exploiting
the multilevel properties of a plant beneficial fungus. Sci Hortic 196:109–123
Lorito M, Woo SL, Fernandez IG, Colucci G, Harman GE, Pintor-Toro JA, Filippone E,
Muccifora S, Lawerence CB, Zoina A, Tuzun S, Scala F (1998) Gene from mycoparasitic
fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci U S A
95:7860–7865
Lowenfels J, Lewis W (2006) Bacteria. In: Teaming with microbes: a gardener’s guide to the soil
food web. Timber Press, Portland, OR
Luu DT, Maurel C (2005) Aquaporins in a challenging environment: molecular gears for adjusting
plant water status. Plant Cell Environ 28(1):85–96
Madrigal C, Melgarejo P (1995) Morphological effects of Epiccocum nigrum and its antibiotic
flavipin on Monilinia laxa. Can J Bot 73:425–431
Madrigal C, Tadeo JL, Melgarejo P (1991) Relationship between flavipin production by Epicoccum
nigrum and antagonism against Monilinia laxa. Mycol Res 95:1375–1381
Malhi GS, Kaur M, Kaushik P, Alyemeni MN, Alsahli AA, Ahmad P (2021) Arbuscular mycor-
rhiza in combating abiotic stresses in vegetables: an eco-friendly approach. Saudi J Biol Sci
28(2):1465–1476
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 609

Manasa KM, Vasanthakumari MM, Nataraja KN, Uma Shaanker R (2020) Endophytic fungi of salt
adapted Ipomeapes-caprae LR Br: their possible role in inducing salinity tolerance in paddy
(Oryza sativa L.). Curr Sci 118:1448–1453
Manoussopoulos Y, Mantzoukas S, Lagogiannis I, Goudoudaki S, Kambouris M (2019) Effects of
three strawberry entomopathogenic fungi on the prefeeding behavior of the aphid Myzus
persicae. J Insect Behav 32:99–108
Mantzoukas S, Eliopoulos PA (2020) Endophytic entomopathogenic fungi: a valuable biological
control tool against plant pests. Appl Sci 10:360
Mantzoukas S, Grammatikopoulos G (2019) The effect of three entomopathogenic endophytes of
the sweet sorghum on the growth and feeding performance of its pest, Sesamia nonagrioides
larvae, and their efficacy under field conditions. Crop Prot 127:104952
Mantzoukas S, Lagogiannis I (2019) Endophytic colonization of pepper (Capsicum annuum)
controls aphids (Myzus persicae Sulzer). Appl Sci 9:2239
Mantzoukas S, Chondrogiannis C, Grammatikopoulos G (2015) Effects of three endophytic
entomopathogens on sweet sorghumand on the larvae of the stalk borer Sesamia nonagrioides.
Entomol Exp Appl 154:78–87
Marquez LM, Redman RS, Rodriguez RJ et al (2007) A virus in a fungus in a plant: three-way
symbiosis required for thermal tolerance. Science 315:513–515
Marschner P (2012) Marschner’s mineral nutrition of higher plants. Academic, London
Martin CA, Stutz JC (2004) Interactive effects of temperature and arbuscular mycorrhizal fungi on
growth, P uptake and root respiration of Capsicum annuum L. Mycorrhiza 14:241–244
Matsubara Y, Hirano I, Sassa D et al (2004) Alleviation of high temperature stress in strawberry
plants infected with arbuscular mycorrhizal fungi. Environ Control Biol 42(2):105–111
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence-a practical guide. J Exp Bot 51:659–668
Maya MA, Matsubara Y (2013) Influence of arbuscular mycorrhiza on the growth and antioxidative
activity in cyclamen under heat stress. Mycorrhiza 23:381–390
Maya MA, Ito M, Matsubara Y (2014) Tolerance to heat stress and anthracnose in mycorrhizal
cyclamen. In: Chomchalow N, Supakamnerd N, Sukhvibul N (eds) international symposium on
orchids and ornamental plants Mej’ıa LC, Rojas EI, Maynard Z et al. endophytic fungi as
biocontrol agents of Theobroma cacao pathogens. Biol Control 46:4–14
Mejia LC, Rojas EI, Maynard Z, Van Bael SA, Arnold EA, Hebbar P et al (2008) Endophytic fungi
as biocontrol agents of Theobroma cacao pathogens. Biol Control 46:4–14
Mestre MC, Fontenla SB, Bruzone MC, Fernández NV, Dames J (2016) Detection of plant growth
enhancing features in psychrotolerant yeasts from Patagonia (Argentina). J Basic Microbiol 56:
1098–1106
Miransari M, Bahrami HA, Rejali F et al (2008) Using arbuscular mycorrhiza to alleviate the stress
of soil compaction on wheat (Triticum aestivum L.) growth. Soil Biol Biochem 40:1197–1206
Mitchell AM, Strobel GA, Moore E et al (2010) Volatile antimicrobials from Muscodor crispans,a
novel endophytic fungus. Microbiology 156:270–277
Molina-Montenegro MA, Acuña-Rodríguez IS, Torres-Díaz C et al (2020) Antarctic root
endophytes improve physiological performance and yield in crops under salt stress by enhanced
energy production and Na
+
sequestration. Sci Rep 10:5819
Moreno AB, Peñas G, Rufat M, Bravo JM, Estopà M, Messeguer J, Segundo BS (2005) Pathogen-
induced production of the antifungal AFP protein from aspergillus giganteus confers resistance
to the blast fungus Magnaporthe grisea in transgenic rice. MPMI 18:960–972
Mougin C, Boukcim H, Jolivalt C (2009) Soil bioremediation strategies based on the use of fungal
enzymes. In: Singh A et al (eds) Advances in applied bioremediation, soil biology, vol 17.
Springer-Verlag, Berlin, Heidelberg, pp 123–149
Murphy BR, Doohan FM, Hodkinson TR (2015) Persistent fungal root endophytes isolated from a
wild barley species suppress seed-borne infections in a barley cultivar. BioControl 60:281–292
Nadai C, Fernandes Lemos WJ Jr, Favaron F, Giacomini A, Corich V (2018) Biocontrol activity of
Starmerella bacillaris yeast against blue mold disease on apple fruit and its effect on cider
fermentation. PLoS One 13(9):e0204350
610 S. Sahay

Nordbring-Hertz B (2004) Morphogenesis in the nematode-trapping fungus Arthrobotrys
oligospora—an extensive plasticity of infection structures. Mycologist 18(3):125–133
Okongo RN, Puri AK, Wang Z, Singh S, Permaul K (2019) Comparative biocontrol ability of
chitinases from bacteria and recombinant chitinases from the thermophilic fungus Thermomyces
lanuginosus. J Biosci Bioeng 127:663–671
Ortas I (2003) Effect of selected mycorrhizal inoculation on phosphorus sustainability in sterile and
non-sterile soils in the Harran plain in South Anatolia. J Plant Nutr 26:1–17
Ortas I (2012) The effect of mycorrhizal fungal inoculation on plant yield, nutrient uptake and
inoculation effectiveness under long-term field conditions. Field Crop Res 125:35–48
Ozimek E, Hanaka A (2021) Mortierella species as the plant growth-promoting fungi present in the
agricultural soils. Agriculture 11:7
Paradis R, Dalpé Y, Charest C (1995) The combined effect of arbuscular mycorrhizas and short-
term cold exposure on wheat. New Phytol 129:637–642
Peña-Peña AJ, Santillán-Galicia MT, Hernández-López J, Guzmán-Franco AW (2015)
Metarhizium pingshaense applied as a seed treatment induces fungal infection in larvae of the
white grub Anomala cincta. J Invertebr Pathol 130:9–12
Pimenta RS, Morais PB, Rosa CA, Correa A (2009) Utilization of yeasts in biological control
programs. In: Satyanarayana T, Kunze G (eds) Yeast biotechnology: diversity and applications.
Springer, Berlin, pp 170–199
Poosapati S, Ravulapalli PD, Tippirishetty N et al (2014) Selection of high temperature and salinity
tolerant Trichoderma isolates with antagonistic activity against Sclerotium rolfsii. SpringerPlus
3:641
Powell WA, Klingeman WE, Ownley BH, Gwinn KD (2009) Evidence of endophytic Beauveria
bassiana in seed-treated tomato plants acting as a systemic entomopathogen to larval
Helicoverpa zea (Lepidoptera: Noctuidae). J Entomol Sci 44:391–396
Qayyum MA, Wakil W, Arif MJ, Sahi ST, Dunlap CA (2015) Infection of Helicoverpa armigera by
endophytic Beauveria bassiana colonizing tomato plants. Biol Control 90:200–207
Qiang X, Weiss M, Kogel KH et al (2012) Piriformospora indica—a mutualistic basidiomycete
with an exceptionally large plant host range. Mol Plant Pathol 13:508–518
Quesada-Moraga E, Munoz-Ledesma FJ, Santiago-Alvarez C (2009) Systemic protection of
Papaver somniferum L. against Iraella luteipes (hymenoptera: Cynipidae) by an endophytic
strain of Beauveria bassiana (Ascomycota: Hypocreales). Environ Entomol 38:723–730
Rabiey M, Ullah I, Shaw MW (2015) The endophytic fungus Piriformospora indica protects wheat
from fusarium crown rot disease in simulated UK autumn conditions. Plant Pathol 64:1029–
1040
Ramakuwela T, Hatting J, Bock C, Vega FE, Wells L, Mbata GN, Shapiro-Ilan D (2020)
Establishment of Beauveria bassiana as a fungal endophyte in pecan (Carya illinoinensis)
seedlings and its virulence against pecan insect pests. Biol Control 140:104102
Reddy NP, Khan APA, Devi UK, Sharma HC, Reineke A (2009) Treatment of millet crop plant
(Sorghum bicolor) with the entomopathogenic fungus (Beauveria bassiana) to combat infesta-
tion by the stem borer, Chilo partellus Swinhoe (Lepidoptera: Pyralidae). J Asia Pac Entomol
12:221–226
Redman RS, Sheehan KB, Stout RG et al (2002) Thermotolerance generated by plant/fungal
symbiosis. Science 298:1581
Redman RS, Kim YO, Woodward CJDA, Greer C, Espino L, Doty SL, Rodriguez RJ (2011)
Increased fitness of rice plants to abiotic stress via habitat adapted symbiosis: a strategy for
mitigating impacts of climate change. PLoS One 6:e14823
Reyes-Perez JJ, Vero S, Diaz-Rivera E, Lara-Capistran L, Noa-Carrazana JC, Hernandez-Montiel
LG (2019) Application of chlorine dioxide (ClO
2
) and marine yeasts to control postharvest
anthracnose disease in mango (Mangifera indica L.). Cien Inv Agr 46:266–275
Rodriguez R, Redman R (2008) More than 400 million years of evolution and some plants still can’t
make it on their own: plant stress tolerance via fungal symbiosis. J Exp Bot 59:1109–1114
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 611

Rondot Y, Reineke A (2018) Endophytic Beauveria bassiana in grapevine Vitis vinifera (L.)
reduces infestation with piercing-sucking insects. Biol Control 116:82–89
Ruotsalainen AL, Kytöviita M (2004) Mycorrhiza does not alter low temperature impact on
Gnaphalium norvegicum. Oecologia 140:226–233
Russo ML, Scorsetti AC, Vianna MF, Allegrucci N, Ferreri NA, Cabello MN, Pelizza SA (2018)
Effects of endophytic Beauveria bassiana (Ascomycota: Hypocreales) on biological, reproduc-
tive parameters and food preference of the soybean pest Helicoverpa gelotopoeon. J King Saud
Univ Sci 31:1077–1082. https://doi.org/10.1016/j.jksus.2018.04.008
Russo ML, Scorsetti AC, Vianna MF, Cabello M, Ferreri N, Pelizza S (2019) Endophytic effects of
Beauveria bassiana on corn (zeamays) and its herbivore, Rachiplusia nu (lepidoptera:
Noctuidae). Insects 10:110
Saad MMG, Ghareeb RY, Saeed AA (2019) The potential of endophytic fungi as bio-control agents
against the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae). Egypt J
Biol Pest Control 29:7
Sahay S (2021) Cold-active microfungi and their industrial applications. In: Yadav AN, Rastegari
AA, Yadav N (eds) Microbiomes of extreme environments. CRC, Taylor & Francis, Boca
Raton, FL
Sahay S, Chauhan D, Chourse V (2019) Laccase from aspergillus nidulans TTF6 showing Pb
activation for smaller substrates and dyes remediation in all climates. Proc Natl Acad Sci 90(1):
1–8
Saikkonen K, Faeth SH, Helander M, Sullivan TJ (1998) Fungal endophytes: a continuum of
interactions with host plants. Annu Rev Ecol Syst 29:319–343
Saxena B, Shukla K, Giri B (2017) Arbuscular mycorrhizal fungi and tolerance of salt stress in
plants. In: Wu QS (ed) Arbuscular mycorrhizas and stress tolerance of plants. Springer,
Singapore
Schisler DA, Janisiewicz WJ, Boekhout T, Kurtzman CP (2011) Agriculturally important yeasts:
biological control of field and postharvest diseases using yeast antagonists, and yeasts as
pathogens of plants. In: Kurtzman PC, Fell JW, Boekhout T (eds) The yeasts, a taxononic
study. Elsevier BV, Amsterdam, pp 45–52
Seibold M, Wolschann P, Bodevin S, Olsen O (2011) Properties of the bubble protein, a defensin
and an abundant component of a fungal exudate. Peptides 32:1989–1995
Serfling A, Wirsel SGR, Lind V et al (2007) Performance of the biocontrol fungus Piriformospora
indica on wheat under greenhouse and field conditions. Phytopathology 97:523–531
Smith SE, Read DJ (1997) Mycorrhizal symbiosis, 2nd edn. Academic Press, London
Spurgeon DJ, Keith AM, Schmidt O et al (2013) Land-use and land-management change:
relationships with earthworm and fungi communities and soil structural properties. BMC Ecol
13:46
Srimathi Priya L, Kumutha K, Arthee R, Pandiyarajan P (2014) Identification of arbuscular
mycorrhizal multiplicity in the saline-sodic soils. Int J Agric Biol Eng 7(2):56–67
Steudle E, Peterson CA (1998) How does water get through roots? J Exp Bot 49:775–788
Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products
bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67:
491–502
Strobel GA, Miller RV, Miller C et al (1999) Cryptocandin, a potent antimycotic from the
endophytic fungus Cryptosporiopsis cf. quercina. Microbiology 145:1919–1926
Strobel GA, Dirkse E, Sears J et al (2001) Volatile antimicrobials from Muscodor albus, a novel
endophytic fungus. Microbiology 147:2943–2950
Strobel G, Ford E, Worapong J et al (2002) Isopestacin, an isobenzofuranone from Pestalotiopsis
microspora, possessing antifungal and antioxidant activities. Phytochemistry 60:179–183
Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep 18:
448–459
Tian DL, Peng YY, Yan WD, Fang X, Kang WX, Wang GJ et al (2010) Effects of thinning and
litter fall removal on fine root production and soil organic carbon content in Masson pine
plantations. Pedosphere 20:486–493
612 S. Sahay

Timling I, Taylor DL (2012) Peeking through a frosty window: molecular insights into the ecology
of Arctic soil fungi. Fungal Ecol 5:419–429
Tóth L, Kele Z, Borics A, Nagy LG, Váradi G, Virágh M et al (2016) NFAP2, a novel cysteine-rich
anti-yeast protein from Neosartorya fischeri NRRL 181: isolation and characterization. AMB
Express 6:1–13
Tu C-Y, Chen Y-P, Yu M-C, Hwang I-E, Wu D-Y, Liaw L-L (2016) Characterization and
expression of the antifungal protein from Monascus pilosus and its distribution among various
Monascus species. J Biosci Bioeng 122:27–33
Varughese T, Rios N, Higginbotham S et al (2012) Antifungal depsidone metabolites from
Cordyceps dipterigena, an endophytic fungus antagonistic to the phytopathogen Gibberella
fujikuroi. Tetrahedron Lett 53:1624–1626
Vega FE, Posada F, Aime MC, Pava-Ripoll M, Infante F, Rehner SA (2008) Entomopathogenic
fungal endophytes. Biol Control 46:72–82
Vega FE, Simpkins A, Aime MC et al (2010) Fungal endophyte diversity in coffee plants from
Colombia, Hawai, Mexico and Puerto Rico. Fungal Ecol 3:122–138
Verma S, Varma A, Rexer K-H et al (1998) Piriformospora indica, gen. et sp. nov., a newroot-
colonizing fungus. Mycologia 90:896
Vidal S, Jaber LR (2015) Entomopathogenic fungi as endophytes: plant–endophyte–herbivore
interactions and prospects for use in biological control. Curr Sci 109:46–54
Villarreal P, Carrasco M, Barahona S et al (2018) Antarctic yeasts: analysis of their freeze-thaw
tolerance and production of antifreeze proteins, fatty acids and ergosterol. BMC Microbiol 18:
66
Wahid A, Gelani S, Ashraf M et al (2007) Heat tolerance in plants: an overview. Environ Exp Bot
61:199–223
Walker DA, Kuss P, Epstein HE et al (2011) Vegetation of zonal patterned-ground ecosystems
along the North America Arctic bioclimate gradient. Appl Veg Sci 14:440–463
Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M et al (2005) The endophytic
fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and
higher yield. Proc Natl Acad Sci 102:13386–13391
Wang F-Y, Liu R-J (2001) A preliminary survey of arbuscular mycorrhizal fungi in saline alkaline
soil of the Yellow River Delta[J]. Biodivers Sci 09(4):389–392
Wang H, Liang L, Liu B, Huang D, Liu S, Liu R, Siddique KHM, Chen Y (2020) Arbuscular
mycorrhizas regulate photosynthetic capacity and antioxidant defense systems to mediate salt
tolerance in maize. Plants (Basel) 24:1430
Wei Y, Zhang SH (2019) Haloalkaliphilic fungi and their roles in the treatment of saline-alkali
soil. In: Tiquia-Arashiro S, Grube M (eds) Fungi in extreme environments: ecological role and
biotechnological significance. Springer, Cham
Worapong J, Strobel G, Ford EJ et al. (2001) Muscodor albus anam. Gen. et sp. nov., and endophyte
from Cinnamomum zeylanicum. Mycotaxon 79:67–79
Wright SF, Upadhyaya A (1998) A survey of soils for aggregate stability and glomalin, a
glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant and Soil 198:97–107
Wu QS, Zou YN (2010) Beneficial roles of arbuscular mycorrhizas in citrus seedlings at tempera-
ture stress. Sci Hortic 125:289–293
Xu H (2015) Major intrinsic proteins of Laccaria bicolor: characterization, transcript profiling and
functions in ectomycorrhizal associations with Picea glauca. Ph.D. thesis, University of
Alberta, Edmonton
Xu H, Zwiazek JJ (2020) Fungal Aquaporins in ectomycorrhizal root water transport. Front Plant
Sci 11:302
Young A (1991) The photoprotective role of carotenoids in higher plants. Physiol Plant 83:702–708
Zajc J, Džeroski S, Kocev D, Oren A, Sonjak S, Tkavc R, Gunde-Cimerman N (2014) Chaophilic or
chaotolerant fungi: a new category of extremophiles? Front Microbiol 5:1–15
Zhang Y, Zhong CL, Chen Y et al (2010) Improving drought tolerance of Casuarina equisetifolia
seedlings by arbuscular mycorrhizas under glasshouse conditions. New For 40:261–267
Zhang X, Li SJ, Li JJ, Liang ZZ, Zhao CQ (2018) Novel natural products from extremophilic fungi.
Mar Drugs 16(6):194
25 Extremophilic Fungi: Potential Applications in Sustainable Agriculture 613

Zheng YH, Xu XB, Wang MY, Zheng XH, Li ZJ, Jiang GM (2009) Responses of salt-tolerant and
intolerant wheat genotypes to sodium chloride: photosynthesis, antioxidants activities, and
yield. Photosynthetica 47:87–94
Zheng Y, Zhang T, Lu Y, Wang L (2021) Monascus pilosus YX-1125: an efficient digester for
directly treating ultra-high-strength liquor wastewater and producing short-chain fatty acids
under multiple-stress conditions. Bioresour Technol 331:125050. https://doi.org/10.1016/j.
biortech
Zhou WN, White JF Jr, Soares MA, Torres MS, Zhou ZP, Li HY (2015) Diversity of fungi
associated with plants growing in geothermal ecosystems and evaluation of their capacities to
enhance thermotolerance of host plants. J Plant Interact 10:305–314
Zhu JK (2001) Over expression of a delta-pyrroline-5-carboxylate synthetase gene and analysis of
tolerance to water and salt stress in transgenic rice. Trends Plant Sci 6:66–72
Zhu XC, Song FB, Xu HW (2010a) Arbuscular mycorrhizae improves low temperature stress in
maize via alterations in host water status and photosynthesis. Plant and Soil 331:129–137
Zhu XC, Song FB, Xu HW (2010b) Influence of arbuscular mycorrhizae on lipid peroxidation and
antioxidant enzyme activity of maize plants under temperature stress. Mycorrhiza 20:325–332
Zhu XC, Song FB, Liu SQ et al (2011) Effects of arbuscular mycorrhizal fungus on photosynthesis
and water status of maize under high temperature stress. Plant and Soil 346:189–199
Zhu XC, Song FB, Liu FL, Liu SQ, Tian CJ (2015) Carbon and nitrogen metabolism in arbuscular
mycorrhizal maize plants under low-temperature stress. Crop Pasture Sci 66:62–70
Zou WX, Meng JC, Lu H et al (2000) Metabolites of Colletotrichum gloeosporioides, an endo-
phytic fungus in Artemisia mongolica. J Nat Prod 63:1529–1530
614 S. Sahay

Extremophilic Fungi for the Synthesis
of Nanomolecules 26
Harshita Shukla and Shyamji Shukla
Abstract
Since the past few decades, metal nanoparticles have attracted enormous attention
in the scientific world as a result of their anti-cancer, catalytic, optical, electronic,
and magnetic properties. Conventionally, the synthesis of metal nanoparticles is
carried out by diverse physical and chemical methods. But these conventional
methods are accompanied with a number of drawbacks, viz. elevated energy
consumption, increased cost, and the use of toxic chemical substances. The
application of various microbial species for biological synthesis of nanoparticles
has recently presented an alternative synthetic platform in an eco-friendly and
cost-effective way. Extremophiles are one of the most unexplored groups of
organisms. Even though environment has provided enormous strength to all
living organisms for their survival, sometimes under extreme environmental
conditions, viz. high or coldness, salinity, pH, pressure, radiation, chemical
extremes, lack of nutrition, osmotic barriers, geological scale/barriers, or
polyextremity, etc. normal survival may not be achievable. However certain
organisms possess unusual characteristics, which make them capable of thriving
in such extreme habitats. The present chapter discusses the role of a variety of
microorganisms in the synthesis of different metal nanoparticles. Such biologi-
cally synthesized nanoparticles by microbes are safe, eco-friendly and have
numerous applications in the fields like agriculture, textile, medicine, drug
delivery, biochemical sensors, and allied areas. Although several researchers
have exploited microorganisms for the biosynthesis of metallic nanoparticles
H. Shukla
Department of Biotechnology, Sri Guru Tegh Bahadur Khalsa College, Jabalpur, Madhya Pradesh,
India
S. Shukla (*)
Department of Biotechnology, Mata Gujri Mahila Mahavidyalaya (Autonomous), Jabalpur,
Madhya Pradesh, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_26
615

but future works need to be carried out for large-scale production, increased
stability, reduced time to get desirable shape and size and their probable
applications in a number of fields. This chapter gives an outline of the promising
use of fungi in the field of metal nanoparticles synthesis, its potential mechanisms
and efficient applications in diverse areas of research.
Keywords
Nanoparticles · Extremophiles · Biosynthesis and eco-friendly
26.1 Introduction
Nanotechnology has emerged as an extremely significant field as a result of its
widespread applications in diverse sectors of science and technology. It is gradually
taking a center stage in the fields of catalysis, optics, biomedical sciences, mechan-
ics, magnetics, and energy science. Nanotechnology is the science which deals with
the design, synthesis, characterization, and applications of particles having diameter
less than 100 nm known as nanoparticles. These nanoparticles are considered as tiny
entity that behaves like a complete unit in terms of its transport and properties. The
word “nano”is derived from Greek word meaning dwarf or extremely small (Rai
et al. 2008). When used as a prefix, it implies 10
9
. A nanometer (nm) is
one-billionth of a meter and is equal to the length of three atoms side by side.
Several biomolecules inside the body of living organisms lie in the range of
nanometer scale including a DNA molecule (2.5 nm wide), a protein molecule
(50 nm), a flu virus (100 nm), and thickness of hair (10,000 nm). The average
diameter of nanoparticles usually ranges between 0.1 and 100 nm and is generally
synthesized via two conventional methods, viz. top-down and bottom-up approach
(Fendler 1998; Thakkar et al. 2010). In top-down approach, the bulk materials are
step by step decreased to nanosized materials, whereas in bottom-up approach,
atoms or molecules are collected to form molecular structures in nanometer range.
The chemical and biological synthetic methods of nanoparticles often employ the
bottom-up approach (Fendler 1998).
Nanoparticles exhibit exclusive properties like physical, chemical, electronic,
electrical, mechanical, magnetic, thermal, dielectric, optical, and biological
properties (Schmid 1992). The physical properties of nanoparticles greatly differ
from most of the other materials because of their decreasing dimensions. These
physical properties are credited to their large surface areas to volume ratio, large
surface energy, spatial confinement, and reduced imperfections. Nanoparticles are
beneficial in comparison to the regular sized bulk materials as a consequence of their
extraordinary properties like phenomenon of surface plasmon resonance (SPR),
increased Rayleigh scattering, and surface-enhanced Raman scattering (SERS).
Nanoparticles are hence considered as building blocks of the subsequent generation
of optoelectronics, electronics, various chemical and biochemical sensors (Wong
and Schwaneberg 2003). The synthesis of nanoparticles possessing a variety of
616 H. Shukla and S. Shukla

chemical compositions, sizes, shapes, and controlled dispersities is the most signifi-
cant area of research in nanotechnology. The development of reliable, cost-effective,
and eco-friendly experimental procedure for the synthesis of nanomaterials is one of
the challenges in nanotechnology. A number of manufacturing techniques
employing atomistic, molecular, and particulate processing during a vacuum or in
a very liquid medium are currently been applied for nanoparticles synthesis (Daniel
and Astruc 2004). But majority of the approaches are capital intensive and moreover
are incapable in efficient production.
Several methods including physical, chemical, biological, and hybrid approaches
are present to synthesize different types of nanoparticles (Mohanpuria et al. 2008;
Liu et al. 2011). Even though a variety of physical and chemical techniques are
comprehensively used for the synthesis of nanoparticles, the involvement of toxic
chemicals in these procedures is a matter of utmost importance. Besides this these
techniques also include the application of toxic chemicals on the surface of
nanoparticles and non-polar solvents which restricts their applications in biotechno-
logical and clinical fields. Therefore, to overcome these drawbacks accompanied
with the physical and chemical methods it is necessary to develop novel, biocom-
patible, non-toxic, and eco-friendly methods for nanoparticles synthesis. Accord-
ingly, researchers working in the field of nanoparticle synthesis and assembly have
inclined their attention toward various biological sources for nanoparticles fabrica-
tion. Numerous biological systems including plants (Shankar et al. 2004), fungi
(Sastry et al. 2003), bacteria (Joerger et al. 2001), actinomycetes (Sastry et al. 2003),
along with some other organisms have the potential of nanoparticles synthesis.
The applications of biological agents for the synthesis of nanoparticles have led to
the emergence of an inherent, clean, non-toxic, and environment friendly field of
science known as nanobiotechnology. Applications of microbial interactions with
metals in various fields including bioremediation, biomineralization, bioleaching,
biocorrosion, and biosynthesis of nanoparticles have been the focus of researchers
around the world (Klaus et al. 2009). With the advent of these applications
nanobiotechnology has developed as a potential research field. This field actually
represents an interaction of field of biotechnology and nanotechnology.
Biosynthesized nanoparticles produced by a biogenic enzymatic method are
much better, in several ways, as compared to those synthesized via chemical and
physical approach. Although physicochemical methods are capable to fabricate huge
amount of nanoparticles with a defined size and shape in a tremendously small
period of time, they are complex, old-fashioned, expensive, and ineffective. More-
over they release a number of hazardous toxic wastes that are dangerous to the
human health as well as the environment. With the development of biological
methods, the involvement of costly chemicals is restricted besides it is not as energy
intensive as the chemical methods and is additionally eco-friendly.
The biosynthetic method is further facilitated by the property of most of the
microorganisms to survive in varying temperature, pH, and pressure conditions. The
biosynthesized nanoparticles possess greater catalytic reactivity, larger specific area,
and enhanced contact between the enzyme and metal salt because of the microbial
carrier matrix (Bhattacharya and Mukherjee 2008). In the microbial synthesis of
26 Extremophilic Fungi for the Synthesis of Nanomolecules 617

nanoparticles, microorganisms attach the target ions on their cell surface from the
surrounding environment and reduce the metal ions into the element metal by
secreting enzymes produced by cellular activities. This approach is divided into
two types: intracellular and extracellular synthesis depending upon the vicinity
where nanoparticles are synthesized (Mann 2001). During the intracellular method
ions are transported inside the microbial cell where they are reduced to
corresponding metal nanoparticles by the enzymes secreted in the cell. Whereas
the metal ions are trapped on the cellular surface of microorganisms during the
extracellular synthesis and reduced thereafter into metal nanoparticles by enzymes
present inside the cells (Zhang et al. 2011a,b).
26.2 Extremophiles
Different groups of extremophilic organisms grow under different optimal
conditions like acidophilic organisms grow best between pH 1 and pH 5, however,
alkaliphilic organisms grow at pH above 9; halophilic organisms require high salt
concentrations for optimum growth; thermophilic grows at temperatures ranging
from 60 to 80 C; hyperthermophilic organisms grow above 80 C; psychrophilic
organisms grow at 15 C or lower temperature, showing a maximum growth at 20 C
and minimal growth at or below 0 C; piezophilic or barophilic organisms exhibit
optimum growth at high hydrostatic pressure; oligotrophic grows well in nutrition-
ally limited environments; endolithic organisms dwell within rock or pores of
mineral grains; and xerophilic organisms grow under dry conditions, in the presence
of tide (Prasanti et al. 2015).
Conversely, some extremophilic organisms are able to survive simultaneously
under multiple stress conditions. Such organisms are called polyextremophiles. Few
common examples of these include thermoacidophilic and haloalkaliphilic
organisms. Extremophilic organisms capable of bearing multiple extreme conditions
are called as polyextremophiles. For example, organisms inhabiting the hot rocks
under the deep Earth’s surface are also thermophilic and barophilic like
Thermococcus barophilus.
Similarly polyextremophile which inhabits high mountains present in the desert is
a radioresistant xerophile, a psychrophile, and an oligotroph. Polyextremophiles are
recognized for their ability to endure high as well as low pH levels. The exploitation
of extremophilic organisms and their biodiversity makes them useful for developing
different techniques and products for human welfare (MacElroy 1974; Rothschild
2007; Tiquia and Mormile 2010; Tiquia-Arashiro and Mormile 2013; Tiquia-
Arashiro 2014a,b). Extremophilic organisms produce enzymes called
extremozymes that are active under extreme environmental conditions. These
enzymes have great biotechnological potential. Extremozymes are known for their
widespread applications in industrial production procedures and research
applications as a result of their capability to stay active even under the severe
conditions (e.g., heat, pressure, and pH) usually employed in these processes. As
reported by several researchers the identification of the extremophiles provided
618 H. Shukla and S. Shukla

opportunities for industrial, biotechnological, and medical use (Adams and Kelly
1998; Niehaus et al. 1999; Tiquia and Mormile 2010; Oren 2012; Gabani and Singh
2013; Pomaranski and Tiquia-Arashiro 2016).
26.3 Metallic Nanoparticles
Metallic nanoparticles have fascinated scientists around the world and are now
profoundly applied in the fields of biomedical sciences and engineering. Due to
their varied applications in different fields, viz. electronics, cosmetics, coatings,
packaging, optics and biotechnology metallic nanoparticles have gained great inter-
est of the scientific community. For example, improved and easy-to-create coatings
for electronics applications are formed by merging nanoparticles into a solid at
comparatively lower temperatures, without melting. In general, nanoparticles have
a wavelength below the critical wavelength of sunshine which makes them transpar-
ent and subsequently useful for applications in cosmetics, coatings, and packaging.
Metallic nanoparticles and single strands of DNA are usually attached together
nondestructively which eventually opens prospects for applications in the field of
medical diagnostics. Nanoparticles can pass through the vasculature and focus on
any organ. This property can lead to novel therapeutic, imaging, and biomedical
applications. Nowadays these nanoparticles after synthesis are modified with a
variety of chemical functional groups which allow them to be conjugated with
antibodies, ligands, and medicines of interest. This could pave the way for large
array of potential applications in biotechnology, magnetic separation,
pre-concentration of target analytes, targeted drug delivery, and vehicles for gene
and drug delivery and more importantly diagnostic imaging (Mody et al. 2010).
26.4 Biosynthesis of Nanoparticles
Metallic NPs are usually synthesized by the conventional physical and chemical
methods out of which the wet-chemical approach is the most commonly used.
During this process nanoparticles are grown in an extremely liquid medium
containing various reactants, specifically reducing agents like sodium borohydride
(Kim et al. 2007), potassium bitartrate (Tan et al. 2003), and methoxypolyethylene
glycol (Mallick et al. 2004). Besides these chemicals certain stabilizing agents like
sodium dodecyl benzyl sulfate (Li et al. 1999) or polyvinyl pyrrolidone (Tan et al.
2003) are mixed to the reaction mixture for preventing the agglomeration of metallic
nanoparticles. In general, the chemical methods are cheaper for prime volume;
though, their disadvantages include contamination from precursor chemicals, use
of toxic solvents, and generation of hazardous by-products. Consequently, the
biological approach for synthesis of nanoparticles has become significant. Moreover
biologically synthesized nanoparticles involving an enzymatic process are much
better, in many ways, in comparison to those particles fabricated by using chemical
methods.
26 Extremophilic Fungi for the Synthesis of Nanomolecules 619

One of the issues in the biosynthesis of nanoparticle is the use of suitable
stabilizing agent to achieve stability and homogeneity of nanomaterials. There are
a number of physicochemical approaches applied for the stabilization of
nanomaterials which function on the basis of any one of these principles, viz.
electrostatic, steric, electrosteric stabilization, and stabilization by a ligand or a
solvent (Roucoux et al. 2002). However, these physicochemical stabilization
procedures possess severe drawbacks, including high capital investment and use of
dangerous chemicals and practices which are in turn harmful for the environment.
Conversely, the biological agents involved in the synthesis of nanoparticles com-
prise an additional benefit of providing an outstanding stability to the synthesized
nanoparticles. Regardless of the actual fact that the equipment and also the
mechanisms for the synthesis of nanomaterials are comprehensively analyzed by
scientists, the stabilizing agents involved during the nanomaterial synthesis
procedures still remain unexplored.
26.5 Microbial Synthesis of Nanoparticles
Synthesis of nanoparticles using microorganisms involves catching the target ions
from their surrounding and then converting them into the metallic nanoparticles by
enzymatic process. Depending on the position of metal ions corresponding to
microbial cell this method is divided into two types, namely intracellular and
extracellular synthesis (Mann 2001). The intracellular method involves synthesis
of nanoparticles inside the microbial cells by reducing enzymes, whereas the extra-
cellular synthesis of nanoparticles takes place outside the microbial cells on their
surface (Zhang et al. 2011a,b). Although microorganisms are well-known as cell
factory synthesizing a variety of biomolecules in cost-effective and eco-friendly
ways, the biosynthesis of nanoparticles has some restrictions like (1) the probable
toxicity of reactants and products against the microorganisms used, (2) expensive
cultivation of microbial cells in sterile conditions, (3) low productivity, and (4) other
economic aspects involved in purification and characterization of biosynthesized
nanoparticles. To overcome these drawbacks, extremophilic microorganisms
possessing ability to grow under extreme environmental conditions, viz. very high
or very low temperature, pH, salt concentration, etc. are being used for the biosyn-
thesis of nanoparticles.
26.6 Nanoparticle Synthesis by Extremophilic Microbes
The extremophilic microbes offer very potential solution to various environmental
challenges by exhibiting remarkable tolerance ability toward extreme conditions.
This property makes them perfect biological agents for the biosynthesis of
nanoparticles. These microorganisms can be cultivated even under non-sterile
conditions by using low-cost media resulting in the synthesis of extremely expensive
biomolecules, biochemicals, bioanalytics, and enzymes having different
620 H. Shukla and S. Shukla

bioactivities. Extremophilic enzymes possess immense potential in several fields’
particularly industrial biotechnology, since they can be used under harsh conditions,
which can result in substrate transformations that are not possible with normal
enzymes (Colombo et al. 1995). In comparison to chemical catalysis, enzyme-
mediated reactions provide more specific stereo-selectivity in organic synthesis
(Koeller and Wong 2001). The most important limitations accompanied by the use
of these enzymes are their stability for long time and recyclability (Schmid et al.
2011). Lately, a number of nanoparticles are in use to improve conventional enzyme
immobilization methods so as to strengthen activity and stability of enzymes which
in turn will minimize the costs in industrial biotechnology (Abad et al. 2005; Yiu and
Keane 2012).
26.7 Nanoparticle Synthesis by Halophilic Fungi
Majority of metal tolerant microbes inhabit the marine environments particularly the
underside of the ocean and play a significant role in the biogeochemical cycling of
inorganic elements. Hence marine environments present a potential source of
microbes with metal tolerance capacity. Moreover, the marine ecosystem is con-
stantly exposed to metallic pollution as a result of volcanic eruptions, natural
weathering of the rocks, anthropogenic activities like mining, combustion of fuels,
industrial, and concrete sewage. However extensive concentrations of metals are
found in estuaries and solar salterns since they serve as efficient traps for river borne
metals (Chapman and Wang 2001). Therefore halophiles residing in these marine
ecosystems are continuously exposed to metals which makes them an effective
source for nanoparticle synthesis. Various research works have reported
nanoparticles biosynthesis by halophiles like bacteria, archaea, fungi, and algae.
Some common species of halophilic yeasts and fungi that are known to synthesize
nanomaterials include Pichia capsulata, which is halophilic yeast isolated from
mangrove. It performs extracellular synthesis of silver nanoparticles (Manivannan
et al. 2010). P. capsulata utilizes a rapid process to synthesize silver nanoparticles
under optimum conditions, viz. pH of about 6.0, 0:3% NaCl concentration, and a
temperature of 5 C. During further study it was revealed that the whole reduction
process was catalyzed by an NADH-dependent (NADH: nicotinamide adenine
dinucleotide) nitrate reductase enzyme which was later partially purified.
In a similar study a metal tolerant yeast Yarrowia lipolytica isolated from polluted
areas containing toxic and unsafe metals is applied for the biotransformation of
organic compounds, production of novel enzymes, cloning and expression of heter-
ologous proteins, bioremediation of hydrophobic substrate contaminated
environments, and the treatment or upgradation of wastes. The metal tolerance
observed in this yeast was attributed to the presence of SOD (a copper tolerating
protein), reductases, CRF1, metallothioneins, efflux mechanisms, and melanin
(Bankar et al. 2009). Likewise a tropical marine isolate of Y. lipolytica (NCIM
3589) obtained from oil-polluted seawater near Mumbai, India was found to synthe-
size gold nanoparticles. This synthetic process occurs at 30 C within 72 h. TEM
26 Extremophilic Fungi for the Synthesis of Nanomolecules 621

analysis confirmed the cell wall associated synthesis of nanoparticles. The size of the
nanoparticles was observed to be affected by the pH. According to study at pH 2.0,
gold nucleation was observed within 15 min. With increasing period of time, these
developed into large triangular and hexagonal plates. Conversely the scale of the
nanostructures at pH 7.0 and at 9.0 was around 15 nm (Agnihotri et al. 2009). This
principle was employed in the custom designing of gold nanoparticles with specific
sizes.
In a study the size of nanoparticle decreased with increase in cell numbers and
same gold salt concentrations. Whereas it was observed that the size of nanoparticles
increased with increasing concentration of the gold salt and the same cell numbers.
The isolation of cell-associated gold nanoparticles can be done by incubation in a
medium at 20 C (Pimprikar et al. 2009). A dark-colored pigment called melanin
was isolated from this yeast which was later proved to be one of the major factors
responsible for nanoparticle synthesis. The synthesis of gold nanostructures was
observed to be facilitated by both the cell-extracted and induced melanin (obtained
by incubating resting cells with L-3,4-dihydroxyphenylalanine (L-DOPA)). Synthe-
sis of gold nanostructures has been reported by another cold-adapted marine strain of
Y. lipolytica (NCIM 3590) also. As the natural amount of melanin in this organism
was low, a precursor L-DOPA was incubated with the yeast to induce the overpro-
duction of melanin. This melanin also aided the rapid fabrication of silver and gold
nanoparticles. Certain functional groups on the cell surface were thought to play a
role in the reductive and stabilization processes (Pawar et al. 2012). The synthetic
ability during this case was also related to the dark-colored pigment, melanin. These
nanoparticles displayed antibiofilm activity against pathogenic bacteria.
One more fungus, Penicillium fellutanum isolated from the rhizosphere of an
Indian endemic mangrove plant (Rhizophora annamala) was examined for silver
nanoparticles synthesis. It was observed that highest biosynthesis of nanoparticles
occurred in the culture filtrate containing 1.0 mM AgNO
3
, 0.3% NaCl at pH 6.0 and
temperature 5 C for 24 h. The nanoparticles were synthesized extracellularly having
spherical shape with size ranging from 5 to 25 nm. During the analysis of cell-free
supernatant a protein of about 70 kDa was reported which was considered to be
responsible for converting the metal ions to their zero valence state (Kathiresan et al.
2009). Preliminary presence of silver nanoparticles within the culture filtrate was
confirmed by absorption peak at 430 nm, through UV-Vis spectroscopic analysis
which was further confirmed by the use of transmission electron microscopy.
Aspergillus species a yet another halophilic fungus is able to survive mostly under
all types of weather conditions found around the world. This fungus is generally
associated with environments possessing high starchy concentrations. As mangrove
ecosystems are made up of debris from leaves, inflorescence, and stems, they are rich
in starch content and therefore Aspergillus sp. inhabit these ecosystems in great
numbers (Seelan et al. 2009). Spherical silver nanoparticles having diameter of
5–35 nm were synthesized by a strain of A. niger (AUCAS 237) isolated from a
mangrove sediment of Vellar estuary, India. These nanoparticles exhibited efficient
antimicrobial activity against various clinically pathogenic microorganisms includ-
ing Gram-negative bacteria and a few fungi. The activity of nanoparticles was
622 H. Shukla and S. Shukla

increased when they were stabilized with polyvinyl alcohol. FTIR analysis also
reported the probability of presence of proteins acting as promising capping and
stabilizing agents (Kathiresan et al. 2010).
In a study silver nanoparticles were synthesized by using a different strain of
A. niger isolated from the Gulf of Cambay, India. These nanoparticles exhibited
potential optical properties and were spherical in shape with a diameter of 5–26 nm
(Vala et al. 2012). Symbiotic association between microorganisms and various
marine animals like sponges are well-known. One such symbiotically associates
strain of A. terreus (MP1) was isolated from a marine sponge and its mycelial extract
was utilized for the synthesis of silver nanoparticles. These particles also showed
potential inhibitory activity against the pathogenic bacterial strains including Staph-
ylococcus aureus,Klebsiella pneumoniae, and Salmonella sp. (Meenupriya et al.
2011).
In similar study marine yeast called Rhodospiridium diobovatum was reported to
synthesize lead sulfide (PbS) nanoparticles intracellulary with the help of nonprotein
thiols (Seshadri et al. 2012). The characterization of PbS nanoparticles was
performed by UV-visible absorption spectroscopy, diffraction (XRD), and energy
dispersive atomic spectroscopy (EDAX). During UV-visible absorption spectros-
copy, a peak at 320 nm was obtained indicating the presence of nanosized particles.
Further XRD analysis established the presence of PbS nanoparticles of cubic shape.
However, the size of these crystalline particles was determined to be in the range of
2–5 nm. However, elemental analysis by EDAX discovered the existence of particles
made up of lead and sulfur in the ratio of 1:2. These observations confirmed capping
of PbS nanoparticles by a sulfur-rich peptide. The yeast grown in the presence of
lead exhibited a striking increase (280 overcome the control) in nonprotein thiols
during the stationary phase. A capping agent consisting of sulfur-rich peptide was
also observed. These were probably concerned in fabrication of the nanoparticles
(Seshadri et al. 2012).
26.8 Nanoparticle Synthesis by Thermophilic Fungi
Thermophiles are organisms which require heat for their growth and development
(thermophile), they can even withstand high temperatures (thermotolerant). A few
thermophiles are also able to tolerate extreme stress conditions like toxicity of heavy
metal ions or metals. Most of them are therefore capable to survive and grow under
high metal ion concentrations and even have probability of binding huge quantities
of metallic cations. Additionally, many of these microorganisms are efficient enough
to synthesize nanoparticles. This outstanding ability of such microorganisms to
survive in the presence of heavy metal ions makes them most appropriate candidates
for the synthesis of nanoparticle. Thermophiles inhabit both natural and man-made
habitats. Natural habitats harbored by such a type of thermophilic microorganisms
include terrestrial geothermal, volcanic areas, and deep-sea hydrothermal vents
(Mehta and Satyanarayana 2013).
26 Extremophilic Fungi for the Synthesis of Nanomolecules 623

