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PankajBhatt
SaurabhGangola
DhanushkaUdayanga
GovindKumarEditors
Microbial
Technology
forSustainable
Environment
Microbial Technology for Sustainable Environment
Pankaj Bhatt •Saurabh Gangola •
Dhanushka Udayanga •Govind Kumar
Editors
Microbial Technology for
Sustainable Environment
Editors
Pankaj Bhatt
State Key Laboratory for Conservation
and Utilization of Subtropical
Agro-bioresources, Guangdong
Laboratory for Lingnan Modern
Agriculture Integrative Microbiology
Research Centre
South China Agricultural University
Guangzhou, China
Saurabh Gangola
School of Agriculture
Graphic Era Hill University
Bhimtal, Uttarakhand, India
Dhanushka Udayanga
Department of Biosystems Technology
Faculty of Technology,
University of Sri Jayewardenepura,
Pitipana
Homagama, Sri Lanka
Govind Kumar
Division of Crop Production
ICAR- Central Institute For Subtropical
Horticulture
Lucknow, India
ISBN 978-981-16-3839-8 ISBN 978-981-16-3840-4 (eBook)
https://doi.org/10.1007/978-981-16-3840-4
©The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
Pte Ltd. 2021
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether
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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
This book is dedicated to all the researchers
working throughout the world to make life
easier and the authors who have contributed
to each of the chapters.
Foreword
It gives me immense pleasure to write a foreword for an upcoming book on
microbiology entitled Microbial Technology for Sustainable Environment, edited
by a learned group of young microbiologists. This book provides some interesting
and straightforward approaches to study complex microbiological themes and the-
ories related to microbial life governing the environment and their role in sustenance
of life under stressed ecosystems in a simple manner. A careful balance of basic and
advanced concepts of microbial systems undertaken in the book provides an insight
of the subject to understand precise networking of microbial life for environmental
benefit. Streamlined updated information on burning topics related to microbial
biotechnology encourages not only students to understand microbial systems and
their physiology like signals, metabolism and metagenomics but also researchers of
various streams to visualize concepts on microbial metabolism/biodegradation of
different pollutants, rhizosphere biology, modern tools for biofertilizer production
and microbiome analysis to address stress environment posed by rapid industriali-
zation. The content of the book related to the theory and practical approaches of
microbiology would also help to inculcate scientific temperament among new
aspirants. To the best of my knowledge, the editors of the book (Dr(s) Pankaj
Bhatt, Saurabh Gangola, Dhanuska and Govind Kumar) have contributed signifi-
cantly to the area of Environmental Microbiology as evident from their quality
research papers. I sincerely congratulate them for their attempt to work on such a
critical subject which would certainly bring a new height in the area of microbial
technology in a simple manner.
Department of Microbiology, GB Pant
University of Ag. and Technology,
Pantnagar, Uttarakhand, India
Anita Sharma
vii
Preface
Microorganisms are highly diverse and ubiquitous on earth. These microorganisms
are able to perform various biological functions in the environment. Microbial
applications are used as biofertilizers, bioremediation, biofortification and other
sustainable approaches of environmental development. Indigenous microbial strains
have the potential to perform various functions that are beneficial to achieve
sustainable goals.
To date, different strains of microorganisms have been commercialized globally,
for industrial and other applications towards the achievement of sustainable devel-
opment. Microbial strains and their consortia have been widely utilized in crop
improvement and protection, bioremediation of xenobiotics and other sustainable
applications in food, agriculture and environment technologies.
In this book, we have compiled a collection of chapters about implementation of
microbes in various sectors in sustainable environment. Therefore, the chapters of
this book cover a vast area of research in the field of microbial biotechnology,
including both traditional and emerging applications. Therefore, the book can be
used as an essential reference source for students, aspiring researchers, industrialists,
entrepreneurs and policy makers in the field of agriculture, food security, environ-
mental engineering and management.
Authors from various fields of expertise have provided their valuable inputs as
chapters complied in this book. Without their expertise contribution, commitment
and dedication, this book could not have ever been accomplished. We would like to
extend our gratitude to Kripa Guruprasad of Springer Nature who worked hard
towards the publication of this book, as well as the families who supported us.
Guangzhou, China Pankaj Bhatt
Bhimtal, Uttarakhand, India Saurabh Gangola
Pitipana, Sri Lanka Dhanushka Udayanga
Lucknow, Uttar Pradesh, India Govind Kumar
ix
Acknowledgements
In the year 2020, the COVID-19 pandemic affected each of the life throughout the
world. We have made a team of four editors from three different countries for the
theme microbial technology for sustainable environment. We all were excited to
complete a book on Microbial Technologies in sustainable environment. The pro-
posal was initially drafted by Pankaj Bhatt and send for revisions and suggestions to
Saurabh Gangola, Dhanushka Udayanga and Govind Kumar. All the editors have
revised the proposal. Furthermore, Pankaj Bhatt has submitted it for review to
Springer Nature. The reviewer reports were positive and we got acceptance for our
book proposal. We all then embarked on this wonderful journey together. This has
been a journey of learning and full excitement. We followed the tireless efforts of
each of the authors who contributed chapters to this book, and their unwavering
determination has made this project from a dream to reality.
We also want to thank the publisher Springer Nature and specially the projector
coordinator Ms. Kripa Guruprasad for their support and guidance. She helped us in
providing a valuable response to each of the query that was raised by the editors and
authors from time to time during the preparation of the manuscript.
We are also very much thankful to the contributing authors from various corners
of the globe to make our project meaningful. The authors shared rich information,
expert insights and collections of informative scientificfigures.
The editors are thankful to Dr. Kalpana Bhatt, researcher at Gurukul Kangri
University, Haridwar; Professor Anita Sharma, Department of Microbiology, G.B
Pant University of Agriculture and Technology, Pantnagar, U.S Nagar, India;
Professor Shaohua Chen, Integrative Microbiology Research Centre, South China
Agriculture University, Guangzhou, China; and Mr. Rakesh Bhatt, Department of
xi
Civil Engineering, Indian Institute of Technology, Kanpur, India, for the inspiration
to complete the book on time.
We are also grateful to each of the members of Springer’s technical staff who
supported us in the completion of the project even during the COVID-19 pandemic.
Pankaj Bhatt
Saurabh Gangola
Dhanushka Udayanga
Govind Kumar
xii Acknowledgements
Contents
1 Microbial World for Sustainable Development ................ 1
Shubhangi Sharma, Raja Singh Rawal, Deepa Pandey,
and Neha Pandey
2 Insights into the Rhizospheric Microbes and Their Application
for Sustainable Agriculture ............................... 13
Ankit Negi, Anchal Giri, Pooja Pant, and Rishendra Kumar
3 Different Biofertilizers and Their Application for Sustainable
Development .......................................... 31
Dharmendra Kumar, Som Dutt, Pinky Raigond, Sushil Sudhakar
Changan, Milan Kumar Lal, Rahul Kumar Tiwari,
Kumar Nishant Chourasia, and Brajesh Singh
4 Microbial Mediated Natural Farming for Sustainable
Environment .......................................... 49
Asha Rani and Beenam Saxena
5 Rhizosphere Manipulations for Sustainable Plant Growth
Promotion ............................................ 61
Pooja Pant, Ankit Negi, Anchal Giri, Pankaj Bhatt,
and Rishendra Kumar
6 Rhizospheric Microbes and Their Mechanism ................. 79
Anuj Chaudhary, Heena Parveen, Parul Chaudhary, Hina Khatoon,
and Pankaj Bhatt
7 Endophytes and Their Applications as Biofertilizers ............ 95
Gaurav Yadav, Rishita Srivastva, and Preeti Gupta
8 Microbial Action on Degradation of Pesticides ................ 125
Hira Singh Gariya and Arun Bhatt
xiii
9 Biofortification of Plants by Using Microbes .................. 141
Ankur Adhikari, Kamal Pandey, Vinita Pant, Tara Singh Bisht,
and Himanshu Punetha
10 Microbial Biopesticides: Development and Application .......... 167
H. T. Mandakini and Dimuthu S. Manamgoda
11 Microbial Consortia and Their Application for the Development
of a Sustainable Environment ............................. 191
Sneha Trivedi, Naresh Butani, Helina Patel, Manoj Nath,
and Deepesh Bhatt
12 Microbial Engineering and Applications for the Development
of Value-Added Products ................................ 203
Ashutosh Paliwal, Abhishek Verma, Ashwini Kumar Nigam,
Jalaj Kumar Gour, Manoj Kumar Singh, and Rohit Kumar
13 Plant Growth-Promoting Rhizobacteria and Their Application
in Sustainable Crop Production ............................ 217
Parul Chaudhary, Heena Parveen, Saurabh Gangola, Govind Kumar,
Pankaj Bhatt, and Anuj Chaudhary
14 Reinstating Microbial Diversity in Degraded Ecosystems
for Enhancing Their Functioning and Sustainability ............ 235
Sachini Wayanthimali Meepegamage, Ambalangodage Thilini
Dhanushka Rathnathilake, Mahesh Premarathna,
and Gamini Seneviratne
15 Recent Advancements and Mechanism of Microbial Enzymes
in Sustainable Agriculture ................................ 247
Pankaj Bhatt, Saurabh Gangola, Charu Joshi, Parul Chaudhary,
Govind Kumar, Geeta Bhandari, Saurabh Kumar, Samiksha Joshi,
Avikal Kumar, Narendra Singh Bhandari, and Samarth Tewari
16 Application of Microbial Technology for Waste Removal ........ 261
Ravi Ranjan Kumar, Chitra Bhattacharya, and Nutan Prakash
Vishwakarma
17 Metagenomics: Insights into Microbial Removal of the
Contaminants ......................................... 293
Dipti Singh, Shruti Bhasin, Anshi Mehra, Manali Singh, Neha Suyal,
Nasib Singh, Ravindra Soni, and Deep Chandra Suyal
18 Methods of Strain Improvement for Crop Improvement ......... 307
Jyoti Rawat and Veena Pande
19 Microbial Technologies in Pest and Disease Management of Tea
(Camellia sinensis (L.) O. Kuntze) .......................... 325
Ganga Devi Sinniah and Padmini Dharmalatha Senanayake
xiv Contents
20 Field Application of the Microbial Technology and Its Importance
in Sustainable Development ............................... 347
Saloni Kunwar, Shristi Bhatt, Deepa Pandey, and Neha Pandey
21 Solubilization of Micronutrients Using Indigenous
Microorganisms ....................................... 365
A. D. Sarangi N. P. Athukorala
22 Synergistic Interaction of Methanotrophs and Methylotrophs
in Regulating Methane Emission ........................... 419
Vijaya Rani, Rajeev Kaushik, Sujan Majumder, A. T. Rani,
Asha Arambam Devi, Pratap Divekar, Priyanka Khati, K. K. Pandey,
and Jagdish Singh
23 Biopesticides: An Alternative to Synthetic Insecticides .......... 439
A. T. Rani, Vasudev Kammar, M. C. Keerthi, Vijaya Rani,
Sujan Majumder, K. K. Pandey, and Jagdish Singh
24 Impact of Pesticides on Microbial Population ................. 467
Sujan Majumder, Anindita Paul, Anup Kumar, Chandan K. Verma,
Pratap A. Divekar, Vijaya Rani, A. T. Rani, Jaydeep Halder,
K. K. Pandey, and Jagdish Singh
25 Microbe-Mediated Removal of Xenobiotics for Sustainable
Environment .......................................... 483
Helina Patel, Sneha Trivedi, Deepesh Bhatt, Manoj Nath,
and Naresh Butani
26 Harnessing the Rhizomicrobiome Interactions for Plant
Growth Promotion and Sustainable Agriculture: Mechanisms,
Applications and Recent Advances ......................... 499
Geeta Bhandari and Niki Nautiyal
27 Fungal Mycelium-Based Biocomposites: An Emerging Source
of Renewable Materials .................................. 529
Dhanushka Udayanga and Shaneya Devmini Miriyagalla
28 An Endophytic Bacterial Approach: A Key Regulator of Drought
Stress Tolerance in Plants ................................ 551
Sudha Bind, Sandhya Bind, and Dinesh Chandra
Contents xv
Chapter 1
Microbial World for Sustainable
Development
Shubhangi Sharma, Raja Singh Rawal, Deepa Pandey, and Neha Pandey
Abstract Increasing population and decreasing sustainability of natural resources is
a global concern; indiscriminate use of natural resources has led to a large-scale
exploitation of nature. Change in lifestyle and urbanisation is also a major cause for
various conditions such as pollution, greenhouse effect etc. It needs an immediate
measure with regard to curb the damage being caused to nature. Sustainability of
natural resources is a major concern. A wise and applicable step at this time could
provide the privilege to upcoming generations to live an efficient life. Microorgan-
isms being ubiquitous have both harmful and beneficial role. Though microbes are a
cause of major pathogenic ailments, efficiently harnessing microbes towards a
developing role could help in achieving the major sustainable development goal
(SDGs). The presence and usefulness of microbes in almost every field like agricul-
tural, industry, health, education, pharmaceutical and environment is undeniable
which can positively regulate nation’s economy, whereas a single outbreak of
pathogenic microbes could destroy the economy. A microscopic creature is potent
enough to cause global disaster, but the misbalance spread by mankind in nature
could be balanced by efficient use of these microscopic creatures. Thus, it depends
on mankind how these microbes need to be handled with efficiency, in order to attain
the best results and help fulfil the goals adopted by United Nations member state to
make this planet a better place for us and upcoming generations.
Keywords Sustainability · Bioenergy · Education · Bioremediation · Ecosystem ·
Economy
S. Sharma · R. S. Rawal · N. Pandey (*)
Department of Biotechnology and Life Sciences, Graphic Era Deemed to be University,
Dehradun, Uttarakhand, India
e-mail: neha.pandey@geu.ac.in
D. Pandey
Department of Zoology, Government Post Graduate College, Ranikhet, Uttarakhand, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_1
1
1.1 Introduction
Life without higher organisms is feasible, but without microbes is not. It is not
exaggeration to mention that life originated from microbes and every one life springs
from microbes (Kuhad 2012; Bhatt et al. 2021a,b; Bhandari and Bhatt 2020; Kumar
et al. 2017). Microbes play an integral role in various aspects of life. One can
consider microbes beyond any imagination altogether the possible regions (Khati
et al. 2018). Microbes if exploited judicially can mark a major effect in overall
development, i.e. sustainable development (Kuhad 2012). Brundtland in 1987 stated
that sustainable development generally meets the needs of the present without
compromising the ability of future generations to meet their own needs (Brundtland
1987; Bhatt and Nailwal 2018).
To collaboratively make an endeavour during sustainable development, around
193 countries agreed to different sustainable development goals (SDG), which is a
UN’s sponsored effort for a sustainable economic development of the planet. These
goals are classified into five (5) subgroups: People, Planet, Prosperity, Peace and
Partnerships (Bhatt and Bhatt 2020). The SDGs goals are the answer that could
permit financial and societal development, however now no longer on the fee of
environmental damage (Bhatt and Maheshwari 2020). Rather, those efforts empha-
sise at the environmental safety with the aid of using stopping and controlling the
illegal exploitation of herbal resources (Akinsemolu 2018).
The World Health Organisation (WHO) has stated certain areas for sustainable
development goals (SDGs) as shown in Fig. 1.1.
•No poverty.
•No hunger.
•Good health and wellbeing.
•Education.
•Clean water and sanitation.
•Affordable clean energy.
•Economic growth.
•Industrial innovation.
•Reduce inequality amongst countries.
•Sustainable cities and community.
•Climate change.
•Life below water.
•Life on land.
•Peace and justice.
•Global partnership for development.
Microbes are capable of fulfilling all the above stated goals of SDGs. Microbes
are omnipresent and also the predominant forms of life on the earth (Goel et al.
2020). Microbes are the backbone of the ecosystem, with many applications that can
contribute in sustainable development. Microbes manifest spectrum of evolutionary,
2 S. Sharma et al.
functional and metabolic diversity (Kumar et al. 2020; Suyal et al. 2019a,b; Bhatt
and Maheshwari 2019).
Microbes omnipresence all over the environment, and therefore, their diverse and
versatile nature makes them vital agents of planetary system. They have the tendency
to facilitate and regulate biogeochemical cycles and consequently use biological
materials and waste products. Microbes are also responsible for producing green-
house gases, viz. carbon dioxide and methane, and are, therefore, necessary deter-
minants of global climate change. In addition to this, they perform essential roles in
soil structure and fertility and within the quality and productivity of land, seas, lakes
and rivers. Microbes, therefore, are also key members of the committee of stewards
of planetary health and property (Timmis et al. 2017).
Industrialisaon
Economic
growth
No poverty
No hunger
Good health
Sustainable
development
Peace and
jusce
Educaon
Reduce inequality
Fig. 1.1 Interlinked sustainable development goal (SDGs): The SDGs are somewhat interlinked;
fulfilment of one will lead to attainment of many other SDGs
1 Microbial World for Sustainable Development 3
1.2 Microbes and the Sustainable Development Goals
1.2.1 No Poverty, Economic Growth and Industrial
Innovation
Eradication of poverty may also help in attaining various other SDGs directly or
indirectly. Mass educating the economically backward class for generating income
utilising microbes may also play a pivotal role in eradication of poverty. Various
techniques such as using microbes to produce fermented food products may help in
raising income, economic development and eradicating hunger. However, the role of
microbes in SDGs is shown in Table 1.1. The economic growth can be efficiently
made via microbes by medicine, vaccine production and lowering disease rate
thereby improving economy (Drexler 2010). Various industries such as food and
beverages and chemical synthesis can efficiently exploit microbial population for
development (Adesulu and Awojobi 2014).
Table 1.1 Role of microorganisms in accomplishing SDGs
SDGs Microorganism Role Reference
Life below water Aspergillus niger Decolourisation of pulp and
paper industry water
Ahmad et al.
(2018)
Bacillus and
Pseudomonas
Reduce metal toxicity Ahmad et al.
(2018)
Life on land Bordetella avium Degrade naphthalene Abo-State et al.
(2018)
No hunger, no poverty,
economic growth
Pseudomonas
fluorescence
Enhance root and shoot
growth
Johansson et al.
(2004)
Bradyrhizobium Enhance soil nutrient content
(N,P,S)
Johansson et al.
(2004)
Vigna radiata ABA production (plant
growth under stress)
Ahmad et al.
(2013)
No hunger, industrial
innovation
Lactobacilli Dairy production Pereg and
McMillan
(2015)
Clean energy Chlorella vulgaris Biobutanol and biohydrogen Srivastava
(2019)
Shewanella
oneidensis
Produce electricity Lal (2013)
Human health, eco-
nomic growth
Streptomyces Aminoglycoside antibiotic
production
Finkelstein
et al. (1996)
Human health, industry
application
Serratia
marcescens
Biotin production Shimizu (2008)
Propionibacterium
shermanii
Vitamin B12 production Shimizu (2008)
4 S. Sharma et al.
1.2.2 Good Health Wellbeing, Clean Water and Sanitation
Humans harbour growth of various microorganisms known as human microbiome.
These microbes play various essential metabolic and physiological roles such as the
intestinal microbiome helps in digestion and absorption of food. Various essential
nutrients that are not synthesised in the body and not included in diet are also
provided by the microbes. Various recent researches also suggest that microbiota
may also influence brain function (Sampson and Mazmanian 2015). By various
activities such as decomposition, environmental cleanup can be efficiently
maintained, and thereby enhancing good health and wellbeing can be strengthened
by intake of probiotics, antibiotics and vaccine.
1.2.3 No Hunger
Microbes play an inseparable part in agriculture by enhancing yield by Bacillus
thuringiensis (Bt) crops. Various fermented products are produced via microbial
activity. Microbes play an exceptionally important role in eradicating hunger posi-
tively regulating agricultural practices. Microbes enhance crop yield and soil fertility
and play vital role in controlling plant pathogens. Soil fertility and in turn crop
productivity can be enhanced by using arbuscular mycorrhizal symbiotic fungi and
phosphate-solubilising and nitrogen-fixing microbes (Johansson et al. 2004).
Various microbes specifically effect various aspects of plant growth, e.g. Strains
of Pseudomonas aeruginosa increases accumulation of dry matter, nodule forma-
tion, grain yield and protein content. Various strains of Azospirillum increase
drought tolerance and enhance root and shoot growth in maize seedlings. Pseudo-
monas fluorescens provides good root and shoot growth and increases tolerance to
salinity for cucumber plant. Bradyrhizobium species enhances nitrogen, phosphate,
sulphur and yield of soybean grain. Microbes also enhance the plant growth by
increasing phytohormone productivity and plant growth regulators by 60 times
(Camerini et al. 2008). Microbial synthesised phytohormone can regulate physio-
logical plant processes both under normal and stress condition. Auxin synthesised by
Pseudomonas and Rhizobium strain helps in tolerating osmotic stress in Vigna
radiate (Ahmad et al. 2013). Abscisic acid (ABA) helps in growth under stress
induction of photoperiodic flowering. Plant growth can also be enhanced by biolog-
ical control of plant pathogens by competition for nutrients, producing antibiotic,
hydrolytic enzymes, siderophores etc. (Glick 2012).
1 Microbial World for Sustainable Development 5
1.2.4 Education
By teaching, research and innovation microbes even play an important role in
education field. Education forms the basis of various other SDGs. Mass educating
people in turn generates growth opportunities, economic development, improvement
in living conditions, good health and research. Education can provide growth
opportunities to economically backward classes and find employment. Steps are
now being taken to establish new teaching methods for numerous technologies such
as environmental technology fermentation technology, food biotechnology and
immunology, so that students can easily understand the present and potential use
of microbiology and biotechnology for better livelihoods and environmental security
(Simonneaux 2000).
1.2.5 Affordable Clean Energy
Bioenergy and biofuel are turning to be good alternate sources of energy, for
example Shewanella oneidensis exploits organic matter to produce utilisable elec-
tricity (Lal 2013). Various wastes such as sewage sludge and municipal solid are
being utilised by numerous fungal species including Trichoderma and Aspergillus to
produce bioenergy (Elshahed 2010).
Fossil fuel burning possesses a great threat to environment and mankind. In order
to curb this, inefficiency biogas and biomass-based energy are good alternatives that
are both cost-effective and environment friendly. The third-generation biofuels can
be developed by using microalgal population and curbing the environmental haz-
ards, e.g. Chlamydomonas reinhardtii produce ethanol. Chlorella vulgaris produce
biobutanol and biohydrogen (Srivastava 2019).
1.2.6 Reduced Inequality
Women, who make up half of the world’s population, often have half the ability to
work and account for more than half of the workforce in fields such as health care
(Kaushik and Kapila 2009). Restricted access to education is a big setback for
women across the globe. Women need sexual and reproductive health and hygiene
knowledge as insufficient education on sexually transmitted diseases such as chla-
mydia, herpes, gonorrhoea, AIDS and syphilis possesses a higher risk of contracting
them (Dehne and Riedner 2001). Therefore, empowerment of girls and women is
urgently needed. Indeed, encouraging the completion of formal education, encour-
aging women to engage in higher education or to learn new skills and raising
women’s awareness of their rights can contribute to their growth (Penner 2015). It
is possible to manage gender disparity by supporting women’s education and
6 S. Sharma et al.
making women qualified enough to earn a living. This can be managed through the
advancement of agricultural, food and dairy and land management activities by
women from rural areas. The general importance of various microorganisms both
in pathogenic and non-pathogenic aspect is to be provided to women, in particular,
for their involvement in agriculture, dairy and medical fields. The knowledge of
microorganisms such as Pseudomonas,Rhizobium,Trichoderma,Bradyrhizobium,
Azospirillum,Lactobacillus, yeasts etc. should be imparted to women in agriculture
in order to increase crop productivity as well as to make various food and dairy
products (Pereg and McMillan 2015).
1.2.7 Sustainable Cities and Communities
Proper solid waste disposal system to avoid clogged drains, floods and the spread of
waterborne diseases is a primary necessity for the durable and sustainable growth of
society. An expensive method is the disposal of agricultural waste. Proper waste
management and dispersal is efficiently maintained by using microbial population.
The development of green concrete wall and bioremediation curl pollution. The
bioconversion of solid waste into useful products such as biofuel, biogas and animal
feedstock, as well as its agricultural uses, is a resourceful, green and sustainable way
to handle waste products. The composting of solid waste is an efficient and eco-
nomically viable process in which different microorganisms such as Pseudomonas,
Bacillus,Microbispora,Actinobifida and Thermoactinomycetes are being used to
convert their organic constituents into usable end products. Compost can be used as
crop manure, thereby improving its productivity and contributing to green growth
(Finstein and Morris 1975).
1.2.8 Global Climate
Global climate can be efficiently controlled by microbes by controlling pollution and
various biogeochemical cycles like nitrogen, carbon and phosphorus cycles. Various
poisonous gases, e.g. released by various human interventions and processes such as
fossil fuels burning and the processes of industrial development, are the main global
climate change players. With different biotic and abiotic variables, microorganisms
are involved in the recycling of elements. Many natural and engineered systems,
such as wastewater treatment, agriculture, remediation, production of biofuels and
metabolite production and mineralisation, are important (Bodelier 2011).
The marine microbial populations are one of the key regulators of carbon dioxide
concentration in the environment. They are even responsible for recycling nutrients
that are further used in marine food webs. Microbes are majorly responsible for
decomposition of organic matter which in turn releases carbon dioxide (CO
2
)
,
methane (CH
4
) and other gases into the atmosphere, thereby indirectly regulating
1 Microbial World for Sustainable Development 7
global climate. Methanogens such as certain archaea produce large amount of CH
4
into environment (Cavicchioli 2019).
1.2.9 Life Below Water
Various industrial effluents discharged into the water bodies and surface run off from
agricultural lands contain harmful chemicals which when reach water bodies may
cause harmful effects to both life on land and life under water. Oil spills are one of
the most common issues prevailing in oceans. Crude oil contains potential carcin-
ogen products. Microbes play an efficient role in bioremediation and removal of
harmful effluents and clearing oil spills, e.g. Aspergillus niger is used for decompo-
sition of pulp and paper and wastewater. Microbes such as Bacillus and Pseudomo-
nas are used at metal contaminated site to reduce toxicity and concentration of
pollutants. Microbes control marine population by controlling pathogenic outburst,
producing oxygen. As the most important contributor to global climate change, the
combustion of fossil fuels may also be controlled by the use of microorganisms as a
source of biofuels or as part of biofuel processing technologies (Ahmad et al. 2018).
1.2.10 Life on Land
Microbes have ubiquitous role on land in almost every field. Microbes stabilise the
soil structure, permit nutrient uptake via way of means of plants, manage pests and
diseases, decompose natural cloth and degrade dangerous chemicals, in addition to
being a hallmark of the soil health. Increasing population and demands of humans
has led to increase in destruction of forest, loss of biodiversity and increased
pollution. Microbes could play an important role in limiting these effects such as
microbes could increase agricultural yield. Microbes increase the soil content or
quality by nitrogen fixation and phosphate solubilisation.
Microbes also help in bioremediation by degrading polyaromatic hydrocarbon,
e.g. Bordetella avium MAM-P22 can degrade naphthalene (Abo-State et al. 2018).
1.2.11 Peace and Justice
In general, one cannot think about the connection of microbes with peace and justice;
however, microbes contribute significantly to the preservation of a stable society.
The occurrence of poverty, insufficient access to food and illiteracy have an adverse
impact on children’s growth. A significant contributing factor to the emotional
wellbeing of children has been implicated in food insecurity (Chilton et al. 2007).
8 S. Sharma et al.
By countering bioterrorism, improving sources of nutrition, improving environ-
mental conditions, introducing green technologies and improving national and
international infrastructure would eventually lead to the growth of society and the
prevalence of peace and justice (Bhatt and Maheshwari 2020).
1.2.12 Global Partnership for Development
Policy mechanisms must contribute to the social, economic and environmental needs
of microbiology in order to achieve a sustainable future. The most important areas
which need urgent attention are the use of microbes in agriculture, pharmaceutical
science, biofuels and fermented food. Without active cooperation and partnership
between nations, sustainable development is not viable. In order to make globalisa-
tion more efficient, more distinct, wider and intercontinental agreements are needed
(Bhatt and Maheshwari 2020). Only by globalisation and breaking land barriers can
the advantages of microbes and microbial technology reach the masses (Chambers
et al. 2004; Finkelstein et al. 1996; Shimizu 2008; Rawat et al. 2019; Suyal et al.
2018; Mishra et al. 2020; Zhang et al. 2020). This green technology must be used to
improve the ideals of equality and social justice.
1.3 Conclusion
It is not an exaggeration to state that the SDGs laid if fulfilled at this point of time
slowly and steadily would definitely make this planet worth living for the upcoming
countless generation. It is not an individual or national concern; it needs a global
effort to curb the gap created in global sustainable development. A carefree approach
towards nature would definitely end the upcoming generations sooner or later.
Though microbes appear to be very insignificant with regard to their size, they are
potent enough to be both a boon and a curse. It just needs a constructive approach,
and microbes alone proves to be a great factor in achieving all sustainable develop-
ment goals. Significantly, microbes contribute to enhance green production technol-
ogies, improve crop productivity and provide earning livelihood to needy people.
However, it is now believed that these perspectives and better knowledge might help
young people to make efforts in achieving sustainable development goals.
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12 S. Sharma et al.
Chapter 2
Insights into the Rhizospheric Microbes
and Their Application for Sustainable
Agriculture
Ankit Negi, Anchal Giri, Pooja Pant, and Rishendra Kumar
Abstract The rhizosphere soil of plant consists of diverse microorganisms. The
study of plant-microbe interactions is necessary for plant health and promotion. The
rhizosphere microbes not only provide important nutrients to plants but also prevent
the growth of harmful pathogenic microbes. The processes that occur in biome
include quorum sensing, nitrogen fixation, nutrients solubilization, volatiles, mobi-
lization, and immobilization of nutrients. The plant allures microbes according to
their requirements by releasing certain chemical compounds needed for microbes. In
case of any pathogen attack, they recruit microbes to suppress pathogens. Some of
the evidences revealed that the recruitment of microbes in rhizosphere shows effects
on plant at physiological and molecular level. This increases the possibility that the
soil microbiota can stimulate the ability of plant to tackle different biotic and abiotic
stresses. In this chapter, we will discuss various mechanisms by which the microbial
communities work for plants and how the plants recruit them for their development
in detail. Altering the microbial population in the rhizosphere either by removing or
adding new species can modify the plant and rhizosphere microbiome. The rhizo-
sphere microbes used in making of agricultural products like biopesticides and
biofertilizers for sustainable agriculture practices. The plant-microbe interaction
can be further studied with the help of bioinformatics, molecular, and modeling
tools.
Keywords Rhizosphere microbes · Rhizosphere plant growth mechanisms ·
Beneficial PGPR · Plant-microbe interactions
A. Negi · A. Giri · P. Pant · R. Kumar (*)
Department of Biotechnology, Sir J.C. Bose Technical Campus, Bhimtal, Kumaun University,
Nainital, Uttarakhand, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_2
13
2.1 Introduction
The rhizosphere is a closed narrow zone around the plant root that consists of large
number of microbial communities. A single plant inhabits a large number of
microbes in their rhizosphere soil and can contain >30,000 prokaryotic species
(Mendes et al. 2011) and up to 1011 microbial cells per gram of root (Egamberdieva
et al. 2008). In fact, the soil microbial communities are the greatest reservoir of
biological diversity till now (Buee et al. 2009). The complete genome of the plant
rhizosphere microbial communities is much more than that of the host plants itself,
thus termed as the second genome of the plant. The functions performed by the
rhizosphere microbial communities are very much similar to that of human gut (Bron
et al. 2012). The microbial species present in the soil changes with time according to
the plant requirements from thousands to millions (Nihorimbere et al. 2011; Sulbhi
et al. 2021; Bhandari et al. 2021; Bhatt et al. 2021a,b,c).
2.2 Rhizosphere Microbes: Role in Plant Health
and Growth Promotion
The plant-microbe interactions play vital roles in plant life like carbon and nitrogen
sequestration and nutrient cycling (Singh et al. 2004). These interactions are mostly
positive because plant attracts selectively only those microbes which are needed for
their growth promotion such as plant growth-promoting rhizobacteria (PGPR),
mycorrhizal fungi, and epiphytes. They can be beneficial or pathogenic and may
live freely in the soil or in mutual or commensal associations (Philippot et al. 2013).
Plants provide the microbes the root exudates which they use as a substrate, and in
turn, they help plants in disease suppression (Haas and Défago 2005), increasing
immunity to biotic (Badri et al. 2013) and abiotic stresses (Zolla et al. 2013). The
plants release fixed carbon as amino acids, soluble sugars, and secondary metabolites
(Badri et al. 2013; Chaparro et al. 2013) which are then used by microbes in the
rhizosphere. The root exudate composition is determined by plant species, plant
developmental stage, and environmental factors like soil pH, temperature, and the
microorganisms present in the soil (Badri and Vivanco 2009). These factors cause
the specificity of microbes to each plant species. The changing numbers and
diversity of microbes’affects crop yield and soil fertility for developing better
varieties. The root exudates of plant are primary factors for attracting and inhibiting
the growth of certain microorganisms in the rhizosphere. They selectively permit
those microbes which are essential for plant growth among the bulk soil population
of microbes (Grayston et al. 1998). These consist of polysaccharides, amino acids,
mucilage, and many secondary metabolites such as flavonoids, terpenes, and
glucosinolates (Moore et al. 2014). Even the minor alteration in the quantity of
substances released by plant can change the microbial community structure of the
plant rhizosphere (Jones et al. 2004). For good plant productivity, healthy soil is
14 A. Negi et al.
necessary, which is affected by various biotic and abiotic factors among which the
soil microorganisms are among the most dominant biotic components (Bhatt et al.
2020a,b,2021d,e,f,g). Thus, plant-microbe interactions are not only important for
growth, health, and biocontrol of plants but also influence the chemical, biological,
and physical properties of soil (Bhatt et al. 2019a,2020c,d,e,f).
2.3 Plant-Microbe Interactions
The plant and microbes interacted with each other by mutual or commensal associ-
ations, in which either both host and microbe get benefitted or only one of them gets
benefits from associations. Sometimes microbes transit from pathogenic nature to
symbiont nature depending on the environment conditions (Newton et al. 2010).
The rhizobia, is a symbiont nitrogen-fixing bacteria changes from symbiont to
neutral interaction with plant with change in soil nitrogen level (Zahran 1999;
Bhatt et al. 2015a,b,2016a,b,2019b,c).
2.3.1 Plant Growth-Promoting Rhizobacteria (PGPR)
PGPR is a group of bacteria that are found in the rhizosphere colonizing the root of
monocot and dicot plants to enhance the plant growth by various mechanisms
(Ahemad and Khan 2012; Huang et al. 2021). PGPR are classified into two types:
intracellular PGPR which are present in nodules of plant root and extracellular PGPR
which are free-living bacteria (Martínez-Viveros et al. 2010). Common examples of
intracellular PGPR are Frankia,Rhizobium,Bradyrhizobium,Allorhizobium, etc.
(Bhattacharyya and Jha 2012), and extracellular PGPR genera are Erwinia,
Azospirillum,Burkholderia,Caulobacter,Pseudomonas,Serratia,Bacillus, and
Chromobacterium, etc. (Ahemad and Kibret 2014). They increase nutrient, like
micronutrients, phosphorous, and potassium solubilization, uptake in plants (Singh
et al. 2007) or release chemical substances like ethylene, IAA, GA, and cytokinins
(Kloepper 1992) and improve plant growth under stress conditions (Egamberdieva
and Kucharova 2009). PGPR along with other bacterial and fungal partners are
inoculated in soil to enhance fertility (Kumar et al. 2013; Prasanna et al. 2011,2014,
2015). The increase in salt tolerance and leaf water content of Zea mays and decrease
in its electrolyte leakage occur with the co-inoculation of Pseudomonas and Rhizo-
bium (Bano and Fatima 2009). Induced systemic resistance (ISR) is acquired by
plant against pathogens when the PGPR are inoculated; they resist a broad spectrum
of pathogens (van Hulten et al. 2006). Many PGPBs such as Paenibacillus alvei,
Azospirillum brasilense,Bacillus pumilus,Pseudomonas fluorescens, etc. form a
colony on roots and protect vegetables, crops, and trees from foliar diseases in field
trials and greenhouse (Van Loon 2007).
2 Insights into the Rhizospheric Microbes and Their Application for... 15
2.3.2 Mycorrhizae
Mycorrhizal symbiotic relationship is a non-disease-producing mutual association
between higher plants and fungi in which both plant and the fungus benefit (Morgan
et al. 2005; Bhatt et al. 2019d). The fungus invades the plant root to absorb nutrients,
while 90% of land plants depend on mycorrhizal fungi for minerals mainly phos-
phorus (Bhatt et al. 2019d; Sharma and Bhatt 2016). In winter season, the plants do
not get sufficient exposure to sunlight which they get dependent on fungi for
nitrogenous compounds, sugar, and other nutrients that fungi absorb from soil. In
lowland forests, mycelia networks are formed that connect various trees; together
with the help of them, trees and their seedlings use that network to exchange
chemical messages and nutrients (Bhatt et al. 2019d; Sharma et al. 2016; Bhatt
and Nailwal 2018; Khati et al. 2018a; Gangola et al. 2018a). Mycorrhizae are of two
types: ectomycorrhiza and endomycorrhiza. Ectomycorrhiza are found in trees,
while endomycorrhiza are mostly found on agricultural crops (Bhatt et al.
2019d,e; Bhatt 2018; Bhatt and Barh 2018; Bhandari and Bhatt 2020; Bhatt and
Bhatt 2020). The examples of mycorrhizae fungi are Scutellospora,Glomus,
Entrophospora, and Gigaspora along with providing minerals to plant mycorrhizae
provide tolerance from drought, heavy metals, and pathogens to plants (Bago et al.
2003).
2.3.3 Nitrogen-Fixing Microbes
Nitrogen-fixing microbes play a great role in plant life by providing them soil
nitrogen through nitrogen fixation process. They may live freely or in symbiotic
association to plant depending on the type of host plant (Deaker et al. 2004). Some of
the examples of symbiotic nitrogen fixers are Photorhizobium,Rhizobium,
Sinorhizobium, and Bradyrhizobium; they form nodules on plant root or stem
(Moreira 2008). Rhizobia represent Proteobacteria; they are not only the one that
form nodules on plant, but also some other examples of α-Proteobacteria such as
Ochrobactrum and Phyllobacterium and β-Proteobacteria such as Devosia and
Burkholderia form nodules (Daniel et al. 2007). Actinobacteria, Frankia (Prasanna
et al. 2009), and few Cyanobacteria like Nostoc, Anabaena, etc. also take part in the
fixation process (Unkovich and Baldock 2008). Common free-living nitrogen-fixing
bacteria are Pantoea,Bacillus,Azotobacter,Burkholderia, etc. Gluconacetobacter
and Herbaspirillum are endophytes that also fix nitrogen. Clostridium,Enterobacter,
Klebsiella, and Desulfovibrio are facultative and obligate anaerobes that fix nitrogen
only in the absence of oxygen (Arnold 2007).
16 A. Negi et al.
2.3.4 Endophytes
Endophytes are the fungus and bacteria that reside inside the tissue of plant cells
without harming them. They intake nutrition from their host plant and provide them
various benefits. Their main functions include the plant growth promotion, yield
enhancement, proper nutrient cycling, and pathogen suppression. They form colo-
nies in different parts of plant like stem, root, leaves, bark, etc. They also produce
phytohormones that protect plant from abiotic stresses (Ganley et al. 2004). A single
plant microbiome consists of diverse endophytes which help plant including the
modulation in gene expression (Coombs and Franco 2003). Common bacterial
endophytes include Azoarcus,Azospirillum,Pseudomonas,Gluconacetobacter,
Achromobacter, etc. (Jumpponen 2001). Common fungal endophytes basidiomy-
cetes, Harpophora, Rhizoctonia/Ceratobasidium complex, Periconia macrospinosa,
and Exophyla are involved in plant growth and promotion (Fierer and Jackson
2006).
2.4 Effect of Rhizosphere Microbes on Root Development
Beneficial rhizosphere microbes induce plant growth by modifying root develop-
ment. Soil consists of huge microbial diversity like fungi, bacteria, archaea, etc.
(Ferguson and Mathesius 2014). The microbes interact with the host plants and
improve their health by direct and indirect mechanisms (Verbon and Liberman
2016). PGPR are most common among them; they modulate the significant deter-
minant of crop yield, i.e., root system architecture. Though it is reviewed that it
modulates the root system, the mechanism by which it influences the cell division,
proliferation, and differentiation in root initiation sites is unknown. The recent
findings also suggest that PGPR play role in root hair formation and lateral root
development (Zamioudis et al. 2013). For studying the effect of bacterial and fungal
species on root development, Arabidopsis root is taken as host. Pseudomonas simiae
WCS417 or Bacillus megaterium UMCV1 colonizes around Arabidopsis root and
causes the transition from proliferation to differentiation in the root (Ortíz-Castro
et al. 2008; Zamioudis and Pieterse 2012). Hence, their effects are different,
P. simiae increases the cell division, whereas B. megaterium decreases it, in the
meristematic zone. These bacterial species decrease cell elongation, thus decreasing
primary root length (Ortíz-Castro et al. 2008). The density of root hairs gets
increased due to their colonization and grows longer. The volatile compounds
released by P. simiae WCS417 enhance lateral root formation without inhibiting
the primary growth (Zamioudis and Pieterse 2012).
2 Insights into the Rhizospheric Microbes and Their Application for... 17
2.5 Impact of Rhizosphere Microbes on Host Immune
System
The beneficial rhizosphere microbes enhance the defensive activity of plants by
activating the induced systemic resistance (ISR) in which the host immune system
gets activated for defense (De Vleesschauwer and Höfte 2009; Millet et al. 2010). In
case of Arabidopsis, the induction of ISR is well studied by PGPR Pseudomonas
fluorescens WCS417. WCS417 secretes low molecular weight molecules that sup-
press the flagellin-triggered immune response (Pineda et al. 2010), while an immune
signaling cascade gets initiated that provides resistance to a broad spectrum of
pathogens and insects (Van Oosten et al. 2008; Van der Ent et al. 2008). This
initiation in Arabidopsis root is regulated by transcription factor MYB72 (Van der
Ent et al. 2009). ISR response causes then the increased deposition of callose at the
pathogen entry site (Pozo et al. 2008). Jasmonic acid, ethylene hormones, and
transcriptional co-activators NPR1 and MYC2 help in the process of WCS417-
ISR activation (Pozo et al. 2008; Segarra et al. 2009). Not only bacteria but also
some fungus like Trichoderma sp. (Trivedi et al. 2012) and mycorrhizal fungi
(Segarra et al. 2009) induce ISR. The rhizosphere microbiome structure also gets
changed when a plant gets infected by a pathogen. For example, the infection caused
by Candidatus Liberibacter asiaticus on citrus plant results in the alteration in the
composition of citrus rhizosphere community (Zhang et al. 2011). Similarly, the
infection caused by Verticillium dahlia alters the rhizospheric microbial composition
of cotton (Bais et al. 2002). These changes may occur due to the release of some type
of antimicrobial compounds by infected roots. Like in the case of Ocimum
basilicum, infection by Pythium causes the release of rosmarinic acid, a caffeic
acid ester with antimicrobial activity (Lanoue et al. 2010). Fusarium graminum
infection on Barley causes the release of phenol compounds with antifungal activity
(Meyer 2000).
2.6 Mechanisms Involved in Plant-Microbe Interactions
The microbes interact with plant through various mechanisms including quorum
sensing, volatile production, microbial signaling, plant hormones, and siderophore
synthesis.
2.6.1 Siderophore Production
Iron is a highly needed nutrient for all living being except for certain like Legionella,
Neisseria, and S. cerevisiae (Atzorn et al. 1988). To satisfy the need of iron,
microorganisms have evolved many pathways that include the low molecular weight
18 A. Negi et al.
iron chelators called siderophores. They are secreted to solubilize the iron from
surrounding environments and form a complex of ferric-siderophore that move by
diffusion and return to the cell surface (Rajkumar et al. 2010). Based on their
structural features, iron-coordinating functional groups, and types of ligands, bacte-
rial siderophores have been classified into four main classes: pyoverdines, carbox-
ylate, phenol catecholates, and hydroxamates (Rodríguez et al. 2006). Hundreds of
siderophores have been identified and reported for cultivable microorganisms; some
of which are widely recognized and used by different microorganisms, while others
are species-specific (Misra et al. 2012). Siderophore production confers competitive
advantages to PGPR that can colonize roots and exclude other microorganisms from
this ecological niche (Bhandari and Bhatt 2020). Under highly competitive condi-
tions, the ability to acquire iron via siderophores may determine the outcome of
competition for different carbon sources that are available as a result of root
exudation or rhizodeposition (Rodríguez et al. 2006). Most organisms require iron
as an essential element; it serves as a cofactor for a wide variety of cellular processes,
such as electron transport chain, oxygen transport, cellular respiration, chlorophyll
biosynthesis, and thylakoid biogenesis and chloroplast development (Neilands
1995). More than 100 enzymes involve in primary and secondary metabolic reac-
tions contain ferric residues such as iron-sulfur clusters (Oves et al. 2013). Although
iron is abundantly present in the environment, the low solubility and slow dissolu-
tion rates of iron-containing minerals often limit the bioavailability of iron. The
rhizoremediation of soils by PGP microorganisms is believed to reduce chemical
fertilizers in agriculture practices (Philippot et al. 2010). Plant growth promotion by
siderophore-producing rhizobacterial inoculations have been reported in various
studies. Siderophore-producing bacteria Pseudomonas strain GRP3 has been
shown to enhance chlorophyll content and iron nutrition in Vigna radiata plants.
Fe-siderophore complex, which is produced by rhizosphere microorganisms, can
deliver iron to plant through specific transporter channels under iron starvation
(Bhatt et al. 2016a). Moreover, chelation of trace elements by bacterial siderophores
in the rhizosphere have employed as natural biodegradable chelators (Moreira 2008).
Some siderophores, e.g., desferal, desferrioxamine B, dexrazoxane, O-trensox,
desferri-exochelins, desferrithiocin, and tachpyridine, are found useful in sickle
cell disease, thalassemia, malaria, haemochromatosis, and cancer therapy.
2.6.2 Quorum Sensing
The small signal molecules that can diffuse easily are termed as autoinducers and
mediate quorum sensing (QS) by regulating the gene expression of the population
(Hooshangi and Bentley 2008; Von Bodman et al. 1998). In case of Gram-negative
bacteria, the N-acyl homoserine lactones (AHL) act as the signal molecules and
regulate the density of population (Rinaudi and González 2009). QS plays a very
important role in legume symbiosis (Fray 2002). Bacterial QS is a type of cell
density-dependent population behavior, in which the production, detection, and
2 Insights into the Rhizospheric Microbes and Their Application for... 19
response to a molecule regulate gene expression. Only proteobacteria among all
bacteria exhibit QS by signaling molecule N-acyl homoserine lactone (AHL) (Elasri
et al. 2001). A huge number of AHL-producing proteobacteria are found in rhizo-
sphere (DeAngelis et al. 2007). Two studies show direct evidence of QS in natural
soil (Burmølle et al. 2005) and compost soil (Passador et al. 1993), although the role
of QS in control of soil processes has not been investigated in biologically intact
soils. QS control of extracellular enzyme activity has been studied almost entirely
within the context of pathogenesis and is known mostly in pathogenic
Gammaproteobacteria like Pseudomonas aeruginosa PAO1 (Worm et al. 2000),
Pseudomonas fluorescens (Rasch et al. 2005), Enterobacteria spp. (Swift et al.
1999), Aeromonas hydrophila (Jones et al. 1993), Erwinia carotovora (Pirhonen
et al. 1993;Eberl et al. 1996), Serratia spp. (Croxatto et al. 2002), and Vibrio spp.
(Aguilar et al. 2003) and Betaproteobacteria Burkholderia cepacia (Chernin et al.
1998) and Chromobacterium violaceum (Von Bodman et al. 2003). The prevalence
of QS-controlled secretion of enzymes among pathogens (Berg et al. 2005) and the
prevalence of pathogens in soil (Dweck et al. 2015) suggest that bacterial QS may
also be important in the context of soil nitrogen cycling.
2.6.3 Volatile Organic Compounds
The chemicals that carry out the communication across all kingdoms of life are
termed as volatile metabolites (Schmidt et al. 2015). They are able to alter the
physiological processes in other bacteria, fungi, and plants (Blom et al. 2011). In
some studies, it has been shown that the bacterium-bacterium and bacterium-host
interactions also get facilitated by bacterial volatiles (Lowery et al. 2008). The
biofilm formation and the motility are some of the bacterial processes that are
regulated by producing quantitative and qualitative differences in the volatiles
(Groenhagen et al. 2013). In Pseudomonas aeruginosa,Burkholderia ambifaria,
and Streptomyces sp., the synthesis of QS-regulated volatile, i.e., 2-amino
acetophenone, has been reported (Kai et al. 2016), where the pattern of volatile
emission is influenced by plant-microbe interactions (Rosier et al. 2016).
2.6.4 Plant-Mediated Signaling
Plants are greatly responsible for assembling the rhizosphere microorganisms
(Walling 2000). When plant get affected by biotic stress, i.e., pathogens, the defense
system called induced systemic response (ISR) gets activated by plants (Glazebrook
2005). The most dominating signaling pathways in plants are based on salicylic acid-
dependent systemic acquired resistance (SAR) (Giron et al. 2013) and jasmonic acid/
ethylene-dependent ISR (Glazebrook 2005). Other hormones like abscisic acid,
cytokinin, auxin, and gibberellins also take part in this signaling network (Stam
20 A. Negi et al.
et al. 2014). The activation of these pathways depends on the stress type (Doornbos
et al. 2011) and influences the rhizospheric microbial community differently (Lee
et al. 2012). In sweet pepper Rhizosphere, Bacillus subtilis GB03 population in soil
are attracted by plants when they get feed by aphids (Lebeis et al. 2015). Salicylic
acid promotes the colonization of very selective bacterial groups in the root
(Lakshmanan et al. 2012). The root exudate composition gets affected by foliar
infection in plant that facilitates the colonization of beneficial rhizobacteria in the
roots (Lakshmanan et al. 2014; Singh et al. 2021; Zhang et al. 2020a,b; Mishra et al.
2020). From the previous research, it was concluded that rhizospheric and indige-
nous soil microbial strains perform various important role in sustainable environ-
mental development (Zhang et al. 2020b; Feng et al. 2020; Lin et al. 2020; Zhan
et al. 2020; Ye et al. 2019; Huang et al. 2019). These strains can be applied for
greater agriculture production and bioremediation of xenobiotics from the soil and
water environments (Huang et al. 2019,2020; Fan et al. 2020; Pang et al. 2020;
Gangola et al. 2018b). Furthermore, more validation of processes of these microor-
ganism could be more beneficial for the resource recovery and sustainable agricul-
tural environments (Bhatt et al. 2021f; Gangola et al. 2018b; Gupta et al. 2018; Khati
et al. 2017a,b,2018b; Kumar et al. 2017).
2.7 Conclusion
The world is facing a lot of issues regarding food quality, soil deterioration, crop
production, soil fertility, and many more. These issues could be overcome with the
help of beneficial microbes that include bacteria, fungi, and archaea. The plant-
microbe interactions pay a great role in promoting plant growth and health. Still the
understanding on this field is in its infancy. As discussed above, the root microbiome
boosts the defense system of plant and also facilitates nutrient solubilization. It is
also suggested that the plants assemble the group of microorganisms in their
rhizosphere according to their requirements and necessity by alluring microbes
with chemical compounds. Due to this selective interaction with beneficial microbes,
the specificity between host plant and microbes has been increased. The beneficial
microbes present in the soil for a long period make the soil disease suppressive and
healthy for crop growth and yield. In future, it is expected that many more mecha-
nisms would be revealed by which the plant and microbes interact. Thus, it will
ultimately helpful in increasing crop productivity and quality.
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2 Insights into the Rhizospheric Microbes and Their Application for... 29
Chapter 3
Different Biofertilizers and Their
Application for Sustainable Development
Dharmendra Kumar , Som Dutt, Pinky Raigond,
Sushil Sudhakar Changan, Milan Kumar Lal, Rahul Kumar Tiwari,
Kumar Nishant Chourasia, and Brajesh Singh
Abstract The excessive use of chemical fertilizers causes many serious negative
impacts on the agriculture production system and natural resources. Therefore, we
need to have alternative options of chemical fertilizers sustainably. Biofertilizer has
become increasingly important in agriculture due to its potential role in food security
and environmentally friendly methods. Organic farming is not possible without the
use of biofertilizers. The biofertilizers are living and latent cells of microbes that
supply nutrients for crop production. The present book chapter highlighted different
biofertilizers such as nitrogen-fixing microbes, phosphorus-solubilizing and
phosphorus-mobilizing microbes, potassium solubilizer microbes, blue-green algae
and Azolla, etc., uses in crop production, their method of production, and illustration
of beneficial microbes which are used in biofertilizer industries.
Keywords Biofertilizers · Sustainable agriculture · Rhizobium · Organic farming
3.1 Introduction
Present day’s world human population is rapidly increasing and is now 7.7 billion.
India’s population is rapidly growing, putting pressure on the agricultural production
system and our natural resources, both of which are required to feed this huge
population on limited land (FAO 2020). The agricultural sector is under pressure
to meet the demand for food security as a result of the increasing human population,
which forces farmers to use modern intensive farming methods with intensive
application of chemical fertilizers, insecticides, fungicides, nematicides, and
D. Kumar (*) · S. Dutt · P. Raigond · S. S. Changan · M. K. Lal · R. K. Tiwari ·
K. N. Chourasia · B. Singh
Division of Crop Physiology, Biochemistry and PHT, ICAR-Central Potato Research Institute,
Shimla, Himachal Pradesh, India
e-mail: sushil.changan@icar.gov.in;milankumarlal@naarm.org.in;rahul.tiwari@icar.gov.in;
kumar.chourasia@icar.gov.in
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_3
31
pesticides for increasing food production and productivity. As a result, the continued
use of agrochemicals for increased soil fertility and plant growth often has negative
environmental effects, such as soil, groundwater, and aquifer pollution. In this
context, environmentally friendly and sustainable crop production that makes effi-
cient use of natural resources is becoming increasingly common as a means of
meeting food security demands. Biofertilizers play a vital role in delivering better
agricultural production in modern crop production systems (FAO 2020; Sharma
et al. 2020a; Bhattacharyya and Jha 2012; Dal Cortivo et al. 2020; Bargaz et al.
2018).
The nutritional content of soil enriched with biofertilizers is improved by prod-
ucts containing living cells of various types of microbes. Bacteria, fungi, and
cyanobacteria are the most important sources of biofertilizers (blue-green algae).
Biofertilizers that fall into one of the categories mentioned above include nitrogen-
fixing microbes, phosphate-solubilizing and phosphate-mobilizing microbes,
potassium-solubilizing microbes, micronutrient-solubilizing microbes, and plant
growth-promoting rhizobacteria (Chittora et al. 2020; Kour et al. 2020a; Mahanty
et al. 2017).
Some major biofertilizers are given in Table 3.1. Rhizobium fixes nitrogen in
symbiotic association with the root nodules of legume plants, and other microbes
may fix nitrogen associatively or independently (Bennett et al. 2020). By dissolving
rock phosphate and tricalcium phosphate, phosphorus-solubilizing microorganisms
that secrete organic acids assist plants in consuming more phosphorus (Alori et al.
2017a).
The most common phosphorus-mobilizing microbes are arbuscular mycorrhizal
fungi types that are omnipresent (Berruti et al. 2016). Plant growth-promoting
rhizobacteria (PGPR) are a type of bacteria that aid plant growth by fixing nitrogen,
solubilizing phosphorus, or producing plant growth-promoting metabolites, as well
as producing antibiosis or antibiotics compounds for disease control. The crop
Table 3.1 Major biofertilizers and target crops
Different biofertiliser Biofertilizer application crops
Rhizobium sp. Leguminous crops (specific for pulses)
Azotobacter sp. Wheat, barley, oats, cotton, mustard, and tomatoes, also some of the
most common crops (potato, chili, okra, cucumber, onion, tomato,
brinjal, and others)
Azospirillum sp. Maize, wheat, barley, oat, Kodo, finger millets, bajra and other
millets, sorghum, and sugarcane are all cereal crops
Cyanobacteria/BGA Rice
PSM All crops
Arbuscular mycorrhiza Crops cultivated in nurseries and orchard trees
K solubilizing microbes All crops
Micronutrient-solubiliz-
ing microbes
All crops
PGPRs All crops
Azolla Rice
32 D. Kumar et al.
productivity enhanced by using potential PGPR is used as microbial inoculants
(Glick 2012).
The chemical fertilizer application in agriculture crops in the last three decades
has been drastically increasing in the use of production, and it is causing serious
concern. The quality of soil and groundwater is adversely affected by the effect of
excessive fertilizers. The amount of of chemical fertilizers used can be decreased by
the application of environmentally friendly biofertilizers that can definite advantage
over chemical fertilizers. Biofertilizers are a cheap source of nutrients in economical
use compared to synthetic chemical fertilizers. Chemical fertilizers are harmful to
life at higher concentrations, while biofertilizers have no toxic effects (Singh et al.
2016; Kong et al. 2018; Vessey 2003).
3.2 Types of Biofertilizers
Integrated plant nutrient management has the combination of different sources of
nutrients; one of the most important is the biofertilizers. Biofertilizers are important
for crop growth, soil productivity, and water sustainability, as well as ecosystem
protection. The renewable source of plant nutrients is biofertilizers which are
sustainable to the agricultural ecosystem.
Biofertilizers are items that contain living latent cells of microbes when applied to
seed, nursery, soil, or plant canopy and are found in the interior or rhizospheric zone
of the plant. Therefore, agriculture production improves through the biological
process such as BNF, solubilization of unavailable form of phosphorus, or mobili-
zation of phosphorus or solubilization of other elements which are important for
crops (Kour et al. 2020b; Thomas and Singh 2019).
The additional protection of crops and plants from pest and pathogens will
improve plant growth and development. Many researchers across the world have
been reviewed for the sustainable development of agriculture by the application of
different soil microorganisms. Various biofertilizer are described below.
3.2.1 Nitrogen-Fixing Bacteria
The plants cannot utilize the atmosphere nitrogen which is nearly 78% in the
environment in free form because of its inert nature. The ammonia or nitrate forms
of N are uptake by plants.
However, the source of an available form of nitrogen is many such as lightening
small relatively amount of ammonia produced and by the Haber-Bosch process,
industrially ammonia produced on very high pressures and fairly high temperature
by using the iron-based catalyst. However, through nitrogenase enzyme activities,
the major conversion of atmospheric nitrogen into ammonia which is the available
form of N is achieved using the BNF process which is performed by
3 Different Biofertilizers and Their Application for Sustainable Development 33
microorganisms. The nitrogenase enzyme converts nitrogen to ammonia and further
by transamination process into proteins; this process is known as nitrogen fixation or
dinitrogen fixation (Islam et al. 2016; Laguerre et al. 2007; Raklami et al. 2019).
The prokaryote group only belongs to nitrogen-fixing microbes. The nitrogen
fixing of diazotroph microorganisms belongs to the different groups of prokaryotes
in nature.
The diazotrophs are classified mainly into the following categories:
1. Mutualistic microorganism: Rhizobium sp.—legume symbiosis Rhizobium-
Parasponia (non-legume) symbiosis Frankia—Trees (e.g., Alder,Casuarina)
Azolla-Anabaena.
2. Free-living microbes: Azotobacter paspali—Paspalum notatum.
(a) Aerobic: Azotobacter,Beijerinckia, Cyanobacteria (e.g., Nostoc,Anabaena,
Tolypothrix,Aulosira).
(b) Facultative: Klebsiella pneumonia,Bacillus polymyxa.
(c) Anaerobic: Clostridium,Desulfovibrio,Rhodospirillum,
Rhodopseudomonas,Desulfotomaculum,Desulfovibrio,Chromatium,
Chlorobium.
3. Symbiosis in associative microbes (Kour et al. 2020a; Laguerre et al. 2007;
Govindarajulu et al. 2005): Azospirillum,Herbaspirillum.
(a) Gluconobacter diazotrophicus,Azoarcus.
Some important types of nitrogen-fixing related biofertilizers can be considered
for the agriculture production system.
3.2.1.1 Rhizobium
Rhizobia is a category of microbes that includes Rhizobium,Bradyrhizobium,
Mesorhizobium,Sinorhizobium, and Azorhizobium, among others. These rhizobia
genera are symbiotic associations with leguminous plants for fixation nitrogen to
ammoniacal form (Mutch and Young 2004; Keet et al. 2017). The rhizobial colonies
are cultivated on the yeast extract mannitol agar (YEMA) medium and appear raised,
wet, shining, translucent, or opaque with a smooth margin. The nitrogen-fixing
rhizobia are found in the root or stem nodules of rhizobium-legume symbiosis.
Therefore, these alternatives to the nitrogen-fixing process are agronomically struc-
tural unique for reducing energy-expensive ammonium and nitrate biofertilizers. All
legume crops cannot fix atmospheric nitrogen. The mainly three families of legumes
fix nitrogen such as Caesalpiniaceae, Mimosaceae, and Fabaceae. However, while
all members of the Caesalpiniaceae family cannot form nodules, all members of the
Mimosaceae and Fabaceae families do. The legume-rhizobia symbiosis relations
repair nearly 75 million metric tons of nitrogen per year. The nitrogen fixation
depends on the species of rhizobia, legume, soil, and environmental conditions
(Dal Cortivo et al. 2020; Laguerre et al. 2007; Andrews and Andrews 2017; Boivin
34 D. Kumar et al.
et al. 2020; Cavassim et al. 2019). Therefore, the legume inoculation in the legumes
is a very important aspect for the manipulation of rhizobial microflora in the
agriculture field for improving soil fertility and crop productivity. Due to high levels
of mineral nitrogen and the presence of adequate native rhizobia, rhizobial inocula-
tion often fails in tropical areas. Therefore, it is very necessary to identify the
situation where rhizobia inoculation can do. Many researchers suggested different
diagnostic tools to measure the rhizobia inoculation in legumes (Missbah El Idrissi
et al. 2020; Rocha et al. 2019).
If rhizobial inoculation is needed, the following criteria should be followed;
•Species-specific rhizobia should have a low population density.
•The previous crop should be not grown as the same legume fields.
•The unproductive or waste soil should be reclaimed before inoculation of
rhizobia.
•The crop rotation should be followed in legume and non-legume manners.
•The nitrogen status of soil should not be high level.
•The soils should not be acidic, alkaline, and saline (Rocha et al. 2019; John et al.
2011).
3.2.1.1.1 Rhizobial Strain Selection for Inoculant Development
The ideal inoculant strain for different legume crops should be screening, identify-
ing, and classifying at a large scale. To differentiate legume inoculants, use acidic,
sodic, saline, nitrate-rich, or heavy metal-polluted soil types (John et al. 2011;
Vassilev et al. 2020; Mosa et al. 2016a).
The following are some of the characteristics that an inoculant strain should have
in order to be suitable for commercial inoculants:
•It should have the ability to shape nitrogen-fixing modules in target legumes.
•The inoculant strain can compete with native rhizobia populations in the soil for
nodule formation.
•They must be able to fix nitrogen in a number of environmental conditions.
•In artificial media, inoculant carriers and, in the soil, the inoculant strain must be
able to expand.
•The inoculant strain should be able to persist in soil, particularly for perennial
legumes such as alfalfa.
•The inoculant strain should be able to migrate away from the initial
inoculation site.
•In the absence of a legume host, the inoculant strain should be able to colonize
the soil.
•The strain used for inoculation should be able to withstand environmental
stresses.
•With a wide variety of host genotypes, the inoculant strain should be able to fix
nitrogen.
3 Different Biofertilizers and Their Application for Sustainable Development 35
•The inoculant strain should have genetic stability.
•The inoculant strain should have compatibility with agrochemicals (Rocha et al.
2019; Vassilev et al. 2020).
3.2.1.2 Azotobacter
Azotobacter is a nitrogen-fixing bacteria that lives in the rhizosphere of a wide range
of plants and forms cysts. Examples are A. niger,A. chroococcum,A. vinelandii,
A. beijerinckii,A. nigricans,A. armeniacus, and A. paspali. The other members are
mostly soil-borne and rhizospheric, except for a few last rhizoplane bacterium.
A. chroococcum and A. paspali are well-known for their ability to serve as a
biofertilizer for a number of non-legume crops (Thomas and Singh 2019; Noar
and Bruno-B Arcena 2018). Azotobacter is a Gram-negative aerobic rod-shaped
bacteria that can be found singly, in chains, or in clumps. It does not produce
endospores, but it does contain cysts with thick walls. Desiccation and some harmful
chemical and physical agents do not affect these cysts. They, on the other hand, are
unable to resist extreme temperatures. They do not fix nitrogen and are optically
refractile during the cyst stage of their life cycle. It may be motile or non-mobile due
to peritrichous flagella. It can be used to produce a water-soluble pigment that is
yellow-green, fluorescent, or red-violet/brownish-black. It thrives in a temperature
range of 20 to 30 C and a pH of 7.0 to 7.5. They may expand on a variety of
carbohydrates, alcohols, and organic acids, among other aspects (Das 2019;
Khosravi and Dolatabad 2020). Azotobacter can be found in the rhizosphere and
phyllosphere of neutral and alkaline soils. Plant growth is influenced by antifungal
metabolites and phosphate solubilization’s ability to fix molecular nitrogen; and
plant growth is influenced by growth-promoting substances like IAA, gibberellin, or
gibberellin-like compounds, and vitamins’ability to fix molecular nitrogen (Das
2019; Van Oosten et al. 2018). Seed inoculation and carrier-based Azotobacter
inoculant Azotobacter mass multiplication are equivalent to rhizobial inoculation.
The Jensen’s N-free medium is widely used in mass multiplication. Seed inoculation
with A. niger increased field crop yields by about 10% and cereal yields by about
15–20%. A. chroococcum is a type of chroococcum. Azotobacter co-inoculation
with other bio-inoculants including Rhizobium,Azospirillum, P-solubilizers, and
vesicular-arbuscular mycorrhizae has been shown to increase legume, cereal, and
vegetable crop growth and yield (Das 2019; Mosa et al. 2016b). Many experiments
have shown that Azotobacter chroococcum inoculation has beneficial effects on a
variety of cereal, vegetable, oilseed, legume, and others (Table 3.2). Higher nitrogen
levels (0 to 30 kg N ha
1
) only improved yield efficiency in Azotobacter inoculation
experiments. These diazotrophic bacteria need a lot of available carbon to survive in
the soil. When farmyard manure (FYM), compost, and other organic additives are
applied to agricultural soils, Azotobacter production increases, and the plant grows
and yields more (Thomas and Singh 2019; Das 2019; Mosa et al. 2016b; Wu et al.
2005).
36 D. Kumar et al.
3.2.1.3 Azospirillum
Beijerinck described the nitrogen-fixing bacterium Spirillum lipoferum for the first
time in 1925. Azospirillum isolation necessitates the surface sterilization of roots
with 70% alcohol and the formation of microaerophilic (low oxygen requirements)
conditions in the medium. The Azospirillum is a soil organism that can colonize both
the roots and the above-ground sections of a wide variety of plants, forming an
associative symbiosis (Cassán and Diaz-Zorita 2016). Azospirillum is a type of
bacteria that lives in the soil. Isolation from half-centimeter-long root fragments
surface-sterilized for 3–5 s in 70% alcohol. Phosphate buffer is used to wash roots
(pH 7). A semi-solid, nitrogen-free sodium malate medium is used to extract
Azospirillum from roots. Before being plated in, the root fragments are washed
several times. The tubes are incubated for 48 h at 35 C. The formation of a white
pellicle 2–4 mm beneath the medium’s surface is associated with Azospirillum
growth (Alexandre 2017).
3.2.1.3.1 Characterization of Azospirillum Strain
Azospirillum is a Gram-negative, motile, and usually vibroid bacterium that contains
poly-hydroxybutyrate granules. The Azospirillum genus has peritrichous flagella that
allow it to swarm on surfaces, is extremely motile, and has a long, polar flagellum
that needs swimming. Additionally, cysts are formed when the shape and size of the
cells change with time in the culture. The Azospirillum can develop in
microaerophilic (N
2
or NH
3
as nitrogen sources), anaerobic (NO
3
as the electron
acceptor, denitrification), and totally aerobic conditions (NH
3
,NO
3
, amino acids)
and needs the cultivation of organic acids like malate, succinate, lactate, and
pyruvate. On Rojo-Congo red medium, Azospirillum forms distinct scarlet red,
dry, and wrinkled colonies (Mosa et al. 2016a; Alexandre 2017; Lin et al. 2016).
Azospirillum is a type of alpha Proteobacterium. Five species of Azospirillum group
include A. brasilense,A. lipoferum,A. amazonense,A. halopraeferens, and
A. irakense (Ayyaz et al. 2016).
Table 3.2 Effect of Azotobacter on crop yield
Agriculturally some important crop Yield (%) increases Other crops Yield (%) increases
Food grains Other
Wheat 10–15 Potato 10–18
Rice 10–25 Carrot 5–20
Maize 10–20 Cauliflower 20–30
Sorghum 15–20 Tomato 9–24
Sugarcane 11–24 Cotton 11–27
3 Different Biofertilizers and Their Application for Sustainable Development 37
3.2.1.3.2 Crop Response to Azospirillum Inoculation
Wheat, sorghum, pearl millet, finger millet, barley, and corn are among the
Gramineae plants that Azospirillum inoculants use to fix nitrogen. Sorghum (Sor-
ghum bicolor), pearl millet (Pennisetum americanum), and finger millet (Eleusine
coracana) were all consistently responsive to Azospirillum in many places across
India (Veresoglou and Menexes 2010). Azospirillum strains increase the yield of
agriculturally important crops in a variety of soil and climate conditions. The seed
inoculants with Azospirillum sp. saved 20–30 kg N/ha equivalents. However,
Azospirillum’s main impacts extend far beyond providing nitrogen to host plants
(Fukami et al. 2018; Steenhoudt and Vanderleyden 2000a). When Azospirillum was
inoculated onto plant roots, the entire root system’s morphology and behavior
changed drastically. The average density and length of the root system increases
as hairs at the root tip become more distinct. Root hairs are root epidermal cells that
help bind the root to its surroundings while also transporting water and nutrients. The
plant roots increase the diameter and length of both lateral and adventitious roots
when inoculated with Azospirillum sp., which typically contributes to further
branching of the lateral roots.
These root system changes are important because they increase the plant’s
absorptive area and the volume of soil substrate available to it (Cassán and Diaz-
Zorita 2016; Steenhoudt and Vanderleyden 2000b). Azos strains contain
siderophores as well. The plant roots increase the diameter and length of both lateral
and adventitious roots after being inoculated with Azospirillum sp., which normally
leads to more lateral root branching. Microorganisms excrete siderophores, which
are low molecular weight iron-binding compounds produced and excreted into the
culture medium when iron levels are low.
The siderophores are transported through the bacterial envelope from the culture
medium, where they are bound with metal ions. By extracting iron, plants can
improve their iron uptake and protect themselves from pathogens by Azospirillum
(Fukami et al. 2018; Raffiand Charyulu 2021). Azospirillum has also been impli-
cated in the biosynthesis of antifungal, antibacterial, and growth-promoting com-
pounds such as phytohormones and vitamins. The most well-known growth
promoters are IAA, gibberellins, cytokinin-like compounds, and vitamins
(Alexandre 2017). Azospirillum strains differ in their ability to produce antibiotic
substances. Azospirillum controls a wide range of phytopathogenic fungi through
fungistatic activity; for example, certain Azospirillum protect cotton plants from
Thielaviopsis basicola and Fusarium oxysporum. By inoculating crops like wheat,
sorghum, pearl millet, and corn with Azospirillum, they can increase root numbers
while also boosting yields. Sorghum plants used moisture accumulated in soils from
winter precipitation better than uninoculated plants by inoculated Azospirillum in a
field experiment in Israel. Azospirillum-inoculated plants are more successful than
uninoculated plants at absorbing nitrogen, phosphorus, potassium, and other micro-
elements from the soil in both field and greenhouse experiments (Cassán and Diaz-
Zorita 2016; Alexandre 2017; Steenhoudt and Vanderleyden 2000b; Bashan and
Levanony 1990; Venkateswarlu and Rao 1985). In recent years, plant-microbe-
38 D. Kumar et al.
microbe interactions have changed from plant-microbe interactions. Many studies
have shown that when Azospirillum is co-inoculated with other microorganisms like
Rhizobium,Azotobacter, and PSB, the beneficial effects on plants are improved. The
stimulation of lateral roots and root hair branching has resulted in a rise in lateral root
and root hair branching development. Azospirillum is predominantly responsible for
the production of phytohormones and other growth factors. At low application rates
of mineral N, the researchers discovered that inoculating non-legumes with
Azospirillum brasilense and Azotobacter chroococcum had a positive effect on
associative nitrogen fixation and crop yield (Mosa et al. 2016a; Cassán and Diaz-
Zorita 2016; Venkateswarlu and Rao 1985).
3.2.1.4 Gluconoacetobacter diazotrophicus
On a semi-solid N-free sugar medium acidified with acetic acid to pH 4.5, the
Gluconoacetobacter diazotrophicus was isolated from washed roots and stems of
sugarcane. This nitrogen-fixing microorganism is Gram-negative, microaerobic, and
motile by lateral or peritrichous flagella and grows best at temperatures about 30 C.
Azolla forms a thick mat on the water’s surface in 15–20 days under ideal condi-
tions. Just about two-thirds of it is harvested, leaving the remainder to grow. It
multiplies again in 2–3 weeks and forms a dense mat. A 100 m
2
nursery can produce
about 100 kg of fresh Azolla inoculum per week. This bacterium uses sucrose as a
carbon source, but it also uses glucose, fructose, galactose, mannitol, and other
sugars. In the pH range of 3.8 to 5.8, Gluconacetobacter diazotrophicus has excellent
nitrogenase activity and development. Sugar concentrations of 10 to 30% are
favorable for this organism’s nitrogen fixation in sugarcane (Suman et al. 2005;
Muñoz-Rojas and Caballero-Mellado 2003). Endophytic diazotrophic bacteria like
Gluconoacetobacter diazotrophicus can be found in sugar-rich plants like sugar-
cane, sweet potato, Cameroon grass, sweet sorghum, and coffee. This bacterium can
colonize the stem, roots, and leaves of the host plant, among other places.
Gluconoacetobacter diazotrophicus contributes more than half of the biologically
fixed nitrogen in sugarcane for the first time in Brazil.
By fixing atmospheric nitrogen, this bacterium excreted ammonia into the
medium. It was also reported that Gluconoacetobacter strains produce a substantial
amount of IAA. When sugarcane, sweet potato, and sweet sorghum plants were
inoculated with Gluconobacter diazotrophicus and AM fungi, they showed syner-
gistic effects on plant growth and yield (Suman et al. 2005; De Oliveira et al. 2016;
Muthukumarasamy et al. 2006; Saravanan et al. 2007).
3.2.1.5 Blue-Green Algae and Azolla
Blue-green algae are found all over the world (cyanobacteria). BGA cells can be
single-celled or can be made up of branched or unbranched filaments. BGA is a free-
living group of microorganisms that fix biological nitrogen in paddy fields in large
3 Different Biofertilizers and Their Application for Sustainable Development 39
amounts. The BGA that fix nitrogen from the atmosphere biologically have a special
structure known as a “heterocyst:, and all heterocystous forms are capable of
biological nitrogen fixation. However, due to newly identified special conditions
in low oxygen environments, some BGA can fix atmospheric nitrogen without
heterocyst. The BGA such as Scytonema,Aulosira,Tolypothrix,Nostoc,Anabaena
and Plectonema as a mixture applied in the field for fixation of atmospheric nitrogen
(Dhar et al. 2007; Lumpkin and Plucknett 1980). The BGA is the only organism on
the biosphere with both photosynthesis and biological nitrogen fixation abilities.
Cyanobacteria contain a wide range of plant growth-promoting compounds, includ-
ing growth hormones, amino acids, small proteins and peptides, carbohydrates,
complex polysaccharides, and vitamins, all of which have played an important role
in crop development.
In the paddy field, the BGA fix biological nitrogen in the range of 25–40 kg
nitrogen per hectare per season and supplied 5–20 tons of fresh weight of
cyanobacteria. This is due to a large amount of organic matter that is commonly
accessible, which greatly benefits succeeding crops (Dhar et al. 2007; Prasanna et al.
2003).
3.2.1.5.1 Production of Algae for Field Application
To grow these algae in large quantities, a simple rural-oriented open-air method
based on the natural ecology of these algae has been developed. The underlying
assumption is to grow them in natural sunshine in rice fields with promoting
conditions. Various agricultural universities and private dealers backed a starter
culture consisting of a soil-based mixture of productive BGA strains (Chatterjee
et al. 2017; Mahapatra et al. 2018).
BGA biofertilizer is made of shallow stays (15 cm 7.5 cm 22.5 cm) made of
galvanized iron sheet, brick and mortar, or pits lined with polythene sheets. The size
of the pit for processing algal biofertilizer may also be increased. First, about 10 kg
of soil is inserted in the trays and mixed with 0.2 kg of superphosphate; then, the
trays are filled with water based on local conditions such as evaporation rate; the pH
of the soil should be about neutral (5–20 cm).
After the soil has settled, the surface of the standing water is sprayed with sawdust
and the starter culture. The whole setup should be in full light. The algae will
continue to grow, and a thick algal mat will form on the soil’s surface in about a
week, and it may even float up in the hot summer months.
More water is applied intermittently if the regular rate of evaporation is high due
to the high temperature during the summer season. The water should be drained and
allowed to dry out in the sun until the algal growth has thickened enough. The dried
algal flakes are collected on the surface and stored in bags to be used later in the
fields. Inoculum is added in the form of a small amount of dry algal flakes, and the
trays are refilled with water.
The operation is repeated in the same manner as before. When the soil in the tray
is depleted (usually after three to four harvests), new soil is added, combined with
40 D. Kumar et al.
superphosphate, and the process is repeated. To prevent insect reproduction, mala-
thion (0.00075 ppm) or carbofuran (3% granule) should be used (Chatterjee et al.
2017; Win et al. 2018).
3.2.1.5.2 Method of Algae in Field Application
One week after paddy transplantation, cyanobacteria is applied at a rate of 10–15 kg/
ha over standing water in the region. After algal treatment, the field is kept damp for
at least a couple of days. The establishment and operation of these algae in the fields
are unaffected by recommended pest control measures and other management
activities. The sun-dried algal material can be processed and used in the field for a
long time. Chemical fertilizers or other agricultural chemicals should not be
processed in close proximity to algal material (Mahapatra et al. 2018).
3.2.1.5.3 Azolla-Anabaena Symbiosis
Azolla is a small aquatic fern that grows in almost any climate. Azolla has a thick
aerial dorsal lobe, two lobes on each leaf, and a thin ventral lobe that is sometimes
larger. The dorsal lobe is green, with a central cavity containing a blue-green algal
symbiont (Anabaena azollae). The site of nitrogen fixation in the symbiont
Anabaena’s heterocyst. Azolla is a nitrogen-fixing plant that also provides nutrients.
Azolla fixes nitrogen, feeds Anabaena, and adds a protective leaf cavity for the fern
(Singh et al. 1984,1988; Prasanna et al. 2008).
Azolla can be found in temperate and tropical wetlands, streams, swamps,
ditches, and paddy fields where there is still water. It has been used as a rice fertilizer
due to its rapid growth, high nitrogen content, and ability to grow in still water. This
has been used as a biofertilizer in Vietnam and China for decades, but it is a
relatively new invention in India. Azolla has seven living species: Azolla sp.,
A. pinnata,A. caroliniana,A. filiculoides,A. rubra,A. nilotica, and A. mexicana,
and mexicana microphylla is a type of microphylla. A. pinnata is a native of India,
but many of these species have been imported to the United States (Singh et al. 1984;
Yadav et al. 2014). Azolla’s potential as a rice biofertilizer is determined by its high
N
2
fixation ability, rapid growth, high biomass accumulation, and N content. Via
biological nitrogen fixation, the Azolla-Anabaena complex is thought to be a
promising biological system for increasing rice yield at a low cost. Azolla’sN
2
repair ability is approximately 1.1 kg N/ha/day. The gross biomass ranged from 0.8
to 5.2 t dry matter/ha, with an average of 2.1 t/ha and a 2 to 10 day doubling period
(Prasanna et al. 2003; Singh et al. 1988).
3 Different Biofertilizers and Their Application for Sustainable Development 41
3.2.1.5.4 On the Large-Scale Production of Azolla
The concrete tanks that keep the soil flooded are the potential Azolla listed. Due to
the high rate of evaporation, partial shade is required during the summer months.
Azolla is harvested from these and used as an inoculum in rice-growing villages’
larger plots or small ponds (Lumpkin and Plucknett 1980). It is cultivated on a large
scale in a well-kept field divided into small subplots with sufficient irrigation
(4–50 sq.m. plot with 5–10 cm water depth). Azolla is inoculated at a rate of 0.5
to 1.0 t/ha. Superphosphate, at a rate of 4–8 kg/ha, promotes fern growth. Furadan
(2.5–3.0 kg/ha) and other insecticides are also used. Under optimal conditions,
Azolla forms a dense mat on the water’s surface in 15–20 days. Only about
two-thirds of it is harvested, leaving the rest to multiply. In 2–3 weeks, it multiplies
once more and forms a dense mat. A 100 m
2
nursery produces approximately 100 kg
of fresh Azolla inoculum per week. Superphosphate, at a rate of 60 kg/ha, can be
administered in two or three doses, or at weekly intervals, to boost performance.
There is no need to repeat Azolla multiplication if the results are satisfactory without
adding P (Dhar et al. 2007; Singh et al. 1984; Yadav et al. 2014).
3.2.1.6 Phosphate-Solubilizing and Phosphate-Mobilizing Microbes
Phosphorus is an essential nutrient for plant growth. While soils contain large
amounts of phosphorus, only a small amount is available to plants. Microorganisms
in the soil can solubilize phosphorus that is unavailable to plants and make it
available to them. Phosphate-solubilizing microorganisms (PSM) are what they are
called. A fungus colonizes the roots of higher plants and transports phosphorus from
the soil to the plant system (Khan et al. 2014a).
3.2.1.6.1 Phosphate-Solubilizing Microorganisms (PSM)
Regular applications of phosphatic fertilizer in crops lead to significant phosphorus
reserves in agricultural soils, which are constantly accumulated in the ecosystem.
Many factors contain significant quantities, fixation, and precipitation of phosphorus
in the soil, including pH sensitivity, which results in low-soluble phosphate fertilizer
efficacy. Phosphate precipitates as aluminum and iron phosphates in acidic soils,
while phosphate precipitates as a result of a high calcium concentration in calcareous
soils. The PSM release soluble phosphorus from insoluble phosphates into the soil
habitat through a series of solubilization reactions. These PSM have been described
as bio-inoculants for crops in soils that have been amended with rock phosphate or
tricalcium phosphate and have low available phosphate (Nautiyal 1999; Abd-Alla
1994). Bacteria, fungi, and actinomycetes are phosphate-solubilizing microorgan-
isms that help in the transformation of insoluble inorganic phosphate into simple and
soluble forms. Pikovaskaya medium is now being used to isolate, identify, and
classify phosphorus-solubilizing bacteria (PSB). The suspended phosphorus source
42 D. Kumar et al.
is insoluble phosphates such as tricalcium phosphate (Alori et al. 2017a,b; Khan
et al. 2007). Phosphate-solubilizing species are being identified by the formation of
clearing zones around their colonies. Microorganisms have been found to mineralize
organic phosphorus to a soluble state due to enzymatic activity (Kour et al. 2020a,b;
Sharma et al. 2013; Kumar et al. 2018). The ability of the effective isolates to
generate organic acids has shown that they can solubilize insoluble inorganic
phosphates including rock phosphate, tricalcium phosphate, iron, and aluminum
phosphates. Seed or seedling inoculation with PSM improves crop grain yield.
They are said to contribute 30–35 kg P
2
O
5
per hectare (Alori et al. 2017b; Khan
et al. 2014b). Acidification, chelation, exchange reactions in the growth medium,
and proton transfer during ammonium assimilation all require inorganic phosphate
solubilization by microbes (Sharma et al. 2020b).
3.2.1.6.2 Phosphate Mobilizing Microbes: Mycorrhizae
Fungi and plant roots form symbiotic relationships called mycorrhizae. Mycorrhiza
is a Greek term that combines the words myces and rhizo, with myceas referring to
fungi and rhizo referring to roots. Under natural conditions, mycorrhizae can be
found in virtually all soils, from mine spoils to agricultural soils, as well as the soil
under horticultural or field crops. Mycorrhizal associations are recognized by over
95% of plant taxa. The fungi receive carbon from the host, while the latter benefits
from increased nutrient absorption as a result of the fungi’s soil nutrient transfer.
Endomycorrhiza, ectomycorrhiza, and ectoendomycorrhiza are the three morpho-
logically distinct types of mycorrhizae, depending on whether or not fungal pene-
tration of root cells occurs. Of the three classes, endomycorrhizal fungi are the most
effective biofertilizers (Berruti et al. 2016; Hodge and Storer 2014). Nearly 90% of
land plants grow endomycorrhizae.
The fungi form external hyphal networks in the soil and thrive extensively within
the cells of the root cortex in this relationship. The Hartig net is a network of fungal
hyphae found within the root cortex. Different forms of endomycorrhizal interac-
tions include basidiomycetes, ascomycetes, and zygomycetes fungi.
The Ericaceae (Ericoid mycorrhizae) and Orchidaceae (Orchidaceous mycorrhi-
zae) families include endomycorrhizae, but arbuscular mycorrhizae are the most
common (earlier referred to as vesicular-arbuscular mycorrhizae). It is made up of
120 different zygomycetes species, all of which are members of the Glomales order
(Glomus, Acaulospora, Gigaspora, Sclerocystis, Entrophospora, and Scutellospora).
Mycorrhiza are yet to be successfully axenic or pure cultured (Wu et al. 2005;
Ritika and Utpal 2014). Numerous researchers have reported the role of mycorrhizae
in promoting plant growth in a variety of plants. The increase in nutrient uptake,
especially phosphorus, has been attributed to mycorrhizae’s beneficial effect on
plant development.
Mycorrhizal fungi improve soil phosphorus supply by solubilizing inorganic
sources of phosphorus or mineralizing organic phosphorus. By solubilizing
3 Different Biofertilizers and Their Application for Sustainable Development 43
inorganic sources of phosphorus or mineralizing organic phosphorus, mycorrhizal
fungi significantly increase phosphorus supply.
Other nutrients, such as NH
4
+
,NO
3
,K
+
,Ca
2+
, SO4
2
,Cu
+
,Zn
2+
, and Fe
+3
, can
be taken up and delivered to plants by mycorrhizal external hyphae. AM’s external
hyphae have been shown to produce up to 80% of plant P, 25% of plant N, 10% of
plant K, 25% of plant Zn, and 60% of plant Cu in experimental chambers. Mycor-
rhizal fungi produce ectoenzymes that allow host plants to access organic nitrogen
and phosphorus that would otherwise be unavailable to AM fungi or
non-mycorrhizal roots (Bargaz et al. 2018; Kumar et al. 2018; Hodge and Storer
2014; Ritika and Utpal 2014; Wahbi et al. 2016).
3.3 Conclusion
Biofertilizers are important part of integrated nutrients management and renewable
source of plant nutrients as an alternative to chemical fertilizers in a sustainable
agricultural system. As environmentally friendly and cost-effective inputs for
farmers, biofertilizers are important in organic crop production and ecosystem
protection, as well as protecting the environment. Therefore, we need to understand
the direct interactions between different microbes and crops which will ultimately
benefit the development and growth of the plants and soil health. First of all, we will
access affordable potential biofertilizers from laboratory and greenhouse to the
farmer’sfield. Therefore, we will require some novel approaches such as the
application method of biofertilizers, storage of different biofertilizer, transportation
and creation of awareness among farmers for particular biofertilizers for specific
crops, etc. So we need more focus on interdisciplinary research such as microbial
genetics, biotechnology, and agriculture extension in order to finally flourish the
biofertilizer industries.
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3 Different Biofertilizers and Their Application for Sustainable Development 47
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48 D. Kumar et al.
Chapter 4
Microbial Mediated Natural Farming
for Sustainable Environment
Asha Rani and Beenam Saxena
Abstract India is an agriculture-based country, and agriculture is the backbone of
Indian economy. More than half of the population depends upon agriculture. The
majority of the farmers rely on conventional farming in comparison to natural or
organic farming. To fulfil the food requirement, it is necessary to increase yield and
production of crops. Different types of chemical fertilizers are used to increase total
yield. Due to the large use of these fertilizers, heavy metal ions increased in the soil
which may be toxic to animals and humans. The heavy metals are also present in city
waste water (CWW) in toxic amount, and when this polluted water reaches to
adjoining areas of the city, it contaminates the soil. When these heavy metals are
absorbed by the plants, it may lead to some adverse effect on different growth
parameters which directly affects the total yield of the crop. The quantity of these
chemicals can be reduced with the help of microbes present in soil or by use of
biofertilizers. This book chapter describes the importance of organic farming to
maintain sustainable agriculture.
Keywords Biofertilizers · Conventional farming · Heavy metal · Soil microbes
4.1 Introduction
Soil is very important and an essential factor for plant growth. However, by the use
of enormous number of chemical fertilizers, it can be contaminated (Chao et al.
2014). Continuous use of chemical fertilizers and regular addition of heavy metals
may cause the different other types of pollution in soil and water environment (Bhatt
et al. 2019a,b; Pankaj et al. 2015a,b,2016a,b). Soil can be contaminated by heavy
metals which reache through city waste water and other industrial wastes to the
A. Rani (*)
Department of Botany, Bareilly College Bareilly, Bareilly, Uttar Pradesh, India
B. Saxena
Department of Zoology, Bareilly College Bareilly, Bareilly, Uttar Pradesh, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_4
49
agricultural fields. The heavy metal ions and other chemicals may also cause
decrease in nutrients in the soil. Heavy metal-contaminated soil cannot be
remediated (Verma et al. 2021). Due to heavy metal pollution, size, composition,
and activity of the microbial community are also adversely affected along with plant
quality and yield (Wang et al. 2016). Heavy metals can interfere with the enzymatic
activity of microbes so organic matter decreases in soil (Shun-hong et al. 2009).
Human exposure to these metals occurs through ingestion of contaminated food or
water. The high cost of chemical fertilizers and their adverse effect on environment
have encouraged scientists to develop alternative method to increase soil productiv-
ity (Huang et al. 2021; Singh et al. 2021; Zhang et al. 2020,2021; Mishra et al. 2020;
Feng et al. 2020; Lin et al. 2020; Vaxevanidou et al. 2015). Microorganisms play
very important role to increase soil fertility which is contaminated by heavy metals.
Phytoremediation is another aspect for the treatment of polluted soil. In this, plants
are used to reduce soil contamination. Some plants have the capacity to absorb heavy
metals when they are planted at the boundary of fields. Highly resistant plants like
sunflower (Helianthus annuus), Indian mustard (Brassica juncea L.), willow (Salix
alba), popular tree (Populus deltoids), vetiver (Chrysopogon zizanioides), etc. can
be used for a remediation of the pollution site. For phytoremediation, molecular
mechanisms of resistance to heavy metals should be studied in different types of
plants. It will be helpful in the near future to find out more plant species having
heavy metal resistance. Effected bioremediation of heavy metal-polluted soils can be
possible by using combination of both microorganisms and plants. However, suc-
cess of this approach will depend on species of organisms involved in the process.
Bioremediation is very economical in comparison to the other techniques for
remediation of contaminated soil. However, it has been found that growth of
different plant growth-promoting (PGP) microbes was proper in the organic soil.
This was due to the frequent use of green manure. The soil health depends upon the
diversity of microbes present in the soil. The productivity of the crop directly
depends upon soil health (Bhatt and Maheshwari 2019,2020a,b,c).
To reduce the amount of chemicals, conventional farming should be replaced by
organic or natural farming with use of soil microbes. Indigenous microbial consor-
tium inhabits the soil and has potential to improve soil fertility. By increasing natural
farming and use of biofertilizers or organic fertilizer, the food quality can be
improved. An “organic fertilizer”can be derived from non-synthetic or organic
sources such as plant or animal, microbes, and rock powders; by different processes
like drying, cooking, composting (Dadi et al. 2019), chopping, grinding, and
fermenting (Mario et al. 2019); or other method (Thanaporn and Nuntavun 2019).
The soil enriched with microbes is considered as healthy, and it helps in plant growth
and makes them resistant against stress. Although the maintenance of organic soil
quality is quite tough in Indian agricultural practices and expensive too. India has
1.94 million hectares of organic farmland in 2018–2019 (Fig. 4.1) accounted for
1.08% of total agricultural land, and certified organic production for all crop
categories stood at 2.6 million metric tons (MT). According to World of Organic
Agricultural Report 2018, India produces 30% of total organic production and has
maximum number of organic producers in world, i.e., about 835,000. In the year
50 A. Rani and B. Saxena
2018–2019, India exported 6.389 lakh MT, and total earning was around INR 4686
crore (Li et al. 2019).
In India, approximately 3.67-million-hectare agriculture area are used for the
organic farming. Among the Indian provinces, Madhya Pradesh represent the large
land for organic farming followed by Rajasthan, Maharashtra, Gujarat, Karnataka,
Odisha, Sikkim, and Uttar Pradesh. In 2016, Sikkim converts its entire land for
organic farming production. Globally, the USA, Canada, Switzerland, Australia,
Japan, UAE, New Zealand, etc., pay more attention for organic farming (Li et al.
2019). Despite of development in this field, organic farming has not yet so popular,
and it is not an easy task for Indian farmers to switch to organic farming as there is no
policy for encouraging the spirit of farmers to opt organic agriculture. Still, it is
necessary to promote organic farming over the conventional farming as it is the need
of the hour (Bhatt and Nailwal 2018; Khati et al. 2018; Gangola et al. 2018; Bhatt
2018; Bhatt and Barh 2018; Bhatt et al. 2019c; Bhandari and Bhatt 2020; Bhatt and
Bhatt 2021).
4.2 Effect of Heavy Metals on Different Crops
Heavy metals are present in toxic amount in the city waste water which goes to the
adjoining areas of different cities. It contains considerable quantities of toxic ele-
ments. Many unused electronic instruments and heavy metal containing batteries are
discarded which also serve as a source of heavy metals in groundwater resources.
The various elements Cd, Cu, Zn, and Pb are most likely to cause phytotoxicity
when waste water is applied to agricultural field or land where different types of
crops are growing (Bhatt and Maheshwari 2019,2020b,c). However, heavy metals
are required for growth and upkeep of plants, but their excessive amounts become
toxic to plants. Accumulation of essential metals in plants enable them to acquire
Australia
Argentina
China
Spain
Uruguay
France
United States of America
Italy
India
Germany
010 20 30 40
Milion hectares
3.63
3.14
2.25
2.15
2.04
2.02
1.96
1.94
1.52
35.69
Fig. 4.1 Farmland under organic cultivation of ten top countries in 2018. (Source FiBL 2020)
4 Microbial Mediated Natural Farming for Sustainable Environment 51
other nonessential metals (Zhou et al. 2008). Some heavy metals in the soil also have
an effect on the growth of soil microbes (Gulser and Erdogan 2008).
Increased application of agrochemicals and inorganic fertilizers is more in prac-
tice which has caused agricultural pollution leading to degradation of the ecosystem
and the environment (Malik et al. 2017). Industrial development also caused nega-
tive impact on the environment (Dhami et al. 2013); however, due to industrializa-
tion, there is rise in global economy over the last century, but it has led to a dramatic
increase in production and release of hazardous metals to the environment (Gerhardt
et al. 2009; Gallego et al. 2012; Burger 2008; Central Pollution Control Board
[CPCB] 2007).
Among the heavy metals, zinc and copper are very essential for plant growth, but
when present at elevated levels in soils, they become toxic and can ultimately cause
the death of plants. When the effect of these heavy metals studied, it is found
phytotoxic to mung bean and have adverse effect on different growth parameters
such as seedling height (Narwal et al. 1992), chlorophyll content (Khandelwal
1993), and nitrogen content (Singh 1999; Rani 2011). Reduction in all these
parameters ultimately affects the total grain yield of the crops. Except this, Pb and
Cd are also found in very low concentration. These are not beneficial for the plants,
but even their low concentration has adverse effects on plant growth. High concen-
tration of arsenic showed inhibitory effect on seed germination and seedling growth
of wheat (Zhang et al. 2010) as well as on length of plumule and radicle of
Helianthus annuus (Imran et al. 2013). The vegetable crops production at the
heavy metal-contaminated soil showed variability in heavy metal accumulation.
The vegetables can be successfully grown into the zinc- and copper-contaminated
soils, where some of them such as mustard, soybean, and spinach cannot be
cultivated (Singh et al. 2012). The accumulation of the heavy metals into the
vegetable crops affect the human health directly due to their entry via food chain
(Fu et al. 2008; Bonanomi et al. 2016).
The occurrence of heavy metals in groundwater is reported from western Uttar
Pradesh, India, and all four districts Shahjahanpur, Bareilly, Moradabad, and Ram-
pur have excessive presence of cadmium (Idrees et al. 2018). Status of different
heavy metals like As, Cd, Pb, and Hg has been investigated in most commonly used
cereals and legumes of Bareilly district of Uttar Pradesh (India). Among cereals, rice
contains the highest levels of all these heavy metals; however, As, Pb, and Hg
accumulation is also found in wheat and maize at lower level. Cd level remains
significantly higher in maize than wheat, and levels of arsenic remain similar among
different legumes (Lipismita and Garg 2012). Growth reduction as a result of
changes in physiological and biochemical processes in plants growing on heavy
metal-polluted soils has been recorded (Chatterjee and Chatterjee 2000; Oncel et al.
2000; Oancea et al. 2005).
It has been clear that heavy metal contamination causes loss of bacterial species
richness and a relative increase in soil actinomycetes or even decreases in both the
biomass and diversity of the bacterial communities in soil (Karaca et al. 2010). By
using microbial-based fertilizers, the soil health can be improved, and by doing so,
sustainability of environment can be maintained.
52 A. Rani and B. Saxena
4.3 Soil Health in Non-organic and Organic Farming Sites
Soil health is affected by the presence of microorganisms which play an important
role for crop production and final yield. Soil bacteria and fungus increase soil
fertility. 1gm. of fertile soil may have around 400,000 fungi (Griffiths et al. 1999).
During the comparative study of organic and inorganic sites, it has been found that
the organic soil has enormous amount of microorganism than inorganic sites. This is
due to the frequent use of green manure in organic soil (Khanna et al. 2010). Due to
the presence of richness of nutrients in the organic soil, growth of microorganism is
directly affected. It has been consistently reported that soil organic matter favours the
growth of bacteria present in soil. The studies have revealed that bacterial diversity
in soil is approximately 100 times greater than the other microbial diversity (Barns
et al. 1999). Pseudomonas and Bacilli however are found in both types of farming
sites, but the richness is much higher in organic sites. Nitrogen (N) is a very essential
element for the growth of leaves and stem which also plays an important role in the
formation and proper functioning of chloroplast. The organic field has high nitrogen
content as compared to non-organic farming site. The higher amount of nitrogen in
organic site is due to addition of compost and green manure which increases soil
fertility. Although chemical compounds as urea and nitrogen fertilizers are also use
in non-organic site, they are not available for plants due to their precipitation (Barns
et al. 1999; Sharma and Bhatt 2016).
Soil organic carbon (SOC) of organic farming site was found to be higher as
compared to non-organic farming site. Soil acts as a main reservoir of carbon, and
the higher SOC value is the direct indication of level of soil health. Soil organic
matter (SOM) present in the soil adds more nutrients to the soil. Good soil fertility
increases aeration, water holding capacity, proper root growth, and soil microflora
which finally affects crop yield. According to a global review, the soils in organic
cropping systems have significantly higher levels of SOC than those in conventional
systems (Sharma et al. 2016).
Phytohormones also play an important role in plant growth and directly affect the
final yield of any crop. Indole acetic acid (auxin) has many physiological roles in
plant development. Low concentration of auxin induces the root growth which
increases the water absorption. The environmental factors and soil microflora affect
the auxin activity.
4.4 Role of Microbes in Treatment of Soil Polluted
with Xenobiotics
Agriculture plays an important role in Indian economy. India holds the second
largest position in growing wheat and rice, the staple food of the world. It is the
need of the hour to increase the soil fertility and productivity of crops to fulfil the
food requirements of the large population. Different types of fertilizers and
4 Microbial Mediated Natural Farming for Sustainable Environment 53
pesticides are being used to increase production; thus further, intensive utilization of
chemical fertilizers and pesticides for higher crop production may become destruc-
tive and detrimental for soil and food quality (Gattinger et al. 2012). Soil rich in
microorganism directly affects the agricultural productivity. Number of microorgan-
ism can be increased by use of biofertilizers and biopesticides. With the help of
microorganism, plants absorb nutrients at a promising speed. These microorganisms
get food from the waste by products of plants.
Plant growth-promoting (PGP) microbes and PGPR play very important role to
cope up with heavy metal pollution of soil. They increase soil fertility, bioremedi-
ation, and stress management for development of eco-friendly sustainable agricul-
ture. Different types of bacteria such as Bacillus,Pseudomonas,Azotobacter, etc. are
beneficial for plant growth (Table 4.1). The bacterial count remains higher in the
organic farming site in comparison to non-organic farming site. Regular use of
chemicals in fields decreases the C—compound in the soil which is necessary for
microbial growth. High CFU counts in organic farming soil may be due to nutrient
richness and absence of high concentration of heavy metal ions that are inhibitory for
microbial growth (Kang et al. 2016). Organic manure increases the carbon source in
the soil which is beneficial for the microbes as it increases the growth and activity.
By increasing microbial count, bioremediation of the soil can be done as this is the
way to treat heavy metal-polluted soil. Several comparisons of organic and conven-
tional farming systems have indicated significant impact of soil microbial commu-
nity on agricultural practices (Smith et al. 2012; Liao et al. 2018; Hartmann 2015;Li
et al. 2012).
Table 4.1 Microbial diversity in soil
Microorganism Plant Plant growth regulation References
Bacillus amyloliquefaciens
5113 and Azospirillum
brasilense NO 40
Wheat Enhance plant growth under
drought condition and increase
enzyme activity in wheat
Edwards
and Lofty
(1974)
Pseudomonas aeruginosa
FP6
Chili Siderophore produced by
bio-control strain to reduce metal
pollution
Amir and
Fouzia
(2011)
Bacillus and Pseudomonas
spp.
C. annum L Plant growth enhancement and
bio-control management to control
plant disease
Kasim
et al.
(2013)
Mesorhizobium spp. Chickpea Increase nodulation, enhance, and
uptake of nutrient yield
Sasirekha
and
Srividya
(2016)
Bacillus thuringiensis Wheat Decrease volatile emissions and
increase photosynthesis
Kumar
et al.
(2014)
Trichoderma harzianum
Tr6 and Pseudomonas
sp. Ps 14
Cucumber
and
Arabidopsis
thaliana
Induced systemic resistance Verma
et al.
(2013)
54 A. Rani and B. Saxena
Naturally available technologies for enhancement of agriculture and management
of agricultural waste are being aimed by scientist. Indigenous microorganism (IMO)-
based technology is being applied in the eastern part of the world for the extraction of
minerals, enhancement of agriculture, and waste management (Rajeshwari 2017).
Bacteria are helpful in nitrogen fixation and many other biological processes.
Rhizobia are found in symbiotic association in root nodules of legumes.
Cyanobacteria are helpful in binding the soil molecules as they act as cementing
material. Pseudomonas sp. are used for remediation. Secondary metabolites have
very effective and vital role in plant growth. Microbes are also helpful in production
of such metabolite which stimulates the growth and development in plants. Micro-
organism may also be protective towards the plants, and rhizosphere soil microbes
form a physical barrier around the roots of plants and reduce the invasion of
pathogens and pests by providing healthy micro-ecological environment
(Table 4.1) (Wu and Lin 2003).
Vermiculture is also a very important tool for organic farming. It is low input
farming in comparison to conventional farming. Many researchers reported that
vermiculture in organic farming sites is more benefited than in conventional farming
site (Timmusk et al. 2014). It is reported that biodegradation process is enhanced
when earthworms and microbes work together and produce vermicompost, which is
worm fecal matter with worm casts. Vermicompost provided macro-elements such
as N, P, K, Ca, and Mg and microelements such as Fe, Mo, Zn, and Cu (Lim and
Kim 2013).
The indigenous microbial strains are able to remediate the xenobiotic compounds
from the soil and water system (Bhandari et al. 2021). The bacterial and fungal
strains are able to degrade the pesticides, antibiotics, endocrine disrupting chemicals,
and other organic compounds from the environment (Bhatt et al. 2021a). These
microbial strains accelerate the residual level of toxic chemicals from the environ-
ment and enhance the sustainable developments (Bhatt et al. 2021b). These potential
microbial strains are used throughout the globe for the remediation of the toxic
xenobiotics from the contaminated sites (Bhatt et al. 2021c).
4.5 Conclusion
It is the need of the hour to fulfil the food requirements of the huge Indian
population. Due to the increasing population and industrialization, the discharge of
polluted waste water and agricultural waste is also increasing. As a result, the heavy
metals are adversely affecting soil health due to their toxic and non-biodegradable
nature. An ideal agriculture system should be developed to improve soil health and
for sustainable environment. To increase the yield of any crop, chemicals and
pesticides are frequently used by Indian farmers; as a result, soil health is continu-
ously deteriorating. There are many techniques to improve the soil health. Microor-
ganisms play very important role for improving soil health contaminated by heavy
metals and for the sustainable environment. It has been proved that they are
4 Microbial Mediated Natural Farming for Sustainable Environment 55
beneficial for society and environment. By using them, we can get social, economic,
and environmental benefits. It is well understood that by increasing microbial
communities in the soil and by detection of heavy metals present in the soil, total
yield can be enhanced. Organic green manure is well suited for the proper growth of
PGPRs and other microbes. The frequent use of the chemicals and pesticides in
inorganic fields is harmful. Although it is bitter truth that natural and organic farming
is costly as compared to conventional farming and the farmers adopting organic
farming face difficulty to survive and market the organic products, but to improve
soil health and for development of sustainable environment, farmers should be
motivated for organic farming as it can provide quality food without any harmful
effect on soil health. Organic farming can be done with proper planning for the
betterment of mankind and upcoming generations, and economically sustainable
organic farming is the prerequisite for ensuring affordability of organic products at
consumer’s end.
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60 A. Rani and B. Saxena
Chapter 5
Rhizosphere Manipulations for Sustainable
Plant Growth Promotion
Pooja Pant, Ankit Negi, Anchal Giri, Pankaj Bhatt, and Rishendra Kumar
Abstract A plant keeps a mesmerizing diversity of microbes inside and outside of its
root surface, known as rhizosphere microbiome. This plant-associated microbial
community is among the most complex communities found on earth. It is also termed
as the second genome of plant. Plants modulate their microbiome by various mech-
anisms like modifying their structures and releasing the secondary metabolites in order
to maximize their fitness. Manipulating the rhizosphere microbiome is an artificial
method but eco-friendly. In this review, we have focused on the great role of beneficial
microorganisms and their interactions with plants. The role of rhizobacteria on
promoting plant growth and the effect of their inoculation over resident bacteria
have been discussed with few examples. We have highlighted the contribution of
biotic and abiotic factors over rhizosphere microbiome shape alteration. Finally, the
process and significance of synthetic or artificial microbial community construction for
sustainable development of plant and some of the techniques developed for harnessing
the plant and the beneficial microbes with some example have been discussed.
Keywords Microbiome engineering · Plant growth promotion · PGPRs ·
Sustainable plant growth promotion · Rhizosphere microbiome
5.1 Introduction
The rhizosphere concept is not a newly generated one, but it has been defined over a
century ago. The German agronomist and plant physiologist Lorenz Hiltner in 1904
was the first one who coined the term “rhizosphere”and centered the basic idea that
P. Pant · A. Negi · A. Giri · R. Kumar (*)
Department of Biotechnology, Sir J. C. Bose Technical Campus, Bhimtal, Kumaun University,
Nainital, Uttarakhand, India
P. Bhatt
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,
Guangdong Laboratory for Lingnan Modern Agriculture Integrative Microbiology Research
Centre, South China Agricultural University, Guangzhou, China
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_5
61
somehow the plant health is related to the microbial composition on the root soil
(Hartmann et al. 2008). The narrow zone of soil around the plant root is known as
rhizosphere. It is consisting of a large number of microorganisms which include
bacteria, fungi, nematodes, etc. Some of the microorganisms are the good ones
which give a huge contribution toward the plant growth and development, while
some of the them are the bad ones that are harmful not only for plants but also for the
mankind. Rhizosphere is basically consisting of three components that interact with
one another: rhizoplane, rhizosphere, and root. The rhizosphere is comprised of large
number of microorganisms that are affected by the release of substrate from root, the
rhizoplane is a closely adhering soil on the surface of the root, while the root is the
main component responsible for assembling of microorganisms. Root also contains
many endophytes inside the root tissues (Barea et al. 2005).
5.2 Plant Growth-Promoting Rhizobacteria (PGPR)
Sustainable agriculture for plant growth promotion is important and a much better
practice as compared to the conventional methods for future agricultural demands.
The rhizospheric soil contains a huge number of useful bacteria which are referred as
“plant growth-promoting rhizobacteria (PGPR)”that gives plant a healthy and
promoting environment. The basic characteristics required for being a good PGPR
are it must compete, survive, and multiply with other microbes for plant growth
promotion and must be able to form colony on root surface. These are present
naturally in the soil and get attracted toward the plant roots and fulfill their require-
ments from the root excretion and secretions. The rhizobacteria lodged around the
plant roots are more capable of solubilizing and mobilizing the nutrients as com-
pared to the rest of the bacterial populations. Thus, they are the crucial ones needed
to be focused for fertile soil (Glick 2012). Various mechanisms have been explained
by scientists for plant development which involves rhizoremediation,
biofertilization, disease suppression, etc. (Lugtenberg et al. 2001; Raaijmakers
et al. 2009). The mechanisms of Pseudomonas and Bacillus, belonging to
Proteobacteria and Firmicutes, and the fungi Trichoderma and Gliocladium, belong-
ing to Sebacinales order, have already been documented (Qiang et al. 2012). PGPRs
not only enhance immunity and growth of a plant but also provide “induced systemic
tolerance”to certain abiotic stresses like salt and drought. They help in uptaking the
nutrients through roots to the plant and help in fulfilling the deficiency of phospho-
rous, nitrogen, etc. It has already been reported that plants release certain compounds
that selectively recruit microorganisms in rhizosphere soil, which are beneficial for
plant health (Reinhold-Hurek et al. 2015). The beneficial PGPRs are deliberately
inoculated in the rhizosphere for checking their effects on the host plant and on the
already present indigenous microbial population. Certain rhizobacteria show a
positive result and suppress the disease-causing pathogens. The effective functioning
of these bio-inoculates can be obtained by exploring the large population pool of
indigenous soil microbes (Hill et al. 2000). These rhizobacteria can be co-inoculated
62 P. Pant et al.
with other fungi or bacteria for their synergistic response. Generally, a bio-control is
consisting of a mixture of antagonists rather than a single one for effective outcome.
These are used as inoculants in biofertilizers, phytostimulators, and bioremediators.
Their mechanism of suppression involves either releasing harmful chemicals against
the pathogen to restrict their growth or by in taking all the nutrition available in their
surrounding and letting the pathogens die with hunger. Some of the very common
and abundant PGPRs in soil are Pseudomonas,Flavobacterium,Rhizobium,Pseu-
domonas,Azotobacter,Bacillus, etc. They are widely used as biocontrol agents for
suppressing pathogenic diseases. The diseases like common leaf spot, Fusarium root
rot, anthracnose, and rust caused by Pseudomonas syringae pv. syringae,Fusarium
oxysporum,Colletotrichum sp., and Uromyces striatus, respectively, (Nyvall 2013)
are certain examples of diseases which affect many crop plants such as legumes like
horse gram (Macrotyloma uniflorum), tomato (Solanum lycopersicum), potato (Sola-
num tuberosum), and fruits like banana, etc. In evidence, activity of GBO3 (Bacillus
subtilis) and PGPR strains INR 7 (Bacillus pumilus) were observed against
P. syringae,C. orbiculare, and E. tracheiphila (causing cucurbit wilt disease)
(Raupach and Kloepper 1998). Bacillus velezensis isolates (Y6 and F7) have poten-
tial antagonist activity against Fusarium oxysporum and R. solanacearum (Cao et al.
2018).B. amyloliquefaciens DGA14 showed antagonism against Colletotrichum
gloeosporioides (causing anthracnose) in mango (Alvindia and Acda 2015), etc.
This review is an effort to focus on the urgent need of bio-control practices and
explains the rhizosphere manipulation benefits on plant growth with recent updates
(Fig. 5.1).
5.3 The Rhizosphere Microbiome
The rhizosphere microbiome consists of large number of actively metabolizing soil
microbial communities; they enhance plant health by either providing nutrition,
playing as an antagonist against harmful pathogens, producing siderophores,
HCN, or many other direct and indirect ways beyond our imagination. This is the
reason why scientists are attracting toward this hotspot concept of plant and micro-
bial interactions. The rhizosphere microbiome of each plant varies from species to
species according to their requirement. The rooted plant soil contains a large number
of microorganisms as compared to the non-rooted ones (Foster et al. 1983). This can
be explained with the help of the concept of the “rhizospheric effect.”According to
this concept, the growth and the increase in the number of microorganisms within the
rhizosphere depends on the excretions and the organic carbon released from the roots
of a plant (Brahmaprakash et al. 2017). The microorganisms not only intake the
nutrients from the plant roots but also help them in stimulating plant growth and
health. For example, rhizobacteria help plant by fixing atmospheric nitrogen and
improve soil nitrogen deficiency. Symbionts like mycorrhizal fungi translocate
minerals and nutrients from soil to the plant (Johnson and Graham 2013) and
suppress the soil-borne plant pathogens (Whipps 2001), and these are well
5 Rhizosphere Manipulations for Sustainable Plant Growth Promotion 63
documented and explained (Johnson and Nielsen 2012). Bacteria like Burkholderia,
Rhizobium,Bradyrhizobium, and Achromobacter species can form a nodule on
cowpea and perform effective nitrogen (Guimarães et al. 2012). Some bacteria
regulate the concentrations of intracellular iron, in plants by releasing siderophores
(Andrews et al. 2003; Hider and Kong 2010). R. arrhizus produces a siderophore
called rhizoferrin that is a carrier of iron much efficient than synthetic chelates
(Yehuda et al. 2000). Some rhizobacteria produce hormones for plant growth.
These rhizobacteria are often termed as PGPR.
Fig. 5.1 Benefits of plant-microbe interactions to plant
64 P. Pant et al.
5.4 The Rhizosphere Microbiome-Mysterious Members?
Does complete rhizosphere microbiota have been identified? Do plants require only
the known bacteria? Does removing the uncultured microbiota would affect plant
growth adversely? These are certain questions which are still needed to be answered.
However, some of the evidences from culture-independent methods have shown that
the diverse microbial population of plant root soils has been highly underestimated
for a long period. Till now only up to 5% of bacteria phyla have been cultured; still a
large proportion is unculturable, and different approach is required for them to
culture. In a soil metagenomic study, the rarefaction curve, prepared from
16SrRNA gene sequencing data, got failed to reach saturation. From that, the soil
clone library was made by taking 150,000 sequencing reads, less than 1% among
them exhibited overlapping with the sequencing reads of other independent clone
libraries (Tringe et al. 2005). In a study, 1 g of boreal forest soil contains c. 10,000
number of bacteria sp. (Torsvik et al. 2002). Further, the researchers showed that
1 gram of soil contains >one million different bacteria, much higher compared to the
previous researches (Gans et al. 2005). In a study from four distinct soils, around
9340 crenarchaeotal and 139,819 bacterial rRNA gene sequences were obtained.
Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes were abundantly pre-
sent bacteria group among them (Roesch et al. 2007). Till now, in the field of
rhizosphere studies, scientists work more on the diversity and number of bacteria
populations and less on other rhizosphere inhabitants (Mendes et al. 2013).
5.5 Rhizosphere Microbiome Support Over Abiotic
and Biotic Stresses
Plants are affected largely by many abiotic and biotic stresses in their complete life
cycle. This directly or indirectly triggers the recruitment of specific microorganisms
to the rhizosphere microbiome. For example, plants intake phosphorous only in the
form of inorganic orthophosphate, present in the soil which activates phosphate
starvation responses (PSRs) thus causing the synthesis of organic acids,
glucosinolates (Castrillo et al. 2017), etc. which attracts the specific microbial
population to fulfill the phosphorous deficiency. The bacteria belongs to Bacillus
decolorationis,Halobacillus [uncultured], and Cesiribacter sp. JJ02 isolated from
the atrazine contaminated soil (Xu et al. 2018). Similarly, glyphosate sprayed on
maize leaves causes enrichment of Fusarium around the maize roots (Kremer and
Means 2009). Diclofop-methyl treatment causes excess release of amino acids, fatty
acids, and organic acids in rice seedlings ultimately enriching the population of
Massilia and Anderseniella genera (Qian et al. 2018). There are many more envi-
ronmental contaminants that alter the rhizosphere community either directly with
their presence or causing root exudates to release certain molecules. Moreover, plant
pathogen also sometimes causes the rhizosphere alteration, for example,
5 Rhizosphere Manipulations for Sustainable Plant Growth Promotion 65
Pseudomonas syringae pv tomato enriches the presence of Roseiflexus genus in soil
(Yuan et al. 2018). Similarly, Fusarium culmorum infection causes the enrichment
of Carex arenaria in plant roots (Schulz-Bohm et al. 2018). The rhizosphere
microbiome gives support to the plant against various types of stresses, and it is
also the primary defense system to the plants against soil-borne pathogens. The
important mechanisms against the biotic stresses involves competition for food,
nutrients (Maloy and Murray 2001), antibiosis (Raaijmakers and Mazzola 2012),
ISR (Pieterse 2012), and parasitism (Mela et al. 2011). A study revealed that
Paenibacillus lentimorbus and Paenibacillus polymyxa have antifungal activities
against Meloidogyne incognita and Fusarium oxysporum pathogens of tomato plants
(Son et al. 2009). Rhizosphere microbiome members not only suppress the patho-
gens and enhance the plant growth but also can modulate the plant immune system
(De Vleesschauwer and Höfte 2009; Berendsen et al. 2012). Achromobacter
piechaudii ARV8, obtained from saline soil environment, enhanced the pepper
biomass and tomato seedlings under drought stress (Mayak et al. 2004a,b).
Rhizobacteria supports plant growth in flooding stress (Grichko and Glick 2001)
(Upadhyay et al. 2009). revealed that about 24 rhizobacterial isolates out of 130 iso-
lates were 8% tolerant to high salt concentration levels. Some of them produced
siderophores, some produced gibberellins, and some were able to solubilize phos-
phorous. The dominant genus found was Bacillus. There are many more evidences
to prove that rhizosphere microbiome members are primarily responsible for pro-
viding support to the plant from every day stresses.
5.6 Manipulating the Rhizosphere for Sustainable Plant
Growth Promotion
The rhizosphere is among the most complex habitat on the earth. It is a large
reservoir of plant roots, soil, and diverse microorganisms including bacteria, fungi,
viruses, etc. Many studies have been done in this field are still much more needed to
be explored. Plants have co-evolved with microbes. Plant domestication and crop
breeding fertilization practices have created certain disturbances among the rhizo-
sphere microbiome which has caused loss of important interactions. Thus, by
re-engineering the beneficial rhizosphere microbiome to the agricultural cropping
systems will work in getting back the lost microbe-plant interactions. Predicting and
then controlling the function and structure of rhizosphere will help in understanding
the plant microbial interactions and also improve the plant response to the climate
change effects and various other environmental stresses. Enhancing the plant health
and growth is not only the problem to be tackled, but another big problem that affects
the plant development is the diseases that are caused by harmful pathogens in crop
plants. Using the chemical fertilizers makes them worse. Thus, for the sustainable
plant growth, alternative method called biological control is required. Microbiome
manipulations are a new approach for this problem. Probably, the first changes that
66 P. Pant et al.
occurred in rhizosphere microbiome assemblage were involuntary while performing
plant domestication and breeding practices. Genetic alterations in plant caused the
reduction of many beneficial microbial interactions (Weese et al. 2015). By manip-
ulating the genetic structure of plants in the process of creating improved varieties,
many plants do not support the beneficial microbe as much as their ancestors
(Philippot et al. 2013). In case of wheat, landraces assemble more complex
microbiome as compared to the modern cultivars (Rossmann et al. 2020). However,
this is not only the case in some cases, but the domestication causes less or negligible
impacts on the microbial communities. For example, in Helianthus annuus (common
sunflower), the effects were shown in fungus only (Leff et al. 2017) (Schuhegger
et al. 2006). explained that Serratia liquefaciens and P. putida produce the [N-acyl
homoserine] AHL signal molecules in plant against the pathogen Alternaria
alternata, by inducing the systemic resistance. Similar approach was done for
A. thaliana (Liu et al. 2012). In the rhizosphere of Lolium perenne, the effects of
Pseudomonas on the phosphate availability were investigated (Zyśko et al. 2012).
This shows that the root exudates affect the bacterial gene expression. The gene
expression of hydrogen cyanide (HCN) biosynthesis in Pseudomonas sp. LBUM300
got increased by the infection caused by Verticillium dahlia on strawberry plant
rhizosphere (DeCoste et al. 2010). Thus, biocontrol was stimulated. There are many
more evidences to prove that manipulating the rhizosphere microbiome structure can
solve problems related to the plant growth. Till now a large proportion of soil
microbiome is unculturable; yet metagenomics is a field for providing a complete
picture to the rhizosphere microbiome. Exposing these microorganisms and their
interactions with plant root will give a better insight toward the crop yield and health
in adverse environmental conditions which are the major problems of today’s world.
