(PDF) Use of Genetic Engineering Approach in Bioremediation of Wastewater

Maulin P. Shah Editor

Modern

Approaches

in Waste

Bioremediation

Environmental Microbiology

Modern Approaches in Waste Bioremediation

Maulin P. Shah

Editor

Modern Approaches in Waste

Bioremediation

Environmental Microbiology

Editor

Maulin P. Shah

Environmental Microbiology Laboratory

Bharuch, Gujarat, India

ISBN 978-3-031-24085-0 ISBN 978-3-031-24086-7 (eBook)

https://doi.org/10.1007/978-3-031-24086-7

Switzerland AG 2023

This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether

the whole or part of the material is concerned, speciﬁcally the rights of translation, reprinting, reuse

of illustrations, recitation, broadcasting, reproduction on microﬁlms or in any other physical way, and

transmission or information storage and retrieval, electronic adaptation, computer software, or by similar

or dissimilar methodology now known or hereafter developed.

The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication

does not imply, even in the absence of a speciﬁc statement, that such names are exempt from the relevant

protective laws and regulations and therefore free for general use.

The publisher, the authors, and the editors are safe to assume that the advice and information in this book

are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or

the editors give a warranty, expressed or implied, with respect to the material contained herein or for any

errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional

claims in published maps and institutional afﬁliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG

The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Much technological advancement has already been made to check the limits but

still there is a long way to go. Integrated Bioremediation Technologies in Industrial

waste treatment is the most effective innovative technology that uses live naturally

occurring microorganisms to degrade environmental pollutants or to prevent contam-

ination. Microorganisms like bacteria, algae, fungi, and yeast carry the ability to

utilize dye as their sole source of carbon and nitrogen thus playing a signiﬁcant

role in bioremediation. This is a multidisciplinary approach, but its central focus

depends on microbiology. This technology includes several techniques such as bio-

stimulation, bio-generation, bioaccumulation, biosorption, physical correction, and

rhyming-emission.

The aim of this book is to describe the limitations and challenges associated with

some generally accepted bioremediation strategies and evaluate the possible applica-

tions of these corrective strategies to eliminate toxic pollutants from the environment

through integrated technologies in Industrial wastewater treatment. Remediation of

polluted sites by the means of microbial process (bioremediation) has been estab-

lished effective and dependable due to its environmentally friendly characteristics.

The use of microorganisms, plants, or microbial or plant enzymes to remove contam-

inants in the soil and other environments is known as bioremediation. Biodegrada-

tion refers to the part of, and sometimes total, transformation or detoxiﬁcation of

contaminants.

Mineralization is a more qualiﬁed term for the absolute conversion of an organic

contaminant to its inorganic constituents by a single species or a consortium of

microorganisms. Co-metabolism is one more restrictive term for referring to the

transformation of a contaminant devoid of the provision of carbon or energy for

the degrading microorganisms. The process of bioremediation increases the rate of

the natural microbial degradation of contaminants by providing the native microor-

ganisms (bacteria or fungi) with nutrients, carbon sources, or electron donors (bio

stimulation and bio restoration) or by adding up an enriched culture of microorgan-

isms. This helps the microbes to develop speciﬁc characteristics that allow them

to degrade the desired contaminant at a faster rate (bio augmentation). It helps to

bring the level within limit as set by regulatory agencies or, ideally, to entirely

v

vi Preface

mineralize organic pollutants to carbon dioxide. This process depends on invigo-

rating the growth of certain microbes that use contaminants like oil, solvents, and

pesticides as a source of food and energy. These microbes devour the contaminants,

converting them into small amounts of water and harmless gases like carbon dioxide.

Successful bioremediation involves combination of the right temperature, nutrients,

and food. If not provided, it may take much longer for the clean-up of contami-

nants. If conditions are not encouraging for bioremediation, they can be improved by

supplementation of “amendments” to the environment, like molasses, vegetable oil

or simply air. These amendments generate the best conditions for microbes to ﬂourish

and complete the bioremediation process. The new approaches that the book high-

lights are Bio augmentation, Bioﬁlters, Biosparging, Bio stimulation, Bioventing,

Bioreactors, Composting and effective Land farming.

The book will picture new aspects of bioremediation that are often tailored to

the requirements of the polluted site in question and therefore the speciﬁc microbes

needed to interrupt down the pollutant are encouraged by selecting the limiting factor

needed to promote their growth.

Bharuch, India Maulin P. Shah

Contents

Emerging Pollutants from the Industries and Their Treatment ......... 1

A. Inobeme, A. I. Ajai, C. O. Adetunji, J. Inobeme, M. A. Adekoya,

M. Maliki, B. I. Onyeachu, T. Kelani, C. A. Eziukwu, and S. Okonkwo

Bioremediation: The Remedy to Expanding Pollution ................. 13

Shreya Anand and Padmini Padmanabhan

Exploration of Plant Growth Promoting Rhizobacteria

(PGPRs) for Heavy Metal Bioremediation and Environmental

Sustainability: Recent Advances and Future Prospects ................ 29

Sumita Mondal, Samir Kumar Mukherjee, and Sk Tofajjen Hossain

Decontamination Strategies and Technologies for Tackling

COVID-19 Hospitals and Related Biomedical Waste .................. 57

Rishav Sharma, Pinakiranjan Chakraborty, and Shraman Roy Barman

Bioremediation of Chlorinated Compounds .......................... 101

Abel Inobeme, Charles Oluwaseun Adetunji, Mathew John Tsado,

Alexander Ikechukwu Ajai, Jonathan Inobeme,

and Bamigboye Oyedolapo

Removal of Heavy Metals Using Bio-remedial Techniques ............. 117

John Tsado Mathew, Charles Oluwaseun Adetunji, Abel Inobeme,

Musah Monday, Yakubu Azeh, Abdulfatai Aideye Otori,

Elijah Yanda Shaba, Amos Mamman, and Tanko Ezekiel

Municipal Wastewater as Potential Bio-reﬁnery ...................... 131

Shipra Jha and Nahid Siddiqui

Phytoremediation of Metals and Radionuclides ....................... 151

Kanchan Soni, P. Priyadharsini, S. S. Dawn, N. Nirmala, A. Santhosh,

Bagaria Ashima, and J. Arun

vii

viii Contents

Anaerobic Biotechnology: Implementations and New Advances ........ 165

Samir I. Gadow, Hatem Hussein, Abdelhadi A. Abdelhadi,

and Abd El-Latif Hesham

Remediation of Soil Contaminated with Heavy Metals

by Immobilization with Organic and Inorganic Amendments .......... 181

Izabela Michalak and Jolanta Warchoł

Poly-γ-Glutamic Acid and Its Application in Bioremediation:

A Critical Review .................................................. 211

Valeria Bontà and Cinzia Calvio

Metagenomics Analysis of Extremophiles and Its Potential Use

in Industrial Waste Water Treatment ................................ 227

Ashok Kumar Shettihalli, Saisha Vinjamuri,

S. Divijendra Natha Reddy, Renu Pai, and Prathibha Narayanan

Prospects of Nanobioremediation as a Sustainable

and Eco-Friendly Technology in Separation of Heavy

Metals From Industrial Wastewater ................................. 251

Prathibha Narayanan, S. Divijendra Natha Reddy, and Praphulla Rao

Nanotechnology for Bioremediation of Industrial Wastewater

Treatment ........................................................ 265

Harshala S. Naik, Parvindar M. Sah, Swapnali B. Dhage,

Smita G. Gite, and Rajesh W. Raut

Plant Mediated Nanomaterials: An Overview on Preparation

Strategies, Characterisation, and Their Potential Application

in Remediation of Wastewater ...................................... 299

Neha Kumari, Lakhan Kumar, and Navneeta Bharadvaja

Bacteria and Pollutants ............................................ 339

Sonia Kaura, Akansha Mathur, and Aakanksha Kalra

Bioﬁlms in Porous Media ........................................... 365

Esha Garg, Ajit Varma, and M. S. Smitha

Removal of Heavy Metals from Industrial Wastewater Using

Bioremediation Approach .......................................... 377

Pooja M. Patil, Abhijeet R. Matkar, Vitthal B. Patil, Ranjit Gurav,

and Maruti J. Dhanavade

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery

Wastewater Treatment ............................................. 409

C. Raja, J. Anandkumar, and B. P. Sahariah

Nanobioremediation: A Sustainable Approach for Wastewater

Treatment ........................................................ 429

Sougata Ghosh, Bishwarup Sarkar, and Sirikanjana Thongmee

Contents ix

Bioremediation of Textile Dyes for Sustainable

Environment—A Review ........................................... 447

Rajalakshmi Sridharan and Veena Gayathri Krishnaswamy

Microbial Contamination of Environmental Waters

and Wastewater: Detection Methods and Treatment Technologies ...... 461

José Gonçalves, Israel Díaz, Andrés Torres-Franco, Elisa Rodríguez,

Priscilla Gomes da Silva, João R. Mesquita, Raúl Muñoz,

and Pedro A. Garcia-Encina

Use of Genetic Engineering Approach in Bioremediation

of Wastewater ..................................................... 485

Jutishna Bora, Saqueib Imam, Vardan Vaibhav, and Sumira Malik

Nanotechnology for Bioremediation of Heavy Metals ................. 515

Anu Kumar, Bhanu Krishan, Shivani, Sunny Dhiman,

and Akshita Sharma

Emerging Pollutants from the Industries

and Their Treatment

A. Inobeme, A. I. Ajai, C. O. Adetunji, J. Inobeme, M. A. Adekoya,

M. Maliki, B. I. Onyeachu, T. Kelani, C. A. Eziukwu, and S. Okonkwo

Abstract Environmental pollution due to various anthropogenic activities associ-

ated with industrialization and urbanization are issues of global concern in recent

times. There are numerous Emerging Contaminants (EC) of concern, with the major

one being: personal care products, pesticides, pharmaceuticals, surfactants and other

industrial chemicals. Various methods have been employed for their removal with

some of the methods having their inherent limitations. Bioremediation has been iden-

tiﬁed a reliable solution for the problems associated with emerging pollutants. There

are numerous microorganisms which are vital for the remediation of the polluted

environment. Bioremediation is an active process involved during the immobiliza-

tion, eradication and degradation of various groups of toxic substances found within

the environment. Due to the limitations of bioremediation in that it is restricted to only

substances that can be acted upon by microorganisms; other advanced approaches

such as membrane technologies and advanced oxidation process have been devel-

oped. This chapter discusses the various emerging pollutants from industries and

their treatment. It highlights some of the limitations of some of the technologies.

Keywords Anthropogenic activities ·Emerging contaminants ·Bioremediation ·

Advanced oxidation ·Membrane technology

A. Inobeme (B

) · M. Maliki · B. I. Onyeachu · T. Ke la ni · C. A. Eziukwu · S. Okonkwo

Department of Chemistry, Edo State University Uzairue, Okpella, Nigeria

e-mail: inobeme.abel@edouniversity.edu.ng

A. I. Ajai · S. Okonkwo

Department of Chemistry, Federal University of Technology Minna, Minna, Nigeria

C. O. Adetunji · S. Okonkwo

Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Department of

Microbiology, Edo State University Uzairue, Okpella, Nigeria

J. Inobeme

Department of Geography, Ahmadu Bello University Zaria, Zaria, Nigeria

M. A. Adekoya

Department of Physics, Edo State University Uzairue, Okpella, Nigeria

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_1

1

2 A. Inobeme et al.

1 Introduction

Environmental contamination has increased rapidly in recent times as a result of

increasing anthropogenic activities and industrialization. Amongst the various pollu-

tants of concern include nuclear wastes, pesticide residues, insecticides, herbicides,

green house gases and heavy metals. Discharge of contaminants into the environment

occurs primarily from industrial activities. Various approaches have been adopted in

time past for the removal of these toxic contaminants from the environment. Some of

the processes involve complete excavation of the site which is an expensive approach.

Much later, other processes such as venting of soil and vapor extraction emerged and

also showed their inherent limitations such as cost and limitations with regards to

high concentration of pollutants hence an incomplete solution to the identiﬁed prob-

lems. More recently, the occurrence of numerous emerging contaminants has been

reported (Briffa et al. 2020; Manisalidis et al. 2020).

Emerging contaminants (EC) refer to naturally occurring or synthetic compounds

or even any microbe that are not usually monitored in the environment but have

the tendency of resulting to suspected or known adverse impact on the health of

humans or the environment at large. Such substances include surfactants, pharma-

ceuticals, and personal care products which are continually rising in surface water,

ground water, drinking water, food materials and municipal wastewater. Some other

substances belonging to this class are analgesics, hormones, antibiotics, antidia-

betis, antiepileptic and anti-inﬂammatory drugs (Masindi and Muedi 2017). ECs

also has broad meanings and has some other terms which have subtle reﬁnement

in their deﬁnition, like pharmaceuticals, contaminants of emerging concern, as well

as organic wastewater compounds. The compounds tend to be of greater concern

recently because most of them have not yet been fully investigated and some of them

cannot be tested for in municipal waste water. The environment is increasingly being

polluted by numerous emerging contaminants which are released from industrial,

urban, agricultural and other anthropogenic activities (Ofrydopoulou et al. 2022).

This chapter discusses the various emerging pollutants from industries and their

treatment. It highlights some of the limitations of some of the technologies.

2 Technologies for Treatment of Emerging Contaminants

Various approaches have been adopted in the treatment of various waste and contam-

inants released from industrial activities. Some of these methods range from phys-

ical, chemical, biological and more recently the used of various advanced techniques

such as nanotechnology, advanced techniques of oxidations and membrane tech-

nologies. Bioremediation technologies have also been found highly promising in the

remediation of various emerging contaminants (Patel et al. 2019) (Fig. 1).

Emerging Pollutants from the Industries and Their Treatment 3

Treatment of

EC

Advanced Oxidation

techniques

Oxidaon

techniques

Membrane

technology Bioremediaon

technique

Fig. 1 Technologies for treatment of EC

3 Oxidation Techniques

Oxidation involves the transfer of electron from a donor to chemical specie. This

process of electron transfer brings about a change in the oxidation number. Processes

of oxidation that involve the formation of reactive intermediates usually have other

oxidation steps between the oxidizing and reducing agents until the ﬁnal product

which is stable thermodynamically is formed. The oxidation potential is an indication

of the extent to which the oxidizing agent can initiate the process of oxidation and

this is a characteristic feature of each oxidizing agent (Krystynik 2021). Some of

the most common oxidizing agents that have been used in the treatment of industrial

contaminants found in waste water include hydroxyl radicals, ﬂuorine, chlorine and

ozone. There are various parameters that affect the processes of chemical oxidation

which include the content of the contaminants in the given environmental matrix,

the rate of reaction between the contaminants and the oxidizing agent employed.

The oxygen concentration, pH and temperature of the media. Speciﬁc optimum

conditions have been put in place for speciﬁc oxidation reactions for organic and

inorganic pollutants (Ghime and Ghosh 2019; Cuerda-Correa et al. 2020).

4 Advanced Oxidation Processes

These are oxidation processes that involve the formation of various categories of

radicals and other reactive intermediates such as hydroxyl radicals. Some of the

processes of advanced oxidation employed for the treatment of environmental pollu-

tants involve the application of hydrogen peroxides, ozone, and ultraviolent radiation.

Most AOPs processes are dependent on the formation of the hydroxyl and other reac-

tive intermediates in situ. These intermediates are the most active oxidants that can be

adopted in the treatment of various organic contaminants most especially in aqueous

media. The hydroxyl radicals that are formed go through non selective reactions

which involve the degradation of the pollutants and their subsequent transformation

4 A. Inobeme et al.

into simple inorganic materials that are environmentally friendly (Cuerda-Correa

et al. 2019). It has been documented that the processes of advanced oxidation are

capable to reduce the contents of environmental pollutants to extremely low concen-

tration that would not constitute deleterious effects on the environment. Studies

have also reported the potential of AOPs in the treatment of various biodegradable

pollutants such as pesticides, petroleum products, aromatic compounds and volatile

organic compounds in various environmental matrices. Due to the cost of operation,

there is limited commercialization of AOPs in the treatment of contaminants such as

those present in waste water. The use of AOPs has however gain outstanding popu-

larity due to the remarkable oxidation potential hence highly promising in the treat-

ment of organic compounds, inorganic compounds and highly recalcitrant (Krishnan

2017).

Principle of AOP

Various techniques are involved in the use of AOPs in the treatment of pollutants.

These include ozonation, Fenton, photo catalytic process and photo Fenton reactions.

For the efﬁcient removal of various contaminants, the continual generation of the

reactive intermediates throughout the process is necessary; this is usually achieved

through photochemical reactions. The formation of these radicals is achieved through

the integration of various oxidizing agents such as ozone, ferrous salts, UV and

hydrogen peroxides. Speeding up of the processes can also be achieved by the use

of various radiation sources such as electron beams, visible rays, solar, microwaves

and ultrasound (Buthiyappan et al. 2016).

5 Bio-remediation

This is a technique that involves the utilization of microorganism, fungi and plants

in the enhancement of the conversion of contaminants and harmful substances into

harmless substances. This is an approach that is safe, cheap and environmentally

friend. The use of this technique in contaminant remediation attracted public

attention around the 1960s after numerous experimentations had been done using

various samples. Several achievements were documented in the 1970s in the use of

bioremediation which was championed by Robinson George. The microorganisms

that are used rely on environmental contaminants and it brought about the reduction

in the contents of the contaminants. As the microorganism consumes the contam-

inants, the population of microorganism available for the bioremediation reduces

continually. This is so due to the reduction in the quantity of food (contaminants)

those are available for supporting the original number of the bioremediating

microbes (Moenne-Loccoz et al. 2015).

This technique of waste management includes the utilization of various living

organisms for the eradication or neutralization of the contaminants from the polluted

site. It is a technique of treatment which utilizes organisms naturally for the break-

down of the harmful contaminants into simple inorganic materials. The process for the

Emerging Pollutants from the Industries and Their Treatment 5

removal of contaminants is dependent primarily on the nature of the pollutants which

include heavy metals, chlorinated compounds, agrochemicals, pesticides, xenobiotic

compounds, nuclear wastes, dyes, green house gases, and hydrogen gas. Bioremedi-

ation is actively involved in breakdown, eradication, detoxiﬁcation, immobilization

diverse waste substances and physical toxic substances from the environment through

the action of microorganisms (Das et al. 2017).

Microorganisms involved in bioremediation

The microorganisms that are employed in bioremediation of contaminants are

classiﬁed into two main groups.

i. Indigenous microorganism:

These are microorganisms which are already found around the site of the contami-

nants. However for the purpose of stimulating the growth of this group of microor-

ganisms, there is need for sufﬁcient oxygen contents, efﬁcient soil temperature, and

adequate nutrients that are essential for the growth of the microorganisms.

ii. Exogenous microorganisms:

The microorganisms that fall into this group are those that were introduced into the

soil to be remediated externally. The introduction is due to the absence of the neces-

sary biological activities required for the degradation of the contaminants present

in the soil (Talabi and Kayode 2019). Aside the supply of the oxygen and nutrients

required for the survival of the exogenous microorganisms, the conditions of the site

may require some adjustments for ensuring that the microorganism survives in the

new area. It is however worth noting that the types of wastes and conditions of sites

are comparable. There is need to speciﬁcally test each of the particular site that is

to be remediated and thoroughly investigated for the optimization of the outcome of

bioremediation (Azubuike et al. 2016).

There are various factors that determine the peculiar bioremediation technique

which include: the kind of microorganisms present, the conditions of the site and

the amount and toxic effect of the contaminants. Different types of microorganisms

act on speciﬁc types of contaminants and are capable of surviving under varying

conditions (Fig. 2).

Types of bio-remediation

i. Microbial bioremediation

This type of bioremediation relies on the activity of microbes, for the transforma-

tion of toxic contaminants into non harmful forms. This is achieved through the

interactions between the contaminants and the microorganisms bringing about the

compartmentalization, immobilization and concentration of the contaminants.

ii. Phytoremediation

This is a cheap cleanup technology that is driven by solar energy. This involves

the utilization of plants for in situ degradation, elimination or the containment of

6 A. Inobeme et al.

Bioremediation

Techniques

In situ

Bioremediation

Ex situ

Bioremediation

Intrinsic

Engineered

Solid Phase

Slurry phase

Fig. 2 Techniques in bioremediation of EC

the contaminant in soil, sludge, sediments and ground water. There are popular

plants that are specially employed for such purpose. However, if the contents of the

contaminants are high, the plants that is employed for the purpose of the remediation

might die. For efﬁcient bioremediation, there is need for a larger surface area of plant

due to the need for a large surface area.

iii. Mycoremediation

This involves the use of fungi for the purpose of bioremediation. The group most

commonly employed is the mushrooms hence the approach is dependent on the

efﬁciency of the enzymes that are produced by the mushroom during the degradation

of different substrates. Aside the enzymatic form of bioremediation in biosorption,

fungi could also be used. Biosorption is an approach during which contaminants are

taken in by the mushrooms into their mycelium thereby making the mushrooms not

edible. The following are fungi that have been found useful in this regard: Aspergillus,

Pleurotus, and Trichoderma used for the removal of lead, nickel, cadmium, arsenic,

iron, mercury in marine environment (Abatenh et al. 2017).

Techniques of bioremediation

i. In-situ technique

This approach does not need the excavation of the polluted soils; hence it is a cheaper

technique in bioremediation as opposed to ex-situ approach. The polluted soil is

treated within through the use of speciﬁcally selected bioremediation. Even though

this approach is cheaper and generates very few quantity of dust in comparison to

the ex situ approach, it may be difﬁcult and slower to manage. It is the most efﬁcient

on site with soils that are permeable (Sharma 2019).

Emerging Pollutants from the Industries and Their Treatment 7

In aerobic in situ technique, oxygen and nutrient are supplied to the organism that

is involved during the bioremediation. The speciﬁc techniques that are commonly

employed in achievement of adequate supply of oxygen and nutrient are Injection of

hydrogen peroxide and bioventing.

ii. Ex-situ technique

This involves the removal of polluted soil or the pumping of the underground contam-

inated water before the treatment process. This technique is easier, cheaper and has

been successfully adopted for various categories of contaminants (Talabi and Kayode

2019). The ex-situ technique consists:

Slurry phase bioremediation: It is very important when quick remediation is highly

needed. The solid phase bioremediation is comparably easier to operate and needs

more space whereas the cleanup process require more time when compared to the

slurry phase technology.

Application of bioremediation technologies

i. Treatment of oil contaminants: Oil spillage is one of the most common pollution

around countries that have a high reserve of oil. Such pollution brings about the

death of aquatic life. It involves the introduction of bacteria that consume the oil

thereby inducing the depletion of the oil spilled in the water bodies. The process

is usually aided using dispersants (Cordes et al. 2016).

ii. Treatment of estuaries, streams and rivers: This technique is used in the removal

of pollutants such pesticides, fertilizers etc. from water bodies.

iii. Treatment of sewage: Sewage is a mixture of chemicals, and wastes which can

be treated in the process of recycling. This process proffers the most cheapest

and efﬁcient technique.

iv. Compost bioremediation: The removal of pollutants in contaminated soils sites

could also be achieved through the mixing of the composts. The pollutants are

removed by the microbes that are present within the compost. This is a very

efﬁcient method of bioremediation.

v. Bioaugmentation: Bioremediation is paramount for the development of highly

effective decomposers. The development of such decomposers aids the fast

removal of environmental contaminants. This is readily achieved through the

application of genetic engineering manipulations on the natural decomposers

for the production of super-decomposers.

Factors affecting bioremediation

Enzymatic metabolic routes of microorganisms aid the progress of chemical reactions

that aid in the degradation of the contaminants. The microorganisms act on the

contaminants only when they come in contact with the compounds which aid the

generation of nutrients and energy for the nutrient multiplication. The efﬁciency of the

bioremediation is dependent on several factors such as concentration of contaminants,

chemical nature of the contaminants, and their ease of accessibility to the original

8 A. Inobeme et al.

microorganisms. The major factors include the population of the microorganism,

nature of the environment, soil pH, temperature, nutrients and oxygen (Talabi and

Kayode 2019).

i. Biotic factors

These are useful in the breakdown of various organic contaminants by microorgan-

isms having less sufﬁcient sources of carbon, antagonistic association among the

protozoa or microorganisms and bacteriophages. The rate of degradation of this is

mostly dependent on the contents of the pollutants and the quantity of catalyst in the

biochemical process. The primary biological factors include activity of the enzymes,

mutation, and biomass production, horizontal transfer of genes, compositions and

size of the population (Manisalidis et al. 2020).

ii. Abiotic factors

The successful interaction existing between the pollutants and microbes is dependent

on moisture, pH, soil structure, nutrients, oxygen content, water solubility, redox

potential, solubility, toxicity, types and concentration.

The association of the environmental pollutants with metabolic activities, physic-

ochemical properties of the microbes targeted in the process are all vital during the

remediation process. The successful association between the microbes and contami-

nants is affected by temperature, moisture, deﬁciency, oxygen content, physicochem-

ical, concentration, chemical structure, solubility, toxicity and degradation kinetics

(Prasad and Yadav 2022).

Biodegradation of pollutants can take place under a pH range of 6.5–8.5 which is

basically optimal for biodegradation in most terrestrial environment. Moisture affects

the breakdown of pollutants because it is dependent on the nature and quantity of the

soluble components that are accessible together with the osmotic pressure and pH

(Azubuike et al. 2016).

Types of in situ bioremediation

There are two major types of in situ bioremediation technique. These are the

engineered and intrinsic bioremediation.

i. Intrinsic bioremediation

This is also known as the natural reduction process which is an in situ bioreme-

diation technique; it involves a passive remediation of contaminated area with any

external inﬂuence or intervention by humans. The process involves the stimulation

of the local or natural population of microbes. The process is based on anaerobic

and aerobic processes i n the biodegradation of the contaminating constituents that

contain the portions that are recalcitrant. The non availability of the external forces

therefore implies that the approach is not expensive when compared to the other

in situ techniques (Abetenh et al. 2017).

Emerging Pollutants from the Industries and Their Treatment 9

ii. Engineered in situ bioremediation

In this approach, certain microorganisms are introduced into the site of pollution.

The genetically engineered microbes that are employed aid in the acceleration of

the remediation process through the enhancement of the physicochemical factors to

induce the growth of microbes.

iii. Bioventing

In this technique there is controlled stimulation of the ﬂow of air through delivering

of oxygen to the unsaturated area so as to increase the activity of the localized

microorganisms for the bioremediation. The amendments used in this process are

produced through the addition of moisture primarily to enhance the bioremediation

process. Among the various in situ techniques this technique has gain remarkable

attention (Prasad and Yadav 2022).

iv. Bioslurping

In this technique soil vapor extraction, vacuum aided pumping and bioventing are

integrated in achieving ground water and soil remediation through indirect provision

of oxygen and inducing of the pollutant breakdown. This approach is designed for

products recovery. It is suitable for the remediation of soils that are polluted by semi

volatile and volatile organic compounds. Though this approach is not reliable for

less permeable soil remediation, it is however efﬁcient in cost advantage as a result

of lower amount of ground water, treatment, minimization of storage and cost of

disposal (Prasad and Yadav 2022).

v. Biosparging

It is a technique that is close in principle to bioventing. It involves the injection

of air into the contaminated soil sub layer for the improvement of the microbial

degradation thereby inducing the removal of the contaminants within the area. The

efﬁciency of biosparging is dependent on two primary factors which are contaminant

biodegradability and permeability of the soil. This technique has been employed

generally in the treatment of aquifers polluted by kerosene and diesel.

vi. Reactive barrier

It is commonly seen as a physical approach for the remediation of polluted ground

water. The biological phenomena involved are sorption of contaminants after precipi-

tation degradation. It is an in situ approach utilized for the remediation of chlorinated

and heavy metal polluted sites.

Bioremediation has numerous merits. These include its functionalities with

respect to cost when compared to the traditional techniques that involve the general

cleanup of the harmful substance during the process. Bioremediation also makes

it possible to achieve a high removal of the contaminants. This technique does not

10 A. Inobeme et al.

require much effort and can easily be done at the site without altering the usual activ-

ities of the microorganisms. This therefore saves the cost of conveying the waste off

area and the potential threats to the environment and human health. This approach is

also fast, requiring less time. It is a nonintrusive approach hence permits the incessant

use of the site (Azubuike et al. 2016).

Bioremediation in spites of its numerous advantages also has certain disadvan-

tages. It can only be employed for contaminants that can be biodegraded. Not all

pollutants are amenable to microbial action. In some other cases, the contaminants

are converted to intermediate compounds that could constitute more harm than the

original compounds. Also, bioremediation is also considered to be time consuming

in comparison to other methods such as total excavation of the polluted soil. Scaling

process for bioremediation is also difﬁcult from the various batches and small scales

to larger scales application (Abatenh et al. 2017).

6 Conclusion and Future Trends

The concept of emerging contaminants has now become trendy and fashionable

research area. The increasing number of these compounds has constituted a serious

challenge for regulatory agencies. Various approaches have been employed for the

treatment and remediation of the compounds. The approach of bioremediation has

proven to be efﬁcient in the restoration of areas that have been contaminated by

various classes of pollutants. Microorganisms have been identiﬁed as the major

driving force for this process. The achievement of remarkable bioremediation is

affected by the degradation potential of the microbes, availability of nutrients and

population of the microorganisms. There is need for more researches for the develop-

ment and advancement of site speciﬁc bioremediation techniques, for complex groups

of contaminants. Bioremediation is limited in that it is only applicable to compounds

that can be acted upon by microorganisms. Other reliable technologies include

oxidation processes, advanced oxidation processes and membrane technologies.

References

Abatenh E, Gizaw B, Tsegaye Z, Wassie M (2017) The role of microorganisms in bioremediation—a

review. Open J Environ Biol 2(1):038–046. https://doi.org/10.17352/ojeb.000007

Abatenh E, Gizaw B, Tsegaye Z, Wassie M, The role of microorganisms in bioremediation—a

review. Open J Environ Biol

Azubuike C, Chikere C, Okpokwasili G (2016) Bioremediation techniques–classiﬁcation based

on site of application: principles, advantages, limitations and prospects. World J Microbiol

Biotechnol 32(11):180. https://doi.org/10.1007/s11274-016-2137-x. Epub 2016 Sep 16. PMID:

27638318; PMCID: PMC5026719

Emerging Pollutants from the Industries and Their Treatment 11

Briffa J, Sinagra E, Blundell R (2020) Heavy metal pollution in the environment and their toxico-

logical effects on human 6(9):e04691. https://doi.org/10.1016/j.heliyon.2020.e04691Get rights

and content

Buthiyappan, Aziz A, Raman A, Daud W, Mohd Ashri W (2016) Recent advances and prospects

of catalytic advanced oxidation process in treating textile efﬂuents. Rev Chem Eng 32(1):1–47.

https://doi.org/10.1515/revce-2015-0034

Cordes E, Jones D, Schlacher T, Carney R (2016) Environmental impacts of the deep-water oil

and gas industry: a review to guide management strategies. Front Environ Sci. https://doi.org/10.

3389/fenvs.2016.00058

Cuerda-Correa E, Alexandre M, Gonzalex (2020) Advanced oxidation processes for the removal

of antibiotics from water: an overview. J Water 12.https://doi.org/10.3390/w12010102

Cuerda-Correa E, Alexandre M, Gonzalez F (2019) Advanced oxidation processes for the removal

of antibiotics from water. an overview. Water Rev 7(8):345

Das RK, Pachapur VL, Lonappan L et al (2017) Biological synthesis of metallic nanoparticles:

plants, animals and microbial aspects. Nanotechnol. Environ Eng 2:18. https://doi.org/10.1007/

s41204-017-0029-4

Ghime D, Ghosh P (2019) Advanced oxidation processes: a powerful treatment option for the

removal of recalcitrant organic compounds.https://doi.org/10.5772/intechopen.90192

Kamal P, Yadav K (2022) Evaluation of normal size of lacrimal glands in subset of popula-

tion at Liaquat University of medical and health sciences Jamshoro by Multiplanar computed

tomography. Aditum J Clinical Biomed Res 4(3). 03.2022/1.1076

Krishnan S (2017) Comparison of various advanced oxidationprocesses used in remediation of

industrial wastewater laden with recalcitrant pollutants. IOP conference series: materials science

and engineering. Mater Sci Eng 206:012089

Krystynik P (2021) Advanced oxidation processes (AOPs)—Utilization of hydroxyl radical and

singlet Oxygen. https://doi.org/10.5772/intechopen.98189

Manisalidis I, Stavropoulou E, Bezirtzoglou E (2020) Environmental and health impacts of air pollu-

tion: a review front. Public Health 20 February 2020 Sec. Environmental health and Exposome.

https://doi.org/10.3389/fpubh.2020.00014

Masindi V, Muedi K (2017) Environmental contamination by heavy metals. https://doi.org/10.5772/

intechopen.76082

Moënne-Loccoz Y, Mavingui P, Combes C, Normand P, Steinberg C (2015) Microorganisms

and biotic interactions. In: Bertrand JC, Caumette P, Lebaron P, Matheron R, Normand P.,

Sime-Ngando T (eds) Environmental microbiology: fundamentals and applications. Springer,

Dordrecht. https://doi.org/10.1007/978-94-017-9118-2_11

Ofrydopoulou A, Nannou C, Evgenidou E, Lambropoulou D (2022) Assessment of a wide array

of organic micropollutants of emerging concern in wastewater treatment plants in Greece:

occurrence, removals, mass loading and potential risks. https://doi.org/10.1016/j.scitotenv.2021.

149860

Patel M, Kumar R, Pittman C (2019) pharmaceuticals of emerging concern in aquatic systems:

chemistry, occurrence, effects, and removal methods. Chem Rev 119(6):3510–3673. https://doi.

org/10.1021/acs.chemrev.8b00299

Sharma I (2019) Bioremediation techniques for polluted environment: concept, advantages,

limitations, and prospects.https://doi.org/10.5772/intechopen.90453

Talabi A, Kayode T (2019) Groundwater pollution and remediation. J Water Resour Protection

11(1). https://doi.org/10.4236/jwarp.2019.111001

Bioremediation: The Remedy

to Expanding Pollution

Shreya Anand and Padmini Padmanabhan

Abstract The worldwide population is growing at an astounding rate, with evalu-

ations suggesting the increase to approximately 9 billion by 2050. The exhaustive

agro-system and the industrial systems required to upkeep this huge number of soci-

eties will unavoidably become the basis of pollution (air, water, soil) buildup. Hydro

systems have slight improved fare, having an approximation of 70% industrial waste

that are discarded into nearby water bodies. The global generation of garbage is

1.3 billion tons per year, the mainstream trash is deposited in the sites of landﬁll

or discarded in the oceans. The microorganisms are commonly acknowledged for

its capability to disrupt the enormous variety of organic compounds and engross the

inorganic substances. Presently, microorganism are used in treatment of pollution

treatment through a process known as ‘bioremediation’. Bioremediation is the effec-

tive green process of removing stubborn contaminants from the environment through

microorganism to decrease the level of pollution using the approach of biological

degradation of pollutants into non-toxic substances.

Keywords Agricultural waste ·Waste water ·Contaminants ·Microorganism ·

Bioremediation

1 Introduction

Bioremediation is a waste management technique that includes the use of living organisms

to eradicate or neutralize pollutants from a contaminated site.

The pollution dilemma has become a big issue all across the world. Every year, it has

a negative impact on millions of people, resulting in numerous health problems and

deaths. Although urban regions are typically more polluted than rural ones, pollution

can spread to far-ﬂung locations; pesticides and other chemicals have been discovered

under the Antarctic ice sheet, for example. The Great Paciﬁc Garbage Patch is a

S. Anand · P. Padmanabhan (B

)

Department of Bioengineering and Biotechnology, Birla Institute of Technology, Mesra,

Ranchi 835215, Jharkhand, India

e-mail: padmini@bitmesra.ac.in

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_2

13

14 S. Anand and P. Padmanabhan

vast concentration of microscopic plastic particles discovered in the centre of the

northern Paciﬁc Ocean. Pollutants can be transported from one location to another by

land, water, and the atmosphere, progressively aggravating the situation. Pollution is

carried by air and water currents, ocean currents and migrating ﬁsh, and the wind can

pick up and spread radioactive material mistakenly discharged from a nuclear reactor

or smoke from a factory from one country to another (Doney et al. 2012). As a result,

pollution is unconcerned about geographical boundaries. Oil spills, fertilizers, waste,

sewage disposals, and poisonous chemicals are only a few of the major pollution-

causing substances found around the world. They all contribute to global pollution by

polluting the soil, air, water, and marine environments. Among 87,000 commercial

compounds, the US Environmental Protection Agency has designated 53 substances

as persistent, bio-accumulative, and hazardous (USEPA 2007).

Contamination is deﬁned as the presence of high quantities of substances in the

environment that are either detrimental to society or not. Pollution, on the other

hand, is the deliberate introduction of harmful elements into the environment by

humans, resulting in a hazardous effect. These pollutants can be produced by a

variety of natural and anthropogenic activities, such as large-scale chemical synthesis,

processing, and handling. Pollution levels are rapidly rising over the world, posing

a huge threat to both industrialized and developing countries. Despite the fact that

statistical data differs widely between countries and years, most contaminants have a

long residence duration and can travel considerable distances. As a result, they change

into very poisonous compounds, exacerbating the problem. Pollutants can easily

travel vast distances since they don’t respect geographical boundaries, as evidenced

by the presence of xenobiotics in Arctic regions where no local sources of pollution-

causing agents can be detected. As a result, pollution has become a worldwide issue

that must be addressed regardless of political or geographical boundaries.

Microorganisms, in this perspective, play a critical role in the maintenance and

sustainability of any ecosystem since they are more capable of quickly adapting

to environmental changes and deterioration. Microorganisms are thought to be the

ﬁrst life forms to evolve; they are adaptable to a variety of harsh environmental

circumstances. Microorganisms are everywhere, and they have a huge impact on the

environment. They are important regulators of biogeochemical cycles in a variety of

habitats, including cold environments, acidic lakes, hydrothermal vents, deep ocean

bottoms, and animal small intestines (Seigle-Murandi et al. 1996). Microorganisms

govern global biogeochemical cycling by performing carbon ﬁxation, nitrogen ﬁxa-

tion, methane metabolism, and sulphur metabolism (Das et al. 2006). They produce a

variety of metabolic enzymes that can be used to safely remove contaminants, which

can be done either by destroying the chemical directly or by transforming the toxins

into a less harmful intermediate (Dash and Das 2012).

Microbes are particularly helpful in remediating the contaminated environ-

ment; bioremediation and natural reduction are also seen as solutions for emerging

contamination problems. The bioremediation process involves a variety of microbes,

including aerobic and anaerobic bacteria as well as fungi. Through the all-inclusive

and action of microorganisms, bioremediation is heavily involved in the degrada-

tion, eradication, immobilization, or detoxiﬁcation of various chemical wastes and

Bioremediation: The Remedy to Expanding Pollution 15

physical dangerous chemicals from the surrounding. The fundamental premise is

to degrade contaminants and transform them to less harmful forms. The pace of

deterioration is determined by two types of factors: biotic and abiotic environments.

Bioremediation is now carried out using a variety of technologies and tactics.

Increased human activities such as population explosion, unsafe agricultural

practices, unplanned urbanization, deforestation, rapid industrialization, and non-

judicious use of energy reservoirs, among other anthropogenic activities, have

resulted in increased environmental pollution in recent decades. Chemical fertil-

izers, heavy metals, nuclear wastes, pesticides, herbicides, insecticides, greenhouse

gases, and hydrocarbons are among the pollutants that cause environmental and

public health concerns due to their toxicity. Thousands of hazardous waste sites

have been identiﬁed, with more expected to be discovered in the future decades.

Illegal dumping by chemical businesses and industry releases contaminants into the

environment. Many of the previous site cleanup approaches, such as digging up the

contaminated soil and shipping it away to be landﬁlled or burnt, were prohibitively

expensive and did not provide a long-term solution. Recent solutions like vapor

extraction and soil venting are less expensive, yet they’re still insufﬁcient.

2 Bioremediation

Bioremediation is a ‘treatment techniques’ that uses naturally occurring organisms to break

down harmful materials into less toxic or non-toxic materials.

Bioremediation is a metabolic process that uses biological organisms to remove or

neutralise an environmental pollution. Microscopic organisms such as fungi, algae,

and bacteria are included in the “biological” organisms, as is the “remediation”—the

treatment of the issue. Microorganisms thrive in a diverse range of environments

across the biosphere. They thrive in a variety of environments, including soil, water,

plants, animals, the deep sea, and the frozen ice. Microorganisms are the ideal candi-

dates to function as our environmental stewards because of their sheer numbers and

desire for a wide spectrum of pollutants.

Bioremediation technologies became widely used and are still increasing at

an exponential rate today. Because of its environmentally benign characteristics,

bioremediation of polluted places has proven to be effective and trustworthy. Recent

advancements in bioremediation techniques have occurred in the last two decades,

with the ultimate goal of successfully restoring damaged areas in an economical

and environmentally beneﬁcial manner. Different bioremediation approaches have

been developed by researchers to recover polluted ecosystems. Bioremediation

can involve either indigenous or non-indigenous microorganisms supplied to the

contaminated site.

Most of the issues connected with pollution biodegradation and bioremediation

can be solved by indigenous microorganisms found in disturbed areas (Khan

et al. 2015). Bioremediation has a number of advantages over chemical and

16 S. Anand and P. Padmanabhan

Fig. 1 Various approaches of bioremediation involved in control of pollution

physical remediation approaches, including being environmentally benign and

cost-effective. Bioremediation works by reducing, detoxifying, degrading, mineral-

izing, or transforming more hazardous contaminants into less toxic ones. Pesticides,

agrochemicals, chlorinated compounds, heavy metals, xenobiotic compounds,

organic halogens, greenhouse gases, hydrocarbons, nuclear waste, dyes, plastics,

and sludge are among the pollutants that can be removed. Toxic waste is removed

from a polluted environment using cleaning techniques. Bioremediation is highly

involved in degradation, eradication, immobilization, or detoxiﬁcation diverse

chemical wastes and physical hazardous materials from the surrounding through the

all-inclusive and action of microorganisms (Fig. 1).

Most of the issues connected with pollution biodegradation and bioremediation

can be solved by indigenous microorganisms found in disturbed areas (Khan et al.

2015). Bioremediation has a number of advantages over chemical and physical

remediation approaches, including being environmentally benign and cost-effective.

Bioremediation works by reducing, detoxifying, degrading, mineralizing, or trans-

forming more hazardous contaminants into less toxic ones. Pesticides, agrochem-

icals, chlorinated compounds, heavy metals, xenobiotic compounds, organic halo-

gens, greenhouse gases, hydrocarbons, nuclear waste, dyes, plastics, and sludge are

among the pollutants that can be removed. Toxic waste is removed from a polluted

environment using cleaning techniques.

3 Factors Involved in Cleaning Pollution Through

Bioremediation

The bioremediation process involves bacteria, fungus, algae, and plants degrading,

eliminating, altering, immobilizing, or detoxifying various chemicals and physical

contaminants from the environment. Microorganisms’ enzymatic metabolic path-

ways aid in the progression of biochemical events that aid in pollution breakdown.

Bioremediation: The Remedy to Expanding Pollution 17

Tabl e 1 Factors affecting the bioremediation and the role of the associated factors

Factors Role

Microbial factors • Growth of microorganisms take place till the

biomass has reached its critical production

• Mutation and horizontal gene transfer

• Enzyme induction

• Enrichment of the capable microbial populations

• Production of toxic metabolites

Environment factors • Depletion of preferential substrates

• Lack of nutrients

• Inhibitory environmental conditions

Substrate factor • Too low concentration of contaminants

• Chemical structure of contaminants

• Toxicity of contaminants

• Solubility of contaminants

• Biological aerobic versus anaerobic process

• Oxidation/reduction potential

• Availability of electron acceptors

• Microbial population present in the site

Growth of microbes, and co-metabolism • Type of contaminants

• Concentration

• Alternate carbon source present

• Microbial interaction

Bioavailability of pollutants • Equilibrium sorption

• Irreversible sorption

• Incorporation into humic matters

Mass transfer limitations • Oxygen diffusion and solubility

• Diffusion of nutrients

• Solubility/miscibility in/with water

Only when microorganisms come into touch with substances that assist them generate

energy and nourishment to multiply cells do they act on pollution. The chemical

composition and quantity of contaminants, as well as the physicochemical properties

of the environment and their accessibility to existing microorganisms, all inﬂuence

the success of bioremediation (Fantroussi and Agathos 2005). The key contributors

include the microbial population’s ability to degrade pollutants, contaminants’ acces-

sibility to the microbial population, and environmental factors like type of soils, pH,

temperature, oxygen and nutrients (Table 1).

3.1 Biological Factors

Biotic factors aid in the breakdown of organic compounds by microorganisms

with limited carbon sources, antagonistic interactions between microorganisms,

and protozoa-bacteriophage interactions. The pace of contaminant degradation is

18 S. Anand and P. Padmanabhan

frequently inﬂuenced by the amount of catalyst present in the biochemical reaction as

well as the concentration of the pollutant. Enzyme activity, interaction (competition,

succession, and predation), mutation, horizontal gene transfer, biomass production

growth, population size, and composition are among the major biological parameters

(Naik and Duraphe 2012; Boopathy 2000).

3.2 Environmental Factors

Environmental pollutants interact with metabolic activity and the physicochemical

properties of the microorganisms targeted throughout the procedure. The success of

the microbe-pollutant interaction is determined by the environmental conditions.

Temperature, pH, moisture, soil structure, water solubility, nutrients, site condi-

tions, oxygen content and redox potential, resource deﬁciency and physico-chemical

bioavailability of pollutants, concentration, chemical structure, type, solubility, and

toxicity are all factors that inﬂuence microbial growth and activity. The dynamics of

degradation are controlled by the components listed above (Adams et al. 2015).

In most aquatic and terrestrial environments, contaminant biodegradation can

occur in a pH range of 6.5–8.5, which is generally ideal for biodegradation. Moisture

affects the metabolism of contaminant because it depends on the kind and amount

of soluble constituents that are accessible as well as the pH and osmotic pressure of

terrestrial and aquatic systems (Cases and Lorenzo 2005).

4 Techniques Used in Bioremediation

There are various types of bioremediation or technologies or strategies used in biore-

mediation, but the following are the most common ways in which it is used are

bio-pile, windrows, land-farming, bioreactor, bioventing, bio-slurping, bio-sparging,

phytoremediation, and permeable reactive barrier.

Bio-pile: To increase bioremediation by microbial metabolic activities, above-ground

stacking of dug toxic soil is followed by aeration and nutrient replenishment. Aera-

tion, irrigation, fertilizers, leachate collection, and treatment bed systems are all

part of this technique. This unique ex-situ technology is increasingly being evalu-

ated due to its cost-effective properties, which allow for effective control of operative

biodegradation variables such as pH, nutrient, temperature, and aeration. The bio-pile

is utilized to address low-molecular-weight contaminants that are volatile, and it may

also be used to repair polluted very cold harsh situations (Gomez and Sartaj 2014;

Dias et al. 2015; Whelan et al. 2015). The versatility of the bio-pile provides for a

faster remediation period since a heating system can be added into the bio-pile design

to promote microbial activity and pollutant availability, speeding up biodegradation

(Aislabie et al. 2006).

Bioremediation: The Remedy to Expanding Pollution 19

In order to promote better bioremediation, warm air can be fed into the bio-pile

design to deliver air and heat simultaneously. Bulking agents such straw sawdust,

bark or wood chips, and other organic materials have been added to a biopile construct

to speed up the restoration process. Although bio-pile systems are linked to other

ex-situ bioremediation techniques in the ﬁeld, such as land farming, bioventing,

and bio-sparging, robust engineering, maintenance and operation costs, and a lack of

power at remote sites, which would allow for constant air circulation in contaminated

piled soil via an air pump Furthermore, high air heating can cause soil drying during

bioremediation, which inhibits microbial activity and promotes volatilization rather

than biodegradation (Sanscartier et al. 2009).

Land farming: Due to its low cost and low equipment requirements, land farming

is one of the most basic and effective bioremediation strategies. It’s most common

in ex-situ bioremediation, although it can also happen in in-situ bioremediation.

This factor is taken into account because of the treatment location. In land farming,

which can be done ex-situ or in-situ, pollutant depth is critical. Polluted soils are

excavated and tilled on a regular basis in land farming, and the form of bioremedia-

tion depends on the treatment site. Ex-situ bioremediation occurs when toxic soil is

removed and treated on-site, since it has more in common with other ex-situ biore-

mediation processes. In general, excavated polluted soils are carefully put above

the ground surface on a ﬁxed layer support to facilitate aerobic biodegradation of

pollution by autochthonous microorganisms (Silva-Castro et al. 2012). Overall, land

farming bioremediation is a straightforward design and implementation technology

that requires little capital investment and may be utilized to treat huge volumes of

dirty soil with minimal environmental impact and energy consumption (Maila and

Cloete 2004).

Windrows: Windrows are a bioremediation technique that involves rotating piled

polluted soil on a regular basis to increase bioremediation by enhancing microbial

degradation activities of native and transient hydro-carbonoclastic in the polluted

soil. Periodic turning of polluted soil improves aeration and equal distribution of

nutrients, contaminants, and microbial degradation activities, increasing the pace

of bioremediation, which can be accomplished by acclimatization, biotransforma-

tion, and mineralization. Windrow treatment exhibited a higher rate of hydrocarbon

removal than bio-pile treatment, but the effectiveness of the windrow for hydro-

carbon removal from the soil (Coulon et al. 2010). Periodic turning in conjunction

with windrow treatment, on the other hand, is not the optimum strategy for bioreme-

diation of soil contaminated with harmful volatile chemicals. The use of windrow

treatment has been associated in greenhouse gas (CH4) release due to formation of

anaerobic zone inside piled polluted soil, which frequently reduced aeration (Hobson

et al. 2005).

Bio-slurping: This technology combines vacuum-assisted pumping, soil vapor

extraction, and bioventing to achieve soil and ground water remediation through

indirect oxygen delivery and pollutant biodegradation stimulation (Gidarakos and

20 S. Anand and P. Padmanabhan

Aivalioti 2007). This method will be used to recover goods from capillary remedia-

tion, light non-aqueous phase liquids, unsaturated and saturated zones. This method

is used to clean up polluted soils with volatile and semi-volatile organic substances.

The approach employs a “slurp” that spreads into the free product layer and draws

liquids up from there. The pumping machine uses upward movement to carry light

non-aqueous phase liquids to the surface, where they are separated from air and

water. In this method, soil moisture reduces air permeability and oxygen transfer

rate, lowering microbial activity. Despite the fact that this technique is not ideal for

low permeable soil restoration, it is a cost-effective operation process since it uses

less ground water and thus reduces storage, treatment, and disposal expenses.

Bioreactor: A bioreactor is a vessel that converts raw materials into particular prod-

ucts through a sequence of biological reactions. Batch, fed-batch, sequencing batch,

continuous, and multistage bioreactors all have different operational modes. Biore-

actors provide ideal conditions for bioremediation growth. For the cleanup process,

a bioreactor is loaded with polluted samples. When compared to ex-situ bioremedi-

ation approaches, bioreactor-based treatment of polluted soil offers various advan-

tages. Bioremediation time is reduced by using a bioreactor-based bioremediation

process with superior control of pH, temperature, agitation and aeration, substrate

and inoculum concentrations. In a bioreactor, the ability to regulate and alter process

parameters indicates that biological responses may be controlled and manipulated.

Bioreactor designs are versatile, allowing for maximum biological degradation while

reducing abiotic losses (Mohan et al. 2004).

Bio-sparging: This method is similar to bioventing in that air is injected into the

subsurface of the soil to increase microbial activity, which stimulates pollution

removal from polluted areas. Bioventing, on the other hand, involves injecting air

into the saturated zone, which can aid in the upward migration of volatile organic

molecules to the unsaturated zone, so accelerating the biodegradation process. Bio-

sparging efﬁciency is determined by two primary factors: soil permeability and pollu-

tant biodegradability. Bio-sparing is a closely related approach in bioventing and

soil vapor extraction (SVE) known as in-situ air sparging (IAS), which relies on high

air-ﬂow rates for pollutant volatilization, whereas bio-sparging encourages biodegra-

dation. It has mostly been used to treat aquifers that have been contaminated with

fuel and kerosene.

Permeable reactive barrier: This technique is widely used to remediate contami-

nated groundwater using a physical manner. Precipitation degradation and sorption

of pollutant removal, on the other hand, are biological mechanisms utilized in the

PRB approach. To accommodate the biotechnology and bioremediation aspects of

the technique, substitute terminology such as biological PRB, bio-enhanced PRB,

and passive bio-reactive barrier have been suggested. PRB is an in-situ approach

for removing heavy metals and chlorinated compounds from polluted groundwater

(Silva-Castro et al. 2012; Obiri-Nyarko et al. 2014).

Bioventing: In order to improve the activity of indigenous bacteria for bioremedi-

ation, bioventing techniques require regulated stimulation of airﬂow by supplying

Bioremediation: The Remedy to Expanding Pollution 21

oxygen to the unsaturated zone. To promote bioremediation, bioventing amendments

are made by adding nutrients and moisture. As a result, contaminants will be micro-

bially transformed into a harmless condition. Among other in-situ bioremediation

approaches, this one has gained traction (Höhener and Ponsin 2014).

Phytoremediation: Phytoremediation is the process of cleaning up polluted soils.

This strategy uses plant interactions such as physical, chemical, biological, microbi-

ological, and biochemical interactions to reduce the harmful characteristics of pollu-

tants in contaminated locations. In phytoremediation, numerous methods such as

extraction, degradation, ﬁltration, accumulation, stability, and volatilization are used,

depending on the amount and nature of the pollutant. Extraction, transformation, and

sequestration are standard methods for removing pollutants such heavy metals and

radionuclides. Degradation, rhizo-remediation, stabilization, and volatilization are

the main methods for removing organic pollutants like hydrocarbons and chlori-

nated chemicals, with mineralization being possible when plants like willow and

alfalfa are utilized (Meagher 2000; Kuiper et al. 2004).

Plant root system, which may be ﬁbrous or tap depending on the depth of the

pollutant, above ground biomass, pollutant toxicity to plant, plant existence and

adaptability to predominant environmental conditions, plant growth rate, site moni-

toring, and above all, time required to achieve the desired level of cleanliness are

all important factors to consider when using plants as phytoremediators. In addi-

tion, the plant must be disease and insect resistant (Lee 2013). Pollutant removal in

phytoremediation comprises uptake and transfer from roots to shoots. Furthermore,

transpiration and partitioning affect translocation and accumulation (San Miguel

et al. 2013). However, depending on other aspects such as the nature of the pollutant

and the plant, the method could change. Phytoremediators are usually plants that

ﬂourish in contaminated areas. As a result, any phytoremediation method’s success

is largely dependent on increasing the remediation potentials of native plants growing

in polluted areas, either by bio-augmentation using endogenous or exogenous plants.

One of the most signiﬁcant beneﬁts of utilizing plants to clean up polluted sites is

that some precious metals can bio-accumulate in speciﬁc plants and be retrieved after

remediation, a process known as phyto-mining.

5 Approaches of Bioremediation

Superﬁcially, bioremediation techniques can be carried out ex-situ and in-situ site of

application (Fig. 1). The type of pollutant, the depth and volume of contamination, the

type of ecosystem, the location, the cost, and environmental policies are all factors to

consider when choosing a bioremediation technique. The efﬁcacy of bioremediation

processes is determined by oxygen and nutrient concentrations, temperature, pH, and

other abiotic variables (Frutos et al. 2012; Smith et al. 2015).

22 S. Anand and P. Padmanabhan

Ex-situ bioremediation techniques

It entails excavating pollutants from polluted places and delivering them to a treat-

ment facility. Ex-situ bioremediation procedures are frequently chosen depending

on the depth of contamination, the type of pollutant, the degree of pollution, the

cost of treatment, and the location of the contaminated site. Ex-situ bioremediation

procedures are likewise governed by performance requirements.

•Solid-phase treatment

Solid-phase bioremediation is an ex-situ technique that involves excavating contam-

inated soil and stacking it. Organic waste, such as leaves, animal manures, and

farm wastes, as well as home, industrial, and municipal wastes, are included. Bacte-

rial growth is facilitated by pipelines positioned throughout the piles. Ventilation

and microbial respiration require air to ﬂow through the pipes. When compared to

slurry-phase procedures, solid-phase systems demand a lot of area and cleanup takes

a long time. Bio-piles, windrows, land farming, composting, and other solid-phase

treatment methods are examples (Kulshreshtha et al. 2014).