Both culture-dependent and culture-independent approaches have been applied
so far for the isolation and identification of extreme thermophiles and
hyperthermophiles from their natural habitats. Geothermal and volcanic areas
mainly include habitats like terrestrial fumaroles (e.g., solfataras), terrestrial hot
springs, and geysers. Whereas some other natural habitats consist of geothermally
heated oil and petroleum reservoirs and sun-heated soils/sediments (Greene et al.
1997; Engle et al. 1995; Ward et al. 1987; Zillig et al. 1980; Mehta and
Satyanarayana 2013). The temperature of these habitats is comparatively lower as
that of other natural habitats and is therefore appropriate for the isolation of moderate
and extreme thermophiles containing enzymes functional at high temperatures and
also aid in their survival.
According to a study by Syed et al. (2013) the biosynthesis of silver nanoparticles
was performed by the thermophilic fungus Humicola sp. This fungus extracellularly
reduces the Ag + ions present in the reaction mixture resulting in the synthesis of
silver nanoparticles. During this study the researchers demonstrated that dimensions
of these nanoparticles could be controlled within the size range of 5–25 nm, so that
they can be applied in the field of biomedicine. Formation of silver nanoparticles was
reported by the change in color of reaction mixture from pale yellow to brown. It was
further confirmed by the application of advanced characterization techniques.
The biosynthesized silver nanoparticles are found to be non-toxic toward cancer-
ous and normal cells up to concentrations of 50 μg/ml. Therefore, they find a variety
of applications in targeted drug delivery systems (Syed et al. 2013). Likewise
biosynthesis of biomedically important cerium oxide (CeO
2
) nanoparticles using
the thermophilic fungus Humicola sp.was reported. When this fungus was exposed
to aqueous solutions of oxide precursor cerium (III) nitrate hexahydrate
(CeN
3
O
9
6H
2
O) extracellular formation of CeO
2
nanoparticles containing Ce (III)
and Ce (IV) in mixed oxidation states was confirmed by X-ray photoemission
spectroscopy (XPS). These biosynthesized nanoparticles were found to be naturally
capped by proteins secreted by the fungus which prevents them to agglomerate. A
thermostable enzyme or protein is the one when a high defined unfolding (transition)
temperature (Tm) or an extended half-life at a particular temperature is observed. A
hot temperature for growth is generally above the thermophile boundary that is
greater than 55 C. Except for a few of them most of the proteins from thermophiles
are thermostable.
26.9 Nanoparticles by Psychrophilic Fungi
Psychrophiles are organisms, which need low temperatures for their growth and
survival and can even tolerate very low temperature conditions (psychrotolerant).
They are also divided into two classes, namely stenopsychrophiles and
eurypsychrophiles. Stenopsychrophiles, also known as obligate psychrophiles, can
only stay alive only at temperatures below 15 C; however, eurypsychrophilic also
known as mesotolerant organisms cultivate best below 15 C but can also withstand
and grow at higher temperatures. Thus several studies have suggested that
624 H. Shukla and S. Shukla

psychrophiles have an optimum growth temperature below 15 C but cannot grow at
temperature greater than 20 C. However, psychrotolerant microorganisms, also
known as psychrotrophs are those which might tolerate cold conditions but have
an optimum growing temperature of about 20 C. Numerous organisms remain
metabolically active at temperatures below freezing conditions (Koshima 1984). A
number of psychrophiles have established the potential to tolerate extreme stress
conditions, viz. toxicity of heavy metal ions or metals. Besides this they have also
exhibited the potential of nanoparticles synthesis.
Psychrophilic fungus Humicola marvinii was found to be able to metabolize and
reproduce best at temperatures below 15 C, while they are reported to survive even
with greatly reduced metabolism all the way down at a temp of about 20 C (Von
Klein et al. 2002; Auman et al. 2006; Kumar et al. 2007; Weinstein et al. 1997).
These cold-loving microorganisms are generally present in Polar region, deep sea,
mountains, glaciers, fresh and marine waters, polar and high alpine soils. These
fungi face many problems under such environments including membrane rigidity,
protein misfolding, and slower reaction rates.
26.10 Nanoparticles by Alkaliphilic and Acidophilic Fungi
Acidophiles and alkaliphiles are another group of microorganisms exploited for
nanoparticles biosynthesis. The biomolecules present in these fungi are not only
involved in the biosynthesis of nanoparticles but they also provide excellent stability
to the biosynthesized nanomaterial. These biomolecules including proteins,
peptides, and a special class of metal-binding molecules are called as phytochelatins
that are used for the in vitro stabilization of synthesized nanomaterials. Organisms
that grow at the extreme pH conditions are divided into two classes, viz. acidophiles,
showing maximum growth below pH 3, and alkaliphiles, exhibiting optimum
growth at pH greater than 9 (Rothschild and Mancinelli 2001; Wiegel 2011).
Fungi can collect metals by different mechanisms including physicochemical and
biological mechanisms. It involves binding of metabolites and polymers, to specific
polypeptides extracellularly and metabolism dependent accumulation. Mukherjee
et al. (2001) reported the formation of gold nanoparticles having diameter of 20 nm
when the acidophilic fungus, Verticillium sp. (AAT-TS-4), was exposed to aqueous
AuCl
4
ions resulting in the reduction of the metal ions. The synthesis of gold
nanoparticles occurred both extracellularly that is on the surface and intracellularly
that is within the fungal cells with slight reduction of the metal ions in solution.
The presence of diffuse rings with lattice spacings was confirmed by the applica-
tion of selected area diffraction analysis of one gold particle. Bulk of the gold
nanoparticles were found on the membrane surface which is surprising because
usually no more than 10 15 nanoparticles in one cell of bacteria are reported
(Klaus et al. 1999). Further it has also been demonstrated that the scale of distribu-
tion of gold nanoparticles produced using Verticillium sp.is way narrower in
comparison to that observed for silver nanoparticles synthesized by bacteria
(Klaus et al. 1999). Mostly spherical AuNPs were observed on the cytoplasmic
26 Extremophilic Fungi for the Synthesis of Nanomolecules 625

membrane along with a few triangular and hexagonal particles as well. A big, quasi-
hexagonal gold particle was reported within the cytoplasm (Mukherjee et al. 2001).
The acidophilic fungus Verticillium sp.when exposed to silver nitrate solution
reduction and accumulation as silver nanoparticles within the fungal cell was
observed (Sastry et al. 2003). The change in the color of fungal biomass to dark
brown after reaction with Ag
+
ions indicated the reduction of the metal ions and
synthesis of silver nanoparticles inside the fungal cell. In case of this fungus the
formation of silver nanoparticles was observed only inside the fungal cell and not
outside. The absorption maxima of normally harvested fungal mycelium was not
found at 400–800 nm, whereas that of the fungal cells exposed to Ag + ions was
found at 450 nm which was also used as indicator of reactivity.
The occurrence of the broad resonance revealed an aggregated structure of the
silver nanoparticles within the film. The extracellular reduction of the Ag
+
ions in
solution followed by their precipitation onto the cells could be a probable mecha-
nism for detecting the presence of silver nanoparticles within the fungal biomass.
The exact mechanism involved in the intracellular formation of silver nanoparticles
is not yet completely described. The mechanism revealed by Sastry et al. (2003)
demonstrated that the nanoparticles are formed on the surface of the mycelia and not
in solution, the chief step includes trapping of the Ag + ions on the surface of the
fungal cells perhaps via electrostatic interaction between the Ag + and charged
carboxylate groups in enzymes present within the semipermeable membrane of the
mycelia. It is followed by the reduction of the silver ions by enzymes present inside
the cytomembrane resulting in the formation of silver nanoparticles. However few
Ag + ions disperse through the cytomembrane and are reduced by enzymes present
on the cytoplasmic membrane and within the cytoplasm. Further possibility of
diffusion of smaller silver nanoparticles across the cytomembrane and entrapment
within the cytoplasm was declared by several researchers (Sastry et al. 2003).
26.11 Bipolaris nodulosa
Examination of mycelia free media (MFM) of Bipolaris nodulosa indicated their
potential to synthesize anisotropic silver nanoparticles (Saha et al. 2010). After
exposure to aqueous silver nitrate solution (1 mM), the mycelia free media exhibited
a steady change in color with time from yellowish to light pink, brown, and finally to
dark brown within 24 h. The formation of silver nanoparticles was further confirmed
by using the UV–Vis spectroscopic analysis during which an absorbtion peak at
420 nm characteristic for silver nanoparticles was obtained. These nanoparticles
were found to be monodisperse in nature having dimensions ranging from 10 to
60 nm via laser diffraction analysis. Moreover antimicrobial potential of these
nanoparticles was examined against Bacillus,Bacillus cereus,Pseudomonas
aeruginosa,Proteus vulgaris,Escherichia, and Micrococcus luteus wherein they
showed potential antimicrobial activity at a concentration of 100 μg/ml (Saha et al.
2010).
626 H. Shukla and S. Shukla

26.12 Fusarium sp.
Fusarium oxysporum is the most highly explored and exploited for the formation of
silver nanoparticles out of all types of fungi. Extracellular synthesis of numerous
nanoparticles, viz. gold, silver, bimetallic Au–Ag alloy, silica, titania, zirconia,
quantum dots, magnetite, strontianite, Bi
2
O
3
and barium titanate has been reported.
In 2002, the synthesis of spherical and triangular gold nanoparticles ranging from
20 to 40 nm was revealed by Mukherjee et al. The FTIR spectrum indicated the
presence of amide (I) and (II) bands from carbonyl and amine stretch vibrations in
proteins, respectively. In addition presence of a protein having molecular mass
between 66 and 10 kDa responsible for nanoparticles stabilization was also
observed, via. electrophoresis. In another study synthesis of Zirconia nanoparticles
was demonstrated when cationic proteins produced by F. oxysporum were incubated
with ZrF6-2 anions. The protein having molecular mass of about 24–28 kDa was
declared to be involved in the formation of zirconia nanoparticles (Bansal et al.
2004). These nanoparticles were in particular quasi-spherical in shape having size in
between 3 and 11 nm.
F. oxysporum exhibited the extracellular synthesis of silver nanoparticles and
bimetallic gold–silver (Au–Ag) alloy nanoparticles. Biomedical applications of Au–
Ag alloy nanoparticles have also been demonstrated. Likewise, production of silica
and titania nanoparticles with SiF
6
2
and TiF
6
2
anionic complexes results in the
synthesis of crystalline titania and calcinations at temperature of 300 C (Bansal
et al. 2005). Moreover, ternary oxide, barium titanate nanoparticles (BaTiO
3
)of
irregular quasi-spherical morphology with an average size of 4 1 nm were also
synthesized. After the mixing of salts K
3
[Fe(CN)
6
] and K
4
[Fe(CN)
6
] and incubating
for 24 h, it resulted in the formation of crystalline magnetite nanoparticles with
single-domain characteristics. The size of these particles was 20–50 nm and they
were quasi-spherical in shape (Bharde et al. 2006).
Lately, extracellular production of optoelectronic material Bi
2
O
3
nanocrystals of
size between 5 and 8 nm with quasi-spherical morphology and good tunable
properties by F. oxysporum was reported. On addition of bismuth nitrate as a
precursor, nanocrystal synthesized were in monoclinic and tetragonal phases
(Uddin et al. 2008). Similarly Kumar et al. (2007) have revealed the enzymatic
synthesis of silver nanoparticles using NADPH-dependent nitrate reductase purified
from F. oxysporum. It was later confirmed by protein assays that an NADH-
dependent reductase is that the chief factor involved in biosynthesis processes.
Besides this the enzymatic method of in vitro synthesis of silver nanoparticles by
NADPH-dependent nitrate reductase isolated from F. oxysporum with capping
peptide, phytochelatin was reported in a recent study (Durán et al. 2007).
26 Extremophilic Fungi for the Synthesis of Nanomolecules 627

26.13 Aspergillus sp.
Kumar et al. in the year 2008 demonstrated the biosynthesis of silver nanoparticles
by using Aspergillus niger isolated from soil. Cell-free filtrate of A. niger was
exposed to 1 mmol/L nitrate and rotated on a rotary shaker at 120 rpm and 25 C.
After 72 h of treatment with nitrate solution Aspergillus flavus started gathering
silver nanoparticles on their cell surface. The average size of these silver
nanoparticles (Ag NPs) was observed to be around 8.92 nm. Further these Ag NPs
were reported to exhibit a characteristic absorption peak at 420 nm and emission
peak at 553 nm (Vigneshwaran et al. 2006). In a similar study mycosynthesis of
silver nanoparticles by Aspergillus clavatus was demonstrated extracellularly
(Saravanan and Nanda 2010; Verma et al. 2010). During another research work
reduction of aqueous Ag ion was done by using culture supernatants of Aspergillus
terreus resulting in the formation of silver nanoparticles. These mycosynthesized
silver nanoparticles were found to be polydispersed spherical particles ranging in
size from 1 to 20 nm and also showed potential inhibitory activity against most of the
plant pathogenic fungi and bacteria. Disc-diffusion technique was applied to exam-
ine the antibacterial action of Ag NPs against pathogenic bacteria, viz. E. coli,
Candida albicans, and Pseudomonas fluorescence. Likewise, antimicrobial activity
against pathogenic fungal and bacterial strains of silver nanoparticles was reported in
a recent study (Jaidev and Narasimha 2010).
26.14 Mechanism of Mycogenesis of Metal Nanoparticles
Nature is a great reservoir of microorganisms which act as biofactories for the
synthesis of metal nanoparticles having potential bio-prospective applications
(Azizi et al. 2017). Recently, the novel biosynthetic pathway and varied applications
of nanobiotechnology have drawn the attention of researchers to work on this field.
Microbial synthesis of nanoparticles has great benefits and huge potential to humans,
because they eradicate the use of toxic chemicals and involve cost-effective synthetic
procedure. One of the major advantages of biosynthesis of metal nanoparticles is
their enormous role in the environment protection which makes it an eco-friendly
approach and a significant approach toward green technologies (Azizi et al. 2017;
Ottoni et al. 2017). Lately, mycosynthesis of nanoparticles has attracted majority of
researchers; however, the correct mechanism linked to the synthesis has not been
explored yet.
Fungal kingdom comprises multicellular eukaryotic organisms that are particu-
larly heterotrophic and play vital role in nutrient cycling in an ecosystem. Fungi
maintain symbiotic associations with plant and bacteria and reproduce both sexually
and asexually. Major groups of fungi include molds, mildews, yeasts, rusts, and
mushrooms. Fungi secreted a variety of enzymes in comparison to bacteria and
therefore synthesize huge number of nanoparticles. They also generate different
biomolecules which are involved in the bioreduction and stabilization process of
nanoparticles. Out of all fungal enzymes especially the reductase enzymes are
628 H. Shukla and S. Shukla

probably responsible for the nanoparticle fabrication and stabilization (Fig. 26.1).
Since the fungal cell surface possesses negative charge and contains sticky
substances, the metal ions adhere on the cell surface via electrostatic interaction. It
is well-known fact that fungi performs both extracellular and intracellular synthesis
of nanoparticles. Intracellular synthesis approach is more appropriate for synthesis of
composite films. However, during extracellular synthesis, the metal ions are
immobilized in a suitable carrier or support (Duhan et al. 2017). The metabolites
and enzymes secreted by the fungi are the chief components and play important role
in conversion of toxic materials into non-toxic materials; these compounds may also
be also involved in the synthetic mechanism of nanoparticles (Owaid and Ibraheem
2017).
Reports confirmed the secretion of proteins and enzymes by a fungus named
Trichoderma reesei outside the hyphae which are known to play a key role in silver
nanoparticles synthesis. Usually the nanoparticles synthesized by fungi are
monodispersed with definite size and shape having diverse chemical compositions.
Since they secrete diversity of enzymes inside and outside of the cells, fungi become
most suitable choice over other microbes for nanoparticles synthesis (Velhal et al.
2016). Moreover the research work carried out in the field of mycogenesis of
nanoparticle has led to emergence of a new field of science called
nanobiotechnology with major potential due to wide range diversity and availability
of fungi. In comparison to bacteria, fungi possess greater metal tolerance and metal-
binding capability which aids in their uptake capacity, hence fungal biomass is an
attractive biological agent for industrial production of nanoparticles. Moreover the
downstream processing involved with fungal biomass is simpler which makes their
Fig. 26.1 Mechanism of nanoparticles synthesis by extremophilic fungi
26 Extremophilic Fungi for the Synthesis of Nanomolecules 629

employment efficient and cost-effective in the synthesis of metal nanoparticles
(Yadav et al. 2015). A variety of fungal species are already known for the synthesis
of different metal nanoparticles, viz. Au, Ag, Pd, Cu, Zn, Ti, Fe, and Pt. Fungi
exhibit slower growth kinetics, as a consequence they provide enhanced regulation
of shape, size, and long term stability of nanoparticles (Zhao et al. 2018).
Studies conducted on fungus-mediated synthesis of nanoparticles mainly focus
on their possible mechanism, action of nitrate reductase, electron shuttle quinones.
These studies suggest that mainly nitrate reductase and α-NADPH-dependent
reductases are responsible for nanoparticle synthesis in both bacteria and fungi.
Extracellularly synthesized nanoparticles are stabilized by the proteins and enzymes
secreted by the fungal cell. During a research, it was revealed that minimum four
proteins including NADH-dependent reductase having relative molecular mass are
involved in nanoparticles synthesis. The analysis of fluorescence spectra of these
enzymes demonstrates the presence of native type of these proteins within the
aqueous extract or solution similar to those binded on the surface of nanoparticles
(Shah et al. 2015).
Further research work indicates that the metal ions reduction and the surface
binding molecule to the nanoparticles comprise protein molecules. Thus it could be
concluded from the studies that fungus contains polypeptides and enzymes which act
as a reducer as well as stabilizing agents. As a result of the cellular complexity of
fungi, further more research is needed to explore the detailed mechanism of synthe-
sis. Moreover extensive research works are required to produce nanoparticles of
definite shape and size and also to increase the speed of synthesis of nanoparticles.
With the advent of recent techniques much information has been generated which
has enhanced the applications of nanoparticle biosynthesis in various fields. How-
ever, the awareness about concrete mechanism of the mycosynthesis of
nanoparticles requires more study and experimental trials.
26.15 Characterization Techniques for Nanoparticles
Characterization studies of nanoparticles include the analysis of shape, size, and
structure of different types of nanoparticles which is usually significant for material
research. According to Liu et al. (2010) nanomaterials are found in unique structure
and size and are estimated to possess diverse bioactivities. New findings in the field
of materials research, processes, and phenomenon at the nano-scale would offer
possible applications for fabrication of novel nanosystems and nanostructures. A
variety of characterization techniques, namely UV–visible spectroscopy, diffraction
technique (XRD), Fourier transform infrared spectroscopy (FTIR), atomic force
microscopy (AFM), scanning electron microscopy (SEM), transmission electron
microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), etc. are involved
in the characterization of metal nanoparticles. Details of these techniques are
discussed below.
630 H. Shukla and S. Shukla

26.16 UV–Visible Spectroscopy
UV–visible spectroscopy is most consistent and efficient technique for the prelimi-
nary characterization of synthesized nanoparticles which is designed not only to
monitor the synthesis but also to analyze the stability of nanoparticles. This tech-
nique is a potential approach to characterize metal nanoparticles since they require
brilliant color which can be visualize by oculus. Metal nanoparticles have elevated
extinction coefficient and their surface plasmon resonance is dependent on the size
and shape. Therefore, this method is the most suitable for the qualitative assessment
of the nanoparticles. Absorption of the suspended nanoparticles is particularly
calculated by the Beers law wherein absorption value (A) mainly depends on
concentration of nanoparticles, path length (l) of measuring cell, and extinction
coefficient of nanoparticles which is depicted by the following formula:
A¼εcl
Particle size, shape, morphology, nature of stabilizing agent, pH, temperature,
type of adsorbate that are present on the surface of nanoparticles and nature of the
encompassing medium affect the position of the optical phenomenon. The width of
optical phenomenon increases with decreasing size of nanoparticle within the
intrinsic size region and also increases with increase within the extrinsic size region
(Sudha et al. 2013; Aziz et al. 2015). A Doppler effect within the extrinsic region in
the SPR is visible as the size of the nanoparticles increases. As the size of the gold
nanoparticles increases it exhibits change in color from ruby red to purple and finally
blue. It was revealed particularly that when the space between the particles becomes
equal to their average diameter, the particles get aggregated; consequently, the
plasmon resonance of each and every particle couples and their absorbance is red
shifted. These optical characteristics of metal nanoparticles are employed for making
sensors. In addition to this, UV–Vis spectroscopy is fast, easy, simple, and sensitive
for numerous types of nanoparticles. It requires only a relatively short period for
quantification and therefore a calibration is not needed for characterization of
nanoparticles present in the colloidal suspensions (Tomaszewska et al. 2013).
26.17 Fourier Transform Infrared Spectroscopy (FTIR)
Fourier transform infrared spectroscopy is applied for the identification of specific
type of chemical bonds or functional groups depending on their definite unique
absorption patterns. It works by measuring the stretching of chemical bond and
bending patterns by absorption of energy through infrared spectroscopy. This energy
lies within the infrared (IR) region of electromagnetic spectrum. FTIR is a significant
technique as it possesses various advantages over the conventional IR spectroscopy.
FTIR therefore provides the information about surface chemistry of nanoparticles
via identification of the specific functional groups attached to the surface of metal
26 Extremophilic Fungi for the Synthesis of Nanomolecules 631

nanoparticles since it depicts different absorption patterns as compared to those of
respective free groups.
26.18 X-Ray Diffraction Technique (XRD)
X-ray diffraction technique is a non-destructive technique employed for the identifi-
cation of the crystalline phase of nanoparticles. For analysis through this method the
crystalline or powdered sample is positioned over a sample holder and X-rays of a
set wavelength are allowed to pass through them, thereby illustrating their structural
characteristics. Thereafter the intensity of the reflected radiation is recorded by using
a goniometer (Wang et al. 2002). Data obtained are then analyzed by Bragg’s
equation, to compute inter atomic spacing for the reflection angle. Following
equation is applied:
nλ¼2dsin θ
where nis an integer, λis wavelength, dis the distance between atomic planes, and θ
is the angle of incidence of the X-ray beam and therefore the atomic planes. Broad
peaks of metal nanoparticles are observed on XRD. The broadening provides
information about crystalline nature and size by Debye–Scherrer equation:
d¼κλ=βcos θ
In this equation, кis the shape factor, λis X-ray wavelength, βis the line
broadening at half the utmost intensity (FWHM) in radius, and θis the Bragg’s
angle.
26.19 Atomic Force Microscopy (AFM)
Atomic force microscopy is a significant tool used for the analysis of the surface
morphology and phase via generation of a 3D map of the sample surface. The
vertical resolution of this instrument is less than 0.1 nm, while the lateral resolution
is of around 1 nm. A cantilever having a fine pointed tip which is fixed perpendicular
to the longitudinal direction of the cantilever is mounted on the sample. From the
back of the cantilever a laser beam is reflected into a spot sensitive to photodiode and
the deflection of the cantilever caused by Vander Waal’s force acting between the
sample and tip is recorded. As a result of these interactions signals are generated.
These signals in turn are further processed to produce the topographical information
of the nanoparticles surface.
632 H. Shukla and S. Shukla

26.20 Transmission Electron Microscopy (TEM)
Transmission electron microscopy is applied for the study of the shape, size, and
dispersity of the metal nanoparticles. Smaller molecules particularly having size less
than 1 μm generate diffraction effects and hence cannot be observed by optical
microscopy. Monograph related information depends on the resolution applied,
whereas the resolution is chiefly dependent on the wavelength of the radiation
beam chosen for the imaging. Application of a short wavelength beam results in
higher resolution. TEM is most extensively used technique for the characterization
of the metal nanoparticles. This instrument is similar to a slide projector except that it
uses an electron beam of around 100–300 kV and this transmitted beam is further
projected on to a phosphor screen resulting in the formation of the final image. It
usually gives information about characters like topography, monodispersity, com-
position, and crystallinity of the synthesized nanoparticles.
26.21 Scanning Electron Microscopy (SEM)
Currently, very advanced high resolution microscopic techniques are being applied
in the field of nanoscience and nanotechnology. It has aided in the analysis and
illustration of nanomaterials very deeply by using a ray of high-energy electrons to
probe. SEM is mainly employed for the study of the morphology and topology of the
metal nanoparticles. In SEM, the surface of specimen is scanned by passing the
electron beam of accelerated voltage through the sample surface. The pictures or
images of the surface are thereafter obtained by collection of backscattered and the
secondary electrons via the detector followed by their analysis. It is principally a
surface imaging technique, which is fully competent of resolving different sized
nanoparticles, their distributions, shapes, and the surface topology (Wang et al.
2002). The only restriction of SEM is that it can only generate useful information
related to the purity and the degree of particle aggregation but it is not able to
determine their internal structure. This technique is particularly appropriate for the
morphological identification of nanoparticles having dimensions below10 nm.
26.22 Electron Energy Loss Spectroscopy (EELS)
Electron energy loss spectroscopy is a technique applied for the examination of the
component elements of nanomaterials. During this process the energy transferred via
such an interaction is in direct relation to the ionization potential of the atom and
hence, the spectrum obtained can be compared to that of known samples or standard
samples. A nanomaterial is initially subjected to a ray of kinetic energies because of
which some of the electrons lose energy by inelastic scattering, which is first and
foremost an interaction of the sample. The inelastic scattering consequence is both
an energy loss and a momentum change. Various interactions including plasmon
26 Extremophilic Fungi for the Synthesis of Nanomolecules 633

excitations or inner shell ionization, phonon excitation, and inter- or intra-band
transitions are observed (Wang et al. 2002).
26.23 Energy Dispersive X-Ray Spectroscopy (EDS or EDX)
Energy dispersive X-ray spectroscopy (EDS or EDX) is a potential investigating tool
that is specifically used for chemical characterization of the nanoparticles. The basic
principle of this technique is that each element of the periodic table has a unique
electronic structure representing the fundamental properties of the elements. There-
fore, the response of every element toward electromagnetic waves is unique. This
type of examination depends on the interaction between matter and light performed
via X-rays penetration through the sample.
26.24 Thermo-Gravimetric/Differential Thermal Analyzer
(TG/DTA)
The TG/DTA is an instrument formed by combination of TG, which measures
continuously utilizing a horizontal differential type of balance beam, with the
extremely flexible feature of DTA. Through this instrument mostly reaction velocity
is measured, acceleration degradation tests are performed, as well as water contami-
nation is analyzed, detection of heavy metal and measurement of ash content in
reaction velocity and acceleration decomposition are carried out (Fulias et al. 2013).
The light weight organization of the balance beam enhances the stability with respect
to fluctuations in temperature and extremely susceptible balance and also stability of
the differential balance to deal with disturbance like oscillation. After carrying out all
measurements, the instrument is automatically cooled to a set temperature, by the use
of a cooling unit which further increases the efficiency of measurements.
26.25 Optimization of Silver Nanoparticles Synthesis
Even though the biosynthesis of silver nanoparticles using fungi is uncomplicated
and cost-effective, the factors used in this method should be optimized so as to attain
good quality monodispersity, stability, and biocompatibility of the nanoparticles
(Balakumaran et al. 2015). From the different research works it could be inferred that
a vast diversity of fungi have potential for use in the biosynthesis of nanoparticles.
Besides this, it is also significant to optimize the synthesis conditions according to
their individual characteristics (Ottoni et al. 2017). To achieve the preferred nano-
particle yield and characteristics, parameters including agitation, temperature, light,
and culture and time of synthesis need to be adjusted since they vary depending on
the type of fungus used. Factors involved for both fungal cultivation and process of
synthesis are adjusted for regulating the size and shape of nanoparticles (Birla et al.
2013). Recent studies have revealed that modifications in temperature, meal
634 H. Shukla and S. Shukla

concentration, pH, culture medium, and amount of biomass can be used to obtain
nanoparticles possessing diverse physicochemical properties (Birla et al. 2013;
Rajput et al. 2016; Saxena et al. 2016; Liang et al. 2017).
26.26 Effect of Temperature
The temperature used during the mycosynthesis of silver nanoparticles can influence
parameters such as the synthesis speed and also the size and stability of the
nanoparticles (Elamawi et al. 2018). In a similar study synthesis of nanoparticles
was conducted by using the cell-free filtrate of Trichoderma harzianum. Ahluwalia
et al. (2014) reported that the rate of synthesis increased with the increase in
temperature up to 40 C, which in turn was declared as the optimum temperature.
In a similar work the filtrate of Fusarium oxysporum was used to obtain huge protein
secretion by the fungal biomass at temperatures ranging between 60 and 80 C. It
was observed that with gradual increase of the temperature, the synthesis rate and
surface plasmon absorbance also increased (Birla et al. 2013). In another study
during the synthesis using the endophytic fungus Colletotrichum sp. ALF2-6, it
was revealed that the reaction rate increased at higher temperatures and synthesis
completed within 20 min at temperatures above 50 C (Azmath et al. 2016).
Likewise Phanjom and Ahmed (2017) used Aspergillus oryzae (MTCC ## 1846)
and found that temperature increased the speed of synthesis. At different
temperatures of 30, 50, 70, and 90 C the synthesis was completed in 6 h, 1 h,
45 min, and 20 min, respectively, whereas no synthesis occurred at temperature of
10 C.
During a research work conducted by AbdelRahim et al. (2017), no synthesis of
silver nanoparticles was observed when the filtrate of leak fungus at 80 or 10 C was
employed. This result was considered due to denaturation or inactivation of enzymes
and other molecules. Even though most of the researches have revealed rapid rates of
synthesis at higher temperatures, it is important to realize the need for
standardization of these parameters during nanoparticle synthesis. In addition to
affect the synthesis rate, the temperature also produces profound affects on the size
and stability of nanoparticles. AbdelRahim et al. (2017) synthesized nanoparticles of
different sizes, viz. 86, 25.89, and 48.43 nm, at temperatures of 40, 20, and 60 C,
respectively, with the smallest size obtained at the intermediate temperature. In a
similar work Shahzad et al. (2019) fabricated nanoparticles of 322.8 nm size at 25 C
using the fungus Aspergillus fumigatus BTCB10. Increasing in size with increasing
temperature was observed with the biggest size reaching 1073.45 nm at 55 C. In
another study the fungus Fusarium oxysporum was employed for nanoparticles
synthesis, wherein the size of biosynthesized nanoparticles decreased to 30.24 nm
with the temperature increasing up to 50 C (Husseiny et al. 2015).
These diverse results point toward the fact that the effect of temperature on the
dimensions and stability of the biosynthesized nanoparticles varies depending upon
the fungus species used. Banu and Balasubramanian (2014) declared 30 C to be the
optimum temperature for synthesis of highly stable silver nanoparticles by using
26 Extremophilic Fungi for the Synthesis of Nanomolecules 635

Isaria fumosorosea. Similarly, Balakumaran et al. (2015) also reported the same
optimum temperature for the synthesis of nanoparticles using the fungus Guignardia
mangifera. These variations in the effects of temperature on the nanoparticles
synthesis take place even within the identical genus of fungus. Trichoderma
longibrachiatum synthesized nanoparticles at a temperature of 28 C, while no
production of nanoparticles was seen at 23 or 33 C (Elamawi et al. 2018), however,
Trichoderma viride exhibited potential synthesis at temperatures of 10, 27, and
40 C (Fayaz et al. 2010).
The biosynthesis of nanoparticles by few fungal species at higher temperatures
corresponds to the fact that electron transfer occurs from free amino acids to silver
ions. Conversely, very high temperatures, between 80 and 100 C, lead to the
denaturation of proteins that create the nanoparticle capping. This denaturation
modifies the nucleation of Ag
+
ions, followed by the nanoparticles aggregation
and increasing size (Birla et al. 2013). Husseiny et al. (2015) reported that increased
nanoparticle size and loss of stability occur at unsuitable temperatures, as a conse-
quence of the low enzymatic activity concerned within the synthesis.
26.27 Effect of pH
Alteration of the pH of nanoparticles biosynthesis process is usually carried out to
regulate some specific features of the metal nanoparticles. Nayak et al. (2011)
observed that changes are induced in the conformation of nitrate reductase enzymes
so as to maintain the concentration of protons in the reaction medium, which in turn
produces a characteristic alteration in the morphology and size of the nanoparticles.
Further it is reported that since there is a big competition between protons and metal
ions for forming bonds with charged regions at higher pH; therefore, alkaline pH
results in higher synthesis of nanoparticles (Sintubin et al. 2009). It was also
observed that a higher alkaline pH led to the synthesis of smaller nanoparticles
during a very lesser time with low polydispersity index values. These specific
features point toward enhanced stability, because of the electrostatic repulsion
between the anions present within the dispersion (Gurunathan et al. 2009). In another
study Colletotrichum sp. ALF2-6 was used for biosynthesis of nanoparticles
employing alkaline pH. It was observed that synthesis was faster at alkaline pH
and at temperature of 50 C as compared to that at lower pH. The whole process was
accomplished in around 20 min (Azmath et al. 2016). Similarly, in a research work
performed by Birla et al. (2013), utilizing Fusarium oxysporum, highest nanoparticle
synthesis was obtained between pH 9 and 11, whereas lower synthesis occurred at
pH 7 including the aggregates formation between pH 3 and 5.
Conversely, Husseiny et al. (2015), during their study with the same fungus,
observed that the rate of nanoparticle synthesis decreased with the increase in the pH
because of the lower activity of the reductases enzyme involved in the synthesis at
higher pH. Additionally few other studies have revealed appreciable synthesis of
nanoparticles at neutral or slightly alkaline pH. Banu and Balasubramanian (2014)
reported nanoparticles synthesis using Isaria fumosorosea at pH 8.5 with better
636 H. Shukla and S. Shukla

physicochemical characteristics, in comparison to nanoparticles synthesized at
pH 4.5 and 6.5. Likewise during a synthetic procedure using Guignardia
mangiferae, change in color was not observed between pH 1 and 4; however,
color change began to appear at pH 5 and 6. With the increase in pH, the amount
of the dispersion increased, with the nanoparticles possessing higher
monodispersion and stability at pH 7 (Balakumaran et al. 2015).
26.28 Effect of Silver Nitrate (AgNO
3
) Concentration
Most of the studies have examined the effect of silver nitrate (AgNO
3
) concentration
on the extracellular synthesis of silver nanoparticles employing fungi particularly at
a concentration of 1 mM (Saxena et al. 2016; Xue et al. 2016). In a few cases, a lesser
metal precursor concentration led to the synthesis of smaller sized nanoparticles and
an enhanced dispersion (Kaviya et al. 2011; Phanjom and Ahmed 2017).
AbdelRahim et al. (2017), while using the fungus Rhizopus stolonifer, produced
the nanoparticles of smallest size (2.86 nm) at 10 mM concentration of AgNO
3
.
Further nanoparticles of 54.67 and 14.23 nm size were synthesized at 100 and 1 mM,
respectively. Same conclusions were recorded by Husseiny et al. (2015) while using
the fungus Fusarium oxysporum. Phanjom and Ahmed (2017) studied the synthesis
of nanoparticles using Aspergillus oryzae and different AgNO
3
concentrations
between 1 and 10 mM.
It was observed that at AgNO
3
concentrations up to 8 mM, the nanoparticles
presented sizes between 7.22 and 17.06 nm, while the size increased to 45.93 and
62.12 nm at AgNO
3
concentrations of 9 and 10 mM, respectively. This effect was
attributed to the lack of functional groups available for the reaction when the metal
precursor concentration was increased. In a study employing Fusarium oxysporum,
it was found that the quantity of nanoparticles increased as the precursor concentra-
tion was increased between 0.1 and 1.5 mM, while no differences were observed at
higher concentrations (Birla et al. 2013). These findings suggest that there is a limit
to the concentration of AgNO
3
used, in order to obtain nanoparticles with satisfac-
tory physicochemical characteristics. The addition of excess amounts of metal ions
results in very large nanoparticles with irregular morphology (AbdelRahim et al.
2017), due to competition between the silver ions and functional groups from the
fungus filtrate (Shahzad et al. 2019). As the concentration of the metal precursor
increases, so also does the intensity of color of the dispersion (Ahluwalia et al. 2014;
Phanjom and Ahmed 2017).
26.29 Effect of the Culture Medium
It is well-known that microorganisms respond in different ways, depending upon the
culture medium and the conditions of cultivation. Adjustments in these conditions
cause the production of various metabolites and proteins (Costa Silva et al. 2017).
During the synthesis of nanoparticles by fungi, substrate specific for the enzymes is
26 Extremophilic Fungi for the Synthesis of Nanomolecules 637

added in the medium which in turn induces their production and release by the
fungus. Thus they enhance the reduction of silver and resulting in the formation of
silver nanoparticles (Husseiny et al. 2015). Similarly Hamedi et al. (2017), during
their work cultivated Fusarium oxysporum on a medium manipulated to enhance the
nitrate reductase enzyme activity. The nanoparticle suspension formed by employing
the fungal filtrate of the fungus grown in the enzyme inducing medium depicted
higher concentrations and particularly smaller sized nanoparticles. This change was
credited to the enhancement of the enzymatic activity through the modification of
nitrogen source in the medium, thereby elevating the rate of nanoparticle synthesis.
Different responses were recorded in studies involving the use of different media for
the fungal cultivation. In a similar study Saxena et al. (2016) performed synthesis of
silver nanoparticles, via cultivated Sclerotinia sclerotiorum, in various broths
resulting in the highest production of nanoparticles within the potato dextrose
medium. The chitin supplemented filtrate was found to contain around three times
more protein and depicted increased production of nanoparticles. Likewise Birla
et al. (2013) examined ten different media for cultivation of Fusarium oxysporum,
thereby observing huge production of silver nanoparticles by utilizing the filtrate
from the fungus cultured in MGYP medium.
26.30 Effect of the Quantity of Biomass
The quantity of biomass employed has an effect on the silver nanoparticles synthesis
and characteristics. On the one hand where few studies have indicated increased
nanoparticle synthesis in the presence of lower biomass concentrations, on the other
hand many have found elevated synthesis rates in the presence of higher biomass
concentrations (Birla et al. 2013; Korbekandi et al. 2013; Balakumaran et al. 2015;
Elamawi et al. 2018). In one such study Balakumaran et al. (2015) suspended 10, 20,
and 30 g quantities of Guignardia mangiferae biomass in 100 mL of water and
obtained their filtrates, respectively. These filtrates were separately subjected to
silver nanoparticles synthetic procedures resulting in the synthesis of silver
nanoparticles with better physicochemical characteristics in the filtrate of lower
biomass concentration. Similarly Shahzad et al. (2019) utilized filtrates of 1, 4,
7, and 10 g biomass of Aspergillus fumigatus BTCB10 observing larger production
of silver nanoparticles with smaller size and better dispersion in the filtrate obtained
from mixture containing 7 g of biomass. During the research it was recorded that
while using a higher biomass concentration of Penicillium oxalicum, increased
nanoparticle production was obtained which was considered due to better release
of the nitrate reductase enzyme by the mycelium. In a similar study Saxena et al.
(2016) obtained higher silver nanoparticle production in the presence of increased
Sclerotinia sclerotiorum biomass. Correlation between the biomass and the release
of biomolecules responsible for the synthesis nanoparticles has been reported by
Birla et al. (2013).
638 H. Shukla and S. Shukla

26.31 Applications of Biosynthesized Nanomolecules
Nanoparticles synthesized using fungi particularly the silver nanoparticles have a
variety of potential applications in the areas of medicine, agriculture, optics, elec-
tronics, etc. As a consequence of large amount of metabolites produced from fungi,
they present to be an attractive source for nanoparticle synthesis. An additional
factor under consideration is the capability of fungi to produce antibiotics which
remain present within the capping, thereby playing a significant role in the nanopar-
ticle stabilization. Several studies related to biological synthesis of nanoparticles
employing fungi have revealed outcomes that show promising applications in
controlling pathogenic fungi and bacteria, combating cancer cells and viruses, and
providing larvicidal and insecticidal activities.
26.32 Health Applications
A number of studies have demonstrated the applications of biogenic silver
nanoparticles in the sectors of health, especially the control of pathogenic bacteria
and fungi. The growth of bacterial cells is directly inhibited by the nanoparticles,
which make contact with the cytomembrane and lead to stepwise metabolic
responses, resulting in the assembly of reactive oxygen species (Gudikandula et al.
2017). The size of nanoparticles directly affects their antimicrobial potential,
because smaller nanoparticles produce larger effects (Lu et al. 2013). Small sized
nanoparticles can pass through the semipermeable membrane of bacterial cells and
harm the respiratory chain, modify permeability, cause DNA and RNA damage,
affect biological process, and ultimately result in necrobiosis (Morones et al. 2005;
Rai et al. 2009).
Antifungal compounds (such as biogenic silver nanoparticles) that are
synthesized from sustainable biological sources are often cost-effective and secure
alternative for the systemic treatment of external and internal fungal infections, thus
enabling the control of resistant fungi (Ashajyothi et al. 2016). The poisonous ions
attach to sulfur containing proteins, disturbing the permeability of cell, thereby
leading to modifications of the DNA replication mechanism. Additionally the
inhibition of enzymatic activity is because of the binding of nanoparticles with
thiol groups. As a result of this inactivation an oxidative stress develops, which in
turn affects electron transport and protein oxidation (Rai et al. 2012). The potential
activity of silver nanoparticles synthesized using the fungus Guignardia mangiferae
against gram-negative bacteria was reported by Balakumaran et al. (2015). The
adverse effects produced by nanoparticles included increased permeability, alter-
ation of membrane transport, and release of nucleic acids.
Sometimes the peptidoglycans constituting the cytomembrane act as an obstacle
and inhibit the internalization of nanoparticles, thereby lowering their side-effects
toward gram-positive bacteria. While analyzing the antimicrobial activity of silver
nanoparticles synthesized using Aspergillus niger, Gade et al. (2008) recorded
efficient inhibitory activity toward the bacteria E. coli and S. aureus similar to that
26 Extremophilic Fungi for the Synthesis of Nanomolecules 639

observed in case of antibiotic gentamicin. Use of silver nanoparticles in combination
with antibiotics and antifungal substances also represents a probable solution to the
emerging resistance toward these drugs. Biosynthesized silver nanoparticles using
candida were tested for their inhibitory activity along with the antibiotic ciprofloxa-
cin against Staphylococcus aureus, Escherichia coli, Bacillus cereus, Vibrio
cholerae, and Proteus vulgaris. It was observed that the combination of antibiotic
improved the activity of nanoparticles, as compared to that when used alone.
Similarly it was examined the antimicrobial and antifungal potential of silver
nanoparticles biosynthesized by using the filtrate of Aspergillus flavus. These
mycosynthesized nanoparticles were efficient in inhibiting the activity of bacteria
Bacillus cereus, Bacillus globigii, Enterobacter aerogenes, escherichia, and Staph-
ylococcus aureus. Out of these the highest activity was exhibited against B. subtilis
and E. coli. It was also observed that the activity was dependent on concentration,
with optimum outcomes obtained while employing the nanoparticles in combination
with the antibiotic tetracycline, in spite of using alone. Likewise concentration
dependent activity of the nanoparticles was also obtained against fungi Aspergillus
niger and Trichoderma harzianum. Silver nanoparticles synthesized by using the
fungi Trametes ljubarsky and Ganoderma enigmaticum for the control of gram-
positive and gram-negative bacteria, namely Bacillus subtilis, Staphylococcus
aureus, Micrococcus luteus, Bacillus cereus, Bacillus megaterium, E. coli,
Enterobacter aerogenes, Klebsiella pneumoniae, Salmonella typhimurium, Proteus
vulgaris, Pseudomonas aeruginosa, and Salmonella paratyphi Gudikandula
et al. (2017).
Both types of nanoparticle possessed potential antibacterial activity. Moreover
biologically synthesized silver nanoparticles have depicted effective activity even
against the multidrug resistant microorganisms. For example, an optimized process
for the synthesis of silver nanoparticles wherein Penicillium sp. was used for the
synthesis of silver nanoparticles having potential activity against the multidrug
resistant bacterial species, viz. E. coli and S. aureus was developed. Besides their
antimicrobial activity, biosynthesized silver nanoparticles are also reported to induce
profound effects on tumor cells. In a similar study Husseiny et al. (2015) recorded
the antibacterial and antitumor activity of silver nanoparticles synthesized using
Fusarium oxysporum. These nanoparticles were efficient in inhibiting the growth of
E. coli and S. aureus, as well as a tumor cell line. After the exposure of tumor cells to
the nanoparticles, the coffee IC50 value (121.23 μgcm
3
) for MCF-7 cells (human
breast adenocarcinoma) was obtained which revealed elevated cytotoxicity and also
the potential tumor control activity of biosynthesized nanoparticles. These activities
were attributed to the participation of the silver nanoparticles in disruption of the
mitochondrial respiratory chain, which in turn causes the congregation of reactive
oxygen species, thereby leading to the hindrance of the nucleotide (ATP) synthesis,
following the damage of the nucleic acids (Husseiny et al. 2015).
640 H. Shukla and S. Shukla