5.7 Induction of Soil Suppressive Ability Against Plant
Pathogens Through Alterations
The organic fertilizers are widely used to suppress the plant pathogens and enhance
plant health in present days (Bonanomi et al. 2007; Cao et al. 2011). The soil
suppressiveness against soil-borne pathogens can be induced by introducing the
beneficial microbes in rhizosphere soil that release certain compounds that inhibit
the growth of pathogen. Some of the pathogenic fungi that affect plant growth are
Verticillium dahlia,Sclerotinia spp., R. solani, and Fusarium spp., and a pathogenic
bacterium is R. solanacearum (Bonanomi et al. 2007). Another interesting technique
that is being used nowadays is solarization technique. The organic matter along with
solarization technique helps in changing the biological structure of plant root soil.
This technique induces the suppressiveness of soil and reduces the growth of some
of the pathogens (Klein et al. 2011). The solar energy is used to raise the temperature
of soil at that extent at which the soil pathogens are unable to survive or become
weak to show any harmful effects. This technique is beneficial because it shows
5 Rhizosphere Manipulations for Sustainable Plant Growth Promotion 67
effective results without removing the soil microorganisms, by modifying the soil
environment in the support of useful microbial community (Raaijmakers et al. 2009).
Next strategy involves the use of synthetic biology by applying probiotics in plants.
The microbiome is manipulated in this strategy. Vegetal probiotics costs low and
increases growth and production more (Kim and Anderson 2018; Picard et al. 2008).
The synergistic characteristics of different microorganisms in the consortium have
more potential as compared to the isolated microbe, thus increasing biocontrol
efficiency in consortium. Different microbes in the consortium use different niches
and thus prevent competition among them. For example, Bradyrhizobium japonicum
when inoculated with Azospirillum brasilense enhance the stabilization of arsenic
(Armendariz et al. 2019). Similarly, R. tropici along with C. balustinum enhance
common bean growth in normal and saline conditions (Estévez et al. 2009). Many
more evidences have been given in various studies that prove that the microbiome
plays an important role in raising the soil disease suppressiveness (Srivastava et al.
2010). Many companies are using the products made based on mixture of
Trichoderma and Bacillus species (Agrofit2020). Another important strategy is
based on controlling the microbiome functions in which the regulation of antibiotics,
gene, and their expressions are manipulated through signaling introduction of
required molecules (Bakker et al. 2012). The organisms’abilities are studied for
developing selective enrichment techniques to be used for long-term manipulations
(Bakker et al. 2012).
5.8 Effects of PGPR Inoculation on Resident Rhizosphere
Microbes
The rhizosphere is a complex habitat consists of plant roots, biotic components like
resident microorganisms, and abiotic soil components. These all function together
for plant growth. Any disturbance can change the whole scenario. The PGPRs are
exogenous bacteria that are inoculated deliberately to the new environment for better
performance of plants. It is necessary to check their interactions with the plant and
their effects on already present resident bacteria. They must be inoculated properly.
Large amount of PGPR inoculation can affect the functioning of resident bacteria;
some groups may be enhanced, some may be inhibited, while some may not be
affected at all (Dobbelaere et al. 2003; Nacamulli et al. 1997). PGPR Azospirillum
brasilense inoculation in maize caused root volume increase as compared to the
uninoculated maize plant (Dobbelaere et al. 2003). PGPR Pseudomonas fluorescens
F113Rif (producing 2,4-diacetylphoroglucinol) inoculation causes the reduction in
rhizobium microbial diversity (Walsh et al. 2003) (Robleto et al. 1998). reported that
Rhizobium etli (producing trifolitoxin) inoculation reduced the population of
trifolitoxin-sensitive bacteria of the Alphaproteobacteria. The PGPR indirectly
affects the resident microorganisms by releasing some chemicals or antimicrobial
compounds. In evidence, Bacillus subtilis strain 330–2 produces some lytic enzymes
68 P. Pant et al.
like laminarase, cellulase, etc. that inhibit Rhizoctonia solani Kuhn growth by
degrading the pathogenic fungi cell wall (Ahmad et al. 2017). B. subtilis strains
PCL1612 secrete iturin A to resist Rosellinia necatrix and Fusarium oxysporum
(Cazorla et al. 2007).B. amyloliquefaciens and B. mojavensis release antifungal
compounds that inhibit the growth of F. oxysporum and F. verticillioides, respec-
tively (Wu et al. 2019).
5.9 The Process of Using Synthetic Communities for Plant
Promotion
The microbial consortia successfully inoculated to enhance plants health were firstly
tested in labs according to their potentials and then were transferred to the field
(Sessitsch et al. 2019). This is based on two strategies: either the closely related
strains are tailored for increasing diverse resources that the strains would use (Wei
et al. 2015) or the distant-related microbes combined to suppress pathogens, tolerate
diverse plant genotypes, and promote plant growth (Compant et al. 2019). In
evidence, when the Pseudomonas consortia was introduced in tomato plant soil
rhizosphere, it reduced the growth of R. solanacearum suppressing the disease in
tomato plants (Hu et al. 2018). Synthetic community made up of Bacillus isolates
enhances tomato growth (Tsolakidou et al. 2019). The complex inoculation of
different bacteria has shown effectively increased plant health and growth as
compared to the single strains (Tsolakidou et al. 2019;Niu et al. 2017).
Constructing a large synthetic community and managing each member of the
mixture is quite a difficult task to perform. A machine learning computational
approach has been developed recently, based on “cry for help”theory to design
the bacterial synthetic community. This method is useful for creating a community
with predictable plant phenotypes. Another method developed by (Berendsen et al.
2018) was more plant-dependent approach based on the same theory, in which plant
attracts bacteria consortium to produce desirable plant phenotypes. This experiment
showed that the microbes that show response on plant stress signals can be used as
reliable predictors for exploring useful microbes (Pascale et al. 2020). Basically, the
assembling of rhizospheric microbiota is a result of series of events that can produce
stable and diversified microbial consortia (Philippot et al. 2013). The factors like
environmental (abiotic) and interactions among microbes and plants (biotic) act as
modulators in the process of assemblage. Competitive and cooperative interactions
promote diversity and microbial co-occurrence and determine the invasive pathogen
fate (Hacquard et al. 2017).
5 Rhizosphere Manipulations for Sustainable Plant Growth Promotion 69
5.10 Framing the Artificial Rhizosphere Microbiome
The rhizosphere is a large pool of microorganisms which include a diverse number
of bacterial and fungal populations. They may be beneficial or harmful for plants or
may change their activities with the environment. Thus, it is required to develop the
new strategies for framing the rhizosphere microbiome of a plant for growth
promotion. Synthetic or artificial microbial community is a new approach in this
perspective. It is a method in which the pure strains are transferred into an axenic
system mimicking the natural system to assemble microbial communities (Vorholt
et al. 2017). Researchers can study the rhizosphere environment in controlled
conditions according to their needs, and they can add, remove, or replace organisms
at the strain level according to the needs. A study showed that the phosphate
starvation mutants and the wild-type Arabidopsis thaliana had a different root
microbiome in natural soils as well as in synthetic community experiments (Castrillo
et al. 2017). Plant-microbe interaction can be understood more easily in synthetic
community experiments than the natural process. In evidence, under the same
nitrogen condition, indica-enriched synthetic microbial community promotes the
rice growth much more than the japonica-enriched ones (Zhang et al. 2019). Thus,
there is a lot more work to explore in the field of artificial rhizosphere microbiome
field. Firstly, for constructing a rhizosphere microbiome, it is highly necessary to
have an expert knowledge about the members in the microbiome. The advanced
molecular and “omics”tools can help in better understanding the plant-microbe
interactions. Those microbes which have a high capability of promoting plant
growth in every aspect must be isolated, characterized, and assembled for further
use. On the other hand, the microbes that are harmful for plant as well as human
welfare must be avoided for the reconstructed microbiome. There are many bacterial
and fungal populations that have been included as the beneficial ones like Bacillus,
Pseudomonas,Azotobacter,Flavobacterium, etc. and fungus like Trichoderma.
Many studies have suggested that the components that decide the rhizosphere
microbiome shape are the soil type and plant genotype (Bakker et al. 2012). The
plant roots along with their general roles of support and nutrient uptake also perform
some specialized roles like synthesis, accumulation, and secretion of diverse array of
compounds (Flores et al. 1999). Microbes get attracted toward the chemicals or
excretions that the plant roots secrete. In evidence, the beneficial microbe Bacillus
subtilis got attracted toward the malic-acid secretion from a plant (Rudrappa et al.
2008). Pseudomonas putida is attracted to DIMBOA, the allelochemical released
from young maize seedling roots (Neal et al. 2012). It has been reported that
Arabidopsis thaliana produces a swathe of chemicals belonging to a family
triterpene, and about half of the microbes attracted towards its root are due to the
network of these chemicals (Huang et al. 2019). Certain cases have been reported
where some of the pathogens also get attracted toward the chemical secretions along
with the beneficial bacteria, e.g., isoflavones released from soybean roots attract
symbionts B. japonicum along with the pathogen Phytophthora sojae (Morris et al.
1998; Zhalnina et al. 2018). showed in a recent study that the root exudates released
70 P. Pant et al.
during the growth cycle of Avena barbata showed variations like high sucrose
content at early age and more defense molecules and amino acids at later develop-
mental stages. Later on, using exometabolomics, it was studied that the aromatic
organic acids like salicylic, cinnamic, IAA, shikimic, and nicotinic cause the inhi-
bition and proliferation of desired microbes during plant life cycle. The secreted
molecules, Arabinogalactan proteins, regulate Agrobacterium and Rhizobium pres-
ence on roots (Xie et al. 2012). Thus, one can construct a rhizosphere microbiome
according to one’s choice keeping the genetic appearance and the nature of a plant in
mind. The plant’s genetic makeup can be altered with the help of genetic engineering
which is another interesting topic to discuss.
5.11 Techniques Involved in Harnessing Plants
and Engineer Beneficial Microbiomes
The concept of engineering microbiomes is a promising approach toward the plant
health and fitness for sustainable future; however, it is not easy to apply. For
overcoming the limitations, certain mechanisms and considerations have been
given by various scientists (Bhatt et al. 2021g). One of the recent reviews has been
given by (Lawson et al. 2019). Metatranscriptomics, metagenomics, metabolomics,
and plant transcriptomics are the approaches that extricate the complexity in the
connections happening among the holobiont members (Stringlis et al. 2018) com-
bined the metabolomics and shotgun metagenomics on an order of Arabidopsis
mutants and said that the rhizosphere microbiome can be reshaped by the coumarin
root exudation. Similarly, the combined analysis of amplicon-based metagenomics
and metabolomics was performed on wild and benzoxazinoid precursor mutant of
maize genotype by (Hu et al. 2018). He explained that benzoxazinoid metabolites
can structure fungal and bacterial community of maize rhizosphere microbiome.
Another approach in this field is system biology approach which is based on the
discovery of microbial associations with the help of correlation networks. For
example, the antagonist and synergist interactions can be analyzed from negative
and positive correlations in system biology approach (Poudel et al. 2016). With the
help of this, the most interactive nodes in the network called microbial hub taxa can
be identified (Pascale et al. 2020) (Agler et al. 2016). identified plant pathogens
Dioszegia and Albugo in Arabidopsis phyllosphere microbiome as microbiome hub.
Metagenome-wide association study [MWAS] is another very important and pow-
erful approach nowadays (Sulbhi et al. 2021; Bhandari et al. 2021; Bhatt et al. 2020a,
2021a,b,c,d,e,f). It is helpful in identifying the associations between the several
plant pathogens and the rhizosphere microbiome (Bhatt et al. 2015a;b,2016a,b,
2019a,b,c,2020b,c,d,e,f, Ahemad and Khan 2012; Huang et al. 2021).
5 Rhizosphere Manipulations for Sustainable Plant Growth Promotion 71
5.12 Concluding Remarks
With the increase in concern about the chemicals used as fertilizers and their harmful
effects over mankind and environment, it is no doubt to promote the ecofriendly
practices. The relationships between the plant and the microbiome are co-evolved.
There are many possibilities and missing links that must be expected. Rhizosphere
manipulation is one of the best ecofriendly approaches. The PGPR used as inocu-
lants are one of the members of soil microbiome from another plant and thus are easy
and harmless to experiment which have already been proved by effective results
obtained from various researches discussed above. The understanding of plant and
microbes is still needed to be explored for more innovative findings regarding plant
health and fitness. Most of the microbes in the soil are still not explored maybe they
are holding some secrets regarding the mechanisms involved in plant-microbe
associations. The knowledge of various fields like metabolomics, plant genetics,
plant transcriptomics, metagenomics, and metatranscriptomics must be gained more
for further development of techniques regarding plant and microbes harnessing. The
synthetic microbial community construction is a novel approach, and the results are
showing that it has a bright future in the field of rhizosphere microbiome.
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5 Rhizosphere Manipulations for Sustainable Plant Growth Promotion 77
Chapter 6
Rhizospheric Microbes and Their
Mechanism
Anuj Chaudhary, Heena Parveen, Parul Chaudhary, Hina Khatoon, and
Pankaj Bhatt
Abstract With constantly rising global population, requirement for higher agro-
nomic productivity is increasing. There is a need to adopt safe and effective
strategies contributing toward precision farming and sustainable crop production.
Rhizosphere is the region of soil where root of plants has large population of
microbes and makes its environment highly complex. The pattern of root exudates
affects microbial activity in the soil. Beneficial and harmful interactions occur in
rhizosphere microbes and plants which ultimately affect root functions and plant
development. A better understanding of this process is needed to manage the
microbes and plant productivity, and maintaining the soil health is important. Plant
growth-promoting activities of PGPRs help to overcome challenges such as low crop
productivity, overuse of agrochemicals, and nutrient loss. This chapter discusses the
rhizospheric microbes and how they facilitate the nutrient solubilization and uptake
by plants and helps in agriculture productivity.
Keywords Rhizosphere · Microbes · Agriculture · Sustainable · Environment
A. Chaudhary
School of Agriculture and Environmental Sciences, Shobhit University, Gangoh, Uttar Pradesh,
India
H. Parveen
Department of Dairy Microbiology, NDRI, Karnal, Haryana, India
P. Chaudhary (*)
Department of Animal Biotechnology, NDRI, Karnal, Haryana, India
H. Khatoon
Uttarakhand Pollution Control Board, Roorkee, Uttarakhand, India
P. Bhatt
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,
Guangdong Laboratory for Lingnan Modern Agriculture Integrative Microbiology Research
Centre, South China Agricultural University, Guangzhou, China
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_6
79
6.1 Introduction
The soil has the possible capacity of plant cultivation, in order to produce food crops
for animal and human consumption (Sulbhi et al. 2021; Bhandari et al. 2021; Bhatt
et al. 2021a,b,c). The health of human being and animals depends solely on
consistency of the soil, since it serves as the primary means of fibrous crops and
food production (Bhatt et al. 2020a,2021d,e,f,2020b,c,d,e,f,2019a). The
qualitative content of directly ingested air and water depends on the conditions of the
soil, as it is an important relation to the overall climate (Bhatt et al. 2019b,c,
2016a,b,2015a,b; Huang et al. 2021). The quality of the land is therefore directly
proportional to the wellbeing of the living beings and the environment (Odelade and
Babalola 2019). Soil is an area where various factors interact with each other either
in a favorable or in a harmful way. However, the most important and highly focused
area is called rhizospheric zone, which is a hot spot, the richest zone where plant
roots interact with various rhizospheric microbes. This zone becomes a multiplex
ecosystem on this planet where multitrophic interactions have been occurred
(Mendes et al. 2013). Broadly three different interconnected participants
documented in the rhizospheric soil, viz., rhizoplane, rhizosphere, and root itself.
Rhizosphere is the soil region affected by the roots, frequently by releasing the
substrates that influence soil microbial activity. Including the firmly adhering soil
particles, the rhizoplane is the root surface. Root is the part of system itself as
different microbes; endophytes are capable to inhabit root tissues (Kennedy 1998;
Bowen and Rovira 1999).
Rhizosphere is affected by the activities of root like exudation of carbon com-
pound, nutrient uptake, and water absorption (George et al. 2006). Rhizospheric
microbial community responds to these plant root exudates through chemotaxis and
affects the rhizospheric microbial population that eventually changes in response to
plant age and seasonal change (Hartmann et al. 2009). These exudates serve as a
basis of carbon for soil heterotrophic microbiota either as a growth substrate or as
signal molecules that elicit plant-microbe signaling pathways. Microbial groups
participated as the major player in plant functioning via affecting their metabolism
and growth. Among the various associates of rhizospheric microbiota, some are
valuable toward plant, and others are pathogenic ones, which inhabit the
rhizospheric zone and cause disease to plant. The main rhizospheric microbial
community comprises of bacteria, fungi, nematodes, viruses, algae, and protozoa.
Majority of the members of microbiome are fraction of a dynamic food web and
utilized the nutrients secreted by plants (Raaijmakers et al. 2009). These organisms
are classified according to their effect on the plant system: the beneficial microbiota
(nitrogen-fixing bacteria, arbuscular-mycorrhizal fungi, and the most prominent and
active group called as plant growth-promoting rhizobacteria) and the harmful
microbiota (pathogenic fungi, pathogenic bacteria, and nematodes) (Yang et al.
2009). These microorganisms are the key player in the improvement of sustainable
agriculture and help in maintaining the growth of plants as well as the soil structure.
With the emergence of molecular studies on the soil microbiota, it has resolved many
80 A. Chaudhary et al.
queries with regard to their role and behavior in this most complex zone of the plant.
Metagenomics approach, the most acceptable technique which has the ability to
discover the unculturable microbiota with all the functions and genes with the
purpose of improving agricultural productions. Hence, in this chapter, the
rhizospheric microbiota has been discussed with their effect on plant as a crop
producer or as a crop destructor. The interaction, role, and application are also
reviewed for the development of a sustainable environment in terms of agriculture
(Bhatt et al. 2019d,e; Sharma and Bhatt 2016; Sharma et al. 2016; Bhatt and Nailwal
2018; Gangola et al. 2018a; Bhatt 2018; Bhatt and Barh 2018; Bhandari and Bhatt
2020; Bhatt and Bhatt 2020).
6.2 Organisms in the Rhizosphere
Various microorganisms colonize plants, which can reach cell densities much higher
than the amount of plant cells. This colonization is a kind of biochemical commu-
nication among plants and microorganisms, in which molecules are released by
plant/microbe system and responsible for the harmful and beneficial activities in soil
system. This underground communication is increasingly recognized in improving
crop productivity as well as giving an understanding of the effect of microbe on plant
health. For the betterment of farming environment health, stability, and sustainabil-
ity, the interspecies or inter-kingdom communication in the rhizospheric zone that is
created during plant-plant/plant-microbe interactions is important. The most consid-
ered microbial relationships for plant productivity are those produced by PGPR
(plant growth-promoting rhizobacteria), NFB (nitrogen fixing bacteria), AMF
(arbuscular- mycorrhizal fungi), pathogenic fungi/bacteria, nematodes, and invasive
plant species.
6.3 Beneficial Microbial Community
6.3.1 Plant Growth-Promoting Rhizobacteria
A population of free-living bacteria, entering in the rhizospheric zone and supporting
plant root growth. Roots of plants are capable to attract valuable soil bacteria from a
large range of genera, called PGPRs, viz., Bacillus,Acinetobacter,Pseudomonas,
and Rhizobium (Agri et al. 2021; Chaudhary et al. 2021c,d). These bacteria are
important because of their position as producers of growth regulators and phospho-
rus solubilizers that help in abiotic and biotic stress tolerance (Bhattacharyya and Jha
2012; Bakker et al. 2007). Colonization of root entails the capacity of bacteria, in the
presence of the indigenous microflora, to develop on or in the plant root and to
reproduce, thrive, and spread along the growing root. Besides with the activity of
plant growth promoters, the PGPR group also involved in controlling
6 Rhizospheric Microbes and Their Mechanism 81
phytopathogens as BCAs (bio-control agents), enhancing fertilizer’sefficiency and
rhizoremediation process (Podile and Kishore 2007). As an off-shoot of biological
control of soil-borne pathogens, the significance of PGPR was realized in other
aspects of plant promotion which divided PGPRs into two groups, according to the
mode of action, i.e., PGPBs and PGPBs as BCAs. Inseparable from the plant
growth-promoting activity, it has been observed that the identified PGPRs have
defense ability of bacterial-inoculated seedlings against soil-borne pathogens. As a
matter of fact, PGPR were considered as protectants for soil-borne pathogens
(Vessey 2003). With the growing value of organic culturing, the requirement for
PGPR as biofertilizers has been re-discovered in recent years with minimum or no
inputs. Biofertilizers are used to enhance the plant growth by providing essential
nutrients to the plants (Kumari et al. 2021). There are different biofertilizers such as
Rhizobium,Allorhizobium spp., Trichoderma spp., and Pseudomonas fluorescens
which enhance the plant development (Badar and Qureshi 2012; Shen et al. 2013). It
was observed that plants require optimal nutrient input and resilience under stress
conditions in order to have the highest crop productivity (Koller et al. 2013). This
enables plants to use soil nutrients effectively by maximizing root/rhizosphere
efficiency in the mobilization and acquisition of nutrients. Such mobilization is
carried out by rhizosphere bacteria at a high rate. Various previous and recent studies
reported the plant growth promoter activity of PGPRs on different plant system.
Pieterse and Van Loon, 1999 introduced P. fluorescence WCS417, a rhizobacterial
strain under in vitro conditions in autoclaved soil, and observed an increase in
growth promotion of Arabidopsis accession Col-O by 33%. Application of Pseudo-
monas taiwanensis,Pantoea agglomerans, and Bacillus spp. improved the plant
health parameters in a pot experiment which shows the plant growth-promoting
activities like IAA and siderophore production (Chaudhary and Sharma 2019; Khati
et al. 2019a,2017). Bioinoculation of Bacillus spp. also improved the plant health
parameters, enhanced the yield of maize crop, and improved the beneficial popula-
tion of soil (Khati et al. 2018,2019b; Chaudhary et al. 2021a,b; Rudrappa et al.
2008).
PGPRs also favor plant development by releasing some signal molecules under
infection/pathogen attacking condition that act as an alarming call for beneficial
bacteria. In a report, it was observed that Pseudomonas syringae pv tomato causes
infection to Arabidopsis and induced the expression of L-malic acid transporter and
elevates the release of malic acid in roots. Once the concentration of malic acid was
increased in the rhizosphere region at some instance, it recruits the beneficial
B. subtilis, which endorse the biofilm formation on Arabidopsis roots (Lakshmanan
et al. 2013; Asaka and Shoda 1996), developing a general resistance reaction toward
the disease-causing microbes. A number of bacteria release antimicrobial metabo-
lites, viz., surfactin and iturin A, that provide a protective guard to roots against
pathogenic fungi and bacteria such as Rhizoctonia and P. syringae (Bais et al. 2004;
Kumari et al. 2020). Kumari et al. (2020) found that application of Bacillus spp.
increased the growth of Fenugreek plants and improved the soil health too. Kukreti
et al. (2020) also observed the positive impact of bioinoculants on maize crop.
82 A. Chaudhary et al.
6.3.2 Nitrogen-Fixing Bacteria
Plant roots provide a position for the growth of soil bacteria that flourish on root
exudates and lysates. The rhizospheric microbiota consume these exudates as nutri-
ent factor for their development, and in response to the plant root exudation, these
bacteria excrete some metabolites into the rhizosphere, which can act as signaling
molecules that help in plant promotion. One of the best examples of this interaction
is Rhizobium and Legume symbiosis. Plant secretes flavonoid which acts as signal
molecule for Rhizobium, and in response to this, the bacterium starts secreting Nod
factors which are received by plant roots and inducing root nodule formation. In
these nodules, the Rhizobium initiates fixing atmospheric nitrogen and promoting
plant growth under nitrogen-poor environments. Bacteria rise at the expense of the
carbohydrates of the host and in exchange provide fixed nitrogen for the biosynthesis
of amino acid (Brencic and Winans 2005; Gray and Smith 2005; Herridge et al.
2008). This mutual symbiotic association has resulted in fixing 50–70 10
6
tons of
nitrogen annually into the soil which results in a great reduction on the application of
chemical fertilizers in agriculture systems (Graham 2008). The atmospheric
nitrogen-fixing rhizobacteria fixed nitrogen either in a symbiotic or non-symbiotic
association and have been employed in agricultural practices for the development of
growth and yield of many crops. The bacteria which develop root nodule formation
in leguminous plants and convert atmospheric nitrogen into utilizable form of
nitrogen are collectively called rhizobia. Nowadays, the rhizobia genera also include
other genera, such as Sinorhizobium,Rhizobium,Bradyrhizobium,Mesorhizobium,
Azorhizobium, and Allorhizobium (Kurrey et al. 2018). Other category of nitrogen-
fixing bacteria belongs to free-living bacteria (Azotobacter and Azospirillum), and
these free-living diazotrophs are recognized to utilize nitrogen from the atmosphere
for their cellular protein biosynthesis which is mineralized in the soil, maintaining a
large portion of nitrogen available from the soil supply to crop plants. Research into
Azotobacter chroococcum in crop production has shown that it is important to
improve plant nutrients and improve soil fertility (Gonzalez-Lopez et al. 2005).
Numerous Azotobacter strains are found to be capable of producing amino acids
supplemented with different sources of carbon and nitrogen when grown in culture
media (Vikhe 2014). They have the ability to fix nitrogen and release some phyto-
hormones, namely, GA3, IAA, and cytokinins that could improve plant growth and
increase the supply of plant root nutrients (Brakel and Hilger 1965). In vitro studies
showed that indole-3-acetic acid (IAA) is released by Azotobacter when tryptophan
is added to the medium (Hennequin and Blachere 1966). Small amounts of IAA
were present in old cultures of Azotobacter that have no supplement with tryptophan
(Jeffries et al. 2003). Besides, auxin and gibberellins are also found in A. chroococcum.
6.3.3 Arbuscular-Mycorrhizal Fungi (AMF)
AMF also enhance plant growth by expanding their hyphal networks into the soil to
obtain nutrients, such as phosphorous, which are then delivered to their hosts (Salam
6 Rhizospheric Microbes and Their Mechanism 83
et al. 2017). They promote the vigorous growth of host plants under stressful
conditions by mediating a variety of complex contact events between the plant and
the fungus, resulting in increased photosynthetic rate and other characteristics
associated with gas exchange, as well as increased water absorption (Kumar et al.
2021). Several studies identify improved tolerance to a range of stresses due to
fungal symbiosis including drought, salinity, temperature, and metals (Redecker
et al. 2013). Basically, AMF develops arbuscules, vesicles, and hyphal network
with plant roots that significantly augment the accessibility of plant nutrient uptake,
causing improvement of plant growth. Fungal hyphae can accelerate the degradation
process of soil organic matter. Arbuscular-mycorrhizal fungi are classified into four
different orders including Glomerales, Archaeosporales, Paraglomerales, and
Diversisporales (Zeng et al. 2014). Better contents of flavonoids, sugars, organic
acids, and minerals enhanced the quality of citrus fruit due to Glomus versiforme
(Chen et al. 2017). AMF inoculation can significantly increase the absorption of
various macro-/micronutrients, resulting in increased production of photosynthate
and thus increased accumulation of biomass (Mitra et al. 2019; MacLean et al.
2017). The distribution of phosphate is one of the most significant advantages for the
host in AM symbiosis, and collective knowledge indicates that the arbuscules are the
position of phosphate transfer from the fungal partner to its host (Wu et al. 2008).
Thus, the plant growth-promoting rhizospheric microorganisms are the key players
in soil ecosystem that enhance the plant growth by protecting them from various
phytopathogens and also providing essential nutrients to the plant, which augment
the crop productivity by providing a sight of sustainable agriculture system.
6.4 Harmful Microbial Community
In the above section, the beneficial rhizospheric microbiota are reviewed, but as we
know, soil is a dynamic ecosystem which interacts with the useful and deleterious
subjects that directly or indirectly affect the plant growth. Besides good community,
bad community of rhizospheric microbes also exists which is mainly responsible for
the development of diseases and causes major reduction of food and fuel crops. Two
foremost communities come under this category, namely, the pathogenic fungi and
bacteria and nematode.
6.4.1 Pathogenic Fungi
Phytopathogenic fungi have multifaceted life cycles of pathogenesis in rhizospheric
region for plant disease development. The initial infection process usually proceeds
by fungal spores’development into conidia that further grows and starts penetrating
into the host plant. Sections of the plants or the whole plant are destroyed by the
pathogens, and the pathogenesis depends upon the type of plant tissue that is affected
84 A. Chaudhary et al.
by the pathogen and the nature of the infection. Finally, in the contaminated plant
tissue, the fungal pathogens develop spores and begin the disease cycle in their host
system. For the dispersal of fungal spores, environmental factors participated in an
active way which includes wind, rain, or insect vectors and finally settles on their
new hosts. For the processing of pathogenesis cycle, various surrounding factors are
involved for the germination, development, and establishment in rhizospheric part of
the soil. Various reports have concluded that, for triggering dormancy of fungal
spores, different factors such as soil pH and root exudation play pivotal role. In a
report by Gonzalez et al. (2011), gallic, salicylic, and ferulic acid stimulates the
conidial germination of pathogenic fungi. Major groups of rhizospheric pathogenic
fungi include Fusarium,Rhizoctonia,Pythium,Phytophthora, and many others
(Rasmann et al. 2012).
6.4.2 Nematodes
Nematodes are free-living soil creature that feed on the plant roots either as an
ectoparasite or by penetrating inside the root cells and reproduce as a sedentary
endoparasite. The plant exudates have a major role in chemotaxis of nematodes
toward plant roots as these compounds have the ability to attract these organisms.
Most of the recent studies have reports on different plant-derived exudates to attract
nematodes (Johnson and Nielsen 2012). Among the various volatile root exudates,
CO
2
is the predominant long-distance chemotaxis molecule that is released by the
plant roots for positioning of parasitic nematode, with a potential of up to 1 m action
radius for one root and for full root system around 2 m of action radius required
(Turlings et al. 2012). CO
2
acts as “response activator”that alarms other species to
the general existence of entomopathogenic nematodes and can increase their respon-
siveness to more specific indications (Ormeño-Orrillo et al. 2013). In addition to
CO
2
, chemotaxis in nematodes is induced by 2,4-dihydroxy-7-methoxy-1,
4-benzoxazine-3-one and ascorbic acid (Zhu et al. 2011).
6.5 Mechanism of PGPRs
Their mode of action can be categorized into two:
6.5.1 Direct Mechanisms
6.5.1.1 Nutrient Acquisition
This mechanism supports the plant growth by providing the essential nutrients like
nitrogen fixation, zinc, potassium, iron, and phosphorus solubilization.
6 Rhizospheric Microbes and Their Mechanism 85
•Fixation of Nitrogen: Nitrogen plays an essential role for plant growth. Seventy
eight percent of total nitrogen is available in the atmosphere and remains
unavailable to plants. N
2
-fixing bacteria and fungi are involved in fixing the
atmospheric nitrogen with the help of nitrogenase enzyme. Thirty percent to
50% of biological nitrogen is fixed using bacteria, a major contribution to the
nitrogen in agriculture crop (Hussain et al. 2015).
•Phosphorus Solubilization: P is also a necessary constituent for the growth of
plants. It makes 0.2% dry weight of plants, and only 0.1% of P is available to
plants and limits their growth (Sattar et al. 2019). Phosphorus-solubilizing bac-
teria and fungi mineralize the insoluble soil P into soluble form by releasing the
organic acids along with decrease in pH.
•Zinc Solubilization: Zinc is the main micronutrient for plant development and
present in bound form as insoluble complexes and minerals. Zinc solubilizing
bacteria have the ability to solubilize the zinc using acidification process and by
organic acid production and decrease the pH of soil (Hider and Kong 2010).
•Potassium Solubilization: Potassium functions as a cofactor to regulate the
enzymatic reaction and helps in plant development. Lowering pH, acidolysis,
and production of organic acids by soil microbes solubilize the K by conversion
of insoluble form of potassium into soluble form which is easily up taken by
plants (Cassán et al. 2014).
•Iron Sequestration: Iron is abundantly found in soil. Its assimilated form (Fe
3+
)
is less predominant in nature because it chelates with several compounds and its
bioavailability becomes very low. Therefore, rhizospheric microbes that synthe-
size siderophores (low molecular weight compounds) can help in sequester (Fe
3+
)
with great affinity. Siderophores have binding affinity toward microbial mem-
brane receptors which can hold iron-siderophore complex and help in the uptake
of iron, favored in stress conditions (Lugtenberg and Kamilova 2009).
•Production of Phytohormones: They are known as plant growth regulators that
help the plants to combat various abiotic stresses and increase the plant growth
and productivity. These include the IAA, Gibberellins, ABA, and cytokinins
(Waweru et al. 2014). Indole acetic acid is an auxin produced by microbes
which promotes plant development such as cell division and elongation. It has
been found that more than 80% of auxin are produced by Rhizobium sp. Many
bacterial species and fungal species produced growth hormones such as Bacillus
megaterium,Pseudomonas fluorescens, and Trichoderma harzianum.
6.5.2 Indirect Mechanism
•Production of Lytic Enzymes: PGPRs are well-known for antibiotics produc-
tion and lytic enzymes which help in the suppression of the growth of phyto-
pathogens. There are various antifungal compounds like phenazines, pyrrolnitrin,
cyclic lipopeptides, glucanases, lipases, cellulases, and chitinases which directly
inhibit the growth of phytopathogens (Gamalero and Glick 2015).
86 A. Chaudhary et al.
•Competition: PGPR and PGPF protect the plants from destructive phytopatho-
gens and help in enhancing the growth of plants. Inoculation of Fusarium
oxysporum significantly suppressed nematode pathogen in banana plants and
increases their yields (Van Loon et al. 1998).
•ACC Deaminase: PGPR produces 1-aminocyclopropane-1-carboxylic acid
deaminase. This enzyme cleaves ethylene precursor into ammonia and
α-ketobutyrate and controls growth of plants by cleaving the ACC and minimiz-
ing the level of ethylene, when present in high amount. This enzyme is encoded
by acdS gene in several bacterial and fungal species. ACC production was also
observed in several fungi such as Issatchenkia occidentalis and Penicillium
citrinum (Itelima et al. 2018).
•ISRs (Induced Systemic Resistance): Rhizobacteria suppress diseases via
inducing the resistance mechanism in plants known as ISR (Singh et al. 2021;
Zhang et al. 2020a; Mishra et al. 2020). It involves ethylene and jasmonate
signaling which help in the stimulation of defense response in host plant against
a large number of pathogens.
6.6 Challenges of Using PGPR as a Bioinoculant
The use of PGPRs as a bioinoculant is very old. PGPR strains selected in laboratory
result in low performance of a variety of activities, while do not for all time results
under field conditions. So, there is need to improve the methods. Developing
bioinocula containing vastly efficient microbes with a lengthy shelf life and high-
rhizospheric colonization rate poses a most important dispute for commercialization
of bioinoculants. PGPR and PGPF are often used in an inappropriate carrier that does
not allow the efficient colonization under field conditions due to competition with
resident soil microbial flora. Creation of bioinoculants for specific soil type and to
train farmers to well apply them to crops are very important in the improvement of
more beneficial inocula (Zhang et al. 2020b; Feng et al. 2020; Lin et al. 2020; Zhan
et al. 2020; Ye et al. 2019; Huang et al. 2019,2020; Fan et al. 2020; Pang et al. 2020;
Gangola et al. 2018b; Gupta et al. 2018).
6.7 Conclusion
Today, concern arises due to the harsh impact of agrochemicals; there is a rising
attention in improving our knowledge to understand the function of rhizospheric
microorganisms in agriculture. The plant rhizospheric microbes show outstanding
prospective for wider application in sustainable agriculture as they improve plant
health and productivity in an ecofriendly and commercial manner. Using
bioinoculants is a potential way to enhance nutrient use efficiency in soil and can
be a good alternative to agrochemicals used in the agricultural fields. However, more
6 Rhizospheric Microbes and Their Mechanism 87
examination is needed in different disciplines to recognize the exact mechanisms of
bioinoculants under different set of conditions.
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6 Rhizospheric Microbes and Their Mechanism 93
Chapter 7
Endophytes and Their Applications
as Biofertilizers
Gaurav Yadav, Rishita Srivastva, and Preeti Gupta
Abstract Endophytes are microorganisms residing inside plant tissues. The endo-
phytes are not harmful for the plant health, and besides this, they provide nutrients to
plants as well as protect plants from stress conditions and from plant pathogens. The
endophytic microbial community includes different genus of bacteria, fungi, algae,
actinomycetes, and transgenic microbes like Bacillus sp. and Piriformospora indica.
These microbial strains can be used as consortium or single species as biofertilizers.
These biofertilizers are free from chemicals and have a number of benefits for
agricultural crops such as they help in plant growth, act as biocontrol agents, protect
plants from stress, and also help in the recovery of diseased plant, N
2
fixation, and
IAA production, etc. This compendium will accentuate on the different types of
endophytic microorganisms and their extensive role as a biofertilizer in the field of
agriculture.
Keywords Endophytes · Agriculture · Biocontrol · Biofertilizers
7.1 Introduction
There are some microorganisms, especially bacteria and fungi, which can thrive on
their plant host externally, and are commonly known as epiphytes, i.e., present on
leaf surfaces, and some live internally and are known as endophytes (Afzal et al.
2019). Endophytes are the microorganism which resides in the inner part of the
plants’body such as xylem vessels without vitiating or infecting the plant, and there
are approximately 300,000 plant species present on earth which carry one or two
endophytes (Afzal et al. 2019; Fadiji and Babalola 2020; Maela and Serepa 2019).
Endophytes are of two types: obligate endophytes and facultative endophytes.
G. Yadav (*)
Amity Institute of Microbial Technology, Noida, Uttar Pradesh, India
R. Srivastva · P. Gupta
Dolphin PG Institute of Biomedical and Natural Sciences, Dehradun, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_7
95
Obligate endophytes are those which require more specific nutrients and strict
conditions to grow in in vitro conditions; without them they cannot be cultivable,
while facultative are those endophytes which do not need any specific nutrients or
conditions to grow, i.e., non-fastidious in nature; they can harbour themselves in
soil, artificial medium, and inside plants (Maela and Serepa 2019). These salutary
microbes can be transmitted to various generation of plants through sexual units such
as seeds and spores (Soldan et al. 2019). Wild species of dicotyledonous and
monocotyledonous plants are also a hub of various endophytes. On the basis of
various studies on endophytes, they are classified as bacteria, fungi, algae, actino-
mycetes, and transgenic endophytes as shown in Fig. 7.1. Archaebacterial and
mycoplasma endophytes also exist in plants, but there are not enough studies on
them (Maela and Serepa 2019; White et al. 2019).
These endophytes may perform various roles in plants such as adaptation and
resistance against temperature stress and environmental stress and overcome the
salinity, and they also help in stimulating plant growth, which may be direct and
indirect. Direct roles are biological nitrogen fixation (BNF), phosphorus solubiliza-
tion, potassium solubilization, siderophore production, IAA production, ACC utili-
zation, ammonia excretion, lytic enzyme secretion, and phytohormone production,
and indirect roles may be designated as production of metabolite, induction of plant
resistance, and promotion of plant growth (Fadiji and Babalola 2020; Sansanwal
et al. 2017). An endophyte can be a good candidate for biofertilizers. A biofertilizer
is a consortium of viable microbes which when used on seeds, plants, and soil will
colonize the rhizosphere and internal structures of plants, seed, etc. Furthermore,
when these endophytes colonize the inner part of plants or its reproductive bodies,
they exert direct beneficial effects. This exercise will obliterate the use of chemical
fertilizers because of the adverse effect of chemical fertilizer on crops and on human
health. They not only affect crops or humans but also deteriorate the quality of soil
and damage the environment (Panpatte et al. 2017; Sansanwal et al. 2017). Endo-
phytes also help in phytoremediation of heavy metals from soil, and they also
produce gluconic acid (GA) (Oteino et al. 2015).
Endophyte
Bacteria Fungi Algae Actinomyces Transgenic
Obligate Facultative
Fig. 7.1 Classification of an endophyte
96 G. Yadav et al.
7.2 Mode of Transmission of Endophytes
Transmission is important for the survival of endophytes as well as for plant species
because endophytes help plants in certain ways; thus, endophytes can be transmitted
from parents to progeny and from one individual plant to another in the same
community. Therefore, based on this view, the mode of transmission can be of
two types (Kuzniar et al. 2019).
7.2.1 Vertical Mode of Transmission
It is a direct mode of transmission in which endophytes are transmitted from parents
to offspring via seeds and through pollens.
7.2.1.1 Vertical Transmission Through Seeds
In the surface-sterilized seeds of various plant species such as alfalfa, maize, rice,
coffee, tobacco, barley, pumpkin, and quinoa, various bacterial species are found to
be more dominating than other microorganisms. These endophytic bacterial species
have been also detected in a range of wild plant species, including Pachycereus
pringlei,Lolium rigidum,Eucalyptus spp., and Picea abies. These endophytes are
found to be present in different parts of seeds such as embryonic tissues, coat, and
endosperm. Bacillus,Pseudomonas,Staphylococcus,Acinetobacter, and Micrococ-
cus are examples of some common bacterial genera which are present as endophytes
in seeds. Endophytes are comparable to biofertilizers as they are known to impart
beneficial properties to plants. For example, in rye grass, they produce cytokines
which help in releasing seed dormancy. Endophytes help in fixing nitrogen and help
plants to survive in extreme environments. In vitro tests demonstrated that they also
have antifungal properties against fungal plant pathogens. It was observed that in
rice seed if endophytes are removed, seedling of the rice seed will become restricted
(Frank et al. 2017). Those endophytes present inside the seeds protect plants against
diseases and abiotic stress. They also help in better germination and preserve seeds
for a longer period of time (Girsowicz et al. 2019).
7.2.1.2 Vertical Transfer Through Pollens
Male gametes are a possible way for transmission of endophytes, but pollens are also
being a method of horizontal way of transmission because they can easily be
colonized by the atmosphere, animals, and through pollinators. Enterobacter spp.
were isolated from fertilized P. brutia ovules. 10
6
–10
9
numbers of bacteria are found
on per gram of pollen; they are present as clusters, biofilms, and single cells. There
7 Endophytes and Their Applications as Biofertilizers 97
are different types of endophytes and it depends upon plant species, plant-specific
endophytes, antimicrobial content of pollen or plants, and nutritional composition of
plants.
7.2.2 Horizontal Mode of Transmission
It is an indirect mode of transmission which occurs between two individual plants in
the same community. This mainly happens in fungal species in which fungal spores
can be easily dispersed among plants through air, animals, and flying insects. Soil is
the main source of microorganisms for both below and above the ground because
soil contains most of the vital nutrients which are needed to survive in the environ-
ment. In the horizontal mode of transmission, endophytes can colonize seeds and
roots via soil, and from soil, endophytes first colonize spermosphere, and in this
endophytes have beneficial effects on the germination process. Secondly, they can
also colonize through the root endosphere region via rhizosphere where vast num-
bers of microorganisms are present which promote plant growth and also protect
against plant pathogens. Flowers and fruits and plant insects can also be a horizontal
mode of transmission and colonization of leaves through stomata (Frank et al. 2017).
7.3 Bacteria
Previously isolated endophytic bacteria from Napier grass and characterized by
molecular description include Sphingomonas,Bacillus,Enterobacter sp., and
Pantoea sp. Endophytes were described after sequence analysis and after analysis
of the phylogenetic relationship. In this study, representative isolates were selected
for the purpose of observing their sequence-based plant developmental capacity and
development relationship. Lately, four iso-member endophytic bacteria are capable
of colonizing the plant root more rapidly. Plant growth-enhancing features such as
production of IAA, production of siderophores, production of ammonia, solubility of
phosphates, fixation of nitrogen, and ACC deaminase are the properties of endo-
phytic bacteria and endophytic bacteria also produce some chemicals which have
antagonist properties as shown in Table 7.1.
Endophytic bacteria have been able to cause an increase in shoot and length in
hybrid pennisetum relative to inoculation controls in both saline and natural envi-
ronments and to colonize host plant roots. Up to 200 mM NaCl were retained. These
PGP attributes are therefore adorable to endophytic bacteria and favourable for
growth of plants and higher yields in unfruitful soil, salt ground, and infertile
areas. With this peculiarity of the endophytic population, the bioinoculants of
agriculture will obviously be used as better biofertilizers. The growth of plants
encourages endophytic bacteria and fuels plant growth by discrete mechanisms.
They encourage phytohormone production, nutrient absorption (Bibi et al. 2012;
98 G. Yadav et al.
Mei et al. 2014; Sturz et al. 2000), and biocontrol by reduction of phytopathogens.
Throughout the plant life cycle, it is created at various times, through various
mechanisms, to promote growth and produce endophytic bacteria (Glick 2003).
The bioinoculant preparation can be used as a biofertilizer in the field in conforma-
tion of endophyte bacterial consortia by using these endophytic bacteria. The use of
mixed useful endophytes in agriculture increases the consistency of the soil and
eventually helps grow plants. Besides other PGP attributes, ACC deaminase is
present in endophytic bacteria activity, and thus the plant root is capable of reducing
ethylene levels compared to other endophytes which was recorded earlier with ACC
deaminase activity. ACC (ethylene precursor) is converted to ammonia and alkaline
butyrates using ACC deaminase enzymes in a stressed setting such as acidic soil
conditions (Jha and Kumar 2009; Jha et al. 2012; Glick 1995; Alexander and
Zuberer 1991; Burd et al. 2000; Nabti et al. 2010) and promotes the expansion of
plants in adverse conditions. High deaminase activity (about 225.2–1106.6 nmol-
KB/h/mg) in the endophytic bacterial population has been documented in contrast to
non-endophytic deaminase activity (approximately 20 nmol-KB/h/mg) (Glick
2003). The use of plant-building endophytic bacteria as biofertilizer in a region,
however, requires attention to maximize the gain within the host plant. In order to
increase crop size, and growth use of different endophtes will be beneficial. Pro-
moting low-dose chemical fertilizer and increased use of different biofertilizer has
proven to be effective (Wolinska et al. 2017; Singh et al. 2021; Zhang et al. 2020a,b;
Mishra et al. 2020; Feng et al. 2020; Lin et al. 2020; Zhan et al. 2020; Ye et al. 2019;
Huang et al. 2019,2020; Fan et al. 2020; Pang et al. 2020; Gangola et al. 2018;
Gupta et al. 2018). By increasing the absorption of nutrients in plants and also by
preserving soil microbial floral dynamics, biofertilizers are increasing soil fertility
(Bhatt et al. 2020a,b,c,d,e,f,2021a,b,c). Plants are immune to harmful conditions
and pesticides by biofertilizers. By plant nutrient intake and also retaining the
microbial dynamics of soil, biofertilizers improve soil fertility to enhance their
health. Endophytic microbials are discussed in the current sense of how they can
Table 7.1 Benefits and some chemicals produced by endophytes which help plants to grow and
survive in stress condition, and these chemicals also have antagonist properties
Benefits of endophytes
Chemicals produced by
endophytes Bacterial spp.
Antibiotics Antioxidants enzymes Acinetobacter
Antiviral Volatile compounds Cellulomonas,
Azotobacter
Immunosuppressive and anticancer
compounds
Phytohormones Clavibacter,
Azospirillum
Antibacterial IAA (indole acetic acid) Pseudomonas,Bacillus
species
Antifungal Cytokinin Curtobacterium,
Pseudomonas
Antiprotozoal GA (Gibberellic acid) Microbacterium
Bio-insecticides
7 Endophytes and Their Applications as Biofertilizers 99
make better crop production. Studies show that PGPP (plant growth promotion)
attributes are present in both Rhizobium and Pseudomonas and that they make plant
nutrients accessible through metabolic modifications, phosphate solubilization, iron
chelation, and many more attributes (Bhatt et al. 2015a,b,2016a,b,2019a,b,c;
Huang et al. 2021).
7.4 Nitrogen-Fixing Endophytes
In the agricultural outlook, some important advantage of endophytes is nitrogen
fixation, siderophore production and antagonism against phytopathogens (Franche
et al. 2009). In soil, diverse kinds of microorganisms are found including bacteria,
fungi, protozoa actinomycetes, and algae. Among all these spp., Rhizobia were
studied extensively in various aspects like physiological, biochemical, and molecu-
lar level to facilitate plant growth and to increase plant productivity without fertil-
izers, and their applications are widely used in maize, wheat, etc. Diazotrophic
endophytic bacteria not only promote plant growth; besides they produce a variety
of compounds that have antagonistic properties and are used as a defense system as
shown in Fig. 7.2. Endophytic microorganisms not only remove nitrogen from the
environment but also provide nitrogen to plants for their stable growth.
The relation between nitrogen fixation and bacteria is very important but how
endophytes fix nitogen is needed to understand and these are some organisms which
help in fixing the nitrogen, Azoarcus,Azospirillum brasilense,Burkholderia spp.,
Klebsiella,Gluconacetobacter,Herbaspirillum, and Serratia, and many others
Diazotrophic
endophytes
1.Plant disease
control
2.Pathogen control
Applications
Antioxidants
Biofertilizers(Azoarcus,Achromobac
ter,BurkholderiaGluconoacetobacter,
Herbaspirillum,Klebsiella, and v
Serratia
Endophytes plant and
biomass
N₂fixation
Fig. 7.2 Diazotrophic endophytes as biofertilizers
100 G. Yadav et al.
availability much easily (Rothballer et al. 2008; Ahmad et al. 2008). Various bacteria
such as Azoarcus spp. produce siderophores to deplete the deficiency of iron
(Newton 2000) by provided iron to plants by soil bacteria help in several situations
like heavy metal pollution. However, the concentration of siderophores affects the
growth of plants. Nitrogen attachment mechanism is performed in the nif genes, and
a total of 16 ATP is consumed throughout the process. As compared to the free-
living bacteria, symbiotic association fixes more nitrogen. Oxygen works as an
inhibitor for enzyme nitrogenase and acts as a negative regulator of nif gene
expression. The nitrogenase is responsible for catalysing reduction of (N
2
)to
ammonia (NO
3
) (Gupta and Nath 2015). Nitrogenase enzyme is sensitive to oxygen
that irreversibly inactivates diazotrophs (N
2
)fixation which, on the other hand,
permits supply of oxygen required for every regeneration and protects from delete-
rious effect of O
2
. In this situation, bacterium Rhizobium spp., which populate
internal plant tissue niche with or without minor complaint to host plants, switch
on a process that is known as biological nitrogen fixation (Gupta et al. 2012). The
ongoing research assuming that some endophytes such as diazotrophic
Gluconacetobacter,Serratia marcescens, and Azoarcus sp. will be good as
biofertilizers in agricultural areas (Annapurna et al. 2018). They are also suitable
for use.
Information is also available on N fixation by non-legumes and bacteria other
than rhizobia (Tanjung et al. 2017; Ohyama et al. 2014). Sphingomonas azotifigens
were first identified in India as a novel bacterium when they were N fasteners
(Naylor et al. 2017; Singh et al. 2017). The earlier documented or identified plant
pathogens were Stenotrophomonas maltophilia and Herbaspirillum
rubrisubalbicans that showed high N fixation and potentials for auxin production.
Diazotrophic endophytes bacteria colonize the palm leaves and also found that
Bacillus cereus do excessive nitrogen fixation (Aryantha and Hidiyah 2018; Sharma
and Roy 2017). For Exiguobacterium, the ability to attach N has also confirmed N4
strong strain of Amaranthus spinosus colonization (Rodriguez et al. 2009).
7.5 Fungal Endophytes
Microorganisms have provided a large variety of biologically active compounds,
which have broad applications in human health and diseases. Fungus is ubiquitous in
nature, and it lives inside the host plant for being a part of their life cycle without
causing visible proclamation. In 1928, antibiotic penicillin from Penicillium notatum
was discovered by Alexander Fleming, which was used later during the Second
World War. After that, various antibiotics were discovered, such as Chloramphen-
icol, Streptomycin, Tetracycline, Griseofulvin, Cyclosporine, and Taxol from dif-
ferent fungal species, and therefore, this period is known as the Antibiotics Era. After
that in the twenty-first century, novel antibiotic compounds were isolated from
different fungal species as it has the ability to produce potential pharmaceutical
7 Endophytes and Their Applications as Biofertilizers 101
products which will be used for various purposes (Naylor et al. 2017; Singh et al.
2017; Aryantha and Hidiyah 2018; Sharma and Roy 2017; Rodriguez et al. 2009).
7.5.1 Classification of Endophytic Fungi
Fungi are classified into two major groups on the basis of their evolutionary
relatedness, taxonomy, plant hosts, and ecological functions.
1. Clavicipitaceous endophytes
2. Non-clavicipitaceous endophytes
Apart from these two main groups, there are four classes of endophytic fungi:
•Class I endophytic fungi
•Class II endophytic fungi
•Class III endophytic fungi
•Class IV endophytic fungi
7.5.1.1 Class I Endophytic Fungi
Clavicipitaceous endophytes are the class I endophytes reported in the late nine-
teenth century by European investigators from seeds of Lolium arvense,Lolium
temulentum, and Lolium remotum (Arnold et al. 2002).
7.5.1.2 Class II Endophytic Fungi
The class II endophytic fungi contain a great diversity of species belongs to
Dikaryomycota, which consists of Ascomycota and Basidiomycota. In 1915, Rayner
reported a phoma species called Calluna vulgaris. The crucial point is that they
always colonized all parts of the plant including the seed coat. Now recently, they
become a common root endophyte that confers benefit to plant.
7.5.1.3 Class III Endophytic Fungi
This class is differentiated on the basis of occurrence, that is, above-ground tissue;
this class transmits horizontally and is extremely high in plant biodiversity. This
class includes the hyper/high endophytic fungi that are associated with the leaves of
tropical trees (Suryanarayanan et al. 2005). Class III endophytes span from above-
ground tissues of non-vascular plants to the seedless vascular plants, conifers, and
herbaceous angiosperms in biomes and differ from tropical forests to Boreal and
Arctic/Antarctica communities (Arnold et al. 2002).
102 G. Yadav et al.
7.5.1.4 Class IV Endophytic Fungi
The discovery of class IV endophytes is concluded during the study of
ectomycorrhizal fungi, where Naik Shankar et al. 2008 remarks a pigmented fungus,
which is brown to blackish in appearance, and it is associated with terrestrial plant
roots that are called as MRA. This species is found with mycorrhizal fungi and
implied as pseudomycorrhizal fungi. Recently, these fungi are mentioned as DSE
(dark septate endophytes) and come under class IV endophyte. A century has passed
since it was discovered, but the role of these queer fungal symbionts is still unknown.
Apart from diseases, fungi are beneficial to humans as they produce numerous
alkaloids (ergot alkaloids from Claviceps), some enzymes (cellulose, lipase,
ligninolytic enzymes), and also some pigments (anthraquinone, betalains, aroma,
and flavours); besides these, they also play an important role in the control of
nematodes. Some fungi are edible like mushrooms and they are useful to our health
as they are abudant in minerals, vitamins and proteins like selenium, potassium,
riboflavin, niacin, vitamin D, proteins. Endophytes are reported from plants that can
be found in various environments including trophic, temperate, aquatic oceans,
xerophytic deserts, Antarctic, geothermal soils, rainforests, mangrove swamps, and
also coastal forests (Naik Shankar et al. 2008). Endophytic fungi from 15 shrubby
medicinal plants grow in Malnad region and southern India (Raghukumar 2008).
She also reported that the isolation of a greater number of endophytic fungi happens
in the winter season rather than in monsoon and the summer season. In India less
number of discoveries are available or we can say that few reports are available on
endophytic fungi, blooming in mangrove plants. It is interesting that mangrove
plants have shown to adapt to anaerobe soil, muddy saline waters, and brackish
tidal activation. Narayanan et al. (2014) reported on cell wall enzymes such as
pectinases, proteases, and pectate transeliminase from endophytic fungi and their
leaf litter degradation activity when exposed to extreme conditions in terrestrial and
marine environment such as high temperature at tropical areas, elevated hydrostatic
pressure, low temperature in deep sea, and aloft hydrostatic pressure. Like all other
endophytes, endophytic fungi with mangrove plants protect mangrove from adverse
environment conditions (Jeffery 2008). Many species of fungi include Aspergillus
(Narayanan et al. 2014; Jeffery 2008), Beauveria bassiana,Trichoderma harzianum,
Lecanicillium lecanii,Metarhizium anisopliae, and Fusarium spp. According to
reports, most common endophytes are Aspergillus,Phomopsis,Wardomyces,Pen-
icillium, and many unidentified fungi. Species Colletotrichum gloeosporioides,
Fusarium solani,Pestalotia, and Phomopsis were predominant endophytes though
Colletotrichum gloeosporioides establish as endophyte and it is a pathogenic fungus
for Cashew tree, which is reported by some authors during studying different hosts.
A substantial research was go through using several other fungi isolated mainly a
epiphytic in an attempt to Jurisdiction pathogen C. gloeosporioides being a
thrichoderma strain, the most hopeful one. Fungal endophytes have the most unique
adaptation in nature; with respect to future prospects that how endophytes
7 Endophytes and Their Applications as Biofertilizers 103
communicate with each other in contrast to their pathogenicity for such innovative
purposes, endophytic fungi should be an effective topic to study.
7.6 Actinomycetes Endophytes
Actinomycetes are one of the most abundant groups of microbes which is exten-
sively distributed in nature. Predominantly, they are found in dry alkaline soils
(Fouda et al. 2018). Endophytic population was greatly impacted by climatic
conditions and locality where their host plant grows (Waheeda and Shyam 2017).
Endophytes reside within plant tissue to finalize their life cycle, despite no delete-
rious effect, and play a noteworthy role in aggravate growth of host plant by
producing phytohormone and build plant tolerant to various stresses, other growth-
encouraging factors in order they are compensation with nutrients and shields within
host plant as shown in Fig. 7.3.
Bacteria present in the rhizosphere is different from endophyte because endo-
phytes are aunthetic and specific to its habitat as they resides within the plant tissue
(Kumar and Jadeja 2016). Endophytic actinobacteria are examined to be a substitute
to combat multidrug-resistant human pathogens as they serve as a latent source of
novel antimicrobial compounds (Limaye et al. 2017). Plant diseases can be reduced
by actinomycetes using distinct mechanisms. A method known as pyrosequencing is
used to disclose a wide range of bacteria that live in and around roots of plants such
as Actinobacteria,Bacteroide,Verrucomicrobia, and Proteobacteria (Limaye et al.
2017). Some actinomycetes secrete a range of enzymes that can entirely degrade all
components of lignocelluloses such as lignin, hemicelluloses, and cellulose
(Andreote et al. 2014). Because of their ability to secrete enzymes, they are virtual
in attacking raw beer. Bioactive molecules from actinobacteria and their biosynthetic
Possess Antimicrobial Activities
Secrete Various Enzymes Promote Plant Growth
ACTINOMYCETES SPECIES
Produced Bioactive Secondary
Metabolites Tolerant to Abiotic
and Biotic Stress
Capable of Reducing Growth
of Pathogen
Fig. 7.3 Actinomycetes species and their various applications
104 G. Yadav et al.
genes have been less studied; bioprospecting of actinobacteria for bioactive mole-
cules holds great promise.
There is an increasing necessity for discovery of new drugs, due to the increasing
threats day by day which incorporate drug-resistant pathogens. In order to
achieve this, novel strategies are needed to be applied to search for a new mole-
cules to fight against resistan microorganism. One of which is “Renaissance”in
antibacterial discovery from actinomycetes. Internally colonizing microbes play
various roles in widening and plant fitness. A few of them also cause diseases
(Yang et al. 2015) but the interaction is said to be beneficial. Antibacterial
compound-producing strains belonging to genera Streptomyces,Nocardiopsis,
Pseudonocardia,Agrococcus, and Isoptericola have been reported from mangrove
plants in Beilun River, Beilum Estuary National Nature Reserve, China
(Purushotham et al. 2018). Actinomycete strains have genetic ability to produce
10–20 secondary metabolites. Pseudowintera colorata (Horopito) is an indigenous
medicinal plant of New Zealand. In the study of Pseudowintera colorata endophytes
it was observed that microbial communities in roots and stems are not diverse but in
leaves microbial communities were found to be spare diverse on the basis of DGGE
pattern. Profusion of actinobacteria taxa was steeper in stems (39%) and roots (27%).
However, three clones among them were identified as uncultured bacteria (Oskay
et al. 2004). Compounds from the rare organisms include Teicoplanin and
Actinoplanes teichomyceticus (Kaaria et al. 2012). In vitro actinomycetes isolated
from Turkey’s farming soil have shown the ability to inhibit Erwinia amylovora
bacteria that cause fire blight to apple and Agrobacterium tumefaciens, a causal agent
of crown gall diseases. In view of the increasing threats day by day which include
drug-resistant pathogens, there is an increasing need for discovery of new drugs. In
order to, this challenge, novel strategies are to be applied to searching new
molecules.
7.7 Algae Endophytes
We all know that endophytes have a symbiotic association with plants, and some-
times they act as a biocontrol agent because they protect plants from animals to
produce certain compounds which help plants to become safe. When we heard about
the word endophyte, automatically in our mind bacterial endophytes strike. In
various aspects of life, algal endophytes also play an important role (Bacon and
White Jr. 2000). Marine algae is found in coastal regions including some
cyanobacteria and other microbes. The marine microbes were isolated from red
algae, green algae, and brown algae as shown in Fig. 7.4.
In red algae, brown algae and green algae, red algae has highest bioactivity
among all. It is found that marine algae harbour some epiphytic and endophytic
microbes that produce antimicrobial substances that can inhibit human pathogens.
They also produce bioactive compounds that can be used by plants (Fremlin et al.
2009). Besides this, they produce some antiprotozoal, antiparasitic, antiviral, and
7 Endophytes and Their Applications as Biofertilizers 105
antitumour activities (Vesterlund et al. 2011). It is shown that host plant tolerance to
various stress, such as abiotic, is increased by endophytic infection (Gonzalez-
Bashan et al. 2000) and biotic. There are some naturally grown microalgae which
are found to be associated with bacteria (Suminto and Hirayama 1997). Currently it
is seen that the plant growth-promoting bacterium (PGPB) Flavobacterium spp. was
built to aid the growth of a marine alga (diatom Chaetoceros gracilis) which is used
as a feed in oyster hatcheries (Correa et al. 1987). With the help of the above
example, we understand how bacteria and microalgae are mutually beneficial for
each other.
Microalgae
Supplied exogenous oxygen for growt
h
7.7.1 Chlorella vulgaris
There are several advantages of PGPB in which environmental application is most
important, For example, Azospirillum species add in wastewater for its treatment with
microalgae to enhancing the development microalgae metabolites. Microalgae have
numerous uses such as water bioremediation. Chlorella vulgaris and Scenedesmus
dimorphus is a unicellular microalgae, which is used for separating the phosphate
and nearly all of the ammonium from dairy industry and pig farm wastewaters. Now
in such studies, microalgae were in a shelving. Although these crisp populations of
such algal endophytes are pigmented (Plumb 1999; Correa et al. 1988). Some
endophytes have been capable of nutritional independence; this indicates that they
have cultured separately from their host Acrochaete heteroclada (Nielsen 1979;
Kumar et al. 2015) have previously been allocated to green algal Chondrus
Red Algae
Green algae
Brown algae
Fig. 7.4 Pie chart indicating the bioactivity (%) based on class of algae
106 G. Yadav et al.
crispus-associated endophytes. A variety of endophytic strains are host to
Stackhouse Red Algae Chondrus Crispus, including pigmented algae, Multicellular
species (Chlorophyta) and pigmented brown (Heterokontophyta) respectively.
7.8 Methanogens as Endophytes
Methylotrophs are those, which can grow by using reduced carbon sources such as
methanol or methane can grow by apply the diminish carbon substrates (Kumar et al.
2016; Meena et al. 2012) some are found advantageous and non-pathogenic for
plants (Pirttila et al. 2005; Holland and Polacco 1992) during study of soybean
seedling. Endophytic methylotrophic bacteria have been marked to increase seedling
growth closely with root biomass growth (Dourado et al. 2012). Some
methylotrophic endophytic reported are Methylobacterium sp., Methylovorus
mays, mesophilic methylobacterium extorquens and methanotropics, directly or
indirectly, plant growth plants directly or indirectly (Ferreira et al. 2008;
Raghoebarsing et al. 2005). Some methanotrophs are described as biofertilizers to
assembled the agricultural development up to the agricultural fields (Keerthi et al.
2015; Rekadwad 2014; Dourado et al. 2015). Ubiquity and worldwide appendage of
genus Methylobacterium sp. is found as Epiphytic and Endophytic bacteria and
PPFM of Methylobacterium sp. are found (pink pigmented facultative methylotroph)
the biotechnology and agronomic future group is reported (Baldani et al. 2000). In
the current conclusion, PPFM and the spinal pseudomonas Biofertilizers have
differed and the intensification of plant growth has been shown in the field to be
continuously forward with positive microbial soil production. Endophytic
Methylobacterium species NPFM-SB3 was found to be remote from the Sesbania
stem nodules, a symbiotic company with rice plant can be systemized (Gyaneshwar
et al. 2005; James et al. 2000). Recently during Fe famine, Methylobacterium
phyllosphere (insulated from the lowland rice phyllosphere) triggered the
hydroxamate of the form siderophores, as well as tryptophan and tyrosine during
Fe famine. The main forebear for methane producing by methanogenesis is fre-
quently acetate, which is one of the largest products from anaerobic digestion of
organic compound by bacterial metabolism (Karakashev et al. 2011; Smith and Mah
1978; Thauer 1998; Ardanov et al. 2011). Methylobacterium fertilizer and the
priming ability of Methylobacterium sp. have been used in potato development.
Also, IMBG290 was observed. The priming of plants with beneficial bacteria and
advantage of using beneficial bacterial strain is that they induce host plant rescue
energy and decrease growth and growth time. Plant priming the host saves energy
and reduces the time taken by non-pathogenic bacteria during a pathogen attack for
the development of the defense reaction (Doty 2008).
7 Endophytes and Their Applications as Biofertilizers 107
7.9 Transgenic Endophytes
Formerly research was conducted on plants to improvise its ability to reduce
environmental pollution. Genes from many plants, microbes, and animals were
inserted into plants to enhance their pollution degrading abilities, known as trans-
genic plants (Yang et al. 2017). For extensive use of endophytes in the field of
agriculture and forestry, some useful genes can be inserted into endophytes to
introduce new features into the microbes. In the study conducted by Glandorf
et al. (2001) to control lepidoptera larvae, in which the insecticidal protein gene of
Bacillus thuringiensis was inserted bacterial endophytes Burkholderia pyrrocinia
JK-SH007 to control lepidopetra larvae. Cloning of cry218 gene was done using
PCR, and PHKT2 expression vector was used for the introduction of gene into
JK-SH007 (Glandorf et al. 2001). Pseudomonas putida WCS358r was genetically
modified. Mini-Tn5 lacZ1 transposon was used as a delivery vector (size 6.8 kb), in
this BglII-XbaI fragment was incorporated that carrying the phzABCDEFG genes
from Pseudomonas fluorescens 2–79 and inserted into Pseudomonas putida to
produce phenazine-I-carboxylic acid (PCA), which shown antifungal action, when
released into wheat plant rhizosphere (Patra et al. 2017). Transgenic endophytes will
have bright future in the field of agriculture because they can be used as
biofertilizers, biocontrol agent and for plant growth promotion also by inserting
the gene of interest into the endophytes.
7.10 Role of Endophytes as Biofertilizers
Biofertilizer is a consortium of living microorganisms together with minerals plus
nutrients that help in plant growth promotion without changing soil properties; they
also secrete secondary metabolites and bioactive compounds; biosynthesis of bac-
tericides and fungicides protects plants from environmental stress (Kuzniar et al.
2019; Lobo et al. 2019). Biofertilizers provide plants a great amount of minerals such
as potassium, phosphorous, nitrogen, and siderophores as well as
exopolysaccharides (Lobo et al. 2019). Optimal condition must be followed to
produce low cost-efficient inoculant consortium which can promote plant growth
(Patle et al. 2018).
7.11 Plant Growth-Promoting Endophytes
Plant growth-promoting endophytes have vast functions like IAA production, phos-
phate solubilization, siderophores, N
2
fixation, and ACC utilization as shown in
Fig. 7.5.
108 G. Yadav et al.
7.12 IAA Production
There are a lot of endophytic bacteria and fungi present, which can synthesize many
phytohormones such as auxin, cytokines, and gibberellins, in which indole-3-acetic
acid is very common and it contains the carboxyl group which is joined to the third
carbon of indole group (Maela and Serepa 2019; Zahir et al. 2003; Valérie et al.
2007). IAA helps in cell division and enhances the root length and hair, thus
increasing the number of sites for infection and nodulation. This change will help
in better absorption of nutrients and in turn stimulate plant growth (Zahir et al. 2003;
Susilowati et al. 2018). IAA helps in stimulation of tuber and germination of seed, it
also increases the rate of root and xylem development, it stimulates pigment forma-
tion, and it also mediates the responses to gravity, florescence, and light and also
affects photosynthesis (Sansanwal et al. 2017). It is the direct mechanism of PGPR
that stimulated the growth and yield of plants but a high level of IAA can lead to
abnormalities in plants during its development stage, and the low level of IAA
stimulates root elongation (Maela and Serepa 2019). The production of IAA is
stimulated by the amino acid known as L-tryptophan which acts as a physiological
precursor for IAA production in plants and microorganisms. It was observed that the
production of IAA can be increased up to 2.7 times in the presence of L-tryptophan
amino acid (Audipudi et al. 2017). An increase in the amount of nitrogen in soil leads
to the production of auxin that softens the cell wall, thus increasing the water
retention capacity by increasing the cell size, and the addition of these cells can
increase the weight of rice. Biofertilizers containing a mixture of Azospirillum,
Pseudomonas, and Bacillus increase nutrient uptake and plant growth and also
increase the size of the rice grain (Audipudi et al. 2017). Bacteria can convert
tryptophan to IAA through four pathways, which include Indole-3-acetamide
(IAM), Indole-3-pyruvate (IpyA), Tryptamine (TAM), and Indole-3-acetonitrile
(IAN) pathways. The indole-3-acetamide pathway forms directly IAA without any
intermediate compound. IAA-producing bacteria through the indole-3-pyruvate
(IpyA) pathway are known, and the pathway is illustrated below in Fig. 7.6
(Audipudi et al. 2017).
PGP
Endophytes
IAA production
Phosphate
solubilization
Siderophore
N2fixation
ACC utililization
Fig. 7.5 Different roles of
PGP endophytes
7 Endophytes and Their Applications as Biofertilizers 109
In one study the authors showed that 15 endophytes isolates were positive for
IAA production out of 65 endophytes, and they produce 25 μg/mL of IAA.
Acetobacter diazotrophicus and Herbaspirillum seropedicae produce IAA in chem-
ically defined culture media. They tested ten isolates of Typha australis, of which
seven isolates were positive for IAA production (Matos et al. 2017). In a study
genera Bacillus,Micrococcus,Escherichia,Staphylococcus, and Pseudomonas were
tested with wild herbaceous flora for the level of IAA production and growth of
Triticum aestivum. Results were obtained using Gas Chromatography and Mass
Spectrometry (GC-MS), which shows enhanced root length and seed weight by 16%
and 70%, respectively, by bacterial endophytes (Maela and Serepa 2019).
7.13 Phosphate Solubilization
Phosphate is the second most essential macronutrient which plants need for their
growth and development after nitrogen, and the average amount of phosphorous
found in soil is 400–1200 mg/kg of soil. Soil types and its pH determine the
precipitation and adsorption of phosphorous (Gull et al. 2004). It is hard to be
utilized by plants itself because phosphate basically presents either as a mineral or
in an insoluble form, which has poor mobility, and this is due to the reactivity of
phosphate ion with other constituents present in the soil (Maela and Serepa 2019;
Lin et al. 2013; Wei et al. 2018). Endophytic microorganisms secret some organic
acids such as citric, acetic, succinic, and oxalic acid, and they also secret phosphate
and also use mechanisms like acidification, chelation, and ion exchange, thus
Tryptophan
aminotransferase
Indole-3- Pyruvic acid (IpyA)
Indole-3-pyruvate decarboxylase (IPDC)
Indole-3-acetaldehyde (IAAId)
Oxidization
Indole-3- acetic acid
Fig. 7.6 IAA production
through IpyA pathway
110 G. Yadav et al.
making phosphorous available to plants by converting it into soluble mono- and
dibasic forms. Some environmental factors like pH, oxygen, humidity, and temper-
ature can affect the whole process of phosphate solubilization (Afzal et al. 2019;
Maela and Serepa 2019).
In one study, different bacterial endophytes were isolated from the two types of
medicinal plants such as Zingiber officinale and Azadirachta indica on different
culture media. After the isolation, isolates were screened for various activities like
IAA, phosphate solubilization, siderophore production using biochemical character-
ization, morphological identification, and molecular ribotyping; fives isolates were
characterized as Bacillus tequilensis (AAU K1), Bacillus endophyticus (AAU K2),
Beijerinckia fluminensis (AAU K3), Bacillus safensis (AAU K4), and Pseudomonas
aeruginosa (AAU K5). From the wheat root Penicillium radicum was isolated which
is a phosphate solubilizing fungus; it shows plant promoting activity in vitro.
Phosphate solubilizing endophytic bacteria can play a very important role in the
field of agriculture as the demand for biofertilizers is increasing (Maela and Serepa
2019; Lacava et al. 2008).
Some of the compounds present in acidic soil such as weak aluminium,
oxyhydroxides, and iron oxide can retain phosphorus and that results in a low
amount of phosphorous. Phosphate fertilizers are of great importance, but in alkaline
soil calcium leads to less efficient solubilization of phosphate fertilizers (Sansanwal
et al. 2017; Panpatte et al. 2017). In one study, a total of 40 endophytic isolates were
isolated from the root of banana tree and evaluated to check the phosphate solubi-
lizing activity, in which 67.5% isolates solubilized phosphorus from tricalcium
phosphate in solid medium and 7.5% isolates in soy lecithin solubilized phosphorus.
In iron phosphate containing medium no isolates show P solubilization activity.
Isolates Aneurinibacillus sp. and Lysinibacillus sp. showed best solubilization activ-
ity, and other genera and species that exhibited positive results were Acetobacter sp.,
Agrobacterium tumefaciens,Bacillus amyloliquefaciens,Aneurinibacillus sp.,
Bacillus subtilis,Bacillus pumilus,Streptomyces sp., Micrococcus luteus, and Bacil-
lus sp. (Panpatte et al. 2017). Endophytes also prevent the absorption and fixation of
phosphate when its amount is not sufficient (Afzal et al. 2019). Phosphate solubi-
lizing bacteria (PSB) can solubilize inorganic phosphate, which is present in the
form of Ca
3
(PO
4
)
2
, FePO
4
, and AlPO
4
by producing hydroxyl PGPB, organic acid,
and siderophores in soil (Sansanwal et al. 2017).
7.14 Siderophore Production
Siderophores are the low molecular weight iron-containing complex present on the
bacterial membrane in the form of Fe
3+
and then get reduced to Fe
2+
released into the
cell from siderophore complex through the gating mechanism. When there is a high
level of contamination of heavy metals and when plants starve from iron availability,
bacterial endophytes make it available to plants (Fadiji and Babalola 2020;
Sansanwal et al. 2017). Siderophores sometimes also serve as a great biocontrol
7 Endophytes and Their Applications as Biofertilizers 111
agent such as phenolate, hydroxymate, and catecholate type which are produced by
endophytes species, and they also help in fixing atmospheric nitrogen in
diazotrophic organisms because for the functioning and biosynthesis of enzyme
nitrogenase (key enzyme in N
2
fixation) diazotrophic organisms require Fe
2+
and
Mo factor (Fadiji and Babalola 2020). In the absence of heavy metal contamination,
absorption of iron can be performed by other mechanisms like iron chelates directly
lead to siderophores absorption or through ligand exchange. Pseudomonas strain
GRP3 which is a siderophore-producing strain was tested for iron nutrition on Vigna
radiata. Forty-five days of evaluation with endophytic organism, showed betterment
in iron availability and in chlorotic symptoms and there is also an increment in the
amount of chlorophyll a and b as compared to the control results in plants (Fadiji and
Babalola 2020; Sansanwal et al. 2017). Actinomycetes endophytic species like
S. acidiscabies E13, Streptomyces sp. Mhcr0816, Nocardia sp., Streptomyces
sp. UKCW/8, and Streptomyces sp. GMKU 3100 are great producers of
siderophores (1). In one study conducted on Methylobacterium sp. which belongs
to the same niche as X. fastidiosa subsp. pauca (Xfp) isolated from citrus variegated
chlorosis (CVC) showed that catechol-type siderophore is not produced by
Methylobacterium sp. and it is the producer of hydroxamate-type siderophores
(Tang et al. 2019).
7.15 N
2
Fixation
Endophytes perform various vital functions for plants, in which nitrogen fixation is
one that abolishes the use of chemical fertilizer to prevent crops as well as the
environment from harmful effects. Basically, nitrogen fixation is done by legume
plant rhizobium. For the first time in India Sphingomonas azotifigens is a novel
bacterium reported to be N
2
fixing bacteria. Bacillus cereus showed highest N
2
fixing ability in oil palm leaves. Herbispirillum rubrisubalbicans and
Stenotrophomonas maltophilia were considered as plant pathogens but were effi-
cient to do biological nitrogen fixation. Exiguobacterium profundum strain N4
which colonizes Amaranthus spinosus,Azospirillum,Rhizobium,Agrobacterium,
and Sphingomonas in wild rye tissue also performs nitrogen fixation (Kuzniar et al.
2019). One non-rhizobial endophytic consortium which shows very promising
results in BNF includes Achromobacter,Burkholderia,Herbaspirillum,Azoarcus,
Gluconacetobacter,Serratia, and Klebsiella. In sugarcane Gluconacetobacter
diazotrophicus is an endophytic organism which has the ability to fix nitrogen up
to 150 kg N ha
1
year
1
. In kallar grass, obligate endophytic diazotrophs known as
Azoarcus fix nitrogen (Sansanwal et al. 2017). A fungus known as Phomopsis
liquidambari in rice promoted better functioning of nitrogen and phosphorus by
inhibiting or promoting various indigenous soil microbes (Card et al. 2016).
112 G. Yadav et al.
7.16 1-Aminocyclopropane-1-Carboxylate (ACC)
Utilization
Ethylene is the most essential plant hormone or metabolite which every plant has and
that helps in the growth and development of the plant. It is affected by different
abiotic and biotic stresses, and it also controls many activities in plants like cell
elongation, leaf senescence, root nodulation, abscission, auxin transport, and fruit
ripening (Afzal et al. 2019; Fadiji and Babalola 2020). Many stress conditions like
drought, high salinity, pathogenicity, and the presence of extreme concentration of
heavy metals cause the elevation of ethylene levels which lead to inhibition of root
elongation, formation of root hairs, and alteration in cellular processes. In these
stress conditions, endophytic organisms which reside inside the plant produce an
enzyme known as 1-aminocyclopropane-1-carboxylate (ACC) deaminase; it is a
pyridoxal phosphate-independent enzyme which breaks down the ACC (precursor
of ethylene). ACC-degrading genera include Agrobacterium,Enterobacter,Bacil-
lus,Achromobacter,Acinetobacter,Pseudomonas,Alcaligenes,Ralstonia,Serratia,
Rhizobium, and Burkholderia. These organisms can bind to roots of plants and break
down the ACC into α-ketobutyrate and ammonia and use ammonia as a nitrogen
source, thus promoting plant growth under extreme conditions (Afzal et al. 2019;
Fadiji and Babalola 2020). Pseudomonas putida HS-2, which has purified ACC
enzyme, was used to enhance the growth of tobacco plants (Maela and Serepa 2019).
7.17 Biocontrol Activity by Endophytes
Protection of plants from plant pathogens or phytopathogens which can retard plant
growth and can make plants diseased and sabotage the agricultural crop is known as
biocontrol activity. Chemical fertilizers can prevent plants from phytopathogens but
these chemicals have harmful effects on soil, environment, and microflora of plants
and soil, so endophytic organisms are a great way to control phytopathogens. Some
endophytes release bioactive compounds in their specific host plant species as
mentioned in Table 7.2.
Endophytes that act as BCA have shown four mechanisms to control pathogens:
(1) antibiosis, (2) competition, (3) host-induced resistance, and (4) direct parasitism.
Some bacterial endophytes have shown plant protection from various diseases such
as Fusarium, Verticillium, Eggplant, and Verticillium wilt, and these endophytes are
Erwinia persicina,Pantoea agglomerans,Achromobacter piechaudii,Enterobacter
cloacae,P. fluorescens,Serratia plymuthica,S. marcescens,B. amyloliquefaciens,
Paenibacillus, sp., Enterobacter sp., Bacillus subtilis, etc. (Lobo et al. 2019; Agrillo
et al. 2019). Siderophores play an important role to fight against phytopathogens by
endophytes because endophytes release siderophores such as salicylic acid, chelate
iron, and pyochelin which bind all available iron, so that other organisms starve for it
and eventually die. 2,4-diacetylphloroglucinol (DAPG) is an antimicrobial
7 Endophytes and Their Applications as Biofertilizers 113
compound which is released by endophytes, which inhibit disease causing microor-
ganisms. Beauveria bassiana is an entomopathogenic fungi for borer insects in
coffee seedlings. Various fungi that have shown great biocontrol activity in the
field of agriculture are Trichoderma koningiopsis,Gibberella fujikuroi,Aspergillus
tubingensis,A. flavus,Galactomyces geotrichum,P. simplicissimum,Eupenicillium
javanicum, and P. ochrochloron (Lobo et al. 2019). The most studied endophytic
BCA are Bacillus subtilis,Beauveria bassiana,Asexual Epichloe spp.,
Lecanicillium lecanii,Piriformospora indica,Rhizobia, and Trichoderma spp.
Beauveria bassiana works against a wide range of pests such as aphids, beetles,
caterpillars, termites, thrips and whitefly; to use Beauveria bassiana fungi, regular
spray of fungi should be done on infected plants. Asexual Epichloe belong to the
family clavicipitaceae which are not found in the roots of plants. They have strong
mutualism with grass species. Epichloe include E. festucae var. lolii and
E. coenophiala which produce alkaloids—secondary metabolites during symbiosis
and that work against invertebrate pests. Lecanicillium lecanii is a ubiquitous
entomopathogenic fungi, which horizontally transmit from one plant to another
plant such as from cotton plants to an insect known as Aphis gossypii and from
insects to leaves. L. lecanii strain 41185 is pathogenic to aphides spp., such as Myzus
persicae,A. gossypii, and Aphis craccivora.L. lecanii also has anti-fungal activity
against Sphaerotheca macularis and Hemileia vastatrix and it also possesses anti-
algae activity against soil-borne pathogens like Pythium ultimum by producing host-
induced resistance and some structural barriers in the plant roots. Piriformospora
indica is a root-colonizing fungus which is found in the root of xerophytic plants
Table 7.2 Examples of endophytic strains and their bioactive compounds which help plants in
different biotic and abiotic stress conditions (modified from Bhatt et al. 2019a)
S. No Endophytes Bioactive compounds References
1Trichoderma harzianum Phytohormones and degradation of cell
wall
Glick
(2003)
2Bacillus amyloliquefaciens Antifungal compounds Yang et al.
(2015)
3Epichloe festucae var. lolii Alkaloids Glick
(2003)
4Arthrobacter endophyticus
SYSU 33332,Nocardiopsis
alba SYSU 333140
Genes for water and potassium ion
uptake, survival in stress condition
Yang et al.
(2015)
5Aeromicrobium ponti 1-Acetyl-β-carboline, indole-3-
carbaldehyde,3-(Hydroxyacetyl)-
Indole, Brevianamide F, and Cyclo-
(L-Pro-L-Phe)
Burd et al.
(2000)
6Serendipita vermifera Hydrolytic enzyme genes, protection
from plant pathogen infection
Yang et al.
(2015)
7Pseudomonas putida Trichloroethylene Burd et al.
(2000)
8Gibberella moniliformis Lawsone Yang et al.
(2015)
114 G. Yadav et al.
present in Indian Thar dessert. It also colonizes both mono- and dicotyledonous
plants such as barley, tobacco, and Arabidopsis thaliana.P. indica has plant growth-
promoting activity and function in both abiotic stress such as salinity, drought,
water, cold, high temperature, and heavy metals and biotic stress like antagonism
against root pathogens. Trichoderma is a fungal species which has a variety of
advantages like anti-fungal properties, can survive in various conditions, simple
nutritional requirement for growth in vivo and in vitro, fast growth, etc. Trichoderma
genus involves many species like T. atroviride,T. asperellum,T. harzianum,
T. polysporum,T. viride,andT. hamatum. These Trichoderma species can be used
to target different soil fungal pathogens such as Phytophthora,Rhizoctonia,
Sclerotinia,Pythium, and Verticillium and also work against foliar fungal pathogens
like Botrytis and Alternaria (Valérie et al. 2007). Erwinia carotovora is a bacterial
pathogen which causes food spoilage and is inhibited by the bacterial endophytes
such as Pseudomonas sp., Pantoea agglomerans and Curtobacterium luteum.A
fungal species Cryphonectria parasitica causing chestnut blight is inhibited by
bacterial endophytes, i.e., Bacillus subtilis.Clavibacter michiganensis subsp.
Sepedonicus was inhibited by bacterial endophytes isolated from potato stem tissues
(Audipudi et al. 2017; Matos et al. 2017). Some bacterial endophytes have anti-
nematode properties like Bacillus megaterium BP17 and Curtobacterium luteum
TC10, which work against Radopholus similis Thorne. Bacterial species like Bacil-
lus thuringiensis and Serratia marcescens produce toxin and enzyme chitinases
which target the Eldana saccharina larvae (sugarcane borer). Some bacterial
genus like Pseudomonas,Bacillus, and Serratia can protect plants by a mechanism
known as induced systemic resistance (ISR). ISR can be initiated by three of the
pathways: (1) salicylic acid [SA], (2) ethylene [ET], and (3) jasmonic acid [JA].
Actinobacteria was inoculated in a plant, i.e., A. thaliana showed that bacterial
endophytes protected the plant from Erwinia carotovora and Fusarium oxysporum
(Afzal et al. 2019). Pseudomonas protegens N has antifungal properties against the
genus Alternaria and stops spore germination (Gull et al. 2004; Lin et al. 2013; Wei
et al. 2018; Lacava et al. 2008; Tang et al. 2019; Card et al. 2016; Agrillo et al. 2019;
Walitang et al. 2017).
7.18 Role of Endophytes in Overcoming Oxidative Stress,
Salinity, Drought, and Temperature Stress
Endophytes play an important role in the prevention of crop loss by preventing
different types of abiotic stress such as extreme heat, salinity, oxidative stress,
drought, and temperature. Extreme conditions cause change in phenotype and
genetic changes (Sansanwal et al. 2017). Endophytes produce osmoprotectant com-
pounds such as proline, trehalose, exopolysaccharides, and volatile organic mole-
cules (Kuzniar et al. 2019). Pseudomonas spp. help Asparagus spp. do better
seedling and seed germination in extreme water stress. In coastal areas where high
7 Endophytes and Their Applications as Biofertilizers 115
saline condition is present, Pseudomonas fluorescens MSP-393 has proven to act as
PGPR for many crops in that area. Pseudomonas putida RS-198 has proven to
promote cotton seedling in extreme salt presence by inhibiting the absorption of Na
+
and increasing the uptake of Mg
2+
,K
+
, and Ca
2+
. Some species of bacterial
endophytes such as Bacillus polymyxa,Mycobacterium phlei,Alcaligenes sp., and
Paenibacillus sp., produce some compounds such as calcisol which has proven to
promote the growth of maize plant under extreme temperature and saline conditions
(Sansanwal et al. 2017). Pseudomonas migulae 8R6 and Pseudomonas fluorescens
YsS6 can reduce salt stress in tomato plants by secreting ACC deaminase, and it was
observed that ACC deaminase activity helps in the production of a greater amount of
chlorophyll and increase in dry and fresh biomass. Plants infected with fungal
endophytes, i.e., Neotyphodium lolii increase the survival chance of plants in
drought conditions. Endophytic fungi belong to the group Ascomycota and
Basidiomycota, which increase the tolerance of plants in drought and heat condi-
tions. Endophytic fungi belongs to the genus Penicillium,Trichoderma,Aspergillus
sydowii,Myxotrichum stipitatum, and Acremonium variecolor, which exhibit saline
resistance (Kumar et al. 2016). During an environmental stress in plants that can
trigger the production of reactive oxygen species such as superoxide, hydroperoxyl
radicals, hydrogen peroxide, and hydroxyl radicals, these can cause damage to plant
proteins, nucleic acids, and membranes. White et al. (2019) showed that colonization
of bacteria at an early stage causes upregulated transcript level of ROS degrading
genes which include the genes of SOD and glutathione reductase. Festuca
arundinacea infected by the endophytic fungi, i.e., Epichloe coenophiala has greater
concentration of mannitol. Osmoprotectants and other fungal carbohydrate which
helps plant to survive in oxidative stress. Metagenomics analysis of rice crop
endophytes has proven to have the presence of numerous genes which encode
enzymes for protection from ROS (White et al. 2019). Xanthomonas sp.,
Microbacterium sp., and Flavobacterium sp. help plants to survive in osmotic and
salinity stress conditions (Walitang et al. 2017).
7.19 Conclusion
A vast majority of endophytic microorganisms have been isolated from a variety of
plants; they help plants in various aspects like plant growth, prevention to pathogen,
phytoremediation, and nutrient availability; besides this, they also help plants to
adapt to different biotic and abiotic stresses. The understanding of plant-endophyte
mutual association has to be better, to give consistent results under field conditions.
However, PGPR has an advantageous impact on the quality of plants by means of
various action mechanisms. Endophytes synthesize a collection of bioactive meta-
bolic compounds which are used to prevent viral infections as effective medicine.
Secondary metabolites are also synthesized by some endophytes including flavo-
noids, alkaloids, steroids, tannin, quinones, benzopyrones, etc. divided according to
their functional groups. Sometimes, it is difficult to identify some rare endophytes
116 G. Yadav et al.
microorganisms that have beneficial characteristics, and these have a huge role to
bring about less and minimal consumption of diverse types of agrochemical sub-
stances which could include pesticides, chemical fertilizers, and so on. If it is studied
in more details, then it is very surprising, and interesting facts will come out, which
will increase the interest of other people. The current compilation shows the numer-
ous forms of advantageous endophytic microbes that are used to increase soil fertility
as biofertilizers in the field and better crop yield and crop production.
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7 Endophytes and Their Applications as Biofertilizers 123
Chapter 8
Microbial Action on Degradation
of Pesticides
Hira Singh Gariya and Arun Bhatt
Abstract The ultimate end fate of chemical pesticide degradations in the environ-
ment depends on microbial activity. The degradation of any biomaterial depends on
the decomposers for their necessary recycling. So, in the same way, pesticide
degradation is one of the major tasks to prevent their accumulation in the food
chain known as bioaccumulation or may be somewhat disastrous known as
biomagnification at each trophic level of the ecosystem. The extensive implication
of pesticides globally causes a serious imbalance in the soil, air, and finally the
potable water. Pesticide degradation does not only safe as after degradation the
resultant products are more harmful and noxious. Instead, it forms a new chemical
product that may be more or less toxic than the original chemical compound.
Generally, they are broken into resultant smaller and smaller pieces until only the
formation of carbon dioxide, water, and minerals is left. Microbes often play a large
role in this process of harmful product formation. Some of the pesticides also cause
restriction in the growth of algae via the release of various biochemicals that are
important for the growth of algae known as an algal bloom. Worldwide, a large
fraction of pests causes loss, damage to crops, and subsequently productivity.
Pesticides have been used extensively from ancient times, but due to increased
application up to date, their release by a various chemical processes in the environ-
ment causes serious ecological problems. Use of pesticides in an unregulated way
causes adverse effect to humans, animals, and non-targeted plants. The
non-biodegradable and recalcitrant pesticides persist in the environment and cause
serious health hazards. Despite of their restriction and ban by the government, their
continuous use is ever increasing. So, it is mandatory to restrict their use to reduce
risk related to the environment as well as to humans and animals. It means the
indiscriminate application of pesticides causes an adverse effect on different life
forms.
Keywords Bioaccumulation · Pesticides · Biodegradable · Hazards ·
Biomagnification · Toxic · Recalcitrant
H. S. Gariya (*) · A. Bhatt
Biotechnology Department, G.B.P.I.E.T, Pauri Garhwal, Uttarakhand, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_8
125
8.1 Introduction
Pesticides are useful for controlling weeds, pests, and various diseases for the
increased productivity of crops (Sulbhi et al. 2021). Nowadays, these pesticides
have become one of the major tools of farmers to get rid out of various diseases and
also the harmful pest that hampers the crop’s productivity (Bhandari et al. 2021).
Pesticides are useful for repelling and mitigating any kind of pest, but due to their
huge application and non-biodegradable nature cause several harmful problems to
the environment (Bhatt et al. 2021a,b,c). So, it is obvious to regularly monitor the
effects of these harmful pesticides by the government and by the farmers. These
pesticides provide various nutrient supplementation for soil microorganisms (Torres
2003; Bhatt et al. 2020a).
The adaptation of microorganisms to biodegradation is a very common phenom-
enon due to the rapid use of chemical pesticides (Bhatt et al. 2021d,e,f,2020b,c).
There is a lot of severe harmful cases of health hazard due to accumulation of
pesticides in the agricultural system, and their quantification is very difficult to
analyze and predict (Bhatt et al. 2020d). It is very important to know that the inbuilt
soil fertility and the half-life is decreasing due to the persistence of the chemical
compound in the soil is the major cause of reduction in soil texture, fertility, half-life,
etc. (Bhatt et al. 2020e,f,2019a,b,c,2016a,b,2015a,b; Huang et al. 2021). There
are three pathways in which a chemical pesticide compound may undergo:
•Pesticides have no transformation, no chemical structure alternation, and direct
compartmentalization
•Pesticides get transformed and from this may go physical transformation or
chemical transformation and subsequently compartmentalization.
•Pesticides through the biotic transformation may go under co-metabolism or
mineralization.
The ultimate fate of above all three pathways is compartmentalization, deposit in
soil, and get sedimented. The exo-toxicological effect of several pesticides has been
not yet analyzed with precise extent, so ultimately soil is the main reservoir of
various pesticides chemical compounds and their resultant product that can be
detectable (Bhatt et al. 2019d). It is a well-known fact that the uniformity of
pesticides degrading microorganisms lacks at every polluted site so, with the com-
bination of advancement in technologies and conventional procedures to get rid out
of pesticides, the effect on different biological system can be severely decreased
(Sharma and Bhatt 2016). Pesticides cause some of the species to restrict their
growth in the environment. Pesticides are classified in two major classes depending
upon microbial degradation: degradative and non-degradative known as recalcitrant
or microbial resistant. Pesticides include the wide category that involves herbicides,
insecticides, fungicides rodenticides, molluscicides, and nematicides (Sharma et al.
2016).
A diverse group of bacteria including the genera of Flavobacterium,Pseudomo-
nas, and Rhodococcus can metabolize pesticides rapidly. Microbial metabolism to
126 H. S. Gariya and A. Bhatt
degrade various classes of pesticides depends on environmental parameters like
light, pH, moisture, etc. Microbial metabolic activity can also be enhanced via
supplying the necessary supplements so that degradation rate can be enhanced
(Bhatt and Nailwal 2018). Microorganisms have the capability to bio-transform
the pesticide. A major and wide range of bacterial genera has the potential that has
been reported to degrade the pesticides depending upon the pesticide’s chemical
nature, microbial action on pesticide, and pesticide composition (Khati et al. 2018a).
Pesticides that are proven to be effective and widely used are DDT, BHC (commonly
known as 666 or gammexane or lindane), aldrin, dieldrin, and 2–4D (Table 8.1).
Pesticides cause the contamination of ground and surface water. By the process of
leaching, pesticides contaminate the ground water in a way that is not potable and
cause serious health-related disorders (Gangola et al. 2018a; Bhatt 2018; Bhatt and
Barh 2018; Bhatt et al. 2019e; Bhandari and Bhatt 2020; Bhatt and Bhatt 2020). The
elimination of pesticides from the environment in a safe way is very important to
reduce the risk mentioned earlier because of the rapid solubility of pesticides,
bioaccumulation, and biomagnification in a non-targeted microorganisms (Agrawal
et al. 2010). The precise concentration of pesticides is difficult to estimate in
the environment as they have been used ancient before and also due to lack of
documented information (Vischetti et al. 2008). So, the remediation of these chem-
ical compounds is necessary for a way to maintain the kinetics of the ecosystem.
Bioremediation is one of the emerging fields to cope up with this problem. Now in
present time, biologist from different interdisciplinary field related to biology is
continuously seeking and exploring to develop the organism via the help of genetic
engineering with increased efficiency to degrade these contaminants rapidly with
a high rate of degradation. The application of pesticides can kill a certain group of
microorganisms that are useful for agro-farming industry (Hussain et al. 2009).
Detection through voltammetry analysis of the electrode modification increases its
ability to quantify uM or nM and up to pM concentrations of a specific analyte, for
metals and metal oxide (Barbosa and Fernanda 2019).
There are several types of chemical-mediated reaction that is used to degrade the
pesticides. Chemical compounds like atrazine are degraded by the elemental
removal of chlorine with the help of the hydroxylation process, i.e., incorporating
hydroxyl group (Singh et al. 2021; Zhang et al. 2020a; Mishra et al. 2020). The
atrazine is finally converted into a nontoxic one (Zhang et al. 2020b). Enzymatic and
Table 8.1 Classification of
the major groups of pesticides Groups Examples
Organochlorine DDT, Aldrin, 666, Chlordane, Mirex
Organophosphate Malathion, diazinon
Carbamates Sevin
Pyrethroids Pyrethrins
Thiocarbamates Ferbam
Organosulfur CHEBI
Biological Dispel, foray
Urea derivative Diuron-desmethyl, diuron
8 Microbial Action on Degradation of Pesticides 127
nonenzymatic processes can be used for pesticide degradation depending upon the
suitable one, which one is easier and relevant to non-harmful. The nonenzymatic
process is also linked to some disadvantages like the chemical to treat pesticides can
cause a biological hazard in the ecosystem (Feng et al. 2020). The nonenzymatic
chemical component can leach out or their release into the water system is a
disastrous event causing the Minamata due to mercury release. The second major
sulfonylurea derivative herbicides are degraded by simply soil pH. Soil acidic
conditions convert the compound into a hydroxylated intermediate product which
is easier to cleave. Some of the cases are reported where the degradation process is
slower as the acidity of soil increases (Lin et al. 2020; Zhan et al. 2020; Ye et al.
2019; Huang et al. 2019,2020; Fan et al. 2020; Pang et al. 2020).
Photodecomposition method of degradation is efficient for cleaving the internal
chemical bond. The molecules which are multicyclic and contain carbon nitrogen are
vulnerable to be attacked by sunlight radiation. The electromagnetic radiation range
between 290 and 450 nM is efficient for the reaction like oxidation, reduction, and
hydroxylation. The main problem for photodecomposition is that if the toxic chem-
ical compound is leached deep into the soil or released deep into the water system,
then the sunlight radiation penetration becomes ineffective, and photodecomposition
rate becomes almost negligible. Pesticides in the environment have several fates
according to the prevailing condition. Some of them are following—run off by
surface water, volatilization in the atmosphere, leaching by ground water, adsorption
and desorption (surface phenomena) by soil particle, degradation by the microorgan-
ism, and photodecomposition (Gangola et al. 2018b; Gupta et al. 2018; Khati et al.
2018b).
The hot and humid prevailing conditions like the tropical regions, where sunlight
has a direct influence, exist among the majority of countries. The hot and humid
condition is favorable for the growth of microorganisms. The metabolic rate of
microorganism is very high during these conditions and can efficiently degrade the
pesticides when they encounter it. Pesticides reduce the waste of crops and food
resources annually. Aerobic bacteria cleave the aromatic benzene ring and make it
prone to be breakdown by further chemical processes. The anaerobic metabolism of
the aromatic compound-cleaving mechanism is slightly different. In the absence of
oxygen, benzene is disrupted by reduction. We can say these chemical processes are
not exclusive for any aerobic or anaerobic bacteria (Kumar et al. 2017; Khati et al.
2017a,b; Van Eerd et al. 2003). Some of the techniques that biologist relies to tackle
with toxic and harmful pesticides have been enlisted in Table 8.2.
Globally, the agriculture sector is the primary and major user of pesticides. On the
positive aspect, pesticides have resulted in the enhanced crop productivity.
Depending upon the chemical composition, the major classification of pesticides
has been proposed by the scientist. Bioremediation is nowadays a novel technique
and the least expensive to handle with these toxic pesticides. The conversion of
harmful and bio-hazardous toxicants simply by breakdown into non-toxic com-
pounds is known as biotransformation or biodegradation. This technique in modern
times is globally hot spot for the researcher. Biodegradation has several advantages
over the traditional one mentioned earlier (Van Eerd et al. 2003). The number of
128 H. S. Gariya and A. Bhatt
pesticides is usually very large that is being used by farmers, and their consequences
are almost proportional to their number. The pesticides are linked to various effects
on humans as well as on the ecosystem. It can cause pulmonary and hematological
morbidity as well. The various types of birth disorders are also related to these
pesticides. The farmers are more prone and vulnerable to the special risk associated
by coming in contact of pesticides and their byproducts. These contaminants can
contaminate the food in the food chain. Fishes are also prone to be contaminated by
the pesticides because pesticides are very harmful in the native as well as byproduct
form. Ultimately, the consequences are disturbed food chain, reproductive failure,
deformities in children, poor health, and teratogenic effects. Before going to the
microbial mechanism of action, it is important to know about the isolation and
screening of pesticides degrading bacteria from nature (Gangola et al. 2018b;
Gupta et al. 2018; Khati et al. 2018b).
8.2 Isolation and Enrichment of Pesticide-Degrading
Microbes
The bacterial and fungal strains have the potential to degrade the pesticides from
the contaminated environment. The contaminated environment mainly includes the
soil and water system. Pesticides acted as the sole source of carbon, nitrogen, and
phosphorous for the growth and reproduction of bacteria. Media provide the nutri-
ents to bacteria in optimal quantity. Both solid and liquid mediums in the laboratory
can be used for the cultivation of pesticide-degrading microbes. Making any material
contamination free is known as the sterilization. Sterilization can be done by heat or
steam and by other methods also. But generally, autoclave is used to sterilize any
material, e.g., media, petri plates, beaker, flask, etc. The media which is selected for
the desired organism is allowed to grow and suppression of undesirable microor-
ganisms with suitable modification in the composition of media (Zhang et al.
2020a,b; Mishra et al. 2020; Feng et al. 2020; Lin et al. 2020; Zhan et al. 2020).
The soil sample is serially diluted to reduce over crowdedness, and isolation of CFU
Table 8.2 Techniques for the remediation of pesticides as well as for the xenobiotics
Treatment process Examples
Physical treatment Adsorption, percolation with variable size pore filters
Chemical treatment Strong oxidizing agents
Photocatalysis Titanium oxide
Incineration High temperature
Photodegradation Light
Acid hydrolysis Strong acid
Alkaline hydrolysis Strong base
Microorganism Pseudomonas, flavobacterium, alcaligenes
GMO (genetically modified organisms) Via RDT-modified organisms
8 Microbial Action on Degradation of Pesticides 129
is possible. Dilution factor is also very important in the calculation of bacterial
numbers. The sample is then poured on to the media with the specific concentration
of the pesticides, enabling to support the growth of pesticide-resistant bacteria. The
concentration of the pesticides can be varied to check the up-to-what concentration
the target bacteria can degrade it and resist it (Gangola et al. 2018b; Gupta et al.
2018; Khati et al. 2018b,2017a; Kumar et al. 2017).
8.3 Characterization of the Pesticide-Degrading Microbes
The microbiological, biochemical, and morphological methods have been used for
the characterization of the pesticide-degrading microbes. The surviving bacteria can
be then isolated in form of pure colonies and can be inoculated to the specific
contaminant sites. Several microbial floras such as bacteria, fungi, actinomycetes,
algae, and plants are useful in degradation process. Fungi and bacteria are involved
in the biodegradation process that releases hydrolytic enzymes, peroxidases, and
oxygenases (Van Eerd et al. 2003). Microbial action on the toxic chemical pesticide
breaks down them and in reciprocation takes carbon source for their efficient
metabolism. The microorganism can be isolated from the soil and inoculated to
contaminated sites that help to get rid out of intoxicants. Various parameters are
important for the microbial activity to degrade them. So, by providing the appropri-
ate supplements and various appropriate influencing supplements, biodegradation
can be enhanced up to such a desirable extent that is not harmful. Few microorgan-
isms are specialized to degrade the specific toxic chemical compound; then micro-
organisms can be isolated, cultured in a suitable feeding media, and inoculated to the
contaminated sites or in a place where there is high exposure to harmful chemicals
pesticides. Microorganisms can be categorized according to their specific biode-
gradability to a compound so that biodegradation efficiency can be accelerated.
Accelerating the rate of biodegradation is done by the following methods: addition
of the surfactant, supplementation with appropriate inoculant nutrients, and control-
ling the environmental factor.
8.4 Microbial Activity Mechanism to Degrade Pesticides
8.4.1 Degradation of Parathion
In the aerobic pathway of degradation, parathion in the presence of phosphodiester-
ases (cleavage of phosphodiester bond) hydrolyzes to form the diethyl-thio-phos-
phoric acid and para-nitro-phenol. In other sets of aerobic pathways, the parathion
undergoes the oxidation process in the presence of an oxidoreductase enzyme. This
pathway results into the di-ethyl-phosphoric acid and para-nitro phenol as same as
above pathway, but the intermediate compounds are different. In the anaerobic
130 H. S. Gariya and A. Bhatt
pathway, parathion undergoes the reduction process to produce amino parathion as
an intermediate which after the process of hydrolysis results into the benzoquinone.
8.4.2 Atrazine Degradation
There are several methods to degrade the toxicant from the environment enlisted
above. The Pseudomonas species used the compound atrazine as a carbon source
and via the help of several enzymes for efficient degradation. Enzymes used by
Pseudomonas ADP strain are AtzA, AtzB, and AtzC. AtzA helps in hydrolysis and
dechlorination. The protein AtzB performs the dehydrochlorination reaction that
converts the hydroxyatrazine to the N-isopropyl cyanuric acid. AtzC catalyzes the
cyanuric acid produced in the above step. Finally, the end product remnants are
carbon dioxide and ammonia. It had been strongly suggested that genes encoding
these degradative enzymes are expressed by the extra chromosome known as a
plasmid. For example, the enzymes degrading the 2–4D are absolutely encoded by
the bacterial plasmid. Several genes are retained on the main bacterial chromosome
for the expression of degradative enzymes (Bhatt et al. 2021d).
8.5 Pesticides Used in the Agriculture
Organochlorine pesticides are widely used in the agricultural sector due to their rapid
action and easy to use. These include hexachlorocyclohexane, 111, DDT (dichloro-
diphenyl-trichloroethane), methoxychlor, dieldrin, chlordane, toxaphene, mirex,
kepone, lindane, etc.
8.5.1 Steps in Biodegradation Mechanism
Degradation mechanism is broadly categorized into mainly three parts: adsorption,
penetration, and enzyme catalysis reaction. Adsorption is a physical phenomenon
that is only on the surface accumulation of substrate also known as physisorption.
Penetration includes the entry of the target chemical compound into the desired cell.
Finally, at last, the enzyme catalysis reaction involves the rapid degradation into
the simpler non-toxic product. The bio-stimulation-based degradation process can be
accelerated via the supplementation of appropriate and additional stimulating agents,
e.g., surfactant and electron donor (water, hydrogen, sulfide). The transformation of
the organic compound into the non-toxic or less toxic inorganic compound under the
action of soil biota via microbial activity is partially or completely degraded.
Through this process, it can degrade the various toxic chemical compounds from
the environment. Co-metabolism also known as the co-substrate inoculation, the
8 Microbial Action on Degradation of Pesticides 131
exogenous addition of suitable biomass or supplementation, rapidly breaks down the
toxic chemical pesticides. Synergistic effect consortium of microbial biomass has
increased efficiency rather than the single microbial degradation (Sulbhi et al. 2021;
Bhandari et al. 2021; Bhatt et al. 2021a,b,c). Synergistic or additive effects can
degrade the toxic chemical compound rapidly comparable to single microbial
degradation of pesticides. For example, the insecticide parathion is degraded syner-
gistically with the combined effect of P. aeruginosa and P. stutzeri. Genetically,
modified organisms are used to increase the rate of degradation. RDT is the possible
way to manipulate the appropriate gene and subsequently degradative process.
Bioventing is a process of bio-stimulation by which stimulating agents are added
into the soil to increase the microbial catabolic activity (Van Eerd et al. 2003;
Nadeau et al. 1994).
8.5.2 Factors Affecting Pesticide Degradation
There are various parameters that influence the efficient degradation. Providing
appropriate condition has increased microbial activity to degrade the pesticides
(Table 8.3).
8.5.3 Chemistry of Pesticide Degradation
The following are some of the reasons with respect to chemistry for the degradation
of pesticides (Table 8.4).
•Linear or aliphatic hydrocarbon compounds are easy to degrade rather than
aromatic compounds.
•Ring restricts the ease of its biodegradation.
•High molecular weight, larger chain compounds are difficult to be degraded.
•Branching decreases the efficiency of degradation.
•Molecular position of functional substituent group has large effect on
degradation.
Table 8.3 Factors influencing the microbial degradation
Factors Examples
Types of microbial
species
Pseudomonas strain specific to atrazine
Effect of pesticide
chemistry
Functional groups, molecular weight, chemical nature
Environmental factor pH, salinity, humidity, nutrition, oxygen availability, substrate
concentration
132 H. S. Gariya and A. Bhatt
8.5.4 Successful Biodegradation Process of Chemical
Compound
•Microorganism catabolic activity on the target substrate.
•The toxic target compound must be bioavailable.
•Soil conditions must favor microbial growth and activity.
•The cost of remediation should be less expensive.
8.6 Microbial Enzyme System
Intracellular and extracellular enzymes play an important and key role in the
degradation of xenobiotics. Various enzymes have been reported to degrade the
organochlorine such as the dioxygenases (Nadeau et al. 1994), cytochrome P450
(the great variety of reactions catalyzed by P450s was well recognized, ligninases)
(Pang et al. 2020), dehydrogenases (Bourquin 1977), esterases, and glycosidases.
These enzymes have been documented for the degradation and detoxification of
xenobiotics. Carbamate degradation (Parekh et al. 1995) has been reported by a wide
genus of bacterial species (Table 8.5).
Table 8.4 Microorganism specific to the chemical pollutant
Microorganism Toxic chemical compound
Pseudomonas sp. Linear (aliphatic) and aromatic hydrocarbons
Corynebacterium sp. Halogenated hydrocarbons
Bacillus sp. High molecular wt. hydrocarbons
Candida sp. PCBs
Fusarium sp. Propanil
Nocardia sp. Naphthalene
Table 8.5 Microorganism
with specialized
catabolic gene
Microorganism Catabolic gene
E. coli pepA
Bacillus cereus phn
SMSP-1 opdB
Nocardia sp. adpB
Pseudomonas monteilii hocA
A. radiobacter opdA
8 Microbial Action on Degradation of Pesticides 133
8.7 Enzyme System for Organophosphate Degradation
Organophosphate compounds are present everywhere in the surroundings and used
in high quantity in the agricultural sector. It can also be used as a weapon tool to
threaten other nation as well. So, there is a need to completely remove these toxic
compounds or convert them to simpler non-toxic. The phosphate ester of alcohol and
the phosphoric acid combines and resulting in the formation of organophosphate. A
wide range of pesticides contains this organophosphate compound, and that can also
affect our nervous system severely. OP can cause chronic or acute symptoms of
diseases depending upon its concentration and exposure to the organs. Organophos-
phate can be removed from the environment by the use of chemical decontamina-
tion, physical adsorbent, and finally an efficient microbial system. In human
neuromuscular junction or synapse, an enzyme acetyl-choline-esterase found
which break acetyl choline (Ach) and in similar way organo-phosphate compound
bind irreversibly with the acetyl choline. Organophosphate chemical compounds can
be degraded through the nucleophilic attack of the phosphorous center of compound.
Some of the common enzymes are peroxidases, laccase, esterase, and oxidoreduc-
tase. Pesticide degradation characteristics are analyzed by following techniques GC–
MS, GC–MS/MS, and LC–MS/MS for two filed trials, high-resolution MS-based
methods, liquid chromatography (LC) with ultraviolet, and diode array (Mishra et al.
2020; Zhang et al. 2020b; Feng et al. 2020; Lin et al. 2020; Zhan et al. 2020; Ye et al.
2019; Huang et al. 2019,2020).
8.8 Cyclic Wave Voltammetry
It is the combination of the square wave potential and staircase potential applied to a
static electrode. It is a form of linear sweep voltammetry. In a simple word, it is the
combination of a square wave superimposed onto a staircase potential. This SWV
involves sweeping the potential linearly with time at a rate between 10 and 100 V/s.
The advantageous properties of this technique are fast scan rate, speed analysis,
sensitivity, low detection limits, and reduced analysis time and reject background
signal (Khati et al. 2017a,b; Van Eerd et al. 2003).
8.8.1 Amperometry
It determines diffusion current; potential fixed electrode and reducible and
non-reducible agents can be determined. Amperometry is based on the principle of
polarography with the exception that the voltage is maintained constant during
titration (Castro et al. 1985; Parte et al. 2017; Pizzul et al. 2009). Here the
electroreducible and electro-non-reducible analytes can be analyzed (Table 8.6).
134 H. S. Gariya and A. Bhatt
8.8.2 Methodology
•The potential of the instrument is fixed.
•Take sample solution and add supporting electrode.
•The titrant is added and current flow is measured in mA.
•It can determine concentration up to 0.0001 molars of analyte in the target
sample.
The other voltammetry techniques for the pesticide’s detection are differential pulse
voltammetry, electrochemical impedance spectra (EIS), differential pulse polarog-
raphy (DPP), anodic stripping voltammetry, Kaloussek’s switcher, and
oscillopolarography.
8.9 Conclusion
As the population natality rate is exponentially increasing, to meet their demands
agricultural sector has to take care and deal to meet their demands. The agricultural
sector relies on technological advancement to decrease the productivity loss and
enhance productivity to prevent a food crisis. Thus, fulfilling these above require-
ments, modern methods of agro-farming using chemical compounds are taking
place. These modern methods that practice to use pesticides besides harmful effects
on the ecosystem can also cause short- and long-term effects of diseases and
disorder, with acute and probably chronic symptoms. The only way to cope up
with the harmful impacts of modern chemical compounds is by monitoring their use
and analyzing their negative effects on the environment. Government and the
agricultural sector should take care of the use of modern chemical compounds and
also their constant monitoring.
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with esterase: bioremediation potential and mechanism. Bioresour Technol 315:13845. https://
doi.org/10.1016/j.biortech.2020.123845
Bhatt P, Verma A, Verma S, Anwar MS, Prasher P, Mudila H, Chen S (2020b) Understanding
phytomicrobiome: a potential reservoir for better crop management. Sustainability 12:5446
136 H. S. Gariya and A. Bhatt
Bhatt P, Huang Y, Rene ER, Kumar AJ, Chen S (2020c) Mechanism of allethrin biodegradation by
a newly isolated Sphingomonas trueperi strain CW3 from wastewater sludge. Bioresour
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Bhatt P, Zhang W, Lin Z, Pang S, Huang Y, Chen S (2020d) Biodegradation of allethrin by a novel
fungus Fusarium proliferatum strain CF2, Isolated from Contaminated. Soils. Microorganisms
8:593
Bhatt P, Huang Y, Zhang W, Sharma A, Chen S (2020e) Enhanced cypermethrin degradation
kinetics and metabolic pathway in Bacillus thuringiensis strain, SG4. Microorganisms 8:223
Bhatt P, Bhatt K, Huang Y, Ziqiu L, Chen S (2020f) Esterase is a powerful tool for the biodegra-
dation of pyrethroid insecticides. Chemosphere 244:125507
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biodegradation of organophosphate, carbamate and pyrethroid group pesticides. J Hazard Mater
411:125026
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oxidoreductase and C-P lyase: molecular docking and molecular dynamics simulation studies. J
Hazard Mater 409:124927
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microbial consortia for the removal of pesticides from the environment. Crit Rev Biotechnol
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Bhatt P, Sethi K, Gangola S, Bhandari G, Verma A, Adnan M, Singh Y, Chaube S (2021d)
Modeling and simulation of atrazine biodegradation in bacteria and its effect in other living
systems. J Biomol Struct Dyn. https://doi.org/10.1080/07391102.2020.1846623
Bhatt P, Gangola S, Bhandari G, Zhang W, Maithani D, Mishra S, Chen S (2021e) New insights
into the degradation of synthetic pollutants in contaminated environments. Chemosphere
268:128827. https://doi.org/10.1016/j.chemosphere.2020.128827
Bhatt P, Sharma A, Rene ER, Kumar AJ, Zhang W, Chen S (2021f) Bioremediation mechanism,
kinetics of fipronil degradation using Bacillus sp. FA3 and resource recovery potential from
contaminated environments. J Water Process Eng 39:101712
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Microbiol 33:356–362
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polyhalomethanes. Biochemistry 24:204–210
Fan X, Ye T, Li Q, Bhatt P, Zhang L, Chen S (2020) Potential of a quorum quenching bacteria
isolate Ochrobactrum intermedium D-2 against soft rot pathogen Pectobacterium carotovorum
subsp. Carotovora. Front Microbiol 11:898
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degradation: current status and future perspective. Front Microbiol 11:389
Gangola S, Bhatt P, Chaudhary P, Khati P, Kumar N, Sharma A (2018a) Bioremediation of
industrial waste using microbial metabolic diversity. In: Pankaj, Sharma A (eds) Microbial
biotechnology in environmental monitoring and cleanup. IGI Global, Hershey, PA, pp 1–27.
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phytic fungus from Centella asiatica (L.) Urban. World J Microbiol Biotechnol 34:111
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current achievements and perspectives. Front Microbiol 10:1754
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the microbial degradation of D-cyphenothrin in contaminated water/soil environments. Micro-
organisms 8:473
Huang Y, Zhang W, Pang S, Chen J, Bhatt P, Mishra S, Chen S (2021) Insights into the microbial
degradation and catalytic mechanism of chlorpyrifos. Environ Res 194:110660
8 Microbial Action on Degradation of Pesticides 137
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nanocompounds on soil microbial activity: an indicator of soil health. Soil, Air, Water 45
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with soil health maintenance. 3 Biotech 7(1):81
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fertilizers and sustainable development. Arch Microbiol 203:1259. https://doi.org/10.1007/
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138 H. S. Gariya and A. Bhatt
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quorum quenching candidate against bacterial pathogen Xanthomonas campestris
pv. Campestris. Front Microbiol 10:2867
Zhan H, Huang Y, Lin Z, Bhatt P, Chen S (2020) New insights into the microbial degradation and
catalytic mechanism of synthetic pyrethroids. Environ Res 182:109138
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acid precursors from various environmental system: advances and perspectives. Environ Pollut
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(γ-Hexachlorocyclohexane) using a microbial system. Front Microbiol 11:522
8 Microbial Action on Degradation of Pesticides 139
Chapter 9
Biofortification of Plants by Using Microbes
Ankur Adhikari, Kamal Pandey, Vinita Pant, Tara Singh Bisht, and
Himanshu Punetha
Abstract In the twenty-first century, a key obstacle is meeting the hunger needs of
rising population every day. Thus, the efficiency of cultivated land and food
fortification needs to be improved. The sole objective of the latest agronomic
practices, particularly with regard to food crops, is to maximize grain size and
weight, even at the price of nutritional value, aiding in few cases to scarcity in
micronutrients (termed as hidden hunger). Life-threatening health situations and
ailments such as birth defects, heart disease, cancer, nerve disorders, and several
others can be caused by hidden hunger. Micronutrient deficiencies are caused by the
lessen amount of micronutrients in food crops and are one of the major threats
affecting more than two million people worldwide. Plant growth-promoting
microbes (PGPM) are categorized in two major groups: plant growth-promoting
rhizobacteria (PGPR) and plant growth-promoting fungi (PGPF), which are feasible
ways to solve this issue. In staple foods, the use of biofortified nutrients and mineral
content can be enhanced. These microbes help to increase micronutrient uptake (e.g.,
phosphorous, iron, selenium, and zinc). Plant-microbe relationships and our
expanding understanding of these interactions can help to biofortify crops with
micronutrients in a sustainable and environmentally friendly manner.
Biofortification of food crops is indeed espoused as a novel method not only to
raise micronutrient concentration in edible food crops but also to boost crop pro-
ductivity on scarce fertile soils. PGPR are soil bacteria that are responsible of
colonizing the rhizosphere and strengthening the development of plants through a
A. Adhikari (*) · H. Punetha
Department of Biochemistry, College of Basic Sciences and Humanities, Gobind Ballabh Pant
University of Agriculture and Technology, Pantnagar, Uttarakhand, India
K. Pandey
Department of Biotechnology, Kumaun University, Bhimtal, India
V. Pant
Indian Council of Agricultural Research-Directorate of Cold Water Fisheries Research, Bhimtal,
Uttarakhand, India
T. S. Bisht
Patanjali Research Institute, Haridwar, Uttarakhand, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_9
141
wide range of different mechanisms, such as organic matter mineralization, biolog-
ical control of soil-borne pathogens, N
2
biological fixation, and root growth devel-
opment. The nutrients are mobilized by microorganisms through multiple methods,
such as chelation, acidification, exchange reactions, and organic acid release. In
addition, several pieces of information reveal that plant inoculation at both physio-
logical and molecular levels with PGPR will have significant effects on plants.