•Slurry-phase bioremediation

When compared to alternative treatment methods, slurry-phase bioremediation is a

faster process. In the bioreactor, contaminated soil is mixed with water, nutrients,

and oxygen to produce the ideal environment for microorganisms to breakdown the

pollutants in the soil. Separation of stones and rubbles from polluted soil is part

of this process. The amount of water added is determined by the amount of pollu-

tants present, the rate of biodegradation, and the soil’s physicochemical parameters.

The soil is removed and dried when this process is completed using vacuum ﬁlters,

pressure ﬁlters, and centrifuges. The next step is to dispose of the soil and treat the

resulting ﬂuids in advance.

In-situ bioremediation techniques

These methods entail treating polluted substances at the source of the pollution.

It does not necessitate any excavation and causes minimal or no soil disturbance.

In comparison to ex-situ bioremediation approaches, these procedures should be

quite cost effective. Some in-situ bioremediation procedures, such as bioventing,

biosparging, and phytoremediation, may be improved, while others, such as intrinsic

bioremediation and natural attenuation, may progress without improvement. Chlo-

rinated solvents, heavy metals, dyes, and hydrocarbons have all been successfully

treated using in-situ bioremediation approaches (Folch et al. 2013; Frascari et al.

2015; Roy et al. 2015). There are two types of in-situ bioremediation: intrinsic and

engineered bioremediation.

•Intrinsic bioremediation

Natural reduction, also known as intrinsic bioremediation, is an in-situ bioremedia-

tion process that involves the passive remediation of polluted places without the use

of any external force (human intervention). The encouragement of an indigenous or

Bioremediation: The Remedy to Expanding Pollution 23

naturally occurring microbial population is the goal of this technique. The biodegra-

dation of contaminating elements, including those that are refractory, is dependent on

both microbial aerobic and anaerobic processes. Because there is no external force,

the process is less expensive than other in-situ techniques.

•Engineered in-situ bioremediation

The second method entails introducing a speciﬁc bacterium to the contaminated

area. In-situ bioremediation using genetically engineered microbes accelerates the

degradation process by improving the physicochemical conditions to stimulate

microorganism development.

6 Recent Researches on Bioremediation

6.1 Bioinformatics Approaches in Bioremediation

From a bioinformatics standpoint, the ﬁeld of bioremediation offers many undiscov-

ered and appealing options that require a large amount of data from various sources,

such as protein sequence, biology and physiology, comparative genomics, chem-

ical structure, reactivity of organic compounds, and environmental biology. It’s an

interdisciplinary ﬁeld of study that straddles the line between computer science and

biology (Kour et al. 2021).

Computers are used in bioinformatics to store, manipulate, recover, and allo-

cate information related to DNA, RNA, and proteins. Bioremediation technolo-

gies based on genomics Bioremediation can be studied using omics-based methods

such as genomics, transcriptomics, interactomics, proteomics, and metabolomics.

This approach aids in the correlation of DNA sequences with mRNA, protein, and

metabolite abundance, resulting in a more accurate and complete picture of in situ

bioremediation.

Genomics: For the study of microbial strains involved in bioremediation, genomics

is an merging subject. This method is based on a concept that analyses all genetic

information in a microbe’s cell. Bioremediation has been documented using a wide

spectrum of microorganisms (Khardenavis et al. 2007; Qureshi et al. 2007). Here,

genomic tools are used to explain biodegradation pathways using PCR, microassay

analysis, DNA hybridization, isotope distribution analysis, molecular connectivity,

and metabolic engineering and metabolic foot printing, as well as metabolic engi-

neering and metabolic foot printing to improve the biodegradation process. For

microbial communities scrambled, a number of genotypic ﬁngerprinting techniques

based on the PCR are available, including ampliﬁed fragment length polymorphisms

(AFLP), automated ribosomal intergenic spacer analysis (ARISA), ampliﬁed ribo-

somal DNA restriction analysis (ARDRA), terminal-restriction fragment length poly-

morphism (T-RFLP), single strand conformation polymorphism (SSCP), randomly

24 S. Anand and P. Padmanabhan

ampliﬁed polymorphic DNA analysis RAPD could be utilised for genetic ﬁnger-

printing, structural and functional interpretation of soil microbial communities, and

assessing essentially allied bacterial species (Gupta et al. 2020).

Within microbial communities, LH-PCR could be utilised to detect natural length

variations of various SSU rRNA genes. T-RFLP can be used to proﬁle microorgan-

isms from many taxonomic groups at the same time in an environment (Singh et al.

2006). Furthermore, a combination of molecular methods such as genetic ﬁnger-

printing, FISH, microradiography, stable isotope probing, and quantitative PCR can

be utilised to investigate microbial interactions with natural variables in the soil

microenvironment. In the study of soil microbial communities, quantitative PCR

is used to detect the abundance and expression of taxonomic and operational gene

markers (Bustin et al. 2005). Using ampliﬁed PCR products, genetic ﬁngerprinting

techniques perform direct investigation of certain molecular biomarker genes. Cluster

aided analysis, which analyses ﬁngerprints from numerous samples, could be used

to investigate the link between varied microbial populations.

Proteomics and metabolomics: Proteomics is concerned with the total amount of

proteins expressed in a cell at a certain location and time, whereas metabolomics is

concerned with the quantiﬁcation and characterization of total metabolites produced

by an organism at a given time or under certain conditions (Rawat and Rangarajan

2019). Proteomics-based research has proved effective in detecting the number of

proteins and changes in their composition, as well as identifying important proteins

implicated in microorganisms’ physiological responses to anthropogenic contami-

nants (Desai et al. 2010). In comparison to genomics, functional study of microbial

communities is more informative and has larger promise.

Metabolomics research employs two main methodologies for evaluating biolog-

ical systems. The ﬁrst considers a broad, untargeted investigation in which no prior

knowledge of the biological system’s metabolic pathways is necessary. This tech-

nique aids in the detection and recovery of a wide range of metabolites present in

the sample, yielding a massive amount of data that can then be compared across

samples to determine metabolic pathway interconnectedness. The second technique

is to conduct a tailored investigation based on prior information to identify speciﬁc

metabolic pathways or metabolites (Hussain et al. 2009). For the detection and

measurement of a wide range of cellular metabolites, the microbial metabolomics

toolbox comprises several approaches such as foot printing and metabolic ﬁnger-

printing, target analysis, and metabolite proﬁling. The combination of proteome and

metabolome data will help in identiﬁcation of the active molecules essential for

cell-free bioremediation.

Transcriptomics and metatranscriptomics: A transcriptome is a collection of genes

that are transcribed under speciﬁc conditions and at speciﬁc times, and it serves as a

vital link between the cellular phenotype, genome, interactome, and proteome. Gene

expression regulation is an important process for adjusting to changes in the envi-

ronment and consequently for survival. Transcriptomics analyses this process across

the entire genome. DNA microassay analysis is a very powerful method in transcrip-

tomics for determining the level of mRNA expression (Dias et al. 2015). Extraction

Bioremediation: The Remedy to Expanding Pollution 25

and enrichment of total mRNA, cDNA synthesis, and either whole cDNA transcrip-

tome sequencing or microarray hybridization of cDNA are all part of transcrip-

tomics study. DNA microarray is a useful transcriptomics technique for analysing and

studying the expression of practically every gene in an organism’s mRNA (Pandey

et al. 2019).

Transcriptomics and metatranscriptomics are useful for gaining functional

insights into the activities of environmental microbial populations by examining tran-

scriptional mRNA patterns (McGrath et al. 2008). Metatranscriptomics, in combina-

tion with metagenomics and genome binning, has been shown to offer information on

microbial associations, syntrophism, and complementing metabolic pathways during

the biodegradation process (Ishii et al. 2015). Metatranscriptomics (Giovanella et al.

2020) is a powerful tool for obtaining quantitative and qualitative gene expression

data.

6.2 Nano-Technological Approaches in Bioremediation

Norio Taniguchi, a professor at Tokyo University of Science, coined the term

nanotechnology in 1974 (Taniguchi 1974). Nanotechnology is concerned with items

with dimensions on the order of a nanometer. They are quickly recognized for

removing diverse hazardous chemicals due to their speciﬁc action against various

recalcitrant pollutants. Nanotechnology has brought a fresh viewpoint to technology,

particularly in the realm of water treatment. Under the headings of photocatalysis and

nanoﬁltration (Prasad et al. 2018), it is now feasible to collect ecologically favorable

approaches.

Effective microorganisms (EM) technology and nanotechnology: Effective

microbes (EM) technology uses effective microbes to remediate waste water, which

is then recycled for irrigation (Leahy and Colwell 1990). Water contaminants can be

reduced using nanotechnology and EM technologies (Shrivastava et al. 2007). The

number of locations contaminated with resistant organic pollutants, such as PAHs

(polycyclic aromatic hydrocarbons) containing numerous benzene rings, is enor-

mous, posing widespread environmental issues. PAHs are mutagenic and relatively

non-biodegradable.

Engineered polymeric nanoparticles for bioremediation of hydrophobic contam-

inants: Organic contaminants, such as PAHs and petroleum hydrocarbons, are

absorbed into soil, limiting their solubility and mobility. Phenanthrene solubility

is increased by polymeric nano-network particles, which improves phenanthrene

release from polluted aquifer material. Poly(ethylene)glycol modiﬁed urethane acry-

late (PMUA) precursor chain is used to make polymeric nanoparticles. PMUA

nanoparticles are designed to maintain their characteristics in the presence of a

heterogeneous active bacterial population (Bhandari 2018).

Enhanced degradation for hazardous waste treatment using cell immobilization

technique: Immobilized cells have proven to be successful in the bioremediation of

26 S. Anand and P. Padmanabhan

a variety of harmful substances. Lee and Lee (2004) used free cells and cells immo-

bilised in Calginate gel beads to study chlorophenol breakdown. They discovered

that ﬁxed cells decomposed chlorophenols considerably faster than free cells and

minimised the lag phase for the extraction of chlorophenols (Lee and Lee 2004).

7 Conclusion

Environmental contamination is the biggest challenge of the twenty-ﬁrst century, and

research communities are paying close attention to it. Because bacteria adapt quickly

to changing and hostile surroundings, bioremediation using microbes is a powerful

strategy for clearing up pollution by increasing natural biodegradation processes.

For the development of ecologically stable, new, and feasible bioremediation tech-

niques, a full understanding of microbial communities and how they respond to the

natural environment and in the presence of contaminants is critical. The extraordi-

nary signiﬁcance of extremophiles in bioremediation highlights the need for more

research so that new species can be discovered and the processes they utilize to

survive in such harsh settings can be investigated. Multi-omics studies are still insuf-

ﬁcient, and additional research is needed to bridge gaps in our knowledge of the

ecology, gene expression, and metabolism of bacteria participating in bioremedi-

ation. Several microbes have important metabolic genes that could be transferred

to other organisms. Microbes that have been genetically modiﬁed to improve their

ability to degrade contaminants will undoubtedly have a bright future in this sector.

Expanding our understanding of microbial genetics in order to improve our ability

to breakdown contaminants and conducting ﬁeld studies will undoubtedly lead to

progress in this subject. It would also be fascinating if bioremediation products were

developed for large-scale application.

References

Adams GO, Fufeyin PT, Okoro SE, Ehinomen I (2015) Bioremediation, biostimulation and

bioaugmention: a review. Int J Environ Bioremed Biodegradation 3(1):28–39

Aislabie J, Saul DJ, Foght JM (2006) Bioremediation of hydrocarbon-contaminated polar soils.

Extremophiles 10(3):171–179

Bhandari G (2018) Environmental nanotechnology: applications of nanoparticles for bioremedia-

tion. In: Approaches in bioremediation. Springer, Cham, pp 301–315

Boopathy R (2000) Factors limiting bioremediation technologies. Biores Technol 74(1):63–67

Bustin SA, Benes V, Nolan T, Pfafﬂ MW (2005) Quantitative real-time RT-PCR–a perspective. J

Mol Endocrinol 34(3):597–601

Cases I, Lorenzo VD (2005) Genetically modiﬁed organisms for the environment: stories of success

and failure and what we have learned from them. Int Microbiol 8:213–222

Coulon F, Al Awadi M, Cowie W, Mardlin D, Pollard S, Cunningham C, … Paton GI (2010) When

is a soil remediated? Comparison of biopiled and windrowed soils contaminated with bunker-fuel

in a full-scale trial. Environ Pollution 158(10):3032–3040

Bioremediation: The Remedy to Expanding Pollution 27

Das S, Lyla PS, Khan SA (2006) Marine microbial diversity and ecology: importance and future

perspectives. Current Sci, 1325–1335

Dash HR, Das S (2012) Bioremediation of mercury and the importance of bacterial mer genes. Int

Biodeterior Biodegradation 75:207–213

Desai C, Pathak H, Madamwar D (2010) Advances in molecular and “-omics” technologies to

gauge microbial communities and bioremediation at xenobiotic/anthropogen contaminated sites.

Biores Technol 101(6):1558–1569

Dias RL, Ruberto L, Calabró A, Balbo AL, Del Panno MT, Mac Cormack WP (2015) Hydrocarbon

removal and bacterial community structure in on-site biostimulated biopile systems designed for

bioremediation of diesel-contaminated Antarctic soil. Polar Biol 38(5):677–687

Doney SC, Ruckelshaus M, Emmett Duffy J, Barry JP, Chan F, English CA, … Talley LD (2012)

Climate change impacts on marine ecosystems. Ann Rev Marine Sci 4:11–37

El Fantroussi S, Agathos SN (2005) Is bioaugmentation a feasible strategy for pollutant removal

and site remediation? Curr Opin Microbiol 8(3):268–275

Folch A, Vilaplana M, Amado L, Vicent T, Caminal G (2013) Fungal permeable reactive barrier to

remediate groundwater in an artiﬁcial aquifer. J Hazard Mater 262:554–560

Frascari D, Zanaroli G, Danko AS (2015) In situ aerobic cometabolism of chlorinated solvents: a

review. J Hazard Mater 283:382–399

Frutos FG, Pérez R, Escolano O, Rubio A, Gimeno A, Fernandez MD, ... Laguna J (2012) Reme-

diation trials for hydrocarbon-contaminated sludge from a soil washing process: evaluation of

bioremediation technologies. J Hazardous Mater 199:262–271

Gidarakos E, Aivalioti M (2007) Large scale and long term application of bioslurping: the case of

a Greek petroleum reﬁnery site. J Hazard Mater 149(3):574–581

Giovanella P, Vieira GA, Otero IVR, Pellizzer EP, de Jesus Fontes B, Sette LD (2020) Metal and

organic pollutants bioremediation by extremophile microorganisms. J Hazard Mater 382:121024

Gomez F, Sartaj M (2014) Optimization of ﬁeld scale biopiles for bioremediation of petroleum

hydrocarbon contaminated soil at low temperature conditions by response surface methodology

(RSM). Int Biodeterior Biodegradation 89:103–109

Gupta K, Biswas R, Sarkar A (2020) Advancement of omics: prospects for bioremediation of

contaminated soils. In: Microbial bioremediation & biodegradation. Springer, Singapore, pp 113–

142

Hobson AM, Frederickson J, Dise NB (2005) CH4 and N2O from mechanically turned windrow

and vermicomposting systems following in-vessel pre-treatment. Waste Manage 25(4):345–352

Höhener P, Ponsin V (2014) In situ vadose zone bioremediation. Curr Opin Biotechnol 27:1–7

Hussain S, Siddique T, Saleem M, Arshad M, Khalid A (2009) Impact of pesticides on soil microbial

diversity, enzymes, and biochemical reactions. Adv Agron 102:159–200

Ishii SI, Suzuki S, Tenney A, Norden-Krichmar TM, Nealson KH, Bretschger O (2015) Micro-

bial metabolic networks in a complex electrogenic bioﬁlm recovered from a stimulus-induced

metatranscriptomics approach. Sci Rep 5(1):1–14

Khan MY, Swapna TH., Hameeda B, Reddy G (2015) Bioremediation of heavy metals using

biosurfactants. Adv Biodegrad Bioremediat Ind Waste

Khardenavis AA, Kapley A, Purohit HJ (2007) Simultaneous nitriﬁcation and denitriﬁcation by

diverse Diaphorobacter sp. Appl Microbiol Biotechnol 77(2):403–409

Kour D, Kaur T, Devi R, Yadav A, Singh M, Joshi D, ... Saxena AK (2021) Beneﬁcial microbiomes

for bioremediation of diverse contaminated environments for environmental sustainability:

present status and future challenges. Environ Sci Pollution Res 28(20):24917–24939

Kuiper I, Lagendijk EL, Bloemberg GV, Lugtenberg BJ (2004) Rhizoremediation: a beneﬁcial

plant-microbe interaction. Mol Plant Microbe Interact 17(1):6–15

Kulshreshtha A, Agrawal R, Barar M, Saxena S (2014) A review on bioremediation of heavy metals

in contaminated water. IOSR J Environ Sci Toxicology Food Technol 8(7):44–50

Leahy JG, Colwell RR (1990) Microbial degradation of hydrocarbons in the environment. Microbiol

Rev 54(3):305–315

28 S. Anand and P. Padmanabhan

Lee JH (2013) An overview of phytoremediation as a potentially promising technology for

environmental pollution control. Biotechnol Bioprocess Eng 18(3):431–439

Lee WC, Lee KH (2004) Applications of afﬁnity chromatography in proteomics. Anal Biochem

324(1):1–10

Maila MP, Cloete TE (2004) Bioremediation of petroleum hydrocarbons through landfarming: are

simplicity and cost-effectiveness the only advantages? Rev Environ Sci Bio/technology 3(4):349–

360

McGrath KC, Thomas-Hall SR, Cheng CT, Leo L, Alexa A, Schmidt S, Schenk PM (2008) Isola-

tion and analysis of mRNA from environmental microbial communities. J Microbiol Methods

75(2):172–176

Meagher RB (2000) Phytoremediation of toxic elemental and organic pollutants. Curr Opin Plant

Biol 3(2):153–162

Mohan SV, Sirisha K, Rao NC, Sarma PN, Reddy SJ (2004) Degradation of chlorpyrifos contam-

inated soil by bioslurry reactor operated in sequencing batch mode: bioprocess monitoring. J

Hazard Mater 116(1–2):39–48

Naik MG, Duraphe MD (2012) Review paper on-Parameters affecting bioremediation. Adv Res

Pharmaceuticals Biol 2(3)

Obiri-Nyarko F, Grajales-Mesa SJ, Malina G (2014) An overview of permeable reactive barriers

for in situ sustainable groundwater remediation. Chemosphere 111:243–259

Pandey A, Tripathi PH, Tripathi AH, Pandey SC, Gangola S (2019) Omics technology to study

bioremediation and respective enzymes. In: Smart Bioremediation technologies. Academic Press,

pp 23–43

Prasad R, Nayak SC, Kharwar RN, Dubey NK (eds) (2018) Mycoremediation and environmental

sustainability. Springer, Cham

Qureshi A, Verma V, Kapley A, Purohit HJ (2007) Degradation of 4-nitroaniline by

Stenotrophomonas strain HPC 135. Int Biodeterior Biodegradation 60(4):215–218

Rawat M, Rangarajan S (2019) Omics approaches for elucidating molecular mechanisms of

microbial bioremediation. In: Smart bioremediation technologies. Academic Press, pp 191–203

Roy M, Giri AK, Dutta S, Mukherjee P (2015) Integrated phytobial remediation for sustainable

management of arsenic in soil and water. Environ Int 75:180–198

San Miguel A, Ravanel P, Raveton M (2013) A comparative study on the uptake and translocation

of organochlorines by Phragmites australis. J Hazard Mater 244:60–69

Sanscartier D, Zeeb B, Koch I, Reimer K (2009) Bioremediation of diesel-contaminated soil by

heated and humidiﬁed biopile system in cold climates. Cold Reg Sci Technol 55(1):167–173

Seigle-Murandi F, Guiraud P, Croize J, Falsen E, Eriksson KL (1996) Bacteria are omnipresent on

Phanerochaete chrysosporium Burdsall. Appl Environ Microbiol 62(7):2477–2481

Shrivastava S, Bera T, Roy A, Singh G, Ramachandrarao P, Dash D (2007) Characterization of

enhanced antibacterial effects of novel silver nanoparticles. Nanotechnology 18(22):225103

Silva-Castro GA, Uad I, Gónzalez-López J, Fandiño CG, Toledo FL, Calvo C (2012) Application

of selected microbial consortia combined with inorganic and oleophilic fertilizers to recuperate

oil-polluted soil using land farming technology. Clean Technol Environ Policy 14(4):719–726

Singh BK, Nazaries L, Munro S, Anderson IC, Campbell CD (2006) Use of multiplex terminal

restriction fragment length polymorphism for rapid and simultaneous analysis of different

components of the soil microbial community. Appl Environ Microbiol 72(11):7278–7285

Smith E, Thavamani P, Ramadass K, Naidu R, Srivastava P, Megharaj M (2015) Remediation trials

for hydrocarbon-contaminated soils in arid environments: evaluation of bioslurry and biopiling

techniques. Int Biodeterior Biodegradation 101:56–65

Taniguchi N (1974) On the basic concept of nanotechnology. Proceeding of the ICPE

US Environmental Protection Agency (USEPA) (2007) Treatment technologies for mercury in soil,

waste, and water

Whelan MJ, Coulon F, Hince G, Rayner J, McWatters R, Spedding T, Snape I (2015) Fate and

transport of petroleum hydrocarbons in engineered biopiles in polar regions. Chemosphere

131:232–240

Exploration of Plant Growth Promoting

Rhizobacteria (PGPRs) for Heavy Metal

Bioremediation and Environmental

Sustainability: Recent Advances

and Future Prospects

Sumita Mondal, Samir Kumar Mukherjee, and Sk Tofajjen Hossain

Abstract Environmental contamination of toxic heavy metals accumulated by fast

industrialization, agricultural and other anthropogenic actions, induces a drastic

harmful effect on living beings, modify the soli characteristic and its biological

action. Among many other existing procedures, microbial mediated remediation

of heavy metals is an eco-friendly and much potent method. Diverse soil micro-

biomes have been perceived as a dominant tool for the sustainable agriculture and

the environment, which contribute an important function in biogeochemical cycles.

The rhizomicrobiome have a wide range of activities for biotic and abiotic stress

tolerance, which can alter the growth and developmental rate of plants. Plant growth

promoting rhizobacteria (PGPR) is a prime cluster to exhibit synergistic and antag-

onistic communications with the soil and involve in an array of pursuit of ecological

signiﬁcance. The present review emphasises current scenario and future research

requirements about their r ole in plant growth promotion and remediation of various

environmental stresses exerted pollutants for agro-environmental sustainability.

Keywords Soil microbiomes ·PGPRs ·Biofertilizers ·Heavy metal ·

Bioremediation ·Sustainable agriculture ·Environmental sustainability ·

Plant–microbe interactions

1 Introduction

With the advent of the industrial revolution, rapid growth of urban areas and anthro-

pogenic liveliness, heavy metal induced environmental pollution become a serious

hazard and challenge in present days. Environmental pollution and toxicity of these

heavy metal components are of considerable ecological concern because of their

non-biodegradable nature and detrimental effect on accumulation. Heavy metals

S. Mondal

Department of Botany, Vivekananda Mahavidyalaya, Burdwan 713103, WB, India

S. K. Mukherjee · Sk T. Hossain (B

)

Department of Microbiology, University of Kalyani, Kalyani 741235, WB, India

e-mail: sktofajjen.hossain@gmail.com

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_3

29

30 S. Mondal et al.

exist naturally in earth environment, however, with the advancement of industrial-

ization throughout the world, the concentration increase beyond of their pestilential

level in nature. These elements move into the ecosystem through the food chain and

accumulates in every trophic level, resulting emerging health risk because of their

mutagenic, carcinogenic, teratogenic effect (Ali et al. 2013; Ahamed 2019).

Heavy metals are inorganic elements with relatively high atomic weight, atomic

numbers, density, and toxic even at very low concentration. Heavy metals are cate-

gorized into three classes: (a) Toxic heavy metals (Mercury, Zinc, Lead, Cobalt,

Arsenic, Tin, Cadmium, Bismuth, Selenium, etc.), (b) Precious heavy metals (Silver,

Gold, Palladium, Platinum, Ruthenium, etc.), and (c) Radionuclides (Uranium,

Thorium, Radium, Cerium, etc.), and also includes other transition metals, metal-

loids, lanthanides, and actinides. Some of them like cobalt, iron, copper, molyb-

denum, zinc, etc. are vitally important in living organism as they act as cofactors of

diverse enzyme and participating in various metabolic pathways as trace elements.

There are numerous conventional physical and chemical remediation methods

like ion-exchange, chemical precipitation, chelation therapy, reverse osmosis, land

ﬁlling, bio-slurries, bio-piles employed for heavy metal remediation in soil and water

bodies. These methods have comprised some negative sides like low efﬁciency,

high-cost chemicals, extended procedure, high energy consumption, soil degradation

and secondary pollution. Nowadays an inclination towards execution of biological

approaches has been launched and become admired due to its relative efﬁciency,

cost effectiveness and eco-friendly nature. This emerging biotechnology has been

integrated with diverse conventional physical and chemical systems for comprehen-

sive management of heavy metal mediated pollution that seems to be a sustainable

approach. Nowadays, the term ‘Bioremediation’ is used to describe such practice.

Bioremediation is a collective process to annihilate toxic contaminants from soil

or water bodies or any other medium, induced by biological means. Various biolog-

ical living organisms likes bacteria, algae, fungi, lichens and as well as plants are

employed in bioremediation method. Heavy metal contamination in soil extremely

affects the biodiversity of plant community of a speciﬁc location. It affects plants

by alter their metabolic processes such as respiration, photosynthesis, cell division,

reproduction, fruit ripening capacity, etc. Moreover, the presence of such harmful

metals in the cell causes an impediment of antioxidant enzyme activity and thereby

generating enormous amounts of reactive oxygen species (ROS) and drastic oxidative

stress in plants. There are some plants that possess the capability to accumulate heavy

metals in high percentages (Brooks 1977). The uses of such hyper-accumulators

plant in the highly contaminated region is now being regular execution proce-

dure throughout the world. A broad range of microorganisms like, Flavobacterium,

Pseudomonas, Bacillus, Arthrobacter, Corynebacterium, Rhodococcus, Methosinus,

Mycobacterium, Stereum hirsutum, Nocardia, Methanogens, Pleurotus ostreatus,

Rhizopus arrhizus, Azotobacter, Alcaligenes, Phormidium valderium, Ganoderma

applanatum, Aspergilus niger, etc. are used in the bioremediation technique (Verma

and Kuila 2019; Shah 2020). The potency of microorganism mediated bioremediation

of soil depends on various components like soil proﬁle, the structure of microﬂora

in the soil, the concentration of heavy metals and types of heavy metals etc.

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 31

Bioremediation carried out in two ways- In-situ process and Ex-situ process.

In in-situ bioremediation methods, the removal of toxic metals takes place at the

site of their origin. Whereas in ex-situ bioremediation methods the contaminated

medium is excavated from the site of origin to other places and then remediation

operation takes place (Paul et al. 2021). In in-situ removal process naturally inhabited

microbes are used to grow in medium containing heavy metals and convert them into

less toxic substance. However, occasionally either new microbial strains are being

instigate within the natural microbe community or provide optimal physical and

chemical environment externally for enrichment of microbial growth to improvise

and accelerate the mechanism of toxic metals r emoval. In many instances microﬂora

is supplemented with oxygen and nutrient materials externally to prop up the growth

and development of microbes in the medium.

Bacteria play signiﬁcant roles in bioremediation as they possess various resistance

mechanisms against heavy metal toxicity. The morphological feature of bacteria like

presence of cell wall, capsule and slime layers obstruct the entry of heavy metals

within cell interior. Another mechanism of active transport of metal ions assisted by

ATPase or non-ATPase types of proteins help to efﬂux metal ions from the cytoplasm.

Diverse extracellular polymeric substances (EPS) produced by bacteria play crucial

role in the adsorption of heavy metal ions like cobalt, mercury, copper, cadmium

(Saraswat et al. 2020).

Plant growth promoting rhizobacteria (PGPR) are a family of plant root colonizer

bacteria that enhance plant growth through diverse mechanism. These are mostly

soil bacteria that capable to undergo symbiotic relationship with plants or sometime

exist as free-living bacteria. PGPR can change the ROS mediated oxidative stress in

plants through producing various antioxidant molecules in plants (Bumunang and

Babalol 2014; Shah Maulin 2021a, b). A variety of PGPR like Rhizobium sp. RP5,

Pseudomonas sp. CPSB21, etc. increase the production of antioxidant enzyme like

superoxide dismutase, catalase, glutathione reductase in plants under stress condition

(Gupta et al. 2018). Some PGPR can transform heavy metals into its less toxic forms

by various oxidation–reduction process and accumulate them within the cell interior

(Mallick et al. 2014). Additionally, through phytovolatilization process some PGPR

convert less toxic heavy metal forms within the plant body (Matsui et al. 2016).

The present review article focusses on the current scenario and future research

requirements about the toxic effect of heavy metals on environment and their

bioremediation with a special emphasises on the role PGPR in plant growth

promotion and remediation of various environmental stresses exerted pollutants for

agro-environmental sustainability.

2 Heavy Metals Toxicity and Environmental Signiﬁcance

Uses of heavy metal in various aspects is escalating day by day to meet the demands

of increasing population. Subsequently, heavy metal mediated pollution becomes a

great threat for environmentalists because these cannot be transformed into non-toxic

32 S. Mondal et al.

or biodegradable substances and as a consequence deposited within the environment

as hazardous waste. Most of the heavy metals present in the environment naturally

and derived from pedogenetic procedures, such as erosion, volcanic activity and

weathering of rocks. Though, various human anthropogenic activities such as mining,

electroplating, smelting and use of pesticides, different chemical fertilizer, biosolids,

intensely rising the deposition of diverse toxic heavy metals in the environment

and interrupt the nature sustainability. Soil, water and air are the major parts of

the environment are contaminated potently due to heavy metal deposition and as

a consequence, homeostasis within biota of ecosystem is hampered signiﬁcantly.

Heavy metals such as, Cd, Hg, As, Cr, Ni, Cu, Pb, and Zn are the most toxic in

the context of environmental pollution. Hazardous impacts of heavy metals in living

organisms depend on the concentration and duration of exposure (Fig. 1).

Effects on soil: Heavy metal mediated soil pollution become a serious concern in the

present days as it has direct consequences on biotic elements of the ecosystem.

Unplanned anthropogenic activities cause rapid changes in soil parameters like

organic matter, clay contents, pH etc. Heavy metals are emerging from mine tailings,

various chemical industrial wastes, improper uses of gasoline and paints, inappro-

priate application of fertilizers and pesticides, sewage sludge, wastewater irrigation,

etc. and get deposited in soil and alter biological and biochemical properties of soil.

Sometimes such modiﬁcations in the soil have a great impact on the maturation and

development of plants as well as soil microbes. Metals may exist either as indi-

vidual elements or in compound form with various soil constituents. Heavy metals

bioaccumulate in the living organisms and their amount increase as they transfer

from creatures of bottom trophic level to upper trophic level, an episode known as

biological magniﬁcation.

The primary effect of heavy metal upon deposit in the soil is to bring an enormous

change in microbial community both qualitatively and quantitatively. A heavy metal

can easily alter the bio-chemical properties of soil, which in turn modify the popula-

tion of microorganism and their activities. Long term exposure to heavy metals can

alter the naturally inhabited microbial community with heavy metal resistant strains

and thereby altering the soil microbial properties like soil respiration rate and enzy-

matic activity. It is found that CO2 can be released in soil exert low contamination

whereas due to high metallic concentrations, soil respiration reduces signiﬁcantly

Fig. 1 Effect of heavy metals and its possible remediation strategies

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 33

by lowering microbial activity. With the increase of heavy metal concentration there

is a sharp decline in enzymatic activity on soil. Heavy metals interfere enzymatic

activity by the altering structure or destroying the group present on the enzyme active

site. By reducing the metabolism, growth, reproduction and biomass of microbes,

heavy metal contamination, also diminish the production and secretion of various

microbial enzymes and metabolites. There is a very intimate connotation between

soil microorganisms and soil enzymes, and most of the living microbes and microbes

secreted enzymes take part in the movement of various elements of soil ecosystems.

Presence of various heavy metals like Cd, Cr, Zn, Pb, Mn has a direct detri-

mental effect on the function of a number of enzymes, such as protease, arylsulfatase,

urease, alkaline phosphatase etc. Arsenic reduces activity of phosphatase and sulfa-

tase whereas Pb has a direct effect on catalase, urease, invertase and acid phosphatase

remarkable. Soil microbes play a signiﬁcant role in recycling and storage of minerals

in nature. Composition of microbial community in the soil had a large impact on the

rate of decomposition and transformation of nutrients. Prolonged duration of heavy

metal exposure changes the decomposition nature of microbes as well as the replaces

microbial ﬂora, which in turn effect on decomposition rate and recycling of mate-

rials in nature. Long exposure of Cr ( VI) causes a huge change in microbial ﬂora in

the soil by effecting on metabolic activity of microbes. Zn and Cd also exert detri-

mental effects on microbial metabolism, therefore can reduce bacterial population

size, diversity and activity (Huang et al. 2009; Hossain et al. 2012; Hossain and

Mukherjee 2012).

Effects on Plants: Plants cannot get away from the undesirable environmental changes

like other organisms due to its sessile nature. Heavy metal exposure increases an array

of physiochemical and metabolic alteration in the plant body. Huge releases of toxic

metals in land due to industrial waste deposition, agricultural malpractices, rapid

urbanization adverse effects on plant proliferation and physical ﬁtness in terms of

reducing biomass, the rate of photosynthesis, yielding capacity, increasing chlorosis,

altering water balance capacity and nutrient assimilation. Some metals such as, Fe,

Co, Cu, Mn, Mo, Zn, and Ni are needed in low amount for plant growth, but at the

concentration rise beyond the optimal level, can lead to toxicity and harmful effects.

Absorption and accumulation of heavy metals in plant depend on temperature, water

content, organic materials, soil pH and nutrient quantity. It has been reported that

heavy metals like Zn, Cd, Mn and Cr accumulates in higher amounts in Beta vulgaris

(Spinach) throughout summer time, whereas higher accumulation of Cu, Pb and Ni

observed at winter time (Sharma et al. 2007).

Toxic effect of heavy metals in plant exhibited by the following mechanisms.

Due to structural similarities, heavy metals compete with other essential nutrient

molecules on root surfaces for being absorbed, such as, As competes the P. Different

heavy metals interact with the functional groups of proteins and disrupt their struc-

tures and function. Generally, most of the heavy metals, generating reactive oxygen

species and impaired the function various biomolecules. Sometime heavy metals can

replace the essential ions from the speciﬁc binding site due to structural similarity

(Sharma and Dietz 2006).

34 S. Mondal et al.

Root is the parts that ﬁrst exposed the excess heavy metal toxicity and as a conse-

quence the root cell division and growth are hampered, resulting altered root length

and biomass. It has been reported that cadmium possesses toxic effect on the expres-

sion of regulatory protein cyclin-dependent kinase (CDK), which play an essential

role in G1-to-S phase transition (Pena et al. 2012). Excess copper has an effect

of auxin redistribution in meristematic zone mediated by PIN1 protein and inhibit

primary root growth found in Arabidopsis thaliana (Yuan et al. 2013). Root defor-

mity was found in plants like the bean (Phaseolus vulgaris) due to high aggrega-

tion of Cu in root (Cook et al. 1997). High accumulation of Mn, Cu, Cd, Zn, and

Ni markedly affect the amount of chlorophyll and carotenoid, which reduces the

photochemical activity of PSII as well. Zn has an adverse effect on Ribulose-1,5-

bisphosphate carboxylase-oxygenase (RUBISCO) activity by replacing the Mg2+ ion

in the active site of the enzyme thus regulating the rate of photosynthesis. Therefore,

heavy metals have substantial impacts on pigment content, photosynthesis rate, the

quantum yield of PSII system, gas exchange through stomata, CO2 assimilation,

nitrogen metabolism, etc. (Maleva et al. 2012).

Greater protease functioning in the cell has been observed due to heavy metal

accumulation and thus reduced essential metabolic activity resulted from lessening

the half-life of various essential enzymes, such as nitrate reductase, nitrite reductase,

glutamine synthetase, Glutamine oxoglutarate aminotransferase, glutamate dehy-

drogenase etc. Cellular nitrogen metabolism is greatly obstructed due to Cd accu-

mulation, which affect directly in nitrate uptake and transportation resulting inhi-

bition in nitrogen assimilation procedure. Excess Hg2+ ions hinder mitochondrial

activities and generate ROS, which distort bio membrane structure and cellular func-

tioning. Due to mercury contamination in plant resulted reduce plant height, inﬂores-

cence formation, low yielding capacity on rice seedlings and tomato. Chromium can

damage the chloroplast structure of therefore inhabiting the electron transport system

and also has an adverse effect on the enzyme of Calvin cycle (Asati et al. 2016).

Cobalt accumulation in plant resulted i n reduction of shoot and root length, low

antioxidant enzyme activity and reduce the amount of sugar, amino acid and protein

contents in various plants like tomato, radish, mung bean etc. Manganese accumulates

in the root and shoot and thereby reduce plant growth was observed due to reduced

chlorophyll a and b, carotenoid content and subsequently decrease the PSII activity.

Heavy metals are able to alter hormonal activities and thereby, responsible for various

physiological changes in plants. On the other hand, arsenic can alter the hormonal

level like, IAA, IBA, NAA etc. as well as alter expression of about 69 microRNAs

in plant body, which directly or indirectly affects on the growth and development of

plants (Singh et al. 2015).

Effects on Human

Some heavy metals like iron, zinc, manganese, etc. are useful for various biological

activities in plant and human body, but a high concentration accumulation of these

elements causes enormous harmful consequences. Generally heavy metals are uptake

by plants and then distributed among trophic levels via the food chain. After entering

in biological system in excessive amount, heavy metals exert severe damages on

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 35

structural integrity of cellular components like Protein, DNA and organelles like cell

membrane structure, mitochondria, lysosome, endoplasmic reticulum, nucleus, etc.

Heavy metals can bind with the protein active site by replacing the active metals

and changes their activities, causing cellular malfunction. Heavy metals produce a

number of reactive oxygen species, thereby creating oxidative stress, which leads

to cell apoptosis. Prolong heavy metals exposure can lead to slowly developing of

degenerative conditions in muscular, and neurological systems and ultimately that

initiate the diseases like, multiple sclerosis, Alzheimer’s disease, Parkinson’s disease

and muscular dystrophy. Regular longer duration vulnerability of some heavy metals

and their combinations may even cause carcinogenic situation.

Effects on Waterbody

Heavy metals from various sources are released continuously in waterbodies creating

a major problem. Manmade activity like industrial sewage efﬂuent, mining, farming,

electronic waste, etc. are major causes for heavy metal discharge. Contaminant metals

are getting dissolved partially of fully in water and accumulated in the sediments of

waterbodies and thus creating a devastating consequence on the ecological equilib-

rium of the aquatic habitat. As these metals are not completely dissolved and there-

fore, they react with particulate matter and precipitate into water sediments. Once

macrophytes and other aquatic phytoplanktons uptake the heavy metals, these toxic

contaminants enter into the food chain and affect every trophic level of an ecosystem.

Organisms at the top level, including human obtain such toxicants by consuming

aquatic animals specially ﬁsh. Consequently, a sharp escalate in the concentration

of heavy metal in the trophic levels of the food chain is observed and the process

termed as biomagniﬁcation.

Heavy metals react with diverse water component such as sulphate, carbonate,

nitrate, etc. and precipitated in the bottom layer as insoluble salt complex. With the

change in the pH level of water bodies due to acid rains or various other reasons, the

metals can exist as their toxic form in water and do contamination. Such pollutants

have great impact in altering the growth, development and reproductive ability of

ﬁshes and other water micrograms. Heavy metals have effect in the development and

functioning of speciﬁc enzyme that can alter the metabolic activity in ﬁshes and mani-

fest cellular toxicity and ultimately leading to death. Different type of toxic metal

form deposited in the sediments can vastly affect the benthic organisms and conse-

quently lessen the food availability for bigger animals such as ﬁsh. Heavy metals

develop various ROS molecules that causes oxidative damages on the organism. A

toxic chemicals compound like methylmercury, produced from the organic mercury

by bacterial methylation, have great hazardous effect on ﬁsh growth. These toxicants

can cause gill necrosis and liver fatty acid degradation in ﬁshes and crustaceans, as

well as inhibit the activity of enzymes involved in cellular respiration, protein produc-

tion (Mishra and Mohanty 2008). The high concentration of heavy metals hampers

the oxygen level of water, which in turn affect the development of phytoplanktons,

zooplankton, mollusks and their reproduction process.

36 S. Mondal et al.

3 Remediation Strategy of Heavy Metals

Heavy metal pollution is a world-wide concern and continuous attainment are given

on this matter. Various chemical, physical and biological methods have employed

to eliminate such pollutants from the environment. A few examples of physical

and chemical methods are: ion exchange, coagulation and ﬂocculation, adsorption,

membrane ﬁltration, solvent extraction, chemical precipitation, electro-chemical

treatment, immobilization, etc. There are some imperfections on applying these

physico-chemical approaches to combat pollutants because of high cost, inefﬁciency

at very low concentration, and contaminant permanent changes of soil properties, etc.

Nowadays, biological remediation or green remediation procedures are implemented

to remove the contaminants, which is considered to be the safest, eco-friendly and

cost-effective approach. In this method various microorganisms like bacteria, fungi,

algae and heavy metal hyper accumulator plants are used.

Physical methods

Membrane Filtration: This technique is applied in waste water treatment. In this

process an appropriate porous membrane such as cellulose acetate, polyamide, poly-

sulfone etc. are being employed through which the solution ﬂow out under different

pressure. This process is categorized into three types: reverse osmosis under high

pressure, reverse osmosis under low pressure and ultraﬁltration. Membrane pore

size, distribution of pores, t he nature of hydrophilicity, amount of surface charge,

solution ﬂow rate and the presence of functional groups are the principal parameters

applied to membrane ﬁltration techniques. The efﬂux rate of solution and speciﬁcity

of the membrane determines the performance of this procedure. Molecules with high

molecular weight, heavy metals, suspended particles are removed by ultraﬁltration

method. It has been reported that reverse osmosis successfully eliminates various

heavy metals and anionic toxicants from metal-plating wastewater. Although this

is an effective process, but has some limitation like, membrane fouling, membrane

longevity and membrane dissolution problem due to oxidizing elements, solvents

and organic compounds (Hube et al. 2020).

Coagulation and ﬂocculation: Coagulation and ﬂocculation are an electro-

static repulsion process that can eliminate heavy metals from solution by neutral-

izing the surface charge of colloidal particles. In this procedure alum (aluminum

sulfate), PACL (polyaluminium chloride), MgCl2 (magnesium chloride), PEI

(polyethyleneimine) and aluminum hydroxide oxides, etc. are required as coagu-

lants. Flocculation involves slow mixing of destabilized particles to agglomerate

into larger particles which can be easily removed by ﬁltration, ﬂotation, or straining

(Tripathy and De 2006).

Ion Exchange: Ion exchange is a pH sensitive method in which the soluble i ons are

attracted from solution to solid phase. It is a cost-effective method and highly appli-

cable for eliminating heavy metals from a liquid solution at very low concentration

of pollutants. Inorganic zeolites or organic ion exchange resins can be used as solid

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 37

phase ion exchanger. Once the resin bed gets saturated by ions, this can be regen-

erated by treating the resin beds with alkaline or acidic medium. It is a reversible

process as resins absorb cations or anions in return release other ions of the same

charge (D¸abrowski et al. 2004).

Adsorption: Adsorption is a very effective surface phenomenon which applied to

remove heavy metals and ions from polluted water by using a suitable adsorbent.

In this method the impurities get adsorbed on the surface layer of adsorbent rather

enter into the internal structure. Impurities get bound tightly with the solid surfaces

by van der Waals forces of attraction or hydrogen bonding. The advantages of this

method are cost effective, eco-friendly, high efﬁciency, minimum sludge formation,

reuses of adsorbent by removing deposited pollutants, etc. Activated carbon is used

profusely to remove organic pollutants from waste water. Photocatalyst beads, red

mud, coal, metals and metal-nanoparticles, activated sludge biomass, zeolite etc. are

being used as adsorbents to remove heavy metals and ions (Chai et al. 2021).

Vitriﬁcation: It is the process of transformation of waste material into the glass like

substances by applying high voltage about 3600°F. The waste present in the soil,

melt down and get embedded into a glassy matrix by chemical bonding, therefore

will not leak out further. The glassy matrix is chemically inert and has a low leaching

characteristic. This method is used to remove hazardous radioactive and organic

waste from soil (Moustakas et al. 2005).

Soil washing: Soil washing is a waste reducing technique which can be applied to the

excavated soil or on-site soil. During this procedure the contaminants are removed

from bulk soil by physical separation method and separated by applying aqueous

chemical. Lastly the solution is recovered by chemical extraction of waste. This is a

cost-effective, fast process and clean up the soil completely (Gusiatin et al. 2020).

Chemical Methods

Chemical immobilization: In this technique, pollutants are trapped by employing

chemical agents and convert them into immobilize form. Through, in this process the

pollutants are not physically removed from the soil, but the mobility of the pollutants

through water current is blocked and thereby minimize their accumulation in plants

and soil microorganisms and water bodies (Tajudin et al. 2016).

Solidiﬁcation: It is a solidiﬁcation technique of contaminated soils by applying

various binding agents like asphalt, cement, ﬂy ash, clay, etc. and transformed the

soil into a solid block. This solid soil block is impermeable to water, thus entrapping

the contaminants and stop being leached out over a long time (Tajudin et al. 2016).

Stabilization: Stabilization is an in-situ ﬁxation process where various stabilizing

agents are incorporate in the soil to initiate physiochemical interactions between

contaminated heavy metals and stabilizing agents and to check their mobile nature.

Various chemical substances can be used as stabilizing agents such as carbonates and

phosphates groups containing material like, bone meal, hydroxyapatite, ammonium

phosphate, apatite etc., alkaline agents like, calcium hydroxide, ﬂy ash, etc., iron rich

38 S. Mondal et al.

minerals and clay like, goethite, bauxite, silica gel, red mud, greensand, vermiculite,

zeolites, etc., and different organic matter like, xanthate, chitosan, starch, compost,

manure, biochar, activated carbon, etc. Among the various materials phosphates

and carbonates containing compounds are the most propitious representative for

effectively stabilizing heavy metal contaminants in polluted soils (Liu et al. 2018).

Neutralization: This is basically a precipitation reaction based neutralization process,

where common alkaline reagents like lime, limestone, ferrous compound and other

salt materials such as CaCl2, Mg(OH)2,MgCO

3,BaCl

2 etc. are used to eradicate

heavy metal pollutants. The main function of these agents is to raise the pH level

of water and thereby neutralize the heavy metal ions. The precipitated compounds,

especially phosphate salts of various heavy metals like magnesium, iron, manganese,

copper, etc. are further used as fertilizer in agricultural ﬁeld. Calcium Oxide is an

effective reagent than other lime because its temperature insensitive nature and CaO

can neutralize cations by formation of hydroxyl ions through partial dissolution

(Hermansson and Syaﬁie 2015).

Solvent Extraction: This is a hydrometallurgical treatment which has been employed

in the operation of various metals like, gallium, cobalt, copper, uranium, nickel, zinc,

molybdenum, hafnium, indium, germanium, platinum, boron, vanadium, tungsten,

zirconium, niobium etc. Organic compounds which are almost insoluble in water

used as extractant in this procedure. This treatment process depends on the extractant

types, anions present in water and pH of water. Organophosphorus is often used in the

process of metal separation. Di-(2-ethylhexyl)-phosphoric acid (D2EHPA) is used as

an excellent extractant to recover zinc and copper at very low pH, as well as chromium

and nickel at pH 6–7. Organophosphorus like Cyanex 272 is also employed for the

removal of various metals like, Fe, Zn, Cr, Cu, Ni etc. in a solution of sulfuric and

sulfate form. It was also reported that the fruitful removal of cobalt from nickel can

be achieved by these compounds around 99.4% at pH 6 (Mubarok and Hanif 2016).

Chemical Precipitation: It is a widely practiced method where pH sensitive transfor-

mation of various heavy metals into sulﬁde, hydroxide, carbonates, or any other less

water soluble compounds takes place. These transformed component compounds

are then easily eliminated by sedimentation, ﬁltration, or ﬂotation techniques. The

efﬁciency of this technique stands on the chemical nature, size, density and surface

charge of the pollutants. Various precipitants materials such as caustic soda, soda

ash, lime, sodium bicarbonate, sodium sulﬁde, sodium hydrosulﬁde, etc. are Some

of the largely used for different heavy metal precipitation. Various kind of chem-

ical precipitation have been employed such as carbonate precipitation by using soda

ash or sodium carbonate, hydroxide precipitation by adding alkali or lime), sulﬁde

precipitation by using sodium sulﬁde or sodium hydro sulﬁde, xanthate precipitation

to eradicate different metals like, Cd, Cr, Hg, Zn, Ni etc. In combined precipita-

tion treatment more than one precipitant are used in combined form such as uses of

carbonate and hydroxide precipitation (Raﬁque et al. 2022).

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 39

Biological Methods

Biological remediation methods are also termed as phytoremediation, green reme-

diation, botanoremediation, agroremediation, which is an eco-friendly remediation

procedure, carried out by using vegetation, microbiota, soil amendments, and agricul-

tural methods to remove environmental contaminants. Phytoremediation generally

is an energy proﬁcient procedure for heavy metal remediation in place containing

little to medium levels of toxicants, and can be employed in together with other

more conventional remedial systems as a complete step in the remedial procedure.

The phytoremediation techniques have so much superiority in comparison with

various physical or chemical remediation procedure such as, eco-friendly nature,

cost-effectiveness, no need of disposal site, no need to excavate and transport the

polluted media and at a time more than one pollutant can be cleaned. There are

various types of technically different biological remediation methods are exploited

to eradicate heavy metals from environment like, phytoextraction, phytostabilization,

phytovolatilization, phytoﬁltration, phytodegradation, phytostimulation, etc.

Phytostabilization: In this technique heavy metals are transformed into their immo-

bile form by metal-tolerant plant species and thereby reducing bioavailability and

leaching. Phytostabilization process accelerates the reactivity and precipitation of

heavy metals present in the rhizospheric soil, and thereby induce bio-adsorption on

the root cell wall and compartmentalization of heavy metals within root tissue, there-

fore hindering the entry of metal contaminants into the food chain. Plants through

their root system can prevent soil erosion and leach out of toxic metals through

ground water ﬂow and, therefore, selection of plants for phytostabilization process

is very crucial. Some important features are essential for plants chosen for this

technique such as, it must have a dense root system, high metal-tolerant, easy to

plant and maintenance, rapid growth, longevity and easy to propagate (Alkorta et al.

2010). Various organic and inorganic materials like biosolids, litter, Zinc, phosphate,

limes are used as soil amendments to enhance the phytostabilization process. These

matters can alter metal solubility and reactivity by changing the soil pH. Moreover,

the organic amendment acts as a nutrient source for soil microorganism and induce

their ﬂourishment. Sometime these microorganisms also assist metal stabilization

process by adsorbing metals onto their cell surface, improving precipitation process

and chelation by secreting chelators of heavy metals (Göhre and Paszkowski 2006).

Phytoextraction: In this process hyperaccumulator plants absorb heavy metals from

rhizospheric soil and transport them to the ground above tissue. It is a permanent reso-

lution for complete eradication of pollutants from soil and therefore ecofriendly and

more commercially relevant. The phytoextraction of heavy metal contaminants using

hyperaccumulator plants performed by some stepwise procedure such as, transloca-

tion of heavy metals in the rhizosphere, then uptake of heavy metals through plant

roots, and after translocation from the roots to aerial parts of plant, the heavy metal

ions sequester and accumulate in the plant tissues (Ali et al. 2013). The effectiveness

of this procedure depends on plant category, health of the plant, concentration of

heavy metals and also various physical and biochemical properties of the soil. The

40 S. Mondal et al.

exploited plants must have the higher level tolerance ability, huge extraction capa-

bility, great metal accumulation capacity, considerable root and shoot growth, high

root absorbance capacity, large biomass production ability, substantial disease resis-

tance power and highly adaptive nature with changing environmental conditions

(Ali et al. 2013). Mainly there are two types of phytoextraction mechanism like,

natural phytoextraction and induced phytoextraction. In the natural phytoextraction

process, the hyperaccumulators plants are readily extract heavy metal contaminants

from the soil. Generally, hyperaccumulators are those plant species that possess the

capability to accumulate heavy metals at a rate more than 100-fold greater than

those of the normal plants. There are about more than 400 species belonging to 45

different families have been so far contemplate as hyperaccumulators. Plants belongs

to Brassicaceae, Fabaceae, Euphorbiaceae, Asteraceae, Lamiaceae, and Scrophulari-

aceae families are considered as good hyperaccumulators (Ali et al. 2013). In case of

induced mechanism, different chelating agents are used in soil for phytoextraction.

Chelating agents like EDTA easily gets bound with the metals to form metal-chelator

complexes and easily uptake by the plants.

Phytovolatilization: It is a process where toxic heavy metal contaminants, absorbed

by the root system and then are converted into the less toxic volatile form, which are

released by the stomatal opening and stem. Through this method various toxic organic

materials and heavy metals such as As, Se and Hg can be released. A few members

of the Brassicaceae family like Brassica juncea, tobacco plants and populus plants

are capable to volatilize different heavy metals and dispersed in the atmosphere.

Inorganic Se is converted into selenomethionine, which is a less toxic volatile form

and released in the air. Tobacco plants are able to vaporize ionic Hg into its less toxic

form. The most advantage of this mechanism is that there is no need to harvest the

plant separately and no need of disposal (Terry et al. 2000; Limmer and Burken 2016).

Phytoﬁltration: Phytoﬁltration is a special type of ﬁltration procedure for separation

of pollutants from water surface by using the root (rhizoﬁltration), shoots (cauloﬁl-

tration), or seedlings (blastoﬁltration). In rhizoﬁltration process heavy metals are

adsorbed or absorbed by the root system. Some chemicals are secreted from roots

that changes the pH of the rhizospheric soil environment, causes heavy metal precip-

itation and change the mobility of heavy metals. Mostly hyperaccumulator plants

are exploited for this technique. A number of aquatic species such as water hyacinth,

duckweed, Azolla, poplar and cattail are usually employed for eradicating toxicants

in water bodies. Various terrestrial plants like Indian mustard and sunﬂower are

the best uses in this technique as because of their dense root system (Olguín and

Sánchez-Galván 2012).

Phytodegradation: Phytodegradation is a process performed by the plants, where all

the steps like, transformation, breakdown, mineralization, mobilization of heavy

metals happen by using various self-producing enzymes. This efﬁciency of this

technique depends on various factors like heavy metals composition and concen-

tration, characteristic of plant species being used, soil proﬁle. Depending upon

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 41

such factors, pollutants may escape through rhizospheric soil or may entrapped by

phytosequestration or phytodegradation (Muthusaravanan et al. 2018).

Mycoremediation: In this process various fungal strains are exploited to remove

heavy metal contaminants. The fungus produces various kinds of degradative enzyme

which is utilized to eradicate heavy metal contaminants from water and soil. Various

fungal species such as Pleurotus ostreatus, Agaricus bisporus, Lentinus squarrosulus,

Phanerochaete chrysosporium, P. ostreatus, P. pulmonarius, Trametes versicolor etc.

are being employed in this technique. Generally, heavy metal polluted soil profuse

growth of the Ascomycetous, Basidiomycetous and Arbascular mycorrhizal fungal

strain in rhizosphere have been reported. Fungal hyphae penetrate deeply through

soil and secretes various chelating compounds to adsorb heavy metals. Mycorrhizal

fungus can produce oxaloacetate crystal through which they can immobilize and

detoxify heavy metal toxicants (Gadd et al. 2014).

Phycoremediation: Several micro- and macro-algae belonging to chlorophycean and

cyanophycean algae are being employed in the bioremediation process for its eco-

friendly and less expensive nature. Wide numbers of blue green algae are widely

used to remove heavy metal contaminants from waterbodies. Metals are gener-

ally taken up by the adsorption process and then transported into the cell inte-

rior through chemisorption, where these pollutants get bounded by poly phosphate

bodies (Dwivedi 2012). Microbial polyphosphate inclusions can easily sequester

various heavy metals like Hg, Ti, Pb, Mg, Zn, Cd, Sr, Co, Ni and Cu. There are

many algal species like Lyngbya putealis, Sargassum myriocystum, Scenedesmus sp.,

Enteromorpha intestinalis, Cladophora glomerata, Ulva lactuca, Euglena gracilis,

Chlorella vulgaris, Phormidium sp. Spirogyra sp., Oscillatoria sp., etc., could easily

remove various heavy metals such as Cu, Co, Cd, Cr, Pb, Ni, Mn, Zn, As, etc. from

the medium (Singh et al. 2017).