26.33 Agriculture and Pest Control Applications
Several research works have been carried out to assess the potential of
biosynthesized silver nanoparticles for the regulation of and other pests harmful
for agriculture. Silver nanoparticles synthesized using the fungus Aspergillus
versicolor exhibited inhibitory effects against the phytopathogenic fungi Sclerotinia
sclerotiorum and Botrytis cinerea causing disease in strawberry plants. These
nanoparticles demonstrated concentration dependent activity against both pests,
with the highest activity against B. cinerea (Elgorban et al. 2016). In a similar
study Qian et al. (2013) mycosynthesized silver nanoparticles utilizing the fungus
Epicoccum nigrum and recorded their activity against some pathogenic fungi,
namely C. albicans, Fusarium solani, Sporothrix schenckii, Cryptococcus
neoformans, Aspergillus flavus. Recent studies have investigated the potential
activities of mixture including biogenic nanoparticles and conventional
biopesticides. Likewise the potential activity of mycosynthesized silver
nanoparticles by the fungus Alternaria alternata combined with the antifungal
compound fluconazole, against the deadly phytopathogenic fungi Phoma glomerata,
Phoma herbarum, and Fusarium semitectum was recorded.
26.34 Environmental Applications
Besides a number of medical applications biosynthesized nanoparticles (BNPs) have
also been investigated by many researchers for their application in the sector of
environmental remediation (Njagi et al. 2011; Hussain et al. 2016). Recent
researches illustrate two major ways for clean-up and rejuvenation of contaminated
sites by employing BNPs, the first one includes adsorption of the pollutant and the
second one involves contaminant degradation or dehalogenation. Adsorption is also
a dreadfully attractive approach to stimulate strong contaminants, since it is charac-
teristically extremely competent, leading to the decrease in chemical sludge
concentrations without the requirement of any strong technological knowledge.
However researchers were able to eliminate heavy metals using cellulose BNPs
and nanoparticles synthesized from microbial biomass. Lately bacterial cellulose
nanofibers (BCF), researchers have drawn attention of most of the researchers
because of their unique structure and properties. This BNP consists of a nanofibrillar
structure, possessing elevated mechanical strength, increased surface area to volume
ratio, inherent environmental inertness, and will be easily functionalized by
incorporating chemical moieties which is able to increase the binding efficiency of
pollutants.
These properties are favorable for adsorbents used in the remediation of environ-
mental contaminants (Shah and Brown Jr 2005). Carboxylation is the conventionally
employed method to increase the adsorption capacity of cellulose. Srivastava and
collaborators depicted one more case of successful carboxylation which caused
enhanced heavy metal removal. It was indicated further that the capacity of removal
of heavy metals, viz. Ni
2+
,Cr
3+
,Cd
2+
, and Pb
2+
after incorporation of carboxylic
26 Extremophilic Fungi for the Synthesis of Nanomolecules 641

groups into cellulose was increased by 3–10 times as compared to that of the
unmodified NPs (Srivastava et al. 2012). Similarly, it was observed that the presence
of carboxylate groups within the cellulose generates a material capable of attaining
2–3 times greater removal capacity of UO
2
2+
than conventional adsorbents (i.e. silica
particles, hydrogels, polymer particles, and montmorillonite). Conversely,
researchers also revealed that the integration of cysteine, containing thiol groups,
was efficient for the removal of Cr (VI) and Pb (II) (Yang et al. 2014).
Some studies have also depicted the removal of pollutants from aqueous
environments by employing non-active (dead) microbial mass for biosorption
(Gupta and Suhas 2009; Ngah et al. 2011). The exceptionally high surface area to
volume ratio of BNPs and a number of functional groups (carboxyl, hydroxyl,
sulfate, phosphate, and others) within these nanoparticles having high affinity for
these pollutants are being considered favorable properties behind their potential
activity. In addition to adsorption, researchers have also demonstrated the applica-
tion of BNPs for the successful removal of contaminants from the water sources,
soils, and sediments.
In a recent research BNPs of Pd have been investigated for remediation purposes
as a consequence of widespread application of Pd as a catalysts in chemistry for
processes like dehalogenation, reduction, etc. (Mabbett et al. 2004; De Windt et al.
2006). The majority research works have aimed on commercial use of
biosynthesized Pd nanoparticles for the dehalogenation of contaminants from waste-
water, groundwater including soil remediation.
26.35 Concluding Remarks and Future Challenges
Microbes possess an outstanding capacity for the synthesis of metal nanoparticles,
having widespread applications in different areas. Therefore in the present scenario,
mycogenesis of nanoparticles has become a center of attraction as a biological source
of various metal nanoparticles. Mycosynthesized nanoparticles have exhibited effi-
cient and promising potential applications in a number of fields, namely agriculture,
food, textile, medicine, cosmetics, optical, and electronics. The advantages of fungi
mediated synthesis of nanoparticles include easy handling and downstream
processing. Recently, nanoparticles have been designed using metals, some metal
sulfides, and only a few oxides. This has led to the emergence of the need to develop
procedures for noble synthesis of nanostructures using other metal oxides, nitrides,
carbides, etc. Biosynthesis of nanoparticles utilizing different biological agents
presents a secure, comparatively cost-effective, and eco-friendly approach. Among
all the biological agents fungi are reported to be considerably good medium for the
synthesis of nanoparticles applicable in several fields. Moreover development of
smart drug delivery system to be delivered to a particular site has proven to be
helpful in the field of disease diagnosis. Construction of smart biosensors and
detection systems is indeed advantageous to protect agricultural crops from the
harmful effects of insects and pathogens.
642 H. Shukla and S. Shukla

Precisely it can be concluded that myconanotechnology is still a developing field.
The applications of nanoparticles are continuously increasing but studies on their
toxic effects, collection within the environment and their effect on human health and
animals need to be carried out in near future. In addition the biosynthesized
nanoparticles can also be applied for the treatment of deadly diseases which will
pave the ways for the new opportunities in the field of biomedicine. Also, these metal
nanoparticles would provide a probable answer for the prevailing energy crisis by
presenting themselves as efficient energy-driven devices. A review of past
researches have also revealed that a significant work has been done for testing the
in vitro applications of nanoparticles but considerably less data is available on
in vivo applications. Further, a detailed study is still required to clarify and intricate
the knowledge and functions of nanomaterials to achieve various landmarks in the
fields of medicines, agriculture, cosmetics, electronics, environment, etc. In spite of a
vast range of advantages associated with fungal-mediated synthesis of metal
nanoparticles, there are certain variety of limitations and challenges also. Conversely
more research is required to achieve optimized reaction conditions to obtain
nanoparticles of better size, shape, and monodispersity.
Therefore according to the reports available from several research works carried
out it could be inferred that the biogenic synthesis of silver nanoparticles using fungi
offers several advantages and has promising applications in number of areas includ-
ing health and agriculture. Moreover these nanoparticles possess extraordinary
stability because of the bioactive compounds present as capping agents derived
from the fungal biomass. Hence fungal systems present a cost-effective,
eco-friendly, and green synthetic approach for the synthesis of variety of metal
nanoparticles. But at the end of the day extensive research works need to be
performed to explore more information about the vast biodiversity of fungal flora
and the mechanism by which they synthesize nanoparticles.
References
Abad JM, Mertens SFL, Pita M, Fernandez VM, Schiffrin DJ (2005) Functionalization of thioctic
acid-capped gold nanoparticles for specific immobilization of histidine-tagged proteins. J Am
Chem Soc 127:5689–5694
AbdelRahim K, Mahmoud SY, Ali AM, Almaary KS, Mustafa AEZMA, Husseiny SM (2017)
Extracellular biosynthesis of silver nanoparticles using Rhizopus stolonifer. Saudi J Biol Sci 24:
208–216
Adams MW, Kelly RM (1998) Finding and using hyperthermophilic enzymes. Trends Biotechnol
16:329–332
Agnihotri M, Joshi S, Kumar AR, Zinjarde S, Kulkarni S (2009) Biosynthesis of gold nanoparticles
by the tropical marine yeast Yarrowia lipolytica NCIM 3589. Mater Lett 63:1231–1234
Ahluwalia V, Kumar J, Sisodia R, Shakil NA, Walia S (2014) Green synthesis of silver
nanoparticles by Trichoderma harzianum and their bioefficacy evaluation against Staphylococ-
cus aureus and Klebsiella pneumoniae. Ind Crop Prod 55:202–206
Ashajyothi C, Praburajeshwar C, Harish KH, Chandrakanth KR (2016) Investigation of antifungal
and anti-mycelium activities using biogenic nanoparticles: an eco-friendly approach. Environ
Nanotechnol Monit Manage 5:81–87
26 Extremophilic Fungi for the Synthesis of Nanomolecules 643

Auman AJ, Breezee JL, Gosink JJ, Kämpfer P, Staley JT (2006) Psychromonas ingrahamii sp. nov.,
a novel gas vacuolate, psychrophilic bacterium isolated from Arctic polar sea ice. Int J Syst Evol
Microbiol 56:1001–1007
Aziz N, Faraz M, Pandey R, Shakir M, Fatma T, Varma A, Prasad R (2015) Facile algae-derived
route to biogenic silver nanoparticles: synthesis, antibacterial, and photocatalytic properties.
Langmuir 31(42):11605–11612
Azizi M, Sedaghat S, Tahvildari K, Derakhshi P, Ghaemi A (2017) Synthesis of silver nanoparticles
using Peganum harmala extract as a green route. Green Chem Lett Rev 10(4):420–427
Azmath P, Baker S, Rakshith D, Satish S (2016) Mycosynthesis of silver nanoparticles bearing
antibacterial activity. Saudi Pharm J 24:140–146
Balakumaran MD, Ramachandran R, Kalaicheilvan PT (2015) Exploitation of endophytic fungus,
Guignardia mangiferae for extracellular synthesis of silver nanoparticles and their in vitro
biological activities. Microbiol Res 178:9–17
Bankar AV, Kumar AR, Zinjarde SS (2009) Environmental and industrial applications of Yarrowia
lipolytica. Appl Microbiol Biotechnol 84:847–865
Bansal V, Rautaray D, Ahmad A, Sastry M (2004) Biosynthesis of zirconia nanoparticles using the
fungus Fusarium oxysporum. J Mater Chem 14:3303–3305
Bansal V, Rautaray D, Bharde A, Ahire K, Sanyal A, Ahmad A, Sastry M (2005) Fungus-mediated
biosynthesis of silica and titania particles. J Mater Chem 15:2583–2589
Banu AN, Balasubramanian C (2014) Optimization and synthesis of silver nanoparticles using
Isaria fumosorosea against human vector mosquitoes. Parasitol Res 113:3843–3851
Bharde A, Rautaray D, Bansal V, Ahmad A, Sarkar I, Yusuf SM, Sanyal M, Sastry M (2006)
Extracellular biosynthesis of magnetite using fungi. Small 2:135–141
Bhattacharya R, Mukherjee P (2008) Biological properties of “naked”metal nanoparticles. Adv
Drug Deliv Rev 60:1289–1306
Birla SS, Gaikwad SC, Gade AK, Rai MK (2013) Rapid synthesis of silver nanoparticles from
fusarium oxysporum by optimizing physicocultural conditions. SciWorld J 2013:796018
Chapman PM, Wang F (2001) Assessing sediment contamination in estuaries. Environ Toxicol
Chem 20:3–22
Colombo S, Toietta G, Zecca L, Vanoni M, Tortora P (1995) Molecular cloning, nucleotide
sequence and expression of a carboxypeptidase-encoding gene from the Archaebacterium
Sulfolobus solfataricus. J Bacteriol 177:5561–5565
Costa Silva LP, Oliveira JP, Keijok WJ, Silva AR, Aguiar AR, Guimarães MCC et al (2017)
Extracellular biosynthesis of silver nanoparticles using the cell-free filtrate of nematophagus
fungus Duddingtonia flagans. Int J Nanomedicine 12:6373–6381
Daniel MC, Astruc D (2004) Gold nanoparticles: assembly, supramolecular chemistry, quantum-
size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem
Rev 104:293–346
De Windt W, Boon N, Van den Bulcke J, Rubberecht L, Prata F, Mast J, Hennebel T, Verstraete W
(2006) Biological control of the size and reactivity of catalytic Pd(0) produced by Shewanella
oneidensis. Antonie Van Leeuwenhoek 90:377–389
Duhan JS, Kumar R, Kumar N, Kaur P, Nehra K, Duhan S (2017) Nanotechnology: the new
perspective in precision agriculture. Biotechnol Rep 15:11–23
Durán N, Marcato PD, De Souza G, Alves OL, Esposito E (2007) Antibacterial effect of silver
nanoparticles produced by fungal process on textile fabrics and their effluent treatment. J
Biomed Nanotechnol 3:203–208
Elamawi RM, Al-Harbi RE, Hendi AA (2018) Biosynthesis and characterization of silver
nanoparticles using Trichoderma longibrachiatum and their effect on phytopathogenic fungi.
Egypt J Biol Pest Control 28:28
Elgorban AM, Aref SM, Seham SM, Elhindi KM, Bahkali AH, Sayed SR et al (2016) Extracellular
synthesis of silver nanoparticles using Aspergillus versicolor and evaluation of their activity on
plant pathogenic fungi. Mycosphere 7:844–852
644 H. Shukla and S. Shukla

Engle M, Youhong L, Carl W, Juergen W (1995) Isolation and characterization of a novel alkali
tolerant thermophile, Anaerobranca horikoshii gen. nov., sp. nov. Int J Syst Bacteriol 45:454–
461
Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R (2010) Biogenic
synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against
gram-positive and gram-negative bacteria. Nanomed Nanotechnol Biol Med 6:103–109
Fendler JH (1998) Nanoparticles and nanostructured films: preparation, characterization and
applications. Wiley, New York, p 488
Fulias A, Vlase G, Grigorie C, Ledeţi I, Albu P, Bilanin M, Vlase T (2013) Thermal behaviour
studies of procaine and benzocaine. J Therm Anal Calorim 113(1):265–271
Gabani P, Singh OV (2013) Radiation-resistant extremophiles and their potential in biotechnology
and therapeutics. Appl Microbiol Biotechnol 97:993–1004
Gade AK, Bonde P, Ingle AP, Marcato PD, Durán N, Rai MK (2008) Exploitation of Aspergillus
niger for synthesis of silver nanoparticles. J Biobaased Mater Bioenergy 2:243–247
Greene AD, Patel BKC, Sheehy AJ (1997) Deferribacter thermophilus gen. nov., sp. nov., a novel
thermophilic manganese and iron-reducing bacterium isolated from a petroleum reservoir. Int J
Syst Bacteriol 47:505–509
Gudikandula K, Vadapally P, Charya MAS (2017) Biogenic synthesis of silver nanoparticles from
white rot fungi: their characterization and antibacterial studies. Open Nano 2:64–78
Gupta VK, Suhas (2009) Application of low-cost adsorbents for dye removal–a review. J Environ
Manag 90:2313–2342
Gurunathan S, Kalishwaralal K, Vaidyanathan R, Deepak V, Pandian SRK, Muniyand J et al (2009)
Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli.
Colloids Surf B Biointerfaces 74:328–335
Hamedi S, Ghaseminezhad M, Shokrollahzadeh S, Shojaosadati SA (2017) Controlled biosynthesis
of silver nanoparticles using nitrate reductase enzyme induction of filamentous fungus and their
antibacterial evaluation. Artif Cells Nanomed Biotechnol 45:1588–1596
Hussain I, Singh NB, Singh A, Singh H, Singh SC (2016) Green synthesis of nanoparticles and its
potential application. Biotechnol Lett 38:545–560
Husseiny SM, Salah TA, Anter HA (2015) Biosynthesis of size controlled silver nanoparticles by
Fusarium oxysporum, their antibacterial and antitumoral activities. Beni Suef Univ J Basic Appl
Sci 4:225–231
Jaidev LR, Narasimha G (2010) Fungal mediated biosynthesis of silver nanoparticles, characteriza-
tion and antimicrobial activity. Coll Surf B 81:430–433
Joerger TK, Joerger R, Olsson E, Granqvist CG (2001) Bacteria as workers in the living factory:
metal-accumulating bacteria and their potential for materials science. Trends Biotechnol 19:15–
20
Kathiresan K, Manivannan S, Nabeel MA, Dhivya B (2009) Studies on silver nanoparticles
synthesized by a marine fungus, Penicillium fellutanum isolated from coastal mangrove sedi-
ment. Colloids Surf B Biointerfaces 71:133–137
Kathiresan K, Alikunhi NM, Pathmanaban S, Nabikhan A, Kandasamy S (2010) Analysis of
antimicrobial silver nanoparticles synthesized by coastal strains of Escherichia coli and Asper-
gillus niger. Can J Microbiol 56:1050–1059
Kaviya S, Santhanalakshmi J, Viswanathan B (2011) Green synthesis of silver nanoparticles using
Polyalthia longifolia leaf extract along with D-sorbitol: study of antibacterial activity. J
Nanotechnol 2011:152970
Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, Kim SH, Park YK, Park YH, Hwang CY, Kim
YK, Lee YS, Jeong DH, Cho MH (2007) Antimicrobial effects of silver nanoparticles.
Nanomed Nanotechnol Biol Med 3:95–101
Klaus T, Joerger R, Olsson E, Granqvist CG (1999) Silver-based crystalline nanoparticles,
microbially fabricated. Proc Natl Acad Sci U S A 96:13611–13614
Klaus T, Joerger R, Olsson E, Granqvist CG (2009) Silver-based crystalline nanoparticles,
microbially fabricated. Proc Natl Acad Sci U S A 96:13611–13614
26 Extremophilic Fungi for the Synthesis of Nanomolecules 645

Koeller KM, Wong CH (2001) Enzymes for chemical synthesis. Nature 409:232–240
Korbekandi H, Ashari Z, Iravani S, Abbasi S (2013) Optimization of biological synthesis of silver
nanoparticles using Fusarium oxysporum. Iran J Pharm Res 12:289–298
Koshima SA (1984) A novel cold-tolerant insect found in a Himalayan glacier. Nature 310:225–227
Kumar S, Arya S, Nussinov R (2007) Temperature-dependent molecular adaptation features in
proteins. In: Gerday C, Glansdorff N (eds) Physiology and biochemistry of extremophiles. ASM
Press, Washington, DC, pp 75–85
Li Y, Duan X, Qian Y, Yang L, Liao H (1999) Nanocrystalline silver particles: synthesis,
agglomeration, and sputtering induced by electron beam. J Colloid Interface Sci 209:347–349
Liang M, Wei S, Jian-Xin L, Xiao-Xi Z, Zhi H, Wen L et al (2017) Optimization for extracellular
biosynthesis of silver nanoparticles by Penicillium aculeatum Su1 and their antimicrobial
activity and cytotoxic effect compared with silver ions. Mater Sci Eng C 77:963–971
Liu T, Liu J, Sonshine DA, Shervani S, Hurt RH (2010) Controlled release of biologically active
silver from nanosilver surfaces. ACS Nano 4:6903–6913
Liu J, Qiao SZ, Hu QH, Lu GQ (2011) Magnetic nanocomposites with mesoporous structures:
synthesis and applications. Small 7:425–443
Lu Z, Rong K, Li J, Yang H, Chen R (2013) Size-dependent antibacterial activities of silver
nanoparticles against oral anaerobic pathogenic bacteria. J Mater Sci 24:1465–1471. https://
doi.org/10.1007/s10856-013-4894-5
Mabbett AN, Yong P, Farr JP, Macaskie LE (2004) Reduction of Cr(VI) by “palladized”biomass of
Desulfovibrio desulfuricans ATCC 29577. Biotechnol Bioeng 87:104–109
MacElroy M (1974) Some comments on the evolution of extremophiles. Biosystems 6:74–75
Mallick K, Witcomb MJ, Scurell MS (2004) Polymer stabilized silver nanoparticles: a photochemi-
cal synthesis route. J Mater Sci 39:4459–4463
Manivannan S, Alikunhi NM, Kandasamy K (2010) In vitro synthesis of silver nanoparticles by
marine yeasts from coastal mangrove sediment. Adv Sci Lett 3:1–6
Mann S (2001) Biomineralization: principles and concepts in bioinorganic materials chemistry.
Oxford University Press, Oxford
Meenupriya J, Majumdar A, Thangaraj M (2011) Biogenic silver nanoparticles by Aspergillus
terreus MP1 and its promising antimicrobial activity. J Pharm Res 4:1648–1650
Mehta D, Satyanarayana T (2013) Diversity of hot environments and thermophilic microbes. In:
Satyanarayana T, Satyanarayana T, Littlechild J, Kawarabayasi Y (eds) Thermophilic microbes
in environmental and industrial biotechnology: biotechnology of thermophiles. Springer,
Dordrecht, pp 3–60
Mody VV, Siwale R, Singh A, Mody HR (2010) Introduction to metallic nanoparticles. J Pharm
Bioallied Sci 2:282–289
Mohanpuria P, Rana NK, Yadav SK (2008) Biosynthesis of nanoparticles: technological concepts
and future applications. J Nano Res 10:507–517
Morones JR, Elichiguerra JL, Camacho A, Holt K, Kouri JB, Ramírez JT et al (2005) The
bactericidal effect of silver nanoparticles. Nanotechnology 16:2346–2353
Mukherjee P, Ahmad A, Mandal D, Senapati S, Sainkar SR, Khan MI, Ramani R, Parischa R,
Ajayakumar PV, Alam M, Sastry M, Kumar R (2001) Bioreduction of AuCl4ions by the
fungus, Verticillium sp. and surface trapping of the gold nanoparticles formed. Angew Chem Int
Ed 40:3585–3588
Nayak R, Pradhan N, Behera D, Pradhan K, Mishra S, Sukla L, Mishra B (2011) Green synthesis of
silver nanoparticle by Penicillium purpurogenum NPMF: the process and optimization. J
Nanopart Res 13:3129–3137
Ngah WWS, Teong LC, Hanafiah MAKM (2011) Adsorption of dyes and heavy metal ions by
chitosan composites: a review. Carbohydr Polym 83:1446–1456
Niehaus F, Bertoldo C, Kähler M, Antranikian G (1999) Extremophiles as a source of novel
enzymes for industrial application. Appl Microbiol Biotechnol 51:711–772
Njagi HH, Stafford L, Genuino H, Galindo HM, Colins JB (2011) Biosynthesis of iron and silver
nanoparticles at room temperature using aqueous sorghum bran extracts. Langmuir 27:264–271
646 H. Shukla and S. Shukla

Oren A (2012) Industrial and environmental applications of halophilic microorganisms. Environ
Technol 31:825–834
Ottoni CA, Simões MF, Fernandes S, Santos JG, Silva ES, Souza RFB et al (2017) Screening of
filamentous fungi for antimicrobial silver nanoparticles synthesis. AMB Express 7:31
Owaid MN, Ibraheem IJ (2017) Mycosynthesis of nanoparticles using edible and medicinal
mushrooms. Eur J Nanomed 9(1):5–23
Pawar V, Shinde A, Kumar AR, Zinjarde S, Gosavi S (2012) Tropical marine microbe mediated
synthesis of cadmium nanostructures. Sci Adv Mater 4:135–142
Phanjom P, Ahmed G (2017) Effect of different physicochemical conditions on the synthesis of
silver nanoparticles using fungal cell filtrate of Aspergillus oryzae (MTCC no. 1846) and their
antibacterial effects. Adv Nat Sci Nanosci Nanotechnol 8:1–13
Pimprikar PS, Joshi SS, Kumar AR, Zinjarde SS, Kulkarni SK (2009) Influence of biomass and
gold salt concentration on nanoparticle synthesis by the tropical marine yeast Yarrowia
lipolytica NCIM 3589. Colloids Surf B Biointerfaces 74:309–316
Pomaranski E, Tiquia-Arashiro SM (2016) Butanol tolerance of carboxydotrophic bacteria: isolated
from manure composts. Environ Technol 37:1970–1982
Prasanti B, Anuj C, Singh K, Om V (2015) Extremophiles and their applications in medical
processes. Springer, New York, p 54
Qian Y, Yu H, He D, Yang H, Wang W, Wan X et al (2013) Biosynthesis of silver nanoparticles by
the endophytic fungus Epicoccum nigrum and their activity against pathogenic fungi.
Bioprocess Biosyst Eng 36:1613–1619
Rai M, Yadav A, Gade A (2008) Current trends in photosynthesis of metal nanoparticles. Crit Rev
Nanotechnol 28:277–284
Rai M, Yadav A, Gade A (2009) Silver nanoparticles as a new generation of antimicrobials.
Biotechnol Adv 27:76–83
Rai MK, Deshmukh SD, Ingle AP, Gade AK (2012) Silver nanoparticles: the powerful nanoweapon
against multidrug-resistant bacteria. J Appl Microbiol 112(5):841–852
Rajput S, Werezuk R, Lange RM, Mcdermott MT (2016) Fungal isolate optimized for biogenesis of
silver nanoparticles with enhanced colloidal stability. Langmuir 32:8688–8697
Rothschild LJ (2007) Defining the envelope for the search for life in the universe. In: Pudritz RE
(ed) Planetary systems and the origin of life. Cambridge University Press, New York, pp
697–698
Rothschild LJ, Mancinelli RL (2001) Life in extreme environments. Nature 409:1092–1101
Roucoux A, Schulz J, Patin H (2002) Reduced transition metal colloids: a novel family of reusable
catalysts? Chem Rev 102:3757–3778
Saha S, Sarkari J, Chattopadhyay D, Patra S, Chakraborty A, Acharya K (2010) Production of silver
nanoparticles by a phytopathogenic fungus Bipolaris nodulosa and its antimicrobial activity.
Digest J Nano Biostruct 5:887–895
Saravanan M, Nanda A (2010) Extracellular synthesis of silver bionanoparticles from Aspergillus
clavatus and its antimicrobial activity against MRSA and MRSE. Coll Surf B 77:214–218
Sastry M, Ahmad A, Khan I, Kumar R (2003) Biosynthesis of metal nanoparticles using fungi and
actinomycete. Curr Sci 85:162–170
Saxena J, Sharma PK, Sharma MM, Singh A (2016) Process optimization for green synthesis of
silver nanoparticles by Sclerotinia sclerotiorum MTCC 8785 and evaluation of its antibacterial
properties. Springerplus 5:861
Schmid G (1992) Large clusters and colloids. Metals in the embryonic state. Chem Rev 92:1709–
1727
Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolt M, Witholt B (2011) Industrial biocatalysis
today and tomorrow. Nature 409:258–267
Seelan JSS, Faisal AAK, Muid S (2009) Aspergillus species isolated from mangrove forests in
Borneo Island, Sarawak, Malaysia. J Threat Taxa 1:344–346
Seshadri S, Prakash A, Kowshik M (2012) Biosynthesis of silver nanoparticles by marine bacte-
rium, Idiomarina sp. p R58–8. Bull Mater Sci 35:1201–1205
26 Extremophilic Fungi for the Synthesis of Nanomolecules 647

Shah J, Brown RM Jr (2005) Towards electronic paper displays made from microbial cellulose.
Appl Microbiol Biotechnol 66:352–355
Shah M, Fawcett D, Sharma S, Tripathy SK, Poinern GEJ (2015) Green synthesis of metallic
nanoparticles via biological entities. Materials 8(11):7278–7308
Shahzad A, Saeed H, Iqtedar M, Hussain SZ, Kaleem A, Abdullah R (2019) Size-controlled
production of silver nanoparticles by Aspergillus fumigatus BTCB10: likely antibacterial and
cytotoxic effects. J Nanomater 2019:5168698
Shankar SS, Rai A, Ankamwar B, Singh A, Ahmad A, Sastry M (2004) Biological synthesis of
triangular gold nanoprisms. Nat Mater 3:482–488
Sintubin L, Windt WD, Dick J, Mast J, Ha DVD, Verstraete W et al (2009) Lactic acid bacteria as
reducing and capping agent for the fast and efficient production of silver nanoparticles. Appl
Microbiol Biotechnol 84:741–749
Srivastava S, Kardam A, Raj KR (2012) Nanotech reinforcement onto cellulose fibers: green
remediation of toxic metals. Int J Green Nanotechnol 4:46–53
Sudha SS, Rajamanickam K, Rengaramanujam J (2013) Microalgae mediated synthesis of silver
nanoparticles and their antibacterial activity against pathogenic bacteria. Indian J Exp Biol
51(5):393–399
Syed A, Saraswati S, Kundu GC, Ahmad A (2013) Biological synthesis of silver nanoparticles
using the fungus Humicola sp. and evaluation of their cytotoxicity using normal and cancer cell
lines. Spectrochim Acta Part A Mol Biomol Spectr 114:144–147
Tan Y, Dai Y, Li Y, Zhua D (2003) Preparation of gold, platinum, palladium and silver
nanoparticles by the reduction of their salts with a weak reductant–potassium bitartrate. J
Mater Chem 13:1069–1075
Thakkar MS, Mhatre SS, Rasesh Y, Parikh MS (2010) Biological synthesis of metallic
nanoparticles. Nanomed Nanotechnol Biol Med 6:257–262
Tiquia SM, Mormile MR (2010) Extremophiles–A source of innovation for industrial and environ-
mental applications. Editorial overview. Environ Technol 31:823
Tiquia-Arashiro SM (2014a) Thermophilic carboxydotrophs and their biotechnological
applications. In: Springer briefs in microbiology. Extremophilic microorganisms. Springer,
New York, p 131
Tiquia-Arashiro SM (2014b) Biotechnological applications of thermophilic carboxydotrophs. In:
Thermophilic carboxydotrophs and their applications in biotechnology, Chap. 4. Springer,
New York, pp 29–101
Tiquia-Arashiro SM, Mormile MR (2013) Sustainable technologies: bioenergy and biofuel from
biowaste and biomass. Editorial overview. Environ Technol 34:1637–1638
Tomaszewska E, Soliwoda K, Kadziola K, Celichowski G, Cichomski M, Szmaja W, Grobelny J
(2013) Detection limits of DLS and UV-Vis spectroscopy in characterization of polydisperse
nanoparticles colloids. J Nanomater 2013:313081
Uddin I, Adyanthaya S, Syed A, Selvaraj K, Ahmad A, Poddar P (2008) Structure and microbial
synthesis of sub-10 nm Bi2O3 nanocrystals. J Nanosci Nanotechnol 8:3909–3913
Vala AK, Chudasama B, Patel RJ (2012) Green synthesis of silver nanoparticles using marine-
derived fungus Aspergillus niger. Micro Nano Lett 7:859–862
Velhal SG, Kulkarni SD, Latpate RV (2016) Fungal mediated silver nanoparticle synthesis using
robust experimental design and its application in cotton fabric. Int Nano Lett 6(4):257–264
Verma VC, Kharwar RN, Gange AC (2010) Biosynthesis of antimicrobial silver nanoparticles by
the endophytic fungus Aspergillus clavatus. Nanomedicine 5:33–40
Vigneshwaran N, Kathe AA, Varadarajan PV, Nachane RP, Balasubramanya RH (2006) Biomi-
metics of silver nanoparticles by white rot fungus, Phaenerochaete chrysosporium. Colloids
Surf B Biointerfaces 53:55–59
Von Klein D, Arab H, Völker H, Thomm M (2002) Methanosarcina baltica, sp. nov., a novel
methanogen isolated from the Gotland deep of the Baltic Sea. Extremophiles 6:103–110
648 H. Shukla and S. Shukla

Wang ZL, Liu Y, Zhang Z (eds) (2002) Handbook of nanophase and nanostructured materials,
vol. I: synthesis, vol. II: characterization, vol. III: materials systems and applications I, vol IV:
materials systems and applications II. Springer, New York
Ward DM, Tayne TA, Andersonf KL, Bateson MM (1987) Community structure and interactions
among members of hot spring cyanobacterial mats. Symp Soc Gen Microbiol 41:179–210
Weinstein RN, Palm ME, Johnstone K, Wynn-Wiliiams DD (1997) Ecological and physiological
characterization of Humicola marvinii, a new psychrophilic fungus from fell field soils in the
maritime Antarctic. Mycologia 89:706–711
Wiegel J (2011) Anaerobic alkaliphiles and alkaliphilic poly-extremophiles. In: Horikoshi K
(ed) Extremophiles handbook. Springer, Tokyo, pp 81–97
Wong TS, Schwaneberg U (2003) Protein engineering in bioelectrocatalysis. Curr Opin Biotechnol
14:590–596
Xue B, He D, Gao S, Wang D, Yokoyama K, Wang L (2016) Biosynthesis of silver nanoparticles
by the fungus Arthroderma fulvum and its antifungal activity against genera of Candida,
Aspergillus and Fusarium. Int J Nanomedicine 11:1899–1906
Yadav A, Kon K, Kratosova G, Duran N, Ingle AP, Rai M (2015) Fungi as an efficient mycosystem
for the synthesis of metal nanoparticles: progress and key aspects of research. Biotechnol Lett
37(11):2099–2120
Yang R, Aubrecht KB, Ma HY, Wang R, Grubbs RB, Hsiao BS, Chu B (2014) Thiol-modified
cellulose nanofibrous composite membrane for chromium (vi) and lead (ii) adsorption. Polymer
55:1167–1176
Yiu HHP, Keane MA (2012) Enzyme–magnetic nanoparticle hybrids: new effective catalysts for
the production of high value chemicals. J Chem Technol Biotechnol 87:583–594
Zhang X, Yan S, Tyagi RD, Surampalli RY (2011a) Synthesis of nanoparticles by microorganisms
and their application in enhancing microbiological reaction rates. Chemosphere 82:489–494
Zhang X, He X, Wang K, Yang X (2011b) Different active biomolecules involved in biosynthesis
of gold nanoparticles by three fungus species. J Biomed Nanotechnol 7:245–254
Zhao X, Zhou L, Riaz Rajoka MS, Yan L, Jiang C, Shao D, Zhu J, Shi J, Huang Q, Yang H, Jin M
(2018) Fungal silver nanoparticles: synthesis, application and challenges. Crit Rev Biotechnol
38(6):817–835
Zillig W, Stetter KO, Wunderl S, Schulz W, Priess H, Scholz I (1980) The Sulfolobus- “Caldariella”
group: taxonomy on the basis of the structure of DNA-dependent RNA polymerases. Arch
Microbiol 125:259–269
26 Extremophilic Fungi for the Synthesis of Nanomolecules 649

Fungal Extremozymes: A Potential
Bioresource for Green Chemistry 27
Imran Mohsin and Anastassios C. Papageorgiou
Abstract
Green chemistry is a rapidly evolving area that aims to reduce the hazards
released to the environment during various chemical processes and improve the
efficiency of bioconversion. Application of green chemistry could therefore lead
to less environmental pollution and better economic outcomes. Enzymes, as
biocatalysts, are expected to play a central role in green chemistry owing to
their reusability, catalytic efficiency, and specificity. Besides, biocatalytic
reactions result in no toxic waste in contrast to chemical processes that require
careful disposal. However, the use of enzymes in chemical reactions presents
various challenges, including stability and unwanted side-reactions. Fungi have
drawn significant attention in recent years as a new source of enzymes that could
be used in harsh conditions to improve various industrial processes, such as
biofuel production and biomass conversion. Combined with modern bioengineer-
ing techniques, fungal extremozymes have emerged as promising tools in future
applications. Also, structural information has provided new insights into the
function and stability of various fungal extremozymes. This review is focused
on latest progress in fungal extremozymes, in particular their structural features as
well as the current research efforts to improve their properties for better use in
green chemistry applications.
Keywords
Fungus · Biocatalysis · Crystal structure · Biomass degradation · Enzyme
improvement · Enzyme stability
I. Mohsin · A. C. Papageorgiou (*)
Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland
e-mail: anapap@utu.fi
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_27
651

27.1 Introduction
Green chemistry has become a worldwide approach toward sustainable growth. New
processes using materials which do not emit pollutants and toxic waste to the
environment are being pursued. The development, however, of new safer methods
that produce less harmful products is not an easy task and presents many challenges.
Enzymes have become a great tool for scientists in the field of green chemistry
(Table 27.1) (Anna Calarco 2015). As enzymes act as biocatalysts, they require mild
conditions to function; thus, they can save key resources such as energy or water
(Sheldon and Woodley 2018). Also, enzymes are attractive alternatives owing to
their minimal impact on the environment and low costs. Taking into account the
benefits of green chemistry, enzyme biocatalysis is now found in various traditional
chemical processes in several fields (Cipolatti et al. 2019). This change is going to
expand to even more areas owing to new emerging technologies in enzyme
engineering.
Fungi have received significant attention in recent years (de Cassia Pereira et al.
2015; Hyde et al. 2019), especially as a reservoir of extremozymes for use in many
biotechnological applications (Table 27.2). Thermophilic fungi, in particular, have
drawn considerable interest owing to their ability to grow at a temperature of 50 C
or above and at a minimum of 20 C or above (Maheshwari et al. 2000). Various
thermophilic fungi have been isolated in recent years and the enzymes they produce
have been investigated at functional and structural level. In this review, an up-to-date
information on structure–function aspects of fungal extremozymes is presented and
the current research efforts to improve their properties for better use in green
chemistry applications are discussed.
27.2 Cellulases
The enzymatic hydrolysis of cellulose to its constituent monosaccharides for the
low-cost production of food and biofuels has attracted considerable attention in
recent years. Cellulose is the most abundant and renewable non-fossil carbon source
on Earth and accounts for 20–50% in dry weight of the plant cell wall material.
Compared to current industrial procedures such as heat, mechanical, and acid
treatment of cellulose, cellulose degradation by enzymes is considered a more
environment-friendly process (Wilson 2009). However, cellulose is the most refrac-
tory carbohydrate polymer to enzymatic degradation among all polysaccharides of
the plant cell wall. Thus, efficient enzymatic degradation of cellulose to glucose
requires the synergistic action of endocellulases (E.C.3.1.1.4), exocellulases
(cellobiohydrolases, CBH, E.C.3.2.1.91), and β-glucosidases (E.C.3.2.1.21).
Endocellulases initiate hydrolysis by cutting internal glycosidic linkages in a random
fashion, resulting in a swift decrease of polymer length and a gradual increase in the
reducing sugar concentration. Exocellulases act upon either the reducing or the
non-reducing ends to release cello-oligosaccharides and cellobiose units. In the
652 I. Mohsin and A. C. Papageorgiou

Table 27.1 Enzymes with potential applications in green chemistry
EC
number Enzyme class Catalyzed reactions Representative enzymes
Polymer
synthesis
Polymer
modification
1 Oxidoreductases Redox reactions by electron transfer Peroxidase, laccase, tyrosinase, glucose
oxidase
Yes Yes
2 Transferases Transfer of a functional group from one
compound (donor) to another compound
(acceptor)
Phosphorylase, glycosyltransferase,
acyltransferase
Yes Yes
3 Hydrolases Hydrolysis of various bonds Glycosidase (cellulase, amylase, xylanase,
chitinase, hyaluronidase), lipase, protease,
peptidase, feruloyl esterase, nitrilase
Yes Yes
4 Lyases Cleavage of C–C, C–O, C–N, and other bonds
by using other than hydrolysis or oxidation
Decarboxylase, aldolase, dehydratase No No
5 Isomerases Either racemization or epimerization of chiral
centers; isomerases are subdivided according
to their substrate specificity
Racemase, epimerase, isomerase No Yes
6 Ligases Linking of two molecules with concurrent
hydrolysis of the diphosphate-bond in ATP or
a similar triphosphate
Ligase, synthase, acyl CoA synthase No No
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 653

last step, β-glucosidases cleave cellobiose to release glucose molecules (Vlasenko
et al. 2010).
Owing to the difficulties for its breakdown, cellulose is subjected to higher
temperatures to swell and become more susceptible to breaking. Besides, the use
of higher temperatures in industrial processes offers additional advantages such as
substrate and product solubility, reduced hydrolysis time, and minimum risk of
microbial contamination. Enzymatic hydrolysis by thermophilic cellulases has there-
fore become a key step for efficient biomass degradation (Atalah et al. 2019).
Cellulases are classified into 13 glycoside hydrolase (GH) families (http://www.
cazy.org): 1, 3, 5, 6, 7, 8, 9, 12, 26, 44, 45, 48, and 61. Thermophilic fungal
cellulases are found in families 1, 3, 5, 6, 7, 12, 45, and 61 (Li and Papageorgiou
2019). GH61 family members are now identified as Cu(II) ion-dependent lytic
polysaccharide monooxygenases (LPMOs) and have been included in the auxiliary
activity families of the CAZy database (Busk and Lange 2015).
Table 27.2 Fungal enzymes with industrial importance
Enzyme
Preferred
Properties Representative fungi Applications
Cellulases Thermostable Melanocarpus albomyces,
Chaetomium
thermophilum, Humicola
insolens
Biofuel, biorefinery, paper
and pulp industry
Xylanases Alkaliphilic,
thermophilic
Humicola insolens,
Melanocarpus albomyces,
Sporotrichum thermophile
Fuels, chemicals, paper,
brewing industry
Feruloyl
esterases
Thermophilic Talaromyces
cellulolyticus,
Sporotrichum thermophile
Food, cosmetics, chemical
synthesis
Laccases Thermophilic,
alkaliphilic
Sporotrichum
thermophile,
M. albomyces,
C. thermophilum,
T. versicolor
Bioenergy, pulp and denim
treatment, food and
beverages, wastewater,
bioenergy
Lipases Thermotolerant Candida sp., Penicillium
sp., Aspergillus sp.
Detergents, therapeutics,
food supplements
Nitrilases Thermostable Ascomycota Organic synthesis,
bioremediation
Transaminases Thermostable Thermomyces stellatus Organic synthesis
Tyrosinases Thermostable Agaricus bisporus,
Neurospora crassa,
Aspergillus oryzae,
Aspergillus niger
Organic synthesis,
bioremediation
Keratinases Thermotolerant,
alkaliphilic
Aspergillus fumigatus,
Myceliophthora
thermophila
Animal feed, medicine,
detergents
654 I. Mohsin and A. C. Papageorgiou