Keywords Biofortification · Plant growth-promoting microorganisms ·
Micronutrients · Siderophores · Rhizosphere
9.1 Introduction
A lot of pressure has been put on agriculture to support the continually growing
human population, by combating not only the hunger but also the “hidden hunger.”
Given the increased food supply-demand and abiotic stresses related to climate
change, plant’s growth, productivity, and nutritional value are getting affected to a
great extent (Roriz et al. 2020). Till now, the concern was to increase the overall
yield to mitigate the food insecurity issues which was significantly resolved by green
revolution. Another upcoming global issue which needs to be addressed is of
malnutrition due to insufficient dietary intake of micronutrients which is regarded
as hidden hunger. Various symptoms manifested by humans, resulting from
micronutrient-deficiency, are stunted growth, respiratory infections, anemia,
impaired mental development, and increased risk of mortality during childbirth
(Singh et al. 2020). It is predominant in poor developing countries because their
major staple food is deprived of some essential micronutrients and they cannot afford
dietary diversification, so the focus has now been shifted to producing food that is
nutritionally rich in a sustainable way. The State of Food Security and Nutrition in
the World has penned in its latest edition that around 690 million people went
hungry in 2019, up by ten million from 2018, and by ~60 million in the last
5 years, according to a WHO 2020 press release. A warning has been put forth by
the heads of five principal agencies, namely, FAO, UNICEF, IFAD, WFP, and
WHO, “five years after the global commitment to end hunger, food insecurity and
all forms of malnutrition, this goal is still off-track by 2030.”It is also reported that
COVID-19 pandemic will intensify this issue. There is a dire need to respond in a
sustainable manner to meet the current societal obstacles of ensuring the production
of foods with an elevated nutritional content associated with a low
environmental diet.
Biofortification has been deployed as a common agricultural practice that will
curb hidden hunger, fulling all the constraints mentioned above. Current strategies of
biofortification like organic and chemical fertilizer application, elite germplasm
breeding, and metabolic engineering come with various drawbacks. Breeding and
CRISPR/Cas9 technologies, for example, can be exploited to improve the micronu-
trient value of crops; however, they are time-consuming and costly and encounter
142 A. Adhikari et al.
multiple challenges with genetically modified organisms (GMOs). In addition, the
utilization of microbes for biofortification has been advocated as a “greener”sub-
stitute for sustainable agriculture due to the environmental hazards from the unnec-
essarily high usages of chemical fertilizers. Given their nutrient mobilization
potential, microbes-based biofortification is the most inexpensive, quickest, and
sustainable way to increase the bioavailability of micro- and macronutrients in our
diets while simultaneously restoring soil health (Singh et al. 2011).
9.1.1 Plants’Hidden Hunger
There are 17 essential elements, required by plants in order to complete their life
cycles, categorized into two groups: macronutrient, required in large quantities, and
micronutrient, required in trace amounts. Macronutrient consists of nine elements N,
P, K, C, O, H, Ca, S, and Mg, whereas micronutrient consists of eight elements, viz.,
Fe, Mo, B, Zn, Mn, Ni, Cl, and Cu (Blevins 2009). The micronutrient is an integral
part of plant’s metabolic pathways as they facilitate catalytic reactions, function as
protein cofactors, and/or stabilize structural domains. Various studies have shown
that even a deficiency of any of the soil’s micronutrients can restrict the ideal plant
yield and contribute to low food products, amidst the adequate presence of all other
nutrients. Plants suffering from hidden hunger become vulnerable to various biotic
and abiotic stresses (Ahmad and Prasad 2011). It is predicted that the issue of hidden
hunger is projected to be amplified by climate change (Ku et al. 2019). Climate
change, typically rising temperatures, changing precipitation patterns, varying levels
of soil water, and elevated atmospheric concentration (CO
2
) have long been
established to have intense repercussions for the nutritional content of crop products
(Nakandalage and Seneweera 2018).
9.1.2 Biofortification
Biofortification is the strategy of strengthening the nutritional efficiency of food
crops through agronomical practices, traditional plant breeding, or modern biotech-
nology, as defined by the WHO (WHO 2016). Plant biofortification is pertinent, as
micronutrients are inadequate in the three staple crops, rice, maize, and wheat, which
make a significant contribution almost half of the calories devoured by humans.
Conventional and genetic breeding are the prime approaches; the objective is to
maximize the bioavailable micronutrient content in principal food crops such as
maize, rice, wheat, pearl millet, etc. (Velu et al. 2014; Prasanna et al. 2016).
The insight of the complicated plant-microbe rhizospheric relationships has
fueled studies into the role and application of PGPB in the crop biofortification.
The role of PGPB in plant growth and defense was first stated by Kloepper
(Kloepper et al. 1980), and since then, a lot of studies has been conducted. They
9 Biofortification of Plants by Using Microbes 143
are now popularly being considered as an efficient and environmental-friendly
alternatives to chemical pesticides and fertilizers (Olivares et al. 2015). Exploiting
this plant-microbe interaction can further benefit in achieving biological substrate
enrichment, and this is imperative as the micronutrients are inadequate in three staple
crops rice, maize, and wheat, which help in providing almost half of the calories
ingested by humans. Biofortification can be achieved through various approaches:
1. Gene manipulation
2. Transgenic approaches
3. Organic fertilizer
4. Chemical fertilizer
5. PGPB mediated
In 1999 came the first biofortified staple crop, “golden rice,”the genetically
modified rice that additionally contains beta-carotene. One of the most important
systematic and symbolic breeding programs, the Harvest Plus program, was initiated
in 2004 to mitigate micronutrient malnutrition in Africa and Asia. The scheme
centered on enriching rice, beans, cassava, maize, sweet potatoes, and pearl millet,
focusing on three main nutrients, Fe, Zn, and vitamin A (Pfeiffer and McClafferty
2007). Carvalho and Vasconcelos (2013) and Garg et al. (2018) stated that the
efficiency of any food fortification programs depends heavily on the acquiescence
of farmers and the public and also on political assistance to determine their cost/
benefit. This lack of public acceptance is a sole reason why golden rice is not yet
commercialized, in spite of its clearly identified benefits.
9.1.3 Plant Growth-Promoting Bacteria (PGPB)
PGPB are those bacteria that intensify the growth of plant and also protect it from
various diseases and abiotic stresses by inducing number of compounds such as
phytohormones (cytokines, gibberellic, ethylene, and indole-3-acetic acid), atmo-
spheric nitrogen fixation, siderophores, organic acid, and phosphate solubilization
and by producing antibiotics which help to decrease the detrimental effect of
pathogens (Ji et al. 2019; Glick 2012; Felestrino et al. 2017). These ecofriendly
and beneficial bacteria inhabit near the plant roots area which is known as rhizo-
sphere. Plant release numerous nutrients (sugar, organic acid, amino acids, vitamin,
flavonoid, and lipid), which is known as root exudates, into the rhizosphere that
leads to relocation of soil bacteria toward these nutrients (as these root exudates
attract the bacteria), and therefore, plethora of microorganism are found inhabited in
rhizosphere (Schillaci et al. 2019). Proteobacteria (Pseudomonas,Acinetobacter,
Serratia,Pantoea,Psychrobacter,Enterobacter,andRahnella) and Firmicutes
(Bacillus sp.) are the two phylum to which PGPB belongs (Ramakrishna et al.
2019). They encourage plant growth by two mechanism, i.e., direct and indirect.
Direct mechanism includes N
2
fixation, siderophore production, phosphate
144 A. Adhikari et al.
solubilization, phytohormone production, and 1-Aminocyclopropane-1-carboxylate
(ACC) deaminase, whereas in indirect one, PGPB create hindrance in the growth of
pathogen (Ahemad and Kibret 2014).
N
2
plays important role in the photosynthesis of plant as it is the vital component
of chlorophyll pigment (Wagner 2011). Even though abundant amount of N
2
is
present in the atmosphere, plants utilize only reduced form of it. Biological nitrogen
fixation (BNF) is a process in which prokaryotic microorganisms convert the
atmospheric N
2
into plant usable reduced form, i.e., ammonia (NH
3
)(O’Hara
1998; Cheng 2008). Bacteria that carried out nitrogen fixation are known as
diazotrophs, and they carry a nitrogenase enzyme that acts as catalyst, hence fasten
the transformation of N
2
into ammonia (Santi et al. 2013). Genes which control the
symbiotic N
2
fixation in rhizobia are nod,nif, and fixgenes (Bano and Iqbal 2016).
N
2
-fixing bacteria are classified as symbiotic N
2
-fixing bacteria such as rhizobia in
leguminous plant and Frankia in non-leguminous plant and non-symbiotic N
2
-fixing
bacteria (Cyanobacteria,Azospirillum,Azotobacter, etc.) that are free living and
endophytes (Ahemad and Kibret 2014). Legume crop supply food and energy to
rhizobia and in lieu of that rhizobia provide ammonia to crop (Lindström and
Mousavi 2020), and this ammonia is used by plant for the synthesis of biomolecules
such as nucleic acid and proteins that is required for the proper growth and devel-
opment of plant (Soumare et al. 2020; Babalola et al. 2017). Siderophores are iron
(Fe)-chelating compound having low molecular weight produced by soil organism.
Its function is to encourage plant growth and hamper the phytopathogens by
arresting Fe from the environment (Maheshwari et al. 2019). Phosphate-solubilizing
bacteria (Pseudomonas,Rhizobium,Bacillus, etc.), another direct mechanism, con-
vert the insoluble form of phosphate into plant utilizable form, i.e., H
2
PO
4
and
HPO
42
via process of chelation, exchange reaction, acidification, as well as pro-
ducing gluconic acid (Rodríguez et al. 2006). Phytohormones (auxin, cytokinins,
gibberellins, and inhibiting ethylene production) are produced by bacteria and
liberated in the rhizosphere by rhizobacteria or in plant tissues by endophytes.
These hormones protect plant from biotic as well as abiotic stresses. Apart from
that, it also helps in the growth, development, and physiological activity of plant
(Esitken et al. 2010; Shilev 2020). High concentration of ethylene can inhibit the
plant growth and can even kill the plant. ACC deaminase enzyme degrades the
precursor of plant ethylene, i.e., ACC into ammonia and α-ketobutyrate. As a result,
concentration of ethylene lowers (Glick 2014; Hao et al. 2007) that helps in
encouraging the growth of plant in adverse conditions.
9.2 Microbe-Mediated Biofortification
In order to improve yield and soil fertility, biofortification of crops via the effects of
PGPM can be seen as a successful concomitant indicator which increased micronu-
trient concentrations in the food crop may occur along with transgenic cultivars. In
addition to strengthening soil fertility and crop yield, PGPM has been observed to
9 Biofortification of Plants by Using Microbes 145
biofortify the micronutrient content in food crops (Rana et al. 2012). In addition to
insoluble phosphorus solubilization, through N
2
fixation, phytohormone synthesis,
reduction of ethylene concentration, synthesis, and induction of systemic resistance
to antibiotics and antifungal metabolites, plant growth will be enhanced by PGPM as
well (Singh and Prasad 2014). Thus, biofortification of crops utilizing PGPMs can be
seen as a potential additional way to enhance micronutrient concentrations through-
out the cultivation of wheat, in addition to improvement of soil fertility and yield
alongside breeding varieties (Singh et al. 2017). All the more as of late, a compre-
hension of the perplexing interaction among microbial and plant networks in the
rhizosphere has fueled the exploitation of soil microorganisms for the crop
biofortification. Bacteria and mycorrhizal fungi have the significant contribution in
the assimilation of the micronutrients in plants which is reported in the literature
(Wu et al. 2015; Berruti et al. 2016). When the connection between beneficial
bacteria and plants is utilized, the rhizosphere, a crucial layer among plant roots
and the soil, can make a substantial contribution to agriculture. In particular, this
chapter will be focusing on biofortification of crop through mycorrhizal and bacterial
mobilization of the rhizosphere-based micronutrients (Prakash and Verma 2016; Jha
and Subramanian 2016). The biofortification of major crops by exploiting these
PGPR has been the focus of plant-microbe interactions. To ensure that the human
body works properly, micronutrients such as Se, Zn, and Fe are vitally important,
and these constitute a major part of PGPR-mediated biofortification (Priyadharsini
and Muthukumar 2016; Kumar et al. 2017; Raghavendra et al. 2016; Zahedi 2016;
Dominguez-Nuñez et al. 2016; Dotaniya et al. 2016).
Colonized plants of arbuscular mycorrhizal fungi (AMF) can efficiently procure
nutrients from a greater volume of soil, well outside the nutrition depletion region to
which the roots of the plant unable to reach. AMF then stimulates the plant to
improve and enhance its efficiency. As a result, the host plant supplies AMF along
the sugar necessary to finish its life cycle. The host plant, in exchange, supplies AMF
with the carbohydrates in order to accomplish its life cycle (Zhang et al. 2015). In
process of extracting pivotal nutrients from the soil, such as Fe, K, Cu, P, N, and Zn,
the relationship of AMF with roots provides plants with exposure to absolutely vital
nutrients; this contributes to the solubilization, mobilization, and uptake of such
principal nutrients needed for development (Clark and Zeto 2000; Chen et al. 2020).
Incorporation of AMF under field condition, in the soil of Cicer arietinum plants,
enhanced the biomass, yield, and dietary component of the legume and
biofortification of zinc and iron (Pellegrino and Bedini 2014). The AMF also
accelerates the relationships accompanied by rhizobacteria, along with valuable
rhizobacteria that can directly induce AMF spore germination and root colonization.
Hyphal exudates of AMF greatly impact microbial communities and therefore can
accelerate beneficial bacterial-plant interactions and therefore strengthen the health
and plant growth (Qin et al. 2016; Cruz and Ishii 2012; Schillaci et al. 2019).
Co-inoculation of AMF and advantageous bacteria like Pseudomonas and
selenobacteria vastly enhances wheat production and phosphorous, iron, manganese,
copper, potassium, zinc, and selenium mineral nutrient concentrations in wheat
grains (Mäder et al. 2011; Durán et al. 2013). In the soybean/maize intercropping
146 A. Adhikari et al.
system, twofold supplementation of arbuscular mycorrhizal fungi and rhizobium
substantially accelerated the pace of soybean N
2
fixation and strengthened the
transport of N
2
from soybean to maize, contributing to improved yields for both
crops (Meng et al. 2015). An even more significant benefit of arbuscular mycorrhizal
fungi is the discharge of large molecular weight glycoprotein that stabilizes soil
aggregation, aggrandizes a healthier soil and plant-soil network, and improves
phytoextraction of aluminum, arsenic, cadmium, mercury, and lead (Wu et al.
2015; He and Nara 2007). AMF’s primary function in nutrient supply and crop
production is the main determinant in the biofortification of crops utilizing soil-
based essential mineral components. Via symbiotic relationship with AMF, crops
benefit immensely from plant-microbial interactions, and this could assist in reduc-
ing the utilization of chemical fertilizers and raising nutrient availability. As a
consequence, AMF could perform a major contribution in the biofortification of
micronutrient-containing crops, while also contributing to better nutrition and sus-
tainable crops that can tackle global malnutrition. Agriculture integrating AMF as
well as other valuable microbes is essential, which include increased biofortification
of Fe, Zn, provitamin-A, carotenoids, aminoacids, and proteins in the crops that are
fiscally valuable include rice, wheat, sorghum, cassava, potatoes, maize, etc. (Mayer
et al. 2008; Khush et al. 2012).
9.2.1 General Mechanisms
In attempt to maximize plant growth and yield, multiple PGPM pathways had also
been reported. Selenium is categorized as a metalloid and is available as elemental
Se(0), selenide(2
), selenite(4
+
), and selenate(6
+
) in different valences in the envi-
ronment, and plant roots do have tendency to process Se as compounds of selenate,
selenite, or oganoselenium, such as selenocysteine and selenomethionine (White
et al. 2004,2007). Selenate is perhaps the most frequently used source of Se in
plants. The ability of PGPR to respond as Se-biofortification agents has been
analyzed on the basis of research carried out over the past two decades. To improve
selenium accumulation and volatilization, Brassica juncea could well be assisted by
PGPR (De Souza et al. 2002). In axenic plant experiments, rhizobacterial strains
classified as BJ15 and BJ2 had been employed to evaluate the influence of
rhizobacteria in accumulation and volatilization of Se. While these two strains had
been applied in the rhizosphere of axenic Brassica juncea plants, the intensity of Se
volatilization from selenite (4
+
) was significantly four times significantly higher than
those of the axenic control plants, and when comparison to axenic controls, plants
managed to accumulate more Se in their tissues (De Souza et al. 2002). Isolates
either from Bacillus and Klebsiella (strains E5 and E1), Bacillus (strain E8.1), or
Acinetobacter (strain E6.2) genera impeded in vitro propagation of
Gaeumannomyces graminis variety tritici mycelia by ~100%, 50%, and 30%,
respectively, increasing the efficiency of PGPR for biofortification agents which
not only improve the accumulation of selenium in the plant but also act as feasible
9 Biofortification of Plants by Using Microbes 147
crop protection agents (Durán et al. 2014). PGPM-synthesized compounds are
siderophores (low molecular weight compounds (<10 kDa)) which are eventually
used by crops escorting the Fe molecule to the plant cells. Through optimizing Fe
ingestion, and hindering plant pathogen through competition, microbial
siderophores can boost plant growth eventually resulting in Fe biofortification of
plants (Srivastava et al. 2013). A broad spectrum of bacterial species, viz., Azoto-
bacter,Azospirillum,Burkholderia,Bacillus,Enterobacter,Arthrobacter,
Rhodospirillum,Pseudomonas,Serratia, and Rhizobium, and fungal species, viz.,
Rhizopus,Syncephalastrum,Aspergillus, and Penicillium, produce siderophores
(Leong and Neilands 1982; Das et al. 2007; Durán et al. 2016; Srivastava et al.
2013). Masalha et al. (2000) experimented that the sterile soil and non-sterile soil
contribute significant affects in the uptake of iron in Helianthus annuus
L. (Sunflower). The concentration of iron in aseptic soil was ~248 29 μgg
1
,
but in non-sterile soil (~1748 48 μgg
1
), the uptake was significantly high,
showing that the iron transport from the soil to the plant was enhanced by microbes.
In a biocontrol capacity, siderophores can also be engaged, with the exception of the
rhizospheric soil, absorbing Fe and making it inaccessible to any other pathogen
(Siddiqui 2005). By triggering Fe-deficient reaction in the plant, microbes provoke
hormonal compounds similar to plant hormones that strengthen Fe increase. By
inducing transcriptional upregulation of FIT1 (Fe deficiency-induced transcription
factor 1), Bacillus subtilis GB03 has the capability to exacerbate Fe procurement in
Arabidopsis. The FIT1 is required for the acquisition of iron at the plant for the
commencement of ferric reductase FRO2 and IRT1 Fe transporters (Zhang et al.
2009). In the mere existence of Acinetobacter isolates, Cicer arietinum L (chickpea)
has the capability for iron biofortification, which greatly increased the mineral
content in PGPR inoculated Cicer arietinum L. comparison to un-inoculated con-
trols, but post-harvesting and cooking can impair grain micronutrient concentrations
by limiting from 5% to 30% and in some cases achieving ~21% (Sathya et al. 2016;
Gopalakrishnan et al. 2016). Therefore, we can conclude that exploiting PGPM-
producing siderophores is a suitable strategy than other traditional methods like
chemical fertilizers to significantly raise Fe content in plants and grains. Staple grain
processing, for example, wheat and rice, reduces the grain micronutrient, particularly
in case of wheat Zn concentration (Kutman et al. 2011; Zhang et al. 2010). Like other
micronutrients, Zn is also one of the most essential micronutrients which is involved
in the various mechanisms. Microorganisms which are beneficial for the plants
support various mechanisms for the zinc solubilization by chelation, soil pH decre-
ment, or ameliorating root growth and absorptive area. The presence of
“antinutrient”phytic acid reduces the uptake of heavy metals because it can form
insoluble compounds to essential nutrients like Fe
3+
and Zn
2+
, and hinders Zn grain
concentration (Urbano et al. 2000; Srivastava 2016). The consortium of two strains
of Azospirillum lipoferum, two strains of the Pseudomonas sp., and one strain of the
Agrobacterium sp. have the potential to increase Zn concentration in rice (Tariq et al.
2007). Zn-solubilizing bacteria are said to enhance the concentration of Zn in
soybean and wheat shoots and roots. The consortium of Bacillus sp. SH-10 and
Bacillus cereus SH-17 provoked the zinc translocation in basmati by ~31 mg/kg as
148 A. Adhikari et al.
compared to control (~18 mg/kg) (He et al. 2010; Shakeel et al. 2015). Microbes
generate chelating compounds that, when bonded with Zn, form complexes. Fur-
thermore, they discharge chelated Zn just at root surface and inevitably improve the
accessibility of Zn to result to biofortification of Zn in plants.
9.2.2 Gene Upregulation
To facilitate growth and development, various biochemical and molecular mecha-
nisms are utilized by microbes. Plant species live in soil, participating in a vast array
of situations with soil microorganisms. Those very interaction may involve an
advantage, vulnerability, or null impact on plant development and nutrient absorp-
tion, and this impact also relies on soil properties, the accessibility of nutrients for
plants and microorganisms in specific. The vital aspects of the microbial communi-
ties are AMF and PGPR. Different nutrient acquisition pathways can also be
provided by AMF, which, in particularly, are significant for plant growth if nutrient
accessibility is limited. The monobasic H
2
PO
4
is one of the plants’most commonly
acquired phosphorus components (Marschner et al. 2011). Pi transport, for example,
is limited, particularly in alkaline soils, and its absorption quickly escorts to the
formation of depletion zones around the roots, even farther limiting the take-up of
phosphorus (Schachtman et al. 1998). The acquisition of Pi in plants is maintained
by plasma membrane members of the Pi/H
+
symporter belonging to the phosphate
transporter family 1 (Pht1), which would be primarily exhibited in the roots,
particularly in the root hair, rhizodermis, and in the outer cortical cells (Liu et al.
1998; Kobae et al. 2010; Daram et al. 1998; Schünmann et al. 2004; Mudge et al.
2002; Karthikeyan et al. 2002; Chiou et al. 2001). Soils may accommodate huge
quantities of phosphorous, but they are not very accessible for need on plants, both
inorganic and organic P being compounds that are extremely insoluble. Various
bacteria of the Azospirillum,Bacillus,Azotobacter,Pseudomonas,Burkholderia,
Rhizobium, and Serratia genera are being identified as microorganisms that solubi-
lize P. Pi migration between plant cells and tissues and even some Pi remobilization
from senescent to novel onset organs were engaged (Lambers et al. 2008). In several
species of plants, homologous Pht1 genes were identified, which include
Arabidopsis thaliana (Misson et al. 2004), tomatoes (Daram et al. 1998), Zea
mays L. (Nagy et al. 2006), and Triticum aestivum (Liu et al. 2013). With regard
to the Pi uptake root pathways, there is still no evidence that PGPR directly affects
Pi. Interestingly, a complicated exchange of signals among host and fungal plants is
triggering the emergence of root/AMF symbiosis which also causes cell
reprogramming. In fact, AMF-colonized plants can take advantage of an added
pathway to acquire P, which occurs in non-identical types of cells depending on
multiple molecular setup (i.e., transporters) and accesses phosphorous in distinct soil
areas. In colonized cortical cells, Pi absorption is accomplished mostly by expression
of Pi transporters that will either be explicitly triggered by AMF symbiosis or
9 Biofortification of Plants by Using Microbes 149
strenuously stimulated all through symbiosis, and basal activation is also present in
non-mycorrhizal roots (Chen et al. 2007; Nagy et al. 2005).
Utilizing mechanisms that are distinct among both monocots and dicots, plants
are able to receive Fe (Kobayashi and Nishizawa 2012). The acquisition of Fe is
entirely focused on a plasma membrane level mechanism that really incorporates
Fe
3+
to Fe
2+
reduction and the accumulation of Fe
2+
through the electrochemical
transmembrane gradient assured by the activity of plasma membrane H
+
-ATPase in
dicots, categorized as plant strategy I (Marschner and Römheld 1994). Strategy I
plants significantly boost the proton transfer in the rhizosphere in the particular
instance of Fe deficit, triggering the Fe concentration to raise in the roots close
vicinity (Colombo et al. 2014). Additionally, the strategy I forms significantly raise
the Fe
3+
reduction activity performed by the Fe-regulated transporting protein-like
(IRT) of FRO (ferrochelate-reductase oxidase) and the transport of Fe
2+
across the
membranes (Connolly et al. 2003). Furthermore, grasses, also referred to as Strategy
II plants, have a fierce chelation affinity for Fe
3+
and are capable of taking Fe from
the biosynthesis and exudation of phytosiderophores (PSs) (Schaaf et al. 2004). In
the rhizosphere, PSs are discharged via the mugineic acid family phytosiderophores1
(TOM1) transporter (Nozoye et al. 2011), while the Fe
3+
-PS aggregates are therefore
transported via specific transporters, the YS1 (yellow stripe1) and YSL (YS1-like)
transporters, into root cells.
In the soil, Zn is usually found in Fe-Mg minerals such as sulfide in crystalline
form (ZnS). The Zn is slightly mobile in plants, and mobility may vary between
species. In the rhizosphere, Zn
2+
is drawn by the plant roots from the soil with an
expenditure of energy after solubilization. The absorption of Zn
2+
takes place in
plasmalemma via the production of intermediates of transiently dissociable ion
carriers (Bowen 1986; Gupta et al. 2016). In compartments of the apoplast and
symplast, Zn absorbed by the roots is racked up in three separate fractions: the
interchangeable one, accumulated to its negatively charged pectin matrix including
its cell wall in the apoplast; the labile one, which is associated to the cytoplasm
nutrient; and the non-labile one, in the vacuole-deposited nutrient, not translocated in
plant (Wang et al. 2014). Two physiological schemes are engaged in Zn absorption
based on the ligand secreted by roots: (Abbaspour et al. 2014) entail efflux of
reducing agents, H
+
ions, and organic acids, which enhance Zn bioavailability for
root epidermal cell uptake, and (Acuña et al. 2013) require phytosiderophore efflux
forming stable complexes with Zn
2+
. Proteins that pass Zn across membranes and
are thus suitable for nutrient homeostasis are clustered in plants into three families
required to carry metal cations (Abbaspour et al. 2014) Zrt/Irt-like protein (ZIP) or
Zn-Fe permease, proteins which promote cytosol Zn entry and likely arbitrate the
absorption of Zn accessible in the soil (White and Broadley 2011; Olsen and
Palmgren 2014), Heavy metal ATPases (HMAs) or P-type ATPase held accountable
for the stacking of xylem, regulation of plastid efflux Zn, and vacuolar scavenging
(Hussain et al. 2004; Hanikenne et al. 2008; Kim et al. 2009; Nagy et al. 2005). At all
levels of phylogenetics, together with bacteria, fungi, plants, and mammals, ZIP
proteins are expressed (Eide 2006). The specific mechanism that controls the
150 A. Adhikari et al.
transporter’s response to the reduction or rise of Zn in the soil solution still remains
obscure.
Se absorption, transfer, and allocation varied from the species to the physiological
conditions (soil and salinity pH), the activities of membrane carriers, and plant
translocation mechanisms (Renkema et al. 2012; Li et al. 2008; Zhao et al. 2005).
Transporters available in root cell membrane initiate the absorption of Se in plants.
Selenite (SeO
2
3
) is observed to be transferred through the transport mechanism of
phosphate (Li et al. 2008). In contrast, selenate (SeO
2
4
) is transported and
channeled through sulfate (Zhang et al. 2003). SULTR1;2 and SULTR1 transporters
were identified to convey (SeO
2
4
) inside the plant in Arabidopsis thaliana sulfate
transporters (El Kassis et al. 2007). Sulfur deprivation improved in the Triticum
aestivum Se absorption (Bowen 1986). In younger leaf tissues, the concentration of
Se was observed to be higher compared to the older ones throughout seedling growth
(Cappa et al. 2014; Harris et al. 2014). Selenium is often accrued in its vacuoles
within the plant cells and thus can be effluxed via sulfate transporters available in the
tonoplast (Gigolashvili and Kopriva 2014; Mazej et al. 2008). The very initial step in
selenium accumulation is to convert inorganic selenium to SeO
2
3
. It requires two
enzymes, APS (ATP sulfurylase) and APR (APS reductase), to perform sequentially.
APS catalyzed ATP hydrolysis to produce adenosine phosphoselenate, which has
been reduced by APR to SeO
2
3
. SeO
2
3
is then transformed by enzymatic sulfite
reductase to selenide (Se
2
) (Sors et al. 2005). Through coupling with O-
acetylserine (OAS) in the presence of the enzyme cysteine synthase, selenide is
then converted to Se-Cys. Se-Cys can then be processed to elemental Se in the
availability of the enzyme Se-Cys lyase, or can be methylated to methyl-Se-Cys by
selenocysteine methyltransferase, or can be switched to selenomethionine by a series
of enzymes, based on plant species and environmental conditions (Fig. 9.1).
Fig. 9.1 General mechanisms for the uptake of micronutrients from the soil with different trans-
porters involved (PS phytosiderophores, MSeCys methyl selenocysteine)
9 Biofortification of Plants by Using Microbes 151
9.3 Common Nutritional Deficiency and Corresponding
Biofortification Strategies
9.3.1 Biofortification for Iron Deficiency
A huge amount of iron present in the earth’s crust is available in the form of Fe
3+
which cannot be accessed by plants. Thus, despite the abundance of iron in soil, its
uptake by plants is hampered due to very low bioavailability. Iron plays important
role in human health as it affects multiple metabolic functions, including oxidative
metabolism, oxygen transportation, DNA synthesis, hemoglobin and myoglobin
synthesis, etc. (Abbaspour et al. 2014). Low iron also affects the agricultural yield
due to lower chlorophyll production, which ultimately leads to necrosis of leaves and
retards the growth of plants (Zhang et al. 2015). Variety of microbes produce
siderophores that help in alleviation of iron deficiency (Patel et al. 2018). The
siderophores form siderophore-Fe
3+
complex that help in increasing the bioavail-
ability of free Fe
3+
present in the soil (Jin et al. 2006). Plant growth-promoting
actinobacteria when inoculated with chickpea seed were found to increase the iron
content by 10–38% in the processed seeds postharvest. Real-time PCR results
supported the hypothesis for siderophore production being responsible for the
increased iron levels in the seeds. Genes responsible for siderophore production
were found to be upregulated by 1.4–25 times as compared to the corresponding
control (Sathya et al. 2016). Similarly, multiple plant growth-promoting bacteria
isolated from organic soils not only enhanced the crop yield but also produced
biofortified chickpea and pigeon pea grains with higher contents of micronutrients
including iron, zinc, copper, and calcium (Gopalakrishnan et al. 2016). Providencia
sp. bacteria isolated from the rhizosphere of wheat plant also exhibited plant growth
characteristics in field trials. Furthermore, it was also found to increase the protein
content by 18.6% and iron, manganese, and copper by 105.3%, 36.7%, and 150%,
respectively (Rana et al. 2012). Later, Providencia sp. when used either individually
or in combination with three different strains of cyanobacteria showed synergistic
benefits with promising results in the crop yield. Furthermore, a better crop yield and
micronutrient levels were observed when the bacterial isolate was inoculated in
wheat crops as compared to rice plants (Rana et al. 2015). Combination of microbial
endophytes with foliar spraying of micronutrients enhanced plant height, area of
leaf, plant biomass, and iron and zinc concentrations 10.14% and 37.93%, respec-
tively, as compared to control without foliar spraying of micronutrients (Yaseen
et al. 2018). Arthrobacter sulfonivorans, a siderophore-producing endophytic bac-
terial treatment to wheat, enhanced the iron content by overexpression of TaZIP3
gene (Singh et al. 2018). Furthermore, combination treatment of native growth-
promoting bacteria alone and in combination with arbuscular mycorrhizae provokes
significant rise in the levels of iron in wheat plants as compared to the controls
(Yadav et al. 2020). Thus, plant growth-promoting bacteria, arbuscular mycorrhiza,
and endophytes alone or in combination could provide future perspectives to deal
with low bioavailability of iron.
152 A. Adhikari et al.
9.3.2 Biofortification for Zinc Deficiency
The bioavailability of zinc being very low in the soil affects its concentration in the
crops and also affects the human health. Zinc deficiency in the plants directly
impacts the growth rate and yield of the crops. Zinc-solubilizing bacteria have
been essential in alleviating zinc deficiency by enhancing the assimilation of zinc
in the seeds and simultaneously maintaining the productivity. Zinc-solubilizing
Bacillus strains exhibited both enhancement of zinc and also increased the crop
yield in soybean and wheat plants (Khande et al. 2017). Similar results with
increased crop productivity and zinc assimilation were observed when for the first
time Bacillus aryabhattai-related bacterial isolates were studied in soybean and
wheat (Ramesh et al. 2014). In another study conducted in wheat plants, Bacillus
subtilis enhanced zinc amount in grains by 24% relative to untreated control
(Moreno-Lora et al. 2019). Biofortification of maize with enhanced levels of zinc
was also studied using Bacillus strains, which produced ammonia, hydrogen
cyanide,siderophores, exopolysaccharides, and cellulase (Goteti et al. 2013;
Mumtaz et al. 2017). Zinc-solubilizing bacteria isolated from the rhizosphere region
of rice plants revealed similar conclusions regarding their plant growth-promoting
potential and zinc solubilization. The isolates were identified as Pseudomonas
aeruginosa,Ralstonia pickettii,Burkholderia cepacia, and Klebsiella pneumoniae
(Gontia-Mishra et al. 2017). Bacterial isolates were screened from rhizosphere of
wheat plants, and out of the five selected strains, Enterobacter cloacae and Pantoea
agglomerans had the highest amount of zinc when the wheat plants were harvested
after a month, while after a 3-month harvesting, Pseudomonas fragi exhibited the
highest amount of zinc in the grains of the plants, estimated using atomic absorption
spectroscopy (Kamran et al. 2017). Burkholderia and Acinetobacter species isolated
from zinc-deficient soil were tested for their plant growth promotion and zinc
solubilization. Zinc levels were estimated with the straw and grain samples of rice
and saw an increase in the zinc uptake by 52.5% (Vaid et al. 2014). Five endophytic
bacteria inoculated in rice plants grown using hydroponics were studied for their
plant growth and zinc biofortification. The endophytes were able to colonize the
plant roots in 72 h and showed increase in zinc concentration by 44.4% and 51.1% in
shoots and by 73.6% and 83.4% in roots treated with Sphingomonas and
Enterobacter sp., respectively. However, when grown in soil, the endophytes
Sphingomonas and Enterobacter sp. increased zinc concentrations by 20.3% and
21.9%, respectively (Wang et al. 2014). In another study with endophytes, two
bacterial isolates (Bacillus subtilis and Arthrobacter sp.) were used for the treatment
of wheat genotypes that had either low or high zinc accumulation rates in soils
sufficient and deficient in zinc. The treatment with endophytes doubled the amount
of zinc present in the grains as compared to the untreated controls regardless of the
zinc-accumulating genotypes (Singh et al. 2017). Biofortification of zinc can also be
done by easily priming the plant seeds with zinc and Pseudomonas sp. in combina-
tion. Moreover, soil or foliar application of zinc resulted in the highest amount of
grain yield and also increased the bioavailability of zinc (Rehman et al. 2018). Thus,
9 Biofortification of Plants by Using Microbes 153
application of zinc-solubilizing bacteria enhances and promotes the growth of plants,
and additionally, these bacteria could help in zinc biofortification and as zinc
translocators for feasible agricultural practice.
9.3.3 Biofortification for Selenium Deficiency
Selenium is an important micronutrient necessary for the vital functions of the
humans and mammals. Golubkina et al. studied the impact of selenium
biofortification and arbuscular mycorrhiza fungi on the yield, quality and antioxidant
properties of onion variety. They showed that the inoculation of both selenium and
mycorrhiza enhanced the overall quality, yield, and selenium content of onions
(Golubkina et al. 2019). In a recent research, volatile organic compounds (VOCs)
extracted from Bacillus amyloliquefaciens were found to be very beneficial in
enhancing the selenium and iron uptake by plants inoculated with VOCs. They
were also shown to enhance the growth and photosynthesis of Arabidopsis plants
and upregulate the expression of genes responsible for iron uptake, sulfur, and amino
acid transport. Surprisingly, when plants with sulfur transport mutants were inocu-
lated with VOCs, no significant change in the selenium absorption was observed.
Thus, upregulated expression of sulfate transporter genes was essential in increasing
the uptake of selenium by plants (Wang et al. 2017). Selenobacteria isolated from the
rhizosphere also serve as potential tool for enhancing the plant growth, yield, and
selenium biofortification. Multiple selenium-tolerant bacteria (Stenotrophomonas,
Bacillus,Enterobacter, and Pseudomonas) have been found to be beneficial in wheat
crops. Acuña et al. demonstrated that inoculation of these salt-tolerant bacteria under
greenhouse conditions increased the translocation of selenium in the leaves of the
wheat plants (Acuña et al. 2013). Another study also reported salt-tolerant bacteria
from Bacillus,Paenibacillus,Klebsiella,andAcinetobacter genera which helped in
selenium biofortification as well as the biocontrol of Gaeumannomyces graminis,a
soil-borne pathogen in wheat plants (Durán et al. 2014). Similarly, Bacillus
pichinotyi isolated from the Roohi Nala drain increased the selenium concentration
in wheat kernels (167%) and stems (252%) as compared to the un-inoculated plants.
The inoculated plants also showed a significant enhancement in the acid phosphatase
activity which might be responsible for the enhanced growth of wheat crops (Yasin
et al. 2015). Furthermore, combinatorial utilization of arbuscular fungi
(Funneliformis mosseae and Glomus versiforme) and selenium fertilizer synergisti-
cally enhanced the bioavailable selenium in the soil and its accumulation in the rice
grains (Chen et al. 2020). In another combination study, inoculation of
selenobacteria (Acinetobacter or Bacillus sp.) and arbuscular fungi (Rhizophagus
intraradices) enhanced the growth of lettuce under drought conditions and also
promoted the uptake of selenium as compared with the un-inoculated controls
(Durán et al. 2016). Recently, Trivedi et al. demonstrated the utilization of endo-
phytic selenobacteria MGT9 in soybean for biofortification of selenium. Moreover,
154 A. Adhikari et al.
Table 9.1 Microorganisms employed to combat common nutritional deficiencies
S. No.
Nutrient
deficiency Crop Microbes utilized
Contribution to
biofortification References
1. Iron Chickpea Enterobacter
ludwigii SRI-229
Increased iron con-
tent up to 18% and
plant growth-
promoting traits
Gopalakrishnan
et al. (2016)
Pigeon
pea
Enterobacter
ludwigii SRI-229
Increased iron con-
tent up to 12% and
plant growth-
promoting traits
Gopalakrishnan
et al. (2016)
Maize Arthrobacter
globiformis
Increased iron and
phosphate uptake
and protein and
chlorophyll
contents
Sharma et al.
(2016)
Pseudomonas
chlororaphis,
Pseudomonas spp.
Increase in germi-
nation percentage
and plant growth
Sharma and
Johri (2003)
Pseudomonas
aeruginosa
Increased iron
transportation
Sah et al. (2017)
Wheat Arthrobacter
globiformis
Increased iron and
phosphate uptake
and protein and
chlorophyll
contents
Sharma et al.
(2016)
Arthrobacter
sulfonivorans
Upregulation of
TaZIP3 and TaZIP7
genes resulted in
higher iron and zinc
translocation
Singh et al.
(2020)
Bacillus pichinotyi Increase in Fe con-
tent and increase in
acid phosphatase
activity, which con-
tributed to the
enhanced growth
Yasin et al.
(2015)
Providencia sp. Increased iron
concentration
Rana et al.
(2015)
Pseudomonas
fluorescens,Pseu-
domonas putida
Enhanced iron con-
centration,
enhanced seed ger-
mination, root
length and shoot
length
Sayyed et al.
(2005)
2. Zinc Rice Acinetobacter sp.,
Burkholderia sp.
Enhanced zinc con-
tent as well as shoot
and root length and
weight
Vaid et al.
(2014)
(continued)
9 Biofortification of Plants by Using Microbes 155
Table 9.1 (continued)
S. No.
Nutrient
deficiency Crop Microbes utilized
Contribution to
biofortification References
Burkholderia
cepacia,Klebsiella
pneumonia,Pseu-
domonas
aeruginosa,
Ralstonia pickettii
Zinc solubilization,
ACC utilization,
EPS production,
Ammonia produc-
tion, production of
lytic enzymes
Gontia-Mishra
et al. (2017)
Wheat Aeromonas sp.,
Arthrobacter sp.,
Trabulsiella sp.
Increased in zinc
and iron percentage
Shaikh and
Saraf (2017)
Arthrobacter sp.,
Bacillus subtilis
Zinc solubilization
enhanced the trans-
location and enrich-
ment of zinc
Singh et al.
(2018)
Providencia sp. Zinc solubilization;
increase zinc
concentration
Rana et al.
(2015)
Pseudomonas sp. Increase grain yield
and zinc uptake
Rehman et al.
(2018)
Soybean Bacillus
amyloliquefaciens,
Bacillus firmus
Increase zinc
concentration
Sharma et al.
(2011)
Bacillus anthracis,
Bacillus cereus
Increased yield as
well as zinc assimi-
lation in soybean
Khande et al.
(2017)
Bacillus
aryabhattai
Improved growth,
yield and zinc
concentration
Ramesh et al.
(2014)
Maize Bacillus
aryabhattai,Bacil-
lus subtilis,Bacil-
lus aryabhattai
Promoted growth
and zinc uptake
Mumtaz et al.
(2017)
Sugarcane Enterobacter cloa-
cae,Pantoea
agglomerans,
Pantoea dispersa,
Pseudomonas fragi
Increased zinc con-
tent and growth
promotion
Kamran et al.
(2017)
Gluconacetobacter Solubilization of
insoluble zinc
compounds
Saravanan et al.
(2007)
Chickpea Rhizophagus
irregularis,
Funneliformis
mosseae
Increased zinc and
iron concentration,
fungal root coloni-
zation, as well as
plant biomass and
yield
Pellegrino and
Bedini (2014)
3. Selenium Wheat Increased selenium
concentration,
Durán et al.
(2014)
(continued)
156 A. Adhikari et al.
selenobacteria also enhanced the growth of soybean under water stressed conditions
(Table 9.1) (Trivedi et al. 2020).
9.4 Role of Biofortified food in human health
Although micronutrients are required in trace amounts, their role in the human health
is very prominent (Imtiaz et al. 2010). The depletion of micronutrient from diet
causes deficiency of micronutrients or hidden hunger in humans, which has dramat-
ically affected over two billion people globally. Surprisingly, more than 60% of the
global population are iron, 30% zinc and 15% selenium deficient. In South African
continent children below 9 years are at the risk of having iron, zinc deficiency of
11% and 45.3% respectively (Siwela et al. 2020). Biofortification allows to increase
the concentration of selectively essential nutrients in the food that can be beneficial
to minimize micronutrient deficiency and health problems associated with. Con-
sumption of biofortified food has found to be beneficial for positive restructuring of
the gut microbiota. Reed et al. demonstrated that those humans which consumed
wheat biofortified with zinc in their diet had enhanced microbial βdiversity, with a
naturally associated increase short chain fatty acid producing lactic acid bacteria
(Reed et al. 2018). Deficiency of a particular micronutrient has shown to develop
Table 9.1 (continued)
S. No.
Nutrient
deficiency Crop Microbes utilized
Contribution to
biofortification References
Acinetobacter sp.,
Bacillus sp., Kleb-
siella sp.
auxin and
siderophore pro-
duction, phytate
mineralization, and
tricalcium phos-
phate solubilization
Bacillus sp.,
Enterobacter sp.,
Pseudomonas,
Stenotrophomonas
Increased selenium
content
Acuña et al.
(2013)
Bacillus pichinotyi Elevated selenium
and increase in acid
phosphatase activ-
ity resulting in plant
growth
Yasin et al.
(2015)
Lettuce Acinetobacter sp.,
Bacillus sp.,
Rhizophagus
intraradices
Enhanced the
growth of lettuce
and uptake of
selenium
Durán et al.
(2016)
Rice Funneliformis
mosseae,Glomus
versiforme
Enhanced the bio-
available selenium
Chen et al.
(2020)
9 Biofortification of Plants by Using Microbes 157
multiple health-related concerns. In the initial stage of pregnancy, the low levels of
selenium in the blood of mother might result in low birth weight of a newly born
child (Pieczyńska and Grajeta 2015). Foods enriched in micronutrients have also
been useful in fighting against viral infections. Zinc and selenium supplements
against SARS-CoV-2-infected people with reduced selenium or zinc in their blood
could serve as an alternative natural remedy against the virus (Zhang and Liu 2020).
In this context, the development of biofortified food to enhance the availability of
micronutrients is very essential for the improvement of human health.
9.5 Conclusion and Future Outlook
Numerous researchers to date have illustrated the potential risks to food security.
PGPMs interact with plants and promote plant development activities and strengthen
the plant’s potential to absorb micronutrients from the soil. The development of
PGPM all across agriculture is a substantial benefit of cost-effective and being
environmentally harmless. In order to tackle the challenge of micronutrient defi-
ciency, the expansion of crops with high concentrations of micronutrients is enor-
mously and promptly deemed necessary. In the future, biofortification tactics to
address the problem of hidden hunger may be used to formulate microorganisms
with multiple beneficial characteristics. PGPMs communicate with plants and pro-
mote plant developmental activities and strengthen the plant’s capacity to absorb
micronutrients from the soil. In the different edible segments of crop plants, Zn
solubilization and siderophore secretion microbes significantly raise the concentra-
tion of Zn and Fe and offer an efficient approach to fortify micronutrients and
produce food prosperous in micronutrients. In addition to genetic modification,
other effective techniques have been used to biofortify crops, such as traditional
breeding strategies. The implementation of inoculants from such microorganisms
lessens dependence on expensive strategies to biofortification, i.e., agronomic and
genetic approaches. In this perspective, the utilization of PGPR as bio-inoculants
could be a very successful technique, given that the both plant growth (root and
shoot biomass enhancement, more branched root system) and nutrient bioavailabil-
ity could be enhanced. Even after such empowering findings, there are still many
elements to be investigated so that all interactions among plants and PGPRs can be
better understood, with a view to improving the effectiveness of nutrients. The rise of
innovative agricultural fertilization practices focused on biotechnological method-
ologies for rhizosphere engineering and management could allow the molecular
mechanisms underlying the implications of microbes on plants to be clarified and
vice versa, as well as the physiological and ecological effects that are mediated.
158 A. Adhikari et al.
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Chapter 10
Microbial Biopesticides: Development
and Application
H. T. Mandakini and Dimuthu S. Manamgoda
Abstract Application of bio-based pesticides has been a growing trend in organic
agriculture globally. The use of natural or genetically modified microorganisms as
biopesticides can be considered as an effective and sustainable approach in disease
control. The discovery and the development of a microbial biopesticide is a process
of two major phases consisting experimental component and commercialization of
the product. The experimental component begins with the field collection of the
potential microbial samples and isolation of potential microbes and to evaluate their
bio-control efficiency. Once a potential candidate microorganism is selected for the
production of biopesticides, the candidate should be accurately identified and char-
acterized. Genetic modifications can be done to improve the efficiency of the
organism. The commercialization of the product includes mass production, formu-
lation, and field testing and safety evaluation. Finally, a biopesticide can be regis-
tered and introduced to the market upon the completion of safety evaluation and
regulatory approval. This chapter summarizes the discovery and development of
biopesticides with special reference to nematophagous fungi and their applications.
Keywords Entomopathogens · Formulation · Mass production · Pesticides
10.1 Introduction
Chemical pesticides had been used to fight effectively against pests and diseases for
a long period of time. However, long-term wide use of chemical-based pesticides has
resulted to several adverse effects such as pesticide contamination of soil and water
leaving residues on crop produce, development of insecticide resistance, and
biomagnifications of insecticides. As a result, regulatory measures are currently
H. T. Mandakini · D. S. Manamgoda (*)
Department of Botany, Faculty of Applied Sciences, University of Sri Jayewardenepura,
Nugegoda, Sri Lanka
e-mail: dsmanamgoda@sjp.ac.lk
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_10
167
established globally to restrict the export of chemical-based pesticides. Thus, there is
a tremendous demand for biopesticides around the world (Leng et al. 2011).
Biopesticides can be defined as the types of pesticides derived from natural
materials as bacteria, plants, animals, and certain minerals (US Environmental
Protection Agency, Regulating Pesticides 2008). During the last three decades,
development in the fields of molecular biology, genetics, protein engineering, and
genome sequencing has improved the production procedures of biopesticides. As a
result, biopesticides are gradually replacing toxic chemical pesticides in the market
(Leng et al. 2011). The numbers of newly developed and registered biopesticides are
increasing at a rate of 4% annually. Similarly, the annual market share of
biopesticides is rising approximately 30% (Cheng et al. 2010). Biopesticides can
be classified as botanical pesticides, microbial pesticides, and zooid pesticides.
Among them, microbial pesticides can be considered as the first developed formu-
lations and widely used biopesticides (Leng et al. 2011).
Many studies are available focusing on screening the pesticide activity of
microbes. However, comparatively a few of them are leading toward successful
commercialization of a microbial biopesticide. The discovery, development, and
commercialization of a microbial biopesticide is a long-term process which should
be carefully handled and managed. This chapter summarizes the discovery, devel-
opment, and application of microbial pesticides in commercial scale with special
reference to nematophagous fungi that have the potential to be used as biopesticides.
10.2 Discovery and Development of Microbial-Based
Pesticides
The development of microbial-based biopesticide is a complex, time, and money-
consuming multistep process. It can be divided into two major phases in which
Phase 1 consists of extensive experimental and research works, where Phase 2 is
mainly considered the commercialization of microbial-based pesticides (Fig. 10.1).
Phase 1 contains six major steps which are screening for suitable sampling site and
sampling, isolation of potential microbial candidates, evaluating bio-control efficacy
of isolates, identification and characterization of isolates, strain improvement, and
selecting the microbial candidate of interest.
After selecting a suitable microbial candidate for biopesticide production, the
next steps would involve the production of biopesticides on a commercial scale. The
first phase (Phase 1) can be carried out inside a laboratory, whereas the second phase
(Phase 2) will be carried out in an industrial-scale facility.
168 H. T. Mandakini and D. S. Manamgoda
10.3 Screening of Suitable Sampling Sites and Sampling
The first step in developing a microbial-based biopesticide is the selection of a
suitable sampling site to isolate potential microbial candidates. It is important to
identify the biology and the nature of the pest problem in a particular area to which a
microbial biopesticide is expected to be developed. Then, the information of pest
biology including the most susceptible stages of the host, host range, nature of the
symptoms, and the most susceptible period of the year must be explored
(Ravensberg 2011).
The background information of the pest problem narrows down the blind isola-
tion of microorganisms and increases the probability of obtaining useful candidates.
After elaborately defining the host, pathogen, and epidemiology stages of the
disease, the next step is to collect potential microbial candidates (usually bacteria
or fungi) from proper sampling sites. The potential microbial candidate may sup-
press the pest by producing secondary metabolites or it can cause serious diseases on
the pest. Keeping this in mind, healthy plants from the field with the disease, disease
suppressive soil, and also the dead insects in the field are considered as good
sampling sites and materials for potential candidates (Glare and Moran-Diez
2016). Besides those, areas with fewer disease developments or no disease devel-
opment at all even with the presence of susceptible hosts are also considered to be
Fig. 10.1 Steps involved in developing microbial-based bio-control agent
10 Microbial Biopesticides: Development and Application 169
potential grounds to isolate prospective biological control agents (Schisler and
Slininger 1997). Once the sampling site is selected, sampling must be carried out
in a manner where it covers a wide area in the selected sampling site or sampling
materials to avoid biased collection and missing any opportunities (Montesinos
2003; Köhl et al. 2011). Collected soil samples are considered as a source for free-
living strains of beneficial microorganisms while plant parts are sources for benefi-
cial endophytes (Glare and Moran-Diez 2016).
10.4 Isolation of Potential Microbial Candidates
After a successful sampling, the next step is to isolate microbial candidates on
natural or synthetic microbiological media or to a biological system like cell cultures
and trap organisms (Montesinos 2003). To isolate all the types of potential micro-
organisms representing each microbial taxon, isolation needs to be carried out on
different media that facilitate the growth of all of them.
As applying microbial isolation techniques can affect and favor some character-
istics of microorganisms, isolation techniques have to be designed carefully. It is
recommended to isolate them under suitable environmental conditions, from correct
plant parts without the use of selective enrichment media (Schisler and Slininger
1997). In some cases, the use of highly selective media and selective incubation
conditions can promote certain types of microbial growth over others and will result
in losing the chance to isolate many potential microbial candidates (Köhl et al.
2011). In some cases, generalized media are used for initial isolation. For an
example, potato dextrose agar (PDA) can be used to isolate entomopathogenic
fungi like Beauveria and Metarhizium (Tuininga et al. 2014). However, microbial
candidates with beneficial traits that are most important in the later stages of the
biopesticide development process can be preselected by carefully selecting appro-
priate isolation techniques. For instance, microorganisms with the ability to grow on
a commercially inexpensive medium can be preselected by using an isolating
medium more similar to a commercially feasible medium which uses at later stages
the biopesticide production process (Schisler and Slininger 1997).
10.5 Evaluating Bio-control Efficacy
Followed by the isolation of microbial candidates, the next step is to experimentally
evaluate their efficacy as bio-controlling agent, against pathogens under both in vitro
and in vivo conditions. This is one of the critical steps of biopesticide development
as the potent strains can be separated from a large number of microbial candidates
(Glare and Moran-Diez 2016). In this step, a large number of microbial isolates are
screened for potential bio-control agents by subjecting to bio-control bioassays.
Bio-controlling ability of microorganisms can be achieved through diverse
170 H. T. Mandakini and D. S. Manamgoda
mechanisms as antibiosis, parasitism, competition, and induction of plant systemic
resistance (Montesinos 2003; Köhl et al. 2019). It is required to know the mode of
action of potential microbial candidates as it is important in achieving optimal
disease controlling and also to address potential risks for humans and the environ-
ment (Köhl et al. 2019).
According to Harvey and Spurr, two types of bioassays are designed to evaluate
bio-control activity: primary in vitro bioassays and secondary in vivo bioassays
(Spurr 1985). In the process of development of new bio-control, isolates are first
screened for bio-control activity based on their potential to inhibit pathogen of
interest in in vitro through antagonistic effect. In vitro bioassays are comparatively
fast, resource-efficient, and easy to perform, and results can be easy. In many cases,
if the isolates showed no in vitro efficacy, those species would not be promoted for
in vivo testing (Besset-Manzoni et al. 2019). Some of the in vitro bioassays are
in vitro petri plate antagonism tests which are performed on agar, such as dual
culture plate test, volatile and nonvolatile compound tests (Glare and Moran-Diez
2016), or spore germination and mycelia growth-inhibitory test performed in liquid
media (Besset-Manzoni et al. 2019).
However, the literature suggests that results of in vitro bio-control efficacy tests
are not always correlated with results of in vivo bio-control experiments. One of the
main reasons behind this is that in vitro assays only evaluate the bio-control potential
mediated mainly through antibiosis mode of action in an artificial environment. The
other mechanisms like competition, hyperparasitism, and induced systemic resis-
tance are not able to be detected by that means (Schisler and Slininger 1997; Köhl
et al. 2019). Also, another drawback of in vitro bio-control efficacy test is the
inability of mimicking the natural environment where the proposed bio-control is
truly expected to act. Generally, nutrient concentration, composition, and the porous
nature of the media result in comparatively higher production and higher diffusion
rate of antibiotic compounds which are responsible for bio-controlling activity (Köhl
et al. 2019).
In vivo bioassays, also known as secondary bioassays, are considered to be more
realistic as they test the bio-control activity of candidates in the presence of a host
plant, grown on field soil or an environment that is close to the field environment.
They are designed to closely mimic the field environment. Literature reveals many
diversely designed in vivo bioassays. In the study of Schisler and Slininger, antag-
onists of Fusarium dry rot were selected on wounded potato tubers incubated at
15 C. Aqueous soil pastes which were prepared by enrichment of potato tuber
periderm and inoculated with Gibberella pulicaris were applied onto wounded
tubers (Schisler and Slininger 1997). Verhaar et al. used a bioassay to select potential
bio-control of cucumber powdery mildew on detached cucumber leaves that had
previously been inoculated with Sphaerotheca fuliginea. They tested the
Verticillium lecanii isolates with high antagonistic potential at different humidity
(Verhaar et al. 1998). Another successful in vivo bioassay performed by Clarkson
et al. is the use of onion seedlings to select potential bio-control of white rot caused
by sclerotium-forming Sclerotium cepivorum. Onion seedling was potted in a soil
mixture that has been previously inoculated with conditioned sclerotia of
10 Microbial Biopesticides: Development and Application 171
S. cepivorum and potential bio-control fungi isolated either as wheat bran cultures or
spore suspension (Clarkson et al. 2002). After performing a successful bioassay, the
next immediate step is to exclude the candidates that lose bio-control efficacy after
repeated laboratory cultivation. Therefore, candidates who pass bio-control efficacy
testing are repeatedly sub-cultured and subjected to bioassays testing of bio-control
efficacy (Köhl et al. 2011). However, it is known that both the knowledge on the
mode of action of bio-control agent and the activity related gene/s together can
improve high-throughput screening approaches. Some of the rapid screening
methods are use of phenotypic or genetic markers, DNA arrays, or specific second-
ary metabolites (Glare and Moran-Diez 2016; Köhl et al. 2011).
10.6 Identification and Characterization
Accurate identification of the isolates is extremely important before commercializa-
tion. Isolated candidates should be characterized and identified up to species level by
macroscopic and microscopic morphologies, physiology, carbon utilization and
growth rates, biochemical characteristics, secondary metabolites profiling, and
molecular methods. Taxonomic literature, morphological keys, guides, and illustra-
tions can be used to identify species using morphological techniques. Patterns of
carbon utilization and relative growth rates on different media have been used to
characterize fungi and bacteria. Biochemical characterization is commonly used in
bacterial identification, and there are kits that are developed for easy biochemical
characterization of bacterial strains. Fatty acid methyl ester analysis (FAME) and
secondary metabolite profiles on high-performance TLC (HPTLC) and high-
performance liquid chromatography (HPLC) have been used for both bacterial and
fungal identification. DNA extraction, PCR, and sequencing can be considered as a
gold strand of microorganism identification. Once sequences are generated, they can
be identified through the BLAST comparison with the data on public databases.
Phylogenetic analysis is always preferred in species identification since true phy-
logeny represents the correct taxonomy. Other than that, the polyphasic approach of
combining all the methods mentioned above has been also practiced in fungal and
bacterial identification.
Once the species are identified, they are screened in numerous databases for
safety information to assess potential risks of allergy, toxicity, and pathogenicity to
humans, animals, and crops, and also to find out the candidates’bio-control appli-
cation is patent protected or not (Köhl et al. 2011). The candidates associated with
potential risks and already patent protected are then discarded from further
screening.
172 H. T. Mandakini and D. S. Manamgoda
10.7 Strain Improvement
If a microbial strain is suitable for a commercial level production, it would lead to the
possibility of improving strains’performance. Due to the main concern on the
economic setting in developing a biopesticide, it is always challenging to obtain
the biologically perfect strain. The most promising candidate is the one that shows
the best compromise between bio-control efficacy and economic factors
(Ravensberg 2011). Hence, improvement of strains to achieve positive production
aspects such as the quick speed of bio-control action, high mass production, and
environmental tolerance is beneficial.
Until the last two decades, there were no many studies involved in the strain
improvement of the pathogenic fungi due to a lack of available information on the
genes that control either pathogenesis or specificity (St Leger and Screen 2001).
With the development of molecular biological techniques, this situation changed
drastically. These techniques helped to elucidate pathogenic process in important
bio-control agents; pathogenicity related genes could be cloned and express just as
microorganisms are induced by physical or chemical stimuli to change its saprobic
habit to pathogenic or parasitic phase. Some of these pathogenic genes encode
hydrolytic enzymes and toxins which can act on the target pathogens. This would
allow them to develop transgenic microorganisms with improved pathogenic
qualities.
10.8 Selecting Microbial Candidate of Interest
In the final selecting process, many criteria are taken under consideration other than
the efficacy and consistency of results in repeated bioassays. Some of these addi-
tional criteria are positive growth kinetics in the field, favorable toxicological pro-
files of microorganisms, the requirement of low effective doses for bioactivity, the
specificity of action to a target pest or range of pests, tolerance to market available
pesticides, and estimated cost-effectiveness of technologies for mass production
(Köhl et al. 2011). These additional limiting conditions further narrow down a
large number of candidate isolates and result in only a fewer isolate. Literature
reveals that only less than 1% of candidate isolates satisfy all those criteria and make
it a successful product (Montesinos 2003).
10.9 Commercialization of Microbial-Based Biopesticides
In Phase 2 of the development of biopesticides, mass scale production, storage, and
perseveration of selected bio-control agents and also the formulation are considered
as major initial steps in the commercialization process (Powell and Jutsum 1993). In
10 Microbial Biopesticides: Development and Application 173
the selection of production process, it is required to pay attention to the nature of the
organisms which are expected to be produced and expected active final product as
the required conditions can differ from one another (Glare and Moran-Diez 2016).
Generally, there are two types of production processes: liquid phase fermentation or
solid phase fermentation. Usually, liquid phase fermentation is widely used for
bacteria and yeast production, while some other fungi are fermented in solid state.
10.10 Mass Production
Production of inoculum is considered as the major step in making a biopesticide
commercially feasible. The goal of mass production is to achieve the highest yield of
stable, quality biomass of bio-control agent under the lowest possible cost. The end
product of the production process can be either microbial cells or biological struc-
tures as conidia, microsclerotia, or specific secondary metabolites like toxins which
are expected to be used as bioactive agents (Glare and Moran-Diez 2016).
The production process should be designed as a reliable, cost-effective one with a
higher production rate. The ideal production process for commercial level produc-
tion should be obtained by experimenting with changing parameters. Key aspects to
be considered here are the type of fermentation, growth media which is intended to
be used, and the choice of the suitable equipment and conditions; for production,
downstream processing, and storage of the end product (Ravensberg 2011).
The type of fermentation and the growth medium greatly depends on the nature of
the expected bio-control agent to be produced. Normally, liquid phase fermentation
is chosen for the production of bacteria and yeast, while solid-phase fermentation is
preferred for the production of filamentous fungi (Köhl et al. 2011). However, in
many cases, solid-state fermentation uses the inoculum produced from prior sub-
merged fermentation (Glare and Moran-Diez 2016). Bacillus thuringiensis is a
commercially available, successful biopesticidal agent and industrially produced in
a larger scale via liquid-state fermentation. However, Devi et al. show the semi-solid
and solid-state fermentation of B. thuringiensis in a small scale in developing
countries for home usage (Devi et al. 2005). Liquid-state fermentation is considered
as the most suitable choice for large-scale biomass production for industrial purposes
due to easy manipulation of growth parameter gradients such as temperature, pH,
and dissolved oxygen, wherein the solid-state fermentation faces difficulty in con-
trolling due to deprived level of water availability. However, the production of
fungal spores by aerial hyphae in small-scale solid-state fermentation is considered
as the best method (Glare and Moran-Diez 2016). At the end of the fermentation
process, produced inocula are recovered and purified by downstream processing, and
the end product is called “technical product”which may be composed of pure
inoculum or inocula with the medium remnant, secreted metabolites, and com-
pounds added during downstream processing (Ravensberg 2011).
However, before scaling up the mass production, preliminary experiments on
mass production are performed on a small laboratory scale using a set of chosen
174 H. T. Mandakini and D. S. Manamgoda
microbial candidates from the end of Phase 1. During these preliminary experiments,
inexpensive growth media and fermentation conditions (temperature, pH, dissolved
oxygen concentration, etc.) are evaluated and optimized. Also, the viability and the
bioactivity of the inocula after several downstream processes are determined. This
allows producers to have a rough estimation of production costs. The selected strains
from research and experiments are further screened, and strains producing low
inconsistent yield which are greatly dependent on specific growth conditions are
excluded (Köhl et al. 2011). Köhl et al. suggest performing the step of evaluating
bio-control efficacy and pilot mass production step parallel to each other, as it can
reduce estimated costs for each step considerably (Köhl et al. 2011). During a
parallel performance of both steps, candidates that are unable to produce sufficient
biomass can be discarded avoiding further efficacy testing.
10.11 Formulation of a Product
Once the biopesticide inoculum is produced, maintaining the stability during stor-
age, distribution, and at the target site is a prerequisite. The formulation allows
stabilization of the technical product along with persistence, safety, high efficacy,
and user-friendliness (Ravensberg 2011; Kala et al. 2020). Hence, it is considered a
vital step in developing high quality, viable biopesticide. During formulation, the
active ingredient (produced inoculum) is mixed with inert ingredients such as
carriers, surface active ingredients (stickers, spreaders), and other additives (stabi-
lizers, coloring agents, etc.) (Kala et al. 2020).
There are different types of formulations of biopesticides available in the market.
The choice of a formulation is mainly determined by the type of active ingredients
and the environment where it is applied (Glare and Moran-Diez 2016).
Physiochemical properties and biological activity of active ingredients, method of
intended delivery, and safety in usage, cost-effectiveness, and market preferences are
further taken into account when selecting the type. Once all the factors have been
determined, the final formulation type is selected with the most appropriate inert
ingredients where formulated biopesticides have at least 2 years of shelf life under
dynamic climatic conditions (Knowles 2008).
With respect to its physical state, biopesticide formulation can be categorized as
liquid formulation and dry formulations which are further divided according to their
properties. Each and every type of formulation can have its own challenges and
benefits. Generally, dry formulations are manufactured by adding wetting agents,
dispersants, and binding agents (Knowles 2008). They are available in the form of
direct application such as dusts (DP), powder for seed dressing (DS), granules (GR),
micro granules (MG) or in the form of concentrates such as water dispersible
granules (WG) and wettable powders (WP), which require dilution in water prior
to use (Kala et al. 2020).
10 Microbial Biopesticides: Development and Application 175
Dust formulation: Dusts are made up by mixing the active ingredient with fine, dry,
inert powder carriers like talc, clay, with anticaking agents, and ultraviolet protectant
as additives. Percentage of the active ingredient is comparatively low and 10% or
less by weight. The particle size of dust can be in the range of 50–100μ. They can be
delivered to the target site either directly or mechanically. However, due to possible
inhalation exposure risk to handlers, usage is limited (Kala et al. 2020).
Granular formulation (GR): Except for large size and heavyweight, granular
formulations are also the same as the dust formulation but made of coarse particles
of carriers. Granule particle size can range from 100 to 1000μm. Minerals like silica,
kaolin, and ground plant residues as corn cobs and walnut shells can serve as
carriers, while the active ingredient (5–20% by weight) either coated to the outer
surface or absorb into them. Once the active ingredients adhere to the granules, they
are coated in a layer of inert material which facilitates the slow release of the active
ingredients (Kala et al. 2020; Singh and Arora 2016). After granules are applied,
some require the presence of water to release the active ingredient, while others do as
they gradually decompose (Knowles 2008).
Wettable powder (WP): Dry, finely ground solid mineral carriers, wetting, and
dispersing agents blended with active ingredients and are generally applied as a
spray after being diluted in water. It can contain 5–95% of active ingredients,
generally more than 50%. Application is convenient using spraying equipment
with agitation. Therefore, such application is known to possess significant residual
activity and pose no harm for treated surfaces.
Water dispersible granules (WG): This is an advanced form of a wettable powder
that is compressed into dust free granules. Once dissolved in water, granules are
broken apart into a fine powder and form dust-free uniform suspension. Compared to
other dust formulations, WG poses a low handler exposure risk and is convenient in
usage.
Liquid formulations can be based on water, oil, polymer, or a combination where
water-based formulations need some additives such as surfactants, coloring agents,
and antifreezing materials (Kala et al. 2020). They are formulated in the forms of
emulsions: suspension concentrates (SC), oil dispersions (OD), suspo-emulsions
(SE), capsule suspension (CS), and ultra-low volume formulations (Kala et al.
2020; Knowles 2008).
Emulsion (EC or E): Emulsion comprises droplets of a liquid active ingredient
(0.1–10μm in size) which is oil-soluble dispersed in an immiscible liquid, usually a
petroleum-based solvent. Emulsions are multipurpose formulations and highly
adaptable as an application of ECs can be done from small portable sprayers to
aircraft sprayers. Emulsions have comparatively lower shelf-life (Knowles 2008).
Suspension concentrate (SC): SC is a solid active ingredient dispersed in a liquid,
usually water. The solid active ingredient should be insoluble and is ground finely
before it is being suspended in water. Other inert ingredients are as follows: wetting
agents, dispersants, viscosity modifiers (thickeners), preservatives, and antifreeze.
176 H. T. Mandakini and D. S. Manamgoda
They often require agitation before use and leave a residue on the treated surfaces.
SCs have low formulation cost and are popular since they are easy to use and
comparatively safe (Knowles 2008).
Capsule suspension: A capsule suspension (CS) formulation is a combination of an
active ingredient encapsulated in a polymer shell suspended in a liquid medium with
a dispersant and wetting agent. These are needed to be diluted before use. The
residual stability of these is enhanced by using living cells for the encapsulation. CSs
are an advanced type of formulation which is highly beneficial, but the production is
complex and expensive (Knowles 2008).
Ultra-low volume liquids: These are usually used as it is without dilution and
comprises high concentrations of the active ingredient (mostly 100%). ULVs are
easy to handle, and the residues on treated surfaces are almost invisible as these are
applied at very low rates in fine droplets (Knowles 2008).
At this stage of biopesticide development, candidates with sufficient biomass
production that show disease control ability repeatedly under control conditions in
bioassays are selected. They are further screened in the formulation step. Shelf-life
studies of the formulated product are performed for differently produced and for-
mulated inocula with sufficient viability. Formulated inocula are stored under
various expected storage temperatures such as room temperature and in freezer
and refrigerators for at least 1 year time duration with regular testing of the viability
of the product (Ali et al. 2013). The stress tolerance of the product is also evaluated
by applying experimental stress conditions. After that, diversely produced, formu-
lated, and stored products are subjected to bioassays under laboratory conditions to
compare each of their actions and to compare with the action of fresh inoculum
which is produced at Phase 1 to select the most promising formulated products (Köhl
et al. 2011).
10.12 Field Testing
After the production and formulation, the end product is needed to successfully
deliver to the target pest under field conditions favorable for disease development.
There are various application and delivery techniques based on the type of formu-
lation of the product including direct delivery, seed dressing delivery, delivery by
dilution in water, etc. (Mishra et al. 2019). In some cases, the pathogen is artificially
inoculated to the field, and growth conditions are controlled to facilitate the disease
development. At this stage, several treatments such as different microbial candidates,
formulations, and concentrations and applying times are tested and compared in field
conditions (Ali et al. 2013).
The success of field trials depends on the intensity of disease during the exper-
iments which frequently vary in accordance with the season of the year and location
of the field. The efficacy of bio-control products is varying with environmental
10 Microbial Biopesticides: Development and Application 177
conditions. Hence, evaluating the efficacy of a developed product under multiple
locations and different seasons is required to predict the consistency of the product in
terms of disease control (Köhl et al. 2011).
10.13 Evaluating Safety
Assessment of biosafety of microbial pesticide is a compulsory aspect in the
biopesticide development process to make sure there is no negative effect on
non-target organisms and also on the environment. Global regulatory authorities of
biopesticide production have posed strict procedures for toxicological assessment of
produced biopesticides. Hence, thorough toxicological assessment at strain level is
essential. Possible toxicity of microbial candidates can be identified at earlier stages
of screening microbial candidates by database mining and then discard toxicologi-
cally suspected microbial candidates from further screening. If the microbial candi-
date is a well-studied one with the absence of clinical or veterinary history, it can be
excluded from the development process without further thorough toxicological
studies (Köhl et al. 2011).
After data mining for potential risks of microbial isolates, primary toxicological
studies can be carried out with non-human model organisms such as Caenorhabditis
elegans in order to get an idea of toxicological profiles of selected microbial
candidates (Zachow et al. 2009).
Furthermore, the detailed experiments on human toxicology and ecotoxicology
data of developed biopesticides are carried out as requested by some regulatory
bodies. These toxicological data can be derived from numerous acute, subchronic,
and chronic studies. The toxicology studies of biopesticide on mammals are a
prerequisite to safeguard the handlers and consumers. One of the well-known
toxicological tests is the oral acute test in which the objective is to find out the
median lethal dose (LD50) and to minimize the lethality. When considering bacterial
bio-control agents, LD50 is considered to be more than 10
11
of colony-forming units
per 1 kg of animal weight (Montesinos 2003).
10.14 Registration and Marketing
Many of the global regulatory authorities concern about the production of biopesti-
cide and information about the composition, performance, and safety. The major
aims of regulation of biopesticides are to safeguard consumers and the environment
and also to characterize products to ensure a continuous supply of consistent quality
products by manufacturers.
Legislation background on regulating the use and development differs from one
country to another. The US Environmental Protection Agency (EPA) and European
Union (EU) are considered as the largest regulatory bodies worldwide and show a
178 H. T. Mandakini and D. S. Manamgoda
contrasting difference in the biopesticide regulation process. International Organi-
zation for Biological Control (IOBC), European and Mediterranean Plant Protection
Organization (EPPO) Economic, and Organization for Co-operative Development
(OECD) are few global regulatory agencies developed to give some flexibility for
the biopesticide regulation process (Arora et al. 2016).
Once the biopesticide is developed, registration is done in the relevant regional or
national regulatory body. However, Glare and Moran-Diez (2016) state that the lack
of specifically developed guidelines for microbial-based biopesticides by most
regulatory bodies results in unfitting registration procedures that are mainly derived
from synthetic chemical-based pesticides (Glare and Moran-Diez 2016). Hence,
registration is known to be a very complex, time- and money-consuming, cumber-
some process that significantly differs from country to country. Major characteristics
of the registration processes of EPA and EU are briefly described below.
The registration process of EPA: The Biopesticide and Pollution Prevention
Division (BPPD) that comes under EPA is responsible for all regulatory activities
of biopesticides. The process of registration starts with a pre-submission consultation
meeting, in which the applicant is informed about guidelines and types of study
required and the labeling. A dossier is then compiled with required data and formally
submitted to BPPD for further evaluation. Upon the formal submission, the content
will initially screen for completeness followed by preliminary technical screening to
determine further adequacy and completeness of data. If sufficient data is present
with no other issues, registration is granted after reviewing. Generally, the full
process takes up to 12–18 months (Arora et al. 2016).
The registration process of EU: In the EU, registration process is a comparatively
complex and long procedure which is composed of two stages where both the active
ingredient and product undergo separate evaluation. In contrast to EPA, the EU does
not identify biopesticide as a separate regulatory category but under the plant
protection product.
Registration of active ingredients, which is the first stage of EU registration, is
composed of three subsequent phases: Rapporteur Member State phase (RMS
phase), risk assessment phase, and risk management phase.
In the RMS phase, a compiled dossier containing all the information on active
substance and at least one representative PPP is submitted to designate RMS for
registration. If the application is admissible, then RMS proceed with the evaluation
and produce a draft assessment report which is then submitted to other responsible
European Committee (EC) and European Food Safety Authority (EFSA) for risk
assessment on the active compound. After the peer-reviewing by EFSA, they
released a conclusive scientific risk assessment report. Using the risk assessment
report, EC presents a review report to the Standing Committee for Food Chain and
Animal health (SCFCAH), and voting is carried out for approval or non-approval of
the active substance. This is called the risk management phase. Depending on the
weight and complexity of the dossier, the process will take up to 2.5–3.5 years.
The second stage or the registration of PPP is carried out by the relevant MS after
the applicant specifies the MS. Once the multiple dossiers for the product, every
active ingredient is submitted; the relevant MS evaluates and grants the authorization
10 Microbial Biopesticides: Development and Application 179
if it is acceptable. Then the application for mutual recognition within one of the three
geographical zones (North, Central, and South) with the same use under similar
conditions can be done by the applicant company (Frederiks and Wesseler 2019).
Although registration processes of both EPA and EU show contrasting differ-
ences including required data for dossier preparation, Glare and Moran-Diez (2016)
present generalized data requirements for dossier compiling as follows.
Data requirements for an active substance are as follows:
Identity and purity, physical and chemical or biological properties, information
on usage and production process, analytical methods for identification of active
substance(s), human and domestic animals’health effects, residue chemistry, fate
and behavior in the applied environment, and possible non-target effects.
Data requirements for the formulated product are as follows:
Type of formulation and composition details, physical and chemical properties,
application techniques, labeling requirements, and packaging details; analytical
methods used, efficacy data, and toxicology and exposure data; residues, fate, and
the behavior of the product in applied environment; and possible effects on
non-target organisms (Glare and Moran-Diez 2016).
10.15 Development and Application of Nematophagous
Fungi as Biopesticides
Plant parasitic nematodes cause significant damage to vegetables and other agricul-
tural crops throughout the world. It was estimated that plant parasitic nematodes
could cause much more damage annually compared to other insect pests. The
estimated annual crop loss due to nematodes is around 12.3% (157 billion dollars)
worldwide (Singh and Kumar 2015). Based on the data generated by the All India
Coordinated Research Project (AICRP) on nematodes, plant-parasitic nematodes
may cause 21.3% crop losses amounting to Rs. 102,039.79 million (1.58 billion
USD) annually in India (Kumar et al. 2020).
During the last decades, concerns about using nematicidal chemicals were raised,
and as a result, biological control agents to control nematodes have been discussed.
Nematophagous fungi as natural enemies of the nematodes offer a propitious
eco-friendly approach in the control of plant parasitic nematodes.
10.16 Nematophagous Fungi as Biopesticides
Nematophagous fungi naturally can be found on soil. These are carnivorous fungi
that are specialized to trap and digest nematodes. Thus, nematophagous fungi can be
used to control the soil nematodes attacking crops and other plants. The activity of
nematodes controlled by several ways as described further:
180 H. T. Mandakini and D. S. Manamgoda
1. Fungi produce specialized structures (adhesive or mechanical traps) that can trap
nematodes.
2. Fungi can produce mycotoxins that immobilize nematodes before invasion.
3. Endoparasitic and egg parasitic fungi can attack all life stages of nematodes.
A nematode trapping fungus Arthrobotrys oligospora was first described in 1852;
however, by the time, its important ability in control nematode diseases was not
discovered.
Nematode trapping fungi are taxonomically heterogeneous; they belong into class
Ascomycota, Basidomycota, and Zygomycota. Around 350 different species are
recorded around the world. Some of the examples are Arthrobotrys,Cystopage,
Dactylellina,Dactylella,Drechslerella,Hohenbuehelia,Hyphoderma,
Monacrosporium,Nematoctonus,Orbilia,Stylopage,Tridentaria,Triposporina,
and Zoophagus.
Nematodes trapping fungi infect their hosts through a sequence of events
(Nordbring-Hertz et al. 2001). First, the nematodes were isolated and trapped by
the fungi. According to the literature, fungi produce traps constitutively. However,
the trap formation is initiated as a response to the prey (Nordbring-Hertz 1977). The
recognition of the presence of the nematodes and increment of producing trapping
devices depend on a molecular mechanism. Lectins is reported to be involved in the
recognition process. However, recent studies based on A. oligospora suggested that
these fungi may be capable of compensating even for the absence of the lectin by
expressing other proteins with similar function(s) to the lectin (Jiang et al. 2017).
The trapping devices of these fungi are derived from the vegetative mycelium.
Trapping adheres nematode or traps the nematode with non-adhesive mycelia so that
the nematode cannot move. Types of trapping devices can be described as adhesive
networks, adhesive knobs, constricting rings, non-constricting rings, adhesive
branches, undifferentiated or unmodified adhesive hyphae, stephanocysts, spiny
balls, and acanthocytes (Fig. 10.2). The type of trapping device varies with the
type of fungi. There is a correlation of the morphology of the trapping device with
the phylogeny of nematode trapping fungi (Jiang et al. 2017).
Just like other pathogenic fungi, after recognition and adhesion, nematode trap-
ping fungi enter the host by enzymatic degradation and by adding mechanical
pressure on the nematode body. Extracellular hydrolytic enzyme produced by the
nematode itself is a key virulence factor in nematode trapping fungi. After the
penetration, fungi obtain nutrients decomposing the nematodes. Proteases are
involved mainly in the penetration of the fungi into the nematodes. The first study
on nematophagous proteases was done based on Arthrobotrys oligospora by Tunlid
et al. (1991). Later, Lopez-Llorca (1990) was able to purify and identify the first
nematophagous serine protease from the nematode egg parasite Verticillium
suchiasporium. After that, many different serine proteases were detected, character-
ized, and cloned.
Several nematophagous fungi are reported to produce nematotoxins that immo-
bilize or kill nematodes. For example, Pleurotus species such as Pleurotus
cystidiosus,P. cornucopiae,Pleurotus djamor,P. levis,P. populinus,
10 Microbial Biopesticides: Development and Application 181
P. tuberregium, and P. subareolatus are reported to produce toxins that can kill or
immobilize nematodes (Lopez-Llorca et al. 2006; de Freitas Soares et al. 2018).
Some fungi in the order Agaricales (Megacollybia platyphylla,Cyathus striatus,
Kuehneromyces mutabilis) and order Polyporales (Daedalea quercina,Fomitopsis
pinicola,Gymnopilus junonius) are reported to produce toxins that would paralyze
the nematodes (Balaeșand Tănase 2016). Other than the one mentioned above, there
are numerous other fungi that are capable of producing toxins against nematodes as a
defense mechanism to prevent the consumption of fungal colony by the nematodes.
Li et al. (2007) reviewed 179 fungal producing chemical compounds with nemati-
cidal action.
Some nematophagous fungi prey on eggs. For example, Myrothecium verrucaria
strain X-16 has been demonstrated to prey on eggs, second stage juveniles, and also
adult nematodes of Meloidogyne hapla (Dong et al. 2015).
Fig. 10.2 Trapping devices of nematophagous fungi. (a) Stalked adhesive knobs, (b) sessile
adhesive knobs, (c) non-constricting rings, (d) adhesive columns Ei and Eii: constricting rings,
(f) adhesive nets. (Source: Redrawn based on Jiang et al. 2017)
182 H. T. Mandakini and D. S. Manamgoda
10.17 Productions of Biopesticides Using
Nematophagous Fungi
The first attempt to use nematophagous fungi as a bio-control agent was carried out
in 1930s (Linford and Oliveira 1937). According to Soares (2006) biological control
of nematodes has many benefits over chemical control. Some of these advantages are
easy application, less or no harm to the environment, not leaving any residue on the
crop, not favoring the emergence of resistant nematode verities, not adversely
affecting soil microbiota, and the potential ability to convert conducive soil into a
suppressive one.
When considering the development of biopesticides, Phase 1 begins with the
isolation of potential nematophagous fungi. Fungi can be isolated from soil, nema-
tode body, and eggs (Aminuzzaman et al. 2013). The in vitro predatory activity of
the fungi can be evaluated by estimating the reduction of nematode population on
assay plates after a given incubation time (Aminuzzaman et al. 2013). Also, assays
are developed to detect the activity of extracellular hydrolytic enzymes of the fungi
(Yang et al. 2007).
After selecting a suitable candidate with a great predatory activity, the next steps
in Phase 1 will continue. As well as the other fungi, nematophagous fungi can be
identified using morphological and molecular methods. Morphological structures of
these groups of fungi are mostly identified using the keys prepared by Cooke and
Godfrey (1964) and Philip (2002). Sequencing the fungal barcode (nuclear ribo-
somal internal transcribed spacers and 5.8S), ITS region is commonly practiced.
DNA sequences are initially identified using NCBI blast comparison, and later
phylogenetic analysis using only ITS or combining the sequences data of protein
coding gene regions has been carried out (Falbo et al. 2013).
Phase 2 of the biopesticide development starts with mass production and formu-
lation. The most favorable temperature for the growth of nematophagous fungi is
25 C (Velvis and Kamp 1996; Castro et al. 2000). Nematophagous fungi can be
mass produced on solid or liquid media. The amount of biomass produced is
generally higher in the liquid media. The formulation step can be carried out by
several means. One of the common formulation techniques used in the literature is
listed below.
10.18 Capsule Formulation Using Sodium Alginate
This technique would encapsulate the conidia of nematophagous fungi. Fungal
strains will be multiplied on solid or liquid media until conidia are produced. The
conidia are quantified on sterile water. In order to capsule formation, clay (5%
sodium bentonite), sodium alginate (1%), and streptomycin sulfate (1%) were used
according to the methodology mentioned by (Carneiro and Gomes 1997). Conidia
were then mixed with a homogenized mixture of above-mentioned three ingredients.
10 Microbial Biopesticides: Development and Application 183
The formulation concentration depends on the added concentration of conidia there
for concentration should be adjusted accordingly.
After that, a CaCl
2
solution (0.25 M) is added to induce the formation of solid
aggregates or pellets. These solid aggregates were then submitted to a drying process
in a laminar flow chamber for about 6 h. According to the literature, process will
yield approximately 3.4 g pellets for each 100 mL of suspension (Carneiro and
Gomes 1997). These formulated aggregates can be stored at room temperature, away
from light, for about 12 months. The viability of the conidia may vary according to
the strain of the fungi.
10.19 Formulation Using Rice and Sorghum Grain
Formulation of fungi on shell rice or sorghum grains is regularly practiced. The
technique can be used for different nematophagous fungi such as Paecilomyces,
Pochonia,Arthrobotrys,Monacrosporium,Dactylella, and Dactylaria. Grains
should be soaked overnight and then cleaned and sterilized in 121 C for 15 min
at 15 PSI. If there is excess water remain after sterilization, it can be drained, and
inoculation of the fungi can be carried out once the grains are cooled.
10.20 Formulation Using Sugarcane Bagasse and Rice Bran
Sugarcane bagasse which is a byproduct of sugar cane production can be enriched
with rice bran to be used as formulation material. A sterilized mixture of sugarcane to
rice bran 2:1 ratio is generally used. After cooled to room temperature, fungi can be
inoculated.
10.21 Cocktail Formulation
Since there are different parasitism/predation forms of nematophagous fungi,
researchers came up with an idea of formulating of some nematophagous fungi
with different capturing strategies (dos Santos and Ferraz 2000). dos Santos and
Ferraz (2000) named this mixture as “nematophagous fungi cocktail.”Instead of
developing formulation from one strain of fungi, nematophagous fungi are sepa-
rately multiplied and then mixed in equal proportions. The first such
“nematophagous fungi cocktail”was comprised of the species of P. lilacinus,
A. musiformis,A. oligospora,D.leptospora, and Monacrosporium robustum, and
it was used against root-knot nematodes, Meloidogyne spp. (dos Santos and Ferraz
2000).
184 H. T. Mandakini and D. S. Manamgoda
Another cocktail containing fungi M. eudermatum,D. leptospora,A. musiformis,
A. conoides, and P. lilacinus was tested against phytopathogenic nematodes of
T. semipenetrans and P. jaehni on citrus plants (Martinelli 2008; Martinelli et al.
2012). A significant reduction in the nematode populations was observed compared
to the use of chemicals against these nematodes on citrus.
10.22 Application of Nematophagous Fungi Formulations
Previous studies indicate that before application of formulations under field condi-
tions, it is extremely important to evaluate some characteristics on soil such as
organic matter content, relative humidity, temperature, and the presence of nema-
todes in the area (Barbosa et al. 2011). Time of application is also critical, because
microorganisms would not survive in dry soil. Application during the midday under
direct sunlight may lead to death of nematophagous fungi prior to its action (dos
Santos and Ferraz 2000). Several studies using cocktail of nematophagous fungi on
crops of chrysanthemum, lettuce, peppers, and okra were conducted by Soares
(2006), and those experiments presented very impressive results in controlling
nematodes. However, the efficacy of nematophagous fungi is inversely proportional
to the longevity of the crop so that a long duration crop requires more than one
application of the fungal formulation (Martinelli et al. 2015).
10.23 Commercialization of Nematophagous Fungi
Formulations
Although there are a large number of fungi with nematicidal activity, only several
fungi can be actually used in the commercial level production. Screening for suitable
fungal strains and strain improvement strategies to develop high virulent strains has
been researched by academic scientists (Casas-Flores and Herrera-Estrella 2007).
The development of these biopesticides and commercialization have been tried by
enraptures; however, still only a few products are successfully introduced to the
market. The main challenges faced by these newly introduced biopesticides are the
low productivity and the inability to establish under a wide range of soil conditions
(Moosavi et al. 2011). However, suppressive soil demonstrates that these bio-control
agents could generate durable and robust nematode control even though genetic
factors that contribute to this development are not fully understood. Genomic studies
on nematophagous fungi and biology of the infection procedure into the phytopath-
ogenic nematodes would provide more evidence about these pathogenic factors.
Several other intrinsic, ecological, and environmental factors may affect the
efficacy of the nematophagous fungi when they are applied on a larger scale. Even
though some nematophagous fungi show excellent activity under a small scale, after
10 Microbial Biopesticides: Development and Application 185
the applied a larger commercial scale under field conditions, it does not provide
expected results. Therefore, it is recommended to start research with few potentially
effective strains without restricting into one because only a highly virulent
nematophagous fungus with highly competitive saprobic ability may provide the
actual acceptable control level. The major environmental factors that affect the
ability of the nematophagous fungi are soil temperature, pH, and the nutritional
state of the soil. Also the biotic factors such as the host plant and soil microflora may
affect the activity of the fungi under the field conditions. A better overall under-
standing of all of these factors would help to develop a successful bioproduct.
Considering the commercialization of the product, favorable traits of the fungi are
the ability to mass produce, persistent and stable formulations, the easy applicability,
and the high productivity. The new scientific and technological developments would
provide easy screening and development procedures for nematophagous fungi. On
the other hand, farmers should also be informed about the environmental benefits
and economic benefits of organic farming for switching into bio-based nematicidal
products (Moosavi and Askary 2015).
10.24 Conclusion
Microbe-based biopesticides are eco-friendly and less hazardous to the environment.
Therefore, these formulations are considered as an integral part of sustainable
agricultural practices. However, the development pathway is challenging due to
high research and production costs and time- consuming legislative requirements.
However, considering the rate of rising market value and global demand and future
of microbe-based biopesticides will be promising alternatives to hazardous
chemicals. Thus, with the development of technology, there is a greater potential
in the research areas focusing on isolation, characterization, and development of
suitable bio-based pesticides.
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10 Microbial Biopesticides: Development and Application 189
Chapter 11
Microbial Consortia and Their Application
for the Development of a Sustainable
Environment
Sneha Trivedi, Naresh Butani, Helina Patel, Manoj Nath, and Deepesh Bhatt
Abstract Abundant diversity of microbial communities existing in nature follows
the rule of coexistence demonstrating interspecies and interkingdom interactions for
their proliferation. These communities exhibit highly complex and interwoven web
of metabolic communications. They also perform division of labor managing their
metabolic load for the survival of different co-inhabitants of that area. These
microbial consortia are involved in various interactions such as mutualism and
syntropy, commensalism, and antagonistic behavior as a part of their struggle for
existence. Due to their unique properties of division of labor, diversified metabolic
capacities, and ability of co-survival, microbial consortia can be exploited for
approaches applied for maintaining sustainability of the environment. They can be
utilized in agricultural practices that can reduce the use of chemical fertilizers and
pesticides. Microbial mixed cultures also have potential for waste management and
bioremediation. Additionally, they can prove to be an attractive alternative as a
source of biofuel and bioenergy generation.
Keywords Arbuscular mycorrhizal fungi (AMF) · Microbial consortia · Microbial
interactions · Plant growth-promoting rhizobacteria (PGPR) · Soil ecosystem
S. Trivedi · N. Butani · H. Patel
Department of Microbiology, Shree Ramkrishna Institute of Computer Education and Applied
Sciences, Sarvajanik University, Surat, Gujarat, India
M. Nath
ICAR-Directorate of Mushroom Research, Solan, Himachal Pradesh, India
D. Bhatt (*)
Department of Biotechnology, Shree Ramkrishna Institute of Computer Education and Applied
Sciences, Sarvajanik University, Surat, Gujarat, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_11
191
11.1 Introduction
Due to enormous human activities which have caused massive impact on environ-
ment, its sustainability has become a matter of great concern. Increased use of
chemical fertilizers and pesticides has created questions regarding sustainability of
soil ecosystems. Increasing efforts are being made to develop such processes which
can lead to sustainable agriculture practices. Along with that, there is an increasing
awareness about solving pollution issues through sustainable approach. Addition-
ally, bioproduction sector also has their own problems regarding the sustainability of
processes, which can cause economic load on the industries. Microbial consortia can
prove to be an attractive alternative to solve these issues.
Microorganisms are the most diverse forms of living beings that thrive in any
ecosystem ranging from extreme environments to water and soil. In all these
ecosystems, different microbial communities flourish simultaneously creating com-
plex web of biochemical interactions that function as a key to ecological architec-
ture. Enormous diversified species in large number forms complex interactive webs
of metabolic activities in natural ecosystems such as soil, gastrointestinal system,
etc. (Shahab et al. 2020). Hence, it leads to a fact that “Mother Nature”has designed
the microbial existence as a large consortium.
Microbial consortia refers to the coexistence or co-cultivation of two or more
species in terms of growth, metabolism, and division of labor, creating conditions for
their own composition (Jia et al. 2016). Even though, in recent times, microbial
consortia have attracted the attention of bioprocess engineers by offering certain
advantages, it was also used in the ancient eras, from wineries in Africa to biogas
generation from undefined microbial mixtures used for heating bath in Assyria (Peng
et al. 2016). These multiple microbes together possess the capacity to perform
difficult metabolic tasks and provide a miraculous outcome. It is advantageous
over single species-operated bioprocesses in certain aspects. Firstly, combining
suitable species can fulfil complex tasks without much difficulty. Secondly,
co-adaptability of multiple strains leads to co-stability and can resists environmental
fluctuations. This is because of their internal beneficial interactions (Nuti and
Geovannetti 2015). Additionally, metabolic requirements of different species can
be fulfilled by the fellow microbial members in the consortia. This occurs when a
metabolite produced by one member of consortia is utilized as limiting nutrient for
other species, thereby manifesting complete mineralization of the components along
with favoring their coexistence. Also, through gene regulation via quorum sensing,
these microbial consortia demonstrate multiple unexplored biochemical mechanisms
that contribute in their functionality (Odoh et al. 2020).
There is an enormous array of mechanisms that occurs continuously among the
members of the consortia which ranges from highly beneficial or synergistic to
extreme damaging or antagonistic. These interactions occur as a part of processes
microbes develop while thriving for their existence in the natural environment, while
dealing with space limitations, nutrient depletion, and other natural or co-inhabitant
generated stressful environment. Hence, these interactive mechanisms can be studied
192 S. Trivedi et al.
and exploited as a tool for the search of newer metabolites, novel enzymes, and other
components. Furthermore, study of these interactions can provide an insight into
gene regulation which assists in altering the production yields of certain components
at the industrial level.
Microbial consortia can be categorized broadly in two types: (1) natural commu-
nities and (2) man-made co-cultures (Canon et al. 2020). The former types are
present in the natural ecosystem and are known to establish a spontaneous associ-
ation where the dynamics are governed by the laws of natural selection. They are
substantially complex, and the species flourishes according to the release of metab-
olites by one member which can be subsequently utilized as substrate by other
members of the community. Natural communities are employed in number of
processes such as methane production in activated sludge process, fermentation
such as cheese or kimchi production, as well as waste management (Canon et al.
2020). The second type of microbial consortia includes artificially mixed cultures as
well as synthetic co-cultures that are developed by human conciliation. They are
customized mixtures framed according to the process requirements. Hence, it can be
deciphered that these mixtures of microorganisms hold enormous applications in
variety of fields which include agriculture (Sekar et al. 2016), bioproduction (Canon
et al. 2020), bioremediation, and even in waste management (Al-Dhabi et al. 2019).
In addition, the potentiality of these microbial consortia had recently opened up the
gates for a newer aspect of synthetic biology that deals with designing and analyzing
synthetic microbial consortia, having applicability in the field of bioprocessing.
They are also added as a part of ingredients which are supplied as plant
bio-stimulants (Woo and Pepe 2018).
Traditional bioprocesses are focused on exploiting the potentiality of monocul-
ture for fermentation; however, when it comes to complex biotransformations, the
mentioned approach faces some of the drawbacks, namely, extensive metabolic
burden, cellular space limitation, and toxic intermediates (Shahab et al. 2020).
This has diverted microbial engineers toward exploring the possibilities of utilizing
microbial consortia to get the job done.
11.2 Microbial Interactions
Cellular interactions among microbial species are ubiquitous in nature and are prime
sculptors of ecosystem dynamics (Kong et al. 2018). Interaction between members
of microbial consortia, either natural or artificially designed, is a pivotal factor that
plays a decisive role in the success of any bioprocess. Furthermore, emergence and
sustainability of metabolic interactions depend on factors such as nutrient availabil-
ity, diffusion constraints, and microbial community spatial structure and structure. In
their habitat, even though microbes tend to demonstrate individualism, the consortia
respond to environmental stress as a unique organism (Odoh et al. 2020). Positive
interactions are mutualistic and improve the fitness among co-cultures and favor
product formation (Canon et al. 2020). When microbial species flourish in their
11 Microbial Consortia and Their Application for the Development of a.. . 193
natural habitat, they may have to overcome through negative interactions such as
competition, parasitism, etc. However, when polycultures are applied purposefully,
the designing is done to selectively enhance beneficial interactions, excluding the
members involved in damaging mechanisms (Fig. 11.1).
11.2.1 Mutualism
Established as one of the most common interactions found in biosphere, mutualism
refers to the interaction in which different species are dependent on each other for
fulfilling their nutritional requirements or need for space (Kouzuma et al. 2015).
Also termed as symbiotic relationship, such interactions are widespread in nature.
The word “symbiose”was coined by De Bary in 1879, for dissimilar organisms that
are living together, and his definition covered both mutualists and parasites (Hirsch
2004).
In agriculture, microbial consortia use symbiotic interactions which can contrib-
ute for enhancing soil fertility. In soil ecosystem, where bacteria accounts for 95% of
the microbial community (Odoh et al. 2020), multiple microbial species colonizes
around the rhizosphere either as free living, epiphytes, or endophytes. They establish
close interactions with root system which may prove beneficial to plants and
microbes both. The signature example of such interactions can be understood in
terms of rhizobium-root legume relationship, where rhizobium is involved in nitro-
gen fixation benefiting plants and in return uses inhabiting space and biochemical
metabolites released by plant roots as carbon and energy source. Additionally, there
are members of plant growth-promoting rhizobacteria (PGPR) community that is
involved in such co-relations (Khan et al. 2020). Members of genera Bacillus,
Paenibacillus,Burkholderia,Azotobacter,Rhizobium, and Pseudomonas (Ahmad
et al. 2018) inhabit plant rhizosphere where they indulge in diversified mechanism
which benefits the plants by solubilization of minerals, production of phytohor-
mones, releasing antagonistic substances that inhibit the growth of pathogenic
organisms and many other. In return, they reap the benefits in the form of exudates
released by plant roots as their nutrient and energy source (Menéndez and Paço
2020), have termed beneficial bacteria of rhizosphere as plant probiotic bacteria
(PPB), and have classified them into two subgroups: (1) rhizobial bacterial endo-
phytes (RBEs) and (2) non-rhizobial bacterial endophytes (NRBEs). Their studies
suggested that those symbiotic interactions between plants and microbes are highly
complex, and microbial consortia in the form of PPBs have ensured good results in
terms of plant benefits. They have also stated improvement in symbiotic effective-
ness between RBEs and NRBEs in the form of consortia along with mycorrhiza
increased crop yields and reduced the usage of chemical fertilizers.
Another popular mutualism is demonstrated by mycorrhizal fungal species. They
are named as arbuscular mycorrhizal fungi (AMF) and belong to subphyla
Glomeromycotina. Under symbiotic association with plants, they acquire lipids
and carbohydrates from the plants through special structures such as arbuscules
194 S. Trivedi et al.
Fig. 11.1 Interactive mechanisms observed among the members of microbial consortia: The figure
deciphers the release of growth factors from one of the members can help in better utilization of the
substrate by other member (Section A). Other possibility is of synergistic relationship where both
the species are interdependent for their substrate requirements as shown in section B of the figure.
Section C explains the favors extended from one member through removal of inhibitors of the
reaction, thereby making substrate utilization possible for co-member. Antagonism is also a major
interaction observed in microbial communities in which competition for resources can inhibit other
organism or else this can divert the organism to produce novel metabolites for the purpose of their
survival (Section D). Also, biotransformations can occur as a result of interactions in which the
intermediate produced by one microbe can act as starting metabolite for fellow member as shown in
Section E
11 Microbial Consortia and Their Application for the Development of a.. . 195
and vesicles. As a return gift, these fungal species are involved in rapid uptake of
plant nutrients mainly phosphorous, nitrogen, and potassium, thereby increasing
crop yields. Mixture of such species provides better outputs compared to single
mycorrhizal species (Paredes et al. 2020).
In addition to the above examples, one more popularized example of symbionts is
lichens. They can be considered as tripartite or quadripartite symbionts which
comprise of photosynthetic organisms as cyanobacteria or algae, fungus as another
member, and a nitrogen-fixing root symbiont which also includes mycorrhiza
(Hirsch 2004).
Kouzuma et al. (2015) have reported findings on specific type of mutualism
known as “syntrophy,”which is referred to as the interspecies interactions in
which tropic benefits are procured by both the partners. They have studied the
establishment of interspecies communication in syntrophic microbial consortium
related with methanogenesis and stated that the rate-limiting step of methanogenesis
is the mutualistic interactions between secondary fermentative bacteria and
hydrogenotrophic methanogens in which intercellular exchange of reducing equiv-
alents occurs. In this metabolic event, H
2
is generated by propionate-oxidizing
bacteria as a part of energy generation through oxidation of propionic acid.
Hydrogenotrophic methanogens trap this hydrogen for methane production thereby
reducing the product concentration, favoring the reaction to move in forward
direction.
11.2.2 Commensalism
Commensalism is an interaction between microbial communities in which one
member termed as commensal can have substances released or captured by the
host due to special proximity (Bogitsh et al. 2018). In this relationship, host remains
unaffected and releases the exudates as a part of its normal metabolic activities.
Microbial communities in the biofilms may be governed by such interactions. Use of
high-end technologies such as fluorescent in situ hybridization (FISH) and micro-
electrodes applications has demonstrated the clustering of nitrate-oxidizing bacteria
around ammonia-oxidizing bacteria in nitrite-oxidizing zones in the waste water
treatment where nitrifying biofilm is involved (Christensen et al. 2002). Hirsch
(2004) has suggested that commensalism has resulted in increased bacterial survival
in rhizosphere as well as bulk soil.
Kong et al. (2018) demonstrated ecosystem dynamics of commensalism using
synthetically designed two strain consortia. They created one strain CmA from
L. lactis which had full nisin pathway and constitutively expressed tetracycline
resistance. It was co-cultured with CmB which contained nisin inducible tetracycline
resistance power. Their studies determined that when two strains were grown as
mono-cultures in the presence of tetracycline, CmA, because of its constitutive
expression of tetR gene, was able to grow; in contrast, CmB could not grow by
itself as its resistance was not self-dependent. However, when both the strains were
196 S. Trivedi et al.
combined, CmB grew well, suggesting that the presence of CmA benefited CmB for
its growth. Studies of detailed mechanism conferred that tetracycline resistance was
the mechanism of commensalism for CmB which was achieved using nisin released
by CmA as a part of its normal metabolism.
Hirsch (2004) has suggested that microbial interactions such as mutualism,
commensalism, and parasitism should be considered in continuum. Mutualists like
rhizobium species initially flourish in the soil or plant roots as commensals and can
form biofilms on inanimate surfaces. This may be for the survival of the bacterium
until the suitable host is found.
11.2.3 Antagonism
Antagonism is the mechanism of causing harm to other organisms primarily for the
purpose of self-survival or as a product of normal metabolism. It has a substantial
contribution for framing the bacterial community by inhibiting one community
while opening the doors for other bacterial niches (Long et al. 2013). Competitive-
ness between different species in the consortia usually occurs due to nutrient
depletion and space requirements which result in the synthesis of secondary metab-
olites, synthesis of enzymes that can combat the situation or activation, and expres-
sion of silent genes present in the genome of the species. Under such situations,
microbial residents of the same niche excrete such substances that can cause cell
damage or oxidative stress in the surroundings. The target species may generate
substances that can combat those stressful situations as a part of microbial defense
mechanism (Zhang et al. 2018). Hence forth, these mechanisms can provide enor-
mous potential in search of newer secondary metabolites or study of gene expression
and regulation or improvement of product yields. It can also be favorable for
combating plant pathogens in agricultural practices.
Maiyappan et al. (2010) have studied the effect of microbial consortia Omega on
the growth promotion of black gram and found its antagonistic effect on pathogenic
fungi like M. phaseolina,S. rolfsii,F. oxysporum,andR. solani. Extracellular
antagonistic substances secreted by bacteria in combination with plant exudates
may be responsible for keeping pathogens in check. Long et al. (2013) conferred
the existence of antagonistic interactions between self-sustaining microbial commu-
nities in the microbial mats of hypersaline lake and suggested that antagonism which
is driven by chemicals is a major contributor in designing of bacterial communities.
They supported their hypothesis by co-culturing assay and determined that 57% of
bacterial isolates expressed damaging behavior toward one or more species, while
5% of the isolates showed antagonism toward 80% of the isolates.
11 Microbial Consortia and Their Application for the Development of a.. . 197
11.3 Applications of Microbial Consortia
There are number of important areas where unique potential of microbial consortia is
exploited for various aspects. Majorly microbial consortia are used for sustainable
agricultural practices (Al-Dhabi et al. 2019; Paredes et al. 2020; Maiyappan et al.
2010), bioproduction processes (Canon et al. 2020; Sabra et al. 2010; Ghosh et al.
2016), and bioremediation strategies (Paerl and Pinckney 1996). Furthermore, they
are now being explored as a tool to unravel the secrets of coexistence in microbial
ecology as well as synthetic biology (Haruta and Yamamoto 2018).
11.3.1 Sustainable Agriculture
In the wave of urbanization, there has been a drastic increase in the usage of chemical
fertilizers and chemical pesticides that has raised serious environmental concerns.
Also, many of these components have entered the food chain causing several health
issues in the people consuming such food. Overall, such harmful chemicals have
entered the water bodies and deteriorated soil quality thereby impacting the biodi-
versity of that particular habitat. Microbial consortia can prove a remedy to such
problem providing an eco-friendly alternative (Sekar et al. 2016; Paredes et al.
2020).
Plant growth depends on the availability of various nutrients such as nitrogen,
phosphorus, and potassium available in solubilized form as well as growth-
promoting hormones, namely, indole acetic acid, gibberellic acid, and others. One
of the prime characteristics of microbial consortia is to act synergistically among
each other and to secret the components that support plant growth promotion. Plant
growth-promoting microorganisms are heterogenous flora encompassing diverse
species including bacteria, actinomycetes, fungi, etc., demonstrating cross kingdom
interactions. Additionally, those pool of microbes provide immunity to plants by
keeping phytopathogenic organisms at bay through various antagonistic
mechanisms.
Maiyappan et al. (2010) have isolated, identified, and evaluated the plant growth-
promoting efficiency of nine stains of microorganisms that were the members of
Bacillus sp., Streptomyces sp., Azotobacter sp., and Fruateria sp., developed a
consortia Omega, and studied its effect of plant growth of black gram (Vigna
Mungo L.). Pot culture studies demonstrated significant increase in shoot length of
the plant compared to chemical fertilizer-added plants and control plants. Also, the
root volume as well as total dry mass was higher in the consortium-fed plants
compared to chemical fertilizers.
The potentiality of Streptomyces sp. associated with tomato plant roots and the
effect of microbial consortia developed from composting the waste were studied by
Al-Dhabi et al. (2019). They stated that use of such approach increased the tomato
198 S. Trivedi et al.
weight up to 15%. The presence of phytohormone such as indole acetic acid and
siderophores was also analyzed.
Sekar et al. (2016), Woo and Pepe (2018), and Vishwakarma et al. (2020) have
summarized outcomes of various studies carried out in the field of analyzing effects
of PGPR and microbial consortia on plant growth and agricultural practices. They
have stated that co-inoculation of rhizobia with other microbial species and PGPR
proved to be favorable for nodulation as well as below-ground and above-ground
development of plant species. Furthermore, such consortia were able to reduce the
occurrence of as well as severity of certain plant diseases. Inoculation of AMF
(arbuscular mycorrhizal fungi) along with PGPR increased plant yields both in
nursery and field studies. They have also suggested that application of microbial
cocultures can provide a solution to alleviate plant abiotic stress and enable more
growth and productivity of plants under stressful environment such as high salinity,
dryness, and rising temperature. This approach can improve the deteriorated soil
quality thereby increasing crop production that can combat food shortage in the
future.
However, the influence of management practices in agriculture decides the
direction of the crop productivity as plants are major selectors of the specific taxa
in their phyllosphere. In addition to microbial inoculants, practices such as organic
farming, crop rotation, intercropping, automation, and technology implementation
are key co-factors that determine the sustainability of agriculture.
11.3.2 Waste Treatment and Pollution Control
A major challenge the world is facing in the present time is rotten fruits of
industrialization, in the form of pollutants that has accumulated in soil, water, and
air. This is causing serious threat to the sustainability of environment along with
risking human health. The processes used currently for reducing toxic compounds
involves burning, recycling, land-filling, and pyrolysis. However, such processes are
releasing even more toxic and non-degradable compounds in the environment
alongside creating difficulty in execution for environmental decontamination
(Ahmad et al. 2018).
Microbial communities as weapons of green technology are more efficient in
metabolizing chemically complex and toxic metabolites compared to single species.
The explanation of this potential of co-cultures lies in the enormous interactions
occurring among the multiple species in which metabolite released by one species
can be utilized by other co-inhabitant of the community. Hence, this capability of
microbial mixed cultures can be utilized in bioremediation of complex polluted sites
such as dye polluted water, heavy metal polluted sites, oil spilled sites, and many
others (Ahmad et al. 2018; Ghosh et al. 2016). Furthermore, artificial designing of
ecological niche is a newer concept emerging in this area where resource specific
niche can be sculptured, thereby achieving desirable biotransformations of the target
substrates (Shahab et al. 2020).
11 Microbial Consortia and Their Application for the Development of a.. . 199
11.3.2.1 Dyes
Dyes are present in industrial effluents discharged from textile industries. Dyes
comprise of recalcitrant compounds, mainly azo dyes that are hard to degrade due
to their strong bonding capacity, causing serious concerns. There are certain bacte-
rial species that produces enzymes such as azo-reductase that can act upon azo dyes.
Some species produce enzymes that can degrade the intermediate components
formed during azo dye reduction reaction. There are number of reports and studies
proving potential of different microbial species including fungi, algae, and bacteria
for dye degradation and other such xenobiotics excreted in the effluents of paper
pulp industries (Ahmad et al. 2018).
11.3.2.2 Organic Domestic Wastes
Domestic waste generated in households also covers a major section of the waste
generation. The usual procedure applied for such waste disposal including dumping
on landfills which can contaminate the soil of that site in a long run. Household-
generated waste is a rich source of organic nutrients, and if treated with microor-
ganisms, they can be transformed into biofertilizers and source of biofuels or animal
feed. Microbial communities have physiological diversity that can be manipulated
and designed for the customized treatment of such organic wastes which can reduce
the treatment time and provide with desired output in waste management.
Sarkar et al. (2011) have developed eleven different consortia which were
analyzed for their capacity to produce suitable cocktail of hydrolytic enzymes,
namely, amylase, lipase, protease, and cellulase, that can breakdown those compo-
nents present in kitchen wastes. In their studies, they found effective reduction of
kitchen waste in reduced time span, that too without the generation of foul smell.
11.3.3 Bioenergy Generation
Biofuels and bioenergy resources such as bioethanol and biogas are equally impor-
tant options for sustainable energy generation alongside the renewable energy
sources like wind energy, hydro energy, and solar energy. These biological pro-
cesses depend on the biochemical activities performed by diverse species of micro-
organisms. As lignocellulosic wastes are major substrate used for biofuel production,
monoculture of microbes cannot degrade them efficiently. A single bioreactor
equipped with correct combination of microbes can convert complex feedstocks to
simpler sugars that can be utilized for the production of bioenergy. For example,
when pair of Bacillus and Clostridium sp. were co-cultured in a bioreactor on
lignocellulosic wastes, Bacillus sp. produced hydrolytic enzymes that acted upon
200 S. Trivedi et al.
complex polymeric substrate and released simple sugars which were further utilized
by Clostridium sp. for efficient generation of hydrogen (Ghosh et al. 2016).
Methanogenic microbes demonstrate a type of mutualistic relationship termed as
syntropy, where reducing equivalents such as formate and hydrogen are transferred
between syntrophic partners. Microbial species involved in such communication
have developed mechanisms at molecular level for the establishment of interspecies
partnership. There are evidences that suggest that these transfers can occur in the
form of electric current which can be exploited for bioenergy generation (Kouzuma
et al. 2015).
11.4 Future Aspects
Deciphering the mysterious potentials of microbial consortia has opened new ave-
nues for its applications in the areas of applied microbiology. Tailor-made designing
of microbial co-cultures has enabled the bioprocesses to achieve custom-made
desirability; hence continuous attempts are carried out for upgradation of such
systems for achieving increased product yields. Engineering of microbial consortia
has lot a greater number of future prospects in environmental restoration, in
improvement of soil and water quality, as well as in biosystem analysis and
industrial biotechnology. Synthetic microbial consortia can be applied in unraveling
the intricate mechanisms involved in gene circuits and quorum sensing. Microbial
consortia engineering and in silico approach hold huge potential that can reframe
ecological niche and can provide novel biomolecules for better environmental
sustainability and ecosystem management.
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202 S. Trivedi et al.
Chapter 12
Microbial Engineering and Applications
for the Development of Value-Added
Products
Ashutosh Paliwal, Abhishek Verma, Ashwini Kumar Nigam,
Jalaj Kumar Gour, Manoj Kumar Singh, and Rohit Kumar
Abstract Downstream is a very affluent process for fermentation. It usually
involves complicated equipment and processes to obtain desired chemicals or
materials from intra- and/or extracellular spaces of microorganisms. Recently, it
becomes possible to simplify the microbial cell separation processes by morpholog-
ically engineering the shapes of small microorganisms. Biologically engineered
entities have enabled discoveries in the past decade and a half, spanning from
novel routes for the syntheses of drugs and value-added products to carbon capture.
The precise cellular reprogramming has extended to the production of nanomaterials
owing to their ever-growing demand. Additionally, nutraceuticals are important
natural bioactive compounds that confer health-promoting and medical benefits to
humans. Globally, growing demands for value-added nutraceuticals for prevention
and treatment of human diseases have rendered nutraceuticals a multi-billion dollar
market. However, supply limitations and extraction difficulties from natural sources
such as plants, animals, or fungi restrict the large-scale use of nutraceuticals.
Metabolic engineering via microbial production platforms has been advanced as
an eco-friendly alternative approach for production of value-added nutraceuticals
from simple carbon sources. Microbial platforms like the most widely used
A. Paliwal · A. Verma
Department of Biotechnology, Kumaun University, Sir J. C. Bose Technical Campus, Bhimtal,
Uttarakhand, India
A. K. Nigam
Department of Zoology, Udai Pratap College, Varanasi, Uttar Pradesh, India
J. K. Gour
Department of Biochemistry, Faculty of Science, University of Allahabad, Prayagraj, Uttar
Pradesh, India
M. K. Singh (*)
National Centre for Disease Control, New Delhi, India
R. Kumar
Cell and Tumor Biology Group, Advanced Centre for Treatment Research & Education in
Cancer, Tata Memorial Centre, Navi Mumbai, Maharashtra, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_12
203
Escherichia coli and Saccharomyces cerevisiae have been engineered as versatile
cell factories for production of diverse and complex value-added chemicals such as
phytochemicals, prebiotics, polysaccharides, and poly amino acids. This chapter
highlights the recent progresses in biological production of value-added
nutraceuticals via metabolic engineering approaches.
Keywords Microbial engineering · Microbes · Nutraceuticals · Phytochemicals
12.1 Introduction
Microbial engineering involves the use of biological, chemical, and engineering
aspect of biotechnology that results in manipulations and development of microbes
to get the desired products in different fields (Peralta-Yahya et al. 2012). The
technology which is employed in microbiological systems and their derivatives to
transform products used in daily need are highly beneficial for humankind (Okafor
2007). India is a country where more than half of the population is engaged in
agricultural practices. India is also considered the second-largest producer of agri-
cultural products worldwide (Gulati and Juneja 2018). Statics data reported that
India produces approximately 81.285 million metric tonnes of fruits and 162.187
million tonnes of vegetables, respectively, in year 2013 (Negi 2014). Most of the
production is consumed fresh; however, a larger quantity which accounts for
approximately 25–40% gets rotten due to unavailability of proper postharvest
facilities. This wastage causes a huge loss to crop yield and also exhibits great
impact on economy. Henceforth, reducing the postharvest wastages requires utmost
consideration for making a chain among consumption and supply. Microbial bio-
technology has been used in handling food since ancient times such as in making
bread or beverage. Though various metabolites are produced by microbes due to the
introduction of modern biotechnology, microbial molecular structures possess
strong potential that could be used in food industry particularly in fermentation of
foods, enzymes, ingredients of food, testing of food, and postharvest administration
of agricultural yields. However, microbial biotechnology in food processing division
represents collection and advancement of microbes by ideas of refining regulated
production, effectiveness, as well as the quality, safety, and consistency of
bioprocessed foodstuffs. Microbes have a pivotal role in the production of fermented
food. Microbial cultures could be genetically modified by traditional and molecular
tactics.
A deliberate breakdown practice which is induced by microorganisms for the
transformation of carbohydrates to alcohols or organic acids is generally referred to
as fermentation (Battcock and Azam-Ali 1998). Fermentation is worldwide func-
tional in the conservation of various raw agricultural products including vegetables,
fruits, tubers and cereals, fish, milk, meat, etc. Some microorganisms accompanying
the fermented foodstuffs such as Lactobacillus sp. are probiotic used as live micro-
bial food complements or food constituents that help in improvement of the
204 A. Paliwal et al.
metabolic process of gastrointestinal tract’sflora. Therefore, microbes are believed
to be advantageous in maximum fermentations. Nowadays, nanotechnology is
considered as innovative field of science which deals with the synthesis and use of
material with nanoscale size in numerous aspect of life. However, the number of
microbes is naturally proficient in generating nanoparticles either intracellularly or
extracellularly while confronted with various metal salts. Accessibility of many
biotechnological tools including synthetic biology, genetic, and protein engineering
increases usage of microbial systems to up-skill synthesis of nanoparticles.
12.2 Nanoparticles Synthesis by Microbial Engineering
The idea of the natural synthesis of nanoparticles was first started in the 1960s and
recently has seen an evolution in the last one and a half decade. Biological nano-
particle synthesis represents an extensive range of biological methods for generating
nanoparticles through biotic hosts which are not restricted to bacteria, yeast, fungi,
algae, and plants. Efficient synthesis of nanoparticle does require a compatible host
that comprises molecular machinery to convert the raw material into the nanoparticle
and can efficiently accommodate the end product, i.e. synthesized nanoparticle.
Majorly few cellular proteins that have a protective role may interfere in the cellular
metabolism and hinder the uptake of metallic ions and their conversion into
nanoparticles with precise size and morphology. Therefore, the manipulation of
such host and proteins should be achievable. The ultimate benefit of the biological
synthesis of nanoparticles is their synthesis at ambient temperature and pressure, and
no involvement of chemicals are required in the synthesis which could perhaps be
hazardous.
Biosynthesis of nanoparticle through microbes occurs in two ways that further
comprise two sub-modes, i.e. (a) intracellular in non-template or template mode and
(b) extracellular in culture or membrane adherent mode. During intracellular syn-
thesis, the cell culture is incubated with the metal salt solution where metal ions pass
across the cell membrane and synthesis takes place inside the cells. Subsequently,
the cells are lysed and nanoparticles are purified. On the other side, extracellular
synthesis as the name suggests involves the synthesis of nanoparticle on the cell
membrane or in the culture broth. Therefore, extracellularly synthesized
nanoparticles are easier to retrieve and require lesser downstream processing phases.
The very first report on biosynthesis of nanoparticle using genetically engineered
bacteria was documented in 2006–2007 (Sambhy et al. 2006; Vigneshwaran et al.
2007). Later, Kang et al. (2008) genetically modified E. coli strain JM109 to express
phytochelatin synthase of Schizosaccharomyces pombe along with improved
g-glutamyl cysteine synthetase (GSHI) to synthesize cadmium sulfide (CdS)
nanocrystals. The GSHI is responsible for catalysing the glutathione synthesis
which is also a precursor of metal-binding peptide phytochelatin that in turn assists
as capping agent for CdS nanocrystals. Phytochelatin synthesis of S. pombe is the
best characterized natural defence mechanism towards cadmium toxicity. Further
12 Microbial Engineering and Applications for the Development of Value-Added... 205
development in the above-mentioned approach was achieved in another strain,
E. coli R189, where uniform CdS quantum dot (QDs) nanocrystals of 3–4 nm size
were synthesized (Kang et al. 2008). Mi et al. (2011) expressed the transgene
encoding CdS-binding histidine-rich peptide (CDS7) reported to bind with CdS
(Peelle et al. 2005) to induce the formation of CdS QDs. Noble nanoparticles from
silver and gold, alkali-earth (Cs, Sr), magnetic (Fe, Co, Ni, Mn), semiconducting
(Cd, Se, Zn, Te) metals, as well as rare earth fluorides (Pr, Gd) were successfully
synthesized using genetically engineered E. coli-expressing recombinant
metallothionein from Pseudomonas putida and phytochelatin from Arabidopsis
thaliana (Park et al. 2010; Ashraf et al. 2021). Some of the extremophiles including
Antarctic bacteria have also been exploited to synthesize the fluorescent nanoparticle
due to their natural resistant to cadmium and tellurides (Plaza et al. 2016). Several
strains of E. coli have a CusCFBA silver/copper system that promotes the synthesis
of silver nanoparticle in periplasmic space (Lok et al. 2008). Shi et al. (2007) also
used a similar type of strain for the synthesis of silver nanoparticle in periplasmic
space using anaerobic conditions. The procedure generates reduced metal nanopar-
ticle using oxidized metal ions as electron acceptors with the assistance of
cytochrome-c (Shi et al. 2007; Suresh et al. 2010).