Bioaugmentation: Bioaugmentation is an approach of using microbes indigenously

or exogenously to polluted media and subsequent eradication of heavy metals.

Successful application of a consortium of different strain over a single strain is

found to be more effective in the removal of various metal pollutants like, Al, Cd,

Cr, Fe, Ni, Pb, Zn from the medium.

4 Plant Growth Promoting Rhizobacteria (PGPR)

Rhizosphere is the soil zone immediate to root surface and is over ﬂooded by the

various nutrients. Plant roots exudate a large number of amino acids, monosaccha-

rides, organic acids, that support the growth of microﬂora in the vicinity of the root.

Rhizosphere is inhabited by a wide array of microorganisms that execute potential

effect on plant health. Some rhizospheric soil microorganisms are pathogenic in

nature and show a negative impact on the plant health, whereas some are advan-

tageous for plant proliferation. PGPRs belong to the second type of microbes that

42 S. Mondal et al.

have an immense positive impact on the health and enlargement of the plants. These

are root colonizing bacterial groups which can undergo symbiotic relationship with

the plant or may remain as a free-living microbe (Fig. 2). There are mainly two

types of PGPRs exist in the environment such as, ePGPR or extracellular plant

growth promoting rhizobacteria and iPGPR or intracellular plant growth promoting

rhizobacteria. Extracellular PGPR group of bacteria reside in the outside the cell,

whereas iPGPRs inhabit within the cell, formed a specialized structure form called

nodules.

An ideal PGPR possess some characteristic such as, it should inhabit in

rhizosphere, should be ecofriendly, should possess efﬁcient colonizing ability

with plant roots, should able to promote plant growth, should exhibit a

broad spectrum of action, should demonstrate better competitive skills over the

existing rhizobacterial communities and should be tolerant of physicochemical

factors like heat, desiccation, radiations and oxidants. Some good examples of

ePGPR include are Azotobacter, Azospirillum, Bacillus, Caulobacter, Chromobac-

terium, Erwinia, Serratia, Agrobacterium, Flavobacterium, Arthrobacter, Micro-

coccus, Pseudomonas and Burkholderia, whereas Allorhizobium, Bradyrhizo-

bium, Mesorhizobium and Rhizobium are some candidates of the iPGPR category.

On the basis of functions PGPR can also be classiﬁed into some category, such

as, biofertilizer, phytostimulators, rhizoremediators and biopesticides. Phytostimu-

lators PGPR have the capability to stimulate plant growth by phytohormones produc-

tion, whereas rhizoremediators PGPR are able to remediate heavy metal pollution.

Biopesticides PGPR are able to control the pathogen attack by secreting various toxic

metabolites and lytic enzymes (Ahemad 2019).

PGPRs also include nitrogen ﬁxing rhizobacteria that undergo symbiotic rela-

tionship with leguminous plants and provide nitrogen to the growing plants. These

bacteria either reside within the plants in a specialized structure called nodule or

may present on the root surfaces. There are two mechanisms by which PGPRs effect

on the plant growth as direct method or indirect method. In the direct method the

PGPRs improve plant health and development by increasing nutrient uptake, nitrogen

ﬁxation, phosphate solubilization, phytohormone regulation, etc., thereby exerting

Fig. 2 Contributory roles of plant growth promoting rhizobacteria in sustainable development

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 43

effects directly on the plant growth. Where as in indirect method the PGPRs enable

plants to ﬁght against phytopathogen and induce resistance power, which are helpful

to overcome stress condition. PGPRs can synthesize various kinds of phytohormones

like IAA, cytokinin, gibberellin and also alter the production of these hormones in

the plant body, which are a direct effect on the crop yield and biomass formation.

The i nteraction between rhizosphere soil and microbes has a direct relation to photo-

synthetic yield of plants, which meet global food demand and green evolution with

respect to increase the population on earth. There are several PGPRs that are reported

to increase the crop yield, seedling vigor, antibiotic, salt and fungicide resistance,

drought resistance, nutrient uptake capability of rice.

Biofertilizers can be described as materials that contain living microorganisms

which can be spread on seeds, plant body, or soil, resulting colonization into the

rhizosphere or the interior of the plant, and enhance plant proliferation by esca-

lating the availability of various main nutrients to the host plant. Uses of PGPRs as

biofertilizer not only a beneﬁcial effect on the plants to collect the proper amount of

nutrient uptake like N, K and P from soil, but also balanced the recycling of nutri-

ents in the environment. Biostimulants are substances that stimulate plant growth

and maintaining plant health without affecting nutrient supply, soil quality, pesti-

cide resistant. Some PGPRs act as biostimulants by enhancing the phytohormone

synthesis and assisting to overcome various biotic and abiotic stresses. Now a day in

agricultural ﬁeld various PGPRs like, Pseudomonas, Bacillus, Enterobacter, Kleb-

siella, Azobacter, Variovorax, Azosprillum, etc. used as biostimulants. PGPRs are

also involved in bioremediation process and prove to be highly effective in heavy

metal eradication from contaminated soil. PGPRs can alter the bioavailability of

metal-ions by affecting on their mobility in soil and also can modify toxic metals

to less toxic forms. PGPRs also used as biopesticides that accelerate plant prolifera-

tion by inhibiting various phytopathogenic representatives. PGPRs can able to resist

pathogenic attack by producing diverse secondary metabolite compounds as well as

inducing systemic resistance in plant body (Basu et al. 2021).

5 How do PGPR Scrimmage with Heavy Metal Toxicity?

PGPRs are utilized in the bioremediation procedure because of their broad-spectrum

resistance or tolerance mechanism against nonessential heavy metal. These mech-

anisms are mostly regulated by genes present either in chromosomes or plasmid

DNA. There are several mechanisms through which PGPRs can resist heavy metal

toxicity like, permeability barrier for metal exclusion, active transport of metals from

microorganisms, extracellular sequestration, intracellular sequestration, altering the

cellular target of metals, detoxiﬁcation of heavy metals and passive transport system

(Bruins et al. 2000). Permeability barrier for metal exclusion is exerted by bacterial

cell wall, extracellular layers, exopolysaccharide layers, capsule, directly prevent

the entry of toxic metal. Some time by genetically changing the protein channels

present on cell membrane PGPRs can operate selective permeability of heavy metals

44 S. Mondal et al.

within the cell interior. Heavy metals get bind non-speciﬁcally with the various extra-

cellular polymeric substances, outer lipopolysaccharides, protein molecules present

in cell membranes, outer membrane envelope and subsequently are prevented to

interfere with various metal sensitive cellular biochemical processes (Bruins et al.

2000). Biosorption is a passive uptake process of metal ions, which takes place in a

metabolic independent pathway by biologically dead or inactive materials. A number

of bacterial genera like Bacillus, Pseudomonas, Streptomyces etc. can act as biosor-

bent and widely used in pharmaceutical and food industries (Vijayaraghavan and

Yun 2008). Biosorption of heavy metals at extracellular layers and cell matrix takes

place via various anionic functional groups such as, carboxyl, sulfonate, sulfhydryl,

hydroxyl, amine and amide etc. and thereby inhibit the intracellular entry of diverse

toxic metal ions. It has been reported that bioﬁlm formation of microorganisms can

be an efﬁcient measure of multi metal resistance by combining the action of chemical,

physical and physiological processes (Harrison et al. 2007). A consortium of microbe

can help to colonize bacteria and produces bioﬁlms in a moist environment where

nutrient molecules are present sufﬁciently. Such colonization of microbes helps them

to tolerate heavy metal toxicity. Various macromolecules present in bioﬁlm struc-

ture can exhibit electrostatic interactions, hydrogen bonding and London dispersion

forces and the extracellular polymeric substances present in the bioﬁlm carried out

an active role in biosorption of metal ions.

Active transports or efﬂux systems of metallic ions are either ATPase dependent

or independent and responsible for the largest category of metal resistance systems in

microorganisms. A large number of bacteria possess efﬂux transporter, which have

high afﬁnity for metal ions at very low concentration and excrete them outside the

cell, thereby keep the cell interior completely free from toxicants. This resistance

system can be mediated by chromosomal or plasmid encoded proteins. Chromosomal

or plasmid mediated resistance to arsenate [As(V)] and arsenite [As(III)] has been

observed in E. coli, Staphylococcus and various other bacteria. Arsenic resistance is

solely dependent on the expression of ars operon, which is made up of three to ﬁve

genes, namely arsR, arsA, arsD, arsB, and arsC which are responsible for encoding

an ATPase efﬂux pump and detoxifying enzymes arsenate reductase and arsenic

oxidase (Ben Fekih et al. 2018). In Bacillus sp. S. aureus, cad operon mediated and

in Alcaligenes eutrophus, czc operon mediated cadmium efﬂux pump is found. In E.

coli, Pb(II) resistance mediated by the gene ZntA (Etesami 2018). P-type ATPases,

another example of an ATPase transport of metal ions is cop operon mediated efﬂux

of Cu(II) ions in Enterococcus hirae. This operon is consisted of four genes, namely

copA, copB, copZ, copY. The Cu(II) uptake mediated by copA gene encoded ATPase

and a P-type efﬂux ATPase encoded by the copB gene (Etesami 2018). In E. coli a

multi protein complex CusCBA assists in Cu efﬂux. A RND protein encoded by CusA

is controlled by the proton motive force and export Cu out of the cell interior with

the help of CusB and CusC gene products (Cha and Cooksey 1991). In extracellular

sequestration mechanism, metal ions are accumulated in the periplasmic space or

complexes with various cellular compounds as insoluble forms. Metal precipitation

is an example of extracellular sequestration. Zinc ion is an essential trace element for

cells, but excessive accumulation causes toxicity for cell viability. Once this ion has

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 45

been excreted out from the cell, re-entry is strictly controlled by the post efﬂux control

mechanism in several resistance strains. The mechanism involved precipitation of

various metal ions, attaching to speciﬁc binding proteins within the periplasmic space

(Choudhury and Srivastava 2001).

In recent years, using various biosurfactant for the removal of metals and metal-

loids from contaminated medium has gained huge interest in the ﬁeld of bioremedi-

ation due to their low toxic nature and biodegradable properties. Biosurfactants can

be deﬁned as the surface-active biomolecules produced by diverse microorganisms,

which have the property of a metaloid complex formation. It has been established

that P. aeruginosa and B. subtilis can remove heavy metals Cu and Zn with the

production of biosurfactant surfactin, rhamnolipid, and sophorolipid. Biosurfactant

produced by bacteria possess selectivity and species-speciﬁc metal binding capacity,

can efﬁciently remove metalloids via ion exchange, counter binding and precipita-

tion process. The ionic character of biosurfactants may be positive or negative, which

can bind strongly anionic or cationic metal-ions respectively. Another biosurfactants

lipopeptide, produced by Bacillus subtilis has been applied to decontaminate polluted

soil by removing various contaminated heavy metals and hydrocarbon compounds.

Sophorolipids, a biosurfactant compound, produced by Torulopsis bombicola,isalso

able to remove more than 60% of Zn and 25% of Cu from contaminated soil (Mulligan

and Yong 2004).

Intracellular sequestration is another mechanism where bacteria can accumulate

toxic metals within the cell interior, so as to prevent metal ions from exposure to metal

sensitive cellular components and biochemical pathways. Some PGPRs can convert

some metals into innocuous form and accumulate within the cell. A metal resistant

strain of Synechococcus sp. possess smtA gene, which encode metallothionein, is

a class of cysteine rich protein that binds metals in cell cytoplasm and maintain

the concentration of metals intracellularly, thereby regulate various metal sensitive

metabolic pathways. A number of PGPRs like P. aeruginosa, P. putida and Anabaena,

can produce metallothionein under certain stress condition and sequester diverse

heavy metals. It was reported that Rhizobium leguminosarum exposed to Cd stress,

the production of glutathione, which is a metallothionein protein, increases in several

fold (Blindauer et al. 2002).

By producing siderophores, are low molecular weight iron binding chelators

molecules, microbes can tolerate heavy metal stress and convert the toxicant into non

available form. P. stutzeri KC can precipitate various metals like, Co, Cu, Ni, Pb and

Zn by producing siderophores. It has also been reported that siderophores can increase

chlorophyll production and thereby support plant growth under heavy metal stress.

Psychrobacter sp. SRS8 can produce catechol and hydroxamate type siderophores

and can enhance the growth and development of certain plants like, castor and

sunﬂower under nickel and other metal stress condition (Ma et al. 2011). By the

mechanism of siderophore production PGPRs can grow in multi-metal contaminated

mine tailing soil. Bacterial siderophore can increase iron absorption capacity of plants

and reduce toxic metal concentration in cell by binding them speciﬁcally. P. aerugi-

nosa decreases the Al toxicity by producing pyoverdine and pyochelin siderophores.

In Streptomycetes, siderophore production stimulates iron uptake simultaneously

46 S. Mondal et al.

decreases Cd uptake. Therefore, evidences showed that by siderophore production

PGPRs can resist metal induced toxicity and also prevents plants from being chlorotic

(Bruins et al. 2000;Etesami 2018).

Methylation is another process adopted by PGPRs to eradicate metal toxicity

by volatilization of metallic ions. PGPRs are able to convert various metallic ions

like Se, Sn, Te, As, Pb, etc. into their methylated volatile form and thus promote

their diffusion from the cell. Various bacterial species like, Bacillus sp., Clostridium

sp., and Pseudomonas sp., are able to transform Hg (II) to gaseous methylated Hg.

Various evidences have been reported for bio-methylation such as, arsenic (As)

to gaseous arsines, lead (Pb) to dimethyl lead, selenium (Se) to volatile dimethyl

selenide (Park et al. 2011). Several PGPRs can detoxify metal ions by enzyme assisted

oxido-reduction process. Several gram positive and gram negative bacteria possess

mercury resistance genes cluster mer operon in the chromosome. These operons

mainly encode two enzymes such as, mercuric ion reductase and organomercurial

lyase. The C-Hg bond can breached by organomercurial lyase and yield Hg(II) form,

which is then again reduced to Hg(0) by the another enzyme mercuric ion reduc-

tase. Hg(0) is basically inert and water soluble, can easily release through the cell

membrane (Bruins et al. 2000). Some other natural remedies have been adopted

by PGRPs to reduce metal toxicity. Microorganisms by altering their cellular reac-

tivity for metal ions can by-pass the deleterious effect of heavy metals, but such

decreases of cellular response may not hamper the basic function of cells. Some time

by increasing production of a particular cellular component to keep ahead of metal

inactivation.

6 Direct and Indirect Impact of PGPR on Plant Health

PGPRs have great impacts on plant growth and development. PGPRs can impro-

vise plant growth and yielding capacity either by direct mechanism or by indirect

mechanism. PGPRs directly promote the plant growth and development by either

helping in nutrient element like, nitrogen, phosphorus and essential mineral acqui-

sition. PGPRs can also indirectly enhance the plant growth and development either

by regulating diverse plant hormone levels, or by reducing the effect of various plant

growth inhibitory pathogens.

Direct impact of PGPRs

Phosphate solubilization: The phosphorus (P) is a macronutrient which is found in

insoluble form in the soil and soil microbes play an important role in its recycling

process. PGPRs transform soil insoluble phosphorus into a soluble form and then

plant can easily uptake from the soil. Phosphorus has played several crucial roles

in the plant metabolism process like, respiration, photosynthesis, membrane forma-

tion, carbon metabolism and energy transfer process. Phosphate also plays a vital

role in binding of heavy metals on cell wall in the form of metal-phosphate complex,

thereby decreasing in heavy metal intake by the plants and subsequently increase

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 47

the heavy metal tolerance. Phosphate solubilizing bacteria (PSB) secrete various

enzymes that needed to convert soil insoluble phosphorus into solubilizing form. PSB

can also improvise nutrient availability in soil by phosphorus mobilization via phytase

enzyme secretion A number of genera, are i solated from different rhizospheric

soil are found to be PSB group, like Pseudomonas, Stenotrophomonas, Bacillus,

Cupriavidus, Agrobacterium, Acinetobacter, Arthrobacter, Pantoea, Rhodococcus

etc. (Billah et al. 2019).

Sequestering Iron: Though iron is very important nutrient for plants’ growth

and development, but neither plants nor microbes can readily uptake iron from

rhizospheric soil. Most of the iron is present in the ferric ion form in the soil but is

poorly soluble in water, and therefore, the assimilation rate of plants and microbes is

very low. Under these circumstances, some bacterial species can synthesize certain

low molecular weight compounds designated as siderophore to trap iron efﬁciently

from soil by forming Fe-siderophore complex. Inoculation of P. ﬂuorescens C7

in Arabidopsis thaliana plants, leads to the formation of Fe-pyoverdine complex,

which is responsible for increasing iron concentration within plant tissues (Vansuyt

et al. 2007).

Potassium solubilization: In nature most of the potassium exists in three different

forms viz., readily exchangeable form, slowly available form or unavailable form.

Readily available potassium remains mixed with as oil, water and 1–2% present on

clay surface. Potassium plays several vital roles in the plant metabolism process like,

controlling cellular osmotic pressure, cell turgidity, water movement, nutrient move-

ment, stomatal opening and closing, activation of various plant enzymes and provide

protection against pathogens. Potassium solubilizing bacteria can solubilize insoluble

potassium through various processes such as, acidolysis, altering pH, by organic acid

production, capsule absorption and enzymolysis reactions. Bacteria by producing

diverse organic acids like, tartaric acid, oxalic acid and citric acids, easily reduces

the soil pH level. Acidiﬁcation of soil causes dissolution of potassium from micas,

illite and orthoclase, and thereafter make it available for plant uptake. In another

mechanism some bacteria secrete slime or acidic polysaccharides, which can form

bacterial mineral complex and assist in releasing potassium from silica. A number

of microbes like, Paenibacillus glucanolyticus, Agrobacterium tumefaciens, Rhizo-

bium pusense, Burkholderia cepacia, Enterobacter aerogenes, Pseudomonas azoto-

formans, P. orientalis, Microbacterium foliorum, Myroides odoratimimus, Pantoea

agglomerans, Bacillus licheniformis, B. subtilis, Pantoea agglomerans, Rahnella

aquatilis, etc. are reported to be potassium solubilizing bacteria (Sattar et al. 2019).

Zinc solubilization: Zinc is a micronutrient and act as cofactor of various metabolic

enzymes of plant like tryptophan synthetase. Zinc is available as an insoluble form in

a very low concentration in rhizospheric soil. Various bacteria have been reported to

improvise zinc uptake by plants, as for example Acinetobacter sp., Bacillus aryab-

hattai, B. subtilis, B. cereus, B. Megaterium, B. tequilensis, Pseudomonas aeruginosa,

P. fragi, Pantoea dispersa, P. agglomerans, Agrobacterium tumefaciens, Rhizobium

48 S. Mondal et al.

sp., etc. Soil microbes help in zinc absorption either by lowering soil pH or by chela-

tion process. Soil microbes solubilize zinc by secreting various organic acids like 2-

ketogluconic acid, 5-ketogluconic acid etc. (Kaur et al. 2021).

Siderophores production: Siderophores are low molecular weight compound having

iron binding capability. On the basis of moiety that donates oxygen ligands for Fe3+

coordination and mainly there are three categories of siderophores like, hydroxam-

ates, catecholates or carboxylates and mixed type. Iron is an essential element plant

cellular metabolism, which is needed to carry out electron transport chain, oxidative

phosphorylation, photosynthesis, tricarboxylic acid cycle, and biosynthesis of

various essential biomolecules like, nucleic acids, vitamins, antibiotics, toxins,

pigments, cytochrome and porphyrins. In iron limiting condition, by secreting

siderophores, microbes help to convert insoluble form Fe3+ form into a soluble Fe2+

form and make available for plants. Additionally, microbial siderophore produc-

tion accelerates the heavy metal movability in contaminated rhizospheric soil by

capturing t hem and increase the metal availability in the rhizosphere through a series

of complex reaction. Siderophore producing microbes reported so far includes all

known genera like, Achromobacter sp., Arthrobacter sp., Bacillus sp., Streptomyces

sp., Pseudomonas sp., Staphylococcus sp., Curtobacterium sp., Plantibacter sp., etc.

Siderophore producing bacteria can enhance the plant growth and development in

heavy metal contaminated area by increasing chlorophyll contents and also reduce

the other toxic heavy metals uptake by the plants. Thus, siderophore producing

PGPRs can alleviate and promotes plant growth and development by increasing

nutrient uptake, particularly Fe and reducing other heavy metal absorption, results

reduced oxidative stress. Siderophore producing PGPRs are also playing a role in

controlling the activity of different plant hormones under metal stress condition

(Kaur et al. 2021).

Nitrogen ﬁxation: Nitrogen is an important element as it is the structural components

of nucleic acids, proteins, and other biomolecules. But plant cannot ﬁx nitrogen itself

from the atmosphere directly. Direct ﬁxation of nitrogen from the atmosphere is

mediated by microbes only. Nitrogen ﬁxation takes place in two ways like, symbi-

otic nitrogen ﬁxation and non-symbiotic nitrogen ﬁxation. Atmospheric nitrogen

needs to be transformed into ammonia to make it available for plants. Microbes

possess an enzyme complex nitrogenase that converts nitrogen to ammonia. Bacteria

can ﬁx atmospheric nitrogen by several mechanisms like, ammoniﬁcation, nitriﬁca-

tion, denitriﬁcation and thereby complete the biological nitrogen cycle. A symbiotic

association of Rhizobium in the legume plants can able to ﬁx more than 50% of

total biological nitrogen ﬁxation. Rhizobium is species speciﬁc strain and induce

nodulation on speciﬁc plant legume. Therefore, application of bacteria in soil as

bio-fertilizer must be selective. It has been reported that PGPRs improves ROS

scavenging activity by up taking nitrogen under heavy metal stress environment by

increasing the synthesis of enzyme glutathione reductase. Diazotrophs are a class of

PGPR that establish a non-obligate relationship with non-leguminous plant and ﬁx

nitrogen. The most exploited PGPRs are rhizobia including Allorhizobium, Bradyrhi-

zobium, Mesorhizobium, Azorhizobium, Sinorhizobium and Rhizobium. There are

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 49

some bacteria like Nitrosomonas, Nitrococcus, and Nitrobacter that contribute in

nitriﬁcation procedure by converting ammonia to nitrite and then to nitrate. Free

living heterotrophic diazotrophs like Azospirillum sp., Azotobactor vinelandii, A.

chroococcum, Pseudomonas sp., etc. that can trap nitrogen for non-legume plants like

rice, wheat and others crop. Combine application of potassium solubilizing bacteria

like, Bacillus mucilaginosus and nitrogen ﬁxing bacteria like, A. chroococcum can

increase in biomass and higher nutrient uptake by various plants. Sphingomonas

trueperi, Psychrobacillus psychrodurans and Enterobacter oryzae are found to be

effective in increasing nutrient uptake like N, Ca, S, B, Cu, and Zn as well as promote

plant growth. If these strains were inoculated in seedlings of maize and wheat grown

under greenhouse conditions, are resulting high nutrient uptake and plant growth

promotion (Xu et al. 2018).

Photosynthesis: PGPRs play an important role in enhancing photosynthesis rate

by increasing photosynthetic pigment chlorophyll in plants. It has been reported

that Rhizobium sp., and Bradyrhizobium sp. increases the photosynthesis activity

in various plants under stress conditions. Planomicrobium chinense, Bacillus cereus

and Pseudomonas ﬂuorescens increases the photosynthesis efﬁciency of wheat grown

under salt and drought conditions (Kaur et al. 2021).

Phytohormone regulation: Different types of plant growth regulators such as, auxin,

cytokinin, gibberellin, abscisic acid, etc. are endogenously produced by plants which

regulates the plant growth and developments. It has been documented that microbe

plays a critical role in regulating the phytohormone production as well as microbe

itself can able to synthesized several phytohormones under normal or stressed condi-

tion. Application of PGPRs as biofertilizers on plants under drought and other stress

conditions, resulting increases stress tolerance by the plant due to modifying effect

of various phytohormones like, IAA, gibberellic acid, cytokinins, ethylene etc.

Indirect impact of PGPRs

EPS production: Exopolysaccharides (EPS) are high molecular weight polymers

secreted by microorganisms. EPS are consisting of homo or hetero polysaccharide

and exist as a capsule or slime layer on bacterial cell surfaces providing protection

to them against various stresses. EPS form complex with heavy metals and decrease

their mobility, therefore, reduces the availability for plants. Microbial exopolysac-

charide also plays a critical function in a different environment situation such as, act

as a signal molecule during nodulation, biocontrol activity, soil particle aggregation,

change the soil structure and proﬁle, bacterial bioﬁlm formation on root and enhance

plant growth (Manoj et al. 2020).

Induction of plant production of antioxidant enzymes: Under various stress condition

both biotic and abiotic, several reactive oxygen species (ROS) like, superoxide ions,

peroxides, hydrogen peroxide, singlet oxygen, hydroxyl radicals are produced in

plant body which causes severe damage to biological membrane and other important

biomolecules. To cope up with the situation plants can produce various antioxidant

50 S. Mondal et al.

enzymes like, catalase, dehydroascorbate reductase, peroxidase, superoxide dismu-

tase, glutathione peroxidase, glutathione reductase, etc. Plant also can produce a

number of non-enzymatic antioxidant component such as ascorbate, glutathione,

proline, tocopherol, glycine betaine etc. for oxidative stress tolerance. It has been

reported that application of PGPRs on plants under stress condition can enable plants

to tackle the deleterious effects of stress by increasing the production of various

antioxidant enzyme and antioxidant components. It is reported that the inoculation of

Zn resistant P. aeruginosa with wheat seedlings under Zn stress condition, resulting

increased antioxidant enzyme like SOD, POD, CAT and increase biomass (Islam

et al. 2014).

Hydrogen cyanide (HCN) production: Rhizobacteria are able to produce volatile

compound hydrogen cyanide (HCN) which inhibits mycelial growth of pathogenic

fungus and thereby provide a defense mechanism against disease development. HCN

can also induce plant growth and entrap the heavy metal movability in the soil.

Biosurfactants production: Biosurfactants are amphiphilic, low molecular weight

molecules produced by microbes and found to be attached on the cell surface. A

biosurfactant usually made up of glycolipids, fatty acids, phospholipids, lipoprotein,

or lipopeptide and mycolic acid. Bacterial biosurfactant provide heavy metal toler-

ance in plants and remove toxic metals from the soil. These substances can bind

pollutants with strong afﬁnity due to their amphiphilic nature than normal cations.

Organic acids production: PGPRs secretes various kind of low molecular weight

organic acids like, oxalic acid, gluconic acid, citric acid, succinic acid, etc. These

organic acids help to alleviate heavy metal toxicity and promote plant growth by

forming metal–acid complex like metaloxalate crystals and turn into their less toxic

form or by acquisition of essential nutrients. Organic acids also play an important

role in enhancing antioxidant enzyme responses of plants and solubilizing mineral

phosphates.

Hydrolytic enzyme productions: PGPRs synthesize a variety of hydrolytic enzymes

like chitinases, protease, lipase, cellulases, glucanases and esterases. These enzymes

provide chemical defense against pathogenic fungal attack by hydrolyzing their cell

wall and thus keep the plants healthy in an indirect way. These enzymes are also

responsible for recycling of nutrient molecules in nature.

Antibiotic production and induced systemic resistance (ISR): PGPRs are reported

to produce antimicrobial compounds, that provide plant protection against harmful

fungus, bacteria, viruses, nematodes etc. via induced systematic resistance mecha-

nism or antagonism. Various kinds of antimicrobial compounds such as, phenazines,

butyrolactones, pyrole-type compounds, 2,4-diacetyl phloroglucinol, kanosamine,

etc. and various peptides are produced by PGPR strains and protect crops and plants

(Manoj et al. 2020).

Synthesizing ACC deaminase: Under heavy metal stress ethylene production increase

drastically, which causes a reduction in root length, accumulation of hydrogen

peroxide and leads to apoptosis situation. To overcome such situation, PGPR

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 51

produces higher amounts of ACC deaminase enzyme to hydrolyse ACC, the

immediate precursor of ethylene. Therefore, enable plants to reduce ethylene level.

7 Synergistic Interconnection of Plants and PGPR

in Heavy Metal Remediation

Plants and microbes interact with each other to get beneﬁted. Sometime plants and

microbes form a symbiotic association with each other. In some circumstances, both

live as independently free-living organism, but interact with each other. The plant

provides nutrients and space to the microbes, and in return of which microbe increase

bioavailability of nutrients from the soil surface, detoxify the toxic heavy metals,

provide resistance against pathogenic attack, thereby vastly inﬂuence on the growth

and health of the plants. Thus, plants and microbe establish a synergistic interaction

between them, which has been exploited nowadays as a successful bioremediation

technique of heavy metals from media (Fig. 3). The efﬁciency of such dynamic inter-

action depends on various factors like the plant species, microbial ﬂora type, type of

pollutants, physical and chemical characters of soil. PGPRs are group of soil residing

bacteria that interact with plants to promote their biomass increase as well as also

assist them to cope up with the heavy metal stress. Co-culture of PGPRs and plants

provide a new path in the ﬁeld of phytoremediation of heavy metals. Plants will store

diverse contaminants in rhizosphere that may later be harvested, whereas microbial

accumulation can even transform contaminants like heavy metals to stable and less

harmful type. Plants secrete various substances like enzymes, amino acids, sugars,

aromatics, aliphatic that stimulate root associated microbial community expansion

in return microbes reduces toxicity of metals and enhance the capability of plants to

degrade and sequester the heavy metals. The process of phyto-extraction by hyper-

accumulators plants i.e., absorption of heavy metals and their translocation to above

ground tissue is accelerated by PGPRs inoculation. PGPRs improvise the mobility of

heavy metals and transportation by changing soil pH, demineralization, biosurfactant

production, siderophore secretion and chelator production. PGPRs strain produces

oxalic acid, citric acid and other organic acids, which increases metal solubility

and mobility by reduction process. Phytovolatilization process is also increased by

PGPRs inoculation. It has been well documented that inoculation with PGPRs can

able to increase heavy metal r esistance of plants by switching on different transcrip-

tion factors, various protein molecules related to stress and thereby provoking the

stress signal pathways and genes to overcome the stressed situation. Under heavy

metal stress a number of signaling pathway like MAPK pathway, ROS signaling path-

ways, Ca-dependent pathway, hormone dependent pathway gets activated which in

turn activates stress related transcription factors and genes (Manoj et al. 2020).

52 S. Mondal et al.

Fig. 3 Plant-PGPR interacting network model

8 Conclusions and Future Prospective

This review revealed that uses of PGPRs become an integral component in the modern

bioremediation technique of heavy metals and subsequently describe the scientiﬁc

gradual progress in the application of this procedure. There are various advance

types of bioremediation approaches that has been started to implement in worldwide

to mitigate the hazardous effect of heavy metals on environment. Though, it is well

established that combining application of PGPRs and plants is the best approach

for metal contaminants bioremediation due to a healthy synergistic interconnection

between microbes and plants. The details molecular mechanism of such synergistic

interaction is yet to be explored more vividly. Although few limited researches have

revealed roles of metal transporter genes, regulatory genes and transcription factors in

some microbial assisted phytoremediation technique, but in future, there is need for

further details study on the regulation process in miRNA and siRNA level, which may

provide us the concrete picture of multicomponent system of PGPR assisted phytore-

mediation. The focus should be given on the application of genetically engineered

microorganism on the demand of plants type and the type of metal contaminant in

different medium and also carefully visualize any negative impact of the engineered

microbes on the ecosystem.

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 53

References

Ahemad M (2019) Remediation of metalliferous soils through the heavy metal resistant plant growth

promoting bacteria: paradigms and prospects. Arab J Chem 12(7):1365–1377

Ali H, Khan E, Sajad MA (2013) Phytoremediation of heavy metals-concepts and applications.

Chemosphere 91(7):869–881

Alkorta I, Becerril JM, Garbisu C (2010) Phytostabilization of metal contaminated soils. Rev

Environ Health 25(2):135–146

Asati A, Pichhode M, Nikhil K (2016) Effect of heavy metals on plants: an overview. IAIEM

5(3):56–66

Basu A, Prasad P, Das SN, Kalam S, Sayyed RZ, Reddy MS, Enshasy HE (2021) Plant growth

promoting rhizobacteria (PGPR) as green bioinoculants: recent developments, constraints, and

prospects. Sustainability 13(3):1140

Ben Fekih I, Zhang C, Li YP, Zhao Y, Alwathnani HA, Saquib Q, Rensing C, Cervantes C (2018)

Distribution of arsenic resistance genes in prokaryotes. Front Microbiol 9:2473

Billah M, Khan M, Bano A, Ul Hassan T, Munir A, Gurmani AR (2019) Phosphorus and phosphate

solubilizing bacteria: Keys for sustainable agriculture. Geomicrobiol J 36(10):904–916

Blindauer CA, Harrison MD, Robinson AK, Parkinson JA, Bowness PW, Sadler PJ, Robinson NJ

(2002) Multiple bacteria encode metallothioneins and SmtA-like zinc ﬁngers. Mol Microbiol

45(5):1421–1432

Brooks RR (1977) Copper and cobalt uptake by Haumaniastrum species. Plant Soil 48(2):541–544

Bruins MR, Kapil S, Oehme FW (2000) Microbial resistance to metals in the Eevironment.

Ecotoxicol Environ Saf 45(3):198–207

Bumunang EW, Babalola OO (2014) Characterization of rhizobacteria from ﬁeld grown genetically

modiﬁed (GM) a nd non-GM maize. Braz Arch Biol Technol 57(1):1–8

Cha JS, Cooksey DA (1991) Copper resistance in Pseudomonas syringae mediated by periplasmic

and outer membrane proteins. Proc Natl Acad Sci USA 88(20):8915–8919

Chai WS, Cheun JY, Kumar PS, Mubashir M, Majeed Z, Banat F, Ho S-H, Show PL (2021)

A review on conventional and novel materials towards heavy metal adsorption in wastewater

treatment application. J Clean Prod 296:126589

Choudhury R, Srivastava S (2001) Zinc resistance mechanism in bacteria. Curr Sci 81(7):768–775

Cook CM, Kostidou A, Vardaka E, Lanaras T (1997) Effects of copper on the growth, photosynthesis

and nutrient concentrations of Phaseolus plants. Photosynthetica 34(2):179–193

D¸abrowski, Hubicki A, Podko´scielny Z P, Robens E (2004) Selective removal of the heavy metal ions

from waters and industrial wastewaters by ion-exchange method. Chemosphere 56(2):91–106

Dwivedi S (2012) Bioremediation of heavy metal by algae: current and future perspective. J Adv

Lab Res Biol 3(3):196–199

Etesami H (2018) Bacterial mediated alleviation of heavy metal stress and decreased accumulation

of metals in plant tissues: Mechanisms and future prospects. Ecotoxicol Environ Saf 147:175–191

Gadd GM, Bahri-Esfahani J, Li Q, Rhee YJ, Wei Z, Fomina M, Lianga X (2014) Oxalate production

by fungi: signiﬁcance in geomycology, biodeterioration and bioremediation. Fungal Biol Rev

28(2–3):36–55

Göhre V, Paszkowski U (2006) Contribution of the arbuscular mycorrhizal symbiosis to heavy metal

phytoremediation. Planta 223:1115–1122

Gupta P, Rani R, Chandra A, Kumar V (2018) Potential application of Pseudomonas sp. (strain

CPSB21) to ameliorate Cr6+ stress and phytoremediation of tannery efﬂuent contaminated

agricultural soils. Sci Rep 8(1):4860

Gusiatin ZM, Kulikowska D, Klik B (2020) New-generation washing agents in remediation of

metal-polluted soils and methods for washing efﬂuent treatment: a review. Int J Environ Res

Public Health 17(17):6220

Harrison JJ, Ceri H, Turner RJ (2007) Multimetal resistance and tolerance in microbial bioﬁlm. Nat

Rev Microbiol 5:928–938

54 S. Mondal et al.

Hermansson AW, Syaﬁie S (2015) Model predictive control of pH neutralization processes: a review.

Control Eng Pract 45:98–109

Hossain ST, Mukherjee SK (2012) CdO nanoparticle toxicity on growth, morphology, and cell

division in Escherichia coli. Langmuir 28(48):16614–16622

Hossain ST, Mallick I, Mukherjee SK (2012) Cadmium toxicity in Escherichia coli: cell morphology,

Z-ring formation and intracellular oxidative balance. Ecotoxicol Environ Saf 86:54–59

Huang SH, Peng B, Yang Z-H, Chai L-Y, Zhou L-C (2009) Chromium accumulation, microorganism

population and enzyme activities in soils around chromium-containing slag heap of steel alloy

factory. Trans Nonferrous Met Soc China 19(1):241–248

Hube S, Eskaﬁ M, Hrafnkelsdóttir KF, Bjarnadóttir B, Bjarnadóttir MA, Axelsdóttir S, Wu B (2020)

Direct membrane ﬁltration for wastewater treatment and resource recovery: a review. Sci Total

Environ 710:136375

Islam F, Yasmeen T, Ali Q, Ali S, Arif MS, Hussain S, Rizvi H (2014) Inﬂuence of Pseudomonas

aeruginosa as PGPR on oxidative stress tolerance in wheat under Zn stress. Ecotoxicol Environ

Saf 104:285–293

Kaur T, Devi R, Kour D, Yadav A, Yadav AN, Dikilitas M, Abdel-Azeem AM, Ahluwalia AS,

Saxena AK (2021) Plant growth promoting soil microbiomes and their potential implications for

agricultural and environmental sustainability. Biologia 76:2687–2709

Limmer M, Burken J (2016) Phytovolatilization of organic contaminants. Environ Sci Technl

50(13):6632–6643

Liu L, Li W, Song W, Guo M (2018) Remediation techniques for heavy metal-contaminated soils:

Principles and applicability. Sci Total Environ 633(2):206–219

Ma Y, Rajkumar M, Vicente JAF Freitas H (2011) Inoculation of Ni-resistant plant growth promoting

bacterium Psychrobacter sp. Strain SRS8 for the improvement of nickel phytoextraction by energy

crops. Int J Phytoremediation 13(2):126–139

Maleva MG, Nekrasova GF, Borisova GG, Chukina NV, Ushakova OS (2012) Effect of heavy metal

on photosynthetic apparatus and antioxidant status of elodea. Russ J Plant Physiol 59:190–197

Mallick I, Hossain ST, Sinha S, Mukherjee S (2014) Brevibacillus sp. KUMAs2, a bacterial isolate

for possible bioremediation of arsenic in rhizosphere. Ecotox Environ Saf 107:236–244

Manoj SR, Karthik C, Kadirvelu K, Arulselvi PI, Shanmugasundaram T, Bruno B, Rajkumar M

(2020) Understanding the molecular mechanisms for the enhanced phytoremediation of heavy

metals through plant growth promoting rhizobacteria: a review. J Environ Manage 254:109779

Matsui K, Yoshinami S, Narita M, Chien M-F, Phung LT, Silver S, Endo G (2016) Mercurry

resistance transposons in Bacilli strains from different geographical regions. FEMS Microbiol

Lett 363(5):fnw013

Mishra AK, Mohanty B (2008) Acute toxicity impacts of hexavalent chromium on behavior and

histopathology of gill, kidney and liver of the freshwater ﬁsh, Channa punctatus (Bloch). Environ

Toxicol Pharmacol 26(2):136–141

Moustakas K, Fatta D, Malamis S, Haralambous K, Loizidou M (2005) Demonstration plasma

gasiﬁcation/vitriﬁcation system for effective hazardous waste treatment. J Hazard Mater 123(1–

3):120–126

Mubarok MZ, Hanif LI (2016) Cobalt and Nickel separation in nitric acid solution by solvent

extraction using Cyanex 272 and Versatic 10. Procedia Chem. 19:743–750

Mulligan CN, Yong RN (2004) Natural attenuation of contaminated soils. Environ Int 30(4):587–601

Muthusaravanan S, Sivarajasekar N, Vivek JS, Paramasivan T, Naushad M, Prakashmaran J,

Gayathri V, Al-Duaij OK (2018) Phytoremediation of heavy metals: mechanisms, methods and

enhancements. Environ Chem Lett 16:1339–1359

Olguín EJ, Sánchez-Galván G (2012) Heavy metal removal in phytoﬁltration and phycoremediation:

the need to differentiate between bioadsorption and bioaccumulation. New Biotechnol 30(1):3–8

Park JH, Lamb D, Paneerselvam P, Choppala G, Bolan N, Chung J-W (2011) Role of organic

amendments on enhanced bioremediation of heavy metal(loid) contaminated soils. J Hazard

Mater 185(2–3):549–574

Exploration of Plant Growth Promoting Rhizobacteria (PGPRs) … 55

Paul A, Jasu A, Lahiri D, Nag M, Ray RR (2021) In Situ and ex situ bioremediation of heavy metals:

the present scenario. J Environ Eng Landsc 29(4):454–469

Shah Maulin P (2020) Microbial bioremediation & biodegradation. Springer

Shah Maulin P (2021a) Removal of refractory pollutants from wastewater treatment plants. CRC

Press

Shah Maulin P (2021b) Removal of emerging contaminants through microbial processes. Springer

Pena LB, Barcia RA, Azpilicueta CE, Méndez AAE, Gallego SM (2012) Oxidative post translational

modiﬁcations of proteins related to cell cycle are involved in cadmium toxicity in wheat seedlings.

Plant Sci 196:1–7

Raﬁque M, Hajra S, Tahir MB, Gillani SSA, Arshed M (2022) A review on sources of heavy metals,

their toxicity and removal technique using physico-chemical processes from wastewater. Environ

Sci Pollut Res 29:16772–16781

Saraswat R, Saraswat D, Yadav M (2020) A review on bioremediation of heavy metals by microbes.

Int J Adv Res 8(7):200–210

Sattar A, Naveed M, Ali M, Zahir ZA, Nadeem SM, Yaseen M, Meena VS, Farooq M, Singh R,

Rahman M, Meena HN (2019) Perspectives of potassium solubilizing microbes in sustainable

food production system: a review. Appl Soil Ecol 133:146–159

Sharma RK, Agrawal M, Marshall F (2007) Heavy metal contamination of soil and vegetables in

suburban areas of Varanasi. India. Ecotoxicol Environ Saf 66(2):258–266

Sharma SS, Dietz K-J (2006) The signiﬁcance of amino acids and amino acid-derived molecules

in plant responses and adaptation to heavy metal stress. J Exp Bot 57(4):711–726

Singh M, Pant G, Hossain K, Bhatia AK (2017) Green remediation. Tool for safe and sustainable

environment: a review. Appl Water Sci 7:2629–2635

Singh S, Parihar P, Singh R, Singh VP, Prasad SM (2015) Heavy metal tolerance in plants: role of

transcriptomics, proteomics, metabolomics, and ionomics. Front Plant Sci 6:1143

Tajudin SAA, Azmi MAM, Nabila ATA (2016) Stabilization/solidiﬁcation remediation method for

contaminated soil: a review. IOP Conf Ser Mater Sci Eng 136:012043

Terry N, Zayed AM, De Souza MP, Tarun AS (2000) Selenium in higher plants. Annu Rev Plant

Physiol Mol Biol 51:401–432

Tripathy T, De BR (2006) Flocculation: a new way to treat the waste water. J Phys Sci 10:93–127

Vansuyt G, Robin A, Briat JF, Curie C, Lemanceau P (2007) Iron acquisition from Fe-pyoverdine

by Arabidopsis thaliana. Mol Plant Microbe Inter 20(4):441–447

Verma S, Kuila A (2019) Bioremediation of heavy metals by microbial process. Environ Technol

Innov 14:100369

Vijayaraghavan K, Yun Y-S (2008) Bacterial biosorbents and biosorption. Biotechnol Adv

26(3):266–291

Xu J, Kloepper JW, Huang P, McInroy JA, Hu CH (2018) Isolation and characterization of N2-ﬁxing

bacteria from giant reed and switchgrass for plant growth promotion and nutrient uptake. J Basic

Microbiol 58(5):459–471

Yuan H-M, Xu H-H, Liu W-C, Lu Y-T (2013) Copper regulates primary root elongation through

PIN1-mediated auxin redistribution. Plant Cell Physiol 54(5):766–778

Decontamination Strategies

and Technologies for Tackling COVID-19

Hospitals and Related Biomedical Waste

Rishav Sharma, Pinakiranjan Chakraborty, and Shraman Roy Barman

Abstract

The complexities of waste management have been enhanced by the arrival of the

novel coronavirus disease-2019. Since the outbreak of COVID-19, biomedical waste

(BMW) is being generating in huge amount worldwide by the isolation ward, insti-

tutional quarantine centres, COVID testing facilities and even household quarantine.

The major contributors to the waste volume include personal protective equipment

(PPE), testing kits, surgical facemasks and nitrile gloves. Discharge of new category

of BMW (COVID-waste) is of great global concern to public health and environ-

mental sustainability if handled inappropriately. It has been established that COVID

R. Sharma · S. R. Barman (B

)

Department of Enviornmental Science, Asutosh College, 92 Shyama Prasad Mukherjee Road,

Jatin Das Park, Patuapara, Bhownipore, Kolkata 700026, West Bengal, India

e-mail: shromonaroybarman123@gmail.com

P. Chakraborty

Department of Zoology, Vijaygarh Jyotish Ray College, 8,2, Bijoygarh, Jadavpur,

Kolkata 700032, West Bengal, India

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_4

57

58 R. Sharma et al.

virus survives up to 7 days on waste like facemasks, which may cause exponential

spreading of this fatal disease with the waste acing as a vector. As vaccines were not

prepared at early phase, to lower the threat of pandemic spread, many protective gears

have been used and discarded. Thus, for sustainable environmental management,

addressing the issue of proper disposal of COVID-19 related waste is an imme-

diate requirement. Henceforth, in the present article, disinfectant technologies for

handling COVID-waste from its separate disposal to collection to various physical

and chemical treatment steps have been reviewed. Furthermore, policy briefs on the

global initiatives for COVID-waste management has been including. The applica-

tion of different disinfection techniques has also been discussed with some potential

examples effectively applied to reduce both health and environmental risks.

Keywords Biomedical waste ·Biomedical waste management ·COVID-19 ·

Infectious waste ·Waste treatment

1 Intrduction

Biomedical waste (BMW) is the by-product of the process including sampling,

testing, diagnosis, treatment, vaccination and surgical treatment of humans, animals,

and in research (Quadar et al. 2018). They are a group of hazardous waste that can

spread infectious disease if not handled and treated properly (Shammi et al. 2021).

The management of BMW is of prime importance to lower public health hazard

especially to sanitation and healthcare personae (Chakraborty and Maity 2020). The

overall wellbeing of a society depends on efﬁcient BMW management and this need

has prompted various legislation and planning (Singh et al. 2018).

Source segregation has undoubtedly been an indispensable step in managing

any type of solid waste (Saraiva et al. 2018). Common Biomedical Waste Treat-

ment Facility (CBWTF) is another vital course of action towards sustainable BMW

management (Devi et al. 2019; Shah 2020). The treated waste is utilized in land-

ﬁll sites and recycled accordingly (Arun and Priya 2020). The concern of medical

administrators regarding handling of this group of waste has now turn into a statuary

necessity with the afﬁrmation of the India governance. The biomedical waste that

are produced from medical care units’ inhabitancies, specialization unit of medical

care, reusable things and their proportion being used, infrastructure and resources

and their accessibility (Dutta et al. 2018).

For execution of industrial plan for medical waste several trial projects have been

done to improve the segregation of hazardous as well as non-hazardous waste that

can be cause decrease of waste that are hazardous in this sector (Dahchour et al.

2020). Different kind of containers (for clinical hazardous wastes) disposal have

been used by different workers of the hospitals to arrange off different kinds of waste

that are harmless (Chakraborty and Maity 2020). The biomedical waste management

concerned has now extended and turned into a humanitarian topic around the world

human and animal health along with the environment are affected by the hazardous

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 59

and poor management of biomedical waste which turned into a concerning matter.

The topic of Biomedical Waste Management and its maintenance has been gaining

expanding importance for as far back as couple of years and uniquely in this situation

of worldwide pandemic Covid-19 (Youseﬁ et al. 2021).

1.1 Deﬁnition

Biomedical waste (BMW) is deﬁned as any waste, generated during the discovery,

treatment or vaccination of humans or animals, or in related research activities, or

in the production of biological experiments and animal waste in slaughterhouses or

other similar construction (Rules for -Biomedical waste (Management and Handling)

Rules 1998 India).

Biomedical waste Management (BMW) has kept on being a cause of arising

toxins essentially brought about by health care practices like medical diagnostics,

therapy and immunization, and/or biological research in animals (Datta et al. 2018).

Drug withdrawal of patients where the dynamic part of the medication and metabo-

lite, substance and drug deposits, similar media containing iodine, etc. As per the

World Health Organisation, healthcare-waste 2018 around 85% of BMW’s total

volume is viewed as non-hazardous waste, and the remaining volume falls under

hazardous waste. Improper disposal of hazardous biomedical waste (HBMW) poses

signiﬁcant risks to public and environmental wellbeing as it goes about as a vari-

ation of pathogenic microorganisms. Bacteria organisms present in HBMW if not

treated appropriately can enter into human’s body through scratching, scratching or

cutting on the skin, mucous layers, smell, and sting. Respiratory contaminations,

gastrointestinal diseases, skin contaminations, fever, microscopic organisms, viral

hepatitis, ﬂu are probably the most widely recognized contaminations carried almost

by HBMW openness (WHO 2020). The most common instance of biomedical/solid

waste collectors can be traced directly to pathogens in contaminated waste revealed

in study directed in Brazil, Greece, India (Singh et al. 2020). Consequently, the

actual administration of HBMW is vital to control the transmission of disease. In

this context, the World Health Organisation (WHO) has set key focuses for June 2017

by accentuating the speculation of proper assets and a total obligation to lessening

wellbeing dangers and contamination (Datta et al. 2018).

1.2 Biomedical-Waste Classiﬁcations

There are various categories of Biomedical Waste, some of which are important

and relevant. According to the World Health Organization (WHO), natural waste

can be divided into eight categories, each indicating a different risk of diffusion of

contagious diseases or adverse health effects due to human contact with this waste.

60 R. Sharma et al.

•General Waste: General waste represents less or less hazardous to human health

as it is mainly composed of household or household waste i.e., kitchens, packaging

materials, laundry and other non-communicable materials that do not require

special treatment.

•Disease waste: Disease waste comprises tissues, organs, organs, human embryos

and animal corpses; and especially blood and body ﬂuids. Apart from the

infectious nature of this waste, its proper disposal is necessary for ethical reasons.

•Radiation: Radiation waste comprises solid, liquid and gaseous pollutants

infested with radionuclides produced by in vitro study of body tissues and ﬂuids,

in vivo body organ imaging and tumour localization processes, as well as proce-

dures. Its signiﬁcance lies in the statistics that several nuclear accidents resulting

from improper disposal of nuclear material are known to have occurred, with a

large number of people suffering from the effects of exposure.

•Chemical Management: Chemical waste comprises of solid, liquid and airborne

disposable chemicals, from diagnostic and testing, cleaning, housekeeping and

disinfection work. These wastes can be either harmful or not.

(i) It is toxic,

(ii) Disabling (acids with pH 2.0 and a base pH 12.0),

(iii) It burns,

(iv) It works (explosive, water works, scared),

(v) Genotoxic (carcinogenic, mutagenic, teratogenic or else) capable of altering

materials, eg, cytotoxic drugs, non-hazardous chemical waste contains

chemicals different from those described above eg, sugar, amino acids,

certain salts of organic and inorganic substances.

•Infectious Waste: Infectious waste is that waste which contains germs with sufﬁ-

cient density or mass exposure to it can cause disease. This category of cultures

and stocks of contagious substances from workshops, waste from surgical oper-

ations and autopsy, wastes from an infective patient, segregated wards, wastes

connected to an infected patient using haemodialysis (eg, gloves and lab coats);

wastes that has been in interaction with animals that have been injected with an

infectious agent or that are suffering from transmittable diseases.

•Sharps: Sharps is a term used to describe waste, which can cause the user to

cut, cut or pierce the skin. This category includes needles, syringes, saws, blades,

broken glass (slides), nails, etc. which can cause cuts or piercings.

•Medical Handling: Medicinal waste includes pharmaceutical products, drugs and

chemicals recovered from wards, disposed of, expired (expired) or soiled, or must

be discarded as they are no longer needed.

•Pressure containers: Pressure containers include those used for display or

teaching purposes, containing hazardous or intrusive gas, and aerosol cans that

may explode or explode accidentally.

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 61

1.3 Health Care Institutions Generating Waste

World Health organization convened a working group at Bergen in 1983 which has

identiﬁed the following types of health care institutions as generating biomedical

wastes (WHO, Bergen 1983).

Primary sources

i. Hospitals

ii. Nursing home

iii. Dispensaries

iv. Maternity home

v. Dialysis centre

vi. Research laboratories

vii. Medical colleges

viii. Immunization centre

ix. Nursing home

x. Blood bank

xi. Industries.

Secondary sources

i. Clinic

ii. Ambulance services

iii. Home treatment

iv. Slaughterhouse

v. Funeral service

vi. Educational institute.

1.4 Categories of Biomedical Waste

According to Biomedical Waste Management and handling rule, 1998, there were

10 categories, but in 2011 Amendment comes with 8 categories (Table 1).

1.5 Covid-19 Pandemic and Increase in Biomedical Waste

A novel virus, similar to Severe Acute Respiratory Syndrome known as SARS-Cov-

2 and later called COVID-19 by World Health Organisation (WHO), was discov-

ered in Hubei province in Wuhan, China, in December 2019. SARS-CoV-2 is a

single-line RNA virus with virion sizes ranging from 50 to 200 nm (He et al. 2020).

SARS-CoV-2 has a 10 to 20-fold higher binding compound than previous respi-

ratory syndromes (Chan et al. 2020). The rapid spread of SARS CoV-2 to human

populations may be due to high cell-ACE2 receptor cell exposure (Wan et al. 2020).

62 R. Sharma et al.

Tabl e 1 Categories of Biomedical wastes (Ministry of Environment, Forest and Climate Change

Government of India 2016)

Waste category Waste class and description

Category No. 1 Human Anatomical Wastes

Category No. 2 Animal Wastes

Animal ﬂesh, organs, corpses, bleeding parts, ﬂuid, blood

and trial animals used in research, waste generated by veterinary hospitals,

colleges, discharge from hospitals, animal houses

Category No. 3 Microbiology and Biotechnology Waste.,

Wastes from research laboratory culture, stocks or specimens of

micro-organisms, live or attenuated vaccines, human and animal cell culture

used in research and infectious agents from research and infectious agents

from research and industrial laboratories, wastes from production of

biologicals, toxins, dishes and equipment’s used for transfer of cultures

Category No. 4 Waste Sharps

Needles, syringes, scalpels, blades, glass etc. that are capable of causing

puncture and cuts. This includes both used and unused sharps

Category No. 5 Rejected Medicine and Cytotoxic Drugs

Category No. 6 Soiled Wastes

Items contaminated with blood and body ﬂuids including cotton, dressings,

soiled plaster casts, beddings and other material contaminated with blood

Category No. 7 Infectious Solid Wastes

Wastes generated from disposable items other than the waste sharps, such as

tubing, catheters, intra venous sets etc

Category No. 8 Chemical Wastes

Chemical used in production of biologicals, chemicals used in disinfection,

as insecticides etc

COVID-19 transmission was detected through human contact shortly after a pneu-

monia outbreak in Wuhan (Hubei province, China), and the disease was proclaimed

a global pandemic by the WHO. COVID-19 becomes a global concern, to date

(01/09/2020) affecting 213 countries and territories and there are 30,350,821 Covid-

19 cases with more than 900,000 registered people worldwide. With the exception

of the income group (low, medium and high income), the COVID-19 epidemic has

highlighted several shortcomings in the socio-economic, health, and environmental

sectors of the world (Owusu et al. 2020). To date, no drugs or vaccines have been

registered to treat COVID-19 patients directly. Mass sampling in rapid trials, segre-

gation of suspects/patient use of preventive measures, social retardation and health-

supporting treatments are well-known ways to combat/prevent this deadly epidemic.