27.2.1 Cellulase Production and Characterization
Heterologous host organisms, such as E.coli, yeast, and filamentous fungi have been
used for the expression of cloned cellulase genes of thermophilic fungi (Li and
Papageorgiou 2019). The majority of the recombinant cellulases expressed in yeast
and filamentous fungi are glycosylated (Li et al. 2009; Takashima et al. 1999).
Glycosylation could be a contributing factor to the thermostability improvement of
cellulases according to previous reports (Meldgaard and Svendsen 1994). The
mechanism, however, is still unknown. It has been reported that N-glycosylation
could increase solubility and reduce aggregation (Ioannou et al. 1998; Kayser et al.
2011). Also, analysis of protein structures deposited in the Protein Data Bank has
also indicated a decrease in protein dynamics upon N-glycosylation without signifi-
cant global or local structural changes (Lee et al. 2015).
Thermophilic fungal cellulases are usually single polypeptides although some
β-glucosidases exist as dimers (Gudmundsson et al. 2016; Mamma et al. 2004). The
molecular weight of thermophilic fungal cellulases has a wide range (30–250 kDa)
with different carbohydrate contents (2–50%). The majority of the purified cellulases
from thermophilic fungi exhibit similar optimal pH and temperature. Indeed, ther-
mophilic fungal cellulases are found active in the pH range 4.0–7.0 and display a
high-temperature maximum at 50–80 C for activity (Li and Papageorgiou 2019).
Also, they exhibit remarkable thermal stability and are stable at 60 C with longer
half-life at 70, 80, and 90 C than those from mesophilic fungi.
27.2.2 Primary and Three-Dimensional (3D) Structure
27.2.2.1 Primary Structure
Most cellulases are characterized by a modular structure. Typically, endocellulases
and cellobiohydrolases consist of four modules: a signal peptide that facilitates
secretion, a cellulose-binding domain (CBD) for the enzyme’s attachment to the
substrate, a catalytic domain (CD) used for the hydrolysis of the substrate, and a
hinge region (linker) which is usually post-translationally glycosylated and rich in
Ser, Thr, and Pro residues.
CBDs consist of less than 40 amino acids and interact with cellulose through a flat
platform-like hydrophobic binding site that is thought to be complementary to the
flat surfaces presented by cellulose crystals (Shoseyov et al. 2006). Studies have
shown that deletion of the CBDs present in T. reesei Cel7A and Cel6A and
Humicola grisea CBH1 reduces significantly enzymatic activity toward crystalline
cellulose (Takashima et al. 1998), suggesting that the efficient hydrolysis of crystal-
line cellulose requires tight interactions to cellulose through the CBDs. Aromatic
residues in CBDs have been suggested to affect the cellulose-binding ability and
enzymatic activity (Takashima et al. 2007).
Variations in the primary structure have been identified. Talaromyces emersonii
CBHII, for instance, has a modular structure (Murray et al. 2003), whereas
T. emersonii CBH1 consists solely of a catalytic domain (Grassick et al. 2004).
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 655

Similar variations are found in Chaetomium thermophilum CBHs (CBH1, CBH2,
and CBH3) where CBH1 and CBH2 consist of a typical CBD, a linker, and a CD,
whereas CBH3 only comprises a catalytic domain and lacks a CBD and a hinge
region (Li et al. 2009). However, cellulases without CBDs can still be efficiently
used (Le Costaouëc et al. 2013; Pakarinen et al. 2014).
27.2.2.2 Three-Dimensional (3D) Structural Details
3D structures of thermophilic fungal cellulases from families 1, 5, 6, 7, 12, and
45 have been reported. Details have been recently reviewed (Li and Papageorgiou
2019).
27.2.3 Improvement of Thermophilic Fungal Cellulases
Two main research approaches are presently in use for improvement and modifica-
tion of enzyme function: structure-based rational site-directed mutagenesis and
random mutagenesis through directed evolution. Detailed knowledge of the 3D
structure of a protein is required for site-directed mutagenesis. In contrast, the
directed evolution approach is not limited by the absence of structural details but
requires an efficient method for high-throughput screening (Labrou 2010).
27.2.3.1 Thermostability Improvement
Generally, the mechanism of protein thermostability has been studied more exten-
sively in thermophilic bacteria and hyperthermophilic archaea (Pack and Yoo 2004;
Trivedi et al. 2006). However, a common thermostability mechanism has not yet
been established. Several contributing factors to protein thermostability have been
proposed. An increase in ion pairs on the protein surface and a more hydrophobic
interior have been put forward as the major factors of improved protein thermosta-
bility (Taylor and Vaisman 2010). Nevertheless, compared with thermophilic
proteins from thermophilic bacteria and hyperthermophilic archaea, the understand-
ing of the nature and mechanism of thermostability in proteins from thermophilic
fungi is relatively poor and additional studies are needed.
Despite that cellulases from thermophilic fungi are already thermostable, addi-
tional increase of their thermostability is desirable for industrial applications. Use of
error-prone PCR, for example, in Melanocarpus albomyces Cel7B resulted in the
improvement of the enzyme and the identification of two positive thermostable
mutants (Voutilainen et al. 2007). Also, introduction of extra disulfide bridges to
the catalytic module of Talaromyces emersonii Cel7A gave rise to three mutants
with improved thermostability as revealed by Avicel hydrolysis efficiency at 75 C
(Voutilainen et al. 2010).
Mutants of three Cys residues of the thermostable Humicola grisea Cel12A were
found to affect the stability of the enzyme (Sandgren et al. 2005). A report of fold-
specific thermostability through variations in amino acid compositions of
endoglucanases has provided new strategies for thermostability improvement
(Yennamalli et al. 2011).
656 I. Mohsin and A. C. Papageorgiou

Random mutagenesis and recombination of beneficial mutations were employed
for the construction of a chimeric Cel6A cellobiohydrolase (Wu and Arnold 2013).
Consequently, increased hydrophobic interactions and reduced loop flexibility by
introduction of Pro residues were found to improve thermostability.
A computational approach, namely SCHEMA, which uses protein structure data
to generate new sequences with minimal structure disruption when they are
introduced in chimeric proteins has been employed to create thermostable fungal
cellulases (Heinzelman et al. 2009). Application of SCHEMA using the high-
resolution structure of Humicola insolens CBHII as a template resulted in a collec-
tion of CBHII chimeras with high thermostability (Varrot et al. 2003).
Improvement of cellulase stability in detergent solutions has also been pursued
(Otzen et al. 1999). The anionic surfactant C12-LAS can inactivate H. insolens
Cel45 endoglucanase because of the positive charges at the surface of the enzyme
(Otzen et al. 1999). Mutation of surface residues R158E/R196E was found to
improve stability, most likely by preventing C12-LAS from binding to the protein.
27.2.3.2 Activity Improvement
Improvement of cellulase activity has been pursued in recent years by using site-
directed mutagenesis and directed evolution. However, the lack of general rules for
site-directed mutagenesis and the limitations of screening methods have resulted in
only a few successful examples of cellulase mutants with considerably higher
activity than the wild-type enzymes (Percival Zhang et al. 2006). Directed evolution
of Chaetomium thermophilum CBHII produced mutants that were able to retain
more than 50% of their activity at 80 C for 1 h, while the wild type abolished all of
its activity under the same conditions (Wang et al. 2012).
Addition or replacement of a CBD to alter the enzyme characteristics and to
improve hydrolytic activity has also been tried (Limon et al. 2001; Shoseyov et al.
2006; Szijarto et al. 2008; Takashima et al. 1999).
27.2.4 Glycosidic Bond Synthesis
Glycosynthases are engineered enzymes able to catalyze the synthesis of glycosidic
bonds (Hayes and Pietruszka 2017). They are best described as retaining GH
mutants in which the catalytic nucleophile (Asp, Glu) has been replaced by a
non-nucleophilic residue, usually a smaller uncharged amino acid. Cellulase engi-
neering to produce glycosynthases by site-directed mutagenesis has been actively
pursued (Shaikh and Withers 2008). The first glycosynthase from thermophilic
fungus was derived from Humicola insolens Cel7B following mutation of catalytic
residue Glu197 to Ala. The resultant Cel7B E197A glycosynthase was able to
catalyze regio- and stereo-selective glycosylation in high yield (Fort et al. 2000).
Three mutants of the H.insolens Cel7B E197A glycosynthase, namely E197A/
H209A and E197A/H209G double mutants, and Cel7B E197A/H209A/A211T
triple mutant, were subsequently produced and characterized (Blanchard et al.
2007). The results suggested that appropriate active site mutations could modulate
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 657

the regioselectivity of the glycosylation reaction. Apart from glycosynthases, use of
β-glucosidases for the synthesis of various glycoconjugates, such as alkyl glucosides
and aminoglycosides, has also been pursued in recent years. A GH3 β-glucosidase
from the thermophilic fungus Myceliophthora thermophila was found to act as an
efficient biocatalyst in alkyl glycoside synthesis (Karnaouri et al. 2013).
27.3 Xylanases
Xylanases (EC 3.2.1.8; endo-β-1,4-xylanases) are the main enzymes that hydrolyze
internal bonds in xylan, a particularly resistant to degradation component of plant
cell walls and the second most abundant polysaccharide in nature after cellulose
(Chadha et al. 2019). Apart from their use in the conversion of lignocellulose
biomass into fermentable sugars, xylanases have also been successfully employed
in the saccharification of agrowaste, such as wheat straw, corn cobs, birch, and
spruce biomass.
Xylanases are found in GH families 3, 5, 8, 9, 10, 11, 12, 16, 26, 30, 43, 44,
51, 62, 98, and 141. Their majority, however, belongs to the GH10 and GH11
families based on sequence considerations. GH11 xylanases are more specific than
GH10 xylanases as the former do not act on substituted forms of xylans.
Sporotrichum thermophile (syn Myceliophthora thermophila), a major thermo-
philic fungus isolated from soil in eastern Russia, is a powerful cellulolytic organism
that produces a variety of enzymes with immense industrial potential (Karnaouri
et al. 2014). S.thermophile synthesizes a complete set of enzymes, including GH10
and GH11 xylanases (Bala and Singh 2016). Characterization of S. thermophile
GH10 and GH11 xylanases has been reported (Basit et al. 2018).
A xylanase from the thermophilic and thermotolerant fungus Myceliophthora
heterothallica F.2.1.4. has been purified and characterized (de Oliveira Simões et al.
2019). The enzyme has 27 kDa MW and displays maximum activity at pH 4.5 and
65–70 C. It maintains more than 80% of its residual activity when exposed to
(1) temperatures between 30 and 60 C for 1 h and (2) pH 5–10 for 24 h at 4 and
25 C.
A GH10 xylanase PspXyn10 produced by the mesophilic fungus Penicillium sp.
has been characterized and found to exhibit thermostability (Shibata et al. 2017). The
molecular weight of PspXyn10 was estimated to be 55 kDa and found to contain a
CBM. Its optimal temperature and pH for xylanase activity were 75 C and pH 4.5,
respectively. PspXyn10 retained more than 80% of its xylanase activity after incu-
bation at 65 C for 10 min.
A purified xylanase produced by T. aurantiacus M-2 was found to be acidophilic
and thermostable (Ping et al. 2018). Its relative molecular mass was approximately
31.0 kDa. The purified xylanase exhibited maximum activity at 75 C and pH 5.0,
and it was stable after treatment at a pH range from 2.0 to 10.0 or a temperature range
from 30 to 80 C for 2 h. A GH10 xylanase from Aspergillus fumigatus var. niveus
(AFUMN-GH10) contains no carbohydrate-binding module. The enzyme was able
658 I. Mohsin and A. C. Papageorgiou

to retain its activity in a pH range from 4.5 to 7.0, with an optimal temperature at
60 C (Velasco et al. 2019).
27.3.1 Structural Details
Homology modeling studies of GH10 and GH11 xylanases from S. thermophile
suggested structural similarities and only minor differences with other fungal
xylanases (Basit et al. 2018). The crystal structure of a GH10 xylanase from the
fungus Fusarium oxysporum (PDB id 3u7b) has been reported (Dimarogona et al.
2012). The structure is similar to that of other GH10 xylanases and is characterized
by a (β/α)
8
-barrel fold. Differences have been identified in the loop regions.
Sequence alignment and homology modeling suggested the presence of a long
loop between strand β6b and helix α6 that may play a role in the catalytic efficiency
of the enzyme.
The GH11 xylanases exhibit a β-jelly-roll fold. The structure of xylanase
XynCDBFV, a GH11 xylanase from ruminal fungus Neocallimastix patriciarum
has been determined and the catalytic residues Glu109 and Glu202 were identified
(Cheng et al. 2014). The structure of a GH11 xylanases from Fusarium oxysporum
has been elucidated to 1.56-Å resolution and deposited to the PDB (pdb ID 5jrm;
Gomez S, Payne AM, Savko M, Fox GC, Shepard WE, Fernandez FJ, Vega MC,
unpublished). The structure of an acidophilic GH11 xylanase (XynC) from Asper-
gillus kawachii,afilamentous fungus used for brewing the Japanese distilled spirit
shochu, has been determined to 2.0 Å resolution (Fig. 27.1). The structure is
characterized by a negatively charged surface, which was postulated to be responsi-
ble for the stability of the enzyme in acidic environments (Fushinobu et al. 1998).
Fig. 27.1 Crystal structure of
Aspergillus kawachii XynC
(PDB id 1bk1). The catalytic
residues Glu79 (nucleophile),
Glu170 (acid-base catalyst),
and Asp37 are shown as sticks
and labeled. N- and C-termini
are depicted. Figures of
crystal structures were created
with Chimera (Pettersen et al.
2004)
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 659

27.3.2 Xylanase Improvement
The GH10 xylanase Xyn10A_ASPNG from Aspergillus niger (Song et al. 2015)
was subjected to iterative saturation mutagenesis (ISM). After four rounds of ISM, a
quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) was generated with improved
thermostability compared to the wild type. The 4S1 mutant retained 30% of its initial
activity after 15 min heating at 65 C and its melting temperature T
m
increased by
17.4 C compared to the wild type. For comparison, the wild-type enzyme retained
0.2% of its initial activity after heat treatment for 10 min at 60 C and was
completely inactivated after 2 min at 65 C. Although each of the five mutations
in 4S1 was found to contribute to thermoresistance, their synergistic action was
suggested to be responsible for the dramatic improvement of the 4S1 enzyme
thermotolerance.
An N-terminal region (NTR) has been identified in XynCDBFV from the ruminal
fungus Neocallimastix patriciarum (Cheng et al. 2014). In the truncated mutant, it
was clearly shown that the NTR plays a role in the catalytic activity of XynCDBFV
and is required for the thermophilic functions of the enzyme. Removal of NTR
resulted in a truncated mutant that retained 61.5% and 19.5% enzymatic activity at
55 and 75 C, respectively, compared with the wild-type enzyme. Elimination of a
disulfide bond in the C4A/C172A mutant resulted in 23.3% activity. These results
suggested that NTR plays a role in XynCDBFV thermostability, and the Cys-4/Cys-
172 disulfide bond is critical to the NTR-mediated interactions. Further
modifications included four single mutants by substituting residues from 87 to
90 by site-directed mutagenesis (Han et al. 2019). Temperature stability
measurements showed promising enhancement of thermostability for all four single
mutants. The mutants retained 50% of their activities after incubation at the optimal
temperature 60 C for 1 h, while the retained activity for wild-type XynCDBFV was
only 20.94% at the same condition. The increase in thermostability was attributed to
a novel hydrogen bonding interaction. However, the enzyme activity of the single
mutants was compromised with their thermostability. Combined mutations
displayed an antagonistic effect due to the closed contact of the mutated residues.
A novel GH11 endoxylanase (Liu et al. 2019) was constructed via DNA shuffling
by using the catalytic domains of two xylanases as parent sequences: Bacillus
amyloliquefaciens xylanase A (BaxA), which is mesophilic and xylanase A
(TfxA) from Thermomonospora fusca, a thermophilic soil bacterium. Notably,
TfxA, one of the most thermostable xylanases, was able to retain 96% of its catalytic
activity after incubation at 75 C for 18 h.
A thermostable GH11 xylanase TlXynA from Thermomyces lanuginosus was
mutated to improve its pH-tolerance using a rational structure-based approach
(Wu et al. 2020).
A thermostable Xyn10A of A. fumigatus Z5, which belongs to GH10 family and
has a CBM1 domain linked to its C-terminus by a Ser/Thr-rich linker was studied
(Miao et al. 2018). Removal of the CBM1 domain had little effect on the thermosta-
bility but further truncation of the linker region significantly decreased its stability at
high temperatures.
660 I. Mohsin and A. C. Papageorgiou

Protein engineering of a GH11 xylanase from Aspergillus fumigatus RT-1 was
performed near the active site and at the N-terminal region to improve the catalytic
efficiency of the enzyme toward pretreated kenaf (Damis et al. 2019). A 13.9-fold
increase in catalytic efficiency for a double mutant showed the most effective
hydrolysis reaction. The enhanced catalytic efficiency resulted in an increase in
sugar yield of up to 28% from pretreated kenaf. In addition, another mutant showed
improved thermostability and acid stability. Notably, these mutations were located at
distances less than 15 Å from the active site and at putative secondary binding sites
away from the active site.
Lytic polysaccharide monooxygenases (LPMOs) are capable of breaking down
xylans. Αxylan-active LPMO from Pycnoporus coccineus PcAA14B LPMO and a
GH30_7 family xylanase (TtXyn30A) from Thermothelomyces thermophila were
found to act synergistically with a family GH11 endoxylanase (AnXyn11) in the
degradation of xylan-containing substrates, resulting in an increase of the released
total oligosaccharides (Zerva et al. 2020).
The replacement of an N-terminal segment in AoXyn11, a mesophilic family
11 xylanase from Aspergillus oryzae, by the corresponding N-terminal of
EvXyn11
TS
, a hyperthermotolerant family 11 xylanase, led to a hybrid xylanase
with improved thermostability (Yin et al. 2013). The new xylanase, NhXyn11
57
, was
overexpressed in Pichia pastoris and its temperature optimum was 75 C, much
higher than that of AoXyn11. AoXyn11 and NhXyn11
57
were thermostable at
40 and 65 C, respectively. A poly-threonine helix from the thermostable GH10
family xylanase XynAF0 from the thermophilic composting fungus Aspergillus
fumigatus Z5 was introduced to the C-terminal of another GH10 xylanase, improv-
ing its thermostability (Li et al. 2019). Thus, the creation of hybrid xylanases can be
another strategy for thermostability modifications in this family of enzymes.
Aspergillus kawachii produces, apart from XynC, a second GH11 xylanase,
namely XynB, which is neutrophilic. Mutants to adjust the pH optimum of xylanases
have been studied. In acidophilic GH11 xylanases, the residue adjacent to the acid/
base catalyst is Asp, whereas in neutrophilic and alkaliphilic GH11 xylanases is Asn.
A D37N mutation in Aspergillus kawachii XynC GH11 xylanase (Fig. 27.1) raised
the pH optimum of XynC from 2.8 to 5.5, whereas an N43D mutation in A. kawachii
XynB GH11 lowered the pH optimum of XynB from 4.2 to 3.6 (Fushinobu et al.
2011).
27.4 Feruloyl Esterases
Feruloyl esterases (FAEs; EC 3.1.2.72) are enzymes that catalyze the hydrolysis of
ester bonds between ferulic (hydroxycinnamic) acid (FA) and plant cell wall
polysaccharides (Dilokpimol et al. 2016). They act as accessory enzymes of plant
biomass degradation to facilitate the access of other enzymes, such as xylanases,
xylosidases, and arabinofuranosidases, to sites of action during biomass conversion.
FAEs act synergistically with xylanases to release FA from cell wall material. Based
on their catalytic properties, FAEs have also attracted a great deal of attention in
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 661

recent years for use in the food, pharmaceutical, and cosmetics industries as syn-
thetic tools of novel hydroxycinnamates with enhanced antioxidant activity and
custom-made lipophilicity (Faulds 2010; Koseki et al. 2009). Recent amino acid
based sequence analysis has classified fungal FAEs in 13 subfamilies (SFs)
(Dilokpimol et al. 2016) as a replacement of the old classification system of
ABCD, which was based on substrate specificity (Crepin et al. 2004). Based on
the analysis of 1000 fungal FAEs, SF5 and SF7 FAEs were found suitable for
biorefinery applications, such as the production of biofuels, where a complete
degradation of the plant cell wall is desired (Underlin et al. 2020). In contrast, SF6
FAEs are promising enzymes for industrial applications that require a high release
of only FA and p-coumaric acid, which are needed as precursors for the production
of biochemicals. Finally, FAEs of SF1, 9, and 13 display an overall low release of
hydroxycinnamates from plant cell wall-derived and natural substrates. A feruloyl
esterase from Aspergillus terreus (Mäkelä et al. 2018) was used to expand the
carbohydrate esterase 1 (CE1) family of the CAZy database. Phylogenetic analysis
showed that the CE1 family can be subdivided into five groups to include more
members of fungal FAEs.
An FAE from the thermophilic fungus Sporotrichum thermophile has shown
stability at the pH range 5.0–7.0 and retained 70% of its activity after 7 h at 50 C
while it lost 50% of its activity after 45 min at 55 C and after 12 min at 60 C
(Topakas et al. 2004). Characterization of an FAE (TcFaeB) from Talaromyces
cellulolyticus, a high cellulolytic-enzyme producing fungus, has been reported
(Watanabe et al. 2015). Thermal stability measurement using differential scanning
calorimetry showed that TcFaeB has a Tm value of 70 C and optimum temperature
of the enzyme was estimated to be 65 C at pH 4.5–6.5, suggesting that this enzyme
may be applicable for biomass saccharification processes.
27.4.1 Structural Features of FAEs
FAEs exhibit an α/βhydrolase fold with a Ser-His-Asp catalytic triad (Hermoso et al.
2004). The FA has been found to bind to a shallow surface pocket able to accom-
modate the methoxy and hydroxyl moieties of the substrate owing to the presence of
hydrophobic and hydrogen bonding specificity determinants (Prates et al. 2001).
Although fungal FAEs show structural similarity to lipases, their catalytic sites are
different with more hydrophobic residues present in the active site of lipases
(McAuley et al. 2004). Fungal and bacterial FAEs show different topology of
secondary structure elements, leading to suggestion that bacterial FAEs diverged
earlier than the fungal FAEs and the lipases from a common ancestor.
The crystal structure of an FAE from Fusarium oxysporum has been reported
(Dimarogona et al. 2020). Similar to other FAEs, the structure revealed a large lid
domain covering the active site with a potential regulatory role (Fig. 27.2). This lid
domain, however, is absent in A. niger FAE (McAuley et al. 2004) and more fungal
structures should be determined to understand its exact role. A disulfide bond brings
together the serine and histidine residues of the catalytic triad. Several differences
662 I. Mohsin and A. C. Papageorgiou

were identified, mainly in the metal coordination site and the substrate binding
pocket.
27.4.2 Improvement Efforts of FAEs
Directed evolution studies have been reported in the filamentous fungus Aspergillus
niger FAE (AnFae) (Zhang et al. 2012). The resultant mutant exhibited 80% residual
activity after heat treatment at 90 C for 15 min and an increase in half-life from
15 min to >4000 min at 55 C. The thermostable mutant displayed significantly
enhanced performance compared to the parental AnFaeA, suggesting it could be
useful in biotechnological applications. Directed evolution has also been applied to
EstF27 identified from a soil metagenomic library (Cao et al. 2015). Structural
analysis showed that a new disulfide bond and hydrophobic interactions formed by
the mutations may play an important role in stabilizing the protein.
The effect of glycosylation has been studied in feruloyl esterase 1a from
Myceliophthora thermophila (Bonzom et al. 2019). Heterologous expression of
the enzyme in three different hosts (M. thermophila,P. pastoris,E. coli) revealed
that the enzyme produced in E. coli had the lowest catalytic efficiency compared to
its glycosylated form. Moreover, differences were found depending on the degree of
glycosylation, suggesting that careful choice of the expression host should be
considered for enzyme optimization depending on the specific biotechnological
application.
Fig. 27.2 Crystal structure of an F. oxysporum FAE (PDB id 6fat). The catalytic triad is shown.
The characteristic lid is colored in yellow. A bound Ca ion is depicted as a green sphere
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 663

Examples of chimeric enzymes have been reported. AnFAEA and a dockerin
from Clostridium thermocellum were connected and the resultant protein was
expressed in A. niger and characterized (Levasseur et al. 2004), showing higher
enzymatic activity than the parental AnFAEA. A type A FAE from A. awamori
(AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside
hydrolase family 54 α-L-arabinofuranosidase of A. kawachii have been used to create
a chimeric enzyme (Koseki et al. 2010). The new enzyme was found to be more
thermostable than AwFaeA.
27.5 Laccases
Laccases (EC 1.10.3.2; p-diphenol:dioxygen oxidoreductases) belong to the family
of multi-copper oxidases and can oxidize a wide range of aromatic and non-aromatic
compounds, such as substituted phenols, non-phenolic compounds, and some inor-
ganic ions (Agrawal et al. 2018). Owing to their ability to degrade and detoxify a
wide range of phenolic and non-phenolic compounds, including lignin, laccases
have received broad attention for use in several industrial and biotechnological
applications, including eco-friendly synthesis of fine chemicals and the gentle
derivatization of biologically active compounds (Mikolasch and Schauer 2009).
Fungal laccases are characterized by higher redox potential compared to the bacterial
ones; thus, they are more suitable for use in a wide range of substrates and act as
“green biocatalysts”in various areas of biotechnology, including bioremediation
(Mäkelä et al. 2020) and bio-bleaching (Baldrian 2006). Fungal laccases have been
purified from wood-rotting white-rot basidiomycetes and to a lesser extent from
other groups of fungi (e.g., other groups of basidiomycetes, ascomycetes, and
imperfect fungi) (Baldrian 2006). Glycosylation (typically between 10 and 25%)
has been detected in fungal laccases and it was suggested that it protects the enzymes
from protease degradation in addition to the structural role it plays (Maestre-Reyna
et al. 2015; Yoshitake et al. 1993).
Fungal laccases require only molecular oxygen as co-substrate, resulting in
significant cost savings and reduced protein inactivation compared to the use of
H
2
O
2
as co-substrate in peroxidases. Moreover, the biocatalytic process products are
water and a corresponding radical derived from the reducing substrate which
afterwards evolves to the formation of insoluble polymers that are easily removable
by filtration or other conventional technologies (Songulashvili et al. 2016).
A laccase from Sporotrichum thermophile exhibits temperature and pH optima of
60 C and 3.0, respectively. The enzyme was found stable in organic solvents, such
as DMSO and ethanol, and has been used in decolorization of six synthetic dyes
(Kunamneni et al. 2008). In the field of bioelectrocatalysis, fungal laccases, for
example, Trametes versicolor laccase (TvL), have been intensively studied because
of their high redox potential compared to the bacterial ones that makes them suitable
for use in enzymatic biofuel cells (EBFCs) (Arregui et al. 2019).
Municipal wastewater is characterized by high alkalinity and high concentration
of metal ions. Thus, laccases from alkaliphilic fungi have been studied for use in
664 I. Mohsin and A. C. Papageorgiou

such environments (Prakash et al. 2019). Metagenomic studies of Soda Lake have
led to the identification of various fungi with laccase-like oxidase activity suitable
for degradation of phenolic compounds.
Thermophilic fungi have been also a source of laccases with thermostability
properties (Hildén et al. 2009). Thermostable laccases have found use in various
industrial applications, such as pulp and denim bleaching, and in the food and
beverage industry as stabilizers (Osma et al. 2010). Among ascomycetes, significant
thermostability has been detected in a laccase from the thermophilic fungus
Melanocarpus albomyces which can retain its activity for 24 h at 50 C and for
2hat60C (Kiiskinen et al. 2002).A laccase produced by C. thermophilum has
been characterized and sequenced (Chefetz et al. 1998). The enzyme retains activity
for 1 h at 70 C and has half-lives of 24 and 12 h at 40 and 50 C, respectively. It is
also stable at a wide pH range, from pH 5 to 10.
27.5.1 Structural Details of Laccases
Fungal laccases are enzymes with 520–550 amino acids and a molecular weight of
60–70 kDa in their glycosylated form. They exist mainly as monomers although
there are cases of homodimeric, heterodimeric, and multimeric laccases. The three-
dimensional structure of several fungal laccases has been reported. A characteristic
of fungal laccases is the presence of 10 histidines with 8 of them belonging to four
HXH motifs (Sitarz et al. 2016).
Laccases are characterized by an active center that contains four copper ions, each
identified based on its spectroscopic properties. The T1 copper (“blue”copper)
exhibits a strong absorption around 600 nm and is paramagnetic, T2 (“non-blue”
copper) is also paramagnetic with an absorbance at 610 nm, and T3 contains a
diamagnetic spin-coupled copper–copper pair with an absorbance at 330 nm. The T1
copper site and the T2/T3 trinuclear copper cluster are connected to each other
through a strongly conserved internal electron transfer pathway. The substrates are
oxidized by the T1 copper and the extracted electrons are transferred, probably
through a highly conserved His-Cys-His tripeptide motif, to the T2/T3 site, where
molecular oxygen is reduced to water (Mehra et al. 2018).
The crystal structure of a laccase produced by M. albomyces has been determined
(Hakulinen et al. 2002). The molecule is divided into three domains (Fig. 27.3).
Domain 1 includes residues involved in the binding of coppers at the trinuclear site.
Domain 2 includes residues that are involved in the binding of reducing substrates.
Domain 3 contains residues that participate in the binding of coppers at the mono-
nuclear and trinuclear sites as well as in substrate binding. Electron-donating organic
substrate molecules are bound in a hydrophobic pocket of domain 2.
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 665

27.5.2 Improvement of Fungal Laccases
Thermostable fungal laccase chimeras have been generated by the SCHEMA-
RASPP computational approach (Mateljak et al. 2019). The most thermostable
variant showed a five-fold increase (up to 108 min) of its thermal inactivation
half-life at 70 C. Interestingly, ancient laccases have recently emerged as a
promising source of novel laccases (Gomez-Fernandez et al. 2020). A fungal
Mesozoic laccase (dated back 250–500 million years) was resurrected and showed
strikingly high heterologous expression and pH stability. Directed evolution, ratio-
nal, and semi-rational approaches to improve laccase function have been reported
(Mate and Alcalde 2015). N-glycosylation sites are found mostly conserved in
fungal laccases and they have been suggested as potential modulators of the laccase
properties (Ernst et al. 2018).
27.6 Lipases
Lipases (EC 3.1.1.3) catalyze the hydrolysis and synthesis of acylglycerol and other
water-insoluble esters. They are important industrial enzymes that have found use in
food, chemical, drug, biodiesel, and detergent industries. As flavor and fragrance
compounds are esters formed by short-chain carboxylic acids and alcohols, the use
of lipases for the enzymatic synthesis of flavor esters offers a more efficient,
economically benign, and promising alternative approach compared to the tradi-
tional methods of chemical synthesis or extraction from natural sources (Dhake et al.
2013; Verma 2019). Biodiesel consists of fatty acid alkyl esters (FAAE) derived
from triglycerides (TGs) by transesterification with alcohols. Thus, fungal lipases
Fig. 27.3 Crystal structure of
M. albomyces laccase (PDB id
1gw0). The three domains are
shown in different colors.
Copper ions are depicted as
brown spheres. The oxygen
atom is colored in red
666 I. Mohsin and A. C. Papageorgiou

have been used in biodiesel production either free and immobilized or as whole cells
and fermented solids (Aguieiras et al. 2015).
Thermophilic fungi have been a promising source of new thermostable lipases.
Lipases from thermophiles are mostly used in wastewater treatments. Using solid-
state fermentation, a thermostable lipase from Thermomyces lanuginosus was
obtained (Avila-Cisneros et al. 2014). The enzyme exhibited thermostability and it
has been used in various applications, from chemical synthesis and biodiesel pro-
duction to transesterification reactions.
Thermophilic lipases are also tools in the pulp industry as the processing of
lignocellulosic material leads to the formation of pitch, a substance rich in esters able
to clog machines. A lipase from Aspergillus oryzae has been used to control pitch
formation (Gutiérrez et al. 2009).
Psychrophilic and alkaliphilic lipases are usually added to the detergent formula-
tion as polymer-degrading agents (Joseph et al. 2008). Talaromyces thermophilus
produces a thermoactive and alkaline lipase which retains activity at pH 9.5
(Romdhane et al. 2010). The enzyme is stable at 60 C and retains 65% of its
enzyme activity after 30 min incubation at 70 C. Its half-activity is retained after
incubation for 40 min at 80 C. The optimum pH for the enzyme activity was 9.0 and
the lipase was stable from pH 8.0 to 12.0. Higher frequency of hydrophobic amino
acids, such as Ala, Val, Leu, and Gly in thermostable lipases from T. lanuginosus has
been suggested for increased thermal stability (Zheng et al. 2011).
A lipase from the biotrophic fungus Sporisorium reilianum SRZ2 (SRL) with
73% amino acid sequence identity to Candida antarctica lipase B (CALB) was
cloned and overexpressed in Pichia pastoris retained 75% of its activity at pH 3–11
for 72 h and it has been suggested to act as a thermophilic fungal lipase (Shen et al.
2020). Moreover, LipG7 from the Antarctic filamentous fungus Geomyces sp.P7
retained 100% of its initial activity after 1 h of incubation at 100 C (Florczak et al.
2013). An acidic and thermostable lipase with a preference for the medium-chain
length p-nitrophenyl esters (C12) rather than short and long-chain length substrates
has been characterized from the thermophilic fungus Neosartorya fischeri P1 (Sun
et al. 2016).
27.6.1 Structural Details of Lipases
Lipases have a common α/β-hydrolase fold, a catalytic triad (Ser-His-Asp/Glu)
similar to that found in serine proteases and a lid covering the active site (Holmquist
2000). The lid is displaced upon activation and opens up the binding pocket, thus
making the active site accessible to the substrate (Stauch et al. 2015) (Fig. 27.4). The
ester hydrolysis mechanism of lipases is similar to that of carboxyl esterases and
serine proteases. It involves a nucleophilic attack on the carbonyl carbon of the ester
bond by the catalytic triad, leading to the formation of an acyl-enzyme intermediate
and alcohol. This acyl-enzyme intermediate in second nucleophilic attack is
hydrolyzed by water and yields carboxylic acid.
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 667

27.6.2 Improvement of Catalytic Efficiency in Lipases
There have been enormous efforts to increase the catalytic efficiency and thermal
properties of the lipases. The Lipase Engineering Database (LED; http://www.led.
uni-stuttgart.de) provides up-to-date information about the updated and engineered
lipases. Lipases are synthetically designed, genetically engineered, cloned, and
expressed using suitable expression systems in recombinant organisms. The
methods employed (Bornscheuer et al. 2002) for this purpose are:
•Physicochemical methods (e.g., immobilization and solubilization).
•Reaction engineering methods (e.g., use of acyl donors and ionic liquids).
•Molecular biology methods (e.g., rational protein design and directed evolution).
Notably, the conversion through directed evolution of the catalytic Ser to Cys in
CALB along with four mutations resulted in 40-fold higher activity lipases (Cen
et al. 2019), suggesting that similar approaches could be useful for the fungal lipases.
27.7 Nitrilases
Nitrilases (NLases; EC 3.5.5.-) catalyze the hydrolysis of different nitriles to
corresponding amides and acids. They can be used in organic synthesis to improve
industrial biocatalysis (Gong et al. 2017). Bacterial NLases have been characterized
Fig. 27.4 Lid displacement
in CALB. The conformational
change of residues 140–147 is
shown after superposition of
the closed conformation
structure (PDB id 5a71; cyan)
onto the open conformation
structure (PDB id 1tca; pink).
The residues of the catalytic
triad are shown as sticks
668 I. Mohsin and A. C. Papageorgiou

in detail. According to their substrate specificity, they are classified as aromatic
NLases, aliphatic NLases, and arylacetoNLases. In contrast, studies of fungal
nitrilases have been slow (Martínková2019). Fungal nitrilases belong to aromatic
nitrilases and arylacetonitrilases. A recent bioinformatics search identified various
nitrilases in fungi (Ruckáet al. 2020). In general, fungal nitrilases are considered
advantageous over the bacterial ones regarding activity, thermostability, and selec-
tivity (Wu et al. 2013).
27.7.1 Structural Details of Nitrilases
No structure of a fungal nitrilase is currently available. Insights into the catalytic
mechanism have been provided by an archaeal nitrilase (Raczynska et al. 2011). A
two-fold dimer symmetry has been identified with each subunit of the dimer
characterized by an αββα sandwich fold, resulting in a super-sandwich αββααββα
structure formed by the association of the two subunits. Dimerization is achieved
through multiple approaches, including interactions of the extended C-terminal of
each subunit and interactions between arginine and glutamate residues that form salt
bridges. The binding pocket lies close to the inter-subunit interface, while a binding
loop assists the binding of the substrate. A Lys residue was suggested as the acid in
the catalytic reaction.
27.7.2 Improvement
Site-directed mutagenesis of a fungal nitrilase from Gibberella intermedia resulted
in mutants with higher catalytic activity and increased stability (Gong et al. 2016).
Notably, point mutations near the active site of an A. niger nitrilase were able to
change the enantioselectivity of the enzyme (Petříčkováet al. 2012). Mutagenesis
studies on bacterial nitrilases to improve their thermostability have been carried out
and may apply to their fungal counterparts as well (Xu et al. 2018).
27.8 Transaminases
Transaminases (TAs), or aminotransferases, are enzymes that catalyze the transfer of
an amino group from an amino donor to an acceptor for chiral amino acid or amine
synthesis (Guo and Berglund 2017). Owing to their excellent enantioselectivity,
environmental friendliness, and compatibility with other enzymatic or chemical
systems, TAs have drawn attention in the area of biocatalysis. The most known
example of use of a TA is in the synthesis of the antidiabetic drug sitagliptin (Savile
et al. 2010). An in silico strategy for sequence-based prediction of substrate speci-
ficity and enantiopreference revealed 17 novel (R)-selective TAs, many of them
from fungi (Höhne et al. 2010).
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 669

27.8.1 Structural Details of TAs
The structure of a pyruvate TA from the fungus Nectria haematococca (Sayer et al.
2014) has been determined and provided initial insights into the R-enantioselectivity
of the TAs. Also, the structure of an (R)-selective amine TA from A. fumigatus has
been elucidated (Thomsen et al. 2014). The enzyme has the typical fold found in
class IV of PLP-dependent enzymes and its overall structure is similar to that of a
branched-chain amino acid aminotransferase from T. thermophilus and D-amino acid
aminotransferase from Bacillus sp. YM-1 (Fig. 27.5). An N-terminal α-helical
extension has been found that was suggested to play a role in the enzyme’s stability.
The structure of an ω-transaminase from Aspergillus terreus has been solved and
found to exhibit also the class IV fold (Łyskowski et al. 2014).
27.8.2 TA Improvement
A thermostable (R)-TA (Huang et al. 2017) from A. terreus (AtRTA) has been
characterized. A homolog of AtRTA from the thermotolerant fungus Thermomyces
stellatus (TsRTA) has been reported (Heckmann et al. 2020). The thermostability of
TsRTA (40% retained activity after 7 days at 40 C) was initially attributed to its
tetrameric form in solution. However, subsequent studies of AtRTA revealed that the
enzyme also exists predominantly as a tetramer but, in contrast to TsRTA, it is
inactivated within 48 h at 40 C. The engineering of a cysteine residue to promote
Fig. 27.5 Crystal structure of
A. fumigatus (R)-selective
amine TA (PDB id 4chi). The
PLP cofactor is depicted in
sticks
670 I. Mohsin and A. C. Papageorgiou

disulfide bond formation across the dimer–dimer interface stabilized both enzymes,
with TsRTA_G205C mutant retaining almost full activity after incubation at 50 C
for 7 days.
27.9 Tyrosinases
Tyrosinases (EC 1.14.18.1, monophenol, o-diphenol:oxygen oxidoreductases)
together with laccases (EC 1.10.3.2) form two subgroups of phenoloxidases. As in
laccases, tyrosinases are also copper-containing enzymes. In tyrosinases, the copper
ions are known as CuA and CuB (Ba and Vinoth Kumar 2017).
Tyrosinases are found in bacteria, fungi, and plants. Fungal tyrosinases from
Agaricus bisporus,Neurospora crassa,Aspergillus oryzae, and Aspergillus niger
have been largely explored (Agarwal et al. 2017). Detailed characterization of a
tyrosinase from T. reesei has also been reported (Selinheimo et al. 2006).
Tyrosinases have been used either in a free or immobilized form for the removal
of micro-pollutants in the environment (Ba and Vinoth Kumar 2017). Besides,
tyrosinases are employed in L-DOPA synthesis and biosensor development (Min
et al. 2019).
27.9.1 Structural Details of Tyrosinases
Crystal structures of an Aspergillus oryzae fungal tyrosinase are available (Fujieda
et al. 2020) and have provided information into the copper movements during the
catalytic reaction (Fig. 27.6). No structures are currently available for tyrosinases
from extremophilic fungi.
27.9.2 Tyrosinase Improvement
The use of D-DOPA in Parkinson’s disease has been proposed as more effective than
the use of L-DOPA. As fungal tyrosinases have lower affinity for D-tyrosine,
improvement of their catalytic activity against D-tyrosine has been pursued (Ali
et al. 2020). Thermostable tyrosinases have been proposed for the removal and
bioconversion of phenol from wastewater (Lee et al. 1996). Immobilization of the
enzyme appears to have beneficial effects. Indeed, an immobilized mushroom
tyrosinase has been found to work at high temperatures (100 C) and in organic
solvents (Wu et al. 2018). Also, immobilized Aspergillus niger tyrosinase has shown
an increased thermal and pH stability (Agarwal et al. 2016). A computational
approach which has been carried out to improve the thermostability of a bacterial
tyrosinase (Guo et al. 2015) may be applicable for fungal enzymes as well.
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 671

27.10 Keratinases
Keratinases are key enzymes used for the degradation of agricultural and industrial
keratin waste. Keratin is the most abundant insoluble structural fibrous protein and a
major constituent of hair, nails, horn, wool, skin, and feather. Chemical and physical
treatment of keratin are currently unfriendly to the environment, leading to a product
with poor digestibility, variable nutrient quality, and destruction of several amino
acids (Mabrouk 2008). Keratinases have been identified in a wide variety of
microorganisms (Brandelli et al. 2010). A crude enzyme preparation from Aspergil-
lus fumigatus (Santos et al. 1996) showed remarkable thermostability by retaining
~90% of its original activity at 70 C for 1.5 h. Besides, a novel thermophilic
M. thermophila strain GZUIFR-H49-1 with potential applications for production
of thermostable keratinase has been identified (Liang et al. 2011). Also, screening of
almost 300 fungi species revealed that Aspergillus flavus was the most productive
fungus in keratinolytic enzymes (Friedrich et al. 1999).
27.10.1 Structural Details of Keratinases
Structural details of keratinases have been obtained from bacterial sources and the
crystal structures of five keratinolytic enzymes are known (Qiu et al. 2020). Phylo-
genetic and structure-based analysis has shown the presence of other keratinases in
Fig. 27.6 Crystal structure of A. oryzae C92A tyrosinase mutant in the presence of peroxide (PDB
id 6jub). Cu atoms are shown as brown spheres and peroxide is colored in red
672 I. Mohsin and A. C. Papageorgiou

various extremophiles, including fungi. The structure of the keratinase fervidolysin
has suggested a distant relationship with pro-subtilisin E using structural alignment
(Kim et al. 2004; Kluskens et al. 2002). The structure also suggested a functional
relationship of fervidolysin to the fibronectin-like domains of the human promatrix
metalloprotease-2 that degrades the fibrous polymeric substrate gelatin. The struc-
ture of a novel heat-stable keratinase (Wu et al. 2017) from the feather-degrading
thermophilic bacterium Meiothermus taiwanensis WR-220 has shown similarities
with the overall fold of the catalytic domain of fervidolysin.
27.10.2 Improvement
Computational techniques to improve the thermostability of bacterial keratinases
have been applied (Liu et al. 2013) and they may be extended to other keratinases of
industrial interest. A synergistic action of keratinases with LPMOs has been pro-
posed (Lange et al. 2016), thus offering additional strategies to improve keratinase
performance.
27.11 Conclusions
Fungal extremozymes are promising alternatives for use in green chemistry. How-
ever, a systematic functional and structural characterization is necessary to under-
stand better their stability, behavior in extreme environments, catalytic mechanism,
synergism, and evolutionary relationships. Currently, the two most preferable
approaches for producing novel enzyme variants are site-directed mutagenesis and
directed evolution. Further improvement of fungal extremozymes will help in
developing better and more versatile enzymes for their use either alone or in
mixtures with other enzymes for green chemistry applications.
Acknowledgement We thank Biocenter Finland for infrastructure support.
References
Agarwal P, Dubey S, Singh M, Singh RP (2016) Aspergillus niger PA2 tyrosinase covalently
immobilized on a novel eco-friendly bio-composite of chitosan-gelatin and its evaluation for
L-DOPA production. Front Microbiol 7:2088
Agarwal P, Singh J, Singh RP (2017) Molecular cloning and characteristic features of a novel
extracellular tyrosinase from Aspergillus niger PA2. Appl Biochem Biotechnol 182:1–15.
https://doi.org/10.1007/s12010-016-2306-2
Agrawal K, Chaturvedi V, Verma P (2018) Fungal laccase discovered but yet undiscovered.
Bioresour Bioprocess 5:1–12. https://doi.org/10.1186/s40643-018-0190-z
Aguieiras ECG, Cavalcanti-Oliveira ED, Freire DMG (2015) Current status and new developments
of biodiesel production using fungal lipases. Fuel 159:52–67. https://doi.org/10.1016/j.fuel.
2015.06.064
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 673