12.3 Microbial Enzymes
Enzymes can be defined as the biotic catalyst which is involved in various biosyn-
thetic reactions and metabolic processes (Li et al. 2021; Kumar et al. 2021).
Microbes serve as a major source of enzymes. Microbes can replicate easily and
rapidly and could be genetically engineered as per the desired requirement of the
product (Bhandari et al. 2021; Verma et al. 2021). Microbial enzymes are relatively
more active and stable compared to that of isolated from plants or animal sources
(Gopinath et al. 2013; Anbu et al. 2017; Bhatt et al. 2021). Various extremophilic
bacterial and fungal strains have been isolated from unfavourable pH and tempera-
ture as well as high salt and heavy metal conditions for the synthesis of different
useful enzymes comprising properties of higher yield (Gopinat et al. 2003; Gopinath
et al. 2005).
Microbial enzymes can be isolated from different microorganisms including
thermophilic (requires a higher temperature for growth), acidophilic (optimally
active in acidic pH range), and alkalophilic (activates at higher pH range) bacteria.
The synthesis of these microbial enzymes can be carried out at extreme conditions
that can decrease the possibility of contamination during large-scale production
(Banat et al. 1992; Cadet et al. 2016). Revolution in enzymes acquired from
microorganisms creates a great opportunity for the enhancement of low liveliness
consuming improvements that could be applied for biotransformation of poultry
waste into beneficial harvests. Enzymatic events may be accommodating to recycle
waste rich in protein unconfined by the poultry industry, besides these lines
protecting environment by declining waste (Atuanya and Aigbirior 2002). There
are some enzymes that have various roles in industrial applications (Table 12.1).
206 A. Paliwal et al.
12.4 Nutraceuticals
Nutraceutical, a fusion of nutrition and pharmaceuticals, is defined as ‘a material
which possesses the nutritional value of a diet and delivers pharmaceutical or health
assistances such as preclusion and disease management’(DeFelice 1995). Further
revision quoted nutraceuticals as ‘a product isolated or purified from foods that are
generally sold in medicinal forms not usually associated with food’(Pandey et al.
2010). Nutraceuticals have been obtained from various sources ranging from
microbes (e.g. poly amino acids), plants (e.g. phytochemicals, vitamins), and ani-
mals (polysaccharides) as well as marine sources (glucosamine and chitosan) (Ras-
mussen and Morrissey 2007; Lordan et al. 2011; Wang et al. 2016). Nutraceuticals
are potentially helpful in health up-gradation and disease prevention especially in
avoiding age-related disorders such as depression, oxidative damages, inflammation,
diabetes, gastrointestinal diseases, and even cancer (Jain and Ramawat 2013). The
increasing demands and benefits of microbial supplements having health benefits
have significantly stimulated advancement in the market of nutraceuticals. The
global nutraceutical market has rapidly grown, and in 2014, it was valued at
$171.8 billion. The market is expected to reach $722.49 billion in the next 6–7
years with a compound annual progress rate (CAGR) of 8.3% over the forecast
period (NMSS&TA 2020). Though, the growing market of nutraceutical could
barely be contented through the efficiency of straight nutraceutical industries. Direct
extraction approaches are restricted with accessibility and price of raw ingredients,
quality check of goods, and less content and pureness of nutraceuticals. While
synthesis by chemicals could be another method, it is unsuitable to generate ade-
quate quantity and quality of biochemicals and certainly not feasible for composite
biochemicals (De Luca et al. 2012). To overcome the issue, metabolic engineering of
microbes is considered a promising methodology that has recently attained prodi-
gious improvement towards production of value-added nutraceuticals. We have
further discussed the recent advances of microbial-based metabolic engineering
and their role in nutraceutical production including phytochemicals, prebiotics,
polysaccharides, as well as poly amino acids.
Table 12.1 Various enzymes and their role in industries
Sr. no. Enzyme Role(s)
1. Protease Breaks proteins into their simple form
2. Keratinase Decomposes keratin found in hairs, nails, etc.
3. Amylase Breaks starch into sugars
4. Xylanase Converts polysaccharides into xylose
Catalytic breakdown of hemicellulose
5. Ligninase Degrades lignin
6. Cellulase Breakdown of cellulose
7. Lipase Hydrolysis of fats, triglycerides
8. Pectinase Breakdown of pectin
12 Microbial Engineering and Applications for the Development of Value-Added... 207
12.5 Phytochemicals
Phytochemicals are the broad spectrum of secondary bioactive metabolites obtained
from different parts of the plants including stem, leaf, fruits, beans, and grains.
Phytochemicals are often involved in plant defence mechanism against adverse
biotic and abiotic conditions or may exert health-promoting or disease-resistant
properties (Jain and Ramawat 2013). Some of the major types of phytochemicals
are discussed further.
12.5.1 Alkaloids
Alkaloids are amino acid-derived nitrogenous complexes with various beneficial
properties including antimalarial to anticancer effects (Marienhagen and Bott 2013).
Due to long biosynthetic pathways and the complex structure, alkaloid production
was limited to the plants for past few years. The most commonly used alkaloids are
(a) monoterpene indole alkaloids (MIAs) derived from tryptophan and
glucosinolates and (b) benzylisoquinoline alkaloids (BIAs) derived from tyrosine.
Due to the recent advancement and knowledge of the BIA biosynthetic pathway,
the synthesis is now carried out in various microorganisms like E. coli and
S. cerevisiae (Nakagawa et al. 2011,2014; Fossati et al. 2014). The (S)-reticuline
biologically synthesized from simple carbon sources is an intermediate of BIA
pathway (Nakagawa et al. 2011). Apart from biosynthesis of (S)-reticuline in
E. coli,S. cerevisiae also facilitate to synthesizes of (R,S)-reticuline that in turn
engineered to produce salutaridine from (R)-reticuline and scoulerine,
tetrahydroberberine, and tetrahydrocolumbamine from (S)-reticuline (Hawkins and
Smolke 2008).
Metabolic engineering of MIA alkaloids in microbes is inadequate and not much
diverse like that of BIA alkaloids. Strictosidine, a de novo MIA alkaloid, has been
successfully produced in yeast by the deletion of three genes and the introduction of
21 new genes in yeast genome (Brown et al. 2015). Yeast has also been
bioengineered by introducing eight genes of plants into its genome for the produc-
tion of tryptophan-derived indolylglucosinolate (IG) (Mikkelsen et al. 2012).
Tryptophan-derived IG is a sulphur-rich, amino acid-derived natural composites of
glucosinolates. For metabolic engineering in microbes and large-scale alkaloid
production, genes isolated from plant platforms are most stable and show promising
potential for biosynthesis of plant-derived complexes (Brown et al. 2015; Mora-Pale
et al. 2013).
208 A. Paliwal et al.
12.5.2 Terpenoids
It serves as the largest class of phytonutrients with various beneficial properties like
anti-infectious, anti-inflammatory, and anticancer properties (Jain and Ramawat
2013; Mora-Pale et al. 2013). It is generally present in cereals, soy plants, and
green foods. Terpenoids are dimethylallyl pyrophosphate (DMAPP) or isopentenyl
pyrophosphate (IPP) derived broad carbon skeleton compounds, like monoterpenes
(e.g. menthol), diterpenes (e.g. paclitaxel), triterpenes (e.g. steroid saponins,
oleanane, ursane), tetraterpenes (e.g. carotenoids), sesquiterpenes (e.g. artemisinin),
and polyterpenes (Marienhagen and Bott 2013). Terpenoids production in microbes
illustrates the success and advancement of metabolic engineering for the synthesis of
terpenoid drugs. Most common terpenoids that are used in pharmaceuticals are
(a) artemisinic acid which is a precursor of antimalarial drug known as artemisinin
and (b) taxadiene which is an intermediate of anticancer drug known as paclitaxel
(Besumbes et al. 2004; van Herpen et al. 2010).
In nutraceutical industries, carotenoids (a tetraterpene) including astaxanthin,
α-carotene and β-carotene, and lycopene act as feed supplements and natural food
colourants (Marienhagen and Bott 2013). For a long time, combinatorial carotenoid
biosynthesis has been done in heterologous non-carotenogenic hosts including
E. coli and S. cerevisiae due to large-scale production of carotenoid so the metabol-
ically engineered efforts are generally focused on it only. Strain improvement
through gene knockout technique generally increases the production of lycopene
to a large extent in E.coli (Lin et al. 2014). Metabolic engineered E. coli have high
supply of ATP and NADPH which help in the production of upgraded β-carotene up
to 2.1 g/L β-carotene and increase the harvest up to 60 mg/g DCW (Zhao et al. 2013).
A high amount of astaxanthin has been produced through harvest of 1.4 mg/g DCW
when the biosynthetic genes of xanthophylls are chromosomally integrated with a
plasmid-free E. coli (Lemuth et al. 2011). Lycopene E. coli has generally used for the
production of carotenoids because it not only is involved in the production of novel
carotenoid like 4-ketozeinoxanthin but also produces some rare carotenoids like
decaprenoxanthin, sarcinaxanthin, and sarprenoxanthin (Netzer et al. 2010; Maoka
et al. 2014).
12.6 Prebiotics
Prebiotics are nonviable components in food that encourage the growth or activity of
useful microorganisms in the gastrointestinal (Pineiro et al. 2008). Prebiotics are
polysaccharide with 3–10 monomeric units of sugar which will not further dissociate
in the body; hence, it is nondigestible. Prebiotics show a beneficial effect on the
metabolic activity and diversity of the gut microbiota, and this leads to major effect
on the immune system of host. Prebiotics can also be used in treatment of diverse
inflammation-induced diseases by improving the gut microbiota using probiotics
12 Microbial Engineering and Applications for the Development of Value-Added... 209
such as Bifidobacteria or Lactobacillus sp. (Lin et al. 2014). A general example of
prebiotics is soluble dietary fibres such as inulin, fructooligosaccharides (FOS), and
lactose-based galactooligosaccharides (GOS). Inulin and fructooligosaccharides are
produced by probiotic Lactobacillus gasseri strain (Anwar et al. 2010), whereas
galactooligosaccharides (GOS) are short-chain and lactose-derived galactose poly-
mer synthesized by Kluyveromyces lactis (Rodriguez-Colinas et al. 2011). Conver-
sion of lactose to GOS is done when the codon-optimized β-galactosidase expresses
from hyper-thermophile Sulfolobus solfataricus in Lactococcus lactis. In terms of
infants and toddler, human milk has been accepted as a best nutritive substance due
to the presence of most abundant oligosaccharide present in it, i.e. 20-fucosyl lactose
(20-FL). 20-FL can also be produced from lactose and glycerol with the help of E. coli
by overexpressing the fucosyl transferase or by increasing the availability of GDP-L-
fructose for the high yield (Lee et al. 2012; Baumgartner et al. 2013).
12.7 Polysaccharides
Polysaccharides are sugar polymers composed of the large number of small mono-
meric sugar units with highly versatile structure. Polysaccharides are produced by
most of the microorganism, e.g. bacteria, fungi, and yeast, or may be extracted from
plant and animal tissues. Due to their health beneficial properties, microbial poly-
saccharides including bacterial polysaccharides and fungal polysaccharides are
referred to as the best source for nutraceuticals. Commercialization production of
bacterial polysaccharides like gellan, dextran, xanthan, and alginate can be carried
out through microbial engineering and refinement, respectively (Giavasis 2013). For
dairy product usage, exopolysaccharides are produced through the metabolic engi-
neering of Streptococcus and Lactococcus species (Jolly et al. 2002). Other than
bacterial and fungal polysaccharide shows various extensive properties like
immunostimulatory, antitumor, antimicrobial, antioxidant, hypocholesterolaemic,
and hypoglycaemic benefits (Giavasis 2014).
Due to these properties, fungal polysaccharides show great potential in pharma-
ceutical and nutraceutical applications (Giavasis 2014). Scleroglucan excreted by
mycelia of the fungus Sclerotiumrol fsii is a potent antiviral and antitumor glucan-
based polysaccharide and the yield of the polysaccharide can easily be increased by
the adding of L-lysine and uridine monophosphate (UMP) (Giavasis 2014).
Hyaluronic acid (HA), chondroitin, and heparosan are animal-based polysaccharides
which have been produced by microbial host instead of extracting it from the animal
tissues. E. coli,L. lactis,andStreptomyces albulus are the major microorganisms
used for the production of hyaluronic acid (Yu and Stephanopoulos 2008; Sheng
et al. 2015; Yoshimura et al. 2015). Some therapeutically essential polysaccharides
like heparosan and chondroitin could be synthesized from engineered E. coli at a
relatively high titter (He et al. 2015; Zhang et al. 2012).
210 A. Paliwal et al.
12.8 Poly Amino Acids
Poly amino acids comprising one or two amino acids are produced by microorgan-
ism through a ribosome-independent enzymatic reaction that differentiates them
from polypeptides which are generally synthesized by translation. There are three
poly amino acid found naturally, viz. poly-γ-glutamic acid (γ-PGA), multi-L-arginyl-
poly (L-aspartic acid), and poly-ε-L-lysine (ε-PL). γ-PGA is a biodegradable polymer
that is soluble in aqueous solutions and therefore used as drug carriers or hydrogels
(Khalil et al. 2017). Few genetically engineered Bacillus species produced γ-PGA in
high quantity ranging from 31.7 to 107.7 g/L, when it feeds on L-glutamic acid
(Hsueh et al. 2017). An equimolar amount of arginine and aspartic acid is used to
produce cyanophycin by cyanobacteria or some chemotrophic bacteria like
Acinetobacter calcoaceticus, etc. Cyanophycin is used as a dipeptide precursor for
therapeutic and nutritional applications (Watzer and Forchhammer 2018).
Cyanophycin can be produced in E. coli by overexpressing the cyanophycin syn-
thetase cphA gene isolated from Synechocystis sp. PCC 6803 with productivity of
120 mg/g CDW (Tseng et al. 2012). Streptomyces species like Streptomyces albulus
are exclusively involved in the production of ε-PL and could reach up 35.14 g/L
when glucose and glycerol are used as carbon source (Chen et al. 2012; Dodd et al.
2018). Poly-ε-L-lysine or ε-PL is a homo poly amino acid that is produced by the
polymerization of lysine via ε-PL synthetase (PLS). ε-PL has been approved as food
preservatives or dietary agents due to having antibacterial anticancer activities in
developed countries like the United States and Japan (El-Sersy et al. 2012).
12.9 Conclusion
Nowadays, due to green manufacturing and sustainable development, microbial
production of different substances is widely used, but this development is limited
because of its high cost. For developing countries, it creates an opportunity for using
microbes and their derivatives from small places like household and village-level
production to large-scale industrial productions. These microbial processes need
more exploration to be exploited with full intensity with their beneficiary effects.
The last decades have recorded extraordinary advancements in production of
nutraceuticals by metabolic engineering of microorganisms. Nutraceuticals own
countless application including strengthening of immunity of human beings. Further
studies are recommended for exploration of different microbial explorations in
which microbes are directly involved in enhancing the productivity of processed
food or food products. The use of metabolically engineered microbes opens a
promising door not only in laboratory-based production but also for the industry-
based production of intricate natural compounds from simple carbon sources. The
emerging role of synthetic biology will promote the progression of this field in
12 Microbial Engineering and Applications for the Development of Value-Added... 211
upcoming years, and hopefully, this will deliver requisite tools for tuneable synthesis
and optimization of nutraceutical synthesis in biological hosts.
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Chapter 13
Plant Growth-Promoting Rhizobacteria
and Their Application in Sustainable Crop
Production
Parul Chaudhary, Heena Parveen, Saurabh Gangola, Govind Kumar,
Pankaj Bhatt, and Anuj Chaudhary
Abstract The world population is increasing at an alarming rate, and to feed such a
huge population from an exhausted arable land with depleted water resources would
be a great concern and challenge in near future for agriculture scientists. Present
agricultural practices are dependent on seed quality, irrigation facilities, and chem-
ical fertilizers that affect the fertility of soil significantly. To overcome from this
problem, some alternative methods must be employed. The use of several
rhizospheric bacteria and fungi as bioinoculant with plant growth-promoting ability
is under practice by some farmers, forming the base of upcoming green revolution.
Plant growth-promoting microbes (PGPM) are usually present around the root zone,
rhizosphere, and inside the plants and found responsible for enhanced plant growth
by way of providing essential nutrition and hormones and controlling pathogens by
producing antifungal and antimicrobial compounds. Soil is the most important
component of a terrestrial ecosystem and essentially required to establish, support,
and enhance plant growth by providing nutrition and water. The cross signaling
between the plants and rhizobacteria for mutual benefits is possible because of the
involvement of different types of root exudates, genes, and several known and
P. Chaudhary (*)
Department of Animal Biotechnology, NDRI, Karnal, Haryana, India
H. Parveen
Department of Dairy Microbiology, NDRI, Karnal, Haryana, India
S. Gangola
Department of School of Agriculture, Graphic Era Hill University, Bhimtal, Uttarakhand, India
G. Kumar
Department of Crop Production, Central Institute for Subtropical Horticulture, Lucknow, India
P. Bhatt
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,
Guangdong Laboratory for Lingnan Modern Agriculture Integrative Microbiology Research
Centre, South China Agricultural University, Guangzhou, China
A. Chaudhary
School of Agriculture and Environmental Sciences, Shobhit University, Gangoh, Uttar Pradesh,
India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_13
217
unknown factors which make a network for a better functioning of plant and
bacteria. Conservation of biogeochemical cycles consisting of carbon, nitrogen,
phosphorus, and sulfur in relation to recycling of essential nutrients in an ecosystem
is only possible by the involvement of numerous soil microbes. Biological activity of
a soil or soil health must be known to get good agriculture produce. In this chapter,
we have undertaken various aspects of rhizospheric microbes including their
involvement in higher food production and maintenance of soil and plant health.
Keywords Biofertilizers · Soil health · Agriculture · Soil fertility
13.1 Introduction
Agriculture, the backbone of Indian economy, provides sufficient food and feed to
satisfy the need of burgeoning human population (Bhatt et al. 2019a,b). Application
of crop protectants, fertilizers, and pesticides to enhance crop production is found to
be a boon for the agriculture system, but indiscriminate use of pesticides not only
contaminates agriculture produce but also affects soil health and quality of ground
water which ultimately affects human health (Pankaj et al. 2015a,b,2016a,b).
Hence, it is important to improve soil health by increasing the appliance of beneficial
microorganisms which are involved in growth and productivity of plants by means
of recycling of essential nutrients via biogeochemical cycles (Huang et al. 2021;
Singh et al. 2021; Zhang et al. 2021). Sustainable agriculture fascinates the increas-
ing demand of biological based natural fertilizers substitute to agrochemicals. Plant
growth-promoting bacteria (PGPB) can be used as a green technology which will
diminish the application of chemical fertilizers and improve soil health by involving
in cycling of biological elements wherein the complex biological is converted into
simpler ones (Di Benedetto et al. 2017). Rhizomicrobiome plays an important part in
improving plant health by influencing the growth of their eukaryotic hosts and
providing essential nutrients to the soil. It is highly influenced through root exudates
of the host plant (Backer et al. 2018). Rhizomicrobiome is composed of diversified
microbial community and highly influenced by plant type, soil type, and environ-
mental conditions. Microbial associations around the roots in soil are complex and
facilitate the plants to get the nutrients from soil through different mechanism.
PGPM are the soil bacteria which make nutrients available, fix atmospheric nitrogen
and are commonly used as biofertilizers which enhanced the productivity of different
crops (Santoyo et al. 2016). Rhizospheric microbes such as bacteria, cyanobacteria,
actinomycetes, and arbuscular mycorrhiza fungi directly or indirectly allied with
plants or soil (Kumar et al. 2016). PGPR that inhabits in the rhizosphere belongs to
Azotobacter,Azospirillum,Bacillus,Pseudomonas,Paenibacillus,Pantoea,
Serratia,Streptomyces, etc. These PGPRs are generally used as bioinoculants for
the biocontrol, biostimulation, and biofertilization (Kaushal and Wani 2016). The
use of biofertilizers not only assures food security but in addition helps in mainte-
nance of the soil microbial diversity.
218 P. Chaudhary et al.
Soil is the most suitable, dynamic, and complex surface environment which
provides substrate for growth of plants, animal, and microorganisms. Microorgan-
isms are metabolically more diverse, and soil offers a variety of microhabitat;
microbial diversity in soil is much higher than other environments. Soil quality
indicators such as microbial population, soil enzyme activities, soil pH, and micro
and macronutrients play a major role in sustenance of soil health status and quality.
Soil physicochemical properties exhibit seasonal variation and alter the structure and
composition of bacterial population (Lopez-Monejar et al. 2015). Among soil
physicochemical properties, pH has been known to influence the bacterial commu-
nities via availability of organic and inorganic nutrients. Soil enzymes are derived
from plant root exudates, animal remains, and soil microorganisms of which soil
microbes have the major involvement (Thomson et al. 2015). They play a vital
function in catalyzing reactions necessary for maintaining microbial life in soil,
stabilize soil structure, disintegrate organic matter, and are involved in nutrient
cycling. Enzyme activities act as biomarker for detecting changes in soil quality
and measure of microbial diversity and community structure. Thus, represent the
functioning of the entire microbial community (Yang et al. 2017). This chapter
reports the prospective use of PGPM for enhancing the crop productivity and
maintaining the soil heath in a sustainable manner.
13.2 Rhizosphere and Plant Growth-Promoting Microbes
Rhizospheric term was given by Hiltner to explain the narrow zone of soil adjacent
to roots where microbial populations are stimulated by root activities and manage
plant growth (York et al. 2016). Rhizosphere is a hot spot for microbial action
contributed generally by native bacteria and fungi. PGPM can be separated into two
major groups: plant growth-promoting bacteria (PGPR) and plant growth-promoting
fungi (PGPF). PGPRs were initially identified by Klopper and Schorth (1978)to
illustrate bacteria which colonize the plant roots and enhance plant growth. Bacteria
are the most dominant microorganism in the soil. There are two groups of PGPR on
the basis of interaction of microbes with plants. Endophytes (symbiotic) colonize
internal plant tissues. Endophytes sense the chemical signal and reach at the root
surface and produce counter signal that makes the plant root vulnerable for microbial
penetration into the root and improve the growth of plants (Defez et al. 2017).
Epiphytes, in which a microbe lives on the surface of plants, are also known as
non-symbiotic. Besides bacterial species, fungal species such as Trichoderma,
Penicillium,Aspergillus, and Alternaria are used as PGPR.
13 Plant Growth-Promoting Rhizobacteria and Their Application in Sustainable... 219
13.3 PGPR and Their Mode of Action
PGPRs are the group of soil bacteria which to be found around the root surface of
plants and support plant health using different mechanism. They affect plant health
parameters by producing various growth-promoting hormones within rhizosphere.
PGPR directly facilitates the plant growth by improving uptake of macro and micro
nutrients like nitrogen, phosphorus, and potassium as well as by modulating phyto-
hormone like IAA, cytokinin, and gibberellins, vitamins, and enzymes and solubi-
lization of different minerals like phosphate, iron, and potassium which are directly
involved in increasing root length (Liu et al. 2017). PGPR protects plant health
indirectly from plant pathogens by producing different bacterial/fungal metabolites
like HCN, phenazines, and tensin and lytic enzymes and boosts plant growth and
productivity (Bhardwaj et al. 2014). Interaction of PGPRs with plant root induces
resistance against pathogens (fungal, bacteria, and viruses) by promoting ethylene
and jasmonate signaling. Different bacteria like Pseudomonas, Bacillus,
Azospirillum,Burkholderia,Enterobacter,Arthrobacter,Serratia,Burkholderia,
and Klebsiella are used as bioinoculants to improve the yield of diverse agricultural
crops (Ghevariya and Desai 2014). Population density of rhizospheric bacteria
depends on the pH, water potential of soil, and plant root exudates. These microor-
ganisms are also helpful in synthesis and degradation of organic matter. Calvo et al.
(2019) reported that Bacillus helps in regulation of ammonium and nitrate uptake
genes in Arabidopsis thaliana which increased the overall growth of plant. Number
of bacterial strains has the capability to induce plant growth and enhance yield in
different cropping systems (Nieto-Jacobo et al. 2017; Chaudhary et al. 2021a).
Pseudomonas taiwanensis and Pantoea agglomerans are used as soil inoculants in
form of biofertilizers in agriculture and enhanced the plant growth by solubilization
of different nutrients (Chaudhary and Sharma 2019). Khati et al. (2019b) reported
that Bacillus spp. (PS2 and PS10) is also involved in IAA and siderophore produc-
tion which improved the plant health parameters. Shen et al. (2015) observed that
application of bioinoculant enhances total bacterial population in soil and helps in
plant growth promotion via way of posing beneficial interactions among
bioinoculants, plants, and microbes. Application of PGPRs enhanced the shoot
and root length of wheat plants by releasing metabolites and mineralization of
nutrients which are simply accessible to plants (Sheirdil et al. 2019). de-Lima
et al. (2019) reported that inoculation with Bacillus subtilis increased leaf area and
water use efficiency in maize and common bean plants.
13.4 Effect of PGPRs on Plant Health Parameters
PGPRs hold promise opportunity for sustainable agriculture. Biofertilizers are
artificially preserved cultures isolated from the soil used as soil inoculants which
improve the soil fertility and enhance the productivity. They colonize the central part
220 P. Chaudhary et al.
of plants and enhanced plant growth by escalating the accessibility of nutrients to
plants and also considered an integrated nutrient management system (Malusa et al.
2012). PGPR has been proven as safe and efficient methods to enhance the crop
yields (Vejan et al. 2016). Agriculture can take advantage of symbiotic relationship
of microbes with plants which helps in maintenance of the particular microbial flora
in the plant rhizosphere (Asei et al. 2015).
Bacillus megaterium helped in growth promotion of tea plants and disease
reduction by producing siderophore (Chakraborty et al. 2006). Use of Pseudomonas,
Micrococcus,Staphylococcus, and Bacillus increases the growth and productivity of
Triticum aestivum (Ali et al. 2009). These microbes showed the production of indole
acetic acid. Mishra et al. (2010) found that Pseudomonas fluorescens and Bacillus
subtilis as bioinoculant (ammonia producers) increased the biomass of Geranium
(a medicinal plant).Genetically, modified strains of Pseudomonas are reported to
improve plant development, yield, and disease resistance in agricultural field (Vessy
2013). Inoculation of seeds using Burkholderia cepacia,Azotobacter chroococcum,
and Bacillus subtilis improved maize and wheat productivity (Zhao et al. 2014).
Application of Pseudomonas putida,Bacillus megaterium, and Mesorhizobium
showed positive effect on Cicer arietinum and significantly enhanced plant biomass
and seed production (Fernández and Alexander 2017). Bioinoculation of Bacillus
sp. in mung bean and rice enhanced plant growth and NPK content in soil which
supported plant growth (Pahari et al. 2017). Rozier et al. (2019) found that
bioinoculation of Azospirillum lipoferum CRT1 enhanced the seed germination,
root surface area, and photosynthetic pigment in maize plants.
Khati et al. (2017,2018) reported that Bacillus spp. improved the maize health
parameters. Bioinoculation of Sinorhizobium meliloti and Pseudomonas fluorescens
enhances the seed weight and morphological and phytochemical parameters of
Fenugreek plants (Sharghi et al. 2019). Rahman et al. (2018) observed that Bacillus
amyloliquefaciens and Paraburkholderia fungorum increased the strawberry fruit
growth, yield, and antioxidant contents. Andrade et al. (2019) reported that
Azospirillum brasilense with Burkholderia cepacia and Enterobacter cloacae
improved the growth of strawberry crop.
13.5 Role of PGPR as Biocontrol Agent
The predominant factor in reducing crop yields is disease in plants. To protect plants
from phytopathogens, farmers rely on pesticides which improve the plant growth
and productivity. The over usage of these chemicals in soil has established resistance
in phytopathogens and poses many environmental issues. Biological control using
PGPRs is an alternative way to control phytopathogens. The use of PGPR as a
biological agent enhanced plant growth and improved health without causing any
toxic effect. There are various mechanism by which plant growth-promoting bacteria
(PGPB) damage the effect of phytopathogens. Production of antibiotics like tensin,
pyrrolnitrin, and cyclic lipopeptides produced by Pseudomonas inhibits plant
13 Plant Growth-Promoting Rhizobacteria and Their Application in Sustainable... 221
pathogens. Ahmad et al. (2017) found that Bacillus subtilis inhibit the growth of
fungal pathogens like Rhizoctonia solani,Botrytis cinerea, and Fusarium
oxysporum. Radhakrishan et al. (2017) reported that Bacillus sp. stimulates plant
growth and prevents pathogen infection. Mokrani et al. (2019) observed that com-
bination of Pseudomonas pseudo and Pseudomonas cichorii hinders the growth of
phytopathogenic fungi. Liu et al. (2018) reported that application of Bacillus
amyloliquefaciens with bio-organic additives suppressed the tomato disease.
Zhang et al. (2019) observed that consortium of Bacillus cereus,Bacillus subtilis,
and Serratia acts as environmentally friendly biocontrol agent on sweet pepper.
These three bacteria reduced the prevalence of phytopthora blight and improved the
quality of fruit. Hashem et al. (2019) found that Bacillus subtilis secrete antibiotics
and hydrolytic enzymes and have potential biocontrol activity against pathogens.
Passera et al. (2017) observed that Paenibacillus pasadenensis have the capabil-
ity to prevent plant diseases. Wu et al. (2016) reported that Pseudomonas
saponiphila increased the disease control of plant pathogens. Sharma et al. (2019)
reported that halotolerant species, Klebsiella species MBE02, regulated the genes
which involved in pathogen defense mechanism and jasmonic acid/ethylene signal
pathways. These bacteria have growth stimulating effect on peanut and controlled
the infection caused by Aspergillus and several other fungal phytopathogens.
13.6 Role of PGPR Under Abiotic Stress Conditions
Stresses have significantly reduced the fertility of soil and adversely influence the
microbial community and therefore affected the plant growth and agriculture pro-
duction. Plant-microbe interactions play significant roles in the maintenance of plant
and soil productivity under stress conditions (Vimal et al. 2019). Under the influence
of stress conditions, plants become vulnerable to osmotic stress and ionic toxicity
and start overproducing reactive oxygen species (ROS) which damages the proteins,
lipids, and other cellular components (Kyei-Boahen et al. 2017). To cope up stress,
use of rhizobacteria as bioinoculants represents powerful techniques to alleviate
various environmental stresses like salinity stress, drought, weed infestation, and
nutrient deficiency. Heydarian et al. (2016) reported that PGPB produce ACC
deaminase in high salt condition and improved the Camelina sativa plant growth
and seed production. Bharti et al. (2016) observed that Dietzia natronolimnaea
protected the wheat plants from salt stress by upregulation of the ABA signaling.
Cura et al. (2017) found that Azospirillum brasilense and Herbaspirillum
seropedicae under drought condition improved the chlorophyll and biomass pro-
duction in corn.Numan et al. (2018) reported that bacteria elicit plants to produce
growth hormones under salt stress condition. Application of Bradyrhizobium in
cowpea plant minimizes the harmful effects of salt stress by stimulation of enzymatic
and non-enzymatic antioxidant enzymes (Santos et al. 2018). Ansari et al. (2019)
reported that Bacillus pumilus tolerated 250 mM salt concentration and enhanced the
plant growth and yield of wheat plant. They observed decline in stress markers like
222 P. Chaudhary et al.
catalase, superoxidase dismutase, proline, and malondialdehyde and reduced salt
stress in wheat. Bidgoli et al. (2019) reported that application of PGPR (Pseudomo-
nas fluorescens) improved the yield of medicinal plant Rosmarinus officinalis under
salinity condition @ 10 g/L NaCl. Curtobacterium albidum inoculation in soil
alleviates the salinity stress and improves the paddy yield (Vimal et al. 2017). He
et al. (2019) observed that Pseudomonas putida Rs-198 increased the shoot/root
length of pepper in slightly saline soil.
13.7 Plant Growth-Promoting Fungi
Different studies showed the beneficial effects of rhizospheric fungi on plant quality
and quantity and their positive role with the ecological environment (Murali et al.
2012). Endophytic fungi are colonizing the interior of plant parts. Plant-microbe
interactions resulted in adaptation, plant growth promotion, and production of
different secondary metabolites and bioactive compounds with potential application
in agriculture (Yadav et al. 2018). Among the PGPF, Penicillium,Aspergillus,
Penicillium, and Chaetomium play a valuable position in promoting plant growth
and disease repression (Kumar et al. 2021). PGPF is involved in production of plant
hormones, organic matter decomposition, and protection of plants from stress
conditions and helps in solubilization of different minerals (Sangamesh et al.
2018). Ripa et al. (2019) reported that Trichoderma aureoviride,T. harzianum,
Aspergillus flavus,A. tenuissima,Alternaria alternate,Fusarium equiseti, and
F. proliferatum produce IAA, ACCD, and phosphate solubilization, tolerate the
abiotic stress, and showed resistance against the antibiotics.
It was observed that Alternaria sp. A13 enhanced the root growth of Salvia
miltiorrhiza and promotes secondary metabolite accumulation (Zhou et al. 2018).
Penicillium enhances the plant growth by production of phytohormones, solubiliza-
tion of phosphate, synthesis of amino acids, and secretion of antioxidants enzymes
and is involved in secondary metabolite production which helps in coping up stress
situation (Chaudhary et al. 2018). Shah et al. (2019) observed that Fusarium sp. and
Hypoxylon sp. increased the shoot/root length and chlorophyll in Rhynchostylis
retusa plants. Wang et al. (2018) reported that Aspergillus niger CS-1 promoted
the plant growth of wheat. Mycorrhizal fungi Rhizophagus intraradices benefits host
plant by improving translocation of mineral nutrients (Chitarra et al. 2016). Tyagi
et al. (2017) found that Rhizophagus intraradices and Piriformospora indica tolerate
drought stress and enhanced the plant biomass and chlorophyll content in finger
millet.
13 Plant Growth-Promoting Rhizobacteria and Their Application in Sustainable... 223
13.8 Bioformulation
In several fields, bioformulation for the promotion of plant growth continues to
encourage research and development. The key goals of the bioformulation industry
are increased soil fertility, promotion of plant growth, and control of phytopatho-
gens, which contribute to the creation of an eco-friendly climate. Bacillus and
Pseudomonas sp. are reported as best PGPRs to enhance the plant growth and
productivity in different cropping systems (Chaudhary et al. 2021c,d; Agri et al.
2021). Bioformulation provides an environmentally sustainable approach to enhance
crop productivity by using microbial bioinoculants and does not affect microbial
activity in soil. PGPRs can be used to enhance the plant productivity under field
condition. It may be an efficient approach to replace the chemical fertilizers with
bioformulation to develop a sustainable agriculture system in India (Aslani et al.
2014).
13.9 Soil Health
Soil is the basis in terms of food, water security, climate alleviation, and protection
of biodiversity (McBratney et al. 2014). Soil health has usually judged in terms of
production and provides overall picture of soil functionality and affects agricultural
production significantly (Haney et al. 2018). Depending on biological and chemical
constituent of the soil that affects the ecosystem and environmental quality, soil
health is characterized as the net product of ongoing conservation and degradation
processes (Bünemann et al. 2018). Healthy soil supports and maintains diverse
microbial community which helps in maintenance of beneficial association of
different microbes with plant roots, controls plant diseases, and recycles plant
nutrients. Soil health is a measure index of soil texture, nutrient cycling, soil protein,
soil respiration, surface hardness, water stable aggregates, and availability of water
capacity (Khati et al. 2019a; Mann et al. 2019).
Soil quality is the ability of a soil to function, within a natural/managed ecosystem
limit to enhance productivity of plants and retain water quality and human health
(Karlen et al. 1997). Number of aspects influence soil fertility, but biological
designators are found as vitally consequential because soil microorganisms directly
manipulate processes of soil ecosystem. Hence, protection of microbial diversity and
biomass in the soil is one of the most important challenges for sustainable resource
use as higher level of microbial diversity leads to more nutrient turnover (Torsvik
and Øvreas 2002). Soil health refers to physical, chemical, and biological texture of
the soil which is vital to get protracted sustainable agricultural productivity with least
environmental impact (Moebius-Clune et al. 2016).
224 P. Chaudhary et al.
13.10 Physical Indicators
Physical indicators such as bulk density, porosity, texture, and aggregate stability of
the soil are involved in erosion and water holding facility (Schoenholtz et al. 2000).
Soil texture is determined by the arrangement of major soil particles like silt, sand,
and clay and can be affected by cropping system(s) (Dexter 2004).
13.11 Chemical Indicators
Different chemicals are considered to provide nutrients to the soil and take part in
determining healthy status of the soil. Organic matter and ion-exchange capacity of
soil help in supplying nutrients like calcium and magnesium, and electrical conduc-
tivity indicates the number of ions in soil. The soil pH affects the solubility of
minerals, activity of microorganisms, and nutrient status of the soil which affects soil
health (Kelly et al. 2009). Chemical indicators are helpful in maintaining nutrient
cycles and organic matter.
13.12 Microorganisms as Biological Indicator of Soil Health
Microorganisms have the capability to provide an integrated evaluation of soil health
that cannot be acquired from higher organisms or by physical/chemical steps.
Microbes respond rapidly to changes and thus adapt quickly to environmental
situations and can be used to analyze the soil health. Population changes and
microbial activities are an indication of changes in soil quality. Microorganisms
respond quickly to environmental stress, because of their close relations. Alteration
in microbial activity in soil can precede an early indication of soil perfection and
degradation (Pankhurst et al. 1995).
13.13 Role of Microbial Enzymes in Maintenance of Soil
Health
Activities of soil enzymes are directly related to microbial population to express
metabolic requirements and available nutrients in a soil system (Nannipieri et al.
2002). These enzymes take part in the disintegration of unprocessed material in the
soil. Enzymes catalyze many critical reactions needed for soil microorganism life
processes and soil structure stabilization, organic waste disintegration, and nutrient
cycling processes (Garcia et al. 2002) (Table 13.1). Application of various
approaches to assess functional diversity in the soil through estimating enzyme
13 Plant Growth-Promoting Rhizobacteria and Their Application in Sustainable... 225
Table 13.1 Soil enzymes used as indicator of soil health
Enzymes
involved Function Microbes involved References
Amylase Helps in breakdown of starch
into simpler form (glucose and
maltose and involved in car-
bon recycling)
Bacteria: Bacillus subtilis,
Pseudomonas sp., Bacillus
amyloliquefaciens,Bacillus
licheniformis
Thomas
et al.
(1971)
Fungi: Aspergillus niger
Arylsulfatase Helps in breakdown of com-
plex sulfate esters into phe-
nols/sulfur and involved in
sulfur cycling
Bacteria: Pseudomonas sp.,
Klebsiella sp., Raoultella sp.
McGill and
Cole
(1981)
Fungi: Trichoderma sp.,
Eupenicillium sp.
β-glucosidase Helps in cellobiose hydrolysis
and involved in C-cycling
Bacteria: Flavobacterium
johnsoniae,Lactobacillus
plantarum, Dyella koreensis
Eivazi and
Tabatabai
(1988)
Fungi: Penicillium
purpurogenum,Trichoderma
Cellulase Helps in cellulose degradation
and involved in C-cycling
Bacteria: Bacillus subtilis,
Acinetobacter junii,
Cellulomonas biazotea,Pseu-
domonas cellulose
Deng and
Tabatabai
(1994)
Fungi: Aspergillus niger,
A. oryzae,Trichoderma
reesei,Phanerochaete
chrysosporium,Agaricus
arvensis
Chitinase Helps in chitin hydrolysis Bacteria: Bacillus,Streptomy-
ces plicatus,S. halstedii
Deshpande
(1986)
Fungi: Aspergillus sp.,
Trichoderma sp.
Dehydrogenase Helps in electron transport
system, soil respiration, and
C-cycling
Bacteria: Pseudomonas
entomophila
Trevors
(1984)
Fungi:
Fluorescein
diacetate
Helps in hydrolysis of lipase,
protease, and esterase in soil
(overall microbial activities)
Bacteria: Pseudomonas
denitrificans
Schnurer
and
Rosswall
(1982)
Fungi: Fusarium culmorum
Invertase Helps in sucrose hydrolysis,
involved in C-cycling
Bacteria: Shi et al.
(2008)
Fungi: Aspergillus niger,
Saccharomyces cerevisiae,
Candida utilis
Phosphatase Helps in conversion of insolu-
ble phosphate to soluble form
and involved in phosphorus
cycling
Bacteria: Bacillus,
Pseudomonas
Eivazi and
Tabatabai
(1977)
Fungi: Aspergillus,
Penicillium
Protease Helps in protein hydrolysis
(amino acids) and involved in
N-cycling
Bacteria: Bacillus subtilis Ladd and
Jackson
(1982)
Fungi: Aspergillus niger
Urease Helps in conversion of urea
into NH
3
and CO
2
and
involved in N-cycling
Bacteria: Bacillus Rotini
(1935)
Fungi: Trichosporon
cutaneum
226 P. Chaudhary et al.
activities supports our perceptive of the relations among accessibility of resources,
microbial community function, and ecosystem processes (Kumari et al. 2021).
Fluorescein diacetate is a nonspecific enzyme assay used for the estimation of
esterase, lipase, and proteases which converts it into fluorescein. Overall, it provides
information about the active microbial activity (bacteria, fungi, and living protist) in
soil (Schnurer and Rosswall 1982). Dehydrogenase is also called DH or DHase. It is
not extracellular but an integral part of intact cells. It provides information about the
biological and microbial population activity in the soil which helps maintain soil
fertility and health (Burns 1982). Phosphatases catalyze the ester and anhydride
hydrolysis and release free phosphate in the soil. Two main types of phosphatases
are found in the soil. Phosphomonoesterases are low molecular weight compounds
and have monoester bond. Acid and alkaline phosphatases play important roles in
plant nutrition. They are involved in mineralization of organic phosphorus to
inorganic phosphorus in soil. Phosphodiesterases are less studied enzymes. They
are occupied in degradation of phospholipids and nucleic acid and provide greater
part of fresh organic P in the soil. Both enzymes are important to release free
phosphate from phosphate diester (Benjamin and Philip 2005). β-glucosidase is
the most important enzyme and plays key role in C-cycle in catalyzing the hydrolysis
of a variety of β-glucosides present in plant waste (Eivazi and Tabatabai 1988). This
enzyme is included in the group of disaccharide-hydrolyzing glucosidases. Also
included among the glucosidases is alpha-glucosidase, which catalyzes the hydro-
lysis of alpha-D-glucopyranoside. α-Galactosidase and β-galactosidase (also referred
to as lactase) are other glucosidases. In soil, β-glucosidase is more prevalent as
compared to alpha-glucosidase and β-galactosidases. This enzyme catalyzes cellu-
lose degradation to glucose, an essential life source of C energy for microorganisms
in soil. Kumari et al. (2020) observed twofold increase in soil enzyme activities
when inoculated with Bacillus spp. as compared to control which improves the soil
quality and helped in plant growth of maize. Kukreti et al. (2020) found that
application of PGPRs improved soil health by increasing the soil physicochemical
analysis and soil FDA, dehydrogenase, and alkaline phosphatase activities.
Chaudhary et al. (2021b) also observed that application of Bacillus sp. improved
the soil health by raising the useful bacteria community of maize rhizospheric soil.
Parul (2019) observed that application of Pseudomonas,Pantoea agglomerans, and
Bacillus spp. improved the soil microbial population and enhanced maize growth
and productivity. The indigenous microbes producing the soil enzymes play an
important role in sustainable development and increased crop production (Goel
et al. 2020; Kumar et al. 2020; Suyal et al. 2019a,b).
13.14 Conclusion
Application of chemical fertilizers and pesticides enhanced plant growth and pro-
tects plants from diseases but affects soil microflora as well as humans. PGPR opens
a new door for the farmers to enhance productivity and protect plants from biotic and
13 Plant Growth-Promoting Rhizobacteria and Their Application in Sustainable... 227
abiotic conditions. They play a vital function in nutrient cycling within the soil and
improve the soil quality and soil health without any harmful effect on microbial
population. Maintenance of the microbial diversity leads to sustainable development
as soil ecosystem forms complex interrelations among the various entities of nature.
For this reason, there is a significant need for research to clearly define the useful and
required bacterial traits for different environmental conditions and plants in order to
be able to select optimal bacterial strains for bioformulation. However, to provide a
better perceptive of the biological efficacy of increased yields in the crop system,
field experiments using bioformulations are required.
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Chapter 14
Reinstating Microbial Diversity
in Degraded Ecosystems for Enhancing
Their Functioning and Sustainability
Sachini Wayanthimali Meepegamage,
Ambalangodage Thilini Dhanushka Rathnathilake, Mahesh Premarathna,
and Gamini Seneviratne
Abstract Biodiversity is the variety of life on earth, starting from genes, individual
species, and communities to the whole ecosystems. The biodiversity and complex
interaction networks of its components play a crucial role in regulating processes in
different ecosystems’functioning and sustainability. Global biodiversity has been
declining rapidly due to human impacts like land-use change, urbanization, envi-
ronmental pollutions, and also the resultant climate change, leading to losing
ecosystem functioning and sustainability. This chapter discusses the biodiversity,
the causes of its degradation, and ways to reverse it. Interestingly, application of
advanced microbial formulations to the soil has been shown to be capable of
reinstating the lost biodiversity in agroecosystems. One such formulation is biofilm
microbial ameliorators [BMAs, e.g., biofilm biofertilizers (BFBFs)]. Once applied to
the soil, they break the dormancy of microbial seed bank formed to circumvent the
stress of agricultural practices, thus re-establishing the biodiversity to a considerable
extent for improved ecosystem functioning and sustainability. The same mechanism
has been shown to be instrumental in environmental bioremediation. Fascinatingly,
the potential of BMAs even in reinstating the biodiversity of disease-proven human
gut microbiota has been reported for improved human health. It is also important to
note that the past social impacts on decreasing biodiversity have now boomeranged
to humans’existence. The application of microbial biotechnologies like developed
BMAs could mitigate such devastating events in the future.
Keywords Biodiversity · Biofilms · Health · Sustainability
S. W. Meepegamage · A. T. D. Rathnathilake · M. Premarathna · G. Seneviratne (*)
Microbial Biotechnology Unit, National Institute of Fundamental Studies, Kandy, Sri Lanka
e-mail: sachini.me@nifs.ac.lk;thilini.ra@nifs.ac.lk;mahesh.pr@nifs.ac.lk;
gamini.se@nifs.ac.lk
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_14
235
14.1 Introduction
Biodiversity is the variety and variability of life on earth at all its levels, starting from
genes, individual species, and communities to whole ecosystems. Genetic diversity
is the variety of genes in a given species. Individuals within a species contain their
genetic compositions and lead to make different populations in a species. The variety
of species within a habitat is known as species diversity. According to similar
characteristics shared by species, they group into genera, families, order, etc.
About 99% of animal species are invertebrates such as insects, worms, snails,
crabs, corals, and sea stars. Most of the invertebrates are insects which are respon-
sible for the pollination of plants and recycling of nutrients. Mammals represent only
less than 1% of the animal species. As a whole, mainly plants and animals constitute
the visible ecosystem diversity, which is the variety of individuals in a particular area
or a place. However, the functional ecosystem is an outcome of both visible and
invisible difference and its interactions with the physical environment.
Soil ecosystem consists of its loose outermost layer of soil and the living and
non-living component in it. The living component includes root of the plant and soil
fauna and flora. The soil fauna is divided into three categories: microfauna,
mesofauna, and macrofauna (Kulasooriya et al. 2017). The microfauna includes
nematodes, protozoa, ciliates, and so on, and mesofauna consists of ants, insects, and
microarthropods. Earthworms, snails, spiders, and mammals belong to macrofauna.
Generally, the soil fauna is involved mainly in manipulating carbon fluxes in the
ecosystem (Lavelle 1996). Microflora includes microscopic alga, archaeobacteria,
eubacteria, actinobacteria, and fungi. The soil microflora continuously interacts with
the plant root system and involves in the process like organic molecular decompo-
sition, nitrogen fixation, phosphorous solubilization, etc.
Primarily, all the functions of ecosystems are influenced by diverse microbial
communities in the soil (Wittebolle et al. 2009). Human impacts on ecosystems like
agricultural and environmental activities tend to decrease soil microbial diversity and
abundance, leading to depleting ecosystem processes. Therefore, restoration of the
degraded soil microbiome is vital for the sustainability of managed ecosystems like
agroecosystems. The conventional method of replenishing the lost soil microbial
diversity is the inoculation of individual microbes as mono or mixed cultures, known
as biofertilization. Except for legume-rhizobium symbiosis, this has not been that
attractive to non-legumes due to low efficacy. However, developed microbial
biofilms have proven the potential to be used as a novel biotechnological means to
reinstate the lost microbial diversity in agroecosystems and possibly in natural
ecosystems (Herath et al. 2017). Therefore, this chapter discusses the effect of the
developed biofilms on re-establishing the lost microbial diversity in agriculture and
the environment and also the health implications of the biofilm use.
236 S. W. Meepegamage et al.
14.2 Ecosystem Networks
Interactions in ecosystems lead to developing biological networks which are
grouped into molecular, cellular, and population levels. Networks in population
level include ecological networks, epidemiological networks, and food-web net-
works (He and Petoukhov 2011). Networks in the cellular level consist of neuronal
networks and immunological networks. Molecular-level networks include gene
regulatory networks, metabolic networks, and protein interaction networks. Gene
regulatory systems include interactions among DNA, RNA, protein, and other
molecules. For example, each mRNA molecule is responsible for the production
of a specific protein or a set of proteins, and the mRNA molecules and proteins
interact with each other, and the outcome of it decides whether they are diffused
around the cell or bound to the cell membrane. In the protein interaction networks,
proteins with the same function interact through protein-to-protein interactions to
connect physically and to make larger macromolecular assemblies. These protein
complexes link to each other via the next level protein-to-protein interactions to form
interaction networks, which are involved in the cellular process. Metabolic systems
consist of chemical reactions of metabolism and regulatory interactions which guide
these chemical reactions. In immunological networks, plant immune signaling
network is stimulated due to pathogen attack, and the stimulus regulates the defense
to the attack (Sato et al. 2010). Ecological systems consist of biotic interactions
between organisms in ecosystems. For example, these biotic interactions can modify
stressor effects, which may be transferred to a distant group of organisms, thus
generating new stressor interactions (Bruder et al. 2019).
Ecological networks can also be seen in microbial community interactions. The
interactions among different taxa within a soil microbial community are linked
directly or indirectly through intermediate species (Lupatini et al. 2014). These
inter-taxa connections allow understanding of soil microbial community structure.
In soil bacterial communities, most of the soil bacterial genera are essential for
network connectance. However, only a few genera like proteobacteria and
actinobacteria play a crucial role as connectors. Moreover, each ecosystem consists
of a different set of keystone genera, and the other genera only show a general
distribution. Species interactions are significant to soil processes, especially in
complex ecosystems.
Biodiversity and their interaction networks play a crucial role in regulating
processes in different ecosystems’functioning and sustainability. In soil ecosystems,
diverse soil organisms are involved in ecosystem functions and sustainability.
Invertebrates contribute to the formation of soil structure which governs hydrologic
processes and gas exchange. Microbes and plant root interaction control soil carbon
sequestration, detoxification, nutrient cycling, organic matter decomposition, and
suppression of pests, parasites, and diseases. The interaction also helps produce
sources of medicines (Bunning and Jiménez 2003). Among soil organisms, micro-
bial diversity plays an essential role in ecosystem functioning. Most of the soil-borne
14 Reinstating Microbial Diversity in Degraded Ecosystems for Enhancing Their... 237
diseases are caused by pathogenic fungi which are suppressed by the high beneficial
microbial biomass, thus generating competition for nutrients.
Moreover, pathogenic species are suppressed by specific antagonists (Brussaard
et al. 2007). Rhizobia, mycorrhizae, actinobacteria, diazotrophic bacteria, other
rhizosphere microorganisms and ants are responsible for symbiotic relationships
with plants and their roots. Different types of litter feeding invertebrates
(detritivores), fungi, bacteria, actinobacteria, and some other microorganisms are
involved in organic matter decomposition (Bunning and Jiménez 2003). Moreover,
soil microbes are involved even in determining plant species diversity in natural
ecosystems like forests (Mangan et al. 2010). In grasslands, high plant species
richness contributes to increased plant productivity and soil nitrogen utilization,
thus lowering nitrogen leaching loss. In deep-sea ecosystems, too, biodiversity is the
key for marine ecosystem functioning and sustainability. Therefore, all the above-
stated evidence supports the fact that biodiversity is the impetus for functioning and
sustainability of any ecosystem.
14.3 Degradation of Biodiversity
Biodiversity in different ecosystems is declining rapidly due to habitat fragmenta-
tion, land-use change, deforestation, climate change, urbanization, agricultural prac-
tices, and environmental pollutions, leading to losing ecosystem functioning and the
sustainability through disrupting all types of the networks. Habitat fragmentation is a
process in which a significant habitat is broken into small patches of areas which are
then not connected as the original habitat. It reduces habitat area and species
connectivity. This can happen through natural or anthropogenic impacts in any
ecosystem. Degradation of forest ecosystem biodiversity is mainly driven by forest
fire, acid rains, and human activities such as deforestation. Acid rains occur due to
climatic change and oil exploration (Nduka et al. 2008; Wei et al. 2017), altering soil
faunal community and vertical distribution of soil faunal groups. To avoid these,
acid stress nematodes and other soil faunas move deep into the soil, which adversely
affects them, thus eventually altering the decomposition of organic matter and
greenhouse gas emission (Wei et al. 2017). Land-use change refers to the transfor-
mation of land to farmlands, grazing areas, human habitation, and urban centers.
These alternations are interconnected with loss of biodiversity and degradation of
land. It has been found that the diversity of indigenes plants and animals and plant
cover are lost due to loss of native vegetation (Maitima et al. 2009). Land-use change
due to agriculture intensification causes the species loss from soil food webs and
reduction of average body mass of soil fauna. Further, alteration of the diversity of a
single group in a food web can adversely effect on the diversity of another group
(Tsiafouli et al. 2014). Eventually, this may lead to an increase in the pressure of
pathogens and pests in agriculture (Plaas et al. 2019).
Growing demand for agricultural produces leads to more and more transforma-
tion of land cover to agroecosystems. In turn, the expansion of croplands with
238 S. W. Meepegamage et al.
intensified agriculture tends to decrease soil fertility and moisture while increasing
soil erosion (Maitima et al. 2009). Here, the degradation of microbial diversity in
agroecosystems is mainly due to tillage and excessive applications of chemical
fertilizers and other agrochemicals. These chemical inputs are dispersed through
abiotic processes such as diffusion or transport by water or biotic processes such as
meta-ecosystem effects, thus changing diversity and composition of natural com-
munities in agroecosystems (Seneviratne and Kulasooriya 2013). Consequently,
degradation of agro-biodiversity influences adversely on ecosystem functioning
and sustainability.
14.4 Reinstating the Lost Biodiversity
There is a renewed interest in searching alternatives to reduce the use of chemical
inputs in agriculture and to reintroduce the lost soil microbiome. Among other
countries, Sri Lankan government has also taken actions to reduce the use of
chemical inputs. However, such actions should be acceptable to farmers in terms
of the yields of the crop and income of the farmer. The non-synthetic, organic
fertilizers named green manure, animal dung, and compost manure which are
prepared on a domestic or industrial level are the traditional alternatives for chemical
fertilizers. Due to bulkiness of those alternatives, they are not much attractive to
farmers who have been using less bulky granulated or liquid forms of chemical
inputs. Transportation, storage, and field application of organic fertilizers would be
time consuming and need additional labor and cost. The organic fertilizers take time
to decompose and release nutrients to the specific crop plants, and the rate of nutrient
release is not enough for the demand of short-term crops. To address this, some
research studies have been conducted in certain countries to increase the decompo-
sition of the organic fertilizers using soil microbes named efficient microbial inoc-
ulants (EMIs) (Kulasooriya et al. 2017). It has been found out that imported EMIs do
not give the predicted outcomes in Sri Lanka (Kulasooriya et al. 2017), and minimal
studies are in progress in this vital field. Optionally, a desirable substitute has been
the establishment and introduction of microbial biofertilizers which are living
microbial formulations that live in close contact with crop plants and increase
nutrient use efficiency and provide other benefits to the ecosystem. One such
formulation is biofilm microbial ameliorators [BMAs, e.g., biofilm biofertilizers
(BFBFs)], a novel concept introduced by the National Institute of Fundamental
Studies (NIFS) in Sri Lanka.
14 Reinstating Microbial Diversity in Degraded Ecosystems for Enhancing Their... 239
14.5 Developed BMAs as Agents of Reinstating Biodiversity
It is a known fact that soil microbes play a crucial role in the ecosystem, even though
they are microscopic. They are not free living, swimming (planktonic) single cells in
nature. Patel et al. (2014) explained that microorganisms tend to live in communities
due to numerous benefits they can acquire in them. They stick together and form
film-like structures, named microbial biofilms. Different microbial genera are capa-
ble of developing biofilms through effective communication. There are three types
of biofilms that can be identified in the soil, viz., bacterial biofilms, fungal biofilms,
and fungal-bacterial biofilms (FBBs) (Seneviratne et al. 2008). Nitrogen-fixing
bacteria which are attached to fungal surface surrounded by extracellular polymeric
substances (EPSs) are generally called FBBs. Seneviratne and Jayasinghearachchi
(2003) for the first time reported the in vitro development of FBBs by colonizing
fungal mycelia using bradyrhizobial and azorhizobial strains.
The EPS that covers the FBBs is less permeable to gases, and hence it generates a
microaerobic state near N
2
-fixing bacteria (Seneviratne et al. 2017). That enables
them to fixN
2
that is moderately dissolved in the EPS. Nutrients generated via N
2
fixation in the N
2
fixers such as ammonium (NH
4
+
), amino acids, and some proteins
may be transferred to neighboring fungal mycelium to fulfill the nutritional require-
ment. In return, carbon sources may be provided by the fungal mycelium as the
energy source to the N
2
fixers. In this way, fungi and bacteria in the FBBs develop a
metabolic relationship in the symbiosis. Moreover, it is also reported that the FBBs
secrete hormones such as IAA, many organic acids, and glycoproteins to the vicinity
(Bandara et al. 2006). Up on decomposition, glycoproteins gradually release their
nutrients in to the soil, and it can be taken up by plants and microbes. Therefore, the
FBBs play an essential role in growth and nutrition of the plant. Also, they are more
effective than monocultures in their activity and function (Bandara et al. 2006). The
FBBs which attach to plant root systems establish root-biofilm associations, and that
benefits both the plant and microbes in the biofilm (Seneviratne et al. 2009). This
association supplies sufficient amount of nitrogen to the soil-plant system and
increases soil nutrient uptake, which results in significant plant growth. It has been
found that some biomolecules of the FBBs exudates are responsible for breaking the
dormancy of soil microbial seed bank which was developed under stress in conven-
tional agricultural practices (Seneviratne and Kulasooriya 2013). This helps to
reinstate the depleted microbial diversity in degraded agroecosystems and conse-
quently to improve ecosystem functioning and sustainability (Fig. 14.1).
It has also been shown that the inoculation of FBBs maintains a higher cell
density of rhizobia on the legume root system than the inoculation of monocultures
of rhizobia (Seneviratne and Jayasinghearachchi 2005). On the roots of
non-legumes, nodule-like structures which are known as “pseudo-nodules”are
formed by FBBs, and they fixN
2
biologically. In addition, these microbial commu-
nities associate with the root system to defend the plant from adverse environmental
conditions and pathogenic infections (Seneviratne et al. 2010).
240 S. W. Meepegamage et al.
14.6 Developed BMAs for Sustainable Agriculture
The BFBFs are essential in solving many issues that directly affect the
agroecosystems’and other terrestrial ecosystems’sustainability. They provide a
variety of biochemicals and physiological benefits to the growth of plant under
stress conditions and enhance soil quality, thus leading to a reduction of chemical
fertilizer such as NPK use even up to 50% of the recommended doses in different
crops (Buddhika et al. 2016). Moreover, reduction of the chemical fertilizers does
not lead to drop crop yields; instead, it increases the crop yields up to ca. 30% on
average. Also, BFBFs contribute to various health, economic, and environmental
benefits in agroecosystems, particularly in regaining of the lost biodiversity. There-
fore, BFBFs lead to restoration of degraded croplands rather than acting just as a
biofertilizer. Hence, their application is essential for sustaining the productivity of
croplands, even in the midst of different nutrient sources, chemical or organic.
Generally, the endophytic microbial colonization is considered to be essential in
environmental stress tolerance of plants and their growth and yield increase. The
endophytes perform this by inducing stress genes and also by producing biomole-
cules like reactive oxygen species scavengers (Lata et al. 2018). Application of
BFBFs to rice plants under soil moisture stress condition illustrated improved
Fig. 14.1 Biofilm microbial ameliorators (BMAs) for improving sustainability in agroecosystems
through increased microbial diversity and ecosystem functioning. (Reproduced from Seneviratne
et al. 2013)
14 Reinstating Microbial Diversity in Degraded Ecosystems for Enhancing Their... 241
seedling and root growth and also a reduction of leaf drying and rolling
(Weerarathne and Seneviratne 2013). Leaf transpiration during dry periods was
significantly reduced by BFBFs in a multi-location experiment of tea cultivation
(De Silva et al. 2014). These results are attributable to enhanced colonization of
endophytic microbes with the BFBF applications, and also a possible endophytic
biofilm formation (Fig. 14.2).
Improving drought-resistant crops through selective breeding and novel molecu-
lar techniques is a common practice at present (Seneviratne et al. 2017). However,
more natural biological techniques such as the use of BMAs, which have promising
effects, have gained little attention thus far.
14.7 Developed BMAs for Environmental Bioremediation
The land and resources of the earth are rapidly losing its overall quality due to the
carelessness and negligence in using them, and that has resulted in contaminated
sites. It is considered that a contaminated site is harmful to human health. For a
considerable period of time, scientists have been seeking novel remedies other than
conventional techniques such as landfill, application of chemical, high-temperature
incineration, etc. (Vidali 2001). Among all, bioremediation is a promising alternative
since traditional methods have some drawbacks. The term bioremediation can be
defined as the degradation of hazardous or contaminated pollutants to non-hazardous
substances by using biological agents (Sasikumar and Papinazath 2003). Bioreme-
diation process can be followed either in contaminated sites in situ or by bringing out
contaminants from the sites, i.e., ex situ, and then applying fauna and flora (Sharma
2012). Some plants are capable of absorbing heavy metals or toxic materials, the
process of which is known as phytoremediation. However, microbial techniques are
much more effective than phytoremediation, because the production of energy by
Fig. 14.2 Rice leaf endophytic bacterial colonies that were isolated on combined carbon medium.
(a) Rice plants treated with biofilm biofertilizer (BFBF) and (b) those treated with chemical
fertilizers alone. Large colonies in plate (a) are due to initial, large colony-forming units, which
reflect the bacterial cell aggregation and endophytic biofilm formation with the BFBF application
242 S. W. Meepegamage et al.
microbes during the formation of nonhazardous substances is utilized for their
metabolic activities, and also the substances are used as nutrient sources (Tang
et al. 2007). Some naturally occurring beneficial bacteria and fungi in the soil
ecosystem are occupied in the bioremediation process. During the transformation
of the contaminants to substances which are non-hazardous, the microbes attack the
pollutants enzymatically (Vidali 2001). However, these microbes can withstand
within a limited range of the chemical contaminant concentrations, because their
utilization varies depending upon the type of contaminants (Prescott et al. 2002).
Moreover, there must be favorable environmental conditions for microbial
growth and activity and practical bioremediation (Kumar et al. 2011). Considerable
attention has been made on microbial biofilms and their ability of bioremediation
(Decho 2000). Therefore, developed BMAs have been tested in environmental
applications (Seneviratne et al. 2017), and this research field is yet in an early stage.
14.8 Perspectives
As explained above, nature is made out of continuously interacting living and
non-living objects. Balance of those interactions determines the stability and hence
sustainability of ecosystems. However, functional strength is predominantly
maintained by diverse microbial communities and their signal-mediated interactions
with plants. Thus, they show a mechanistic relationship between plant diversity and
ecosystem functioning (Seneviratne and Premarathna 2020). Being the focal point of
the biosphere, microbes govern human, animal, plant, and microbial health
(Seneviratne 2020). Therefore, they control diversity and abundance of the organ-
isms, which lead to the functioning and sustainability of the biosphere, including soil
which is the main home and source of diverse microbes. Human impacts on the
world after green and industrial revolutions altered/decreased the natural diversity
and abundance of microbes, which has led to an imbalance of the other living beings
too, again through their decreased diversity or abundance in the biosphere.
Further, increased greenhouse gas emissions, CO
2
, in particular, in the post-green
and industrial revolutions have led to global warming and climate changes. Inter-
estingly, as a remedial measure to mitigate the climate effects, BFBFs have shown
the potential of sequestering a considerable amount of C in the soil in large-scale rice
ecosystems. Other environmental benefits like locking toxic compounds and heavy
metals in the soil organic matter and reduced N
2
O emissions (Seneviratne et al.
2017) also contribute immensely to lower health cost and mitigate climate change,
respectively.
Good health, as the complete physical, mental, and social well-being, is a
dividend that one can obtain in his/her lifespan. However, the health has been widely
vulnerable to diseases such as deadly cancer in the present context. Recent studies
have proven that human gut microbiome is directly related to the conditions and
illnesses encountered by us. This has been attributed partly to the intake of antimi-
crobials like antibiotics, which degrade the native gut microbiota. Sometimes,
14 Reinstating Microbial Diversity in Degraded Ecosystems for Enhancing Their... 243
people use probiotics after intake of antibiotics. They are mixtures of beneficial gut
bacteria, but not in the biofilm mode, which may not be that effective in regaining the
native gut bacterial diversity, as explained above. Thus, in future medicines, devel-
oped microbial communities in biofilm mode and their exudates, known as biofilm
medicines, should be adopted for treating ailments (Seneviratne and Premarathna
2020). Then, they would help increase native biodiversity of gut bacteria, thus
resulting in enhanced functioning of human body ecosystem for a healthy life.
Scientists have found that a microbiome assembling via food web takes place
according to a specific pattern (Premarathna 2019). Generally, the environment gifts
a subset of their microbiome to plants. Thus, the herbivores, including humans, get a
subset of the microbiome from the plants that are associated with the soil-animal-
microbial network interactions in the environment. Therefore, reinstating the
degraded networks using the BFBFs in the soil may lead to an enriched human
microbiome consisting of least number of pathogens and plenty of beneficial
microbes. Ultimately, it may result in improved human health (Premarathna 2019).
It is also important to note here that the past human impacts on decreasing
biodiversity have now boomeranged to the humans’existence. As explained
above, the application of microbial biotechnologies like developed BMAs could
mitigate such devastating events in the future.
14.9 Conclusion
BMAs like BFBF are capable enough to reinstate the degraded ecosystems for
enhancing their function and sustainability. It holds promise for increasing food
security, mitigating climate change, and improving human health. Thus, the appli-
cation of BMAs is a holistic, important approach to make the globe a safer place to
continue life. Doing it is an urgent requirement because options like Mars coloniza-
tion in an abrupt event like a climate calamity on the earth are not near. Therefore,
microbial interventions like BMAs should be researched rapidly and adopted for the
sustainability of the earth system.
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Chapter 15
Recent Advancements and Mechanism
of Microbial Enzymes in Sustainable
Agriculture
Pankaj Bhatt, Saurabh Gangola, Charu Joshi, Parul Chaudhary,
Govind Kumar, Geeta Bhandari, Saurabh Kumar, Samiksha Joshi,
Avikal Kumar, Narendra Singh Bhandari, and Samarth Tewari
Abstract To make the environment sustainable, using microbial technology is an
important aspect. Indigenous microbial strains have been investigated for their
application in bioremediation and sustainable development. The microbial enzymes
are found effective for the bioremediation of the xenobiotic compounds from the
environment. The enzymes can be produced extracellularly and intracellularly by
microbial cells and catalyze the degradation of the toxic chemicals. Catalysis of the
enzymes is performed via the respective amino acids located onto the binding site.
The enzymes belonging to the esterase, laccase, dehydrogenase, oxygenase etc. are
P. Bhatt (*)
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,
Guangdong Laboratory for Lingnan Modern Agriculture Integrative Microbiology Research
Centre, South China Agricultural University, Guangzhou, China
S. Gangola · A. Kumar · N. S. Bhandari · S. Tewari
School of Agriculture, Graphic Era Hill University, Bhimtal, Uttarakhand, India
C. Joshi
Department of Biotechnology, Kumaun University Nainital, Bhimtal, Uttarakhand, India
P. Chaudhary
Department of Animal Biotechnology, NDRI, Karnal, Haryana, India
G. Kumar
Division of Crop Production, ICAR-Central Institute for Subtropical Horticulture, Lucknow,
Uttar Pradesh, India
G. Bhandari
Department of Biotechnology, Sardar Bhagwan Singh University, Dehradun, Uttarakhand,
India
S. Kumar
ICAR-Research Complex for Eastern Region, Patna, India
S. Joshi
Department of Bioscience, Shri Ram Group of Colleges, Muzaffarnagar, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_15
247
found effective for the bioremediation and sustainable development in agricultural
fields. Here in this book chapter, we have discussed the microbial enzymes and their
application for the sustainable development.
Keywords Microbial technology · Agriculture · Sustainable development ·
Enzymes
15.1 Introduction
To fulfill the global food and feed demands, the agriculture is the main component to
nourish the global society (Bhatt et al. 2021a,b). The various crops, vegetables, and
fruits have been used as a sole source of nutrition by humans globally (Bhatt et al.
2020a; Goel et al. 2020; Kumar et al. 2019). The microorganism is the key
component of the agricultural system (Bhatt et al. 2020b; Suyal et al. 2019a).
Microbes are found effective for the transfer of the macro and micro element from
the soil to the plant rhizospheric region (Bhatt and Barh 2018; Bhatt and Bhatt 2021;
Verma et al. 2021). Plant-microbe interaction makes the beneficial effects into the
ecosystem (Ye et al. 2019; Rawat et al. 2019; Kukreti et al. 2020). The earlier
research focuses on the beneficial effects and depth of molecular mechanism
involved into the plant-microbe interaction for the sustainable agricultural develop-
ment (Kumar et al. 2019; Jin et al. 2019; Suyal et al. 2019b). Microbial cells are able
to produce the various extracellular and intracellular enzymes participated into
contrasting biochemical mechanism (Bhatt et al. 2020c,d,e,f). This biochemical
mechanism includes the bioremediation of toxic chemicals, complex reactions into
the plant cells, and biodeterioration (Bhatt et al. 2020b,g,h,i,2021a,b; Lin et al.
2020; Bhandari et al. 2021).
Microbes act as the cellular factory for the production of the enzymes (Bhatt et al.
2020a,2021b). Microbial enzymes catalyze the reactions on the basis of the pre-
ferred substrates. The complex polymer lignin has been degraded by the microbial
cells using the enzymes such as lignin peroxidase and laccase. Bacterial and fungal
strains have been found effective for the degradation of the wood in ecological
niches. Previous researchers investigated the fungi as the potential lignin degrader.
Fungal strains belonging to the Basidiomycota, Aphyllophorales, and Ascomycota
are considered as the potential wood degrading fungi (Bhatt et al. 2020c; Anasonye
et al. 2015; Zhang et al. 2020a).
Bioremediation using microbial enzymes is the most promising approach for the
sustainable agricultural development (Bhatt et al. 2019a,b; Pankaj et al. 2016a). The
organophosphate, carbamate, organochlorine, pyrethroids, and neonicotinoids group
of the pesticides are degraded by using the microbial enzymes such as hydrolases,
dehydrogenases, laccases, monooxygenases, and dioxygenases (Mishra et al. 2020;
Pankaj et al. 2016b). Immobilized enzymes have been documented for their more
potential approach as compared to the individual strains (Bhatt et al. 2019c,2020i;
Pankaj et al. 2015).
248 P. Bhatt et al.
15.2 Microbial Enzymes Used in Bioremediation Process
15.2.1 Microbial Oxidoreductases
Microorganisms (bacteria and fungi) use various oxidoreductase enzymes and
reduce the toxicity of the various organic contaminants by oxidation coupling
reactions. During the reaction, electrons are transferred from the reduced substrate
(donor) to another substrate (acceptor) by cleaving the bonds and generating energy.
So, in that way the toxic compounds are converted into the less toxic compounds
(Mishra et al. 2021). Oxidoreductase enzymes have a wide range of activity for
degradation of different organic substrate such as azo dyes, pesticides, lignin, and
polyhydrocarbons (Karigar and Rao 2011; Park et al. 2006; Bhatt et al. 2015).
15.2.2 Microbial Oxygenases
Oxygenase enzymes belong to oxidoreductase family (Huang et al. 2021). It oxi-
dizes the reduced substrate by adding oxygen from the molecular oxygen in the
presence of co-substrate (FAD/NADH/NADPH). Generally, the oxygenases are
subdivided into two groups on the basis of number of molecules of oxygen used
for the oxidation of substrate. If one molecule of oxygen is added in the substrate, it
is called monooxygenases, while when the two molecules of the oxygen are added in
the substrate, it is called dioxygenases. These are the two enzymes which play key
role in the metabolism of the various organic substances by increasing the substrate
reactivity and solubility in water and help to break down the cyclic ring structure of
the chemical compounds by adding oxygen molecule. Most of the enzymes used in
the bioremediation process are mono-oxygenases or di-oxygenases (Arora et al.
2009; Singh et al. 2021).
15.2.3 Microbial Laccases
Laccase ( p-diphenol:dioxygen oxidoreductase) is a multicopper protein generally
found in plants, fungi, and bacteria. Laccase has a wide range of activity that
catalyzes the oxidation of reduced substrate with concomitant oxidation reaction
coupled to four electron reduction of molecular oxygen to water (Gianfreda et al.
1999; Mai et al. 2000; Zhang et al. 2020b). Laccase has multiple form of isoenzymes
encoded by different genes (Mishra et al. 2020; Giardina et al. 1995). Many
microbial cells are reported to produce extracellular or intracellular laccase enzyme.
Bacillus subtilis produces laccase which help to degrade the pesticides present in
agricultural fields (Gangola et al. 2018). Laccase helps to metabolize the organic
contaminant and provides the nutrients for the microorganisms (Kim et al. 2002).
15 Recent Advancements and Mechanism of Microbial Enzymes in Sustainable... 249
Among all the oxidoreductase enzymes, laccase has the biotechnological and biore-
mediation applications (Karigar and Rao 2011; Gianfreda et al. 1999).
15.2.4 Microbial Peroxidase
Microbial peroxidases are highly oxidizing agents that oxidize lignin, lignocellulose,
and other phenolic substrate into substances devoid of hydrogen atom. Multiple
isoenzymes of the fungal associated peroxidases are found such as lignin peroxidase
and manganese peroxidase (Bansal and Kanwar 2013; Cocco et al. 2017). Degra-
dation of lignocellulose is a very important step for the carbon recycle in land
ecosystem. Fungi generally basidiomycetes are known to degrade lignin and help
in the carbon recycling process. White and brown rot basidiomycetes digest the
lignocellulose as a sole source of carbon. Manganese peroxidase is an extracellular
enzyme, generally found in the basidiomycetes, which also helps in digestion of
lignin and other phenolic compounds. For initiating the activity of manganese
peroxidase, Mn
2+
acts as substrate and helps to oxidize phenolic compounds (Lin
et al. 2020; Zhang et al. 2020a; Chowdhary et al. 2019; Feng et al. 2020).
15.2.5 Hydrolases
Hydrolytic enzymes reduce the toxicity of xenobiotic compounds by breaking the
linkage in between. Hydrolase enzymes are also known to degrade oil spill and
agricultural pesticides degradation (Karigar and Rao 2011). The key feature of the
enzyme is its broad range of activity, availability, and high tolerability. The enzymes
such as lipase, DNases, proteases, xylanases, and pullulanase are the hydrolytic
enzyme generally used in food industry and biomedical sciences. The hemicellulase,
cellulase, and glycosidase are the hydrolytic enzymes and actively participate in
biomass degradation (Peixoto et al. 2011; Thakur et al. 2019).
The microbial systems are found effective for the development of the sustainable
agriculture by using the enzymes. Application of enzymes in agricultural fields
accelerates the agricultural wastes and helps in resource recovery from the environ-
ment. Here we are enlightening the impact of the microbial enzymes for their
resource recovery potential. Accumulation of different kinds of wastes has become
a severe environmental and public concern. It is urgent to develop innovative
approaches for the removal/degradation of disposal. Microbial enzymes are
extremely effective to catalyze the biochemical reaction. Microbial enzymes have
achieved importance for their extensive utilization in food, agriculture, and pharma-
ceuticals. These enzymes are capable of degrading complex compounds into simpler
ones through the process of bioremediation. The researchers focus on the resource
recovery potential of the microbial enzymes. These enzymes could be used directly
for the xenobiotics compound degradation and cleaning of soil and water
250 P. Bhatt et al.
environments Zhan et al. 2020; Ye et al. 2019; Huang et al. 2019; Danso et al. 2018;
Fan et al. 2020).
15.3 Importance and Mechanism of Microbial Enzymes
in Maintenance of Soil Health
Soil enzymes secreted by microbes are natural mediators which catalyze many
processes like organic matter decomposition, soil humus formation, and release of
different minerals involved in different cycles and help in the maintenance of soil
health and plant growth (Wallenstein et al. 2012; Khati et al. 2019). Thus, it is crucial
to find out the enzymatic activities of soil enzymes and their mechanism to illustrate
metabolic prospective of soil fertility (Astner et al. 2020). Activity of enzymes is an
insightful marker; any changes in soil environment can affect their activity in
agricultural farming (Chaudhary et al. 2021). There are different bacterial and fungal
enzymes which involve in the breakdown of complex form of mineral nutrients into
simpler ones which are easily taken up by plants and enhance their growth (Kukreti
et al. 2020; Khati et al. 2017). Kwiatkowski et al. (2020) reported that dehydroge-
nase, urease, and protease activity improves the fertility of soil and biological
properties of soil and involves in nutrient cycling (Kwiatkowski et al. 2020).
Cellulose is a largely abundant polysaccharide which is hydrolyzed by cellulase
enzyme into D-glucose. It consists of three enzymes such as endo-1,4-β-glucanase
which attacks at random on cellulose chain, exo-1,4-β-glucanase which removes
glucose/cellobiose from cellulose chain, and β-D-glucosidase which hydrolyses
cellobiose into glucose. These enzymes are found in various bacteria and fungi
such as Bacillus subtilis,Aspergillus niger,Phanerochaete chrysosporium,Clos-
tridium thermocellum, and Poria placenta (Deng and Tabatabai 1994). Amylase
enzyme is involved in the breakdown of starch into glucose and maltose which also
play an important role in carbon nutrient cycling. This enzyme is secreted by various
bacterial and fungal species in soil such as Pseudomonas,Bacillus spp., and Asper-
gillus niger. Arylesterase enzyme helps in degradation of complex sulfate esters into
sulfur which takes part in sulfur cycle. Klebsiella spp., Raoultella spp., and
Trichoderma sp. release this enzyme in soil and helps for maintenance of soil fertility
and plant development.
Phosphorus is usually entrapped in the complex soil system and becomes
unavailable for plant uptake. To enhance the phosphorus bioavailability, phospha-
tase enzyme is involved. The conversion of organic P to inorganic P by microbial
associated phosphatase activity leads to P cycling. Phosphatase activity can be
measured as acid and alkaline phosphatase at pH range 4–6 and 9–11, respectively.
After the action of such enzymes, the available P (inorganic P) had good correlation
with nutrient transport and plant vigor. Phosphatase enzyme is involved in the
conversion of insoluble form of phosphate into soluble form and helps in phosphorus
cycling (Khati et al. 2019). Phosphomonoesters and phosphodiesters are involved in
15 Recent Advancements and Mechanism of Microbial Enzymes in Sustainable... 251
the breakdown of sugar phosphates and phospholipids and release free phosphate
from phosphate diester (Turner and Haygarth 2005). Alkaline and acid phosphatase
are good indicators of soil microbial activity and correlated to organic content of soil.
These enzymes are secreted by Anabaena oryzae,Bacillus,Pseudomonas, and
Penicillium (Pankaj et al. 2016a; Khati et al. 2019, 2017). Various factors are
involved for phosphatase activity: (1) if pH increases the activity of alkaline, P
increases, while acid phosphatase activity reduces significantly and vice versa;
(2) water content of the soil is better linked with alkaline phosphatase activity as
compared to the acid phosphatase activity; and (3) salinity stress is better correlated
with alkaline phosphatase activity in all seasons.
Urease enzyme takes part in the conversion of urea into ammonia and CO
2
with
rise in pH and helps in nitrogen cycle which is produced by fungi, bacteria, and yeast
such as Pseudomonas and Trichosporon cutaneum. These enzymes are used as
biological indicators because they are influenced by soil factors like organic matter
content, heavy metals, and nanocompounds and due to the cropping history
(Fig. 15.1). Β-glucosidase enzyme is the most common enzyme in soil which
catalyzes the hydrolysis of glucosides present in soil. This enzyme activity was
observed in various plant species and microbes such as Flavobacterium,
Trichoderma spp., and Lactobacillus plantarum (Kwiatkowski et al. 2020).
For the health and sustainable soil system, the soil enzymatic activity plays a vital
role in nutrient cycling. To establish system for organic matter decomposition to
channelizing available nutrient to the plant system, the soil enzyme system plays a
key role. Different enzymes are available with the soil system that showed overall
health of the soil system. Dehydrogenase (DHA) enzyme is one of them that plays a
significant role to indicate the oxidative power of the soil system associated with the
existed microbiome (Zhang et al. 2020a). Dehydrogenase is a subclass of oxidore-
ductase enzymes that catalyze the oxidation and reduction process. DHA activity is
directly linked with the soil organic matter (SOM) that is available for microbial
activity.
Fig. 15.1 Mechanism of different soil enzymes in nutrient cycling
252 P. Bhatt et al.
The enzymatic activity of the microbes in soil can be measured by fluorescein
diacetate (FDA) hydrolysis assay. In FDA analysis, membrane-bound nonspecific
kind of enzymes like esterases, proteases, and lipases hydrolyzes the 3,6-diacetyle
fluorescein (FDA). After hydrolysis, a yellow green color is produced that can be
quantified by using spectrophotometer at 490 nm (Gilan et al. 2004). The total
enzymatic activity (TEA) including oxygen utilization, microbial biomass, amount
of ATP, etc. can be directly correlated with the FDA hydrolysis (Dzionek et al.
2020). Soil protease enzyme is one of the major enzymatic actions involved in
N-cycle due to availability of huge protein content in the soil system. The protein
components of organic nitrogen in the soil system are usually hydrolyzed by
protease enzyme action. In nitrogen cycle, degradation of protein is an essential
component. Protease enzyme is mainly identified in microorganisms that are respon-
sible for conversion of protein to polypeptide to amino acids (de Morais et al. 2018).
15.4 Circular Economy for SPs and Sustainable
Development
Synthetic pollutants (SPs) have a detrimental effect on the environment and long-
term economic aspects. The existing unsustainable “linear”material and energy flow
model are also prominent contributing factors to these issues (Blomsma and Brennan
2017). However, in recent years, the sustainable development strategies have been
designed for the reduction of the SPs from the environment that favors economic
security without simultaneously degrading the environment. The concept of the
circular economy has become a promising alternative model for sustainable devel-
opment by reducing the SPs and promoting ecological design. The circular economy
aims at minimizing waste production, enhancing product life, optimizing reuse, and
utilizing energy sources such as biomass (Saldarriaga-Hernandez et al. 2020).
The methods used for bioremediation of the SPs can be based on the cyclical flow
of the resources. The bacteria, fungi, and algae are used in a circular perspective for
the bioremediation of the SPs from soil and water systems. Microbial strains can use
the SPs as a source of nutrition for their growth and development and convert the
toxic metabolites into the environmentally accepted form. The metabolic end prod-
ucts can be exploited for the production of useful metabolites and as a resource for
recovery for value-added products. Aerobic and anaerobic digestion of SPs gener-
ates humus and digestate which could be applied to soil as a fertilizer. During the
anaerobic digestion process, biogas that is rich in methane and carbon dioxide is
produced, which could be used as a fuel for combustion in transport or energy
production (Soobhany 2019). Various volatile fatty acids are also formed as inter-
mediates during the anaerobic degradation process and can find various industrial
applications (Singh et al. 2012). Bio-electrochemical systems (BESs), integrating
microbial–electro-chemical removal mechanisms, have been intensively investi-
gated for the organic compound removal in wastewater and simultaneous generation
15 Recent Advancements and Mechanism of Microbial Enzymes in Sustainable... 253
of electricity and biofuel. Different microbial strains can also synthesize biopoly-
mers by utilizing various synthetic pollutants (Pagliano et al. 2017). New emerging
strategies are also allowed production of value-added products such as enzymes
(Steiner and Schwab 2012), single-cell oil (de Oliveria Finco et al. 2017), and
building block chemicals (FitzPatrick et al. 2010) from biodegradation end products.
The methods used for the conversion of the SPs from the environment when aligned
in a sustainable model can generate more fruitful results in the long-term future.
However, still, it is a big challenge to convert the SPs into the environmentally
accepted form and use them for the production of the value-added products.
15.5 Conclusion
Many xenobiotic compounds are present in our surrounding environment which are
released from the different source such as textile industry releases many azo dye and
excess application of pesticides in agricultural field. The physical and chemical
techniques are available to remove these xenobiotic compounds from the environ-
ment. However, these techniques are very costly and expensive. Using the microbial
system for the degradation of these xenobiotic compounds is ecofriendly and cost-
effective technique. Application of indigenous microorganism transforms the toxic
xenobiotic compounds into less toxic or nontoxic and environmental accepted form.
These microorganisms produce different class of extracellular and intracellular
enzymes which help to metabolize the environmental pollutant and use them as a
source of energy. Generally, the enzymes such as lipase, esterase, laccase, dehydro-
genase, and oxygenase play a key role in bioremediation processes. In a similar way,
microbes are also playing key role in the agricultural fields. They produce different
plant growth-promoting hormones, solubilize the insoluble compounds, and make
them available for the plants and enhance the plant productivity and quality.
Therefore, the use of microbial enzymes is important for the development of
sustainable environment. In addition to that, this microbial technique is efficient,
cost-effective, and ecofriendly.
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Chapter 16
Application of Microbial Technology
for Waste Removal
Ravi Ranjan Kumar, Chitra Bhattacharya, and
Nutan Prakash Vishwakarma
Abstract The continuous rise of waste in the environment becomes a global burden
as it decreases the natural balance of waste recycle. It has further accelerated due to
quality and amount of waste added in the environment in the last century. Increasing
human population, introduction of xenobiotic compounds, overexploitation of nat-
ural resources and alarming increased waste generation rate are major threats to
environmental safety. Several waste management practices have been implemented
to decrease the harmful impacts of waste. Microorganisms are inhabitants of nature
that play a major role in biodegradation, bioremediation, nutrient cycling and
detoxification to maintain a sustainable environment. Microbial technology utilizes
a wide range of selective microorganisms in specific condition for removal of waste
from the environment. The utilization of microbes is only limited to culture-
dependent method, and the majority of undiscovered microbes has also been
explored using culture-independent techniques. Technological advancement has
increased the exploration of microbial diversity for their utilization in solid and
liquid waste management. Traditional and advanced techniques such as composting,
anaerobic digestion and bioremediation techniques have been implemented in solid
waste management. Waste from wastewater has been successfully removed using
fixed-film processes, activated sludge, biosorption technology and microbial elec-
trochemical technology. Notorious chemicals such as synthetic dyes and oil spillage
have been also removed from wastewater using microbial technology. Microbial
technology has been magnificently implemented around the world for removal of
waste from the environment. This chapter represents traditional and advanced
microbial technology in both solid and liquid waste treatments.
Keywords Microbes · Microbial technology · Waste disposal · Aerobic processes ·
Anaerobic processes Solid and liquid waste management
R. R. Kumar · N. P. Vishwakarma (*)
Department of Biotechnology, Atmiya University, Rajkot, Gujarat, India
C. Bhattacharya
Department of Microbiology, Atmiya University, Rajkot, Gujarat, India
e-mail: chitra.bhattacharya@atmiyauni.ac.in
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_16
261
16.1 Introduction
Waste management and environmental sustainability are major global concerns of
the present society. In the past, there was a balance between the biosphere’s natural
cycle and mankind activity. The amount and quality of waste were not a burden,
hence recycled by natural phenomenon. Human activity always generated waste
products either as a by-product of their activity or generation of product which is
assumed to be not useful for their life. Despite the generation of waste throughout
their life, nature has treated the waste using their own treatment process such as
waste degradation, dispersion and dilution. But, the activity of modern society
creates disturbance in the equilibrium that continuously accelerates the burden of
waste. There are three primary causes that play a synergistic effect on the continuous
increase of waste: exponential growth of human population, extensive utilization and
diffusion of toxic metals into the environment and tremendous utilization and
dispersion of notorious chemicals such as xenobiotic compounds (Gandolla and
Aragno 1992). The amounts of different wastes rise in such a way that they create
intense damage to the environment and possibility of return to the environment is
also reduced. Rapid consumption rate of raw materials leads to an upsurge of huge
quantities of physical, chemical and radioactive wastes in the environment, which
damage the biosphere rapidly. The accumulation of waste is the core factor for
change of environmental consequences and loss of materials (Kumar et al. 2017).
There is an urgent interference needed to tackle this critical situation.
Waste is difficult to address, as it becomes useful for someone and not for others.
It was also defined as “any material or product which is useless to the producer is
considered as waste”. According to Dijkema, peoples want to dispose the generated
materials even if it requires them to pay for their disposal (Dijkema et al. 2000).
Brunner and Rechberger mentioned that despite the fact that waste is always a major
concern for the environment and mankind, most human activities generate waste
(Brunner and Rechberger 2015). Waste can be classified on the basis of some
common characteristics such as physical states, reusable and recyclable potentials,
physical properties, source of production, biodegradable potentials and degree of
environmental impact (Demirbas 2011). It can be further classified on the basis of
source as domestic waste, industrial waste, commercial waste, agriculture waste,
mining waste and construction waste. Waste is also classified on the basis of
environmental impact as hazardous waste and non-hazardous waste.
Waste has been categorized into various categories in different countries and their
treatment is also based on its quality. The reduction and disposal of wastes have
become a global concern that needs to be managed. All the waste treatment strategy
is based on the return of waste mass to the environment. Disposal of waste into the
environment can be done by either concentration of waste at a target site or dispersal
of the waste substance into the biosphere. Dispersal is a good strategy but only
environmentally acceptable substance can be dispersed. Therefore, waste treatment
is aimed to either produce dispersible derivatives or concentrate the harmful ingre-
dients. According to United Nations Environment Programme, waste management
262 R. R. Kumar et al.
programmes should focus on the 3R concept which is expanded as “Reduce, Reuse
and Recycle”. The proper waste management planning and control is necessary for
the prevention of the harmful impacts of waste on the environment (Ghiani et al.
2014).
16.2 Waste Management Practices
There are three categories of waste management practices: physical, chemical and
biological. Physical methods comprise of incineration, compacting and sorting for
solid waste management, whereas sediment dredging, artificial aeration, mechanical
algae removal and water diversion are methods of waste water treatment (Wang et al.
2016). These traditional methods require a lot of time, material, effort and financial
sustenance. These methods are able to remove large-size particles but not all
pollutants from land or waterbodies competently.
The second technique is chemical method that includes gaseous emissions,
gasification and pyrolysis for solid waste treatment. Liquid waste is chemically
treated by flocculation, precipitation, chemical oxidation and chemical algae
removal (Ma et al. 2015). Chemical methods require the support of a lot of chemical
agents that include oxidizing agents, precipitants and coagulant salts. These methods
are considered as emergency treatment, as they are not much efficient for the
treatment of organic matter. Chemical methods have several other disadvantages
such as consumption of high energy, foul odour and generation of toxic methane gas
which are hazardous for the ecosystem.
The third approach is biological process that includes anaerobic digestion,
composting, vermicomposting, biofiltration, microbial technology, biological-
ecological methods, plant purification technology, combinatorial biotechnology,
artificial wetland technology and biofilm technology (Xu et al. 2017). Biological
methods are environmental-friendly that improve the natural process of waste
removal and self-purification process of the polluted water ecosystem (Ravikumar
et al. 2017). Technical supports are needed to overcome minor difficulties and cost of
waste removal process in the future. Among various biological methods, microbial
technology positions its own advantages and is considered as a highly efficient
method.
16.3 Microbial Technology
Microorganisms have long since been explored in the food and beverage industry,
pharmaceuticals, healthcare products, agriculture and industrial applications and for
environment protection. A large number of microbes still need to be discovered and
explored for their ecological application. Microbes play an important role in envi-
ronmental protection and sustainability. Proper selection and effective utilization of
16 Application of Microbial Technology for Waste Removal 263
microbes are crucial steps for the development of microbial technology. Microbial
technology has been utilized for nutrient cycling, biodegradation, bioremediation,
environmental detoxification, production of biocatalysts, bio-detergent, biomass fuel
production, bioaccumulation, bioleaching, biomonitoring and so on. It has several
advantages such as highly competent degradation ability, free from secondary
pollution, reduced energy consumption, enduring viability, ease in technical opera-
tion and low capital investment.
Microbial technology has been magnificently implemented in several countries
for environmental safety and waste management. Microorganisms especially bacte-
ria, fungi, yeasts and their products were effectively used to remove different quality
of waste and returned back into the atmosphere. Microbial technology was also used
for the production of energy and biofuel from waste; biotreatment of pulp, oil and
textiles; as well as production of valuable end products by fermentation process.
They were also used for treatment of sewage water using aerobic and anaerobic
microbes sequentially. Engineered microbes were recently used as additive for
treatment of heavily polluted tidal river in China which shows improvement in
treatment up to 70% (Sheng et al. 2012). Advanced microbial technology uses
microalgae-bacteria consortium for treatment of nitrogen and phosphorus contami-
nation from surface water (Liu et al. 2017). These photosynthetic bacteria were
easily grown, more viable, more environment friendly, economical and highly
effective in bioremediation process (Idi et al. 2015). The salient features of applica-
tion of microbial technology seem to be highly effective in waste removal, and
therefore, the present chapter emphasizes primarily on effective microorganisms and
recent methods of microbial technology for removal of waste substance from
terrestrial and wastewater.
16.4 Effective Microorganism
Microorganisms are cosmopolitan, present dominantly in soil where they a play vital
role for sustainability of the environment. They are also residing in fresh- and marine
water, plant, animals as well as air. These microbes are highly diverse, considered to
be the largest unexplored reservoir. They perform numerous functions in the bio-
sphere. Effective microorganisms play several roles in the biosphere such as
recycling of waste from soil and water, plant growth-promoting activity, inhibition
of soil-borne pathogens, enhancement of anti-oxidation capability in soil, nutrient
recycling and so on (Shalaby 2011). They have functional diversity; some are
known, while most need to be explored. Attention has been paid for the exploration
of desired microorganisms from unique and less explored habitats. Selection of
effective microorganisms is a fundamental part of microbial technology. It is usually
followed by strain improvement using mutagenesis or genetic engineering to make
them more capable for desired purposes.
The microbial activity can be further accelerated by application of microbial
accelerating agents. Microbial accelerating agents are formulation of microbial
264 R. R. Kumar et al.
growth promoters such as trace elements, amino acids, humic acid enzymes and
vitamins in proportionate amount. These agents are always harmless to soil envi-
ronment. These nutrients can accelerate microbial growth and stimulate them for
biological reactions. They promote the proliferation and activity of indigenous
microorganisms while inhibiting the anaerobic decomposition of pollutants
(Wu and Xie 2012).
16.5 Microbial Technology for Waste Removal
Microbial technology is a scientific technique which utilizes a wide range of
microorganisms in meticulous condition without distressing the ecosystem. An
eclectic variety of microorganisms have been effectively used for waste removal
practices. Microbial technology is fairly used for waste management practices in
very effective way. It is eco-friendly, cost-effective and a better substitute compared
to expensive physico-chemical remediation processes. The efficient methods
adopted for waste removal process using microorganisms are composting, bioreme-
diation, biodegradation, bioaccumulation, biotransformation, anaerobic digestion
and wastewater treatment.
16.6 Microbial Technology for Solid Waste Treatment
16.6.1 Composting
Composting is an aerobic microbial decomposition process in which organic matter
hydrolyzes into stable residue (Wei et al. 2017). This process is generally used
through metabolic activity of microbial consortium to produce safe and stabilized
form of organic compost for various agricultural practices. During the process
temperature increases spontaneously; it would help to eradicate the pathogenic
organisms; hence, finally generated compost becomes safe for usage (Rastogi et al.
2020). Several composting divisions exist at various locations in India such as
Mumbai, Bengaluru, Indore, Vadodara, Kanpur and Delhi. These units have 150–-
300 tonnes/day as installation capacity (Sharholy et al. 2008).
16.6.1.1 Framework of Composting Process
The following phases are included in compositing process:
1. Mesophilic phase: In this phase mesophilic fungi and bacteria degrade the
complex compounds such as carbohydrate and amino acids into simple one by
rapidly elevating the temperature.
16 Application of Microbial Technology for Waste Removal 265
2. Thermophilic phase: This is the second phase of composting in which thermo-
philes degrade the organic matters (lignin, cellulose, fats and hemicellulose).
During this phase, thermophilic microbes would utilize the organic carbon
content for their feedstock and metabolic activities.
3. Cooling phase: In the last phase microbial activity is diminished and temperature
has also decreased. During this stage mesophilic microorganisms degrade the
residual substances such as cellulose, hemicellulose, sugars and humus (Albrecht
et al. 2010).
16.6.1.2 Factors Affecting the Composting Process
Some physiochemical factors such as nutrient balance, C/N ratio, pH, particle size,
moisture, temperature, porosity, oxygen, moisture content and nutrient availability
play a foremost role in various phases to determine the development of microbial
populations during composting (Leow et al. 2018).
16.6.1.2.1 C/N Ratio
As a nutritional parameter, the optimum balance of carbon-nitrogen ratio is essential
for the formulation of compost. Ideally, C/N ratio essentially range between 25 and
35, declaring that microbial requisite is around 30 parts carbon to 1 part nitrogen
(Kutsanedzie 2015). However, some authors state that C/N value at the range of
20–50 also gives a worthy result (Petric et al. 2015). During the composting process,
carbon is transformed to carbon dioxide during organic degradation. Due to higher
C/N ratio, there is deficiency in nutrient level to microbiota and composting speed
decreases. Lower C/N gives rise to increased nitrogen content compared to degrad-
able carbon and that results in loss of ammonia in soil through leaching or volatil-
ization (Zhang and Sun 2016). Generally, declined C/N ratio indicates higher waste
degradation over the mineralization process (Wang et al. 2016).
16.6.1.2.2 Temperature
Composting is an exothermic technique, which includes organic matter degradation
by bio-oxidative microbial activity. In this process a huge magnitude of energy is
produced. But microorganisms utilize only 40–50% of the generated energy for the
synthesis of ATP; the rest of the energy is lost in the form of heat. The huge quantity
of heat is the basic reason for increased temperature during the process and it can
reach up to 70–90 C. This process was named as “microbial suicide”by Finstein
(Waszkielis et al. 2013). This high temperature inhibits the microbial growth and
slows down the biodegradation process. The organic matter decomposition rate is
dependent on the temperature of the raw material. In the higher temperature (above
70 C), only few thermophilic bacteria can survive and show metabolic activity. So,
266 R. R. Kumar et al.
for the destruction of pathogens, weed seeds and fly larvae, maximum temperature
would be maintained at least 3–4 days (Garg and Tothill 2009). Increased microbial
diversity will be necessary to obtain higher biodegradation rate and the required
temperature should be in the range of 30–45 C (Finstein et al. 1983). The set point
for feedback temperature control ranged between 30 and 45 C during the process of
composting.
16.6.1.2.3 Aeration
For the aerobic decomposition of raw material, sufficient oxygen supply is required.
Anaerobic condition could be developed in the deficiency of oxygen in the environ-
ment that will create pungent odour of methane (CH
4
) gas. Oxygen would be refilled
in the waste materials by using perforated pipes (Garg and Tothill 2009).
16.6.1.2.4 Moisture Content
Water is the most essential part of composting cycle and microbial activity. The
maintenance of optimal moisture content is dependent on particle size, physical state
of the initial material and the type of composting system. Usually, 60% of moisture
content is considered as suitable for starting material. Because different materials
have different water-holding capacities, an exact generalization of moisture levels
cannot be made. If the moisture content is too low, early dehydration occurs which
arrests the microbial activity, giving physically stable but biologically unstable
compost. In modern composting systems, water can be added during the process.
In newly designed plant system having a capacity to remove a large amount of water,
evaporative cooling system and high rate of heat generation, this dried material
required the addition of water for sustainability of microbial activity. This will be
only possible in conjunction with mechanical turning. The biological activity of
stabilized end material can be prevented by lowering (about 30%) the moisture
content of composting process (Diaz and Savage 2007).
16.6.1.2.5 Time
The period of active composting: dairy waste can be converted into compost around
1–14 weeks; this is followed by 3–4 weeks of curing period (Garg and Tothill 2009).
16.6.1.2.6 pH
The estimated pH range for the decomposition of organic matter is 5.5–8.0 (Zhang
and Sun 2016). If the pH is more than 8.0, lime or bleaching powder is used at the
collection/storage points. If the pH decreases less than 5.5, then microbial
16 Application of Microbial Technology for Waste Removal 267
nitrification and volatilization occur, which results in the production of enormous
quantity of acids and CO
2
(Wang et al. 2016). Volatilization of ammonia leads to
disappearance of protein mineralization and slowdown of nitrogen during conse-
quent stages of composting (Guo et al. 2012). Alkalization of compost is associated
with preeminent pH (more than 9). It may obstruct the survivability of pH-sensitive
microbes that have plentiful contribution in compost sanitation (Hachicha et al.
2009). Herbocel or sanitreat process is applied to control flies and foul odour
(Raza and Ahmad 2016).
16.6.1.3 Composting Methods
There are numerous composting methods that suit the goal of the researchers and the
nature of waste materials to be composted can be adopted for their purpose of
utilization. Some of the composting methods are enumerated below.
1. Vessel Composting
This method depends on the variety of forced aeration and mechanical turning
techniques for the enhancement of the composting process. A modified version of
in-vessel composting refers to confining the materials within an enclosed area
such as a container, building or vessel (Gonawala and Jardosh 2018). This
method is too costly and labour-intensive.
2. Windrow Composting
In this composting process, raw materials are employed in a long narrow piles
or windrows that curved frequently. Aeration is utilized for the mixing of
materials into the setup. In the windrow composting process, system arrangement
should be started from 3 ft. height for manures (dense materials) and 12 ft. height
for fluffy materials such as leaves. It is a rapid process due to the materials’heat
holding capacity and expensiveness in nature (Ayilara et al. 2020).
3. Vermicomposting
Vermicomposting is a fundamentally accomplished process for digesting
organic matter by combined action of earthworms and microorganisms to trans-
form the organic waste material into a compost for soil amendment. Earthworms
are insatiable consumers that biodegrade matter as vermi-castings or excreta.
Earthworms are key players in vermicomposting but they also stimulate microbial
activity through fragmentation of organic matter and aeration, and they increase
the surface area for microbes. During composting, microbial colonization begins
that leads to succession of microbial community composition (Rogayan et al.
2010). Vermicompost provides growth-promoting hormones and nutrients to
plants and also plays a responsive role in improving the soil structure by increas-
ing nutrient capacities and moisture content of the soil. With the usages of
vermicompost, fruits, flowers, vegetables and other plant products are grown in
better quality (Arumugam et al. 2017).
268 R. R. Kumar et al.
16.6.1.4 Advantages and Disadvantages of Composting
Despite the advantages of compost, some disadvantages also occur (Beffa 2002).
Advantages
•It is cost-effective and very sophisticated.
•Humus and plant litter nutrients can be recycled into the soil.
•It increased the microbial diversity into the soil.
•Compost can be used as biofertilizer as well as biocontrol agent, as they compete
with phytopathogens.
•It would be beneficial for biodegradation of toxic compounds and pollutants.
Disadvantages
•Pungent odour nuisances.
•Propagation and dispersal of potential pathogens or allergenic microorganisms.
•If heavy metal content is too high in compost, it could affect the soil.
•This process could be used only for sewage sludge that originate from non-
industrial sources.
16.6.2 Aerobic Digestion
In the aerobic biological treatment system, organic matter in waste material is
digested under humid and warm condition present. Activated sludge, trickling
filters and oxidation ponds are the foremost categories of aerobic systems. Aerobic
digestion system works by employing the basic biochemical reaction applied for all
the microorganisms (McKinney 1957).
Food þMicroorganisms þOxygen !Increased Microorganisms
þCarbon dioxide þWater
þWaste products þEnergyðÞ
All the digestion system operates on the fundamentals of biochemical ethics, but
some vary from each other by oxygen transfer method. Compressed air is utilized in
activated sludge for mixing of oxygen source. Generally, in trickling filter microor-
ganisms are attached to the stones and attained their oxygen from the diffused air.
Details of aerobic digestion treatment process have been included in wastewater
treatment process in this chapter.
16 Application of Microbial Technology for Waste Removal 269
16.6.3 Anaerobic Digestion
Anaerobic absorption is a fermentative decomposition technique which converts the
organic waste into composites that can be utilized as biofertilizers and soil condi-
tioners (Rastogi et al. 2020;D’Annibale et al. 2006). Anaerobic absorption is defined
as a sequence of organic developments, where microbes can catalyze organic waste
material in anaerobic condition. Stages included in anaerobic digestion are
acidogenesis, hydrolysis, methanogenesis and acetogenesis.
16.6.3.1 Hydrolysis
Hydrolysis is the preliminary juncture of anaerobic absorption, in which insoluble
organic substances such as polymers of amino acids, carbohydrates and fats are
converted into soluble matter such as long chain of fatty acids, amino acid and sugar
molecules (carbohydrates). Hydrolase enzymes are supported in the hydrolysis
reactions. The microbes which synthesize these enzymes are termed as hydrolytic
microorganisms such as Bacteroides,Clostridium,Bacillus,Proteus vulgaris,vibrio
and Micrococcus and Staphylococcus bacteria (Amani et al. 2010).Some hydrolase
enzymes are utilized in the form of peptidases, esterase and glycosidases. During
anaerobic digestion process, hydrolysis is considered as a rate-limiting stage where
insoluble complex organic substance is slowly depolymerized.
16.6.3.2 Acidogenesis
The second stage of anaerobic digestion process involves acidogenesis, where acid-
gas-producing bacteria can hydrolyze the soluble molecules to alcohols, lactate,
volatile fatty acids and carbon dioxide. Numerous fermentation pathways are
involved in this process. Different bacterial genera are responsible to carry out
acidogenesis. Some bacterial species that perform fermentation under anaerobic
digestion are as follows: for the production of alcohol, Saccharomyces is used; for
lactate fermentation Lactobacillus and Streptococcus were utilized; Butyribacterium
species are employed for butyrate fermentation; and Clostridium is used for propi-
onate fermentation. The group of bacteria such as Clostridium,Sporomusa,
Acetobacterium and Eubacterium is utilized for the production of acetate. These
acetate-forming fermentative bacteria are distinctive species for acidogenesis
(Amani et al. 2010).
16.6.3.3 Acetogenesis
Acetogenesis is a process involved in the third stage of anaerobic digestion method.
Acetogenic bacteria called acetogens can transform the alcohols and volatile acids
270 R. R. Kumar et al.
into acetate in the form of by-product hydrogen and carbon dioxide. Acetogenic
bacteria such as Syntrophobacter wolinii and Smithella propionica are acetate-
producing microbes that abrade the butyrate and propionate, respectively. On the
other hand, some bacterial species such as Pelotomaculum thermopropionicum and
Syntrophobacter fumaroxidans convert volatile fatty acid into formic acid with the
liberation of CO
2
and H
2
.Clostridium aceticum has the ability to produce acetate
from H
2
and CO
2
(Amani et al. 2010).
16.6.3.4 Methanogenesis
Methanogenesis is the ultimate step of anaerobic digestion. In this phase
methanogens play a significant role in the consumption of acetogenesis products.
It can convert the acetate and molecular hydrogen to methane gas and terminate the
activity of acetate-forming bacteria. Few pathways are involved for the production of
methane from acetate:
•Acetoclastic methanogenic pathway: In this pathway bacterial species
Methanothrix concilii and Methanosaeta soehngenii are able to transform acetic
acid to carbon dioxide and methane.
•Hydrogenotrophic methanogenic pathway: Hydrogen and carbon dioxide are
used for the production of methane by employing Methanobacterium bryantii,
Methanobrevibacter arboriphilus and Methanobacterium thermoautotrophicum.
Some another bacterial species such as Methanococcus voltae,
Methanobacterium formicicum and Methanobrevibacter smithii are applied for the
production of methane from methanoate, hydrogen and carbon dioxide (Amani et al.
2010).
16.6.3.5 Factors That Affect the Anaerobic Digestion Process
Temperature: Temperature is the most significant cause for the continued existence
and microbial growth in anaerobic digestion. There are two ranges of temperature
for the growth of microorganism such as mesophilic (30–40 C) and thermophilic
(50–60 C) bacteria that optimize the digestion of organic matter. In mesophilic
environment (35 C), the organic part of municipal solid waste can easily be
assimilated in the atmospheric condition. Maximum microbial growth rate,
microbial activity and production of methane are accomplished by anaerobic
digestion under thermophilic conditions (Mata-Alvarez 2002).
Moisture: Moisture content is necessary for controlling the cell turgidity, transport
of nutrients, enzymes and hydrolysis of complex organic matters (Khalid et al.
2011). The methanogenic activity increased in high-solid sludge at 90–96%
moisture content of mesophilic anaerobic digestion process (Jiunn-Jyi et al.
1997).
16 Application of Microbial Technology for Waste Removal 271
Retention Time: In mesophilic condition, 8–40 days of retention time is necessary
for anaerobic digestion. According to the report of Fdez, thermophilic anaerobic
digestion process of solid organic matter is also accomplished in the range of
8–40 days (Fdez-Güelfo et al. 2011).
pH: The ideal pH range for anaerobic digestion process is 6.8–7.2. Hydrolysis and
acidogenesis process enhanced at pH 5.5–6.5, whereas methanogenesis requires
pH 7.0 for optimum activity (Ward et al. 2008).
Carbon and Nitrogen Content:Carbon is the energy source for the growth of
microbial community, whereas nitrogen assists in the enhancement of microbial
growth. The requirement of carbon source by microorganisms is 25–30 times
more greater than that of nitrogen, chiefly at a ratio of 20–30:1 (Ward et al. 2008).
Elsewhere, for methanization process, the required elements C/N/P/S (carbon/
nitrogen/phosphorus/sulphur) have a nutrient ratio of 600:15:5:3 for microbial
growth (Khalid et al. 2011).
16.6.4 Bioremediation
Bioremediation is distinct as a technique involved in the degradation of organic
matter primarily by microorganisms that can utilize for the transformation of less
toxic substances. In this process, naturally occurring microorganisms such as bacte-
ria and fungi and chlorophyll-containing plants can detoxify hazardous substances
which are harmful for human health and the environment. The microorganisms are
isolated from the contaminated site by the physiological factors that influence the
optimization of bioremediation method. Some of the environment factors such as
type of soil, temperature, pH, aeration and nutrients are incorporated for the avail-
ability of contaminants to the microbial inhabitants. Based on the utilization of
microbes, it can subcategorize microorganisms into various groups:
Aerobic bacteria: Microorganisms which survive in the existence of oxygen and
utilize the contaminant carbon as the energy source for their growth. Some of the
aerobic bacterial species such as Alcaligenes sp., Mycobacterium sp.,
Rhodococcus sp. and Pseudomonas species have the abilities to degrade under
aerobic condition. Aerobic bacterial species can also degrade the chemical con-
stituents such as hydrocarbons and pesticides, both poly-aromatic compounds
and alkanes.
Anaerobic bacteria: For the degradation of polychlorinated biphenyls (PCBs),
anaerobic bacteria can be employed for the bioremediation process of river
sediments. Dechlorination process is involved in the bioremediation by anaerobic
bacteria. In this process, solvent trichloroethylene (TCE) and chloroform are
degraded in the absence of oxygen molecule.
Ligninolytic fungi: Fungi such as Phanerochaete chrysosporium are involved in the
process of degradation of the extremely miscellaneous range of toxic
272 R. R. Kumar et al.
environmental pollutants. They utilize some easily available substrates including
straw, sawdust or corn cobs for their growth and survival.
Methylotrophs: Methylotrophs are a group of aerobic bacteria that utilizes methane
and converts it to carbon as energy source for their augmentation and survival. In
this pathway the initial enzyme, i.e. methane monooxygenase, is used as a
catalyst to produce chemical compounds such as chlorinated aliphatic trichloro-
ethylene and 1,2-dichloroethane.
16.6.4.1 Bioremediation Strategies
Various methods are involved in the process of bioremediation and are employed
depending on the degree of saturation and aeration of an area as follows.
16.6.4.1.1 Culture-Dependent Approach
In the process of culture-dependent method, microorganisms are cultured into the
nutrient growth media and further processed maintaining an objective. This tech-
nique involves microbes for the degradation of poisonous composites and manufac-
ture of by-products (Gupta et al. 2019). There are two categories of bioremediation
process.
In Situ Remediation
In this technique, generally site-specific microflora plays a significant role in the
bioremediation process of the polluted site, which involves without any excavation
and transport of contaminants. This method is eco-friendly and cost-effective (Singh
2014). Generally, three methods are used for in situ remediation: bioventing,
biosparging and bioaugmentation. These methods are favourable for less polluted
sites (Gupta et al. 2019).
1. Biosparging
Petroleum hydrocarbons containing benzene, toluene, ethylbenzene, and
xylene isomers (BTEX) are the major components of hazardous fuel that can be
accidentally spilled into the environment with the release of gasoline products
from the leakage of pipelines and fuel storage tanks. This is one of the primary
sources of groundwater impurity. Intrinsic bioremediation process is considered
as a potential method for the cleanup and removal for the sites of petroleum-
hydrocarbon-contaminated areas (Chen et al. 2006). In the process of
biosparging, indigenous microorganisms are used to degrade the organic sub-
stances in the saturated zone. Oxygen and nutrient are introduced into the
saturated zone for the enhancement of microbial activity. Biosparging technique
is utilized for the reduction of petroleum components which are dissolved in
groundwater and within the capillary fringe. Biosparging differs from air sparging
16 Application of Microbial Technology for Waste Removal 273
techniques in the sense that in the air sparging process contaminants are removed
through volatilization method, while biosparging stimulates biodegradation of
ingredients. If any volatile constituent is present in the form of contaminants,
biosparging is frequently combined with soil vapour extraction and other reme-
dial technologies. Whenever combinational process of biosparging and extraction
of vapour occurs, the vapour extraction system can produce negative pressure in
the unsaturated zone of groundwater through the extraction wells, which could
also control the vapour plume migration.
2. Bioventing
This is one of the important in situ remediation processes in which microor-
ganisms can be utilized for the degradation of organic matter which is usually
adsorbed with soil in the unsaturated zone. Bioventing system essentially has
similar components as present in the soil venting system. It consists of a series of
air induction (influent), blower and venting (effluent) wells. Bioventing usually
occurs at specific site where air is injected into the soil or extracted from the soil.
It can also be accomplished with excavated soils. In the sparging method, air is
utilized to provide oxygen for the biodegradation and it can transfer the volatile
contaminants from groundwater to the vadose zone in the soil. In the bioventing
process, withdrawn air (off-gas) needs treatment (Hoeppel et al. 1991). The
off-gas is usually treated by activated carbon, adsorption against thermal pressure
and catalysis in a biofilter. During carbon adsorption the air treatment contains a
very high cost of venting project (Hoeppel et al. 1991); therefore, as an alternate
option, the vented air is used as a biofilter that can pass through unsaturated and
uncontaminated soil (Miller and Bartha 1989). With the usage of bioventing
process, the cost remediation process can be minimized and the need for
off-gas treatment is also eliminated (Lee and Swindoll 1993).
3. Bioaugmentation
Bioaugmentation is an in situ method for the removal of undesired hazardous
compounds through the genetically modified microorganisms (hydrocarbon-
degrading microbes) from soil and groundwater (Mrozik and Piotrowska-Seget
2010). The foundation of this approach is the augmentation of pollutant-
degrading microorganisms for degradation of complex pollutants (Omokhagbor
Adams et al. 2020). In this process microorganisms containing the specific
metabolic activity are augmented to enhance the rate of waste degradation at
the contaminated site. Contamination of chlorinated ethenes, such as
tetrachloroethylene and trichloroethylene in the groundwater or soil, can be
completely eradicated using in situ microbes. These contaminants are converted
to non-toxic substances such as chloride and ethylene (Singh 2014).
4. Biostimulation
Biostimulation is a cost-effective, eco-friendly and extremely efficient biore-
mediation process. It refers to the addition of micronutrients such as phosphorus,
nitrogen, oxygen and electron donors in harshly polluted area for stimulation and
to improve the efficient degradation ability of inhabitant microorganisms.
Biostimulation technique is applied to accelerate the rate of decontamination
and degradation of the toxic contaminants and hazardous compounds. The rate
274 R. R. Kumar et al.
of biostimulation is influenced by various environmental conditions such as
moisture content, pH and temperature which predominate the ecological physi-
ology (Abdulsalam and Omale 2009). Biostimulation technique plays an impor-
tant role in the degradation of petroleum products, hydrocarbons and their
derivatives (Kaouther Zaafouri 2014). In the petroleum-contaminated areas, the
microbial population and their activity are very low. Poor metabolic activity of
microbial population can be remediated through the process of biostimulation
(Tyagi et al. 2011).
Ex Situ Remediation
In ex situ remediation, the process is carried out in any other place from the
contaminated site (Singh 2014). By using this method, it gives favourable result in
decreasing the total load of toxic xenobiotic compounds from the waste (Kumari
et al. 2014). Ex situ bioremediation technologies can be facilitated by different
methods such as biopiling and landfarming and bioreactors.
1. Biopiling
Biopiling-mediated remediation process is employed for the piling of exca-
vated polluted soil above the ground and nutrient modification, followed by
aeration. It enhances the remediation process by increasing the microbial activ-
ities. Ex situ technique has been always considered for their cost efficiency.
Biopiling can control the nutrient condition, aeration rate and temperature during
the process (Whelan et al. 2015). Biopile can also be useful for the limited
volatilization of low molecular weight pollutants. It can be effectively utilized
for the remediation of pollutants of psychrophilic environments, i.e. cold regions
(Dias et al. 2015). In the biopile heating system, augmentation of microbial
activity is combined with contaminant availability which enhances the rate of
biodegradation. Moreover, heated air is inserted into biopile design to deliver the
air and heat in tandem, to facilitate enhanced bioremediation (Aislabie et al.
2006). In the processing of contaminated soil, sieving and aeration are provided
by biopile (Delille et al. 2008). However, biopile systems have disadvantages
over ex situ bioremediation process that include robust engineering, lack of
power supply, maintenance cost, and land farming, especially at remote areas.
Further, excessive heating of air can lead to drying of soil undergoing bioreme-
diation, which results in the inhibition of microbial activities, reduction of
biodegradation process and promotion of volatilization (Sanscartier et al. 2009).
2. Landfarming
Landfarming is useful mainly for the control of pollutions from the soil by
pesticides. In the landfarming process, a bilayer of fresh soil, clay or concrete are
prepared from the fresh soil and combination of clay and concrete. The fresh soil
is placed at the bottom part and the concrete layer is always placed in the superior
layer. Then, natural microbial deprivation is permitted. In this process air, nutrient
and humidity are necessary to exhibit the protocol by maintaining the 7.0 pH
through liming (Singh 2014).
16 Application of Microbial Technology for Waste Removal 275
3. Bioreactors
In the ex situ treatment, slurry bioreactors are used for the removal of stained
soil and water pumped up which contaminated the column. Bioremediation by
bioreactors involves the removal of contaminated solid materials such as soil,
residues and sludge. In this reactor system the rate of biodegradation is higher
than in situ remediation, because the contained environment is more convenient,
predictable and manageable. The disadvantages of bioreactor is pretreatment of
contaminated soil by excavation or vacuum extraction method (Mary 2011).
16.6.4.1.2 Culture-Independent Approach
In this approach, sequencing technique is being utilized for the analysis of genomes
of all nucleotides such as DNA, RNA and protein. Various processes are involved in
this culture-independent method like processing of whole DNA sequence from
specific environmental condition known as metagenomics, whole-genome sequenc-
ing, whole-transcriptome sequencing (isolation of RNA from a single and pure
culture), metatranscriptomics (RNA isolated from a sample of ecological unit) and
so on (Rathour et al. 2017). In nature microorganisms are omnipresent; therefore, to
explore their microbial diversity, “next-generation sequencing”would be in assis-
tance to overcome the concealed potential of microbiome functionally, taxonomi-
cally and morphologically (Gupta et al. 2019).