According to the WHO, COVID-19 virus is transmitting mainly through saliva drops

or from the nose and mouth when an infected person coughs or sneezes (Otter et al.

2020). After these droplets fall into items and the regions surrounding a person, some

persons become infected when they touch their eyes, nose, or mouth after coming

into contact with contaminated regions. As a result, the WHO and the Ministry of

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 63

Health and Family Welfare of the Government of India recommended that indi-

viduals and paramedics (doctors, nurses, cleaners, and police ofﬁcers) should wear

personal protective equipment (PPE), surgical (protective) equipment, aprons/gowns,

and nitrile gloves when exposed to germs and pollution (Singh et al. 2020).

In addition to guidelines on the rational usage of COVID-19 protective gears,

healthcare sectors are facing greater demand for PPE across all levels of health

workers for fear of infection. Fear often leads to the misuse of PPE which often

exacerbates the problem by producing large quantities of BMWs that are difﬁcult to

maintain and transport with limited resources and staff available during a disaster

(Garg et al. 2020). The amount of biomedical waste (BMW) produced by COVID-19

patients is steadily growing. Due to the stockpile of gloves, gowns, masks and other

protective equipment, there appears to be an emergency waste due to the unusual form

of families and health facilities (Ma et al. 2020). Also, with increasing number of

tests for COVID-19, and widespread vaccination drives, infectious waste are further

increasing with time. India produces approx. 2,00,000 tons of biomedical waste

(BMW) per year are treated by 198 Common Biomedical Waste Treatment Facilities

(CBMWs) and 225 burn captives, so the daily generation of BMW is imminent. 550

tons. It is estimated that hospitals produced six times as much medical waste during

the COVID-19 outbreak.

COVID waste can be controlled by regulation but there are other concerns that

the disease is spreading beyond hospitals. Some people with minor symptoms have

asymptomatic, who may not know that the waste they dispose of can be contaminated.

This means that people are more likely to produce more contaminated waste and

that this will contaminate sanitation workers as the virus is said to persist for days

on cardboard, metal and plastic (Ranjan et al. 2020). The enormous growth in the

number of persons infected with COVID-19 has demonstrated that the world will be

inundated by COVID waste in the future years, and the impact of this glut will have

a profound inﬂuence on the on-going waste process (Cutler 2020).

SARS-CoV-2 can survive in COVID-waste like needles and syringes used to

draw blood samples, surgical masks, and personal protective equipment (PPE) kits.

Exposure to COVID-Waste has the potential to boost the virus’s transmission by

raising the birth rate (R0) from its predetermined range of 2.2–3.58. (Li et al. 2020;

Zhao et al. 2020). Without antibiotics, improper discharge of COVID-Waste can put

ordinary people and health personnel at danger of infection. To limit the spread of

the infection, proper COVID-Waste management, including suitable disinfection and

disposal procedures, is required.

2 Biomedical Waste Treatment Methodologies

Phases in Waste Management for the effective management of biological wastes,

various steps must be followed from collection through disposal, which are

summarised below.

64 R. Sharma et al.

2.1 Waste Segregation

Segregation is the main factor in a waste management framework. Reliant upon the

treatment alternative and the elimination of different waste things, compartments

of speciﬁc tones should be isolated and put away in a brief stockpiling region for

removal inside 48 h. Trash that should be covered further should be gathered in a

plastic bag or yellow bin. Waste should be collected in a red or blue container or bag

for autoclaving, microwaving, chemical treatment, and ﬁnally disposal or recycling.

Trash used to sterilise and destroy or cut off needles, s harp edges, and other sharp

objects should be gathered in a white container that will be inserted or thrown for

reuse as a ﬁnal disposal. Chemical waste (strong), expired pharmaceuticals, and

cytotoxic drugs that need to be stored safely should be gathered in a black container

and labelled as such. Other than a black cabin or bag in which a cytotoxic label

should be marked, all containers and bins should have biohazard labels.

This steps also includes waste the board of different sorts for a few recycling

bins (reduce, recycling and minimization). Reuse of chemicals, medical equipment

etc. Recycling certain items such as sterile and shredded plastic helps the second

industry to reduce waste production which decreases the price of waste disposal.

The transmission of infection is detected by segregation thus decreasing the chances

of infection by health care workers. Scratching or piercing damage that causes waste

needs to be discarded as “sharp” and should be separated from other waste. A mixture

of sharp metals and broken mirrors is allowed but not with uncomplicated debris.

Blending of glass or plastic waste containing ﬂammable waste, organic waste or

other laboratory waste should avoided.

2.2 Waste Storage

Waste must be stored as required in according with the Biomedical Waste (Manage-

ment and Handling) Rules, 1988. Landﬁll sites takes place between the land disposals.

Before safe disposal natural waste can be temporarily sealed under the refrigerator

without creating a problem with nature. A storage area is located near the garbage

dumps. There should no drain pipes containing the spill and must be reverted to

the liquid storage elevator. Stiffness of the liquid is required on the ﬂoor and walls,

in simple compliance with cleaning procedures. Regular disinfection is mandatory.

Refrigerator is required to have rot and other contaminants alive for a period. There

must be a post in the last place displaying signs of ‘EXPLICIT’ (Da Silva et al. 2005).

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 65

2.3 Containers and Their Label

Non-invasive containers must be used in conjunction with good labelling and

integrity, as l ong as the treatment is performed chemically and physically. The

bio hazardous material content should be properly closed. The usage of leaking

containers that are strong against heat and chemical treatments ought to be used

chemically. Sturdy containers that are resistant to piercing and not tightly closed

should be used to sharpen the metal for proper disposal and should be able to with-

stand a pressure of 40 psi without cracking. For non-hazardous materials heavy plastic

bags or other such containers should be used in conjunction with the Biohazard logo.

Biohazard bags in red or orange should not be used for non-hazardous materials.

Sturdy and perforated containers would be used for Pasteur pipes and broken glass

(plastic, heavy cardboard or metal) and sealing should be done in “Biohazard bags”

made of heavy plastic and these are easily accessible. No label is required unless

there is a possibility of waste recycling and in such cases the container should be

marked ‘Do not recycle’. Bags should not used for open injections or other forms

of injection. Each bag or container should have a label based on adherence to the

generator information included in the bags containing medical waste. Construction

services should provide special labels with location for recording dates and contact

persons and those labels should be used in all containers packaged inside medical

trash cans. A clear diagnosis of all containers of untreated bio-hazardous waste and

their appropriate label and biohazard label should be made.

2.4 Organic Waste Management

Organic waste should be collected and transported in such a way that it poses

no damage to human health or the environment. Only highly qualiﬁed technical

employees should manage or dispose of bio hazardous material that remains unpro-

cessed. Following the manufacturing of rubbish separators in containers or bags

designated in a bright colour should be done as soon as possible. During the treat-

ment of this waste, it is vital to reduce the risk of needle wounds and infection.

Biomedical waste must not be combined with any other sort of garbage. If biological

waste is left unprocessed on site, it must be transferred from the manufacturing plant

to a treatment and disposal facility.

The following points need to be follow for the transportation of BMW:

1. The carrier must be provided with separate chambers and containers of natural

waste.

2. Ensuring the foundation of the recyclable quality waste disposal cabinet.

3. The waste disposal room should be designed in such, that it can be simply kills

the germs, and help to keep containers.

66 R. Sharma et al.

2.5 Treatment and Disposal Method

The basic criterion governing the disposal of anatomical wastes is that mutilation or

shredding must be capable of preventing unauthorised recycling. Chemical treatment

with 1% hypochlorite is recommended in the basic form. On the other hand, there is no

pre–treatment in the incineration process. The process of deep burial is only required

in cities with populations of less than ﬁve lakhs. Waste management should also be

performed as close to the site of origin as possible. With all of these considerations

in mind, the treatment and disposal of various types of wastes must be carried out

with caution and precision.

1. Animal carcasses and body parts: Incineration, biodegradation or landﬁll.

2. Solid waste (bedding, manure, etc.,) from animals:

i. Animal waste (Biohazards): Thermal or chemical treatment for incineration

and disinfection.

ii. Animal waste (Non–hazardous): Using as compost or fertilizer.

3. Chemical waste: It should be disinfected using a 1% sodium hypochlorite solution

or another chemical agent of the same type. Following the treatment of ﬂuids

and the acquisition of landﬁlls for solids, sewage discharge is required.

4. Materials containing recombinant DNA or organism that have been genetically

altered must be disposed of according to National Institutes of Health (NIH)

rules.

5. Human pathological waste:

i. Cremation or burial for a deceased corpse with identiﬁable body parts.

ii. Incineration or disinfection for disposal of other solids.

iii. Body ﬂuids—thermal or chemical disinfection before to discharge into the

drain system.

6. Metal sharps: To avoid laboratory, storage, and landﬁll worker injuries, metal

sharps should be disposed together with encapsulation. After cleaning or capping

in the original container, needles, knives, and other sharp objects pose a biohazard.

If autoclaving is required, an autoclave marking tape strip should be placed in the

container prior to the autoclaving process. Gas chromatography needles should

be rinsed to remove chemicals (hazardous) and disposed of with broken glassware

(non–contaminated).

7. Microbiological waste: It must be thermally or chemically processed before being

discharged into the sewage system.

8. Non–hazardous biological waste:

i. Even if the materials are non-hazardous, all microbiological components

must be autoclaved or thermally treated for improved laboratory procedures.

ii. Solid–solid waste should be placed in trash dumpsters.

iii. Liquid–liquid pollutant should discharge into large sewer system directly.

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 67

9. Plastic waste, Pasteur pipets; glassware (broken): If the equipment’s got contami-

nated by biohazards materials, then it should be Sterilized thermally or chemically

or encapsulation. And in case of non-contaminated wastes, they should be trash

in dumpster. And plastic and glassware should always be incinerated (Ravi Kant

et al. 2002).

Schedule I of Biomedical Waste (Rule 6) (Table 2).

Schedule II of Biomedical Waste (Rule 6) (Table 3).

According to Biomedical waste management and handling rule, 2016, MoEFCC.

Government of India:

Tabl e 2 The classiﬁcation of biomedical wastes, as well as their treatment and disposal (Rule

6, Biomedical waste management and handling rule, 2016., Ministry of environment, forest and

climate change, Government of India)

Waste category no Waste category (type) Treatment and disposal optiont

Category No. 1 Waste from the human body

(human tissues, organs, body parts)

Incineration**/deep burial*

Category No. 2 Waste from animals (waste of

veterinary clinics and practical’s)

Incineration**/deep burial*

Category No. 3 Microbiology and Biotechnology

waste and extra laboratory wastes

Disinfection at the site of origin by

chemical treatment**, autoclaving, or

microwaving, followed by

mutilation/shredding***, and ultimate

disposal in secure landﬁll or recycling

through authorized recyclers

Category No. 4 Waste sharps (needles, scalpel,

scissors etc.)

Chemical treatment** or needle and

tip cutter distribution, autoclaving or

microwaving, followed by mutilation

or shredding***

Category No. 5 Rejected medicines and cytotoxic

drugs

Disposal is seemed landﬁll and

Incineration**

Category No. 6 Soiled waste Incineration**

Category No. 7 Contagious solid waste (disposal

materials other than waste sharps)

(IV tubes, oxygen masks, saline

bottle, surgical gloves)

Chemical treatment**, autoclaving,

or microwaving, followed by

mutilation and shredding***

Category No. 8 Chemical waste (chemical access

in making medicines)

Chemical treatment** then release it

into drains

**Chemical treatment with a 1% hypo chloride solution or any other chemical reagent that is similar.

It is critical to guarantee that treatment results in disinfection

***Mutilation or shredding should be done in such a way that unauthorised re-use is prevented

**Before incineration, there is no chemical pre-treatment. Incineration of chlorinated plastics is

prohibited

*Only municipalities with populations of less than ﬁve lakhs and rural regions would have the

option of deep burial

68 R. Sharma et al.

Tabl e 3 Colour coding and type of bins for discarding of Biomedical Waste (Rule-6, Biomedical

waste management and handling rule, 2016., Ministry of environment, forest and climate change

(MoEFCC), Government of India)

Colour coding Type of container Waste category no

Yell ow Non-Chlorinated plastic bags Category NO.1, 2, 5, 6

Red Non-Chlorinated puncture proof plastic bags Category No. 3, 4, 7

Blue Non-Chlorinated plastic bags Category No. 8

Black Non-Chlorinated plastic bags Municipal Solid Waste

Fig. 1 Label for Biomedical Waste Containers/Bags (Rule 6, Biomedical waste management and

handling rule, 2016, Ministry of environment, forest and climate change, Government of India)

1. Depending on the treatment option chosen, which must be as stated in Schedule I,

colour coding of waste categories with numerous treatment choices as established

in Schedule I shall be selected.

2. Chlorinated plastics should not be used in the trash collecting bags for

incineration waste.

Schedule III of Biomedical Waste (Rule 6) (Fig. 1).

3 Disinfection and Reprocessing Techniques of Covid-19

Waste

3.1 Disinfection Using Incineration

This is described as a thermal process that requires a high temperature under regulated

waste burning circumstances in order to convert waste into non-reactive products and

gases. Worldwide, three types of incinerators are utilised to dispose of biomedical

waste: multiple hearth types, rotary kilns, and air types (controlled). Each main

and secondary combustion chamber is available in three different conﬁgurations,

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 69

providing optimal combustion (Chan et al. 2020). It is a frequently utilised disposal

method that is safe, simple, and effective, particularly in industrialised countries

(Ghodrat et al. 2018). The incinerator’s temperature must be more t han 1000 °C. This

high temperature could not only entirely kill microorganisms, but also incinerate and

not only burn the greatest amount of microorganisms, but also incinerate and burn

the majority of organic matter, converting it to inorganic dusts. Once the material is

incinerated, the amount of waste produced might be decreased by 80–85% (Wang

et al. 2020). Other biomedical wastes, aside from radioactive and explosive wastes,

can be incinerated.

The majority of COVID waste is transported to incinerate at a temperature >

11000C, according to BGL private limited (a typical biomedical waste treatment

facility recognised by Jharkhand Pollution Control Board, India). Depending on the

volume reduction of COVID waste, the leftover mass is sometimes re-incinerated

with fresh charge. However, pollutants such as product of incomplete combustion

(PIC) and dioxins are created during the process (Singh et al. 2020). Waste compo-

nents dissolve and recombine during incineration and post-combustion cooling,

generating new hazardous particles known as PIC (Vilavert et al. 2020). Dioxins

are an accidental by-product of trash combustion that occurs in incinerators. This

is a collection of 75 chemicals that live alongside another group of poisons known

as furans. Toxins have a proclivity for accumulating in fatty tissues and travelling

up the food chain. The most signiﬁcant dioxin producer in the environment is the

burning of medical equipment manufactured of Polyvinyl Chloride (PVC). Further-

more, metals found in medical waste act as a catalyst for the synthesis of dioxins.

These are very dangerous, having been identiﬁed as carcinogenic, and cause harm to

the human immune and nervous systems. In January 2017, recognising the impor-

tance of dioxins in the environment, the Council of Scientiﬁc and Industrial Analysis

and the National Institute for Interdisciplinary Science and Technology launched a

cooperative research to investigate the presence of dioxins in Thiruvananthapuram

(Subramaniam et al. 2020). Furthermore, incinerator ash is dangerous and must be

tested for toxin levels before being transferred to a secure landﬁll. As a result, most

nations are turning to alternate ecologically acceptable BMW disposal procedures,

taking these factors into account.

3.2 Disinfection Using Alternative Thermal Techniques

To deal with COVID-waste, there are primarily two types of thermal technologies

available and in use: (i) high temperature pyrolysis and (ii) medium temperature

microwave technology.

(i) High temperature pyrolysis technique

Pyrolysis, also known as controlled air incineration or double-chamber incinera-

tion, is the most reliable and often used treatment process for health-care waste. In

70 R. Sharma et al.

comparison to incineration, pyrolysis is a more technically sound technique. It has

a pyrolytic chamber and a post-combustion chamber, and it performs the following

activities (WHO, biomedical waste treatment):

•The wastes were t hermally destroyed in a pyrolytic chamber, creating solid ashes

and gases through an oxygen-deﬁcient, medium-temperature burning process

(500–850 °C). A fuel burner is included in the pyrolytic chamber, which is used to

initiate the process. The garbage is deposited in a dumpster or a rubbish container.

•In the post-combustion chamber, gases generated by a fuel burner are burnt at

high temperatures (900–1200 °C) with an abundance of air to reduce smoke and

smells.

Plasma pyrolysis t echnique is state–of–the–art for medical waste disposal. It’s

an environmentally beneﬁcial technique that converts organic waste into economi-

cally useful byproducts. Disposal of solid wastes, such as biomedical and hazardous

wastes, produces a lot of heat (Surjit et al. 2020). It has pyrolysis-oxidation, plasma

transformation, induction-based transformation, and laser-based transformation and

operates at a temperature range of 540–8300 C (Datta et al. 2018). The air measured

below the theoretical chemical reaction is equipped to a ﬁxed level of the ﬁrst chamber

in pyrolysis-oxidation The organic solid and liquid wastes are vapourised at a temper-

ature of 600 °C in the presence of air turbulence, leaving ash, glass, and metallic

pieces behind. The ﬂammable gaseous vapour is combusted at high temperatures

between 982° to 1093 °C in the second phase of combustion, completely destroying

harmful substances such as dioxins and generating clean exhaust steam. Given the

fast spread of SARS-CoV-2, it is advised that plasma energy be used to quickly

decompose COVID-waste rather than the typical laser/gaseous combustion (Wang

et al. 2020). This process produces a low emission rate, an inert residue, a volume

reduction of up to 95%, and a mass reduction of up to 90% (Ilyas et al. 2020). To

get plasma energy, plasma pyrolysis uses plasma torches. The ionising gas will carry

electrical current in the plasma form, however the electrical energy will be born-

again to energy due to its high resistance. Carbon, black, vitriﬁed glass aggregates,

and metallic residues are among the residues produced. Infectious waste, sharps,

plastics, dialysis waste, hazardous trash, chemotherapeutical waste, chemotherapy

waste, and low-level radioactive waste are all often eliminated in plasma-based tech-

nologies (with the exception of mercury, which plasma systems do not handle) (Datta

et al. 2020) (Fig. 2).

(ii) Medium temperature microwave technique

Microwaves are electromagnetic waves with wavelengths ranging from 1 to 1000 mm

and frequencies ranging from hundreds to 3000 MHz. 2450 ± 50 MHz and 915

± 25 MHz are the most common microwave frequencies used for sterilisation.

Microwaves move through the medium and are absorbed by it, causing heat to be

generated. The heat was created by the material molecule vibrating and rubbing

billions of times per second, creating the effect of high temperature disinfection.

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 71

Fig. 2 From BMW/hospital waste creation through disinfection and disposal processes, here’s a

schematic. Adopted from Ilyas et al. (2020)

When disinfected, this method is known for its energy efﬁciency, low action temper-

ature, slow heat loss, quick action, light damage, and minimal environmental pollu-

tion, with no residues or harmful waste product. It has a broad bacterial disinfection

spectrum and may destroy a variety of pathogens. In terms of pathogen destruction,

there is now strong conﬁrmation t hat speciﬁcally designed microwave systems may

effectively kill inert bacteria; nevertheless, the process must be closely regulated by

particular microwave equipment. Though certain improved microwave technologies

with precise measurements are now employed primarily for the treatment of bio-

hazardous wastes, it must be determined if they would be beneﬁcial for methods

requiring the management of water content, such as drying bio-therapeutic products.

This technique works at temperatures ranging from 177° to 540 °C and comprises

reverse polymerisation using high-energy microwaves in an inert environment to

break down organic materials. As a result of the vibration and rubbing of molecules

caused by the absorption of electromagnetic waves (with a wavelength of 1 mm to 1 m

and a frequency of several hundreds of megahertz to 3000 MHz). Although an inert

atmosphere created by nitrogen prevents combustion with oxygen, high-temperature

disinfection is demonstrated. Microwave technology has a number of advantages,

including lower energy and action temperatures, less heat loss, and less environmental

load with no hazardous residue after disinfecting. SARS-CoV-2 will be rendered

inactive by speciﬁcally constructed microwave devices used in a tightly regulated

manner. According to a report by the Chinese Ministry of Ecology and Environment,

this disinfection technology may achieve logarithmic values of killing hydrophilic

viruses (Wang et al. 2020) and is recognised to be highly effective for COVID-waste

transportation on-site disinfection that saves time (Resilient Environmental Solutions

72 R. Sharma et al.

2020). In the instance of COVID-waste disinfection, the microwave approach is

employed in conjunction with autoclaving where steam is used for s terilisation (in

temperature vary from 93 to 177 °C).

3.3 Chemical Disinfection Technique

Chemical disinfection, which is often used in health care to kill bacteria on medical

equipment, ﬂoors, and walls, is now being extended to the treatment of medical waste.

Chemicals applied to pathogens deactivate or kill them, thus this treatment results in

disinfection rather than sterilisation. This method is effective for dealing with liquid

wastes such as blood, urine, faeces, and hospital sewage. Although solid and even

severely hazardous health-care wastes, as well as microbiological cultures, sharps,

and other items, may be chemically disinfected (WHO, medicine waste manage-

ment 2018). Chemical disinfection of human waste and animal carcasses is not

recommended. If no other options for disposal are available, they will be shredded

and subsequently subjected to chemical disinfection (B.B.R Panel 2012). Chemical

disinfectants should be disposed of in the proper manner. And, if the procedures are

not correctly implemented, they will result in serious environmental issues.

Chemical disinfection is commonly used to pre-treat COVID waste after it has

been mechanically shred. To prevent the generation of aerosols during shredding, the

exhaust air is routed through a high efﬁciency particle absolute ﬁlter. The bulk of the

crushed trash is combined with chemical disinfectants and maintained in a closed

system or under negative pressure for a set period of time. The contagious germs

are destroyed or inactivated by the orezarganic degraded material in this procedure.

Chemical disinfectants have several advantages, including low effective concentra-

tions, steady performance, quick action, and a broad sterilising spectrum, as well

as minimal residual risks. They not only successfully kill germs but also render

bacterial spores inert (Wang et al. 2020). COVID-waste treatment is frequently sepa-

rated into chlorine and non-chlorine-based systems. The disinfection medium in a

chlorine-based treatment system is NaOCl or ClO2, where the tendency of chlorine

aids in oxidising peptide linkages and denaturing proteins that follow penetration

of cell layers even at neutral pH. Although NaOCl is one of the principal chemical

disinfectants that releases halo acetic acid, dioxins, and chlorinated biocide, it is

only employed on-site due to its unstable nature. It also decomposes to produce salt

and less-toxic compounds that aren’t affected by alcohol or ammonia (Ilyas et al.

2020; Shah 2021a, b). On the other hand, in non-chlorine-based treatment systems,

H2O2 is commonly employed as a disinfection medium. It oxidises and denatures

proteins and lipids, resulting in membrane disorganisation due to the inﬂammation

of saturated H+-ions. This approach is favourable because of its high reactivity and

lack of toxicity associated with chlorinated systems.

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 73

3.4 Disinfection Technique for Processing of Personal

Protective

Surprisingly, the potential application of disinfection technology will not be restricted

to the degree of safety; rather, due to the global scarcity of personal protective equip-

ment, its value will be huge (Mallapur 2020; Singh et al. 2020). Gloves, aprons,

long-sleeved gloves, goggles, ﬂuid repellents, eyes, nose, and mouth protection, face

visor, and breathing mask are among the COVID-19 pandemic protective equipment.

As a result, several governments are pursuing unplanned ways to recycle discarded

personal protective equipment, despite the severe health hazards involved with incor-

rect decontamination (Barcelo 2020). There has been some attention on airborne

particles emitted by COVID-19 infected patients’ breathing or sneezing, which may

travel several metres, remain suspended for approximately 30 min, and survive on

the environment for many days. Because the coronavirus does not puncture mate-

rials, disinfection or PPE sterilisation of the surface or contact area will be sufﬁcient

(Rowan and Laffy 2020). The most difﬁcult task, however, is the re-use of personal

protective equipment that is employed at the same time as ensuring materials are

functional after treatment. As a result, in addition to the replacement of personal

protective equipment, an effective disinfection method is also necessary. The above-

mentioned disinfection temperature techniques are not acceptable due to thermal-

sensitive qualities, resulting in reprocessing; nevertheless, the chemical disinfectant

spray is f ound to damage personal protective equipment (Rowan and Laffey 2020).

The use of vaporised hydrogen peroxide (vH2O2) instead of an aqueous disinfectant

solution has demonstrated encouraging results in the sterilisation of bacteria, prion,

and viruses (Barcelo 2020). The key beneﬁt of low temperature vH2O2 is polymetric

signiﬁcant compatibility, while processing time is reduced (from 10 to 15 h using

ethylene oxide to less than six hours in the conventional vH2O2 process) under both

atmospheric and vacuum settings. However, due to the reduction in H2O2 energy

in the presence of cellulose, compatibility with cellulose-based compounds and the

capacity to penetrate into speciﬁc regions are reduced, limiting the application of

vH2O2 (McEvoy and Rowan. 2019). The N95 mask was recently disinfected using

two methods: I dry heat (using hot air at 750 C for 30 min) and (ii) ultraviolet germi-

cidal irradiation (UVGI, at 254 nm and 8 W for 30 min) (Price et al. 2020). Several

studies have found that hot air treated N95 masks used for more than 5 cycles do

not degrade mask ﬁt (change equally, 1.5%; p-value, 0.67), whereas UVGI treated

N95 masks used for more than 10 cycles signiﬁcantly degrade mask ﬁt and do not

pass quantitative ﬁt testing in a human model (change in ﬁt factor, −77.4%; p-value,

0.0002). However, even if decontamination applies to all layers of the trapped virus I

particles, there remain outstanding questions that must be addressed before COVID

waste may be reprocessed.

74 R. Sharma et al.

3.5 Common Safety Measures on Disinfection Process

COVID-waste has the potential to transmit hospital-acquired infections. However,

as part of overall COVID-waste management, certain safety issues must be adhered

to (Yang et al. 2020).

•For household cleaning and disinfection: Use household cleansers and disinfec-

tant containing 2 g/L chlorine for 30 min on regularly handled surfaces such as

tables, doorknobs, lights, switches, handles, desks, and toilets. For cleaning and

disinfection of electronic gadgets, follow the manufacturer’s instructions. When

it comes to devices, consider utilising a washable cover. If the manufacturer’s

instructions are not available, alcohol-based wipes or sprays containing at least

70% alcohol can be used to disinfect the touch screen. To avoid liquid pooling,

make sure all surfaces are completely dry.

•Cleaning and disinfection of homes with persons isolated in home care (e.g.,

COVID-19 suspected or conﬁrmed): COVID-19 symptoms and how to prevent

the spread of COVID-19 in the home should be educated and understood by all

members of the household. Spray high-touch surfaces four times a day with a

disinfectant containing 75% ethyl alcohol to clean and disinfect them (C2H5OH).

Personal cleaning materials may be provided by the caregiver for a sick person

who has been occupied by a kid or another individual for whom such supplies

are not acceptable. Tissues, paper towels, cleansers, and disinfectants are among

these items. After collecting COVID-waste in double-layered ﬁrmly zipped yellow

bags, the same cleaning process must be used on the waste released by patients.

•White-coats, N95 facial masks, surgical masks, surgical hats, protective goggles,

shoe covers, isolation gowns, gloves, protective suits, and another pair of gloves,

protective hoods, and boot covers should all be worn in the following order: white-

coats, N95 facial masks, surgical masks, surgical hats, protective goggles, shoe

covers, isolation gowns, gloves, protective suits, and another pair of gloves, protec-

tive hoods, and boot covers. The main disinfection procedure while removing

protective clothing is to spray ethyl alcohol in the buffer room, and then they can

be permitted to take off the hood, PPE, goggles, and surgical mask successively

in another room. After that, in a semi-contaminated room, remove the isolation

gown, surgical cap, N95 face mask, and gloves, as needed. After hand washing

and donning a clean-surgical mask, the healthcare worker can be authorised to

enter the clean area.

All used objects must be collected as COVID-waste (even after disinfection

spraying) and processed for the next level of treatment, such as incineration or

microwave (as deﬁned in above section) (Table 4).

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 75

Tabl e 4 Each disinfection technology’s overall Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis (ACR Plus 2020)

Disinfection technology Strengths Weakness Opportunities Threats References

Incineration technique Simple operation,

complete destruction of

BMW/COVID-waste

Energy-intensive, high

capex, release of toxins

and solid residual waste

−90% reduction of

waste volume

Release of secondary

pollutants like dioxin,

furans and bottom ash

Dutta et al. (2018),

Wang et al. (2020)

Pyrolysis technique Complete destruction of

toxins like furan and

dioxins

High investments costs

and strict demand for

heat value

Energy saving and

complete decomposition

of waste volume

Not known and taken as a

safe technology

Dutta et al. (2018),

Wang et al. (2020)

Microwave technique Low action temperature

conserves energy and

reduces pollutant

discharge while avoiding

gaseous emissions

Because of the rather

narrow disinfection

spectrum, autoclaving is

occasionally required

On-site trash treatment is

facilitated by the

construction of a

transportable microwave

treatment unit

Disinfection has a

number of complex effect

elements

Datta et al. (2018),

Wang et al. (2020)

Chemical technique Stable and rapid

performance, as well as a

wide range of sterilizing

options

BMW’s volume and

bulk are not reduced

Disinfectants used

in-house/on-site have the

capacity to kill viral

spores, effectively

controlling virus

transmission

Inhalation of

anthropogenic aerosols

can reach alveoli,

whereas skin absorption

of atomized disinfectants

promotes cancer

Mallapur (2020),

Rowan and Laeffy

et al. (2020),

Singh et al. (2020)

Vaporized hydrogen Low-temperature

heat-sensitive application

In the presence of

cellulose molecules,

concentration decreases

After a thorough

cleansing, protective

objects can be

reprocessed and reused

Fogging creates atomized

aerosols to inﬂict serious

health harm to alveoli,

skins, and mucosa

Barcello (2020),

McEvoy and Rowan

(2019)

Dry heat technique Compatibility of

polymeric materials with

the ability to be

reprocessed

How decontamination

works across all of the

layers of virus trapped in

the particles remains

unresolved

Reuse of N95 masks and

PPE are possible that can

mitigate the risk of

supply-chain

It’s debatable if all layers

of trapped virus in

particles can be

decontaminated

Price et al. (2020)

76 R. Sharma et al.

4 Present Scenario of COVID Waste Management

Worl dwi de

With COVID-19’s expanding worldwide reach, the world is confronted with new

difﬁculties that will put leadership and community resolve to the test. Despite the

fact that governments accept stay-at-home orders and varied degrees of state crises,

households continue to generate garbage. As a result, waste management services

must guarantee that urban base services promote residents’ health and virus contain-

ment to the greatest extent feasible. In this regard, a set of new rules for dealing with

COVID-19-related trash has been issued by a number of organisations and govern-

ments. The new recommendations are precautionary measures to ensure that no addi-

tional health hazards arise as a result of the epidemic. In addition to the standards

governing biological waste disposal, these guidelines must be followed. Although it

is the responsibility to update this guidance as needed, it is based on existing knowl-

edge and COVID-19 protocols for the management of infectious waste created in

hospitals when treating viral and other infectious disease (Fig. 3 and Tables 5, 6).

4.1 On-Site Treatment of COVID-Waste in China

The COVID-19 pandemic and legislative approaches to end the outbreak of the

infection have prompted a worldwide monetary downturn and furthermore produced

9,862,228

1,665,403

8,507,754

902,490

1,171,441

1,382,832

5,653,651

5,653,561

668,114

521,640

237,113

39,861

126,121

41,063

48,888

38,833

162,269

11,306

7,636

TOTAL COVID-19 CASES TOTAL DEATHS

Fig. 3 Graph showing the countries where COVID-19 effected most and number of individuals

deceased due to this (Gutierrez and Clarke 2020)

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 77

Tabl e 5 Global distribution

of conﬁrmed COVID-19(Top

10 countries) (Gutierrez and

Clarke 2020)

Country Total COVID-19 Cases Total deaths

US 98,62,228 2,37,113

France 16,65,403 39,861

India 85,07,754 1,26,121

Italy 9,02,490 41,063

United Kingdom 11,71,441 48,888

Spain 13,82,832 38,833

Brazil 5,653,651 162,269

Russia 56,53,561 1,62,269

Germany 6,68,114 11,306

Poland 5,21,640 7,636

a l ot of clinical waste. This segment was impacted by dispensable plastic-based indi-

vidual defensive hardware (PPEs) and single-utilized plastics by buying shopping

on the web for most essential requirements. PPE during pandemics and the utiliza-

tion of single-use plastics builds the volume of clinical waste as well as changes

the normal thickness of clinical waste (Xuo 2020). Coronavirus is an ecological

and general wellbeing risk, particularly among waste creating PPEs and single-use

plastics, predominantly in creating economies and experiencing signiﬁcant change

nations. Safe strong waste administration is as of now a signiﬁcant worry for nations

where there is an absence of protected and supportable practices and medical services

waste isn’t sufﬁciently controlled (Singh et al. 2020). The current fast development

of medical care waste because of the COVD-19 pandemic is worsening the issue

and there’s an impending risk that the impacts of risky removal of medical care

waste will spread to the danger of ecological contamination (Jiangtao and Zheng

2020). Risky removal of medical services waste contaminates the climate, but at

the same time is related to the spread of transmittable illnesses similar to hepatitis,

HIV/AIDS, cholera, typhoid and respiratory intricacies, fundamentally through the

reuse of removal clinical hardware. Given the unexpected ascent in medical services

waste age, powerful encounters and exercises of COVID-19-drove waste the board

in Wuhan, China, those are possibly useful procedures for some agricultural nations

where COVID-19 is as yet growing and inﬂuencing a lot of medical services waste

age. By and large, dismissed medical care wastes and different types of clinical waste

are arranged off in clean land—or burned as waste energy recuperation. Furthermore,

wellbeing waste alongside city strong waste is unloaded in the open or in inadequately

oversaw land in many agricultural nations—where the development of waste pickers

and animals like canines, goats and cows is regularly noticed (Nezedigu and Chang

2020). A few nations have likewise utilized cutting edge innovation to treat their

clinical waste by steam-sanitized or compound sanitizers, yet this is extraordinary.

Albeit many created nations have exhibited great administration of COVID-19-drove

clinical waste administration, China, like other developing economies and coun-

tries undergoing major transformation, has demonstrated compelling and successful

78 R. Sharma et al.

strategies to combat COVID-19-driven clinical waste administration. More than 30

administrative orders and crisis board orders on ecologically solid administration of

clinical waste have been executed in China since 2003, after the outbreak of the Severe

Acute Respiratory Syndrome (SARS) in the area (Wee and Wang 2020). Exercises

and powerful measures in Wuhan and different districts of China might be important

for signiﬁcant data for some non-industrial nations despite an unforeseen ﬂood in

Tabl e 6 Different countries and agencies have updated their COVID-19-related waste rules. (WHO

2020; EU 2020; SNPA 2020; US OSHA 2020)

Countries/agencies New guidelines to handle biomedical waste at the time OF COVID-19

WHO All biomedical waste generated during the care of COVID19 patients

should be collected in designated containers and bags, processed, and

then securely disposed of or treated, or both, preferably on site,

according to t he WHO. Waste can be transferred off-site with only

suitable treatment and disposal facilities in place. It has also been

emphasised that all personnel participating in health care waste

management should wear adequate PPE (boots, apron, long-sleeved

gown, thick gloves, mask, and goggles or a face shield) and wash their

hands after each usage

European Union (EU) The EU has released the following guidelines/recommendations for

household waste management: (EU 2020). Paper tissues and face masks

should be thrown away right away in the trash bag that was provided in

the patient’s room speciﬁcally for that reason. A second bag should be

kept separate from the ﬁrst to store the caretaker’s and cleaner’s gloves

and face masks. The collected bag must be kept closed at all times and

should never be dumped into another bag. All of these bags should be

gathered and kept in a clean garbage bag (doing so makes the waste to

be collected in a double-layered bag). If the aforementioned actions are

carefully performed, however, these bags can be placed immediately in

the unsorted garbage, with no further collection or disposal methods

required

ITALY In Italy, a government agency has attempted to distinguish between two

types of municipal garbage created by homeowners. T1: Households

with COVID-19 positive persons in isolation or under forced quarantine

create municipal garbage. T2: Municipal garbage produced by families

that do not have COVID-19 positive persons in isolation or who are

required to be quarantined. T1 type waste should be categorised as

contagious medical waste and treated as such, in accordance with

applicable laws. In most cases, just a few ﬁrms deal with this type of

garbage, and they collect it in uniform bags before sterilising it. T2

waste, on the other hand, is collected in accordance with the existing

separate collection system. Tissues, masks, and single-use gloves should

all be part of the residual waste stream, which must be transported in

two sealed bags. Personal protective equipment (PPE) should be worn

by personnel who handle such trash. The rules suggest that the elderly

should not deal with T1 waste, but they can deal with T2 waste if they

take the required measures

(continued)

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 79

Tabl e 6 (continued)

Countries/agencies New guidelines to handle biomedical waste at the time OF COVID-19

USA The Occupational Safety and Health Administration (OSHA) of the

United States has stated that waste that is suspected or known to contain

or be contaminated with COVID-19 does not require any additional

safeguards beyond those already in place to protect workers from the

hazards they face in their daily work in solid waste and waste water

management. Furthermore, they have stated that municipal solid waste

with possible or known SARS CoV-2 contamination should be managed

similarly to non-contaminated municipal solid waste, with strict

engineering and administrative controls, safe work practises, and

personal protective equipment (PPE), such as puncture-resistant gloves

and face and eye protection, in place to protect workers from being

exposed to recyclable materials they manage, including any

contaminants in the materials

clinical waste acquired through SARS and COVID-19-drove clinical waste adminis-

tration. At the peak of the plague, roughly 247 tonnes of clinical waste are generated

every day in the Chinese city of Wuhan, which is multiple times higher than the

pandemic. The peak came in between 15 February and 15 March (Singh et al. 2020).

Before the Covid-19 ﬂare-up, the city had a clinical garbage removal limit of 50 tons

each day with a normal yield of 45 tons (Zuo 2020). This limit depended entirely on

a protection plant that worked typically day in and day out without the capacity to

discard any extra saves or capacity for clinical waste administration. With the rise of

COVID-19 cases in the city, clinical waste production increased by 110–150 tonnes

per day in mid-February and continued to rise to 247 tonnes per day by the conclusion

of the episode on March 15. Later it slowly got back to business as usual in mid-

May (Singh et al. 2020). At the point when neighbourhood specialists understood

that clinical waste was running out of existing ability to securely discard the devel-

oping measure of clinical waste after third seven day stretch of January, they chose

to discover master direct techniques and remember four exceptional organizations

for Solid Waste Management including Giant, which professes to need assembled a

crisis therapy plant with a limit of 30 tons/day by February 22, treated about 25%

of the absolute clinical waste created in the city during the COVID-19 plague (Wei

2020). From January 23 to March 8, 2020, the neighbourhood government author-

itatively recorded lockdown and actual distance approaches in the city. During this

time, the creation of clinical waste has surpassed the current therapy/removal plant

limit in the city, there was an enormous amount of clinical waste accumulated in

wellbeing establishments and put away for a couple of days for guaranteed removal.

This drove the nearby waste administration position to convey versatile incinera-

tors in the city of 11 million individuals to discard disposed of PPEs, for example,

face covers, gloves and other tainted single-utilize defensive stuff. Moreover, neigh-

bourhood specialists took some intense measures to securely discard the developing

measure of clinical waste as per public laws and guidelines, and if any demonstration

was ignored by the administration framework, the comparing join was ﬁned likewise

80 R. Sharma et al.

(Chen et al. 2020). As per government reports, around 50,333 instances of COVID-

19 have been accounted for in Wuhan city. Information delivered by organizations

engaged with the city’s clinical waste administration shows that there are around

90,000 beds in medical clinics and centres in the city, remembering 54,000 beds for

signiﬁcant clinics, 14,000 beds utilized by COVID19 patients alone and 20,000 beds

in recently constructed impermanent emergency clinics (Wei 2020). Waste created

from isolate focuses and self-separation zones was not treated as clinical waste yet

waste produced from possibly dubious family and isolate regions was securely gath-

ered and discarded appropriately as clinical waste. The report shows that China’s

public clinical garbage removal limit expanded to 6066.8 tons/day on March 21,

2020, contrasted with 4902.8 tons/day before the pandemic. In the city of Wuhan,

it arrived at 265.6 tons/day from the initial 50 tons every day prior to the pandemic.

(Chen et al. 2020). A signiﬁcant proportion of involvement and exercises learned

in clinical waste administration during COVID-19 ﬂare-ups in Wuhan is: (i) All the

regions of the city can utilize different crisis removal hardware like protection gadget,

portable therapy gadgets, family incinerators, and mechanical furnaces for removal

of clinical waste. Notwithstanding satisfactory capacity and hold limit of clinical

waste treatment ofﬁces is vital, which can forestall the stripping up of waste created

during crises, for example, COVID-19.

(ii) Changes in clinical garbage removal procedures. Three key shifts were

observed during the ﬂare-up in Wuhan: from decentralisation to centralization, from

occasional to regular activity, and, for the most part, from cremation enlistment

removal innovation. Therapy ofﬁces for clinical waste ought to be more mechanized

and with negligible specialist association, the Internet ought to be founded on the

innovation of the Internet of Things (IoT). Through IoT innovation, the whole cycle

of removal of clinical waste was made in the city of Wuhan constant following

and control measure. IoT’s innovation additionally understood the objectives of

computerizing cycles and utilizing insigniﬁcant specialists for irresistible waste,

remembering data for detecting gadgets, area frameworks, ﬁltering gadgets and video

reconnaissance, and Internet access with every gadget. Enormous limit of versatile

ofﬁces ought to be kept up, particularly during pestilences, which can be vital for

agricultural nations where clinical garbage removal ofﬁces are restricted. Portable

highlights are appropriate for crisis circumstances, yet can likewise be utilized as

essential reinforcement ability for the state later on.

4.2 COVID-Waste Management in South Korea

On January 28, 2020, shortly after COVID-19 was discovered in South Korea,

the environment ministry strengthened the current “Waste Management Act” by

declaring “Extraordinary Measure Way for Safe Waste Management against COVID-

19” (Ministry of Environment, Republic of Korea (MoE-RoK) 2020). COVID waste

may no longer be retained for more than 24 h and must be destroyed on the same day

of collection, as opposed to the previous legislation, which allowed for seven days

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 81

for the last time to be burned within two days after delivery. COVID garbage will also

include indoor domestic waste, which will be subject to conventional COVID waste

disposal procedures. (United Nations Economic and Social Commission for Asia and

the Paciﬁc (UNESCAP 2020). After elevating health warnings to level 4, the guide-

lines were revised to state that garbage disposed of by home-based patients would

be placed in bags and containers provided after spraying. If COVID-19 positivity is

discovered, stored waste should be kept near to the resin box. Local trash disposal

facilities are ordered to address medical waste created by the patient’s isolation from

home and other municipal garbage ﬁrst, in order to treat waste collected on an urgent

basis (within 24 h). The help of the desired waste management organisations is aided

in this effort by the distribution of almost 84,000 units of PPE and masks to project

participants (UNESCAP 2020).

Until mid-July, more than 2600 tonnes of medical waste were gathered from 91

COVID hospitals, 8 residential centres, 24 temporary centres, and isolation houses

that were set on ﬁre. COVID waste is generated not only by hospitals, health facilities,

and isolation, but also during disinfection in a public place or when an infected person

is visited, and such waste is instructed to be treated as medical waste, with recycling

being made twice as mandatory before being sent to the hot spot. COVID-19, a waste

generated by health personnel and medical waste examiners, has also been chosen

as an alternate treatments.

The South Korean government’s prompt, ﬂexible, and transparent steps to iden-

tify COVID waste can help avoid dangerous circumstances and diseases caused by

medical waste. Closing COVID waste output at home quarantine, designated hospi-

tals, and health care institutions has been discovered to assist track their disposal and

treatment within a 24-h period. Local environmental ofﬁcials can conduct a thor-

ough investigation to guarantee that disposal requirements in bags and containers are

strictly followed. Furthermore, as illustrated in Fig. 4a, b collaborative approach for

COVID waste management might be useful in developing a straightforward decision-

making process (MoE-RoK 2020). In order to solve the issues, organised and priori-

tised collaborations between the agencies engaged under the direction of the central

disaster headquarters and the control of the Ministry of Environmental Affairs and

local government have been proven to be successful.

4.3 Using Resources to Handle COVID Waste Efﬁciently

in Spain

Spain is one of the countries that has been the most affected by the SARS-CoV-2

pandemic. Spain had registered 255,953 COVID-19 patients as of J uly 14, 2020, with

28,406 passing. The nation was afﬂicted with 8271 new instances on March 26, 2020,

resulting in the recovery of 1648 people. As a result, the country has seen an increase

in COVID usage, with treatment being a particularly difﬁcult task due to a scarcity of

treatment facilities. Clinical waste, such as protective gloves, face veils, and personal

82 R. Sharma et al.

Fig. 4 In South Korea, public standards for the treatment of trash for self-quarantine and COVID-19

patients have been created, as a cooperative framework for the proper management of COVID-waste

(UNESCAP 2020)

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 83

protective equipment (PPE), increased by 350% in mid-March 2020, according to

the Catalonia Waste Agency. On average days, a regular clinical container of 275

tons/month produced almost 1200 tonnes of COVID (Ilyas et al. 2020).

To help the quick and powerful treatment of gigantic measures of infectious waste,

the organization has used to reinforce the overall administration of three approved

plants. The burning of a bit of clinical waste (the one considered as generally safe)

is approved by some treatment places getting waste from wellbeing ofﬁces and

changed inns. Existing civil waste incinerators have placed work in any case for

clinical garbage removal. Despite the fact that it needed some expenditure, around

700 tonnes of clinical waste from a total of 1200 tonnes was handled in Catalonia

begin on April 15, 2020, using metropolitan rubbish removal agencies (Ilyas et al.

2020). According to the Association of Cities and Regions for Sustainable Resource

Management’s assessment, the most difﬁcult issue is separating COVID waste from

strong city/nearby garbage, particularly family-owned waste. Given the intricacy

and restricted assets that don’t have clear rules during the main ﬂare-up, the present

circumstance has been seen as not comparable to South Korea. Notwithstanding,

taking into account the abrupt attack of COVID-19 and the energizing European

nations, for example, Spain their reaction to the COVID embarrassment is to a great

extent disregarded.

It very well may be seen that the COVID waste assortment is controlled related to

MSW, notwithstanding, the guidelines are obvious to twofold close waste compart-

ments and keep them separate from people who are independent and don’t blend

them in with standard family garbage removal (Association of urban communities

and Region for Sustainable Waste Management 2020). The primary disadvantage of

the framework was found in the hotly anticipated conveyance of the last removal.

Because of the long-term industriousness of SARS-CoV-2 in diverse places (Chin

et al. 2020; Doremalen et al. 2020), there is a risk of contamination dropping into the

COVID waste creation hole and being sent to a mechanised cremation ofﬁce (Fig. 5).

4.4 COVID-19 Waste Management Strategies in Kenya

In April 2020, Kenya’s Ministry of Health (MOH) issued a special process titled

“Safety management and disposal of safety items in the prevention of COVID-19

transmission,” which applies to communities, public places, healthcare i nstitutions,

and COVID-19 isolation centres. COVID-19-related waste handling is strictly regu-

lated. All garbage generated by houses where COVID-19 cases are suspected or

conﬁrmed must be separated and placed in leak-proof liner bags/containers labelled

“infectious waste.“ The leak-proof liner bags/container will be provided by the public

health ofﬁcer from the nearest health facility to the above-mentioned families. The

public health ofﬁcer is responsible for ensuring that infectious trash from suspected

or conﬁrmed COVID-19 households is properly managed. According to Ministry

of Health (MOH) guidelines, infectious waste from suspected or proven COVID-19

must be cleaned daily by the household. When the waste liner bag is two-thirds

84 R. Sharma et al.

Fig. 5 After the breakout of COVID-19 in March 2020, a schematic of waste management in

European nations is shown (Ilyas et al. 2020)

(2/3) full, disinfect it according to MOH guidelines, tie it up correctly, mark it as

infectious trash, and deposit it in a designated area for collection (Olukanni et al.

2018). According to National Environmental Management Authority (NEMA) regu-

lations, infectious trash from suspected or proven COVID-19 households must be

transferred to the nearest public health centre. The public health ofﬁcer is respon-

sible for receiving garbage and managing it in accordance with MOH guidelines and

NEMA regulations.

Commercial, ofﬁce, factory, and industrial waste, as well as waste from

other public places: All trash created by the aforementioned entities is consid-

ered potentially contagious (Wainaina 2020). According to MOH guidelines,

the owner/occupier/manager/caretaker provided suitable coloured leak-proof liner

bags/containers to such public spaces. In conjunction with appropriate actors, the

public health ofﬁcer oversees the safe treatment of infectious waste generated in

such public spaces, as well as ensuring that garbage is handled by registered BMW

handlers in accordance with NEMA regulations. According to MOH guidelines, the

infectious waste was cleaned everyday by the owner/occupier/manager/caretaker.

Infectious waste was potentially transported, handled, and disposed of in accordance

with NEMA regulations. In accordance with MOH guidelines, all trucks, conveyors,

containers, and receptacles used in the holding, storage, transportation, treatment,

and disposal of potentially contagious material were disinfected.

In terms of garbage created by quarantine centres, all waste generated by

these facilities was considered as contagious waste. To quarantine centres, admin-

istrators and supervisors were given enough correctly coloured leak-proof liner

bags/containers. The Ministry of Health also oversees the proper disposal of infec-

tious material from quarantine centres. The administrators and managers were

assured that, in accordance with NEMA regulations, garbage would be handled by

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 85

licenced BMW handlers. When the bags were ﬁlled to two-thirds (2/3), the garbage

was disinfected according to MOH guidelines, correctly knotted, labelled as infec-

tious waste, and put in a designated collecting area. Infectious waste from quarantine

centres was collected, transported, processed, and disposed of in accordance with

NEMA regulations.

4.5 COVID-19 Waste Management Strategies in Australia

The National Biohazard Waste Industry (BWI) advisory group, a division of the

Waste Management and Resource Recovery Association of Australia (WMRR), has

developed table direction to assist in providing guidance to medical clinics, matured,

and medical services suppliers overseeing Coronavirus-inﬂuenced materials, as well

as to assist those who are in charge of waste management, both inside and outside

of these ofﬁces. In the wake of the revelation of Coronavirus as a pandemic by

the World Wellbeing Association (WHO), numerous partners including emergency

clinics, matured and medical care suppliers, are thinking about the extra measures to

guarantee the suitable administration of waste from patients, afﬁrmed or suspected,

contaminated with Coronavirus. WMRR said that the articulation looks to offer

general direction to partners. BWI likewise suggests that associations contact and

work with their waste administration suppliers in the event that they have particular

inquiries or require additional data. The BWI added that its assertion is adjusted

from the 3 march 2020 WHO directing record—Water, sterilization, cleanliness and

waste administration for Coronavirus. The WHO’s specialist brief’s content is based

on the data now available for SARS-CoV-2 and the ingenuity of previous Coro-

navirus diseases. It incorporates information and advice from microbiologists and

virologists, contamination control experts, and others with practical knowledge of

water, sanitation, and hygiene (WASH) and infection prevention and control (IPC)

in crisis and sickness situations.

Nobody knew of any proof that Coronavirus was not transmitted through unpro-

tected human contact during the treatment of biomedical waste until recently, and

Coronavirus is not considered a ‘category A’ infectious illness (World Healthcare

Organisation 2020). BWI likewise comprehends that the WHO and same Australian

healthcare authorities have indeed, proclaimed that clinical waste from tainted

patients ought to be treated as would be expected clinical waste with no extra

measures, BWI feels it is reasonable to propose the reception of extra measures.

It was suggested from BWI that those proposed measures ought to be received

close by current PPE and other signiﬁcant practices. Individuals dealing with clinical

waste should wear appropriate PPE (boots, covers, long-sleeved suits, thick gloves,

covers, and goggles) and complete proper hand hygiene following waste removal.

Once more, BWI would rehash the signiﬁcance of all ofﬁces proceeding to work and

draw in with their waste administration suppliers on the suggested extra measures,

which incorporates Execution of “twofold sacking” of waste from patients afﬁrmed

as Contaminated with Coronavirus. This was effectively be accomplished by ﬁrst

86 R. Sharma et al.

covering all clinical waste. Mobile Garbage Bins (MGBs) with clinical waste canister

liners. For canisters or compartments that have been utilized in segregation rooms or

in nearness to patients afﬁrmed as contaminated with Covid-19, the outside surface

were cleaned off as per WHO rules preceding assortment.

4.6 Strategies for Management of COVID-19 Waste in Sri

Lanka

In March 2020, the Sri Lankan government plans to issue a “Interim rule for the board

of solid garbage formed by families and spots under self-isolation due to the COVID-

19 occurrence.“ The interval regulation is set up in accordance with the existing strong

waste management strategy, guideline, and standards; nevertheless, provisions have

been made to accommodate the speciﬁc needs of the present health crisis situation,

with local specialists in Sri Lanka overseeing garbage. Neighbourhood specialists

and partners should follow these general rules. The ﬁrst phase in the COVID-19

waste management strategy is to identify families, places, and individuals who are

at risk of self-isolation, and to provide extraordinary assistance to such places and

families. Local experts allocated after assets to provide varying rubbish collection

services. They arranged for a different truck to transfer and arrange the rubbish.

Having a sufﬁcient number of waste management groups and a supply of appropriate

trash collection bags. General wellbeing ofﬁcials were relegated to exhort family

units on location removal and furthermore to prepare and oversee exercises of waste

taking care of by occupants and assortment groups in the neighbourhood authority.

Guidelines were given to family units undergoing self-isolation not to practise illegal

open unloading (stressing the possibility of making a legal move against such cases)

and the acceptance of waste for reusing centres or shops was temporarily halted

until the COVID-19 pandemic ended if a patient or deﬁlement was recorded in the

assigned region. Furthermore, until the COVID-19 pandemic ended, the preparatory

programmes, site inspections, and study trips were temporarily suspended to put

the executive destinations that were being worked on by local authorities to waste.

Individuals and beasts were not allowed to go to dumpsites or rummage through

waste. Capable ofﬁcials were selected to actualize waste the board exercises.

Guidance for the family unit garbage removal incorporates age of insigniﬁcant

waste and restricted use of pressing material, instruments utensils and so forth Further

these family units have additionally been told to carefully submit to the waste decrease

direction and guidelines given by the nearby position and general wellbeing ofﬁcial

(Durate and Santana 2020). MSW from families, as well as one’s own isolating loca-

tions with COVID-19 positive cases or those suspected of being tainted, was divided

into at least three kinds. side-effects and room (natural waste)—Waste produced

from food measure, extra once utilization, ruined/disposed of food sources, Non-

biodegradable waste, Special waste—Waste and without a doubt polluted things

like face veils, covers, gloves, cloths, tissues, cleanly cushions, diapers, and elective

materials debased by body liquids of inhabitants.

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 87

For word related wellbeing and security of waste controllers: Local specialists

will supply proper PPE for all people engaged with waste assortment, transport,

and removal. The PPE incorporates unique work wear (overalls), veils, gloves,

shoes/boots and a cover or dispensable work dress. Clean specialists ought to be told

to utilize PPE appropriately and will be checked. The waste overseers and managers

should be appropriately informed and upheld with respect to the idea of the waste

and the essential security strategies to help them to complete their errands. They

should wear adequate PPE, and a new set of PPE should be worn every day of

unusual help. After thorough cleaning and sanitation, overalls can be reused. It is the

power’s responsibility to provide basic PPE. Waste managers should be instructed

to maintain a social distance of at least one metre when performing door-to-door

combination exercises. After emptying the trash, the unique waste collection vehicle

will be sanitised at the ﬁnal disposal location (showering a sanitizer arrangement,

i.e. 70% alcoholic sanitizer/clothing cleanser arrangement/ﬂuid/bathroom cleaners).

For each assortment trip, the clearing will be accomplished. Rubbish collection

workers will be given a sufﬁcient supply of hand sanitizers and cleaning profes-

sionals (cleaner/sanitizers) to wash their hands every time they collect distinct waste

from a separate house/location. During the collection of rare wastes, assortment

groups will be encouraged to minimise their interaction with the outside environ-

ment (carefully instructing them not to drink tea/water, bite betel nut, smoke, or visit

stores). They should be reminded not to touch their faces or the veil when collecting

various wastes.