Ali M, Ishqi HM, Husain Q (2020) Enzyme engineering: reshaping the biocatalytic functions.
Biotechnol Bioeng 117:1877–1894. https://doi.org/10.1002/bit.27329
Anna Calarco ADS (2015) A review on extremozymes biocatalysis: a green industrial approach for
biomaterials production. J Biomol Res Ther 04:1–10. https://doi.org/10.4172/2167-7956.
1000121
Arregui L et al (2019) Laccases: structure, function, and potential application in water bioremedia-
tion. Microb Cell Factories 18:200. https://doi.org/10.1186/s12934-019-1248-0
Atalah J, Cáceres-Moreno P, Espina G, Blamey JM (2019) Thermophiles and the applications of
their enzymes as new biocatalysts. Bioresour Technol 280:478–488. https://doi.org/10.1016/j.
biortech.2019.02.008
Avila-Cisneros N, Velasco-Lozano S, Huerta-Ochoa S, Córdova-López J, Gimeno M, Favela-
Torres E (2014) Production of thermostable lipase by Thermomyces lanuginosus on solid-
state fermentation: selective hydrolysis of sardine oil. Appl Biochem Biotechnol 174:1859–
1872. https://doi.org/10.1007/s12010-014-1159-9
Ba S, Vinoth Kumar V (2017) Recent developments in the use of tyrosinase and laccase in
environmental applications. Crit Rev Biotechnol 37:819–832. https://doi.org/10.1080/
07388551.2016.1261081
Bala A, Singh B (2016) Cost-effective production of biotechnologically important hydrolytic
enzymes by Sporotrichum thermophile. Bioprocess Biosyst Eng 39:181–191. https://doi.org/
10.1007/s00449-015-1502-8
Baldrian P (2006) Fungal laccases - occurrence and properties. FEMS Microbiol Rev 30:215–242.
https://doi.org/10.1111/j.1574-4976.2005.00010.x
Basit A, Liu J, Miao T, Zheng F, Rahim K, Lou H, Jiang W (2018) Characterization of two
endo-β-1, 4-xylanases from Myceliophthora thermophila and their saccharification efficiencies,
synergistic with commercial cellulase. Front Microbiol 9:233. https://doi.org/10.3389/fmicb.
2018.00233
Blanchard S et al (2007) Unexpected regioselectivity of Humicola insolens Cel7B glycosynthase
mutants. Carbohydr Res 342:710–716. https://doi.org/10.1016/j.carres.2006.12.017. pii:
S0008-6215(06)00561-1
Bonzom C et al (2019) Glycosylation influences activity, stability and immobilization of the
feruloyl esterase 1a from Myceliophthora thermophila. AMB Express 9:126. https://doi.org/
10.1186/s13568-019-0852-z
Bornscheuer UT, Bessler C, Srinivas R, Hari Krishna S (2002) Optimizing lipases and related
enzymes for efficient application. Trends Biotechnol 20:433–437. https://doi.org/10.1016/
S0167-7799(02)02046-2
Brandelli A, Daroit DJ, Riffel A (2010) Biochemical features of microbial keratinases and their
production and applications. Appl Microbiol Biotechnol 85:1735–1750. https://doi.org/10.
1007/s00253-009-2398-5
Busk PK, Lange L (2015) Classification of fungal and bacterial lytic polysaccharide
monooxygenases. BMC Genomics 16:368. https://doi.org/10.1186/s12864-015-1601-6
Cao LC, Chen R, Xie W, Liu YH (2015) Enhancing the thermostability of feruloyl esterase EstF27
by directed evolution and the underlying structural basis. J Agric Food Chem 63:8225–8233.
https://doi.org/10.1021/acs.jafc.5b03424
de Cassia Pereira J et al (2015) Thermophilic fungi as new sources for production of cellulases and
xylanases with potential use in sugarcane bagasse saccharification. J Appl Microbiol 118:928–
939. https://doi.org/10.1111/jam.12757
Cen Y et al (2019) Artificial cysteine-lipases with high activity and altered catalytic mechanism
created by laboratory evolution. Nat Commun 10:3198. https://doi.org/10.1038/s41467-019-
11155-3
Chadha BS, Kaur B, Basotra N, Tsang A, Pandey A (2019) Thermostable xylanases from thermo-
philic fungi and bacteria: current perspective. Bioresour Technol 277:195–203. https://doi.org/
10.1016/j.biortech.2019.01.044
674 I. Mohsin and A. C. Papageorgiou

Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium
thermophilium and its role in humification. Appl Environ Microbiol 64:3175–3179. https://doi.
org/10.1128/AEM.64.9.3175-3179.1998
Cheng YS et al (2014) Structural analysis of a glycoside hydrolase family 11 xylanase from
Neocallimastix patriciarum: insights into the molecular basis of a thermophilic enzyme. J
Biol Chem 289:11020–11028. https://doi.org/10.1074/jbc.M114.550905
Cipolatti EP, Cerqueira Pinto MC, Henriques RO, da Silva Pinto JCC, de Castro AM, Freire DMG,
Manoel EA (2019) Enzymes in green chemistry: the state of the art in chemical
transformations. In: Advances in enzyme technology. Elsevier, Amsterdam, pp 137–151.
https://doi.org/10.1016/B978-0-444-64114-4.00005-4
Crepin VF, Faulds CB, Connerton IF (2004) Functional classification of the microbial feruloyl
esterases. Appl Microbiol Biotechnol 63:647–652. https://doi.org/10.1007/s00253-003-1476-3
Damis SIR, Murad AMA, Diba Abu Bakar F, Rashid SA, Jaafar NR, Illias RM (2019) Protein
engineering of GH11 xylanase from Aspergillus fumigatus RT-1 for catalytic efficiency
improvement on kenaf biomass hydrolysis. Enzym Microb Technol 131:109383. https://doi.
org/10.1016/j.enzmictec.2019.109383
Dhake KP, Thakare DD, Bhanage BM (2013) Lipase: a potential biocatalyst for the synthesis of
valuable flavour and fragrance ester compounds. Flavour Fragr J 28:71–83. https://doi.org/10.
1002/ffj.3140
Dilokpimol A, Mäkelä MR, Aguilar-Pontes MV, Benoit-Gelber I, Hildén KS, de Vries RP (2016)
Diversity of fungal feruloyl esterases: updated phylogenetic classification, properties, and
industrial applications. Biotechnol Biofuels 9:231. https://doi.org/10.1186/s13068-016-0651-6
Dimarogona M, Topakas E, Christakopoulos P, Chrysina ED (2012) The structure of a GH10
xylanase from Fusarium oxysporum reveals the presence of an extended loop on top of the
catalytic cleft. Acta Crystallogr D Biol Crystallogr 68:735–742. https://doi.org/10.1107/
S0907444912007044
Dimarogona M, Topakas E, Christakopoulos P, Chrysina ED (2020) The crystal structure of a
Fusarium oxysporum feruloyl esterase that belongs to the tannase family. FEBS Lett 594:1738–
1749. https://doi.org/10.1002/1873-3468.13776
Ernst HA et al (2018) A comparative structural analysis of the surface properties of asco-laccases.
PLoS One 13:e0206589–e0206589. https://doi.org/10.1371/journal.pone.0206589
Faulds CB (2010) What can feruloyl esterases do for us? Phytochem Rev 9:121–132. https://doi.
org/10.1007/s11101-009-9156-2
Florczak T, Daroch M, Wilkinson MC, Białkowska A, Bates AD, Turkiewicz M, Iwanejko LA
(2013) Purification, characterisation and expression in Saccharomyces cerevisiae of LipG7 an
enantioselective, cold-adapted lipase from the Antarctic filamentous fungus Geomyces sp. P7
with unusual thermostability characteristics. Enzym Microb Technol 53:18–24. https://doi.org/
10.1016/j.enzmictec.2013.03.021
Fort S et al (2000) Highly efficient synthesis of beta(1->4)-oligo- and -polysaccharides using a
mutant cellulase. J Am Chem Soc 122:5429–5437. https://doi.org/10.1021/ja9936520
Friedrich J, Gradišar H, Mandin D, Chaumont JP (1999) Screening fungi for synthesis of
keratinolytic enzymes. Lett Appl Microbiol 28:127–130. https://doi.org/10.1046/j.1365-2672.
1999.00485.x
Fujieda N et al (2020) Copper–oxygen dynamics in the tyrosinase mechanism. Angew Chem Int Ed
59:13385–13390. https://doi.org/10.1002/anie.202004733
Fushinobu S, Ito K, Konno M, Wakagi T, Matsuzawa H (1998) Crystallographic and mutational
analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic
residues and importance of Asp37 for catalysis at low pH. Protein Eng 11:1121–1128. https://
doi.org/10.1093/protein/11.12.1121
Fushinobu S, Uno T, Kitaoka M, Hayashi K, Matsuzawa H, Wakagi T (2011) Mutational analysis
of fungal family 11 xylanases on pH optimum determination. J Appl Glycosci 58:107–114.
https://doi.org/10.5458/jag.jag.JAG-2011_001
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 675

Gomez-Fernandez BJ, Risso VA, Rueda A, Sanchez-Ruiz JM, Alcalde M (2020) Ancestral
resurrection and directed evolution of fungal Mesozoic laccases. Appl Environ Microbiol 86:
e00778–e00720. https://doi.org/10.1128/AEM.00778-20
Gong J-S et al (2016) Engineering of a fungal nitrilase for improving catalytic activity and reducing
by-product formation in the absence of structural information. Cat Sci Technol 6:4134–4141.
https://doi.org/10.1039/C5CY01535A
Gong JS, Shi JS, Lu ZM, Li H, Zhou ZM, Xu ZH (2017) Nitrile-converting enzymes as a tool to
improve biocatalysis in organic synthesis: recent insights and promises. Crit Rev Biotechnol 37:
69–81. https://doi.org/10.3109/07388551.2015.1120704
Grassick A et al (2004) Three-dimensional structure of a thermostable native cellobiohydrolase,
CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus,
Talaromyces emersonii. Eur J Biochem 271:4495–4506. https://doi.org/10.1111/j.1432-1033.
2004.04409.x
Gudmundsson M et al (2016) Structural and functional studies of the glycoside hydrolase family
3β-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii. Acta
Crystallogr D Struct Biol 72:860–870. https://doi.org/10.1107/S2059798316008482
Guo F, Berglund P (2017) Transaminase biocatalysis: optimization and application. Green Chem
19:333–360. https://doi.org/10.1039/C6GC02328B
Guo J, Rao Z, Yang T, Man Z, Xu M, Zhang X, Yang S-T (2015) Enhancement of the thermosta-
bility of Streptomyces kathirae SC-1 tyrosinase by rational design and empirical mutation.
Enzym Microb Technol 77:54–60. https://doi.org/10.1016/j.enzmictec.2015.06.002
Gutiérrez A, del Río JC, Martínez AT (2009) Microbial and enzymatic control of pitch in the pulp
and paper industry. Appl Microbiol Biotechnol 82:1005–1018. https://doi.org/10.1007/s00253-
009-1905-z
Hakulinen N, Kiiskinen LL, Kruus K, Saloheimo M, Paananen A, Koivula A, Rouvinen J (2002)
Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper
site. Nat Struct Biol 9:601–605. https://doi.org/10.1038/nsb823
Han N, Ma Y, Mu Y, Tang X, Li J, Huang Z (2019) Enhancing thermal tolerance of a fungal GH11
xylanase guided by B-factor analysis and multiple sequence alignment. Enzym Microb Technol
131:109422. https://doi.org/10.1016/j.enzmictec.2019.109422
Hayes M, Pietruszka J (2017) Synthesis of glycosides by glycosynthases. Molecules 22:1434.
https://doi.org/10.3390/molecules22091434
Heckmann CM, Gourlay LJ, Dominguez B, Paradisi F (2020) An (R)-selective transaminase from
Thermomyces stellatus: stabilizing the tetrameric form. Front Bioeng Biotechnol 8:707. https://
doi.org/10.3389/fbioe.2020.00707
Heinzelman P et al (2009) SCHEMA recombination of a fungal cellulase uncovers a single
mutation that contributes markedly to stability. J Biol Chem 284:26229–26233. https://doi.
org/10.1074/jbc.C109.034058
Hermoso JA, Sanz-Aparicio J, Molina R, Juge N, González R, Faulds CB (2004) The crystal
structure of feruloyl esterase A from Aspergillus Niger suggests evolutive functional conver-
gence in feruloyl esterase family. J Mol Biol 338:495–506. https://doi.org/10.1016/j.jmb.2004.
03.003
Hildén K, Hakala TK, Lundell T (2009) Thermotolerant and thermostable laccases. Biotechnol Lett
31:1117. https://doi.org/10.1007/s10529-009-9998-0
Höhne M, Schätzle S, Jochens H, Robins K, Bornscheuer UT (2010) Rational assignment of key
motifs for function guides in silico enzyme identification. Nat Chem Biol 6:807–813. https://doi.
org/10.1038/nchembio.447
Holmquist M (2000) Alpha/Beta-hydrolase fold enzymes: structures, functions and mechanisms.
Curr Protein Pept Sci 1:209–235
Huang J, Xie D-F, Feng Y (2017) Engineering thermostable (R)-selective amine transaminase from
Aspergillus terreus through in silico design employing B-factor and folding free energy
calculations. Biochem Biophys Res Commun 483:397–402. https://doi.org/10.1016/j.bbrc.
2016.12.131
676 I. Mohsin and A. C. Papageorgiou

Hyde KD et al (2019) The amazing potential of fungi: 50 ways we can exploit fungi industrially.
Fungal Divers 97:1–136. https://doi.org/10.1007/s13225-019-00430-9
Ioannou YA, Zeidner KM, Grace ME, Desnick RJ (1998) Human alpha-galactosidase A: glycosyl-
ation site 3 is essential for enzyme solubility. Biochem J 332:789–797
Joseph B, Ramteke PW, Thomas G (2008) Cold active microbial lipases: some hot issues and recent
developments. Biotechnol Adv 26:457–470. https://doi.org/10.1016/j.biotechadv.2008.05.003
Karnaouri A, Topakas E, Paschos T, Taouki I, Christakopoulos P (2013) Cloning, expression and
characterization of an ethanol tolerant GH3 beta-glucosidase from Myceliophthora thermophila.
PeerJ 1:e46. https://doi.org/10.7717/peerj.46
Karnaouri A, Topakas E, Antonopoulou I, Christakopoulos P (2014) Genomic insights into the
fungal lignocellulolytic system of Myceliophthora thermophila. Front Microbiol 5:281. https://
doi.org/10.3389/fmicb.2014.00281
Kayser V, Chennamsetty N, Voynov V, Forrer K, Helk B, Trout BL (2011) Glycosylation
influences on the aggregation propensity of therapeutic monoclonal antibodies. Biotechnol J
6:38–44. https://doi.org/10.1002/biot.201000091
Kiiskinen LL, Viikari L, Kruus K (2002) Purification and characterisation of a novel laccase from
the ascomycete Melanocarpus albomyces. Appl Microbiol Biotechnol 59:198–204. https://doi.
org/10.1007/s00253-002-1012-x
Kim J-S, Kluskens LD, de Vos WM, Huber R, van der Oost J (2004) Crystal structure of
fervidolysin from Fervidobacterium pennivorans, a keratinolytic enzyme related to subtilisin.
J Mol Biol 335:787–797. https://doi.org/10.1016/j.jmb.2003.11.006
Kluskens LD, Voorhorst WG, Siezen RJ, Schwerdtfeger RM, Antranikian G, van der Oost J, de Vos
WM (2002) Molecular characterization of fervidolysin, a subtilisin-like serine protease from the
thermophilic bacterium Fervidobacterium pennivorans. Extremophiles 6:185–194. https://doi.
org/10.1007/s007920100239
Koseki T, Fushinobu S, Ardiansyah, Shirakawa H, Komai M (2009) Occurrence, properties, and
applications of feruloyl esterases. Appl Microbiol Biotechnol 84:803–810. https://doi.org/10.
1007/s00253-009-2148-8
Koseki T, Mochizuki K, Kisara H, Miyanaga A, Fushinobu S, Murayama T, Shiono Y (2010)
Characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate-
binding module. Appl Microbiol Biotechnol 86:155–161. https://doi.org/10.1007/s00253-009-
2224-0
Kunamneni A, Ghazi I, Camarero S, Ballesteros A, Plou FJ, Alcalde M (2008) Decolorization of
synthetic dyes by laccase immobilized on epoxy-activated carriers. Process Biochem 43:169–
178. https://doi.org/10.1016/j.procbio.2007.11.009
Labrou NE (2010) Random mutagenesis methods for in vitro directed enzyme evolution. Curr
Protein Pept Sci 11:91–100
Lange L, Huang Y, Busk PK (2016) Microbial decomposition of keratin in nature—a new
hypothesis of industrial relevance. Appl Microbiol Biotechnol 100:2083–2096. https://doi.
org/10.1007/s00253-015-7262-1
Le Costaouëc T, Pakarinen A, Várnai A, Puranen T, Viikari L (2013) The role of carbohydrate
binding module (CBM) at high substrate consistency: comparison of Trichoderma reesei and
Thermoascus aurantiacus Cel7A (CBHI) and Cel5A (EGII). Bioresour Technol 143:196–203.
https://doi.org/10.1016/j.biortech.2013.05.079
Lee S-G, Hong S-P, Sung M-H (1996) Removal and bioconversion of phenol in wastewater by a
thermostable β-tyrosinase. Enzym Microb Technol 19:374–377. https://doi.org/10.1016/S0141-
0229(96)00001-4
Lee HS, Qi Y, Im W (2015) Effects of N-glycosylation on protein conformation and dynamics:
Protein Data Bank analysis and molecular dynamics simulation study. Sci Rep 5:8926. https://
doi.org/10.1038/srep08926
Levasseur A et al (2004) Design and production in Aspergillus Niger of a chimeric protein
associating a fungal feruloyl esterase and a clostridial dockerin domain. Appl Environ Microbiol
70:6984–6991. https://doi.org/10.1128/AEM.70.12.6984-6991.2004
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 677

Li D-C, Papageorgiou AC (2019) Cellulases from thermophilic fungi: recent insights and biotech-
nological potential. In: Tiquia-Arashiro SM, Grube M (eds) Fungi in extreme environments:
ecological role and biotechnological significance. Springer, Cham, pp 395–417. https://doi.org/
10.1007/978-3-030-19030-9_20
Li YL, Li H, Li AN, Li DC (2009) Cloning of a gene encoding thermostable cellobiohydrolase from
the thermophilic fungus Chaetomium thermophilum and its expression in Pichia pastoris.J
Appl Microbiol 106:1867–1875. https://doi.org/10.1111/j.1365-2672.2009.04171.x
Li G et al (2019) Improvement of GH10 family xylanase thermostability by introducing of an extra
α-helix at the C-terminal. Biochem Biophys Res Commun 515:417–422. https://doi.org/10.
1016/j.bbrc.2019.05.163
Liang JD, Han YF, Zhang JW, Du W, Liang ZQ, Li ZZ (2011) Optimal culture conditions for
keratinase production by a novel thermophilic Myceliophthora thermophila strain GZUIFR-
H49-1. J Appl Microbiol 110:871–880. https://doi.org/10.1111/j.1365-2672.2011.04949.x
Limon MC, Margolles-Clark E, Benitez T, Penttila M (2001) Addition of substrate-binding
domains increases substrate-binding capacity and specific activity of a chitinase from
Trichoderma harzianum. FEMS Microbiol Lett 198:57–63. pii: S0378-1097(01)00124-0
Liu B, Zhang J, Fang Z, Gu L, Liao X, Du G, Chen J (2013) Enhanced thermostability of keratinase
by computational design and empirical mutation. J Ind Microbiol Biotechnol 40:697–704.
https://doi.org/10.1007/s10295-013-1268-4
Liu M-Q, Li J-Y, Rehman AU, Xu X, Gu Z-J, Wu R-C (2019) Laboratory evolution of GH11
endoxylanase through DNA shuffling: effects of distal residue substitution on catalytic activity
and active site architecture. Front Bioeng Biotechnol 7:350. https://doi.org/10.3389/fbioe.2019.
00350
Łyskowski A, Gruber C, Steinkellner G, Schürmann M, Schwab H, Gruber K, Steiner K (2014)
Crystal structure of an (R)-selective ω-transaminase from Aspergillus terreus. PLoS One 9:
e87350. https://doi.org/10.1371/journal.pone.0087350
Mabrouk MEM (2008) Feather degradation by a new keratinolytic Streptomyces sp. MS-2. World J
Microbiol Biotechnol 24:2331–2338. https://doi.org/10.1007/s11274-008-9748-9
Maestre-Reyna M et al (2015) Structural and functional roles of glycosylation in fungal laccase
from Lentinus sp. PLoS One 10:e0120601. https://doi.org/10.1371/journal.pone.0120601
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes.
Microbiol Mol Biol Rev 64:461–488
Mäkelä MR, Dilokpimol A, Koskela SM, Kuuskeri J, de Vries RP, Hildén K (2018) Characteriza-
tion of a feruloyl esterase from Aspergillus terreus facilitates the division of fungal enzymes
from carbohydrate esterase family 1 of the carbohydrate-active enzymes (CAZy) database.
Microb Biotechnol 11:869–880. https://doi.org/10.1111/1751-7915.13273
Mäkelä MR, Tuomela M, Hatakka A, Hildén K (2020) Fungal laccases and their potential in
bioremediation applications. In: Schlosser D (ed) Laccases in bioremediation and waste
valorisation. Springer, Cham, pp 1–25. https://doi.org/10.1007/978-3-030-47906-0_1
Mamma D, Hatzinikolaou DG, Christakopoulos P (2004) Biochemical and catalytic properties of
two intracellular [beta]-glucosidases from the fungus Penicillium decumbens active on flavo-
noid glucosides. J Mol Catal B Enzymatic 27:183–190. https://doi.org/10.1016/j.molcatb.2003.
11.011
MartínkováL (2019) Nitrile metabolism in fungi: a review of its key enzymes nitrilases with focus
on their biotechnological impact. Fungal Biol Rev 33:149–157. https://doi.org/10.1016/j.fbr.
2018.11.002
Mate DM, Alcalde M (2015) Laccase engineering: from rational design to directed evolution.
Biotechnol Adv 33:25. https://doi.org/10.1016/j.biotechadv.2014.12.007
Mateljak I, Rice A, Yang K, Tron T, Alcalde M (2019) The generation of thermostable fungal
laccase chimeras by SCHEMA-RASPP structure-guided recombination in vivo. ACS Synth
Biol 8:833–843. https://doi.org/10.1021/acssynbio.8b00509
678 I. Mohsin and A. C. Papageorgiou

McAuley KE, Svendsen A, Patkar SA, Wilson KS (2004) Structure of a feruloyl esterase from
Aspergillus niger. Acta Crystallogr Sect D 60:878–887. https://doi.org/10.1107/
S0907444904004937
Mehra R, Muschiol J, Meyer AS, Kepp KP (2018) A structural-chemical explanation of fungal
laccase activity. Sci Rep 8:17285. https://doi.org/10.1038/s41598-018-35633-8
Meldgaard M, Svendsen I (1994) Different effects of N-glycosylation on the thermostability of
highly homologous bacterial (1,3-1,4)-beta-glucanases secreted from yeast. Microbiology 140
(pt 1):159–166
Miao Y, Kong Y, Li P, Li G, Liu D, Shen Q, Zhang R (2018) Effect of CBM1 and linker region on
enzymatic properties of a novel thermostable dimeric GH10 xylanase (Xyn10A) from filamen-
tous fungus Aspergillus fumigatus Z5. AMB Express 8:44. https://doi.org/10.1186/s13568-018-
0576-5
Mikolasch A, Schauer F (2009) Fungal laccases as tools for the synthesis of new hybrid molecules
and biomaterials. Appl Microbiol Biotechnol 82:605–624. https://doi.org/10.1007/s00253-009-
1869-z
Min K, Park GW, Yoo YJ, Lee J-S (2019) A perspective on the biotechnological applications of the
versatile tyrosinase. Bioresour Technol 289:121730. https://doi.org/10.1016/j.biortech.2019.
121730
Murray PG, Collins CM, Grassick A, Tuohy MG (2003) Molecular cloning, transcriptional, and
expression analysis of the first cellulase gene (cbh2), encoding cellobiohydrolase II, from the
moderately thermophilic fungus Talaromyces emersonii and structure prediction of the gene
product. Biochem Biophys Res Commun 301:280–286. pii: S0006291X02030255
de Oliveira Simões LC, da Silva RR, de Oliveira Nascimento CE, Boscolo M, Gomes E, da Silva R
(2019) Purification and physicochemical characterization of a novel thermostable xylanase
secreted by the fungus Myceliophthora heterothallica F.2.1.4. Appl Biochem Biotechnol 188:
991–1008. https://doi.org/10.1007/s12010-019-02973-8
Osma JF, Toca-Herrera JL, Rodríguez-Couto S (2010) Uses of laccases in the food industry.
Enzyme Res 2010:918761–918761. https://doi.org/10.4061/2010/918761
Otzen DE, Christiansen L, Schülein M (1999) A comparative study of the unfolding of the
endoglucanase Cel45 from Humicola insolens in denaturant and surfactant. Protein Sci 8:
1878–1887. https://doi.org/10.1110/ps.8.9.1878
Pack SP, Yoo YJ (2004) Protein thermostability: structure-based difference of amino acid between
thermophilic and mesophilic proteins. J Biotechnol 111:269–277. https://doi.org/10.1016/j.
jbiotec.2004.01.018. pii: S0168165604001841
Pakarinen A, Haven MO, Djajadi DT, Várnai A, Puranen T, Viikari L (2014) Cellulases without
carbohydrate-binding modules in high consistency ethanol production process. Biotechnol
Biofuels 7:27. https://doi.org/10.1186/1754-6834-7-27
Percival Zhang YH, Himmel ME, Mielenz JR (2006) Outlook for cellulase improvement: screening
and selection strategies. Biotechnol Adv 24:452–481. https://doi.org/10.1016/j.biotechadv.
2006.03.003
PetříčkováA, Sosedov O, Baum S, Stolz A, MartínkováL (2012) Influence of point mutations near
the active site on the catalytic properties of fungal arylacetonitrilases from Aspergillus niger and
Neurospora crassa. J Mol Catal B Enzymatic 77:74–80. https://doi.org/10.1016/j.molcatb.
2012.01.005
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004)
UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem
25:1605–1612. https://doi.org/10.1002/jcc.20084
Ping L et al (2018) Production and characterization of a novel acidophilic and thermostable
xylanase from Thermoascus aurantiacus. Int J Biol Macromol 109:1270–1279. https://doi.
org/10.1016/j.ijbiomac.2017.11.130
Prakash O, Mahabare K, Yadav KK, Sharma R (2019) Fungi from extreme environments: a
potential source of laccases Group of Extremozymes. In: Tiquia-Arashiro SM, Grube M (eds)
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 679

Fungi in extreme environments: ecological role and biotechnological significance. Springer,
Cham, pp 441–462. https://doi.org/10.1007/978-3-030-19030-9_22
Prates JAM, Tarbouriech N, Charnock SJ, Fontes CMGA, LsMA F, Davies GJ (2001) The structure
of the feruloyl esterase module of xylanase 10B from Clostridium thermocellum provides
insights into substrate recognition. Structure 9:1183–1190. https://doi.org/10.1016/S0969-
2126(01)00684-0
Qiu J, Wilkens C, Barrett K, Meyer AS (2020) Microbial enzymes catalyzing keratin degradation:
classification, structure, function. Biotechnol Adv 44:107607. https://doi.org/10.1016/j.
biotechadv.2020.107607
Raczynska JE, Vorgias CE, Antranikian G, Rypniewski W (2011) Crystallographic analysis of a
thermoactive nitrilase. J Struct Biol 173:294–302. https://doi.org/10.1016/j.jsb.2010.11.017
Romdhane IB-B, Fendri A, Gargouri Y, Gargouri A, Belghith H (2010) A novel thermoactive and
alkaline lipase from Talaromyces thermophilus fungus for use in laundry detergents. Biochem
Eng J 53:112–120. https://doi.org/10.1016/j.bej.2010.10.002
RuckáL et al (2020) Plant nitrilase homologues in fungi: phylogenetic and functional analysis with
focus on nitrilases in Trametes versicolor and Agaricus bisporus. Molecules 25:3861. https://
doi.org/10.3390/molecules25173861
Sandgren M, Stahlberg J, Mitchinson C (2005) Structural and biochemical studies of GH family
12 cellulases: improved thermal stability, and ligand complexes. Prog Biophys Mol Biol 89:
246–291. https://doi.org/10.1016/j.pbiomolbio.2004.11.002. pii: S0079-6107(04)00167-1
Santos RMDB, Firmino AAP, de SáCM, Felix CR (1996) Keratinolytic activity of Aspergillus
fumigatus fresenius. Curr Microbiol 33:364–370. https://doi.org/10.1007/s002849900129
Savile CK et al (2010) Biocatalytic asymmetric synthesis of chiral amines from ketones applied to
Sitagliptin manufacture. Science 329:305. https://doi.org/10.1126/science.1188934
Sayer C, Martinez-Torres RJ, Richter N, Isupov MN, Hailes HC, Littlechild JA, Ward JM (2014)
The substrate specificity, enantioselectivity and structure of the (R)-selective amine : pyruvate
transaminase from Nectria haematococca. FEBS J 281:2240–2253. https://doi.org/10.1111/
febs.12778
Selinheimo E, Saloheimo M, Ahola E, Westerholm-Parvinen A, Kalkkinen N, Buchert J, Kruus K
(2006) Production and characterization of a secreted, C-terminally processed tyrosinase from
the filamentous fungus Trichoderma reesei. FEBS J 273:4322–4335. https://doi.org/10.1111/j.
1742-4658.2006.05429.x
Shaikh FA, Withers SG (2008) Teaching old enzymes new tricks: engineering and evolution of
glycosidases and glycosyl transferases for improved glycoside synthesis. Biochem Cell Biol 86:
169–177. https://doi.org/10.1139/o07-149
Sheldon RA, Woodley JM (2018) Role of biocatalysis in sustainable chemistry. Chem Rev 118:
801–838. https://doi.org/10.1021/acs.chemrev.7b00203
Shen J-W, Cai X, Dou B-J, Qi F-Y, Zhang X-J, Liu Z-Q, Zheng Y-G (2020) Expression and
characterization of a CALB-type lipase from Sporisorium reilianum SRZ2 and its potential in
short-chain flavor ester synthesis. Front Chem Sci Eng 14:868–879. https://doi.org/10.1007/
s11705-019-1889-x
Shibata N, Suetsugu M, Kakeshita H, Igarashi K, Hagihara H, Takimura Y (2017) A novel GH10
xylanase from Penicillium sp. accelerates saccharification of alkaline-pretreated bagasse by an
enzyme from recombinant Trichoderma reesei expressing Aspergillus β-glucosidase.
Biotechnol Biofuels 10:278. https://doi.org/10.1186/s13068-017-0970-2
Shoseyov O, Shani Z, Levy I (2006) Carbohydrate binding modules: biochemical properties and
novel applications. Microbiol Mol Biol Rev 70:283–295. https://doi.org/10.1128/mmbr.
00028-05
Sitarz AK, Mikkelsen JD, Meyer AS (2016) Structure, functionality and tuning up of laccases for
lignocellulose and other industrial applications. Crit Rev Biotechnol 36:70–86. https://doi.org/
10.3109/07388551.2014.949617
680 I. Mohsin and A. C. Papageorgiou

Song L, Tsang A, Sylvestre M (2015) Engineering a thermostable fungal GH10 xylanase, impor-
tance of N-terminal amino acids. Biotechnol Bioeng 112:1081–1091. https://doi.org/10.1002/
bit.25533
Songulashvili G, Flahaut S, Demarez M, Tricot C, Bauvois C, Debaste F, Penninckx MJ (2016)
High yield production in seven days of Coriolopsis gallica 1184 laccase at 50 L scale; enzyme
purification and molecular characterization. Fungal Biol 120:481–488. https://doi.org/10.1016/
j.funbio.2016.01.008
Stauch B, Fisher SJ, Cianci M (2015) Open and closed states of Candida antarctica lipase B:
protonation and the mechanism of interfacial activation. J Lipid Res 56:2348–2358. https://doi.
org/10.1194/jlr.M063388
Sun Q et al (2016) Heterologous production of an acidic thermostable lipase with broad-range pH
activity from thermophilic fungus Neosartorya fischeri P1. J Biosci Bioeng 122:539–544.
https://doi.org/10.1016/j.jbiosc.2016.05.003
Szijarto N, Siika-Aho M, Tenkanen M, Alapuranen M, Vehmaanpera J, Reczey K, Viikari L (2008)
Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of
Melanocarpus albomyces. J Biotechnol 136:140–147. https://doi.org/10.1016/j.jbiotec.2008.
05.010
Takashima S, Iikura H, Nakamura A, Hidaka M, Masaki H, Uozumi T (1998) Isolation of the gene
and characterization of the enzymatic properties of a major exoglucanase of Humicola grisea
without a cellulose-binding domain. J Biochem 124:717–725
Takashima S, Iikura H, Nakamura A, Hidaka M, Masaki H, Uozumi T (1999) Comparison of gene
structures and enzymatic properties between two endoglucanases from Humicola grisea.J
Biotechnol 67:85–97
Takashima S, Ohno M, Hidaka M, Nakamura A, Masaki H, Uozumi T (2007) Correlation between
cellulose binding and activity of cellulose-binding domain mutants of Humicola grisea
cellobiohydrolase 1. FEBS Lett 581:5891–5896. https://doi.org/10.1016/j.febslet.2007.11.
068. pii: S0014-5793(07)01222-7
Taylor TJ, Vaisman II (2010) Discrimination of thermophilic and mesophilic proteins. BMC Struct
Biol 10(suppl 1):S5. https://doi.org/10.1186/1472-6807-10-s1-s5. pii: 1472-6807-10-S1-S5
Thomsen M, Skalden L, Palm GJ, Höhne M, Bornscheuer UT, Hinrichs W (2014) Crystallographic
characterization of the (R)-selective amine transaminase from Aspergillus fumigatus. Acta
Crystallogr D Biol Crystallogr 70:1086–1093. https://doi.org/10.1107/S1399004714001084
Topakas E, Stamatis H, Biely P, Christakopoulos P (2004) Purification and characterization of a
type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile.
Appl Microbiol Biotechnol 63:686–690. https://doi.org/10.1007/s00253-003-1481-6
Trivedi S, Gehlot HS, Rao SR (2006) Protein thermostability in Archaea and Eubacteria. Genet Mol
Res 5:816–827
Underlin EN, Frommhagen M, Dilokpimol A, van Erven G, de Vries RP, Kabel MA (2020)
Feruloyl esterases for biorefineries: subfamily classified specificity for natural substrates.
Front Bioeng Biotechnol 8:332. https://doi.org/10.3389/fbioe.2020.00332
Varrot A et al (2003) Structural basis for ligand binding and processivity in cellobiohydrolase
Cel6A from Humicola insolens. Structure 11:855–864. pii: S0969212603001242
Velasco J et al (2019) Heterologous expression and functional characterization of a GH10
endoxylanase from Aspergillus fumigatus var. niveus with potential biotechnological applica-
tion. Biotechnol Rep 24:e00382. https://doi.org/10.1016/j.btre.2019.e00382
Verma ML (2019) Biotechnological applications of lipases in flavour and fragrance ester
production. In: Arora PK (ed) Microbial Technology for the Welfare of society. Springer,
Singapore, pp 1–24. https://doi.org/10.1007/978-981-13-8844-6_1
Vlasenko E, Schülein M, Cherry J, Xu F (2010) Substrate specificity of family 5, 6, 7, 9, 12, and
45 endoglucanases. Bioresour Technol 101:2405–2411. https://doi.org/10.1016/j.biortech.
2009.11.057
Voutilainen SP, Boer H, Linder MB, Puranen T, Rouvinen J, Vehmaanperä J, Koivula A (2007)
Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random
27 Fungal Extremozymes: A Potential Bioresource for Green Chemistry 681

mutagenesis to improve its thermostability. Enzyme Microbiol Technol 41:234–243. https://doi.
org/10.1016/j.enzmictec.2007.01.015
Voutilainen SP, Murray PG, Tuohy MG, Koivula A (2010) Expression of Talaromyces emersonii
cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its
thermostability and activity. Protein Eng Des Sel 23:69–79. https://doi.org/10.1093/protein/
gzp072. pii: gzp072
Wang XJ, Peng YJ, Zhang LQ, Li AN, Li DC (2012) Directed evolution and structural prediction of
cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum. Appl Microbiol
Biotechnol 95:1469–1478. https://doi.org/10.1007/s00253-011-3799-9
Watanabe M, Yoshida E, Fukada H, Inoue H, Tokura M, Ishikawa K (2015) Characterization of a
feruloyl esterase B from Talaromyces cellulolyticus. Biosci Biotechnol Biochem 79:1845–
1851. https://doi.org/10.1080/09168451.2015.1058700
Wilson DB (2009) Cellulases and biofuels. Curr Opin Biotechnol 20:295–299. https://doi.org/10.
1016/j.copbio.2009.05.007
Wu I, Arnold FH (2013) Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases
hydrolyze cellulose efficiently at elevated temperatures. Biotechnol Bioeng 110:1874–1883.
https://doi.org/10.1002/bit.24864
Wu Y et al (2013) Isolation and characterization of Gibberella intermedia CA3-1, a novel and
versatile nitrilase-producing fungus. J Basic Microbiol 53:934–941. https://doi.org/10.1002/
jobm.201200143
Wu W-L et al (2017) The discovery of novel heat-stable keratinases from Meiothermus taiwanensis
WR-220 and other extremophiles. Sci Rep 7:4658. https://doi.org/10.1038/s41598-017-
04723-4
Wu L et al (2018) Characterization of immobilized tyrosinase –an enzyme that is stable in organic
solvent at 100 C. RSC Adv 8:39529–39535. https://doi.org/10.1039/C8RA07559J
Wu X, Zhang Q, Zhang L, Liu S, Chen G, Zhang H, Wang L (2020) Insights into the role of
exposed surface charged residues in the alkali-tolerance of GH11 xylanase. Front Microbiol 11:
872. https://doi.org/10.3389/fmicb.2020.00872
Xu Z, Cai T, Xiong N, Zou S-P, Xue Y-P, Zheng Y-G (2018) Engineering the residues on “A”
surface and C-terminal region to improve thermostability of nitrilase. Enzym Microb Technol
113:52–58. https://doi.org/10.1016/j.enzmictec.2018.03.001
Yennamalli RM, Rader AJ, Wolt JD, Sen TZ (2011) Thermostability in endoglucanases is fold-
specific. BMC Struct Biol 11:10. https://doi.org/10.1186/1472-6807-11-10
Yin X, Li J-F, Wang J-Q, Tang C-D, Wu M-C (2013) Enhanced thermostability of a mesophilic
xylanase by N-terminal replacement designed by molecular dynamics simulation. J Sci Food
Agric 93:3016–3023. https://doi.org/10.1002/jsfa.6134
Yoshitake AK, Katayama YO, Nakamura MA, Iimura YO, Kawai SH, Morohoshi NO (1993)
N-linked carbohydrate chains protect laccase III from proteolysis in Coriolus versicolor.
Microbiology 139:179–185. https://doi.org/10.1099/00221287-139-1-179
Zerva A, Pentari C, Grisel S, Berrin JG, Topakas E (2020) A new synergistic relationship between
xylan-active LPMO and xylobiohydrolase to tackle recalcitrant xylan. Biotechnol Biofuels 13:
142. https://doi.org/10.1186/s13068-020-01777-x
Zhang SB, Pei XQ, Wu ZL (2012) Multiple amino acid substitutions significantly improve the
thermostability of feruloyl esterase A from Aspergillus niger. Bioresour Technol 117:140–147.
https://doi.org/10.1016/j.biortech.2012.04.042
Zheng Y-Y, Guo X-H, Song N-N, Li D-C (2011) Thermophilic lipase from Thermomyces
lanuginosus: gene cloning, expression and characterization. J Mol Catal B Enzymatic 69:
127–132. https://doi.org/10.1016/j.molcatb.2011.01.006
682 I. Mohsin and A. C. Papageorgiou

Fungal Extremozymes in Green Chemistry 28
Ajay Nair, Archana S. Rao, K. Nivetha, Prakruthi Acharya,
Aneesa Fasim, Veena S. More, K. S. Anantharaju, and Sunil S. More
Abstract
An endeavor to accommodate sustainability within chemical manufacturing led
to the recently popularized green chemistry. For a chemical process to be green, it
must meet the following criteria: avoiding usage of non-biodegradable materials,
all industrial processing transformations should lead to minimal waste accumula-
tion, the processes must be cost-effective, both energetically and economically,
etc. This is why biocatalysis using enzymes brought about a monumental shift
toward a greener chemical process. Since its initial introduction, enzymes have
mediated almost all industrial sectors, from pharmaceutics to food industry, pulp,
textile, agro-waste management, bioremediation, etc. Recently, metagenomic
approaches have led to the discovery of enzymes from extremophilic organisms
that thrive under conditions considered optimal for most life on earth. Most of
these extreme-loving organisms are bacterial or fungal in origin. Regardless of
their origin, it has been demonstrated that extremozymes produced by them are
far more efficient and resilient under the harsh industrial conditions. This makes
them preferred candidates to be used as biocatalysts. Recently, several
extremozymes of fungal origin have gained interest as potential industrial
biotransformants. This chapter discusses the applications of fungal
extremozymes, improves on the synthesis, bioconversions, and bioremediation
processes.
A. Nair · A. S. Rao · K. Nivetha · P. Acharya · A. Fasim · S. S. More (*)
School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, Karnataka, India
V. S. More
Department of Biotechnology, Sapthagiri College of Engineering, Bangalore, Karnataka, India
K. S. Anantharaju
Department of Chemistry, Dayananda Sagar College of Engineering, Bangalore, Karnataka, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_28
683

Keywords
Bioremediation · Fungal extremozymes · Biocatalysis · Extremophiles
28.1 Introduction
Microorganisms are by far the most prevalent species inhabiting our planet. To
understand them is to understand the evolutionary timeline of biological causation;
to understand them is to realize the boundary conditions that define life. Now there
are microorganisms (such as mesophiles) that thrive within the prescribed life-
promoting parameters. But there are others (such as extremophiles) which thrive
under conditions that are usually considered optimal. Of the several strategies
employed for survival under extreme conditions, the production of specialized
enzymes called extremozymes is ranked very high. In recent times, it has therefore
been a persistent endeavor to explore extremophiles toward the functional character-
ization of more and more extremozymes. The largely unexplored variety of
microbes- the extremophiles and the mechanisms involved in their production of
extremozymes under extreme conditions supporting their survival.
Enzymes play a significant role commercially as an ecofriendly alternative to
synthetic chemicals, in a wide variety of industries such as pharmaceuticals, food
and beverage production, biofuels, and chemical production. The inherent effects of
the microbial enzymes produced by mesophiles (moderate temperature-dwelling)
are found to be of profound importance in several processes such as antibiotic
production, organic acid production, flavor enhancement, chiral compound produc-
tion, as emulsifiers, and many more. Likewise, there are several industrial protocols
involving harsh conditions which can be endured by specific enzymes such as
extremozymes that are found to be produced by the extremophiles. Several
properties of the extremozymes such as reproducibility, consistency, and high yields
must be explored to understand their wide range of applications in several industries
(Sarmiento et al. 2015). Such extremozymes demonstrated a wide range of activity,
efficiency, and stability under extreme conditions of temperature, pH, salt concen-
tration, etc. (Fig. 28.1).
The industrial processes involve reactions carried out under extreme conditions of
temperature, pressure, pH, etc. Enzyme catalysis, in the recent past, has been
preferred over chemical reagents as far as bioprocessing is concerned. This is due
to the ability of enzymes to carry out processes, which are far greener and more cost-
effective. However, the enzymes secreted by microorganisms thriving within the
realms of normal growth parameters fail to withstand the harsh industrial conditions.
This is why chemical processes are preferred over enzyme catalysis. But using
synthetic chemicals would have inclusions such as high cost, toxicity, environmental
pollution from industrial effluents, etc. Therefore, a desperate need to make indus-
trial processes more sustainable, green, without compromising on the efficacy of the
process led to the discovery of extremozymes.
684 A. Nair et al.