16.6.4.1.3 Bioremediation of Rubber Waste
The use of rubber is increasing day by day in vehicles (Holst et al. 1998). Tire is
composed of high-grade black carbon and synthetic polymers for maintaining the
strength of the rubber tire (Larsen et al. 2006). About 12% of rubber is considered as
solid waste. With regard to its physical composition, rubber can neither be degraded
easily nor recycled (Conesa et al. 2004). A major environmental problem arises due
to the burning of rubber; it produces a large number of toxic chemical components
such as zinc oxides and carbon monoxide (Zabaniotou and Stavropoulos 2003). The
toxic component from rubber has been preliminarily eliminated by using Recinicium
fungi. In the second step, rubber can be devulcanized by sulphur-oxidizing or
sulphur-reducing bacteria like Thiobacillus ferrooxidans and Pyrococcus furiosus.
Devulcanized rubber can be recycled (Stevenson et al. 2008). The best way of waste
management is to control the combustion of rubber and the liberated heat is also
utilized for the generation of energy (Conesa et al. 2004).
276 R. R. Kumar et al.
16.7 Roles of Microorganisms in Wastewater Treatment
Application of microbial technology is now being also focused on the removal of
heavy metals, toxic substance, dissolved inorganic dyes and nutrients. Microorgan-
isms in sewage treatment are manifested through their metabolic activities, degra-
dation potential as well as detoxification capacity. Microorganisms regulate a series
of chemical reactions, and metabolize the pollutants by utilizing organic matter as a
nutrient source present in wastewater. Microbial technology also utilizes degradation
of organic matter into inorganic substance and detoxification. On the basis of
metabolic capacity of microbes under anaerobic or aerobic conditions, microbial
process is divided into aerobic and anaerobic biological treatment.
Wastewater treatment process is associated with the following group of
microorganisms:
1. Bacteria: Bacteria play a pulsating role in the treatment of wastewater. They hold
the principal responsibility for the removal of organic compounds. Organic
matters are utilized by them to get energy and they use this energy for their
growth. Most common bacteria present in wastewater are Achromobacter sp.,
Arthrobacter sp., Acinetobacter sp., Alcaligenes sp., Bacillus sp.,
Chromobacterium sp., Cronobacter sp., Citramonas sp., Enterobacter sp.,
Escherichia coli,Flavobacterium sp., Klebsiella sp., Kosakonia oryzae,
Leclercia sp., Pseudomonas sp. and Serratia sp. (Silva-Bedoya et al. 2016).
2. Filamentous bacteria: They are normally associated with biomass present in
activated sludge. Their presence is noteworthy for the formation of floc. Their
population size varies with nutrient conditions such as the amount of nutrients,
DO, sludge age, pH and temperature of wastewater system. Common filamentous
bacteria found in activated sludge are Alcanivorax sp., Beggiatoa sp., Microthrix
sp., Sphaerotilus natans and Thiothrix sp. (Paillard et al. 2005).
3. Algae: They are photosynthetic organisms that have a substantial role in nutrient
(nitrogen and phosphorous) removal and accumulation of some xenobiotic com-
pounds, toxic substances (both organic and inorganic) and heavy metals. Some
common algae present in wastewater are Chlamydomonas sp., Euglena sp.,
Limnothrix,Lyngbya,Microcystis,Oscillatoria,Phormidium autumnale and
Synechocystis (Martins et al. 2010).
4. Protozoa: Protozoans are unicellular eukaryotic organisms that digest and elim-
inate free-swimming bacteria and other suspended particles from wastewater.
Requirement of oxygen varies among different species of protozoan for their
survival. Common ciliated protozoans found in wastewater are Aspidisca sp.,
Carchesium sp., Chilodonella sp., Opercularia coarctata,Trachelophyllum
sp. and Vorticella sp. (Amaral et al. 2004).
5. Fungi: They are multicellular eukaryotes having ability to hydrolyze organic
matter even at low pH. They secrete hydrolytic enzymes to degrade substrate and
adsorb suspended solids through mycelia to accomplish their nutrient require-
ments during wastewater treatment. Common fungi in waste removal process are
16 Application of Microbial Technology for Waste Removal 277
Aspergillus,Absidia,Fusarium,Sphaerotilus,Penicillium and so on (Akpor et al.
2013).
16.8 Microbial Technology for Wastewater Treatment
The strategy for treatment of wastewater begins in the early twentieth century.
Technology application for wastewater treatment has been focused on the quality
of waste present in sewage. The major pollutants present in wastewater are biode-
gradable and volatile organic compounds, nutrients (nitrogen and phosphorus),
suspended solids, toxic metals, recalcitrant xenobiotics as well as microbial patho-
gens. Microbial technologies are environment friendly and sensible choice for
hazardous waste from wastewater. Waste present in water is usually treated primarily
by physical treatment methods to remove the physical pollutants. Microbes were
usually applied as a secondary treatment to remove organic matter present in waste.
The choice of technique is based on the quality of waste, aerobic or anaerobic
method of degradation and several other parameters. The British Royal Commission
declares their goal for waste water treatment to decrease BOD of wastewater up to
20 mg/L and the final yield of effluent and suspended solids should also decrease to
30 mg/L. BOD (biochemical oxygen demand) is defined as “the amount of oxygen
required by microbes present in waste water for the oxidation of organic nutrients”.It
measures the strength of the organic waste present in wastewater; the more organic
matter present, the higher the BOD value. We included some common and advanced
technology used for wastewater treatment.
16.8.1 Fixed-Film Processes
Fixed-film microbiological processes are based on attachment of high concentration
of microorganisms on a solid support material such as plastic, gravels, sand or stone
particles. Microbial growth is influenced on the substratum by factors such as
geometric configuration of particles and the flow rate of wastewater. There are
several advantages of fixed-film processes such as low specific growth rate of
microorganisms, suitability for small size reactor and lower operation cost. The
disadvantage associated with this process is overgrowth of microorganisms on the
solid substratum and biofouling which affects heat exchange process unpleasantly.
Two major categories of fixed-film reactors, trickling filters and rotating biological
contactors, are described here.
1. Trickling Filters
The treatment of wastewater by trickling filters is conducted in a rectangular or
circular reservoir filled with filter medium with a depth of approximately
100–250 cm. Large surface area available in filter medium is suitable for growth
of microorganisms and also provides adequate void space for diffusion of air.
278 R. R. Kumar et al.
Usually crushed stones, granite, hard coal, ceramic material, plastic and treated
wood are used in filter media. The size of support matrix is based on microbial
attachment and void space. Smaller sized matrix has higher surface area for
microbial attachment, but void space is less. Wastewater is sprayed upon the
surface of the bed with sprinklers that allow uniform hydraulic load. Wastewater
containing organic matter is dripped above the filter media that provides nutrients
for the growth of microorganisms on filter surface. During the flow, the waste-
water undergoes aerobic decomposition by microorganisms. An underdrain sys-
tem is also connected that collects the treated wastewater as well as sloughed off
microbial biomass. A final clarifier separates the microbial biomass from the
treated wastewater. Depending on the amount of treated water, the trickling filters
may be subdivided into flushing and percolating filters.
Microbes in trickling filter: Biofilm formation occurring at filter surface is
called zoogleal film which consists of algae, bacteria, protozoa and fungi. Biofilm
formation on the surface of filter media is similar to naturally occurring aquatic
environments. Common bacteria found in zoogleal film are Achromobacter,
Alcaligenes,Flavobacterium,filamentous bacteria, nitrifying bacteria
(Nitrosomonas and Nitrobacter), Pseudomonas and Zooglea. Bacteria adsorb
and anchor on the substratum using polymer-containing matrix, glycocalyx.
After acclimatizing to the substratum, the filter surface colonized by bacteria is
also further occupied by successional life forms (Rani et al. 2019). Common
fungi found in zoogleal film are Aspergillus,Geotrichum,Fusarium, yeasts,
Mucor and Penicillium. The growth of fungal hyphae supports the transfer of
oxygen at the depths of the biofilm. Several types of algae also flourish in biofilm
such as Anacystis,Chlorella,Euglena,Phormidium and Ulothrix. Algae produce
oxygen and some of them also fix nitrogen. The protozoa occurring in biofilms
are amoeba, arcella, ciliates (Colpidium,Vorticella) and flagellates (Bodo,
Monas). Microbial biofilm degrades the organic matter present in wastewater
and continues to grow (Arezoo et al. 2017). The increase in thickness of biofilm
acts as a limiting condition for diffusion of oxygen in the deeper layers, hence
creating an anaerobic environment. It also reduces supply of organic substrates
into the deeper layer. These biofilms are later sloughed off and further a new
biofilm forms.
Advantages and Disadvantages: Trickling filters are reliable, are easy to
operate and have little maintenance costs. Both domestic and toxic industrial
effluents can be treated. Its disadvantage is associated with the clogging of filter
under higher organic load. It results into excessive growth of slime bacteria that
cause restriction in air circulation, low oxygen availability and hence foul odour.
2. Rotating Biological Contactors (RBCs)
This is a fixed-film bioreactor in which the disk surface is used for adsorption
of microbes. A thin biofilm of 1–4 mm thickness is accountable to decrease BOD
level. RBC comprises a horizontal shaft with a series of microbial immobilized
disks straddling on it. Disks are around 40% submerged and rotate slowly in the
wastewater. The rotation enhances oxygen transfer required for microbial activ-
ity. It also improves contact between immobilized biomass and wastewater that
16 Application of Microbial Technology for Waste Removal 279
reduces BOD at a faster rate. There is successional growth of microorganism on
the rotating disk. Biofilms on RBCs encompass diverse microbial community
such as filamentous and eubacteria, metazoan, protozoa and filamentous algae
such as Oscillatoria (Aguilera et al. 2007). RBCs mostly remove organic mate-
rials and also oxidized ammonia. Several advantages associated with RBCs are
low operation cost, short residence time, low maintenance costs and release of
dewatered sludge.
16.8.2 Activated Sludge
This is the most extensively used suspended-growth wastewater treatment process. It
utilizes microbial culture to degrade organic matter under aerobic condition. The
treatment consists of oxidation of organic matter to NH
4
,CO
2
and H
2
O with the
formation of cellular biomass. Activated sludge forms floc for separation of solid in
settling tank during the aeration of wastewater. Activated sludge process is
constructed with two major objectives:
1. By using aerobic oxidation process, soluble organic matter is converted into new
biomass of biodegradable organic matter
2. Activated sludge technique is applied for the separation of biomass from treated
effluent
Activated sludge system consists of aeration and sedimentation tank. Aerobic
oxidation of organic matter using microorganism is carried out in aeration tank.
Wastewater treated after preliminary method is pass in the tank and assorted with
sludge or return activated sludge (RAS) to form mixed suspended solids. Aeration is
provided by mechanical method. Air is utilized by microorganisms to develop a
biological floc. During return activated sludge, a huge amount of the microbial
biomass is recycled, maintaining an enormous number of intial microbes for effec-
tive oxidation of organic matters in a reasonably short time. The total content
activated sludge present in aeration tank is called mixed liquor, whereas the total
amount of microorganisms, mineral and organic suspended solids in the assorted
with liquor is called mixed liquor suspended solids (MLSS). MLSS possess both
organic and inorganic portions. The organic portion comprises live or dead micro-
organisms, lacking microbes in the organic matter, and cellular debris is denoted by
MLVSS.
The MLSS is shifted from the aeration tank to the sedimentation tank for the
separation of sludge from the treated effluent. Sedimentation tank is utilized for the
sedimentation of microbial sludge formed in aeration tank during oxidation of
organic substance. The amounts of regimented solids can be varied by waste
activated sludge (WAS) and returned activated sludge (RAS). A rational settled
activated sludge is returned back to the aeration tank for the treatment of incoming
raw wastewater (RAS). The remnants or excess sludge is removed to balance the
ratio of food for the microorganisms (F/M). The food to microorganism ratio is
280 R. R. Kumar et al.
significant to maintain the balance between organic loads to biomass generation in
activated sludge system conveyed in terms of kilogram BOD per kilogram of MLSS
per day. Mean cell resident time (MCRT) or solid retention time (SRT) is an
important component of activated sludge that measures the contact time of micro-
organism with substrate. Activated sludge process is controlled by several parame-
ters including food to microorganism ratio, sludge volume index, mean cell
residence time, sludge age, dissolved oxygen and biochemical oxidation demand
(Johnston et al. 2019).
The solid part of flocs absorbs impurities present in wastewater, while microor-
ganisms oxidize the absorbed substances. Activated sludge has a loose and porous
structure. These microbial cells occur as flocs or agglomerates whose density is
abundant for sedimentation in the sedimentation tank. Sedimentation is monitored
by using “secondary clarification”for the separation of microorganism and solids
from treated wastewater.
Microbes in Activated Sludge: The activated sludge flocs contain numerous
microorganisms but mostly bacteria, organic substance and inorganic particles.
The size of floc varies between <1 mm and >1000 mm. Microelectrodes are used
to measure microbial activity and estimate the concentration of oxygen, ammonia,
nitrate, redox potential, pH or sulphide profiles within flocs. The major bacterial
genera present in flocs are Achromobacter,Acinetobacter,Alcaligenes,Bacillus,
Brevibacterium,Corynebacterium,Comamonas,Flavobacterium,Pseudomonas
and Zooglea. Fungal growth is usually not favoured; however, some genera such
as Alternaria,Cladosporium,Cephalosporium,Geotrichum and Penicillium are
present in activated sludge (Yang et al. 2020). Several conditions such as oxygen
deficiency and overloading of aeration tanks do not favour proper floc formation;
this phase is called as active-sludge swelling. There are two discrete types of sludge
swelling: fibrous and non-fibrous swelling. The growth of Sphaerotilus,Beggiatoa
or Thiothrix is observed during fibrous swelling and the amount of mucous secretion
is increased during non-fibrous swelling.
16.8.3 Biosorption Technology
Microorganism-based sorbent materials have been recently implicated for the
retrieval of heavy metals from wastewater. Biosorption is a specific property of
some microbes that concentrate the organic or inorganic substances from liquid
solutions. Though biosorption techniques are principally applied to sequester heavy
metals, metalloids, radionuclides and rare earth elements, recently it has been
implicated for the removal of organic dyes (Kaushik and Malik 2009). Microbial
EPS (extracellular polymeric substance) and cell wall play an important role in
absorption and act as an alternative of synthetic adsorptive substances.
EPS is a high molecular weight natural polymer secreted by several microorgan-
isms, and their chemical composition also varies with microbial genera. EPS is
utilized by microbe primarily to protect themselves from metal toxicity. Diverse
16 Application of Microbial Technology for Waste Removal 281
structure, chemical stability, metal binding property, selectivity and high reactivity
of microbial EPS advocate them as great competitor. Factors for biosorption include
the type of metal and its ionic form, metal binding site and external environmental of
microbes. Biomass of algae, bacteria, cyanobacteria, filamentous fungi and yeast
was commonly used for biosorption.
These microbes could be isolated from their natural habitat or obtained from
waste by-product of fermentation industries to make the process economic. The
surface area of bacteria and yeast has been increased for enhanced absorption using
genetic engineering methods. Water inhabitants as algae and cyanobacteria were
reported to absorb a variety of toxic metals in natural water and wastewater. They
show various degrees of binding and specificity with Ni, Pb, Cd, Zn, Co, Cu, As,
Mn, Mg and Zn. Anabaena doliolum Ind1, having surface group’s carboxyl,
hydroxyl, carbonyl, sulphate and amide groups, has great ability to bind with Cd
(II) (Goswami et al. 2015). Similarly, Providencia vermicola strain SJ2 and
Paenibacillus peoriae strain TS7 have shown specificity to bind with Pb
(II) (Arumugam et al. 2017). Streptomyces rimosus and Rhodococcus opacus also
show their ability to bind with Al (III) (Cayllahua and Torem 2010). Rhizobium
radiobacter strain VBCK1062 which is commonly found in contaminated soil is a
highly specific strain that binds with (V).
Metal ions were extraordinarily fixed by bacterial cell wall. Two mechanisms are
conveyed in metal binding capacity of microbial EPS and cell wall:
1. Ion exchange performed by high quantity of negatively charged functional group
in microbial EPS.
2. Complex formation with the charged group present in EPS or cell wall.
A thick layer of peptidoglycan found in gram-positive bacterial cell wall and
lipopolysaccharide in gram-negative bacteria plays an important role in binding with
metal ions (Flemming and Wingender 2001). Gram-positive bacteria and
actinobacteria are comparatively more capable of adsorbing metal on their cell
wall when exposed to selective metals. Copper was efficiently removed by
desulphurization bacteria from wastewater. White-rot fungi and yeasts were also
used to absorb toxic substances such as chromium, lead and other constituents of
wastewater in China. Microbial technology for adsorption was also integrated with
activated sludge treatment.
Advantage: Biosorption process offers several advantages such as low operating
cost, high efficiency for even low metal concentrations, minimization of chemical
uses, free from nutritional requirements, free from disposal of organic or inorganic
sludge and avoidance from metal toxicity issues.
16.8.4 Microbial Electrochemical Technology
Microbial electrochemical technology (MET) is an emerging technology that amal-
gamates microbiology with electrochemistry (Schröder et al. 2015). In MET method,
282 R. R. Kumar et al.
electroactive bacteria are capable of using a solid electrode as electron acceptor or
electron donor (Rabaey et al. 2006). This electrode acts as an alternative to tradi-
tionally used nitrate/oxygen as electron acceptor or hydrogen/organic matter as
electron donor (Karanasios et al. 2010). Depending on the quality of groundwater
or pollutant present in them, MET system can be operated as microbial electrolysis
cell (MEC) or as microbial fuel cell (MFC) (Schröder et al. 2015). MFC device
differs from MEC in the sense that in MFC, energy can be extracted, while in MEC
energy is supplied to allow or enhance bio-electrochemical process. MFC differs
substantially from the conformist fuel cells. For conversion of fuel cell microorgan-
isms act as biocatalysts for the cathodic and anodic substrate to catalyze the
electrochemical reactions. Direct electron transfer in MFC was initially demon-
strated in Saccharomyces cerevisiae, where it was grown in enriched medium, for
the separation the platinum cathode and anode electrodes; porous cylinder is utilized
in the field.
Microbial fuel cells emerged as a new bioremediation technology that is primarily
used to recover toxic metal or mobilize pollutants present in wastewater. Several
groups of bacteria, yeast, algae and fungi were found to remediate the heavy metal
ions (Pous et al. 2017). Microbial electrochemical technologies are effectively
applied for in situ or ex situ treatment of groundwater contamination. However, ex
situ MET is widely applicable. During ex situ treatment, wastewater or groundwater
has to be pumped to the other location, where intensive treatment is applied for
reckless removal of pollutants. Different MET patterns have been utilized for the
treatment of different groundwater pollutants such as aromatic hydrocarbons, chlo-
rinated hydrocarbons (Aulenta et al. 2007) and metals (Huang et al. 2013).
16.8.5 Wastewater Treatment Using Oleaginous
Microorganisms
Oleaginous microorganisms are well-known sources to produce microbial biofuels
of comparable fatty acid configuration present in higher plants and animal oils.
However, a major limitation of synthesis of biofuel from oleaginous microorganisms
is the high cost of raw material (Azócar et al. 2010). Therefore, nutrient-rich
wastewater was employed as cheaper substrates for oleaginous microorganisms. It
was not only feasible economically but also important for environment-friendly
biodiesel production (Huang et al. 2013). Different oleaginous microorganisms
have been studied for simultaneous biofuel production along with simultaneous
wastewater treatment. Certain groups of microbes such as bacteria, yeast microalgae
and fungi are acting as single cell oils (SCOs) or oleaginous microorganisms (Arous
et al. 2019).
Yeast: Oleaginous yeast Debaryomyces etchellsii is able to accumulate a substantial
quantity of lipids from agroindustrial wastewaters such as wastewater obtained
from olive mill, expired soft drinks and confectionary industries. Wastewater
16 Application of Microbial Technology for Waste Removal 283
obtained from milk candy was effectively used by Rhodosporidium toruloides to
produce sufficient quantity of reserve lipids (Zhou et al. 2013). Strain
Rhodotorula glutinis TISTR 5159 was found to convert corn starch wastewater
into lipids under semicontinuous fermentation conditions with higher efficiency
of 65% reduction of COD value. Oleaginous yeasts belonging to genus
Trichosporon also showed their high ability to remove pollutants, removing
more than 55% COD from industrial wastewaters. Wastewater obtained from
bioethanol industry was efficiently utilized by oleaginous yeast Rhodosporidium
toruloides Y2 to decrease 72% BOD level. Other group of oleaginous yeast such
as Cryptococcus sp. (Fernandes et al. 2014), Lipomyces starkeyi,
Rhodosporidium toruloides and Yarrowia lipolytica (Louhasakul et al. 2016)
was found to be highly efficient for utilization of wastewater.
Algae: Oleaginous microalgae show significant advantages over yeasts such as
natural habitat to grow in wastewater and the requirement of low nutrition
owing to their autotrophic characteristics (Cai et al. 2013). Some oleaginous
microalgae such as Neochloris,Arthrospira,Botryococcus,Chlorella and
Scenedesmus are able to remove pollutants from wastewater along with the
production of microbial lipids (Perez-Garcia et al. 2011).
Fungi: An oleaginous fungus Aspergillus oryzae is able to produce biodiesel using
starch-rich industrial wastewater (Muniraj et al. 2013).
Bacteria: An oleaginous bacterial strain of Alcanivorax was able to solve environ-
mental pollution by removal of spilled petroleum. Their population significantly
increased in oil spilled water by utilizing them as nutrients. Researchers also
observed that soil-borne bacteria Geobacter are able to electroplate uranium,
rendering it in insoluble form; therefore, it cannot dissolve and contaminate
groundwater. There will be a wide application of Geobacter at the uranium
contamination sites such as nuclear plants and mines in order to limit the
catastrophic spillages.
16.8.6 Removal of Synthetic Dye Using Microbial Technology
Synthetic dyes with various structural diversities are frequently used in textile, paper,
leather tanning, agricultural research and photochemical research. They cause exten-
sive environmental pollution, deteriorate the quality of wastewater, increase toxicity
and are also hazardous for health. Several wastewater treatment methods have been
adopted to handle artificial dyes. The solicitation of microbial technology for
degradation of synthetic dye is highly effective, economic and environment friendly.
Different wastewater treatment methods using microbes have been adopted for the
removal of synthetic dye. Rhodamine B and methyl violet have been removed during
activated sludge process using cattle dung derived from microorganisms (Kanekar
and Sarnaik 1991). Certain microbial culture was used for degradation of Acid
Orange 7 dye in biofilm. For the assimilation of immobilization and decomposition
of azo dye, multistage rotating biological reactor is used (Ogawa and Yatome 1990).
284 R. R. Kumar et al.
Removal of synthetic dye tartrazine and azo dye from wastewater was done by
some bacterial species under anaerobic conditions. Both individual strains as well as
mixed cultures were used under aerobic and anaerobic conditions for removal of
synthetic dye. The combined aerobic and anaerobic sequential method has been
effectively engaging on the disintegration of anthraquinone and monochlotriazine
dyes (Panswad and Luangdilok 2000). More than 100 fungal and some bacterial
laccase producers such as Bacillus subtilis,Coprinus cinereus,Melanocarpus
albomyces,Pycnoporus cinnabarinus,Thielavia arenaria and so on have been
reported that efficiently decolourize dye from wastewater (Mishra et al. 2019).
Some dye decolourizer and degrader group of microorganisms have been mentioned
below.
Fungi: White-rot fungi have attracted researchers in recent years due to their potent
biodegradation capacity of highly stable natural molecule such as cellulose, lignin
and hemicellulose. They are able to synthesize the wide range of extracellular
enzymes such as laccase, phenol oxidase, lignin peroxidase, manganese-
dependent peroxidase and manganese-independent peroxidase. They have been
extensively investigated for decolourization of wastewater (Young and Yu 1997).
A white-rot fungus, Phanerochaete chrysosporium, has been employed for the
decolouration of Azure B, Congo red, Orange II and Tropaeolin O dye under
aerobic conditions. This fungus is able to remove these dyes from wastewater
between 6 and 9 days (Cripps et al. 1990). Trametes versicolor were able to
decompose azo, anthraquinone and indigo-based dyes. Highly sensitive azo dyes
have been efficiently removed from the wastewater by Aspergillus foetidus.
Another dye Remazol Brilliant Blue R has been successfully decolourized by
fungus Pycnoporus cinnabarinus using packed-bed bioreactor (Schliephake and
Lonergan 1996). Anthraquinone dyes and triarylmethane and indigoid dye pre-
sent in environment were efficiently degraded by Trametes hirsute (Abadulla
et al. 2000). One of the efficient fungi Kurthia sp. is capable to decolourize the
synthetic dyes such as brilliant green, pararosaniline, crystal violet, magenta and
malachite green (Sani and Banerjee 1999).
Bacteria: Bacillus subtilis is able to degrade p-aminoazobenzene to produce p-
phenylenediamine and aniline compound under anoxic condition (Zissi and
Lyberatos 1996). Under fixed-film reactor conditions, Pseudomonas mendocina
are used for the decolourization of methyl violet in the textile industrial effluent
wastewater. Pseudomonas luteola and Klebsiella pneumoniae bacteria were used
to decolourize reactive azo dyes from wastewater.
Algae: Reactive azo dyes have been also degraded by algae such as Chlorella
vulgaris,Chlorella pyrenoidosa and Oscillatoria tenuis. An enzyme “azo reduc-
tase”found in some algae is responsible for the breakdown of azo linkage, hence
converting azo dyes into aromatic amines.
16 Application of Microbial Technology for Waste Removal 285
16.9 Conclusions and Future Prospects
Hazardous waste is a major threat for environmental safety that needs to be eradi-
cated in various ways. Microbial technology is the amalgamation of microbial
process with technology in which selective microbes are used by scientific methods.
Microbial technology has been extensively used for the removal of waste from
terrestrial area and wastewater. Removal of waste using microbes is not only cost-
effective, efficient but also environment friendly. A major section of the waste is
organic matter which can be properly handled by suitable microorganisms using the
phenomena of reduction, reutilization and recycling of waste. Besides the organic
matter, several microorganisms have been explored that are able to remove oil,
synthetic dyes, toxic heavy metals and xenobiotic compounds. Modern techniques
such as biosorption and microbial electrochemical technologies were implemented
for toxic metal recovery. Exponential increase of hazardous waste still creates
pressure to further explore newer microbial technology or extend the existing
techniques with underexplored microbes to solve the global problem. Therefore,
genetically engineered microbes and high-throughput screening of specific micro-
organism have been promoted for microbial technology as more convenient and
effective for waste removal. Careful monitoring of multiple strain-based inoculums
could also reduce waste in rapid rate. Generation of energy using microbes during
waste management is an economic way that needs to be implemented in the future.
The potentials of hidden domains of microbes have great prospect that can be further
explored for the treatment of waste.
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16 Application of Microbial Technology for Waste Removal 291
Chapter 17
Metagenomics: Insights into Microbial
Removal of the Contaminants
Dipti Singh, Shruti Bhasin, Anshi Mehra, Manali Singh, Neha Suyal,
Nasib Singh, Ravindra Soni, and Deep Chandra Suyal
Abstract Metagenomics has changed the microbial world completely. It has pro-
vided new insights to analyze microbial genes and metabolites. In metagenomics, the
influence of genomics is applied to the entire communities, by avoiding the require-
ment of their isolation and selection. It requires several interconnected approaches
and methods to get the maximum information. It offers an outstanding way to
characterize the microbes, their genes, proteins, and metabolic pathways, which
can be explored in the bioremediation of various contaminants. Recently, this
technique is being explored to identify the unique microbial groups in an ecosystem
which are later utilized in the development of microbial consortia for biodegradation.
With the emergence of new sequencing techniques, the field has completely revo-
lutionized. Moreover, new bioinformatic and statistical tools will always be in
demand to analyze the huge metagenomic data and transformed it into meaningful
results.
D. Singh
Department of Biochemistry, Govind Ballabh Pant University of Agriculture and Technology,
Pantnagar, India
S. Bhasin
Department of Biotechnology, Banasthali Vidyapith, Tonk, Rajasthan, India
A. Mehra · M. Singh
Department of Biotechnology, Invertis Institute of Engineering and Technology (IIET), Invertis
University, Bareilly, India
N. Suyal
Government Nursing College, Haldwani Nainital, Uttarakhand, India
N. Singh · D. C. Suyal (*)
Department of Microbiology, Akal College of Basic Sciences, Eternal University, Sirmaur,
Himachal Pradesh, India
R. Soni
Department of Agricultural Microbiology, College of Agriculture, Indira Gandhi Krishi Vishwa
Vidyalaya, Raipur, Chhattisgarh, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_17
293
Keywords Bioremediation · Shotgun metagenomics · DNA sequencing ·
Bioinformatics · Gene prediction
17.1 Introduction
The term metagenome, introduced by Handelsman et al. (1998), describes the
genome that is directly extracted from an environmental sample. It corresponds to
all the cells present in that particular sample. Its isolation and characterization
constitute the field of metagenomics that is being explored for energy production,
human health, and food security (Joshi et al. 2017; Goel et al. 2018). Traditional
microbiology relies upon the cultivation of the microbes, while metagenomics
requires genomic sequences in order to characterize molecular taxonomy. It is a
well-known fact that the majority of the microbial population is still uncultured;
therefore, traditional microbiology fails to explore them properly (Goel et al. 2017;
Yadav et al. 2019). The alternative technology is microbial community analysis,
called metagenomics (Suyal et al. 2014; Soni et al. 2016,2017). It involves the direct
extraction of the DNA from environmental samples, their cloning, and screening for
diversity or the functions. Nowadays, the function-driven approach becomes popular
for searching of newer metabolites. The combination of the metagenomic data with
diversity analysis, meta-transcriptomics, meta-proteomics, and environmental
parameters has opened a new way for integrated ecosystem studies.
Metagenomics has overcome the issues related to the genomic diversity and
uncultivability of several microbial groups, the major obstructions to proceed in
environmental and clinical microbiology. The combination of metagenomics and
environmental parameters has also been used to screen stress-tolerant microorgan-
isms. Moreover, metagenomics has also shown its potential for bioremediation,
antibiotic discovery, biodegradation, etc.
17.2 Structural and Functional Metagenomics
Metagenomics depend on the characteristics of the native microbial communities
and their adjacent environmental conditions. It has two ideas: structural and func-
tional metagenomics. Structural metagenomics deals with the diversity analysis of
the microbial communities by using phylogenetic markers. It focuses on the micro-
bial community structure and unculturable microbial groups. While functional
metagenomics analyzes the gene functions, enzymes, and proteins of the
microbiome, it targets the role of microbial communities in the ecology and biogeo-
chemical cycles. It employs nucleic acid and protein databases for interpreting the
functional attributes. It is considered low throughput and time-consuming. Besides
these, metagenomics employs two different strategies: shotgun metagenomics and
targeted metagenomics. Shotgun approach analyzes the complete metagenome with
294 D. Singh et al.
the help of restriction digestion and/or sequencing methods. Contrary to it, targeted
metagenomics involves the analysis of a specific gene(s) and/or enzyme(s) in the
metagenome with the help of PCR techniques (Suyal et al. 2015a,b). Both strategies
have their own limitations—targeted metagenomics is dependent on the PCR
primers, whereas shotgun metagenomics is dependent on the strength of sequencing
technologies (Yadav et al. 2019).
Metagenomics starts with DNA extraction from the environmental sample and
represents the blueprint of the whole community. The contaminants must be
removed from the DNA as they can hinder the sequencing and other analytical
applications. The main challenge of the metagenomics is to extract maximum
information from the huge DNA libraries. New bioinformatics and biotechnological
technologies will make metagenomics easier.
17.3 Steps of Metagenomics
The major steps of metagenomics are:
•Metagenome extraction directly from the environmental sample.
•PCR amplification of the marker genes (marker-based metagenomics) or restric-
tion digestion of the metagenome (shotgun metagenomics).
•Cloning of the amplicons or DNA fragments in the molecular vectors.
•Transformation of the recombinant vectors into a suitable host strain.
•Preparation of the metagenomic library.
•Sequencing of the clones and diversity analysis (structural metagenomics).
•Screening of the clones for important metabolites, enzymes, proteins, antibiotics,
etc. (functional metagenomics).
The combined use of meta-transcriptomics, meta-proteomics, metabolomics, and
other meta-approaches in association with metagenomics could be promising to
identify novel genes and metabolic pathways for bioremediation of the
contaminants.
17.3.1 Designing of the Metagenomic Experiments
To get accurate and high-quality reliable data, the researchers need to focus on the
proper experimental design (Fig. 17.1). It must employ updated, cost-effective,
replicative, and accurate technologies. More specifically, during experiment design-
ing, one must consider the replicates of samples, budget for sequencing, paramount
protocols for DNA extraction, and updated sequencing platforms (Cooke et al.
2017).
17 Metagenomics: Insights into Microbial Removal of the Contaminants 295
17.3.2 Sampling
Sampling is an important step in the metagenomics. Any environmental sample can
be used as the sample to isolate the DNA, viz., plant tissues, animal tissues, water,
soils, and air. The contaminated samples need to be sampled properly. The quality of
the sample defines the DNA quality and thus is a crucial step. Moreover, sampling
should be done in such a way that it represents whole population of the ecosystem
and must be reproducible. The details should be mentioned on the samples properly,
viz., date, time, season, conditions, etc. The statistical methods and experimental
designs should be defined before starting the experiments.
17.3.3 Sample Fractionation
It is a procedure to treat the cells for lysis and metagenomic DNA extraction. It is a
preparatory step and depends upon the type of the cell wall and cell membranes. This
process can be done by using physical methods, chemical methods, and enzymatic
methods. The suitable nuclease degradation reagents should be used in the fraction-
ation buffer to avoid DNA lysis.
Fig. 17.1 The major steps in metagenomic studies
296 D. Singh et al.
17.3.4 DNA Extraction
It is the most crucial step in metagenomics. For getting the good quality and yield of
DNA, it is important to select proper DNA extraction protocols. The DNA may
present in the free form or in the bound form (within the cells/tissues). Therefore,
selection of extraction protocols is crucial to get the maximum DNA. Two strategies
can be used for DNA extraction: direct and indirect. In direct strategy, cell lysis and
DNA extraction occur in situ (within a sample), while during indirect strategy,
separation of sample is done from non-cellular material before lysis, and thus, it is
ex situ. By the indirect method, the yield of DNA product comes very low,
approximately 100 times; however, it covers higher microbial diversity.
17.3.5 Preparation of the Inserts and Gene Cloning
The extracted DNA can be PCR amplified (PCR cloning) or restricted digested
(shotgun cloning) as per the requirements and objectives. The DNA inserts thus
formed are ligated in the suitable molecular vector. These recombinant vectors are
transformed in the host cells especially in the bioengineered E. coli cells. In that way,
one can prepare a gene library that can be screened or sequenced further.
17.3.6 DNA Sequencing
Two different approaches can be employed for sequencing DNA molecules, which
are shotgun sequencing (restriction digestion-based sequencing) and amplicon
sequencing (PCR-based sequencing). Amplicon sequencing utilizes PCR techniques
and is frequently used for the microbial identification and characterization. It
employs phylogenetic markers, viz., 16SrRNA gene, for microbial diversity and
community structure analysis (Kumar et al. 2019). On the other hand, shotgun
sequencing explores the ability of restriction enzymes to cut the DNA into smaller
fragments. These fragments are then sequenced individually and later assembled
again to get the whole sequence. Advancement in the next-generation sequencing
technologies has tried to answer the above-raised questions, therefore identifying the
microbial world with new dimensions.
17.3.7 Quality Control
It typically involves recognition and filters out those sequencing which are contam-
inating reads and low in quality. In metagenomic data, sequences of high eukaryotic
17 Metagenomics: Insights into Microbial Removal of the Contaminants 297
species are typically measured as contaminations, which should be identified and
filtered out before further analysis to avoid incorrect results and interferences. For
quality control analysis, various tools are available, viz., FastX-Toolkit, PRINSEQ,
NGS QC Toolkit, FastQC, etc.
17.3.8 Marker Gene Analysis
Marker gene analysis is the best way for the characterization of the taxonomic
diversity of the microbial communities. This will help in making out the richness
and profusion about microbe’s population in a particular community (Suyal et al.
2019a,b). Taxonomic diversity can be analyzed by examining the marker genes,
grouping sequences into a distinct taxonomic class called binning, and assembling
sequences into separate genomes. It helps in the comparison of gene families,
phylogenetic resemblance, and taxonomic annotation of the homologs. The ribo-
somal genes that are present in a single copy in the microbial genomes are preferred
as a marker gene. Two methods can be used to annotate metagenome with the help of
the marker genes: MetaPhyler (Liu et al. 2011) and AMPHORA (Wu and Scott
2012). MetaPhyler is based on sequence similarity between the sequences and the
marker genes. The alignment and comparative analysis between the metagenomic
sequences and available databases provides fruitful information. Moreover,
sequence attributes are analyzed to get an idea about the taxonomy. Further,
AMPHORA is based on phylogenetic information, which takes much time to
calculate, but it provides higher accuracy. Further, it employs hidden Markov
models (HMMs) for homolog recognition and taxonomy.
17.3.9 Assembly
In this process, small DNA fragments that are generated from the sequencer are
reconstructed. It is used to obtain full-length protein-coding sequences (CDS) or full
genomes of the uncultivable microorganisms. It provides longer genomic contigs.
Assembly can be done by using two approaches: de novo assembly or comparative
assembly. In the first approach the reads data are directly used to reconstruct the
genome, while in the latter case, the reference genomes are needed. Sometimes,
variations may be observed in the metagenome due to polymorphism, insertion,
and/or deletion. It results in the fragmented assembly and error-full results.
298 D. Singh et al.
17.3.10 Gene Prediction
Gene prediction is an important step in metagenomics. It utilizes the following three
approaches.
(a) De Novo Gene Prediction
It employs the different properties of the genes, viz., GC content, length,
codon usage, etc. to predict the genes. It can recognize the diverged genes in the
metagenome.
(b) Protein Family Classification
This method identifies the protein-making ability of the gene. The sequences
are converted into protein-coding frames and compared with the existing data-
base. From this method, novel proteins can be identified by comparing with the
reference database. Moreover, it helps in identifying the protein homologs which
are diverged during the evolution.
(c) Gene Fragment Recruitment
This gene prediction method is based on a reading of algorithm mapping that
quickly evaluates whether a genomic fragment is almost similar to a sequence of
database. But this can be concluded after analysis of different homologs of a
known gene. Various tools are available nowadays that help in gene prediction,
viz., MetaGene, GeneMark, MetaGeneAnnotator, Gliommer MG, etc.
17.3.11 Annotation
Annotation step represents the analysis of gene locations, functions, homologs,
metabolic pathways, and sequence-specific biological information. It involves the
prediction of structure and functions of the metagenome based on the available
information and databases. Several metagenome annotation tools are available
online, viz., RAST (Aziz et al. 2008) or IMG (Markowitz et al. 2009). This process
involves two sub-steps: structural annotation and functional annotation. Structural
annotation identifies the gene of interest and its coding sequences (CDS). It is
followed by functional annotation in which the functions of the respected genes
are predicted. WebMGA, IMG/M, MG-RAST, STAMP, CoMet, RAMMCAP, and
CAMERA are some online tools that can be explored for functional annotation.
17.3.12 Binning
In this process, metagenomic sequences from the population are arranged in the
groups and assigned to the particular individual. Such groups represent the tempo-
rary metagenome of the whole population. This step is important as it has potential to
17 Metagenomics: Insights into Microbial Removal of the Contaminants 299
identify the novel non-homologous genes and proteins. It may be achieved by using
FAMeS (Mavromatis et al. 2007).
17.4 Meta-science and Bioremediation
The metagenome sequencing is not enough to provide the answer “who is doing
what,”because a lot of genes have unknown functions. The meta-transcriptome tells
only about the expression of a gene in a particular sample at a certain instant and only
in certain circumstances by executing the total mRNA. Now it is advanced in
metagenomics to carry out meta-transcriptomic analysis. This meta-approach can
deliver the functional and expression profile of the microbial communities. It can use
two approaches, viz., first, de novo assembly of the transcripts and, second, using a
reference transcriptome. The first approach is restricted by the capability of software
programs to collect the transcripts, while the second approach is restricted by the
availability of the reference transcriptome. Similarly, meta-proteomics offers an
additional way to characterize the native microbiome along with metagenome. In
it, environmental proteins are extracted, analyzed, and compared for identifying the
functional attributes of the population. Metabolomics is another technique that
complements metagenomics. It involves the study of all the metabolites that are
identified and quantified which are released by the organism into the instantaneous
environment. Due to various environmental stresses, microbial cells released various
kinds of primary and secondary metabolites. The metabolomics strategy is based on
the analysis of the practical values of these low-molecular-weight metabolites.
Through the analysis of metabolome, it is directly told about the conditions of an
environment or of any changes in homeostasis. Any change in the signature metab-
olites represents the deviation in the metabolic pathways and can be linked to the
environmental factors. It involves metabolite detection, extraction, analysis, and
characterization. However, one of its main limitations is complexity.
17.5 Role of Metagenomics in Bioremediation
As urbanization has increased around the globe by human activities, the environment
gets polluted to a great extent from various sources. Further, industrialization has
also disturbed environmental balance and has resulted in severe health issues that are
enhanced by large-scale environmental pollutions. Bioremediation offers an
eco-friendly way to remediate environmental contaminants ex situ or in situ (Bhatt
et al. 2020a,b,c). Bioremediation is usually based on the identification of which
microbial communities can degrade pollutants efficiently. It can be used for func-
tional screening of the potential microorganisms.
Ex situ and in situ experiments were conducted to compare hydrocarbon-
degrading efficiency of Arctic soil metagenome with others (Bell et al. 2011).
300 D. Singh et al.
Further, metagenomic analyses showed that mixed culture is more efficient than
monocultures (Debbarma et al. 2017). Through this analysis, it could make recog-
nition of genes that are concerned in the degradation of specific pollutants possible.
Sequencing is also very selective with this method because the sequencing of rRNA
and housekeeping genes is restricted (Bell et al. 2015). In most cases, bioremediation
aims to identify novel genes, proteins, and metabolic pathways that could remove the
contaminants (Table 17.1). In many bioremediation analyses, various oxidizing,
reducing, and definite catabolic genes are the topics of concern. In that situation, it
is enviable to merely amplify and sequence these targeted genes. Various studies
revealed the role of metagenomics in bioremediation under a variety of environ-
mental conditions like soil and marine environments. Xenobiotics are human-made
chemical compounds that get accumulated in the environment due to their lower
degradation rate (Bhatt et al. 2021a,b). Microorganisms respond to those chemicals
through different mechanisms: (1) Due to the random mutation, toxic xenobiotic is
formed. (2) These mutations could increase the microbial capability to degrade a
xenobiotic. (3) Through horizontal gene transfer, it attains new genes encoding
catabolic enzymes. Some species of microbes are known to neutralize a huge amount
of xenobiotics, like halogenated, polyaromatic, and polyester compounds (Jeffries
et al. 2018). Iwai et al. (2010) have analyzed novel dioxygenase genes in the
contaminated soils. Similarly, Bell et al. (2011) have analyzed alkane
monooxygenases from the Arctic soil and revealed that monoammonium phosphate
affected the microbial population to a greater extent. Recently, Jaswal et al. (2019)
have identified novel Burkholderia and other proteobacterial phyla that were able to
degrade environmental contaminants from uraniferous soils. In another study,
Table 17.1 Exploration of metagenomics in the isolation of the biodegradation-related enzymes
Enzymes DNA source
Screening
method References
Carboxylesterase Seawater Function-
based
Zhang et al. (2017)
β-Glucosidase Soil Function-
based
Matsuzawa and Yaoi
(2017)
Lipases, EstATII
Esterase
Red Sea Atlantis II brine
pool
Function-
based
Yasmine et al. (2013)
Proteases
Serine protease
Tannery sludge Function-
based
Devi et al. (2016)
Dioxygenase Soil Function-
based
dos Santos et al. (2015)
Oxygenases Artificially polluted soil Function-
based
Nagayama et al. (2015)
Amylases
Amylopullulanase
Insect gut Function-
based
Lee et al. (2016)
Chitinase Soil Function-
based
Thimoteo et al. (2017)
Phytase Peat soil Sequence-
based
Tan et al. (2016)
17 Metagenomics: Insights into Microbial Removal of the Contaminants 301
Kachienga et al. (2018) have analyzed the microbial diversity in African petroleum-
contaminated water samples. These analyses have given enhanced reorganization in
different fields like climate change, agriculture, and bioremediation. In a character-
istic hydrocarbon-polluted site, a metagenomics molecular strategy has been
performed to describe the bacterial population. It has revealed the dominance of
proteobacteria and Clostridia on those sites (Costeira et al. 2019). From industrial
wastewater, various harmful chemicals has been observed like dibutyl phthalate,
benzeneacetamide, resorcinol, benzene-1,2,4-triol, and benzoic acid. Since these
chemicals are very harmful and caused genotoxicity, it is not safe to discharge
them openly into the river or aquatic systems (Yadav et al. 2019). Such harmful
compounds can be managed by using a metagenomics mechanism. The help of a
group of diverse microbes that act as active biomass contributed a chief role in this
mechanism (Jadeja et al. 2019). Additionally, the metagenomic approach has also
been used for isolation of the potential genes toward wastewater treatment. Cabral
et al. (2019) studied metagenomic methods to understand the degradation process
through the genes which are responsible for the biotransformation of antibiotic
resistance and metals in the oil mangrove microbiome.
In bioremediation-related studies, different samples and the various approaches
are essential to attain a rich metagenomic library. However, the collection of samples
and preparations for metagenome extraction differ on the type of environments, viz.,
water, soil, and air. Sampling from the contaminated water samples involves the
proper filtration and separation. The excessive use of agrochemicals, industrial
waste, and sewage contaminates the water and poses a serious threat to humankind.
These contaminants can be removed by using bioremediation techniques, especially
microorganisms. In this perspective, water metagenome plays a very important role
to identify the novel genes and/or enzymes. Hamner et al. (2019) have performed a
metagenomic study on the polluted water to analyze the microbial diversity and
search for novel genes.
The soil system perhaps has the maximum quantity of microbial diversity in the
environment. Soil microbial communities have been explored for agricultural and
environmental purposes especially in the degradation of xenobiotics. In recent years,
this approach has given wide knowledge of the contaminated soil metagenomes. For
examining the contaminated soils, samples should be collected from different layers
and transported immediately in the sterile bags and under cold conditions. It prevents
any damage to the samples. The soil DNA should be extracted by using appropriate
methods in good quality and quantity. The collected metagenome then further be
analyzed by using any metagenomic approach either to analyze the microbial
diversity or to identify the bioremediation-associated genes/enzymes. Metagenomics
has also been used to analyze the microbiome of contaminated air. Moreover, the
role of air microbial diversity on climatic variations has also been assessed (Schloss
et al. 2016).
302 D. Singh et al.
17.6 Clinical Waste Management and Metagenomics
Any type of waste material generated during the work of healthcare personnel at the
hospitals and laboratories comes under clinical waste. It may include both biode-
gradable and non-biodegradable materials, viz., culture plates, plasticwares, glass-
wares, gloves, needles, swabs, bandages, tissues, etc. These waste materials may be
infectious and hazardous. Therefore, it is very important to manage these wastes
properly to avoid any environment as well as health risks. In view of the limitations
and environmental concerns associated with traditional methods, bioremediation
offers an eco-friendly and effective way for clinical waste management (Wen et al.
2021). In this perspective, metagenomics has proven itself a crucial technique to
identify the potential microorganisms and their enzymes (Anitha and Jayraaj 2012).
Mwaikono et al. (2015) have analyzed the microbial diversity in the biomedical
waste to isolate the bacteria having biodegradation potential. Further, Marathe et al.
(2018) have revealed the novel antibiotic resistance genes in the biomedical-waste-
contaminated rivers by using functional metagenomics.
17.7 Future Perspective
Metagenomics has the immense possibility in the field of bioremediation. Integrated
use of metagenomics with pure-culture techniques will be more effective for the
screening of novel microbes, genes, enzymes, and proteins toward environmental
cleaning. Exploration of microorganisms for bioremediation requires the under-
standing of their diversity, community structure, and mutual interactions. Further,
prediction of degraded intermediates, metabolites, and their pathways is the major
concern in bioremediation. This can only be achieved with the help of metagenomic
technologies. Further, processing and analysis of the huge metagenomic data are
among the major challenges in the field. New and updated bioinformatic softwares,
tools, and databases are inevitable for the success of meta-technologies.
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306 D. Singh et al.
Chapter 18
Methods of Strain Improvement for Crop
Improvement
Jyoti Rawat and Veena Pande
Abstract Biofertilizers are suitable substitutes to chemical-based fertilizers and
pesticides, which cause serious environmental problems around the world. There-
fore, there is an important requirement to implement ecological regulation using
native microorganisms. These beneficial microorganisms are inexpensive, consis-
tent, and more effective than synthetic fertilizers in terms of plant protection against
pathogens. These beneficial microorganisms protect plants against pathogens and
enhance nutrient availability. Hence, to achieve this goal, better quality strains are
needed. Crop improvement relies on the modulation of genes and genomic regions
that underlie crucial characteristics, either directly or indirectly. Recombinant bio-
technology intends to benefit in reducing the use of synthetic fertilizers; for this
function genetically improved microbes could be used. By using recombinant DNA
technology, genes of microbes are improved via several genetic modifications
depending on the recognition and selection of the desirable characteristics or genes
of interest. The current investigation is focused on different strategies used to
improve beneficial strain for crop productivity.
Keywords Agriculture · Crop improvement · Gene information · Molecular
approaches
18.1 Introduction
Agriculture relies heavily on the use of chemical or synthetic fertilizers and insec-
ticides to achieve higher yields. Issues such as environmental pollution, health
threats, disruption of the natural cycle of ecological inputs, and the destruction of
biological ecosystems that otherwise support agricultural production are correlated
with this reliance. There is a growing use of biological resources to replace chemical
J. Rawat · V. Pande (*)
Department of Biotechnology, Sir J. C. Bose Technical Campus Bhimtal, Kumaun University,
Nainital, Uttarakhand, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_18
307
fertilizers and pesticides. Agricultural development and pest and disease manage-
ment must therefore be carried out with fewer harmful inputs at shorter periods. In
this sense, plant growth-promoting rhizobacteria (PGPR) are potential resources to
bring significant benefits to agriculture. Studies have shown that PGPR have great
potential to improve crop growth and yield. Cereals are the primary source of food
for human nutrition and constitute more than two-fifths of the world population’s
staple diet. Environmental and genetic factors influence crop productivity
(Radhakrishnan et al. 2017). The usage of beneficial microbes alone or as microbial
consortia to selected plants with multifunctional properties is a good method to
stimulate strength and crop productivity (Ahmad et al. 2018; Maron et al. 2018).
Investigation on isolation and characterization of advantageous microbes to plants
has been extensively cited, but some of them have been commercialized. It has been
demonstrated that many commercial bioinoculants were not effective in the agricul-
ture field but they were effective in laboratory or greenhouse experiments (Vassilev
et al. 2015; Arora and Mishra 2016; Sulbhi et al. 2021; Bhandari et al. 2021) due to
their low stability and quality of formulation (Bhatt et al. 2021a,b,c). Newly,
selective use of beneficial microbiome plants and their combinations to combat
biotic and abiotic stress is gaining traction and becoming a stimulating research
frontier (Malusáet al. 2016; Bashan et al. 2016; Baez-Rogelio et al. 2017;
Stamenkovic et al. 2018). Biofertilizers are formulated from nitrogen-fixing
rhizobacteria naturally present in the legume nodules or microbes that are responsi-
ble for plant growth promotion. However, these bio-formulations would not be
proficient enough for supplying nitrogen to non-leguminous plants. In that circum-
stances, the practice of genetic engineering is of particular significance, for devel-
oping efficient management systems is needed. Consequently, the non-leguminous
plants could be cultivated with symbiotic rhizobia root nodules without applying
external nitrogen fertilizers (Santi et al. 2013). Foreign genes used to transform
microorganisms could be integrated into the genome of the host. For that, the
regulatory region of the gene is altered at the promoter or the terminator sites to
augment the inserted gene function in the host. The addition of a particular gene that
can confer biological control capacity could improve the biological control capacity
of microbes that lack these genes (Dash et al. 2016). Various rhizobacteria possess
biological control activity that simultaneously produces chitinases. However, few
rhizobacteria such as P. putida and R. meliloti are root colonizer but devoid of
chitinase synthesis (Bagwan et al. 2010). Hence, the chitinase gene assimilation into
their genome made them competent in the defense of the plants against fungi (Huang
et al. 2001). Biofertilizers, when formulated using molecular tools, can enhance
cellular pathways for phytohormone production, such as cytokinin, auxin, etc. as
well as help in plant growth and development (Fuentes-Ramirez and Caballero-
Mellado 2005). Most breeding approaches for biotic and abiotic stress resistance are
based on the insertion of a single resistant gene into plants, and therefore crop
resistance only lasts for a short duration (Kottapalli et al. 2010; Bhatt et al.
2020a,b,2021d,e,f). Therefore, the development of multi-stress resistant genotypes
is now demonstrated by combining multiple genes from different sources in a
single plant (Bhatt et al. 2019a,2020c,d,e,f). The process of manipulating and
308 J. Rawat and V. Pande
improving microbial strains to enhance their metabolic capabilities is called strain
improvement.
Protoplast fusion is an important tool in the selection of strains to provide genetic
recombination and develop hybrid strains in filamentous fungi (Steiner et al. 2019).
It is used to produce interspecific or even intergeneric hybrids. It has become an
important tool for genetic manipulation, as it breaks down obstacles to the genetic
exchange performed by conventional mating systems. This technique has great
potential for genetic analysis and strain improvement. The stress tolerance
capacity in crops has been explained in many studies using the pyramid of various
resistance genes (Suresh and Malathi 2013; Muthurajan and Balasubramanian
2010). Abiotic stress also affects the growth and yield of the crop (Pancaldi and
Trindade 2020) and can even disturb the survival of plants (Rana et al. 2019; Verma
et al. 2008). Salinity is one of the major problems for crop productivity, and
maximum crops are subtle to salt during their lifespan and particularly at the seedling
period (Bai et al. 2018). Certain varieties of crops that are salt resistant express salt-
sensitive genes to tolerate excess salts, and the quantitative trait locus (QTL) linked
to these genes can be mapped by microsatellite markers for the selection of salt-
tolerant lines (Ruengphayak et al. 2015; Llorens et al. 2020). Numerous drought-
tolerant genes have been well discovered and modified in various crops to develop
drought resistance (Yu and Yang 2016; Waqas et al. 2020). In many plants, cold
resistance genes (OsRAN1 and QTL) have also been acknowledged which is further
used in developing cold tolerance in plant varieties using molecular marker enhance-
ment tools (Thitisaksakul et al. 2015; Tiwari et al. 2016; Bimpong et al. 2016). Plant
tissue cultures (PTC) also have an important part in modern biotechnology. They are
widely used in studies of plant development processes (Sandhu et al. 2019), genetic
function (Rai et al. 2018), micropropagation (Zhang et al. 2014), and generation of
transgenic plants with specific industrial and agronomic characteristics (Shinada
et al. 2014). In this chapter, various techniques involved in beneficial microbes/
strain improvement, for the production of biotic and abiotic stress resistivity in
different plant varieties, are described. Molecular biological applications for crop
improvement like genetic engineering (GE)/recombinant DNA technology (RDT) to
adopt better traits of agronomic importance are too elaborated (Almeida et al. 2016;
Firn et al. 1994; Bhatt et al. 2015a,b,2016a,b,2019b,c; Deng et al. 2010; Kumar
2011; Loyola-Vargas and Ochoa-Alejo 2018).
18.2 Crop Improvement by Genetic Engineering
For many decades, gene transfer among distinct species of plants has played a
fundamental role in crop improvement. By transforming genes, many useful traits,
such as insect, stress, and disease resistance, have been shifted to many varieties of
non-cultivated plant crops (Akhtar et al. 2014; Amin et al. 2014; Dar et al. 2014;
Tariq et al. 2014). Recombinant DNA methods and many other methods are used for
the transformation of genetic information. Genetic engineering is a technique that
18 Methods of Strain Improvement for Crop Improvement 309
has made possible the transfer of genes between different genera or species using
recombinant DNA. This method is an exceptional selection method of expanding the
genetic base as compared to conventional breeding. Additionally, because it avoids
the skidding problem associated with conventional farming, it is more efficient and
takes less time (Khan et al. 2015a). Until now, many genetically modified crops have
been developed and commercialized, resulting in higher production efficiency, a
greater focus on the market, and better conservation of the environment. These crops
include longer postharvest storage tomatoes, insect-resistant cotton and corn, virus-
resistant potatoes, herbicide-resistant soybeans, and canola, and many others
(Puspito et al. 2015). To improve crops through genetic engineering, an efficient
processing system is needed. Different approaches are used to transform different
cultures such as recombinant DNA technology, which is used to manipulate genes of
microbes via various genetic modifications (Tabashnik et al. 2011). Also, many
Pseudomonas spp. chelate Fe ions by producing siderophores, thus increasing Fe
uptake in plants. S. meliloti (RMBPC-2), a genetically modified strain, was made by
introducing the genes that drive the plant nitrogenase enzyme to the bacteria (Boccia
and Sarnacchiaro 2015). T. harzianum is a very effective colonizer that is widely
present in soil and also can parasitize pathogenic fungi. In fact, many extracellular
enzymes like chitinases, proteases, and glucanases are synthesized by Trichoderma
which are enhanced by adding chitinase genes. Many extracellular enzymes such as
glucanases, chitinases, and proteases synthesized by Trichoderma have been
enhanced by the addition of chitinase genes (Tabashnik et al. 2011; Boccia and
Sarnacchiaro 2015; Awais et al. 2010). Therefore, these genetically altered strains
could effectively act as biofertilizers and improve crop yield and quality.
18.2.1 Genetically Modified Microbes
GM microbes provide better access to nutrients for crops and therefore increase plant
development. The most important beneficial microbes that are used as biofertilizers
are nitrogen-fixing bacteria, such as Rhizobium and Azospirillum.Rhizobium and
Sinorhizobium are the symbiotic bacteria that form root nodules in legumes and fix
nitrogen. It has been reported that these bacteria can stay in soil alive for a long time
and in certain cases even without a defined host (Ngwako 2008). These microbes
have been widely used as bioinoculants to enhance the growth and yield of crops.
However, the improvement in yield is variable and the success of the inoculants
appears to depend on the competition with the native strains which are generally the
least effective (Qaim 2009). Mycorrhizal fungi signify the group of fungi that form a
symbiotic association with plants. An investigation has been carried out to identify if
transgenic Rhizobium strains enhance nodulation or interfere with a symbiotic
association in plants. It was noticed that the strain GM S. meliloti does not interfere
with the formation of mycorrhizae but improves nodulation. GM sweet clover
increased colonization units of arbuscular mycorrhiza and increased the nutrient
acquisition capability of mycorrhizal plants (Papagianni 2004; Van Loon 2007).
310 J. Rawat and V. Pande
Azospirillum is recognized for its capability of plant growth promotion by
augmenting nitrogen uptake, through phytohormone production (Gonzalez et al.
2015). Sinorhizobium meliloti has been genetically modified to promote nodulation
in alfalfa roots. This genetic modification includes modification of the expression of
nifA gene which is responsible for the management of all other nitrogen fixation (nif)
genes (Bakshi 2003). It is assumed that nifA regulates the gene expression other than
nif cluster that aids in nodule development (Beyer et al. 2002). In the rhizosphere
region of Pisum sativum,GMRhizobium leguminosarum strains, labeled with HgCb
resistance genes (mer genes) and lacZ genes, were inoculated. In order to observe its
impact on crop productivity, Alcaligenes faecalis, a non-nodule-forming bacterium,
has been genetically engineered and introduced into rice fields in China. By intro-
ducing a constitutively expressed nifA regulatory gene, A. faecalis was genetically
modified and nitrogen fixation got increased as compared to treated fields (Gray and
Smith 2004; Huang et al. 2021).
18.3 Intraspecific and Interspecific Gene Transfer
In the nineteenth century, plant breeding began with discoveries about how plant
traits are inherited. Plant breeding could be achieved by selecting plants with
interesting attributes and manipulation in cross-fertilization. An improved variety
with the desired characteristics is formed when a cultivated variety is backcrossed
with a wild variety (Goodman et al. 1987; Khan et al. 2015b). In the twentieth
century, plant breeders used interspecies hybridization to transfer genes from a
non-cultivated plant species to other convertible crop species. For example, Avena
sativa (oats) and Beta vulgaris (sugar beet) were processed and resulted in increased
yields of 25–30% and resistance to sugar beet nematodes, respectively (Sharma and
Gill 1983). In the 1940s, methods for transferring DNA directly from one organism
to another organism were developed as DNA was established as a chemical base of
genetic inheritance. Genes can be obtained from plant, animal, bacterial, and viral
sources and injected into crops. Tissue specificity, timing, and expression level of
genes are under control and they can be modified by gene modification into a fresh
host. These methods provide the basis of diversity and permit the regulation of
expression of genes (Qamar et al. 2015). In recent times, the expansion of molecular
methods has generated different options for the assortment and genetic improvement
of livestock (Godrat et al. 2005).
18 Methods of Strain Improvement for Crop Improvement 311
18.4 Genetic Modification Through Somatic Hybridization
18.4.1 Protoplast Fusion
Somatic hybridization is the best technique aimed at the production of interspecific
and intergeneric hybrids for plant breeding and crop improvement. In this technique
fusion of protoplasts from two different genomes followed by the selection of the
desired somatic hybrid cells is carried out for regeneration of hybrid plants (Evans
and Bravo 1988). Therefore, it is accepted as an effective approach to generate
hybrids by joining two different protoplasts from different plant species or varieties,
and hybrids produced via this method are called somatic hybrids. Protoplast fusion is
a commonly used method for introducing a group of biosynthetic genes or entire
chromosomes into a recipient cell for subsequent genetic manipulation or directed
evolutionary approaches. It facilitates the transmission of mitochondrial genomes
among taxonomically associated species (Vincelli 2016). This is one of the impor-
tant or widely studied approaches as a technique to improve fungal strains (Assefa
2018; Nagoshi et al. 2018). In physiology, genetic study and genetic manipulation
fungal protoplast are important tools that can be successfully carried by fusing
protoplasts into filamentous fungi that lack sexual reproductive ability (Kage et al.
2016; Sharifzadeh et al. 2018). It is admitted as one of the recombinant DNA
technologies that provide the tools to increase gene dosage and gene expression
from strong promoters, remove unwanted genes from the fungal genome, manipulate
the metabolic pathways, and develop fungal strains for the production of heterolo-
gous proteins. Several reports have confirmed the isolation and regeneration of
protoplasts in different fungi. Protoplast fusion is found to be good for improvement
of Trichoderma spp. and development of hybrid strains in other filamentous fungi
(Atique et al. 2018; Mwobobia et al. 2020). The isolation, fusion, and regeneration
of protoplasts were carried out in the genus Trichoderma primarily to improve its
cellulolytic activity (Federico et al. 2019; Pandeya et al. 2018) and chitinase
production (Bowman and Zilberman 2013). However, partial attempts have been
done to improve Trichoderma species and increase enzyme production (Pandeya
et al. 2018; Waddington et al. 2010). Ogawa and his team (Ogawa et al. 1989)
revealed an increased cellulase production in Trichoderma reesei through interspe-
cific protoplast fusion, while Prabavathy et al. reported an increase in chitinase and
biological control activity in Trichoderma harzianum through protoplast auto-
fusion; nevertheless, little research has been done on the application of chitinase in
the degradation of shellfish waste applying this method (Prabavathy et al. 2006).
18.4.2 Agrobacterium-Mediated Gene Transfer
Agrobacterium tumefaciens is a phytopathogenic bacterium capable of transferring
part of its genetic material to other plant species through a simple process called
312 J. Rawat and V. Pande
transformation. The genes are encoded in a region of the Ti plasmid called T-DNA.
This causes the growth of a tumor termed “crown gall”disease in plants (Gordon and
Christie 2015). This bacterium is altered in the laboratory and transfers the gene of
interest to plants without causing disease symptoms. The Agrobacterium system is
quite attractive due to the easy protocol that is associated with minimal cost in terms
of equipment and also the resulting transgenic plants have single-copy insertion
(Gordon and Christie 2015; Hansen and Wright 1999). With this method, genes for
resistance to insects and diseases were transferred. Using recombinant DNA tech-
nology, many plant and bacterial genes encoding enzymes have been engineered to
make crop plants tolerant of broad-spectrum herbicides and safer for the environ-
ment. Because this bacterial gene is designed in such a way that its enzyme is
insensitive to the herbicide and then transfers it to the plant, it can also be done by
having plants express genes that detoxify the herbicide. The genes obtained from
Bacillus thuringiensis have been modified and transferred to plants that act as
insecticides (Shahid et al. 2016).
18.4.3 Non-Agrobacterium-Based Gene Transfer
Four decades before it was identified, some members of the Rhizobiaceae family
also can transfer the gene to the host. Ensifer adhaerens,Ochrobactrum
haywardense, and Rhizobium etli are some of the Agrobacterium-related species
that have been used in gene transfer but have the disadvantage of a limited host range
(Mullins et al. 2006).
18.4.4 Viral-Mediated Gene Transfer
Viruses carry complex arrangements and life cycles; many are pathogenic but act as
very efficient vehicles in gene delivery (Patel and Misra 2011). RNA and DNA
viruses that infect plants can be used as a vector to transfer genes to the target. The
gene to be transferred is integrated into the viral genome, and at this instant, the virus
acts as a vector to transfer the gene. The virus with the transferred gene infects the
target cell and results in a successful transformation. The main disadvantage is the
high number of copies per cell, and virus-mediated gene transfer can only produce
transient transfer and not stable transformation—that means they cannot be trans-
ferred to the offspring. Some of the viral vectors used are a retrovirus, an adenovirus
(Chailertvanitkul and Pouton 2010), adeno-associated virus, herpes virus, smallpox
virus, human moss virus (HFV), and lentivirus (Patel and Misra 2011; Fiandaca and
Federoff 2014).
18 Methods of Strain Improvement for Crop Improvement 313
18.5 Mutagenesis and Crop Improvement
18.5.1 Site-Directed Mutagenesis
In a study, chemical mutagenesis was used to attain fungicide benomyl-resistant
strains of Trichoderma harzianum (Ahmad and Baker 1987). Remarkably, the
mutant strains were better colonizers of the rhizosphere than wild-type strains. The
mutation technique will undoubtedly contribute to the upgradation of biological
control agents. Genetic engineering proposes stimulating possibilities for the genetic
manipulation of fungi both to improve biological control strains and to understand
how biological control works. Transformation of filamentous fungi was first reported
in the laboratories of Tatum (Mishra and Tatum 1973) and Case (Case et al. 1979).
Since then, molecular techniques have become more accessible for use by possible
biological control fungi (Fincham 1989; Bhatt et al. 2019d; Sharma and Bhatt 2016;
Sharma et al. 2016; Bhatt and Nailwal 2018). There is no doubt that the expansion
and use of molecular practices will persist to advance rapidly (Khati et al. 2018a;
Gangola et al. 2018a; Bhatt 2018; Bhatt and Barh 2018; Bhatt et al. 2019e; Bhandari
and Bhatt 2020; Bhatt and Bhatt 2021).
18.6 Bioinformatics Tools in Crop Improvement
Bioinformatics resources, in addition to various web databases, provide extensive
information on genomic data that is widely needed for research purposes. Improving
crops using bioinformatics tools is more promising these days (Singh et al. 2021;
Zhang et al. 2020a,b; Mishra et al. 2020; Feng et al. 2020; Lin et al. 2020; Zhan et al.
2020; Ye et al. 2019; Huang et al. 2019,2020). Over time, technology has improved
to a surprising level, bioinformatics provides crucial information about crop geno-
mic data, and this technology explores the sequence of many genes. This could help
us to sequence the economically important crop and the more beneficial traits.
Whole-genome comparisons are accelerating the pace of competent research (Fan
et al. 2020; Pang et al. 2020; Gangola et al. 2018b; Gupta et al. 2018; Khati et al.
2017a,2018b; Kumar et al. 2017). Projects of genome sequencing of economically
important crops have been accomplished and are seen as the access to new research.
Database of specific data sets in a compiled form with enriched annotations helps to
study gene families with greater precision. Genomic comparisons of different crops
help pinpoint the conserved regions between crops, providing common adaptation
strategies for plants (Nagoshi et al. 2018). After completing the sequencing of the
cultures, the data generated was used to create modeled proteomic data that helped to
understand the content of certain gene families. Major events, such as gene dupli-
cation, as well as other abnormalities, are manipulated using bioinformatics tools
(Khati et al. 2017a,b,2018b; Kumar et al. 2017). Additionally, access to critical data
to improve crop traits is positively simplified at a great end by using advances in
314 J. Rawat and V. Pande
technology and data acquisition sites. Therefore, efficient use of genetic data sup-
ports sustainable crop improvement. Different techniques, such as high-throughput
sequencing, generate a stack of crop data. Omics research works on the prediction of
candidate genes and, therefore, on the predicted functions (Mochida and Shinozaki
2010; Lockhart and Winzeler 2000). Data on transcriptomics and metabolomics
have also elucidated the regulatory networks that are crucial against plant stressors.
As a result, several crops were protected from biotic and abiotic stressors and yield
was restored.
18.7 Plant Tissue Culture in Crop Improvement
Advancements in tissue culture methods have very important part in breeding
various crops. These in vitro tissue culture techniques offer cloning, screening,
micropropagation, micrografting, organogenesis, etc. to assist plant breeders in
several ways. In tissue culture practices, the phenomenon of totipotency capacity
of the plants (explants) is exploited to introduce variance in genetic organization of
plants (Brown and Thorpe 1995). Explants or plants are treated with appropriate
treatments such as thermotherapy to eradicate viruses and diseases and allowed to
divide to forms a colorless undifferentiated mass of cells (callus) (Jain 2001). The
epigenetic alterations induced during tissue culture processes are known as
somaclonal variations. Together with molecular and biotechnological interventions,
several techniques have been developed to transfer necessary genetic traits that are
commercially favored. Clonal multiplication ornamental crop industries operate
massively and thus greatly increase cultivars. Plant traits are thus evaluated against
different plants in plant breeding (Tazeb 2017). Several genetically modified plants
have been established during the last 20 years utilizing technological advancements
in genetic engineering (Bawa and Anilakumar 2013). These plants have been
developed such that often use either a transforming vector or other techniques that
require chemical and enzyme action coupled to favor transformation such as use of
liposomes, biolistic particle gun, microinjection, and electroporation techniques
(Bhalla 2006). Transformation vector such as Agrobacterium tumefaciens induces
tumors with its Ti plasmid and subsequently transfers T-DNA (transfer DNA) into
host plant parts (typically leaves). The DNA segment of interest was inserted into the
T-DNA (transfer DNA), eliminating the nonessential part (the portion of the plasmid
that is not required for the act of transfer) (Gheysen et al. 1998; Gelvin 2003).
Transformation success modifies the cells, followed by cell harvesting and finally
regenerated in vitro into complete plantlets. However, assembly of necessary and
advantageous crop traits for any crop enhancement program is certainly most critical
and is usually performed by genetic transformation or hybridization program. Single
genes are favored for transfer by most molecular and genetic methods. Hybridization
is preferred for the successful transfer of more genes in a single reaction time. Tissue
culture techniques facilitate the hybridization process when the embryo is aborted
and therefore does not favor plant establishment. Tissue culture embryo rescue has
18 Methods of Strain Improvement for Crop Improvement 315
been used successfully to overcome the problem of embryo abortion or the inability
of seeds to develop (Tazeb 2017).
There are so many important advantages of plant tissue culture over crops. A
wide variety of cultures have been recovered by IVF using pistil pollination and self-
pollination and cross-pollination of ovules. A wide series of plants have been
recovered by IVF using pistil pollination and self-pollination and cross-pollination
of ovules, such as tobacco, corn, clover, poppy, canola, cabbage, cotton, etc.
Another type used to give value to cultures is embryo culture and orchids, roses,
and bananas are formed by embryo culture. Several other varieties are also success-
fully formed, such as stress, drought, and heat-tolerant varieties. In vitro propagation
by meristem, cell organ and tissue culture, organogenesis, and somatic embryogen-
esis are presented. These techniques certainly may make breeding programs simpler
and overcome some important economic and agronomic factors that might have
never occurred with conventional plant breeding and improvement methods (Wang
et al. 2016; De Filippis 2013). The method of plant tissue culture plays a dominant
role in the second green revolution in which plant biotechnology is considered
desirable crops. The yield and quality of the crops are greatly increased through
the extensive use of this technology. Increasing nutrition and food safety are the
basic points to consider before implementing tissue culture techniques.
18.8 Immobilization of Microbes to Improve Soil Health
and Crop Yield
The use of beneficial microbes as bioinoculant increases their number in soil, which
in turn increases the availability of nutrients to the plants. Yet, complications in
technical handling are often observed with fungal cells when employed as
bioinoculants for practical purposes, since satisfactory results are observed during
in vitro conditions, but not typically realized in natural agricultural systems (Jain
et al. 2010). A number of factors attribute to poor survivability and colonizing ability
in rhizosphere, such as competition with native microbiota and abiotic stresses.
Encapsulation of the cells in biodegradable capsules can be useful to overcome
such hindrances.
18.8.1 Encapsulation of Bacterial Cells
Cell encapsulation facilitates sustainability and stability of biological functions;
hence, enhanced cellular activities are realized (Juarez-Jimenez et al. 2012). Besides
stability, encapsulation also aids in protecting the cells against all contrary ecological
factors and facilitates slow release of cells into the soil in a controlled method, thus
316 J. Rawat and V. Pande
improving the efficiency of microbial fertilizers or biofertilizers (Vassilev and
Vassileva 2003).
18.9 Conclusion
The world population is growing rapidly. Thus, in the next few years, it will be the
biggest challenge to feed a huge population. Global warming, restricted environ-
mental conditions, and biotic factors limit crop yields. The main challenge for
researchers working on different crops is to increase agricultural productivity to
counter the demand for foodstuff supply to a rapidly expanding global population.
Therefore, crop improvement is the main element of agricultural progress, and there
are still a lot of zones left to work on in the field of crop improvement. Applications
of RDT or genetic engineering to crop improvement are well suited to deciphering
the problem of world hunger and depriving sustainable intensification.
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324 J. Rawat and V. Pande
Chapter 19
Microbial Technologies in Pest and Disease
Management of Tea (Camellia sinensis (L.)
O. Kuntze)
Ganga Devi Sinniah and Padmini Dharmalatha Senanayake
Abstract Tea, the natural beverage prepared from Camellia sinensis (L).
O. Kuntze, is the most consumed drink next to water. Commercially grown tea
plants are attacked by pest and diseases leading to considerable crop loss. Synthetic
pesticides have been the preferred method of pest and disease control in tea cultiva-
tion. The emerging consumer demand on pesticide-free tea, environmental and
human impacts felt due to continuous pesticide use, forced us to look for alternatives.
Microbial biopesticides have been identified as one of the alternatives in the inte-
grated pest management strategies. This chapter discusses about the microbe-based
techniques used in pest and disease management of tea and highlights the opportu-
nities for further development.
Keywords Biopesticides · Entomopathogens · Microbial metabolites ·
Nanoparticles
19.1 Introduction to Tea Pest and Diseases
Tea (Camellia sinensis (L.) O. Kuntze) is a non-alcoholic natural beverage prepared
from infusion of tea leaves. Due to its economic value, tea is cultivated as a
commercial plantation crop mainly in tropical and subtropical areas of Asia, Africa
and America. The world tea production in 2018 accounts to six million tonnes to
which China, India, Kenya, Sri Lanka, Vietnam, Argentina and Indonesia alone
contributed to about 90% in production (Anonymous 2019). Tea is grown as
monoculture in large contiguous area under specific climatic conditions of temper-
ature of 10–30C, minimum annual rainfall of 1250 mm, acidic soils of 4.5–5.5 pH,
G. D. Sinniah (*)
Plant Pathology Division, Tea Research Institute of Sri Lanka, Talawakelle, Sri Lanka
P. D. Senanayake
Entomology and Nematology Division, Tea Research Institute of Sri Lanka, Talawakelle, Sri
Lanka
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_19
325
0.5
–10slopes and elevations up to 2000 m above the sea level. Tea is a perennial
plant. Under commercial cultivation, it is subjected to continuous agronomic prac-
tices. Climatic and anthropogenic factors make tea plant host for several disease-
causing organisms and insect pests. It has been reported that over 350 fungal
diseases, 1 algal disease, 1034 species of arthropods and 82 species of nematodes
are associated with tea plants worldwide (Chen and Chen 1989).
19.2 Important Diseases of Tea
Fungi, bacteria, algae and viruses are known to be associated with diseases in tea.
Among them fungal pathogens are considered as the most important causes of tea
diseases. Blister blight (Exobasidium vexans Massee) (Fig. 19.1a), brown blight/
anthracnose (Colletotrichum camelliae and other Colletotrichum spp.) and grey
blight (Pestalotiopsis spp.) (Fig. 19.1b) are important fungal diseases which attack
foliage of a tea plant. Bacterial blight caused by Pseudomonas syringae and an algal
disease caused by Cephaleuros parasiticus Karsten and C. mycoidea Karsten are
also important in causing considerable crop loss in tea (Fig. 19.1c). Stems of a tea
plant are attacked by different types of cankers. Fungi that belong to family
Botryosphaeriaceae (Macrophoma theicola Petch, Lasiodiplodia theobromae
(Pat.) Griffon & Maubl.) (Fig. 19.1d), Poria hypobrunnea Petch, Phomopsis theae
Petch and members of Fusarium solani species complex are the important canker-
causing fungi in tea. Root rots caused by Poria hypolateritia Berk. ex Cooke (red
root rot, Fig. 19.1e, f), Rosellinia arcuata Petch (black root rot), Phellinus noxius
(Corner) G. Cunn.(brown root rot), Ustulina deusta (Hoffm.) Maire (charcoal root
rot) and Armillaria mellea (Vahl) P. Kumm.are the important diseases which affect
the root system of tea plants.
19.3 Important Pests of Tea
Several insect and mite pests cause damage to foliage, stem and root systems of tea
plants. These tea pests can be classified as perennial pests, seasonal pest and
occasional pests depending on their occurrence in tea (Fig. 19.2). Shot-hole borer
(SHB, Euwallacea fornicatus Eichhoff) and live wood termites (Glyptotermes
dilatatus Bugnion & Popoff, Postelectrotermes militaris Desneux, Neotermes greeni
Desneus) are adapted to tea-growing environments and considered as perennial
pests. The seasonal pests infest tea plants only during specific environmental con-
ditions favourable for them. Leaf-eating caterpillars such as Tea tortrix (Homona
coffearia Nietner), looper caterpillar (Buzura suppressaria Guenée), tea mosquito
bug (Helopeltis theivora Waterhouse), fringed nettle grubs (Macroplectra nararia
Moore), red slug (Eterusia aedea cingala Moore) and different mite species
(Oligonychus coffeae Nietner, Acaphylla theae Watt, Calacarus carinatus Green,
326 G. D. Sinniah and P. D. Senanayake
Fig. 19.1 Common diseases of tea. (a) Blister blight (Exobasidium vexans). (b) Grey blight
(Pestalotiopsis theae). (c) Red rust (Cephaleuros parasiticus). (d) Canker on main stem
(Macrophoma theicola). (e) Death of bush due to root infection. (f) Red root rot (Poria
hypolateritia)
19 Microbial Technologies in Pest and Disease Management of Tea (Camellia... 327
Brevipalpus californicus Banks, Hemitarsonemus latus Banks) are important sea-
sonal pests in tea. Occasional pests such as red borer (Zeuzera coffeae Nietner),
grubs (Holotrichia disparalis Arrow and Microtrichia costata Wlk.), etc. only cause
damage under specific environmental conditions or anthropogenic influences.
19.4 Microbes in Integrated Pest Management of Tea
Integrated pest management (IPM) in tea focuses on sustainable pest and disease
management by combining available management tactics such as cultural practices,
use of resistance plant materials, chemical application and biological control
methods. Though application of synthetic pesticides has been a promising method
of pest and disease management in tea, the detrimental effects of chemicals in soil,
Fig. 19.2 Common pests of tea. (a) Shot-hole borer (Euwallacea fornicates). (b) Tea tortrix
(Homona coffearia.(c) White grubs (Holotrichia disparilis). (d) Fringed nettle grubs
(Macroplectra nararia). (e) Live wood termites (Postelectrotermes militaris)
328 G. D. Sinniah and P. D. Senanayake
water, humans, microbes and all other living and non-living component in the
environment have been felt. Thus, reduction of chemicals is promoted in the tea
plantations and in agriculture sector as a whole. Biological control methods captured
the attention of scientists and growers due their eco-friendly nature, specificity,
biodegradability and negligible effect on host plants, environment and humans.
Broadly, biological control is managing a target pest or pathogen using its natural
antagonists or enemies by suppressing its growth, infection or reproduction. Micro-
bial inoculants, parasites and predators are generally used as biological control
agents. Several virulent bacterial, fungal and viral microbial inoculants (microbial
pesticides) are used in crop protection purposes. This chapter mainly focuses on
microbial inoculants and other technological application of microbes against dis-
eases and insect pests of tea.
In the past decades researches and commercialization of microbial biopesticides
have been given more importance globally. Cernava et al. (2019) showed that
phyllosphere bacterial diversity of tea has been altered with the use of chemical
application. They elucidated non-target effects by showing distinct microbial finger-
prints between plants that were treated with synthetic chemical pesticides and plants
treated with a Piriformospora indica spore solution. Increasing consumer awareness
on chemical residues and technical advancements in analytical methods for detecting
very low levels of chemical residues lead many countries to establish maximum
residue limit (MRL) set for pesticides as food safety standard. Tea being a major
export-oriented crop in major producing countries is challenged by strict regulations
on MRLs in the global export market. Therefore, several tea-producing countries
adopt microbial pesticides as one of the potential non-chemical alternatives to
minimize or replace the use of synthetic chemicals in a sustainable manner to ensure
food, environmental and health safety.
Microbial biopesticides employ different modes of actions to suppress target
organisms. They can directly antagonize organisms by hyperparasitism and preda-
tion. Hyperparasitic biocontrol agents directly kill the target organism or its propa-
gules (Köhl et al. 2019). Hypoviruses and obligate bacterial and fungal organisms
are the examples of hyperparasites. Several fungal and bacterial biocontrol agents
produce antibiotics, which are antimicrobial substances that can suppress or kill
other microorganisms and their action is dose dependent. Pseudomonas fluorescens,
Bacillus subtilis and Trichoderma spp. are known antibiotic-producing microbial
biocontrol agents (Lo 1998). Many microorganisms release lytic enzymes targeting
polymers such as chitin, proteins, cellulose, hemicellulose and DNA of the target
organisms (Pal and Gardener 2006). Microbial biocontrol agents also compete for
physical space and nutrients with target pathogenic organism, thereby minimizing
the damage caused to the host plants. Non-pathogenic microorganisms known to
trigger induced systemic resistance (ISR) in plants (Yang et al. 2008a). Induction of
host resistance mechanisms and plant growth promotion are reported when plants are
colonized by plant growth-promoting rhizobacteria (PGPR). Direct parasitism and
production of toxin and hydrolytic enzymes are mainly employed by
entomopathogens against insect hosts (Sandhu et al. 2012).
19 Microbial Technologies in Pest and Disease Management of Tea (Camellia... 329
19.5 Microbial Biopesticides Used in Disease Management
in Tea
19.5.1 Biopesticides of Fungal Origin
Trichoderma based products are the most common biopesticides used in the man-
agement of diseases of tea. Trichoderma harzianum,T. atroviride and T. asperellum
are the commonly used microbial biopesticides. Trichoderma has shown antagonis-
tic ability against leaf, stem and root pathogens which infect tea plants. Foliar
application of T. harzianum formulations has been used to control bird’s-eye spot
caused by Cercospora theae (Gnanamangai and Ponmurugan 2012). Trichoderma
viride,T. asperellum and T. harzianum have been used in India for managing
Fusarium dieback (Kumhar and Babu 2015; Kumhar et al. 2015,2020; Sarmah
et al. 2017). Trichoderma spp. have also been successfully tested against grey blight
caused by P. theae (Sarkar et al. 2015) and brown blight caused by Glomerella
cingulata (Kuberan et al. 2012).
Trichoderma atroviride (Anita and Ponmurugan 2011) and
T. harzianum +Gliocladium virens (T. virens) (Ponmurugan et al. 2002) are reported
to control Phomopsis canker in India. Both wood dressing with a paste of talc
formulation and soil application of T. harzianum +G. virens were effective in
protecting from collar canker (Ponmurugan et al. 2002). Hypoxylon serpens,a
wood rot-causing fungus in tea, was successfully controlled by T. viride (Nepolean
et al. 2014). Balasuriya and Pradeepa (2005) reported T. harzianum to suppress root
rot pathogen P. hypolateritia (red root rot) and horse hair blight (Marasmius
equicrinis). Recently T. asperellum isolated from tea soil in Sri Lanka showed
high efficacy against P. hypolateritia,R. arcuata (black root rot) and P. noxius
(brown root rot) (Fig. 19.3). Biocontrol of brown root and charcoal stump rot with
Trichoderma was effective in India (Hazarika et al. 2000).
Fig. 19.3 Biocontrol activity of Trichoderma asperellum on brown root rot pathogen Phellinus
noxius
330 G. D. Sinniah and P. D. Senanayake
In addition to Trichoderma-based biocide, Phong et al. (2014) reported that
Chaetomium cupreum strain significantly inhibited grey blight. Gliocladium virens
was found to be better than T. harzianum in managing Phomopsis canker
(Ponmurugan and Baby 2007). Baby et al. (2004) showed G. virens was effective
in controlling red root disease in new planting area and protected the young plants.
Aspergillus niger has been evaluated against black rot disease caused by Corticium
theae and algal red rust disease under field conditions (Premkumar et al. 2009).
19.5.2 Biopesticides of Bacterial Origin
In Northeast India, B. subtilis and two strains of actinomycetes (MM AC/02 and 05)
were found to give satisfactory control of blister blight disease. The disease protec-
tion achieved with B. subtilis was 40% (Sarmah et al. 2020), while actinomycetes
strains gave above 50% protection when compared to 84% protection achieved with
standard copper oxychloride (COC) treatment (Premkumar et al. 2009). Bacillus
subtilis have been used in India for the potential reduction (74–87%) of Fusarium
dieback (Kumhar et al. 2020; Kumhar and Babu 2019) and algal red rust under field
conditions (Sarmah et al. 2020). Bacillus subtilis has also been tested against
P. theae under in vitro conditions. Hong et al. (Hong et al. 2005) reported that
B. subtilis TL2 isolated from C. sinensis cv. Tie-guanyin in China showed 83.6%,
83.3%, 90.3% and 86.5% inhibitory effect against Phyllosticta gemmiphliae,
P. theae,Gloeosporium theae-sinensis and Neocapnodium theae, respectively.
The bacterium Micrococcus luteus showed antagonism to G. cingulate, a causal
agent of brown blight (Chakraborty et al. 1998). Ochrobactrum anthropi
(1.9 10
8
CFU/ml) was found to be effective against blister blight (Sowndhararajan
et al. 2013a,b) and brown root rot disease (Chakraborty et al. 2009). Rhizosphere
inhabitant Serratia marcescens strain ETR17 was effective in suppressing root rot
disease caused by Rhizoctonia solani in tea plants. Broad-spectrum antifungal
activity, production of lytic enzymes such as HCN, IAA, siderophore and antibiotics
production by S. marcescens inhibited growth of R. solani (Purkayastha et al. 2018).
Application of bioformulations of P. fluorescens and B. subtilis at the rate of 100 g/
planting pit is also equally effective to Trichoderma in controlling primary root
diseases of tea (Premkumar and Baby 2005).
Plant growth-promoting rhizobacterial strain-mediated induced systemic resis-
tance has been reported against brown root rot and charcoal stump rot (Mishra et al.
2014). Islam et al. (2018) reported Bacillus,Pseudomonas and Streptomyces strains
are capable of 10–14% reduction of black rot disease severity.
19 Microbial Technologies in Pest and Disease Management of Tea (Camellia... 331
19.6 Microbial Biopesticides in Insect Pest Management
in Tea
19.6.1 Entomopathogenic Viruses
Nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) (baculoviruses) are the
most commonly used viral biopesticides in tea. Nucleopolyhedroviruses mainly
occur on lepidopteran, dipteran and hymenopteran hosts, while GVs are more
specific to lepidopteran hosts.
Viruses isolated from natural infection of insect pests such as B. suppressaria
NPV (BusuNPV), Ectropis obliqua NPV (EcobNPV), Euproctis pesudoconspersa
NPV (EupeNPV), Andraca bipunctata GV (AnbiGV) and Adoxophyes orana GV
(AdorGV) have been used as large-scale biocontrol agents in tea in China (Roy and
Muraleedharan 2014). Among the above viruses, EcobNPV and EupeNPV formu-
lations have been accepted by the Ministry of Agriculture of China as commercial
insecticides against tea geometrid E. obliqua and tea caterpillar E. pseudoconspersa,
respectively (Ye et al. 2014; Sun 2015). EupeNPV has shown 80% mortality in
E. pseudoconspersa (Sun et al. 1988,1996). A biopesticide based on A. orana GV
(AdorGV) and H. magnanima GV (HomaGV) has been registered as the commercial
biopesticide, Hamaki-Tenteki, in Japan (Takahashi et al. 2015).
Application of BusuNPV has resulted in more than 90% mortality of the first and
second generations of B. suppressaria in China (Peng et al. 1998). Outbreak of NPV
disease in B. suppressaria has also been reported in India (Hazarika and Puzari
2001). In Japan, NPVs, GVs and entomopoxviruses (EPVs) have been tested against
Adoxophyes honmai and H. magnanima (Kodomari 1987; Ishii et al. 2002; Nakai
2009). An entomopoxvirus isolated from A. honmai (AHEV) has shown broad host
range including H. magnanima and A. insularis (Takatsuka et al. 2010).
Nucleopolyhedroviruses have also been isolated and characterized from
A. bipunctata (Hazarika et al. 1995), Arctornis submarginata (Mukhopadhyay and
De 2009), Hyposidra talaca NPV (HytaNPV) (Mukhopadhyay et al. 2011) and
H. infixaria NPV (HyinNPV) (Antony et al. 2011) in India. Natural populations of
entomopathogenic viruses are prevalent in H. coffearia in Sri Lanka. These reports
show widespread occurrence of viruses, and so far, 82 species of viruses have been
reported from insects associated with tea plant (Ye et al. 2014). Antony (2014) has
shown that H. infixaria virus can be passed on to the next generation, and thus it may
be useful for the long-term pest control.
19.6.2 Entomopathogenic Fungi
Entomopathogenic fungi commonly used in tea are Beauveria bassiana (Bals.)
Vuill., Metarhizium anisopliae (Metschn.) Sorokin, Verticillium lecanii
(Lecanicillium lecanii), Paecilomyces spp., Hirsutella thompsonii,Cladosporium
332 G. D. Sinniah and P. D. Senanayake
sp., A. niger,A. flavus,Cephalosporium sp., Aschersonia aleyrodis Webb., Aegerita
webber Fawcett, etc. (Table 19.1). Beauveria bassiana has been used to control a
few pests of tea successfully in the global tea industry (Table 19.1). Beauveria
Table 19.1 Entomopathogenic fungi reported against tea pests
Biocontrol
agents Target pests References
Aegertia
weberri
Black spiny whitefly,
Aleurocanthus spiniferus
Quaint.
Chen et al. (1997)
Aspergillus sp. Aphids Barua (1983)
Tea mosquito bug, H. theivora Bordoloi et al. (2012)
Beauveria
bassiana
Brown weevil,
M. aurolineatus
Wu and Sun (1994)
Tea mosquito bug Kumhar et al. (2020)
SHB E. fornicatus Selvasundaram and Muraleedharan (2000),
Senanayake and Kulathunga (2015)
Tea looper, B. suppressaria Ghatak and Reza (2007)
Tea termites Microtermes
obesi
Microcerotermes spp.
Singha et al. (2011), Roy et al. (2020)
Beauveria
brongniartii
H. picea Yaginuma et al. (2006)
Metarhizium
anisopliae
Red spider mite Oligonychus
coffeae
Kumhar et al. (2020)
Termites Singha et al. (2011), Roy et al. (2020)
Tea thrips Shanmugrapriyan and Mathew (2011),
Shanmugrapriyan et al. (2010)
Entomophthora
sp.
Scale insects Barua (1983)
Hirsutella
thompsonii
Red spider mite Muraleedharan (2001), Babu (2010)
Tea aphid Toxoptera aurantii Wahab (2004)
Scale insect Coccus viridis Hazarika and Puzari (2001), Barua (1983)
Paecilomyces
fumosoroseus
Red spider mite Muraleedharan (2001)
P. carneus Mole cricket Gryllotalpa
africana
Hazarika et al. (1994)
P. cinnamomeus WhiteflyAleurocanthus
camelliae
Saito et al. (2012)
P. tenuipes Flushworm Cydia leucostoma,
Laspeyresia leucostoma
Debnath (1986)
P. lilacinus Bunch caterpillar Andraca
bipunctata
Debnath (1998)
Red spider mite Debnath (1997)
Tea termite Odontotermes spp. Debnath (1997)
Verticillium
lecanii
Tea thrips Babu et al. (2008)
Red spider mite Muraleedharan (2001), Mamun et al. (2014)
19 Microbial Technologies in Pest and Disease Management of Tea (Camellia... 333
bassiana (strain 871) gave more than 95% mortality of the brown weevil
(Myllocerus aurolineatus) (Wu and Sun 1994) and 80% control of the weevil in
the field (Wu et al. 1995) in China. Ghatak and Reza (2007) observed that the
bioefficacy of B. bassiana against tea looper, B. suppressaria, was comparable to
that of the synthetic insecticides. Beauveria amorpha (Hiromori et al. 2004) and
B. brongniartii (Yaginuma et al. 2006) have been reported as alternatives to chem-
ical insecticides for Heptophylla picea larvae in a tea plantation of Japan. Effective
strains of B.brongniartii have also been isolated from H. disparilis (white grub)
from Sri Lanka (Fig. 19.4).
Metarhizium anisopliae was found to be effective in controlling red spider mite
(Kumhar et al. 2020), tea termite (Microtermes obesi) (Singha et al. 2011) and tea
thrips (Shanmugrapriyan and Mathew 2011; Shanmugrapriyan et al. 2010). Appli-
cation of M. anisopliae (2 10
7
cfu/g) at 10% in September to October during initial
infestation of termite for organic gardens has been recommended by Tocklai Tea
Research Institute. Commercial formulation of M. anisopliae var. anisopliae (BIO
1020) has been evaluated against white grubs and found to be effective in controlling
white grubs in tea in Sri Lanka (Vitarana et al. 1997).
Paecilomyces fumosoroseus from south Indian tea fields was found effective
against red spider mite, O. coffeae, and is now being marketed as a commercial
formulation (Muraleedharan 2001). The potentiality of utilizing V. lecanii has also
been tested against red spider mite (Muraleedharan 2001) and tea thrips (Babu et al.
2008). Natural infection of Entomophthora sp. and Verticillium sp. has been reported
in O. coffeae in Sri Lanka (Vitarana 2000).
Fig. 19.4 Beauveria
brongniartii infection on a
white grub larva
(Holotrichia disparilis)
334 G. D. Sinniah and P. D. Senanayake
19.6.3 Entomopathogenic Bacteria
Bacillus thuringiensis (Bt) is the mostly used bacterial biopesticide used solely or in
combination with other biopesticides and chemicals in tea. Bacillus thuringiensis has
been tested against B. suppressaria,D. baibarana,E. pseudoconspersa and
E. obliqua in China and H. magnanima,Adoxophyes sp., Agromyza theae and
Gracillaria theivora in India (Ye et al. 2014; Hazarika et al. 1995). Two kinds of
B. thuringiensis preparations, TOAROW-CTR (spore-dead B. thuringiensis) and
BACILEXR (living spore crystal mixture), are registered for use in tea plantations in
Japan (Kodomari 1987).
Bacillus subtilis formulation at 5% is recommended for tea mosquito bug in India.
The Enterobacter sp. has been identified as an effective biocontrol agent of leaf
rollers (De et al. 2008). Roobakkumar et al. (2011) showed efficacy of P. fluorescens
against O. coffeae and also reported that P. fluorescens produced bacterial chitinases
which had been reported to be effective in controlling mites. Serratia marcescens
was reported to be pathogenic to two species of scale insects, P. tubulorum and
C. ficus (Wang et al. 2010).
19.7 Bioconsortium and Compatibility of Biopesticides
with Chemicals
Different species of microbial biopesticides are used in combination (bioconsortium)
or as a cocktail mixture with synthetic pesticides and/or botanicals in order to
enhance bioefficacy and maintain their efficacy under different environmental con-
ditions. For example, a consortium of Pseudomonas,Trichoderma and VAM fungi
Glomus fasciculatum was used, and the level of disease control achieved against
U. zonata was higher than the application of individual biocontrol agents (Hazarika
and Phookan 2003). The highest reduction of Macrophoma canker size was reported
with an integrated treatment of Companion (carbendazim 12% + mancozeb 63%
WP) + B. amyloliquefaciens and COC + B. amyloliquefaciens than application of
Companion and COC alone (Jeyaraman and Robert 2018). Application of two
rounds of 5% of M. anisopliae +B. bassiana spore suspension (1:1) and
P. fumosoroseus 2 kg/ha + jaggery 2 kg/ha at 7-day interval showed promising
control of red spider mite in mild to moderately infested tea fields in North India
(Roy et al. 2018). Disease and pest control can be improved when biocontrol agents
are combined with synthetic chemicals at a dose which is not harmful to the
biological agents. These treatment combinations may have the potential to develop
new strategies for integrated pest management by lowering the chances of resistance
development and reduce the chemical dose compared to conventional treatment with
single pesticides (Ons et al. 2020).
19 Microbial Technologies in Pest and Disease Management of Tea (Camellia... 335
19.8 Microbe-Synthesized Nanoparticles in Tea Disease
Management
The use of nanotechnology in agriculture is getting attention of both researchers and
farmers following its successful application in other fields such as medicine, phar-
macology, automobile, etc. Nanoparticles are materials that range from 10 to 100 nm
in size and they can be produced with unique physical, chemical and biological
properties (Yang et al. 2008b). Silver, gold, copper, zinc oxide and titanium oxide
nanoparticles have shown to possess an excellent biocompatibility and low toxicity,
and they have antibacterial/antifungal/antiviral properties (Ali et al. 2020).
Nanoparticles themselves can act as plant protectant directly or they can be used
as carrier materials for existing or new pesticides (Worrall et al. 2018). The use of
microorganisms in the synthesis of nanoparticles is considered as an eco-friendly
biological approach (Ali et al. 2020).
The nanoparticles synthesized from antagonistic microbes have been tested in
controlling tea diseases under both laboratory and field levels. Silver and gold
nanoparticles synthesized by T. atroviride showed the maximum growth inhibition
of 75.7% and 80.3%, respectively, in P. theae on the seventh day of incubation
(Ponmurugan 2017). The shelf-life of both nanoparticles synthesized from
T. atroviride was 3 months. Inhibition of P. theae by silver nanoparticle was about
75%. The maximum disease control was observed at 2 ppm dosage when compared
with untreated control.
Trichoderma atroviride and S. sannanensis were evaluated for copper and silver
nanoparticle synthesis against Cercospora theae isolates (Gnanamangai et al. 2017)
causing bird’s-eye spot disease. Initially, the freshly prepared extracellular silver
nanoparticles showed high disease control (59.42–79.76%), but the stability of
antagonistic property in stored nanoparticles was significantly high in copper
nanoparticles (58.71–73.81%). Ponmurugan et al. (2016) tested field effectiveness
of copper nanoparticles biosynthesized extracellularly by using S. griseus through
soil application in controlling P. hypolateritia. The maximum disease control was
observed at 2.5 ppm dosage with 52.7% disease reduction when compared to copper
fungicide application which resulted in 45.3% disease reduction. In addition, max-
imum leaf yield, improvement in soil macronutrients and difference in population
dynamics of microbes were noted in plants treated with nano-copper in comparison
to synthetic copper and carbendazim fungicide-treated plants. These studies show
the potential of using green-synthesized nanoparticles in pest and disease manage-
ment of tea. However, toxic potentials of synthesized nanoparticles, if any, to plants
and beneficial microbes, human and environment are to be evaluated before large-
scale adoption of this technique.
336 G. D. Sinniah and P. D. Senanayake
19.9 Microbial Metabolites in Tea Pest and Disease
Management
As discussed above, microbial pesticides produce an array of secondary metabolites
including enzymes, hormones and toxins to exploit the pathogenic microbes and
insect pests. Antimicrobial metabolites from fungi and bacteria involve in antibiosis
on phytopathogens and also trigger the plant’s defence systems (Ongena et al. 2007).
Lipases, chitinases and protease enzymes that digest the insect’s body shells help
entomopathogens to penetrate, and other secondary metabolites such as cyclic
depsipeptides, peptides, amino acid derivatives, polyketides, peptide hybrids and
terpenoids cause paralysis and disrupt the host’s physiological processes enabling
the microbes to colonize the hosts (Litwin et al. 2020).
Therefore, researchers have targeted microbe-based metabolites alone as pesti-
cides in plant protection. Microbial metabolites tested against tea pathogens include
volatile compounds of Trichoderma isolate T4, which inhibited the growth of
G. cingulate Gc3 by 50.0%. The nonvolatile compound of Trichoderma cell-free
culture at 10% concentration showed 74.28% growth inhibition of the same isolate
Gc3 (Kuberan et al. 2012). Trichoderma atroviride has shown to produce volatile
and nonvolatile secondary metabolites to suppress the Phomopsis canker-causing
pathogen (Anita and Ponmurugan 2011). Cell-free culture filtrate of non-pathogenic
C. gloeosporioides CgloTINO1 isolated from the tea garden of Assam in India had a
strong antagonistic effect on the tea pathogens P. theae and C. camelliae. The
chitinase and protease enzymes released by C. gloeosporioides played a role in the
process of antagonism (Rabha et al. 2014).
19.10 Endophytic Organisms in Tea Pest and Disease
Management
Endophytes are microorganisms (fungi, bacteria) associated with plants which do
not cause any damage to the host plant (Zhang et al. 2019). Endophytes have
attracted attention of researchers as they promote plant growth and protect the host
plants from herbivores/insect pest and diseases. Several fungi and bacteria reported
as biocontrol agents have shown endophytic colonization in tea and other plants.
Beauveria bassiana,M. anisopliae,Cladosporium spp., Acremonium spp.,
V. lecanii,Bacillus sp. and Streptomyces sp. have been isolated as endophytes
(Xie et al. 2020).
Utilization of endophytes in plant protection as indirect biocontrol mechanisms
has been considered as a novel approach. Entophytic colonization of B. bassiana and
L. lecanii on cotton leaves were found to reduce feeding and reproduction of Aphis
gossypii (Gurulingappa et al. 2010). Endophytic colonization of B. bassiana in
banana plants significantly reduced the survival of banana weevil larvae and
protected them from damage (Akello et al. 2008). Similarly, M. anisopliae was
19 Microbial Technologies in Pest and Disease Management of Tea (Camellia... 337
successfully established as endophyte in tea by Kaushik and Dutta (2016). They
recorded less infestation of aphid (6.3%), tea mosquito bug (13.8%) and red spider
mite (12.5%) in foliar application and less infestation of termites and carpenter worm
in soil application of M. anisopliae. This reveals the influence of mode of endophytic
fungal application in targeting pests. It is evident in other crops that B. bassiana
applied via seed treatment in tomato and cotton induced protection from Pythium
myriotylum and R. solani, pathogens that cause damping off of seedlings and root rot
of older plants (Ownley et al. 2008).
Shan et al. (2018) reported 13 genera of endophytic actinomycetes including
Streptomyces,Actinomadura,Kribbella,Nocardia,Kytococcus,Leifsonia,
Microbacterium,Micromonospora,Mobilicoccus,Mycobacterium,Nocardiopsis,
Piscicoccus and Pseudonocardia belonging to 11 families in 15 tea cultivars col-
lected from Fujian province in China. These endophytic actinomycetes have shown a
high potential for producing antimicrobial metabolites, and their growth-promoting
ability was proven by indoleacetic acid (IAA) production and 1-aminocyclopropane-
1-carboxylic acid (ACC) deaminase activities. Groups of Herbaspirillum spp.,
Methylobacteria spp. and Bacillus spp. are reported as the most important endo-
phytic bacteria for tea cultivars of Zijuan and Yunkang-10 in China (Yan et al.
2018). Forty three strains of endophytic fungi belonging to 14 genera were isolated
and morphologically identified from the roots, stems and leaves of tea (You 2008).
The endophytic community, their abundance and their activity depend on tea
cultivars, age of the plants and environmental conditions (Yan et al. 2018). Use of
biocontrol agents and entomopathogens as endophytes could be a novel alternative
for management of insect pests and plant pathogens in tea.
19.11 Conclusions and Future Prospects
Potential use of rhizospheric, phyllospheric and entomopathogenic microorganisms
for managing pests and disease-causing pathogens is increasing in global tea indus-
try. Trichoderma sp., B. subtilis and Pseudomonas spp. are the common microbial
pesticides used in disease management. Entomopathogenic fungi B. bassiana and
M. anisopliae, entomopathogenic bacterium B. thuringiensis and NVPs are the
common microbial inoculants in insect pest control. In addition to direct use of
microbial biopesticides, microbial metabolites, microbe-synthesized nanoparticles
and endophytic colonization have been tried in pest and disease management in tea.
Most of the techniques discussed above are so far restricted to experimental stage
and field studies. Hence, adoption and commercial application of microbiological
techniques including the use of microbial biopesticides in global tea industry are
limited. Commercial application of microbial biopesticides is relatively high in
China and Japan followed by India. Commercial-scale utilization of microbial
pesticides in other major tea-growing countries such as Sri Lanka, Indonesia,
Malaysia, Kenya, etc. is scarce.
338 G. D. Sinniah and P. D. Senanayake
Recent studies and reviews on rhizosphere, phyllosphere and endophytic
microbiome (Pandey and Palni 2002; Bhattacharyya and Sarmah 2018; Tanti et al.
2016; Wu et al. 2020; Borah and Thakur 2020; Xu et al. 2020) unravel information
on microbial dynamics and interaction of microorganism with tea plants. The new
information has opened new horizons for research and shows the potential use of
microbes in most beneficial ways in plant protection.
Though microbial pesticides have been considered as potential alternatives in
crop protection, variability in their efficacy, environmental influence on efficacy,
limited availability, attitude of growers towards microbial biopesticides and govern-
ment policies and registration constrain have hampered the wider use of microbial
biopesticides in global tea industry. Advancement in molecular genetics and bio-
technology has allowed improving the efficacy of wild strains of biocontrol agents.
Mutation, protoplast fusion, genetic modification and transformation allow genetic
manipulation of microbial biopesticides. Protoplast fusion enhanced
carboxymethylcellulase activity in Trichoderma reesei (Prabhavathi et al. 2006).
Genetically modified P. fluorescens F113Rif (pCUGP) increased
2,4-diacetylphloroglucinol (Phl) production for biocontrol efficacy against
Polymyxa betae on sugar beet (Resca et al. 2001). Genetic modifications change
the properties of these biological agents to enhance production of toxins and
secondary metabolites, survive under stress conditions, grow under varied environ-
mental condition and enhance their efficiency and spectrum of biocontrol activity.
Lengthy, complicated data requirement and the high cost dissuade registration
and commercialization of microbial biopesticidal products. Laws and policies reg-
ulating biopesticidal development and use vary from country to country (Arora et al.
2016). Simpler and harmonized regulatory policies would encourage commerciali-
zation of new products. Further, as discussed by Verma et al. (2020), microbial
metabolites could be used as primary molecules for the synthesis of plant protective
chemicals opening up new vistas for entrepreneurs and industrialists. Ready avail-
ability at affordable price and convincing large-scale farmer field trials promoting
the use of microbial biopesticide could encourage the practical use of these products.
Implementation of other IPM practices is also equally necessary to overcome the
limitations and problems associated with microbial technologies and ensure sustain-
able crop protection in tea.
Acknowledgements The authors sincerely thank Ms. R.D.S.M. Gamlath and the staff of Plant
Pathology and Entomology and Nematology Divisions, Tea Research Institute of Sri Lanka, for
their support and field photographs.
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19 Microbial Technologies in Pest and Disease Management of Tea (Camellia... 345
Chapter 20
Field Application of the Microbial
Technology and Its Importance
in Sustainable Development
Saloni Kunwar, Shristi Bhatt, Deepa Pandey, and Neha Pandey
Abstract Microorganisms are ubiquitous in nature and are a rich source of primary
as well as secondary metabolites. The uniqueness of microorganisms and their
unpredictable nature attracts them for more and more exploration for the welfare
of the humans and society. Products formed by microbes are natural and have the
ability to reduce problems like high cost of synthetic chemicals, environmental
pollution, hazards to human health, etc., and are helpful in sustaining the environ-
ment by applying different microbial technologies and sustainability goals. These
indigenous microorganisms are involved in biotechnological field applications such
as sustainable agriculture (biofertilizers and PGPR), food technology, chemical
technology, recombinant technology, and sustainable environment (wastewater
treatment, micro- and nanoparticle synthesis, oil remediation, and radioactive treat-
ment). Apart from this, various strains of microbes are also being modified geneti-
cally for defending many environmental sustainability aspects. This review focuses
upon the applications of microbial technologies for sustainable development of
environment that meets the needs of current generations without compromising the
ability of future generation to meet their own needs. Microorganisms like Micro-
coccus,Pseudomonas,Chromobacterium,Bacillus, and many others play a major
role in the development of a sustainable environment.
Keywords Microorganisms · Sustainable development · Microbial technologies ·
Environment
S. Kunwar · S. Bhatt · N. Pandey (*)
Department of Biotechnology and Life Sciences, Graphic Era Deemed to be University,
Dehradun, Uttarakhand, India
e-mail: neha.pandey@geu.ac.in
D. Pandey
Department of Zoology, Government PG College, Ranikhet, Uttarakhand, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_20
347
20.1 Introduction
Microorganisms are small creatures and consist of protozoa, fungi, viruses, micro-
algae, and bacteria. These microorganisms live in water, animal intestines, food, soil,
and different environments (Mosttafiz and Rahman 2012). Microbes can survive
extreme environmental challenges. There are many reasons why microorganisms are
important, especially because what they produce is valuable to us (Liu 2020). These
substances may be very immense substances (such as nucleic acids, proteins,
carbohydrate polymers, and cells), or they may be smaller molecules. They are
usually divided into metabolites (mainly) essential for vegetative growth and non-
essential metabolites (minor). There are many kinds of microorganisms. They play
an important contribution to develop sustainable environment and also play a vital
role in a series of green processes and cleaner technologies (from biogeochemical
cycles to various industrial productions) (Kuhad 2012).
The advanced era provides various means of technology in various fields, and the
emergence of the microbes establishes themselves as an advantage for this century.
Though the microbes came forefront during the 1660s, their uses in association with
technologies marked the beginning of the microbial technology dimension. A
microbial technology is defined as a technology that uses microbial system or the
organisms or its derivatives for the manufacturing of a product or its modification for
any specific purpose. The current situation of the biosphere drives the attention of
using any newer technology for its sustainability, and since microbes are a part of the
ecosystem, their application for the sustainable development has surrey advantage
and acts as a feasible remedy for the global eradication of problems in agriculture
and environment. Various microbial technologies including GMOs (genetically
modified organisms), plant growth-promoting rhizobacteria (PGPR), biopesticides,
and biofertilizer have the potential to resolve many environmental and agricultural
issues such as in bioremediation of nutrients in soil, promoting healthy plant growth
and maintaining good health of the soil. The deterioration of the biosphere calls for
an urgent need to replenish it in a feasible manner.
20.2 Microbial Biotechnology and Its Applications
The application of Microbial Biotechnology in science aims at treatment of materials
by microorganisms to produce useful products or processes. The technologies along
with their applications are mentioned below.
348 S. Kunwar et al.
20.2.1 Agriculture Technology
The widespread use of microorganisms in sustainable agriculture is due to the
genetic dependence of plants on the beneficial functions provided by symbiotic
inhabitants (Noble and Ruaysoongnern 2010). Plant microbe symbiosis is carried
out through the analysis of their ecological effects, which is the best research method
for fixing nitrogen (N
2
) (Franche et al. 2009). Industrial microbiology has revolu-
tionized agriculture through genetic engineering and related disciplines. The com-
monly used bacteria, Bacillus thuringiensis (insecticidal bacteria) and
Agrobacterium tumefaciens usually produce corona choloma in dicots. The tumor-
forming gene of Agrobacterium tumefaciens exists in tumor-inducing plasmid
(Ti plasmid). These genes instruct plants to form opines (nutrient factors that bacteria
cannot produce by themselves). Ti vector has extremely important value for intro-
ducing foreign genes into dicot plants to produce transgenic plants. However, Ti
plasmids cannot successfully transfer genes into monocots. For example, they
bypassed a problem and developed particle accelerator, which injects metal particles
encapsulated with DNA into the host cells or plant cell, thereby avoiding this
problem. Along with this, different techniques for microbial applications in agricul-
ture sector are as follows.
20.2.1.1 Biofertilizers
Biofertilizers consist of microorganisms that facilitate the growth of the plant and is
also required to meet the growing demands for food by protecting the crops from
pathogens with the use of naturally derived fertilizers in soil (Youssef and Eissa
2014). Organic farming is one of the practices that allow microbes to maintain the
biodiversity of the soil. Biofertilizers aid in nutrient uptake such as nitrogen and
phosphorus and recycle the nutrients back to the soil via various mechanisms
including nitrogen uptake, phosphate solubilization, mineralization, production of
antibiotics, or degradation of compounds (Gopal et al. 2013). A metagenomic study
revealed a core microbiome transfer therapy which provides resistance to crops from
the diseases. The technique involves complete transfer of microorganisms by mixing
disease-inhibiting soil with the diseased favorable soil. For example, in an experi-
ment by Mendes and colleagues, soils suppressive to Rhizoctonia solani were mixed
with diseased conducive soil in the ratio of 9:1 which proved to be effective against
sugar beet infection. Other successful examples of this treatment include infection of
common scab of potato and tobacco black root rot infection (Rosenzweig et al.
2012).
20 Field Application of the Microbial Technology and Its Importance in.. . 349
20.2.1.2 Plant Growth-Promoting Rhizobacteria (PGPR)
They are always in a symbiotic relationship with plant- and root-related bacteria.
PGPR improve the utilization of nutrients by dissolving unusable forms of nutrients
and by producing siderophores, thereby contributing to the transportation of nutri-
ents. There are some examples how PGPR work as a microbial technology:
1. PGPR as Disease-Suppressive Agents. To enhance the disease suppression ability
of the soil, PGPR secrete metabolites that aid in the protection of plants from
various diseases. Bacillus subtilis GBO3 produces salicylic acid and jasmonic
acid for defense pathway (Ryu et al. 2004). PGPR with B. amyloliquefaciens
937b are effective for tomato mottle virus (Murphy et al. 2003). B. megaterium
IISRBP x17 from black pepper acts against Phytophthora capsici (Aravind et al.
2009).Bacillus subtilis N11 together with composts controls Fusarium infection
on banana roots (Zhang et al. 2011). B. subtilis (UFLA285) shows resistance
against R. solani and induces leaf and root growth of cotton plants (Medeiros
et al. 2011). Paenibacillus polymyxa SQR-21 controls the Fusarium wilt in
watermelon (Ling et al. 2011). PGPR plays a vital role in restoring plants from
blight virus of tomato,cucumber mosaic virus and pepper mottle virus and banana
bunchy top virus (Harish et al. 2009). Glomus mosseae is found to be used against
Fusarium oxysporum, the pathogen of basil plant root rot disease (Toussaint et al.
2008). Psuedomonas fluorescens and arbuscular mycorrhiza (fungi) reduced root
rot disease and also assist in increasing the productivity of Phaseolus vulgaris
(common bean) (Neeraj 2011).
2. PGPR as Plant Growth inducers
PGPR showed much effective results when applied alone but was effective
much more than usual when used in combination with arbuscular mycorrhiza
(fungi) like Glomus intraradices; it leads to better nutrient absorption and
improves physiological processes in lettuce under stress conditions. Trifolium
alexandrinum is inoculated with Rhizobium trifolii; it increased nodulation under
saline stressed condition (Antoun and Prevost 2005). Paul and Nair found that
Pseudomonas fluorescens (MSP-393) have the ability to overcome the influence
of soil with the production of osmolytes and salt-stress-induced proteins.
P. putida can withstand high alkaline and saline condition by uptake of K
+
,
Mg
+
, and Ca
2+
and lowering the uptake of Na
+
which gained effectiveness in
cotton production (Yao et al. 2010). Arbuscular mycorrhiza (fungi) along with
nitrogen-fixing bacteria was effective for legumes in drought conditions, and also
inoculation of rice crops with AM increased antioxidant and photosynthetic
efficiency (Ruiz-Sanchez et al. 2010). Pseudomonads sp. improved the photo-
synthetic pigments and antioxidants in basil plants under drought condition. They
increase the enzymes catalase, glutathione peroxidase, and ascorbate peroxidase
activity and chlorophyll content in leaves under drought conditions (Heidari and
Golpayegani 2012).
350 S. Kunwar et al.
20.2.1.2.1 Mechanisms of Plant Growth PGPR
•Direct mechanism (by promoting nutrient acquisition such as nitrogen, phospho-
rus) or secretion of plant hormone levels.
•Indirect mechanism (by interfering with plant pathogen and promoting plant
growth and development). The flowchart of PGPR mechanism is shown in
Fig. 20.1.
20.2.1.3 Nitrogen Fixation
Plants cannot utilize atmospheric nitrogen directly, so it needs to be converted by a
process called nitrogen fixation, where nitrogenase enzyme is used to produce
bacteria such as Rhizobium and Cyanobacteria.Rhizobium forms a symbiotic
association with leguminous plant roots by the formation of nodules which act as
colonizing sites of rhizobia (Giordano and Hirsch 2004). In non-leguminous plants,
rhizobacteria called as diazotrophs form a non-mandatory interaction with the host
(Glick 2012).
20.2.1.4 Phosphate Solubilization
Phosphorus occurs in insoluble form in soil which is not assimilated by plants. So
insoluble phosphorus needs to be converted into soluble form to be utilized by
plants. Soluble phosphorus as monobasic (HPO
4)
and dibasic (H
2
PO
4
) is utilized by
plants. Phosphate is provided to soil in fertilizers that is converted into insoluble
form in soil, causing phosphorus deficiency in soil; only 0.1% of phosphorus is
available for plant usage. Bacteria such as Flavobacterium,Bacillus,Azotobacter,
Erwinia,Microbacterium,Enterobacter,Beijerinckia,Pseudomonas,Burkholderia,
PGPR
Direct
Nitrogen
ixation
Phosphate
solubilization
Indirect
Siderophore
production
Fig. 20.1 Mechanism
of PGPR
20 Field Application of the Microbial Technology and Its Importance in.. . 351
Rhizobium, and Serratia are phosphate-solubilizing bacteria (Bhattacharyya and Jha
2012). These bacteria solubilize phosphorus by two mechanisms: solubilization
(hydrolyzation of organic and inorganic insoluble phosphorus compounds to soluble
phosphorus) and mineralization (conversion of organic phosphorus into inorganic
phosphorus). Phosphate-solubilizing bacteria (PSB) secrete phosphatase enzyme
that converts insoluble phosphorus into soluble phosphorus by dissolving it. PSB
promote plant growth and improve the utilization of other trace elements in the soil
which yields a good variety of plants (Zaidi et al. 2009).
20.2.1.5 Siderophore Production
Iron is important for all organisms. Iron (Fe
3+
) in the environment is present in form
of insoluble hydroxides and oxyhydroxides that makes it difficult for plants to
uptake them from soil. Bacteria secrete water-soluble siderophores (low-molecular
mass iron chelators) which complex with iron and form siderophore-Fe complex.
This complex reduces Fe
3+
to Fe
2+
and it is introduced into cell through channels in
membrane and ultimately siderophore gets destroyed or used up once again
(Rajkumar et al. 2010). Bacterial siderophores also reduce heavy metals such as
aluminum, cadmium, copper, gallium, indium, lead, zinc, uranium, and neptunium
(Neubauer et al. 2000).
20.2.2 Food Technology
Microorganisms have two different roles. First, they play a prime role in fermenta-
tion (in this case, genetically modified organisms are not allowed). Second, they
become absorbed in the industries to produce food ingredients. Genetic engineering
is used to modify the yeast and improve its performance in the fermentation process.
Yeasts are optimized to changes in temperature, pH, and high yields on a wide range
of products. Amylases are acquired from fungus Aspergillus niger (Adejuwon et al.
2015) or bacteria Bacillus subtilis; for example, they were used to replace the
chemical additives for processing wheat flour, improving dough preparation for
baking foods to be possessed (Bueno et al. 2016). The protein extracted from SCP
is used as supplements in whole foods, replacing valuable traditional sources and
solving the protein deficiency problem. In animal and human food, the single cell
protein is used as a source of protein (Sadiku et al. 2019).