4.7 COVID-19 Waste Management from Bangladesh’s

Perspective

Biomedical waste poses a concern to worldwide public health, particularly in low-

and middle-income nations like Bangladesh. Each year, at least 5.2 million people,

including 4 million children, die from illnesses linked to uncontrolled medical waste

throughout the world. Human health can be harmed by sharp items, infectious

illnesses can be spread to humans, and the environment can be contaminated by

poisonous and dangerous substances if medical waste is not properly disposed of.

Bangladesh has 460 Upazilla level hospitals, 9722 community level clinics, and

1449 outdoor health facilities that are covered by the DGHS at the national level.

According to the research of Biomedical Waste Management in Covid-19 (Rahman

et al. 2020), there are approximately 117 hospitals operating at the district level. There

are around 2501 recognised private hospitals and 5122 registered diagnostic centres

throughout the country. There are also several clinics, including over 5000 govern-

ment and non-governmental organization-run clinics, as well as doctor’s chambers,

where medical waste is created. There are over 1200 hospitals, clinics, and diag-

nostic centres in Dhaka City alone. According to a Bangladeshi research (Haque

et al. 2020), medical waste is not correctly managed in most Bangladeshi hospitals

88 R. Sharma et al.

due to a lack of proper guidelines, commitment, and training of healthcare profes-

sionals. The biomedical waste management and disposal guidelines were enacted

by Bangladesh’s Ministry of Environment and Forest in 2008. The development of

various boards with their responsibilities, the order of various biomedical waste and

their speciﬁc removal techniques, the use of various shading coding frameworks

and images in waste administration, directions for organisations engaged in conclu-

sive removal of biomedical waste while ensuring natural safety and security, and

ﬁnally punishments for breaking these standards are all detailed in these guidelines.

The Ministry of Health and Family Welfare (MoHFW) is collaborating closely with

the Ministry of Local Government to ensure that medical clinic trash is properly

disposed of. BMW’s executive exercises were ﬁrst remembered for the MoHFW’s

5-yearly HPSP in 1998 for medical clinics providing optional services. In HPNSDP

2011–16, the Health Service organised an Environmental Assessment and Action

Plan. DGHS and DGFP devised a plan to form an Infection Prevention and Control

Committee that would bring all of the emergency clinics together under the leader-

ship of the association’s president to care for medical clinic trash across the board.

Other than the government, a few NGOs are currently working on this issue. For the

time being, the government is just providing coordination to public clinics for the

BMW board. To obtain their operations authorization, private clinics must follow to

government runs on BMW executives. Bangladesh’s government is implementing

BMWM in collaboration with non-governmental organisations. Currently, ﬁve non-

governmental organisations (NGOs) are operating in various parts of the nation to

collect and dispose of medical clinic trash. Only PRISM is using all of the tactics for

removing BMW f or good, including burning, autoclaving, and unloading. Various

organisations rely only on unloading. Before the COVID-19 epidemic, Bangladesh

was dealing with hapless clinical waste administration, and it has now been impacted

severely by a rapid increase in the volume of clinical waste. In Dhaka, Bangladesh’s

capital, the usual clinical waste age rate is 1·63–1·99 kg per bed per day (Miah

and Rashid 2020). Because of Coronavirus, at least 14,500 tonnes of trash from

clinical consideration were created across the nation in April 2020 (Chartier et al.

2020), which has probably increased as a result of the increasing tainting rate. Every

day, 206 tonnes of clinical waste are generated in Dhaka alone due to Coronavirus

(Business standard report, Dhaka 2020). This incapably directed waste addresses a

colossal regular risk and may make a drawn out and unwanted general wellbeing

peril and be a possible wellspring of returning illness. Clinical beneﬁts workplaces

in Bangladesh present gigantic peril to prosperity and environment by uprightness

of insufﬁcient waste administration. Appropriate waste administration strategy is

needed to guarantee wellbeing and ecological security. Basic changes in arrangement

and arranging and backing from government and coordinated effort among public

and private areas would acquire signiﬁcant changes medical care wastes the exec-

utives. Notwithstanding, the medical care waste the executives rule, arranging and

strategy ought to be under the shadow of enactment, accentuation ought to be given

in the advancement of instructive preparing program, record continuing, checking,

audit of existing circumstance and there ought to be cooperation between various

services, emergency clinic specialists, and dynamic investment from the local area.

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 89

4.8 Identiﬁcation and Isolation of COVID-Waste for a Safer

Treatment in India

Although no speciﬁc rules for dealing with COVID-waste were provided till the

middle of March 2020, as COVID-19 expanded across India, an acceptable COVID-

waste management framework was supplied. The major notable step ahead was the

passage of the Epidemic Disease Act of 1897, which allowed the Indian Central

Government to directly impose its policies on state governments (Somani et al.,

2020). On March 18, 2020, the Central Pollution Control Board (CPCB), which is

part of the Ministry of Environment, Forestry, and Climate Change, issued the speciﬁc

rules (CPCB 2020). The “Rules for taking care of, treating, and removing waste

produced during treatment/ﬁnding/isolation of COVID-19 patients” were issued to

reduce COVID-garbage removal at medical facilities, which included isolate camps,

home-care, test collection centres, testing labs, state contamination control sheets,

and bio-waste therapy ofﬁces (CPCB 2020). Despite the fact that the Bio-clinical

Waste Management Rule 2016 was passed, the requirements were maintained clear

to ensure that COVID-waste was removed in a sensible manner (Aggarwal 2020).

As of late, the two most inﬂuential urban centres in India are Delhi and Mumbai.

In Delhi, more than 40 sanitation workers have tested positive for the disease, with

15 of them dying. In Mumbai, 10 experts and two security screens were infected

with COVID-19 and retrieved from the city’s two landﬁlls, Deonar and Kanjurmarg.

These are only two of the most heavily hit cities in the country today (Mallapur

2020). India is on the verge of a COVID-sanctioned trash emergency, and experts

are concerned. Similarly, used veils, tissues, head covers, shoe covers, expanding

material outﬁts, non-plastic, and semi-plastic overalls were to be disposed of in a

yellow bag recommended for burning at a standard biological waste treatment facility

(CBWTF).

There are 200 biomedical waste therapy facilities in the country, two of which

are in Delhi and one in Mumbai. Furthermore, according to CPCB data, these

work settings are currently operating at 60% capacity, a 15% increase since March.

In light of the several occurrences that have occurred in Delhi and Mumbai, the

public normal is low. According to the CPCB and the Maharashtra Pollution Control

Board, the CBTWFs in these two metropolitan areas are operating at 70–75% and

70% cut-off limits, respectively. Prior to the COVID-19 incident, a charity or a

private calamity centre would provide 500 g of biological waste each bed, every day.

According to SMS Water Grace BMW Private Limited, one of Delhi’s CBWTFs,

which collects waste from labs, segregation centres, and crisis facilities, including

one of the city’s COVID-19 government workplaces, the Lok-Nayak Jai Prakash

Hospital, that number has risen to between 2.5 and 4 kg per bed per day. Every day,

a massive COVID-19 government ofﬁce may generate between 1800 and 2200 kg

of biological waste. At the moment, double this by the number of Covid-19 crisis

facilities in the nation, which is 2900. When you add in biomedical waste generated

by 20,700 isolate centres, 1,540 example assortment centres, and biomedical trash

collected by districts (Delhi alone has 12,000 home seclusion ofﬁces), you get a

90 R. Sharma et al.

sense of the magnitude of the problem (ICMR 2020). Per day, Delhi generates 11

tonnes of COVID-related garbage (CPCB 2020); Mumbai has supplied 9 tonnes of

COVID-related rubbish every day (assessed by Brihanmumbai Municipal Corpora-

tion). If Covid-19 instances continue to climb in the coming months and testing limits

remain skewed, several urban zones, including Delhi, may be forced to ship their

Covid-waste to neighbouring states for disposal, according to CPCB experts. Not only

in Delhi and Mumbai, but also in Kerala, the progress in biomedical waste is focusing

on specialists. Kerala has remunerated over 100 tonnes of garbage from Coronavirus

treatment centres within two months after the state began regulating Coronavirus

biomedical waste based on new CPCB standards (Ramteke and Sahu 2020).

Due to the Coronavirus pandemic, India produces around 600 metric tonnes of

biomedical waste each day, which is approximately 10% more garbage than before

the outbreak. Because Covid-waste is transported at such a high rate and volume, it

requires considerably greater scrutiny to ensure that it is managed without generating

further clinical concerns (ICMR 2020). It is estimated that 2 tonnes of Covid-waste

is generated in each state as a result of the inspection, separation, and treatment

of sickness. This is exorbitantly low when compared to the 240 tonnes of garbage

produced each day in Wuhan, the pandemic’s conﬂuence point (CPCB 2020).

On March 18, 2020, the Central Pollution Control Board, Government of India,

issued a set of guidelines for supervising, treating, and expelling waste communicated

throughout treatment, as well as determining and isolating Coronavirus sufferers.

Disengagement wards in medical clinics must maintain segregated concealing

coded canisters for waste detachment under these conditions (Datta et al. 2020).

A provided holder labelled ‘ Coronavirus’ should have been stored in a separate,

small extra area and should only be considered by authorised personnel. In these

wards, a separate sanitization worker strategy for biological waste organisation was

presented. The CPCB educated collecting of biological waste in yellow packs and

the canisters holding this should be sent over to certiﬁed professionals for seclude

camps and home thinking of the predicted patients. According to the requirements,

people handling such garbage should be provided with suitable preparation and

personal protective equipment (PPE), such as three-layered coverings, s prinkle

conﬁrmation covers, gloves, gumboots, and safety goggles (CPCB 2020;Ramteke

and Sahu 2020) (Table 7).

The National Green Tribunal’s (NGT) Status Report documents an increase in

improper biomedical waste segregation from COVID-19 isolation units, quarantine

centres, and quarantine houses (Singh et al. 2020). According to the BMWM Rules

2016, all isolation wards for the diagnosis and treatment of COVID-19 patients must

separate their trash into various color-coded containers or bags. While Urban Local

Bodies (ULBs) may give color-coded yellow bags for hazardous COVID-waste and

manage it from quarantine houses, Indian families do not follow the segregation

process. For example, an uninfected mask or glove should be shredded and wrapped

in paper for 72 h before being discarded as trash (CPCB 2020). In this case, ULBs

serve a critical role in sanitation worker training and monitoring of BMW disposal in

a safe and scientiﬁc manner. While some governments have chosen private garbage

collection organisations and deployed cars until CBTWFs are ready, others have

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 91

given CBTWFs the responsibility for waste collection. ULBs must conﬁrm that

biological waste is removed from hospitals and isolation centres as soon as possible

by an authorised CBTWF. They must give yellow color-coded bags to quarantine

households and guarantee that household medical waste is collected door-to-door

by trained waste workers or CBTWFs directly. According to the recommendations

and the BMWM Rules 2016, home hazardous waste might be deposited at collection

Tabl e 7 Guidelines for COVID waste management in India from the Central Pollution Control

Board (CPCB 2020; Ilyas et al. 2020)

Directions to Guidelines for the handling of COVID-waste

Isolation ward of COVID-19 • Use of different coloured bins/bags in wards

and proper waste segregation as per BMWM

Rules 2016

• In the case of COVID-waste, use double-layer

yellow-colored waste

• After disinfecting the inside and exterior

surfaces of the bags with a 1% NaOCl solution,

the COVID-waste is stored in a special

collecting container labeled “COVID-19”.

COVID-trash must also be labeled as

“COVID-19 waste” to guarantee priority

disposal at treatment facilities

• General garbage, excluding COVID waste,

should not be combined and should be

disposed of as common solid waste

• Maintain a separate record for COVID waste

generated in isolation wards

• Separate collection staffs for COVID-waste

and other solid trash to enable timely garbage

collection and disposal

• SPCBs recording waste creation, collection,

and treatment data

Testing labs and sample collecting centers • State pollution control boards are reporting the

opening of collection facilities and testing labs

to monitor COVID-waste records

• All isolation ward guidelines should be

followed at sample collecting facilities and

testing labs

COVID-19 patients’ quarantine camps and

home care

• Solid waste treatment of ordinary collected

garbage (non-medical)

• Separately collect BMWs in yellow colour

bags/bins if any are present

• As soon as the BMW is created, the quarantine

camps must notify the CBWTF operator so that

COVID-waste may be collected on time

• Waste generated by self/home-quarantine

suspects/patients should be collected separately

in yellow bags and given over to the local

bodies authorized collectors

(continued)

92 R. Sharma et al.

Tabl e 7 (continued)

Directions to Guidelines for the handling of COVID-waste

Common biomedical waste treatment facility • Notifying the appropriate SPCBs when

COVID-waste is received from isolation wards,

quarantine facilities and households, and

testing centres

• Sanitation of waste collectors on a regular basis

• Providing personal protective equipment (PPE)

such as nitrile gloves, three-layer masks, splash

proof aprons, safety boots, and goggles

• Use a designated vehicle for COVID waste

collection, with vehicle branding and

sanitization with 1% sodium hypochlorite as

needed.

• COVID-waste must be disposed of as quickly

as possible after receipt, and the facility’s

operator must keep separate records for

COVID-waste collection, treatment, and

disposal

• If a worker exhibits sickness signs, he or she

should be given enough absence without being

paid less

centres by the generator, however due to the lack of such centres, real implementation

is hampered. The rules also allow ULBs to appoint a nodal person to be in charge of

data feeds on a daily basis in the ‘COVID-19 BMW Tracking App’.

India now has 200 CBTWFs operating for the treatment and disposal of BMW

(usually for BMW generated in HCFs), with the exception of ﬁve states/UTs

(Andaman and Nicobar Island, Arunachal Pradesh, Goa, Mizoram, Nagaland, and

Sikkim), which have none. Aside from that, there are 15,281 HCFs with captive

treatment facilities (installed in the absence of CBTWFs within 75 km of the HCFs).

According to the NGT Status Report, the states have already used 55% of the total

incineration capacity. Even before the pandemic, India’s infrastructure treatment

capacity was inadequate. BMW disposal has grown much more difﬁcult now that

the number of coronavirus infections has surpassed four million. The number of

instances registered and the available disposal capacity appear to be proportionate.

Around half of the states and UTs have insufﬁcient disposal facilities. States that

use 70% of total incineration capacity have the most positive examples. Because of

a lack of waste segregation methods and technical advancements in machinery, the

majority of garbage is burned in incinerators, resulting in the production of poisonous

fumes that are dangerous to employees and residents living near these facilities.

The CPCB standards allow the use of at least 100 feet-deep burying trenches

as a last option in the case of BMW generation if the required disposal facility is

lacking. Furthermore, according to the WHO data cited earlier, 56% of BMW is

discarded publicly, and there have been reports of unlawful dumping in agricultural

ﬁelds. Such practises harm soil health and contaminate groundwater, rendering it

unsuitable for human consumption. According to the WWF, if 1% of all masks are

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 93

disposed of wrongly, 10 million masks might be spread in the environment each

month. Environmentalists have warned that the pandemic would result in a new

environmental disaster (Table 8).

Tabl e 8 Details on generation of COVID-19 waste and Facilities for Disposal (NGT Status report

2020)

State/UT COVID waste

generated (in

tonnes per

day)

Details of facilities for disposal of

covid-19 waste

Adequacy of existing

traetment facility

No. of

CBTWFs

Engaged

Captive

fcailities

(Yes/No)

Deep burial

pits (Yes/No)

Maharashtra 17.494 29 No Yes Adequate, Stand-by

arrangement also

made with TSDFs in

Mumbai, Pune,

Nagpur cities

Gujarat 11.693 20 No No Adequate

Delhi 11.114 2No No 70% of existing

capacity of 2

incineration utilised.

Need to ensure

proper segregation

Tamil Nadu 10.41 8No No 91% of incinerator

capacity utilised.

Need to ensure

proper segregation

and identify alternate

incinerator/disposal

options

Madhya

Pradesh

7.486 11 No No Adequate

Uttar Pradesh 7 18 No No Adequate

Wes t B en ga l 6.5 6No No Adequate

Rajasthan 5.9 8No No Adequate

information not

submitted

Andhra

Pradesh

5.516 0Yes No Adequate with

captive facilities

Kerala 4.71 1Yes No All COVID-waste

sent to CBTWF.

Capacity of CBTWF

not adequate for total

BMW. Hence captive

facilities need to be

operated

94 R. Sharma et al.

5 Recommendation

COVID-waste is clearly at the top of the BMW priority list. The only way to cope

with this seductive abuse of high danger is to collect, transport, and remove COVID-

waste in a timely and efﬁcient manner while adhering to safety precautions. The

segregation of wards/emergency clinics/isolate focuses/home-isolates should prac-

tise separate variety in twofold seal allocated sacks/receptacles. For a convenient

assortment and removal, the job of metropolitan local bodies is somewhat funda-

mental regardless of the way that inside the interment sum a few waste treatment

ofﬁces are confronting the workers emergency. Along these lines, the specialists

associated with this work should be taken as a piece of a fundamental assistance

(Prata et al. 2020). Local governments and CBWTF ofﬁcials should be in charge of

authorised medical services and wellness assessments. It is recommended that no

COVID-waste be dumped by mixing it with household garbage and stored in closed

compartments/containers. The human-to-human contact as well as openings with the

other potential transporters like cell phones, consoles, and so on ought to be kept

away from. Additionally, following each cycle of COVID-waste assortments, the

cars should be cleaned with a 1% Sodium hypochlorite arrangement. After putting

on the evacuation PPE and facemasks, the personnel should avoid touching their

faces, noses, mouths, and eyes, especially after using a 70% liquor sanitizer (Borg

et al. 2020). Individual mindfulness may be a panacea for more secure COVID-waste

treatment, thus the government, local governments, and waste treatment facilities

should spearhead the mindfulness programme, using a variety of media to directly

reach people. The regular usage of protective facemasks and hand gloves is often

consumed to a large amount, and because of their small size and light weight, there

is a chance that these wastes will be disposed with strong trash. It is recommended

that such trash be handled with care, since they might be highly contagious for up

to 7 days. As a result, the rules to keep waste covers falling for at least 72 h must

be observed, as “prevention is better than treatment”. The large number of applied

analysts that must address the successful control of pandemic ﬂare-up because of the

encompassed novel Coronavirus highlights the large number of the applied analyst

that must address the successful control of pandemic ﬂare-up because of the encom-

passed novel Coronavirus (Wigginton and Boehm 2020). As a result, environmental

engineers, medical professionals, healthcare workers, and researchers may use their

extraordinary talents and expertise with multidisciplinary research to answer the

urgent demand. For example, disinfectant spray (H2O2/NaOCl) is frequently advised

to render the encapsulated virus inactive on the waste surface (Gallandet et al. 2017),

however this does not always work. Blood on fomites would necessitate much higher

sanitising assessments (Wood et al. 2020), which can be better sensed for a few

distinct scenarios through multidisciplinary studies.

Because of the global shortage of personal protective equipment, reprocessing,

rather than uncontrolled recycling methods, can help to alleviate the shortage. VH2O2

and hot air disinfection measures can potentially apply for the reprocessing of

COVID-waste, notwithstanding, convenient defeat from existing constraints like

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 95

decrease in oxidant concentration in the present, as reprocessing technology will not

only help to reduce infection spread and environmental health but also increase the

accessibility of personal protectives by their conceivable re-use. On this premise, it is

suggested that an integrated strategy including environmental engineers, healthcare

professionals, and researchers be used to overcome the challenges of COVID-waste

management.

Apart from that, there are a few policy proposals for policymakers that might aid

in the planning of a framework to deal with pandemics in the future.

i. Sanitation employees must be safeguarded, and all governments must acknowl-

edge their importance. For example, the UK government has awarded trash

specialists ‘Keyworker’ status, which implies that education and care arrange-

ments for their children and families would be maintained throughout the

COVID-19 emergency in order for them to continue with their services.

ii. Waste management should be included in disaster preparation, which is now

focused on debris. To cope with and respond to the dynamics of waste gener-

ated during a future pandemic, response measures and guidelines should be

calculated. While bringing states and centres together, such a charter should

be entrenched alongside the regulation for catastrophe waste management

planning.

iii. While solid waste management is incorporated into disaster waste management

planning, it is necessary to guarantee that those involved are well-prepared

to deal with hazardous biological waste, which should be accomplished by

establishing a global standard information sharing platform.

iv. For an efﬁcient and practical biological waste management system, a national

policy framework with guidelines and specialised norms is essential. Most

nations, whether developed or developing, use color-coded BMW segregation

according on their legal structure. Universal coding based on the kind and nature

of trash, as well as training for healthcare workers, would aid in the accurate

classiﬁcation of infectious waste and reduce waste creation.

v. New and sustainable improvements in the reuse of blended and other compli-

cated types of plastics should be prioritised. Incorporating AI into the reuse

process’s organising and handling phases might result in greater recyclability

rates and subjective goods. Complex and ﬁnancially unviable to reuse objects,

as well as fancier and multi-layer plastic packing, should be handled. Incen-

tives for homogeneous plastics, environmentally friendly bio-plastics, and round

developments should also be developed and implemented effectively.

vi. A mechanism should be put in place to distribute additional monies in order to

educate people about circular economy concepts, which should be achievable

through a combination of public and private investments. Greener goods, such

as bio plastics and biodegradable materials with increased recyclability, should

be rewarded and promoted. It should be obvious that the devastation caused by

the COVID-19 emergency should not be addressed at the expense of solving

the longer-term issue of environmental emergency (Climate Action Tracker

2020). Outﬂows might rebound if monetary upgrade packages responding to

96 R. Sharma et al.

the COVID-19 pandemic recovery include low-carbon improvement strategies

and arrangements, such as for greener and more practical items. According to a

handful of recent environmental change reports, the chances are that it will even

exceed lately expanded levels by 2030, notwithstanding decreased monetary

growth (Climate Action Tracker 2020). In this vein, a frameworks level tech-

nique on a global size would be required in the post-COVID-19 world to handle

the issue of robust waste administration and maintain our existing situation.

6 Conclusion

Handling COVID waste has a larger risk than handling regular BMW. To deal with

this extremely infectious waste, it is critical to collect, handle, and dispose of COVID

waste in a timely manner while adhering to thorough safety procedures. Wards, hospi-

tals, detachment centres, and home quarantines should all have their own collec-

tions of double-seal designated bags/containers. Even though many waste treatment

institutions are experiencing personnel challenges during the shutdown period, the

involvement of urban bodies is critical for timely collection and disposal. As a result,

workers who perform this activity should be viewed as part of a vital service. Local

organisations and operators of the Common Biomedical Waste Treatment Facility

shall be responsible for taking appropriate health and safety precautions (CBWTF).

It is not suggested to dispose of COVID waste by combining it with household

garbage and storing it in a sealed container or bin. Personal touch, as well as expo-

sure to other potentially harmful things such as cell phones, keyboards, and other

electronic devices, should be avoided. In addition, vehicles used in COVID waste

collection should be disinfected after each collection by spraying a 1% solution of

sodium hypochlorite on them. Employees should avoid contacting their face, nose,

mouth, and eyes after using 70% alcohol sanitizer shortly after removing PPE and

face work. Because public awareness can be a safe alternative to COVID waste treat-

ment, the government, local groups, and waste treatment facilities should undertake

an awareness campaign utilising various media means to reach out to the general

public. Face masks and gloves are commonly used, and due to their small size and

low weight, there is a signiﬁcant likelihood that this trash will be discarded in solid

garbage. It is suggested that such garbage be handled with caution because it might

be infectious for up to 7 days. As a result, “prevention is better than cure,” and stan-

dards for keeping paper waste disposal for at least 72 h should be observed. The need

for applied researchers to deal with an effective pandemic outbreak as a result of a

coronavirus-covered novel by reducing the threat of SARS-CoV-2 using COVID-

waste forms, its release by the host, and persistence in carrying areas highlights the

number of applied researchers who need to deal with an effective pandemic outbreak

as a result of a coronavirus-covered novel (Wigginton and Boehm 2020). Environ-

mental engineers, medical physicians, health care professionals, and scientists may

now combine their distinct abilities and expertise with a range of studies to solve the

demand for an hour through an integrated strategy. For example, it is recommended to

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 97

use an antiseptic spray (H2O2/NAOCI) to inhibit the surface-sprayed virus (Gallandat

et al. 2017), however this does not always succeed. Blood in fomites may necessitate

a very high antibiotic dosage (Wood et al. 2020), which can be better understood in

a variety of different situations through multidisciplinary study.

References

ACR Plus (2020) Association of cities and regions for sustainable resource management. https://

www.acrplus.org/en/municipal-waste-management-covid-19#data_catalonia

Adly AS, Adly MS (2020) Approaches based on artiﬁcial intelligence and the internet of intelligent

things to prevent the spread of COVID-19. Scoping review. J Med Int Res 22(8):e19104

Aggarwal M (2020) Pollution watchdog releases guidelines to handle COVID-19 biomed-

ical waste. https://india.mongabay.com/2020/03/pollution-watchdog-releases-guidelines-to-han

dle-covid-19-biomedical-waste/

Arun V, Priya BS (2020) Biomedical waste: safe disposal and recycling, a primer biomedical waste.

Retrieved June, 30, 2020

Barcelo D (2020) An environmental and health perspective for COVID-19 outbreak; meteorology

and air quality inﬂuence, sewage epidemiology indicator, hospitals disinfection, drug therapies

and recommendations. J Environ Chem Eng 8:104006

Chakraborty I, Maity P (2020) COVID-19 outbreak: Migration, effects on society, global

environment and prevention. Sci Total Environ, 138882

Chan JF, Kok KH, Zhu Z, C hu H, To KK, Yuan S (2020) Genomic characterization of the 2019

novel human-pathogenic coronavirus isolated from a patient with a typical pneumonia after

visiting Wuhan. Emerg. Microbes Infect 9:221–236

Chartier Y, Emmanual J, Pruss A, Rushbrook P, Stringer R (2020) Safe management of wastes from

healthcare activities, 2nd edn. WHO Press Geneva, Switzerland

Chen M, Yang H, Yin X, Gai R (2020) Current status of medical wastes disinfection and disposal

technologies. Chin J Disinfect 33:171–174

Chin A, Chu J, Perara M, Hui K, Yen HL, Chan M, Poon L (2020) Stability of SARS-CoV-2 in

different environmental conditions. The Lancet Microbe 1(1):e10

CPCB (2020) Guidelines for handling treatment and disposal of waste generated during treat-

ment/diagnosis/quarantine of COVID-19 patients: https://cpcb.nic.in

Cutler S (2020) Mounting medical waste form COVID-19 emphasizes the need for a sustainable

waste management strategy. https://ww2.frost.com/frost-perspectives/managing-the-growing-thr

eat-of-covid-19-generated-medical-waste/

Da Silva CE, Hoppe AE, Ravanello MM, Mello N (2005) Med Waste Manag 25:600–605

Dahchour A, Hajjaji SE (2020) Management of solid waste in MENA regions. Springer Water.

Springer, Cham. 978-3-030–18350

Datta P, Mohi GK, Chander J (2018) Biomedical waste management in India: critical appraisal. J

Lab Physicians 10:6–14

Devi A, Ravindra K, Kaur M, Kumar R (2019) Evaluation of biomedical waste management

practices in public and private sector of health care facilities in India. Environ Sci Pollut Res

26(25):26082–26089

Durate P, Santana VT (2020) Disinfection measures control of SARS-COV-2 transmission. Global

Biosecurity 1(3)

Gallandet K, Wolfe MK, Lantagne D (2017) Surface cleaning and disinfection: efﬁcacy assessment

of four chlorine types using Escherichia coli and the Ebola surrogate Phi6. Environ Sci. Technol

51:4624–4631

Garg S, Lindsay K, Fry A (2020) Hospitalization rates and Charcteristics of patients hospitalized

with laboratory-conﬁrmed coronavirus disease. MMWR Morb Mortl Wkly Rep 69(15):458–464

98 R. Sharma et al.

Ghodrat M, Rashidi M, Samali B (2018) Lifecycle assessment of incineration treatment for sharp

medical waste. Energy Tech, 131–143

Goswami M, Goswami PJ, Nautiyal S, Prakash S (2021) Challenges and actions to the environmental

management of Biomedical Waste during COVID-19 pandemic in India. Heliyon 7(3):e06313

He F, Deng Y, Li W (2020) Coronavirus disease 2019; what we know? J Med Virol 92:719–725

Ilyas S, Srivastava RR, Kim H (2020) Disinfection strategies for Covid-19. Sci Total Environ, 749

Jiangtao S, Zheng W (2020) Coronavirus: China struggling to deal with mountain of medical

waste created by epidemic. https://www.scmp.com/news/china/society/article/3065049/corona

virus-china-struggling-deal-mountain-medical-waste-created

Li Q, Guan X, Wu P (2020) Early transmission dynamics in Wuhan, China of novel coronavirus-

infected pneumonia. N Engl J Med 382:1199–1207

Mallapur C (2020) Sanitation worker at risk from discarded medical waste related

to COVID-19. https://www.indiaspend.com/sanitation-workers-at-risk-from-discarded-medical-

waste-relatedto-covid-19/

McEvoy B, Rowan NJ (2019) Termination sterilization of medical devices using vapourised

hydrogen peroxide: a review of current methods and emerging opportunities. J Appl Microbial

127(5):1403–1420

MoE-RoK (2020) Ministry of Environment. The extraordinary measures for safe waste manage-

ment against COVID-19. https://me.go.kr/home/web/board/read.do?pagerOffset=0&maxPageIt

ems=10&maxIndexPages=10&searchKey=createName&searchValue=%EA%B9%80%EC%

9C%A0%EA%B2%BD&menuld=286&orgCd=&BoardId=1338400&boardMasterId=1&boa

rdCategoryId=&decorator=

Olukanni DO, Aipoh AO, Kalabo IH (2018) Recycling and reuse technology: waste to wealth

initiative in a private tertiary institution. Nigeria. Recycling 3(3):44

Otter JA, Donskey C, Yezli S, Douthwaite S, Goldenberg SD, Weber DJ (2016) Transmission of

SARS and MERS coronavirus and inﬂuenza virus in healthcare settings: the possible role of dry

surface contamination. J Hosp Infect 92:235–250

Owusu PA, Sarkodie SA (2020) Impact of COVID-19 pandemic on waste management. Environ-

ment development and sustainibility. Springer, 020–00956-y

Panel BBR (2012) Guidelines for safe work pratices in human and animal medical diagnostic

laboratories. Morbidity Mortality Weekly Report 61:357–63

Price A, Cui Y, Liao L, Xiao W, Yu X, Wang H, Zhao M, Wang Q, Chu S, Chu L (2020) Is the ﬁt

of N95 facial masks effected by disinfection? A study of heat and uv disinfection methods using

the OSHA protocol ﬁt test. medRxiv

Quadar J, Bhat AH, Gurdekar DK (2018) Quantitative characterization of biomedical waste

generated from some healthcare units of Rewa City. Int J Eng Sci Invent 7(9):85–87

Ranjan MR, Tripathi A, Sharma G (2020) Medical aste generation during COVID-19 (SARS-CoV-2)

pandemic and its management an Indian perspective. Asian J Environ Protcetion 12(5):328–344

Ravi Kant CV, Jaiswal SP, Vaidya K, Chitnis DS (2002) Efﬂuent treatment plant; why and how?

Acad Hospital Admin 14(1):33–37

Rowan NJ, Laffey JG (2020) Challenges and solutions for addressing critical shortage of supply

chain for personal and protective equipment (PPE) arising from Coronavirus disease (COVID-19)

pandemic-case study from the Republic of Ireland. Sci Total Environ 725:138532

Shah MP (2020) Microbial bioremediation & biodegradation. Springer

Saraiva AB, Souza RG, Mahler CF, Valle RAB (2018) Consequential lifecycle modelling of solid

waste management systems-Reviewing choices and exploring their consequences. J Clean Prod

202:488–496

Shammi M, Behal A, Tareq SM (2021) The escalating biomedical waste management to control

the environmental transmission of COVID-19 pandemic: a perspective form two south Asian

Countries. Environ Sci Technol 55(7):4087–4093

Shah MP (2021a) Removal of refractory pollutants from wastewater treatment plants. CRC Press

Singh N, Tang Y, Ogunsietan OA (2020) Environmentally sustainable management of used personal

protective equipment. Environ Sci Technol 54(14):8500–8502

Decontamination Strategies and Technologies for Tackling COVID-19 Hospitals … 99

Singh R, Singh K, Singh G (2018) A study of the biomedical waste management in a teaching

hospital (NCMC and Hospital, Panipat). J Dental Med Sci

Shah MP (2021b) Removal of emerging contaminants through microbial processes. Springer

Somani M, Srivastava AN, Gummadivali SK, Sharma A (2020) Indirect imlications of COVID-19

towards sustainable environment: an investigation in Indian context. Bioresource Technol Reports

11:100491

UNESCAP-United Nation Economic and Social Commission for Asia and the Paciﬁc

(2020) The safe waste treatment for COVID-19, lessons from the Republic of

Korea. https://www.unescap.org/sites/default/ﬁles/200514%20waste%20management%20for%

20COVID-19%28edited%29%20FINAL.pdf

van Doremalen N, Bushnaker T, Morris DH, Hollbrook MG, Gamble A, Williamson BN, Lloyd-

Smith JO (2020) Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1.

N Engl J Med 382:1564–1567

Vilavert L, Nadal M, Schunhmacher M, Domingo JL (2020) Two decades of environmental surveil-

lance in the vicinity of a waste incinerator: human health risks associated with metals and

PCDD/Fs. Arch Environ Contam Toxicol, 69–241–53

Wan Y, Shang J, Graham R, Baric RS, Li F (2020) Receptor recognition by the novel coronavirus

from Wuhan: an analysis based on decade-long structural studies of SARS coronavirus. J Virol,

94

Wang J, Shen J, Ye D, Yan X, Zhang Y, Yang W, Li X, Wang J, Zhang L, Pan L (2020) Disinfec-

tion technology of hospital waste and waste water: suggestion for disinfection strategy during

coronavirus disease 2019 (Covid-19) pandemic in China. Enviorn Pollut 262:114665

Wee SL, Wang V (2020) Here’s how Wuhan plans to test all 11 million of its people for coronavirus.

https://www.nytimes.com/2020/05/14/world/asia/coronavirus/testing-wuhan-china.html

WHO (2020) Shortage of personal protective equipment endangering health workers

worldwide. https://www.who.int/news-room/detail/03-03-2020-shartage-of-personal-protective-

equipment-endangeringhealth-workers-worldwide

Wigginton KR, Boehm AB (2020) Environmental engineers and scientists have important roles

to play in stemming oubreaks and pandemic caused by enveloped viruses. Environ Sci Technol

54:3581–3590

Wood JP, Richter W, Sundarman M, Calfee MW, Serre S, Mickelsen L (2020) Evaluating the

environmental persistence and inactivation of MS2 bacteriophage and the presumed Ebola virus

surrogate Phi6 using low concentration hydrogen peroxide vapour. Environ Sci Technol 54:351–

3590

Yang Y, Wang H, Chen K, Zhou J, Deng S, Wang Y (2020) Shelter hospital mode: how do we prevent

COVID-19 hospitals-accquired infection? Infection Control Hospitals Epidemiology 41:872–873

Youseﬁ H, Moshuri L, Alhari SK (2021) Detection of SARS-CoV-2 viral particles using direct,

reagent-free electrochemical sensing. J Am Chem Soc 143(4):1722–1727

Zhao S, Lin Q, Ran J (2020) Preliminary estimation of the basic reproduction number of novel

coronavirus (2019-nCov) in China from 2019 to 2020: a data-driven analysis in the early phase

of the outbreak. Int J Infect. Dis 92:214–217

Zuo M (2020) Coronavirus leaves China with mountains of medical waste. https://www.scmp.com/

news/china/society/article/3074722/coronavrus-leaves-china-mauntains-medical-waste

Bioremediation of Chlorinated

Compounds

Abel Inobeme , Charles Oluwaseun Adetunji, Mathew John Tsado,

Alexander Ikechukwu Ajai, Jonathan Inobeme, and Bamigboye Oyedolapo

Abstract Environmental contamination by various emerging organic and inorganic

pollutants has become an issue of global concern in recent times. Amongst the various

emerging contaminants known, chlorinated compounds constitute a major group of

interest due to their deleterious effect on humans and the environment. Chlorinated

compounds are widely utilized for different purposes in various areas of modern

society. These contaminants are present in pesticides, petroleum derivatives and

solvents that are used on daily basis in different sectors. Some of these compounds

tend to be persistent in the environment and have the tendencies to bio accumulate

as they are passed across the food chain. Remediation of chlorinated compounds in

contaminated areas from various environmental matrices is therefore of signiﬁcant

priority. Various approaches have been employed for the remediation of this group

of compounds some of which have their inherent limitations. Bioremediation tech-

nology is a promising approach in this regards due to its cost effectiveness, ease

of execution and environmental friendly nature. This chapter critically reviews the

bioremediation of chlorinated compounds. It discusses the source, distribution and

types of chlorinated compounds found in the environment, their effects on humans

and the environment, the role of aerobic and anaerobic microbes in biodegradation

A. Inobeme (B

)

Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Department of

Microbiology, Edo University Iyamho, PMB 04, Auchi, Edo State, Nigeria

e-mail: inobeme.abel@edouniversity.edu.ng; abelmichael4@gmail.com

C. O. Adetunji

Department of Chemistry, Edo University Iyamho, PMB 04, AuchiEdo State 312101, Nigeria

M. J. Tsado

Department of Chemistry, Ibrahim Badamasi Babangida University, Niger State, Lapai 911101,

Nigeria

A. I. Ajai

Department of Chemistry, Federal University of Technology Minna, Minna, Nigeria

J. Inobeme

Department of Geography, Ahmadu Bello University, Zaria, Nigeria

B. Oyedolapo

Department of Chemistry, Kings University, Odeomu, Nigeria

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_5

101

102 A. Inobeme et al.

and the various classes of enzymes that are involved in the bioremediation of chlo-

rinated compounds. An attempt is also made in highlighting the future trends in this

regards.

Keywords Bioremediation ·Chlorinated compounds ·Contamination ·Enzymes

and solvents

1 Introduction

Organic and inorganic pollutants present in the environment are threat to drinking

water and other natural resources such as soil and atmosphere. Amongst the various

organic contaminants, chlorinated compounds mostly in the form of their solvents

constitute a major group of concern due to their deleterious health impacts on the

environment and humans. They are therefore the primary pollutants of ground water

in most countries of the world. Different physical and chemical processes have

been employed for the removal of these contaminants from various environmental

matrices. Some of these methods have their inherent limitations in that they also

generate undesired by products, are highly expensive and non feasibility for large

scale applications (Ali et al. 2019).

One of the most recent approaches that have been widely considered to be efﬁcient

with various promising modiﬁcations for better results is the use of bioremediation.

Bioremediation i s a complex phenomenon that involves various biological processes

that utilize bio-systems for the restoration and clean-up of polluted areas. Studies

have documented that about 400,000 sites around the United States of America are

polluted with chlorinated compounds mostly in the form of solvents. Chlorinated

compounds have been employed widely as degreasing agents in most industrial

processes for a long time. There are various methods of disposal that have been

employed and these have contributed to the broad spread of such compounds in

ground water and soils. One of the most common practices was the returning of such

used chlorinated solvents back to a drum. In such a case, immediately the drum was

ﬁlled, it was then covered and buried underground with other drums. Such drums

corroded eventually and the contents inside escaped into the soil and eventually end

up in the underground water (Sharma 2020).

The microbial communities play an indispensable role in bioremediation tech-

nologies. There are various groups of indigenous microbes that are indispensable

in the process of environmental restoration. The microbial population achieves this

through processes such as immobilizing, oxidizing and transformation of the contam-

inants. The primary role of the microbes is to bring about a reduction in the levels

of the contaminants to non-toxic, undetectable and within permissible range set

by various regulating agencies. Most recent approaches to bioremediation involve

the search for novel microbial groups from the polluted site. The microorganisms

isolated are considered to have a unique ability to induce the remediation of the

contaminants (Mishra et al. 2021, Shah 2020). Several studies have reported the

Bioremediation of Chlorinated Compounds 103

potential of bioremediation in the restoration of environments contaminated with

chlorinated compounds. In one of the most recent studies, Cichocka et al. (2010)

assed the reduction removal of the chlorine substituents present in tetrachloroethene

to ethane. The study involved the use of culture containing Dehalococcoides, which

dechlorinated the compounds to ethane. In their investigation, the microcosms were

made from ground water and then modiﬁed using hydrogen or lactate as electron

acceptor and donor respectively.

Poritz et al. (2013) showed the potential of two Dehalococcoides strains in

coupling all the reductive dehalogenation processes for the conversion into ethane.

They revealed that the genome of Dehalococcoides spp was capable of encoding

for over 20 reductive dehalogenases and this was documented to be the ﬁrst case

of genome that has three different enzymes that are vital for the coupling of the

contaminant to ethane. Kranzioch et al. (2015) observed a rise in the gene copy of

the various intermediates during the process of dechlorination. Matteucci et al. (2015)

observed that trichloroethene and perchloroethene and other chlorinated compounds

are widely distributed in ground water contaminants. They used a microcosm anaer-

obic approach for the assessment of the potential for enhanced or in situ bioreme-

diation. Positive results were obtained for most of the microcosm with regards to

dechlorination, most especially those that were inoculated with mineral medium.

This showed the presence of an active indigenous dechlorinating group within the

ground water. Butyrate and lactate were shown to enhance the process of dechlorina-

tion among the electron donors investigated. Mundle et al. (2012) documented that

chlorinated ethenes are one of the most common contaminants in ground water.

He also opined that there are limited studies details on the isotopic enrichment

factors connected to the various processes. Hence in their study, they determined the

enrichment factor connected with microbial degradation of ethane under anaerobic

microcosms using cultures obtained from River Side in Georgia.

This chapter critically reviews the bioremediation of chlorinated compounds. It

discusses the source, distribution and types of chlorinated compounds found in the

environment, their effects on humans and the environment, the role of aerobic and

anaerobic microbes in biodegradation and the various classes of enzymes that are

involved in the bioremediation of chlorinated compounds. An attempt is also made

in highlighting the future trends in this regards.

2 Concept of Bioremediation

This is a fast emerging technological process which can be utilized alongside together

with various chemical and physical processes of treatment for the effective removal

of various classes of organic and inorganic contaminants in the environment. This

is a remarkable and promising technique for efﬁcient environmental management of

contaminants. Bioremediation is a remediation technology that involves the utiliza-

tion of various microorganisms, fungi or plants for the degradation of contaminants

thereby enhancing the transformation or utilization of substances. There are various

104 A. Inobeme et al.

groups of microorganisms that play role in the process of bioremediation with bacteria

being one of the most prominent. Bacteria are ubiquitous and are found in various

environmental media such as soil, water and air. Processes that involve the utilization

of localized bacteria for the degradation of contaminants under existing conditions

of subsurface are known as the natural attenuation or passive bioremediation process

(Yan et al. 2020, Shah 2021a, b). Natural attenuation processes are most common

within the subsurfaces where the population of the bacteria responsible for the degra-

dation is high. The enhanced process of bioremediation is the type in which the

indigenous bacteria community is stimulated through the addition of electron donors

or substrates so as to bring about an increase in the growth of the bacteria enhancing

faster rates of biodegradation. The substrate introduced is depended on the nature of

the bacteria that are being stimulated for the degradation of the contaminants (Xu

et al. 2018).

Various agencies, industries and researchers have utilized some organic substrates

for the promotion of anaerobic reductive dechlorination of numerous chlorinated

compounds into their ﬁnal innocuous end results. Various remarkable and promising

outcomes have been documented in ﬁeld anaerobic bioremediation applications.

Phytoremediation and bioremediation have advanced progressively and proved efﬁ-

cient most especially in the area of treatment of contaminated areas. Sites polluted

with recalcitrant and chlorinated compounds have been shown to prove more resis-

tances to these techniques, but there are however remarkable progress in the ﬁeld

and laboratory. Some of the most recent breakthroughs in the area of bioremediation

of chlorinated compounds include advancement in various anaerobic and aerobic

processes, bio reductive dechlorination processes, biomonitoring, bioaugmentation

and phytoremediation. Various advanced processes of bioremediation which involve

destructive and non destructive approaches are being employed in the process of

bioremediation. Some of the approaches include phytoremediation, biodegradation,

thermal incineration, reductive dechlorination and advanced oxidation processes

(Sharma 2020).

3 Sources and Distribution of Chlorinated Compounds

in the Environment

Chlorinated compounds have been utilized in various industries as solvents for

degreasing machinery. Common chlorinated compounds are derivatives of ethanes,

methanes and ethane. Most of the known chlorinated compounds show minimal

solubilities in water, variable vapor pressure, and are also denser than water.

Hence some of these chlorinated solvents are commonly described as dense

non aqueous state solvents. There are various chlorinated pollutants that are

discharged into the environment with the prominent being carbon tetrachloride

(CT), trichloromethane (TCM), methylene chloride (MC), dichloromethane, tetra-

chloroethene (PCE) and trichloroethane (TCA). Some other chlorinated compounds

Bioremediation of Chlorinated Compounds 105

include: chlorinated pesticides such as chlordane, chlorinated cyclic compounds,

polychlorinated biphenyls (PCBs) and others (Lin et al. 2021). These chlorinated

compounds are of serious concern because of their deleterious impact on the environ-

ment and human health as well as their remarkable resistance to biological processes

of degradation. Since most of these compounds are usually found in their oxidized

form, there are there not vulnerable to aerobic processes of oxidation, except in

the case of co-metabolism. Chlorinated compounds such as polychloroethylenes,

polychloromethanes and polychloroethane are commonly employed as degreasing

agents and solvents in various commercial and industrial products. They are ubiq-

uitous hence found in water, soil and atmosphere. These compounds have serious

health implications as they also affect the ecosystem at large. There are various

sources of chlorinated compounds which primarily include the use of volatile chlo-

rinated solvents products, the process of disinfection using chlorinated compounds,

emission from various industrial processes as well as improper disposal and storage

methods (Marcon et al. 2021).

There are various groups of chlorinated compounds such as trichlorethene and

perchloroethene as well as other chlorinated solvents which are very common as

underground contaminants. They constitute a dense liquid phase that is non aqueous

and sink through ground water aquifers that are permeable until a zone of nonper-

meability is reached. Chlorinated compounds are known to be persistent in the envi-

ronment and one of the well known contaminants at various industrial sites. The

ubiquitous nature of these compounds is as a result of their wide applications in dry

cleaning, metal degreasing and other production processes (Matteucci et al. 2015).

4 Types of Bioremediation Techniques for Chlorinated

Compounds

There are various strategies involved in the process of bioremediation of chlorinated

compounds.

Two major approaches for the bioremediation of chlorinated solvents present in

ground water have been identiﬁed.

In the ﬁrst approach, the water is pumped to the surface and treatment is carried out

above the ground inside a bioreactor. In another approach, the aquifer is remediated

within (in situ).

i. In situ bioremediation of chlorinated compounds

In situ approach of bioremediation is a better and more reliable approach when

compared to such traditionally employed methods. In situ bioremediation was ﬁrst

utilized as a natural approach for petroleum based compounds in contaminated under-

ground aquifers. Further studies later reported the potential of microbial population

in the degradation of chlorinated compounds as well as other organic and inorganic

contaminants.

106 A. Inobeme et al.

The in situ bioremediation approach is further classiﬁed into two. The ﬁrst is

intrinsic in situ bioremediation while the other is engineered in situ bioremediation.

In intrinsic in situ bioremediation, the primary interest is monitoring the process

of degradation which is already on going so as to ensure that the contamination plume

does not expand.

For the engineered in situ bioremediation, natural degradation is not taking place in

the environment and in some other cases it occurrence is very slow, hence the subsur-

face environment is ﬁrst manipulated so as to stimulate the process of biodegradation

and the rate of the process is enhanced. In the engineered approach, the primary

strategies involve the supply of nutrients such as electron acceptors, phosphorus and

nitrogen to the subsurface. One of the most prominent acceptor of electron employed

is oxygen. However as a result of the low solubility of oxygen gas in water and

the accompanied high biomass production, anaerobic processes have been applied

recently (Philp 2015).

This approach involves the treatment of contaminated substances at the area of

contamination. Hence it does not need the process of excavation; therefore, it is

usually followed by small or no disturbance to the structural composition of the soil.

Normally this approach is supposed to be relatively cheaper in comparison to the ex

situ technique of bioremediation since there is no extra cost required for the excava-

tion of the soil around the site. However the economic constrains of this approach is

the additional cost that is required for the onsite installation and design of some of

the complex equipment required for the technique. Some of the in situ approaches

could be aided (phytoremediation, biosparging and bioventing) while some others

could proceed without the need for any form of enhancement (natural attenuation

and intrinsic bioremediation). Intrinsic bioremediation technique has been efﬁciently

employed for the treatment of site contaminated by chlorinated compounds. However

an effective and successful application of various conditions such as pH, moisture

content, temperature and availability of nutrients must be suitable for the process

(Ashraf et al. 2013).

ii. Ex situ bioremediation of chlorinated compounds

In this approach, the contaminants are removed through excavation from the site

of pollution and then conveyed to another area for the purpose of treatment. This

approach is commonly considered on the basis of extent of pollution, economic

implication of treatment, performance criteria as well as the geographical location.

4.1 Biopile

The biopile aided bioremediation involves the pilling of excavated contaminated soil

above ground which is then accompanied by the amended of nutrients and in some

cases aeration to aid bioremediation through the improvement of microbial activities.

This technique includes some primary components which are irrigation, aeration,

collection systems for leachate and nutrients and bed for treatment. The application

Bioremediation of Chlorinated Compounds 107

of ex situ technique for chlorinated compounds is considered increasingly as a result

of its constructive characteristics together with cost efﬁciency, which aid effective

breakdown on the condition that temperature, nutrients and aeration are controlled

effectively (Wuana and Okieimen 2011).

4.2 Windrows

Windrows is an ex situ technique that depends on the periodic turning of the piled

contaminated soil so as to aid bioremediation through the increase of degradation

processes of the indigenous hydrocarbonoclastic microbes that are present within

the soil. The continuous turning of the contaminated soil, alongside the addition of

water gives rise to an increase in the uniformity of the contaminants, and aeration

hence favoring the process of degradation.

4.3 Bioreactors

As the name suggests, it involves the use of a vessel where the raw material conversion

take place. Several biological reactions are involved. There are various modes of

operation for bioreactors some of which include: fed batch, continuous, multistage

and batch. The selection of a speciﬁc mode of operation is depended on cost and

market economy. The conditions of the bioreactor are capable of supporting natural

processes of cells through mimicking and maintenance of their natural environmental

conditions (Das and Chandran 2011)(seeFig.

1).

5 The Role of Microorganisms in the Bioremediation

of Chlorinated Compounds

Subsurface microbes have the potential for the degradation of most of chlorinated

contaminants. In some instances, the process of biodegradation takes place natu-

rally without any necessary engineering process, in some others, electron acceptors

and nutrients need to be introduced into the subsurface, or some speciﬁc micro-

bial groups have to be stimulated for the purpose of creating some suitable condi-

tions. Bacteria involved in the process of bioremediation are grouped based on their

usage of oxygen: anaerobic, aerobic and facultative anaerobes. The aerobes need

oxygen while the anaerobes do not require oxygen in their environment while the

108 A. Inobeme et al.

Bioremediation

In situ bioremediation Ex situ bioremediation

Aided or engineered

in situ

Intrinsic in situ

Biosparging

Bioventing

Fig. 1 Types of bioremediation technologies

facultative anaerobes are able to survive in both anaerobic and aerobic environ-

ments. Enhanced bioremediation involves the addition of exogenous microbes to

a polluted site. Bioaugmentation basically is employed together with nutrient and

substrate injection approach during the remediation of an aquifer that is polluted

with chlorinated compounds (Nikolova and Gutierrez 2020).

Although several aerobic bacteria posses the ability to degrade chlorinated

compounds, aerobic processes has been shown to be less promising for the remedia-

tion of the subsurface. Composting, which is an aerobic treatment has been employed

for ﬁeld scale treatment of polluted soils. The application of this approach is however

restricted by a rise in the volume of the waste substance, the end products that are

not well characterized and the potential of residual toxicity which is still possible

after treatment makes it less suitable.

Bioremediation under anaerobic environment was found to be favorable. The

creation of an anaerobic condition in a slurry of soil through the addition of

byproducts of potatoes processing was effective for the stimulation of an anaerobic

bacteria population capable of degrading the chlorinated solvent contaminants (Jing

et al. 2018).

Bioremediation of Chlorinated Compounds 109

6 Human Exposure and Health Impact of Chlorinated

Compounds

There are various routes of human exposure to these compounds which inhalation,

dermal contact and ingestion. Various studies have documented the toxicological

impacts of these compounds and the ﬁndings from the study show the possibility of

association of these with cancer incidence in humans. Most chlorinated compounds

of environmental concern have been enlisted as pollutants of priority by various

environmental agencies.

Humans and other organisms within the environment are exposed to broadly

complex mixtures of toxic chlorinated compounds which also include various deriva-

tives of organic and inorganic chlorinated compounds. The various routes for expo-

sure differ depending on the compounds and the nature of the organism under

consideration. The absorption of volatile organic compounds could take place mainly

through inhalation. There are cases were these chlorinated contaminants are absorbed

directly from the environment. For instance, the ﬂatﬁsh commonly found lying

on polluted sediments may absorb a high amount of chlorinated compounds. For

classes of chlorinated compounds that are highly volatile, their intake is usually

through inhalation. Plants could absorb soluble derivatives of these compounds when

dissolve and washed into the environment. Some of the most investigated chlori-

nated compounds due to their inherent toxicity and environmental impact are the

organochlorines, most especially pesticides. The most vital pathway for biological

exposure to some of these volatile chemicals is through the consumption of food.

There is also occupational exposure of humans to these chemicals at work places

(Olutona et al. 2016).

7 Processes and Mechanisms in Bioremediation

of Chlorinated Compounds

The bioremediation of chlorinated compounds occurs through aerobic and anaerobic

processes. Among the class of chlorinated compounds, the chlorinated ethenes are

the most commonly detected within the ground water contaminants and soil. Tetra-

chloroethane (PCE) is capable of resisting aerobic process of degradation and it’s

the only member with this speciﬁc property. Vinyl chloride and the three isomers

of dichloroethene are mineralized by phenol-oxidising bacteria and methanotrophic

microbes through aerobic co-metabolic processes. The oxygenases with wide group

of substrates are responsible for the metabolic oxidation. Some other bacteria also

use vinyl chloride as a source of electrons and carbon for their growth. All the chlo-

rinated derivatives of ethane are dechlorinated reductively under anaerobic envi-

ronment giving rise to harmless compounds as by products such as ethane and

ethane. There are existing evidences which show that the oxidation of vinyl chlo-

ride takes place in the presence of iron (III) reducing conditions. Perchloroethane is

110 A. Inobeme et al.

dechlorinated to tetrachloroethane through the action of methanogens, homoaceto-

gens, sulfate reducers amongst others in a co-metabolic process. The dechlorination

process is catalyzed by various enzymes which contain tetrapyrrole cofactors such as

porphyrins, corrinoids and factor F430. Also, the tetra chloroethane in most bacteria

acts as end electron acceptor during respiration process. Most of these isolates dechlo-

rinate these compounds into cis-1, 2-dichloroethene, although there are also instances

where there is complete dechlorination into ethane (Ferguson and Pietari 2000).

There are various electron donors for the dechlorination of tetrachloroethene

and perchroethene which include formate, pyruvate, acetate, lactate, ethanol and

molecular hydrogen. The Dehalospirillum multivorans posses a rather wide substrate

spectrum, whereas Dehalobacter restrictus only uses hydrogen.

Chlorinated compounds when discharged into the environment are vulnerable

to natural processes of microbial breakdown. They commonly act as acceptor of

electrons due to the presence of substituent that are highly electronegative. The

redox characteristics of the organo halogenated compounds are determined by the

nature of the halogen present and the conditions of the reaction. On a general note,

the greater the number of halogen present as substituent with higher oxidation state,

the more the ease with which reduction occurs. Particularly, the reductive process of

dehalogenation is the pathway through which an halogen atom present is reduced and

there is replacement of a chlorine atom with an hydrogen atom. This occurs mainly

in compounds which have a large number of halogens as substitutes and are totally

not affected by aerobic microbes. In the natural route of microbial breakdown, they

produce 1,2-cis dichloroethene which is then converted to vinyl chloride, a compound

known to be highly carcinogenic. Some of the available studies have reported the

potential of Dehalococcoides spp, a bacteria genus in the complete dechlorination

of the chlorinated compounds to terminal products (Weigold et al. 2016).