Several extremozymes have been extensively studied and employed for various
reactions. Most of the extremozymes known are bacterial in origin. There are again a
large number of fungal extremophiles which can be explored more to employ them
successfully for green chemistry. Many researches carried out to explore fungal
extremophiles indicate the production of extremozymes of various factors, which are
beneficial for chemical conversions and hence used in different industries. The table
below summarizes the use of various extremozymes fungal in origin for different
industrial processes (Table 28.1).
Since their discovery, extremozymes have created a monumental shift in the area
of bioprocessing using ecofriendly alternatives. Not only do these extreme enzymes
are more efficient catalytically than their mesophilic counterparts (optimal enzymatic
activity for a given condition) but also each extremozyme also seems to possess an
innate plasticity that allows it to be functional at varying physicochemical
parameters. Therefore, it is worthwhile to continue the pursuit of characterizing
more and more extremozymes which can be further added to the list of industrially
important enzymes. The following sections discuss the applications of fungal
extremozymes in different reactions involved in various industrial processes.
Fungal
Extremozymes
Polyextremophillic
Enzymes
Halophillic
Enzymes
Psychrophillic
Enzymes
Thermophillic
Enzymes
Peizophillic
Enzymes
Acidophillic
Enzymes
Alkalophillic
Enzymes
Fig. 28.1 Fungal enzymes from different extremophilic conditions
28 Fungal Extremozymes in Green Chemistry 685

Table 28.1 Applications of fungal extremozymes
Fungal
extremozymes Producer
Extreme
condition Applications Reference
Amylases and
cellulases
Cystofilobasidium
capitatum,Mrakia
blollopis,
Rhodotorula
glacialis,
Tetracladium sp.,
Saccharomyces
cerevisiae
Low
temperature
Bioethanol
industries
Wine
production
Food industries
Tiquia-
Arashiro and
Grube (2019)
Cell wall-
degrading
enzymes,
antifungal
metabolites
Aureobasidium
pullulans and
Rhodotorula
mucilaginosa,
Papiliotrema
laurentii
Low
temperature
Cold
preservation of
fruits and
vegetables
Tiquia-
Arashiro and
Grube (2019)
Conversion of
albendazole
(a prodrug) to
albendazole
sulfoxide
Rhizomucor
pusillus
High
temperature
Pharmaceutical
industries
Tiquia-
Arashiro and
Grube (2019)
Ferulic acid to
guaiacol
Sporotrichum
thermophile
High
temperature
Pharmaceutical
industries
Tiquia-
Arashiro and
Grube (2019)
Biotransformation
of steroids, steroid
production
Acremonium
alabamensis and
Talaromyces
emersonii
High
temperature
Pharmaceutical
industries
Tiquia-
Arashiro and
Grube (2019)
Cellulases Myceliophthora
thermophile and
Thielavia
terrestris
High
temperature
Biofuel, food
industries
Tiquia-
Arashiro and
Grube (2019)
Xylanases S. thermophile High
temperature
Biomass
degradation,
biofuels, paper,
pulp
Tiquia-
Arashiro and
Grube (2019)
Cellulases,
xylanases,
esterases,
glucosidases,
mannanases, and
phytases
Chaetomium
thermophilum,
Malbranchea
cinnamomea,
Paecilomyces
thermophile,
Thermoascus
aurantiacus, and
Thermomyces
lanuginosus
High
temperature
Bioconversions Tiquia-
Arashiro and
Grube (2019)
Proteases Aspergillus flavus High salt
concentration
Food, textile,
paper and pulp
industries
Tiquia-
Arashiro and
Grube (2019)
(continued)
686 A. Nair et al.

28.2 Fungal Extremozymes in Green Chemistry
28.2.1 Psychrophilic Enzymes
Psychrophilic fungi are microorganisms that are known to efficiently adapt at very
cold temperatures. These microorganisms are able to grow from 20 to 10 C and
are termed as Psychrophiles. However, they cannot grow above 15 C, and
psychrotolerants grow around 20–25 C, but have been shown to be metabolically
active even at 0 C. Among the psychrophilic fungal species, certain varieties of
lichenised fungi Lecidia acarospora and Umbilicaria and other cold-adapted yeasts
such as Debaryomyces hansenii, Cryptococcus sp. and Rhodotorula mucilaginosa,
Crystobasidium larynges, Vishniacozyma victoriae, and Papiliotrema laurentii on a
major portion and on a minor portion Candida, Dioszegia, Makia, and Naganishia,
Table 28.1 (continued)
Fungal
extremozymes Producer
Extreme
condition Applications Reference
Amylases Engyodontium
album, A. gracilis,
A. penicillioides
High salt
concentration
Food, textile,
paper and pulp
industries
Tiquia-
Arashiro and
Grube (2019)
Cellulases A. terreus,
Penicillium sp.
High salt
concentration
Food, textile,
paper and pulp
industries
Tiquia-
Arashiro and
Grube (2019)
Beta mannanases Penicillium
oxalicum GZ-2,
Aureobasidium
pullulans, Bispora
antennata
Acidic Food, feed, and
detergent
industries,
paper
Liao et al.
(2014), Li
et al. (1993)
and Liao et al.
(2012)
Hemicellulase Teratospaeria
acidotherma,
A. niger
Acidic Food, feed and
detergent
industries,
paper
Hatzinikolaou
et al. (2005)
and Isobe et al.
(2013)
α-Amylases Thermomyces
lanuginosus,
Saitozyma
Acidic Industrial
processes
Kunamneni
et al. (2005)
Laccases Trametes hirsuta
Bm-2
Acidic Pulp and paper
industry,
biosensors, fuel
cells, stabilizers
Ghindilis
(2000), Ben
Younes et al.
(2007) and
Zapatae
Castillo et al.
(2012)
Proteases A. jilinensis Alkaline Food and
beverages
Wu et al.
(2006)
Cellulase and
hemicellulase
P. citrinum Alkaline Paper, pulp,
polymer
industries
Raghukumar
et al. (2004)
28 Fungal Extremozymes in Green Chemistry 687

have been reported for study and to understand their extremophilic mechanisms
responsible for them to strive in a very cold environments (Tiquia-Arashiro and
Grube 2019). Metagenomics since its inception has offered a culture-free platform to
characterize and isolate microorganisms that were once not cultivable. This helps in
identifying specific extremophilic fungal species that are of great importance from an
industrial point of view. Metagenomic analysis in extremely cold regions has
identified less than 10% of microbial eukaryotes (predominantly occupied by the
genera Ascomycetes and Basidiomycetes fungal species) in comparison with the
prokaryotes in most of the extremophilic regions around the world. Meta-barcoding,
by utilizing internal transcribed spacers (ITS) and D1/D2 domain of large rDNA as
barcode sequences, is said to be a significant molecular technique in successfully
identifying fungal species from Antarctic regions (Tiquia-Arashiro and Grube 2019).
The cold-adapted enzymes are involved in many chemical transformations that
need to be made at lower temperatures. Low temperature bioprocesses are far more
economical as they cut down on the energy cost. The low temperature processes also
avoid the growth of unwanted mesophilic microbes.
The hydrolytic enzymes of the psychrophilic yeasts such as amylases, lipases, and
proteases are found to play a potential role in several industrial processes. Amylases
and cellulases produced by psychrophiles such as Cystofilobasidium capitatum,
Mrakia blollopis, Rhodotorula glacialis,Tetracladium sp. are found to play a
significant role in bioethanol industries. This is mainly due to the ability of these
enzymes to remain highly active at very low temperatures, which helps in making
the process cost-effective. Psychrophilic fungi are of major importance as they can
be potentially used during fermentation processes at very low temperatures, which
helps in improving the flavor of fermented foods. They can be also used in the
production of alcoholic beverages, bread, and dairy products. There is an increasing
demand in recent times for alcoholic beverages with lower alcoholic content as they
are found to have lesser negative impacts on the aroma and flavor. Although this
could be done by several physical and chemical techniques, yet an organic approach
to reduce the alcohol content in wines is to use psychrophilic Saccharomyces and
other yeasts, which are potentially used in the process of must fermentation at
decreased temperatures (Tiquia-Arashiro and Grube 2019).
Spanish wineries were found to use this technique by using psychrotolerant yeast
Candida sake to ferment concentrated natural must at 12 C. Further, it was noticed
that this process had avoided the typical lag phase of the psychrophilic S. cerevisiae
during the fermentation process, thereby reducing the ethanol and glycerol content in
the wines. Also, the use of psychrophilic fungi in fermentation processes not only
improved the flavor but has also proved to avoid microbial contamination. This was
proved by observing successful continuous fermentation at 5–15 C, while using a
psychrophilic strain S. cerevisiae being immobilized onto apple cuts. S. cerevisiae
(AXAZ-1) is also used in beer production as it is known to improve the quality of
beer, as it is responsible to provide acceptable quantities of volatile acids and
decreased diacetyl and polyphenol contents in the beer, when compared to other
commercial beers (Tiquia-Arashiro and Grube 2019).
688 A. Nair et al.

Psychrophilic fungi also play a significant role in degrading a wide range of
hydrocarbons. Psychrophilic yeasts such as Cryptococcus terreus and Rhodotorula
sp. identified from Alps regions are found to degrade phenol and phenol-related
mono-aromatic compounds. This is also done by utilizing immobilized yeast cells at
low temperatures, around 10 C. Mrakia blollopis, an Antarctic yeast is used in the
treatment of contaminant materials as it is known to possess high biological oxygen
demand (BOD) removal rate in wastewaters at low temperatures. M. blollopis is also
known to ferment cellulosic biomass, in fermentation industries. They also may play
a role in the bioremediation of contaminated water and soils (Tiquia-Arashiro and
Grube 2019).
Different psychrophilic yeast species have been potentially used for the biological
control of fungal phytopathogens, due to various control mechanisms offered by the
yeasts. They mainly include induction of host resistance, exhibition of
mycoparasitism, microbial competition for space and nutrients and production of
antifungal compounds and cell wall-degrading enzymes. Psychrophilic yeasts also
play a role in post-harvest biocontrol from various phytopathogens. Aureobasidium
pullulans and Rhodotorula mucilaginosa are known to thrive in extremely cold
temperatures. Antifungal mechanisms of these psychrophilic fungal species help
them to reduce fruit decay to about 33% produced by Penicillium expansum in
refrigerated packages. Other psychrophiles such as R. mucilaginosa and Cryptococ-
cus laurentii (now Papiliotrema laurentii) are found to be promising isolates in
reducing the decay of fruits and vegetables, especially cherry tomatoes, by acting
against Botrytis cinerea and Penicillium expansum, which are the common fungal
contaminants (Tiquia-Arashiro and Grube 2019).
Psychrophilic oleaginous yeasts isolated from Tibetan Plateau are known as
potential biodiesel sources as they are capable of producing lipids from several
economical substrates by the process of fermentation under aerobic conditions and
decreased temperatures. Some of these psychrophiles include Yarrowia lipolytica,
Cryptococcus species (accumulates over 30% of lipid content), Rhodotorula
glacialis (with 68% of lipid biomass) R. glutinis and R. glacialis (Tiquia-Arashiro
and Grube 2019).
Candida albicans produced lipase with optimal temperature of 15 C (Lan et al.
2011). Another yeast Glaciozyma antartica living in temperature range of 4–200 C
produces many cold-adapted enzymes, viz., chitinase, protease, etc., and antifreeze
proteins (Boo et al. 2013). Yeasts like fungus Guehomyces pullulans produce β-D-
galactosidases active at cold temperatures.
Several fungal species (such as Penicillium, Alternaria, Phoma, etc.) isolated
from the Antarctic have also shown, though minimally, to produce psychrophilic
xylanases (Bradner et al. 1999). Some of the characteristic features underlying
psychrophilic xylanases include: a low temperature optimum with improved cata-
lytic efficiency, increased flexibility, etc. (Collins et al. 2003). Protein quenching
studies demonstrated conclusively that the cold-adapted xylanases have increased
flexibility and increased low temperature activity. The study therefore proposed that
perhaps xylanase activity under psychrophilicity requires a tradeoff between enzy-
matic flexibility and stability of the molecular structure. This is achieved by reducing
28 Fungal Extremozymes in Green Chemistry 689

the number of salt bridges and enhanced exposure of the hydrophobic residues (Van
Petegem et al. 2002).
Hence, exploring, identifying, and understanding the metabolic potential of
psychrophilic fungal communities aid in discovering several species and their
novel genes that are required in wide varieties of applications.
28.2.2 Thermophilic Enzymes
Thermophilic fungi belong to a small group of mycota that are capable of growing at
an increased temperature of at or beyond 50 C, while thermotolerant fungi grow at
an optimum of 20–55 C. Thermophily in these fungi is comparatively less extreme
when compared to other archaeal and eubacterial species that survive at areas where
the temperature goes beyond 100 C, such as hot springs, hydrothermal vents, and
solfatara fields. Thermophilic fungi grow in various natural habitats such as animal/
municipal refuse, plant/ mushroom compost, bird nest materials storage products,
desert soils, alkalescent thermal springs, and other organic matter accumulations/
piles of agricultural and forestry products. Thermophilic fungi are capable of
surviving under extreme stress conditions such as desiccation and oxygen levels.
They are the primary components of microflora in these diverse range of locations,
as they provide an aerobic, warm, and a humid environment that are found to be ideal
for fungal growth (Tiquia-Arashiro and Grube 2019).
Saprophytic mesophiles exhibit exothermic reactions, which increase the temper-
ature of the substratum to about 40 C. This elevated temperature provides a
favorable environment for the germination of spores of the thermophilic fungi.
Consequently, it is observed that these thermophiles to outgrow the mesophilic
microflora. The earliest known thermophilic fungus that was identified on bread
was found to be Mucor pusillus. Further, Thermomyces lanuginosus, another ther-
mophilic fungus was isolated from potato. Later, a wide range of thermophilic fungi
were isolated and were found to belong to a wide variety of genera such as
Ascomycotina, Deuteromycotina, and Mastigomycotina (Tiquia-Arashiro and
Grube 2019).
Cellulose is the most abundant non-fossil carbon source available on earth. Its
primary constituents are of great importance in biofuel production and food
industries. Although several options such as mechanical treatment, heat treatment,
or acid treatment are industrially available for the breakdown of cellulose into its
principal constituents, yet enzymatic breakdown is better favored on a large-scale,
since it is an ecofriendly process. Several microbial cellulases are capable of
breaking down cellulose, yet thermophilic enzymes from thermophilic fungi hold
a better advantage over the other enzymes. This is because, during the breakdown
process as the temperature increases, cellulose tends to swell up. Cellulose break-
down is much easier than when performed at a lower temperature. Hence, the
thermophilic enzymes are favorable for cellulose breakdown, whose activity is
high at higher temperatures. Thus, several thermophilic fungi have been isolated
from various locations and their cellulases are characterized at structural and
690 A. Nair et al.

functional levels. Studies suggest that the genomes of a few thermophilic fungi
(Myceliophthora thermophile and Thielavia terrestris) are sequenced and their
enzymes were found to be highly efficient in hydrolyzing several major
polysaccharides (Tiquia-Arashiro and Grube 2019).
Thermostable enzymes are highly desirable commodities and are well exploited
in industrial bioprocesses due to several advantages. Some of the commonly
explored thermostable enzymes are produced from thermophilic fungi such as
Sporotrichum thermophile, Scytalidium,Thermotoga, and Thermoascus. Several
strains of S. thermophile (thermophilic mold) are known to produce highly active
xylanases that are capable of catalyzing the hydrolysis of several types of substituted
and non-substituted xylans. Xylanases from thermophilic fungi are of great demand
in the industrial processes such as biofuel, food, paper and pulp production pro-
cesses. This is due to its thermostability at higher temperatures, during which the
time taken for hydrolysis is relatively reduced. Also, at higher temperatures, the
viscosity of the medium decreases, which favors the process of hydrolysis. Thermo-
stable xylanases are potentially used for the biomass conversion of lignocellulosics
into fermentable sugars that are useful for several bioprocesses and for the sacchari-
fication of agrowastes such as corn, cobs, bruce, straw, spruce biomass, etc. Several
other examples of thermophilic fungi that are capable of producing industrially
important enzymes such as cellulases, xylanases, esterases, glucosidases,
mannanases, and phytases include Chaetomium thermophilum,Malbranchea
cinnamomea,Paecilomyces thermophile,Thermoascus aurantiacus, and
Thermomyces lanuginosus(Tiquia-Arashiro and Grube 2019).
Much of that we understand about Xylanases has come from fungi and bacteria
that are majorly found in and around the mesophilic ranges of life; be it temperature
or pH (Subramaniyan and Prema 2002). However recently, the overall effort to
characterize extremophiles has produced a range of extremophilic xylanases that are
far more resilient and efficient than their mesophilic counterparts. Xylanases isolated
from thermophilic molds have been shown to possess amplified kinetic efficiency
and stability at high temperatures, with complementary activity profiles (Ghatora
et al. 2007). A recent study suggested that using a combinatorial approach involving
mannanolytic enzymes, galactosidases, acetyl glucomannan esterase, mannosidase,
glucosidase for a comprehensive hydrolysis of sugars (Luonteri et al. 1998).
Investigations looking at the structural conformations, sequence alignments between
extremophilic and mesophilic xylanases revealed that there is a lot of similarity
between the two except for a few differences. Xylanases isolated from thermophilic
fungi have been shown to improve thermostability by undergoing few necessary
structural modifications. These include: alterations to the number of salt bridges and
hydrogen bonds, increase in the number of charged surface residues, presence of
tandem repeats, etc. (Turunen et al. 2002).
Hence, thermophilic fungi can be well-exploited commercially in pharmaceutical
industry for various applications, especially in an ecofriendly and an economic point
of view.
28 Fungal Extremozymes in Green Chemistry 691

28.2.3 Halophilic Enzymes
The halophiles are microorganisms which can thrive in areas of extremely high salt
concentrations such as oceans and brackish salt lakes. Halophiles and halotolerants
inhabit such regions of high salt concentrations (3–5% NaCl). They also play a major
role in participating in biogeochemical cycles such as carbon, nitrogen, phosphorus,
sulfur at areas under extreme conditions. Industrially, salted foods are produced with
the addition of increased amount of salts, mainly for the flavor and preservation of
the food products. To these industries, halophilic microorganisms appear to be of
great importance especially due to their ability to survive under these extreme
hypersaline conditions and to produce metabolites that are useful for various bio-
technological applications. Hortaea werneckii (halotolerant fungi), Sulfolobus
solfataricus, Haloarcula sp., Cladosporium sp., Eurotium,Emerciella, Aspergillus,
Penicillium, Wallemia ichthyophaga (obligate halophile), and other melanized and
non-melanized fungal species are some of the common halophilic fungal species
available in brackish hypersaline environments and some of their genomes have
been explored for a variety of industrial applications. The proteomic analysis of
halophilic fungi indicates lower hydrophobicity, lower number of cytosine residues,
and increased repetition of acidic residues (Tiquia-Arashiro and Grube 2019).
The survival mechanism of prokaryotes in high saline conditions is mainly due to
hyperaccumulation of potassium ions in hypersaline environments. On the other
hand, halophilic fungi are incapable of tolerating high intracellular ion
concentrations, and hence their survival mechanism varies from that of halophilic
prokaryotes. They tend to produce high amounts of organic solutes, glycerol, and
trehalose, which helps them to maintain positive turgor pressure at increased salinity.
Also, the fungal metabolites are extracellular unlike that of halophilic prokaryotes
that exhibit better performance in both quantity and quality. Thus they are better
preferred for several industrial applications. There are several parameters based on
which the growth and viability of halophilic fungi are assessed when used in
industries that include sampling time, geographical distribution/location, dissolved
oxygen levels in the surrounding, availability of the water activity along with organic
and inorganic nutrients essential for its survival (Tiquia-Arashiro and Grube 2019).
Studies suggest that halophilic fungal species produce highly specific bioactive
metabolites with biological activities of industrial importance, such as antibacterial
potential, hemolysis, etc. It was observed that decreased temperature and lower
water activity improved hemolytic activity of halophilic fungi, thereby improving
its stress response. Similarly, the extracellular metabolites produced by halophilic
fungi are found to be antibacterial in nature, which when used in industries helps in
avoiding bacterial contamination. Some of the examples of halophilic fungi with
antibacterial potential include Aspergillus flavus,A. gracilis, and A. penicillioides.
Increase in salt concentrations causes increase in the antioxidant capacity of halo-
philic fungi and this is assessed by performing various antioxidant enzyme assays
such as catalase assay, superoxide dismutase, glutathione S transferase assay, and
guaiacol peroxidase assay, followed by thin layer chromatography and total phenolic
content assay. Hence, they can contribute to the production of antioxidants with
692 A. Nair et al.

commercial viability when subjected to high concentrations of salt levels (Tiquia-
Arashiro and Grube 2019).
Fructooligosaccharide possesses a number of commercial applications by their
use as artificial sweeteners and prebiotics, by reducing high cholesterol levels, by
curing traveler’s diarrhea and constipation. Fructooligosaccharide can be produced
by certain halophilic fungi (Cladosporium cladosporioides) from sucrose. Studies
suggest that several species of thermohalophilic fungi are capable of producing a
number of industrially important enzymes (both themostable and halostable) in an
economic and an ecofriendly manner, since they are capable of being highly active at
high temperature and increased salt concentrations. Some of these enzymes include
proteases (Aspergillus flavus), amylases (Engyodontium album, A. gracilis,
A. penicillioides), and celluloses (A. terreus, Penicillium sp.). These are of great
importance in food, textile, paper and pulp industries. Certain halophilic fungi such
as Aspergillus sp.nov. F1 have been found to produce secondary metabolites that act
as cytotoxic compounds, which when extracted and purified exhibited anti-
cancerous properties. This shows the importance of halophilic fungi in pharmaceu-
tical industries (Tiquia-Arashiro and Grube 2019).
Several other applications of halophilic fungi include
1. phenol degradation (Debaryomyces sp.)
2. bioremediation of halite on sandstones,
3. treatment of salt damage on building materials due to dampness (Cladosporium
sphaerospermum, Aureobasidium pullulans, Aspergillus nidulans, and Wallemia
sebi).
4. heavy metal removal by obligate halophilic fungal species (A. flavus, A. gracilis,
A. penicillioides, A. restrictus, and Sterigmatomyces halophilus).
Hence, exploring several halophilic fungi from diverse locations helps in under-
standing their potential in industrial applications and thereby helps in promoting
research in this field (Tiquia-Arashiro and Grube 2019).
28.2.4 Acidophilic Enzymes
Acidophilic fungi are very important in the field of biotechnology. The fungi
inhabiting acidic niches are a source for acid stable enzymes which can be employed
in many industrial processes and products. Acidophilic environments are created on
earth naturally and also by anthropogenic activities. The microorganisms living in
such regions have adapted and evolved naturally to dwell in high acidic conditions.
The enzymes produced by acidophiles are exploited for many industrial processes.
Acidophilic fungi are very important for the field of biotechnology. The fungi
inhabiting the acidic niches are the source for acid stable enzymes which can be
employed in many industrial processes and products. Acidophilic environments are
created on earth naturally and also by anthropogenic activities. The microorganisms
living in such regions have adapted and evolved naturally to dwell in high acidic
28 Fungal Extremozymes in Green Chemistry 693

conditions. The enzymes produced by acidophiles are exploited for many industrial
processes.
Mannan is the main constituent of hemicellulose in plants with β-1,4 backbone of
mannose and glucose residues. Beta mannanases are the enzymes which help in
cleaving this linkage and are applicable in many industries like paper, pulp, animal
feed, food, and oil industries. Most of the beta mannanases produced from fungi are
acidic in nature which has optimal pH range of 2–3. Fungi like Penicillium
sp. 40 (Kimura et al. 2000), Aureobasidium pullulans (Li et al. 1993), Penicillium
oxalicum GZ-2 (Liao et al. 2014), Bispora antennata (Liao et al. 2012), etc.
Mannanases with xylanases in combination are effective bleaching agents for pulp
processing (Woodcock et al. 1989).
Xylan is the second most important constituent of hemicellulose of monocots and
woody plants. Xylan degrading enzymes are hence very useful in industries like
paper, pulp, textile, and agriculture. Xylanase can break down 1–4 glycosidic
linkage between the units of xylopyranol. The fungal xylanases from extreme
environments are having high economical value for such industrial processes. In
pulp industry for pre-bleaching protocols, in bakeries to process dough, in feed
processing, etc., the enzyme is useful. Li et al. (1993) reported xylanases with
activity at pH 2–5 from Penicillium sp., and with activity at 4.8 from Aureobasidium
pullulans.
Hemicellulases are involved in bioconversion of lignocellulosic biomass in food,
feed, and detergent industries. The use of hemicellulase in paper industries enhances
the quality of paper. Fungal mannanases are acidic in nature with optimal pH range
of 2–6 and hence becomes industrially important. Filamentous fungi like Aspergil-
lus, Penicillium, and Trichoderma are promising sources of mannanases (Morreira
and Filho 2008).
D-galactosidases, (lactase) which hydrolyzes lactose into its monomers, are
required by food and dairy industries for whey processing, to produce food
supplements for lactose intolerant individuals, for low lactose food milk products,
cheese, sweet yoghurt production, etc. (Wang et al. 2009; Baumgartner and Hinrichs
2000). A. niger with high thermal and low pH stability, Teratospaeria acidotherma
AIUBGA-1 with optimum pH of 4 are reported (Hatzinikolaou et al. 2005; Isobe
et al. 2013). β-galactosidases are used as digestive supplements in many products,
which need to be active at low pH of stomach. Hence the enzyme with optimum pH
of acidic range is highly useful (Lin et al. 1993).
Laccases degrade lignin and hence are used for the delignification in paper and
pulp industries and also used for synthesis of biosensors and fuel cells (Ghindilis
2000). Also used in beer as stabilizers, in dye and effluent treatment from industries
(Ben Younes et al. 2007). Laccases from fungi with optimum pH in acidic range are
hence useful. Trametes hirsute Bm-2 produced laccase with optimum pH of 4–4.5
(Zapatae Castillo et al. 2012), T. versicolor with laccase of pH 4–5 (Minussi et al.
2007), Ganoderma lucidum with optimum pH 3.5 (Ko et al. 2001), etc.
Therefore, fungi producing enzymes which are active under low pH can be
explored more for the industrial uses.
694 A. Nair et al.

28.2.5 Alkaliphilic Enzymes
Industrial bioprocessing requires several chemical transformations and most of these
transformations take place under alkaline conditions. pH-tolerant enzymes produced
by mesophiles have been an ecofriendly bedrock for these chemical processes
majorly. However, due to limitations in catalytic efficiency it became prudent to
look for extremozymes with optimum biocatalysis at alkaline pH.
Proteases are specialized enzymes produced by the salt-loving Haloalkaliphiles
which thrive under high salt conditions. Microbial proteases in general are among
the most extensively studied hydrolytic enzymes. With a biotechnological potential
that extends into several industrial processes, from detergent, leather, textile, dairy,
etc., this group of enzymes represent roughly 60% of the net enzyme sales in the
world (Zambare et al. 2011). Considering fungi can grow on cheaper substrates,
without compromising on the enzyme yield, in recent times, fungal proteases have
grown as potential source for industrial proteases (Anitha and Palanivelu 2013).
Proteases secreted by alkaliphilic Aspergillus species (A. jilinensis) have been
extensively studied because of their innate ability to produce the enzyme in huge
amounts. These proteases find special purpose in food and beverage industry
(Wu et al. 2006). Lately, fungal proteases are being used as laundry detergent
additives, providing for almost 25% of the world’s net requirement (Demain and
Adrio 2008). Incorporation of these extremozymes has improved the detergent’s
ability to remove tough proteinaceous stains. Alkaline proteases from A. flavus and
C. coronatus are also being used religiously as ecofriendlier alternative to using
hazardous chemicals during the leather tanning process (Laxman et al. 2005). Fungal
proteases have found their way into pharmaceutical industries, where they are used
in the elimination of keratin in acne, elimination of callus, and also improving drug
delivery (Brandelli et al. 2010).
Almost all of the industrially used cellulases and hemicellulases are primarily
purified from well-studied non-extremophilic bacteria and fungi. But recent studies
have shown alkaline thermophilic fungi to be much preferable sources of cellulases
in textile and detergent industries (Ito 1997). Cellulases and hemicellulases extracted
from P. citrinum have shown to be functionally efficient at extreme alkaline
conditions (Raghukumar et al. 2004). Evaluation of several thermophilic fungal
species, such as Thermomyces, Myceliophthora, Aspergillus for their ability to
produce cellulolytic and hemicellulolytic enzymes, demonstrated superior cellulo-
lytic and xylanolytic activity. The α-arabinosidase activity in these enzymes was
particularly high. Also, the capacity to hydrolyze lignocellulose in the substrate with
an improved thermal stability has further added to its potential as enzymes highly
sought after in polymer industries (Cantarel et al. 2009).
Although most of the natural environments on this planet remain predominantly
neutral in pH, habitats with varying degrees of pH (alkaline or acidic) are not that
uncommon. Geothermal regions, soils saturated with carbonate, soda deserts and
lakes, etc. have been shown to exhibit extreme ranges of pH (Kimura et al. 2000).
Alkaliphilic xylanases, especially from Penicillium sp., and Aspergillus sp. are
hugely dependent on pKa of the catalytic residues that are in turn dependent on
28 Fungal Extremozymes in Green Chemistry 695

the immediate ambient conditions. Interestingly it was noted that some acidophilic
xylanases have an aspartate residue hydrogen bonded to a general acid/base catalyst
which is flexible and can be substituted with an asparagine when the enzyme is
exposed to alkaline conditions. With this inherent plasticity to work efficiently under
varying pH, it was observed that stability of the extremozymes at extreme pH is
accounted for by the biased distribution of charged residues (Fushinobu et al. 1998).
Xylanases have application in several industrial processes, covering every sector
of commercial enzyme market (Bhat 2000). From baking, to textile, to pulp, the
industrial applications are boundless. Some of the other less studied applications
include: in brewing, coffee extraction, in protoplastation of plant cell, synthesis of
compounds that are potentially pharmacologically active (Christakopoulos et al.
2003).
28.3 Fungal Extremozymes in Specific Green Industrial
Processes
28.3.1 Food Processing
Fungal extremozymes are being rigorously used in the food industry, due to their
ability to remain functionally versatile under extreme processing conditions.
Extremophilic proteases, lipases, and amylases have shown to biocatalyze with
minimal energy and more endurance. As a result, they found their way into
industries on confectionery and dairy. Viticulture and processing of juice primarily
depends on laccases belonging to the Basidiomycetes and Ascomycetes. Special
class of laccases are applied to bottle corks to minimize tart flavor during extended
wine storage. Laccases also play hugely in improving the baking structure by
stabilizing gluten in the dough. Additionally, by reducing the viscosity of the
dough, it improves on the workability. Lipases extracted from filamentous fungi
such as Aspergillus,Mucor have been actively used to catalyze the synthesis esters
that impart flavor and fragrance. These lipases significantly enhance flavor in Italian
cheeses by catalyzing mild lipolysis to help them mature aster (Jooyandeh et al.
2009).
Another biocatalyst that falls under the category of hydrolases is Pectinase that
has been lately sought after to allow fruit juice pressing, in particular, during the
production of apple juice. Not only this, but also extremophilic pectinases help to
minimize the turbidity in wines and remove mucous coating from coffee beans
during its fermentation process (Chandra and Enespa Singh 2020).
Microbial proteases in general are among the most extensively studied hydrolytic
enzymes. With a biotechnological potential that extends into several industrial
processes, from detergent, leather, textile, dairy, etc., this group of enzymes
represents roughly 60% of the net enzyme sales in the world (Zambare et al.
2011). Proteases secreted by alkaliphilic Aspergillus species (A. jilinensis) have
been extensively studied because of their innate ability to produce the enzyme in
huge amounts. These proteases find special purpose in food and beverage industry
696 A. Nair et al.

(Wu et al. 2006). Fungal extremophilic proteases are being actively used for the
production of milk substitutes. In the past, use of proteases in dairy industry would
fail to correct the bitter taste in milk. However, fungal proteases when used appro-
priately help with the rectification of this unwanted trait. Precipitation of casein
protein has been greatly improved with the use of acid proteases from the Mucor
species. Additionally, for the production of soy sauce which involves the hydrolysis
of soy proteins has been catalyzed using proteases extracted from the A. niger and
A. oryzae (de Souza et al. 2015).
28.3.2 Textile and Pulp Industry
In textile industry, bleaching of cotton and bio-stoning process are key to the
production of cotton. Fungal laccases help improve whiteness in cotton during its
conventional production process. Also, during the bleaching of textile dyes, the use
of laccases instead of physical and chemical methods leads to the proper degradation
of dyes with next to no entering of the synthetic in the industrial waste and later to
the environment. This is because synthetic dyes are resistant to fading under
exposure to light, water, and other chemicals. However, laccases catalyze the
complete elimination of dyes from fabrics during the bleaching process. Dyeing
fabric with deeper colors has been found to be economically unprofitable. But by
using biocatalysts such as laccases which enhance the fixation of the dye, one might
not need to apply the dye in excess. Alternatively, to prevent wool from shrinking,
the traditional method involves chlorination. But doing so impacts the environment
negatively. Instead, treatment of wool with proteinases hugely reduces the deleteri-
ous environmental impact, in addition to preventing the wool from shrinking.
Deinking is a process in paper industries for reducing the brightness of the paper.
The process is performed by chemical modes using hazardous chemicals like
surfactants, chelating agents, sodium hydroxide, hydrogen peroxide, sodium carbon-
ate, sodium silicate, etc. These can be replaced by microbial enzymes which are
ecofriendly. White rot fungi are best source for hydrolytic enzymes like manganese
peroxide, laccase, and xylanase for degradation of lignin and hemicellulose.
Bio-bleaching involves cellulose hydrolysis facilitating ink detachment. Increased
brightness and dirt removal are the advantages of using enzymes. Extremozymes
like alkaliphilic xylanase and mannase increase paper quality and hence used in
bio-bleaching protocols. Recycling of the old paper involves deinking of newspaper
pulp, where combination of xylanase and laccase was found to be performing better
in enhancing the paper quality and decreasing the chemical consumption. Laccase
helps in removing the lignin content and hence used in lignin-rich pulp. Either
lipases or laccase and hemicellulase together can be used in deinking for the old
newspapers. The enzymes like amylase, cellulase, lipase, pectinase, and xylanase are
important for pulp processing at different stages like deinking, draining, fiber
modification, debarking, bleaching, etc. As all these processes involve many harsh
conditions like alkaline pH, high salt concentration, temperatures, the extremozymes
are very important.
28 Fungal Extremozymes in Green Chemistry 697

28.3.3 Pharmaceutical Industry
Enzymes for hot temperatures are important for many industrial processes. Industri-
ally, hot extremozymes bear a wide range of applications in industries such as
pharmaceuticals, pulp and paper, food and beverages, and many more. These
extremozymes benefit in various biotechnological processes as they can
1. undergo separation from other heat-labile enzymes during production steps,
2. shorten fermentation processes due to high growth rate at increased temperatures
and low concentration at decreased nutrient content,
3. lower energy consumption owing to lower viscosity of the liquids at high
temperature,
4. retain high efficiency at extreme conditions,
5. alleviate the constraints of industrial processes.
(Sarmiento et al. 2015; Pabulo and Rampelotto 2016).
Apart from a wide range of biotechnological applications, thermophilic fungi are
found to be useful in pharmaceutical industries, especially in the process of biotrans-
formation of organic compounds for bioactive compound synthesis. The use of
thermophilic fungi makes the process hazard-free that helps to overcome the
disadvantages of other chemical processes such as minimizing the complications
of isomerization, racemization, and epimerization. Thermophilic enzymes display
thermostability that offers better advantages than the enzymes from mesophiles
during industrial processes. It is found that thermophilic enzymes cloned and
expressed in mesophilic hosts can easily be purified by heat treatment and are
capable of withstanding high amounts of chemical solvents. Such reactions are
favorable in pharmaceutical industries since there exists a high demand for
enantiomerically pure compounds. Thermophiles during biocatalytic reactions help
in improving transfer rates and substrate solubility, decrease the viscosity of the
medium, and avoid the risk of mesophilic microbial contamination. Thermophilic
fungi are well-known to resist denaturants present in extreme alkaline and acidic
conditions (Tiquia-Arashiro and Grube 2019).
Some of the biotransformation reactions performed in pharmaceutical industries
using thermophilic fungi includes:
(a) albendazole (a prodrug) to albendazole sulfoxide by Rhizomucor pusillus. This
when performed chemically is a difficult process as it is a site-specific reaction
that may also lead to environmental pollution. This process with thermophilic
fungi offers a better yield at a high temperature when compared to mesophilic
fungi.
(b) ferulic acid to guaiacol by Sporotrichum thermophile.
(c) biotransformation of steroids by thermophilic fungi is said to produce effective
steroids in an ecofriendly and economic manner. Acremonium alabamensis and
Talaromyces emersonii are commonly used thermophilic fungi for steroid
production in pharmaceutical industries (Tiquia-Arashiro and Grube 2019).
698 A. Nair et al.

Thermophilic fungi are also known to help in predicting mammalian drug
metabolism and metabolite toxicity, as microbial models. This is a very crucial
step during drug innovation as well as to establish the safety and efficacy of the
drug prior to human consumption. Using thermophilic fungi offers several
advantages such as ease of handling, cost-effective process, potential to decrease
animal use for the same and to improve scale-up capacity. Some of them that are
commonly used for this approach are Rhizomucor pusillus NRRL 28626,
Rhizomucor pusillus NRRL 28626, and Thermomyces lanuginosus NCIM-1934.
Thermophilic fungi have also been proven to be potential in the production of
novel value-added metabolites, such as Losartan as Human Peroxisome Proliferator
Activated Receptor-Gamma (PPAR-γ) and Human angiotensin Receptor (AT1R)
Binders, Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B inhibition
potentials of albendazole and its biotransformed metabolites. Thermophilic fungi
also aid in antibiotic production, e.g. Myriocin, a new crystalline antifungal com-
pound that acts against Candida sp., Trichophyton granulosum and Microsporum
gypseum. Other antibiotics such as thermozymocidin penicillin G,
6-aminopenicillanic acid, sillucin, miehein, and vioxanthin are known to be pro-
duced by thermophilic fungi (Tiquia-Arashiro and Grube 2019).
Industrial bioprocessing requires several chemical transformations and most of
these transformations take place under alkaline conditions. pH tolerant enzymes
produced by neutrophils have been an ecofriendly bedrock for these chemical
processes majorly. However, due to limitations in catalytic efficiency it became
prudent to look for extremozymes with optimum biocatalysis at alkaline pH. Certain
halophilic fungi such as Aspergillus sp.nov. F1 have been found to produce
secondary metabolites that act as cytotoxic compounds, which when extracted and
purified exhibited anti-cancerous properties. This shows the importance of halophilic
fungi in pharmaceutical industries (Tiquia-Arashiro and Grube 2019).
28.3.4 Bioconversion
Fungi, among all microorganisms, have been identified as key mediators during
biofuel production and other bioconversion processes. The supremacy of fungi in all
areas of bioconversion processes is attributable to their ability to specifically degrade
lignocellulosic materials with enzymes possessing superior redox potentials and
themostability. Lignin biodegradation is one of the key steps involved in biofuel
production and during the conversion of other biorefinery products. Lignin
modifying enzymes (LMEs) comprise a wide collection of enzymes ranging from
peroxidases, to laccases, to oxidases that are capable of disrupting lignin
components, or oxidizing various phenolic compounds. Most the LMEs work
synergistically during the lignification and depolymerization of lignin polymers.
Thermostable cellulases are being preferred lately as suitable candidates for
bioprocessing industries because of their ability to efficiently hydrolyze lignocellu-
losic substrates by improving on the reaction rate, rate at which organic compounds
become bioavailable, while lowering the viscosity and economic cost. In the recent
28 Fungal Extremozymes in Green Chemistry 699

times, adopting an enzyme-assisted technology has increased the economic viability
during the production of biofuels. Cellulases hold a key role in the hydrolysis of
cellulosic polymers toward the secretion of fermentable sugars, which eventually
lead to the production of biofuels.
Biofuels made from lignocellulosic non-food waste biomass represent second-
generation biofuels. Fungal lignocellulosic biomass offers a renewable source of
carbon. It can be also converted into several value-added products. At the same time,
the waste biomass contributes hugely to the fermentation process, wherein the waste
produced can also be used as animal feed with high nutritional quality (Srivastava
et al. 2018).
28.3.5 Bioremediation
The industrial revolution has transformed the socioeconomic status of our society. It
has impacted every facet of human growth from food to energy, production, sanita-
tion, and manufacturing technologies. While it led to prosperity and improved
quality of life, it has adversely affected the ecosystem and the environment posing
serious threats to humankind and its survival. The earlier section of this chapter
discussed the applications of fungal extremozymes for industrial processes. The
same array of extremozymes is also being employed for the bioremediation of
industrial wastes as discussed below (Fig. 28.2).
Increased industrialization has led to the depletion of natural resources, global
warming, and the accumulation of inorganic and organic pollutants in soil, air, and
Applicaons
Fungal Extremozymes
Industrial Processes, Synthesis,
Bioconversions etc.
Bioremediaon
Fig. 28.2 Applications of fungal extremozymes for green chemistry
700 A. Nair et al.

water (Singh et al. 2020). Solid and chemical waste management has become the
foremost concern to conserve the environment. Pollutants like polyaromatic
hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-
p-dioxins (PCDDs), and heavy metals have contaminated the soil causing the quality
of soil to decrease drastically. Many physical and chemical treatments were
introduced to remove these pollutants; however, large-scale application of these
treatments was not achievable (Deshmukh et al. 2016). Bioremediation is recognized
as an effective alternative to combat these issues. It is a procedure that converts
hazardous compounds into non/less-hazardous compounds by the action of microbes
like fungi, bacteria, algae (Singh et al. 2020). It is an environmentally friendly,
economical, and efficient method in the disposal of these toxic chemicals.
Biostimulation, bioaugmentation, and natural attenuation are certain major
techniques that can be followed to carry out the technique of bioremediation
(Deshmukh et al. 2016).
28.3.6 Mycoremediation
The application of fungi in bioremediation is termed as “mycoremediation.”Certain
fungi can grow and survive in polluted environments as they are adapted to metabo-
lize and utilize the chemicals, as a nutritional resource. They produce hydrolytic
enzymes such as protease, lipase, nuclease, amylase, etc. that degrade complex
chemicals into simple less toxic compounds or transform metals, metalloids, and
other organic materials into harmless ions via redox reactions. Fungi can also
immobilize and store the metalloids or metals in mycosphere or various other parts
of the cell. Another distinct characteristic of fungi is that they can degrade
compounds that are not intoxicated by bacteria (Singh et al. 2020). Extremophilic
fungi have been isolated from highly contaminated environments such as acidic
mining wastewaters, hot volcanic geothermal regions, etc. (Chan et al. 2019).
However, bioremediation is a recent development and there is limited data available
on the use of extremophilic fungi in this process. Hence this chapter concentrates on
the diverse role of extremophilic fungal groups in examining the technique of
bioremediation.
Extremophilic organisms, counting marine fungi, are imperative resources of
steady and treasured enzymes defined as “extremozymes .”Numerous marine
extremozymes have been employed for biotechnological studies. However, only a
few extremozymes are presently being produced and utilized at the industrial level.
Therefore, additional scientific trials are needed to understand the possible
applications of extremozymes (Barone et al. 2019).
28.3.6.1 Marine Extremophilic Fungi in Mycobioremediation
Salinity predominates oceanic environment. The adaptive strategies employed by
extremophiles for a successful halotolerance make them ideal candidates to be
employed in the bioremediation process.
28 Fungal Extremozymes in Green Chemistry 701

Salt pans are a significant resource for extremophilic microorganisms. However,
not many fungi species can survive in such extreme saline conditions as it adversely
affects the protein structure and hampers the enzyme function. Few fungi, such as
black yeasts, Hortaea werneckii, Phaeotheca triangularis, Aureobasidium
pullulans, and Trimatostroma salinum have been isolated from salt pans having
15–30% salinity is reported. Phaeotheca triangularis and black yeasts are described
as obligate halophiles, whereas other species are facultative halotolerants (Damare
et al. 2012). Marine fungi have been found to tolerate high concentrations of heavy
metals such as lead and copper and their interaction with metal ions is one such
property that can be used in bioremediation.
Trichoderma viride Pers NFCCI-2745 was isolated from an estuary polluted with
phenolics and it was reported to produce a halotolerant laccase enzyme. This enzyme
was utilized in the bioremediation and removal of phenolics. Related applications of
enzyme facilitated bioremediation were verified for decolorizing remazol brilliant
Blue-R dye. Three basidiomycetes isolated from marine sponges were used. Simi-
larly, C. unicolor, marine white rot basidiomycete was employed to degrade anthra-
quinone reactive blue 4 dye. Reports also suggest that marine fungi belonging to the
genus Penicillium and Trichoderma harzianum aid in the biotransformation of
persistent organic pollutants like PCB 118 and pentachlorophenol, respectively.
Additional marine-derived fungi including Mucor,Aspergillus, and slime mold
confirmed bioremediation possibility for water-soluble crude oil fractions. However,
higher concentrations caused toxicity to the microbes (Singh et al. 2020).
Marine species of Aspergillus sclerotiorum CBMAI 849 (isolated from
cnidarians) were proficient in reducing 99.7% of pyrene and 76.6% benzo[a] of
pyrene after 8 and 16 days, respectively. They also reported considerable quantities
of benzo[a]pyrene (>50.0%) reduction by a Mucor racemosus CBMAI 847. The
mechanism of hydroxylation was studied by coupling with sulfate ions which are
reduced by a cytochrome P-450 monooxygenase enzyme produced by the marine
extremophilic fungi. Another study reported the use of a marine fungus Aspergillus
candidus that could grow in the presence of arsenic (25 and 50 mg/L) and decreased
the volume of the metal by the bioaccumulation process (Damare et al. 2012).
28.3.6.2 Other Extremophilic Fungi in Mycobioremediation
Numerous extremophile fungi such as Coniochaeta fodinicola, Teratosphaeria
acidotherma, Hortaea acidophilia, and Acidomyces acidophilus were known to
produce metabolites and novel enzymes to endure severe environments. These
fungi are utilized as bioremediation agents to remove toxic metalloids from water
and soil.
A. acidophilus WKC-1 was tested for biosorption of arsenic and antimony. It was
observed that PO
4
,SO
3
,–OH, –NH, –CH are few of the functional groups that are
recognized as the significant biosorption binding sites for As
5+
and Sb
5+
. The isolate
WKC-1 showed a high percentage of As
5+
removal (around 70.30%). The ability of
the fungi to tolerate low pH and high arsenic concentration together makes it a
potential candidate to be used in the bioremediation of arsenic (Chan et al. 2019).
702 A. Nair et al.