Emerging need of the increase in production of food allowed the use of agricul-
tural farming techniques with chemical-based fertilizers, but this did not prove itself
to be beneficial for the environment as it offered itself a large number of drawbacks
with its extensive and prolonged use. Here the microbial technology stepped forward
and in comparison to conventional methods is a much safer practice; it showed a
10–20% increase in economically important crop production. Despite its great
advantage, limits itself to the need of the strain of the organism, the selection, and
352 S. Kunwar et al.
the application of the technology required for a better understanding of the relation-
ship between the inoculants and the microbiome. The understanding of their rela-
tionship is important for the use of new technologies involving microfluidics-based
technologies like ‘microbiome on a chip’; this foregrounds multitrophic plant-
microbiome interactions with the expression of environmental parameters and the
host response against the treatments (Stanley and van der Heijden 2017). It can
induce long shelf-lives and also enhance the effectiveness of the microbial product.
One way of accomplishing this goal is by modifying the plant at an early stage
(seedling stage) by incorporating the desired bacteria (Mitter et al. 2017). This
technique is more reliable and has great advantages including the expression of
desired and favorable traits and protection of the plants from another microbiota. The
use of the synthetic microbial communities promotes early flowering, enhances
nutrient acquisition by plants, and induces resistance to plants (Gopal et al. 2013),
but the maintenance of the microbe in plant is achieved by genetic engineering and
plant breeding techniques. The plant interacts with the microbes in soil by the release
of components which are specific in nature and allows interaction with only the
required organism, but the incorporation of the desired organism makes it easy for
plants to interact and come up with expected outcomes.
20.2.3 Chemical Technology
Chemicals like organic acids via activity of microbes are very bright. Most organic
acids are natural products present in important metabolic pathways or intermediates
of microbial metabolism (Sauer et al. 2008). For example, global annual industrial-
scale production of citric acid is demanded by the market as a food additive is
through glucose fermentation using Aspergillus niger to form cane molasses, corn
starch, or beet molasses (Wang et al. 2016). In addition to this, the lactic acid
fermentation process has recently received more attention due to the increasing
demand for new biomaterials, such as biocompatible polylactics and biodegradable
products (Gao et al. 2011). The production of butanol and acetone, which was
effectively carried out by the genus Clostridium, was one of the industrial fermen-
tation processes of global importance, but this production led to chemical synthesis.
Likewise, the inability of a chemical compound to compete with a petrochemical has
affected the microbial composition of glycerol feedstocks.
20.2.4 Recombinant Technology
The main microbial hosts for the production of recombinant proteins are Hansenula
polymorpha,Bacillus subtilis,Aspergillus niger,S. cerevisiae,E. coli, and Pichia
pastoris. The recombinant microorganisms have provide the methods for the host to
produce glycosylated recombinant proteins with high bacterial content, such as
20 Field Application of the Microbial Technology and Its Importance in.. . 353
mammalian and insect cell cultures, as well as transgenic animals and plants. Plant
breeding along with genetic engineering has become a common practice for the
development of in-demand product, and its establishment has contributed to elimi-
nate hunger and poverty on a global scale. The introduction of the GM crops was
first developed in the mid-1990s in the USA. Today this technique is adopted
globally to meet the increasing demand of the population and a way to move forward
toward attaining sustainable production of crops. The GM crops are regulated by
three agencies: EPA (Environmental Protection Agency), FDA (Food and Drug
Administration), and USDA (US Department of Agriculture). The development of
GM crops decreased the use of chemically originate herbicides and pesticides.
Besides, the newly modified crop was herbicide resistant naturally. The administra-
tion of a soil bacterium called Bacillus thuringiensis (Bt) allowed modification in the
plants to make them insect resistant; such plants are popularly known as Bt crops.
Globally today many forms of Bt plants are available in the market; the advantage of
such engineering is that it produced plants of superior quality with the extension of
its shelf-life. Some of the crops considered under GMO crops are rice, tomato,
cotton, soybean, maize, etc. The crops to be modified are targeted with the bacterium
containing the desired gene; primarily the gene is introduced in the crop in vitro, and
once the crop attains the growth, it is subjected to the field and allowed to grow in
natural conditions. Such crops are usually herbicide tolerant and insect resistant; the
commonly used bacteria are Agrobacterium tumefaciens and Bacillus thuringiensis.
20.2.5 Environmental Health and Microbial Technology
Environmental microbiology is the study of the composition and physiology of the
microbial community in the environment. The various microorganisms have been
recorded during the solid waste composting process, including autotrophic or het-
erotrophic aerobic bacteria, fecal coliform bacteria, thermophiles, yeast, actinomy-
cetes, and other fungi (Tiquia et al. 2002). Treatment of waste is based on enzymatic
processes and is inexpensive; however, these enzymes are biodegradable, so further
research is needed for microbial enzymes that are thermally stable or resistant to
significant changes in pH (Hasan et al. 2006).
20.2.6 Wastewater Treatment
Water is essential to sustain the life-forms on earth. Water accounts for 71% of the
surface of earth; about 96.5% of water found in the oceans is unfit for the sustain-
ability of life-forms. About 0.3% water is available as suitable for human consump-
tion, which is obtained from rivers and lakes, and only 0.61% of water is available in
groundwater. According to a study, 321 billion gallons of water is consumed per day
by human beings and 77 billion gallons of water is taken up from groundwater.
354 S. Kunwar et al.
Wastewater contains organic and inorganic pollutants which are lethal to the biota
system; therefore, prior to discharge the wastewater requires treatment. Modern
approach used to treat wastewater is microbial fuel cell (MFC) (Rabaey et al.
2010). MFC has several advantages such as:
1. It restores electrical energy and valuable products.
2. It generates nontoxic effluents.
3. It is easy to monitor and control and provides easy monitoring of the system.
MFC uses microbes at the electrode (either cathode or anode) to catalyze oxido-
reduction and generate electric current. The reducing microbes reduce the electrode
and donate electrons to the negative electrode (anode). These microbes reduce
substrate from the wastewater required for the reaction by microbes to produce
energy and provide a healthy biosphere (Clark and Pazdernik 2016).
20.2.7 Oil Remediation
With the increase in the world’s population, the demand for the resources also
increases; therefore, there is a subsequently increased need for petroleum and
petroleum-based products, but accidents related to petroleum and petroleum-based
products are also hazardous to all life-forms including marine lives and humans and
plants (Strong and Burgess 2008). There have been numerous incidents pertaining to
petroleum products as the 1971 incident in Pennsylvania where a gasoline pipeline
was damaged and it released 100,000 gallons of the gasoline into the nearby water
supply.
The first application of the use of this technique was the Exxon Valdez oil spill
which proved to be effective to clean up the petroleum-contaminated ground system;
this gained the interest in the use of bioremediation to clean up the environment in a
safer way. Petroleum is a natural resource obtained beneath the earth layer which is
an admixture of nitrogen, sulfur, and oxygen. It constitutes a variety of compounds
such as aromatic compounds, some metals (iron, copper, nickel, vanadium), alkanes,
and cycloalkanes as shown in Table 20.1.
The permeation of these compounds into the groundwater or surface water can
impose serious health hazards to the biosphere, and the uptake of these compounds
by the living organisms can affect their health. Gasoline is considered as a cancer-
causing agent to humans by the International Agency for Research on Cancer
(IARC) (2000) and tends to cause irritation in the eyes and mucous membrane.
Table 20.1 Composition of
compounds present in petro-
leum by weight
Hydrocarbon Amount present (%)
Alkanes 30
Naphthalenes 49
Aromatics 15
Asphaltics 6
20 Field Application of the Microbial Technology and Its Importance in.. . 355
Table 20.2 shows various compounds of petroleum and their harmful effects on
humans.
20.2.7.1 Remediation of the Petroleum Products
The conventional method of the remediation of petroleum products includes disper-
sion, sorption, volatilization, recovery, dilution, and abiotic modification of hydro-
carbon removal. These require high capital investment and large machineries and
also dispose the residues into the environment (Matsumiya and Kubo 2007). On the
other hand, bioremediation generates ecofriendly residues and does not require much
capital; bioremediation techniques of petroleum products make the affected site free
of every contaminant.
20.2.7.2 Petroleum Hydrocarbon Degradation Mechanism
The initial step of the degradation in petroleum hydrocarbons is the oxygenation
process in which the organic pollutants are attacked intracellularly and the oxygen is
transferred within the cell by the enzymes like oxygenase and peroxidases, followed
by the conversion of the pollutants into various intermediates through a number of
degradation pathways such as TCA (tricarboxylic cycle). This method is mediated
by the enzymes and hence is considered as an enzymatic-dependent mechanism.
Below are some of the examples that show the enzymes produced by various
microorganisms:
1. Methylococcus produces soluble methane and particulate methane that act on C1
to C8 of cycloalkanes, alkenes, and alkanes (McDonald et al. 2006).
2. Burkholderia species produces alkanes that act on C5–C16 of fatty acids, alkanes,
cycloalkanes, and alkyl benzenes. Rhodococcus and Mycobacterium produce
hydroxylases that act on C5–C16 of alkyl benzenes, fatty acids, cycloalkanes,
and alkanes (Jan et al. 2003).
Table 20.2 Illustrates various petroleum compounds and their effects with the concentration
Petroleum
compounds Effect on humans
Concentration (parts per
million)
Benzene Leukemia <1
Toluene Memory loss and coordination impair-
ment
Palpations
200–500
500–1000
Gasoline Cancer >2000
Cyclohexane Polyneuropathy <1000
Other hexanes Narcosis 1000
356 S. Kunwar et al.
3. Acinetobacter produces dioxygenases that act on C10–C30 of alkanes (Maeng
et al. 1996).
4. Species of Caulobacter,Mycobacterium, and Acinetobacter produce bacterial
oxygenase P450 that acts upon C5 to C16 of cycloalkanes and alkanes (Van
Beilen et al. 2006).
Other methods include the production of biosurfactants. Biosurfactants are agents
produced by some microorganisms that form micelles by reducing the surface area
given in Table 20.3. In this method the cell surface is encapsulated with
microdroplets that absorb the petroleum products and degrade them. It was observed
that 90% of hydrocarbons was degraded within a time period of 6 weeks in vitro
(Cameotra and Singh 2008); with further alteration it was shown that most of the
hydrocarbons is degraded with the application of crude biosurfactant (Muthuswamy
et al. 2008).
The recent advancement offers application of plasmid DNA incorporation for the
biodegradation of hydrocarbons. Plasmid DNA is a mobile DNA that can be
transferred by the processes of conjugation and transformation and has the ability
to express the quality of the incorporated DNA and change the phenotypic expres-
sion of the host. This has been successfully exhibited on Pseudomonas sp. by
incorporating plasmid DNA for the nutrition of various compounds such as octane,
naphthalene, toluene, camphor, salicylate, and xylene. Further incorporation of
plasmid DNA has been able to degrade the recalcitrant compounds of petroleum.
The plasmid DNA enhances the potential of the recipient after getting incorporated
inside the recipient (Okoh 2006).
20.2.8 Radioactive Waste
Radioactive compounds are widely used in every field like industry, research and
development, and medical sectors as an innovative technique but their disposal is
very heedful. Accumulation of radioactive waste poses a threat to all life-forms. To
remediate the pollutant in soil and water, bioremediation has become a successful
method. Microbial-associated techniques aim to reduce radionuclides significantly;
the genetically engineered (GE) microorganisms affect the properties of
Table 20.3 Microorganisms producing biosurfactants
Biosurfactant Microorganism References
Lipomannan Candida tropicalis Ilori et al. (2005)
Glycolipid Aeromonas sp. Youssef et al. (2007)
Surfactin Bacillus subtilis Daverey and Pakshirajan (2009)
Sophorolipids Candida bombicola Kumar et al. (2008)
Rhamnolipids Pseudomonas aeruginosa Das and Chandran (2011)
20 Field Application of the Microbial Technology and Its Importance in.. . 357
radionuclides and thereby reduce their concentration. The following are the common
radionuclides that are released by industrial and biomedical wastes:
Cobalt-60
Uranium-238
Thorium-232
Radium-226
Radon-222
Plutonium-239
Technetium-99
Among this technetium is the most commonly used isotope in medical imaging
and has a shelf-life of 6 h, but other isotopes like Uranium-238 have a half-life of
1600 years (Kurnaz et al. 2007). The radioactive wastes not only affect the health of
life-forms but also deteriorate the nutrient level in soil if remained for a longer
period.
A common way by which soil gets contaminated with radioisotopes is by
dumping of radionuclides into the environment along with other wastes. The
physiochemical ways are practiced for a long time but failed to be resolved;
therefore, bioremediation came forth to act as an alternative way to remediate the
soil from radionuclides. In view to this, Fig. 20.2 gives details of different methods
for bioremediation of radionuclides. Some microorganisms act directly on the
polluted site and convert the radionuclides into soluble product by either oxidation
or reduction, allowing their fast removal from soil. Bacteria like Rhodanobacter
sp. and Desulfuromusa ferrireducens act directly on the pollutant (Green et al.
2012).
20.3 Importance of Microbial Technology in Sustainable
Development
The microorganisms play important roles in the environment. In recent studies,
every possible industrial process, the use of chemicals, the increased use of
nonrenewable energy, and the uncontrolled production of waste products pose a
Biosorpon
Bioaccumulaon
Biomineralisaon
Microbial
approach to
reduce
radionuclides
Direct enzymac
reacon
Indirect enzymac
reacon
Fig. 20.2 Various microbial techniques for bioremediation of radionuclides
358 S. Kunwar et al.
huge threat to environmental sustainability. Now, the world has a greater responsi-
bility to adopt cleaner production, green technologies, and sustainable measures in
order to protect the earth’s ecology for future generations (Kuhad 2012).
Microorganisms play an important role in the urban ecosystem. There are various
resident microorganisms in the urban ecosystem, which play an important role like
waste management, soil management, industrial productivity, bioremediation, health
and disease and marine pollution management, etc. (King 2014).
•By using wastes such as municipal waste, agricultural waste, and sewage sludge,
microorganisms can also be used to produce bioenergy (Appels et al. 2011).
Microorganisms such as Penicillium,Aspergillus,Trichoderma, and Clostridium
are highly effective (Elshahed 2010).
•Solid waste management is the biological conversion of its organisms into useful
products such as biofuels and biogas. Using microbial Thermoactinomyces,
Pseudomonas,Actinobifida,Microbispora, and Bacillus to compost solid waste
as an economically feasible method to convert its organic compounds into useful
products. Compost is used as fertilizer for the production of crops, thereby
increasing their product activity and developing a sustainable environment.
•Microorganisms such as Rhizobium are used as biological synthetic materials and
increase the productivity of industrial agriculture.
•The algae Nostoc and Azolla are used as economical viable sources of bioreme-
diation, which can then be used to produce biofuels.
•GM E. coli produces large amounts of insulin.
•Microorganisms show their role in infant health. Bifidobacterium and Lactoba-
cillus are microbes which are important for the regulation of human immune
system (Romano-Keeler and Weitkamp 2015).
•Microorganisms are also used to generate clean electricity. For example,
Geobacter sulfurreducens and Shewanella oneidensis are used to generate usable
electricity (Lal 2013).
•Microorganisms such as Micrococcus,Pseudomonas,Chromobacterium,Bacil-
lus,Arthrobacter,Candida, and Burkholderia degrade hydrocarbons and crude
oil through a method called intrinsic repair without any artificial enhancement
(Kumar and Gopal 2015).
•Microorganisms like Marinobacter,Pseudomonas,Bacillus,E. coli, and Strep-
tomyces help in the remediation of heavy metals (arsenic, mercury, and lead) from
waterbodies and control the pollution in marine waters.
20.4 Conclusion
Microbial technology has unique applications in majority of areas including its
effectiveness in achieving a sustainable environment. It has not limited its applica-
tion only in healthcare but has evolved in other sectors too. The present scenario
regarding the issues on agricultural and environmental sustainability has increased
20 Field Application of the Microbial Technology and Its Importance in.. . 359
abruptly with the advancement of urbanization. Meeting the challenges of modern-
ization has greatly deteriorated the conditions of the biosphere. In agriculture,
microbial technology has contributed to the production of improved crop varieties.
It produces crops with an admixture of different types of crops, resulting in improved
version of the crops containing the dominant and admirable traits of the different
crops. It has also contributed in increasing nutritional content of the product by
manipulating the genes of the product through genetic engineering. In environment,
microbial technology has contributed very much in eliminating the pollutants from
the contaminated sites which includes the removal of oil spills from massive oceans
without generation of any additional toxic residues and also in significantly remov-
ing heavy metals from unfertile land.
Resources are also replenished more conveniently by microbial technology than
conventional methods since it used microbes that naturally conserve resources or
generate such product. Although microbial technology has some drawbacks due to
which it has not come forward more distinctively, with the pace in the biotechnol-
ogy, it may become the only possible, reliable, and ultimate method to resolve all the
issues relating to sustainability in agriculture and environment and accomplish the
sustainability goals of the UN to meet the demands of the developing urbanization.
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20 Field Application of the Microbial Technology and Its Importance in.. . 363
Chapter 21
Solubilization of Micronutrients Using
Indigenous Microorganisms
A. D. Sarangi N. P. Athukorala
Abstract Out of the 17 elements essential for plant growth and reproduction, 8 are
micronutrients. The soil supplies relatively large amounts of nitrogen, phosphorus,
potassium, calcium, magnesium, and sulfur as macronutrients and relatively small
amounts of iron, manganese, boron, molybdenum, copper, zinc, chlorine, and cobalt,
as micronutrients. Both deficiency and excess of micronutrients negatively impact
the growth and productivity of plants and therefore should be supplied in sufficient
amounts in appropriate ratios. A number of biotic and abiotic factors and their
relationships affect the appropriate balance of macro- and micronutrient pool in
the soil. The focus on addressing micronutrient deficiencies in soil in relation to
agriculture has not been adequate in comparison to that for macronutrients. How-
ever, with the recent recognition on their impact on crop productivity and the
efficiency of NPK uptake by plants, much attention was drawn to regulating
micronutrient content in soil with chemical supplements. Environmental concerns
encountered with the use of chemical supplements have directed the world into
eco-friendly and sustainable approaches in addressing issues in many fields includ-
ing agriculture. The use of naturally inhabiting microorganisms, “indigenous micro-
organisms,”has been one of such eco-friendly approaches in agriculture. This
review discusses the approaches that have been researched and used with indigenous
microorganisms having micronutrient solubilization ability to regulate micronutrient
availability in soil and the potential of developing them to optimize the crop
productivity while maintaining a sustainable environment.
Keywords Micronutrients · Solubilization · Bioavailability · Indigenous
microorganisms
A. D. S. N. P. Athukorala (*)
Department of Botany, Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka
e-mail: sarangiathu@pdn.ac.lk
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_21
365
21.1 Introduction
The nutrients which are required by organisms in comparatively small quantities are
called micronutrients. They are required in plant tissues at concentrations of less than
100μg
1
dry weight (Welch and Shuman 1995). The attention to micronutrients has
increased in the recent past with the understanding of their important role in disease
resistance in plants and stress resistance in roots especially in crop plants (Welch and
Shuman 1995; Graham and Webb 1991; Miller et al. 1991; Van Campen 1991;
Nielsen 1992). Micronutrients are as equally important as macronutrients for plant
growth, yield, and quality (Maurya et al. 2018; Yadav et al. 2018). There are eight
micronutrients which have currently been recognized to be essential for higher
plants, namely, boron (B), iron (Fe), copper (Cu), zinc (Zn), manganese (Mn),
chlorine (Cl), molybdenum (Mo), and nickel (Ni) (Welch and Shuman 1995).
However, an exact number of the micronutrients vital for higher plants cannot be
strictly proposed since intense molecular and physiological studies are required for
each and every nutrient before such conclusions and for elimination of controversies.
One such example is Si where there are arguments whether it is to be categorized as
an essential or a beneficial element (Maathuis 2013; Mengel et al. 2001; Barker and
Pilbeam 2015). Micronutrients play a role in primary as well as in secondary
metabolism, energy metabolism, cell defense, signal transduction, hormone percep-
tion, and gene regulation (Maathuis 2013; Barker and Pilbeam 2015; Maathuis and
Diatloff 2013; Vatansever et al. 2017). They also enhance the chemical composition
and the quality of plants including crops and are known to act as catalyst in various
organic reactions in plants (Karthick et al. 2018). Since these play an important role
in plant growth and development, the deficiencies result in several physiological
disorders and diseases in plants that reduce the yield and quality of plant produce
(Sharma 2006). It should be taken into account that a number of physiological events
in relation to plant metabolism are directly or somewhat related to the mineral
elements. Deficiencies of mineral elements or toxicities intensely affect the life
cycle of plants, and their availability to plants especially crops should be closely
looked at in order to address the food safety and food security issues that would
upswing in the near future. With the United Nations sustainability goals to be met by
2030 with the already identified and not yet identified ecological, environmental, and
health problems associated with chemical fertilization, eco-friendly alternatives for
soil augmentation are being investigated from which mineral-solubilizing microor-
ganism is one. Deficiencies in micronutrients in soil will ultimately be related to
human and animal health risks by crops not having the required amount of
micronutrients present in crop produce. This chapter will be focused on different
mechanisms and methods by which soil indigenous microflora increases the micro-
nutrient availability in soil, enhancing the soil fertility and crop production and
thereby human health through a sustainable approach. When considering a sustain-
able environment, the role of microbes in other processes such as bioremediation and
bioleaching in addition to biofortification and biofertilization should also be
366 A. D. S. N. P. Athukorala
discussed. Therefore, the chapter will also discuss situations where indigenous
microbes have been used in the above processes in relation to eight micronutrients.
21.2 Micronutrient Deficiencies
Deficiency of micronutrients in soil is a global issue with slight variations in
particular to different micronutrients (Monreal et al. 2015; Voortman and Bindraban
2015; Dimkpa and Bindraban 2016). In addition, their low crop use efficiency (i.e.,
low crop response per unit of micronutrient, relative to no micronutrient applica-
tion), typically <10% in comparison to 20% and 80% for N, P, and K (Baligar et al.
2001), has also contributed to their deficiency in global agro-ecosystems. Many
arable lands are affected by deficiencies of more than one micronutrient (Monreal
et al. 2015; Voortman and Bindraban 2015; Oliver and Gregory 2015). This issue
has further been made complicated with extensive extraction by high yielded crops
supplemented with NPK fertilizers in addition to inadequate micronutrient fertiliza-
tion. Yield increments reported with the addition of micronutrient supplements in
different crops both with and without NPK fertilizers (Dimkpa and Bindraban 2016;
Katyal and Ponamperuma 1974; Kanwar and Youngdahl 1985; Rietra et al. 2015)
suggest the importance of micronutrients in crop productivity and for effectiveness
of NPK fertilization (Dimkpa and Bindraban 2016). An amount ranging from 0.01 to
4.9 kg ha
1
of micronutrients has been estimated to be collectively removed from the
soil annually by different crops (Rietra et al. 2015) and the type of micronutrient
(Mallarino et al. 2011; Marschner 2012). A normal growth of a plant requires an
amount of each micronutrient ranging between 0.1 and 100 mg kg
1
with mean
levels of 90 mg kg
1
present in DTPA-extractible form (Samourgiannidis and Matsi
2013; Sobral et al. 2013). Therefore, soils do not fulfill the micronutrient require-
ment of a normal healthy plant. These deficiencies in soil result in low crop
productivity and nutritional quality which will ultimately affect human health
(Marschner 2012; Alloway 2009; Itelima et al. 2018; Dhaliwal et al. 2019; Kaura
et al. 2020).
Since human nutrition is directly or indirectly based on plants, any micronutrient
deficiency in food crops could cause micronutrient deficiency in humans, referred to
as “hidden hunger”(Oliver and Gregory 2015; Kaura et al. 2020; White and
Broadley 2009; Joy et al. 2015; Riaz et al. 2020). For example, Zn deficiency in
soil has been shown to cause Zn-deficient symptoms in humans, such as stunting and
child death (Monreal et al. 2015; Cakmak 2008).
21 Solubilization of Micronutrients Using Indigenous Microorganisms 367
21.3 Micronutrients in Plants
The ability of a plant to obtain sufficient levels of vital minerals is also a function of
specific characteristics in the plasma membranes of root cells such as the presence of
relevant transport proteins and related acquisition mechanisms of ions (Vatansever
et al. 2017; Dimkpa and Bindraban 2016; Kochian 1991). Modification of the
rhizosphere by plant roots can also affect nutrient availability, through the release
of protons, chelators, phytosiderophores, and/or chemical reductants and also by
elaboration of extensive root systems (Dimkpa and Bindraban 2016; White et al.
2013; Keuskamp et al. 2015). Micronutrient availability for plants is not entirely
dependent on the amount of mineral present in the soil matrix but also depends on
the molar fraction existing in soil solution and on the variation of the ions of a
particular mineral (Vatansever et al. 2017; Lindsay 1991). Speciation and solubility
of a particular mineral are influenced by abiotic factors such as redox state, pH, and
temperature as well as by biotic factors such as phenolic compounds and organic
acids which are metabolically generated or released through degradation of soil
organic matter by indigenous microorganisms. In the soil, some micronutrients react
with compounds such as phosphates and carbonates, to form chemical precipitates or
interact with clay particles and other mineral complexes, making them unavailable
(Dimkpa and Bindraban 2016; Marschner 2012; Allen 2002).
Factors such as plant species, genotype, growth conditions, and different organs
and tissues of the same plant species affect the micronutrient content of a plant.
Genetic makeup together with physiological and environmental factors changes the
concentrations of micronutrients inside the plants, deficiencies, or toxicities
(Table 21.1). Environmental variables and differences between plant species, as
well as genetic variation within a plant species, can affect micronutrient concentra-
tions in higher plants (Luber and Taureau 1990; Benton-Jones 1991; Benton-Jones
et al. 1992). Micronutrients play a significant role in both general and specific
physiological processes in plants. Fe, Cu, Mn, and Cl are involved in different
metabolic processes like photosynthesis acting as cofactors. Fe, Mn, Zn, Cu, Ni,
Mo, and Cl contribute to the activity of different enzymes such as DNA/RNA
polymerases, N-metabolizing enzymes, dismutases, catalases, superoxide, dehydro-
genases, oxidases, ATPases, and enzymes involved in redox processes (Broadley
et al. 2012). Zn specifically plays a role in the enzymatic biosynthesis of auxin
(Hossain et al. 1997; Fageria 2002) which enhances root growth. Ni is involved in N
metabolism of plants by transforming urea to ammonia (Polaccao et al. 1999; Sirko
and Brodzik 2000). Mo is used by both symbiotic and free-living N-fixing bacteria
for N fixation since it is a component of the nitrogenase enzyme system (Barron et al.
2009). The role of micronutrients as cofactors is crucial for enzyme and nonenzyme
activities in plant metabolism depending on the environment especially in abiotic
and biotic stress mitigation by plants. Zn is shown to modulate the activity of
membrane-bound NADPH oxidase (Cakmak 2008) in homeostasis of reactive
oxygen species which regulates defense and signaling by the host during drought
or other abiotic stresses (Bagci et al. 2007; Golldack et al. 2014). Cu is an essential
368 A. D. S. N. P. Athukorala
Table 21.1 Summary of major functions, deficiency, and toxicity levels and symptoms of micronutrient defficiency and toxicity
Element Major functions
Deficient concentration
in plant Deficiency symptoms
Toxic concentration in
plants Toxicity symptoms References
Fe Biological redox sys-
tems (i.e., electron
transport chains in pho-
tosynthesis and respira-
tion), enzyme
activation, carrier in N
fixation (i.e.,
leghemoglobin in bacte-
roids of legume roots)
Less than
50–100μgg
1
Interveinal chlorosis in
young leaves (caused
by decreased chloro-
phyll synthesis),
retarded/stunted growth
and reduced activity of
hill reaction
Above 50–100μgg
-1
Growth inhibition,
reduced chlorophyll
synthesis, inhibition of
photosynthesis
Welch and
Shuman
(1995),
Tripathi
et al.
(2015)
Mn Enzyme activation, bio-
logical redox systems
(e.g., electron transport
reactions in photosyn-
thesis), detoxification of
oxygen free radicals,
secondary plant metab-
olite synthesis, struc-
tural constituent of
ribosomes, disease
resistance
Less than
10–100μgg
1
Interveinal chlorosis in
young tissue, appear-
ance of greenish-gray
specks at the lower base
of monocots, develop-
ment of brown necrotic
spots on the cotyledons
of legume plants, pre-
mature leaf fall, white-
gray spots of leaf and
delayed maturity
Above 10–100μgg
1
Interferes with absorp-
tion and utilization of
other mineral elements,
affects the energy
metabolism, decreases
photosynthetic rates,
causes oxidative stress
Welch and
Shuman
(1995),
Tripathi
et al.
(2015)
Zn Membrane integrity,
enzyme activation, gene
expression and regula-
tion, carbohydrate
metabolism, anaerobic
root respiration, protein
synthesis, structural
integrity of ribosomes,
detoxification of
Below 15 mg Zn kg
1
dry weight
Impaired stem elonga-
tion in tomato, root
apex necrosis (“die-
back”), interveinal
chlorosis (“mottled
leaf”), development of
reddish-brown or
bronze “bronzing,”
internode shortening
Above 20 mg Zn kg
1
dry weight
Reduced yields and
stunted growth. Leafy
vegetable crops are
sensitive to Zn toxicity.
Soybean and rice have
been recognized as Zn
sensitivity crops in
which Zn toxicity insti-
gates genetic variation,
Welch and
Shuman
(1995),
Tripathi
et al.
(2015)
(continued)
21 Solubilization of Micronutrients Using Indigenous Microorganisms 369
Table 21.1 (continued)
Element Major functions
Deficient concentration
in plant Deficiency symptoms
Toxic concentration in
plants Toxicity symptoms References
superoxide radicals,
phytohormone activity
(e.g., IAA and
gibberellic acid), gene
structure (Zn finger
motif), disease
resistance
(“rosetting”), epinasty,
inward curling of leaf
lamina “goblet”leaves
and reductions in leaf
size (little leaf)
increased lipoxygenase
activity and lipid per-
oxidation enhancing
antioxidative activity in
plants
Cu Physiological redox
processes (e.g., photo-
synthetic electron trans-
port, respiration),
detoxification of super-
oxide radicals, lignifica-
tion, disease resistance,
pollen viability
Below 5μgg
1
dry
weight
Improper growth rate
and distortion or whit-
ening (chlorosis) of
young leaves, decrease
in cell wall formation
lignification in several
tissues and curling of
leaf margins, damages
apical meristem, pollen
development, the fruit
and seed production,
wood production
Above 20μgg
1
dry
weight
Chlorosis and necrosis,
stunting, and inhibition
of root and shoot
growth inhibit enzyme
activity and protein
function, which later
produces highly toxic
hydroxyl radicals lead-
ing to oxidative damage
of plant cell
Welch and
Shuman
(1995),
Tripathi
et al.
(2015)
Ni Urea and ureide metab-
olism, iron absorption,
seed viability, nitrogen
fixation, reproductive
growth
0.05–10 mg kg
1
of dry
weight
Leaf tip necrosis
(legumes), chlorosis
and patchy necrosis
(Gramineae)
>10 mg kg
1
dry
weight (DW) in sensi-
tive species like barley,
water spinach, and
wheat
>50 mg kg
1
DW in
moderately tolerant
species and
>1000 mg kg
1
DW in
Inhibition of mitotic
activities, reductions in
plant growth and
adverse effects on fruit
yield and quality.
Extremely high soil Ni
concentrations have left
some farm land
unsuitable for growing
Chen et al.
(2009),
Alloway
(2008)
370 A. D. S. N. P. Athukorala
Ni hyper-accumulator
plants
crops, fruits, and
vegetables
Cl Osmoregulation, charge
compensation (i.e.,
counter ion in cation
uptake), reactivity of
enzymes, photosynthe-
sis, disease resistance,
stomatal regulation
Can vary from 0.1 to
6mgg
1
(dry matter) or
between 0.03 and
0.17 mmol L
1
of the
plant tissue water con-
tent for different species
Wilting of leaves, espe-
cially at margins, shriv-
eling and necrosis of
leaves, frond fracture
and stem cracking in
coconut, sub-apical
swelling in roots
Crops with high Cl
endurance, such as
corn, sugar beet, grain
sorghum, cotton, and
spinach, can endure the
Cl of >600 mg kg
1
with no visible negative
effects
The crops with mid-Cl
endurance, such as
wheat, rice, cucumber,
tomato, cabbage, pea-
nut, and grape seedling,
can endure the Cl of
300–600 mg kg
1
The crops with low Cl
endurance, such as soy-
bean, lettuce, sweet
potato, strawberry, and
apple seedling, can’t
endure the environmen-
tal Cl when it exceeds
300 mg kg
1
Reduced yields Chen et al.
(2009),
Alloway
(2008)
B Cell wall formation and
stabilization, lignifica-
tion, xylem differentia-
tion, membrane
Less than 0.3 ppm and
0.14 mg kg
1
(gener-
ally required in greater
amounts by
Stunted growth inhibi-
tion of cell expansion,
cracking or rotting of
fruits, wilted or curled
Above 0.3–1 ppm and
3–100μgg
1
dry
weight
Yellowing of the leaf
tips and distorted shoot
growth, chlorotic and
necrotic patches in the
Welch and
Shuman
(1995),
Tripathi
(continued)
21 Solubilization of Micronutrients Using Indigenous Microorganisms 371
Table 21.1 (continued)
Element Major functions
Deficient concentration
in plant Deficiency symptoms
Toxic concentration in
plants Toxicity symptoms References
integrity, carbohydrate
utilization, pentose
phosphate metabolism,
phenol metabolism and
auxin activity, pollen
germination and growth,
inhibition of callose
formation, stomatal
regulation
dicotyledonous plant
species than by
monocotyledons)
leaves, water-soaked
petiole
margin/older leaves,
spots on fruits
et al.
(2015)
Mo Electron transfer reac-
tions, nitrate reduction,
nitrogen fixation, ureide
metabolism, sulfate oxi-
dation, pollen forma-
tion, protein synthesis
0.2–2 ppm in forage
legumes
Chlorosis of leaf mar-
gins, “whiptail”of
leaves and distorted
curding of cauliflower;
“fired”margin and
deformation of leaves
due to NO3 excess and
destruction of embry-
onic tissues
100–1000 mg kg
1
Stunted growth with
yellow-brown leaf
discolorations
Maathuis
(2013),
Baligar
et al.
(2001)
372 A. D. S. N. P. Athukorala
element for lignin synthesis needed for cell wall strengthening (Yruela 2009; Ryan
et al. 2013) which facilitates withstanding abiotic stresses such as wilting, wind, and
rain. B facilitates cross-linking of pectic polysaccharides serving cell wall function-
ing and maintaining the structural support of the cytoskeleton (Miwa et al. 2008).
Chloride plays a role in stomatal regulation and protects plants from wilting and
death (Broadley et al. 2012).
When considering crop plants, the role of micronutrients in combating abiotic and
biotic stresses, improving nutritional quality, increasing yield, and enhancing uptake
of essential macronutrients, NPK, is substantial in addition to their physiological
role. Micronutrients assist plants to mitigate biotic stresses by means of developing
resistance to plant diseases either directly affecting the pathogens in the rhizosphere
or inducing different types of physiological responses in the plant during pathogen
attack through mechanisms such as siderophore production (Kloepper et al. 1980;
Lim and Kim 1997; Fernandez et al. 2005; Vansuyt et al. 2007; Dimkpa et al. 2009,
2015a; Radzki et al. 2013), inducing cellular activity, disease resistance (Shirasu
et al. 1999; Datnoff et al. 2007) and microbial biocontrol agents to produce antimi-
crobials (Dimkpa et al. 2012,2015a; Duffy and Defago 1999) and acidification of
soil (Dimkpa et al. 2013a,b). Zn and Mn were reported to suppress diseases (Huber
and Wilhelm 1988), and Cu, Ni, Mn, Mo, and B have been reported through
mechanisms such as inducing the production of antioxidants, strengthening cell
walls through the production of lignin and suberin, and controlling N metabolism
in the plant (Huber and Wilhelm 1988; Römheld and Marschner 1991; Boyd et al.
1994; Bai et al. 2006; Evans et al. 2007; Stangoulis and Graham 2007; Taran et al.
2014; Servin et al. 2015) which make the crop more resistant to diseases and drought
conditions.
Even though there should be a remarkable contribution of micronutrients for the
nutritional value of crop produce with a number of physiological roles governed by
them in plants, their relationship has been inconsistent (Dimkpa and Bindraban
2016). Some studies demonstrated a positive outcome with the addition of
micronutrients (Rietra et al. 2015; Dimkpa et al. 2015a,b; Kumar et al. 2009),
especially through positively modulating the uptake of other micronutrients. Some
studies reported that the levels of other micronutrients can be reduced by the addition
of a specific micronutrient perhaps because of the competition for uptake among
micronutrients (Dimkpa and Bindraban 2016). The positive effects of micronutrients
in the yield, quality, earliness, fruit setting, postharvest life, and biotic and abiotic
stresses in vegetable crops have been documented by Sidhu et al. (2019). In addition
to crop nutritional quality, they have been reported to enhance seed vitality and
thereby good seed emergence and vigorous seedling growth (Brodrick et al. 1995;
Nestel et al. 2006; Eggert and von Wirén 2013; Velu et al. 2014). The role of
micronutrients in improving the agronomic quality including yield has been
overserved with the supplement of individual element (Dimkpa and Bindraban
2016) as well as in combined applications (Yaseen et al. 2013; Vanlauwe et al.
2014). The significance of fortification of micronutrients into the soil through
different means in order to improve the quality and quantity of food crops, has
been discussed only recently and its real value will be persuaded in the future.
21 Solubilization of Micronutrients Using Indigenous Microorganisms 373
21.4 Micronutrients, Ecosystems, and Environment
The major ecological role micronutrients play in soils is increasing the NPK fertilizer
efficiency use by plants that would otherwise be lost via leaching, fixation, and/or
volatilization. Additionally micronutrients in soils may improve water use efficiency
of crops under water-deficient conditions (Movahhedy-Dehnavy et al. 2009; Molden
et al. 2010; Ashraf et al. 2014), and when coupled with organic matter in soils, they
have been observed to enhance the ion exchange capacity, soil structure, and water
storage capacity, improve drainage and aeration, and decrease soil salinity (Dhaliwal
et al. 2019). These benefits together with the suppression of plant diseases will
enhance the sustainable agricultural production systems. However, most of the
micronutrients are heavy metals; therefore, nonstrategic applications would exert
ecological and environmental challenges. Further, contaminations with high levels
of micronutrients can also occur through water irrigation, carrying pesticides and
heavy metal-containing wastewaters, as biosolid accumulates in soil (Alloway and
Jackson 1991; Wuana and Okieimen 2011) which ultimately can cause micronutri-
ent toxicity to soil flora and fauna. Despite both beneficial and detrimental
agronomical, environmental, ecological, and health effects of micronutrients to
living beings, increasing their availability in soil strategically will offer great poten-
tial in mitigating some challenges related to food security and “hidden hunger”
through enhancing the quality and quantity of food produce.
21.5 Addressing Micronutrient Deficiencies
Since micronutrient fertilization is not a prevailing cultural farming practice, miti-
gating their deficiencies in soil would require vigilant fortification intervened after
prior evaluations of crop and soil conditions (Joy et al. 2014; Kumssa et al. 2015)
with systematically determined nutrient ratios and antagonistic interactions among
the micronutrients, as well as between micronutrients and macronutrients. This will
lead to the need of vigorous experimentation on the micronutrient applications in
combination with fertilizer regimes to ensure more plant-specific and balanced ratios
of micronutrient in fertilizer formulations (Dimkpa and Bindraban 2016; Rietra et al.
2015; Kaura et al. 2020). Micronutrient can be fortified through a number of
methods which have their own limitations. The most common and direct method
is the agronomic fortification through soil applications (Cakmak 2008; Velu et al.
2014; Duffner et al. 2014), as foliar sprays or as seed treatments (Farooq et al. 2012;
Mondal and Bose 2019) in which foliar sprays have been more effective in yield
improvement and grain enrichment, but are restricted by high cost (Johnson et al.
2005). Soil application would require higher doses due to low nutrient-use efficiency
since 60–90% of the total applied fertilizer is lost and only the remaining 10–40% is
taken up by plants. Seed treatment with its easiness to apply would be a better option
both economically and environmentally as less micronutrient is needed while
374 A. D. S. N. P. Athukorala
improving seedling growth (Farooq et al. 2012; Mondal and Bose 2019; Singh et al.
2003). However, this is under thorough investigation in terms of optimization of
formulation, application protocols, and storage methods (Farooq et al. 2012). As
alternatives to agronomic fortification, methods such as plant breeding, genetic
engineering (biofortification), and postharvest biofortification of food are used in
different countries. These methods are usually time consuming, and several tedious
optimization trials such as screening of germplasm, crossing between varieties,
molecular marker-assisted selection, and new crop breed phenotyping and high
technical skills are needed (Velu et al. 2014; Waters and Sankaran 2011). In
addition, transformation of micronutrients to available forms through building up
of soil organic matter content has gained attention recently (Dhaliwal et al. 2019);
however, it is yet to be further investigated with field trials done in large scale and
stimulation models to better understand the relationship and to formulate manage-
ment strategies (Dhaliwal et al. 2019). With these practical issues encountered with
other alternatives, soil applications as chemical fertilizers have been the most
common method of micronutrient fortification; however, they would result in chem-
ical residues in soil due to their low use efficiency which will lead to severe
environmental problems and toxicities to plants if not duly addressed. Further,
imbalanced application can enhance the micronutrient deficiency levels in soils
(Dhaliwal et al. 2019). The use of chemical fertilizers causes soil acidification
(Chun-Li et al. 2014) and groundwater and air pollution (Youssef and Eissa 2014).
More importantly, concerns over the contribution of chemical fertilizer for global
warming and climate change have led the world toward sustainable fortification
strategies in the recent past. Using indigenous microorganisms which are capable of
promoting growth (plant growth-promoting rhizosphere microorganisms, PGPMs)
and disease resistance (biocontrol agents) in crops through different mechanisms
including converting nutritionally significant elements from unavailable to available
form (mineralization/solubilization) has gained attention in the recent past as a
substitute to chemical fertilizer in sustainable farming (Kaura et al. 2020; White
and Broadley 2009; Bhardwaj et al. 2014; Fomina et al. 2005b). It has also been
identified as an eco-friendly and cheaper approach in maintaining a sustainable
environment with their potential to conserve and increase the soil biodiversity
(Vessey 2003; Raja 2013) and to reduce environmental pollution including heavy
metal contamination. When considering a sustainable environment and increasing
bioavailability of micronutrients, the role of indigenous microbes in bioleaching and
bioremediation process should also be considered since they exist as ores mainly in
insoluble forms, while some (Fe, Cu, Zn, Ni) are categorized as heavy metals.
Therefore, there is a recent advancement of research on micronutrient-solubilizing
indigenous microorganisms toward developing sustainable environments (Cai et al.
2013; Kumar and Gopal 2015).
21 Solubilization of Micronutrients Using Indigenous Microorganisms 375
21.6 Indigenous Microorganisms (IMOs)
Indigenous microorganisms refer to a group of beneficial microorganisms that are
native to a given area which are different from effective microorganisms that are
laboratory-cultured mixture of microorganisms (Kumar and Gopal 2015). Ideally
indigenous microorganisms are a mixture of a variety of beneficial microorganisms
yet can also be considered as organized microbial communities. Their ability of
microbial biofilm formation and their microbiome networks in various activities in
the soil have been discussed for the last few years (Mandakovic et al. 2018; Horton
et al. 2019; Akkaya et al. 2020). Their potentiality in plant growth promotion
through processes such as biodegradation, nitrogen fixation, soil fertility improve-
ment, and mineral solubilization has been observed for decades (Umi and Sariah
2006). In addition, their role in bio-composting, biodegradation, bioremediation,
bioleaching, and natural farming has gained attention in the recent past and has been
the focus of many researchers (Dhaliwal et al. 2019; Kumar and Gopal 2015; Gadd
2010; Sangeetha et al. 2020; Saravanabhavan et al. 2020; Sarker and Rahman 2020;
Sharma et al. 2020). These have made them potential tools in developing sustainable
approaches in agriculture, environmental restoration, and safeguarding targets since
they are composed of a natural microbiome (Kumar and Gopal 2015). Depending on
the purpose, a variety of terms are being used to refer to indigenous microorganisms.
In agriculture, they are mostly being termed as “biofertilizers”referring to the
products containing a combination of different types of microorganisms which are
applied to crops in order to increase their quality and quantity. In some other context,
they are named as “plant growth-promoting microorganisms (PGPMs)”which
inhabit in root rhizosphere and considered as bioprotectants of plants (Akkaya
et al. 2020; Yang et al. 2009; Ahmad et al. 2018; Pitiwittayakul and Tanasupawat
2020). As biofertilizers and PGPMs, they lead to crop productivity through decom-
position of organic matter, nutrient acquisition, absorption of water, nutrient
recycling,weed control and bio-control of plant pathogens (Bhardwaj et al. 2014;
Vessey 2003; Sangeetha et al. 2020; Ahmad et al. 2018; Berg et al. 2013) improving
the soil structure and function. Mineralization and solubilization have been identified
as two main methods by which IMOs increase the bioavailability of nutrients by
increasing solubilization which ultimately promote growth and yield of plants
(Vessey 2003). Further, the role of IMOs in bioremediation and bioleaching of
metals has also been investigated and might have an impact on regulating nutrient
contents in soil. Solubilization of nutrients, mainly NPK and to some extent other
macro- and micronutrients, by solubilizing IMOs has widely been researched and
reviewed recently (Djajadi and Hidayati 2020).
376 A. D. S. N. P. Athukorala
21.7 Nutrient-Solubilizing IMOs
Both macro- and micronutrients are originated from minerals deposited under the
Earth’s crust as ores. Many nutrients are metals from which some are considered as
potentially hazardous metals or heavy metals when in high concentrations in soil,
water, and biological tissues. The majority of metals exist as minerals in soil with a
number of mineral forms for each metal element having varying distribution in the
environment with different physicochemical properties (Gadd 2010; Ehrlich and
Newman 2009). The minerals in soil are subjected to various geological processes
such as chemical cycling of elements including mineral formation (mineralization),
mineral deterioration, and chemical transformations of metals, metalloids, and
radionuclides (solubilization/mobilization). Solubilization refers to “the preparation
of a thermodynamically stable isotropic solution of a substance normally insoluble
or very slightly soluble in a given solvent by the introduction of an additional
amphiphilic component or components”(Yadav et al. 2018) so that its availability
is increased. Mineralization refers to the conversion of organic compounds (metals)
into inorganic compounds through various decomposition procedures. Microorgan-
isms significantly contribute to all of these geological processes. Microbes are in
continuous interaction with metals and minerals under natural and artificial environ-
ments. Their interactions alter the physical and chemical state of metals and min-
erals, while microbial growth, activity, and survival are in return affected by the
characteristics of metals and minerals (Gadd 2010). As a result many minerals are
biogenic in origin (biomineralization) and some make structural components for
many organisms such as diatoms (Ehrlich 1996; Gadd and Raven 2010). Most
biominerals are in the form of silicates, calcium carbonates, and iron oxides or
sulfides (Baeuerlein 2000; Bazylinski 2001). All kinds of microbes (bacterial,
fungi, protists) and their symbiotic associations such as lichens and mycorrhizae
contribute actively to the above geological processes (Macalady and Banfield 2003;
Bottjer 2005; Chorover et al. 2007; Konhauser 2007; Gleeson et al. 2007; Gadd
2008), especially metal and mineral transformations (Ehrlich 1996). Specific groups
of microbes that are directly involved in geochemical transformations include both
pro- and eukaryotes such as manganese-oxidizing and manganese-reducing bacteria,
iron-oxidizing and iron-reducing bacteria, sulfate-reducing bacteria, and sulfur-
oxidizing and sulfur-reducing bacteria that can form or degrade silicates, carbonates,
phosphates, and other minerals (Gadd 2007,2010; Ehrlich 1996; Kim and Gadd
2008). In addition, soil microorganisms, especially mycorrhizal fungi (Tao et al.
2008), are solely responsible for nutrient cycling through decomposition of soil
organic matter and also by making chemically fixed nutrients such as phosphorus
(P), zinc (Zn), potassium (K), and iron (Fe) available (Ahmad et al. 2018). In
addition, early stages of soil formation are supported by the activity of microbes
such as lichens through weathering process (Purvis and Pawlik-Skowronska 2008;
Gilmour and Riedel 2009; Uroz et al. 2009). General metabolic activities of all
microbes affect metal distribution and bioavailability through cellular accumulation,
decomposition, or biodeterioration of organic and inorganic substrates (Gadd 2007;
21 Solubilization of Micronutrients Using Indigenous Microorganisms 377
Warren and Haack 2001; Huang et al. 2004). However, mineral and metal solubi-
lization in other terms may have negative contribution when they are potentially
hazardous/heavy metals in certain context such as contaminated soil including solid
wastes (Sayer et al. 1999; Fomina et al. 2004,2005a,b).
21.8 Role of Nutrients in Microorganisms
Microbial growth, metabolism and differentiation require nutrients (Gadd 1992).
Microbes interact with minerals containing nutrients in several ways depending on
the type of nutrient, organism, and environment. All microbes use nutrients for
structural functions and/or catalytic functions (Ehrlich 1997). The structure and the
function of microbes also affect metal speciation and thereby solubility, mobility,
bioavailability, and toxicity of nutrients (Gadd 2010). When these elements are
metals, they particularly interact with microbes in different ways. Firstly, microbes
incorporate trace metals into metalloenzymes or utilize enzyme activation (Wackett
et al. 1989) such as nitrogenase (Mo/Fe or sometimes V/Fe or Fe only), cytochromes
(Fe) and cytochrome oxidase aa3 (Fe, Cu), superoxide dismutases (Fe, Mn, Cu, or
Zn), bacteriochlorophyll (Mg), iron-sulfur proteins, CO dehydrogenase with Ni in
anaerobic bacteria and Mo in aerobic bacteria, NADP-dependent formate dehydro-
genase (W/Se/Fe), and formate dehydrogenase H (Mo/Se/Fe) (Wackett et al. 1989;
Fridovich 1978; Yamamoto et al. 1983; Robson et al. 1986; Scheer 1991; Orme-
Johnson 1992; Boyington et al. 1997). Some, especially by eubacteria and archaea,
use certain metals/metalloids as electron donors or acceptors in energy metabolism
(Ehrlich 1996). The entire energy demand of chemolithotrophs like eubacteria
Thiobacillus ferrooxidans and Leptospirillum ferrooxidans and the archaea
Acidianus brierleyi and Sulfolobus acidocaldarius can be satisfied by oxidizable
metals or metalloids, particularly through oxidation (FeII) to Fe(III) (Ehrlich 1996,
1997). Thirdly, microbes can enzymatically detoxify harmful metals or metalloids
by oxidation or reduction or, when cannot be detoxified, removed from cell interior
using efflux systems (molecular pumps) (Gadd 2010). Anaerobes such as sulfate-
reducing bacteria enzymatically catalyze biocorrosion through cathodic depolariza-
tion. Anaerobic biocorrosion is thought to be regulated by biofilms consisting of a
consortium of a variety of bacteria, often including aerobic, facultative, and anaer-
obic bacteria, each with specific locations in the biofilm (Ehrlich 1997).
Non-enzymatic usage of metals by microbes occurs with accumulating and even
with dead cells, binding them as cations to the cell surface with a passive process
(Gadd 1993).
Despite the positive interactions of microbes and nutrients, their toxicities for
microorganisms can occur through natural geochemical events and anthropogenic
contamination in aquatic and terrestrial ecosystems by domestic, agricultural, and
industrial activities. However, many microbes grow and survive in locations pol-
luted with metals by different mechanisms which contribute to resistance (Avery
2001; Holden and Adams 2003; Verma and Kuila 2019; Tarekegn et al. 2020).
378 A. D. S. N. P. Athukorala
Changes in metal speciation affect the survival of microbes. These changes can be
redox transformations, efflux and intracellular compartmentalization with cell walls,
production of metal-binding peptides and proteins (e.g., metallothioneins,
phytochelatins), active transport, organic inorganic precipitation, and other constit-
uents with metal-binding abilities (Gadd 2010). They also can convert the pollutants
into metabolic intermediates and be utilized as primary substrates for cell growth
(Verma and Kuila 2019). In addition, the presence of plasmids containing resistance
genes (Rosen et al. 2005; Silver and Phung 1996) also affects the activity of bacteria
and fungi toward certain nutrients (Van Ho et al. 2002). Many microbial processes
such as energy generation, cell adhesion, biofilm formation, and nutrient acquisition
(Hochella 2002; Brown et al. 2008) are influenced by minerals and nutrients.
Further, some mineral surface properties such as surface composition,
microtopography, surface charge, and hydrophobicity affect thigmotropism, micro-
bial attachment and detachment, and thereby colonization and biofilm formation
(Brown et al. 2008; Vaughan et al. 2002; Bowen et al. 2007; Gleeson et al. 2010).
Oxides of some micronutrients such as Fe significantly influence microbial activity
by altering soil behavior through soil physical, chemical, and biological processes
(Huang et al. 2005).
21.9 Mechanisms of Solubilization/Mobilization Nutrients
Nutrient solubilization/mobilization from different substrates such as rocks, min-
erals, soil, and others can occur by different processes and can result in volatilization
through protonolysis, complexation by excreted metabolites and Fe(III)-binding
siderophores, chemical oxidation or reduction, indirect Fe(III) attack, and methyla-
tion (Gadd 2010). Other metabolites that are excreted with metal-complexing prop-
erties such as amino acids, phenolic compounds, and organic acids may also play a
role. For example, oxalic acid can form soluble oxalate complexes with nutrients
such as Al and Fe (Strasser et al. 1994). Microbes play a major role in all of these
mechanisms which affect their bioavailability and toxicity. Extracellular compounds
such as enzymes and other metabolic products such as gluconic acid and its
derivatives (Gadd and Sayer 2000; Saravanan et al. 2007; Khan et al. 2013), H
2
S,
formate, or other secondary metabolites produced by microbes regulate redox
processes (Fe) (Ehrlich and Newman 2009). Metal chelators of microbial origin
related to Fe(III) solubilization include oxalate, citrate, humic acids, and tannins.
Methylation is another mechanism by which some microbes such as methanogens,
clostridia, and sulfate-reducing bacteria under anaerobic conditions and fungi (Pen-
icillium and Alternaria), under aerobic conditions, solubilize nutrients (Gadd 2010).
For example, the production of siderophores is the key mechanism by which Fe
assimilation occurs in fungi and bacteria (Kalinowski et al. 2000; Glasauer et al.
2004). Since this chapter is focused on micronutrients, a detailed review of their
importance to plants, deficiency and toxicity symptoms, availability, experimental
records on solubilizing microorganisms, their role in microbial growth and function,
21 Solubilization of Micronutrients Using Indigenous Microorganisms 379
and mechanisms of solubilization is presented in the following section and summa-
rized in Tables 21.1 and 21.2.
21.10 Iron-Solubilizing IMOs
With a significant role in some life-sustaining processes of microbes and plants, iron
is considered to be an essential, multifunctional micronutrient. It is required for the
different physiochemical processes in plants and plays a vital role in the activation of
chlorophyll, photosynthesis, structural component of the chloroplast membrane,
respiration, and synthesis of many iron-sulfur (Fe-S) clusters and heme proteins as
cofactors of proteins that function in the life of plants.
Iron mostly occurs in two oxidation states (+2 and +3) in nature. Plants absorb
iron as Fe
2+
and must be in the general range of >7.7-10 mol L
-1
to avoid any
deficiency (Lindsay and Schwab 1982). The functions of iron are mainly based on
the reversible redox reaction of Fe
2+
(ferrous) and Fe
3+
(ferric) ions. The biosynthe-
sis ALA, which is a precursor in the formation of chlorophyll, might need an
intermediate that contains iron in the electron transfer chain, ferredoxin. This
could control the reduction and activation of one or more enzymes responsible for
ALA formation (Miller et al. 1984). Deficiency symptoms in plants include
interveinal chlorosis in young leaves and stunted growth, while toxicity causes
growth inhibition, reduced chlorophyll synthesis, and inhibition of photosynthesis
(Table 21.1).
Iron is the fourth most prevailing element after O, Si, and Al in the crust and soils.
The forms of Fe in the soil can be categorized into four types as Fe
II
in primary
minerals, Fe
III
in secondary minerals, Fe crystalline minerals and poorly ordered
crystalline (hydro) oxides, soluble and exchangeable Fe, and organic matter-
bounded Fe in soluble or insoluble forms (Colombo et al. 2014).
Iron release by weathering of soil mineral deposits is a very slow process and it is
regulated by pH value, O
2
concentration, and the dissolution-precipitation process of
both crystalline and poorly ordered Fe-hydroxide minerals (Lindsay 1988). Once
mobilized in weathering processes, occurrence of redox reaction and pH conditions
of the soil environment affect the destiny of Fe
II
. Although there is a more than
enough iron (Fe) content in soils for plant requirement, especially in calcareous soils,
bioavailability of Fe is often severely limited. Those types of soils are mainly found
in dry areas of the earth. Plants grown in calcareous soils usually show iron
deficiencies. These plant species have evolved various strategies to enhance their
uptake of iron. However, usually these strategies are not sufficient to avoid Fe
deficiency completely. Hence, soil microbial community plays a significant role in
influencing plant Fe uptake.
The close relationship between microbes and oxides of Fe coexists in soils, and
they provide adequate opportunities for mutual interactions. Primary minerals may
provide Fe as well as many other important nutrients such as K, P, and S
while accommodating microbes in mineral cycling (Lowenstam 1981; Lower et al.
380 A. D. S. N. P. Athukorala
Table 21.2 Summary of micronutrient-solubilizing microorganisms and the mechanisms of
solubilization
Element
Solubilizing
microorganism
Mechanism(s) of
solubilization References
Mo Escherichia coli,
Enterobacter cloacae
strain, Pseudomonas sp.,
Serratia spp.,
Enterobacter sp.,
Acinetobacter
calcoaceticus
Production of
siderosphores
Halmi et al. (2013)
Thiobacillus ferrooxidans
(now Acidithiobacillus
ferrooxidans)
Bioleaching by changing
the soil pH
Frascoli and Hudson-
Edwards (2018)
Klebsiella,Bacillus,
Rhodobacter
Possess NADH-dependent
nitrate reductase caterlizers
Schaechter (2009)
Azotobacter vinelandii Siderophore production,
protochelin and
azotochelin
Hänsch and Mendel
(2009)
Rhizobium bacteria Cofactor for the enzyme
nitrate reductase which is
involved in nitrogen
assimilation
Hänsch and Mendel
(2009)
Rhodobacter capsulatus Possess Mo and Fe nitro-
genases. Mo-nitrogenases
exhibit higher specific
activities than the alterna-
tive nitrogenases
Cu Aspergillus niger Production of organic
acids such as oxalic, citric,
malic, and tartaric acids
Mulligan et al. (2004)
Pseudomonas lurida Promote Cu uptake by
roots and leaves in plants
Kumar et al. (2020)
Bacillus toyonensis Exhibited a considerable
capacity for Cu
2
(OH)
2
CO
3
solubilization, increased
the soluble Cu concentra-
tion in the soil
Sheng et al. (2012)
Penicillium bilaji Chelating mechanisms by
lowering the solution/soil
pH to 4.0
Asea et al. (1988)
Herbaspirillum sp. By metabolic products of
the strain
Govarthanan et al. (2014)
Phosphorus-solubilizing
bacteria (PSB)
Production of low-
molecular-weight organic
acids
Li and Ramakrishna
(2011)
Mycorrhizal colonization Redoxolysis, acidolysis,
and complexolysis
Nouren et al. (2011)
PGPR (plant growth-
promoting bacteria)
Secreting siderophores and
organic acid and by
Ke et al. (2020)
(continued)
21 Solubilization of Micronutrients Using Indigenous Microorganisms 381
Table 21.2 (continued)
Element
Solubilizing
microorganism
Mechanism(s) of
solubilization References
increasing soil organic
carbon content
Zn Aspergillus niger,
A. oryzae,A. nomius
Secretion of gluconic acid
and its 2- and 2,5-keto-
derivatives during growth
via decreasing soil pH
White et al. (1997)
Aspergillus niger Production of citric and
oxalic acid
White et al. (1997)
Aspergillus terreus Decrease in pH through the
production of gluconic
acid
Anitha et al. (2015)
Trichoderma harzianum
Rifai
Releasing Zn
2+
ion via
oxidative dissolution
process
Altomare et al. (1999)
Beauveria caledonica Process of acidolysis,
complex lysis, and metal
accumulation
Fomina et al. (2004)
Ericoid mycorrhizal fun-
gus Oidiodendron maius
Arbuscular mycorrhizae
Production of organic
acids (solubilize ZnO and
Zn
3
(PO
4
)
2
)
Martino et al. (2003),
Subramanian et al. (2009)
Bacillus sp. alone or in
combination
Bacillus pumilus
Bacillus sp. AZ6
Organic acid production
Production of amino acids,
plant hormones, chelating
ligands, and organic acids
via oxido-reductive sys-
tems and proton extrusion
Yadav et al. (2018),
Monreal et al. (2015),
Mahdi et al. (2010), Jha
(2019), Hussain et al.
(2015), Saravanan et al.
(2004)
Bacillus aryabhattai Production of organic
acids
Vidyashree et al. (2018)
Pseudomonas sp.
Pseudomonas
pseudoalcaligenes
P. putida
Organic compound pro-
duction, keto-D-glutarate,
propionic acid, formic
acid, lactic acid, gluconic
acid acetic acid, glycolic
acid, citric acid, fumaric
acid, succinic acid, malic
acid, and oxalic acid.
Auxin production
Soluble carbohydrate
production
Yadav et al. (2018), Jha
(2019), Patten and Glick
(2002), Vazquez et al.
(2000)
P. fragi Production of siderophores Kamran et al. (2017)
Pseudomonas taiwanensis Production of gluconic and
2-keto-gluconic acid
Fasim et al. (2002)
Gluconacetobacter
diazotrophicus
Solubilize insoluble zinc
source especially ZnO,
ZnCO
3
, and Zn
3
(PO
4
)
2
Saravanan et al. (2007)
Azotobacter,Azospirillum Production of chelating
agents
Biari et al. (2008)
(continued)
382 A. D. S. N. P. Athukorala
Table 21.2 (continued)
Element
Solubilizing
microorganism
Mechanism(s) of
solubilization References
Burkholderia,
Acinetobacter
Production of organic
acids
Vaid et al. (2014)
Consortia of Azospirillum
lipoferum Pseudomonas
sp., Agrobacterium sp.
Production of organic
acids
Mengel et al. (2001)
Pantoea agglomerans Auxin production
Extracellular enzyme
production
Kamran et al. (2017)
Enterobacter cloacae Extracellular enzyme
production
Kamran et al. (2017)
Providencia sp.
Anabaena sp.
Calothrix sp.
Anabaena sp.
Rana et al. (2011)
Ni Sphingomonas
macrogoltabidus
Microbacterium
liquefaciens
Microbacterium
arabinogalactanolyticum
Those rhizobacteria are
shown to play an important
role in increasing the
availability of Ni in soil,
thus enhancing Ni accu-
mulation by Alyssum
murale
Abou-Shanab et al. (2003)
PGPR (Pseudomonas sp.) Siderophore production Tank and Saraf (2009)
Aspergillus niger,Asper-
gillus fumigatus,
Acidithiobacillus
ferrooxidans
Solubilize nickel at room
temperature 30–37 C,
whereas organism unable
to solubilize nickel at
higher temperatures 45 C
Mohapatra et al. (2007)
Pseudomonas sp. SRI2,
Psychrobacter sp. SRS8,
Bacillus sp. SN9
Production of indole-3-
acetic acid (IAA),
siderophores, utilization of
1-aminocyclopropane-1-
carboxylic acid (ACC)
Ma et al. (2009)
Azotobacter chroococcum
(N-fixing bacteria), Bacil-
lus megaterium
(P-solubilizer), Bacillus
mucilaginosus
(K-solubilizer), and Bacil-
lus sp. RJ16
pH reduction by producing
acids
Arunakumara et al. (2015)
Cl No available records Most of the micronutrients
are present in the form of
chloride complexes of their
cationic forms. Most of the
soil microorganisms pro-
duce acids and
siderophores and release
into the soil. These
(continued)
21 Solubilization of Micronutrients Using Indigenous Microorganisms 383
Table 21.2 (continued)
Element
Solubilizing
microorganism
Mechanism(s) of
solubilization References
chemical components
reduce the soil pH and
cause the changes of the
pH which facilitate the
breaking down metal-Cl
complexes and release
of chloride in to the soil
Fe Fe-reducing bacteria
Gallionella spp.
Leptothrix spp.
(microaerophilic bacteria)
Shewanella alga
Shewanella putrefaciens
Release of low-molecular-
weight Fe-binding
molecules –siderophores
Colombo et al. (2014)
Fe-oxidizing bacteria
Acidithiobacillus
ferrooxidans
Fe-oxide formation onto
extracellular polymers in
order to enhance metabolic
energy generation
The oxidation of Fe
(II) increases the pH gra-
dient across the cell mem-
brane, which in return
increases the proton
motive force and the
energy-generating poten-
tial of the cells
Graham and Webb
(1991), Alloway (2009)
Leptospirillum
ferrooxidans
Catalysis of sulfide oxida-
tion by ferric iron at very
low pH (0.7–1.0)
Pseudomonas and
Trichoderma genera
By the synthesis and
release of siderophores
Singh (2020)
Mn Acidophilic Mn
solubilizers
Enterobacter sp.
Bacillus cereus
Bacillus nealsonii
Staphylococcus hominis
Enzymatic conversion,
metal effluxing, and
reduction in sensitivity of
cellular targets, intra- or
extracellular sequestration,
and permeability barrier
exclusion
Direct solubilization by
utilization of MnO
2
as a
final electron acceptor in
the bacterial respiratory
chain, instead of oxygen
Indirect solubilization by
the formation of metabolic
reductive compounds
Metal anion protonation,
soluble Mn ligand complex
formation, and production
Samourgiannidis and
Matsi (2013)
(continued)
384 A. D. S. N. P. Athukorala
Table 21.2 (continued)
Element
Solubilizing
microorganism
Mechanism(s) of
solubilization References
of bio-generated organic
acids
Mn oxidation
Leptothrix sp.
Pedomicrobium sp.
Hyphomicrobium,
Caulobacter, or common
Gram-positive or Gram-
negative bacteria, e.g.,
Arthrobacter,Micrococ-
cus,Bacillus,
Chromobacterium,Pseu-
domonas,Vibrio,
Oceanospirillum
Indirect oxidation by the
production of hydrogen
peroxide, free radical, or
oxidant
Direct oxidation
(an enzymatic reaction)
catalyzed by Mn binding
and oxidizing proteins
found in crude or purified
extracts
Gounot (1994)
Mn reduction
Pseudomonas spp.
Bacillus spp.
Corynebacterium
Acinetobacter johnsonii
Pseudomonas
fluorescens
Mn oxide reduction
through a drop of pH
and/or redox potential due
to bacterial metabolism
Reduction through direct
or indirect processes
Mn (IV) be reduced by
inorganically or organic
reductants produced by
microorganisms
Enzymatic Mn
(IV) reduction (e.g.,
Acinetobacter
calcoaceticus)
Maathuis and Diatloff
(2013)
Bacillus polymyxa By coupling of metal
reduction with oxidation of
a non-fermentative carbon
source like lactate
Marschner (2012)
Geobacter
metallireducens,
Shewanella (formerly
Alteromonas)
putrefaciens, a facultative
anaerobe and obligate
respire Shewanella
putrefaciens
Lovley et al. (1993)
B Boron-tolerant microor-
ganisms
Lysinibacillus
boronitolerans
Chimaereicella
boritolerans
Gracilibacillus
boraciitolerans
High B efflux and exclu-
sions which actively pump
boric acid from the cells
and are thus able to main-
tain a lower B concentra-
tion in the cell than in the
external medium
Ahmed and Fujiwara
(2010)
(continued)
21 Solubilization of Micronutrients Using Indigenous Microorganisms 385
2001). Microbes especially by bacteria like Thiobacillium and Metallogenium
sp. dissolve primary minerals which contain iron through various processes which
are termed as solubilization or chelation, sorption, accumulation, transformation, and
precipitation (Colombo et al. 2014; Mengel 1994). These mechanisms are even more
complex within the rhizosphere, due to the activity of the plants. The activity of plant
roots can affect the abundance, diversity, and activity of microbes as well as Fe
availability, and the interactions between Fe minerals and microbes (Colombo et al.
2014).
Iron solubilization mediated by PGPR was reported by Kloepper et al. (1980).
Many microbes belonging to bacterial genus Pseudomonas and fungal genus
Trichoderma are found to possess the ability to solubilize iron (Singh 2020). In
line with Jin et al. (2010), an isolated Pseudomonas sp. could grow and produce
siderosphores under Fe-deficient medium (Jin et al. 2010). Their work also showed
that phenolic compounds exuded from plant (red clover) roots under Fe-deficient
conditions favor the rhizosphere microbes to secrete more siderosphores which help
to improve plant iron uptake.
Reduction or oxidation of iron minerals provides energy for anaerobic ferric-
reducing and ferrous-oxidizing bacteria. This apparently plays an important role in
catalyzing iron transformations in anoxic environments. Lithotrophic acidophilic
and neutrophilic bacteria oxidize ferrous iron aerobically (Harrison Jr 1984). The
acidophile, Acidithiobacillus ferrooxidans, is the most widely studied of all iron
oxidizers. Phototrophic purple, non-sulfur bacteria were found capable of anaerobic
ferrous iron oxidation by utilizing ferrous iron as electron donor in the light (Widdel
et al. 1993). Desulfuromusa kysingii,Geospirillum barnesii,Rhodobacter
capsulatus,Desulfofrigus oceanense,Desulfotalea psychrophila,Geobacter
metallireducens,G. sulfurreducens, and Shewanella putrefaciens (now
S. oneidensis) are some of the reported iron-reducing bacteria (Straub et al. 2001).
Table 21.2 (continued)
Element
Solubilizing
microorganism
Mechanism(s) of
solubilization References
Bacillus boroniphilus
Arthrobacter sp.
Rhodococcus sp.
Lysinibacillus sp.
Algoriphagus sp.
Boron accumulators
Variovorax
boronicumulans
Miwa et al. (2008)
Boron uptake promoters
Bacillus pumilus
Masood et al. (2019)
386 A. D. S. N. P. Athukorala
21.11 Mechanisms of Fe Metabolism
The most accepted mechanism for iron solubilization by microbes is by production
of siderosphores under iron-deficient growth conditions. Siderophores are chelating
agents that are secreted by bacteria and fungi with formation constants for ferric iron
in the range of 10
25
to 10
35
and in exceptional cases as high as 10
51
(Hider 1984).
Iron availability in the surrounding environment highly regulates the siderophore
production of microorganisms (Kalinowski et al. 2000). Siderosphores differ in
structure and are low in molecular mass. The main groups of siderophores are
catecholates, hydroxamates, and carboxylates. The catecholate is a main siderophore
which is produced by bacteria, whereas fungi produce hydroxamate (Miethke and
Marahiel 2007; Hider and Kong 2010). Stable soluble complex, made by iron with
siderophore in soil solution and at the mineral surface, makes them available for
uptake by the cell membrane of plant roots. The siderophore is either destroyed or
recycled during this reduction in some cases.
There are three different mechanisms in transporting siderophore combined with
Fe across the cell membranes in microorganisms. Membrane-spanning proteins may
involve in binding these to the substrate which are transported into the cell after
undergoing a conformational change. The location of Fe release mechanisms differs.
Alternatively, Fe may also be removed by hydrolytic destruction of the chelate. The
second mechanism is termed as the direct shuttle (Crowley et al. 1991). In that
mechanism, ferric siderophore binds to a cell surface receptor where Fe is cleaved
and simultaneously transported without concomitant transport of the desferri
siderophore. In the third mechanism, an indirect shuttle (extracellular dissociation)
acquires Fe in which Fe is removed through reduction at a site some distance to the
carrier protein (Crowley et al. 1991).
21.12 Manganese-Solubilizing IMOs
Manganese is an essential plant trace element that plays a significant role in plant
metabolism and development but could be toxic at high concentrations. Mn occurs in
many oxidation states like II, III, and IV in approximately 35 enzymes of a plant cell
(Hebbern et al. 2009). In plant proteins, manganese acts either as a catalytically
active metal or as an activator of enzymes. Superoxide dismutase which contains
manganese protects the cells from the damaging effect of free radicals, oxalate
oxidase, and manganese-containing water splitting system of photosystem
II. Manganese activates PEP carboxykinase, malic enzyme, isocitrate dehydroge-
nase, and phenylalanine ammonia-lyase enzymes (Hänsch and Mendel 2009). Mn
plays an important role in the synthesis of lignin along with Cu and provides
resistance in root tissues to pathogens. Symptoms of Mn deficiency in plants include
chlorosis, premature leaf fall, and delayed maturity, while toxicity causes reduced
yields and stunted growth (Table 21.1).
21 Solubilization of Micronutrients Using Indigenous Microorganisms 387
Manganese is the fifth most abundant metal found on the earth’s surface. In the
Earth’s crust, Mn is mainly found as minor components of rock-forming silicate
minerals such as olivine, pyroxenes, and amphiboles along with Fe. Manganese
oxide (pyrolusite) and Mn carbonate (rhodochrosite) minerals are among the most
important Mn ore resources in the world. Mn is extensively available in deposits of
complex ores, nodules on ocean floors (Patrick 2010), wastewater sludge (Wang
et al. 2011), and municipal solid wastes (Abdulsalam et al. 2011). Reduced soluble
or adsorbed Mn (II) and insoluble Mn (III) and Mn (IV) oxides are the naturally
found forms of Mn in soil. Solubility and availability of Mn in soil are increased with
increasing state of reduction. Oxidation of Mn in soil is basically a biological
process, while reduction of Mn may be either chemical or biological. Mn availability
in the rhizosphere soil depends on the redox condition and the pH ranges (Gounot
1994).
Both oxidation and reduction of manganese in natural environments is domi-
nantly promoted by microbial catalysis, but abiotic converters are often important
too and it may compete with the biological processes (Gounot 1994). Oxidation of
Mn has been reported by many types of microorganisms such as fungi, bacteria, and
algae (Ghiorse 1984). Most of them are bacteria that belong to common Gram-
positive or Gram-negative bacteria or group of sheathed bacteria, Leptothrix, and
budding and appendaged bacteria: Pedomicrobium,Hyphomicrobium, and
Caulobacter (Gounot 1994). The demosponge Suberites domuncula was found to
have a Mn-oxidizing bacterium by Wang et al. (2011) which belongs to Bacillus
strain BAC-SubDo-03. Most Mn-oxidizing bacteria are heterotrophic aerobic bac-
teria that use organic substances as the substrate. Some rhizosphere bacteria like
Bacillus,Pseudomonas, and Geobacter can reduce oxidized Mn
+4
into Mn
+2
which
is the plant metabolite form of Mn.
Effective rhizosphere Mn-reducing bacteria (Pseudomonas sp.) have been
reported by Marschner and Dell (1994). Most of Mn-oxidizing bacteria are hetero-
trophic aerobic bacteria that grow on organic substances. The bacterial isolates
Bacillus anthracis,Acinetobacter sp., Lysinibacillus sp., and Bacillus sp. are capable
of solubilizing Mn in a range of pH (Ghosh et al. 2016). Bacillus thuringiensis has
been found to have the capability to tolerate high concentrations as 4000 mg L
1
of
Mn (II) and the highest removal rate of Mn (II). Hence, Bacillus thuringiensis plays a
significant role in detoxifying and immobilizing excessive Mn in soil (Huang et al.
2020). Many fungi like Acremonium spp. can also take part in manganese oxidation
(Miyata et al. 2004). The white rot fungus Ganoderma lucidum has possessed a good
potential in solubilizing Mn under shaking and non-shaking conditions (Nouren
et al. 2011). The soil fungi Aspergillus niger and Serpula himantioides have shown
the ability to tolerate and solubilize manganese oxides (Wei et al. 2012).
388 A. D. S. N. P. Athukorala
21.13 Mechanisms of Mn Solubilization
The possible mechanisms of Mn oxidation by microorganisms can be described as
direct or indirect. Production of hydrogen peroxide, free radical, or oxidant indicates
the indirect oxidation of Mn which is due to the change of the surrounding environ-
ment. Arthrobacter and Leptothrix like bacterial groups are found to oxidize Mn by
producing hydrogen peroxide as a mechanism of protecting the cells from the
harmful effects of hydrogen. Direct oxidation is an enzymatic reaction which is
facilitated through Mn binding and oxidizing enzymes which are found in crude or
purified extracts. Examples can be found as a spore protein of Bacillus SG-1
(de Vrind et al. 1986) and an intracellular protein of a Pseudomonas sp. (Jung
et al. 1998). G. lucidum was able to solubilize Mn by production of organic acids
such as citric acid, tartaric acid, and oxalic acid (Nouren et al. 2011). In addition,
roots and rhizosphere bacteria produce chelating agents like phenolic compounds
and organic acids and other elements and hence avoid precipitation of Mn
(Marschner and Dell 1994).
21.14 Zinc-Solubilizing IMOs
Zinc is another vital micronutrient for normal growth and development of plants.
The normal concentration range for zinc in plant tissue is 15–60 ppm. Zinc require-
ment in plants is 30–100 mg kg
1
, below which would result in its deficiency. Plants
require Zn for optimum fruit size, crop production, and yield. It is also used in the
carbonic anhydrase activity involved in photosynthetic tissues for biosynthesis of
chlorophyll (Xi-Wen et al. 2013). Further, Zn plays a key role in the synthesis of
protein, activation of enzymes, RNA and DNA synthesis and regulations, and
oxidation and metabolism of carbohydrates and prevents the peroxidation of lipids
and proteins due to reactive oxygen species. Zinc is important for auxin production
and for normal fruit and flower development. In plants, genes responsible for
environmental stress tolerance are Zn dependent (Hafeez et al. 2013). Several studies
reported that the use of zinc-containing fertilizers and micronutrients improves crop
quality (Hussain et al. 2018).
Soil contains considerable amounts of Zn but in insoluble forms. Zinc is easily
mobile in oxidizing acid soils, whereas it is immobile in poorly reducing neutral or
alkaline soils. In the soil solution, Zn is a divalent cation or complexes with ligand
via different transporter systems which is carried by mass flow, diffusion, and root
extension in the direction of roots. A majority of the Zn absorption happens via
active transport system, and it is transported from the root to the shoots via both
xylem and phloem tissues. A little amount of Zn is retained at the basal node which
governs the distribution of Zn in plants (White et al. 1997). The deficiency symp-
toms appear in the new leaves due to its immobility. Symptoms depend on the crop.
Deficiency symptoms are expressed as preliminary in young leaves and could be
21 Solubilization of Micronutrients Using Indigenous Microorganisms 389
visualized as patterns of chlorosis of the new leaves (often interveinal) and necrotic
spots on the margins or leaf tips (Table 21.1). These affected leaves are smaller in
size and form leaf rosette. The shortened internodes give the plant a rosette appear-
ance and poor bud development which result in reduced branching and flowering.
Crops with Zn deficiency may have susceptibility to injury or infection (Ghosh et al.
2014; Gandhi and Muralidharan 2016).
Several studies have found that inoculations of potent strain of Zn mobilizer
rhizobacteria have been found to increase the yield of field crop such as rice wheat
barley and maize (Hussain et al. 2018; Kutman et al. 2010; Tariq and Ashraf 2016).
For example, Zn-mobilizing PGPR inoculation had a significant impact on root
weight (74%), root length (54%), root area (75%), root volume (62%), shoot weight
(23%), and panicle emergence index (96%) (Kutman et al. 2010) which exhibits
potential in mitigating Zn deficiency in soils and crops.
The composition of Zn in polluted soils is dependent on both soil location and
sources of pollution (Kabata-Pendias and Pendias 2001). Zinc distribution in agri-
cultural soils ranges from 10 to 300 mg kg
2
. Under anaerobic conditions, higher
concentration of iron reduces the zinc bioavailability in soil (Hussain et al. 2018).
Geochemical composition and weathering of the parent rock will determine the Zn
content in the developing soil. Environmental pollution or Zn-rich products can add
up and alter the parent rock composition. Zn composition in the Earth’s crust is
78 mg kg
1
which varies in different parent rocks. In soil, active Zn occurs either in
the divalent form (Zn
2+
) or in complex form like ZnOH
+
, ZnHCO
3+
, Zn(OH)
3
, and
ZnO
2
. Zinc exists in five different pools within the soil, namely, water-soluble,
organically bound, exchangeable, chelated, and adsorbed. The strength of these
forms will determine their ability to plant uptake and leaching.
The bioavailability of Zn to plants is influenced by total Zn contents in the soil,
soil pH, elevated concentration of cations (Na, Ca, and Mg), phosphate soluble
forms, anion bicarbonate, soil organic matter, and CaCO
3
content. Zn is strongly
adsorbed on calcium carbonate (CaCO
3
), magnesium carbonates (MgCO
3
), iron
oxide (FeO), or manganese oxide (MnO) (Alloway 2009). Chemical fertilizers
indirectly affect the conversion of soluble Zn into different insoluble Zn fractions.
Therefore, several studies have suggested the use of biofertilizers containing Zn-
solubilizing microbes to increase the soluble Zn concentration in the soil (Kamran
et al. 2017).
Several microorganisms play a significant role in solubilization and mobilization
Zn (Kamran et al. 2017; Fasim et al. 2002; Javed et al. 2018). Several studies have
reported the effectiveness of rhizospheric fungi in solubilizing insoluble Zn com-
pounds both in vitro and in vivo. The production of organic acids by microorganisms
has been found to increase solubilization and release of Zn compounds (Agusto da
Costa and Duta 2001). Some filamentous non-mycorrhizal fungi, namely, Aspergil-
lus niger,A. oryzae, and A. nomius isolated from a Zn mining site at Tak Province,
Thailand, showed that the solubilization of insoluble Zn compounds ZnO,
Zn
3
(PO
4
)
2
, and ZnCO
3
occurs through the secretion of gluconic acid and its 2-
and 2,5-keto-derivatives during growth (White et al. 1997). Release of Zn from
organic complexes and calcium carbonate is facilitated by microbes through
390 A. D. S. N. P. Athukorala
mineralization and solubilization, respectively. It has been reported that Aspergillus
niger solubilizes insoluble ZnO, Zn
3
(PO
4
)
2
, and Ca
3
(PO
4
)
2
to soluble form through
the production of citric and oxalic acid (White et al. 1997). Aspergillus terreus
(ZSF-9) isolated from Tiruppur District, India, was found to solubilize ZnO, ZnCO
3
,
and Zn
3
(PO
4
)
2
through the production of gluconic acid (Anitha et al. 2015).
Trichoderma harzianum Rifai 1295-22 (T-22) converts insoluble Zn present in the
soil into soluble form by releasing Zn
2+
ion via oxidative dissolution process. During
the process, fungus releases a complex compound which segregates Zn
2+
, resulting
in the enhancement of dissolution of metallic Zn in the soil. Fungus Beauveria
caledonica converts insoluble Zn
3
(PO
4
)
2
into soluble Zn through the process of
acidolysis, complex lysis, and metal accumulation (Fomina et al. 2004). Similarly,
ZnO and Zn
3
(PO
4
)
2
can be solubilized by the ericoid mycorrhizal fungus
Oidiodendron maius (Martino et al. 2003).
Bacterial species such as Pseudomonas striata,Gluconacetobacter
diazotrophicus,Thiobacillus thiooxidans,Burkholderia cenocepacia,Pseudomonas
pseudoalcaligenes,P. fluorescens,P. japonica,P. fragi,Acinetobacter,Serratia
marcescens,S. liquefaciens,Enterobacter cloacae, and Pantoea agglomerans and
several cyanobacterial species have been reported to solubilize insoluble Zn
(Kamran et al. 2017; Zaheer et al. 2019; Altomare et al. 1999; Bapiri et al. 2012;
Abaid-Ullah et al. 2015; Hussain et al. 2015). However, some Bacillus sp. (Bacillus
subtilis,Bacillus pumilus,Bacillus thuringiensis,Bacillus aryabhattai) alone or in
combination with cheaper insoluble Zn such as ZnO, ZnCO
3
, and ZnS has been
suggested as an effective alternative to costly ZnSO
4
and found to be more effective
than other Zn solubilizers (Mahdi et al. 2010; Pawar et al. 2015; Mumtaz et al. 2017;
Jha 2019; Zaheer et al. 2019).
21.15 Mechanisms of Zn Solubilization
Microorganisms can solubilize Zn by either a single mechanism or multiple mech-
anisms. As for other micronutrients, soil pH affects the availability of Zn where a
100 times increase in solubility can be achieved by decreasing one unit in pH
(Mumtaz et al. 2017; Havlin et al. 2005). In addition, Pseudomonas, Bacillus spp.
(Saravanan et al. 2004) and arbuscular mycorrhizae (Subramanian et al. 2009) were
observed to reduce pH in the solubilization of ZnS, ZnO, and ZnCO
3
. The main
mechanism by which plant growth-promoting bacteria improve the Zn availability is
by releasing organic acids like gluconate (Saravanan et al. 2011) or the derivatives of
gluconic acids, e.g., 2-ketogluconic acid (Fasim et al. 2002), 5-ketogluconic acid
(Saravanan et al. 2007), and various other organic acids (Tariq et al. 2007) and
extrude protons (Fasim et al. 2002; Wu et al. 2006). Bacillus sp. AZ6 was found to
secrete organic acids like cinnamic acid, ferulic acid, caffeic acid, chlorogenic acid,
syringic acid, and gallic acid in a liquid medium (Hussain et al. 2004). Mycorrhizal
fungi were also observed to secrete organic acids (Martino et al. 2003) to solubilize
Zn from insoluble Zn
3
(PO
4
)
2
and ZnO. Organic acids produced by some Bacillus
21 Solubilization of Micronutrients Using Indigenous Microorganisms 391
sp. and Pseudomonas sp. include keto-D-glutarate, propionic acid, formic acid, lactic
acid, gluconic acid acetic acid, glycolic acid, citric acid, fumaric acid, succinic acid,
malic acid, and oxalic acid. Three ZSB isolates were found to produce 11 organic
acids against ZnO, ZnCO
3
, and Zn
3
(PO
4
)
2
including lactic acid, malonic acid, malic
acid, citric acid, and succinic acid being the major acids. Bacillus aryabhattai
produced many organic acids during Zn solubilization process as compared to
Pseudomonas taiwanensis and other Bacillus sp. Organic acid secretions by Bacillus
and Pseudomonas were dependent on the substrate of Zn minerals (Vidyashree et al.
2018). Some Bacillus strains were found to produce some other compounds in
relation to Zn solubilization such as amino acids and plant hormones (Saravanan
et al. 2004) and glucose or sucrose (Gluconacetobacter diazotrophicus) (Saravanan
et al. 2007). Enhancement of chlorophyll, carotenoid, and antioxidant enzymes
catalase (CAT)- and peroxidase (PO)-related functions by Bacillus pumilus and
Pseudomonas pseudoalcaligenes had been reported to protect plants from salinity
injuries. Further plants inoculated with the above ZMB also accumulated soluble
carbohydrates in leaves, helping plants to overcome osmotic stress under salinity.
Further, both bacterial isolates were positive for auxin production,
P. pseudoalcaligenes showing more than B. pumilus in the presence of Zn in the
medium compared to the control (Jha 2019). Auxin production in response to Zn has
also been observed by Patten and Glick (2002)inP. putida which increased the
length of canola seedling roots.
Zn-chelating compounds released by plant roots facilitate Zn solubilization by
microbes in the rhizosphere (Obrador et al. 2003; Velazquez et al. 2016). Bacterial
metabolites reduce reaction of Zn in the soil by forming complexes with Zn
2+
(Tarkalson et al. 1998). At the root surface, Zn chelates the ligand (Zn
2+
). Pseudo-
monas monteilii,Microbacterium saperdae, and Enterobacter cancerogenesis are
thought to manufacture Zn-chelating metallophores (Whiting et al. 2001). A
biofertilizer containing Pseudomonas sp. (96-51), Azospirillum lipoferum
(JCM-1270, ER-20), and Agrobacterium sp. (Ca-18) produces chelating agent
ethylenediaminetetraacetic acid (Tariq et al. 2007), and Penicillium bilaji was
reported to increase Zn bioavailability to plants through chelating mechanism
(Kucey 1988).
Zinc bioavailability can also be increased by facilitating nutrient uptake from long
distance through improving root growth and surface area. Mycorrhizal fungi can
change the root architecture which enables plant to obtain Zn from a distance of
40 mm from the root surface (Burkert and Robson 1994). An increase in Zn
concentration up to 4% in cereal grains and increased root length by mycorrhizal
fungus were reported (Subramanian et al. 2009). Tariq et al. (2007) reported
significant increase in weight, length, and volume of root with biofortification of
rice with Zn-solubilizing bacteria.
392 A. D. S. N. P. Athukorala
21.16 Copper-Solubilizing IMOs
Copper is another micronutrient needed for the growth of plants. Among many roles,
it involves several enzyme processes and is the key to the formation of chlorophyll.
Its normal range in most tissues is between 3 and 10 ppm. Cu requirement in plants is
3–20 mg kg
1
and below this range would result in deficiency. Although copper
deficiencies or toxicities rarely occur, deficiencies have been reported in several
parts of the world and need to be addressed since either extremes can have a negative
influence on crop growth and quality. Cu ions act as a cofactor in enzymes such as
Cu/Zn-superoxide dismutase (Cu/ZnSOD), cytochrome oxidase, ascorbate oxidase,
amino oxidase, laccase, plastocyanin, biogenesis of molybdenum cofactor, and
polyphenol oxidase (Krämer and Clemens 2006). Cu plays a vital role in the
signaling of the transcription protein trafficking machinery, cell wall metabolism,
iron mobilization, and oxidative phosphorylation and oxidative stress protection at
cellular level (Yruela 2009; Puig et al. 2007). Cu is also required in photosynthesis
and plant respiration electron transport chains and plant metabolism of carbohy-
drates and proteins. Its ethylene-sensing ability supports to intensify flavor and color
of vegetables and flowers. Copper also acts as a structural element in certain
metalloproteins (Pilon et al. 2006).
The deficiency symptoms of copper occur in the newer leaves. Symptoms vary
depending on the crop. The symptoms are slight chlorosis of either in the whole leaf
or between the veins of the new leaves. Small necrotic spots may form within the
chlorotic areas on the leaf margins. The newest leaves are smaller in size, lose their
sheen, and ultimately may wilt. Necrosis occurs on the apical meristems, which leads
to death, inhibiting the growth of lateral branches. Lighter colored flowers than
normal are produced by the plant (Welch and Shuman 1995; Tripathi et al. 2015).
Copper stress condition of the plant can cause burning of the root tips and thereby
causes excess lateral root growth (Franco et al. 2011). When the copper concentra-
tion is higher in the soil, iron and sometimes molybdenum or zinc nutrients have to
compete with copper for micronutrient availability in the soil and plant uptake (Tyler
and Olsson 2001). Affected plants can exhibit symptoms of iron deficiency or other
micronutrient deficiencies. Copper toxicity ultimately can reduce branching.
Legumes have a tendency to be the most sensitive plants to copper toxicity
(Carruthers 2016a,b).
The composition of Cu in polluted soils is dependent on both sources of pollution
and soil location. Cu level in the environment has been increased by the mining of
Cu-containing ores and industrial activities (Engelhardt et al. 2020). In soil, active
Cu occurs mostly in the divalent form (Cu
2+
) or in complex form with soil organic
matter. The largest portion of Cu is usually present in the crystal lattices of primary
and secondary minerals. The Cu ion is adsorbed to organic and inorganic negatively
charged groups and is dissolved as Cu
2+
and organic Cu complexes in the soil
solution. Also it is specifically adsorbed to soil organic matter, carbonates,
phyllosilicates, and hydrous oxides of Fe, Mn, and Al. The strength of these forms
will determine their ability to plant uptake and leaching (Engelhardt et al. 2020).
21 Solubilization of Micronutrients Using Indigenous Microorganisms 393
The bioavailability of Cu in soil is influenced by physical, chemical, and biolog-
ical properties at soil-root interface in rhizosphere. It is affected by different prop-
erties such as organic matter, soil type, pH, soil moisture, clay particles, temperature,
retention, permeability, and different metal ions and their oxides (Hinsinger et al.
2009). In general, the bioavailable form of Cu
2+
in the soil solution is decreased
dramatically with an increasing soil pH. However, organic Cu complexes may
dissolve at higher pH (Kumar et al. 2020). Calcareous or alkaline soils can limit
the phytoavailability of Cu. Hence, the ability of plants to efficiently uptake Cu from
soil solution, and distribution of this among different organs and tissues can strongly
affect the crop growth and yield under Cu-limiting conditions (Migocka and Malas
2018).
Some micronutrients including Cu have limited mobility in soils which are
transported to roots by slow diffusion. Even though Cu is usually present in large
quantities in the bulk soil, the plant-available fraction in the rhizopheric soil solution
can be insufficient to satisfy plant requirements. Copper allocation in the soils was
found to be in the order of strong organic >residual >water soluble >ion
exchangeable >carbonate >reducible >weak organic fractions, indicating that
Cu is more distributed in organic fraction (84.67%). High affinity of Cu to organic
matters make organic bound Cu distributed in the soil in large quantities. Cu in the
crystalline lattice of the residual fraction cannot be easily released (Govarthanan
et al. 2014). As one of the strategies to overcome this problem, microorganisms have
been investigated in many studies.
Both bacterial and fungi have shown Cu-solubilizing ability (Table 21.2). Some
filamentous non-mycorrhizal fungi, namely, Penicillium and Aspergillus, have been
the most active metal-leaching fungi (Burgstaller and Schinner 1993). Several
studies have shown the potential of Aspergillus niger to generate organic acids
such as oxalic, citric, malic, and tartaric acids which resulted in maximum solubi-
lization of Cu, Zn, and Ni (Mulligan et al. 2004). Bioleaching of Cu from ores has
been done with Penicillium simplicissimum (Sukla and Panchanadikar 1993).
Several studies have reported the effectiveness of rhizosphere microorganisms in
solubilizing insoluble Cu compounds both in vitro and in vivo. They mobilize and
degrade organic pollutants. Cu-resistant bacterium, Pseudomonas sp. DGS6 isolated
from a natural Cu-contaminated soil, stimulated root elongation of maize and
sunflower (Yang et al. 2013). Pseudomonas lurida strain EOO26 was found to
increase Cu uptake by 8.6-fold by roots and 1.9-fold by leaves in inoculated plants
(Kumar et al. 2020). The bacteria isolated from the rhizosphere, Elsholtzia
splendens, significantly increased the bioavailability of Cu while stimulating the
other heavy metals like Zn in the soil (Chen et al. 2005). Increasing Cu bioavail-
ability and bioaccumulation with inoculations was observed to be species-specificin
certain cases. The influence of Bacillus spp.on Cu bioavailability and the
bioaccumulation for ryegrass and fescue were different to each other (Ke et al.
2020). Similarly, Liu et al. (2014) found that inoculations of 11 PGPR strains
increased the Cu concentration in Oenothera erythrosepala and Medicago sativa,
but decreased that in Pennisetum purpureum, which suggests the complex interac-
tions between plants, microbes, and the soil (Liu et al. 2014). The addition of
394 A. D. S. N. P. Athukorala
Bacillus toyonensis alone had the maximum effect on Cu
2
(OH)
2
CO
3
solubilization
(Sheng et al. 2012). Penicillium bilaji was able to solubilize cuprous and cupric
oxide, cupric carbonate. This is mainly by chelating mechanisms under a low pH
value as 4.0 (Asea et al. 1988). This mechanism involves the use of organic acids
which have been reported to have phosphorus-solubilizing abilities (Khan and
Bhatnagar 1977). Another study reported Cu leaching ability of Herbaspirillum
sp. from ion-exchangeable, reducible, strong organic, and residual fractions
(Govarthanan et al. 2014). The maximum solubilization (40%) in strong organic
fractions was suggested to be resulted by the metabolic products of the microbe
(Deng et al. 2012). The metabolites of microorganisms can act as indirect reactive
species and solubilize metal sulfides and oxides during the bioleaching process
(Mishra et al. 2008).
21.17 Mechanisms of Cu Solubilization
There are no clear mechanisms defined for Cu solubilization by microbes. Most of
the literature suggest that microorganisms release chemical compound siderosphores
that have ability to oxidize Cu ore, making them available to plants. However, the
principal mechanisms of bioleaching of metal by fungi are documented to be
redoxolysis, acidolysis, and complexolysis. The fungi are also found to produce
organic acids such as citric, oxalic, malic, and gluconic acids during bioleaching
which might contribute to solubilization (Mulligan et al. 2004; Johnson 2006). In
addition, siderophores, organic acids and soil organic carbon content increased by
the activity of PGPR improve soil Cu bioavailability (Ke et al. 2020). Phosphorus-
solubilizing bacteria (PSB) also enhance Cu availability (Li and Ramakrishna 2011)
by secreting low-molecular-weight organic acids. Many organic acids such as malic,
lactic, 2-ketogluconic, citric, oxalic, glycolic, malonic, valeric, piscidic, tartaric,
formic, and succinic have been identified as chemical compounds secreted by
PSB, which have chelating properties.
21.18 Nickel-Solubilizing IMOs
Nickel is considered as an essential micro-element for plant growth since the late
1980s (Brown et al. 1987). The usual range for nickel in most plant tissue is between
0.05 and 5 ppm (Chen et al. 2009). Nickel is required to the plant as a component of
certain plant enzymes like urease, superoxide dismutase, and hydrogenase. Legume
plants use nickel as a catalyst in nitrogen fixation enzymes (Ahmad et al. 2012). It is
required for urease enzyme which metabolizes urea nitrogen into usable ammonia in
the plant. It prevents the accumulation of toxic levels of urea in plant tissues forming
necrotic legions on the leaf tips. Therefore, the deficiency of nickel in plant can cause
urea toxicity (Krämer 2005), reduced leaf size, disruption of amino acid metabolism,
21 Solubilization of Micronutrients Using Indigenous Microorganisms 395
and urea accumulation in leaf (Bai et al. 2006). Nickel provides tolerance to plant
diseases; however, the mechanism is unclear.
Nickel deficiency is unusual and minor and is often difficult to identify due to less
symptom development. In certain cases, it can reduce yield and growth of plants. As
nickel is a mobile element, its deficiency symptoms first appear typically in the
mature leaves of the plants (Chen et al. 2009). In legume plants, deficiency causes
whole leaf chlorosis along with necrotic leaf tips due to the increased levels of urea.
In woody ornamentals, deficiency causes shortened internodes and it gives a rosette
appearance to the plant, weak shoot growth, death of terminal buds, and eventual
death of shoots and branches. The symptoms in pecans include decreased expansion
of the leaf blade and necrosis of the leaf tips (Bai et al. 2006). Nickel turns out to be
less available for plant uptake at higher pH of the soil or in growing media. Some
other micronutrients like magnesium, zinc, iron, copper, cobalt, or cadmium in high
amounts in the growth medium can result in nickel deficiency to the plant. Legumes
(beans and alfalfa), barley, pecans, peach, plum, wheat, citrus, and certain wetland
plants are some plants which are most sensitive to nickel deficiency (Merlot 2020).
The Earth’s crust composition is comprised of approximately 3% of Ni and it is
the 24th most abundant element. Its concentration in plant leaves ranges from 0.05 to
5mgkg
1
, which is equal to 0.05–5 ppm on a dry weight basis. The required content
of Ni in vegetative tissues of plants is between 2 and 4 ng g
1
dry biomass (Dalton
et al. 1988) and up to 90 ng g
1
dry biomass in barley. Nickel concentrations
10 ppm are generally considered to be toxic to sensitive species. Ni
2+
is the
available form of Ni for plant. Rapid oxidation of Ni ion (Ni
2+
) to unavailable
forms in the soil makes total Ni concentration not a useful measure for Ni bioavail-
ability. Thus, plants grown in high pH soils are vulnerable to Ni deficiency (Brown
et al. 1987). In soil, active Ni occurs almost exclusively in divalent form (Ni
2+
)orin
complex form with soil organic matter. High pH soils can cause Ni deficiency. Other
than that, excessive use of Cu and Zn can result in Ni deficiency in soil because Ni,
Cu, and Zn share a common nutrient uptake system in the plant.
Rhizosphere microorganisms play a major role in Ni solubilization. Three bacte-
ria, Sphingomonas macrogoltabidus,Microbacterium liquefaciens, and
Microbacterium arabinogalactanolyticum, isolated from the rhizosphere of Alyssum
murale, were observed to increase Ni uptake into the shoot by 17%, 24%, and
32.4%, respectively, compared to the non-inoculated control (Abou-Shanab et al.
2003). Tank and Saraf (2009) suggested that PGPR (Pseudomonas sp.) positively
influence the growth of plants and also facilitate plant growth in Ni-contaminated
soils. Mohapatra et al. (2007) reported that Aspergillus niger,Aspergillus fumigatus,
and Acidithiobacillus ferroxidans solubilized nickel at room temperature, 30–37 C,
whereas organisms were unable to solubilize nickel at higher temperatures as 45 C.
In a pot experiment, inoculation of plants (Brassica juncea and B.oxyrrhina) with
Ni-mobilizing strains of Pseudomonas sp. SRI2, Psychrobacter sp. SRS8, and
Bacillus sp. SN9 maximized the biomass of the plants. In addition, strain SN9 was
observed to increase Ni concentration in the root and shoot tissues of B.juncea and
B.oxyrrhina (Ma et al. 2009).
396 A. D. S. N. P. Athukorala
21.19 Mechanisms of Ni Solubilization
The possible mechanisms of Ni solubilization by microbes include pH changes in the
soil, siderophore production, and phosphate solubilization (Burd et al. 2000).
Siderophore production in relation to Ni has significantly increased the size and
chlorophyll content of leaf (Tank and Saraf 2009). Bacteria such as Azotobacter
chroococcum (N-fixing bacteria), Bacillus megaterium (P-solubilizer) and Bacillus
mucilaginosus (K-solubilizer), and Bacillus sp. RJ16 were reported to decrease the
pH by producing acids which enhance the bioavailability of Ni in the soil
(Arunakumara et al. 2015). Zaidi et al. (2006) reported a reduction in pH from 7.5
to 4.8 with the solubilizing Bacillus subtilis SJ-101, resulting in increased Ni
availability. In addition, acidic soil conditions created by phosphate solubilization
have shown to increase Ni accumulation in the presence of some bacteria (Rajkumar
et al. 2008). Ma et al. (2009) reported the production of indole-3-acetic acid (IAA)
and siderophores and utilization of 1-aminocyclopropane-1-carboxylic acid (ACC)
by Ni-mobilizing bacterial strains.
21.20 Chlorine-Solubilizing IMOs
Chlorine is another micronutrient which is needed for the proper growth and
processes in plants including osmotic and stomatal regulation, evolution of oxygen
in photosynthesis, and disease resistance. Plant uses the ion (Cl
) rather than the gas
(Cl
2
). In chloroplast, chloride is a structural constitute of photosystem II in the
oxygen-evolving complex which is one of the three important cofactors (Kusunoki
2007). Chloride stimulates the ATPase at the tonoplast. However, excess chloride is
accumulated in certain tissues such as guard cells, and their opening and closing is
regulated by the flux of potassium and anions such as malate and chloride and
therefore important for plant photosynthesis. It also maintains the rigidity of leaves
(Chen et al. 2010).
Reports of Cl deficiency are rare in agriculture (Dordas 2008). Fairly a larger
amount of Cl application was reported to enhance disease resistance in plants. These
amounts are much higher than those required as a micronutrient but far less than
those required to induce toxicity (Mann et al. 2004). Cl has been shown to be
effective on a number of diseases such as stalk rot in corn, stripe rust in wheat,
take-all in wheat, northern corn leaf blight and downy mildew of millet, and septoria
in wheat (Graham and Webb 1991; Mann et al. 2004). The mechanism by which Cl
increases resistance is not well understood. However, it appears to be nontoxic
in vitro and does not stimulate lignin synthesis in wounded wheat leaves. It was
suggested that Cl can compete with NO
3
absorption and influences the rhizosphere
pH by suppressing nitrification and increasing the availability of Mn mediating
reduction of Mn
III,IV
oxides which increases tolerance to pathogens.
21 Solubilization of Micronutrients Using Indigenous Microorganisms 397
As a beneficial micronutrient, Cl
regulates increased fresh and dry biomass,
greater leaf expansion, elongation of leaf and root cells, improved water relations,
higher mesophyll diffusion to CO
2
, and better water- and nitrogen-use efficiency
(Colmenero-Flores et al. 2019). In most cases, deficient leaves exhibit distinct
characteristic and continuous boundaries between the affected and healthy tissue
and appear as blotchy leaf chlorosis and necrosis. In such cases, Cl deficiency may
result in wilting and bronzing of leaves. Chlorine toxicity can occur naturally in
plants grown in coastal soils due to the excess Cl. Chlorine toxicity usually results in
necrosis along the leaf margins. Leaves are smaller than usual. They may be yellow
and drop early. The symptoms first appear on mature leaves. In some species,
chlorosis may also occur. Chlorine toxicity can result from air pollution, in the
form of chlorine gas, or from excess chloride in the soil (Table 21.1).
Generally, soil contains sufficient amount of chloride in the soil. The plant
available form of chlorine is an anionic form which is chloride (Cl
). Anionic
form is the dominant form of chlorine in soils. Chloride is thought to pass through
the root by a symplastic pathway and is mobile within the plant (White and Broadley
2001). The content of Cl
fluctuates greatly in soils. Most soils contain sufficient
levels of chlorine. However, Cl may become deficient in inland soil under frequent
high rainfall and irrigation. Plants may be able to absorb some metal-Cl complex
such as CdCl
+
, but with a minimal percentage (Weggler et al. 2004). Negatively
charged chloride ion tends to be repelled from the surfaces of soil particles, making it
difficult to form complexes readily with negatively charged mineral soils. Therefore,
chloride in the bulk solution contains a higher concentration than in the diffuse layers
surrounding soil particles. Water fluxes, relationship between precipitation, and
evapotranspiration determine the movement of chloride ion within the soil (Chen
et al. 2010).
There is no available literature on Cl-solubilizing microorganisms in the soil.
However, the soil microbes make changes in soil pHs which will ultimately release
chloride into the soils in plant available forms.
21.21 Mechanisms of Cl Solubilization
Most of the micronutrients are present as forms of chloride complexes in their
cationic forms. Soil microorganisms produce acids which reduce the soil
pH. Reduced pHs facilitate breaking down of metal-Cl complexes making them
available for plants.
398 A. D. S. N. P. Athukorala
21.22 Boron-Solubilizing IMOs
Boron is a non-metal micronutrient which essentially optimizes plant growth and
development. The critical concentration of B in plant tissues is 20–25 mg kg
1
(usually 35 mg kg
1
) on a dry mass basis (Ahmad et al. 2012). It plays an important
role in cell wall synthesis and structural integration as well as in protein and
enzymatic functioning of the cell membrane, providing improved membrane integ-
rity (Brown et al. 2002). B is cross-linked with pectin assembly, glycosylinositol
phosphorylceramides (GIPCs), and rhamnogalacturonan-II (RG-II) that control the
tensile strength and porosity of the cell wall (Shireen et al. 2018). Optimum B
concentration in cells enhances the plasma membrane hyperpolarization, while its
deficiency alters the membrane potential and reduces H
+
-ATPase activity. In young
growing tissues, B acts primarily in cell division and elongation, and starvation leads
to the inhibition in root elongation with deformed flower and fruit formation. Boron
is also involved in phenolic metabolism and nitrogen metabolism in plants. The role
of B in rhizobial N fixation, actinomycete symbiosis, and cyanophyceae heterocyst
formation in leguminous crops has been highlighted in previous studies. B defi-
ciency is thought to affect photosynthesis indirectly by weakening the vascular
tissues responsible for ion transport (Rasheed 2009).
Boron affects the availability and uptake of other plant nutrients from the soil. B
application increased the uptake and translocation of P, N, K, Zn, Fe, and Cu in
leaves, buds, and seeds of cotton (Ahmed and Fujiwara 2010). Boron deficiency has
occurred in over 132 crops and 80 countries during last 60 years. After Zn, B is the
second most deficient micronutrient severely affecting the growth of crops on global
scale (Alloway 2008). Deficiency symptoms depend on the age of the plant and
include stunted root growth, restricted apical meristem growth, stunted root growth,
reduced chlorophyll content, brittle leaves, and photosynthetic activity, disruption in
ion transport, increased phenolic and lignin contents, and reduced crop yield
(Shireen et al. 2018).
In the soil, the presence of boron is common as boric acid or borate. Boron is
percolated in the form of uncharged molecules rather than as ions. It is extremely
deficient in soils which are developed from calcareous, loessial, or alluvial deposits
and also in highly leached soils (Borkakati and Takkar 2000). There are various
other factors, including sandy/coarse texture, drought, alkalinity, liming, and inten-
sive cultivation with more nutrient uptake and less fertilizer application, which affect
the availability of B to plants (Ahmad et al. 2012). In many regions of the world such
as Brazil, the USA, China, Japan, and Korea, B availability is limited which is
resulted by its high solubility and leaching off by irrigation water or rainfall in
shallow or coarse-textured soils. In drought conditions and in soils with less organic
matter, the availability of B is low due to alkalization and breakdown of organic
matter (Shireen et al. 2018). Optimal B availability in soils can be achieved by using
several beneficial and eco-friendly techniques.
Boric acid uptake is affected by the transpiration stream. Enhancement of
transpiration-driven water flow can be affected by plant growth-promoting bacteria
21 Solubilization of Micronutrients Using Indigenous Microorganisms 399
which can increase B accumulation in plant. This may also cause B toxicity.
Inoculation of these bacteria under low pH into soil increased the growth of
rapeseed. Further, addition of P enhanced the uptake of B by rapeseed, while
B. pumilus inoculation inhibited the growth of rapeseed under B supply (Masood
et al. 2019). There have not been many studies done on increasing the bioavailability
of B especially with indigenous microorganisms despite it being the second most
deficient micronutrient affecting crops worldwide, therefore warranting
investigations.
21.23 Molybdenum-Solubilizing IMOs
Molybdenum is most common in agricultural soils which can exist in several
oxidation states ranging from zero to VI (Kaiser et al. 2005). In a plant, Mo performs
various physiological and metabolic functions. Despite its requirement in small
amounts for normal plant development, it plays a critical role in the regulation of
various plant functions. The required concentration range in plant tissue for its
normal function is between 0.3 and 1.5 ppm. Mo has been utilized by specific
plant enzymes to participate in reduction and oxidative reactions (Thomas et al.
2017).
Molybdenum is an essential component in nitrogenase enzyme used by symbiotic
nitrogen-fixing bacteria in legumes to fix atmospheric nitrogen. Plants also use Mo
to convert inorganic phosphorus into organic forms in the plant (Beevers and
Hageman 1969). Molybdenum deficiency can affect nitrogen deficiency in plant,
since it is closely linked to nitrogen fixation process. Molybdenum is the only mobile
micronutrient in the plants. Therefore, older and middle leaves show Mo deficiency
symptoms early, but it spreads up to the stem and affects the new leaves (Table 21.1).
Some plants such as poinsettias show thin chlorotic, leaf margins around the leaf
perimeter followed by necrosis (Carruthers 2016a), which restricts plant growth and
flower formation. Molybdenum deficiency or toxicity is uncommon in many plants.
However, crops that are most sensitive to molybdenum deficiency are crucifers
(broccoli, cauliflower, cabbage), legumes (beans, peas, clovers), poinsettias, and
primula (Carruthers 2016a). Research has shown that high sulfates can reduce plant
uptake of molybdenum (Kaiser et al. 2005).
The average concentration of Mo in the lithosphere is 2–3mgkg
1
but can
increase to a concentration like 300 mg kg
1
with significant content of organic
matter (Kaiser et al. 2005; Reddy et al. 1997; Fortescue 1992). Different environ-
mental factors such as soil pH, extent of water drainage, concentration of adsorbing
oxides (e.g., Fe oxides), and organic compounds found in the soil influence the
availability of molybdenum for plant growth. Molybdenum becomes more soluble
and is accessible to plants mainly in its anion form MoO
4
in alkaline soils, while it
decreases in acidic soils (pH <5.5) (Reddy et al. 1997). In agricultural soils, the
complex that molybdenum is present depends on the chemical speciation of the soil
zone. Molybdenite (MoS
2
), wulfenite (PbMoO
4
), and ferrimolybdenite [Fe
2
(MoO
4
)]
400 A. D. S. N. P. Athukorala
are the mineral forms of molybdenum found in rocks (Kaiser et al. 2005).
Weathering releases Mo from solid mineral forms (Kaiser et al. 2005). Molybdenum
is typically added to the soil by fertilization or by the addition of other chemicals
such as sodium or ammonium molybdate.
Molybdate reduction by microbes has been reported from 100 years ago and
includes mainly bacteria (Table 21.2). Potential mechanisms of reduction of molyb-
denum were first reported in Escherichia coli,Thiobacillus ferrooxidans (now
Acidithiobacillus ferrooxidans), Enterobacter cloacae strain, Pseudomonas sp.,
Serratia spp., Enterobacter sp., Acinetobacter calcoaceticus, and Klebsiella
sp. (Halmi et al. 2013). Mo-reducing soil bacteria are reported from Pakistan,
Sudan (Enterobacter sp. strain Zeid-6), Indonesia, and Antarctica (Pseudomonas
sp. strain DRY1). Except Bacillus sp., molybdenum-reducing bacteria are gram
negative (Frascoli and Hudson-Edwards 2018). Many Mo-reducing bacteria isolated
from Pakistani soils were resistant to high Mo concentrations (up to 50 mM) (Khan
et al. 2014). Some plants eliminate Mo from their roots and shoots (e.g., Cistus,
Quercus species), while some take up Mo without any harmful effects (e.g.,
Baccharis species) (Frascoli and Hudson-Edwards 2018). Autotrophic bacteria
Acidithiobacillus ferrooxidans and Thiobacillus thiooxidans isolated from drainage
from Kennecott’s open-pit mine in Bingham Canyon, Utah, USA, were capable of
bioleaching molybdenite (Frascoli and Hudson-Edwards 2018; Bryner and Ander-
son 1957).
Molybdenum is an essential component in nitrogenase enzyme and thereby in
nitrogen fixation process (Hänsch and Mendel 2009). Molybdenum is the cofactor
for the enzyme nitrate reductase during nitrogen assimilation (Hänsch and Mendel
2009). Also Mo is the key regulatory component for nodule initialization and
maintenance of nitrogen fixation in legumes (Franco and Munns 1981), and the
enzyme activity was elevated with a high Mo content. Several microorganisms are
associated with the biofertilization process and they enhanced the activity of the Mo
in plant growth and development. Bradyrhizobium inoculation and Mo fertilization
with at least 50 g ha
1
increased the yield of peanut pods and kernels (Crusciol et al.
2019). Chatterjee and Bandyopadhyay (2017) found that the application of
biofertilizers together with boron and molybdenum enhanced the growth, nodula-
tion, and pod yield of vegetable cowpea in acid soil of eastern Himalayan region.
21.24 Mechanisms of Mo Solubilization
Molybdenum seems to induce the production of iron-chelating compounds such as
dihydroxybenzoic acid (DHBA) and tris(catechol) protochelin and bis(catechol)
azotochelin. Protochelin and azotochelin production also increases at lower concen-
trations of Mo and vanadium (V). Protochelin and azotochelin act as strong
complexing agents for Fe(III), molybdate, and vanadate. Azotochelin (LH5) reacts
with molybdate to form a 1:1 complex with Mo
(VI) (LH
4
+ MoO
42
!MoO
2
L
3
+2H
2
O). Essential metals (Fe, Mo, and V) are
21 Solubilization of Micronutrients Using Indigenous Microorganisms 401
acquired by these compounds while excluding toxic ones (such as W). At low
concentrations, these catechol compounds form siderophore complexes with essen-
tial metals (Fe, Mo, V) and are taken up by the bacteria through specialized transport
systems (McRose et al. 2017).
Microorganisms, those who have a capability of fixing atmospheric nitrogen and
form ammonia, have Mo-nitrogenase or three forms of nitrogenase enzymes.
Diazotrophic organisms such as Klebsiella and Rhizobium have Mo-nitrogenase.
Mo in nitrogenase enzyme can also be combined with other metals depending on the
microbial species. For example, Azotobacter chroococcum possesses Mo and V
nitrogenases, Rhodobacter capsulatus has Mo and Fe nitrogenases, and Azotobacter
vinelandii contains the three enzymes. The most commonly occurring nitrogenases
have Mo in their active center to form the iron-molybdenum cofactor.
Mo-nitrogenases exhibit higher efficacy than the alternative nitrogenases with
respect to N
2
reduction rates. The metal cluster called FeMoco, an abbreviation for
the iron-molybdenum cofactor, is the site of conversion of N
2
into ammonia (Hänsch
and Mendel 2009).
Assimilatory nitrate reductases (Nas) that catalyze the first reaction in nitrate
assimilation are molybdoenzymes. Molybdenum acts as the cofactor in nitrate
reductase. Nitrate reductase in higher plants is proposed to be a homodimer, with
two identical subunits joined and held together by the Mo cofactor. In bacteria, there
are two types of nitrate reductases, first the ferredoxin- or flavodoxin-dependent
enzyme found in cyanobacteria, Azotobacter, and the archaeon Haloferax
mediterranei, and second, the NADH-dependent enzyme present in heterotrophic
bacteria and R. capsulatus. The cyanobacterial nitrate reductase enzyme is an 80 kDa
monomer, encoded by narB. Electrons from ferredoxin or flavodoxin are transferred
to the cluster and the Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD)
cofactor, the nitrate reduction site. NADH-dependent nitrate reductase catalizers are
present in Klebsiella,Bacillus, and Rhodobacter.InKlebsiella, a catalytic subunit
and a small electron transfer subunit are present in the enzyme, while the large
subunit (nasA gene product) binds to Mo-bis-MGD (Schaechter 2009).
21.25 Conclusions and Future Prospects
This review discusses the significance of micronutrients for plants with special focus
on increasing the productivity of crop plants and the role of microorganisms in
sustainable agriculture while maintaining a sustainable environment. Furthermore, it
presents developments in research and their applications in agriculture and environ-
mental management and highlights their potential applications in achieving sustain-
able environments by taking into account their dimensions mainly in processes such
as bio-composting, biodegradation, and other processes such as bioremediation and
bioleaching. This review also highlights the fact that research has not given enough
attention to microbes in terms of micronutrient solubilization and when did, the
focuse has mainly been on bacteria with fungi to some extent. Other groups of
402 A. D. S. N. P. Athukorala
microorganisms such as cyanobacteria and algae have not been investigated much
for micronutrient solubility. Research especially on field trials/application of
micronutrient-solubilizing microbes are currently being restricted to certain regions
of the world mainly to India, Pakistan, and Africa and therefore should be expanded.
With already successful stories and extensive future research, biofortification/
biofertilization of crops with micronutrient-solubilizing microorganisms will open
up new avenues in addressing the “hidden hunger”in a sustainable environment in
years to come while creating a clean and efficient environment for sustainable
developmental goals to be achieved.
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21 Solubilization of Micronutrients Using Indigenous Microorganisms 417
Chapter 22
Synergistic Interaction of Methanotrophs
and Methylotrophs in Regulating Methane
Emission
Vijaya Rani, Rajeev Kaushik, Sujan Majumder, A. T. Rani,
Asha Arambam Devi, Pratap Divekar, Priyanka Khati, K. K. Pandey, and
Jagdish Singh
Abstract The atmospheric methane concentration is increasing rapidly at the rate of
around 10 ppb/year. A concerted effort is required to reduce methane emission.
Methanotrophs possess methane monooxygenase enzyme system and can consume a
major portion of the methane produced in the environment. These microbes play a
major role in the single-carbon-driven microbial food web. Microbial interaction is
an important component of microbial ecology studies, and its role in community
functioning and various biogeochemical cycles still remains unclear. A synergistic
interaction occurs between the methanotrophs and non-methane-utilizing
methylotrophs (NUM) in the natural ecosystem. The intermediates produced by
the methanotrophs can be used as a carbon source by the NUM and support its
existence. On the other hand, NUM consumes toxic intermediates like methanol and
formaldehyde of the methanotrophs and prolongs their growth. The consumption of
the intermediates (methanol, formaldehyde and formate) of the methane utilization
pathway by methylotrophs as a result of cross-feeding enhances the methane utili-
zation rate of that ecosystem. Co-inoculation of methanotrophs and NUM in the
natural habitat particularly paddy ecosystem can aid in the reduction of net methane
emission. This chapter highlights the role of microbial interactions, particularly
between methanotrophs and methylotrophs, that can be harnessed to mitigate meth-
ane emission from the methane-rich environment.
Keywords Methylotrophs · Methanotrophs · Cross-feeding · Methane · Methanol
V. Rani (*) · S. Majumder · A. T. Rani · P. Divekar · K. K. Pandey
Division of Crop Protection, ICAR-Indian Institute of Vegetable Research, Varanasi, India
R. Kaushik · A. A. Devi
Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi, India
P. Khati
ICAR-Vivekananda Parvatiya Krishi Anusandhan Sansthan, Almora, Uttarakhand, India
J. Singh
ICAR-Indian Institute of Vegetable Research, Varanasi, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_22
419
22.1 Introduction
Methane, the principal component of natural gas, is a colourless, odourless greenhouse
gas (GHG) and contributes around 14% to the total greenhouse gas emission.
Methane-rich environments like paddy fields, wetlands, sewage, landfills and digestive
system of ruminants and termites possess huge diversity of methanogen,
methanotrophs and other methylotrophs (Kirschke et al. 2013; Lee et al. 2014).