There are various reaction mechanisms and routes such as bioaccumulation, routes

of biodegradation as well as various modes of adsorption using plants and microbes

that have been reported for the elimination of contaminants.

There are several reactions that are connected with the degradation of chlori-

nated compounds found in the subsurface under both anaerobic and aerobic condi-

tions. However not all the chlorinated compounds are amenable to the process of

degradation through each of these processes. However, the processes of anaerobic

biodegradation may have the potential of inducing the degradation of most common

chloroethanes, chloroethenes and chloromethanes (Peng and Shih 2013).

i. Anaerobic reductive dechlorination process

This is a process of degradation that is targeted by induced anaerobic bioreme-

diation. The introduction of organic substrate to the subsurface brings about the

enhancement of the process converting mildly anoxic aquifer areas to anaerobic zones

that are reactive, making them suitable for the anaerobic degradation of chlorinated

compounds.

Some of the processes that have been identiﬁed to be connected with the degrada-

tion of chlorinated contaminants include abiotic transformation, aerobic oxidation,

Bioremediation of Chlorinated Compounds 111

cometabolic anaerobic reduction, aerobic co-metabolism and anaerobic oxidation

(He and Su 2015).

ii. Direct anaerobic dechlorination

It is a process in which the bacteria gain energy resulting to their growth since one

or more atoms of chlorine are replaced by hydrogen in an anaerobic atmosphere.

In this process the chlorinated compounds act as the acceptor of electrons while

hydrogen atoms act as direct donor of electrons. The hydrogen that is used up during

this reaction comes from the fermentation of the organic substrate.

iii. Cometabolic reductive dechlorination

This reaction involves the reduction of chlorinated contaminants through the action of

a co-factor or an enzyme that is non speciﬁc. The cometabolic process for chlorinated

contaminants does not give rise to growth beneﬁts or energy the microorganisms that

mediated the reaction process.

Abiotic reductive dechlorination is a chemical process of degradation, it is not

connected with biological activity, the reduction of a chlorinated compound takes

place through the action of reactive compounds. Basically, biotic anaerobic processes

take place through the stepwise removal of chloride ions.

Findings from existing studies have shown that under natural settings, some chlori-

nated compounds can be degraded anaerobically into other compounds. For example,

trichloroethylene can be broken down into ethylene, vinyl chloride and dichloroethy-

lene. Studies in small scale of in s itu anaerobic and aerobic co-metabolic processes

of transformation have revealed that local indigenous microorganisms that have been

grown on phenol are more efﬁcient for the breakdown of cis-1,2-dichloroethylene

when compared to other microorganisms that are grown on methane. Results from

modeling investigations also show that the elimination of dichloroethylene is as

a result of the biostimulation of the local microbial population. Modeling studies

and ﬁeld tests show that under certain conditions, the degradation process becomes

limited stoichiometrically (Atashgahi et al. 2018) (see Fig. 2).

8 Advantages and Limitations of Bioremediation

for Chlorinated Compounds

Bioremediation has the advantage for its ease in treating the contaminants in place

without need for removal of large amount of sediments, soil or water do not need

to be removed before treatment for the removal of the compounds. Bioremedia-

tion promising for chlorinated compounds in that it is an environmentally friendly

approach for pollution management.

Bioremediation is however limited in its application for chlorinated compounds

in situation where there is high concentration of the contaminants within the site

hence the need for combination approaches with other processes of remediation.

Like some other existing technologies for the remediation of various chlorinated

112 A. Inobeme et al.

Anaerobic

dechlorination

Bioremediation Processes

Aerobic processes Anaerobic processes

Aerobic

cometabolism

Abiotic

transformation

Aerobic

oxidation

Reductive

dechlorination

Cometabolic

dechlorination

Fig. 2 Bioremediation processes for chlorinated compounds

compounds, bioremediation also has some other inherent limitations in this regards.

Some of the contaminants that are highly chlorinated with a high molecular mass

such as the chlorinated polycyclic compounds are not easily amenable to degradation

by microorganisms. Also in the microbial breakdown of some of these chlorinated

compounds, the intermediates and end products in some cases tend to be also toxic and

in some cases even more toxic than the parent compounds. A typical example is the

reductive dehalogenation of some chlorinated compounds which could result in the

accumulation of toxic by product which is the vinyl chloride which has been reported

to be carcinogenic. Thus bioremediation approach for some groups of compounds

such as the chlorinated compounds requires a comprehensive understanding of the

microbial processes as well as the intermediate compounds and byproducts. Other-

wise the outcome could be deleterious to the ecosystem (Ghattas et al. 2017) (see

Table 1).

Bioremediation of Chlorinated Compounds 113

Tabl e 1 Advantages and limitations of bioremediation for treated of chlorinated compounds

Bioremediation Advantages Limitations

Ease of treatment Not suitable for treating highly

contaminated sites

Environmentally friendly for most

contaminants

Some intermediates products e.g.

vinlyl chloride are more toxic

than starting contaminants

Suitable for different groups of

organic contaminants

Not efﬁcient for highly

chlorinated compounds with a

large molecular mass

In situ bioremediation Does not require excavation

Structural composition of affected

site is maintained

Additional cost for the onsite

installation of required equipment

Engineered in situ

bioremediation

It is a fast process Requires the supply of nutrients

which act as electron acceptors

Ex situ bioremediation Recommendable for small polluted

sites

Cost for conveying and treatment

away from the contaminated sites

9 Conclusion and Future Trend

Bioremediation is a fast growing technology that has proven to be highly reliable

and promising for the remediation of chlorinated compounds. Its unique potential

of being utilized alongside other chemical and physical treatment process makes

it further promising. Its sustainability as an approach for the management of chlo-

rinated compounds has been well documented. There is however need for further

investigations most especially on the intermediates formed during their degradation

and their impact on the immediate environment.

There is need for further investigations in the area of bioremediation applications

for the treatment of chlorinated contaminants. It is also paramount in this regard to

proffer a synergistic relationship between the environmental effect on behavior and

fate of these pollutants as well as the efﬁciencies of the various bioremediation tech-

nologies. Other approaches such as bioﬁltration could also be incorporated further

into bioremediation and adapted for industrial scale application for the treatment of

chlorinated compounds. There is also need for multidisciplinary technologies for the

efﬁcient treatment of various chlorinated compounds using bioremediation. The need

for further studies into the bioremediation of chlorinated compounds is focusing on

the methods that have been known to bring about alteration of various environmental

parameters and conditions as well as improving the mechanisms through which co

metabolic pathway to bioremediation functions. There is also need for evaluating

nutritional needs, suitable environments, degradation rate and lag time for various

classes of chlorinated compounds. Also there should be more studies with focus

on optimization of various environmental conditions, and enhancement of essential

growth conditions within site speciﬁc differences. Finally there is also need for up

114 A. Inobeme et al.

to date and reliable studies into bioaugmentation together with the speciﬁc microor-

ganism responsible for the degradation of particular chlorinated compounds as well

as the detailed mechanism involved.

References

Ali H, Khan E, Ilahi I (2019) Environmental chemistry and ecotoxicology of hazardous heavy

metals: environmental persistence, toxicity, and bioaccumulation. J Chem 2019:14. Article

ID 6730305. https://doi.org/10.1155/2019/6730305

Ashraf M, MOhd J, Yusoff I (2013) Soil contamination, risk assessment and remediation

Atashgahi S, Liebensteiner M, Janssen D, Smidt H, Stams A, Sipkema D (2018) Microbial synthesis

and transformation of inorganic and organic chlorine compounds. Front Microbiol 9:3079. https://

doi.org/10.3389/fmicb.2018.03079

Cichocka D, Nikolausz M, Jan Haest P, Nijenhuis I (2010) Tetrachloroethene conversion to ethene

by a Dehalococcoides-containing enrichment culture from Bitterfeld. FEMS Microbiol Ecol

72:297–310https://doi.org/10.1111/j.1574-6941.2010.00845.x

Das N, Chandran P (2011) Microbial degradation of petroleum hydrocarbon contaminants: an

overview. Biotechnol Res Int 2011:13. Article ID 941810. https://doi.org/10.4061/2011/941810

Ferguson J, Pietari J (2000) Anaerobic transformations and bioremediation of chlorinated solvents.

Environ Pollut 107(2):209–215. https://doi.org/10.1016/S0269-7491(99)00139-6

Ghattas A, Fischer F, Ternes A (2017) Anaerobic biodegradation of (emerging) organic contaminants

in the aquatic environment. Water Res 116:268–295

He Y, Su C (2015) Use of additives in bioremediation of contaminated groundwater and soil. Open

Access Peer-Rev Chapterhttps://doi.org/10.5772/60915

Jing R, Fusi S, Kjellerup B (17 Jul 2018) Remediation of polychlorinated biphenyls (PCBs) in

contaminated soils and sediment: state of knowledge and perspectives. Front Environ Sci. https://

doi.org/10.3389/fenvs.2018.00079

Kranzioch I, Ganz S, Tiehm A (2015) Chloroethene degradation and expression of Dehalococcoides

dehalogenase genes in cultures originating from Yangtze sediments. Environ Sci Pollut Res

22:3138–3148. https://doi.org/10.1007/s11356-014-3574-4

Lin F, Wang Z, Zhang Z, Xiang L, Yuan D, Yan B, Wang Z, Chen G (2021) Comparative investigation

on chlorobenzene oxidation by oxygen and ozone over a MnOx/Al2O3 catalyst in the presence

of SO2. E nviron Sci Technol 55(5):3341–3351. https://doi.org/10.1021/acs.est.0c07862

Marcon L, Oliveras J, Puntes V (2021) In situ nanoremediation of soils and groundwaters from

the nanoparticle’s standpoint: a review. Sci Total Environ 791https://doi.org/10.1016/j.scitotenv.

2021.148324

Shah Maulin P (2021a) Removal of emerging contaminants through microbial processes. Springer

Shah Maulin P (2021b) Removal of refractory pollutants from wastewater treatment plants. CRC

Press

Matteucci F, Ercole C, Gallo M (02 Sep 2015) A study of chlorinated solvent contamination of the

aquifers of an industrial area in central Italy: a possibility of bioremediation. Front Microbiol.

https://doi.org/10.3389/fmicb.2015.00924

Mishra S, Lin Z, Pang S, Shang W, Bhatt P, C hen S (10 Feb 2021) Recent Advanced technologies for

the characterization of xenobiotic-degrading microorganisms and microbial communities. Front

Bioeng Biotechnol. https://doi.org/10.3389/fbioe.2021.632059

Mundle S, Johnson T, Lacrampe-Couloume G, Mora A (2012) Monitoring biodegradation of ethene

and bioremediation of chlorinated ethenes at a contaminated site using compound-speciﬁc isotope

analysis (CSIA). Environ Sci Technol 46(3):1731–1738

Bioremediation of Chlorinated Compounds 115

Nikolova C, Gutierrez T (17 Jan 2020) Use of microorganisms in the recovery of oil from recalcitrant

oil reservoirs: current state of knowledge, technological advances and future perspectives. Front

Microbiol. https://doi.org/10.3389/fmicb.2019.02996

Olutona G, Olatunji S, Obisanya J (2016) Downstream assessment of chlorinated organic

compounds in the bed-sediment of Aiba Stream, Iwo, Nigeria. Springerplus 2016(5):67. https://

doi.org/10.1186/s40064-016-1664-0

Shah MP (2020) Microbial bioremediation & biodegradation. Springer

Peng Y, Shih Y (2013) Microbial degradation of some halogenated compounds: biochemical and

molecular features. Open Access Peer-Rev Chapter. https://doi.org/10.5772/56306

Philp R (2015) Bioremediation: the pollution solution? https://microbiologysociety.org/blog/bio

remediation-the-pollution-solution.html

Poritz M, Goris T, Wubet T, Tarkka MT, Buscot F, Nijenhuis I et al (2013) Genome sequences of

two dehalogenation specialists – Dehalococcoides mccartyi strains BTF08 and DCMB5 enriched

from the highly polluted Bitterfeld region. FEMS Microbiol Lett 343:101–104. https://doi.org/

10.1111/1574-6968.12160

Sharma I (2020) Bioremediation techniques for polluted environment: concept, advantages,

limitations, and prospects. Open Access Peer-Rev Chapterhttps://doi.org/10.5772/intechopen.

90453

Weigold P, Elhadidi M, Ruecker A, Huson D, Scholten T, Jochmann M, Kappler A, Behrens S

(2016) A metagenomic-based survey of microbial (de)halogenation potential in a German forest

soil. Sci Rep 2016(6):28958. https://doi.org/10.1038/srep28958

Wuana R, Okieeimen F (2011) Heavy metals in contaminated soils: a review of sources, chemistry,

risks and best available strategies for remediation

Xu X, Liu W, Tian S, Wang W, Qi Q, Jiang P, Gao X, Li F, Li H, Yu H (03 Dec 2018) Petroleum

hydrocarbon-degrading bacteria for the remediation of oil pollution under aerobic conditions: a

perspective analysis. Front Microbiol. https://doi.org/10.3389/fmicb.2018.02885

Yan A, Wang Y, Tan S, Yusof M, Ghosh S, Chen Z (30 Apr 2020) Phytoremediation: a promising

approach for revegetation of heavy metal-polluted land. Front Plant Sci. https://doi.org/10.3389/

fpls.2020.00359

Removal of Heavy Metals Using

Bio-remedial Techniques

John Tsado Mathew, Charles Oluwaseun Adetunji, Abel Inobeme,

Musah Monday, Yakubu Azeh, Abdulfatai Aideye Otori,

Elijah Yanda Shaba, Amos Mamman, and Tanko Ezekiel

Abstract Heavy metal pollution has become one of the most signiﬁcant environ-

mental problems globally leading to ecological imbalance. Different techniques like

physical, chemical and biological have been used for removal of heavy metal contam-

inants from the environment. Some of these have limitations such as cost, time

consumption, logistical problems, and mechanical involvedness. Biological strate-

gies, unlike other methods of remediation, are unique in that biological strategies are

environmentally friendly and acceptable, the diversity of organisms involved is wide

and of diverse capabilities that have not yet been exhaustively exploited and also

amenable to genetic modiﬁcation for accelerated bioremediation. There are several

techniques entails with the removal of heavy metals. Therefore, this chapter proposes

to present thorough information on some techniques that might be applied for the

removal of heavy metals using bioremediation. The encouraging evidence as to the

usefulness of microorganisms and their constituents for the remediation of heavy

metals from contaminated environment is reviewed in detailed. Recent advances

in the application of removal of heavy metals through bioremediation also were

highlighted.

Keywords Bioremediation ·Remediation ·Heavy metals ·Techniques

J. T. Mathew (B

) · M. Monday · Y. Azeh · A. Mamman

Department of Chemistry, Ibrahim Badamasi Babangida University, Niger State, Lapai 911101,

Nigeria

e-mail: jmathew@ibbu.edu.ng

C. O. Adetunji

Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Department of

Microbiology, Edo University Iyamho, PMB 04, Auchi, Edo State, Nigeria

A. Inobeme

Department of Chemistry, Edo University Iyamho, PMB 04, Auchi 312101, Edo State, Nigeria

A. A. Otori

Department of Chemical Engineering, Federal Polytechnic, PMB 55, Bida, Niger State, Nigeria

E. Y. Shaba · T. Ezekiel

Department of Chemistry, Federal University of Technology Minna, Minna, Nigeria

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_6

117

118 J. T. Mathew et al.

1 Introduction

The soil contamination through metalloids and heavy metals is a worldwide problem

as a result of the accumulation of these compounds in the environment, endangering

plants, human health as well as animals. Metalloids and heavy metals are usually there

in nature, although the increase of industrialization has cause concentrations to rise

compare to the acceptable ones. However, they are toxic and non-biodegradable, this

happen at lower concentrations. Deposits accumulate in living beings in addition

be converted into dangerous each point in time they are incorporated and pile up

more rapidly compared to when they are metabolized. Consequently, the potentially

dangerous effects are as a result of persistence in the surroundings, toxicity as well

as bioaccumulation in the organisms (Tchounwou et al. 2012;Briffaetal. 2020) (see

Fig. 1).

The rigorousness effects depend on the kinds of metalloid or heavy metal.

Certainly, several of the heavy metals (such as, Fe, Mn, Ni and Co) at concen-

trations very low are vital for living organisms, despite the fact that others

(includes Pb, Hg and Cd) are nonessential, moreover they are toxic even in

trace quantity. It is signiﬁcant to scrutinize the concentration of metalloids and

heavy metals in the ecological system and approve techniques to eliminate them.

For this rationale, diverse methods have been created in some years back, these

includes: chemical remediation (which includes catalysis, adsorption, solubiliza-

tion/precipitation, electrokinetic techniques), physical remediation (such as thermal

desorption, washing, solidiﬁcation), biological remediation (viz: phytoremediation,

Type of Heavy Metal

or Metalloid

At Very Low

concentraon

Toxic even at low

concentraon

Essenal

Noneessenal

Mn, Fe,

Co, Ni

Cd, Pb, Pd

E.g

Fig. 1 Type of heavy metal or metalloids concentration

Removal of Heavy Metals Using Bio-remedial Techniques 119

biodegradation, bioventing), and combined remediation (including washing–micro-

bial degradation and electrokinetic–microbial remediation;) (Fig. 2)(Raffaetal.

2021).

In the study of Dhaliwal et al. (2019) revealed the pollution obtained from heavy

metal which is part of the severe problems and infects the environment through

diverse ways amid the blow of manufacturing in numerous nations. Many techniques

such as chemical physical, as well as biological have been utilised for elimination of

heavy metal pollution from the environs. However, several of these techniques have

constraints which include time consumption, cost, mechanical involvedness along

with logistical problems. At the present time, phytoremediation, in situ immobiliza-

tion of metals as well as biological techniques seem to be to the best way out for

removal of metal/metalloids from the soil. They targeted contaminant site for reme-

diation which is to restrict the heavy metal to go through into the soil, food-chain,

along with the introduction to human beings. In the other hand, the sort of technique

applied for a given location depends on the features such as usual developments

take place at the polluted location, type of chemicals, soil type, and the intensity of

polluted site.

Typically, heavy metals are channeled to environment through the disposal and

processing of heavy metal containing manufactured goods. Pollution of the envi-

ronment caused through the heavy metal enhances awareness globally as a result

of their toxicity in animal, human beings as well as plant along with their inade-

quate of biodegradability. As soon as the metals are polluting the ecosystem, they

possibly will continue for some period depending on the kids of metal that that is

present in the site. The process of remediation for heavy metal polluted sites might

be ex-situ or in-situ, biological and off-site or on-site, chemical as well as physical.

Furthermore, many of these methods applied in mixture through each other intended

for more cost-effective and proﬁcient remediation of a heavy metal polluted environ-

ment. Remediation using biological means in biotransformation of heavy metals into

non-harmful type was look into, in the study. The molecular mechanism of heavy

Various techniques for

remediaon

Physical remediation Chemical remediation Biological remediation Combined re mediation

Washin g,

solidification and

thermal de sorption

Adsorption, catalysis,

Precipita tion/solubilization

and electrokinetic

Biodegradation,

Phytoremediation,

bioventing

Electrokinetic–

microbial remediation

and washing–

microbial degradation

Fig. 2 Types of techniques in bioremediation

120 J. T. Mathew et al.

metal buildup has often biotechnological insinuations for bioremediation of polluted

metal sites (Madhuppriya et al. 2020).

Also, Akhtar et al., (2020) in their study, opined that contamination of heavy metal

has turn out to be one of the major momentous environmental problems worldwide

leading to environmental disproportion. Several of the techniques such as biolog-

ical and physicochemical are consider for the elimination of heavy metals. A good

number of the physical and chemical techniques are less cost-effective and less eco-

friendly, at the same time the biological techniques are not fast in reactions. Nanopar-

ticles, in recent times, have been recommended as proﬁcient substitutes to obtainable

treatment techniques, in altogether supply maintenance as well as ecological reme-

diation of compounds generated from anthropogenic activities. Nanotechnologies

are persistent way out vectors in our monetary ecosystem. Synthesis of biological

nanoparticles has developed noticeably to generate novel resources that are cost-

effective, eco-friendly as well as established with enormous signiﬁcance in wider

use in the regions of medicine, agriculture and electronics. Consequently, they focus

on a proportional remediation of heavy metals by means of chemical, biological and

physical techniques. The nano-structured copper iodide is applied as an adsorbent in

the process of eliminating zinc and chromium. The techniques used in the removal of

heavy metal in the study are; chemical (UV photocatalysis by the application of CuI),

physical (UV light irradiation and adsorption studies by means of CuI) and biological

techniques (by means of co-culture bacteria strains). A grouping of biological and

chemical techniques was in addition investigated by means of CuI–polyvinyl alcohol

nano-composite consists of bacterial co-cultures.

Liu et al. (2018) in a related study, used techniques of ex-situ and in-situ reme-

diation to remedy the heavy metal polluted sites, such as encapsulation, surface

capping, soil ﬂushing, landﬁlling, electrokinetic extraction, soil washing, solidiﬁca-

tion, stabilization, phytoremediation, bioremediation and vitriﬁcation. These reme-

diation methods make use of restraint, removal/ extraction, and immobilization

schemes to decrease the pollution effects by chemical, physical, electrical, thermal as

well as biological remedy developments. These methods display precise disadvan-

tages, applicability and advantages. In addition, the technique of in-situ soil reme-

diation tends to be more cost-effective compared to ex-situ treatment, and pollu-

tant extraction/removal is more encouraging than containment and immobilization.

Among the accessible soil remediation methods, chemical stabilization, phytoreme-

diation and electrokinetic extraction are at the advance phase, whereas some of the

others have been adept at complete, ﬁeld scales. Comprehensive evaluation point out

that chemical stabilization proves a momentary s oil remediation method, phytore-

mediation requires enhancement in effectiveness, serious-contamination sites, land-

ﬁlling and surface capping are related to small, while vitriﬁcation and solidiﬁcation

are the most recent remediation alternative. However, treatability studies are vital

to decide on practicable techniques for a soil remediation scheme, with consider-

ations of the degree and type of pollution, site characteristics, remediation goals,

implementation time, public acceptability and cost effectiveness.

Awasthietal. (2022) study the penalty of heavy metal pollution progressively

mortifying soil eminence in this current era of industry. As a result of this reason,

Removal of Heavy Metals Using Bio-remedial Techniques 121

enhancement of the soil eminence is essential. The application of plants to eliminate

toxins from the soil, like trace elements, heavy metals, radioactive substances, and

organic chemicals, is said to be bioremediation. Fly ash and Biochar techniques are

evaluated for efﬁciency in enhancing the quality of polluted soil. They compiles

amelioration methods and how they are applied in the ﬁeld.

With their toxic efﬂuent, municipal wastewater, or slurry comprising a divers of

heavy metals, anthropogenic and industries activities all around us pollute our mineral

resources. These hazardous metals, in turn, are posing new health concerns to

humans, including allergies, infections, deformities, and diseases. As a result, there is

a growing demand for environmentally friendly, systematic, and creative approaches

of removing these harmful heavy metals. In dealing with a polluted ecosystem,

chemical, biological and physical techniques have not shown to be very effective.

These traditional methods have drawbacks in terms of energy consumption, efﬁciency

and cost. Overcoming their limitations, adsorption, a chemical and physical surface

phenomena, has emerged as a far more cost-effective, reactive, ﬂexible, and efﬁcient

method of removing heavy metals such as chromium, cobalt, nickel, lead, arsenic,

mercury, cadmium, copper and uranium. Microbes, industrial waste biomass, ligno-

cellulosic material, metal organic frameworks (MOFs), nanotubes, and nanocom-

posite substance are all used to fabricate a spectacular adsorbent by modifying their

chemical and physical features (Mahendra et al. 2021).

Heavymetalcontamination has been detected insoilsandrivers asaresultof anthro-

pogenic and rapid industrialization activities like the uncontrolled use of fossil fuel

burning, wastewater sludge dumping and agrochemicals. Heavy metals are non-

biodegradable and have a long shelf life. As a result, remediation is needed to prevent

heavy metal mobilization or leaching into the ecosystem, as well as to make heavy

metal removal easier. Microbes are used in bioremediation to remove heavy metals.

Microbes use a variety of bioremediation processes. These mechanisms are one-of-

a-kind in terms of their specialized requirements, beneﬁts, and drawbacks, because

their success is largely determined by the types of organisms and toxins associ-

ated with the process. plants, Heavy metal contamination causes stress to humans,

animals, plants, and other species in the ecosystem. To ensure cost-effective and effec-

tiveprocesses, a thorough understanding of the process along withvariousremediation

options at several stages is required (Kapahi and Sachdeva 2019).

2 Physicochemical and Biological Methods for the Removal

of Heavy Metals

Since the dawn of time, humanity has used plants and natural materials to combat the

threat of heavy metal toxicity in both human health and the ecosystem around them.

Affected by exposure to about thirty-ﬁve metals have been reported as a result of

accidental or occupational exposure. Twenty-three of them are heavy metal bands.

122 J. T. Mathew et al.

The rising use of heavy metals, especially radionuclides, is causing health prob-

lems. The presence of heavy metals in the environment, as well as their impacts on

humans further down the food supply chain, poses a health risk. As a result, the

abolition of heavy metal has become a top priority. Their study shows systematic

of books, patents, and scientiﬁc material from widely recognized scholarly databases

and search engines on plant-based and natural chemicals against heavy metal contam-

ination are presented. It is thought that a variety of phytoconstituents agents, along

with microorganisms, could operate as heavy metal removers in both humans and the

environment. Bacteria, algae, fungi, and yeast are among the microorganisms that

are utilized to remove heavy metals out aquatic environment (Sharma et al. 2016).

Heavy metal contamination has already been recognized as a global problem

since the beginning of the industrial revolution. Because of its poisonous nature,

heavy metal pollution poses major environmental and health problems. Heavy metal

remediation using traditional techniques is inefﬁcient and results in a huge amount

of secondary trash. Biological process, on the other hand, including microorganisms

and plants, provide simple and environmentally friendly methods for removal of

metal ions, and are thus regarded cost effective and substitute metal removal tools.

Reduction, adsorption, or removal of pollutants from the ecosystem using biolog-

ical resources is referred to as bioremediation (both plants and microorganisms).

Microorganisms’ heavy metal remediation abilities are derived from self-defense

strategies including enzyme production, cellular morphological alterations, and so

on. These defense systems include the active participation of microbial enzymes

which including oxygenases, oxidoreductases, and other enzymes that impact biore-

mediation efﬁciencies. Immobilization methods are also enhancing the technique on

a large scale (Jacob et al. 2018).

Toxic heavy metal pollution is one of the most serious environmental challenges,

and it has accelerated considerably as a result of shifting industrial activities. This

review focuses on the most popular heavy metal phytoremediation techniques,

approaches, including biological techniques. It also gives a broad review of the role

of microbes in heavy metal environmental remediation in damaged ecosystems.

Biological and Physicochemical approaches of heavy metals removal are effective

measures, with the latter divided into ex situ and in situ bioremediation. The

in situ practice such as biosparging, bioventing, bioaugmentation, phytoremedia-

tion and biostimulation. Ex situ bioremediation which includes composting, land

farming, bioreactors and biopiles. Bioremediation make use of microorganisms

that naturally occur such as Sphingomonas, Pseudomonas, Alcaligenes, Mycobac-

terium and Rhodococcus. Bioremediation, in general, requires very little work, is

labor-intensive, sustainable, inexpensive, environmentally friendly, long-term, and

relatively simple to apply. The majority of bioremediation’s drawbacks are related

to its slowness and time-consuming nature; also, biodegradation metabolites can

occasionally be more harmful than the initial substance. Bioremediation summative

assessment may be difﬁcult due to the lack of an acceptable end - point. More

research is needed to develop phytoremediation innovations and discover more

biological remedies for the bioremediation of heavy metals pollution in various

ecosystems (Sayqal and Ahmed 2021).

Removal of Heavy Metals Using Bio-remedial Techniques 123

Nanotechnology has engulfed all aspects of life, encompassing industry, medicine

and health, agriculture, environmental challenges, and bioengineering, to name a few.

Nanostructure materials have transformed every industry. Environmental contami-

nation is a major worry in today’s world, affecting both industrialized and advance

nations. To address this issue, a variety of techniques have been implemented. The use

of nanotechnology in environmental pollution bioremediation is an approach beyond

revolution. Several in-situ (bioslurping, bioventing, phytoremediation, biosparging

and permeable reactive barrier) and ex-situ (windrows, biopile, land farming and

bioreactors) methods are used to accomplish this. Nanoparticles are appropriate

for natural applications due to improved qualities such as reduced time utilization,

nanoscale size, high adaptability for Ex-situ and In-situ use, indisputable amount of

surface-region to-volume percentage for probable sensitivity, and protection from

environment components. To cure contaminants, many nanomaterials and nanotools

are available. The qualities of foreign compounds and the pollution location inﬂuence

each of these approaches and nanotools (Hussain et al. 2022).

Aquaglyceroporins, phosphate transporters, and the effective extruded scheme all

take in arsenic, which is then decreased through arsenate reductases via a dissim-

ilatory reduction pathway. Arsenic oxyanions are used by some autotrophic and

heterotrophic bacteria for energy renewal. Arsenate can be used as a nutrient by

some microbes during the process of cellular respiration. In bacteria, decontamina-

tion operons are a prevalent form of arsenic resilience. As a result, bioremediation

may be a viable and cost-effective method of removing this contaminant from the

ecosystem (Lim et al. 2014).

Adsorption on novel adsorbents, membrane ﬁltration, on exchange, reverse

osmosis, electrodialysis, photocatalysis and ultraﬁltration were among the physico-

chemical removal techniques explored. In terms of application, their beneﬁts and

downsides were assessed. Microorganisms have a function in biological methods of

treatment by settling sediments in the solution. Industrial wastewater is treated with

trickling ﬁlters, activated sludge, and stabilization ponds. Bioadsorption is a novel

biological technology that uses a variety of low-cost bioadsorbents (forest waste,

agricultural waste, algae, industrial waste, and so on) to remove heavy metals from

wastewater to the greatest extent possible. Bioadsorption methods, rather than chem-

ical and physical approaches, are the most environmentally acceptable techniques for

removing heavy metals from wastewater. Chemical techniques, on the other hand, are

the best treatments for harmful inorganic chemicals produced by several industries

that cannot be eliminated through biological or physical means (Gunatilake 2015).

Environmental contamination from pesticides and heavy metals has raised worries

about toxic potential to a variety of species, therefore their removal from water

is critical. The goal of this study was to use physical and biological approaches

to remove heavy metals, pesticides, arsenic, Diazinon and Malathion from water.

Particle trapping techniques, which had straws to trap tiny and big particles, were

used to physically remove the contaminants (Arash et al. 2018).

Biosorption, reduction/oxidation, bioaccumulation, precipitation,

leaching, degradation, phytoremediation and volatilization are among the biological

methods currently available or potentially available for removing and detoxifying

124 J. T. Mathew et al.

toxic metalloids and heavy metals from contaminated sediments and water (bioac-

cumulation, biosorption, reduction/oxidation, precipitation, leaching, degradation,

volatilization, and phytoremediation). They also go over the alternatives for recov-

ering metals accumulated through biosorbents (with the right desorbing agents)

as well as plant biomass and microbial (leaching through biological processes

or chemical reagents, and thermal treatment in controlled systems) (Kikuchi and

Tanaka 2012).

3 Transport Mechanisms of Heavy Metals

Due to their environmental permanence, mobility and toxicity in soils, heavy metals

have sparked a lot of attention. Owing to the increase of mining companies, pesticide

use, as well as other sociocultural activities, certain Chinese soils have been poisoned

by heavy metals, causing the agro-ecosystem to have become damaged. Their study

focus to provide light on the current state of contamination in China, as well as the

sources of heavy metals, their transport modes, and the factors that inﬂuence their

mobility. Additional studies on source identiﬁcation and heavy metal movement

features in soil will be presented in the future (Jing et al. 2018).

Although a set of genes encoding putative transporters have since been discovered,

the methods used in the absorption of necessary heavy metal micronutrients are still

unknown. The heavy metal (CPx-type) ATPases, the natural resistance associated

macrophage protein family, and members of the cation diffusion facilitator family

are the three categories of membrane transporters that have been accused in the

transport of heavy metals in a variety of microorganisms and therefore could utilize

such an important roles in plant. They hope to provide an overview of the main

characteristics of these transporters in plants in terms of function, structure, and

regulation, based on research in a variety of microorganisms (Lorraine et al. 2000).

There are three types of mercury fragments: semi-mobile mercury (Hg(0)-metal,

Hg(0)), mobile mercury (EtHgCl, MeHgCl, and other mercuric compounds) and

nonmobile mercury (EtHgCl, MeHgCl), as well as other mercuric compounds) (HgS,

HgSe, and Hg2Cl2)(Yaoetal. 2019).

Membrane transport of non-essential hazardous heavy metals (type 0 heavy

metals) clearly deﬁnes their absorption, excretion from the body, and distribution, as

well as restricts their access to Intracellular target sites. Membranes have a crucial

role Several researchers have focused on the toxicity of class 0 metals, and signiﬁcant

data has been gathered on the mechanism(s) of metal transfer through membranes.

Metal transport features are not exactly equivalent in cell populations, or even on

separate sides with same cell, or under various physiological circumstances, and no

single hypothesis to explain this process in all cells has been proposed. However, it’s

plausible that the various cell mechanisms hypothesized are variants on a few basic

motifs (Foulkes 2000).

Removal of Heavy Metals Using Bio-remedial Techniques 125

Adsorption, desorption and ion exchange, mobilization, aqueous complexa-

tion, and biological immobilization, mineral dissolution, plant uptake, and precip-

itation all inﬂuence lead dispersion in soil. Simultaneously, chemical mechanisms

such as oxidation–reduction reactions, cation sorption on exchange complexes, and

chelation with organic matter are responsible for lead dispersion as well as migration

in soils (Kushwaha et al. 2018; Palansooriya et al. 2020).

Under fed conditions, two critical processes occur: iron oxide is decreased, and

absorbed arsenic is discharged into solution phase; arsenate [As(V)] absorbed on

solid phase is lowered to As(III), which is less effectively absorbed than As(V) and

thus has a higher potential to separation into solution phase (LeMonte et al. 2017).

Various physical and chemical processes regulate cadmium’s behavior in the soil.

Rainfall and adsorption reactions help to keep it in the soil. The fundamental mech-

anism is rainfall, with anions in the f orms of PO4 3, OH, CO2, and S2, with cadmium

adsorption on the surface of soil minerals occurring through both nonspeciﬁc and

speciﬁc mechanisms (Zhang et al. 2018; Wang et al. 2022).

The majority of chromium in soils comes from weathered minerals found in

ultramafc rocks. The chromite weathering process has two phases: a manganese

oxide oxidation reaction to chromium (VI), and a chromium (III) hydrolysis reaction

to Cr(OH)3. Furthermore, chromium is found in signiﬁcant amounts in spinels, clay

minerals, and iron oxides (Christopher et al. 2011; Hausladen et al. 2019).

They leach into subsurface ﬂuids, travelling down water routes and ﬁnally settling

in the aquifer, or they are swept away by run-off into surface waters, causing water

and soil contamination. Toxicity and poisoning are common in ecosystems due to

coordination and exchange processes. They mutilate their structures and obstruct

bioreactions of their functions when consumed, forming stable biotoxic chemicals

(Ruangcharus et al. 2020).

4 Impact of Heavy Metals on Human Health

and the Environment

Heavy metals can enter the body through the use of the intestinal system, the

skin, or breathing. Toxic metals have shown to be a signiﬁcant health risk, owing

to their propensity to harm membranes and DNA, as well as disrupt enzyme

activity and protein function. By attaching to free thiols or other functional groups,

perturbing protein folding, accelerating the oxidation of amino acid side chains,

or/and displacing critical metal ions in enzymes, these metals disrupt native proteins’

activities. The biochemical and physiological implications of hazardous metal inter-

actions with proteins and enzymes were accounted for in their study. Because heavy

metal poisoning of the ecosystem is one of the most serious worldwide issues, certain

detoxifying procedures are also discussed (Witkowska et al. 2021).

126 J. T. Mathew et al.

Toxic heavy metal pollution of terrestrial and aquatic ecosystems is an environ-

mental issue that is a public health risk. Heavy metals accumulate in the ecosystem as

persistent contaminants and harm food systems as a result. The buildup of poten-

tially hazardous heavy metals in biota poses a health risk to their consumers, who

include humans. This article examines the various elements of heavy metals as

hazardous compounds in depth, with a particular focus on their environmental dura-

bility, toxicity for living beings, and bioaccumulative potential. These elements’

trophic transmission in aquatic and terrestrial food chains/webs has signiﬁcant rami-

ﬁcations for animal and human health. The amounts of potentially harmful metalloids

and heavy metals in various environmental components and in the resident biota must

be assessed and monitored (Ali et al. 2019; Madiha et al. 2022).

Cadmium poisoning has been linked to bone and kidney damage. Cadmium

also has been discovered as a human carcinogen that can cause lung cancer. Lead

poisoning affects fetuses, babies, and children’s growth and neurobehavioral devel-

opment, as well as raising blood pressure in adults. Mercury is harmful in both its

inorganic and elemental forms, but the organic molecules, particularly methylmer-

cury, that accumulate in the food chain, i.e. in predatory ﬁsh in lakes and seas, are the

primary routes of human exposure. Long-range transboundary air pollution is really

only one source of exposure to toxic metals, but due to their potential and persis-

tence for global atmospheric transmission, atmospheric emissions have an impact on

even the most remote places (Rafati et al. 2017).

Also, heavy metals can cause toxicity in some organs of the human body

including as neurotoxicity, nephrotoxicity, skin toxicity, cardiovascular toxicity and

hepatotoxicity, among other things (Saikat et al. 2022).

5 Recent Reports on Removal of Heavy Metals Through

Bio-remedial

In the last few decades, rapid industrialization, increased population expan-

sion, hazardous industrial and urbanization practices have all contributed to the

growth of ecological pollution. Heavy metals are one of those contaminants that,

due to its toxicity, are linked to public health and environmental consequences.

Successful bioremediation can be achieved using both “in situ” and “ex situ” tech-

niques, depending on the kind and quantity of contaminants, site conditions, and cost.

Recent advancements in artiﬁcial neural networks and microbial gene modiﬁcation

aid in improving “in situ” heavy metal bioremediation at contaminated areas. For

the efﬁcient elimination of toxic metals using diverse indigenous microorganisms,

multi-omics techniques are used (Oindrila et al. 2021).

Because of the damaging effects of long-term environmental contamination, heavy

metal pollution poses a major threat to all forms of life in the environment. At low

quantities, these metals are very sensitive and can be preserved in food webs, posing

a severe public health danger. Several organic contaminants and metals are still not

Removal of Heavy Metals Using Bio-remedial Techniques 127

biodegradable and can persist for a long period in the ecosystem. Traditional chemical

and physical techniques of remediation are inefﬁcient and result in enormous amounts

of chemical waste. Over the years, there has been a growing and strong interest in

the equilibrium of dangerous metals. Biosensor bacteria are both environmentally

friendly and cost-effective. As a result, microbes have a range of metal sequestration

processes that allow them to have higher metal biosorption capabilities (Tarekegn

et al. 2020).

Due to increased industrialization as well as certain other anthropological prac-

tices, substantial quantities of heavy metals are already being added to the soil and

untreated sewage on a daily basis. Several more heavy metals are already non-

biodegradable, so they continue to stay in circulation after being discharged into

the ecosystem. Certain heavy metals could cause chronic and deadly disorders in

people, as well as impact plants and animals metabolism, if their concentrations

above the threshold level. Many of the current physical and chemical techniques for

removal of heavy metals from industrial efﬂuents, including ion exchange, electro-

chemical treatment, reverse osmosis, and precipitation, have not been proven to be

cost effective, so a biological perspective might also demonstrate to be a substitute

remediation innovation for accumulation of heavy metals. Microbes have developed

techniques to resist, metabolize or detoxify heavy metals, including sequestration or

active efﬂux with insoluble or proteins substances. In t heir, it was observed that, the

relationship of microorganisms and heavy metals, as well as their uses in heavy metal

remediation (Sanjay 2020).

6 Conclusion and Future Trends

Bacteria are one of the most important microbiological options for bioremediation;

nevertheless, only a few studies have been conducted in this ﬁeld, and more compre-

hensive and comprehensive investigations are needed to get the most out of bacterial

systems as “heavy-metal pollution alleviators.” Multidisciplinary methods are also

required for the effective treatment of various heavy metals employing bioremedia-

tion. Further research into heavy metal bioremediation is needed, with an emphasis

on strategies that have been proven to change various environmental characteristics

and conditions, as well as reﬁning the processes by which the co metabolic pathway

to bioremediation works. There’s also a requirement to assess appropriate conditions,

deterioration rates, and lag times for diverse heavy metal genres. In addition, further

research should be done on optimizing diverse environmental circumstances and

improving vital growth conditions within site-speciﬁc variances. Furthermore, there

is a need for current and credible research on bioaugmentation, as well as the speciﬁc

microbe accountable for heavy metal breakdown and the exact factors involved.

128 J. T. Mathew et al.

References

Akhtar FZ, Archana KM, Krishnaswamy VG (2020) Remediation of heavy metals (Cr, Zn) using

physical, chemical and biological methods: a novel approach. SN Appl Sci 2:267. https://doi.org/

10.1007/s42452-019-1918-x

Ali H, Ezzat K, Ikram I (2019) Environmental chemistry and ecotoxicology of hazardous heavy

metals: environmental persistence, toxicity, and bioaccumulation. J Chem 14https://doi.org/10.

1155/2019/6730305

Arash JK, Nooshin JGJ, Paria D, Kiadokht J (2018) Application of physical and biological methods

to remove heavy metal, arsenic and pesticides, malathion and diazinon from water. Turk J Fish

Aquat Sci 19(1):21–28. https://doi.org/10.4194/1303-2712-v19_1_03

Awasthi G, Nagar V, Mandzhieva S, Minkina T, Sankhla MS, Pandit PP, Aseri V, Awasthi KK, Rajput

VD, Bauer T et al (2022) Sustainable amelioration of heavy metals in soil ecosystem: existing

developments to emerging trends. Minerals 12:85. https://doi.org/10.3390/min12010085

Briffa J, Sinagra E, Blundell R (2020) Heavy metal pollution in the environment and their

toxicological effects on humans. Heliyon 6(9):e04691. https://doi.org/10.1016/j.heliyon.2020.

e04691

Christopher TM, Jean MM, Martin BG, Karl JE (2011) Chromium(VI) generation in vadose zone

soils and alluvial sediments of the southwestern Sacramento Valley, California: a potential source

of geogenic Cr(VI) to groundwater. Appl Geochem 26(8):1501. https://doi.org/10.1016/j.apgeoc

hem.2011.05.023

Dhaliwal SS, Singh J, Taneja PK, Mandal A (2019) Remediation techniques for removal of heavy

metals from the soil contaminated through different sources: a review. Environ Sci Pollut Res

32(10). https://doi.org/10.1007/s11356-019-06967-1

Foulkes EC (2000) Transport of toxic heavy metals across cell membranes (44486). https://doi.org/

10.1177/153537020022300304

Gunatilake SK (2015) Methods of removing heavy metals from industrial wastewater. J Multidiscip

Eng Sci Stud 1(1):12–18

Hausladen D, Fakhreddine S, Fendorf S (2019) Governing constraints of Chromium(VI) formation

from Chromium(III)-bearing minerals in soils and sediments. Soil Syst 3:74. https://doi.org/10.

3390/soilsystems3040074

Hussain A, Rehman F, Rafeeq H, Waqas M, Asghar A, Afsheen N, Rahdar A, Bilal M, Iqbal HMN

(2022) In-situ, Ex-situ, and nano-remediation strategies to treat polluted soil, water, and air-a

review. Chemosphere 289:133252. https://doi.org/10.1016/j.chemosphere.2021.133252

Jacob JM, Karthik C, Saratale RG, Kumar SS, Prabakar D, Kadirvelu K, Pugazhendhi A (2018)

Biological approaches to tackle heavy metal pollution: a survey of literature. J Environ Manag

217:56–70. https://doi.org/10.1016/j.jenvman.2018.03.077

Jing F, Chen X, Yang Z, Guo B (2018) Heavy metals status, transport mechanisms, sources, and

factors affecting their mobility in Chinese agricultural soils. Environ Earth Sci 77(3):104. https://

doi.org/10.1007/s12665-018-7299-4

Kapahi M, Sachdeva S (2019) Bioremediation options for heavy metal pollution. J Health Pollut

27;9(24):191203. https://doi.org/10.5696/2156-9614-9.24.191203

Kikuchi T, Tanaka S (2012) Biological removal and recovery of toxic heavy metals in water envi-

ronment. Crit Rev Environ Sci Technol 42(10):1007–1057. https://doi.org/10.1080/10643389.

2011.651343

Kushwaha A, Hans N, Kumar S, Rani R (2018) A critical review on speciation, mobilization and

toxicity of lead in soil-microbe-plant system and bioremediation strategies. Ecotoxicol Environ

Saf 147:1035–1045. https://doi.org/10.1016/j.ecoenv.2017.09.049

Removal of Heavy Metals Using Bio-remedial Techniques 129

LeMonte JJ, Stuckey JW, Sanchez JZ, Tappero RV, Rinklebe J, Sparks DL (2017) Sea level

rise induced arsenic release from historically contaminated coastal soils. Environ Sci Technol.

acs.est.6b06152. https://doi.org/10.1021/acs.est.6b06152

Lim KT, Shukor MY, Wasoh H (2014) Physical, chemical, and biological methods for the removal

of arsenic compounds. Biomed Res Int 1(10):1–9. https://doi.org/10.1155/2014/503784

Liu L, Li W, Song W, Guo M (2018) Remediation techniques for heavy metal-contaminated soils:

Principles and applicability. Sci Total Environ 633(23):206–219. https://doi.org/10.1016/j.scitot

env.2018.03.161

Lorraine EW, Pittman JK, Hall JL (2000) Emerging mechanisms for heavy metal transport in

plants. Biochimica et Biophysica Acta (BBA)-Biomembranes 1465(1–2):126https://doi.org/10.

1016/s0005-2736(00)00133-4

Madhuppriya M, Shyamala GR, Saranya A, Rajarajeswari P, Prabhavathi P, Dinesh KS (2020)

Remediation techniques for heavy metal contaminated ecosystem–a review. J Adv Sci Res

11(2):1–10

Madiha Z, Rashid AY, Ameen A, Yasir S, Ali L, Mahpara F, Khalid AK, Li S (2022) Health and

environmental effects of heavy metals. J King Saud Univ Sci 34(1):101653. https://doi.org/10.

1016/j.jksus.2021.101653

Mahendra K, Aparna S, Alak KS, Manish SR, Mohd S (2021) Remediation strategies for heavy

metals contaminated ecosystem: a review. Environ Sustain Indic 12:100155. https://doi.org/10.

1016/j.indic.2021.100155

Oindrila P, Amrita J, Dibyajit L, Nag M, Ray R (2021) In situ and ex situ bioremediation of heavy

metals: the present scenario. J Environ Eng Landsc Manag 29(4):454–546. https://doi.org/10.

3846/jeelm.2021.15447

Palansooriya KN, Shaheen SM, Chen SS, Tsang DCW, Hashimoto Y, Hou D, Bolan NS, Rinklebe J,

Ok YS (2020) Soil amendments for immobilization of potentially toxic elements in contaminated

soils: a critical review. Environ Int 134(23):105046. https://doi.org/10.1016/j.envint.2019.105046

Rafati RM, Rafati RM, Kazemi S, Moghadamnia AA (2017) Cadmium toxicity and treatment: an

update. Casp J Int Med 8(3):135–145. https://doi.org/10.22088/cjim.8.3.135

Raffa CM, Chiampo F, Shanthakumar S (2021) Remediation of metal/metalloid-polluted soils: a

short review. Appl Sci 11(9):4134. https://doi.org/10.3390/app11094134

Ruangcharus C, Kim SU, Hong CO (2020) Mechanism of cadmium immobilization in phosphate-

amended arable soils. Appl Biol Chem 63:36. https://doi.org/10.1186/s13765-020-00522-0

Saikat M, Arka JC, Abu MT, Talha BE, Firzan N, Ameer K, Abubakr MI, Mayeen UK, Hamid O,

Fahad AA, Jesus SG (2022) Impact of heavy metals on the environment and human health: novel

therapeutic insights to counter the toxicity. J King Saud Univ Sci 34(3):101865. https://doi.org/

10.1016/j.jksus.2022.101865

Sanjay P (2020) Microbial bioremediation of heavy metals: emerging trends and recent advances.

Res J Biotechnol 15(1):164–178

Sayqal A, Ahmed OB (2021) Advances in heavy metal bioremediation: an overview. Appl Bion

Biomech 2021:1609149. https://doi.org/10.1155/2021/1609149

Sharma S, Rana S, Thakkar A, Baldi A, Murthy RSR, Sharma RK (2016) Physical, chemical and

phytoremediation technique for removal of heavy metals. J Heavy Metal Tox Dis 1(2):1–15.

https://doi.org/10.21767/2473-6457.100010

Tarekegn MM, Fikirte ZS, Alemitu II (2020) Microbes used as a tool for bioremediation of heavy

metal from the environment. Cogent Food Agric 6:1. https://doi.org/10.1080/23311932.2020.

1783174

Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ (2012) Heavy metal toxicity and the

environment. Experientia Suppl 101:133–164. https://doi.org/10.1007/978-3-7643-8340-4_6

Wang F, Bao K, Huang C, Zhao X, Han W, Yin Z (2022) Adsorption and pH values determine the

distribution of cadmium in terrestrial and marine soils in the nansha area, pearl river delta. Int J

Environ Res Public Health 19:793. https://doi.org/10.3390/ijerph19020793

130 J. T. Mathew et al.

Witkowska D, Słowik J, Chilicka K (2021) Heavy metals and human health: possible exposure

pathways and the competition for protein binding sites. Molecules 26:6060. https://doi.org/10.

3390/molecules26196060

Yao Y, Fang F, Wu H, Wu M, Kuang Y, Lin Y (2019) Concentrations and speciation of mercury in

soil affected by bird droppings. Pol J Environ Stud 28(3):1451–1459. https://doi.org/10.15244/

pjoes/87104

Zhang X, Zeng S, Chen S, Ma Y (2018) Change of the extractability of cadmium added to different

soils: aging effect and modeling. Sustainability 10(3):885. https://doi.org/10.3390/su10030885

Municipal Wastewater as Potential

Bio-reﬁnery

Shipra Jha and Nahid Siddiqui

Abstract Globally and locally, there is need of acceptable water quality and waste

water treatments are precedence. The various conventional waste water treatment

methods are used for the removal of particulate matter, organic matter and nutrient

load before releasing into river. And these treatment methods include higher cost,

higher energy consumption and impact on environment. With increasing research

evidence for the impact of contaminated water on environment and human health,

wastewater bioreﬁnery is gaining interest. Certain technologies include bioreﬁnery

can convert wastewater into valuable product and reduce economic and environ-

mental burden. Due to the potential, to ﬁll the gap between wastewater treatment

and bioreﬁnery. This chapter will provide wealth of information on new research

on technological interventions on the implementation, design and municipal waste

water for bioreﬁnery and promoting a green and cleaner environment.

Keywords Municipal water ·Environment ·Technologies ·Bioreﬁnery ·Green

technology

1 Introduction

In developing countries the waste disposal becomes big problem due to poor infras-

tructure, budget limitations and lack of facilities to maintain practical standards. The

waste pollutants become the reason behind air, water, and soil pollution, emission

of green house gases and source of infection. Hence with the development of biore-

ﬁneries, waste can be utilized and wide range of valuable products can be produced.

With the increase in population, the demand for development of industrial sector

and infrastructure also increases which in return dispose different waste efﬂuent in

S. Jha (B

)

Healthcare Management, Asia Paciﬁc Institute of Management, New Delhi, India

e-mail: sjanvi79@gmail.com

N. Siddiqui

Amity Institute of Biotechnology, Amity University Uttar Pradesh (AUUP), Gautam Buddha

Nagar, Sector-125, Noida, India

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_7

131

132 S. Jha and N. Siddiqui

environment. The wastewater management is difﬁcult task due to the presence of

excessive nutrients discharge in the environment leads to acidiﬁcation and become

risky for human health (Jerbi et al. 2020; Al-Zboon and Al-Ananzeh 2008;Arora

et al. 2021; Bailey and Ollis 1986).

Globally, municipal bioreﬁnery are gaining attention due to the waste manage-

ment and produce products includes heat, fuel, valuable products and energy. Many

experimental data provided by scientist using wastewater containing heavy metal,

biomass, chemicals, plastics, leather, oil, detergent used in bioreﬁnery (Belinsky

et al. 2005; Bhatia et al. 2017). The scientiﬁc studies show that there are many

operational and technical challenges to achieve economical beneﬁts of bioreﬁnery.

The wastewater bioreﬁnery represents complex network and it’s important to iden-

tify the economical and sustainable development of bioreﬁnery. The research study

reported that agricultural waste, used cooking oil and poultry waste can also be used

in bioreﬁnery (Bhatia et al. 2020).

Conventional wastewater treatment plant were designed for the treatment of

suspended solids, particles and biological oxygen demand even along with trace

elements to release treated waste water to land water. For the purpose of reuse of

wastewater and complete breakdown of pathogenic microbes, tertiary treatment may

be used along with the combination of conventional method. It is important to develop

economically feasible methodology for complete removal of hazardous organic

compounds and trace elements. Because presently municipal sludge constituents

can be controlled by maintaining waste water quality before treatment (Fisher-Jeffes

et al. 2014; Burton et al. 2009).

Considering many scientiﬁc reports contributed by global research team, it is

concluded that wastewater bioreﬁnery has potential to develop various valuable prod-

ucts and provide solution to manage large municipal waste generate daily in urban

areas. This chapter gives overview of the types of treatment, characteristic feature

of Municipal wastewater bioreﬁnery, potential of bioreﬁnery and design of reactor

(Narayanan and Narayan 2019; Carey et al. 2016; Coetzee 2012).

2 Potential of Municipal Waste Water Pollutant

The research studies shows that the municipal waste water contains different pollutant

includes pathogens, toxic contaminants, phosphorous, nitrogen, organic matter and

dissolved minerals. Most of the cities started collecting waste water from commer-

cial places, household etc. and treating municipal wastewater through centralized

treatment plant. The waste pollutant differs from source to source. The composition

of waste belongs similar to same sources. In urban areas varieties of toxic pollu-

tant are discharged into water through human activities (Bruin et al. 2004; Kreuk

et al. 2007a). In household wastewater pollutant contain number of microbes along

with different toxic chemicals due to which chemical and physical methods are used

to remove contaminant. Based on physical and chemical approach contaminant are

categorised into dissolved impurities, settle able pollutant, colloidal pollutant and

Municipal Wastewater as Potential Bio-reﬁnery 133

suspended pollutant. The main objective behind municipal wastewater treatment to

clear off the waste and reuse the water (Kreuk et al. 2007b, 2010).

With the vigorous research, scientist could develop municipal wastewater method

to remove almost all the pollutant from municipal wastewater. The treatment plant

consists of continuous series of system for the removal of complete pollutants (Details

2011).

3 Concept of Wastewater Bioreﬁnery

Bioreﬁnery are effective way to utilize bioenergy in a durable manner to reduce the

usage of chemicals, to secure, to minimize environmental changes. Bioreﬁnery an

essential built-in which can convert various substrates into different usable produce

includes energy, compounds and substances after extraction or further treatments

like chemical or biological (Donofrio et al. 2009).

The operational condition to setup traditional Bioreﬁnery plant considered to

be expensive process in terms of social, environmental and economical concern.

Due to the high cost of feedstock includes biopolymers, vegetables oil, glucose and

diesels, pressurizing the agriculture sector for providing substrate and creating load

on ecosystem by releasing heat, it become essential to explore wastewater bioreﬁnery.

The basic objective behind the construction of Waste water Bioreﬁnery plant is to

generate not only clean water but also the commercial valued products (Donofrio

et al. 2009; Rabelo et al. 2011; Drosg et al. 2015; Sas et al. 2021).

Depending upon the types of raw material and technology for product devel-

opment, Bioreﬁnery are categorized into different seven types which includes

Lignocelluloses Bioreﬁnery, Thermo chemical Bioreﬁnery, whole crop Bioreﬁnery,

conventional Bioreﬁnery, Two-platform Bioreﬁnery, Green Bioreﬁnery and Marine

Bioreﬁnery (European Union 1986, 2009) (see Table 1).