A psychrophilic fungus, Cryptococcus sp. was isolated from deep-sea sediments.
It displayed tolerance and development in the existence of high levels of heavy
metals (up to 100 mg/L) ZnSO
4
, CdCl
2
, CuSO
4
, Pb(CH
3
COO)
2
. This conveyed that
Cryptococcus sp. can be used in the bioremediation process under extreme
conditions with good quality of its activity being exhibited (Deshmukh et al. 2016).
A study was conducted by Bano et al. (2018) to examine the biosorption of
copper, lead, zinc, cadmium, ferrous, and manganese by testing with the following
fungi species: Aspergillus flavus, A. gracilis, A. penicillioides, A. penicillioides,
A. restrictus, and Sterigmatomyces. A. flavus and Sterigmatomyces halophilus
showed a potential for heavy metal removal with a value of 85% and 83%,
respectively.
28.3.7 Extremozymatic Bioremediation
The extremophiles and their extremozymes are one of the most desirable bioremedi-
ation tools.
28.3.7.1 Laccases
Cladosporium oxysporum, Curvularia lonarensis, Aspergillus niger, Fusarium
equiseti, Cladosporium funiculosum, Cladosporium halotolerans were isolated
and identified from a hypersaline lake in Maharashtra. The fungi were highly tolerant
to alkaline conditions and employed to treat the municipal wastewater treatment
plants and sewages having high concentration of metal ions and alkaline pH. Studies
also describe that Cerrena unicolor MTCC 5159 produces halotolerant laccase that
assists in the degradation process of alkaline raw textile mill effluents.
Laccases not only oxidize phenolic and methoxyphenolic acids, but also decar-
boxylate and attack their methoxy groups (demethylation). These enzymes are
involved in the depolymerization of lignin (a constituent of biomass) (Gangola
et al. 2019). It comprises phenylpropanoid units linked by C–C and C–O bonds.
Since fewer organisms produce laccases extracellularly, fungal laccase plays a
potential role in lignin depolymerization. Hence extremophilic fungal laccase
works as an outstanding bioinoculant for bioremediation (Prakash et al. 2019).
28.3.7.2 Peroxidases
Lignin peroxidase (LiP) and manganese peroxidase (MnP) were exposed in the
mid-1980s in Penicillium chrysosporium and defined as true ligninases since they
display high redox potential. LiP and MnP catalyze the oxidation of lignin units by
H
2
O
2
LiP which degrades non-phenolic lignin, whereas MnP generates Mn
3+
, which
acts as a diffusible oxidizer on phenolic or non-phenolic lignin units via lipid
peroxidation reactions. The use of peroxidases for soil cleaning has been considered,
precisely for soils contaminated with aromatic hydrocarbons and detoxified by
autochthonous fungi producing peroxidases (Mougin et al. 2009). Peroxidases are
operative for the biodegradation of oil spills, carbamate insecticides, and
organophosphates. The key benefits of this enzyme are its ready availability,
28 Fungal Extremozymes in Green Chemistry 703

tolerance to water-miscible solvents, and lack of cofactor stereoselectivity that make
them suitable enzymes for biotechnological research (Gangola et al. 2019).
28.3.7.3 Catalases
Accumulation of reactive oxygen species (ROS) results in damage to cellular
macromolecules, which is toxic for cellular integrity. The chief defense mechanism
to ROS in fungi involves catalases and peroxidase. Heavy metals such as copper
(Cu), lead (Pb), cadmium (Cd), and zinc (Zn) have been reported to be among the
major reasons for ROS induction in microbial cells. To date, all studies indicate an
increase in anti-oxidative activity of enzymes in the presence of heavy metals and
ROS. A study reported higher catalase activity when Cu
2+
,Pb
2+
50 (mg/L) were
added in combination or individually to the fungal consortia comprising of A. niger,
Rhizopus sp., and Penicillium sp. Hence catalase activity can be employed as a
screening technique to check the effectiveness of bioremediation in oil-contaminated
soil. Hence, engineering this fungal enzyme can be favorable for bioremediation of
metal-polluted spots (Deshmukh et al. 2016).
Studies on the effects of extremozymes in bioremediation are less understood.
The limited studies on extremophiles are due to their distinct nutritional supplies and
stimulating growth conditions (Jin et al. 2019). Extremozymes from fungal sources
are an important arsenal in the bioremediation process and many techniques and
extreme environments are explored to screen and isolate extremozymes. The biodi-
versity of extremophiles and their adaptations to harsh conditions is one of the
important means that can help reverse the damage and destruction caused by this
environment.
28.4 Conclusion
The endeavor to blend industrial productivity with sustainability has been a constant
source of inspiration to seek newer alternatives. With the addition of biocatalysis in
industrial chemical transformations, the concept of green chemistry has been very
closely bound to enzyme catalysis. This in essence has been seen as an alternative to
the classic chemical catalysis which has predominated majorly since the industrial
revolution. In the recent times, biocatalysis has evolved toward more specialized
extremophilic enzymes, extremozymes as they are termed, which have the ability to
withstand an array of harsh conditions, one that would rival the efficiencies of most
enzymes produced within the mesophilic realm, be it properties of extreme
halotolerance, thermal stability or enduring freezing temperatures. But very little is
known about the manner in which these specialized enzymes work, or the molecular
strategies they adopt to optimally function. Therefore, any further strides made,
either from a bioprocessing point of view or be it the use of these enzymes in the
utilization of industrial wastes or for bioremediation, would require a more compre-
hensive understanding of these enzymes.
704 A. Nair et al.

References
Anitha TS, Palanivelu P (2013) Purification and characterization of an extracellular keratinolytic
protease from a new isolate of Aspergillus parasiticus. Protein Expr Purif 88(2):214–220.
https://doi.org/10.1016/j.pep.2013.01.007
Bano A, Hussain J, Akbar A, Mehmood K, Anwar M, Hasni MS, Ullah S, Sajid S, Ali I (2018)
Biosorption of heavy metals by obligate halophilic fungi. Chemosphere 199:218–222. https://
doi.org/10.1016/j.chemosphere.2018.02.043
Barone G, Varrella S, Tangherlini M, Rastelli E, Dell’Anno A, Danovaro R, Corinaldesi C (2019)
Marine fungi: biotechnological perspectives from deep-hypersaline anoxic basins. Diversity 11:
113. https://doi.org/10.3390/d11070113
Baumgartner C, Hinrichs J (2000) Sweet products without sugar additives. DMZ Lebensmittelind
Milchwirtschaft 121:635–639
Ben Younes S, Mechichi T, Sayadi S (2007) Purification and characterization of the laccase
secreted by the white rot fungus Perenniporia tephropora and its role in the decolourization of
synthetic dyes. J Appl Microbiol 102:1033–1042
Bhat MK (2000) Cellulases and related enzymes in biotechnology. Biotechnol Adv 18:355–383
Boo SY, Wong CMVL, Rodrigues KF (2013) Thermal stress responses in Antarctic yeast,
Glaciozyma antarctica PI12, characterized by real-time quantitative PCR. Polar Biol 36:381–
389. https://doi.org/10.1007/s0030-0-012-1268-2
Bradner JR, Sidhu RK, Gillings M, Nevalainen KM (1999) Hemicellulase activity of Antarctic
microfungi. J Appl Microbiol 87:366–370
Brandelli A, Daroit DJ, Riffel A (2010) Biochemical features of microbial keratinases and their
production and applications. Appl Microbiol Biotechnol 85:1735–1750
Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohy-
drate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids
Res 37(Database issue):D233–D238. https://doi.org/10.1093/nar/gkn663
Chan WK, Wildeboer D, Garelick H, Purchase D (2019) Acidomyces acidophilus: ecology,
biochemical properties and application to bioremediation. In: Tiquia-Arashiro SM, Grube M
(eds) Fungi in extreme environments: ecological role and biotechnological significance.
Springer International, Cham, pp 505–515. https://doi.org/10.1007/978-3-030-19030-9_25
Chandra P, Enespa Singh R (2020) Microbial lipases and their industrial applications: a compre-
hensive review. Microb Cell Fact 19:169. https://doi.org/10.1186/s12934-020-01428-8
Christakopoulos P, Katapodis P, Kalogeris E, Kekos D, Macris BJ, Stamatis H, Skaltsa H (2003)
Antimicrobial activity of acidic xylo-oligosaccharides produced by family 10 and
11 endoxylanases. Int J Biol Macromol 31:171–175
Collins T, Meuwis MA, Gerday C, Feller G (2003) Activity, stability and flexibility in glycosidases
adapted to extreme thermal environments. J Mol Biol 328:419–428
Damare S, Singh P, Raghukumar S (2012) Biotechnology of marine fungi. In: Raghukumar C
(ed) Biology of marine fungi. Progress in molecular and subcellular biology. Springer, Berlin,
Heidelberg, pp 277–297. https://doi.org/10.1007/978-3-642-23342-5_14
de Souza PM, Bittencourt ML, Caprara CC (2015) A biotechnology perspective of fungal proteases.
Braz J Microbiol 46(2):337–346. https://doi.org/10.1590/S1517-838246220140359
Demain AL, Adrio JL (2008) Contributions of microorganisms to industrial biology. Mol
Biotechnol 38:41–55
Deshmukh R, Khardenavis AA, Purohit HJ (2016) Diverse metabolic capacities of fungi for
bioremediation. Indian J Microbiol 56:247–264. https://doi.org/10.1007/s12088-016-0584-6
Fushinobu S, Ito K, Konno M, Wakagi T, Matsuzawa H (1998) Crystallographic and mutational
analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic
residues and importance of Asp37 for catalysis at low pH. Protein Eng 11:1121–1128
Gangola S, Joshi S, Kumar S, Pandey SC (2019) Comparative analysis of fungal and bacterial
enzymes in biodegradation of xenobiotic compounds. In: Smart bioremediation technologies.
Elsevier, Amsterdam, pp 169–189. https://doi.org/10.1016/B978-0-12-818307-6.00010-X
28 Fungal Extremozymes in Green Chemistry 705

Ghindilis A (2000) Direct electron transfer catalyzed by enzymes: application for sensor develop-
ment. Biochem Soc Trans 28:84–89
Ghatora S, Chadha BS, Badhan A, Saini HS, Bhat MK (2007) Identification and characterization of
diverse xylanases from thermophilic and thermotolerant fungi. Bioresources 1:18–33
Hatzinikolaou DG, Katsifas E, Mamma D, Karagouni AD, Christakopoulos P, Kekos D (2005)
Modeling of the simultaneous hydrolysis ultrafiltration of whey permeate by a thermostable
begalactosidase from aspergillus Niger. Biochem Eng J 24:161–172
Isobe K, Takahashi N, Chiba S, Yamashita M, Koyama T (2013) Acidophilic fungus,
Teratosphaeria acidotherma AIU BGAe1, produces multiple forms of intracellular
begalactosidase. J Biosci Bioeng 116:171–174
Ito S (1997) Alkaline cellulases from alkaliphilic Bacillus: enzymatic properties, genetics, and
application to detergents. Extremophiles 1(2):61–66. https://doi.org/10.1007/s007920050015
Jin M, Gai Y, Guo X, Hou Y, Zeng R (2019) Properties and applications of extremozymes from
deep-sea extremophilic microorganisms: a mini review. Mar Drugs 17:656. https://doi.org/10.
3390/md17120656
Jooyandeh H, Amarjeet K, Minhas KS (2009) Lipases in dairy industry: a review. Food Sci Technol
46(3):181–189
Kimura T, Ito J, Kawano A, Makino T, Kondo H, Karita S, Sakka K, Ohmiya K (2000) Purification,
characterization, and molecular cloning of acidophilic xylanase from Penicillium sp. 40. Biosci
Biotechnol Biochem 64:1230–1237
Ko EM, Leem YE, Choi ET (2001) Purification and characterization of laccase isoenzymes from the
white rot basidiomycete Ganoderma Lucidum. Appl Microbiol Biotechnol 57:98–102
Kunamneni A, Permaul K, Singh S (2005) Amylase production in solid state fermentation by the
thermophilic fungus Thermomyces lanuginosus. J Biosci Bioeng 100(2):168–171. https://doi.
org/10.1263/jbb.100.168
Lan DM, Yang N, Wang WK, Shen YF, Yang B, Wang YH (2011) A novel cold-active lipase from
Candida albicans: cloning, expression and characterization of the recombinant enzyme. Int J
Mol Sci 12:3950–3965. https://doi.org/10.3390/ijms12063950
Laxman RS, Sonawane AP, More SV (2005) Optimization and scale up of production of alkaline
protease from Conidiobolus coronatus. Process Biochem 40:3152–3158
Li XL, Zhang ZQ, Dean JF, Eriksson KE, Ljungdahl LG (1993) Purification and characterization of
a new xylanase 68 N. Hassan et al. (APXeII) from the fungus Aureobasidium pullulans
Ye2311e1. Appl Environ Microbiol 59:3212–3218
Liao H, Xu C, Tan S, Wei Z, Ling N, Yu G, Raza W, Zhang R, Shen Q, Xu Y (2012) Production and
characterization of acidophilic xylanolytic enzymes from Penicillium oxalicum GZe2.
Bioresour Technol 123:117–124
Liao H, Li S, Zheng H, Wei Z, Liu D, Raza W, Shen Q, Xu Y (2014) A new acidophilic
thermostable endoe1,4ebemannanase from Penicillium oxalicum GZe2: cloning, characteriza-
tion and functional expression in Pichia pastori. BMC Biotechnol 14:90
Lin MY, Dipalma JA, Martini MC, Gross CJ, Harlander SK, Savaiano DA (1993) Comparative
effects of exogenous lactase (begalactosidase) preparations in vivo lactose digestion. Dig Dis
Sci 38:2022–2027
Luonteri E, Alatalo E, Siika-Aho M, Penttilä M, Tenkanen M (1998) alpha-Galactosidases of
Penicillium simplicissimum: production, purification and characterization of the gene encoding
AGLI. Biotechnol Appl Biochem 28(Pt 2):179–188
Minussi RC, Rossi M, Bologna L, Rotilio D, Pastore GM, Duran N (2007) Phenols removal in
musts: strategy for wine stabilization by laccase. J Mol Catal B: Enzym 45(3–4):102–107
Morreira LR, Filho EX (2008) An overview of mannan structure and mannan degrading enzyme
systems. Appl Microbiol Biotechnol 79:165–178
Mougin C, Boukcim H, Jolivalt C (2009) Soil bioremediation strategies based on the use of fungal
enzymes. In: Singh A, Kuhad RC, Ward OP (eds) Advances in applied bioremediation, soil
biology. Springer, Berlin, Heidelberg, pp 123–149. https://doi.org/10.1007/978-3-540-
89621-0_7
706 A. Nair et al.

Pabulo H, Rampelotto (2016) Biotechnology of extremophiles; grand challenges in biology and
biotechnology. Springer, Berlin, pp 581–595
Prakash O, Mahabare K, Yadav KK, Sharma R (2019) Fungi from extreme environments: a
potential source of laccases Group of Extremozymes. In: Tiquia-Arashiro SM, Grube M (eds)
Fungi in extreme environments: ecological role and biotechnological significance. Springer
International, Cham, pp 441–462. https://doi.org/10.1007/978-3-030-19030-9_22
Raghukumar C, Muraleedharan U, Gaud VR, Mishra R (2004) Xylanases of marine fungi of
potential use for biobleaching of paper pulp. J Ind Microbiol Biotechnol 31(9):433–441.
https://doi.org/10.1007/s10295-004-0165-2
Sarmiento F, Peralta R, Blamey JM (2015) Cold and hot extremozymes: industrial relevance and
current trends. Front Bioeng Biotechnol 3:148. https://doi.org/10.3389/fbioe.2015.00148
Singh RK, Tripathi R, Ranjan A, Srivastava AK (2020) Fungi as potential candidates for
bioremediation. In: Abatement of environmental pollutants. Elsevier, Amsterdam, pp
177–191. https://doi.org/10.1016/B978-0-12-818095-2.00009-6
Srivastava N, Srivastava M, Mishra PK, Gupta VK, Molina G, Rodriguez-Couto S, Manikanta A,
Ramteke PW (2018) Applications of fungal cellulases in biofuel production: advances and
limitations. Renew Sust 82(3):2379–2386
Subramaniyan S, Prema P (2002) Biotechnology of microbial xylanases: enzymology, molecular
biology, and application. Crit Rev Biotechnol 22:33–64. https://doi.org/10.1080/
07388550290789450
Tiquia-Arashiro SM, Grube M (2019) Fungi in extreme environments: ecological role and biotech-
nological significance. Springer, Berlin. ISBN 978–3–030-19029-3 ISBN 978–3–030-19030-9
(eBook)
Turunen O, Etuaho K, Fenel F, Vehmaanperä J, Wu X, Rouvinen J, Leisola M (2002) A combina-
tion of weakly stabilizing mutations with a disulfide bridge in the alpha-helix region of
Trichoderma reesei endo-1,4-beta-xylanase II increases the thermal stability through synergism.
J Biotechnol 88(1):37–46. https://doi.org/10.1016/s0168-1656(01)00253-x
Van Petegem F, Collins T, Meuwis MA, Gerday C, Feller G, Van Beeumen J (2002) Crystallization
and preliminary X-ray analysis of a xylanase from the psychrophile Pseudoalteromonas
haloplanktis. Acta Crystallogr 58:1494–1496
Wang H, Luo H, Bai Y, Wang Y, Yang P, Shi P, Zhang W, Fan Y, Yao B (2009) An acidophilic
begalactosidase from Bispora sp. MEYe1 with high lactose hydrolytic activity under simulated
gastric conditions. J Agric Food Chem 57:5535–5541
Woodcock DM, Crowther PJ, Doherty J, Jefferson S, DeCruz E, Noyer-Weidner M, Smith SS,
Michael MZ, Graham MW (1989) Quantitative evaluation of Escherichia coli host strains for
tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17
(9):3469–3478. https://doi.org/10.1093/nar/17.9.3469
Wu TY, Mohammada AW, Jahim JM (2006) Investigations on protease production by a wild-type
aspergillus terreus strain using diluted retentate of pre-filtered palm oil mill effluent (POME) as
substrate. Enzyme Microb Technol 39:1223–1229
Zambare V, Nilegaonkar S, Kanekar P (2011) Novel extracellular protease from Pseudomonas
aeruginosa MCM B-327: enzyme production and its partial characterization. N Biotechnol 28:
173–181
Zapatae Castillo P, Villalongae Santana LM, Tamayoe Cortes J, Riverae Munoz G, Pereira S (2012)
Purification and characterization of laccase from Trametes hirsuta Bme2 and its contribution to
dye and effluent decolorization. Afr J Biotechnol 11(15):3603–3611. https://doi.org/10.5897/
AJB11.2050
28 Fungal Extremozymes in Green Chemistry 707

Phylogenomics, Microbiome
and Morphological Insights of Truffles: The
Tale of a Sensory Stimulating
Ectomycorrhizal Filamentous Fungus
29
Mohan Das, Ananya Pal, Subhodeep Banerjee, Subhara Dey,
and Rintu Banerjee
Abstract
Truffles are generally considered as subterraneous ascomycete, capable of
forming ectomycorrhizas with plant roots via symbiosis. The fruiting body of
truffles are widely appreciated around the world for its distinctive aroma.
Although the plantation system for truffles is established, it still faces an array
of challenges. The queries are very unique on its own and yet to be resolved. The
taxonomic classification seems to be highly complicated and needs proper sam-
pling to achieve progress using advanced technologies. The truffles being an
extremophile need precise soil and weather conditions for its growth. The unique
feature of truffle is its extreme slow growth rate for rejuvenation under optimum
cultivation conditions. While positioning of truffles is a very crucial criterion to
understand the type of symbiosis it maintains with different plants. Together with
this it is apprehended that it is the plant and the rhizospheric soil which partially
determines the microbial community within truffles. This in turn partially
contributes in producing the distinctive aroma of truffles. Nevertheless, it is
worth mentioning that a truffle renders several pharmacogenetic effects on
human system, which need more investigation to unravel this exorbitant creation
of nature.
M. Das · R. Banerjee (*)
Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur, West
Bengal, India
e-mail: rb@iitkgp.ac.in
A. Pal · S. Dey
P. K. Sinha Centre for Bioenergy and Renewables, Indian Institute of Technology, Kharagpur, West
Bengal, India
S. Banerjee
Advanced Technology Development Centre, Indian Institute of Technology, Kharagpur, West
Bengal, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2022
S. Sahay (ed.), Extremophilic Fungi,https://doi.org/10.1007/978-981-16-4907-3_29
709

Keywords
Truffles · Phylogeny · Microbiome · Molecular characterization · Health benefits
29.1 Introduction
Apicius’Legendary Banquet considers truffles as an Epicurean delicacy honoured
by Greeks and Romans. The first article on the nature of “Truffles”was published by
Ciccarelli in 1564. Hervé This, the discoverer of molecular gastronomy, named
truffles as ‘diamonds’or ‘the best food’. In true sense, truffles are actually hypoge-
ous edible fungus, which possibly undergoes a complicated life cycle, a time period
when a symbiotic relationship between the mycelium and the roots of trees (e.g. Oak,
hazel, willow, poplar) and shrubs (e.g. Cistus) gets established. The truffle or the
fruiting body finally develops due to the aggregation of the hyphae. Generally,
species belonging to the genus Tuber are regarded as “true truffles”and are also
considered as one of the very few ectomycorrhizal Ascomycetes. Although
according to the classical classification system truffles were in the order Tuberales,
which includes hypogeous ascomycetes, today they are under the order of Pezizales
(including both hypogeous and epigeous fungi) and are irrespective of their
saprotrophic or symbiotic nature, since they are all attached to each other
phylogenetically (Antonietta et al. 2006).
Tuber magnatum or the “white truffle”and Tuber melanosporum Vittad or the
“black truffle”are in high demand due to their characteristic taste and aroma, which
actually results from a concocted blend of volatile compounds. The formation of
ascocarp and truffle life is influenced both by biotic (bacteria, fungi, yeasts,
mesofauna) and abiotic (composition of soil, rain, temperature and sunshine).
Most of the truffle species share common features and therefore need calcareous
soil (pH 7–8), with exceptions for Tuber borchii since it needs acidic soils. The
demographic locations define the extreme criteria and conditions of truffle growth
and availability. The entire Europe is mostly rewarded with Tuber borchii and Tuber
maculatum whereas Western and Southern Europe (e.g. Spain, Italy, France) are
especially prized with Tuber melanosporum and handsome collection of Tuber
magnatum is mostly from Italy and in limited quantity from Eastern Europe
(e.g. Croatia, Slovenia, Hungary) (Antonietta et al. 2006; Das et al. 2020).
The report on Tuber melanosporum by Zampieri et al. (2011) emphasizes tran-
scriptional profiling for expressing the specific gene of different traits in different
climatic regions indicates phenotypic characters and specifically mentioned the
profiling in colder climatic conditions (4 C) with other temperature variations.
The validation of experiment has been carried out using standard protocol of
RT-PCR where the genes show better expression in case of heat shock protein,
cell wall production and lipid accumulation profiling of T. melanosporum. Thus, it is
a real challenge to the mycologist to grow truffle ascomata in low temperature and
environmental conditions. As mentioned earlier, T. melanosporum has an interesting
life cycle, where root colonization is mainly taken place in winter that establishes a
710 M. Das et al.

symbiotic relationship with the host that can withstand the harsher climatic
conditions (Martin et al. 2010). Similarly for the growth and maturation of Tuber
borchii, dehydrin-like proteins play a significant role that code the high expression
of the protein under colder condition and osmotic stresses that are needed for proper
growth of Tuber spp. Thomas and Büntgen (2019) reported that summer temperature
and precipitation are the two important factors for reduced growth of truffles as it has
been reported that under low temperature condition and optimum soil and moisture
ratio the growth of truffle can be accelerated. Thus, the environmental issues for
truffle growth are a real challenge to the scientific community due to its extreme low
growth profile. Whereas, the dessert truffles cultivation occurs in semiarid conditions
of the Algerian deserts of the Sahara and are thus extremely well adapted to less
rainfall and harsh climatic conditions such as dry and hot environment, i.e. contrast
to the cold loving truffles as discussed (Bradai et al. 2015).
Truffles are mostly hunted with the aid of trained pigs and dogs. The huge
demand for truffles on one side and dropping productivity on the other have enforced
the scientists to understand the extreme culturable conditions and mimic the system
and for this purpose advanced strategies and approaches have been adopted to
understand the bottlenecks of the system, its environmental microbiology, its molec-
ular definition and phylogenetic lineages to illustrate, explore and search possible
chances to overcome the hurdles of truffle production (Antonietta et al. 2006; Das
et al. 2020; Dash et al. 2016).
29.2 Morphological Diversification of Truffles Through
Evolutionary Approach
The truffles which possess sac-like spore-producing structures are classified as the
morals and cup fungi in the class Ascomycetes. Ascomycetes are characterized by
sexual spores called ascospores, formed in sac-like structures called asci, millions of
which form the fruiting body called ascoma (plural: ascomata) with tightly interwo-
ven hyphae. In evolutionary sequence, truffles are mostly similar to the members of
the order Pezizales which includes family like Pezizaceae,Sarcosomataceae and
Otideaceae. During the course of evolution, family members of Otideaceae show
gradual changes to sequestrate (their fruit-body does not liberate spores at maturity)
or become hypogenous (they produce their ascomata underground). Under this
family, Genea produces closed but hollow ascomata, while Geopora has much
less air space inside the fruit-body and is hypogenous. Although a solid truffleof
the genus Tuber, family Tuberaceae which is evolved by the elimination of airspace
altogether, possesses sequestrated, hypogenous and solid (no air spaces anymore)
ascomata. The ascospores with highly ornamented walls are found within rounded
and thin walled asciin, a highly convoluted hymenium. Their hypogenous nature has
necessitated a new method for passive spore dispersal. The odours released from
mature ascospores lead many mammals to unearth and consume them subsequently
depositing the still viable spores elsewhere. Thus, forcible spore discharge mecha-
nism is replaced by spore dispersal via animal mycophagy (Kendrick 1985).
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 711

True truffles, belonging to the genus Tuber, are characterized by a pattern of
sterile and fertile veins filling the gleba, which darken as the fertile tissue matures
and vary in colour by species. Their stereothecia has a distinct, simple to layered
peridium enclosing a gleba of fertile tissue marbled with sterile, hypha-stuffed veins
that tend to open through the peridium. Ascomata with smooth pubescent surface or
with rounded or angular warts may be white, grey, yellow, olive, brown, reddish
brown, dark brown or black in colour. The gross morphological features that help to
distinguish Tuber species are the texture of the peridium and microscopic
characteristics of the asci and spores. The peridium may bear large warts as in
T. aestivum or it could be rough, scaly, pubescent or glabrous as in T. oregonense.
Asci may be globose or sub-globose or more flask-shaped and bear a stem as in
T. lyonii. Finally, spores may be alveolate-reticulate or spiny, as in T. maculatum and
T. melanosporum, respectively (Trappe et al. 2008,2010). The species Tuber
gennadii is characterized by having locules lined by a palisade of asci and was
hypothesized to be an intermediate form of epigeous and hypogeous fruiting
behaviour.
Besides the members of ascomycetes, some truffle-like members are in the
primitive fungal class Zygomycetes and within Basidiomycetes, named as false
truffles. The false truffles of Basidiomycetes produce underground truffle-like
basidiocarps, evolved by extensive morphological divergence. Being intermediate
species between truffles and mushrooms, they have a vestigial stem, unable to lift
them out of the ground; while the cap remains closed with crowded gills which are
distorted or even replaced by chambers. Such fruit-bodies are named as ‘secotioid’in
reference to genus Secotium. Finally, the losing of vestigial stem or columella occurs
to improve. Selection for animal dispersal (instead of forcible spore dispersal) and
water loss reduction forces the morphological evolution of the false truffle
Rhizopogon from a suilloid ancestor (Trappe et al. 2008).
Most false truffles have a gleba (the internal, spore-producing tissue) that is
divided up into small chambers called locules. Many false truffles also have a
short stipe at the base or a stipe-columella that extends into the gleba as a branching
structure. Aside from the stipe-columella, the gleba of false truffles is fairly regular
in appearance. This easily separates them from true truffles, which appear marbled
when sliced in half. Some examples of false truffles are Rhizopogon luteolus (yellow
false truffles), Alpova trappei,Gastroboletus turbinatus,Hymenogaster boozeri, etc.
(Trappe et al. 2010).
Genus Rhizopogon has white to yellow, salmon, red or brown peridium with
wine-like, cheesy or spicy-pungent odours. Gleba varies from white to yellow in
youth, becomes olive, olive-grey, olive-brown, orange-brown or blackish brown in
maturity. Genus Alpova with yellow to reddish brown peridium has yellow to
ochraceous (in youth) gleba which is gelatinous and sticky to touch. It stains reddish
brown when exposed. The chambers with spores in a gelatinous matrix are walled
off by meandering veins. Genus Endogone under Phylum Zygomycota has solid
grey to bright yellow to brown gleba with a mass of spores and mycelium without an
organized structure. Peridium if present varies from white to bright yellow to brown
in colour with smooth to cottony texture (Trappe et al. 2008).
712 M. Das et al.

29.3 Truffle Microbiome
29.3.1 Bacterial Communities
While investigating the microbial diversity in truffles it has been observed that
bacterial communities tend to colonize in million to billion cells/gram within the
inner region and outer parts of fruiting bodies. Compositional analysis of bacterial
community has also been investigated in context to its life cycle (fruiting body v/s
mycorrhizas), maturation state of fruiting body and the peridium (outer layer) v/s the
gleba (inner layer). Based on both culture-independent and culture-dependent
methodologies, it has been reported that different species of truffles contain complex
communities of bacteria including mostly Actinobacteria,Bacteroides,Firmicutes
and Proteobacteria. There exist considerable differences between bacterial compo-
sition of Tuber borchii with Tuber melanosporum and Tuber magnatum, since it has
been observed that the microbiota of Tuberborchii is dominated by
Betaproteobacteria,Bacteroidetes and Gammaproteobacteria. It has been reported
that a Bacteroidetes strain may exist under axenic conditions within the mycelium of
T. borchii, which suggests strong association of truffles with bacteria. Maturation of
fruiting bodies occurs after melanization of the gleba, which is mainly due to the
formation of spores occurring within the fungal asci. The uniqueness in the aroma of
truffles is mostly dictated by the metabolic activities played by the microbes present
within them. For truffles like T. borchii,T. magnatum and T. melanosporum, which
are mostly harvested in Europe, the process of melanization extends for a few
months and occurs during the season of winter or late autumn. With increasing
maturity a significant decrease in bacterial count has been observed for the afore-
mentioned varieties, as proved by fluorescence in situ hybridization (FISH). Never-
theless, it is worth mentioning that there did not exist any considerable differences in
composition of the community, especially for Actinobacteria,Alphaproteobacteria,
Bacteroidetes,Betaproteobacteria,Gammaproteobacteria and Firmicutes
(Vahdatzadeh et al. 2015).Therefore, it is apprehended that it is the ratio of different
microbes and their metabolic system which is actually determining and dictating for
the unique aroma of truffles.
29.3.2 Yeast Communities
While assessing the presence of yeast community within fruiting bodies of
T. magnatum,T. aestivum,T. melanosporum, soil of truffle orchard and
ectomycorrhizas, it has been observed that fruiting bodies and truffle
ectomycorrhizas are highly colonized by yeasts, reaching 3 10
7
CFU/g of dry
fruiting bodies. The results are based on culturable techniques, which although
provide insights about the system but may overlook the detailed diversity of the
system. The diversity is mostly dominated by Cryptococcus albidus,Debaryomyces
hansenii,Cryptococcus humicola,Saccharomyces paradoxus and Rhodotorula
mucilaginosa. The density and types of species may vary between gleba and
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 713

paridium as well as with change of distinct species. Indeed, isolated yeasts were
detected by culturable methods from the peridium of T. melanosporum and
T. aestivum but not from intact truffles gleba (Vahdatzadeh et al. 2015).
29.3.3 Fungal Communities
Truffles are mostly colonized by guest filamentous fungi, as isolated from Tuber
rufum,Tuber aestivum,Tuber nitidum,Tuber brumale,Tuber magnatum,Tuber
melanosporum,Tuber excavatum and Tuber puberulum. The loose association
between truffles and fungi was depicted from the observation that only 26% of
truffles have guest fungi. Filamentous fungi mostly colonize within the peridium of
truffles, having a density of 10
2
CFU/g of truffle fruiting bodies, whereas they seem
to be mostly absent in the gleba of truffles (Vahdatzadeh et al. 2015).
29.4 Taxon Sampling and Phylogenetic Analyses
The phylogenetic position of hypogeous taxa inside Pezizales is determined by LSU
rDNA (large subunit ribosomal deoxyribonucleic acid) sequences of 134 epigeous
pezizalean species (out of 141 specimens) and 48 hypogeous species (out of
55 specimens) by Laessoe and Hansen (2007). Considering the entire of Pezizales,
three subsets representing three distinct lineages of Pezizales were constructed. The
taxa representing the family Pezizaceae for lineage A, Caloscyphaceae,Tuberaceae,
Discinaceae,Rhizinaceae,Morchellaceae and Helvellaceae for lineage B and
Ascodesmidaceae and Pyronemataceae for lineage C were assessed in the three
alignments.
29.4.1 Phylogenetic Relationship Within Lineage A
Within lineage A, parsimony analysis yielding three MPTs considering
973 characters revealed Pezizaceae as monophyletic having a sister group named
Ascobolaceae. The consensus tree is although resolved but deep root relationships
are yet to be well understood and supported. Out of 14 lineages, five or six housed
17 species of truffles, covering 11 genera. Although during analysis by maximum
parsimony (MP) Eremiomycese chinulatus has been resolved and attached with
Peziza vacini but after maximum likelihood (ML) and Bayesian analyses
E. echinulatus is positioned with Plicaria–Hapsidomyces lineage together with
Peziza phyllogena. The genus Pachyphloeus, apothecial Scabropezia and anamorph
Glischroderma form a highly supported clade, to which Amylacustasmanicus forms
a sister taxon. It has been reported that Sarcosphaera forms a group with two species
of Hydnotryopsis genera. Within all the MP trees, although Mattirolomyces has been
nested inside Peziza s. str. lineage but after ML and Bayesian analyses
Mattirolomyces group with Iodophanus, being a sister group of Peziza s. str. lineage.
714 M. Das et al.

It has been suggested that Iodowynnea has close relation with Kaliharituber. The
species of Peziza,P. whitei and P. ellipsosora along with the genera Tirmania,
Ruhlandiella,Cazia and Terfezia are found to resolve apothecia forming species of
Peziza within the P. depressa–Ruhlandiella lineage (Laessoe and Hansen 2007).
29.4.2 Phylogenetic Relationship Within Lineage B
For determining phylogenetic relationship within lineage B, parsimony analyses
yielded three MPTs considering 699 characters. It has been reported that the
consensus tree is resolved but except Tuberaceae there is a lack of support for
other families. On the basis of Bayesian analyses, Underwoodia columnaris has
been excluded since it is unresolved but Helvellaceae is highly supported. But
maximum parsimony (MP) and maximum likelihood (ML) analyses resolved a
Helvellaceae–Tuberaceae and Morchellaceae–Discinaceae lineage. Although the
exact position is yet to be determined, Fischerula subcaulis and Leucangium are
variedly positioned inside Morchellaceae–Discinaceae lineage. In all the analyses, a
monophyletic group has been formed by two Hydnotyra species, named
H. cubispora and H. cerebriformis which is placed inside Discinaceae. A sister
group of a clade of Wynella silvicola and species of Helvella has been formed by
Balsamia oregonensis and Balsamia magnata. A clade composed of four additional
genera of truffles, namely Choiromyces s. Str.,Dingleya,Labyrinthomyces and
Reddellomyces included 11 Tuber species as a sister group (Laessoe and Hansen
2007).
29.4.3 Phylogenetic Relationship Within Lineage C
For establishing phylogenetic relationship within lineage C, parsimony analyses
have been performed which yielded three MPTs (Most Parsimonious Trees) consid-
ering a total of 894 characters. Based on the studies, it has been reported that the
consensus tree is almost totally resolved but the three MPTs were not capable
enough to explain deep root relationships. It has been suggested that
Pyronemataceae are paraphyletic since Ascodesmidaceae are placed inside it,
which is mostly accepted as monophyletic. After performing the analyses, the
pyronemataceous taxa were recovered within 12 clades, where nine of the species
of truffles included were nested inside three supported clades. A monophyletic group
was formed by five species of hypogeous Genea, which is actually acting as a sister
group to Humaria hemisphaerica; whereas taking five epigeous Geopora species, a
monophyletic group was formed by Geopora cooperi. It has been suggested that
Geopora is actually sister group, where Miladina,Scutellinia and Ramsbottomia are
in the clade. Stephensiais monophyletic and so Stephensi abombycina forms group
with Paurocotylispila,Geopyxis carbonaria and Stephensias hanorii (Laessoe and
Hansen 2007).
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 715

29.5 Life Cycle and Molecular Characterization
With the advancement in different techniques and understanding the reproductive
biology of Tuber spp. at a molecular level, it was hypothesized that these are selfing
organisms. Tuber melanosporum popularly known as black truffles is considered as
the model for the study of the biology of Tuber spp. because till date only the whole
genome of Tuber melanosporum has been sequenced. The existence pattern of Tuber
spp. is partitioned into stages, which starts with the development of filamentous
vegetative mycelia and ends with the association of the fungal hyphae with the host
root to build up a fungal mantle and the Hartig net.
29.5.1 Life Cycle of Tuber sp.
Truffle has been considered as a highly esteemed food since the eighteenth century.
During this period the experts in this field have made various endeavours to
comprehend the riddle behind Truffle’s life cycle. Most recent contemplates made
based upon genetic and genomic approaches have helped mycologists to take a rise
towards a better understanding of the life cycle and sexual mode of Tuber spp. To
gain more knowledge about the Tuber sp.scientists considered T. melanosporum as
the model organism, since the whole-genome sequencing of this species has been
conducted and the information related to the genome sequence was available to
intricate studies. After numerous in-depth molecular studies performed by the
researchers, an overview regarding the life cycle of Tuber sp.was made for better
understanding. In general, the life cycle is divided into (1) the formation of the
underground ascocarp, i.e. the fruiting body (Truffles) (2) the formation of
ascospores which are released into the surrounding environment and (3) the infection
and establishment of a symbiotic relationship made by the ascospores in the roots of
a nearby host plant.
It has been observed that throughout the life cycle of T. melanosporum the
haploid phase predominates as compared to the dikaryotic phase. The ascospores
which are released from the mature ascocarps have been observed to undergo a
symbiotic relationship with the nearby host plant. For the establishment of a
symbiotic relationship between the fungi and the host plant root it was found that
the production of indole-3-acetic acid (IAA) and ethylene which are considered to be
plant hormones should be secreted by the mycelia first, so that the induction of
shortening of the main root and the sideways root formation can occur. An extra
radical pseudo-parenchymatous mantle formation by hyphae around small and
growth-restricted lateral secondary roots takes place, which is followed by the
colonization of hyphae which grows between rhizodermis and cortical cells, which
further leads to the formation of Hartig net. An immature fruiting body is formed due
to the fertilization of uniparental maternal hyphae and dikaryotic hyphae, after the
successful establishment of symbiosis with the host plant. The karyogamy and
meiosis in the ascus mother cells lead to the formation of mature asci containing
haploid ascospores (Rubini et al. 2014). Therefore, it has been noticed that a mature
716 M. Das et al.