Methanotrophs are those bacteria that can consume methane to meet their carbon
and energy requirements before it gets released into the atmosphere and plays a major
role in reducing net methane emission, thereby maintaining global carbon balance. On
the other hand, methylotrophs are a diverse group of bacteria, yeast, fungi and archaea
that can utilize C1 compounds like methanol, monomethylamine, dimethylamine,
trimethylamine, methanesulfonate and dimethylsulfonate including methane as the
sole source of carbon and energy. Traditional methanotrophs of the group Alpha- and
Gammaproteobacteria widespread in Types I, II and X of methanotrophs with the
prefix‘methylo’are well studied and investigated. Members of Betaproteobacteria of
the genera Methylophilus (Madhaiyan et al. 2009), Methylovorus (Govorukhina and
Trotsenko 1991)andMethylibium (Nakatsu et al. 2006) are recently recognized as
methane oxidizers. Besides Proteobacteria, few members of the phylum
Verrucomicrobia belonging to genera Methylacidimicrobium and Methylacidiphilum
can also utilize methane (Op den Camp et al. 2009; Sharp et al. 2013; van Teeseling
et al. 2014). Methylotrophs, on the other hand, cover all the three domains of
microorganisms, viz. Bacteria, Archaea and Eukarya. Methylotrophs are microorgan-
isms with a diverse group that besides utilizing methane (methanotrophs) also include
those that can utilize other carbon substrates with no C–C bonds like methanol and
other methylated compounds like methylamine (Chistoserdova et al. 2009). Since all
methylotrophs cannot utilize methane, it can be said that all methanotrophs are
methylotrophs but all methylotrophs are not methanotrophs. The ability to oxidize
methanol has been reported in both prokaryotes and eukaryotes. Eukaryotic yeast
belonging to genera Candida,Pichia,Hansenula and Torulopsis can utilize methanol
as the sole carbon source (Negruţaetal.2010). The prokaryotic members capable of
oxidizing methanol are spread across Alphaproteobacteria (Methylobacteria,
Hyphomicrobium), Betaproteobacteria (Burkholderia,Methylibium,
Methyloversatilis) and Gammaproteobacteria (Clonothrix fusca,Beggiatoa,Pseudo-
monas), Verrucomicrobia, Cytophagales, Bacteroidetes (Flavobacterium), Firmicutes
(Bacillus methanolicus,Paenibacillus) and Actinobacteria (Microbacterium,
Gordonia,Arthrobacter and Mycobacterium) (Rani et al. 2021b;Kolb2009;
Madhaiyan et al. 2010; Waturangi et al. 2011; Jhala et al. 2014; McTaggart et al.
2015; Macey et al. 2020).
Non-methane-utilizing methylotrophs (NUM) are known to co-occur with
methanotrophs in the natural ecosystem and affect methane utilization rate.
Modern-day techniques like stable-isotope probing have indicated that a synergistic
interaction occurs between the methanotrophic and non-methane-utilizing
methylotrophic community (Shiau et al. 2020; van Grinsven et al. 2020). NUM is
420 V. Rani et al.
known to survive on methane-derived carbon particularly methanol and enhance the
methane oxidation rate (Krause et al. 2017). Moreover, emergent properties like
interaction-induced production of metabolites may arise when microorganisms
interact leading to altered community functions otherwise not possible in the indi-
vidual cells (Watrous et al. 2012; Abrudan et al. 2015). The transfer of metabolites
from methanotrophs is not only restricted to NUM but to a wide range of microbial
taxa as evident from the DNA-SIP study (Beck et al. 2013). These findings suggest
that the assimilation of methane by methanotrophs in the methane-rich environment
provides carbon to a diverse group of microbes (NUM and other heterotrophs) and
sometimes to other life forms as well (Sanseverino et al. 2012; Oshkin et al. 2015;
Yu et al. 2017).
22.2 Pathway for Methane Utilization
The unique ability of the methanotrophs to metabolize methane comes from the
presence of methane monooxygenase (MMO) enzyme system. It’s the first enzyme
in the metabolic pathway of methanotrophs. MMO enzyme can be either housed in
an intracytoplasmic membrane known as particulate MMO (pMMO) or suspended
freely in the cytoplasm known as soluble MMO (sMMO). pMMO, a copper-
containing, membrane-associated enzyme, is found in all the methanotrophs except
for the genera Methylocella and Methyloferula (Theisen et al. 2005) but is less
studied as it is membrane-associated when compared to sMMO. Both sMMO and
pMMO enzyme can act on a wide range of substrates ranging from single carbon
substrate, methane to as long as eight carbon compounds. They can act on alkane,
alkenes, cycloalkanes and even halogenated derivatives (McDonald et al. 2006).
Alkanes can be oxidized by a group of enzymes like cytochrome P450, alkane
hydroxylases, sMMO and pMMO (Beilen and Funhoff 2005). However, among
these, only sMMO and pMMO can act on methane. Some methanotrophs can
produce both pMMO and sMMO, and their expression is regulated by copper
concentration in the environment. pMMO is expressed under high copper-to-bio-
mass ratios, whereas sMMO is expressed when the copper-to-biomass ratio is low
(Murrell et al. 2000).
Methanol produced by the action of MMO is further acted upon by methanol
dehydrogenase to produce formaldehyde. Methanol dehydrogenase (Mdh) is
pyrroloquinoline quinone (PQQ)-containing NAD
+
-dependent oxidoreductase
enzyme (Anthony and Williams 2003). Formaldehyde produced by methylotrophs
can be assimilated either by RuMP pathway (Type I) or by serine pathway (Type II).
RuMP (ribulose monophosphate pathway) was earlier thought to be restricted to
methylotrophic bacteria. However, they are now reported in various prokaryotic
microorganisms and their role in formaldehyde fixation and detoxification has been
established (Nobuo et al. 2006). Anaerobic methane oxidation by archaea differs in
their mechanism to utilize methane. They utilize methane via reverse and modified
methanogenesis pathway. Various intermediates of the methane oxidation pathway,
22 Synergistic Interaction of Methanotrophs and Methylotrophs in Regulating... 421
viz. methanol, formaldehyde, formate, acetate and other organic acids when secreted
by the methanotrophs, can be used as a growth substrate by both non-methane-
utilizing methylotrophs and other heterotrophs as shown in Fig. 22.1. Methane-fed
microbial microcosm study showed the abundance of methanotrophs of the family
Methylococcaceae particularly Methylobacter along with other methylotrophs
(Methylotenera) and heterotrophs, suggesting that there is a distribution of carbon
from methane among diverse bacterial populations rather than a single type of
microbe and thus methanotrophs play an important role in methane cycling (Oswald
et al. 2016).
22.3 Cross-Feeding of Methane by NUM
Non-methanotrophs, particularly methylotrophs, play a major role in combating
climate change in a methane-rich environment. Other heterotrophic forms may affect
the growth of methanotrophic bacteria as a result of its various metabolic activities
(secretion of growth factors or removal of toxic intermediates) (Hrsak and Begonja
Fig. 22.1 Cross-feeding of metabolites produced by methanotrophs. sMMO soluble methane
monooxygenases, pMMO particulate methane monooxygenases, Mdh methanol dehydrogenases,
RuMP ribulose monophosphate pathway
422 V. Rani et al.
2000). NUM is often known to coexist with methane-utilizing bacteria by cross-
feeding on methane-derived carbon, particularly methanol (Takeuchi et al. 2019).
Methane-oxidizing microorganisms possess monooxygenases that catalyze the con-
version of methane to methanol. Methanol produced in the periplasmic space by the
action of MMO enzyme system can easily diffuse out and serve as an alternative
carbon source for other groups of microorganisms (Corder et al. 1986). Methanol-
dependent cross-feeding between methanotrophs and other methylotrophs is largely
dependent upon methanol excreted by the methanotrophic bacteria. Microflora
residing in the rhizosphere, phyllosphere and non-rhizosphere or as endophytes of
plants can utilize methanol and consume a major proportion of it (Kolb 2009; Iguchi
et al. 2015; Chistoserdova and Kalyuzhnaya 2018). Methane-derived carbons (meth-
anol, formaldehyde and formate) particularly methanol from methanotrophs can be
utilized by NUM and enhance methane utilization rate by cross-feeding (Hanson and
Hanson 1996; Qiu et al. 2008).
The findings of various researchers confirm the abundance of NUM along with
methanogens and methanotrophs in the environment as shown in Table 22.1. Syn-
ergistic associations of methane and methanol oxidizers have been reported that
favours the utilization of methane due to the removal of its intermediate methanol by
the other partner (Krause et al. 2017; Jeong and Kim 2019). The coordinated
response of Methylococcaceae (methanotroph) and Methylophilaceae (NUM) to
changing methane and nitrate levels suggests that the two different functional groups
of microbes may be involved in some type of cooperative behaviour (Beck et al.
2013). Similarly, methane oxidation by Methylocystis was found to increase in the
presence of helper organism Hyphomicrobium according to the experiments carried
out by (Jeong and Kim 2019). The transfer of methanol from the methanotrophic
partner Methylobacter tundripaludum to the non-methanotroph methylotrophic part-
ner Methylotenera mobilis has been confirmed in a microcosm model by (Krause
et al. 2017). Their findings indicate that the non- methanotrophic partner induces a
change in the gene expression of the methanotrophic partner causing the synthesis of
less efficient methanol dehydrogenase enzyme (MxaF-type catalysing the conver-
sion of methanol to formaldehyde) resulting in methanol excretion.
In the natural ecosystem, a complex interaction occurs between methanotroph,
NUM and other heterotrophs. The success of single carbon-based microbial food
web is determined by the effective transfer of intermediates from one microbial
group to the other, allowing them to survive in methane-rich environments. A
successful example of cross-feeding is the experiment carried out by Yu et al.
(2017).They made a synthetic community of 50 bacterial cultures comprising of
10 methanotrophs (Methylomonas,Methylobacter,Methylosarcina and
Methylosinus), 28 methanol-utilizing methylotrophs (Methylotenera,Methylovorus,
Methylophilus,Ancylobacter,Hyphomicrobium,Labrys,Methylobacteria,
Methylopila,Paracoccus,Xanthobacter and Methyloversatilis), 8 non-methanol-
utilizing methylotrophs (Aminobacter,Arthrobacter,Mycobacterium and Bacillus)
and 4 heterotrophs (Pseudomonas,Janthinobacterium and Flavobacterium) to study
syntrophy in the aerobic methane-oxidizing environment (Yu et al. 2017).The
metatranscriptomics analysis showed that across all the treatment with varying
22 Synergistic Interaction of Methanotrophs and Methylotrophs in Regulating... 423
Table 22.1 Studies showing cross-feeding of metabolites from methanotrophic to non-methane-
utilizing methylotrophic partner
Methanotroph Non-methanotroph
Substrate
transferred Salient findings References
Methylobacter
tundripaludum
Methylotenera
mobilis
Methanol Transcriptome analysis
showed high expres-
sion of genes involved
in methanol oxidation
in the methylotrophic
partner
Methylotenera mobilis
causes a change in the
expression of
methanotrophic partner
causing it to secrete
methanol
Krause
et al.
(2017)
Methylocaldum
marinum
Methyloceanibacter
caenitepidi (faculta-
tive methylotroph)
Acetate Observed syntrophic
association between
M. caenitepidi and
M. marinum
Under co-culture con-
dition, genes involved
in serine pathway were
downregulated in
M. caenitepidi
Organic compound
probably acetate might
be the major carbon
source for the
methylotrophic partner
M. caenitepidi
Takeuchi
et al.
(2019)
Members of
Methylococcaceae
and others
Methylophaga Not stud-
ied (may
be
methanol)
DNA-SIP experiment
identified members of
Methylococcaceae as
major
13
CH
4
con-
sumers
Microbial mats showed
diverse assemblage of
bacteria, protozoa with
Methylophaga as key
consumers of methane-
derived organic matter
Paul et al.
(2017)
Methylomicrobium Methylophaga,
Hyphomicrobium
and other
unrecognized
methylotrophs
Not stud-
ied (may
be
methanol)
DNA-SIP study indi-
cates that methane-
derived carbon particu-
larly methanol pro-
duced by
methanotrophs may be
consumed by
Methylophaga and
other related
Jensen
et al.
(2008)
(continued)
424 V. Rani et al.
Table 22.1 (continued)
Methanotroph Non-methanotroph
Substrate
transferred Salient findings References
uncultivated
Gammaproteobacteria
Methylococcaceae,
particularly
Methylobacter
Methylophilaceae,
particularly
Methylotenera
Not stud-
ied (may
be
methanol)
Observed coordinated
response of both
methanotroph and
methylotroph to chang-
ing methane and nitrate
levels, suggesting
cooperative behaviour
Beck et al.
(2013)
Methylobacter sp. Methylotenera Not stud-
ied (may
be
methanol)
No physical contact
was required between
the partners for the
transfer of carbon as
was confirmed by
stable-isotope probing
(SIP) and nanoscale
secondary ion mass
spectrometry
(NanoSIMS)
Requires nitrate for
carbon transfer as it is
potentially used by
Methylotenera sp. and
its deficiency may
affect the methane oxi-
dation rate of
Methylobacter sp.
van
Grinsven
et al.
(2020)
Methylococcaceae Methylophilaceae Methanol Made synthetic bacte-
rial communities of
50 isolates including
methanotrophs,
methylotrophs and het-
erotrophs with varying
oxygen and methane
levels
Observed predomi-
nance of the
methanotrophs of the
family
Methylococcaceae and
non-methanotrophic
methylotrophs of the
family
Methylophilaceae
across all the treat-
ments
Vitamin B
12
produced
by Methyloversatilis
Yu et al.
(2017)
(continued)
22 Synergistic Interaction of Methanotrophs and Methylotrophs in Regulating... 425
nitrogen, oxygen and methane concentration, methanotroph of the family
Methylococcaceae and methylotroph of the family Methylophilaceae did
outcompete other species. Heterotrophs of the genera Janthinobacterium and Pseu-
domonas were detected in only a few treatments. Their research shows that methane-
utilizing bacteria support the growth of other NUM and heterotrophs through the
transfer of metabolites.
The bacterial community structure in a methane-rich environment is influenced
by various factors like the existing concentration of methane, oxygen, nitrogen and
other nutrients. The eutrophic lakes have high nitrate concentration and are one of
the major sources of aquatic methane production. The nitrate in the aquatic ecosys-
tem does influence the growth of microbial species and affect the cross-feeding of
metabolites. The transfer of methane-derived carbon between Methylobacter
(methanotroph) and Methylotenera (NUM) is based on the nitrate levels as it is
required by the methylotrophic partner (van Grinsven et al. 2020). It has been
observed that nitrate can cause stimulation in methane oxidation resulting in
increased transfer of associated carbon compounds. Similarly, oxygen level selects
the population of methanotrophs and methylotrophs, thereby determining their
microbial diversity in a particular niche. The effect of oxygen on the conversion of
methane-derived carbon has been studied (Wei et al. 2016). They observed greater
transfer of methane-derived carbon at high O
2
concentration (21%) as compared to
that observed at 2.5 and 5% O
2
concentration. They even reported higher microbial
diversity index at 2.5% O
2
concentration and concluded that more methane-derived
carbon was exuded into the environment and available for the growth of
non-methanotrophs in O
2
-limiting environments. Similar findings were reported
where speciation within Methylococcaceae and Methylophilaceae family at different
oxygen gradient with an abundance of Methylosarcina (methanotroph) and
Methylophilus (NUM) at high O
2
tension (150–225 μM) and Methylobacter
(methanotroph) and Methylotenera (NUM) at low initial O
2
tension (15–75 μM)
was observed (Hernandez et al. 2015). The specific species differentiation observed
within the methanotrophic and methylotrophic members of the Methylococcaceae
and Methylophilaceae family is driven towards niche adaptation to specific oxygen
gradient. The change in the population of methanotrophs and NUM to varying
oxygen and methane concentration has been observed, suggesting that the relative
concentration of methane and oxygen selects microbial community that can thrive
under such situations. A synthetic community model comprising 50 bacterial species
(methanotrophs, methylotrophs and heterotrophs) showed a change in the species
composition with the abundance of methanotrophs of the family Methylococcaceae
Table 22.1 (continued)
Methanotroph Non-methanotroph
Substrate
transferred Salient findings References
may be shared among
other community
members
426 V. Rani et al.
and methylotrophs of the family Methylophilaceae at varying methane and oxygen
concentration (Yu et al. 2017). Lanthanum (Ln), a rare earth metal, also affects the
transfer of methane-derived carbon as it is an important co-factor of XoxF-type
methanol dehydrogenases (MDHs) present in Gram-negative methylotrophs
(Vu et al. 2016; Yanpirat et al. 2020). A shift in the expression of methanol
dehydrogenases from lanthanide-dependent MDH (XoxF) type to the more efficient
calcium-dependent MDH (MxaF) type occurs when non-methanotrophs are cultured
along with methanotrophs, allowing an excess of methanol production that can be
used by the methylotrophic partner (Krause et al. 2017). The presence of lanthanides
allows a partner-induced change in gene expression and influences microbial inter-
actions in the environment. The above finding suggests that the existing concentra-
tion of methane, oxygen, nitrate and other nutrients in the natural ecosystem plays a
major role in determining the community composition of methanotrophs and
methylotrophs, thereby influencing the transfer of methane-derived carbon and
methane oxidation capacity of that particular ecosystem.
22.4 Approaches Used to Study the Interaction
of Methanotrophs and NUM
Techniques involving the cultivation of different microbial groups cannot be very
useful for interaction studies as it is difficult to simulate natural conditions under
laboratory and most of the microorganisms still remain un-culturable due to their
specific growth requirement. A useful approach is to simulate the natural environ-
ment under controlled condition through a microcosm or mesocosm experiment
depending upon the scale of the model ecosystem and use molecular tools to
determine community composition. Microcosms are artificial, controlled, simplified
ecosystem used to simulate natural ecosystems mostly done under laboratory con-
ditions, whereas mesocosms are bounded and partially enclosed outdoor experiment
used to bridge the gap between the laboratory and the real world in environmental
science (Bruckner et al. 1995). Microcosm and mesocosm experiment reduces the
credibility gap and helps us to provide a solution to large-scale environmental
problems. They provide a better understanding of the ecological problems by
bringing them to spatial and temporal scale convenient enough to carry out the
study (Benton et al. 2007). Microcosm experiments have widely been designed to
study the diversity and dynamics of both methanotrophs and methylotrophs in soil
and sediment samples collected from the natural environment (Shiau et al. 2020;
Oshkin et al. 2015; Morris et al. 2002). Research shows that the activity pattern of
methane-oxidizing bacteria and the population structure of methylotrophs follow the
same pattern under field and microcosm condition (Eller et al. 2005). It can be
concluded that the findings of the microcosm study can be extrapolated to field scale
keeping in mind the concerned quantitative changes. Various molecular tools and
22 Synergistic Interaction of Methanotrophs and Methylotrophs in Regulating... 427
techniques are commonly being used to study the interaction of methanotrophs with
NUM. Some of them are mentioned below.
DNA-Based Stable Isotope Probing (DNA-SIP): It is a powerful means to study
the flow of intermediates from microbes with one functional group to the other. In
DNA-SIP study, environmental samples are fed with substrate labelled with a heavy
isotope (
13
C). The labelled isotope then gets incorporated into the cell biomass
including DNA, which can be processed and analysed to determine phylogenetic
affiliations of species with labelled DNA. Isotope labelled
13
CH
4
is used to study the
cross-feeding of intermediates produced by methanotrophs determining the associ-
ation of methanotrophs with methylotrophs and other heterotrophs in the natural
environment. DNA-SIP helps us to establish a direct link between CH
4
oxidation
and taxonomic identity for active methanotrophs and methylotrophs in complex
environments (Shiau et al. 2020). It has been widely used to study metabolic
interactions in methane-fed communities (van Grinsven et al. 2020; Paul et al.
2017; Jensen et al. 2008). DNA-SIP experiments are widely used to uncover the
participants involved in the C1 cycle and give a clear picture of the transfer of
metabolites from one microbe to the other. It provides confirmatory evidence of the
associations of actively interacting microorganisms, sharing carbon derived from a
single-key biogeochemical process.
PCR-Based Method: Functional marker genes unique to the physiology and
metabolism of methanotrophs and methylotrophs can be targeted to study the
diversity of microbes involved in the metabolism of single carbon compound.
Functional genes commonly targeted to study the diversity of methanotrophs and
methylotrophs are those of methane monooxygenases (pmoA and mmoX), methanol
dehydrogenase (mxaF), 16S rRNA region targeting serine pathway and RuMP,
dinitrogen reductase (nifH) and formyltransferase/hydrolase complex ( fhcD)
(McDonald et al. 2008). PCR product can be run on denaturing gradient gel to
separate amplicons even with a single-nucleotide difference. PCR followed by
denaturing gradient gel electrophoresis (PCR-DGGE) will help us to determine the
degree of genetic polymorphism in the target regions within the community
(Bodelier et al. 2005; Piterina and Pembroke 2013). One major limitation of
DGGE methodology is that the size of the amplicon should be between 100 and
500 bp, and therefore, primer set should be carefully designed (Marzorati et al.
2008). Eller et al. (2005) used three universal eubacterial primers set targeting
methylotrophs with RuMP (533F/907R and 197F/533R) and serine pathway
(142F/533R) followed by DGGE to study the community composition of
methylotrophic bacteria in soil samples collected from the paddy field. The advan-
tage of PCR-DGGE over DNA-SIP technique is that it does not require a closed
controlled environment and can be used to determine community composition of
samples directly collected from the natural environment.
Next-Generation Sequencing (NGS): Metagenomic and transcriptomic approach
to study microbial diversity requires sequencing of a large amount of DNA and
transcripts. Next-generation sequencing methods are more sensitive and can detect
428 V. Rani et al.
low-frequency variants. It is a high-throughput process that handles hundreds and
thousands of genes simultaneously and provides a comprehensive gene coverage
(Krishna et al. 2019). Storage, analysis and interpretation of NGS data are the major
rate-limiting steps of NGS technology. A large number of online bioinformatics
tools are available that can process original raw sequencing data to functional
biology (Kulski 2016). Techniques involving the use of NGS technology are widely
used to study the interaction between methanotrophs and NUM (Krause et al. 2017;
Beck et al. 2013; Takeuchi et al. 2019). Whole-genome sequencing and
transcriptomic approach were used to study the interaction between the
Methylocaldum marinum (methanotroph) and Methyloceanibacter caenitepidi
(NUM) and observed that there is non-methanol-based cross-feeding (particularly
acetate) of metabolites between the partners (Takeuchi et al. 2019). Pyrosequencing
of 16S rRNA gene (27F/519R) was done to study the community dynamics in
methane-fed microbial microcosms (Oshkin et al. 2015). The result showed low
species diversity with the predominance of Methylococcaceae species, closely
related to Methylobacter tundripaludum with few members of Methylotenera,
Flavobacterium,Pseudomonas,Janthinobacterium,Achromobacter and
Methylophilus. They also studied the community dynamics through Illumina
sequencing of prepared DNA libraries and observed the predominance of
methanotroph (Methylobacter) followed by NUM of the family Methylophilaceae
(Methylobacter tundripaludum,Methylophilus methylotrophus,Methylotenera
versatilis and Methylotenera mobilis). Both these techniques confirmed the strong
correlation of the population of methanotrophs to that of NUM, suggesting that there
may be the flow of intermediates between the two partners.
22.5 Interaction of Methanotrophs with Microbes
of Different Functional Group
Besides methylotrophs, intermediates of the methanotrophic bacteria also support
the growth of few heterotrophic bacteria. Synergistic interactions occur between the
methanotrophs and heterotrophs where one provides the other with carbon source
and the other produces growth factor or remove toxic intermediates from the
environment (Stock et al. 2013; Ho et al. 2014; Veraart et al. 2018; Singh et al.
2019). Growth stimulation of methane-utilizing Methylovulum miyakonense in the
presence of Rhizobium has been documented (Iguchi et al. 2011). They identified
cobalamin secreted by Rhizobium as the key factor responsible for stimulating the
growth of the methanotroph. Removal of toxic intermediates like organic acids can
also support the growth and proliferation of methanotrophic partners (Singh et al.
2019). The effect of the interaction of methanotrophs with non-methanotrophs
(heterotrophs/ autotrophs) has been summarized in Table 22.2.
Methanotrophic bacteria can grow with other organisms and aid in the removal of
other greenhouse gas (Singh et al. 2019). Co-culture of alkaliphilic methanotrophic
22 Synergistic Interaction of Methanotrophs and Methylotrophs in Regulating... 429
bacteria with microalga Scenedesmus obtusiusculus in the ratio 3:1, 4:1 and 5:1 can
lead to complete CH
4
and CO
2
uptake and thus is a promising strategy for green-
house gas mitigation in a single step (Ruiz-Ruiz et al. 2020). Methanol-independent
cross-feeding occurs in the natural ecosystem and supports the existence of
non-methylotrophic heterotrophic bacteria. A recent study shows that methane-
oxidizing bacteria can undergo mixed acid fermentation under the anoxic condition
and release other products like acetate, succinate and H
2
(Kalyuzhnaya et al. 2013;
Xin et al. 2004). These fermentation products can be used as a substrate by a diverse
group of heterotrophic bacteria. The complex interaction of methanotrophs with
other microbes occurs in the natural environment and thus can greatly influence net
methane emission from these areas.
Table 22.2 Beneficial effect of the interaction of methanotrophs with non-methanotrophs
Methanotroph Non-methanotroph Effect of interaction References
Gammaproteobacteria
(Methylosarcina and
Methylocaldum)
Algae (autotroph) Autotrophs provide O
2
to the
methanotrophs and increase
methane oxidation rate,
whereas methanotrophs pro-
vide them CO
2
for
photosynthesis
Yoshida
et al.
(2014)
Methylocystis Sphagnum mosses
(autotroph)
The autotrophs provide O
2
to
the methanotrophs and
increase methane oxidation
rate, whereas methanotrophs
provide them CO
2
for
photosynthesis
Kip et al.
(2011)
Methylobacter luteus Pseudomonas
mandelii (heterotroph)
Growth stimulation and
increased methane oxidation
Veraart
et al.
(2018)
Methylovulum,
Methyloparacoccus,
Methylomonas
Rhizobium sp.,
Mesorhizobium
sp. and Sinorhizobium
sp. (heterotroph)
Heterotrophs produce vitamin
B
12
and support the growth of
methanotrophs
Hoefman
et al.
(2014)
Methylomonas
methanica
Rhizobium/
Ochrobactrum/Pseu-
domonas/Escherichia
coli (heterotroph)
Growth promotion Ho et al.
(2014)
Methylovulum
miyakonense
Rhizobium
sp. (heterotroph)
Growth stimulation Iguchi
et al.
(2011)
Methylomonas Cupriavidus
taiwanensis
(heterotroph)
Heterotroph could synthesize
quinone, pyridoxine and vita-
min B
12
and supported the
growth of methanotroph
Stock et al.
(2013)
430 V. Rani et al.
22.6 Importance of Interaction of Methane Utilizers
with Non-methanotrophs in the Natural Ecosystem
Methanotrophs allow microbial food web to work at locations where it is difficult for
other microbes to survive and consume methane which is the most reduced form of
carbon. At the oxic-anoxic interface, aerobic methanotrophs survive that consume
methane produced by methanogenic archaea and support the growth of other
methylotrophs as well as heterotrophs. The type of interaction between these micro-
bial functional groups in a methane-rich environment has been shown in Fig. 22.2.
Methylotrophic partner removes toxic intermediates of the methane utilizers like
methanol and formaldehyde and allows sustained growth of the methanotrophs.
Reports on excretion of methanol (up to 100 μM) in the culture medium are available
that suggests a mismatch between the methanol produced and methanol that can be
further assimilated into the cell biomass (Xin et al. 2004; Tavormina et al. 2017). The
release of methanol will decrease the methane oxidation rate and inhibit methanol
production by the methanotrophic culture. The presence of methanol-utilizing
methylotrophs will allow removal of the released methanol and allow the sustained
activity of methane monooxygenase enzyme system. Low methanol concentration in
the environment is associated with low ozone concentration in the atmosphere and
thus plays an important role in atmospheric chemistry (Warneke et al. 1999; Galbally
and Kirstine 2002). Methanol-utilizing methylotrophs thereby play a key role and
consume both plant-derived methanol and those obtained from methanotrophs
Fig. 22.2 The effect of the interaction of methanotrophs with non-methanotrophs (methylotrophs/
heterotroph/autotroph) in a methane-rich environment
22 Synergistic Interaction of Methanotrophs and Methylotrophs in Regulating... 431
before it gets released into the atmosphere. Isolation of proteobacterial methanotroph
requiring lanthanides by enrichment culture technique led to co-isolation of
non-methanotrophic community, belonging to the genera Methylophilus,
Methyloversatilis,Hyphomicrobium,Methylobacteria,Pseudomonas and
Thiobacillus, as they can utilize intermediate compounds of the methane oxidation
like methanol, acetate, and formate (Kato et al. 2020). Mesocosm experiments
showed that there is a relative abundance of methanotrophs and NUM, indicating
that a large part of methane-derived product (methanol, acetate and others) was
being transferred from methanotrophs to non-methane-utilizing methylotrophs
(Kuloyo et al. 2020).
In a natural environment, methanotrophs are found along with other
methylotrophs, heterotrophs and autotrophs. Metabolites produced by each one of
them may support or suppress the growth of other bacteria. Besides methylotrophs,
heterotrophs and autotrophs also affect the activity of methane-utilizing bacteria.
Growth factors (quinone, pyridoxine and vitamin B
12
) produced by these organisms
may support the activity of methanotrophs (Stock et al. 2013; Ho et al. 2014;
Hoefman et al. 2014). Research shows that synergistic interaction exists between
methanotrophs, methylotrophs and heterotrophs. A methane-utilizing mixed culture
composed of a methanotroph, methanol-utilizing methylotroph (Methyloceanibacter
caenitepidi) and a heterotroph was successfully established from the sample col-
lected from marine sediments in Japan (Takeuchi et al. 2014). The stable association
of these three functional groups on a medium with methane as carbon source shows
that the methanotrophs via providing its metabolic intermediates (methanol, form-
aldehyde, acetate and formate) support the growth of methylotrophs as well as other
heterotrophs in the environment. The close association of methane-oxidizing bacte-
ria with autotrophs (macrophytic algae/Sphagnum mosses) suggests that their pho-
tosynthetic activity may provide O
2
to the methanotrophs and support its growth and
proliferation (Yoshida et al. 2014; Kip et al. 2011). In turn, the methanotrophs may
provide fixed nitrogen (NH
4
+
) to the Sphagnum mosses by its N
2
fixation activity
and exert beneficial effect (Larmola et al. 2014). Research suggests that the flow of
methane-derived carbon does not stop at the microbial level but sometimes extend to
the whole aquatic food web, up to the fish level (Sanseverino et al. 2012). Their
findings indicate the importance of methanotrophs in the C1 cycle (particularly
methane) and the role it plays in the food web of aquatic systems. Natural
methane-rich environments possess a diverse group of microflora right from
methanogens to methylotrophs, heterotrophs and autotrophs in close association,
thereby allowing the microbial community to thrive.
22.7 Conclusion and Future Prospects
Studies have emphasized the importance of biotic interactions, particularly microbial
interactions, as key modulators of biogeochemical processes. Methanotrophs along
with other microbes allow methane-based food web to function in various anaerobic
432 V. Rani et al.
ecosystems. Mitigation of methane emission through the use of methane-utilizing
bacteria from various anthropogenic sources (paddy fields, wastewater treatment and
landfills) has gained impetus in recent years (Oswald et al. 2016; Strong et al. 2017;
Davamani et al. 2020). With the increase in anthropogenic methane emissions, the
importance of these bacteria is set to increase as they play an important role in
reducing global methane sink. Artificial inoculation of methanotrophs with plant
growth-promoting traits in paddy field can cause a substantial reduction in methane
emission and an increase in grain yield (Rani et al. 2021a; Davamani et al. 2020).
Removal of methane from anoxic lake waters upon inoculation with
γ-proteobacterial methanotrophs has been reported (Oswald et al. 2016). However,
efforts to harness the synergistic interaction of methanotrophs with other microbial
groups have not been undertaken. We propose that co-inoculation of NUM with
methanotrophs may expedite the methane removal process due to their synergistic
interaction. Studies in this area have still not gained impetus, and the effect of
microbial co-inoculation on the removal of methane has still not been explored
much. This chapter provides enough evidence and confirms the transfer of metab-
olites from methanotrophs to the other microbial groups. This microbial synergistic
interaction can be tapped for reducing methane emission from various anoxic
habitats.
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22 Synergistic Interaction of Methanotrophs and Methylotrophs in Regulating... 437
Chapter 23
Biopesticides: An Alternative to Synthetic
Insecticides
A. T. Rani, Vasudev Kammar, M. C. Keerthi, Vijaya Rani, Sujan Majumder,
K. K. Pandey, and Jagdish Singh
Abstract The modern agriculture is negatively affected due to the rapid exploita-
tion of natural resources, indiscriminate use of pesticide application, and frequent
weather events influenced by climate change. Biocontrol action is a significant tool
for IPM, offers alternative management techniques that are safer for human and
environmental health. It is also worth noting that biological control has worked in a
versatile manner in different agricultural management systems and with different
types of disease causing organisms existing in the nature. Several key pests and
invasive pests were successfully controlled with the application of biological agents.
Although biopesticides very slowly replace the use of pesticide that may be due to
biopesticide exploration and application range. In India the main challenge for
biopesticide is related to their shelf life, narrow host range for pathogens, variation
in the lab to land performances, economic regulation, etc. Integrated approach will
be beneficial for biopesticide application for this private and government sectors that
come together with farmers to the village level and to build confidence in the use of
biopesticides.
A. T. Rani (*)
Agricultural Entomology, ICAR-Indian Institute of Vegetable Research, Varanasi, Uttar
Pradesh, India
V. Kammar
Quality Control Cell, Pune, Department of Food and Public distribution, M/o Consumer Affairs
and Food and Public distribution, Government of India, Pune, India
M. C. Keerthi
Agricultural Entomology, ICAR-Indian Grassland and Fodder Research Institute, Jhansi, Uttar
Pradesh, India
V. Rani
Microbiology, ICAR-Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India
S. Majumder
Agricultural Chemicals, ICAR-Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh,
India
K. K. Pandey · J. Singh
ICAR-Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_23
439
Keywords Biopesticides · Entomopathogens · Success stories · Semiochemicals ·
Bacteria · Neem
23.1 Introduction
Agriculture is critical in fostering people’s food and nutrition welfare, supporting
farmers’livelihoods, and ensuring the long-term growth of countries like India
(Pawlak and Kołodziejczak 2020). Sustainable development requires the mainte-
nance or enhancement of environmental quality through conservation of natural
resources simultaneously (Ansari et al. 2012). Sustainable agriculture therefore
necessitates effective agricultural resource management, with the goal of reducing
pest and disease issues to the point that they do not significantly harm crops by not
disrupting nature’s equilibrium (Kogan 1998). India grows into self-sufficient in
food production after the introduction of modern technologies such as the use of high
yielding crop varieties, irrigation facilities, and chemical inputs including fertilizers
and synthetic pesticides (Rena 2004). Overuse of chemical pesticides resulted in
pesticide resistance, resurgence of pests, and accumulation of residues on non-target
areas, resulting in the eradication of natural predators, threats to farmworkers, and
negative environmental consequences (Gill and Garg 2014).
Considering the negative effect of pesticides, the EU and USA have already
banned few chemical insecticides and also many conventional products have been
withdrawn from use because of raising concern about environment and human
health related issues (Pesticides Safety Directorate 2008; Damalas and
Eleftherohorinos 2011). In India, 32 pesticide active ingredients were banned from
import, manufacture, and use, eight were withdrawn from the market and 13 pesti-
cides were restricted for use as of October 2015 (CIBRC 2017). Remaining insec-
ticides were appeared to be ineffective in controlling insect pests due to development
of insecticide resistance (Kranthi et al. 2002; Mishra et al. 2015). Several other
limitations of synthetic insecticides including low biodegradability associated with
high persistence (Tayade et al. 2013), detrimental effects on soil microbiota lead to
loss of biodiversity and recycling of nutrients (Su et al. 2014), environmental
contamination (soil and water), and harmful effect of insecticide residues on bene-
ficial organisms (bees, spiders, earthworms, plants) (Singh et al. 2014). In such
conditions integrated approach offers different management actions that are
eco-friendly and sustainable for human and environmental health. In integrated
approach, biological control is an important tool that includes utilization of efficient
biocontrol agent to reduce the damage caused by harmful organisms (DeBach and
Rosen 1991). Biopesticide can work in a diversified manner for different agricultural
management systems, landscapes with potential actions.
The basic concept is to use biological species and natural products to control the
properties of an ecological environment and its elements in order to reduce insect
pests’biotic and reproductive capacity (Ruiu 2018). Biopesticides offer solutions to
challenges such as insecticide tolerance, environmental, and public health concerns
440 A. T. Rani et al.
in the age of sustainable agriculture (Chandler et al. 2011). Biopesticides are well-
known for being much cleaner for the environment than natural pesticides, but this
long-term solution is competing for a place in the current synthetic pesticide
industry. To popularize or to promote the biopesticides, immediate attention is
required to address the major issues related to technological challenges and long-
standing sustainability for more adoptability.
23.2 Biopesticides and Its Classification
Biopesticides or biological pesticides are derived from naturally occurring living
organisms including plants, animals, and microbes (fungi, bacteria, virus, nema-
todes, etc.) used as such or as their products or by-products that can control serious
pest and diseases of plants by their non-toxic eco-friendly mechanism. Biopesticides
are described as mass-produced agents obtained from living organisms or a natural
material manufactured and marketed for the control of plant pests, according to the
Organization for Economic Co-operation and Development (OECD). The target
specific biopesticides gaining importance all over the world because as they offer
an effective and ecologically sound solution to the pest problems.
Biopesticides are broadly classified into four different categories based on the
origin of the active substance or the living organism used in the control of target pest.
It includes (1) microbial pesticides; (2) biochemical pesticides (botanical pesticides
and semiochemicals); (3) transgenics (plant-incorporated products); (4) natural ene-
mies (insect predators and parasitoids) (Fig. 23.1).
23.2.1 Microbial Pesticides
Microbial pesticides are the pest control products comprising of several pathogenic
microorganisms such as bacteria, fungi, baculoviruses, protozoa, nematodes, and
nematode-associated bacteria as their active ingredient (Fig. 23.1).
Entomopathogenic viruses, bacteria, and fungi are the most commonly used
among farming community used as alternatives to traditional insecticides. Some of
the examples of commonly used microbial pesticides and their target pests are listed
in Table 23.1. Among various microbial pesticides, B. thuringiensis (Bt) is the most
widely used entomopathogenic bacterial biopesticide. Nearly 90% of the biopesti-
cide market was covered by the different strains/serotypes and subspecies of Bt
(Chattopadhyay et al. 2004; Romeis et al. 2006). Over 6000 isolates are stored in
many repositories distributed around the world (Ansari et al. 2012). These
biopesticides can control diverse range of pests and each separate active ingredient
is specific to a target pest providing safety to the non-target organisms. Most often
these pesticides are applied to crops in a similar way to chemical pesticides and they
may be delivered either in the form of spores, as living organisms or dead organisms.
23 Biopesticides: An Alternative to Synthetic Insecticides 441
Fig. 23.1 Classification of biopesticides
442 A. T. Rani et al.
Table 23.1 Some of the examples of microbial biopesticides for insect pest management
Microbial species/
variety Target pest Selected references
Bacterial
Bacillus thuringiensis
var. kurstaki, var.
aizawai, var. galleriae;
Lepidoptera (armyworms, dia-
mondback moth, Helicoverpa
armigera, Spodoptera litura, etc.)
Pawar and Singh (1993), Mohan
et al. (2009)
Bacillus thuringiensis
var. tenebrionis
Coleoptera (Colorado potato beetle,
Japanese beetle, etc.)
Pawar and Singh (1993), Bravo
et al. (2007)
Bacillus popilliae
Bacillus thuringiensis
var. isralensis;Bacillus
sphaericus
Diptera (mosquitoes and black flies,
etc.)
Pawar and Singh (1993), Bravo
et al. (2007)
Bacillus moritai
Fungal
Beauveria bassiana Broad spectrum of mites and insects Bhattacharya et al. (2003),
Mohammad Beigi and Port
(2015), Tadele and Emana
(2017)
Beauveria brongniartii Root grubs, B. borer, H. armigera Ruiu (2018), Zimmermann
(2007)
Metarhizium
anisopliae
Coleoptera, Diptera, Hemiptera,
Isoptera
Mohammad Beigi and Port
(2015), Erler and Ates (2015)
Lecanicillium lecanii Leafminers, mealybugs, scale
insects, aphids, whiteflies, thrips.
Kim et al. (2008), Nithya and
Rani (2019), Bouhous and
Larous (2012)
Nomuraea rileyi Lepidoptera Ruiu (2018), Pekrul and Grula
(1979)
Hirsutella thompsonii Spider mites Ruiu (2018)
Isaria fumosorosea Whitefly Ruiu (2018), Zimmermann
(2007)
Paecilomyces
fumosoroseus
Insects, mites, nematodes, thrips Ruiu (2018), Siddiqui and
Akhtar (2008)
Baculoviruses
Granulosis virus (GV) Adoxophyes orana, Plutella
xylostella, Cydia pomonella,
Phthorimaea operculella
Ansari et al. (2012), Ruiu (2018)
Nuclear polyhedrosis
virus (NPV)
Helicoverpa armigera, Heliothis
virescens, H. zea, Spodoptera
litura, S. littoralis, S. exigua,
Lymantria dispar
Ansari et al. (2012), Ruiu
(2018), Ramakrishnan (1993)
Entomopathogenic nematode
Steinernema
carpocapsae
Caterpillars, R. ferrugineus,
B. beetles, moth larvae crane fly,
Tipulidae
Ruiu (2018), Shapiro-Ilan et al.
(2012)
Steinernema feltiae Bradysia spp., C. syngenesiae,
codling moth larvae, P.vitalbae,
soil-dwelling pests, sciarids, thrips
Ruiu (2018)
(continued)
23 Biopesticides: An Alternative to Synthetic Insecticides 443
Since the active ingredients are living substances they offer them potentially higher
benefit over chemical pesticides, because of their target specificity, reproducibility,
and hence provide continuous pest control (SP-IPM 2006). The detailed mode of
action of many of these microbial pesticides has already been reported by several
workers (Ansari et al. 2012; Sahayaraj et al. 2011; Senthil-Nathan et al. 2009). These
pesticides suppress insect pests by causing disease or by producing specific toxins
against the target pest or through competition by preventing the establishment of
other organisms (Clemson 2007). The specific and diverse range of properties of
microbial pesticides make them desirable component for integrated pest
management.
23.2.2 Natural Biochemical Pesticides
Naturally occurring biochemical substances are retrieved from plants or insects and
used in pest management by eco-friendly mode of action. Biochemical pesticides are
broadly classified into three categories that include botanical pesticides and
semiochemicals (Fig. 23.1). It is estimated that approximately 2500 plant species
belonging to 235 different families have showed measurable biocidal properties
against various pests (Ansari et al. 2012). The botanical pesticides include plant
extracts and essential oils, natural plant growth regulators, and secondary metabo-
lites (Table 23.2). The natural compounds derived from plant extracts have biolog-
ically active compounds that are employed in pest control. Among different
botanical pesticides neem tree, Azadirachta indica (Meliaceae) is one of the best
known and most effective plant that contains azadirachtin as active ingredient, which
has various effects on insects such as antifeedant, repellent, deterrent (Saxena 1989),
and insect growth regulator because of its activity same as an insect hormone and can
be used in pest control. Semiochemicals are organic compounds produced by an
organism (either plant or insect) that mediate interactions between individuals of
same species (intraspecific communication caused by pheromones) or individuals of
a different species (interspecific communication caused by allelochemicals).
Semiochemicals include pheromones and allelochemicals. They can be used for
behavioral manipulation of insects and can be used in the control of insect pest. The
Table 23.1 (continued)
Microbial species/
variety Target pest Selected references
Heterorhabditis
bacteriophora
M. melolontha,Otiorhynchus spp.,
chestnut moths, black vine weevil,
leaf miners, caterpillars, cutworms,
and soil-dwelling beetle larvae
Ruiu (2018), Shapiro-Ilan et al.
(2012)
Protozoa
Nosema and
Vairimorpha
Lepidopteran and orthopteran
insects
Lewis (2002)
444 A. T. Rani et al.
Table 23.2 Some of the examples of biochemical pesticides for insect pest management
Botanical pesticides
Plant Active compound(s) Target pests Selected references
Neem tree, Azadirachta
indica
Azadirachtin Most of the herbivorous insects –
Chrysanthemum Pyrethrum Most of the herbivorous insects Casida and Quistad (1995)
Thyme, Thymus vulgaris Thymol Varroa mite (Varroa destructor) Floris et al. (2004)
Ageratum houstonianum Precocene I and II Heteroptera, Homoptera, and Orthoptera Bowers et al. (1976), Bowers
(1991), Grafton-Cardwell et al.
(2006)
Saponin IGR Mites, lepidopterans, beetles, and many other
insects
Ishaaya et al. (1969)
Basil, Ocimum basilicum Juvocimenes Several field crop pests Bowers and Nishida (1980),
Lopez et al. (2005)
Garlic, Allium sativum Diallyl disulfide B. brassicae, Sitotroga cerealella, S. littorals,
T. molitor, C. maculatus, P. xylostella
Plata-Rueda et al. (2017),
Lengai and Muthomi (2018)
Insect pheromones
Insect pest Compound(s) Type of pheromone Selected references
Helicoverpa armigera (Z)-11-hexadecenal + (Z)-9-hexadecenal and
(Z)-9-tetradecenal
Sex pheromone Zhang et al. (2012)
Spodoptera litura (Z,E)-9,11- and (Z,E)-9,12-tetradecadienyl
acetate
Sex pheromone Chen et al. (2018)
Leucinodes orbonalis (E)-11-hexadecenyl acetate, (E)-11-
hexadecen-1-ol
Sex pheromone Cork et al. (2001)
Fall armyworm
(Spodoptera frugiperda)
7-dodecen-1-ol acetate, dodecan-1-ol acetate,
(Z)-11-dodecen-1-ol acetate, (Z)-9-
tetradecen-1-ol acetate, (Z)-11-hexadecenal,
(Z)-9-tetradecenal, and (Z)-11-hexadecen-1-
ol acetate
Sex pheromone Tumlinson et al. (1986)
(continued)
23 Biopesticides: An Alternative to Synthetic Insecticides 445
Table 23.2 (continued)
Pink bollworm
(Pectinophora
gossypiella)
(Z,Z)-7,11- and (Z,E)-7,11-hexadecadienyl
acetates
Sex pheromone Foster and Roelofs (1988)
Japanese beetle, Popillia
japonica
(R,S)-5-(1-decenyl)-dihydr0-2-(3H)-
furanone
Sex pheromone Klein et al. (1981)
Cotton boll weevil,
Anthonomus grandis
Grandlure (cyclobutane alcohol (3 parts), a
cyclohexane alcohol (4 parts), and Z-and E-
pair of cyclohexane and acetaldehydes)
Aggregation pheromone Ansari et al. (2012)
Colorado potato beetle,
Leptinotarsa
decemlineata (Say)
(S)-3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol Aggregation pheromone Dickens et al. (2002)
Southern pine beetle,
Dendroctonus frontalis
1,5-Dimethyl-6,8-dioxabicyclo[3.2.1]octane Aggregation pheromone Renwick and Vite (1969)
California five-spined ips,
Ips confusus
(+)-2-Methyl-6-methylene-2,7-octadien-4-ol Aggregation pheromone Silverstein et al. (1966)
Green peach aphid,
Myzus persicae,
(E)-β-farnesene Alarm pheromone Kunert et al. (2010), De-Vos
et al. (2010)
Allelochemicals
Plant species Compound(s) Target pest Selected references
Neem tree, Azadirachta
indica
Azadirachtin (OR, AF, GR) Most of the herbivorous insects –
Lemon grass oil,
Cymbopogon nardus
Methyl eugenol (4-allyl-1,2-
dimethoxybenzenecarboxylate) (A)
Several Bacterocera species Cunningham (1989), Wee et al.
(2002), Khrimian et al. (2006)
Raspberry Cue lure [4-( pacetoxyphenyl)-2-butanone]
(A)
B. cucurbitae Khrimian et al. (2006)
Geraniol, eugenol, and phenethyl alcohol (A) Japanese beetle, P. japonica Hamilton et al. (1971),
Schwartz (1975)
446 A. T. Rani et al.
Cotton, Gossypium
hirsutum
Gossypol (Ab) Tobacco bud worm, Heliothes virescens and
cotton leaf worm, Spodoptera littoralis a
number of herbivorous insects
Parrott (1990), Guo et al.
(2013)
Cucurbits Cucurbitacins (A, Ab) Cucumber leaf beetles (Phyllotreta spp.,
Phaedon spp., and Ceratoma trifurcate); stem
borer (Margonia hyalinata); and red spider
mite (Phyllotreta spp., Phaedon spp., and
Ceratoma trifurcate); and other arthropods
(Phyllotreta spp., Phaedon spp.)
Da-Costa and Jones (1971)
OR oviposition repellent,AFantifeedant,GRgrowth regulation,Aattractant,AbAntibiosis
23 Biopesticides: An Alternative to Synthetic Insecticides 447
most commonly used semiochemicals in insect pest management are sex phero-
mones and aggregation pheromones (Table 23.2). Insect sex pheromones are an
essential component of insect pest control because of their high species specificity
and low toxicity (Goldansaz et al. 2004). Some of the examples of biochemical
pesticides are listed in Table 23.2.
Biochemical pesticides’active ingredients typically disrupt insect pests’growth,
development, reproduction, and overall biology. A single molecule or a combination
of molecules, such as plant secondary metabolites and/or essential oils, or a mixture
of structurally related molecules (isomers), such as insect pheromones, may be used
as the active ingredient (Ansari et al. 2012). While these active ingredients are
normally only in nature, a synthetic analog might be possible. Allelochemicals can
be further grouped into attractants, repellants, deterrents, stimulants, growth regula-
tors, or mating disruptors based on the behavior elicited by them. Methyl eugenol
and cue lure are the most commonly used attractants in the male annihilation
technique (MAT) for the control mango fruit fly, Bactrocera dorsalis and melon
fruit fly, Zeugodacus cucurbitae (Table 23.2; insect pheromones are often used for
tracking or mass trapping (Reddy et al. 2009), lure-and-kill Schemes (El-Sayed et al.
2009), and mating destruction (Witzgall et al. 2010). There are about 122 registered
active ingredients of biochemical pesticides that include 6 insect growth regulators,
20 plant growth regulators, 18 floral attractants, 19 repellents, and 36 insect phero-
mones (Mandula 2008).
23.2.3 Transgenics/Plant-Incorporated Products
Transgenic crops are referred as plant-incorporated protectants (PIPs), which are
grouped under biopesticides as an alternative to chemical insecticides. Plants them-
selves produce protecting substance (toxins) after the introduction of genetic mate-
rial coding for that toxic substance. Plants inserted with such transgene are called
genetically modified crops, plant pesticides, or plant-incorporated protectants. The
best-known example for the PIPs is Bt transgenic plants inserted with gene coding
for the Bt toxin into the chromosome of the crop plants, thus the plants become toxic
to the pest. Examples of some of the transgenic plants developed for management of
insect pests are listed in Table 23.3. In this situation, the Environmental Protection
Agency (EPA) regulates the Bt pesticidal protein and its genetic content, but not the
plant itself (Mazid et al. 2011). Transgenic crops are low cost and eco-friendly
technology for the resource poor farmers to manage pests and diseases as well as
other constraints such as abiotic stress and human vitamin A deficiency (SP-IPM
2006).
448 A. T. Rani et al.
Table 23.3 Some of the examples of transgenics/plant-incorporated products developed for insect
pest management
Plant Gene Target pest Selected references
Tomato cry 1Ab, cry 1Ac Heliothis virescens,
M. Sexta, H. armigera
Mandaokar et al.
(2000), Leng et al.
(2011), Arora and
Shera (2014)
Potato cry 3, cry 3a, cry 3b, cry 2a5,
cry 1Ab, cry 1Ac9, cry 5
Leptinotarsa decemlineata,
Phthorimaea operculella
Arora and Shera
(2014), Rico et al.
(1998), Coombs
et al. (2002)
Soybean cry1A(c) H. zea,H. virescens, ciga-
rette beetle Pseudoplusia
includens
Leng et al. (2011),
Stewart et al.
(1996a)
Maize/
corn
cry 1Ab, cry 9C, cry 3Bb, cry
1F, cry 34Ab1/cry 35 Ab1,
cry 1 Ab + cry 3Bb, cry
1F + cry 34Ab1/cry 35 Ab1
Ostrinia nubilalis, Chilo
partellus, Busseola fusca,
H. zea, Diatraea
grandiosella, D. saccharalis,
S. frugiperda, Diabrotica
undecimpunctata howardi,
D. virgifera virgifera
Leng et al. (2011),
Arora and Shera
(2014), Jansen et al.
(1997)
Rice cry 1 Ab, cry 1 Ac, cry
1Ab/cry1Ac, cry2a
Chilo suppressalis,
Cnaphalocrocis medinalis,
Scirpophaga incertulas
Leng et al. (2011),
Arora and Shera
(2014)
Cotton cry 1Ac, cry 1Ab/cry 1Ac, cry
1Ac + cry2Ab, cry1C
Helicoverpa armigera,
Pectinophora gossypiella,
Earias spp., Heliothis
virescens, H. zea,
Trichoplusia ni, Spodoptera
spp.
Arora and Shera
(2014), Adamczyk
and Hardee (2001)
Canola cry1A(c), cry 1C Diamondback moth, H. zea,
cabbage looper, Susumia
exigua, H. zea, S. exigua
Arora and Shera
(2014), Stewart
et al. (1996b)
Poplar cry 1Aa, cry3Aa Lymantria dispar,
Chrysomela tremulae F.
Arora and Shera
(2014), Cornu et al.
(1996)
Sorghum cry 1Ac Chilo partellus Arora and Shera
(2014)
Sugar
cane
cry 1Ab D. saccharalis Arora and Shera
(2014)
Chickpea cry 1Ac H. armigera Arora and Shera
(2014)
Tobacco cry 3, cry 2a5, cry 1Aa, cry
1Ab, cry 1Ac
H. virescens, Manduca
sexta, H. armigera, H. zea,
Leptinotarsa decemlineata
Arora and Shera
(2014), McBride
et al. (1995)
Brinjal cry 1Ac, cry 3b Leucinodes orbonalis,
Leptinotarsa decemlineata
Arora and Shera
(2014)
Chinese
cabbage
cry 1Ab, cry 1Ac Plutella xylostella Arora and Shera
(2014)
(continued)
23 Biopesticides: An Alternative to Synthetic Insecticides 449
23.2.4 Natural Enemies (Predators and Parasitoids
of Insects)
Plants are protected or vector colonies are reduced by natural enemies such as insect
predators (Coccinellid beetle and lacewings) and parasitoids (hymenopteran wasps
and dipteran flies). Natural enemies are responsible for natural suppression of pest
population. A few of the most common insect predators and parasitoids, respective
target pests and their host plants are provided in Table 23.4.
23.3 Success Stories of Insect Pest Management Using
Biopesticides
In India, several good cases of traditional biological management of insect pests and
weeds have been analyzed in-depth (Singh 2004). Some of the success stories of
agricultural pests management in India including weeds by using biological control
agents are given in brief.
23.3.1 Controlling the Papaya Mealy Virus
Paracoccus marginatus Williams and Granara de Willink (Hemiptera:
Pseudococcidae), the papaya mealybug (Hemiptera: Pseudococcidae) is a dreaded
insect pest native to Mexico and/or Central America (Miller et al. 1999) that was first
described in 1992. It never attained the status of a pest in the native country due to
the existence of endemic natural enemies’complex, however posed a serious threat
to horticultural industry following its invasion into the Caribbean region, south and
southeast Asian countries. Natural enemies from native areas, such as Acerophagus
papayae (Noyes and Schauff), Anagyrus loecki (Noyes and Menezes), Anagyrus
californicus Compere, Pseudleptomastix mexicana, and Pseudaphycus sp.,were
used to monitor the pest in invaded countries (U.S. Department of Agriculture,
Animal and Plant Health Inspection Service 1999,2000; Meyerdirk and Kauffman
2001; Noyes and Schauff 2003). In the Dominican Republic, Guam, and Puerto
Table 23.3 (continued)
Plant Gene Target pest Selected references
Broccoli cry 1C P. xylostella, T. ni, Pieris
rapae
Zhao et al. (2001)
Groundnut cry 1Ac Elasmopalpus lignosellus Arora and Shera
(2014)
Alfalfa cry 1C S. littoralis Arora and Shera
(2014)
450 A. T. Rani et al.
Table 23.4 Some of the examples of natural enemies used in insect pest management
Natural enemy Target pest Host Selected references
Predators
Cryptolaemus
montrouzieri
Coffee green scale, Coccus viridis
(Green); Planococcus citri (Risso),
P. lilacinus (Cockerell), Ferrisia
virgata (Cockerell), Maconellicoccus
hirsutus (Green)
Several plantation and
horticultural crops
Mayne (1953)
Rodolia
cardinalis
Cottony cushion scale, Icerya purchasi Citrus, wattle, and other
Acacia spp. are among the
crops affected
Subramanyam (1955)
Platymeris
laevicollis
Oryctes rhinoceros Coconut Singh (1994)
Phytoseiulus
spp.
Tetranychus urticae Phaseolus vulgaris; Sola-
num lycopersicon;
Fragaria ananassa
Rhodes and Liburd (2006), Silva et al. (2010), Hoque et al. (2010)
Tetranychus evansi
Neoseiulus spp. Tetranychus urticae Vicia faba; Capsicum
annuum; Persea
americana
Elmoghazy et al. (2012), Takano-Lee and Hoddle (2002)
Oligonychus perseae
Amblyseius
swirskii
Scirtothrips dorsalis Capsicum sp. Arthurs et al. (2009)
Parasitoids
Trichogramma
spp.
Many lepidopteran pests Several field and horticul-
tural crops
Sankaran (1974)
Copidosoma
koehleri
Phthorimaea operculella Potato Noyes and Hayat (1994)
Cotesia sp. Chilo infuscatellus, C. sacchariphagus
indicus, Scirpophaga excerptalis
Sugarcane Rao et al. (1971)
Chelonus
blackburni
Cameron
Pectinophora gossypiella, Earias spp.,
Phthorimaea operculella
Cotton, okra, hollyhock,
potato
Singh (1994)
(continued)
23 Biopesticides: An Alternative to Synthetic Insecticides 451
Table 23.4 (continued)
Natural enemy Target pest Host Selected references
Diadegma
semiclausum
Plutella xylostella Cruciferous vegetables Singh (1994)
Encarsia
guadeloupae
Spiraling whitefly, Aleurodicus
dispersus
Several agricultural and
horticultural crops and
ornamentals
Ramani et al. (2002)
Aphidius
colemani
Aphis gossypii Dendranthema grandiflora Vasquez et al. (2006)
Encarsia
formosa
Bemisia tabaci Solanum lycopersicon Dai et al. (2014), Moreno-Ripoll et al. (2012)
Tetrastichus sp. Chilo infuscatellus, C. sacchariphagus
indicus, Scirpophaga excerptalis
Sugarcane Sankaran (1974)
Tachinid flies Caterpillars, beetles, bugs Sugarcane –
452 A. T. Rani et al.
Rico, respectively, the release of parasitoid wasps resulted in a 99.7%, 99.5%, and
97.5% decline in the density of mealybug species (Meyerdirk and Kauffman 2001;
Kauffman et al. 2001). Later during 2008 the pest was first detected in Tamil Nadu,
India subsequently spread to the rest of the country caused serious damage to
papaya. The pest was successfully brought under control through the importation
of three natural enemies, i.e. A. papayae,A. loecki, and P. mexicana. Even though
the aforesaid bio-agents able to parasitize the mealybug, the establishment of
the parasitoids was not uniform over the varied geographic region.In Puerto Rico,
the Dominican Republic, and the Indian subcontinent, Acerophagus sp. has arisen as
the dominant parasitoid group (Meyerdirk and Kauffman 2001).
23.3.2 Management of Sugarcane Wooly Aphid:
Ceratovacuna lanigera Zehnt-ner
The aphid is native to India and was first reported from West Bengal during 1958 and
limited distribution to northern and northeastern region of India. The aphid invaded the
tropical Indian states of Maharashtra and Karnataka in 2002 and later spread to southern
Indian states (Patil et al. 2004; Joshi and Viraktamath 2004; Thirumurugan et al. 2004;
Srikanth 2007). A year after its first report the pest was observed in 3.13 lakh ha of
sugarcane-growing areas in Maharashtra and Karnataka alone. Following its damage the
sugarcane growers resorted to spraying chemical pesticides to control the pest, but in
vain. The chemical gave only temporary reliefandoftenfarmershadtosprayrepeatedly.
Thisresultedinthedestruction of the natural enemy’s complex as well as environmental
hazards. Hence effective bio-agents were selected and released for the management of
wooly aphids and farmers were advised not to apply the chemical pesticides.
Two effective parasitoids, namely Micromus igorotus Banks (Neuroptera:
Hemerobiidae) and Dipha aphidivora (Lepidoptera: Pyralidae), were mass multi-
plied and released under field condition. In addition, farmers have been advised to
conserve existing natural enemies in the sugarcane ecosystem. Within a few years of
release, bio-agents have been successful in reducing the aphid population. An
estimated benefit of approximately 398.23 crores was realized through this biocon-
trol intervention. Continuous surveys and surveillance of wooly aphid in sugarcane-
growing areas in southern states revealed the scanty presence of wooly aphid (up to
5% incidence), indicating the effectiveness of the biocontrol strategy.
23.3.3 Biological Control Cassava Mealybug in Sub-Saharan
Africa
Phenacoccus manihoti, a mealybug, was inadvertently introduced to Africa from
South America in the 1970s and soon became a major threat to cassava, one of
Africa’s most valuable staple crops. In order to manage the P. manihoti, the parasitic
23 Biopesticides: An Alternative to Synthetic Insecticides 453
wasp, Anagyrus lopezi was imported from South America, which is the native place
of Cassava and cassava mealybug. The parasitic wasp reared in the laboratories and
released into the field which began controlling the mealybug almost immediately
after release that caused measurable reduction in the pest damage. The mealybug
population was reduced to 10% of peak numbers within 2–4 years and that continues
till today. This is one of the most active traditional biological control programs in
history (SP-IPM 2006).
23.3.4 Classical Biological Control of Terrestrial and Aquatic
Weeds Using Insect Biocontrol Agents
Via trade and transportation, many weed species were either intentionally or
unintentionally introduced into India. Prickly Pear, Opuntia elatior Miller, Opuntia
Stricta (Haworth), and Opuntia vulgaris Miller (Cactaceae), which entered India
through cochineal trade, have become a serious problem in agriculture in South
India. This weed species was successfully controlled by the introduction of cochi-
neal insect, Dactylopius ceylonicus during 1795. O. Stricta was spectacularly con-
trolled by the introduction of North American species D. Opuntiae in 1926 from Sri
Lanka. Presently D. ceylonicus continues to successfully control O. vulgaris in
southern India and northern Sri Lanka reducing it to the state of virtual extinction
(Singh 2004).
Similarly, in 1982, the arrival of the weevil, Cyrtobagous salviniae, from
Australia, which was endemic to Brazil, was used to biologically kill the water
fern, Salvinia molesta. Water hyacinth, Eichhornia crassipes, a free-floating aquatic
grass, was successfully regulated in India in 1982 by introducing three exotic natural
enemies, namely hydrophilic weevils Neochetina bruchi (Ex. Argentina) and
N. eichhorniae (Ex. Argentina) and galumnid mite Orthogalumna terebrantis
(Ex. South America), and galumnid mite (Singh 2004).
23.3.5 Entomopathogenic Microorganisms: An Unsung
Warrior of Biological Control
Entomopathogenic organisms including bacteria, fungi, and viruses are the environ-
ment friendly and best alternative to traditional insecticides (Usta 2013). Its usage is
not generalized because of high specificity each pest has its own strain. They are
specific to target insects and highly safe to mammals and the environment. Despite
their vulnerability to environmental changes, microbial insecticides are preferable to
conventional insecticides in that they destroy pest colonies while maintaining natural
predator and parasite species under certain conditions (Chattopadhyay et al. 2017).
454 A. T. Rani et al.
Rather the bio-agents are self-perpetuating and only control tactic that increases in
numbers and spread, with increasing pest population.
The bio-agents are the very important component of IPM that helps in successful
control of key insect pest under different ecosystem. Among the biopesticides, the
products based on entomopathogenic bacteria (EPB) are most commonly used
among the farming community (Chattopadhyay et al. 2017). Many public and
private laboratories are producing biopesticides and distributing to farmers.
23.4 Status and Market Scenario of Biopesticides in India
For a very long time, the idea of biological control of insect pests and pathogens has
been in use (Schmutterer 1985). For a long time, seed grain protectants have been
made from the derivatives of the neem tree, Azadirachta indica, such as leaf extract,
fat, and seed cake (Isman 1997; Brahmachari 2004). When chemical insecticides
failed to combat Helicoverpa armigera, Spodoptera litura, and other cotton pests in
India, the value of biocontrol became clear (Kranthi et al. 2002). It was recognized
that biocontrol is the only alternative management approach that can be used to
combat chemical pesticide resistance in insect pests and diseases.
In India, there are 361 biocontrol laboratories and/or units, according to the
Directorate of Plant Protection, Quarantine, and Storage (DPPQS) that includes
private sector laboratories and also those private sector laboratories aided by GOI
grants, state as well as ICAR/SAUs/DBT laboratories and central IPM centers.
Although only a handful of them are engaged in processing (Fig. 23.2). According
Fig. 23.2 The total number of biocontrol laboratories and units currently operating in India.
(Source: Mishra et al. 2020)
23 Biopesticides: An Alternative to Synthetic Insecticides 455
to the data biopesticide use seems to have risen in the last two decades. The share of
biopesticides, however, is only two percent of the overall market for pesticides.
There are currently 970 biopesticide products registered with the Central Insecticides
Board and Registration Committee (CIBRC), which is India’s top governing body
for all forms of biopesticides use. The total number of registered biopesticides and
their formulation types is listed in Table 23.5. Totally 51 biopesticides formulations
Table 23.5 Under section 9(3) of the Insecticides Act, 1968, a list of approved biopesticides and
their formulations for use in the country is available. (As on 01.01.2021)
Name of bio-insecticide Formulation type
No. of
formulation
I. A. Microbial–bacterial biopesticides
Ampelomyces quisqualis 2.00%WP 1
Pseudomonas fluorescens 0.5%WP,1.75%WP, 1%WP,1.5%WP, 2.0% AS,
1.5% LF
6
Bacillus sphaericus 1.3% FC 1
Bacillus subtilis 2.0% AS 1
Bacillus thuringiensis var.
galleriae
1.3% FC 1
Bacillus thuringiensis var.
israelensis
5% AS, 12% AS, 5.0% WP 3
Bacillus thuringiensis var.
kurstaki
5% WP, 2.5% As, 0.5% WP, 3.5% AS 4
I. B. Microbial–fungal biopesticides
Beauveria bassiana 1.15% WP, 1.0% WP,1.15%SC, 10% SC, 5.0%
WP
5
Metarhizium anisopliae
Verticillium lecanii 1.15% WP, 1.5% liquid 2
Verticillium
chlamydosporium
1.00 WP 1
Trichoderma harzianum 0.50% WS, 1.0% WP, 2.0% WP, 2.0% AS 4
Trichoderma viride 1.0% WP, 1.15% WP, 5.0% SC, 1.0% AS, 5.0% L.
F.
5
I. C. Microbial–viral biopesticides
Nuclear polyhyderosis virus
of Helicoverpa armigera
0.43% AS, 2.0% AS. 0.5% AS 3
Nuclear polyhyderosis virus
of Spodoptera litura
0.5% AS 1
II. A. Biochemical–botanical pesticides
Azadirachtin (neem
products)
25%, 10%, 0.03% EC, 0.1% EC, 0.15% EC, 5%
EC, 0.3%EC, 15% extract concentrâtes, 1% EC,
0.1% Gr, 0.15%Gr, 0.03% (300 PPM) w/w min
12
III. B. Biochemical pesticides–pheromones
Gossyplure (PB-RopeL) Dispenser 1
Total 51
Data collected from the DPPQS of the Ministry of Agriculture and Farmers Welfare of the
Government of India.; ppqs.gov.in/divisions/cib-rc/major-uses-of-pesticides
456 A. T. Rani et al.
have been registered for use in the country under the Insecticide Act 1968.
Azadirachtin or Neem products have registered maximum number of formulations
compared to other biopesticide products. The industries are producing bacterial,
fungal, microbial, and other (plant-based, pheromones) biopesticides that account
for 29, 66, 4, and 1% of total production, respectively.
As per the DPPQS data, the area under use of biopesticides is 11%, whereas,
maximum area comes under the use of chemical pesticides with a share of 84% and
only 5% area comes under both chemical and biopesticides (Fig. 23.3). Among
different types of biopesticides, the fungal biopesticides consumption is maximum
with a 50% share as compared to bacterial (21%), viral (15%), and botanical
pesticides (14%), respectively (Fig. 23.4). The data suggests that the consumption
of various biopesticide formulations in different states has showed steady increase
from 5152 MT during 2014–2015 to 7804 MT of technical grade material during
2019–2020 (Fig. 23.5).
Fig. 23.3 Area under chemical and biopesticides in India during 2019–2020. (DPPQS, Ministry of
Agriculture and Farmers Welfare, Government of India data; ppqs.gov.in/statistical-database)
23 Biopesticides: An Alternative to Synthetic Insecticides 457
Fig. 23.5 Consumption of biopesticides formulations in various states during 2014–2015 to
2019–2020. (Data collected from the DPPQS of the Ministry of Agriculture and Farmers Welfare
of the Government of India; ppqs.gov.in/statistical-database)
Fig. 23.4 Pesticide wise consumption of indigenous biopesticides in India during 2019–2020.
(Data collected from the DPPQS of the Ministry of Agriculture and Farmers Welfare of the
Government of India; ppqs.gov.in/statistical-database)
458 A. T. Rani et al.
23.5 Synthetic Pesticides Versus Biological Pesticides
(Table 23.6)
23.6 Limitations/Constraints in the Promotion
and Consumption of Biopesticides
The need for new synthetic insecticide substitutes that are good for the environment
and human health has become a major focus of science research and may lead to the
production of safe foods. While biopesticides will never be able to completely offset
the use of conventional insecticides, they will help to solve some of the problems
associated with their use. The major limitation of use of biopesticides is their limited
shelf life and timely unavailability. In addition, the efficacy of biopesticides shows
Table 23.6 Comparison between synthetic and biological pesticides
Synthetic or chemical pesticides Biological pesticides or biopesticides
Uses non-living substance, synthetic products
in the form of different type of formulations
Derived from naturally occurring living organ-
isms including plants, animals, and microbes,
used as such or as their products or by-products
Quick and easy to use, highly effective as they
are fast-acting
Most of them are slower acting, often less toxic
than conventional pesticides
Broadspectrum of action Very specific to the target pest
Relatively expensive Relatively cheaper
Easily stored for long duration, i.e. more shelf
life
Cannot be stored for long duration, i.e. less shelf
life
Readily available through a long-established
market
Not readily available
Causes serious environmental pollution Eco-friendly; safe to environment
Hazardous to natural predators, pollinators,
and non-target species are all negatively
affected
Pest natural predators, pollinators, and
non-target species are all safer
They work against nature disrupt ecosystems They work with nature, maintain ecological
balance
Pests eventually become resistant Less likely to have resistance issues
Diminishing market Growing market preference
Longer residual activity, provide greater per-
sistent control under field conditions
Effective in small quantities, resulting in lower
operator exposure
The cost effectiveness is low, but the cost of
spraying is higher
Expenses are higher, but the number of appli-
cations is lower
High persistence and long-term impact Low persistence and decompose quickly
Easy handling and bulkiness but danger and
harmful
Bulk: Carrier based, easy: Liquid formulation
Curative nature of control Preventive nature of control
23 Biopesticides: An Alternative to Synthetic Insecticides 459
high variability under different environmental condition. The users prefer consis-
tency and reliability of pest control methods. In the era of commercial agriculture,
the farmers prefer to use conventional insecticides having quick knockdown effect
on the insect pests.
23.7 Future Directions
The modern agriculture is increasingly affected by degradation and overexploitation
of natural resources, frequent occurrence of inclement weather events influenced by
climate change, and excessive application of synthetic inputs on farms including
injudicious use of pesticide or even use of banned or spurious ones. The government
should set stringent safety criteria on conventional insecticides, which result in the
fewer molecules in the market which promote biopesticides. The use of biopesticides
must be a mass movement rather than an individual’s effort. Biopesticides have been
used to protect seeds from rodents and diseases for decades. However, in contrast to
traditional chemicals, their demand and position among agrochemicals are still far
behind.
The effectiveness, shelf life, processing processes, limited range of host or target
pathogens/pests, low field efficiency, distribution system issues, economics, and
regulations are all obstacles to widespread biopesticide use in India. To create trust
in the system, both the private and public sectors will need to collaborate and work
with farmers at the grassroots level.
23.8 Conclusions
The farmers around the globe are majorly using synthetic insecticides for the
management of a pest in their agricultural ecosystem. Even though the application
of chemicals helps in bringing down the insect population, it unfortunately resulted
in posing multiple side effects to the surrounding environment. Hence, there is an
immediate need to develop an appropriate pest management approach like use of
biopesticides. These pesticides are promising alternatives for use in pest manage-
ment tactics. The biopesticides like entomopathogens (bacteria, fungi, viruses, pro-
tozoa, and nematodes), insect growth regulators, semiochemicals (pheromones),
botanicals, plant-incorporated protectants (alkaloids, steroids, terpenoids, essential
oils) have been proposed as safer and ecologically alternative to conventional
synthetic pesticides for sustainable agricultural production.
Acknowledgements The first author is thankful to the editor for inviting to contribute this chapter.
This work was supported by the ICAR-Indian Institute of Vegetable Research, Varanasi.
460 A. T. Rani et al.
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Chapter 24
Impact of Pesticides on Microbial
Population
Sujan Majumder, Anindita Paul, Anup Kumar, Chandan K. Verma,
Pratap A. Divekar, Vijaya Rani, A. T. Rani, Jaydeep Halder, K. K. Pandey,
and Jagdish Singh
Abstract Microbes are constituting elements of the soil environment and their
abundance, enzymatic activity, degradation process, and biodiversity indicate the
balance in the agro-ecological system. It is necessary to keep strengthening the
scientific basis of modern agriculture because pesticides may be purposefully used
only if their persistence, bioaccumulation, and toxicity in agro-ecosystems are
strictly controlled. The use of agrochemicals, such as chemical fertilisers and
pesticides, is important in modern crop management strategies (mainly insecticides
and herbicides). Pesticide poisoning affects three million people worldwide,
according to the WHO. Long-term and indiscriminate pesticide use has serious
negative consequences for soil microbes, the nutrient cycle, the decomposition
process, and the atmosphere, resulting in long-term negative consequences for
food stability, human health, and the environment. Pesticide application can alter
microbial diversity, which can be detrimental to plant growth and development by
decreasing nutrient availability or disrupting the nutrient cycle. Therefore, the
qualitative, innovative, and demand-driven pest management is the need of the
hour. Hence, this chapter covers the positive and negative consequences of pesti-
cides on microbes and their environment and current issues about the extensive use
of pesticides.
Keywords Microbes · Pesticides · Decomposition processes · Nutrient cycle
S. Majumder (*) · C. K. Verma · P. A. Divekar · V. Rani · A. T. Rani · J. Halder · K. K. Pandey ·
J. Singh
ICAR-Indian Institute of Vegetable Research, Varanasi, India
A. Paul
ICAR-Central Tobacco Research Institute, Rajahmundry, India
A. Kumar
ICAR-Indian Grassland and Fodder Research Institute, Jhansi, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_24
467
24.1 Introduction
Pesticides are effective agrochemicals that are used to protect crops from pests in the
agricultural system. Pesticides are chemicals used to deter, eliminate, or control
pests, unwanted species of plants, or animals that are causing harm or interfering
with the cultivation, manufacturing, storage, transportation, or selling of food,
agricultural crops, wood, wood products, or animal feedstuffs or which can be
given to animals to keep flies, mites/spider mites, and other pests out of or on their
bodies (FAO 1989).
Pesticide residue is described as substances that remain in or on a feed or food
product, soil, air, or water after a pesticide has been applied. It contains the parent
compound as well as any toxicologically relevant degradation products, metabolites,
or impurities. Pesticides are often applied in crop to manage the pest during cropping
seasons and non-cropping seasons to regulate the weed in fallow land and those pes-
ticides reach the soil, and water. The non-judicious use of pesticides caused hazard
and toxicity to non-target organism (birds) and environmental pollution. The effect
of pesticides on soil microorganisms is determined by the physical and chemical
characteristics of the soil, as well as the chemical composition and concentration of
the pesticides. Microbes have been shown to be able to thrive in the presence of
pesticides and use the pesticide molecules as carbon and energy sources in numerous
studies. Pollen and nectar poisoned by systemic insecticides (pesticide moves inside
the plant via xylem) can kill bees and other pollinators. Pesticide usage over time can
lead to bioaccumulation and biomagnification in plants and other species. Pesticides
that disrupt microorganisms’activities can have an effect on the nutrient cycle and
soil nutritional quality, resulting in severe ecological imbalance (Handa et al. 1999).
The microbial population of 1 g of soil during a counting is a barometer of a
country’s agricultural prosperity. The total mass of microflora and fauna underneath
the soil is 20 times that of the entire world’s human population. In one gramme of
healthy soil, there are one million to 100 million bacteria involved in organic matter
breakdown, 0.15–0.5 mg of fungal hyphae, 10,000–100,000 protozoa, a few to
several hundred microarthropods, 15–500 nematodes, and a couple of earthworms
(Lavelle et al. 2001). Microbial activity (primarily bacteria and fungi) aids in the
breakdown of soil organic matter and the management of soil aggregates, while other
soil components help to maintain proper number of bacteria and fungi through prey–
predator encounters, assisting in the recycling and preservation of basic nutrients.
These soil food cycle components are in harmony and are masterfully crafted in
interdependent relationships. The structure and performance of the soil food cycle,
i.e. the number, operation, and community structure, serve as a key indicator of
ecosystem health. Directly monitoring the active and total biomass of each organism
group can aid in detecting the dynamics of change that lead to ecosystem harm.
Once pesticides are applied, they dissipate in the soil, water, and environment and
persist for a long time or even year after year and have negative impacts on microbial
populations. As a result of the decrease in microbe numbers, the food chain
468 S. Majumder et al.
conducted by a group is disrupted, as are the components that depend on it. Owing to
the loss of soil organic matter, most microbial groups undergo a series of modifica-
tions, resulting in altered predator–prey modules, which cause changes in soil
aggregation, soil chemistry, pH, and structure. Over the course of a few decades,
the soil becomes barren due to a lack of organic matter to support microorganism
growth and development (Chowdhury et al. 2008).
24.2 Recent Trend of Pesticides in India
Chemicals such as insecticides, herbicides, or fungicides are commonly used for the
control of various pests in agriculture. There are thousands of pesticides of both
biological and chemical origins which are used commonly all over the world to
minimise losses of crop production. In India insecticides contribute a higher share in
total consumption of pesticides. Both per hectare consumption and total consump-
tion of pesticides increase significantly after 2009–2010 (Fig. 24.1).
Pesticide intake per hectare in 2014–2015 was 0.29 kg ha
1
, which is approxi-
mately 50% higher than the consumption in 2009–2010. Due to the rising cost of
manual labour for weed control, herbicides play an important role in the increased
use of pesticides (FICCI 2015). Punjab had the highest per hectare intake in
2016–2017 (0.74 kg), followed by Haryana (0.62 kg) and Maharashtra (0.62 kg)
(0.57 kg). Pesticide consumption per hectare in India is 0.69 kg on average. When
compared to other countries such as China (13.06 kg ha
1
), Japan (11.85 kg ha
1
),
Fig. 24.1 Pesticide (technical grade) consumption in India. (Source:Data from Ministry of
Chemicals and Fertilizers, Govt. of India)
24 Impact of Pesticides on Microbial Population 469
Brazil (4.57 kg ha
1
), and other Latin American countries, this is significantly lower
(FAOSTAT 2017).
24.3 Pesticide Production Scenario
In India, insecticides are preceded by fungicides, herbicides, and rodenticides in
order of development (Fig. 24.2).
The production share of insecticides lower down from more than 70% in the year
2003–2004 to 39% in the year 2016–2017. The shares of other groups of pesticides
such as herbicides, fungicides, and rodenticides grow over a period of time. The
increase in the production of fungicides is mainly due to increased use in vegetables
and fruits.
24.4 Trade in Agro-chemicals
In the year 2016–2017, 377.76 thousand tonnes of pesticides were exported from
India in which fungicide contributes the largest share of 45.94%, followed by
herbicides with a share of 28.19% (Fig. 24.3).
Mancozeb, cypermethrin, sulphur, acephate, and chlorpyrifos were the top five
pesticides exported in 2016–2017, according to the Central Board of Excise and
Customs (CBEC), while glyphosate and atrazine were the top two imported items.
Brazil, the USA, and France are the major countries where pesticides were
exported from India (Table 24.1), while China and Germany were major exporters
to India.
Fig. 24.2 Share of different groups of pesticides (technical grade) in terms of production. (Source:
Ministry of Chemicals and Fertilizers, Govt. of India)
470 S. Majumder et al.
24.5 Effect of Insecticides on Microbes
In plant protection perspective, insecticides are widely used in agriculture. Due to
their xenobiotic properties, the growth of soil microbes and their related soil
bioremediation can be negatively impacted by pesticides. Insecticide-contaminated
soils are found to inhibit nitrogen-fixing and phosphorus-solubilising microorgan-
isms. Recent studies show that certain pesticides impair plant-to-plant molecular
interactions with N-fixing rhizobacteria, thereby retarding the essential biological
nitrogen fixation cycle. Similarly, several studies have shown that insecticides
suppress soil enzyme activity, which is a key indicator of soil health. Several
biochemical reactions can also be affected by pesticides such as mineralisation of
organic matter, nitrification, denitrification, ammonification, methanogenesis, etc.
On the other hand, a few studies show some positive effects of chemicals applied on
soil health.
Pesticides undergo a sequence of degradation, transport, and adsorption/desorp-
tion processes in soil, which are influenced by the pesticide’s chemical composition
(Laabs et al. 2007) and soil properties (Shixian et al. 2018). Interaction of pesticides
with soil microbes and their metabolic activities influence the physiological and
Fig. 24.3 Export and import of major pesticides in India for the year 2016–2017. (Source:
DGCI&S, Ministry of Commerce and Industry taken from Subash et al. 2017)
Table 24.1 Major import and
export countries for pesticide
trade in 2016–2017 (tonnes)
Country Insecticides Fungicides Herbicides
Export Brazil 9437.61 42,898.27 20,457.02
USA 3275.35 8307.82 6095.06
France –7954.77 –
Import China 11,095.18 2220.28 15,243.93
Germany 1065.93 1523.81 –
Japan ––2428.02
Israel ––4732.66
Source: DGCI&S, Ministry of Commerce and Industry, Govt. of
India, taken from Subash et al. (2017)
24 Impact of Pesticides on Microbial Population 471
biochemical behaviour of the microbes (Singh et al. 2006). Microbial biomass is a
key indicator of microbial activity and provides a direct measure of the association
between microbial activities and the transformation of nutrients and further ecolog-
ical processes (Schultz et al. 2008). In general, a decline in soil respiration represents
a decrease in microbial biomass (Klose et al. 2004) or an increase in respiration
(Haney et al. 2000). Some microbial groups are able to replicate using applied
pesticides as a source of energy and nutrients, whereas the pesticide may be
detrimental to other species (Johnson et al. 2001). The use of pesticides can indeed
suppress or destroy certain groups of microorganisms and outnumber other groups
by eliminating them from competition. Various positive and negative effects of
insecticides on microbes are summarised in Table 24.2.
24.6 Effect of Herbicides on Microbes
Use of herbicides is a common practice all over the world to control unwanted plants
in cropped as well as non-cropped areas. Herbicides that were used onto the surface
of soil are more likely to influence the growth of microflora as well as micro-fauna.
Due to excessive addition of herbicides in the soil, qualitative as well as quantitative
alteration in terms of microbial population as well as their enzymatic activities may
occur (Min et al. 2002; Saeki et al. 2004). Application of herbicides may also kill the
sensitive species of bacteria, fungi, and protozoa that compete with the disease-
causing microorganisms, leading to upsetting balance between harmful and benefi-
cial microorganisms. This might lead to a rise of a problem and give opportunities to
the pathogens to infect the main crops (Kalia et al. 2004). It is well known in the
literature that some microorganisms degrade herbicides leading to stimulated growth
of microorganism, whereas some of the other microorganisms were adversely
affected in terms of their growth and population depending on the rate of application
and type of herbicides used in the field and also on microorganism species and
environmental conditions (Sebiomo et al. 2011; Zain et al. 2013).
Many researchers looked into the impact of various types of herbicides on
different types of microbes (Adhikary et al. 2014). The effects of three widely
used herbicides (pendimethalin, oxyfluorfen, and propaquizafop) on soil microbial
species in chilli (total bacteria, fungi, and actinomycetes) were studies and found that
herbicide treatments inhibited the production of all three microbial populations in the
soil, with the degree of inhibition varying depending on the herbicide used during
chilli growth. From the start of the effect until 15 days after application, there was a
growing pattern of inhibition on microbial population growth.
Herbicides (paraquat, glyphosate, glufosinate-ammonium, and metsulfuron-
methyl) have an impact on microbial population growth, with the degree of inhibi-
tion being strongly linked to the rates at which they are applied in oil palm
plantations. To bacteria and actinomycetes, paraquat had the greatest inhibitory
effect, while glyphosate had the greatest effect on fungi, and metsulfuron-methyl
472 S. Majumder et al.
Table 24.2 Effect of insecticides on microbes
Sl.
no. Insecticides Microbes Impact References
1. Chlorpyrifos,
methylpyrimifos,
profenofos
Nitrogen-fixing
bacteria,
denitrifying bacte-
ria, and nitrifying
bacteria
Decreased beneficial
microorganism
populations
Martinez-
Toledo et al.
(1992)
2. Fenvalerate Azotobacter
Azospirillum
brasilense
Azospirillum
lipoferum
Decreased respiration rate
and protein contents of
diazotrophs
Omar et al.
(1992)
3. Profenofos Soil fungi, Penicil-
lium chrysogenum
Reduction in total-N in
fungi P. chrysogenum
Moharram et al.
(1994)
4. Carbofuran Methanotrophs Carbofuran stimulated the
proliferation of
methanotrophs
Kumaraswamy
et al. (1998)
5. Endosulfan,
monocrotophos,
and deltamethrin
Entomopathogenic
microorganisms
Beauveria
bassiana,
Metarhizium
anisopliae, and
Sporothrix
insectorum
Reduced the production
of conidia and vegetative
growth of
entomopathogenic fungi
Filho et al.
(2001)
6. Carbofuran Soil microflora Adversely affected soil
microorganisms
Kalam et al.
(2001)
7. Lindane and
dieldrin
Nitrosomonas,
Nitrobacter, and
Thiobacillus
Toxic Odokuma et al.
(2004)
8. DDT Soil algae A decrease in the diversity
of soil algal forms was
observed, as well as a
decrease in the amount of
viable soil algae
Mallavarapu
(2002)
9. Monocrotophos,
lindane, dichlorvos,
endosulfan, chlor-
pyrifos, malathion
Gluconacetobacter
diazotrophicus
May affect the cell mor-
phology and produced
pleomorphic cells in large
number
Madhaiyan
et al. (2006)
10. Acetamiprid Escherichia coli,
Pseudomonas, and
Bacillus subtilis
Stress enzymes in
microbes: Superoxide
dismutase,catalase, and
ATPase were all nega-
tively affected
Yao et al.
(2006)
11. Methamidophos Soil microorganism Increase in population of
some microbes but
decrease in total biomass
Wang et al.
(2006)
12. DDT, methyl
parathion
Rhizobium Interfere with legume–
rhizobium chemical
Rockets (2007)
(continued)
24 Impact of Pesticides on Microbial Population 473
had the least inhibitory effect. Glufosinate ammonium and metsulfuron methyl also
had similar effects (Zain et al. 2013).
Another study showed the effect of 2,4-dichlorophenoxyaceticacid (2,4-D) and
2,4,5-trichlorophenoxyaceticacid (2,4,5-T) on respiration of Azotobacter and con-
cluded that both inhibited the respiration of Azotobacter but inhibition was more by
2,4,5-T in comparison to 2,4-D (Magee et al. 1955).
24.7 Pesticidal Impact on Processes of Decomposition
The decomposition of organic matter in soil is an integral portion of nutrient cycling
method in soil. During spraying pesticides get in contact with crop residues in the
soil. The nonselective preplant herbicides like, glyphosate and paraquat are exten-
sively used, which contribute to slow decomposition of various plant materials.
There is inhibition of decomposition of paraquat-treated cellulose thread, but very
negligible decomposition of paraquat-treated soil was examined (Grossbard et al.
1978). Further studies on barley and wheat straw also revealed that when straws
were kept on soil surface, greater inhibition occurs with application of paraquat
(Grossbard et al. 1981). Although in some experiments glyphosate has shown its
inhibitory effect, its effect are inconsistent too (Grossbard et al. 1981); (Hendrix
et al. 1985; House GJ et al. 1987). Collectively, these studies indicated that the
recommended doses of paraquat and glyphosate can hinder the crop residue decom-
position. When paraquat and glyphosate were sprayed on crop residues, they
appeared to improve decomposition, but their overall results are mixed. Decompo-
sition was reduced more when residues were left on the soil surface than when they
were added. The effects of those herbicides on crop residue decomposition were
apparently due to high herbicide concentrations remaining after spraying. Paraquat
and glyphosate concentrations that inhibit microorganisms in pure culture were
typically higher than those present in soil after field application (Grossbard et al.
1979; Grossbard et al. 1985), and both herbicides used in the soil have negligible
Table 24.2 (continued)
Sl.
no. Insecticides Microbes Impact References
signalling which results in
reduced nitrogen fixation
13. Cypermethrin Bacteria and fungi In the cucumber
phyllosphere, there was a
rise in bacterial biomass
and a decline in fungal
biomass, as well as a
decrease in the ratio of
gram-positive to gram-
negative bacteria
Zhang et al.
(2008)
474 S. Majumder et al.
harmful effects on microbial species (Roslycky 1982). Both paraquat and glyphosate
are highly adsorbed in soil, which may explain why herbicides applied to soil are
ineffective. Other herbicides, such as 2,4-D and 2,4,5-T (Gottschalk et al. 1979;
Fletcher et al. 1986; Sikka et al. 1982), trifluralin, and its metabolites (Boyette et al.
1988), tend to have little effect on cellulose or plant residue decomposition, at least at
high concentrations. However, the effects of other recently established herbicide
classes, such as sulfonylurea herbicides, which inhibit amino acid synthesis in
microorganisms and are similar to glyphosate, have yet to be evaluated.
Though the reduction rates of crop residue decomposition had not yet been
assessed fundamentally, some of the effects can be drawn from this. The effects of
paraquat and glyphosate will be more in without-tilled crop residue than in other
different residue management systems because of surface retention of different crop
residues which increases the use of those herbicides. Greater amount of surface
pesticide residues may interfere with plant growth as well as weed control, and these
lead to increased inhibition of decomposition process. The residence time of straw or
other crop residues on the soil surface is determined by additional crop residue
coverage in cropping systems. While herbicides can temporarily delay the decom-
position process, there is no evidence that they have any long-term effects. Further-
more, there is no evidence that the use of these herbicides affects long-term nutrient
turnover in crop residues.
24.8 Pesticidal Impact on Nutrient Cycling
24.8.1 Effects on Nitrogen Transformations
One of the most crucial criteria for crop production is the management of soil N. The
conversion of plant nitrogen found in crop residues and soil organic matter to NH
4
and then to NO
3
is known as mineralisation, and it accounts for 40–60% of crop
nitrogen, with fertiliser N accounting for the remainder. The residual nitrogen in crop
residues was returned to the soil, where it was gradually re-mineralised as the
residues decomposed. A wide variety of soil microorganisms participate in the
mineralisation process, but only a few species transform NH
4
to NO
3
. Many studies
have been conducted to determine the effects of pesticides on nitrogen
mineralisation, and it is clear that the contrast of NO
3
and NH
4
nitrogen produced
in pesticide-treated soils to that produced in untreated soils is the most important
factor. The capacity for N production in a soil is influenced by the C/N ratio of soil
organic matter and crop residues, as well as the size of the microbial biomass.
Goring and Laskowski (1982) examined the impact of insecticides, herbicides,
fungicides, and nematicides on N transformations in soil and found that the majority
of pesticides have a marginal effect, inhibiting nitrification and mineralisation by
less than 25%. It was observed that most of the pesticides inhibit those natural
processes only above the recommended rates for field application. Soil fumigants,
e.g. chloropicrin or methyl bromide, particularly have important effects on nitrogen
24 Impact of Pesticides on Microbial Population 475
mineralisation and nitrification (Martin 1972). Initially, decreased populations of all
microorganisms and N mineralisation were observed. Since fumigated soils get
recolonised, nitrogen mineralisation process enhances and progressively may exceed
than that of non-fumigated soil. However, long after fumigation nitrification may get
suppressed, thus newer systemic nematicides and insecticides replaced soil fumi-
gants to a large extent. There was no declination in microbial biomass or soil
nitrogen mineralised in laboratory and field experiments using nematicides such as
fenamiphos and oxamyl (Tu 1980; Ross et al. 1984,1985). Fensulfothion, but not
carbofuran, inhibited the populations of various fungi and bacteria. Moreover Tu
(1972) reported that neither chemical inhibits mineralisation or nitrification. Dithio-
carbamate fungicides may inhibit nitrogen mineralisation under certain conditions.
Single pesticide applications have varying effects on nitrogen availability, but
repeated pesticide applications or single applications above the recommended levels
will reduce both ammonification and nitrification (Jaques et al. 1959; Dubey et al.
1970; Mazur et al. 1975). Maximum 10 ppm of those fungicides can cause reduc-
tions in fungal and bacterial populations, and subsequently increasing rates inhibit
soil respiration (Tu 1980) and glucose metabolism (Boyette et al. 1988), indicating
that those compounds are relatively nonselective towards microbes. Since certain
fungicides are relatively nonpersistent in soil and microbial activity, soil nitrogen
mineralisation ability will easily recover following their degradation. Corke et al.
(1970) discovered that certain pesticide-degraded products had slight effects on
nitrogen transformations, such as a 2- to 4-day lag for nitrification when low
concentrations of 3,4-dichloroaniline were present. This is a popular phenylamide
herbicide and substituted urea degradation product.
When applied at the same concentrations as the metabolites of the herbicides, the
parent herbicides had no effect. The metabolites of insecticides including terbufos
and phorate, such as sulfoxide and sulfone, reduced nitrification in soil to a lesser
degree than the parent compounds (Tu 1980). Other pesticide metabolites have little
attention towards mineralisation. The effect of pesticides on microbial fertiliser
transformations has yet to be thoroughly investigated. Marsh (1985) investigated
the impact of seven herbicides on nitrogen and phosphorus transformations in both
fertilised (triple superphosphate and NH
4
NO
3
) and unfertilised soil. Asulam not only
avoided nitrification in fertilised soil, but it also greatly reduced nitrogen
mineralisation. While glyphosate, chloridazon, paraquat, and isoproturon decreased
phosphorus availability, the effects were very subtle and not agronomically impor-
tant. In general, the above pesticides tend to have a much lower inhibitory effect on
the nitrification process than commercially available nitrification inhibitors (Bundy
et al. 1973; Turner 1979), although some direct comparisons have been made. The
soil enzyme urease converts urea to NH4+, which is a common nitrogen fertiliser.
Monuron, fenuron, diuron, linuron, and neburon, among other substituted urea
herbicides, inhibited soil urease production (Cervelli et al. 1976). According to
one study, conversion of urea to NH4+ was reduced by 8–39% depending on
herbicide concentration (2–10 ppm) and soil type (Maria et al. 2013). Mancozeb, a
dithiocarbamate fungicide, inhibited urease activity as well. Pesticides that inhibit
476 S. Majumder et al.
urease activity could theoretically reduce the conversion of this fertiliser to NO
3
over time, particularly if used in combination or close proximity to urea treated in the
soil. Mild inhibition of the urease enzyme will avoid NH
4
+
toxicity issues on
occasion and limit nitrogen losses from NH
4
volatilisation and nitrate leaching.
Denitrification has been shown to be inhibited by pesticides when used at higher
rates. At low concentrations, the insecticide carbaryl and the herbicide dalapon were
found to be inhibitory (Grant et al. 1982; Weeraratna 1980). Later studies by
Yeomans et al. (1985,1987) found that although dalapon’s inhibitory effects were
not verified, higher rates of metribuzin or dinoseb induced denitrification. Although
inhibiting denitrification can help with efficient nitrogen management, these findings
showed that herbicides used at recommended rates have a fairly consistent impact on
denitrification.
24.8.2 Transformation of Sulphur
The oxidation of elemental sulphur to sulphate and the reduction of sulphite are the
most important transformations since sulphate is the main plant-available nutrient.
Despite previous assumptions that heterotrophic microorganisms are more essential
for nitrification, the oxidation process is carried out by advanced chemoautotrophic
bacteria. Due to sulphur deficiency in different parts of the world, pesticides have a
significant impact on the sulphur oxidation mechanism (Coleman et al. 1966).
Sulphur, which must be oxidised before it can be used by plants, can also be used
to address such shortages. Given the importance of sulphur in crop production, the
literature includes only a few studies on pesticide effects on sulphur oxidation. The
organophosphate group of insecticides had little impact on soil sulphate, while
nematicides such as DDVP, carbofuran, and Vorlex reduced sulphur oxidation
marginally (Tu 1972; Aristeidis et al. 2020). Paraquat, on the other hand, slows
down the process significantly (Tu et al. 1968). Audus (Audus et al. 1970) discov-
ered that when insecticides (DDT, BHC, aldrin, dieldrin, etc.) are applied at field
concentrations, they have no effect on sulphur-oxidising bacteria.
24.8.3 Availability of Trace Elements
The solubility of trace elements in soils has been shown to be impaired by soil
fumigants (Warcup 1957). Fumigants appear to affect manganese in particular, so
steam sterilisation increases toxic levels (Sonneveld et al. 1973). Some pesticides’
effects on trace elements have received much less publicity. Smith and Weeraratna
(Smith et al. 1974) found that the herbicides simazine and ioxynil increased man-
ganese solubility as well as Mg, Ca, Fe, and Cu solubility in acidic and alkaline soil
media.
24 Impact of Pesticides on Microbial Population 477
Wainwright and Pugh (Wainwright et al. 1974) discovered a similar pattern in
their research after fungicide treatment in both laboratory and field treated soils. The
mechanism of the process is unknown; it may be caused by lysis of dead microor-
ganism cells or by microorganisms solubilising the components.
24.9 Conclusions
The primary aim of agricultural growth is to feed and supply enough food, nutrition,
and surplus to the increasing human population while mitigating environmental and
ecological harm. Pesticides are regarded as one of the most effective crop protection
tools in developing countries. Since pesticides and their derivatives stay in the soil
system for such a long time, they pose significant risks to soil health, the soil
microbial ecosystem, and human health. Plant defence chemicals have been shown
to minimise soil bacteria, fungi, and almost all flora and fauna populations, as well as
soil microbial activity, biomass carbon, and nitrogen mineralisation. The use of
natural pesticides and bio-pesticides, as well as judicious application of agrochem-
icals, should be encouraged.
Pesticide use must be limited in order to reduce the negative effects of pesticides
on humans and the environment, which necessitates public awareness campaigns
among farmers and other stakeholders. Long-term impact of pesticides on soil
microbial populations and the soil ecosystem should be researched in detail. Organic
pesticides can aid in the preservation of our environment’s microbial niche at this
early stage of organic agriculture.
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24 Impact of Pesticides on Microbial Population 481
Chapter 25
Microbe-Mediated Removal of Xenobiotics
for Sustainable Environment
Helina Patel, Sneha Trivedi, Deepesh Bhatt, Manoj Nath, and Naresh Butani
Abstract Xenobiotics are man-made chemical compounds widely used in pesti-
cides, dyes, drugs, explosives and other industrial chemicals. Poor waste disposal
practices, intensive agricultural practices and fossil fuel combustion are some of the
reasons that lead to the release of these compounds into natural ecosystem such as
soil and water. These cause serious damage to aquatic and terrestrial ecosystems due
to noxious nature of chemical compounds. Xenobiotics when build up in soil kill
beneficial microbes of soil that are accountable for soil fertility. Degradation of
xenobiotic compounds is being done using physical and chemical means; however,
these methods consequently result in the formation of lethal intermediates and end
products. Therefore, microbial remediation is adopted as a sustainable emerging
technique to eliminate these pollutants from nature. The present study highlights on
the involvement of several bacterial and fungal genera in catabolism of recalcitrant
xenobiotics. Moreover, phytoremediation approach employing plants for treating
chemically contaminated soil is also discussed.
Keywords Xenobiotics · Microbial remediation · Phytoremediation · Myco-
remediation · Bioremediation
H. Patel · S. Trivedi · N. Butani (*)
Department of Microbiology, Shree Ramkrishna Institute of Computer Education and Applied
Sciences, Sarvajanik University, Surat, Gujarat, India
D. Bhatt
Department of Biotechnology, Shree Ramkrishna Institute of Computer Education and Applied
Sciences, Sarvajanik University, Surat, Gujarat, India
M. Nath
ICAR—Directorate of Mushroom Research, Solan, Himachal Pradesh, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_25
483
25.1 Introduction
Xenobiotic chemicals are distant to the biosphere, are effectively lethal and are the
cause of undesirable physiological and/or ecological effects, consequently leading to
disease conditions in humans, other living creatures and pollution, respectively
(Olicón-Hernández et al. 2017). Environmentally predominant synthetic xenobiotic
compounds include hazardous pollutants such as pesticides, fuels, solvents, alkanes,
hydrocarbon derivatives, synthetic polymers, dyes, plastics, etc. (Sharma et al.
2018). Antibiotics, steroids and biomedical waste mixtures also are an example of
man-made xenobiotic compounds. Xenobiotic substances are becoming progres-
sively a major problem as they are relatively new substances that resist degradation,
mainly because of their recalcitrant nature. Being foreign to the organisms, xenobi-
otics are not easily recognized by the microbes in nature, and hence these com-
pounds do not enter common metabolic pathways, thereby remaining persistent in
nature. Such chemical contaminants are referred as persistent organic pollutants
(Bhatt et al. 2019). The accumulation of persistent pollutants affects the environment
and survival of higher as well as lower eukaryotes. Moreover, their recalcitrant
nature is a major risk factor that poses threat to human health as it disrupts normal
cellular pathways that play a significant role in their development and reproduction
(Gangola et al. 2018; Baker et al. 2019). Nevertheless, prolong exposure with these
xenobiotics may even cause neurological damage, immunosuppression and cancer
(Kalyoncu et al. 2009).
Thousands of persistent organic pollutants exist in the atmosphere and hold an
extended half-life of several days in air, or about a decade in soil and in living
organisms. For instance, xenobiotic organic pesticides DDT, BHC, polychlorinated
biphenyls, halogenated aromatic compounds and other such compounds that mainly
partake agricultural and industrial routines are discarded in the environment in huge
amounts globally. The chemical pesticides are being used to eliminate insect-borne
diseases for healthier harvest, so as to meet the growing demand of food. Although
these substances are advantageous to humans, their prolonged presence in the
biosphere poses perilous effects, as such chemical compounds are not easily
biodegraded and hence their concentration gradually upsurges with time (Varsha
et al. 2011). These chemical compounds build up in natural reserves (soil, air and
water) and also found to be accumulated in plants, animals and humans; thus, they
remain present in the ecosystem for decades (Breivik et al. 2004).
25.2 Points of Xenobiotic Discharge
The prime or direct sources of xenobiotics entering into the environment are
effluents from (1) chemical and pharmaceutical industries which include
alkylphenols, octylphenol and biphenyls such as bisphenol A, stilbene, genistein,
estrogens, etc. These pharmaceutically active complexes are controversial endocrine
484 H. Patel et al.
disruptors which disturb the physiological stability in animals and are bothersome to
human health (Asgher et al. 2008). (2) Paper and pulp bleaching are major sources of
release of harmful polychlorinated phenols (PCPs) and other organic compounds
and include dyes like azo dye and crystal violet (D’Souza et al. 2006). Effluent
discharge from textile industries and paper printing where synthetic dyes are majorly
being used, adversely affects the aquatic life (Kumari et al. 2014). (3) Mining
releases heavy metals into biogeochemical cycles. (4) Fossil fuel combustion and
gas plants produce polycyclic aromatic hydrocarbons like naphthalene and benzo
(a)pyrene, which are noxious and possibly carcinogenic xenobiotics (Gojgic-
Cvijovic et al. 2012). (5) Intensive agriculture practices use enormous amount of
chemical fertilizers, herbicides and pesticides such as chlorinated aromatic com-
pounds and their derivatives: DDT, chlordane and lindane. Bioaccumulation and
biomagnification of pesticides lead to toxic behavioural effects on animals and the
human race.
25.3 Classes of Xenobiotic Compounds
On the basis of chemical composition, the recalcitrant xenobiotic compounds that
are released from different industrial residual wastes, viz. paper and pulp remnants,
dye effluents, chemicals, plastics and pharma waste, are categorized into the follow-
ing types.
25.3.1 Halocarbons
These are volatile compounds comprised of different numbers of halogens (Cl, F, Br,
I) in place of hydrogen atoms. These compounds are mainly used in the preparation
of organochlorine pesticides, i.e. insecticides (DDTs and its metabolites [toxaphene,
chlordane, etc.], BHC, lindane, etc.), herbicides (dalapon,
2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, etc.) and fungi-
cides (Qadir et al. 2017). Volatile compounds when escaped into the environment
cause damage to the ozone layer, and when deposited in soil and leach into
waterbodies, they lead to biomagnification (Bharadwaj 2018).
25.3.2 Polychlorinated Biphenyls (PCBs)
PCBs (2-chlorobiphenyl, 4,40-dichlorobiphenyl, 2,20,5,50-tetrachlorobiphenyl, etc.)
are prevalent toxic pollutants, inert in nature and highly stable mixtures resistant to
extreme temperature and pressure (Tigini et al. 2009a). These compounds are
covalently linked with two benzene rings having chlorine in place of hydrogen
25 Microbe-Mediated Removal of Xenobiotics for Sustainable Environment 485
atoms. These are broadly used in plasticisers, in electrical equipment like capacitors
and insulator coolants in transformers, etc.
25.3.3 Synthetic Polymer
Synthetic polymers are high molecular weight compounds also known as plastic
polymers which include polystyrene, polypropylene, polyethylene, polyvinyl chlo-
ride, etc. Polyamide such as nylon, is expansively being used for wrapping materials,
in garments, etc. (Shrivastava 2018).
25.3.4 Alkylbenzylsulfonates
These are anionic surfactants (branched alkylbenzene sulfonates and linear
alkylbenzene sulfonates) broadly used in formulation of detergents. They have
hydrophilic sulfonate group present at one end, as a result of which they are resistant
to degradation by microorganisms, while at another end hydrophobic alkylbenzene
tail is present, making it recalcitrant if it is branched (Bharadwaj 2018).
25.3.5 Oil Mixture
Oil is a natural product, is insoluble in water, possesses some toxic constituents and
thus is a recalcitrant. Microbial degradation of oil has varying rates of degradation
based on the complexes present in it. However, biological means of degradation of
large oil spills over the water surface is ineffective, resulting in severe pollution
problems (Qadir et al. 2017).
25.4 Hazardous Effect of Xenobiotic Compound
Xenobiotic compounds are potentially perilous to both lower and higher eukaryotes
and even to humans. Exposure to xenobiotic pesticides increases the risk of diabetes,
neurological disorder and several skin diseases and extended exposure may even
cause cancer (Qadir et al. 2017). As described in the reports by Kelce et al. (1995),
scientific inferences from environmental impact assessment studies depict that
persistent organic pollutants are a major hazard that causes impairment of brain
function, reproductive dysfunction and endocrine disruption. Besides, their recalci-
trant nature leads to their gradual accumulation in the environment with time,
486 H. Patel et al.
thereby entering into the food chain and hence upsetting the ecosystem (Bharadwaj
2018).
25.5 Microbial Remediation of Xenobiotics
The use of chemicals that are noxious to human beings and that damage the
wilderness of nature is prevalent. Industries manufacture different chemical com-
pounds to satisfy the need of people for better living of life. Although the usage of
pesticides, paints, plastics, pharmaceuticals and textiles that contribute to xenobi-
otics cannot be neglected from our daily needs, steps should be taken to eliminate
these xenobiotic chemicals from the environment. Degradation of organic pollutants
using physical and/or chemical route is economically not feasible; besides, chances
of undesirable toxic intermediates and end products being formed are high. For this
purpose, exploitation of microorganisms is the most competent, sustainable and
feasible way to achieve efficient degradation of xenobiotic pollutants (Fig. 25.1).
Microbes are likely used because of their rapid growth rate and that they possess
complex enzymatic machinery that helps to degrade complex toxic compounds to
innocuous or less harmful degraded products. Microorganisms also have evolved
excellent biochemical control mechanisms so as to utilize pollutants as a source of
carbon and energy and degrade them. However, microorganisms fail to degrade all
chemical compounds for the reason that they are unable to break certain chemical
bonds present in them (Gangola et al. 2019).
25.5.1 Bacterial Remediation
Successful bioremediation requires potent microbial strains which can withstand and
degrade hazardous pollutants. Bioremediation mechanisms include both isolation of
naturally occurring xenobiotic-degrading microbes from heterogenous microbial
population and genetically engineered microorganisms. Biodegradation is affected
by various factors like hydrophobic nature of hydrocarbons, bioavailability and
predominant environmental conditions. Microorganisms can enhance the hydropho-
bicity of the cell surface by inherently changing their outer membrane so as to
facilitate the uptake of hydrocarbons (Shukla and Singh 2020). Microbial commu-
nities get colonized at contaminated sites as they metabolize recalcitrant xenobiotic
compounds (Galvão et al. 2005). Several aerobic (Pseudomonas,Bacillus,
Escherichia,Serratia,Gordonia,Moraxella,Micrococcus,Sphingobium,
Pandoraea,Rhodococcus), anaerobic (Desulfovibrio,Desulfotomaculum,
Methanospirillum,Methanosaeta,Pelatomaculum,Syntrophobacter,Syntrophus)
bacteria, methanotrophic and methanogenic bacteria, cyanobacteria and
sphingomonads possess xenobiotic degradative potential (Varsha et al. 2011;
Sinha et al. 2009).
25 Microbe-Mediated Removal of Xenobiotics for Sustainable Environment 487
A soil bacterium Serratia sp. strain DS001 utilized methyl parathion
(an organophosphate insecticide), 4-nitrocatechol, p-nitrophenol and 1,2,4-
benzenetriol as sole carbon and energy source and effectively metabolized methyl
parathion, signifying the role of enzyme parathion hydrolase in degradation (Pakala
et al. 2007). Yao et al. (2006) reported efficient degradation of phenolic compounds
using H
2
O
2
as oxidizer and an enzyme extracted from Serratia sp. AB 90027 as a
Fig. 25.1 Xenobiotic compounds, its types, effects and various strategies for degradation and
detoxification (BAS branched alkylbenzene sulfonates, LAS linear alkylbenzene sulfonates)
488 H. Patel et al.
catalyst. Using this enzyme/H
2
O
2
treatment, high COD removal efficiency was also
achieved. Kafilzadeh et al. (2015) in their studies collected samples from the
sediments of Kor River, Iran, from the area with high agricultural activity and
isolated five bacterial genera (Klebsiella,Acinetobacter,Alcaligenes,
Flavobacterium and Bacillus)that were able to metabolize endosulfan, a lipophilic
insecticide. Degraded metabolites of endosulfan produced depicted less toxicity
when compared with endosulfan itself.
Extremophiles such as halotolerant and thermotolerant Bacillus sp. strain DHT
also possess the capability of utilizing fuels, crude oil, several pure alkanes and
polycyclic aromatic hydrocarbons as a sole carbon and energy source (Kumar et al.
2007). Thermophilic bacterium Brevibacillus borstelensis degraded polyethylene
xenobiotic compounds for the sole source of carbon (Hadad et al. 2005). Efficient
catabolism of phenanthrene and crude oil was attained by M19F and M16K strains
of Bacillus subtilis on day 28 and day 18 post-inoculation, respectively (Oyetibo
et al. 2017). Furthermore, Bacillus drentensis strain S1 isolated from sewage sample
proved its potential in biodegradation of drug acetaminophen (paracetamol) (Chopra
and Kumar 2020). A pure strain of Pseudomonas sp. YATO411 in an immobilized
and a freely suspended system exhibited biodegradation of benzene and toluene,
indicating its potential to catabolize high concentrations of these xenobiotics (Tsai
et al. 2013). Additionally, bacteria of Pseudomonas species have been testified for
partial and complete decomposition of fungicides, pesticides, aliphatic or polycyclic
aromatic hydrocarbons, recalcitrant dyes, phenolic compounds and hexavalent toxic
heavy metals (Poornima et al. 2010; Wasi et al. 2010; Joe et al. 2011). Rhodococcus
erythropolis bacteria presented its potentiality in bio-desulfurization of crude oil
(Amin 2011). An idea of “Super Bugs”also has been advanced to catabolize an
extensive array of xenobiotic pollutants (Furukawa 2003). A metabolic by-product,
known as “microbial concrete”, is produced by urease-producing bacterial strains,
namely, P. aeruginosa,P. mirabilis and Micrococcus. Microbial concrete is
employed for remediation and re-establishing the buildings (Reddy and Yang
2011). Cyanobacterial mats also were exploited for cleaning up contamination of
oil spinoffs (Bordenave et al. 2009; Raeid 2011). Sarkar et al. (2017) reported 98%
removal of total petroleum hydrocarbons with the aid of microcosms bioaugmented
with Enterobacter,Pandoraea and Burkholderia strains.
Owing to the refractory properties of xenobiotics, their bioavailability is very low
and thus their accessibility might be difficult under subsurface environment, and
therefore they are persistent in the environment, escaping the metabolism by micro-
organisms. Consequently, organisms having degradative potential can be genetically
manipulated to enhance mobility so as to access these pollutants (Díaz 2004). A
major restraint of bioremediation process is optimal physico-chemical conditions
that are obligatory for accurate metabolic working of microorganisms, as a prereq-
uisite for degradation of xenobiotics, which may be tough to achieve in natural
environment (Singh and Ward 2004). In several studies, syntrophic bacterial con-
sortia are being used for degradation of xenobiotics, as single microorganism in
some cases may not be able to perform all metabolic activities required (Díaz 2004).
Therefore, when mixed bunch of bacteria works in combination, the dead-end
25 Microbe-Mediated Removal of Xenobiotics for Sustainable Environment 489
products from one organism will be then broken down by another bacterium (Singh
and Ward 2004). Biodegradation of chrysene, a persistent polycyclic aromatic
hydrocarbon, was achieved by employing bacterial consortium consisting of Bacil-
lus sp., Rhodococcus sp. and Burkholderia sp. Chrysene was utilized as a sole source
of carbon and energy by the consortium (Vaidya et al. 2018). Researches also
reported for possibility of application of a microaerophilic mixed bacterial consor-
tium for complete mineralization of azo dyes methyl orange and Congo red
(Dissanayake et al. 2021). Furthermore, anaerobic microbial consortia also are
reported for anaerobic degradation of levofloxacin (Shu et al. 2021).
25.5.2 Myco-Remediation
Fungi are characterized as one of the most diverse collections of microorganisms that
exhibit a significant part in nature as decomposers, mutualists or pathogens (Schmit
and Mueller 2007). Mineralization exploiting fungus may be accomplished by direct
metabolism, where a xenobiotic component may be totally degraded by fungi to
non-toxic end products like inorganic chemical compounds and CO
2
(Mougin et al.
2009). Amongst diverse groups of fungi, white rot fungi can degrade several
xenobiotics, including polycyclic aromatic hydrocarbons, persistent pesticides,
dyes, chlorinated phenols and pharmaceuticals (Vanhulle et al. 2008). Besides, a
wide variety of fungal species that assist in degradation of recalcitrant xenobiotic
compounds are listed in Table 25.1.
25.5.3 Phytoremediation
Phytoremediation, termed as green remediation, agro- or botano-remediation, is a
technique that employs plants for treating chemically contaminated soils (Wenzel
et al. 1999), thereby reducing the concentration of hazardous compounds (Utmazian
and Wenzel 2006). Plants are used because they can endure reasonably elevated
amounts of xenobiotic chemicals deprived of noxious effects (Briggs et al. 1982).
Moreover, plants have a potential to take up these chemicals and convert them into
less toxic compounds (Bock et al. 2002). Therefore, it is one of best green routes to
target chemical pollutants present in the environment for their removal. Enzyme
secreted within plants aids in degradation of chlorinated compounds, herbicides and
other organic pollutants; this method of removal of pollutants is termed as
phytotransformation (Shukla et al. 2010). Likewise, rhizodegradation also leads to
degradation and detoxification of recalcitrant pollutants present in soil with the aid of
plant roots. The process is employed for catabolism of chlorinated solvents, surfac-
tants, PCBs, petroleum hydrocarbons and various pesticides (Goyal and Basniwal
2017). For elimination of toxic metals from the soil, a method known as
phytomining is employed, in which metal ions are absorbed by plant roots. If
490 H. Patel et al.
Table 25.1 List of fungal species that possess xenobiotic degradation potential
Xenobiotic compounds Fungi degrading xenobiotics References
Pesticides
Diazinon Aspergillus niger MK640786 Hamad
(2020)
Chlorpyrifos, profenofos and methyl
parathion
Aspergillus sydowii CBMAI 935 Soares et al.
(2021)
DDT Consortium of fungus Fomitopsis
pinicola + bacterium Ralstonia
pickettii
Purnomo
et al.
(2020a)
Xerocomus chrysenteron Huang and
Wang
(2013)
P. aeruginosa + P. ostreatus Purnomo
et al. (2017)
Allethrin Fusarium proliferatum CF2 Bhatt et al.
(2020a)
Chlorfenvinphos Penicillium citrinum,Aspergillus
fumigatus,Aspergillus terreus and
Trichoderma harzianum
Oliveira
et al. (2015)
Endosulfan and chlorpyrifos Cladosporium cladosporioides,
Phanerochaete chrysosporium,
Trichoderma harzianum,
Trichoderma virens,Trametes hirsuta
and Trametes versicolor
Bisht et al.
(2019)
PCBs
Aroclor 1254 Phanerochaete chrysosporium Eaton
(1985)
3,30,4,40-Tetrachlorobiphenyl,
2,3,30,4,40-pentachlorobiphenyl,
2,30,4,40,5-pentachlorobiphenyl,
3,30,4,40,5-pentachlorobiphenyl and
2,30,4,40,5,50-hexachlorobiphenyl
Phlebia brevispora Kamei et al.
(2006)
2-Chlorobiphenyl, 4,40-dichlorobiphenyl
and 2,20,5,50-tetrachlorobiphenyl
Aspergillus fumigatus MUT 4026,
Penicillium chrysogenum
MUT 4021, Fusarium solani MUT
4020, Penicillium digitatum MUT
4079, Scedosporium apiospermum
MUT 641 and Scedosporium
apiospermum MUT 631
Tigini et al.
(2009b)
PCBs Pleurotus sajor-caju LBM 105 Sadañoski
et al. (2019)
Dye
Methyl orange Gloeophyllum trabeum Purnomo
et al.
(2020b)
Triphenylmethane dyes Penicillium simplicissimum Chen et al.
(2019)
(continued)
25 Microbe-Mediated Removal of Xenobiotics for Sustainable Environment 491
required, metals can be extracted by incineration of plants to yield ash and assim-
ilation of plant incinerations will help metal reuse (Shukla et al. 2010). Thlaspi
caerulescens,Alyssum murale,Alyssum markgrafii,Bornmuellera baldacii subsp.
markgrafiiand Leptoplax emarginata are examples of some plants involved in
phytomining (Aboudrar et al. 2007; Bani et al. 2007,2009). Trends in
phytoremediation also use transgenic or genetically engineered plants that help to
elevate tolerance and metabolism of xenobiotic chemicals for remediation by plants
(Sonoki et al. 2011). In contrast to conventional mechanical methods being used,
Table 25.1 (continued)
Xenobiotic compounds Fungi degrading xenobiotics References
Acid red 88 Achaetomium strumarium Bankole
et al.
(2018a)
Reactive blue 4, Remazol brilliant blue
and acid blue 129 (AB129)
Trametes hirsuta D7 Alam et al.
(2021)
Scarlet RR dye Peyronellaea prosopidis Bankole
et al.
(2018b)
Polycyclic aromatic hydrocarbons
Mixture of four polycyclic aromatic
hydrocarbons
Fusarium oxysporum Marchand
et al. (2017)
Phenanthrene, anthracene and pyrene Trematophoma genus Moghimi
et al. (2017)
Anthracene Trichoderma harzianum,
Cladosporium sp., Aspergillus
sydowii,Penicillium citrinum and
Mucor racemosus
Birolli et al.
(2018)
Anthracene, anthrone, anthraquinone,
acenaphthene, fluorene, phenanthrene,
fluoranthene, pyrene and nitropyrene
Cladosporium sp. CBMAI 1237
Phenanthrene, anthracene and pyrene Coriolopsis caperata,Fomes
fomentarius,Pluteus chysophaeus
Hadibarata
and
Yuniarto
(2020)
Benzo[a]pyrene Penicillium canescens,Cladosporium
cladosporioides,Fusarium solani and
Talaromyces helicus
Fayeulle
et al. (2019)
Anthracene and dibenzothiophene Penicillium oxalicum Aranda et al.