Municipal wastewater plants passes through primary, secondary and tertiary treat-

ment or even sludge treatment either to further utilize for water reuse purpose or to

dispose. After the treatment of municipal wastewater reuse for crop production, agri-

culture irrigation, and sludge used for landﬁll purpose, store as ground water and for

construction sites (EUROSTAT 2014; Fux and Siegrist 2004; Shizas and Bagley

2014). The wastewater treatment plant consist of preparatory treatment, primary,

secondary treatment and if needed then advanced treatment to safeguard the quality

of environment and public health known as tertiary treatment. A different phase

of development used includes small particles or stone removal, waste screening,

sedimentation and biological treatment (Fava 2012; Finger and Parrick 1980).

134 S. Jha and N. Siddiqui

Tabl e 1 Characteristic feature of Bioreﬁneries

Technology used Sources of raw

material

Types of bioreﬁneries Stage of

development

Cell disruption,

extraction and product

isolation

Seaweed, marine algae Marine Pilot plant, Research

and Development

stage

Biochemical and

thermo chemical

conversion

All types of raw

material

Two platform Pilot plant

Preliminary treatment,

squeezing, partition,

extract, isolate and

digestion

Green crops and

grasses

Green Pilot plant, Research

and Development

stage

Initial treatment,

enzymatic hydrolysis,

fermentation and

isolation

Material containing

lignocellose like Wood,

reed, straw and reed

Lignocelluloses Pilot plant, Demo

stage, Research and

Development

Conversion through

roasting, thermal

decomposing,

gasiﬁcation, isolation,

enzymatic synthesis

All types of raw

material

Thermo chemical Pilot plant, Demo

stage, Research and

Development

Wet or dry grinding,

conversion through

digestion, fermentation

and harvesting or

composting

Maize, rye, wheat and

straw

Whole crop Pilot plant and

Demo stage

Biochemical

conversion: enzymatic

catalytic hydrolysis,

fermentation,

fractionation and

product isolation

Plant oil, carbohydrate,

terpenes,

lignocelluloses rich

material

Conventional Phase-III

(Advanced)

3.1 Initial or Preparatory Treatment

The purpose of initial treatment includes removal of stone, inorganic solid or particles

and screening of wastewater to enhance water quality to ovoid interference during

further processes (Fisher-Jeffes et al. 2014; Fytili and Zabaniotou 2008).

3.2 Primary Treatment

In primary treatment screened sediments, suspended particles, Biochemical oxygen

demands and settled particles are easily removed for economical purpose before

Municipal Wastewater as Potential Bio-reﬁnery 135

secondary treatment. The research study shows that primary treatment may reduce

pathogens, reduce nutrients concentration in wastewater, harmful organic compounds

and trace elements (García-Martíneza et al. 2019).

3.3 Secondary Municipal Wastewater Treatment

The research study shows that chemical and physical method is not effective in case

of secondary treatment. In secondary wastewater treatment, microorganisms used

in activated sludge, pond or trickling ﬁlters known as biological method. In biolog-

ical method oxidation of some portion of organic material takes place to release

products and carbon dioxide, and rest part utilized by microbial growth and devel-

opment (Ginni et al. 2021; Graedel et al. 2009; Harding 2009). The microorganisms

form biological aggregate and separated from sediment tanks known as secondary

sludge. Due to microbial aggregates, wastewater can linked to secondary sludge. The

ammonia can be reduced by secondary treatment method (Guimarães et al. 2016).

3.4 Advanced or Tertiary Wastewater Treatment

Tertiary treatment utilized for municipal wastewater when there is requirement for

high quality water treatment after secondary wastewater treatment. For the removal

of pathogenic microorganisms, trace elements, viruses and organic compounds

advanced treatment method is used (Harding et al. 2007; Jung et al. 2009;Harrison

et al. 2016; Heidrich et al. 2011). By adding coagulant Biochemical oxygen demand

and suspended solid can be reduced. Trace elements and organic compounds can be

removed by using activated carbon. Through chemical precipitation or microorgan-

isms, phosphorous is easily removed and using nitriﬁcation nitrogen content can be

removed from waste water (Henze et al. 2008) (see Figs. 1 and 2).

The characteristic feature of Municipal wastewater bioreﬁnery includes:

•To handle complicated and poorly managed wastewater efﬂuent containing

diversity in nature and concentration.

•Municipal wastewater treatment require less amount of energy for its operation.

•Easily adaptable system which can adjust i nternal and external environment.

•Cheaper Cost of waste treatment in robust environment.

•Comparison to conventional bioreﬁnery, wastewater bioreﬁnery deliver more

effective ecological service.

•Ability to give double beneﬁt includes more important by-products along with

preserving natural legacy.

•Potential to allow easy construction of business model for Eco-Industrial systems.

136 S. Jha and N. Siddiqui

Raw material Operational

Treatment

Biologiacl

Treatment

WASTE BIOMASS

• Organic waste

• Animal manure

• Food waste

• Corn stove

• Crop residue

• Rice straw

•Tree

• Muncipal sludge

Chemical

•Acid hydrolysis

•Alkaline hydrolysis

•Enzymatic

Fermentation

• Biological

•Fungal

•Bacterial

• Physical

•Ultrasonic

•Steam explosion

ANAEROBIC

TRANFORMATION

• Mixed

digester

• Anaerobic

lagnoon

• Anerobic filter

•Dyes

• Solvents

• Adhesives

• Pigments

• Detergent

Chemical

•Renewal

Diesel

• Ethanol

Fuel

•Power

•Electricity

Powder

Fig. 1 Flowchart shows potential wastewater bioreﬁnery

Municipal Wastewater as Potential Bio-reﬁnery 137

Biofuel

Water

Fertilizes

Biorefinery

Combine

d heat &

power

Material

High

Value

chemical

Animal

feed

Material

Fig. 2 Modern Bioreﬁnery concept

4 Application of Wastewater Bioreﬁnery

Municipal wastewater nutrients used for producing biomass of Nannocholoropsis

species. Municipal water nutrients open up new ways of enhancing economy for the

production of valuable product from microalgae. A municipal water nutrient contains

growth promoting nutrients and growth inhibiting pollutant (Huang et al. 2011).

4.1 Different Categories of Raw Material for Wastewater

Bioreﬁneries

To design the reactor for bioreﬁney, it becomes very essential to classify the wastew-

ater as feed. Based on the reactor, wastewater classify into three factors –quantity of

constituents present, variable constituents and their number present in the wastewater

and ﬂow rate. Depending upon three factors, bioreﬁnery scale up to produce desired

products (Jackson et al. 2009; Iranpour et al. 2002). Large volume of waste water

enters into streams per day and may get diluted with minor to major components.

The heavily diluted waste water includes dyes, chemicals, detergent, acids, paints,

cooking oil with changing concentrations enters from various sources becomes mega

138 S. Jha and N. Siddiqui

challenge for their treatment. Waste water Bioreﬁneries can be designed based on

the volume ﬂow (Coetzee 2012).

4.2 Waste Water Source for Production of Valuable Products

The main objective behind bioreﬁnery, to enhance water quality and number of

products which yields after the complete treatment. Due to heavily dilute wastewater,

desired product can be recovered by proving different growth condition through

microbial activity (Jeong et al. 2010). The wastewater bioreﬁnery products can be

classiﬁed into three types: [a] to generate power or electricity, biogas produced under

anaerobic tank [b] break down of large molecule into smaller one includes ethanol

which can further utilized for complex industrial products and [c] third classiﬁed

group includes super molecules with simple puriﬁcation functional based products

include soil conditioners, bioﬂocculants (Kleerebezem and Loosdrecht 2007; Kosaric

et al. 1984). During the course of wastewater treatment, there are various valuable

products includes alginate, pigments, organic acids, volatile substances, enzymes

and polymers produced at the end of the treatment cycle. The behaviour of products

always depends upon the bioactive agent and type of wastewater treatment method

(Koskan et al. 1998; Lalloo et al. 2010).

4.3 Wastewater Bioreﬁneries Source for Irrigation

Globally, waste free irrigation water is main concern due to limited clean water.

The scientiﬁc studies reported that with the advancement in drainage system, crop

development process, waste water which is of low quality can be used for crop

irrigation (Lettinga 1995;Li

2009; Liu and Tay 2004). Low quality water not only

affects crop development but also interfere in soil properties (Libutti et al. 2018).

Many research studies shows advantage and disadvantages of wastewater irrigation

which affects the plant growth includes chlorophyll content, length of leaves, root

length, seed size, soil quality in terms of nutrients and may increase salt concentration.

Limited water resources are forcing to invite more studies to explore waste water

treatment method. And the concept of Municipal waste water bioreﬁnery used for

treating contaminated water to improve water quality. The key aim of wastewater

bioreﬁnery to use distinct unit to release clean water and to produce variable products

after complete treatment of wastewater (Baghel et al. 2018).The research studies

indicated that multiple waste includes poultry waste, used edible oil, agro-industrial

waste can also be used in bioreﬁnery for water treatment and treated water can be

utilize for fast-growing plant irrigation which may help soil to hold nutrients (Mitra

and Sandhya Mishra 2019; McCarty et al. 2011).

Municipal Wastewater as Potential Bio-reﬁnery 139

4.4 Prevent Waterborne Pollution

Water clariﬁcation is essential part of bioreﬁnery treatment system which involves

removal of ﬂoating matter and suspended particles. Through the process of adsorp-

tion, ion-exchange, reverse osmosis, precipitation and oxidation heavy metals are

removed. Adsorption is considered as most efﬁcient in removing heavy metal as

compared to other methods due to high cost and operational limitations. Slit and

sand can also be removed by using equipment and washed water may be reused for

irrigating plants (McCarty et al. 2011; Mohan and Ramesh 2006; Wang et al. 2018).

5 Integration of Waste Water Treatment

Increasing population and urbanization around the world has resulted in scarcity of

water even in areas which were initially rich in water resources and water supply.

This brought up the need to reclaim the wastewater and then reuse it for non drink-

able purposes. Therefore the purpose of management of wastewater from different

sources, gained importance which would decrease the burden of water pollution as

well as use the treated waste water for purposes other than drinking and washing

(Narala et al. 2016; Narayanan and Biswas 2015; Pandey et al. 2010). New tech-

nologies and methods are in place to help treat the waste water coming from

different sources. Combination of the conventional and modern treatment processes

are employed to reclaim the water to its original quality. The use and reclamation

of waste water for irrigation, agriculture and landscaping is the most cost effective

solution for protection of environment and also to mitigate the lack of water resources

around the world. Integration of water and reclaimed water is done effectively. Plan-

ning with respect to the facilities like site of wastewater treatment plant, reliability of

the treatment process, ﬁnancial support and also future use of the reclaimed water,

quality of water, regulatory mechanisms etc. is necessary for this to have a sustainable

method in place (Rabelo et al. 2011).

Treating wastewater is done by the combination of chemical, Physical and biolog-

ical processes. Also, these treatment plants require high-cost infra structure, highly

skilled workers, and lots of energy. Constant efforts are being made to ﬁnd an alter-

native and cost effective approach for the wastewater treatment. Wastewater rich in

nutrients is produced from different industrial sources such as municipality, textile,

pharmaceutical, dairy, food and many others (Ramaswamy et al. 2013; Richardson

2011; Ren et al. 2019). Being rich in inorganic and organic substances, these cause

eutrophication in the environment, which is harmful for the environment. Eutroph-

ication mainly affects the irrigation, agriculture, ﬁsheries and causes the growth

of different microbes and pathogens in the environment (Li 2009; Richmond and

Cheng-Wu 2001). Above all, if left untreated, it will contaminate the ground water,

soil and air as well, adding to the pollution woes of the environment and causing a

140 S. Jha and N. Siddiqui

potential damage to the ecosystem. Traditional methods use the physical and chem-

ical methods of treatment but these are expensive and generate sludge/ slurry, which

is again needed to be, treated (Guimarães et al. 2016).

Using the traditional method of treatment, pose a lot of challenge to the wastewater

industry. Methods have to be adopted to valourize the wastewater, turn it into a

useful resource and at the same time reduce the environmental and economic load.

Researchers are exploring different methods for treatment and valourization of the

components of the wastewater (Ramaswamy et al. 2013). Strategically optimization

to enhance the effectiveness of the waste treatment, and better utilization of the

bioresources with regard to the impact on environment and economy. Therefore,

integration of environmental engineering and Bioprocess technology seemed to be

the need of the hour which would help to produce useful and sustainable products

from the waste and at the same time help improve the environment and help in

the remediation of contaminants. Wastewater used as nutritive substrate followed

by further treatment yields useful bioproducts which can be put to use (Saratale

et al. 2020; Sheik et al. 2014). This can be termed as Wastewater bioreﬁnery. When

put into action, the wastewater bioreﬁneries can extract the valuable components

from the wastewater, valorize them and reinsert them for economic use and at the

same time bring up the remediation of the pollutants. These products must be easily

recoverable and should fulﬁl a role in ecology, for which sturdy treatment system

should be maintained. The bioreﬁneries should be able to produce fewer footprints,

should be strong and resilient, should have the capability to generate more than

one co-product and should need least energy for operation. In addition to these, the

competitiveness of the bioreﬁneries can be improved by considering systems based of

renewable resources such as sunlight, gravity based ﬂow systems for the economic,

social and environmental issues. Waste water bioreﬁnery can aim to enhance the

wastewater industry by improving through industrial ecology. So that environmental

and ecological sustainability can be achieved. Taking care to achieve the better ﬁnal

water stream is of top priority of the bioreﬁneries (Shizas and Bagledy 2004).

One of the effective and sustainable approaches appears to be the integration of

Microalgae in wastewater treatment (Ramaswamy et al. 2013). Microalgae grow

in waste water and convert sunlight and carbon dioxide into biomass. The biomass

produced contains different biomolecules like lipids, carbohydrates and other impor-

tant organic compounds which are further utilized for the production of bio fuels.

And these can have other applications (Bhatia et al. 2020; García-Martíneza et al.

2019) as these cells do not use the energy for the growth and development but store

it within them. This stored energy with the biomass can be used for the produc-

tion of biofuels, so, technologies based on such bioreﬁneries have taken up a lot of

interest of researchers. The Biological waste water treatment using microorganisms

to degrade the pollutants is an integral step of the treatment system. Protozoa, Algae

bacteria, fungi, nematodes are used for the breakdown of unstable organic wastes to

convert them into stable inorganic forms, using aerobic or anaerobic methods (Shizas

and Bagledy 2004). Microalgae based technologies are the most viable methods of

waste water treatment which allows almost 100% of the recovery of the nutrients

from the waste water. The wastewater environment is non-sterile in nature, thus the

Municipal Wastewater as Potential Bio-reﬁnery 141

microorganisms for the product formation should be selected accordingly such that

the microbial ecology is maintained. Also beneﬁcial culture conditions and products

are selected which can have a selective contribution to the microbial community of

interest (Show et al. 2017).

Two-step degradation process is used for the production of biofuels.

1. Microalgae are grown in the wastewater aerobically.

2. Biofuel is produced from the biomass anaerobically.

Biodiesel can be produced from lipid while as fermentation of other compo-

nents of the biomass can be yield other biofuels (both liquid and gas) like as

bioalcohol. Dark fermentation can be used for the production of biohydrogen

and anaerobic co-digestion to yield biomethane (Stafford et al. 2013).

Microalgae can be cultivated in different types of cultivation systems on a small

scale as well as large scale (Stefanakis and Tsihrintzis 2012a, b). Choice of the system

depends upon the type of the microalgae selected, availability of type of nutrients and

the utilization of the biomass thus produced. Open and closed system of cultivation

is most preferred but advanced method so cultivation is also available, but at times

these might overlap. For large scale production, Open System cultivation uses open

spaces such as ponds, tanks for cultivation. These are low cost, and more economical

as compared to the closed systems, but have few disadvantages. Since these are

open, evaporation of water, Co2 diffusing in the atmosphere causing pollution, poor

utilization of light by the cells and requirement available land for the cultivation are

a few disadvantages with this system (Verster et al. 2013). Open systems there are

not preferred for pilot scale production. Closed systems, most appropriately calls

Photobioreactors (PBR) are preferred as there is no direct exchange of gases and no

contaminants in the surrounding environment can affect the system (Berg 2009).

Another study based on the integration of the Willow as bioreﬁnery was evaluated

with primary efﬂuent wastewater irrigation was done. Wastewater irrigation led to

increase as lignin, phytochemical and glucose yield in the biomass. Also this could

treat waste water in a more sustainable manner (Shizas and Bagledy 2004).

6 Bioreactor Design Requirement of Wastewater

Bioreﬁnery Includes

Large volume reactor-

•Semi continuous or Continuous ﬂow.

•Large commodity.

•Decouple hydroluric and sludge retention time.

Complex variable

•Targeted non-sterile or microbial community.

•Create environmental niche and target to product beneﬁts.

142 S. Jha and N. Siddiqui

Environment

•Allow to ﬂow water into environment.

Downstream processing

•Product can formed in different phase.

•Recovery of product.

•Design reactor for load balancing and elimination.

Reactor designing to increase residence time

•Recovering before cell settling.

•Recycle after settling.

•Rectors in parallel.

7 Designs

Bioreactor refers to any device or system which may be manufactured or engineered

which a biological system with active environment, in which growth of microorgan-

isms can take place. Recovery of the useable resources from the wastewater can be

done using a Bioreﬁnery reactor, the design of which is optimized as per the require-

ment. Downstream processes (DSP) are more developed, easily adaptable inagiven

reactor in which the separation mode is depended upon a few properties such a size,

charge, solubility, separation properties and volatility (Sterr and Ott 2004; Stuart and

El-Halwagi 2012). Primary objective of an environment efﬁcient and a cost effective

DSP is to obtain easy recovery from the bulk material and to reduce the amounts of

different unwanted components. Design of the reactor towards product recovery is

done which will reduce the loading on the DSP operation units and at the same time

efﬁciency can be increased. The ﬂow rates of gas (i.e., air, oxygen, nitrogen, carbon

dioxide), pH, temperature, and agitation speed/circulation rate and dissolved oxygen

levels, need to be monitored and controlled (Takkellapati et al. 2018).

A few challenges are encountered with respect to the current design of the reac-

tors. Firstly, Optimization of the system as a single unit will pose a challenge as it

will not ensure desired results, therefore, a systems approach is needed where other

productions are also considered along with the reactor design to have the maximum

utility of the reactor functionally as well as deliver high productivity. Aerobic reac-

tors working at low substrate concentration are the primary source of employment

of energy, with increasing cost of energy supply this design needs to be reconsidered

(Sung et al. 2007; Taniguchi et al. 2005).

Various types of Biological waste water treatment Bioreﬁnery reactors are in

application as below.

Municipal Wastewater as Potential Bio-reﬁnery 143

7.1 Stirred Tank Reactor for Aerobic Treatment of Waste

Water

This process which has undergone large number of diversiﬁcations and modiﬁcations

is one of the oldest methods of biotechnology for wastewater treatment. An activated

sludge process employs a tank which is an agitated vessel in which the inoculum of

the microbial sludge is introduced. Air at high pressure is introduced from the bottom

to provide sufﬁcient amount of dissolved oxygen to the medium in the tank. Due to

large volume of the tank and less solubility of atmospheric oxygen in the medium,

large volumes of gas has to be introduced in the tank, requiring huge compressors so

that aerobic conditions inside the tank are maintained. The constraint with this type

of a system is the high cost of the compressors although the system can be easily

designed and installed (Tsihrintzis et al. 2007).

Oxidation of the dissolved organic matter, denitriﬁcation and nitriﬁcation can

be achieved in this process. Conventionally, the system requires two stirred tanks in

series-in the ﬁrst one has aerobic conditions in which carbon removal and nitriﬁcation

occurs and in the second tank denitriﬁcation is done anoxically. Using a number of

small sized tanks in series or dividing the tank in various compartments are the

different types of modiﬁcations in this technique, where in the cascade remains the

same as in a single tank, but improves performance, as increased BOD destruction

occurs, also reducing the deadzones and bypass streams (Tsihrintzis and Gikas 2010).

Source C.M. Narayanan and Vikas Narayan, Biological wastewater treatment and bioreactor design:

Sustainable Environmental Research (2019)9, Article number: 33 (Kreuk et al. 2010)

144 S. Jha and N. Siddiqui

1. Stirred tank reactor for Anaerobic treatment of waste water:

This process involves the treatment of wastewater using culture of microbes which are

acidophilic, methanogenic or acetogenic. The products thus produced are converted

into useful products such as Biogas. The sludge digested anaerobically can be used

as a fertilizer directly or can be used in the production of phosphate rich fertilizer.

This process this slow and the microbes especially the methanogenic ones can be

sensitive to pH and temperature changes (Berg 2009).

Although not that economically cost effect, this process can be used with ther-

mophilic microbes as well, the cost of the installation of the heating pipes may prove

a constraint (Belinsky et al. 2005) Mesophilic bacteria can also be used.

These can be horizontal, vertical or tube like are the close systems, considered

easiest to scale up. Algae and the growth media are continuously circulated through

the tubes using a mechanical pump. A whole range of algae such as Chlorella,

Porphyridiumetc can be successfully grown on a pilot scale using tubular photo-

bioreactors. Problems like unfavourable Co2 and pH gradients and high levels of

Dissolved oxygen are a few problems encountered with this system (Verster et al.

2013).

7.2 Flatplate Photobioreactors

These are ideal for large scale indoor and outdoor conditions. Less accumulation do

dissolved oxygen, lots of solar light on the plate and easy to use modular designs

make these a better choice. Temperature controls, algal ﬁlm formation on the plates,

hydrodynamic stress are some of the drawbacks with this method (Sung et al. 2007).

7.3 Plastic Bag Photobioreactor

Plastic bags with a diameter of 0.5 m with aerators attached to it are use as photobiore-

actors, vertically hung inside plastic or metal cages and kept exposed to sunlight.

Air is pumped from the bottom and the microbes are continuously mixed with the

air. The drawback with this is the poor mixing of the microbes and air leading to the

destruction of the cultures (Verstraete and Vlaeminck 2011).

Municipal Wastewater as Potential Bio-reﬁnery 145

7.4 Packed Bed Bioﬁlm Reactors

Support particles such as activated carbon particles, silica granules, polymer beds etc.

Microbial cells surround each particle forming a bioﬁlm. The aggregation of particle

and bioﬁlm complexe form a distinct phase in the Bioreactors. Microbial cells in

the bioﬁlm grow and multiply till the thickness of, δ = 0.3–0.5 mm is reached after

which they slough off from the particle surface and get replaced by fresh cells on the

particle. High rate of bioconversion is achieved as the bioﬁlm thickness is low and

the concentrated cell mass on the bioﬁlm is high. Since the unconverted substrate

and the product accumulation in the bioﬁlm does not occur, the substrate and product

inhibition for the growth of microbe is low in such bioreactors (Vymazal 2002).

7.5 Moving Bed Bioreactors

As the name indicated in this system, the bed of the particles is not ﬁxed in the

column rather it is a ﬂuidized type of a reactors in which the particle-bioﬁlms aggre-

gates move against the current of water. The aggregate is fed to the stirred tank

bioreactor and remain suspended in the liquid substrate (wastewater) in the tank.

Under aerobic conditions, Air sparged in at high pressure also keeps these aggre-

gates suspended and moving in the liquid, thus the name moving bed. The microbes

grow attached to the support particles so there is no need of the sludge to be recycled

as the microbes do not leave the bioreactor. Batchwise method of operation of this

system is preferred although it can be operated in the continuous mode as well. The

biomass concentration is substantially high in the bioﬁlms, BOD removal is high but

a little resistance in the transfer of substrate in the bioﬁlms is encountered (Yen et al.

2019).

The denitriﬁcation and aerobic tank bioreactors can be operated for the activated

sludge process using the moving bed system.

7.6 Fluidized Bed Bioﬁlm Reactors

These bioreactors can be operated at high velocity and ﬂow rates. Used for large

scale production, the industrial efﬂuent enters from the bottom of the column at high

velocity due to which all aggregates remain suspended due to the upstream ﬂow of

the ﬂuid. The bioreactor performance is enhanced as all the bioﬁlms are surrounded

by the ﬂuid on all sides making an intimate contact with the aggregates. The total

volume of the reactor increases due to the expansion of the bed. Advantage with this

reactor is that the pressure drop across the bed is close to constant (Show et al. 2017;

Yen e t a l . 2019).

146 S. Jha and N. Siddiqui

Source Neha Baghel*, Satyam Sopori, Bagwan Mohamadazrodin, Ashpak Raﬁk

Saudagar, Saudagar Ajharoodinournal of Advances and Scholarly Researches in Allied Education,

Vol. XV, Issue No.2 (Special Issue) April-2018, ISSN 2230-754 (Stafford et al. 2013)

8 Conclusions

Many wastewater treatment programme and waste segregation at community level

result in the removal of contaminant from sewage water. Treated waste may increase

the load of contaminant in soil which comes from sewage sludge, agricultural

pollutant. The ﬁnal product of municipal wastewater treatment is sewage solid or

mud containing contaminant removed from wastewater efﬂuent. I n sewage mud

containing nitrogen and phosphorous as nutrients similar to other organic compost

which can be used as soil conditioner and improves soil properties. Currently,

ﬁnancial beneﬁts for sewage mud are argumental issue.

With the increasing rise in bioﬁnery technology, municipal wastewater treatment

plant releases improved water quality for non-portable use. It has been found through

many research studies that land irrigated continuously with treated polluted water

which motivates municipal wastewater bioreﬁnery to use for crop irrigation. Munic-

ipal wastewater bioreﬁnery can be new key source to solve irrigation water deﬁcit

problem worldwide. In future with proper reactor designing, improved operational

conditions, varieties in raw material may signiﬁcantly enhance the irrigation water

problem.

Municipal Wastewater as Potential Bio-reﬁnery 147

References

Al-Zboon K, Al-Ananzeh N (2008) Performance of wastewater treatment plants in Jordan and

suitability for reuse. Afr J Biotech 7(15):2621–2629

Arora K, Kaur P, Kumar P, Singh A, Patel SKS, Li X, Yang Y-H, Bhatia SK, Kulshrestha S (2021)

Valorization of wastewater resources into biofuel and value-added products using microalgae

system. Front Energy Res 9:119

Bailey JE, Ollis DF (1986) Biochemical engineering fundamentals, 2nd edn. McGraw Hill,

Singapore

Belinsky M, Rubin H, Agnon Y, Kit E, Atkinson J (2005) Characteristics of resuspension, settling

and diffusion of particulate matter in a water column. Environ Fluid Mech 5(5):415–441. 18

Van den Berg M (2009) The South African wastewater market—business opportunities and export

promotion for Dutch companies. MSc thesis, University of Twenty, Netherlands

Bhatia SK, Gurav R, Choi T-R, Kim HJ, Yang S-Y, Song H-S, Park JY, Park Y-L, Han Y-H, Choi

Y-K et al (2020) Conversion of waste cooking oil into biodiesel using heterogeneous catalyst

derived from cork biochar. Bioresour Technol 302:122872

Bhatia SK, Palai AK, Kumar A, Bhatia RK, Patel AK, Thakur VK, Yang Y-H (2021) Trends in

renewable energy production employing biomass-based biochar. Bioresour Technol 340:125644

Bhatia SK, Bhatia RK, Yang Y-H (2017) An overview of micro diesel. A sustainable future source

of renewable energy. Renew Sustain Energy Rev 79:1078–1090

Burton S, Harrison S, Pather-Elias S, Stafford W, Van Hille R, Von Blottnitz H et al (2009) Energy

from wastewater—feasibility studies. Essence report. Water Research Commission report TT

399-09. ISBN 978-1-77005-847-7

Narayanan CM, Narayan V (2019) Biological wastewater treatment and bioreactor design. Sustain

Environ Res 9. Article number: 33

Carey D, Yang Y, McNamara P, Mayer B (2 Mar 2016) Recovery of agricultural nutrients from

bioreﬁneries. Bioresour Technol. ISSN 0960-8524

Coetzee B (2012) Technical strategic support manager, utility services directorate, City of Cape

Town. Personal communication

De Bruin LMM, de Kreuk MK, van der Roest HFR, Uijterlinde C, van Loosdrecht MCM (2004)

Aerobic granular sludge technology: an alternative to activated sludge? Water Sci Technol 49(11–

12):1–7

De Kreuk M, Kishida N, van Loosdrecht MCM (2007a) Aerobic granular sludge–state of the art.

Water Sci Technol 55(8–9):75–81

De Kreuk MK, Picioreanu C, Hosseini M, Xavier JB, van Loosdrecht MCM (2007b) Kinetic model

of a granular sludge SBR: inﬂuences on nutrient removal. Biotechnol Bioeng 97(4):801–815

De Kreuk MK, Kishida N, Tsuneda S, van Loosdrecht MCM (2010) Behaviour of polymeric

substrates in an aerobic granular sludge system. Water Res 44:5929–5938

Details A (2011) Irrigation with domestic wastewater: responses on growth and yield of ladyﬁnger

abelmoschus esculentus and on soil nutrients. J Environ Biol 32:645–651

Donofrio J, Kuhn Y, McWalter K, Windsor M (2009) Water sensitive urban design: an emerging

model in sustainable design and comprehensive water cycle management. Environ Pract

11(3):179–189

Rabelo S, Carrere H, Maciel Filho R, Costa A (2011) Production of bioethanol, methane and

heat from sugarcane bagasse in a bioreﬁnery concept. Bioresour Technol 102(17):7887–7895.

Dordrecht: Springer

Drosg B, Fuchs W, Al Seadi T, Madsen M, Linke B (2015) Nutrient recovery by biogas digestate

processing, IEA bioenergy task 37 report, June 2015. ISBN 978-1-910154-15-1 (printed). ISBN

978-1-910154-16-8 (eBook)

European Union (1986) Council directive of 12 June 1986 on the protection of the environment,

and in particular of the soil, when sewage sludge is used in agriculture. http://eur-lex.europa.eu/

legal-content/EN/TXT/?uri=CELEX:31986L0278

148 S. Jha and N. Siddiqui

European Union (2009) Directive 2009/28/EC of the European parliament and of the council of 23

April 2009 on the promotion of the use of energy from renewable sources. http://eur-lex.europa.

eu/legal-content/EN/ALL/?Uri=CELEX:32009L0028

EUROSTAT (2014) Landﬁll of sewage sludge from urban wastewater. https://www.quandl.com/

Fava F (2012) Biowaste bioreﬁnery in Europe: opportunities and R&D needs. European Federation

of Biotechnology, Barcelona

Finger RE, Parrick J (1980) Optimization of grit removal at a wastewater treatment plant. J Water

Pollut Control Fed 52(8):2106–2116

Fisher-Jeffes L, Carden K, Armitage N (2014) The future of urban water management in South

Africa: achieving water sensitivity. Water Sci Technol 14(6):1026–1034. Biowaste bioreﬁnery in

Europe: Opportunities and R&D needs. Barcelona: European Federation of Biotechnology

Fisher-Jeffes L, Carden K, Armitage N (2014) The future of urban water management in South

Africa: achieving water sensitivity. Water Sci Technol 14(6):1026–1034

Fux C, Siegrist H (2004) Nitrogen removal from sludge digester liquids by nitriﬁca-

tion/denitriﬁcation or partial nitritation/anammox: environmental and economical considerations.

Water Sci Technol 50(10):19–26

Fytili D, Zabaniotou A (2008) Utilization of sewage sludge in EU application of old and new

methods-a review. Renew Sustain Energy Rev 12:116–140

García-Martíneza JB, Urbina-Suarezb NA, Zuorroc A, Barajas-Solanob AF, Kafarova V (2019)

Fisheries wastewater as a sustainable media for the production of algae-based products. Chem

Eng Trans 76:1339–1344

Ginni G, Kavitha S, Kannah Y, Bhatia SK, Kumar A, Rajkumar M, Kumar G, Pugazhendhi A, Chi

NTL (2021) Valorization of agricultural residues: different bioreﬁnery routes. J Environ Chem

Eng 9:105435

Graedel TE, Allenby BR (2009) Industrial ecology and sustainable engineering. Prentice Hall,

Upper Saddle River

Guimarães NR, Filho SSF, Hespanhol BP, Piveli RP (2016) Evaluation of chemical sludge

production in wastewater treatment processes. Desalin Water Treat 57:16346–16352

Harding K, Dennis JS, von Blottnitz H, Harrison STL (2007) A life-cycle comparison between

inorganic and biological catalysis for the production of biodiesel. J Clean Prod 16:1368–1378

Harding KG (2009) A generic approach to environmental assessment of microbial bioprocesses

through life cycle assessment (LCA) (PhD thesis). University of Cape Town, South Africa

Harrison S, Johnstone-Robertson M, Pott R, Verster B, Rumjeet S, Nkadimeng L et al (2016)

Towards wastewater bioreﬁneries: integrated bioreactor and process design for combined water

treatment and resource productivity. Water Research Commission Report 2580/1/2016

Heidrich E, Curtis T, Doﬂing J (2011) Determination of the internal chemical energy of wastewater.

Environ Sci Technol 45(2):827–832

Henze M, Loodsdrecht M, Ekama G, Brdjanovic D (2008) Biological wastewater treatment:

principles, modelling and design. IWA Publishing, London

Huang J, Du Y, Xu G, Zhang H, Zhu F, Huang L, Xu Z (2011) High yield and cost-effective

production of poly("-glutamic acid) with Bacillus subtilis. Eng Life Sci 11(3):291–297

Iranpour R, Shao YJ, Ahring BK, Stenstrom MK (2002) Case study of aeration performance under

changing process conditions. J Environ Eng 128(6):562–569

Jackson VA, Paulse AN, Bester AA, Neethling JH, Khan S, Khan W (2009) Bioremediation of metal

contamination in the Plankenburg river, Western Cape, South Africa. Int Biodeterior Biodegra-

dation 63(5):559–568. HYBACS Process: Technical Assessment Report, 2010, Department of

Wat er A ff ai rs

Jeong JH, Kim JN, Wee YJ, Ryu HW (2010) The statistically optimized production of poly (γ-

glutamic acid) by batch fermentation of a newly isolated Bacillus subtilis RKY3. Bioresour

Technol 101:4533–4539

Jerbi A, Brereton N, Sas E, Amiot S, Lachapelle T, Comeau Y et al (2020) High biomass yield

increases in a primary efﬂuent wastewater phytoﬁltration are associated to altered leaf morphology

and stomatal size in Salix miyabeana

Municipal Wastewater as Potential Bio-reﬁnery 149

Jung HW, Tschaplinski TJ, Wang L, Glazebrook J, Greenberg JT (2009) Priming in systemic plant

immunity. Science 324:89–91

Kleerebezem R, van Loosdrecht M (2007) Mixed culture biotechnology for bioenergy production.

Curr Opin Biotechnol 18(3):207–212

Kosaric N, Cairns WL, Gray NCC, Stechey D, Wood J (1984) The role of nitrogen in multiorganism

strategies for biosurfactant production. JAOCS 61(11):1735–1743

Koskan LP, Meah ARY, Sanders JL and Ross RJ (1998) Method and composition for enhanced

hydroponic plant productivity with polyamino acids. US Pat. No. 5,783,523

Lalloo R, Maharaj D, Gorgens J, Gardiner N (2010) Functionality of a Bacillus cereus biolog-

ical agent in response to physiological variables encountered in aquaculture. Appl Microbiol

Biotechnol 79(1):111–118

Lettinga G (1995) Anaerobic digestion and wastewater treatment systems. Antonie Van Leeuwen-

hoek 67(1):3–28

Li X (2009) Micro-scale investigation of aerobic granular sludge: formation and stability, PhD

thesis, North-western University, USA

Libutti A, Gatta G, Gagliardi A, Vergine P, Pollice A, Beneduce L, Disciglio G, Tarantino E

(2018) Agro-industrial wastewater reuse for irrigation of a vegetable crop succession under

Mediterranean conditions. Agric Water Manag 196:1–14

Liu Y, Tay J-H (2004) State of the art of biogranulation technology for wastewater treatment.

Biotechnol Adv 22:533–56

McCarty PL, Bae J, Kim J (2011) Domestic wastewater treatment as a net energy producer-can this

be achieved? Environ Sci Technol 45:7100–7106

Mitra M, Mishra S (2019) A bioreﬁnery from nannochloropsis spp. utilizing wastewater resources.

In: Application of microalgae in wastewater treatment

Mohan S, Ramesh ST (2006) Assessment of recirculation ratio impacts on mixed liquor suspended

solids in the activated sludge process. Asian J Water Environ Pollut 3(2):57–61

Narala RR, Garg S, Sharma KK, Thomas-Hall SR, Deme M, Li Y, Schenk PM (2016) Comparison

of microalgae cultivation in photobioreactor, open raceway pond, and a two-stage hybrid system.

Front Energy Res 4:29

Narayanan CM, Biswas S (2015) Computer aided design and analysis of three phase ﬂuidized bed

bioﬁlm reactors for waste water treatment. Asian J Biochem Pharm Res 5:224–249

Baghel N, Sopori S, Mohamadazrodin B, Saudagar AR, Saudagar A (2018) J Adv Sch Res All Educ

XV(2)(Special Issue). ISSN 2230-7540

Pandey A, Soccol C, Larroche C (eds) (2010) Current developments in solid-state fermentation.

Springer, Dordrecht

Rabelo S, Carrere H, Maciel Filho R, Costa A (2011) Production of bioethanol, methane and heat

from sugarcane bagasse in a bioreﬁnery concept. Bioresour Technol 102(17):7887–7895

Ramaswamy S, Ramarao B, Huang H-J (2013) Separation and puriﬁcation technologies in

bioreﬁneries. Wiley, Chichester

Ren H-Y, Zhu J-N, Kong F, Xing D, Zhao L, Ma J, Ren N-Q, Liu B-F (2019) Ultrasonic enhanced

simultaneous algal lipid production and nutrients removal from non-sterile domestic wastewater.

Energy Convers Manag 180:680–688

Richardson C (2011) Investigating the role of reactor design for maximum environmental beneﬁt

of algal oil for biodiesel. MSc dissertation, University of Cape Town, South Africa

Richmond A, Cheng-Wu Z (2001) Optimization of a ﬂat plate glass reactor for mass production

of Nannochloropsis sp. outdoors. J Biotechnol 85:259–269. https://doi.org/10.1016/S0168-165

6(00)00353-9

Saratale GD, Bhosale R, Shobana S, Banu JR, Pugazhendhi A, Mahmoud E, Sirohi R, Bhatia SK,

Atabani A, Mulone V et al (2020) A review on valorization of spent coffee grounds (SCG) towards

biopolymers and biocatalysts production. Bioresour Technol 314:123800

Sas E, Hennequin LM, Frémont A, Jerbi A, Legault N, Lamontagne J, Fagoaga N, Sarrazin M,

Hallett JP, Fennell PS, Barnabé S, Labrecque M, Brereton NJB, Pitre FE (2021) Bioreﬁnery

150 S. Jha and N. Siddiqui

potential of sustainable municipal wastewater treatment using fast-growing willow. Sci Total

Environ 792:148146

Sheik A, Muller E, Wilmes P (2014) A hundred years of activated sludge: time for a rethink. Front

Microbiol 5

Shizas I, Bagledy D (2004) Experimental determination of energy content of unknown organics in

municipal wastewater streams. Journal of Energy Engineering 2(45–53):130

Show PL, Tang MSY, Nagarajan D, Ling TC, Ooi C-W, Chang J-S (2017) A holistic approach to

managing microalgae for biofuel applications. Int J Mol Sci 18:215

Stafford W, Cohen B, Pather-Elias S, Blottnitz H, von Hille R, Harrison S et al (2013) Technologies

for recovery of energy from wastewaters: applicability and potential in South Africa. J Energy

South Afr 24(1):15–26

Stefanakis AI, Tsihrintzis VA (2012a) Effect of various design and operation parameters on

performance of pilot-scale sludge drying reed beds. Ecol Eng 38(1):65–78

Stefanakis AI, Tsihrintzis VA (2012b) Effects of loading, resting period, temperature, porous media,

vegetation and aeration on performance of pilot-scale vertical ﬂow constructed wetlands. Chem

Eng J 181–182:416–430

Sterr T, Ott T (2004) The industrial region as a promising unit for eco-industrial development—

reﬂections, practical experience and establishment of innovative instruments to support industrial

ecology. J Clean Prod 12:947–965

Stuart P, El-Halwagi MM (eds) (2012) Integrated bioreﬁneries: design, analysis, and optimization.

CRC Press, Boca Raton

Sung GM, Lee D-G, Kim SB, Lee D-U, Woo S-H, Koopman B (2007) Dominance of endospore-

forming bacteria on a rotating activated bacillus contactor bioﬁlm for advanced wastewater

treatment. J Microbiol 45(2):113–121

Takkellapati S, Li T, Gonzalez MA (Sep 2018) An overview of bioreﬁnery derived platform

chemicals from a cellulose and hemicelluloses bioreﬁnery. Clean Technol Environ Policy

20(7):1615–1630

Taniguchi M, Kato K, Shimauchi A, Ping X, Nakayama H, Fujita K-I, Tanaka T, Tarui Y, Hirasawa

E (2005) Proposals for wastewater treatment by applying ﬂocculating activity of cross-linked

poly-"-glutamic acid. J Biosci Bioeng 99(3):245–251

Tsihrintzis VA, Gikas GD (2010) Constructed wetlands for wastewater and activated sludge

treatment in North Greece: a review. Water Sci Technol 61(10):2653–2672

Tsihrintzis VA, Akratos CS, Gikas GD, Karamouzis D, Angelakis AN (2007) Performance and cost

comparison of a FWS and a VSF constructed wetland systems. Environ Technol 28(6):621–628

Verster B, Madonsela Z, Minnaar S, Cohen B, Harrison STL (2013) Introducing the wastewater

bioreﬁnery concept: a scoping study of polyglutamic acid production from Bacillus rich mixed

culture using municipal wastewater. Pretoria: Water Research Commission Report TT587/13

Verstraete W, Vlaeminck SE (2011) Zero waste water: short-cycling of wastewater resources for

sustainable cities of the future. Int J Sust Dev World 18(3):253–264

Vymazal J (2002) The use of sub-surface constructed wetlands for wastewater treatment in the

Czech Republic: 10 years experience. Ecol Eng 18:633–646

Wang M, Zhang D, Dong J, Tan SK (2018) Application of constructed wetlands for treating

agricultural runoff and agro-industrial wastewater: a review. Hydrobiologia 805:1–31

Yen H-W, Hu I-C, Chen C-Y, Nagarajan D, Chang J-S (2019) Chapter 10—design of photobiore-

actors for algal cultivation. In: Biofuels from algae, 2nd ed.

Zaharieva RH, Dimitrova E, Buyle-Bodin F (2003) Building waste management in Bulgaria:

challenges and opportunities. Waste Manag 23(8):749–761

Phytoremediation of Metals

and Radionuclides

Kanchan Soni, P. Priyadharsini, S. S. Dawn, N. Nirmala, A. Santhosh,

Bagaria Ashima, and J. Arun

Abstract Heavy metals and radionuclides are t he hazardous pollutants that are

need to be remediated from environment for safer health and well-being. Heavy

metals are leached out into the environment via numerous activities. Industrializa-

tion and modernization of radioactive applications and metal-based compounds have

ended up in environment. Wetlands paves greater advantage via removal of pollu-

tants in a natural process. This chapter highlights the phytoremediation of metals and

radionuclides compounds in a sustainable and economical way. Apart from plants,

microbes were also used in phytoremediation process. Metal binding particles were

also employed for enhanced microbial assisted phytoremediation process. Phytore-

mediation is a chemical free pollutant removal process, eco-friendly and environ-

mentally safe, etc. However further research are needed in effective processing of

Phyto remediated biomass s ource.

Keywords Phytoremediation ·Heavy metals ·Radionuclides ·Microbial

remediation ·Wetlands

1 Introduction

The increased requirement for resources from industry, military, nuclear weapon

industry and agricultural site leads to huge contamination on earth environment,

i.e. major cause of accumulation of the toxic pollutants including heavy metals,

radionuclides, and organic contaminants in terrestrial and aquatic (surface waters,

and groundwater) fauna and ﬂora, this is due to inﬁltration of human activities, lack of

knowledge and carelessness (Thakare et al. 2021; Singh et al. 2022b) responsible for

the highest damage in the ecosystems. Most of the heavy metals i.e. Mercury (Hg),

K. Soni · B. Ashima

Department of Physics, Manipal University Jaipur, Dehmi Kalan, Jaipur, Rajasthan 303 007, India

P. Priyadharsini · S. S. Dawn · N. Nirmala · A. Santhosh · J. Arun (B

)

Centre for Waste Management–International Research Centre, Sathyabama Institute of Science

and Technology, Jeppiaar Nagar (OMR), Chennai, Tamil Nadu 600119, India

e-mail: arunjayaseelan93@gmail.com

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_8

151

152 K. Soni et al.

Lead (Pb), Nickel (Ni), Cobalt (Co) or Chromium (Cr), Zinc (Zn), Arsenic (As),

Copper (Cu) etc. present in ecosystem beyond permissible limit, are toxic in nature

and get accumulated through the food chain; contaminate drinking water reservoirs

and freshwater habitats leading to serious ecological and health effects. It alters the

structure and physiological functioning of all form of living beings (Microorganisms,

human, animal and plants). On account of their adverse effects, the presence of

metals in water and soil poses serious challenge to environmental managers as the

remediation options are not only limited but also expensive. As the heavy metal

takes long time to degrade and there are lot of physical (carbon adsorption, air

stripping, precipitation, extraction and membrane separation technologies), chemical

(chemical extraction and leaching, hydrolysis, etc.) (Jhilta et al. 2021) and biological

(biosorption and bioﬁltration) methods are available for example these are the most

economical method and take more time to implement or accomplish. So, to overcome

this problem researchers attempted to evaluate the potential phytoremediation, it is

an advantageous technique which is comparatively one of the safer and cheaper

for heavy metal removal. There are different types of phytoremediation, they all are

potential approach for the remediation of heavy metals, metalloids, and radionuclides

and most frequently used phytoremediations are phytodegradation, phytoextraction,

phytoﬁltration, phytostabilization, and phytovolatilizationare (Singh et al. 2022b).

Plants have the highest capacity of up taking nutrients and mineral so they are

highly magnetic towards contaminants present in terrestrial and aquatic ecosystem

(Barbosa et al. 2016). Phytoremediation is an in-situ method which involves the

straight use living plants for the degradation of contamination form, land, oil sludges

and aquatic environment, t his is because of plants use the energy from of solar power

and this pollution removal is totally solar energy mediated method with low cost

(Jhilta et al. 2021).

As mention above most of the other technology are economically expensive and

could be applied on mini scale, unlike phytoremediation is proved by researchers for

application in large scale and economical (Jhilta et al. 2021). The use of a biological

material is an emerging and environmentally friendly technology with great prospects

to effectively clean up toxic metals at low concentrations and possible recovery for

re-use in industry. The energy savings and environment beneﬁts associated with

phytoremediation activities are also quite signiﬁcant and novel approach. This is an

emerging technology which will use for the treatment of polluted sites and produce

guidelines on the use of plants for the remediation of contaminated land, enabling

transfer of the approach to other sites.

2 Hazardous Wastes

Hazardous waste is deﬁned as any waste that has the possibility of causing irreversible

harm to human health or environment due to its physical, chemical, biological or

infectious qualities. It is a waste material whose chemical composition renders it

potentially hazardous to humans. These wastes are ﬂammable, corrosive, poisonous

Phytoremediation of Metals and Radionuclides 153

and reactive compounds that pose a threat to safety, human health and the environment

(LaGrega et al. 2010).

Heavy metals are categorized as harmful substances in the environment. Even

at extremely low quantities, non-essential heavy metals are hazardous to plants,

animals, and people. At high doses, even essential heavy metals have negative health

impacts (Huat et al. 2019). Arsenic (Ar), chromium (Cr), zinc (Zn), lead (Pb), mercury

(Hg), cadmium (Cd) and copper (Cu) are examples of common heavy metals (Kim

et al. 2019; Shah 2020). Heavy metals are described as metallic elements with a

higher density than water. Heavy metals are also termed trace elements due to their

appearance in trace amounts (less than 10 ppm) in different environmental matrices

(Kabata-Pendias 2000). Physical parameters such as temperature, adsorption, phase

association and sequestration all have an impact on their bioavailability. It is also

inﬂuenced by chemical parameters such as speciation at complexation kinetics, ther-

modynamic equilibrium and lipid solubility. Biological variables such as species

features, trophic interactions and physiological or biochemical adaptability are also

signiﬁcant (Tchounwou et al. 2012).

Another signiﬁcant aspect that endangers human health is radioactive pollution.

The nuclear power sector has reached a critical stage in its evolution after decades of

tremendous expansion. Nuclear technology’s use in medical, industry and farming

has also accelerated. With the fast advancement of nuclear power and its technical

uses, the disposal of many forms of radioactive waste has become a concerning

issue. However, with the uncertainty of long-term temporary storage, the potential

safety concerns of radioactive wastes rise due to inadequate waste management

(Xu et al. 2021). The most signiﬁcant sources of radioactivity in the environment

include radionuclides from the natural radioactive series of uranium (238U), thorium

(232Th), and actinium (235U) and non-series potassium (40 K) found in the Earth’s

crust. Based on the geological and geochemical structure of the region, the activity

concentrations of these radionuclides differ from one location to another (Kayakökü

and Do˘gru 2020).

2.1 Heavy Metals: Sources and Toxicity

Since the Earth’s origin, these heavy metals have been found naturally in the

Earth’s crust. The massive growth in the usage of heavy metals has resulted in an

impending inﬂux of metallic compounds in both the aquatic and terrestrial environ-

ments (Gautam et al. 2016). Heavy metal pollution has evolved as a result of anthro-

pogenic activity, which is the primary source of pollution, mining, smelting, metal

leaching, waste dumps, excretion, animal and chicken dung and runoffs. Natural

sources of heavy metal contamination include metal corrosion, metal evaporation

from soil and water, volcanic activity, sediment re-suspension, geological weathering

and soil erosion (Briffa et al. 2020; Masindi and Muedi 2018).

Heavy metal pollution in water and soils has increased dramatically in recent

decades as a result of fossil fuel combustion, electronic waste, municipal waste

154 K. Soni et al.

disposal, pesticides, mining and smelting, fertilizer and sewage (Kim et al. 2019).

Heavy metals are non-biodegradable contaminants, and even trace amounts of non-

essential heavy metal ions (As, Hg, Pb, and Cd) may be harmful to living creatures.

If essential metals including Zn, Cu, and Fe are present in amounts beyond their

hazardous thresholds, they can become poisonous (Jaishankar et al. 2014).

Heavy metal increase in the human body causes serious damage to different

organs, including the neurological, reproductive systems, respiratory as well as the

digestive system (Huat et al. 2019). Heavy metals have been attributed to carcinogen-

esis, mutagenesis, and teratogenic effects. They produce reactive oxygenic species

(ROS) and consequently cause oxidative stress. Numerous disorders and patholog-

ical conditions are caused by oxidative stress in organisms. Heavy metals can also be

metabolic toxins. Heavy metal toxicity is caused by their interaction with sulfhydryl

(SH) enzyme systems and consequent inhibition (Csuros and Csuros 2016). Heavy

metals, such as Hg, Pb and Cd, are nephrotoxic, particularly in the renal cortex.

The chemical form of heavy metals plays a crucial role in toxicity. The toxicity of

mercury is primarily determined by Hg speciation. Patients with cancer and diabetes

have increased levels of harmful heavy metals, such as Cd, Cr and Pb, and lower levels

of the antioxidant element Se (Ali et al. 2019; Shaﬁque et al. 2011). Human health

concerns associated with arsenic poisoning include respiratory, dermal, immuno-

logical, reproductive, liver cancer, genotoxic, neurological, and mutagenic conse-

quences (Järup 2003; Shah 2021). Excess Pb consumption can impair children’s

cognitive development and intellectual aptitude, elevate blood pressure, and increase

the risk of cardiovascular disease in adults (Akoto et al. 2019).

2.2 Radionucleotides: Sources and Toxicity

The continual growth of the nuclear industry and other hazardous technologies

required for the broad and ever-increasing usage of radiation and radioactive isotopes

necessitates an evaluation of the baseline of natural radiation in order to identify man

made pollution to secure the population and the ecology (Abd El-mageed et al. 2011).

Radionuclides as they are exposed directly to through the environment, aquatic habi-

tats also get a major portion of natural and artiﬁcial radionuclides that build on the

soil by connecting the rivers (Ligero et al. 2006). The sources of radionuclides are

nuclear power plants, Radioactive waste, nuclear explosions and radioisotopes. Many

radioactive elements such as radium 224, uranium 235, uranium 238, thorium 232,

radon 222, potassium 40 and carbon 14 occur in rocks, soil and water.

The consequences of radioactive nuclide contamination in surface and ground-

water sources are serious health concerns that must be addressed. This is highly

hazardous when they exceed the drinking water’s recommended permissible limit.

According to studies, multiple types of fatal tumors caused by radon, a radium

descendent, consumed via drinking water could be equivalent to total lungs cancer

caused by radon inhalation (Ahmad et al. 2019). Uranium isotopes, radon isotopes,

and radium isotopes are the most signiﬁcant isotopes in ground water. The most

Phytoremediation of Metals and Radionuclides 155

signiﬁcant radium isotopes are 226 Ra and 228Ra, both of which have potential health

implications (Abbasisiar et al. 2004).

Leaching process carries 226 Ra (238U), 228Ra (232Th), and their decay daughters,

as well as the single non-series 40 K, to water from many types of rock formations,

minerals, and ores with large quantities of terrestrial radionuclides. The consumption

of radionuclide-contaminated water exposes human internal organs to alpha, beta,

and gamma radiations (Ugbede et al. 2020).

3 Pollutant Remediation Strategies

There are various phytoremediation strategies involved for treating the contaminants

using plants in order to reduce the heavy metal in soil. Such remediation technolo-

gies include phytostabilization, phytovolatilization, phytoﬁltration and phytoextrac-

tion. Figure 1 provides the different strategic approaches on pollutant removal via

phytoremediation process. Each of the technologies are responsible to remediate

pollutants using plants to extract and remove the heavy metals from soil; by using

plants to absorb and release the heavy metal pollutants into the atmosphere as a

volatile compound; by using hydroponically cultured plants to absorb and adsorb

the heavy metal pollutant from the groundwater; breakdown of organic pollutants by

performing the degradation process (Yan et al. 2020).

The performance of phytoremediation techniques can also be improvised by

choosing a plant species having potential phytoremediation capabilities like slow

growing, plant species should be capable to adapt at any environmental condition

and should also be limited to the large scale applications (Kaur 2020). Genetic engi-

neering has also been recently found to be a promising technique for improvising

the abilities of plants with respect to the phytoremediation of heavy metals. While

comparing genetic engineering with the traditional breeding, genetic engineering

has great advantages in order to manipulate plant species with the desirable traits

Fig. 1 Phytoremediation strategies on removal of toxic pollutants from wastewater environment

156 K. Soni et al.

for phytoremediation in a stipulated time (Singh et al. 2022a). Genetic engineering

can also help to achieve the transfer the speciﬁed genes from hyperaccumulation to

sexually incompatible plant species which was found to be difﬁcult with respect to

the traditional breeding.

4Phytoremediation

Accumulation of heavy metals in soil is increasing rapidly due to various natural

processes and anthropogenic (industrial) activities. Because heavy metals are not

biodegradable, they can remain in the environment, enter the food chain through

crops, and eventually accumulate in the human body through bio expansion. Heavy

metal pollution is toxic and poses a serious threat to human health and ecosystems.

Therefore, remediation of soil pollution is of utmost importance. Phytoremediation

is an environmentally friendly approach and can be an effective mitigation measure

for cost-effectively revitalizing heavy metal-contaminated soil. To improve the efﬁ-

ciency of phytoremediation, it is essential to have a better understanding of the

underlying mechanisms of heavy metal accumulation and tolerance in plants. This

review describes the mechanism by which heavy metals are taken up by plants, trans-

ported and detoxiﬁed. We focus on strategies applied to improve the efﬁciency of

plant stabilization and plant extraction, including the application of genetic engi-

neering, microbial support, and chelation support approaches. Foreword With the

progress of industrialization and urbanization, the abundance of heavy metals in the

environment has increased signiﬁcantly in recent decades, raising serious concerns

around the world (Suman et al. 2018; Ashraf et al. 2019).