fruiting body (truffle) is composed of gleba and based on the data available, it has
been observed that the structure of gleba in the fruiting body is from a haploid
hyphae of uniparental (maternal) source, where the rate of outcrossing is either
potential need or else necessary. Kües and Martin (2011) through their studies
showed that the ascospores which are discharged in the soil from a mature ascocarp
germinates and turns out to be either MAT(+) or MAT()primary haploid mycelia. It
was observed that a single lateral root was not coexisted by different homokaryotic
mycelia rather only a single genotype dominates. The reason behind the dominance
of a single genotype was found to be due to the competition among completely
different strains of mycorrhiza that share similar mating type. Under favourable
surroundings and in the presence of other factors, MAT(+) and/or MAT()mycelia
establishes a successful mycorrhiza on the root of the host plant and follows a
symbiotic relationship whereas some get dispersed in the soil and grow
nonsymbiotically. Contact between the mycelia of opposite MAT locus, which
originates from ascospores or mycorrhizas from adjacent host plants is vital for
fertilization to occur. Fertilization leads to the formation of fruiting bodies/ truffles.
The life cycles of Tuber spp. are still poorly understood and numerous questions
are yet to be answered. Research is still going on with different tools to gather and
learn more about the life cycle of Tuber spp. In the following section, we will learn
more about the MAT locus and also the major role played by it during a particular
segment of the life cycle.
29.5.2 MAT Locus of T.melanosporum
With the aid of advanced molecular techniques, it has been discovered that a single
locus which is found in small regions of the genome is responsible for controlling the
sexual reproduction in filamentous ascomycetes. These small sections of the genome
are now known as the mating type (MAT) loci. It was found that a MAT loci with two
major regulators were responsible for coordinating the sexual reproduction in
ectomycorrhizal fungi. Through transcriptional analysis, it was found that the
MAT1–1-1 and the MAT1–2-1sequence were responsible for encoding an α-box
domain protein and a high-mobility group (HMG) domain macromolecule, respec-
tively. The ascomycetes generally follow two types of reproductive mode,
i.e. heterothallism and homothallism. In the heterothallic mode, two MAT sequences
happen to occur in different strains and are self-sterile, thus crossing among strains
of opposite mating type is necessary. Interestingly, both the versions of the MAT
locus in ectomycorrhizal fungi are not the allelic versions and are known as
idiomorphs. In the case of homothallic mode, both the MAT genes were found
harbouring in a single strain making them capable of undergoing selfing or crossing
with any other of the alike species, as they don’t have any separate sexual identity
(Rubini et al. 2012,2014).
The whole-genome sequencing of T.melanosporum has already been conducted
and it was found that the MAT1–2and MAT1–1segment were approximately
7430 bp and 5550 bp, respectively (Rubini et al. 2014). The MAT locus is flanked
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 717

with genomic regions that are found conserved among ascomycetes. Some of the
genes like APN2 and SLA2 were found to be linked to the MAT locus in
Pezizomycotina and some Saccharomycotina species and are considered as an
indication of a preserved evolutionary source of the MAT locus (Rubini et al. 2012).
For the scientific community, the arrangement of MAT locus in the T.
melanosporum genome offered numerous definitive proof regarding the molecular
basis of sexual reproduction in ectomycorrhizal fungal species and also the knowl-
edge obtained from genome sequencing of T.melanosporum genome has provided
clues for further understanding and categorization of potential sexual reproduction
strategies in use by the other Tuber sp.
29.5.3 Molecular Tools for the Identification of Tuber sp.
Over the years crucial knowledge regarding the diversity and organization of
ectomycorrhizal fungi has been gained on the basis of data obtained from a variety
of techniques such as enzymatic polymorphism analysis, immunological techniques,
etc. Urbanelli et al. (1998) showed in his studies that a specific host plant can be
mycorrhized by various taxa on a very small and precise root area. For the field
studies, work with a single root tip is very important, but the aforementioned
techniques were not efficient enough to provide reliable data from a small number
of samples. With time flow it became obvious that studies using these techniques
aren’t enough to explore more about the Tuber spp.Furthermore, the formation of
fruit-bodies is also reliant upon ecological factors and also knowing the fact that
these organisms don’t deliver fruit-bodies each year. Subsequently, while depicting
ectomycorrhizal fungal diversity, it is obvious that the ectomycorrhizas itself must
be studied to decide the fungi that are well connected with the host plant’s root.
Morphological studies of ectomycorrhizal roots have provided valuable inputs for
distinguishing the fungi; however, most species have not been depicted by this
technique. Through techniques like sporocarp studies and morphological studies
the foundation for understanding truffles has been established, but the utility of
advanced molecular techniques like PCR has helped to understand the
ectomycorrhizal fungal diversity and ecological factors in a bigger picture.
Techniques like morphological techniques have advantages as well as
disadvantages; for example, it was observed that morphological grouping is not
exact enough to precisely portray ectomycorrhizal fungal communities, as morpho-
logical studies are too time-consuming to learn and might be reliable among
laboratories.
In 1990, White along with his co-scientist first developed the universal primer for
PCR to amplify the nuclear ITS segment. Bruns and Gardes (1993) designed (ITS1-
F) and (ITS4-B) primers which were expected to be explicit for organisms and
basidiomycetes, respectively. The rRNA genes like internal transcribed spacer (ITS)
or intergenic spacer (lGS) which are non-coding genes provide exceedingly variable
target amongst morphologically diversified mycorrhizal fungal strains. Grades et al.
in 1991 showed that intraspecific variation in the ITS regions is low. The ITS region
718 M. Das et al.

in the genome was reported to be 600 bp–800 bp which was appropriate for
amplification and also suitable for amplification with universal primers (White
et al. 1990) in PCR method and rDNA, with the ITS region blanketed, is present
within the genome as a pair of copies, allowing amplification from very small as well
as less concentrated samples. Interest in the identification of ectomycorrhizal fungal
community on the roots increased because of the sole reason, i.e. the evaluation
based on ITS location turned into particularly less complicated. A major drawback in
the ITS segment is that the level of intraspecific variation isn’t uniform among
different species and error in identification of the symbiont may occur due to
contaminant DNA of other origins. On the other hand, the ITS segment is in
widespread sufficiently variable to permit clear discrimination among distantly
related species or genera and consequently allowed the construction of the phyloge-
netic tree of Tuber spp. Different molecular markers were used to identify
ectomycorrhizal fungal species, like in restriction fragment length polymorphism
(RFLPs) restriction enzyme digested ITS regions were used for the identification.
PCR technique has been widely used to identify the ectomycorrhizal fungal species
from root tip samples.
Development in the PCR-based method was coming up with the use of random
oligonucleotide primers to spot the bacterial strains. In 1990, Williams and his
research team got up with a similar idea that makes use of autoradiography in
preference to radioactivity in visualizing the amplification effects and thus were
mostly used because of its ease. This method in present days is popularly referred to
as random amplified polymorphic DNA (RAPD). The important elements of the
RAPD technique include: firstly no prior knowledge about the priming sites in the
unknown target genome is needed and secondly only a single primer is needed to
amplify the region located between the priming sites. The advantage of RAPD
markers has made it possible to recognize the strains or isolates of non-sporulating
root with great resolution. It is practically cost-effective, fast and maybe beneficial in
any laboratory geared up for PCR strategies. The bottleneck of this method is
reproducibility because of which it cannot be used for the study of the symbiotic
stage as the random primer applied fails to differentiate and thus amplifies the DNA
from the symbiotic partner (plant and fungus) and sometimes from accidental
contaminants which inhabit the rhizosphere, which led to a generation of complex
DNA fingerprints which is difficult to analyse.
Research community which is dedicated to exploring more about Tuber spp. is
using another PCR-based technique other than RFLP and RAPD, i.e. real-time PCR
technique (RT-PCR). The principle of this method is based upon the quantification
of the fluorescent signals which are generated from a fluorescently labelled
sequence-specific probe or an intercalate dye. The strength of the signal is directly
proportional to the amplified PCR product in a reaction mixture. Thus, it became
possible to observe the PCR during the exponential period by recording the amount
of fluorescence emission per cycle. This method is widely used for quantifying
fungal DNA. This method has wide applications for quantification of mycelia
biomass and extra radical hyphal biomass. Suz et al. (2008) and Rizzello et al.
(2012) used RT-PCR with SYBR Green dye for staining nucleic acid and evaluate
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 719

the amount of soil mycelium in productive against non-productive truffle ranch.
These aforementioned techniques proved to be very beneficial in learning more
about truffles but a lot more study is yet to be done.
29.6 Overcoming the Bottlenecks of Truffle Production
A gourmet food has been the tagline for truffles for decades, priced not only for the
flavour but also for the rarity. The reproductive system of truffles is still not fully
understood and still eludes the biologist to a large scale. The bottleneck for truffle
cultivation remains the inability of humans to make them mate under controlled
conditions. Researchers are now looking for molecular markers to forecast the
population genetics of truffles and thus shade some light into the cultivation of
truffles to type cast the species with morphological similarity (Henrion et al. 1994).
Genetic variability has been studied in species that have an economic interest to
cultivate T. magnatum and T. melanosporum, the species for white and black truffles
extensively used in gourmet foods. T. melanosporum population had a strong
bottleneck due to phylogeographic signal absence during last glaciations (Bertault
et al. 2001). The findings always tend to implicate that a huge environmental factors
are responsible for the difference in taste and morphology for the truffles species
found across different geographical regions. Tuber spp. is proposed to have a self-
reproductive system. This finding has been a great influence on studies of genetics
for T. melanosporum and T. magnatum for sampling and mating model strategies
(Frizzi et al. 2001; Murat et al. 2004; Rubini et al. 2004). The research has led us to
differentiate their population genetics and track the speciation for better cultivation
and maintaining the symbiosis of the species with the environment. It has been found
that some Tuber species like T. aestivum and T. uncinatum outcross following the
studies on T. magnatum (Wedén 2004; Wedén et al. 2004). The genes for mating
type have not been isolated from the fungi in spite of several attempts. More and
more population genetics studies are required to get an insight into the life cycle of
these species. Sampling has become a hurdle for these experiments due to its low
availability throughout a region. The morphology of the fertilization process is still a
great mystery, only hypothesis is made for the fact that absence of male hyphae may
be fulfilled by some detached cells such as ascospores (Urban et al. 2004). Truffle
requires a unique set of ecological parameters unmatched to any other cultivars and
thus the artificial plantations are also required to be set in natural conditions to get
advantage of the climate factors and soil health (Hall et al. 2003). There is thus a
need for representation of local truffle biodiversity as one of the conditions for
improvement of artificial plantations to succeed (Rubini et al. 2004). This includes
the plants that grow and feed on.
The recent draughts have induced a decline in the harvest of Mediterranean truffle
and thus climate change and seasons change in precipitation and summer arids are
one of the factors responsible for change in decline in truffle production which has
led to huge loss in economics for black truffle sales and rise in prices (Büntgen et al.
2012). The change in smell of the truffles due to different conditions has misled the
720 M. Das et al.

pigs and dogs which are used to hunt truffles underground the soils which favour
truffles having dimethyl sulphide compounds (Talou et al. 1990). Truffles require a
strict soil property, for example T. melanosporum requires a soil with C/N ratio of
10.
29.7 Biological Importance of Secondary Metabolites
Like other fungal metabolites, i.e. mycotoxin, phytotoxin or aromatic compounds,
truffle metabolites possess an important role in the ecosystem as well as in its own
life cycle. Being ectomycorrhizas in nature, truffle volatiles have an immense
concern in mycophagal mode of spore dispersal. Besides odorant signals for
mammals and insects, they also participate in microbes and plant interaction to
regulate a complex molecular interrelation among soil fauna and flora. More than
200 volatile organic compounds (VOC) and many non-volatile compounds,
associated with the fruit-body, free-living mycelium and mycorrhiza of truffles are
responsible for the interactions with the host plants and non-host plants (the
so-called brunt, a zone with scarce herbaceous cover). Some common VOCs,
found in fruit-body of all truffles are 1-octen-3-ol, 3-methyl-1-butanol, 2-methyl-1-
butanol, dimethyl disulphide, etc. Structurally they are alcohols, aldehydes, ketones,
esters, aromatic groups and sulphur compounds. Most characterized VOCs can be
classified as fatty acid derived VOCs, terpenoids, sulphur-containing compounds
and aromatic compounds. VOCs like 3-octanone and 1-octen-3-ol derived from the
fatty acid metabolism are responsible for the strong fungal smell typical of T. borchii
and of some other fungi (Abraham and Berger 1994; Chiron and Michelot 2005;
Venkateshwarlu et al. 1999; Wnouk et al. 1983). Compounds like 3-methyl-1-
butanol, 2-methyl-1-butanol and their respective aldehydes, all are derived from
fatty acid catabolism. These compounds along with dimethyl sulphide are consid-
ered as the key contributors to the absolute aroma of T. melanosporum. Although
terpenoids are the minor part of truffles VOCs, they might have ecological impor-
tance. Some monoterpenes and sesquiterpenes, which could be involved in microbial
defence, are identified in T. borchii (Zeppa et al. 2004) and T. brumale (Mauriello
et al. 2004) at different maturation stages. Aromadendrene is rendered as a good
marker for fruit-body maturity in T. borchii as it has limited quantity in every
immature fruit-body of T. borchii. Sulphur-containing compounds like thiols,
sulphides, thioesters, thiophenones and thioalcohols are characteristic of most truffle
species. They might act as fumigants against microbes (Bending and Lincoln 1999)
and as repellents against amphipods (Schnitzler et al. 1998). One sulphur-containing
compound, dimethyl trisulphide has also been detected in pure mycelial cultures of
T. borchii (Tirillini et al. 2000). Among non-VOCs quinonoid and polyphenolic
biopolymers are reported as the major constituents of T. melanosporum’s melanin
(De Angelis et al. 1996). Some aromatic compounds found in truffles are
2-phenylethanol, benzaldehyde and 1-methoxy-3-methylbenzene.
VOCs produced during ectomycorrhiza formation of Tuber borchii with Tilia
americana were investigated by several research groups (Menotta et al. 2004;
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 721

Gioacchini et al. 2002). During their growth, the pre-mycorrhizal stage is reported to
have specific VOCs as hydrocarbons, alcohols, a brominated cholesterol derivative,
ketones and terpenoids including sesquiterpene Germacrene D, as well as
dehydroaromadendrene, longicyclene and β-cubebene which might be responsible
for chemotropism of hyphae towards roots of the host (Menotta et al. 2004).
Truffle metabolites have great impact on the interaction with plants and insects.
Some metabolites are reported to affect the root architecture of plant under labora-
tory conditions resulting in primary root shortening and their elimination (Splivallo
et al. 2007). White truffle mycelia are reported to produce hormone ethylene
(through the KMBA pathway) which was detected to play role in root hair elonga-
tion of the non-host plant. Some truffles like T. melanosporum can form a brunt, a
peculiar zone surrounding the host plan where the herbaceous cover is scarce. Due to
having truffle mycelium in the soil, ethylene or IAA released by truffles might
explain the reason for dying out the herbaceous plants in the brunt. On the other
side, 1-octen-3-ol extruded by both truffle mycelium and fruiting bodies generally
exerts toxic effects on plants including shortening the primary root, loss of chloro-
phyll through oxidative stress (Splivallo et al. 2007). Truffles attract mammals
ranging from squirrel to pig to consume the fruiting bodies for spore dispersal.
Truffle hunter traditionally uses pig and trained dogs to locate the truffle under-
ground. A steroidal pheromone, 5-α-androstenol secreted by black truffles was
reported to be responsible for attracting pig (Claus et al. 1981).
29.8 Aroma of Truffles
Truffles are admired for their unique smells worldwide, making them one of the top
listed luxury food items beside their rarity, limited seasonal availability and high
price. Species variability, maturity of the fruiting body, microbial flora along with
geographical origin are some important factors determining truffle aroma (Zeppa
et al. 2004; Gioacchini et al. 2002). Being one of the most aromatic species, Tuber
melanosporum with complex aroma is commonly known as ‘Black Diamond of
Cuisine’. Another black truffle, Tuber brumale has characteristic musky odour with
‘earthy notes’. The most expensive truffle, T. magnatum with complex aroma of
garlic and cheese is considered as the finest species. The aromatic profile of a single
species usually contains more than 50 volatile constituents (Bellesia et al. 1998;
Mauriello et al. 2004). Beside some common truffle volatiles, there are some
species-specific compounds. For example, thiophene derivatives 2-methyl-4,
5-dihydrothiophene and 3-methyl-4,5-dihydrothiophene are specific for Tuber
borchii while other sulphur volatile 2,4-dithiapentane is a dominant constitute of
T. magnatum (Splivallo et al. 2011). 1-methoxy-3-methylbenzene is specific for the
Black truffle species like T. aestivum,T. melanosporum,T. brumale, etc. (Mauriello
et al. 2004). During the truffle formation numerous microbes might end up trapped
inside fruiting bodies. As a matter of fact, this microbial diversity also plays a
speculative role in truffle aroma formation (Tirillini et al. 2000).
722 M. Das et al.

29.8.1 Biosynthesis
The hypothetical biosynthetic routes of the precursor metabolites for major and
characteristic components of truffle aroma have documented from the sequencing
of the black truffle(T.melanosporum) genome (Martin et al. 2010). Two main
sulphur-containing VOC (S-VOC) biosynthetic pathways executed through L-methi-
onine catabolism have been found in some bacteria and in some ascomycetous
yeasts, leading to form methane thiol (MTL), a bad smelling thiol compound
detected in truffles at low concentration (Arfiet al. 2006; Liu et al. 2008). When
transcript abundance profiles were emphasized for fruit-bodies vs. mycelia, it was
found that during maturation of the fruiting body of T. melanosporum, the relative
concentration of methionine tends to remain constant, as sulphur assimilation and
metabolism are particularly sustained in fruiting bodies (Martin et al. 2010). Beside
methionine C-lyase, cystathionine C-S lyase, which uses cystathionine as substrate
for MTL production has been found overexpressed in T. melanosporum fruiting
body (Harki et al. 2006). Ultimately the complete balanced amount of diverse
compounds creates the specific truffleflavour. It is possible because of the highly
tuned regulation of the genes coding for various flavour biosynthetic enzymes during
the truffle development and maturation. Some of these volatile are derived from free
amino acid catabolism through Ehrlich pathways (Singh et al. 2019). A candidate
gene for the carboxylation and cancellation process of this pathway has been
proposed for T. melanosporum (Martin et al. 2010). Besides these genes for isopren-
oid pathway have been characterized in T. borchii and all genes for mevalonate
pathway have been identified within the genome of black truffle (Guidi et al. 2006).
Nevertheless, it is worth mentioning that via these entire pathways truffle can
potentially synthesize members of the most diverse family of natural products.
29.8.2 Potential Health Benefits
Truffles mostly consist of nitrogenous products of which proteins and mineral salts
particularly that are rich in potassium and phosphorus are available. Comparing
other forms of fungus truffle is 75% water rather than 90% water in other fungi that
are cultivated. For example, truffle aphasia possesses 48 g of carbohydrate, 2 g of
protein and 3.2 g of fat per 100 g of dry weight. There are many studies which
indicate that truffles are easily digested and the value of truffles is due to its unique
fragrance. The aromatic fragrance mainly consists of organic sulphur compounds.
This noted component of truffles has made it an integral part of delicate foods
blended with other components of Mediterranean and French cuisines that the
cooks are very proud to offer. True truffles are mostly cultivated or collected mainly
due to this extraordinary nature but they also offer a valuable medicinal component
that can prove to be having a dual role to both tongue and health.
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 723

29.8.2.1 Antioxidant Properties
Truffles are great source of vitamin A, β-carotene, vitamin C and other phenolic
components, which have a great role to play in scavenging the free radicals and
chelate ferric ions by reducing the reactive oxygen species roaming through our
bodies (Al-Laith 2010; Biswas et al. 2020; Kundu et al. 2020). Methanolic extract of
different truffles species of the Tuber genera states that T. aestivum comprises high
phenolic and ergostrerol compounds that directly correlate to great antioxidant
properties (Das et al. 2020).
Desert truffles are also considered a source of higher phenolic content and thus
have a greater antioxidant potential. According to studies by Hamza et al. in 2016,
ascorbic acid, carotenoids and anthocyanins content of 10.63 mg, 1.17 mg and
29.1 mg, respectively, per 100 g in T. nivea are facts supporting its antioxidant
properties. Catechin present in T. boudieri is responsible for the high radical
scavenging activity, being the principal phenolic component of the truffle (Doğan
and Aydın2013) along with ferulic acid, p-coumaric acid and cinnamic acid.
Chen et al. (2016) demonstrated that Chinese truffles have high antioxidant
properties like the DPPH radical scavenging activity of T. huidongense
polysaccharides. T. latisporum,T. subglobosum and T. pseudohimalayense pos-
sessed antioxidant properties, in particular, T. latisporum and
T. pseudohimalayense had predominant amount of phenolic compounds and thus
possessed more scavenging activities than T. subglobosum (Yan et al. 2017).
29.8.2.2 Anti-Inflammatory Properties
The phenolics present in truffles react with free radicals and mono oxygen species
and serve as antioxidants (Sies and Stahl 1995). Luo et al. (2011) experimented that
T. indicum polysaccharides have proven to have a cytoprotective effect on the PC12
(rat adrenal medulla pheochromocytoma) cells during exposure to hydrogen perox-
ide stressors. The antioxidant properties with all the phenolics, terpenoids and
polysaccharides attribute to the fact that they have also anti-inflammatory properties
(Friedman 2016). Zhang et al. (2018) experimented on hyperglycaemic rats with
aqueous extract of T. melanosporum, which showed reduced glucose levels like
other standard diabetic rat treated with antidiabetic drug, glibenclamide. This glu-
cose reducing effect of rats can be attributed to the correlation with Nrf2 and NF-kB
pathways and variation in superoxide dismutase, catalase and vitamin C metabolism
and regulatory changes. In addition a hepatoprotective effect of T. claveryi extract
was also noticed by Janakat and Nassar (2010). Inhibition of COX-1 and 12-LOX
pathways, 12(S)-hydroxy-(5Z, 8E, 10E)-heptadecatrienoic acid (12-HHT), throm-
boxane B2 (TXB2) and 12(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraenoic acid
(12-HETE), the ones which are usually overexpressed in different inflammatory
diseases was observed by inducing Truffle extract treatment from T. magnatum
(Beara et al. 2014).
724 M. Das et al.

29.8.2.3 Antitumour Activity
Currently, many research works are going on in finding out the anticancer properties
of truffles. Methanolic extracts of various truffles species T. aestivum and
T. magnatum have shown significant in vitro cytotoxic effects on cancer cell lines
(HeLa, MCF-7 and HT-29). Aqueous extract of these truffles has also shown
prominent anticancer activity against breast adenocarcinoma MCF-7. Silver
nanoparticles were synthesized from aqueous extract of truffles that are rich in
flavonoids and amino acids (Khadri et al. 2017); during colorimetric assay with
the help of sulforhodamine B they showed significant cytotoxic effects against
MCF-7 cells with IC50 value of 10 mg/mL. T. claveryi extracts were used in
doing MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide) assay
on defined cancer cell lines and human brain carcinoma cell lines (U-87MG) with
IC50 values of 50.3 μg/mL of dry weight (Dahham et al. 2016). The anticancer
activities of extracts from truffles can be correlated with the use of different bioactive
compounds such as stigmasterol, phytosterol, squalene and lupeol (Dahham et al.
2016). These compounds play a vital role in inhibiting the growth of mutagenesis
together with the stimulation of cell apoptosis (Woyengo et al. 2009),
downregulation of lipid peroxidation and increase in the levels of glutathione
peroxide dismutase which directly causes decrease in tumour size and decrease in
survival rate of carcinoma in mice (Ghosh et al. 2011; Das et al. 2018).
Truffle also contains different polysaccharides that have anticancer activity such
as β-glucan polymers (Friedman 2016). The polysaccharides play a vital role in cell
to cell communication (Zong et al. 2012) and signal recognition, as it has been
reported that derivatives of these play a major role in preventing metastasis including
raising the immunity through creation of cytokines, interferons and
immunoglobulins to act against cancer antigens (Li et al. 2018; Moradali et al.
2007; Wasser 2003). 52 polysaccharides have been isolated from different truffle
species, viz. T. aestivum,T. indicum,T. melanosporum and T. sinense which showed
antitumour activity against A549, HCT-116, HepG2, HL-60 and SK-BR-3 cell lines
(Zhao et al. 2014). Oleic acid content in truffles has the ability to supress the
overexpression of HER2 oncogene; they also induce caspase 3 activity and conse-
quently cancer cell death (Carrillo et al. 2012; Menendez et al. 2006). Aqueous
extract of the T. boudieri showed increasing TH1 cytokines and decrease in TH2
cytokines that can stimulate lymphocyte proliferation and induce phagocytosis
(Al Obaydi et al. 2020). Further work is going on in assessing truffles activities as
antitumour agents.
29.8.2.4 Anti-Microbial Properties
Desert truffle has been reported to possess antimicrobial activity for a few decades
now. Aqueous extract of T. claveryi showed growth inhibition of Staphylococcus
aureus by 66.4% and Pseudomonas aeruginosa by 40% (Janakat et al. 2004;
Casarica et al. 2016). Owaid et al. (2018) demonstrated silver nanoparticles
synthesized from Tirmania spp.truffle possess antibacterial action against gram
negative and gram positive species mainly of the eyes, Pseudomonas aeruginosa.
Doğan and Aydın(2013) reported about antibacterial action of aqueous extract of
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 725

T. boudteri specifically against Candida albicans.Truffle lectins possess the ability
to recognize bacterial exopolysaccharides and destroy them (Elsayed et al. 2014).
Salmonella typhimurium,Escherichia coli,Pseudomonas aeruginosa,Enterococcus
faecalis,Staphylococcus aureus,Staphylococcus epidermis and Bacillus subtilis
species of bacteria were subjected to treatment of T. nivea extract and exhibited
inhibitory activities (Hamza et al. 2016; Das et al. 2017). Ethyl acetate extract of
T. pinoyi containing pyrazines and derivatives proved to be effective against Bacillus
subtilis and Staphylococcus aureus (Dib-Bellahouel and Fortas 2011). The
methanolic extract of the same fungus has shown inhibiting Staphylococcus infec-
tion in chicken soup in refrigerator (Stojkovićet al. 2013). Nadim et al. (2015)
proposed that laccase from T. magnatum catalyses the oxidized phenols releasing
superoxide anion radicals leading to inhibiting pathogenic bacteria.
29.9 Conclusion
Based on the research progress reported till date, it is worth mentioning that truffles
are nature’s artistic creation which needs more engagement of scientific researchers
to step forward for understanding the taxonomic classification and their phylogenetic
relationship, chemical component profile of each edible truffle and its pharmaceuti-
cal importance, artificial cultivation in green houses and detailed structural studies.
Truffles are a kind of aromatic bomb, therefore extraction of such compounds as well
as their importance to chemical ecology needs to be studied in detail. While
considering the pharmacogenic potential of truffles, it has been observed that they
are effectively capable of curing several disorders. But there is a need that different
species and their potential role in curing diseases are to be investigated, so that by
simply feeding this exquisite cuisine one can get rid of diseases.
References
Abraham BG, Berger RG (1994) Higher fungi for generating aroma components through novel
biotechnologies. J Agric Food Chem 42:2344–2348
Al Obaydi MF, Hamed WM, Al Kury LT, Talib WH (2020) Terfezia boudieri: a desert truffle with
anticancer and immunomodulatory activities. Front Nutr 7:38
Al-Laith AAA (2010) Antioxidant components and antioxidant/antiradical activities of desert
truffle(Tirmania nivea) from various middle eastern origins. J Food Compos Anal 23:15–22
Antonietta M, Claude M, Paola B (2006) Truffles: much more than a prized and local fungal
delicacy. FEMS Microbiol Lett 260:1–8
ArfiK, Landaud S, Bonnarme P (2006) Evidence for distinct Lmethionine catabolic pathways in the
east Geotrichum candidum and the bacterium Brevibacterium linens. Appl Environ Microbiol
72:2155–2162
Beara IN, Lesjak MM, Četojević-Simin DD, MarjanovićŽS, RistićJD, MrkonjićZO, Mimica-
DukićNM (2014) Phenolic profile, antioxidant, anti-inflammatory and cytotoxic activities of
black (Tuber aestivum Vittad.) and white (Tuber magnatum Pico) truffles. Food Chem 165:460–
466
726 M. Das et al.

Bellesia F, Pinetti A, Bianchi A, Tirillini B (1998) Volatile compounds of white truffle(Tuber
magnatum Pico.) from middle Italy. Flavour Fragr J 11:239–243
Bending GD, Lincoln SD (1999) Characterization of volatile sulphur containing compounds
produced during decomposition of Barassica juncea tissue in soil. Soil Biol Biochem
31:695–703
Bertault G, Rousset F, Fernandez D, Berthomieu A, Hochberg ME, Callot G, Raymond M (2001)
Population genetics and dynamics of the black truffle in a man-made trufflefield. Heredity 86:
451–458
Biswas P, Das M, Boral S, Mukherjee G, Chaudhury K, Banerjee R (2020) Enzyme mediated
resistant starch production from Indian Fox Nut (Euryale ferox) and studies on digestibility and
functional properties. Carbohydr Polym 237:116158
Bradai L, Bissati S, Chenchouni H, Amrani K (2015) Effects of climate on the productivity of desert
truffles beneath hyper-arid conditions. Int J Biometeorol 59(7):907–915
Bruns TD, Gardes M (1993) Molecular tools for the identification of ectomycorrhizal fungi—taxon-
specific oligonucleotide probes for suilloid fungi. Mol Ecol 2(4):233–242
Büntgen U, Egli S, Camarero JJ, Fischer EM, Stobbe U, Kauserud H, Tegel W, Sproll L, Stenseth
NC (2012) Drought-induced decline in Mediterranean truffle harvest. Nat Clim Change 2:827–
829
Carrillo C, del Cavia M, Alonso-Torre SR (2012) Antitumor effect of oleic acid; mechanisms of
action. A review. Nutr Hosp 27:1860–1865
Casarica A, Moscovici M, Daas M, Nicu I, Panteli M, Rasit I (2016) A purified extract from brown
truffles of the species Terfezia claveryi chatin and its antimicrobial activity. Farmacia 64:298–
301
Chen G, Zhang S, Ran C, Wang L, Kan J (2016) Extraction, characterization and antioxidant
activity of water-soluble polysaccharides from Tuber huidongense. Int J Biol Macromol 91:
431–442
Chiron N, Michelot D (2005) Mushrooms odors, chemistry and role in the biotic interactions—a
review (in French). Cryptogam Mycol 26(4):299–364
Claus R, Hoppen HO, Karg H (1981) The secret of truffles: a steroidal pheromone? Exp Dermatol
37:1178–1179
Dahham SS, Al-Rawi SS, Ibrahim AH, Majid ASA, Majid AMSA (2016) Antioxidant, anticancer,
apoptosis properties and chemical composition of black truffleTerfezia claveryi. Saudi J Biol
Sci 25:1524–1534
Das M, Kundu D, Singh J, Rastogi A, Banerjee R (2017) Physiology and Biochemistry of
Indigenous Tribal Liquor Haria: a state of art. Adv Biotechnol Microbiol 6(2):1–5
Das M, Kundu D, Singh J, Rastogi A, Mukherjee G, Chatterjee A, Banerjee R (2018) Rejuvenation
of metabolic cascades for controlling aging through bioactive compounds: a review. J Nutr Food
Sci Forecast 1:1–6
Das M, Mukherjee G, Biswas P, Mahle R, Banerjee R (2020) Black truffle—an exorbitant creation
of nature. Austin J Nutr Metab 7(1):1–3
Dash A, Kundu D, Das M, Bose D, Adak S, Banerjee R (2016) Food biotechnology: a step towards
improving nutritional quality of food for Asian countries. Recent Pat Biotechnol 10(1):43–57
De Angelis F, Arcadi A, Marinelli F et al (1996) Partial structures of truffle melanins. Phytochem-
istry 43:1103–1106
Dib-Bellahouel S, Fortas Z (2011) Antibacterial activity of various fractions of ethyl acetate extract
from the desert truffle, Tirmania pinoyi, preliminarily analyzed by gas chromatography–mass
spectrometry (GC–MS). Afr J Biotechnol 10:9694–9699
Doğan HH, Aydın S (2013) Determination of antimicrobial effect, antioxidant activity and phenolic
contents of desert truffle in Turkey. Afr J Tradit Complement Altern Med 10:52–58
Elsayed EA, El Enshasy H, Wadaan MAM, Aziz R (2014) Mushrooms: a potential natural source of
anti-inflammatory compounds for medical applications. Mediators Inflamm 2014:805841
Friedman M (2016) Mushroom polysaccharides: chemistry and antiobesity, antidiabetes, antican-
cer, and antibiotic properties in cells, rodents, and humans. Foods 5:80
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 727

Frizzi G, Lalli G, Miranda M, Pacioni G (2001) Intraspecific isozyme variability in Italian
populations of the white truffle Tuber magnatum. Mycol Res 105:365–369
Ghosh T, Maity TK, Singh J (2011) Evaluation of antitumor activity of stigmasterol, a constituent
isolated from Bacopa monnieri Linn aerial parts against Ehrlich ascites carcinoma in mice.
Orient Pharm Exp Med 11:41–49
Gioacchini AM, Menotta M, Polidori E, Giomaro G, Stocchi V (2002) Solid-phase microextraction
gas chromatography/ion trap mass spectrometry and multistage mass spectrometry experiments
in the characterization of germacrene D. J Mass Spectrom 37:1229–1235
Guidi C, Zeppa S, Annibalini G, Pierleoni R, Guescini M, Buffalini M, Zambonelli A, Stocchi V
(2006) The isoprenoid pathway in the ectomycorrhizal fungus Tuber borchii Vittad.: cloning
and characterization of the tbhmgr, tbfpps and tbsqs genes. Curr Genet 50:393–404
Hall IR, Yun W, Amicucci A (2003) Cultivation of edible ectomycorrhizal mushrooms. Trends
Biotechnol 21:433–438
Hamza A, Jdir H, Zouari N (2016) Nutritional, antioxidant and antibacterial properties of Tirmania
nivea, a wild edible desert truffle from Tunisia arid zone. Med Aromat Plants 5:258
Harki EH, Bouya D, Dargent R (2006) Maturation-associated alterations of the biochemical
characteristics of the black truffle Tuber melanosporum Vitt. Food Chem 99:394–400. https://
doi.org/10.1016/j.foodchem.2005.08.030
Henrion B, Chevalier G, Martiw F (1994) Typing truffle species by PCR amplification of the
ribosomal DNA spacers. Mycol Res 98(1):37–43
Janakat S, Nassar M (2010) Hepatoprotective activity of desert truffle(Terfezia claveryi)in
comparison with the effect of Nigella sativa in the rat. Pak J Nutr 9:52–56
Janakat S, Al-Fakhiri S, Sallal AK (2004) A promising peptide antibiotic from Terfezia claveryi
aqueous extract against Staphylococcus aureus in vitro. Phytother Res 18(10):810–813
Kendrick B (1985) The fifth kingdom. Mycologue, Waterloo
Khadri H, Aldebasi YH, Riazunnisa K (2017) Truffle mediated (Terfezia claveryi) synthesis of
silver nanoparticles and its potential cytotoxicity in human breast cancer cells (MCF-7). Afr J
Biotechnol 16:1278–1284
Kües U, Martin F (2011) On the road to understanding truffles in the underground. Fungal Genet
Biol 48(6):555–560
Kundu D, Das M, Mahle R, Biswas P, Karmakar S, Banerjee R (2020) Citrus fruits. In: Valorization
of fruit processing by-products. Academic Press, New York, pp 145–166
Laessoe T, Hansen K (2007) Truffle trouble: what happened to the Tuberales? Mycol Res 111:
1075–1099
Li LF, Liu HB, Zhang QW, Li ZP, Wong TL, Fung HY, Zhang JX, Bai SP, Lu AP, Han QB (2018)
Comprehensive comparison of polysaccharides from Ganoderma lucidum and G. sinense:
chemical, antitumor, immunomodulating and gut-microbiota modulatory properties. Sci Rep
8:6172
Liu M, Nauta A, Francke C, Siezen RJ (2008) Comparative genomics of enzymes in flavor-forming
pathways from amino acids in lactic acid bacteria. Appl Environ Microbiol 74:4590–4600
Luo Q, Zhang J, Yan L, Tang Y, Ding X, Yang Z, Sun Q (2011) Composition and antioxidant
activity of water-soluble polysaccharides from Tuber indicum. J Med Food 14(12):1609–1616
Martin F, Kohler A, Murat C, Balestrini R, Coutinho PM, Jaillon O, Montanini B, Morin E, Noel B,
Percudani R, Porcel B, Rubini A, Amicucci A, Amselem J, Anthouard V, Arcioni S,
Artiguenave F, Aury JM, Ballario P, Bolchi A, Brenna A, Brun A, Buée M, Cantarel B,
Chevalier G, Couloux A, Silva CD, Denoeud F, Duplessis S, Ghignone S, Hilselberger B,
Lotti M, Marçais B, Mello A, Miranda M, Pacioni G, Quesneville H, Riccioni C, Ruotolo R,
Stocchi V, Tisserant E, Viscomi AR, Zambonelli A, Zampieri E, Henrissat B, Lebrun MH,
Paolocci F, Bonfante P, Ottonello S, Wincker P (2010) Périgord black truffle genome uncovers
evolutionary origins and mechanisms of symbiosis. Nature 464(7291):1033–1038
Mauriello G, Marino R, D’Auria M, Cerone G, Rana GL (2004) Determination of volatile organic
compounds from truffles via SPME GC–MS. J Chromatogr Sci 42:299–305
728 M. Das et al.

Menendez JA, Papadimitropoulou A, Vellon L, Lupu R (2006) A genomic explanation connecting
“Mediterranean diet”, olive oil and cancer: oleic acid, the main monounsaturated fatty acid of
olive oil, induces formation of inhibitory “PEA3 transcription factor-PEA3 DNA binding site”
complexes at the Her-2/neu (erbB-2). Eur J Cancer 42:2425–2432
Menotta M, Amicucci A, Sisti D, Gioacchini AM, Stocchi V (2004) Differential gene expression
during pre-symbiotic interaction between Tuber borchii Vittad. and Tilia Americana L. Curr
Genet 46:158–165
Moradali MF, Mostafavi H, Ghods S, Hedjaroude GA (2007) Immunomodulating and anticancer
agents in the realm of macromycetes fungi (macrofungi). Int Immunopharmacol 7:701–724
Murat C, Díez J, Luis P, Delaruelle C, Dupré C, Chevalier G, Bonfante P, Martin F (2004)
Polymorphism at the ribosomal DNA ITS and ITS relation to postglacial re-colonization routes
of the Périgord truffle Tuber melanosporum. New Phytol 164:401–411
Nadim M, Deshaware S, Saidi N, Abd-Elhakeem MA, Ojamo H, Shamekh S (2015) Extracellular
enzymatic activity of Tuber maculatum and Tuber aestivum mycelia. Adv Appl Microbiol 5(7):
523–530
Owaid MN, Muslim RF, Hamad HA (2018) Mycosynthesis of silver nanoparticles using Terminia
sp. desert truffle, Pezizaceae, and their antibacterial activity. Jordan J Biol Sci 11:401–405
Rizzello R, Zampieri E, Vizzini A, Autino A, Cresti M, Bonfante P, Mello A (2012) Authentication
of prized white and black truffles in processed products using quantitative real-time PCR. Food
Res Int 48(2):792–797
Rubini A, Topini F, Riccioni C, Paolocci F, Arcioni S (2004) Isolation and characterization of
polymorphic microsatellite loci in white truffle (Tuber magnatum). Mol Ecol Notes 4:116–118
Rubini A, Belfiori B, Riccioni C, Paolocci F (2012) Genomics of tuber melanosporum: new
knowledge concerning reproductive biology, Symbiosis, and aroma production. In: Edible
ectomycorrhizal mushrooms. Springer, Berlin, Heidelberg, pp 57–72
Rubini A, Riccioni C, Belfiori B, Paolocci F (2014) Impact of the competition between mating types
on the cultivation of tuber melanosporum: Romeo and Juliet and the matter of space and time.
Mycorrhiza 24(Suppl 1):S19–S27
Schnitzler BE, Thebo PL, Mattsson JG, Tomley FM, Shirley MW (1998) Development of a
diagnostic PCR assay for the detection and discrimination of four pathogenic Eimeria species
of the chicken. Avian Pathol 27:490–497
Sies H, Stahl W (1995) Vitamins E and C, beta-carotene, and other carotenoids as antioxidants. Am
J Clin Nutr 62(6 Suppl):1315S–1321S
Singh J, Kundu D, Das M, Banerjee R (2019) Enzymatic processing of juice from fruits/vegetables:
an emerging trend and cutting edge research in food biotechnology. In: Enzymes in food
biotechnology. Academic Press, New York, pp 419–432
Splivallo R, Novero M, Bertea CM, Bossi S, Bonfante P (2007) Truffle volatiles inhibit growth and
induce an oxidative burst in Arabidopsis thaliana. New Phytol 175:417–424
Splivallo R, Ottonello S, Mello A, Karlovsky P (2011) Truffle volatiles: from chemical ecology to
aroma biosynthesis. New Phytol 189:688–699
StojkovićD, Reis FS, Ferreira ICFR, Barros L, Glamočlija J, ĆirićA, NikolićM, StevićT,
Giveli A, SokovićM (2013) Tirmania pinoyi: chemical composition, in vitro antioxidant and
antibacterial activities and in situ control of Staphylococcus aureus in chicken soup. Food Res
Int 53:56–62
Suz LM, Martin MP, Oliach D, Fischer CR, Colinas C (2008) Mycelial abundance and other factors
related to truffle productivity in Tuber melanosporum-Quercus ilex orchards. FEMS Microbiol
Lett 285(1):72–78
Talou T, Gaset A, Delmas M, Kulifaj M, Montant C (1990) Dimethyl sulphide: the secret for black
truffle hunting by animals? Mycol Res 94(2):277–278
Thomas P, Büntgen U (2019) A risk assessment of Europe’s black truffle sector under predicted
climate change. Sci Total Environ 655:27–34
Tirillini B, Verdelli G, Paolocci F, Ciccioli P, Frattoni M (2000) The volatile organic compounds
from the mycelium of Tuber borchii Vitt. Phytochemistry 55:983–985
29 Phylogenomics, Microbiome and Morphological Insights of Truffles: The... 729

Trappe JM, Claridge AW, Arora D, Smit WA (2008) Desert truffles of the African Kalahari:
ecology, ethnomycology, and taxonomy. Econ Bot 62:521–529
Trappe JM, Kovacs GM, Claridge AW (2010) Comparative taxonomy of desert truffles of the
Australian outback and the African Kalahari. Mycol Progr 9:131–143
Urban A, Neuner-Plattner I, Krisai-Greilhuber I, Haselwandter K (2004) Molecular studies on
terricolous microfungi reveal novel anamorphs of two Tuber species. Mycol Res 108:749–758
Urbanelli S, Sallicandro P, Vito ED, Bullini L, Palenzona M, Ferrara AM (1998) Identification of
Tuber mycorrhizae using multilocus electrophoresis. Mycologia 90(3):389–395
Vahdatzadeh M, Deveau A, Splivallo R (2015) The role of the microbiome of truffles in aroma
formation: a meta-analysis approach. Appl Environ Microbiol 81(20):6946–6952
Venkateshwarlu G, Chandravadana MV, Tewari RP (1999) Volatile flavor components of some
edible mushrooms (basidiomycetes). Flavour Fragr J 14:191–194
Wasser S (2003) Medicinal mushrooms as a source of antitumor and immunomodulating
polysaccharides. Appl Microbiol Biotechnol 60:258–274
Wedén C (2004) Black truffles of Sweden. Systematics, population studies, ecology and cultivation
of T. aestivum syn. T. uncinatum. PhD thesis. Uppsala University, Sweden
Wedén C, Danell E, Camacho FJ, Backlund A (2004) The population of the hypogeous fungus
tuber aestivum syn. T. uncinatum on the island of Gotland. Mycorrhiza 14:19–12
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal
RNA genes for phylogenetics. In: Innis MA, Gelfland DH, Sninsky JJ, White TJ (eds) PCR
protocols: a guide to methods and applications. Academic Press, San Diego, CA, pp 315–322
Wnouk S, Kinastowski S, Kaminski E (1983) Synthesis and analysis of 1-octen-3-ol, the main
flavor component of mushrooms. Nahrung 27:479–486
Woyengo TA, Ramprasath VR, Jones PJH (2009) Anticancer effects of phytosterols. Eur J Clin
Nutr 63:813–820
Yan X, Wang Y, Sang X, Fan L (2017) Nutritional value, chemical composition and antioxidant
activity of three tuber species from China. AMB Express 7:136
Zampieri E, Balestrini R, Kohler A, Abbà S, Martin F, Bonfante P (2011) The Perigord black truffle
responds to cold temperature with an extensive reprogramming of its transcriptional activity.
Fungal Genet Biol 48(6):585–591
Zeppa S, Gioacchini AM, Guidi C, Guescini M, Pierleoni R, Zambonelli A, Stocchi V (2004)
Determination of specific volatile organic compounds synthesized during tuber borchii fruit
body development by solid-phase microextraction and gas chromatography/mass spectrometry.
Rapid Commun Mass Spectrom 18:199–205
Zhang T, Jayachandran M, Ganesan K, Xu B (2018) Black truffle aqueous extract attenuates
oxidative stress and inflammation in STZ-induced hyperglycemic rats via Nrf2 and NF-κB
pathways. Front Pharmacol 9:1257
Zhao W, Wang XH, Li HM, Wang SH, Chen T, Yuan ZP, Tang YJ (2014) Isolation and
characterization of polysaccharides with the antitumor activity from tuber fruiting bodies and
fermentation system. Appl Microbiol Biotechnol 98:1991–2002
Zong A, Cao H, Wang F (2012) Anticancer polysaccharides from natural resources: a review of
recent research. Carbohydr Polym 90:1395–1410
730 M. Das et al.