(2017)
Estrogenic xenobiotics
Bisphenol A, estrone, 17β-estradiol,
estriol, 17α-ethinylestradiol, triclosan
and 4-n-nonylphenol
Pleurotus ostreatus HK 35 Křesinová
et al. (2018)
4-t-Octylphenol Fusarium falciforme Rajendran
et al. (2017)
Testosterone and 17α-ethynylestradiol Lentinula edodes Muszyńska
et al. (2018)
492 H. Patel et al.
phytoremediation is an eco-friendly and economically feasible approach to clean up
polluted groundwater (Bhatt et al. 2020b). Nevertheless, it is a time-consuming
process as it is reliant on the growth of the plants. Additionally, for successful
remediation by means of this green technique, it is necessary to look for right
plant for right pollutant (Goyal and Basniwal 2017; Bhandari and Bhatt 2021).
25.6 Conclusion and Future Prospects
Environmental pollution is escalating due to increased global industrialization,
leading to generation of hefty portions of xenobiotic wastes that present potent
health hazard to mankind. Degradation of recalcitrant chemicals is thus the need of
the hour so as to lessen contamination of these environmental pollutants. In this
context, remediation strategies employing microorganisms, plants and their enzymes
have gained substantial attention. As cited in this chapter, microbial remediation
using bacteria and fungi and phytoremediation are capable of mineralization of
xenobiotics to innocuous or less toxic end products. Though microbial-based sys-
tems for removal of pollutants are slow, it offers several pros over physico-chemical
methods of remediation, as it is an economically feasible and eco-friendly method.
Furthermore, comprehensive studies on the utility of these microorganisms and their
enzyme machineries and studies on cloning and expression of genes appear to be
compelling tools to decrease the levels of toxic chemicals and to understand the
mechanism of biodegradation, respectively. Thus, it can be concluded that elimina-
tion of toxic pollutants from the environment through green approach is only
possible with the assistance of microorganisms and plants.
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25 Microbe-Mediated Removal of Xenobiotics for Sustainable Environment 497
Chapter 26
Harnessing the Rhizomicrobiome
Interactions for Plant Growth Promotion
and Sustainable Agriculture: Mechanisms,
Applications and Recent Advances
Geeta Bhandari and Niki Nautiyal
Abstract Rhizomicrobiome is of utmost significance in agricultural sciences on
account of highly diverse, distinct rhizospheric microflora present in direct or
indirect association with plants. It is vital for plant growth promotion due to its
involvement in processes such as nutrient uptake and incorporation; enhancement of
soil characteristics; regulation of the production of plant growth hormones, second-
ary metabolites and antibiotics; regulation of stress tolerance; and rhizoremediation.
The diverse rhizomicrobiomes are significant for maintaining sustainability in agri-
cultural practices. Fulfilment of increasing food demand with employment of min-
imal chemical fertilizers has become a great challenge for both researchers and
farmers. PGPR and their secretions play a pivotal role in efficiently stimulating
plant growth and enhance stress tolerance for the biotic and abiotic stresses. The
versatile PGPR-based biofertilizers have been formulated for use in agricultural
practices, thus minimizing chemical fertilizers and agrochemical input. Therefore,
a deep and comprehensive insight into the plant rhizomicrobiome and their mech-
anism is critical for exploring the aforesaid sustainability of agricultural practices.
Keywords Rhizomicrobiome · PGPR · Biofertilizer · Sustainable agriculture
26.1 Introduction
Soil is a vital, living matrix and is a significant reservoir crucial for agriculture
productivity, food security and sustenance of living beings. It is regarded as a
repository of bacterial metabolism even though microbes occupy not more than
5% of the entire soil space. The area of soil around the plant root is commonly
termed as rhizosphere, which is under chemical, physical and biological influence of
G. Bhandari (*) · N. Nautiyal
Department of Biotechnology, Sardar Bhagwan Singh University, Dehradun, Uttarakhand,
India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_26
499
plant roots. The rhizospheric microbial communities comprise of several organisms
such as bacteria, archaea, fungi, algae and viruses. Rhizospheric microbes are
constituent of microflora that are associated with the plant roots and are regarded
as highly intricate ecosystems on the Earth (Wagg et al. 2014). The plant, the soil and
the microbes in the rhizosphere are all interconnected to one another forming
“microbiome”which allows the occurrence of several processes beneficial for
enhanced plant production (Ahmed et al. 2019). The microbiomes of the
rhizospheric region encompass potential microbes significant for mineral exchange,
recycling, improving soil fecundity and plant protection and production (Jacoby
et al. 2017). The microbes present in the rhizosphere are usually known as plant
growth-promoting rhizobacteria—“PGPR”. These are the useful microbes that
inhabit the rhizospheric zone and augment the plant development directly or indi-
rectly, such as inhibition of plant pathogens, degradation of hazardous pollutants,
stimulation of induced systemic resistance (ISR), release of phytostimulating com-
pounds and furnishing of beneficial nutrients, such as N and P (Glick 2012). Root
exudates produced by the plant roots alter soil physiology (Mukherjee et al. 2018)
and eventually modulate heterogeneity and structure of rhizospheric microbiome
(Huang et al. 2014) and thus selectively stimulate beneficial microbes required for
plant growth and development (Chaparro et al. 2014).
26.2 Plant Growth-Promoting Rhizospheric Microbiome
The valuable results of several traditional sustainable farming practices on plant
development have been reported 100 years ago by the ancient Greeks and Romans.
They found that combining various soil types or adding organic amendments
enhances soil health and fecundity and thus subsequently improves plant yield
(Tisdale and Nelson 1975). Thereafter, Kloepper and Schroth then coined the
word “plant growth-promoting rhizobacteria”(PGPR) for the rhizospheric microbial
population (Kloepper and Schroth 1981). PGPR is a broad term that especially refers
to such microbial strains inhabiting the rhizosphere and they considerably enhance
plant growth, productivity, soil health, pest resistance and synthesis of plant growth
hormones (Prasad et al. 2019; Compant et al. 2019), and thus the PGPR are given
prominence in farming practices. The PGPR are divided into two groups,
i.e. extracellular PGPR (ePGPR) and intracellular PGPR (iPGPR) (Viveros et al.
2010). The ePGPR are usually present in the rhizospheric soil or inhabit the
outermost root cortical region and belong to genera Flavobacterium,Azotobacter,
Caulobacter,Pseudomonas,Serratia,Burkholderia,Erwinia,Chromobacterium,
Azospirillum,Arthrobacter,Agrobacterium,Bacillus and Micrococcus. In contrast,
the iPGPR form symbiotic association with the plant root and reside in the root
nodule and include genera such as Rhizobium,Bradyrhizobium,Allorhizobium,
Mesorhizobium, etc. (Viveros et al. 2010; Bhattacharyya and Jha 2012). The
PGPR enhance plant growth and development in direct and indirect mechanisms
and thus render soil fecundity and enhance plant productivity (Gupta et al. 2015).
Direct mechanism includes phosphate solubilization, biological nitrogen fixation,
500 G. Bhandari and N. Nautiyal
siderophore synthesis and iron acquisition and synthesis of plant growth protomory
compounds. Indirect methods involve biocontrol through antibiotic, antifungal and
volatile organic acid production (Glick 2012; Ahemad and Khan 2012; Jahanian
et al. 2012; Liu et al. 2016). The PGPR should first inhabit and thrive in the
rhizospheric zone so as to work as plant growth promoters. Rhizosphere colonization
by PGPR is dependent on several aspects including soil characteristics, occurrence
of protozoans, synthesis of antibacterial metabolites of plants or microflora and root
exudate usage.
Currently, the employment of nonhazardous substitutes for accomplishing
increased plant productivity in a sustainable manner is of utmost significance since
the use of environment-friendly agricultural practices is the fundamental principle of
sustainable agriculture. Various efforts are being done for screening and exploiting
PGPR to be employed for improving soil fertility and health in place of synthetic
agrochemicals. Thus, various symbiotic (Bradyrhizobium,Rhizobium,
Mesorhizobium) and nonsymbiotic (Flavobacterium,Chromobacterium,Klebsiella,
Pseudomonas,Agrobacterium,Azomonas,Enterobacter,Micrococcus,
Burkholderia,Variovorax,Azotobacter,Bacillus,Serratia,Azospirillum,
Caulobacter,Erwinia and Arthrobacter) rhizobacteria are globally employed as
biofertilizers for improving crop production in various agroclimatic zones (Glick
2012; Ahemad and Khan 2012; Egamberdieva and Lugtenberg 2014). Several
PGPR employed for plant growth promotion are listed in Table 26.1.
26.3 Nutrient Acquisition by Plant Growth-Promoting
Rhizospheric Microbiome
26.3.1 Phosphate Solubilization
Phosphorus (P) is a vital nutrient necessary for optimum development of plants. It
plays a significant role in plant developmental activities such as biomolecule syn-
thesis, cellular respiration, photosynthesis and signalling pathways (Anand et al.
2016). The plants are able to utilize only monobasic (H
2
PO
4
) and dibasic
(H
2
PO
42
) forms of phosphate (Bhattacharyya and Jha 2012). Although phosphate
exists abundantly in the soil, its plant-utilizable form is generally very less since
greater than 95% of phosphorus is insoluble and/or immobile and thus is not
accessible to the plants (Ahemad and Kibret 2014). Phosphorus is rapidly converted
into insoluble metal oxide complexes in soils, thus making it deficit of the available
phosphate (Bhattacharyya and Jha 2012). PGPR have evolved several mechanisms
for converting insoluble soil P into plant-utilizable soluble forms and such microbes
are known as phosphate-solubilizing bacteria (PSMs) (Alori et al. 2017). Solubili-
zation of phosphorus by microbes occurs via (a) production of organic acids
(gluconic and citric acid) which drop the soil pH resulting in the release of insoluble
P; (b) synthesis of extracellular phosphatases and phytases which break down
26 Harnessing the Rhizomicrobiome Interactions for Plant Growth Promotion and... 501
Table 26.1 Plant growth-promoting mechanisms of plant rhizomicrobiomes
Rhizobacteria PGP traits
Crops/plant
rhizosphere References
Mesorhizobium ciceri Phosphate solubilization,
ammonia production
Cicer
arietinum
Ahmad
et al.
(2008)
Bacillus sp. Phosphate solubilizer Zea mays Oliveira
et al.
(2009)
Azospirillum brasilense Az39,
Bradyrhizobium japonicum
E109
Phytostimulation Glycine max Cassan
et al.
(2009)
P. fluorescens Aur6,
Chryseobacterium balustinum
Aur9
Biocontrol agents Oryza sativa Lucas
et al.
(2009)
Chryseobacterium palustre,
Chryseobacterium humi,
Sphingobacterium,Bacillus,
Achromobacter
IAA, HCN, NH
3
, siderophore
production, ACC deaminase
activity
Zea mays Marques
et al.
(2010)
Bacillus sp. PSB10 IAA, HCN, siderophore
production
Cicer
arietinum
Wani and
Khan
(2010)
Rhizobium MRPI Nitrogen fixation and phosphate
solubilization
Pisum
sativum
Ahemad
and Khan
(2011)
Rhodococcus sp. EC35, Pseu-
domonas sp. EAV,
Arthrobacter nicotinovorans
EAPAA
Phosphate solubilizer Zea mays Sofia et al.
(2014)
Pseudomonas fluorescens strain
Psd
Zinc solubilizer, nitrate reducer Triticum
aestivum
Sirohi
et al.
(2015)
Bacillus circulans Potassium solubilization Solanum
lycopersicum
Mehta
et al.
(2015)
Bacillus sp. strain WG4 Antifungal metabolite
production
Zingiber
officinale
Jimtha
et al.
(2016)
Burkholderia sp.
Pseudomonas aeruginosa strain
MAJ PIA 03
Bacillus firmus strain MAJ
PSB12
IAA, GA3, ACC deaminase
activity, HCN production, NH
3
production, siderophore pro-
duction, antagonistic activity,
phosphate solubilizer
Ricinus
communis
Sandilya
et al.
(2016,
2017)
Bacillus,Azotobacter,Pseudo-
monas and Acinetobacter
IAA, NH
3
, HCN, siderophore
production, phosphate
solubilizer, antagonistic activ-
ity, nitrate reducer
Momordica
charantia
Singh
et al.
(2017)
Bacillus megaterium PSB12 Phosphate solubilization Triticum
aestivum
Mukhtar
et al.
(2017)
(continued)
502 G. Bhandari and N. Nautiyal
phosphoric esters, resulting in mineralization of P; (c) biological P mineralization by
substrate degradation; and (d) production of chelating or phosphorus-solubilizing
molecules, e.g. organic acid anions, protons, hydroxyl ions and CO
2
(Glick 2012;
Zaidi et al. 2009). Moreover, these microbes in the existence of unstable C immo-
bilize even very minute concentrations of P, and thus these PSMs act as P source
upon their starvation, predation or death (Butterly et al. 2009).
PSMs generally belong to genera Azospirillum,Achromobacter,Acetobacter,
Acinetobacter,Serratia,Microbacterium,Azotobacter,Rhodococcus,Klebsiella,
Erwinia,Arthrobacter,Rhizobium,Enterobacter,Burkholderia,Pseudomonas,
Beijerinckia,Mesorhizobium,Bacillus and Flavobacterium (Bhattacharyya and
Jha 2012; Kumar and Dubey 2012). Bacillus megaterium is marketed as Bio Phos
(Bio Power Lanka, Sri Lanka) and employed as “P”biofertilizer (Samina 2016).
Other reported P-solubilizing strains by AgriLife (India) are Pseudomonas striata,
B. polymyxa and B. megaterium (Samina 2016).
26.3.2 Nitrogen Fixation
Nitrogen is another vital mineral required for several developmental activities in
plants governing their growth. Nitrogen being present in limiting amount, its bio-
logical fixation is a significant process carried out by several PGPR in the symbiotic
and free-living forms. Biological nitrogen fixation (BNF) is defined as enzymatic
reduction of atmospheric dinitrogen to ammonium using a nitrogenase enzyme
complex (Masson-Boivin and Sachs 2018). Since the utilizable form of nitrogen
Table 26.1 (continued)
Rhizobacteria PGP traits
Crops/plant
rhizosphere References
Bacillus megaterium Phosphate solubilization Vigna
radiata
Biswas
et al.
(2018)
Azotobacter chroococcum Siderophore production Cereals Zhang
et al.
(2019)
Azospirillum brasilense Nitrogen fixation Oryza sativa Thomas
et al.
(2019)
Bacillus siamensis Gibberellin production Arabidopsis
mutants
Hossain
et al.
(2019)
Burkholderia cenocepacia Phosphate solubilization Nicotiana
tabacum L.
Liu et al.
(2019)
Enterobacter ludwigii Zn solubilization Triticum
aestivum L.
Singh
et al.
(2018)
26 Harnessing the Rhizomicrobiome Interactions for Plant Growth Promotion and... 503
(nitrate or ammonium) is available in limiting amounts in comparison to biological
demand, nitrogen fixation is an ecologically significant process for providing fixed
nitrogen in several terrestrial and aquatic ecosystems (Vitousek and Howarth 1991;
Arp 2000). The BNF microbes include symbiotic nitrogen fixers of genera Rhizo-
bium,Bradyrhizobium,Azorhizobium,Sinorhizobium,Allorhizobium,
Mesorhizobium,Azoarcus,Beijerinckia, Frankia,Pantoea,Burkholderia,Klebsi-
ella,Achromobacter and Herbaspirillum and free-living nitrogen fixers of genera
such as Arthrobacter,Acetobacter,Clostridium,Azotobacter,Azospirillum,Bacil-
lus,Pseudomona,Enterobacter,Burkholderia,Gluconacetobacter,Cyanobacte-
rium and Diazotrophicus (Dinnage et al. 2019;Babalola 2010;Pérez-Montaño
2014;Turan et al. 2016). The symbiotic N
2
-fixing bacteria inhabit inside the root
cells of various plants and cause formation of root nodule, which are the site of
transformation of atmospheric nitrogen to fixed forms of nitrogen that can be utilized
by the plants (Ahemad and Kibret 2014). In exchange, bacteria obtain fixed form of
carbon such as dicarboxylates from the plants which enable them to thrive and carry
out the highly energy-demanding process of nitrogen fixation (Udvardi and Poole
2013). All of the nitrogen-fixing microbes contain nif genes responsible for the
production of the enzyme “nitrogenase”. Nitrogenase consists of two multisubunit
metalloproteins, i.e. dinitrogen reductase (Fe) and dinitrogenase (MoFe) (Howard
and Rees 1996). The nifHDK genes code for the enzyme nitrogenase and are
generally arranged contiguously. The component I of the enzyme complex is
translated from nifD and nifK genes and has a molecular weight of approximately
250 kDa and is responsible for reduction of N
2
. The component II having a
molecular weight of 70 kDa is involved in coupling ATP hydrolysis to electron
transfer chain and is coded by the nifH gene. Both these components work in a
coordinated way for reduction of N
2
to NH
3
(Santi et al. 2013). Numerous authors
have described various kinds of nitrogenase complexes with differing metal ions
found in association with the nitrogenase enzyme complex (Ahemad and Kibret
2014).
Nitrogen-fixing microbes find diverse applications in agriculture such as growth
enhancement, pathogen inhibition and supply of fixed nitrogen (Damam et al. 2016).
Azospirillum, a nonsymbiotic nitrogen-fixing microbe, has been reported to improve
growth development of various plants (Lin et al. 2015). Fukami et al. have suggested
that Azospirillum can be employed for improving yield and productivity of sun-
flower by supplying nitrogen (Fukami et al. 2018). Yadav and Verma observed the
impact of native symbiotic N
2
-fixing strain, Rhizobium leguminosarum BHURC04,
and Pseudomonas aeruginosa on the development and production of Cicer
arietinum L. var. C-235 and reported enhanced nitrogen-fixing capacities and
improved plant growth (Yadav and Verma 2014).
504 G. Bhandari and N. Nautiyal
26.3.3 Potassium Solubilization
Potassium is another vital nutrient required for enhancement of plant development.
Greater than 90% of potassium occurs as insoluble forms and thus the plant-
utilizable form is often limited in the soil (Parmar and Sindhu 2013). Potassium
deficiency affects root growth and complete plant development and thus results in
poor crop productivity. Thus, there is a need of exploring alternative resources of
potassium for sustaining plant development without imposing adverse impact on the
environment (Kumar and Dubey 2012). Potassium-solubilizing bacteria are consid-
ered as highly definitive and effective due to their capability to synthesize organic
acids for solubilization of insoluble potassium in rocks and minerals. K solubiliza-
tion occurs due to synthesis of organic and inorganic acids and protons (Meena et al.
2015) which cause the conversion of insoluble K found in biotite feldspar, mica and
muscovite to soluble K by lowering the soil pH and releasing K ions by chelation of
Si
4+
,Al
3+
,Fe
2+
and Ca
2+
ions found in K minerals (Meena et al. 2014; Verma et al.
2017; Yadav et al. 2019). Several acids including oxalic, fumaric, citric, malonic,
succinic, tartaric, glycolic, lactic, gluconic, propionic, malic and 2-keto-gluconic are
responsible for K solubilization (Saiyad et al. 2015). Several potassium-solubilizing
PGPR such as Bacillus mucilaginosus,Pseudomonas sp., Bacillus edaphicus,
Burkholderia sp., Acidithiobacillus ferrooxidans and Paenibacillus sp., are well
known (Liu et al. 2012). Enhanced growth and potassium uptake of vital crops,
e.g. cotton, maize, cucumber, rape, peanut and pepper in the presence of potassium-
solubilizing microbes, have been reported (Ashley et al. 2006). Carboxylic acid
synthesis for potassium solubilization by Bacillus species has been well documented
for enhancing soil fecundity and crop production (Majeed et al. 2018; Saha et al.
2016a).
26.3.4 Siderophore Production
Iron is a beneficial micronutrient for all living beings and works as a cofactor of
many oxidoreductases. Majority of iron occurs in insoluble form of ferric hydroxide
and oxyhydroxides in soil, thus making it a limiting factor for uptake and assimila-
tion by plant and bacteria even in iron-rich soils (Rajkumar et al. 2010). The
available form of iron is usually limiting owing to conversion of Fe
++
to Fe
+++
.In
iron-limiting soil, bacteria synthesize iron-complexing molecules known as
siderophores, which form complexes with Fe
3+
. Siderophores exists in extracellular
and intracellular form and are manifested to be significant iron-solubilizing agents.
In intracellular chelation, Fe
3+
is converted to Fe
2+
having lesser affinity for
siderophores and thus released from the chelation complex inside the cell and is
employed for bacterial growth and development (Boukhalfa and Crumbliss 2002).
Siderophores secreted by plant growth-promoting bacteria (PGPB) possess maxi-
mum affinity for iron in comparison to siderophores synthesized by either plants or
26 Harnessing the Rhizomicrobiome Interactions for Plant Growth Promotion and... 505
fungi (Saha et al. 2016b). Pseudomonads are the chief secretors of siderophores and
thus play a crucial role in the development of plants (Sandilya et al. 2017).
Siderophores contain three Fe-binding domains linked by a flexible backbone with
and two oxygen atoms being joined to each functional group. The functional groups
present are generally hydroxamates or catecholates, carboxylate, citrate or
ethylenediamine (Laschat et al. 2017). Siderophore-producing PGPR such as
Aeromonas,Streptomyces sp., Bacillus,Azotobacter,Serratia,Rhizobium,
Burkholderia and Pseudomonas have been reported (Sujatha and Ammani 2013).
Siderophores are also significant for the existence of both plants and microbes
due to inhibition of pathogenic fungal strains and other bacterial competitors in the
soil (Shen et al. 2013). Siderophores chelate Fe in the rhizosphere and thus limit the
pathogenic organisms, e.g. Fusarium oxysporum and Pythium ultimum, from iron
required for their growth and work as biocidal agent for these pathogens. Moreover,
siderophores are documented to chelate several heavy metals (Cd, Zn, Ga, Al, Pb
and Cu), thus increasing their amount, and assist in the alleviation of heavy metal
stress in plants (Rajkumar et al. 2010; Neubauer et al. 2000). Kloepper et al. have
reported siderophore production by Pseudomonas putida B10 and assistance in
biocontrol against Fusarium oxysporum in iron-limited conditions, but this inhibi-
tion was however terminated on amending iron in the soil (Kloepper et al. 1980).
Siderophores have also been documented in PGPR Chryseobacterium spp. C138
and were found to be efficient in supplying iron to plants (Radzki et al. 2013).
26.3.5 Phytohormone Production
Phytohormones are organic compounds which greatly regulate the biochemical,
physiological and morphological activities of plants or may act as chemical mes-
sengers even in very low concentrations (Fuentes-Ramírez and Caballero-Mellado
2006). Phytohormone production capability is broadly found in various microor-
ganisms, and several PGPR capable of producing plant growth-promoting hormones
such as indole-3-acetic acid, gibberellins, cytokinins and ethylene are well known
(Spaepen et al. 2007). Several PGPR belonging to genera such as Proteus mirabilis,
Azotobacter chroococcum,Escherichia coli,Stenotrophomonas maltophilia,Pseu-
domonas vulgaris,Pseudomonas aeruginosa,Rhizobium leguminosarum,Klebsi-
ella pneumoniae,Mesorhizobium ciceri,Bacillus cereus,Paenibacillus polymyxa,
Klebsiella oxytoca,Pseudomonas putida and Enterobacter asburiae are documented
to synthesize an extensive variety of phytohormones (Ahemad and Kibret 2014)
(Table 26.2).
26.3.5.1 Indole Acetic Acid (IAA)
IAA is a naturally existing auxin and significantly impacts the plant growth promo-
tion activities such as cell division, differentiation and root extension (Miransari and
506 G. Bhandari and N. Nautiyal
Table 26.2 Phytohormone production by plant growth-promoting rhizomicrobiomes
Phytohormone PGPR Crop References
IAA Enterobacter sp. C1D Vigna radiata
L.
Subrahmanyam
and Archana
(2011)
Enterobacter sp. Cicer
arietinum L.
Fierro-Coronado
et al. (2014)
Proteus vulgaris JBLS202 Arabidopsis
thaliana
Bhattacharyya
et al. (2015)
Pseudomonas sp., Bacillus sp. Sulla carnosa Hidri et al. (2016)
Bacillus licheniformis Triticum
aestivum L.
Singh and Jha
(2016)
Bacillus subtilis Acacia
gerrardii
Benth
Hashem et al.
(2016)
Pseudomonas sp. Zea mays Mishra et al.
(2017)
Azospirillum brasilense Cereals Schillaci et al.
(2019)
Cytokinin Micrococcus luteus Zea mays Raza and Faisal
(2013)
Bacillus subtilis Platycladus
orientalis
Liu et al. (2013)
Paenibacillus polymyxa Lens
culinaris
Gupta et al.
(2015)
Pseudomonas fluorescens Arabidopsis
thaliana
Großkinsky et al.
(2016)
Bacillus subtilis Solanum
lycopersicum
L
Tahir et al. (2017)
Advenella kashmirensis –Ibal et al. (2018)
Frankia casuarinae,F. inefficax,
F. irregularis and F. saprophytica
–Nouioui et al.
(2019)
Proteus vulgaris JBLS202 Arabidopsis
thaliana
Bhattacharyya
et al. (2015)
Gibberellin Phoma glomerata,Penicillium sp. Cucumis
sativus
Waqas et al.
(2012)
Pseudomonas putida Brassica
campestris L.
Kang et al. (2014)
Bacillus amyloliquefaciens Oryza sativa Shahzad et al.
(2016)
Proteus vulgaris JBLS202 Arabidopsis
thaliana
Bhattacharyya
et al. (2015)
ACC
deaminase
Methylobacterium fujisawaense Brassica sp. Madhaiyan et al.
(2006)
Ralstonia sp. J1-22-2, P. agglomerans
Jp3-3, Pseudomonas thivervalensis Y1-3-
9
B. napus/B.
juncea
Zhang et al.
(2011)
(continued)
26 Harnessing the Rhizomicrobiome Interactions for Plant Growth Promotion and... 507
Smith 2014; Bhatt et al. 2020g). Patten and Glick have documented that about 80%
of PGPR produce and secrete auxins as a secondary metabolite (Glick 2012;
Spaepen et al. 2007; Patten and Glick 1996). IAA synthesized by the microbes
affects various metabolic processes such as vegetative development, geotropism and
phototropism, apical dominance and lateral root growth, cell division and differen-
tiation, xylem development, photosynthesis, germination, pigment production, resis-
tance against biotic and abiotic stress and establishment of rhizomicrobiome
(Spaepen and Vanderleyden 2011; Grobelak et al. 2015). IAA is also responsible
for microbial signalling and affects bacterial metabolism (Spaepen et al. 2007;
Spaepen and Vanderleyden 2011). The precursor molecule for microbial IAA
biosynthesis has been found to be tryptophan (Etesami et al. 2009) and four
tryptophan-dependent pathways have been reported. The pathways are referred on
the basis of the intermediates formed, e.g. indole-3-acetamide, indole-3-pyruvic
acid, indole-3-acetonitrile and the indole-3-tryptamine (Imada et al. 2017). Innumer-
ous microbes including Erwinia herbicola,Klebsiella,Pseudomonas,
Agrobacterium,Enterobacter,Bradyrhizobium,Azospirillum and Rhizobium have
been well documented for synthesizing IAA (Patten and Glick 1996; Spaepen and
Vanderleyden 2011). The IAA produced by rhizobacteria has been conferred to
magnify the root surface area and length, making it easier for mineral acquisition by
the crops from the soil (Ahemad and Khan 2012). IAA synthesized by PGPR has
been documented to cause induction of transcriptional changes in the hormone and
cell wall genes which result in improved root length and root biomass and reduced
stomata size and density (Backer et al. 2018; Vacheron et al. 2013).
26.3.5.2 Cytokinins and Gibberellins
Cytokinins and gibberellins are essential phytohormones required for regulating
vital metabolic processes of plant development. Several PGPR such as Rhizobium,
Paenibacillus polymyxa,Pantoea agglomerans,Azotobacter,Gluconacetobacter
diazotrophicus,Acinetobacter calcoaceticus,Pseudomonas fluorescens,
Azospirillum,Rhodospirillum rubrum,Bacillus subtilis,Achromobacter
xylosoxidans and Bradyrhizobium have been documented to produce these phyto-
hormones (Glick 2012; Singh 2015; Deka et al. 2015). Cytokinins are generally
responsible for regulating cell division and lateral and adventitious root growth
Table 26.2 (continued)
Phytohormone PGPR Crop References
P. putida (N21), P. aeruginosa (N39),
Serratia proteamaculans (M35)
Triticum
aestivum
Zahir et al. (2009)
Bacillus,Microbacterium,Methylophaga,
Agromyces
Oryza sativa Bal et al. (2013)
Enterobacter sp. C1D Vigna radiata
L.
Subrahmanyam
et al. (2018)
508 G. Bhandari and N. Nautiyal
(Salamone et al. 2005). Azospirillum was reported to metabolize and absorb gibber-
ellins under different conditions while being associated with higher plants (Bottini
et al. 2004). Dobert et al. reported significant internode elongation on inoculation of
Phaseolus lunatus with Bradyrhizobium sp. (Dobert et al. 1992). In a similar study
by Kucey, increased N uptake and root growth were reported on inoculation of
wheat and maize with gibberellin-synthesizing Azospirillum sp. (Kucey 1988).
Lucangeli and Bottini documented the reversion of the dwarf phenotype in rice
and maize on inoculation with Azospirillum sp. (Lucangeli and Bottini 1996). A
genetically modified Sinorhizobium meliloti strain was capable of cytokinin
overproduction and improved tolerance of alfalfa plants towards water stress
(Xu et al. 2012).
26.3.5.3 Ethylene
Ethylene is another essential plant growth hormone involved in regulating plant
growth, development and survival (Bhattacharyya and Jha 2012). It plays an
indispensible role in promoting fruit maturation, defoliation, flower wilt and induc-
tion of some other phytohormones (Glick et al. 2007). Additionally, ethylene
synthesis increases endogenously in abiotic and biotic stress and thus it is implicated
to play an essential role in stress tolerance. Ethylene biosynthesis employs
1-aminocyclopropane-1-carboxylate (ACC) as a precursor and is under tight regu-
lation of several transcription and post-transcription factors controlled by biotic and
abiotic stresses (Hardoim et al. 2008). During stress state, ethylene endogenously
controls plant physiology and causes reduction in root and shoot development.
Diverse PGPR genera such as Achromobacter,Serratia,Azospirillum,Alcaligenes,
Burkholderia,Ralstonia,Enterobacter,Acinetobacter,Rhizobium,Bacillus,
Agrobacterium and Pseudomonas synthesize an enzyme ACC deaminase that
inhibits ACC by transforming it into 2-oxobutanoate and NH
3
, thus restraining the
deleterious impact of ethylene and augmenting plant development by allowing
resistance against drought and salt (Glick et al. 2007; Nadeem et al. 2009). Iqbal
et al. documented the inoculation of Pseudomonas sp. resulting in lowered ethylene
production, thus enhancing root nodule development and straw and grain production
(Iqbal et al. 2012). Actinomycetes Microbispora sp. and Streptomyces sp. were
found to synthesize ACC deaminase and IAA (Glick 2014). A recent study has
reported the effect of ACC deaminase on regulating ethylene content by the ACC
deaminase-synthesizing Pseudomonas fluorescens YsS6 on assisting the nodulation
by rhizobia (Nascimento et al. 2019).
26 Harnessing the Rhizomicrobiome Interactions for Plant Growth Promotion and... 509
26.4 Plant Growth-Promoting Rhizospheric Microbiome
as Biocontrol Agents
The agricultural sector experiences severe economic loss every year owing to
various plant diseases causedby over 60 pathogens resulting in major damage in
plant productivity. However, the utilization of agrochemicals has improved the crop
production but the agroecosystems have become unbalanced (Kremer et al. 2006).
The surge for sustainable development has invigorated interest in search of novel
biocontrol methods as a significant part of Integrated Pest Management (IPM) or
Integrated Plant Disease Management (IPDM) for plant disease control (Sayyed and
Chincholkar 2010). Since the last two decades, PGPR have been employed for their
immense potential to perform both as biocidal and growth promotion agents. The
biocontrol PGPR confer a resistance to plants against various plant pathogens by
synthesizing several allelochemicals, e.g. volatile organic compounds, iron-
complexing siderophores, lytic and detoxification enzymes, antibiotics and
exopolysaccharides (Table 26.3). PGPR work as biocontrol agents by employing
two vital processes which can be divided according to elicitation and regulation
methods employed: (1) systemic acquired resistance and (2) induced systemic
resistance.
26.4.1 Systemic Acquired Resistance (SAR)
SAR is induced or acquired through exposure of the plants to virulent and
nonpathogenic bacterial strains or organic compounds such as salicylic acid,
benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and
2,6-dichloro-isonicotinic acid (INA) (Sticher et al. 1997). Exposure of plants with
“necrotizing”pathogens (eliciting a hypersensitive response) produces enhanced
resistance towards several other pathogens due to a biological phenomenon referred
to as systemic acquired resistance. Activation of group of SAR genes results in the
synthesis of proteins called pathogenesis-related proteins (PR proteins). PR-1 is one
of most documented PR genes and thus is generally employed as a biomarker for
SAR (Van Loon et al. 2006). Several reports on the production of salicylic acid by
PGPB due to activation of SAR are available in literature (Chen et al. 1999).
26.4.2 Induced Systemic Resistance (ISR)
Induced systemic resistance (ISR) is a defined as phenomena involving nonpatho-
genic microbes, including PGPB, which reduce the detrimental impact of pathogenic
organisms by inducing resistance in the plants through production of antimicrobial
agents (Van Loon et al. 1998). The presence of bacterial antagonist in the
510 G. Bhandari and N. Nautiyal
Table 26.3 Plant growth-promoting rhizomicrobiomes as biocontrol agents
Biocontrol agent Crop Pathogen Mode of action References
Burkholderia cepacia
BAM-6,B. cepacia
BAM-12 and Pseudo-
monas fluorescens
BAM-4
Vigna
radiata
–IAA production,
siderophore and
chitinase
production
Minaxi
and
Saxena
(2011)
B. amyloliquefaciens
LJ02
Cucurbits Powdery mildew
disease
ISR-SA
mediated
Li et al.
(2015)
Pantoea
agglomerans strain
ENA1
Glycine max
(L.)
Macrophomina
phaseolina Merrill
Antibiosis by
pyrrolnitrin
Vasebi
et al.
(2015)
Bacillus anthracis Triticum
aestivum L.
–Protease
production
Verma
et al.
(2016)
Bacillus
endophyticus
Curcuma
longa
Fusarium solani Chitinase
production
Chauhan
et al.
(2016)
Bacillus subtilis
RB14
Solanum
lycopersicum
Rhizoctonia solani Antimicrobial
Inturin A
Zohora
et al.
(2016)
P. aeruginosa strain
LV
C. sinensis
cv. Valencia
Xanthomonas citri
subsp. Citri
Antibiosis by
organocopper
compound
De
Oliveira
et al.
(2016)
Aneurinibacillus
aneurinilyticus
CKMV1
Solanum
lycopersicum
Sclerotium rolfsii,
Fusarium oxysporum,
Dematophora
necatrix,Rhizoctonia
solani,Alternaria
sp. and Phytophthora
sp.
Antifungal activ-
ity, IAA, HCN
and siderophore
production
Chauhan
et al.
(2017)
Burkholderia cepacia
JBK9
Piper nigrum F. oxysporum,
R. solani,P. capsici
Pyrrolnitrin Jung et al.
(2018)
Bacillus sonorensis Capsicum
annuum L.
–Chitinase
production
Thilagar
et al.
(2018)
Bacillus
amyloliquefaciens
Solanum
lycopersicum
Agrobacterium
tumefaciens
Antibiotics Abdallah
et al.
(2018)
Bacillus aerius Piper nigrum Phytophthora capsici HCN production San
Fulgencio
et al.
(2018)
Bacillus sp. strain
B25
Zea mays Fusarium
verticillioides
Antibiosis by
chitinases, glyco-
side hydrolases,
siderophores and
antibiotics
Douriet-
Gámez
et al.
(2018)
26 Harnessing the Rhizomicrobiome Interactions for Plant Growth Promotion and... 511
rhizosphere allows the plant to develop induced systemic resistance for a wide range
of plant pathogenic organisms (Lugtenberg and Kamilova 2009). The mechanism of
“ISR”involves fungal cell wall lysis (Maksimov et al. 2011), root inhabitation
(Kamilova et al. 2005), decreased ethylene content (Van Loon 2007) and synthesis
of siderophore and antibiotics (Beneduzi et al. 2012). PGPR induce ISR in plants
resulting in the activation of pathogenetic genes, mediation of phytohormone sig-
nalling mechanisms and production of regulatory proteins which protect plants from
hazardous impact of pathogenic organisms (Pieterse et al. 2014).
26.4.3 Antibiosis
One of the major mechanisms employed by PGPB for countering the detrimental
effects of phytopathogens is the production of antimicrobial agents (Couillerot et al.
2009; Raaijmakers and Mazzola 2012). Several different types of antimicrobial,
antiviral, antihelminthic, cytotoxic, antioxidant and antitumor agents have been
documented, e.g. phenazine, kanosamine, pyoluteorin, cyclic lipopeptides, karalicin,
pyrrolnitrin, pseudomonic acid, cepafungins, tensin, phenazine-1-carboxylic
acid (PCA), tropolone, rhamnolipids, viscosinamide, amphisin,
2,4-diacetylphloroglucinol (DAPG), azomycin, ecomycins, aerugine, hydrogen cya-
nide, oomycin A, cepaciamide A and butyrolactones synthesized by Pseudomonas,
and oligomycin A, xanthobaccin, subtilosin, Tas A, bacillaene, zwittermicin A,
fengycin, kanosamine, chlorotetain, sublancin, iturin, subtilin, bacilysin and
surfactin synthesized by Bacillus,Streptomyces and Stenotrophomonas spp.
(Goswami et al. 2016). Soil actinomycetes have also been found to synthesize an
array of antibiotics and thus are biocidal for a wide range of pathogenic organisms
(Verma et al. 2009). Antibiotic-producing genes such as srf,dfn,fen,bmy,mln,nrs,
bac,dhb and bae have been located in Bacillus subtilis 168 and Bacillus
amyloliquefaciens FZB42 and are involved in the production of peptides and
polyketides through NRPSs and PKS enzymes (Chang et al. 2007).
26.4.4 Production of Enzymes
PGPB also enhance plant development by synthesizing various metabolites, con-
tributing to the antibiosis and antifungal properties. PGPR synthesize various fungal
cell wall hydrolytic enzymes including chitinase (Husson et al. 2017),
β-1,3-glucanase (Vaddepalli et al. 2017), protease and lipase (Friedrich et al.
2012). The cell wall of fungi is generally composed of chitin and β-glucan; therefore,
chitinases and β-glucanase-synthesizing microbes can assist in inhibiting fungal
growth. Serratia plymuthica synthesizes the enzyme chitinase which results in the
inhibition of spore germination and germ tube expansion in Botrytis cinerea
(Frankowski et al. 2001).Serratia marcescens also works as a biocontrol agent
512 G. Bhandari and N. Nautiyal
against Sclerotium rolfsii by producing extracellular chitinases (Ordentlich et al.
1988). Lim et al. reported the production of extracellular enzymes such as
laminarinase and chitinase by Pseudomonas stutzeri capable of degrading Fusarium
solani mycelia (Lim et al. 1991). Pseudomonas spp. are capable of inhibiting two of
the most detrimental crop pathogens Rhizoctonia solani and Phytophthora capsici
by releasing extracellular enzymes chitinase and beta-glucanases. The
β-1,3-glucanase produced by Burkholderia cepacia is capable of degrading the
cell wall of several pathogenic microbes, e.g. Rhizoctonia solani,Pythium ultimum
and Sclerotium rolfsii. The synthesis of these hydrolytic enzymes is under tight
regulation of several regulatory systems such as GrrA/GrrS or GacA/GacS (Ovadis
et al. 2004). Enterobacter asburiae BQ9 mediates tolerance against tomato yellow
leaf curl virus by activation of defence-related genes and production of phenylala-
nine, ammonia lyase, superoxide dismutase, peroxidase and catalase (Yan et al.
2018). The bacteria also synthesize ACC deaminase and contribute to tolerance in
tomatoes caused by Scelerotium rolfsii to southern blight disease. The inoculated
plants demonstrated modulated ethylene metabolism and antioxidant enzyme activ-
ity; the pathogen-related gene expression analysis corroborated systemic tolerance
(Dixit et al. 2016).
26.4.5 HCN Production
Cyanide is an extremely hazardous compound well known for its toxicity and thus
can be employed for inhibition of pathogenic organisms damaging agricultural
crops. HCN is a secondary metabolite synthesized by several PGPB, is generally
employed for weed and pest control and simultaneously does not impose any
harmful impact on the host plants (Aarab et al. 2019). HCN toxicity is mediated
by inhibition of cytochrome C oxidase along with other vital metalloenzymes (Nandi
et al. 2017). Several PGPR such as Rhizobium,Aeromonas,Alcaligenes,Pseudo-
monas and Bacillus are reported to produce HCN and assist in biocontrol of dreadful
pathogenic strains including Pythium ultimum,Fusarium oxysporum and
Agrobacterium (Ahmad et al. 2008; Das et al. 2017; Zachow et al. 2017). Elimina-
tion of Meloidogyne javanica and Odontotermes obesus crop pests in India has been
conferred due to HCN production (Kumar et al. 2015; Siddiqui et al. 2006). HCN
production by PGPB is effective for elimination of crop pests and is also vital for
metal-chelate complex formation necessary for biogeochemical cycling (Kumari
et al. 2018).
26.4.6 Production of Volatile Organic Compounds (VOCs)
The production of VOCs by various microorganisms in the rhizospheric zone is an
additional method of influencing plant development indirectly. Arthrobacter agilis,
26 Harnessing the Rhizomicrobiome Interactions for Plant Growth Promotion and... 513
Azospirillum brasilense,Chromobacterium violaceum,Bacillus spp., Pseudomonas
fluorescens and Burkholderia pyrrocinia are documented for synthesizing several
kinds of VOCs (Yadav and Yadav 2018; Santoro et al. 2011) such as 2-pentylfuran,
tetradecane, jasmonates, 2,3-butanediol, acetonin, methyl decane,
1-chlorooctadecane and cyclohexanes (Zou et al. 2010; Ryu et al. 2003;
Kanchiswamy et al. 2015). VOCs released in the rhizosphere function as a signalling
molecule for triggering the resistance mechanisms in plant towards plant pathogens
and induce systemic resistance (ISR) (Santoro et al. 2011). Cotton (Gossypium
hirsutum) plants on inoculation with Bacillus spp. were reported to secrete gossypol
and jasmonic acid, thus lowering larval feeding by Spodoptera exigua (Zebelo et al.
2016). Higher transcript level of genes responsible for synthesizing alleochemicals
and jasmonates was reported in comparison to pest control in PGPR-inoculated
plants (Zebelo et al. 2016). Khan et al. studied the efficacy of endophytic fungal
strain Penicillium janthinellum LK5 (PjLK5) for production of defence-related
endogenous phytohormone salicylic acid to counter the deleterious impact of
metal stress in Solanum lycopersicum (Khan et al. 2015). Several VOCs such as
dimethyl disulphide, acetaldehyde, β-caryophyllene, 2,3-butanediol, methanol,
dimethylhexadecylamine, geosmin, furfural, camphor, 5-hydroxy-methylfurfural,
camphene, 2-methyl isoborneol, propanoic acid, 1-octen-3-ol, butanoic acid and
α-pinene are secreted by PGPR species including Agrobacterium,Serratia,
Burkholderia,Arthrobacter,Paenibacillus,Bacillus,Rahnella,Enterobacter,Pseu-
domonas,Collimonas and Pedobacter, thus causing inhibition of various fungal
pathogens and resulting in improved soil fertility and health (Kanchiswamy et al.
2015; Chenniappan et al. 2019). These VOCs have significant applications for
biocontrolling plant pathogens, stimulating of immune system in plants and modu-
lating root expansion (Kanchiswamy et al. 2015; Rojas-Solís et al. 2018).
26.4.7 Production of Antifungal Agents
Several PGPB are capable of synthesizing a wide range of antifungal compounds
including phenazines, pyoluteorin, 2, 4-diacetylphloroglucinol (DAPG), HCN,
viscosinamide, tensin and pyrrolnitrin (Bhattacharyya and Jha 2012). Majority of
identified Pseudomonas biocontrol strains synthesizes antifungal metabolites (phen-
azine, DAPG, pyoluterin, tensin, pyrrolnitrin and viscosinamide) able to inhibit
growth of phytopathogenic fungi (Bloemberg and Lugtenberg 2001; Thrane et al.
2000). A pigment prodigiosin reported from rhizospheric microbe Serratia
sp. possesses the potential to act as an antifungal agent (John et al. 2017). John
and Radhakrishnan have also documented the synthesis of an antifungal compound
pyrrolnitrin by Serratia plymuthica isolated from Curcuma amada (John and
Radhakrishnan 2018). Pyrrolnitrin was found to provide tolerance against soft rot
due to Pythium myriotylum to ginger rhizome (John and Radhakrishnan 2018).
514 G. Bhandari and N. Nautiyal
26.5 Rhizoremediation
Bioremediation is defined as implementation of biological agents/processes for the
removal of toxic xenobiotics in the contaminated environment and emphasizes on
combining phytoremediation and bioaugmentation to result into rhizoremediation
(Bhatt 2019; Bhatt et al. 2019a,b; Negi et al. 2014). Rhizoremediation relies on the
application of root exudates derived from the plants for stimulation, survival and
degradation activities of soil microbes, which ultimately allow efficient degradation
of toxic pollutants (Bhatt et al. 2020a). Several PGPB have been reported to be vital
in the geochemical recycling of minerals, thus causing cleanup of the contaminated
ecosystem (Gangola et al. 2018; Bhatt et al. 2020b,2019c). Many PGPR strains
possessing the ability to degrade a wide range of pollutants are well documented and
generally belong to genera such as Pseudomonas,Flavobacterium,Ralstonia,Bacil-
lus,Enterobacter,Corynebacterium,Achromobacter,Agrobacterium,
Rhodococcus,Sphingomonas and Azospirillum (Bhatt et al. 2020c,d,e,f; Gangola
et al. 2016) (Table 26.4). Rylott et al. have described the conversion of
Table 26.4 Bioremediation of xenobiotics by plant growth-promoting rhizomicrobiomes
PGPR Pollutant References
Pseudomonas putida (PML2) Polychlorinated biphenyls Narasimhan
et al. (2003)
Azospirillum lipoferum strains 15 Crude oil Muratova
et al. (2005)
Achromobacter xylosoxidans_Ax10 Copper Ma et al.
(2009b)
Pseudomonas sp. SRA 2,SRA 1,B. cereus SRA
10, B. juncea,Brassica oxyrrhina
Nickel Ma et al.
(2009a)
Serratia sp. SY5 Cadmium and copper Koo and Cho
(2009)
Bacillus sp. PSB10 Chromium Wani and
Khan (2010)
Bacillus pumilus Chlorpyrifos Ahmad et al.
(2012)
Pseudomonas fluorescens Polychlorinated biphenyls Toussaint
et al. (2012)
Burkholderia metalliresistens Cadmium, copper, lead, zinc Guo et al.
(2015)
Chryseobacterium sp. PYR2 Organochlorine pesticides:
hexachlorocyclohexane
(HCH)
Qu et al.
(2015)
Brevundimonas diminuta Arsenic Singh et al.
(2016)
Pseudomonas putida Tannery effluent: lead and
chromium
Nokman
et al. (2019)
Bacillus megaterium Palladium Chen et al.
(2019)
26 Harnessing the Rhizomicrobiome Interactions for Plant Growth Promotion and... 515
2,4,6-trinitrotoluene (TNT) by Enterobacter cloacae PB2 and Pseudomonas
fluorescens IC (Rylott et al. 2011). Ceratobasidium stevensii, an endophyte of
members of Euphorbiaceae, is capable of metabolizing 89.51% of phenanthrene
(Dai et al. 2010). PGPR such as Azospirillum lipoferum,P. fluorescens,
Enterobacter cloacae and Pseudomonas putida possess the ability to degrade several
petroleum hydrocarbons, PAHs and trichloroethylene (Glick 2012). Pseudomonas
putida KT2440, a PGPR, has the potential of degrading naphthalene (Fernandez
et al. 2012). Uhlik et al. have analysed the activated modification of root exudates in
the rhizomicrobiome composition and its bioremediation ability in polychlorinated
biphenyl (PCB)-contaminated soil (Uhlik et al. 2013). Bisht et al. have reported
several PGPR, Kurthia sp., Bacillus circulans,Micrococcus varians and
Deinococcus radiodurans, obtained from the rhizospheric soil of Populus deltoides
capable of degrading naphthalene and anthracene (Bisht et al. 2010). Acinetobacter
calcoaceticus P23, obtained from the rhizospheric zone of duckweeds, has the
potential to degrade phenol (Yamaga et al. 2010). Yang et al. have investigated
the effect of inoculation of VAM in legumes, Robinia pseudoacacia, for the
remediation of lead-contaminated environments (Yang et al. 2016). PGPR
possessing metal tolerance ability are capable of immobilizing various toxic heavy
metals, thus reducing their detrimental impact on plants (Kong and Glick 2017).
Prapagdee et al. have documented cadmium-tolerant microbial strains: Micrococcus
sp. MU1 and Klebsiella sp. BAM1 capable of enhancing cadmium mobilization and
plant growth promotion (Prapagdee et al. 2013). Jing et al. have obtained metal-
tolerant strains of Enterobacter sp. and Klebsiella sp. from the rhizosphere of
Polygonum pubescens cultivated in metal-contaminated soil (Jing et al. 2014). On
inoculating these strains in Brassica napus, hyperaccumulation of heavy metals (Zn,
Pb and Cd) was observed along with improved plant growth. Hassen et al. reported a
biosurfactant-producing PGPR strain Pseudomonas rhizophila S211, capable of
biofertilization, biocontrol and bioremediation (Hassen et al. 2018).
26.6 Conclusions and Perspectives
Unravelling the prospective PGPR mechanisms of growth promotion and relation-
ship with plants is indispensible for augmenting plant growth and production. PGPR
possess enormous potential to contribute towards sustainable agricultural practices
with minimal harm to the environment. Microbial metabolites affect the plant
physiology in complex manner, governing plant development, nutrition and resis-
tance to biotic and abiotic challenges consequently. Plant-microbe relationships have
played a significant impact in development of several biofertilizer, biocontrol and
bioremediation agents. Despite this, there exists a vast difference in progression from
the in vitro conditions to field due to variable environmental conditions, deficiency
in microbial inhabitation and constraints in persistence in the rhizosphere, thus
reducing the possibilities of implication of PGPR for development of sustainable
agricultural practices. PGPR research should shift focus towards understanding the
516 G. Bhandari and N. Nautiyal
genetic mechanisms regulating growth promontory processes. Concomitant investi-
gations regarding genetic modification of the plant, chemical genomics strategy,
rhizospheric engineering and colonizing with large subpopulations of
rhizomicrobiomes may assist in overcoming these constraints and thus can be
significant for evaluating vital microbial molecular components regulating plant
development and executing PGPR in the field. By recognizing and interpreting the
particular processes of plant-microbe interaction, safer, more efficient and sustain-
able PGPR applications may be designed for sustainable agricultural practices.
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528 G. Bhandari and N. Nautiyal
Chapter 27
Fungal Mycelium-Based Biocomposites: An
Emerging Source of Renewable Materials
Dhanushka Udayanga and Shaneya Devmini Miriyagalla
Abstract Fungi are efficiently used to produce a variety of medicinal compounds,
functional foods, and environmentally sustainable raw materials for a wide range of
consumer goods due to their distinctive biological properties. Mycelium, the vege-
tative structure of filamentous fungi, acts as a natural, self-assembling adhesive as it
grows, binding the fragments of organic substrates, leading to the production of
fungal mycelium-based biocomposites (MBCs). These biocomposites are biode-
gradable alternatives for many synthetic polymers, such as polystyrene, and are
therefore considered as a widely applicable, emerging class of renewable materials.
MBCs are excellent examples of circular materials, ensuring a cradle-to-cradle
(C2C) design, in which biodegradable products can be returned to the ecosystem
after its use. Diverse species of fungi can be used to produce MBCs together with a
range of agricultural and other plant-based lignocellulosic substrates. Several busi-
ness start-ups, by innovative investors, are globally leading in mycelium-based
product manufacturing. MBCs, including both mycelium-based foams (MBFs) and
mycelium-based sandwich composites (MBSCs), are known for their potential
industrial applications, such as packaging materials, architectural design, construc-
tion, fashion, and automotive insulation products. Both the mycelium binder and
substrate type have an immense impact on the significant material properties of
MBCs, including their hydrophobicity, acoustic nature, thermal insulation, and fire
resistance. This chapter summarizes the diversity of the fungi used to produce MBCs
as well as their potential feeding substrates, manufacturing process, physical and
mechanical properties, innovative applications, and future directions for related
research endeavours.
Keywords Biodegradation · Biofabrication · Circular economy · Mushrooms
D. Udayanga (*) · S. D. Miriyagalla
Department of Biosystems Technology, Faculty of Technology, University of Sri
Jayewardenepura, Pitipana, Homagama, Sri Lanka
e-mail: dudayanga@sjp.ac.lk
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_27
529
27.1 Introduction
Fungi are important microorganisms due to their vital role as a major decomposer in
natural ecosystems as well as their industrial, food, and medicinal applications
(Blackwell 2011; Hyde et al. 2019). Mycelium, consisting of bundles of thread-
like hyphae, is the vegetative structure of filamentous fungi. Hyphae spreading
through the substratum or an artificial medium absorb nutrients required for the
growth of the mycelium of a fungus. Therefore, mycelium represents the major
component of fungal biomass. Vegetative mycelia of the filamentous fungi and
mushrooms are traditionally well-known for their capability to produce antibiotics,
enzymes, organic acids, nutritious functional foods, high-value health supplements,
and natural food flavouring agents (Rathore et al. 2019). However, recent advances
in environmental engineering and biomaterial research have revealed the potential of
filamentous fungal mycelium as a promising raw material for biocomposites (Attias
et al. 2020; Ghazvinian et al. 2019; Jones et al. 2020). This is due to their possible
use as a substitute for a variety of non-biodegradable, inert synthetic materials, such
as polystyrene and other plastics, including moulded or fabricated expanded poly-
styrene (EPS), expanded polypropylene (EPP), and expanded polyethylene (EPE).
Therefore, MBCs have now become an eco-friendly alternative in various commer-
cial applications, such as architecture, textile, fashion accessories, footwear, auto-
motive insulation padding, electronics, healthcare, and packaging, where EPS-like
synthetic polymers were traditionally used.
Mycelium is primarily comprised of polysaccharide chitin, which makes up the
cell wall of fungi. Additionally, lipids, glycoproteins, minerals, fibre, and other
polysaccharides, like mannans and beta-glucans, are also found in the mycelium
(Bowman and Free 2006). The unique structure and composition of fungal myce-
lium have made it a convenient source of natural composite materials with tuneable
and well-ordered structural and mechanical properties. In some cases, the properties
of mycelium grown on a natural substrate can be engineered by changing the type of
medium as well as other growth conditions. Therefore, fungal mycelium grown in a
large scale can be used as a raw material for the manufacture of various sustainable
products.
All fungi are heterotrophs, which means they require organic substrates for their
growth. MBCs are produced by ceasing the growth of mycelia before its organic
substrate or medium is fully degraded. Among the diverse groups of fungi,
mushroom-forming basidiomycetes are the primary choice of microorganisms
used in MBC production (Cerimi et al. 2019). Therefore, MBCs are also popularly
known as “mushroom-composites”,“mushroom-material”,“mycomaterial”, and
“mycocomposites”by commercial producers and innovators. In MBCs, when the
fibrous substrate material is used, fungal hyphae bind organic fibres or particles
together while colonizing the substrate (Appels and Wösten 2020). The ability of
fungi to bind and digest lignocellulosic plant material produces an inherent bonding
that can be developed into a natural, unique, and lightweight biocomposite. These
MBCs are usually free of synthetic adhesives; therefore, they represent an
530 D. Udayanga and S. D. Miriyagalla
eco-friendly, biodegradable alternative for many industrial applications. The mor-
phology and mechanical properties of the composite material are largely defined by
the feeding substrate and biological nature of the fungus. Based on the composition
of the feeding substrate of fungi, the final fibrous structures exhibit varying structural
compositions.
The global environmental crisis, which stems from the accumulation of synthetic
plastic debris and the depletion of fossil fuels, has caused the global scientific
community to re-consider renewable sources of material in industrial applications.
The major environmental issues in the world have arisen due to the continued
practice of a linear economy based on the “cradle-to-grave”concept in which raw
natural resources are taken, transformed into products, and disposed into the natural
ecosystems after their use. In contrast, a circular economy model, which is based on
the “cradle-to-cradle”design concept, aims to close the gap between production and
the disposal of related waste, turning the waste of a given process into the raw
material for another product. Therefore, biodegradable and compostable MBCs are
ideal solutions to the global demand towards sustainable living through a circular
economy. In this review, we discuss the fungi used in the production, the
manufacturing process, the physical and mechanical properties of MBCs, and the
recent developments of advanced applications of similar materials.
27.2 Fungi Used in the Production of MBCs
The saprotrophic fungal species belong in the phyla Basidiomycota and Ascomycota
have generally been used as binding components in MBCs. Mushroom-forming
Basidiomycota represent the widely used species of fungi in MBCs. Their
prevalence in nature, ability to colonize a large area by making extensive
three-dimensional hyphal networks, and desirable physicochemical properties are
invaluable traits in MBC design, which make them the prime candidates for this up-
and-coming sustainable material design concept (Appels and Wösten 2020).
Fungi are ideal for the partial degradation of the majority of plant-derived filler
materials of MBCs consisting of fibrous or particulate matter with a rich lignocel-
lulose composition. For instance, fungal species belonging to the genera Trametes,
Ganoderma, and Pleurotus are widely used in MBCs, since those fungi can be easily
collected and have a high abundance of extracellular enzymes, which allows them to
specialize in partially degrading more complex nutrients, such as cellulose and lignin
in substrates (Jones et al. 2020).
When selecting a suitable fungal species for composite manufacturing, growth
predictors and the biology of the organism, such as the growth rate, mycelium
density, hyphae nature, biochemical composition, and pathogenicity, must be con-
sidered. Non-obligate pathogens can become versatile colonizers of filler materials
due to their innate ability to aggressively compete with living hosts (Jones et al.
2018a). The mitic system, comprised of mono-mitic, di-mitic, and tri-mitic net-
works, defines the different types of hyphal networks found within the mycelia of
27 Fungal Mycelium-Based Biocomposites: An Emerging Source of Renewable Materials 531
basidiomycete fungi (Pegler 1996). In MBC design, tri-mitic strains, especially
Trametes versicolor and T. multicolor, which are composed of all three hyphal
types, generative, binding, and skeletal hyphae, are favoured. Those fungi with
tri-mitic strains are known to have a better mechanical performance within a
composite structure provided by their often solid, thick-walled hyphal filaments
than mono-mitic strains, such as Pleurotus, that are composed of less complex
generative hyphae (Jones et al. 2020). Trametes versicolor had been used in binding
hemp and hardwood chips in a novel MBC design (Zimele et al. 2020). In a similar
study, T. multicolor was experimented on for beech sawdust, rapeseed straw, and
cotton waste (Appels et al. 2019). Studies on the fire-resistant properties of high
silica agricultural and industrial waste-based composites have been conducted with
T. versicolor as a binder by Jones et al. (2018b).
Fungi belong in the genus Ganoderma form slow-growing, coarse, densely
matted, woody mushrooms. Therefore, the incorporation of the mycelium of
Ganoderma in MBCs may result in mechanically resilient end-products. Two
species of mushroom-forming fungi, G. lucidum and Pleurotus ostreatus, with
sawdust, ground walnut shells, rice hulls, millet hulls, and cardboard have been
evaluated for a potential application as thermal insulators for buildings (Yang 2020).
In a related study, researchers investigated the possibility of using G. lucidum as a
binder for a composite board made from palm sugar fibre and cassava bagasse fibre
(Agustina et al. 2019). Moreover, spent mushroom substrate generated by sawdust,
food waste, and diaper waste has been used along with G. lucidum,P. ostreatus, and
Auricularia polytricha in strong fire- and water-resistant bio-boards (Khoo et al.
2020). The San Francisco-based start-up, MycoWorks, dedicated to introducing new
mycelium materials to the world, primarily uses G. lucidum and P. ostreatus as their
binder fungi.
With a global distribution, the genus Pleurotus has been studied at a fundamental
level in a variety of MBC designs.For instance, P. ostreatus,P. djamor,P. eryngii,
P. ostreatus var. columbinus (Kalisz and Rocco 2011), P. pulmonarius,
P. salmoneostramineus (Attias et al. 2017), and P. citrinopileatus (Silverman
2018) have been explored in many similar studies. Grifola frondosa,Lentinula
edodes,Agrocybe aegerita,Coprinus comatus (Kalisz and Rocco 2011),
Pycnoporus sanguineus,Lentinus velutinus (Bruscato et al. 2019), Fomitopsis
pinicola,Gloeophyllum sepiarium,Laetiporus sulphureus,Phaeolus schweinitzii,
Piptoporus betulinus,Polyporus arcularius,Schizophyllum commune (Lakk et al.
2018), Trametes pubescens,T. suaveolens,Trichaptum abietinum (Wimmers et al.
2019), Polyporus brumalis (Jones et al. 2018a), and Agaricus bisporus (Tacer-Caba
et al. 2020) include some other mushroom species that have been investigated in
MBC design.
When compared to basidiomycete fungi, members of the phylum Ascomycota are
relatively less studied as binding agents for biocomposites, since the reliance on
fungi already limits the rate at which biocomposites can be manufactured. The
characteristics of the fungi such as high hyphal extension rates to effectively
colonize new areas of the substrate and density of mycelium are some contributing
factors when selecting a species to produce MBCs. In ascomycete fungi, the hyphal
532 D. Udayanga and S. D. Miriyagalla
extension rate has been found to be decreased with an increase of branching. When
the colonies branch with a high mycelial density, inhibitory compounds are pro-
duced with the increased utilization of substrates, thus reducing the hyphal extension
rates. This could potentially explain the reason for ascomycetes being an unpopular
option for MBC design (Jones et al. 2018a). However, due to the availability of a
range of enzymes, such as cellulases, pectinases, proteases, and xylanases, as well as
fast growth and colonization rates of the mycelium, Fusarium oxysporum has been
tested for biocomposite production with waste paper and spent coffee substrates
(Iordache et al. 2018). Tacer-caba et al. (2020) evaluated the potential of using
ascomycete fungus, Trichoderma asperellum in oat husk and rapeseed cake sub-
strate fillers. In addition, a recent patent review highlights a MBC based on asco-
mycete, Morchella angusticeps that has been granted a patent (Cerimi et al. 2019).
However, the same review indicated four applications based on a few wood-
associated ascomycete fungi-based (Xylaria polymorpha,X. hypoxylon,
X. filiformis,andX. longipes) MBCs, which have not been granted the patents or
awaiting consideration (Cerimi et al. 2019).
27.3 Feeding Substrates for MBCs
The feeding substrate for MBC formation usually depends on the availability of
excess plant materials from the agricultural waste, food processing, or wood and
landscape industries. In addition to a carbon source, effective mycelium growth
requires micro-nutrients, moisture, an ambient temperature, and oxygen. Therefore,
any cellulose-rich or fibre material in sufficient supply is ideal for the production of
biocomposites if the resulting composite meets the required mechanical property
standards (Lelivelt 2015). Cellulose-rich substrates hinder mushroom-forming fungi
from producing fruiting bodies, and the substrate provides optimum mechanical
properties of the final product (Jones et al. 2017a). Therefore, wood-containing
substrates, such as sawdust or wood chips of common landscape trees (i.e. spruce,
pine, and fir), are commonly used in prototype preparation and industrial applica-
tions. Agricultural waste materials, such as rice straw, rice husk, wheat straw, wheat
bran, corn straw, corn cobs, coconut fibre, sorghum stubbles, flax shive, kenaf fibre,
cotton bur fibre, hemp hurd, and sugarcane bagasse, are also ideal sources of organic
fibrous material, that have been used in various studies (Lelivelt 2015; Arifin and
Yusuf 2013; Jones et al. 2017b; Pelletier et al. 2017). Therefore, typically, any
non-toxic cellulose-rich plant material is an ideal source for the biofabrication of
MBCs, as mycelium can be grown with or without supplements of artificial sub-
strates or other chemicals.
In addition to its wood and fungal components, essential nutrients and artificial
fungal growth media can be added to the substrate to be used in MBC production.
For instance, Haneef et al. used pure cellulose and a cellulose–Potato Dextrose Broth
(PDB) mixture as a fibre-nutrient source to develop MBCs (Haneef et al. 2017).
Moreover, attempts have been made to produce hybrid composites via the
27 Fungal Mycelium-Based Biocomposites: An Emerging Source of Renewable Materials 533
incorporation of cellulose nano-fibrils (CNFs) in novel panel composites in addition
to mycelium and wood components (Sun et al. 2019). Producing a hybrid composite
requires a mycelium-treated wood produced by the growth of mycelia on soft wood,
which is then hybridized with various levels of CNF as a binder (Sun et al. 2019).
Although MBCs are self-grown materials on a substrate, pure mycelium can be
separately grown in a broth culture, or an artificial media can be used to make a
composite by mixing the harvested mycelia with a sterile fibre substrate. Similarly,
an organic substrate can be amended with an appropriate amount of inert material,
such as metals, plastics, or ceramics, in order to produce composites for various
purposes (Appels and Wösten 2020).
27.4 Manufacturing Process of MBCs
The positive environmental impact of MBCs is partially accomplished through their
eco-friendly, sustainable, and circular manufacturing processes (Fig. 27.1). This is
highly beneficial when compared to how their conventional petroleum-based,
non-renewable composite counterparts are manufactured. In MBC production, rel-
atively less energy-consuming, natural, and low-cost production processes are
facilitated by the selection of usually mesophilic, indigenous, or commonly available
fungi, which prefer ambient temperatures. Therefore, unlike in the industrial
processing of synthetic polymers, no additional energy is required for maintenance
or incubation under specific high or low temperatures.
The typical process of manufacturing a MBC starts with the selection of the
substrate material and the fungal strain that is biocompatible with the substrate of
choice. The significant factors that determine the growth performance (growth rate,
mycelial density, and composition) of the fungal mycelium are the nutritional
properties of the substrate, environmental conditions, and the genetic factors of the
species used (Jones et al. 2018a; Appels et al. 2018). Low-cost agricultural wastes
are the popular choice in many available MBC products and prototypes, but some-
times higher-grade, expensive, more nutritious substrates, such as wheat grains, have
also been used (Jones et al. 2018b,c).
The second step in the production is the pre-treatment of the substrate, which
involves soaking (to supply the optimum moisture content for the mycelium that is to
be inoculated) followed by the homogenization of the substrates in order to increase
the surface area for mycelial colonization. The chopping or cutting of feedstock,
grinding, milling, or blending can be employed to macerate the substrates (Elsacker
et al. 2019). Heat treatment of wood fibres to open and expand the air cavities is
suggested to increase the porosity of the MBCs produced (Attias et al. 2017).
Following the pre-treatments, sterilization of the substrates is a crucial step in the
process, preferably achieved through wet heat methods, such as autoclave or pres-
sure cooking, to ensure a contaminant-free medium without drying out of the
substrate (Lelivelt 2015).
534 D. Udayanga and S. D. Miriyagalla
Once the substrate is pretreated and sterilized, the introduction of the fungal
culture into the sterilized substrate should be done via inoculation. The fungal
cultures can be introduced in the form of a spore suspension or through an interme-
diate nutrient-rich substrate, such as grain or sawdust ramified with mycelia, focus-
ing on the even distribution of the inoculum within the substrate. The inoculated
substrate is then filled into moulds to provide a defined shape for the MBCs (Jiang
et al. 2013; Bayer et al. 2008).
During the incubation period, followed by the substrate inoculation, the myce-
lium initiates growth from the points of inoculation and colonizes the filler material,
adhering the particles/fibres together into a dense networked structure while
maintaining the mould shape, a process that can take anywhere from days to months
depending on the strain (Jones et al. 2020). Temperatures around 25–35 C are
preferred, since temperatures above 40 C may induce fruiting bodies. The humidity
Fig. 27.1 A typical cradle-to-cradle (C2C) cycle of mycelium-based biocomposites (MBCs). Both
the fungal mycelium and the natural cellulosic substrate used in biocomposite manufacturing are
returned to the environment as a nutrient-rich ingredient for soil enrichment. Therefore, in the C2C
life cycle, materials flow cyclically in an appropriate, continuous biological or technical nutrient
cycle. All the waste materials are productively re-incorporated into a new sustainable product within
the cycle
27 Fungal Mycelium-Based Biocomposites: An Emerging Source of Renewable Materials 535
levels of 60–65%, proper aeration, and dark conditions are generally considered
ideal for optimum mycelial growth (Silverman 2018; Butu et al. 2020). Although
common practice involves incubation in ambient environmental conditions, con-
trolled environments (e.g. higher temperatures) may also be employed to achieve
rapid growth rates (Jones et al. 2020).
To ensure a contamination-free and properly ventilated incubation, growth cham-
bers are often used in most studies (Silverman 2018). The final and most critical step
in MBC manufacturing is stopping the growth of the fungus on the substrate prior to
the complete degradation of the fillers by neutralizing/dehydrating the mycelium via
heating/drying, thus simultaneously making the material denser (Jones et al. 2018b).
Heating of the fully colonized structures from 2 to 48 h in 60–125 C temperatures in
convectional ovens or hot pressing is used industrially for mycelium deactivation.
Alternatively, air drying for 48 h can be considered a more practical and energy-
efficient method to be used in large-scale, industrial settings (Jiang et al. 2019).
Further processing steps, such as hot pressing, cold pressing (Appels et al. 2019),
hybridization with natural polymers (i.e. cellulose nano-fibrils) (Sun et al. 2019), and
natural resin/glue impregnation (Jiang et al. 2013), can be carried out if desired at the
end of production to obtain a high-quality end-product with enhanced mechanical
properties.
27.5 Physical and Mechanical Properties of MBCs
Apart from the key advantage of being biodegradable, MBCs have several other
desirable physical and mechanical material properties that encourage their use in
various applications. These properties considered here include density, tensile
strength, compressive strength, rigidity and material’s strain response parameters,
water absorption propensities, fire resistance, heat-insulating capability, and acoustic
properties. According to recent studies, the density of MBCs falls within the range of
60–300 kg/m
3
. According to an analysis by Jones et al., MBCs made with lignin-rich
woody substrates are relatively denser than those made with more fibrous, cellulose-
rich agricultural waste (Jones et al. 2020). Compared with similar building materials,
such as plywood (680 kg/m
3
) and particle board (750 kg/m
3
), the apparent disparity
in the density values between these conventional construction materials and MBCs
is remarkable.
Tensile strength, or the maximum load a MBC material can bear once stretched, is
primarily dependent on how strongly the binder fungus adheres the fillers together
with adequately strong interfacial bonding, which is directly influenced by the
nutrient richness of the substrate that can facilitate a rich network of mycelium
(Jones et al. 2020). The compressive strength of the MBC is affected by the porosity
of the substrate, which explains why more porous, fibrous composites have lesser
compressive strengths than particulate ones. Compressive strength values range
from 29 to 567 kPa for MBFs, which is considered relatively lower than that of
standard categories of EPS foams (Girometta et al. 2019). The flexural strengths/
536 D. Udayanga and S. D. Miriyagalla
bending strengths of the structure are determined by the tensile and compressive
stress limits the two opposite surfaces of the composite structure can withstand when
flexed. The formation of a thick, continuous mycelial mat on air-exposed surfaces is
paramount to withstand these conditions, and this is achieved through the utilization
of nutrient-rich substrates that can support the rich growth of mycelium via proper
aeration.
In materials, elastic deformation is usually expressed by means of the shear
modulus (G) and Young’s modulus (E), which are types of elastic moduli that can
be used to determine the rigidity and strain response parameters, respectively.
Young’s modulus, which measures the ability of a material to withstand changes
in length when under lengthwise tension or compression, can be calculated by taking
the slope of the linear part of a stress/strain curve (Appels et al. 2018; Jastrzebski
1959). The shear modulus is defined as the ratio of the shear stress to strain (IUPAC
1997). Both E and G are known to be relatively higher in fibrous MBCs, which are
mechanically more rigid (Haneef et al. 2017) compared to some synthetic counter-
parts, like large lightweight EPS blocks (geoforms). For instance, the Young’s
moduli of MBCs have been reported in the range of 3–11 and 17–27 MPa for
Ganoderma lucidum and Pleurotus ostreatus pure mycelia, respectively (Haneef
et al. 2017), with the average value of the initial elastic modulus of EPS ranging from
5.08 to 3.31 MPa (with a standard deviation of 1.89–1.48 MPa) (Bathurst et al.
2007). However, these numbers might not be directly comparable as they are highly
density-dependent as well as influenced by the specimen size, rate of loading, and
loading path (Zarnani and Bathurst 2007).
27.6 Water Absorption Propensities
The relatively high water absorption propensity of MBCs is one drawback that may
limit their application, except when used as acoustic and thermal insulators within
door cores with dry indoor environments that prevent moisture contact. The increas-
ing of weight when in contact with water for 48–192 h are ranged from 30 to 580%
as reported in several studies experimenting on this quality (Zimele et al. 2020;
Appels et al. 2019; Agustina et al. 2019; Sun et al. 2019; Elsacker et al. 2019; Ziegler
et al. 2016). The hydrophilic nature of the lignocellulosic filler substrates, the
hydrophobic nature of the mycelium binder, and the porosity of the composites
govern the overall extent of the water absorbed by the material. The cellulose-rich
filler materials in general contain ample hydrophilic, hydroxyl (–OH) groups that
readily bind with water. Meanwhile, certain fungal mycelia contain the protein
hydrophobin, which makes them hydrophobic (Appels et al. 2018; Girometta et al.
2019). For instance, the absence of the sc3 gene in Schizophyllum commune affects
the cell wall composition, where the deletion of the sc3 gene increases the total
amount of cell wall polysaccharide schizophyllan as well as decreases the amount of
glucan cross-linked to chitin (Sietsma and Wessels 1977,1981; van Wetter et al.
2000). Haneef et al. reported a high sensitivity to water in mycelia with low chitin
27 Fungal Mycelium-Based Biocomposites: An Emerging Source of Renewable Materials 537
content in the cell walls (Haneef et al. 2017). According to results obtained by Khoo
et al., high water-resistant nature of their tested bio-boards was attributed to a thick
impenetrable skin formed outside the structure by Pleurotus and Ganoderma binders
(Khoo et al. 2020). During swelling, water is absorbed into the pores of the MBCs
through capillary action. Therefore, the lower void volume in particulate fillers like
sawdust and the higher density of the final MBC product can reduce the absorbance
of the water into the material (Jones et al. 2020; Zimele et al. 2020; Elsacker et al.
2019).
27.7 Fire Resistance, Insulating Capability, and Acoustic
Properties
When using MBCs as an innovative, novel material for application in building
construction and packaging, it is paramount to consider their fire-retardant property,
since these composites are unequivocally rich in combustible organic matter that can
easily catch fire. Therefore, research on fire-resistant properties of MBCs needs to be
encouraged to promote their usage in the above applications. Wheat- and rice husk-
based MBCs start degradation at around 250 C, react to fire in three degradation
stages, and form a stable carbonaceous char at 600 C (around 23% by weight),
producing a relatively greater residual yield when compared to other polymers; this
indicates lower amounts of toxic volatiles and smoke being released upon combus-
tion (Jones et al. 2018b). This significant amount of char formation hinders the
oxygen migration in the core and acts as a thermal insulator, thereby limiting
combustion (Mouritz and Gibson 2007). Phosphorus, a commonly used flame
retardant in polymers, had been found extensively within char and acts as a dehy-
dration catalyst in the char generation reaction by forming phosphoric and
polyphosphoric acids. As evident from scanning electron microscopic (SEM)
images of pyrolyzed MBCs, their fibrous structures were preserved post-combustion
due to the presence of mycelial chitin, which is an excellent flame retardant,
thermostable compound (Jones et al. 2018c). The heat release rate, one of the most
important features to be considered in the fire safety of a material, was found to be
between 33 and 107 kW/m
2
(Jones et al. 2018b) in MBCs. Therefore, the heat
release rate of MBCs is also lower than that of synthetic building materials (EPS
foam, 114 kW/m
2
; particleboard, 134 kW/m
2
). Thus, MBCs can be used in the
products in order to significantly ensure the fire security. Utilizing substrates like rice
husks, which are rich in silicon and lignin, can further enhance the fire-related
properties of MBCs. This is because plenty of more stable, silicon-rich residual
char can be generated by the combustion of such material that can act as a better
thermal and oxygen insulator (Jones et al. 2018b).
One of the practical applications of MBCs thus far has been their use in thermal
insulators and acoustic materials, and, according to recent research, they can emulate
commonly used synthetic insulators, such as polystyrene and polyurethane foam, in
538 D. Udayanga and S. D. Miriyagalla
terms of performance. They can be jointly used within walls (intra wall panels) as
thermal and acoustic insulations (Butu et al. 2020). The thermal conductivity (the
ease with which heat can travel across a material) values of MBCs fall within the
range of 0.05–0.07 W/m*K, which is a competitive value when compared with
conventional insulation foams (XPS foam, 0.03º0.04 W/m*K; polyurethane,
0.006–0.18 W/m*K). While there is an optimal density for high insulator perfor-
mance (Yang 2020), the low-density straw and hemp-based substrates also have
shown promising results (Elsacker et al. 2019; Xing et al. 2018). The characteristic
porous nature and the presence of air cavities within the MBCs that can trap large
volumes of air can be noted as a reason limiting the heat conduction across the
material. Low-moisture content is also associated with better thermal insulators, and
completely dry conditions are required for MBCs to perform optimally (Yang et al.
2017). A study by Wimmers et al. discovered that sawdust-based MBCs have
remarkable insulation properties (0.051–0.055 W/m*K) that are better than those
of conventional wood panels (0.12 W/m*K–0.19 W/m*K) (Wimmers et al. 2019).
Ecovative Design’s prototype product Greensulate™is a MBC insulation panel
designed and tested at both residential and commercial levels (US Environmental
Protection Agency 2019).
Pelletier et al. proposed the use of MBCs as acoustic insulators in automotive
panels as well as in installations for acoustic damping (Pelletier et al. 2013).
Acoustic absorbers work by converting sound waves into heat, which prevents
sound accumulation and reflection. An acoustic absorption of over 70–75% at
1000 Hz was achieved in this study, with a 50–50% composition of switchgrass
and sorghum fibres showing the highest absorption levels (Pelletier et al. 2013). A
combination of surface porosity and the fibrous quality of the MBCs determines the
degree of sound absorption. Surface porosity of MBCs causes the sound waves to
enter the material while the fibres present inside generate a resistance to the air flow,
thus damping the sound waves by converting them to heat (Jones et al. 2020).
27.8 Improvement of Material Properties
There are limitations of applications of MBCs partially due to the lack of
documented information available regarding the physical and mechanical properties
(Jones et al. 2020). Physically, after colonization, the surface of the MBC material
appears white due to a mycelial mat formation. Several studies have reported the
formation of a more smooth mycelial skin on top of the material, particularly with
longer periods of colonization. Thus, the incubation time can be used to optimize
physical and mechanical properties during the colonization step.
The overall physical and mechanical properties of MBCs can be improved
through post-processing steps, such as cold pressing, hot pressing, and the addition
of various chemicals. During cold pressing, the structure undergoes compaction due
to the reduction in its porosity, and, when heat-pressed, the moisture inside the
composite evaporates, causing steam to diffuse through the cells and the voids in the
27 Fungal Mycelium-Based Biocomposites: An Emerging Source of Renewable Materials 539
mycelium to increase the plasticity and density of the material. A non-enzymatic
browning reaction that causes cross-linking between amino acids and reducing
sugars is another factor that strengthens MBCs when heat-pressed (Martins et al.
2000).
27.9 Innovative Applications of MBCs
MBCs are increasingly growing in prevalence globally, with primary interest mov-
ing towards renewable alternatives in industrial applications. Many of the recent
successful and potential uses of MBCs are discussed in recent scientific publications,
patent applications, and various news sources regarding science and technology.
Among them, only a limited number of successful business start-ups and industrial-
scale applications are available worldwide, which may not fully satisfy the global
demand. For instance, a few leading companies based in Europe and the USA, such
as Ecovative Design, Mogu, the Magical Mushroom Company, MycoWorks, and
Officina Corpuscoli, provide a range of innovative novel products based on MBCs.
The applications of these composites include artwork, packaging, shipment padding,
construction, textiles, shoe soles, automotive interior padding, model making, inte-
rior design, vertical green walls, raised garden beds, electrical circuit boards, and
many more innovative alternatives replacing petroleum-based synthetic material.
27.10 Architectural Designs Using MBCs
MBCs are known for their potential use as a renewable substitute for architectural
construction constituents. These composites are ideal for specific use as architectural
raw materials due to their properties of insulation, fire resistance, and plasticity that
can be exploited to produce desired shapes and patterns compared to synthetic
petroleum-based materials, such as EPS, XPS (extruded polystyrene), and polyure-
thane (Xing et al. 2018). Mogu, a leading European company based in Italy, has
developed mycelium-based wall panels, ceiling panels, and acoustic panels with
remarkable sound proof properties that can be effectively used in the interior of the
modern architectural designs (Fig. 27.2).
As an initiative of potential construction projects based on mycocomposites, the
Hi-Fi tower, a mycelium brick structure opened in the courtyard of MoMA’s PS1
(Museum of Modern Arts, Public School-1 building) space in New York and
designed by Architect David Benjamin, has been popular in the last few years
(Scott 2014). MycoTree, a spatial branching structure, which was featured at the
Seoul Biennale of Architecture and Urbanism 2017 exhibition in Seoul, Korea, had
been built using Ganoderma lucidum to bind bamboo plates in an innovative,
geometrically weight-bearing architectural design (Heisel et al. 2017). Furthermore,
Mycotecture Alpha, an architectural design by Phil Ross, was grown from the
540 D. Udayanga and S. D. Miriyagalla
mushroom G. lucidum (Reishi), made into MBC bricks, and stacked into an arch
(Stone 2015). It was also confirmed that Schizophyllum commune mycelium can
potentially be used in designing biocomposite building materials for habitats on
Mars and the Moon (Lakk et al. 2018; Wösten et al. 2018). The unique properties of
MBCs have made it possible to use them as thermal insulation panels, acoustic
architectural designs, and other construction materials. Compared to the many
man-made, synthetic materials used to make building materials, MBCs represent
an excellent non-toxic, cleaner alternative for the growing needs in sustainable
alternatives for construction and interior designs.
27.11 MBCs as Packaging Material
The majority of the retail products bought by consumers around the world come in
plastic, polyethylene, or polystyrene packaging. Once the consumer receives the
product, the packaging material is eventually disposed of in the environment,
leading to the global crisis of plastic pollution. MBCs have been developed over
time as various types of packaging material. These products are an ideal substitute
for polystyrene and other synthetic hydrocarbon-based packaging materials
(Abhijith et al. 2018). One of the leading start-up companies based on mycelium-
based products, Ecovative Design, produced MBCs to be used as a wide range of
Fig. 27.2 Mycelium-based interior products by Mogu (https://mogu.bio/). (a) Mycelium resil-
ient—oyster tile and samples. (b) Mycelium-resilient floors. (c) Flooring mycelium panels—
interiors, kitchen. (d) Acoustic mycelium panel—plain (interiors). (e) Acoustic mycelium panel.
(f) Acoustic mycelium panel—interiors. (Courtesy: Images published with the copyright permission
from Mogu Press and Communication team. MOGU S.r.l. Via San Francesco d’Assisi 62, 21020
Inarzo (VA), Italy)
27 Fungal Mycelium-Based Biocomposites: An Emerging Source of Renewable Materials 541
packaging materials as an alternative to polystyrene and other synthetic packaging
materials. Ikea, a leading multinational company founded in Sweden that designs
and sells ready-to-assemble furniture, kitchen appliances, and home accessories
around the world, recently announced their intention to use MBCs in their packaging
materials (Hammon 2020). Apart from commercial providers of packaging material,
various studies have been conducted to develop packaging solutions based on
MBCs. For instance, six blends of processed cotton plant biomass materials were
evaluated as a substrate for the colonization of selected fungi in the manufacture of
moulded packaging materials (Holt et al. 2012). The same study revealed that the
resulted MBCs had more favourable properties when compared to extruded poly-
styrene foam. Ecovative Design (Bayer et al. 2008) and Shenzhen Tech (Lacourse
and Altieri 1989) have received several patents for mushroom packaging solutions
and their production protocols (Cerimi et al. 2019).
Packaging solutions based on MBCs are an excellent example of a circular
economy. These are bio-based materials resulting from a bioprocess similar to that
of solid-state fermentation followed by a few processing steps. Therefore, the
production process requires less energy. Once the synthetic packaging material is
used, the remains are dumped into the environment directly unless it is not properly
recycled. However, the biocomposite-based packaging material will be compost-
able, thus closing the loop, in accordance with the C2C model for materials, by
reducing waste and promoting the continual use of resources. The hydrophilic nature
of some mycelium materials gives them the advantage of superabsorbent materials.
In contrast, the properties of MBCs, including hydrophobicity, low density, insula-
tion, and fire resistance, have made it more promising sustainable future material in
packaging applications.
27.12 Mycelium-Based Sandwich Composites and Their
Applications
One of the most recently identified applications of mycelium materials is their use in
making sandwich composites (Bayer et al. 2008). These composites comprise of a
core made up of a lightweight mycelium-based material sandwiched within two thin,
but relatively stiff, laminates. The uniqueness in its design provides MBSCs with
advanced material properties, like greater shear strength, flexural strength, and
tensile strength, over conventional synthetic materials, in addition to the advantage
of being lightweight overall.
In contrast to the MBSCs, polymer matrix composites (PMCs) are the materials
made up of fibres, embedded in an organic polymer matrix. The PMCs consist of
three constituents including a matrix, reinforcement, and interface, which affect its
overall mechanical properties. The matrices of PMCs are made up of a petroleum-
based polymer component. The reinforcement can be either glass or carbon fibres
with plastic resin, epoxy, polyester, and nylon metal sheets as laminate skins. The
542 D. Udayanga and S. D. Miriyagalla
use of biodegradable materials like sawdust and agricultural waste to reinforce
PMCs could seal them in an inert matrix, preventing the natural degradation process
of those cellulosic components. Therefore, the PMCs reinforced with wood or
agricultural waste are also considered to be less eco-friendly compared to MBSCs
(Jiang et al. 2013,2014a). Attempts have been made in the material technologies to
overcome the disadvantage of PMCs, by seeking a bio-based approach to build
MBSCs with natural fibres as reinforcements and natural glues as matrices in the
laminate skins with MBCs in the cores, which would render the whole structure
biodegradable. Ecovative Design, along with the Center for Automation Technolo-
gies and Systems (CATS) at Rensselaer Polytechnic Institute, pioneered research on
this tech-front (Jiang et al. 2013,2014a). In a series of studies by Jiang et al. (2013,
2014a,b,2016,2017a,b,2019), a seven-step protocol was proposed to manufacture
MBC laminate and MBSC structures with several process optimization stages.
For mycelium-based cores, different textile fabrics with natural fibres are being
tested as a natural fibre reinforcement for the laminate (Jiang et al. 2013). The fabric
can first be cut into the required ply shape using cutting dies, an industrial clicker
press, laser cutters, or hand cutting. The laminate can be made with a single ply of the
fabric or with multiple plies. The cut fabric plies should then be impregnated with a
natural glue; for this, Jiang et al. used a pinch roller impregnator method (Jiang et al.
2013,2014a,2017b,2019), and dip-and-soak, spray coating, and curtain coating
were also tested (Jiang et al. 2013). Corn starch mixed with maltodextrin was used as
the natural reinforcement glue, which acts as a matrix for binding the fabric fibres
(Jiang et al. 2014a). This skin preform is next moulded to give it a defined shape and
then sterilized before the core material is introduced. Moulding and sterilization
were carried out simultaneously, using a matched mould-forming method in which
the mould was heated, thus activating the glue and stiffening the preform at the same
time while creating a gap between the two skin preforms to fill in the core material
(Jiang et al. 2013,2014a).
The selection of the lightweight substrate/filler for the core and the fungal strain
as the binder takes place in the same way as in manufacturing basic MBCs. Loose,
pre-colonized core materials are introduced between the laminates, and the incuba-
tion and mycelium inactivation steps are similar to basic MBC production protocols.
Table 27.1 depicts the main components of the sandwich structures used in a few
selected studies. During incubation, the mycelium colonizes the reinforcement fibres
and core fillers, firmly binding the two skins to the core and forming a unitized
sandwich composite within 5–7 days. In addition to the filler material, the impreg-
nated natural glues in the skins also act as a substrate for the colonizing mycelium
(Jiang et al. 2014a).
Jiang et al. (2014b,2019), further experimented upon infusing bio-resins
(soy-based polyols) into the material, thus forming MBSCs, in order to further
increase their strength, stiffness, and other material properties. Compared with a
resin-less MBSC beam, the resin-infused beams had shown better core shear ulti-
mate stress, core shear yield stress, skin ultimate stress, and flexural strengths.
Ziegler et al. (2016) suggest packaging, shipping, marine flotation, and other
non-structural applications for these sandwich structures due to their comparatively
27 Fungal Mycelium-Based Biocomposites: An Emerging Source of Renewable Materials 543
lower resistance to compression. Moreover, doors, cubicle walls, basement panel-
ling, conventional insulating applications, roof insulation, and table tops were also
suggested as potential applications (Bayer et al. 2008).
27.13 MBCs in the Fashion Industry
Textile and fashion industries have rapidly evolved from traditional plant- and
animal-based natural products to synthetic products over las few dacades. The
annual global production of all apparel and textile fibres is estimated at more than
110 million tons (Ütebay et al. 2020). The accumulation of piles of textile waste and
the release of hazardous effluents from fabric industries have caused a major
environmental threat globally. Additionally, growing concerns regarding consumers
rejecting animal leather have also triggered the fashion industries to seek biological
alternatives for their raw materials.
The project MycoTEX/Mycelium Textile by an artist, Aniela Hoitink from the
Netherlands, investigates the use of pure fungal mycelia as material for dresses
(NEFFA 2004–2020). MycoTEX team has extended the project to create biodegrad-
able MycoTEX fabric grown from mycelium which has gained the attention of the
scientific community, fashion industry, as well as the field of contemporary arts and
crafts (Cairns et al. 2019; Goncu-Berk 2019; Nai and Meyer 2016; Meyer 2021). A
flexible composite product called MycoTEX developed by Aniela Hoitink com-
prised of disc-shaped mycelium and placed them on a mannequin to shape the dress,
retaining the flexibility needed without using traditional textile material (Vasquez
and Vega 2019; Nayak et al. 2020).
Table 27.1 Examples of laminate and sandwich biocomposite structures with mycelium-based
cores
Skin/laminate Core substrate Reference(s)
100% jute burlap
(loose-weave)
100% linen cloth
(tight-weave)
Unspecified agricultural waste Jiang et al. (2013,
2014a,2016)
Biotex jute
Biotex flax
BioMid cellulose
Kenaf/hemp mix (50/50% by weight) and corn
Stover/hemp mix (50/50% by weight)
Jiang et al. (2014b,
2017a,2019)
Recycled jute
Recycled cotton
Cornstarch
Sawdust spawn block mixture with
Flour, feathers, and psyllium husk
Silverman (2018)
Unspecified woven/
non-woven mat
Cotton by-product (ginning waste) and hemp Ziegler et al.
(2016)
Biotex jute (plain
weave)
Biotex flax twill
Kenaf/hemp Jiang et al. (2017b)
544 D. Udayanga and S. D. Miriyagalla
The specific application tested by Jiang et al. was a benchmark product of
Ecovative, i.e. a shoe sole for an outdoor sandal (Jiang et al. 2014a,2017b). Cost
modelling was conducted as well to minimize the cost and maximize the efficiency
in the optimization of the manufacturing system of this product (Jiang et al. 2016).
Mycelium-based cores were also tested for another shoe sole application, in which
the design was an incomplete sandwich with one skin and the core (Silverman 2018).
Its applicability was suggested for the sole of a casual shoe/sandal with less
compressive force given by the wearer. Mushroom MBCs derived from reishi,
oyster, king oyster, and yellow oyster with or without a natural fabric mat have
been tested as a potential source for footwear products (Silverman et al. 2020).
Among them, the material made from king oyster mycelium provided higher
compressive strength when compared to other mushroom species tested in the
same study.
27.14 Future Directions of MBC Research
Although both tropical and temperate species of mushroom-forming fungi have been
used in research and start-ups related to MBC production, only a few species of the
fungi kingdom have been tested and developed into viable products thus far.
Therefore, new fungi from different ecological niches can be explored for the
potential use of mycelium-based product discovery and development (Attias et al.
2019). Despite the approximately similar chemical composition of mycelium com-
pared to diverse groups of fungi, the physical and mechanical properties of myce-
lium can vary among species due to their growth characteristics; nature of
substratum; growth conditions such as temperature, relative humidity, etc.; and
processing techniques (Jones et al. 2019). The genetic and environmental factors
affecting the chemical composition and mechanical properties of mycelium have to
be investigated in detail in order to improve the productivity and quality of the
MBCs (Jones et al. 2018a). Fungi growing under mesophilic conditions which also
produce dense mycelium are ideal for high-quality MBC-based products. Improve-
ments in the mechanical performance of the mycelium have been challenging in
most MBC research in the past decade. The deletion of the hydrophobin gene from a
strain of Schizophyllum commune has resulted in a remarkable increase in the
mechanical properties of mycelium material, such as the Young’s modulus and
maximum tensile strength (Appels et al. 2018; van Wetter et al. 1996; Momenteller
2017). Therefore, an approach based on genetic manipulations as well as rigorous
optimization of environmental factors could be used to improve the properties of
mycelium, thus enhancing the efficiency and cost of production. In addition, the
incorporation of material engineering and nanotechnology in MBC research could
result into major tech breakthroughs in developing healthy, renewable materials that
may eventually resolve many prevailing environmental issues.
27 Fungal Mycelium-Based Biocomposites: An Emerging Source of Renewable Materials 545
27.15 Concluding Remarks
Globally, the “fungal mycelium-based materials”is a topic that experiences a surge
in popularity among researchers working on biocomposites and environmental
sustainability. MBCs are significant, in terms of their capability to attract scientists,
investors, and policy-makers looking forward to overcome challenges in global
environmental issues. This is because MBCs and their variants are eco-friendly
alternatives for various polystyrene foams and similar products that cause a serious
environmental crisis upon large-scale disposal to the environment. Furthermore,
possible rewards of using renewable sources of materials are highly complementary
with the goals of the 2030 agenda for sustainable development adopted by the United
Nations. Therefore, we encourage more research and innovative projects based on
MBCs worldwide, through multidisciplinary collaborations among scientists, tech-
nologists, and prospective investors from various industries.
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550 D. Udayanga and S. D. Miriyagalla
Chapter 28
An Endophytic Bacterial Approach: A Key
Regulator of Drought Stress Tolerance
in Plants
Sudha Bind, Sandhya Bind, and Dinesh Chandra
Abstract Drought is the chief limiting factor for crop productivity around the
world. The continuous rise in temperature due to global climate change leads to
scarcity of water which directly enhances drought frequency. One eco-friendly and
safer approach is to utilize the endophytic bacteria as biofertilizers for agriculture
productivity and sustainability. Plant-endophytic bacteria improve plant health
through three types of interaction; they may be symbiotic, mutualistic and
commensalistic. Endophytic bacteria have direct and indirect working mechanism
for plant growth promotion. Direct mechanism involves making nutrient accessible
to the plants via fixing nitrogen and solubilizing the phosphate, zinc and potassium,
and siderophore, phytohormone and 1-aminocyclopropane-1-carboxylate (ACC)
deaminase production, whereas indirect mechanism involves production of antibi-
otics, exopolysaccharide and hydrolytic enzymes and competition for nutrient and
space to inhibit the pathogen. Thus, the aim of this chapter is to provide a better
understanding of plant-endophytic bacterial interaction in amelioration of drought
stress and determinants produced by endophytic bacteria beneficial in plant growth
promotion under drought stress.
Keywords Endophytes · ACC deaminase · Drought · Sustainability · Nutrient
uptake
S. Bind · S. Bind
Department of Biological Sciences, CBS&H, G.B. Pant University of Agriculture and
Technology, Pantnagar, Uttarakhand, India
D. Chandra (*)
Department of Biological Sciences, CBS&H, G.B. Pant University of Agriculture and
Technology, Pantnagar, Uttarakhand, India
GIC Chamtola, Almora, Uttarakhand, India
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
P. Bhatt et al. (eds.), Microbial Technology for Sustainable Environment,
https://doi.org/10.1007/978-981-16-3840-4_28
551
28.1 Introduction
Drought is a critical abiotic stress throughout the world and its intensity is increasing
due to climate change and global warming. Drought negatively affects the crop yield
and productivity. According to the studies from 1980 to 2015, drought reduced 40%
yield in maize and 21% in wheat (Daryanto et al. 2016). To provide food to the huge
population of the world, productivity of crop must be enhanced under drought
conditions. Drought can be divided into metrological, agricultural and hydrological
drought (Dai 2011). A period of months to years with below normal precipitation is
called metrological drought. In metrological drought, the temperature remains above
the normal temperature, also caused by high temperature and pressure. Hydrological
drought occurs because of low water content in rivers, streams, lakes and ground
water after the long persistence of metrological drought (Van Loon 2015). Drought
disturbs the water potential and turgor of the plant and thus interferes with normal
physiological functioning (Hsiao 2000).
28.2 Responses of Drought in Plant
Drought stress causes reduction in diffusion and supply of nutrients such as nitrate,
calcium, sulphate, magnesium and silicon (Selvakumar et al. 2012). Drought also
increases oxidative stress in plant by generating high quantity of reactive oxygen
species (ROS) such as superoxide radical, hydrogen peroxide and hydroxyl radical
(Chandra et al. 2019a,b). A high concentration of ROS induces destruction of the
lipid, nucleic acid and protein in cells (Sgherri et al. 2000). Under drought stress,
chlorophyll concentration decreases due to photo-oxidation (Rahdari et al. 2012;
Chandra et al. 2018a,b). Drought negatively affects the nitrate reductase function-
ality and thus leads to lower uptake of nitrate from the soil (Caravaca et al. 2005).
Ethylene concentration also increased under drought condition which inhibits plant
growth (Ali et al. 2014).
28.3 Mechanism of Drought Tolerance in Plants
Plants resist drought by four mechanisms: recovery, tolerance, avoidance and
escape. Drought tolerance is the capability of plants to withstand severe dehydration
via osmotic regulation and osmo-protectants. Various methods are being used to
increase the plant tolerance towards drought stress for higher crop production.
Methods being currently used in agriculture are non-renewable and expensive
(Ullah et al. 2019; Chandra et al. 2020). Excessive usage of chemicals as fertilizers
and pesticides creates environmental pollution and generation of greenhouse gases
that negatively affected the soil property, thus causing a hazardous effect on human
552 S. Bind et al.
health (Sulbhi et al. 2021; Bhandari et al. 2021; Bhatt et al. 2020a,b,
2021a,b,c,d,e,f). These xenobiotics enter into the food chain via the process
known as biomagnification (Bhatt et al. 2019a,2020c,d,e,f). Conventional
breeding and genetic engineering were used for the production of drought-tolerant
and high-yielding varieties of various plants (Ngumbi and Kloepper 2016). Con-
ventional breeding has some drawbacks; it requires a lot of labour and time and loses
other useful traits and breeding can be granted one crop at one time (Philippot et al.
2013). On the other hand, genetic engineering is faster but the development of
genetically modified crops brings about time and labour challenges until commer-
cialization (Ullah et al. 2019; Eisenstein 2013). Moreover, due to response of
consumers to the genetically modified crop which varies among various countries,
the success of transgenic crops on the market cannot be guaranteed (Fedoroff et al.
2010). Recently, plant-endophytic bacterial interaction receives much attention
because of their plant growth enhancement ability under drought condition (Finkel
et al. 2017).
28.4 Endophytic Bacteria: A Sustainable Approach
to Reduce Drought Stress
Endophytic bacteria are considered as beneficial bacteria which live inside the plant
tissue without any harmful effect. Plant-endophytic bacterial interaction may be
symbiotic, mutualistic and commensalistic. Endophytic bacteria are present in
almost all the parts of plants; they are isolated and characterized from different
types of plant and plant parts (such as seeds, roots, leaves, stems, fruits, etc.). Roots
contain the highest population of endophytic bacteria as compared to the other
aboveground plant parts (Rosenblueth and Martínez-Romero 2006). Endophytic
bacteria have a broad host range, making them a powerful tool for sustainable
agriculture. Endophytic bacteria present in all plants are studied till now, and plants
without endophytic bacteria are rare exception in nature (Partida-Martinez and Heil
2011). Plants without endophytic bacteria are more susceptible against phytopatho-
gens and stress conditions (Timmusk et al. 2011). Endophytic bacteria having ACC
deaminase confer resistance against various types of abiotic stress (high temperature,
drought, salinity, flood, acidity) by reducing the level of ethylene inside the plant
tissues. They also break down ACC, which is a precursor of ethylene, into
α-ketobutyrate and ammonia, thus eliminating the negative impact of high ethylene
concentration on plant growth. Numerous studies on drought tolerance in plants
mediated by endophytic bacteria are summarized in Table 28.1.
28 An Endophytic Bacterial Approach: A Key Regulator of Drought Stress... 553
Table 28.1 Drought tolerance in plants by endophytic bacteria
Endophytic bacteria
Benefitted
plant
Source of plant
species Effect of bacterial endophytes on plant References
Bacillus sp. (Acb9), Providencia sp. (Acb11),
Staphylococcus sp. (Acb12)
Staphylococcus sp. (Acb13) and Staphylococ-
cus sp. (Acb14)
Ananas
comosus
Leaf IAA, ACC production, nitrogen fixation Jayakumar
et al. (2020)
Staphylococcus sp. Ceb1 Curcuma
longa
Rhizome IAA production
Bacillus megaterium HX-2 Agastache
rugosa
Leaf IAA production, phosphate solubilization Li et al. (2019)
Bacillus cereus,B. amyloliquefaciens Fagonia
mollis
Leaf IAA production, phosphate solubilization, cellu-
lase, gelatinase, pectinase, xylanase
ALKahtani
et al. (2020)
Brevibacillus agri Achillea
fragrantissima
Leaf Ammonia, IAA production
Achromobacter xylosoxidans Helianthus
annuus
Root IAA, jasmonic acid, 12-oxo-phytodienoic acid,
ABA, phosphate solubilization
Forchetti et al.
(2007)
Enterobacter cloacae EB-48, Microbacterium
sp. EB-65
Sorghum
bicolor
Root P-solubilization, siderophores, IAA production,
high expression of sbP5CS 2 gene, N-fixation,
ACC deaminase production
Govindasamy
et al. (2020)
Bacillus subtilis,Paenibacillus illinoinensis Capsicum
annuum
Root Phosphate solubilization, siderophore production,
overexpression of root vacuolar proton pump H+-
PPase (V-PPase)
Vigani et al.
(2019)
Paraburkholderia megapolitana MGT9 Ricinus
communis
Auxin production, phosphate solubilization,
siderophore production enhanced proline, CAT,
SOD, POD and GR antioxidant activity
Trivedi et al.
(2019)
Pseudomonas azotoformans Alyssum
serpyllifolium
Leaf Increased enzymatic activity of POD, SOD, CAT
and proline content
Timmusk
et al. (2014)
Sphingomonas sp. LK11 Tephrosia
apollinea
Leaf ABA, jasmonic acid production, enhanced proline,
glycine, glutathione and glutamine content
Khan et al.
(2014)
554 S. Bind et al.
Bacillus amyloliquefaciens SB-9 Vitis vinifera Root Melatonin, tryptamine, 5-hydroxytryptophan, sero-
tonin and N-acetylserotonin production
Jiao et al.
(2016)
Bacillus subtilis B26 Panicum
virgatum
Leaf Phosphate solubilization, cellulase production Gagne-
Bourgue et al.
(2013)
Pseudomonas frederickbergensis Phoenix
dactylifera
Root Siderophore, auxin production and phosphate
solubilization
Cherif et al.
(2015)
Pantoea alhagi Alhagi
sparsifolia
Leaf IAA, siderophore, EPS, protease, ammonia pro-
duction, phosphate solubilization
Chen et al.
(2017)
B. amyloliquefaciens Euphorbia
trigona
Stem, root EPS production, phosphate solubilization Eke et al.
(2019)
Bacillus licheniformis Lepidium
perfoliatum
Root Catalase, protease, esterase, amylase, biofilm
formation
Li et al. (2017)
Staphylococcus pasteuri strain MBL_B3,
Bacillus sp. strain MBL_B15, Kocuria sp.strain
MBL_B19
Corchorus
olitorius
Leaf, root, seed
and seedling
Phosphate solubilization, siderophores, IAA and
ACC deaminase production
Haidar et al.
(2018)
Arthrobacter sp.
Bacillus sp. TW4
Capsicum
annum
Stem ACC deaminase, IAA production Sziderics et al.
(2007)
Klebsiella sp. Alhagi
sparsifolia
Root IAA, protease, ammonia, acetoin, 2,3-butanediol
production, nitrogen fixation
Zhang et al.
(2017)
Pseudomonas azotoformans ASS1 Trifolium
arvense
Leaves of Alys-
sum
serpyllifolium
Enhanced chlorophyll content; increased Cu, Zn
and Ni uptake; and also enhanced proline, antioxi-
dant enzymes, decreased MDA content
Ma et al.
(2017)
Pseudomonas plecoglossicida Vitis vinifera Root 1AA, siderophore, ammonia production, phosphate
solubilization
Rolli et al.
(2015)
Pseudomonas poae Astragalus
mongholicus
Root IAA production, drought tolerant Sun et al.
(2019)
Bacillus pseudomycoides,B. subtilis subsp.
inaquosorum
Zea mays Seed IAA, ammonia, protease, lipase, esterase, amylase,
cellulase, pectinase
Bodhankar
et al. (2017)
(continued)
28 An Endophytic Bacterial Approach: A Key Regulator of Drought Stress... 555
Table 28.1 (continued)
Endophytic bacteria
Benefitted
plant
Source of plant
species Effect of bacterial endophytes on plant References
Bacillus subtilis WL19, B. thuringiensis WS11 Triticum
aestivum
Leaf, stem IAA, catalase, oxidase, chitinase, HCN and EPS
production, phosphate solubilization, carbonic
anhydrase activity
Aslam et al.
(2018)
Xanthomonas sacchari,Bacillus idriensis,
Pseudomonas geniculate
Dodonaea
viscosa
Root Ammonia, HCN, IAA production phosphate solu-
bilization, protease, pectinase, cellulase, chitinase
Afzal et al.
(2017)
Bacillus subtilis LK14 Moringa
peregrina
Bark IAA production, ACC deaminase, phosphate
solubilization
Latif Khan
et al. (2016)
Pseudomonas putida Zingiber
officinale
Rhizome Siderophore, IAA, and ACC deaminase, production Jasim et al.
(2014)
556 S. Bind et al.
28.5 Endophytic Bacteria Against Drought Stress
Drought stress is most critical among all abiotic stresses. The interaction of plant and
endophytic bacteria increased the tolerance or resistance towards drought stress
(Paul and Lade 2014). Plant-microbe interaction is very effective in plant tolerance
towards drought stress. Endophytic bacteria represent the subclass of rhizospheric
bacteria having multiple traits of plant growth promotion (Bhatt et al. 2015a,b,
2016a,b,2019b,c). These bacteria are better over rhizospheric bacteria in plant
growth promotion because they are having a close association with plant tissue and
exert a direct beneficial effect to plant growth promotion and development under
drought condition (Cooper et al. 2014). Plant shows various morphological, phys-
iological, biochemical and molecular responses towards drought stress, and endo-
phytic bacteria help in the modulation of these responses to improve their tolerance
capacity under drought stress. Endophytes increase stress-responsive gene expres-
sion, osmolytes and antistress metabolite during stress condition (Lata et al. 2018),
thus enhancing plant tolerance and plant growth under drought stress. Endophytic
bacteria increase plant growth and development by direct and indirect mechanism.
Direct mechanism includes making nutrients available to plant by nitrogen fixation;
phosphatesolubilization, zinc solubilization and potassium solubilization; and pro-
duction of phytohormones, siderophore and 1-aminocyclopropane-1-carboxylate
(ACC) deaminase, and indirect mechanism includes the production of antibiotics,
exopolysaccharide, hydrolytic enzymes, and competition for nutrient and space to
inhibit the pathogen. These mechanisms enhance the plant growth and thus help to
cope with different kinds of biotic and abiotic stresses.
28.5.1 Direct Mechanism of Plant Growth Promotion by
Endophytic Bacteria
Endophytic bacteria improve plant growth with direct mechanism by making the
nutrients available (phosphate, potassium and zinc solubilization, nitrogen fixation,
siderophore production), production of phytohormone (IAA, gibberellin, cytokinin,
etc.) and ACC deaminase activity.
28.5.1.1 Nutrient Availability
Soil usually contains insufficient amount of nutrients which are crucial for plant
growth. Some of the nutrients present in the soil are not easily available to the plants.
Endophytic bacteria increase the availability of the limiting nutrients (N, P, Fe) for
better growth of plants.
28 An Endophytic Bacterial Approach: A Key Regulator of Drought Stress... 557
28.5.1.2 Nitrogen
Most of nitrogen in the atmosphere is present in unobtainable form. Endophytic
bacteria help in increasing the nitrogen availability for the plants. They fix the
atmospheric nitrogen with the help of nitrogenase enzyme. Nitrogenase is a complex
enzyme consisting of dinitrogenase and dinitrogenase reductase, which helps in N
2
fixation. Some N
2
-fixing endophytic bacteria are Azospirillum brasilense,
Burkholderia spp., Klebsiella oxytoca,Azoarcus spp., Gluconobacter,Enterobacter
cloacae,Klebsiella pneumoniae and Pantoea sp. and Herbaspirillum.
28.5.1.3 Phosphorus
Phosphorus is the chief micronutrient for various enzymatic reactions that is respon-
sible for many physiological processes of the plant. It is present in large quantity but
in insoluble form which is unavailable for the plants. Seventy-five per cent of
phosphorus applied as fertilizers makes the complex with soil and becomes inacces-
sible to the plant (Ezawa et al. 2002). Endophytic bacteria solubilize the insoluble
phosphate into soluble form by chelation, organic acid production, acidification and
ion exchange (Nautiyal et al. 2000) and also produce the acid phosphatase for
mineralization of organic phosphate. Some phosphate-solubilizing endophytic bac-
teria are Bacillus methylotrophicus,B. megaterium,B. amyloliquefaciens,
B. subtilis,Acinetobacter sp., Pseudomonas spp., Enterobacter asburiae, and
Pantoea spp.
28.5.1.4 Iron
Iron is a crucial element in the entire living organism and is involved in various
important functions such as DNA synthesis, respiration and photosynthesis. It is a
chief content of chlorophyll and essential for maintenance and function of chloro-
plast. In normal state iron is found in Fe
3+
state which is insoluble and cannot be
absorbed by plant. Endophytic bacteria produce siderophore (low-molecular-weight
iron-chelating compound). Siderophore provides iron to the plant under iron-
limiting condition (Ma et al. 2011). Some endophytic bacteria for siderophore
production are Pseudomonas,Bacillus,Azotobacter,Enterobacter,
Methylobacteria,Rhizobium,Burkholderia, etc.
28.5.1.5 Phytohormone Production
Endophytic bacteria increase the growth of plant by accumulating nutrient and
metabolism of the host plant by producing various phytohormones. Five main
phytohormones produced by endophytic bacteria are auxin, gibberellin, abscisic
558 S. Bind et al.
acid, cytokinin and ethylene. IAA is the chief plant hormone produced by endo-
phytic bacteria.
28.5.1.5.1 IAA Production
IAA is a significant plant hormone having a key role in various plant physiological
processes. It plays a chief role in growth and development of plant that includes cell-
cell signalling, cell division and elongation and tissue differentiation. Other roles of
IAA are initiation of adventitious and lateral root formation, response to light and
gravity, apical dominance, mediation of resistance to plant stress and induction of
plant defence. IAA can also modulate the synthesis of ethylene (Woodward and
Bartel 2005; Glick 2012). Production of IAA by endophytic bacteria resulted in
increased root biomass and lateral root in the host plant (Tsavkelova et al. 2007;
Taghavi et al. 2009). According to the study of Lata et al. (2006), Pseudomonas
stutzeri P3 strain can produce IAA. Etminani et al. (2018) found that endophytic
bacteria Bacillus pumilus and Pseudomonas protegens isolated from wild pistachio
tree have potential of IAA production. According to the study of Patten and Glick
(2002), Pseudomonas putida GR12-2 defective in IAA synthesis is not able to
increase the root growth and lateral root formation, indicating the role of IAA in
lateral root formation.
28.5.1.5.2 Cytokinin
Cytokinin has the main role in plant growth, physiology and development. It is a
plant growth regulator playing an important role in apical dominance, seed germi-
nation, leaf senescence and flower and fruit development. Cytokinin also enhances
the resistance against various plant pathogens (fungi, bacteria, pest and insect).
Higher concentration of cytokinin in plants increases the resistance towards the
pathogen. Endophytic bacteria Pseudomonas resinovorans and Paenibacillus
polymyxa isolated from Gynura procumbens have the ability to produce cytokinin
(Bhore et al. 2010).
28.5.1.5.3 Gibberellin
Gibberellins are diterpenoid compound, having an important role in growth and
development of plant which includes stem elongation, seed germination, flowering
fruiting and seed dormancy. According to Gutierrez-Manero et al. (2001), GA1,
GA3, GA4 and GA20 were detected in liquid media of Bacillus pumilus and Bacillus
licheniformis. Forchetti et al. (2007) and Eke et al. (2019) studied the production of
GA in the culture media of Bacillus pumilus and Achromobacter xylosoxidans.
According to Cohen et al. (2009), Azospirillum lipoferum-treated maize plant
increased the GA under drought condition. According to the study of Khan et al.
28 An Endophytic Bacterial Approach: A Key Regulator of Drought Stress... 559
(2014)Sphingomonas sp. LK11 isolated from Tephrosia apollinea leaves produced
the GA4, GA9 and GA20. Piccoli et al. (2011) isolated Arthrobacter from Prosopis
strombulifera root and they showed GA1 and GA3 production.
28.5.1.5.4 ABA Production
ABA is an important plant hormone regulating plant growth, development and stress
responses. ABA plays a significant role in various physiological processes of plants
including stomatal closure, leaf senescence, bud dormancy, seed germination, cutic-
ular wax deposition and osmotic regulation. According to a study of Shahzad et al.
(2017), endophytic bacteria Bacillus amyloliquefaciens RWL-1 isolated from the
seed of rice produced ABA under saline condition. Bacillus pumilus isolated from
sunflower produced ABA under drought condition (Eke et al. 2019). According to
Cohen et al. (Bhore et al. 2010) inoculation of maize with Azospirillum lipoferum
increased the ABA content under drought condition. Endopyhtic bacteria
Curtobacterium spp. SAK1 when inoculated to soybean plant under salt stress
increased the ABA content (Khan et al. 2019).
28.5.1.6 Control of Ethylene Level via ACC Deaminase
Ethylene is a small organic molecule with biological activity and acts as a plant
growth regulator at very low concentration. It is associated with various biological
and developmental processes, e.g. fruit ripening, senescence of leaf, root initiation
and nodulation, abscission, auxin transport and cell elongation (Sun et al. 2016).
During plant growth and development, the concentration of ethylene remains low,
but it enhanced during senescence and fruit ripening. Ethylene concentration
increased due to various biotic (mechanical wounding, disease, pest) and abiotic
(temperature extreme, water stress, UV light) stress and inhibits the elongation of
root, nodulation of legumes, lateral root and root hair formation, defoliation, leaf
senescence, destruction of chlorophyll, leaf abscission and epinasty. Regulation of
ethylene concentration is necessary for proper plant growth.
Although endophytic bacteria use different mechanisms of plant growth, the
possession of ACC deaminase activity is a key trait of endophytic bacteria for
enhancement of plant growth in drought stress. ACC deaminase reduces the ethylene
level by breaking the ACC, the precursor of ethylene. ACC deaminase-possessing
bacteria use ACC as a nitrogen source, bind to the surface of the root and degrade the
ACC by cleavage of ACC into ammonia and α-ketobutyrate (Woodward and Bartel
2005). Thus, by utilizing the ACC deaminase activity, endophytic bacteria alleviate
the stress effect and improve plant growth and development. ACC deaminase is a
pyridoxal phosphate-dependent enzyme, induced in the presence of its substrate
ACC. ACC deaminase induction is complex and slow process. Activity of ACC
deaminase gradually decreases after few hours of induction (Jacobson et al. 1994).
According to the model for functioning of bacterial ACC deaminase proposed by
560 S. Bind et al.
Glick et al. (1998), it has been revealed that an appropriate amount of ACC is
released by the plant’s root and taken up by the bacteria, and ACC deaminase
activity of microbes breaks this ACC into ammonia and α-ketobutyrate. Uptake
and breakdown of ACC by bacteria decreases the ACC level outside the plant root,
thus maintaining the equilibrium between external and internal environments
through exudation of ACC from inside to the rhizosphere. Thus, the maintenance
of low ACC level, to reduce the biosynthesis of ethylene, is important for plant
growth and development.
28.5.1.7 Indirect Mechanism of Plant Growth by Endophytic Bacteria
Endophytic bacteria by producing various compounds, e.g., antibiotics, volatile
compound, HCN, siderophore, hydrolytic enzymes and toxins, inhibit the growth
of phytopathogen, thus increasing the growth of plant indirectly. Endophytic bacte-
ria increase the resistance of plant against pathogen by producing secondary metab-
olites including alkaloids, phenolics, terpenoids, steroids, quinines and peptides
(Yu et al. 2010). Endophytic bacteria inhibit growth and activity of both fungal
and bacterial plant pathogens. Pseudomonas,Bacillus,Enterobacter, Paenibacillus
and Serratia are common genera of endophytic bacteria having antimicrobial activ-
ity. According to the study of Hong-Thao et al. (2016), endophytic bacteria Strep-
tomyces from citrus fruit exhibited antimicrobial activity against Colletotrichum
truncatum,F. oxysporum,F.udum and Geotrichum candidum. Ramesh et al.
(2009) reported in their study that endophytic bacteria Burkholderia cepacia,Pseu-
domonas sp. and Enterobacter cloacae decreased the eggplant wilt by 70% by
inhibiting Ralstonia solanacearum. Liu et al. (2020) studied Bacillus megaterium
isolated from potato tuber and showed its inhibitory effect against Streptomyces
scabies and Erwinia carotovora subsp. Atroseptica. Endophytic bacteria Staphylo-
coccus warneri and Bacillus velezensis isolated from Gnetum gnemon showed
inhibition against Ralstonia solanacearum (Agarwal et al. 2020). Bacillus subtilis
isolated from sugarcane inhibited the growth of red rot causing Colletotrichum
falcatum (Shastri et al. 2020; Bhatt et al. 2019d,e; Sharma and Bhatt 2016; Sharma
et al. 2016; Bhatt and Nailwal 2018; Khati et al. 2018a; Gangola et al. 2018a; Bhatt
2018; Bhatt and Barh 2018; Bhandari and Bhatt 2020; Bhatt and Bhatt 2021).
Jayakumar et al. (2020) isolated Bacillus axarquiensis and Bacillus licheniformis
from sugarcane and showed their antagonistic effect against Colletotrichum
falcatum. Thus, there is an immense need of eco-friendly strategies in agriculture
for crop production under abiotic stresses (Singh et al. 2021; Zhang et al. 2020a,b;
Mishra et al. 2020; Feng et al. 2020; Lin et al. 2020; Zhan et al. 2020; Ye et al. 2019;
Huang et al. 2019,2020). Utilization of endophytes as biofertilizer is an eco-friendly
approach for sustainable agriculture (Huang et al. 2020; Fan et al. 2020; Pang et al.
2020; Gangola et al. 2018b; Gupta et al. 2018; Khati et al. 2018b). Indigenous
microbial strains are able to make the environment sustainable. Both bacteria and
28 An Endophytic Bacterial Approach: A Key Regulator of Drought Stress... 561
fungi played a direct role in resource recovery of the agricultural fields via various
mechanisms (Khati et al. 2017a,b,2018b; Kumar et al. 2017).
28.6 Conclusion and Future Prospects
Drought stress affects the growth and productivity in all crop varieties. However,
tolerance towards drought varies according to different species. Endophytic bacteria
play a major role to adopt and resist drought stress in plants and thus can be a future
tool for solving the future food security. Plant and endophytic bacteria interaction
increases the growth and development of plant via direct and indirect mechanisms
(Fig. 28.1). Direct mechanism includes enhancing nutrient availability, production
of siderophore, regulation of phytohormones and accumulation of several compat-
ible solutes/osmolytes, whereas indirect mechanism increases plant growth by acting
against the phytopathogen by producing antibiotic, lytic enzymes and HCN and
making nutrients unavailable for phytopathogen. Various approaches have been
used for enhancement of drought tolerance in different varieties of plants. The future
research needs to develop an effective microbial formulation for improving plant
productivity under drought conditions. Understanding the molecular level of inter-
action between plant and endophytic bacteria is also necessary for better crop yield.
Fig. 28.1 Conceptual diagram showing different mechanisms of plant growth promotion by
endophytic bacteria
562 S. Bind et al.
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