Heavy metals are a group of metallic chemical elements with relatively high densi-

ties, atomic masses, and atomic numbers. Common heavy metals/metalloids include

cadmium (Cd), mercury (Hg), lead (Pb), arsenic (As), zinc (Zn), copper (Cu), nickel

(Ni) and chromium (Cr). included. These heavy metals/metalloids, sewage sludge

(Farahat and Linderholm 2015), metal mining and smelting (Chen et al. 2016), pesti-

cide use (Iqbal et al. 2016), Electroplating and fossil fuel combustion (Muradoglu

et al. 2015). Heavy metals are not decomposed by biological or physical processes,

remain in the soil for long periods of time, and pose a long-term threat to the envi-

ronment (Suman et al. 2018). Heavy metals can be divided into essential and non-

essential, depending on their role in the biological system. Essential heavy metals

such as Cu, Fe, Mn, Ni and Zn are required for physiological and biochemical

processes during the life cycle of plants (Cempel and Nikel 2006). However, it can

be toxic if present in excess. Non-essential heavy metals such as Pb, Cd, As and

Hg are highly toxic, have no known function in plants (Fasani et al. 2018), cause

environmental pollution and are involved in various physiological and biochemical

processes of crops. Agricultural productivity (Clemens 2006), which has and can

have serious implications. They can enter the food chain through crops, accumulate

in the human body through bioexpansion, and pose a major threat to human health.

Phytoremediation of Metals and Radionuclides 157

4.1 Pollutant Removal via Phytoremediation

Water is an essential resource for human life and is polluted by human inﬂuence.

Industrialization by a more invasive population than other means (Diez-del-Molino

et al. 2018). The collapse of such pollutants is the main reason for ecological

pollution of all. A single key circle, especially the hydrosphere, lithosphere, and

biosphere (Bilal et al. 2018). Waste from the dye industry, power plants, reﬁneries,

mines and pharmaceuticals Industrial sector. These contain both toxic and non-toxic

metal ions and other components Littering directly into waterways, causing water

pollution and causing a lot of health Impact on humans and plants. Cancer, human

infectious diseases like humans Carcinogens, neuronal depletion, cholera, typhoid

fever, gastroenteritis, diarrhoea, vomiting, skin and kidney problems (Haseena et al.

2017). In plants, it acts on seeds Germination, increased production of ROS (reac-

tive oxygen species), cellular components It also alters various metabolic pathways,

affecting plant growth, police yields, and biomass. Production (Stambulska et al.

2018). There are many methods used for treatment Contaminated water for adsorp-

tion, ion exchange processes, nanoﬁltration, agricultural applications, etc. Use of

waste, reverse osmosis, distillation, plant engineering, and biological compounds.

Phytoremediation is an effective way to remove harmful heavy metals from your

body Pollution environment. The genetic term phytoremediation consists of the

Greek preﬁx phyto. Plants attached to Latin root remedies mean to correct or elimi-

nate evil (Tangahu et al. 2011). Because contaminated water contains many compo-

nents that are toxic metals Causes of many diseases and effects in humans and plants.

Toxic metal concentration is determined using a variety of methods, including atomic

absorption spectroscopy (AAS). Calorie measurement and ratio measurement ﬂuo-

rescent probe (Rasheed et al. 2018), different chromatography Methods include high

performance liquid chromatography (HPLC) and gas chromatography (GC), Perkin

Elmer Sciex Elan 6100 ICPMS, Flame Ionization Gas Chromatography Detection

(GCFID) and Gas Chromatography-Mass Spectrometry (GCMS).

Table 1 explains the detailed note and collection of literature note on different

plants and their heavy metal tolerance level. action of plants on these compounds

is manifold: They can be immobilized, stored and evaporated, transformed various

intentions (mineralized) or combinations from these, speciﬁc compounds, environ-

mental conditions, vegetation mental types differ depending on the type of spirit.

As a result, various plant technologies are happening, it will help to relieve organic

contamination. Plant extraction is removal of contaminants from the ground and

accumulation in the subsequent plant tissue (Sashin ﬁltration is a preferred term

that water source occurs it will be treated. Plant contrast agents contain chemicals

correction of pollutants, usually they render harmful, followed by storage or elimi-

nation. Plants Ovolatitration takes contaminants made of soil or water and, they do

not contain plants covering them in the atmosphere or convert them into volatile

compounds. Plant stability reduces the bioavailability of contaminants by immobi-

lization or binding to the substrate matrix. The availability of pollutants is obviously

important Fiter Mediation Success Factors.

158 K. Soni et al.

Tabl e 1 Plants and their tolerance limit tested in literature towards heavy metals

Heavy metal Plant name Concentration (mg/kg) References

Arsenic Pteris vittata 8331 Kalve et al. (2011)

Corrigiola telephiifolia 2110 Garcia-Salgado et al.

(2012)

Eleocharis acicularis 1470 Sakakibara et al. (2011)

Chromium Pteris vittata 20,675 Kalve et al. (2011)

Cadmium Tumip landraces 52.94–149.95 Li et al. (2016)

Phytolacca americana 10,700 Peng et al. (2008)

Prosopis laevigata 8176 Buendia-González et al.

(2010)

Mercury Achiliea millefolium 18.275 Wang et al. (2012)

Silene vulgaris 4.25 Pérez-Sanz et al. (2012)

Cicer arietinum 0.2 Wang et al. (2012)

Copper Pteris vittate 91.975

Eleocharis acicularis 20,200 Sakakibara et al. (2011)

Manganese Alyxia rubricaulis 14,000 Chaney et al. (2010)

The movement of organic compounds in ecosystems depends on it mainly its

physicochemical properties (solubility) underwater, molecular size, charge, vapor

pressure, etc.) interaction with surrounding molecules. Soil properties such as pH,

texture, structure, organic matter Content is related to this context. Rhizosphere

It can also be very inﬂuential (Dzantor 2007). From experience, before applying

comprehensive phytoremediation techniques to contaminated areas, many issues

need to be considered. Of particular importance are: (i) A detailed description of

the location. Including the degree of pollution; (ii) Choosing the right plant Seeds of

this site; (iii) Assessment of total cost Cultivation (planting, irrigation, manage-

ment) and soil Change as needed. And (iv) determine the fate of Pollutants on

plants. Estimated time Repairs that are heavily dependent on pollutants Uptake or

removal rate is also an important parameter. Pilot tests are usually run to support

this Estimate (McCutcheon 2003). Contamination level of each remaining facility

Materials-especially underground materials, that is Expensive and difﬁcult to r emove.

The mechanisms concerned withinside the uptake, translocation and cleansing of

those compounds. Plants absorb xenobiotics mainly thru roots and leaves (Wang and

Liu 2007). Leaf absorption is usually a consequence of agricultural spraying with

organo chemicals, although direct uptake of risky compounds may be additionally

signiﬁcant (Burken et al. 2005). Penetration into roots takes place speciﬁcally through

easy diffusion thru unsuberized mobileular walls, from which xenobiotics attain the

xylem stream. There are manifestly no unique transporters in plant life for those

man-made compounds, so the motion charge of xenobiotics into and thru the plant

depends in large part on their physicochemical properties. Because this motion is

basically a passive bodily process, it’s far as a substitute predictable and amenable to

Phytoremediation of Metals and Radionuclides 159

particularly easy modelling (Fujisawa 2002). Certain agronomic practices can also

additionally beautify the effectiveness of the uptake process. For example, plant roots

may be guided artiﬁcially closer to polluted sectors, and supplemental irrigation,

fertilization or root oxygenation also can be applied. Likewise, plant life may be

selected or engineered for s uitable root architecture (Wang et al. 2006). Trees like

poplar (Populus spp.) or willow (Salix spp.), with large root structures and high

transpiration rates, keep unique promise for phytoremediation (Jansson and Douglas

2007).

The United Nations Environment Program has deﬁned phytoremediation as “the

efﬁcient use of plants to remove, detoxify, or immobilize environmental pollu-

tants”. Phytoremediation An environmentally friendly and beneﬁcial technology

for cleaning Contaminated media. Its mechanism includes absorption of harmful

substances by roots, accumulation in body tissues, and decomposition. Converts

pollutants into less harmful forms. Different phytoremediation techniques are used

in different media Widely discussed by several workers around the world (de Campos

et al. 2019; Favas et al. 2018). This technology is effectively used to clean up

water pollution. It is receiving serious attention from scholars, government agen-

cies and non-governmental agencies. But the use of plants in treatment the amount

of wastewater started about 300 years ago (Carolin et al. 2017).

Various types of aquatic plants have been validated and recognized Efﬁciency in

collecting inorganic and organic pollutants from water through hydroponics or ﬁeld

applications. Many species of aquatic plants like Ranunculaceae, Remna, Kayatsuri-

gusa, Haroraga, Hydrocharitaceae, Pondweed, Pondweed, Najadaceae, Pontederi-

aceae, Juncaceae, Zosterophyllaceae are the most important representative of aquatic

plant phytoremediation Environment (Prasad 2007). The technique of phytoremedi-

ation is an effective tool for tackling unprecedented pollution aquatic environment.

With this technique, the ﬁrst step is Identiﬁcation and screening of highly effective

plants Collect dissolved nutrients, metals and other pollutants (Lu 2009).

Plant selection for phytoremediation technology it should grow fast and be easy to

handle and harvest. Other biological processes of plants such as growth and develop-

ment, photosynthesis are important elements of sustainability. Successful phytosan-

itary system It also depends on the factors related to the seriousness of the pollution

(Jamuna and Noorjahan 2009). In addition, various plant technologies such as plant

degradation, plant stabilization, rhizosphere ﬁltration, rhizosphere degradation, and

plant volatilization have been applied to contaminated ecosystems (Cunningham and

Berti 1993). The decrease in contaminants present in the soil is performed by rash

Plant roots. Stabilize these roots, dismantling, non-moving, and binding Impurities

are called vegetable stability processes. In addition, the root, adsorption, adsorption,

and soil and water precipitation of speciﬁc plant species Immobilization Process.

This is an important way to eliminate Organic and inorganic pollutants present in

soil and sediments and the mud unit (Cunningham and Berti 1993). Plant extraction

is a useful process for different ﬂoors impurity pollutants are also absorbed in the

process As over-accumulated in different parts of the plant (Rulkens et al. 1998).

Plants have an excellent ability to consume and evaporate It releases pollutants

directly into the atmosphere through the plant volatilization process. This process is

160 K. Soni et al.

Tabl e 2 Plants and their phytoremediation responses towards pollutants

Plant name Phytoremediation response References

Pistia stratiotes L. Cell membrane damage, reduced root

volume, less growth rate and

photosynthesis, Increase in enzyme activity

of peroxidase, catalase, superoxide

dismutase

de Campos et al. (2019)

Echinodorus amazonicus Reduce plant height, root length, plant

growth

Sricoth et al. (2018)

Eichhornia crassipes Lesser plant growth, Less plant height and

root length and resulted in death of plant

Ipomonea aquatica Increased root size but reduced root length Chen et al. (2010)

Lemna minor LLess enzymatic activity and

photosynthesis, reduced chlorophyll, root

and shoot growth

Radi´cetal. (2010)

cost effective in removing contaminants from soil, groundwater, tailings and sludge

(Girdhar et al. 2014). Plants partially metabolize pollutants Compounds produced in

plant tissue Plant conversion/plant decomposition process (Chaudhry 1998). Table 2

provides the note on different type of plants and their response to phytoremediation

process. For resoﬁltration, Metal pollutants on surrounding growth substrates Root

zone. In this process, groundwater contamination and surface water are performed

by plant roots (Dushenkov et al. 1995). Plants release tightened different organic

compounds Microbial communities present on the ground help the root of the impu-

rities. This Phytoroma Mediation Technology provides an alternative to replace reha-

bilitation purposes. Bio-solvitation is a physicochemical process used as a method

for. Metal removal (Verma et al. 2008). I used this method As an alternative technique

for removing HMS from wastewater (Ali et al. 2020).

5 Challenges and Conclusion

Unawareness or negligence in knowing that water is essential for all forms of life

to survive in the universe. This water resource is continuously contaminated due to

anthropogenic activities. Government should make stringent rules and ensure that

authorities are in constant surveillance on water localities. Beyond this modern and

hybrid mode of water treatment techniques need to be identiﬁed and experimented

for effective wastewater treatment with focusing on reduce and reuse strategy. This

book chapter was dedicated to focus especially on phytoremediation of wastewater

contaminated with heavy metals and radionuclides. Phytoremediation process helps

in improving the COD and BOD level of water bodies. Physiochemical parameters

like concentration, variety of contaminant, plant type used, experimental design.

Phytoremediation of Metals and Radionuclides 161

In future integrated approaches of mechanical, physical, chemical and biological

approaches need to be engaged in phytoremediation process.

Acknowledgements Author wishes to thank Sathyabama institute of science and technology for

the support.

Conﬂict of Interest None.

References

Abbasisiar F, Hosseini T, Fathivand A, Heravi G (2004) Determination of uranium isotopes (234U,

238U) and natural uranium (U-nat) in water samples by alpha spectrometry

Abd El-mageed AI, El-Kamel AH, Abbady A, Harb S, Youssef AMM, Saleh II (2011) Assessment

of natural and anthropogenic radioactivity levels in rocks a nd soils in the environments of Juban

town in Yemen. Radiat Phys Chem 80:710–715

Ahmad N, ur Rehman J, Rehman J, Nasar G (2019) Effect of geochemical properties (pH, conduc-

tivity, TDS) on natural radioactivity and dose estimation in water samples in Kulim, Malaysia.

Hum Ecol Risk Assess Int J 25:1688–1696

Akoto O, Nimako C, Asante J, Bailey D, Bortey-Sam N (2019) Spatial distribution, exposure, and

health risk assessment of bioavailable forms of heavy metals in surface soils from abandoned

landﬁll sites in Kumasi, Ghana. Hum Ecol Risk Assess Int J 25:1870–1885

Ali H, Khan E, Ilahi I (2019) Environmental chemistry and ecotoxicology of hazardous heavy

metals: environmental persistence, toxicity, and bioaccumulation. J Chem

AliS,AbbasZ,RizwanM,Zaheer IE,Yava¸s ˙

I, Ünay A, Abdel-Daim MM, Bin-Jumah M, Hasanuz-

zaman M, Kalderis D (2020) Application of ﬂoating aquatic plants in phytoremediation of heavy

metals polluted water: a review. Sustainability 12:1927

Ashraf S, Ali Q, Zahir ZA, Ashraf S, Asghar HN (2019) Phytoremediation: Environmen-

tally sustainable way for reclamation of heavy metal polluted soils. Ecotoxicol Environ Saf

174:714–727

Barbosa B, Costa J, Boléo S, Duarte MP, Fernando AL (2016) Phytoremediation of inorganic

compounds. In: Electrokinetics across disciplines and continents. Springer, pp 373–399

Bilal M, Rasheed T, Sosa-Hernández JE, Raza A, Nabeel F, Iqbal H (2018) Biosorption: an interplay

between marine algae and potentially toxic elements—a review. Mar Drugs 16:65

Briffa J, Sinagra E, Blundell R (2020) Heavy metal pollution in the environment and their

toxicological effects on humans. Heliyon 6:e04691

Buendía-González L, Orozco-Villafuerte J, Cruz-Sosa F, Barrera-Díaz CE, Vernon-Carter EJ (2010)

Prosopis laevigata a potential chromium (VI) and cadmium (II) hyperaccumulator desert plant.

Bioresour Technol 101:5862–5867

Burken JG, Ma X, Struckhoff GC, Gilbertson A (2005) Volatile organic compound fate in

phytoremediation applications: natural and engineered systems. Z Naturforsch C 60:208–215

Carolin CF, Kumar PS, Saravanan A, Joshiba GJ, Naushad M (2017) Efﬁcient techniques for

the removal of toxic heavy metals from aquatic environment: a review. J Environ Chem Eng

5:2782–2799

Cempel M, Nikel G (2006) Nickel: a review of its sources and environmental toxicology. Polish J

Environ Stud 15

Chaney RL, Broadhurst CL, Centofanti T (2010) Phytoremediation of soil trace elements. Trace

Elem Soils 311–352

Chaudhry TM (1998) Phytoremediation: focusing on accumulator plants that remediate metal

contaminated soils. Australas J Ecotoxicol 4:3–51

162 K. Soni et al.

Chen B, Stein AF, Castell N, Gonzalez-Castanedo Y, De La Campa AMS, De La Rosa JD (2016)

Modeling and evaluation of urban pollution events of atmospheric heavy metals from a large

Cu-smelter. Sci Total Environ 539:17–25

Chen J-C, Wang K-S, Chen H, Lu C-Y, Huang L-C, Li H-C, Peng T-H, Chang S-H (2010) Phytore-

mediation of Cr (III) by Ipomonea aquatica (water spinach) from water in the presence of EDTA

and chloride: Effects of Cr speciation. Bioresour Technol 101:3033–3039

Clemens S (2006) Toxic metal accumulation, responses to exposure and mechanisms of tolerance

in plants. Biochimie 88:1707–1719

Csuros M, Csuros C (2016) Environmental sampling and analysis for metals. CRC Press

Cunningham SD, Berti WR (1993) Remediation of contaminated soils with green plants: an

overview. Vitr Cell Dev Biol 29:207–212

de Campos FV, de Oliveira JA, da Silva AA, Ribeiro C, dos Santos Farnese F (2019) Phytoreme-

diation of arsenite-contaminated environments: is Pistia stratiotes L. a useful tool? Ecol Indic

104:794–801

Díez-del-Molino D, García-Berthou E, Araguas RM, Alcaraz C, Vidal O, Sanz N, García-Marín

JL (2018) Effects of water pollution and river fragmentation on population genetic structure of

invasive mosquitoﬁsh. Sci Total Environ 637:1372–1382

Dushenkov V, Kumar PBAN, Motto H, Raskin I (1995) Rhizoﬁltration: the use of plants to remove

heavy metals from aqueous streams. Environ Sci Technol 29:1239–1245

Dzantor EK (2007) Phytoremediation: the state of rhizosphere ‘engineering’for accelerated

rhizodegradation of xenobiotic contaminants. J Chem Technol Biotechnol Int Res Process Environ

Clean Technol 82:228–232

Farahat E, Linderholm HW (2015) The effect of long-term wastewater irrigation on accumulation

and transfer of heavy metals in Cupressus sempervirens leaves and adjacent soils. Sci Total

Environ 512:1–7

Fasani E, Manara A, Martini F, Furini A, DalCorso G (2018) The potential of genetic engineering of

plants for the remediation of soils contaminated with heavy metals. Plant Cell Environ 41:1201–

1232

Favas PJC, Pratas J, Rodrigues N, D’Souza R, Varun M, Paul MS (2018) Metal (loid) accumulation

in aquatic plants of a mining area: potential for water quality biomonitoring and biogeochemical

prospecting. Chemosphere 194:158–170

Fujisawa T (2002) Model of the uptake of pesticides by plant. J Pestic Sci Sci Soc Japan-Japanese

Ed 27:279–286

García-Salgado S, García-Casillas D, Quijano-Nieto MA, Bonilla-Simón M (2012) Arsenic and

heavy metal uptake and accumulation in native plant species from soils polluted by mining

activities. Water Air Soil Pollut 223:559–572

Gautam PK, Gautam RK, Banerjee S, Chattopadhyaya MC, Pandey JD (2016) Heavy metals in the

environment: fate, transport, toxicity and remediation technologies. Nov Sci Publ 60:101–130

Girdhar M, Sharma NR, Rehman H, Kumar A, Mohan A (2014) Comparative assessment for

hyperaccumulatory and phytoremediation capability of three wild weeds. 3 Biotech 4:579–589

Haseena M, Malik MF, Javed A, Arshad S, Asif N, Zulﬁqar S, Hanif J (2017) Water pollution and

human health. Environ Risk Assess Remediat 1

Huat TJ, Camats-Perna J, Newcombe EA, Valmas N, Kitazawa M, Medeiros R (2019) Metal toxicity

links to Alzheimer’s disease and neuroinﬂammation. J Mol Biol 431:1843–1868

Iqbal M, Iqbal N, Bhatti IA, Ahmad N, Zahid M (2016) Response surface methodology application

in optimization of cadmium adsorption by shoe waste: a good option of waste mitigation by

waste. Ecol Eng 88:265–275

Jaishankar M, Tseten T, Anbalagan N, Mathew BB, Beeregowda KN (2014) Toxicity, mechanism

and health effects of some heavy metals. Interdiscip Toxicol 7:60

Jamuna S, Noorjahan C (2009) Treatment of sewage waste water using water hyacinth-Eichhornia

sp and its reuse for ﬁsh culture. Toxicol Int 16:103

Jansson S, Douglas CJ (2007) Populus: a model system for plant biology. Annu Rev Plant Biol

58:435–458

Phytoremediation of Metals and Radionuclides 163

Järup L (2003) Hazards of heavy metal contamination. Br Med Bull 68:167–182

Jhilta P, Dipta B, Rana A (2021) Phytoremediation of heavy metals and radionuclides: sustainable

approach to environmental management. In: Phytoremediation for environmental sustainability.

Springer, pp 83–111

Kabata-Pendias A (2000) Trace elements in soils and plants. CRC Press

Kalve S, Sarangi BK, Pandey RA, Chakrabarti T (2011 Arsenic and chromium hyperaccumulation

by an ecotype of Pteris vittate–prospective for phytoextraction from contaminated water and soil.

Curr Sci 888–894

Kaur L (2020) Role of phytoremediation strategies in removal of heavy metals. Emerg Issues Water

Environ Dur Anthr 223–259

Kayakökü H, Do ˘gru M (2020) Radiological hazard assessment of natural radionuclides and heavy

metal pollution in deep mud samples of Van Lake. Turkey J Radioanal Nucl Chem 324:1339–1350

Kim J-J, Kim Y-S, Kumar V (2019) Heavy metal toxicity: An update of chelating therapeutic

strategies. J Trace Elem Med Biol 54:226–231

LaGrega MD, Buckingham PL, Evans JC (2010) Hazardous waste management. Waveland Press

Li X, Zhang X, Yang Y, Li B, Wu Y, Sun H, Yang Y (2016) Cadmium accumulation characteristics in

turnip landraces from China and assessment of their phytoremediation potential for contaminated

soils. Front Plant Sci 7:1862

Ligero RA, Feria F, Casas-Ruiz M, Corredor C (2006) Diffusion of 226Ra and 40K radionuclides

reproduced in underwater sedimentary columns in laboratory. J Environ Radioact 87:325–334

Lu Q (2009) Evaluation of aquatic plants for phytoremediation of eutrophic stormwaters. University

of Florida Florida

Masindi V, Muedi KL (2018) Environmental contamination by heavy metals. Heavy Met. 10:115–

132

McCutcheon SC (2003) Overview of phytotransformation and control of wastes. In: McCutcheon

SC, Schnoor JL (eds) Phytoremediation: transformation and control of contaminants, pp 1–58

Muradoglu F, Gundogdu M, Ercisli S, Encu T, Balta F, Jaafar HZE, Zia-Ul-Haq M (2015) Cadmium

toxicity affects chlorophyll a and b content, antioxidant enzyme activities and mineral nutrient

accumulation in strawberry. Biol Res 48:1–7

Peng K, Luo C, You W, Lian C, Li X, Shen Z (2008) Manganese uptake and interactions with

cadmium in the hyperaccumulator—Phytolacca Americana L. J Hazard Mater 154:674–681

Pérez-Sanz A, Millán R, Sierra MJ, Alarcón R, García P, Gil-Díaz M, Vazquez S, Lobo MC

(2012) Mercury uptake by Silene vulgaris grown on contaminated spiked soils. J Environ Manag

95:S233–S237

Prasad MNV (2007) Aquatic plants for phytotechnology. In: Environmental bioremediation

technologies. Springer, pp 259–274

Radi´cS,Babi´c M, Škobi´c D, Roje V, Pevalek-Kozlina B (2010) Ecotoxicological effects of

aluminum and zinc on growth and antioxidants in Lemna minor L. Ecotoxicol Environ Saf

73:336–342

Rasheed T, Li C, Bilal M, Yu C, Iqbal HMN (2018) Potentially toxic elements and environmentally-

related pollutants recognition using colorimetric and ratiometric ﬂuorescent probes. Sci Total

Environ 640:174–193

Rulkens WH, Tichy R, Grotenhuis JTC (1998) Remediation of polluted soil and sediment:

perspectives and failures. Water Sci Technol 37:27–35

Shah MP (2020) Microbial bioremediation & biodegradation. Springer

Shah MP (2021) Removal of refractory pollutants from wastewater treatment plants. CRC Press

Sakakibara M, Ohmori Y, Ha NTH, Sano S, Sera K (2011) Phytoremediation of heavy metal-

contaminated water and sediment by Eleocharis acicularis. CLEAN–Soil Air Water 39:735–741

Shaﬁque U, Anwar J, Ali SZ (2011) Assessment of concentration of lead, cadmium, chromium and

selenium in blood serum of cancer and diabetic patients of Pakistan. J Chem Soc Pak 33:869

Singh BSM, Singh D, Dhal NK (2022a) Enhanced phytoremediation strategy for sustainable

management of heavy metals and radionuclides. Case Stud Chem Environ Eng 5:100176

164 K. Soni et al.

Singh G, Bhadange S, Bhawna F, Shewale P, Dahiya R, Aggarwal A, Manju F, Arya SK

(2022b) Phytoremediation of radioactive elements, possibilities and challenges: special focus

on agricultural aspects. Int J Phytoremediation 1–8

Sricoth T, Meeinkuirt W, Saengwilai P, Pichtel J, Taeprayoon P (2018) Aquatic plants for

phytostabilization of cadmium and zinc in hydroponic experiments. Environ Sci Pollut Res

25:14964–14976

Stambulska UY, Bayliak MM, Lushchak VI (2018) Chromium (VI) toxicity in legume plants:

modulation effects of rhizobial symbiosis. Biomed Res Int

Suman J, Uhlik O, Viktorova J, Macek T (2018) Phytoextraction of heavy metals: a promising tool

for clean-up of polluted environment? Front Plant Sci 1476

Tangahu BV, Sheikh Abdullah SR, Basri H, Idris M, Anuar N, Mukhlisin M (2011) A review on

heavy metals (As, Pb, and Hg) uptake by plants through phytoremediation. Int J Chem Eng

Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ (2012) Heavy metal toxicity and the

environment. Mol Clin Environ Toxicol 133–164

Thakare M, Sarma H, Datar S, Roy A, Pawar P, Gupta K, Pandit S, Prasad R (2021) Understanding

the holistic approach to plant-microbe remediation technologies for removing heavy metals and

radionuclides from soil. Curr Res Biotechnol 3:84–98

Ugbede FO, Aduo BC, Ogbonna ON, Ekoh OC (2020) Natural radionuclides, heavy metals and

health risk assessment in surface water of Nkalagu river dam with statistical analysis. Sci Afr

8:e00439

Verma VK, Tewari S, Rai JPN (2008) Ion exchange during heavy metal bio-sorption from aqueous

solution by dried biomass of macrophytes. Bioresour Technol 99:1932–1938

Wang CJ, Liu ZQ (2007) Foliar uptake of pesticides—present status and future challenge. Pestic

Biochem Physiol 87:1–8

Wang H, Inukai Y, Yamauchi A (2006) Root development and nutrient uptake. CRC Crit Rev Plant

Sci 25:279–301

Wang J, Feng X, Anderson CWN, Xing Y, Shang L (2012) Remediation of mercury contaminated

sites–a review. J Hazard Mater 221:1–18

Xu T, Wang S, Li Y, Li J, Cai J, Zhang Y, Xu D, Zhang J (2021) Review of the destruction of organic

radioactive wastes by supercritical water oxidation. Sci Total Environ 149396

Yan A, Wang Y, Tan SN, Mohd Yusof ML, Ghosh S, Chen Z (2020) Phytoremediation: a promising

approach for revegetation of heavy metal-polluted land. Front Plant Sci 11:359

Anaerobic Biotechnology:

Implementations and New Advances

Samir I. Gadow, Hatem Hussein, Abdelhadi A. Abdelhadi,

and Abd El-Latif Hesham

Abstract Anaerobic technology has gained widespread acceptance in environ-

mental sustainability as a low-cost alternative for pollution control. The anaerobic

technologies for contaminants treatment have three essential returns, i.e., bioenergy

recovery, energy-saving and low sludge production. Therefore, the anaerobic process

will be the favored green treatment technology for a sustainable environment in years

to come. Currently, anaerobic treatment remains to ﬂourish in several features, such

as reactors development, bio-hythane production, molecular techniques for micro-

bial studies and kinetic modeling and extending applications to a wide range of waste

and wastewater efﬂuents. Therefore, this chapter brings together the most up-to-date

information on the new developments in anaerobic technology. Also, it sheds light

on the current conversion methods and technologies for energy recovery with a focus

on the use of natural materials as sustainable and environmentally friendly sources

for creating new materials used in this regard.

Keywords Anaerobic technology ·Bioenergy recovery ·Pollution control ·

Annamox process ·Emerging pollution

S. I. Gadow (B

)

Department of Agricultural Microbiology, Agriculture and Biology Research Institute, National

Research Centre, 33 EI Buhouth St., Dokki 12622, Cairo, Egypt

e-mail: si.gadow@nrc.sci.eg

H. Hussein

Maskoub International for Contracting Company—Water Treatment and Environmental

Solutions, Cairo, E gypt

e-mail: Hatem.a.hamid@maskoub.com

A. A. Abdelhadi

Faculty of Agriculture, Genetics Department, Cairo University, Giza 12613, Egypt

e-mail: abdelhadi.abdallah@agr.cu.edu.eg

A. E.-L. Hesham (B

)

Faculty of Agriculture, Genetics Department, Beni-Suef University, Beni-Suef 62511, Egypt

e-mail: hesham_egypt5@agr.bsu.edu.eg; hesham_egypt5@agr.au.edu.eg

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_9

165

166 S. I. Gadow et al.

1 Introduction

Anaerobic biotechnology is important for two important challenges in our society:

environmental protection and resource recovery. Today, 7.9 billion people (2022) live

in this global community, sharing an increasingly polluted environment and quickly

dwindling resources. The existence of our global community is further affected by the

ongoing growth of the human population, which may exceed 12 billion by the end of

the twenty-ﬁrst century. Anaerobic technology has been proved as a viable treatment

procedure for numerous types of waste and wastewater after decades of development.

Despite its inherent beneﬁts and demonstrated effectiveness, this biotechnology is

still frequently overlooked by many today. In comparison to the more traditional

aerobic approach, the anaerobic process for waste and wastewater treatment has

three i nherent advantages: energy savings, lower sludge yield, and biofuel genera-

tion. Because of these beneﬁts, as well as recent technological advancements, anaer-

obic treatment technology has become a cost-effective way of pollution manage-

ment, leading in the installation of thousands of full-scale treatment plants across the

world during the last two decades. These beneﬁts also make the anaerobic process

a long-term environmental technology with considerable promise. Today, anaer-

obic technology is thriving in many areas, including the creation of new types of

reactors, the application of molecular tools for microbial investigations, and kinetic

modeling, bio-hydrogen generation by fermentation and a microbial electrolysis cell,

with expanded applicability to municipal wastewater, chemical industrial efﬂuents,

and agricultural wastes with high lignocellulose content. Therefore, this chapter shits

light on identifying inﬂuencing factors on anaerobic sustainable management for

energy and resource recovery. Moreover, encourage further research work that would

help expand and improve the best technologies to ensure affordability, suitability, and

environmental sustainability.

2 Anaerobic Technology as a Tool to Promote a Sustainable

Development

As a result of a paradigm shift in the treated waste/wastewater as renewable resources,

a reduction of dependency on fossil fuels and dropping in the amount of pollu-

tion can be addressed simultaneously. The clean water and energy supplies avail-

able are depleting due to population growth, competition among users and lack of

studies concerning the risk assessment of emerging pollutants (EPs) (Hernando et al.

2011; Patel et al. 2020). Additionally, the wastewater efﬂuent became more complex

and dangerous over time, due to innovation and marketing in the various sectors

(Z˘abav˘aetal.

2019). This leads to higher levels of the EPs (i.e., toxic organics

and metal) in the ecosystem (see Fig. 1). The polluted water is graded according

to its sources and is deﬁned accordingly (Hsien et al. 2019). Generally, wastew-

ater includes three major groups. The ﬁrst is domestic wastewater, which refers

Anaerobic Biotechnology: Implementations and New Advances 167

to ﬂows discharged mainly from residential sources created by food preparation,

washing, cleaning, and personal hygiene. Secondly, it relates to industrial wastewater

produced through production and commercial activities such as everyday products

industries, pharmaceutical industries, and agro-industries. The third is agricultural

wastewater which refers to ﬂows discharged principally from agricultural activities

such as poultry wastewater, dairy farming wastewater, and nutrient runoff. Nowadays,

water resources are becoming increasingly scarce in Middle East Countries (MEC)

and Egypt is expected to be absolute water scarcity by the year 2030 due to the rapidly

increasing population that reached now near 100 million inhabitants and economic

growth (Miralles-Wilhelm et al. 2018). In addition to recalcitrant organic pollutants

(ROPs), the wastewater efﬂuents include potentially toxic substances, along with

various pathogenies. Thus, this will require a lot of effort to be made for more efﬁ-

cient management of wastewater treatment at low cost and sustainable solutions, to

secure sustainability and development for the coming generations. To date, there is

no economically and rapidly attractive solution to treat wastewater efﬂuents due to

socio-economic factors (Foteinis et al. 2018). However, among various technolo-

gies used for wastewater treatment, biological technology is a promising technology

because of its simplicity and a promising net positive energy, even when heating of

the liquid is required, could be produced from wastewater containing recalcitrant

and toxic organics by different operation conditions and bio-systems. Therefore, this

review sheds light on the current conversion methods and technologies for energy

recovery from wastewater sludge treatment with a focus on the use of natural mate-

rials as sustainable and environmentally friendly sources for creating new materials

used in this regard.

Clean and renewable water supplies are essential for establishing and maintaining

a wide range of human activities. Accordingly, the emerging pollutants caused by

human activities such as industrial and agricultural production are a permanent

concern throughout the world. Which requires a lot of effort to ﬁnd and develop effec-

tive treatment technologies for wastewater treatment with energy-saving in mind.

Environmental pollution

risks

Fig. 1 Economic growth and sustainable society

168 S. I. Gadow et al.

Data in the literature show that there are three main technologies for wastewater

management which are physical, chemical and biological treatments. Each of these

technologies is characterized by advantages and disadvantages, beneﬁts and tech-

nological limitations (Cheremisinoff 2001; Bui et al. 2019). The physical/chemical

technologies such as membrane technologies, coagulation & ﬂocculation and chem-

ical transformations, have been studied extensively for pollutants and pathogens

removal from wastewater efﬂuents (Abdullah et al. 2019;Hoetal. 2020; Ahamed

et al. 2019). However, there are still various limitations in the implementation of

the above technologies such as cost and operational difﬁculty (Bello et al. 2019).

The bio-treatment of aerobic and anaerobic technologies, however, presents a pros-

perous technology. In this technology, microbes adapt to toxic pollutants and natu-

rally produce new resistant strains that convert various toxic substances into less

harmful substances. Several reports established that anaerobic technology is one of

the promising and suitable technology to treat a wide range of wastewater efﬂuents

and bioenergy production (Stazi and Tomei 2018). The biodegradation of complex

and toxic pollutants is achieved efﬁciently by enzymatic mechanisms with energy-

saving options and low-cost management, such as those associated with agricultural

wastes (Jiang et al. 2017, 2018), cellulosic wastewater (Gadow and Li 2019; Gadow

et al. 2019a), and recalcitrant textile dyes (Gadow and Li 2020a, b; Gadow et al.

2019b). Consequently, a new classiﬁcation of wastewater treatment technologies has

been proposed, which takes into account using of natural materials as sustainable

and environmentally friendly s ources for creating new materials used in this regard

and the energy-saving option.

Although the energy-consuming technologies for wastewater treatment have been

recommended to eliminate pollutants and pathogenic microorganisms, they still

face some challenges including issues of cost and operational difﬁculties (Farooq

and Ahmad 2017). As for several chemical technologies such as chemical transfor-

mations, the main concern for consuming large amounts of energy and chemicals

and often cause secondary contaminations under ineffective management (Zhang

et al. 2019). Consequently, these methods are economically unfavorable or tech-

nically complicated and are only used in special cases of wastewater treatment.

Energy recovery during wastewater treatment is particularly important because of its

effectiveness in comparison with other techniques, on minimizing treatment costs

and reduction of sludge. Figure 2 shows the potential wastewater-energy recovery

options by using different technologies. A signiﬁcant amount of renewable energy

such as biogas, chemical, liquid fuel, heat, and electricity generation can be recov-

ered from sludge management (Jiang et al. 2018; Gadow and Li 2019; Thanos

et al. 2020). There are four different technologies to extract energy during wastew-

ater treatment including combustion, pyrolysis, gasiﬁcation and anaerobic digestion

(Shukla et al. 2019; Shi et al. 2019; Shareef 2020). The beneﬁts and drawbacks of

these technologies and their technical weaknesses will be discussed in the following

sections.

Anaerobic Biotechnology: Implementations and New Advances 169

Anaerobic

digestion

Combustion

(incineration)

Pyrolysis

Gasification

Minutes

Days Minutes

Thermochemical technologies

Biological

technologies

Organic waste/Sludge

Sludge Pre-treatment

Biogas

H

2,

CH

4,

CO

2

(CO

2

, H

2

O ,CO, N

2

No

x,

So

x

)

Heat

Ash

Bio-char

Bio-oil

CH

4,

CO

2

Bio-char

CO, H

2,

CH

4,

CO

2

Tar

Absence of oxygen

10-75

o

C

Excess air

800- 1150

o

C

Absence of oxygen

300- 900

o

C

Partial oxidation

650- 1000

o

C

Hours

Energy recovery

Fig. 2 Methods of energy recovery from organic waste and/or sludge

3 Anaerobic Granulation and Granular Sludge Reactor

Systems

Anaerobic wastewater treatment is a biological conversion of organic pollutants with

high water content to produce biogas mixtures of hydrogen, methane and carbon

dioxide (Jiang et al. 2018; Gadow and Li 2020a). Recently, anaerobic digestion is

an important and promising industrial technology to treat a wide range of wastew-

ater including industrial, municipal and farming efﬂuents (see Fig. 2). The general

advantage of the anaerobic digestion process is that it can be applied for low-strength

wastewaters and high-strength organic solid waste to the proper reactor conﬁguration

(Xiong et al. 2020). Moreover, it can accommodate high COD loads, which adapt

to remove various toxicant components provided that adaptation time is allowed for

the anaerobic waste/wastewater (Luo et al. 2019). Generally, up to 95% of organic

material will become biogas as a renewable resource by the properly run digester

(Stazi and Tomei 2018;Lietal.

2015). On the other hand, the improper operation of

the anaerobic plant will cause the following drawbacks to become more evident: (1)

low growth by high temperature (Gadow et al. 2012). (2) The process is sensitive to

COD overloads and toxicant shock loads (Xiong et al. 2020).

Especially treating for the high C/N ratio waste/wastewater (Gadow et al. 2019a).

(3) The optimum pH for the process lies in a narrow range near neutrality and

170 S. I. Gadow et al.

with an inapposite operation such as the temperature and substrate concentration,

the process will be inhibited due to intermediates accumulation (Jiang et al. 2018;

Lim et al. 2020). Therefore, the biodegradation of recalcitrant pollutants is always a

permanent concern to achieve environmental suitability with energy-saving in mind.

3.1 Hydrogen Production

Hydrogen is considered to be the promising energy carrier due to its highest heating

value (142 MJ/kg Higher Heating Value (HHV) and leaving pure water as the only

end product. Dark hydrogen fermentation is considered a sustainable approach to

waste/wastewater treatment issues and has the added advantage of yielding bioenergy

(Gadow et al. 2013a;Mekyetal. 2020). The organic pollutants are converted by

anaerobic hydrogen-producing bacteria (HPB) grown. The HPB is able to use energy-

rich hydrogen molecules by hydrogenases to generate H2. During dark hydrogen

fermentation, acetic acid, butyric acid, and ethanol are t he main soluble by-products

as shownbyEqs.

1, 2 and 3, respectively (Balachandar et al. 2020; Gadow et al.

2016). Accordingly, it can be noted that the hydrogen yield is greater when coupled

with acetic acid rather than butyric acid and ethanol.

C6H12O6 + 2H2O −→ 4H2 + 2C2H4O2 + 2CO2(1)

C6H12O6 −→ 2H2 + C4H8O2 + 2CO2(2)

C6H12O6 + 2H2O −→ 2H2 + C2H4O2 + C2H5OH + 2CO2(3)

The ratio of butyrate/acetate is very important and a suitable indicator to evaluate

the hydrogen production efﬁciency.

Table 1 shows the stoichiometry of different reactions to produce hydrogen in the

dark fermentation and the maximum theoretical reached 4 mol H2/mol hexose with

acetate as an only soluble by-product (Gadow et al. 2013a). However, the formation

of other by-products, such as propionic and butyric acids as well as methanol, butanol,

or acetone, lowers the amount of hydrogen produced in fermentation. Several studies

reported that there is a diversity of microorganisms including facultative and strictly

anaerobic such as bacteria and archaea able to produce hydrogen by dark fermenta-

tion. Two bacterial genera that have been studied extensively are enteric and clostridia

(Collet et al. 2004). The importance of using a mixed culture of obligate and facul-

tative bacteria is valuable since facultative bacteria make an anaerobic condition

for the oxygen-sensitive obligate bacteria. For continuous bio-hydrogen production,

the hydraulic resistance time is an important operational parameter in the anaerobic

bioreactors. The hydrogen-producing bacteria have a long speciﬁc growth rate which

reached 0.172 h−1. Therefore, short HRT is suitable for hydrogen production and

eliminating other hydrogen-consuming bacteria. Zhang et al. (2006) conﬁrmed that

Anaerobic Biotechnology: Implementations and New Advances 171

Tabl e 1 The theoretical maximum H2 yield under different reactions

Reaction Stoichiometry References

Acetate production C6H12O6 + 4H2O → 2CH3COO−

+ 4H2 + 2HCO3− + 4H+

Thauer et al. (1977)

Butyrate production C6H12O6 + 2H2O →

CH3CH2CH2COO− + 2H2 +

2HCO3 3H+

Thauer et al. (1977)

Acetate and ethanol production C6H12 O6 + 3H2O → CH3CH2OH

+ CH3COO− + 2H2 + 2HCO3− +

3H+

Hwang et al. (2004)

Lactate production C6H12O6 → 2CH3CHOHCOO− +

2H+

Kim et al. (2006)

Butanol production C6H12O6 + H2O →

CH3CH2CH2OH + 2HCO3− + 2H+

Chin et al. (2003)

Propionate production C6H12O6 + 2H2 →

2CH3CH2COO− + 2H2O + 2H+

Hussy et al. (2003)

Valerate production C6H12O6 + H2 →

CH3CH2CH2CH2COO− + HCO3−

+ H2O + 2H+

Ren and Gong (2006)

Acetate fermentation to H2CH3COO− + 4H2O → 4H2 +

2HCO3− + H+

Stams (1994)

Butyrate fermentation to H2CH3CH2CH2COO− + 10 H2O →

10H2 + 4HCO3− + 3H+

Sawers (2005)

decreasing the hydraulic resistance time from 8 to 6 h led to signiﬁcantly increasing

hydrogen production efﬁciency (Zhang et al. 2006).

3.2 Methane Production

The anaerobic digestion of methane production from waste/wastewater is promising

and growing worldwide due to its economic and environmental beneﬁts. Furthermore,

the other crucial beneﬁts offered by the use of biogas over natural gas are as follows:

(i) it is produced from renewable resources, (ii) it does not add any greenhouse

gases to the atmosphere, (iii) it is produced locally without any dependency on other

energy supplies, (iv) it helps in reducing the pollution produced by the organic wastes,

which account for most freshwater pollution, and, (v) it helps in retarding the waste

management problems.

The anaerobic digestion will begin by hydrolysis of complex organic substances

such as carbohydrates and proteins into sugars, amino acids and respectively (see

Fig. 3). The Acetogenic bacteria and other fermentative bacteria convert the reduced

compounds to short-chain volatile fatty acids (VFAs) and hydrogen. The ﬁnal step in

the anaerobic digestion process is methanogenesis, where the methane was produced

172 S. I. Gadow et al.

by converting the acetic and hydrogen by methanogenic bacteria such as a consortium

of microbes (Ali et al. 2019). Methane fermentation technology is the most efﬁcient

way of handling and energy generation from waste/wastewater, in terms of energy

output/input ratio (28.8 MJ/MJ) in comparison to all other technology of energy

production through thermochemical routes of energy conversion processes (Deublein

and Steinhauser 2011). Since anaerobic digestion of methane production is a biolog-

ical reaction, environmental conditions such as pH, temperature and growth medium

characterization have a signiﬁcant effect. Additionally, operational factors such as the

hydraulic retention time and organic loading rate are the factors affecting the amount

of methane produced. This approach is guaranteed to reduce environmental risks

and create renewable sources. The produced methane can replace natural gas and the

residues resulting from anaerobic digestion can use as fertilizer and bio-conditioner

for agricultural purposes. The hydrolysis steps of anaerobic digestion can achieve at

a wide range of pH, however, the optimum pH value of methanogenesis around is

neutral. Several studies state that the pH below 6.5 and high than 7.5 decreases the rate

of biogas production (Khan et al. 2016). Therefore, in order to preserve the optimal

pH level required for methanogenesis, an adequate amount of bicarbonate alkalinity

in the solution is necessary. As for operational parameters, the loading rate is not

limited by substance supplement, but by the processing capacity of the microﬂora.

Consequently, it is essential to retain a sufﬁcient bacterial mass in the anaerobic

bioreactor. The speciﬁc growth rate of methanogenic bacteria is very low (0.0167–

0.02 h−1), when compared with hydrogen-producing bacteria (0.172 h−1) (Kamyab

et al. 2019). Therefore, in many studies, where a mixed consortium, such as sewage

sludge, is used, long HRT is preferable to increase methanogenic bacteria. An inter-

esting study reported that bio-methane production was increased from 0.11 to 0.24

L/L leachate signiﬁcantly by increasing HRT from 12 to 48 h (Kaparaju et al. 2010).

Fig. 3 Hythane production

pathway

Sludge

Simplifier fee dstock process

Anaerobic Biotechnology: Implementations and New Advances 173

3.3 Bio-hythane Production

Bio-hythane is deﬁned as a mixture of hydrogen and methane gases, which are

produced by a biological process in two and/or single-stage (see Fig. 4) and trade-

marked in 2010 by Eden. It is also has been utilized commercially in India and the

United States of America and several individual companies like Volvo and Fiat have

taken Hythane into account as well. Bio-hythane production has been proposed as

a promising approach for waste/wastewater treatment. Several studies reported that

bio-hythane production is required a short resistance time compared with conven-

tional anaerobic digestion (Krishnan et al. 2019). It also found that high biodegrada-

tion over 95% was achieved with high energy recovery efﬁciency and better process

control and resilience under two-stage treatment. The concentration of hydrogen

in the gas mixer ranged from 5 to 25% in the dark fermentation (Gadow and Li

2019, 2020a). Therefore, the H2/CH4 ratio can be easily regulated under the biolog-

ical process by microbial community structure design and improvement (Ramos

et al. 2019). Recently, there are many efforts to produce bio-hythane in a single-

stage process, thus making the treatment and bioconversion an attractive and suitable

solution for wastewater management (Gadow and Li 2019).

The pH value is a crucial factor for bio-hythane production. The pH value affects

Fe–Fe hydrogenase activity therefore, metabolic products signiﬁcantly change. The

optimum pH for hydrogen production (ﬁrst stage) ranges between 5.0 and 6.5,

however, the optimum pH for methane production (second stage) is around the neutral

condition. Therefore, the recirculation process in the two-stage bio-hythane produc-

tion provides efﬁcient performance in the overall COD degradation efﬁciency and

bioenergy recovery, however, it is still remaining unclear its effects on microbial

community structure in the anaerobic bioreactors.

Fig. 4 Bioenergy

optimization by anaerobic

dark fermentation

Sludge

Single-stage

process

Two- stage

process

Biogas

CH4 50-70% V/V

CO2 30-50% V/V

First stage

Second Stage

Biogas

H2 10-30% V/V

CO2 70-90% V/V

Biogas

CH4 50-70% V/V

CO2 30-50% V/V

H

2

CH

4

Bio-hythane

174 S. I. Gadow et al.

4 Challenge and Recent Advances in Anaerobic Technology

In fact, the pollutants in industrial wastewater become more complex over time,

due to the innovation and marketing in the various industry sectors (Yi et al. 2020).

This leads to higher levels of the emerging pollutants (Eps) (i.e., toxic organics and

engineering nanoparticles (ENPs)) in the ecosystem (Hao et al. 2020). The untreated

agricultural-based industries cause environmental pollution and harmful effect on

human and animal health and create different problems with climate change by

increasing the number of greenhouse gases. Egypt is an agricultural country and agro-

industrial waste products are abundantly available. Therefore, the agro-industrial

waste uses for wastewater treatment is attractive technology because of its good

adsorption potential due to the presence of carboxyl, hydroxyl, and amino groups

over their surfaces as well as their easy availability, low cost (Amor et al. 2019). The

conventional treatment systems are designed and constructed to reduce BOD, COD,

and nitrogen compounds but not for the EPs. They could be washed and released in

the wastewater (domestic or industrial) and able to kill the microorganisms and/or

change the microbial community structure in wastewater treatment plants (WWTP),

which could lead to a threat to drinking water safety (Li et al. 2016). Therefore, the

scientiﬁc communities need to explore innovative, cost-effective, energy-saving and

sustainable solutions to reduce the risks to acceptable levels in the treated discharge.

4.1 Rapid Start-Up Under Ambient Temperature

The recalcitrant, complex and toxic pollutants are the most important limiting factor

for energy optimization and biodegradation efﬁciency in the anaerobic degradation

process. Therefore, the hydrolysis step is a permanent concern in recent studies to

enable fast start-up under ambient temperature (Gadow and Li 2020a; Kong et al.

2019). Aside from the hydrolysis step, there are many parameters such as tempera-

ture, pH, microbial community design and reactor conﬁguration, which would affect

the bio-reaction speed and the end product characterization. Under two-stage tech-

nology, acidogenesis and methanogenesis occur in two separate reactors and each

stage required different environmental conditions. Under the acidogenic phase, the

ﬁrst stage, the recalcitrant, complex and toxic pollutants are hydrolyzed and converted

to simple compounds, which is slow-rate bioconversion. On the other hand, the

HPB is rapid growth compared with methanogenic bacteria which means the bio-

system conﬁguration is very important to improve the energy recovery efﬁciency by

hydraulic retention time (HRT) optimization (Gadow and Li 2020b). In recent times,

different reactor conﬁgurations and operational conditions were applied to validate

the wastewater treatment level and bioenergy recovery improvements. Gadow et al.

(2013a, b) reported that the temperature affects not only the rate of biogas production

but also the microbial community structure (Gadow et al. 2013b).

Anaerobic Biotechnology: Implementations and New Advances 175

4.2 Inhibitors

It is essential to obtain high performance and deal with inhibitory factors for sustain-

able energy production (Hou and Ji 2018) under dark fermentation technology. It

has been proposed that resilience plays an important role in maintaining the process

(Gadow and Li 2020b). Microbial acclimation plays an important role in the sustain-

ability of the process. Strategies to achieve and maintain optimal microbial communi-

ties by acclimation in hydrogen and methane fermentation can enhance its economic

viability and implementation (Zhao et al. 2020; Soares et al. 2020). A variety of

different inhibitors have been reported in the literature for a single or two-stage

process such as toxic pollutants, temperature, pH and metal ions (Hou and Ji 2018;

Habets and Boerstraat 1999). Consequently, only when the composition of wastew-

ater and operational parameters is well-known can the stable activity of the anaerobic

reaction be assured. For example, under acidogenesis, the production of hydrogen

coupled with butyrate and ethanol means the degradation process was not complete

and we still need to remove such unfavorable by-products economically. As for

methane production, two mechanisms for methane formation from acetate have been

reported. Aceticlastic is the ﬁrst one, which is produced by Methanosarcinaceae. The

second reaction involves a two-stage reaction in which acetate is initially oxidized

into H2 and CO2 and then converted into methane with those products (Shah 2020).

4.3 Internal Circulation Reactor

UASB reactor is being used to handle the extremely high loads of COD which needs

high retention time using Aerobic reactor, by mean of keeping a high level of TSS

(100,000 ppm or higher), Keeping this blanket in the bottom of the reactor is the

key of the successful treatment, this can’t be achieved with Up-ﬂow high velocity

(maximum velocity 1 m/s), as a result of this UASB reactor needs high area footprint

and large retention time 24 h. UASB also suffers from bad mixing due to low velocity

inside the reactor, also the poor separation of gas in the bottom part causing the extra

needed volume to avoid solid drift up. The disadvantages of UASB are reclaimed

and minimized in the smart idea of IC UASB reactor, IC tends to Internal circulation

(Shah 2021).

Reactor Process Description

Feed Water ﬂows from the bottom of the reactor in static ﬁns causes cyclone pattern

ﬂow the high velocity in the centre of cyclone eye of cyclone leads to slight vacuum

this make the suction needed for internal circulation, and achieve very good missing

also the rotary motion enhance the granulation of the Sludge blanket components

(Fig. 5). Water ﬂows up to the top of the reactor with relatively high velocity, and

to avoid drifting the biomass up to the reactor top losing its effect, the smart design

takes into consideration 3 phase separator in the 1st bottom 1/3rd part of the reactor

176 S. I. Gadow et al.

Fig. 5 Internal Circulation

Up-Flow Anaerobic Sludge

Blanket (IC-UASB)

and the 2/3rd up part of the reactor, both 3 phase separators helps the achieving

of high up-ﬂow velocity, better mixing, better gas separation and lower retention

time/Footprint. Cycles of gas removal are being increased and this improves the

reaction forward speed by getting red frequently of all biogas generated inside the

reactor. This very efﬁcient ﬂow pattern doesn’t need any type of mixers or moving

parts, energy-saving design, near to Plug ﬂow reactor.

5 Conclusion

Anaerobic technologies have grown in relevance in the ﬁeld of environmental tech-

nology because they are the major mechanism controlling the result of anaerobic

microbial metabolism. Energy recovery during waste/wastewater treatment is partic-

ularly important because of its effectiveness in comparison with other techniques, on

minimizing treatment costs and reduction of sludge. A signiﬁcant amount of renew-

able energy such as biogas, chemical, liquid fuel, heat, and electricity generation

can be recovered from sludge management. The general advantage of the anaerobic

Anaerobic Biotechnology: Implementations and New Advances 177

digestion process is that it can be applied for low-strength wastewaters and high-

strength organic solid waste to the proper reactor conﬁguration. Moreover, it can

accommodate high COD loads, which adapt to remove various toxicant components

provided that adaptation time is allowed for the anaerobic biomass. Generally, up to

95% of organic material will become biogas as a renewable resource by the properly

run digester. However, the complex composition of organic pollutants remains its

main limitation due to the hydrolysis step which affects the safety of treated water,

increases energy input and increases the cost of treatment. All the technologies

discussed in this review demonstrate the need for further research and development

in the operation condition and energy recovery optimization while reducing cost and

emissions. An interesting observation is the lack of data to integrate biological and

thermochemical technologies to improve energy recovery. This is easily conceived as

an integrated approach to bio-reﬁnery that can be appropriately designed to maximize

energy recovery to reduce harmful environmental impacts. Regardless, all of these

processes must be tailored to suit speciﬁc cases and require an in-depth technical

assessment to determine their sustainability in a low-carbon future.

References

Abdullah N, Yusof N, Lau W, Jaafar J, Ismail A (2019) Recent trends of heavy metal removal from

water/wastewater by membrane technologies, 76:17–38

Ahamed MI, Asiri AM, Lichtfouse E (2019) Nanophotocatalysis and environmental applications:

energy conversion and chemical transformations, vol 31. Springer

Ali SS, Al-Tohamy R, Manni A, Luz FC, Elsamahy T, Sun J (2019) Enhanced digestion of

bio-pretreated sawdust using a novel bacterial consortium: microbial community structure and

methane-producing pathways. Fuel 254:115604

Amor C, Marchão L, Lucas MS, Peres JA (2019) Application of advanced oxidation processes for

the treatment of recalcitrant agro-industrial wastewater: a review. Water 11(2):205

Balachandar G, Varanasi JL, Singh V, Singh H, Das D (2020) Biological hydrogen production

via dark fermentation: a holistic approach from lab-scale to pilot-scale. Int J Hydrogen Energy

45(8):5202–5215

Bello MM, Raman AA, Asghar A (2019) A review on approaches for addressing the limitations of

Fenton oxidation for recalcitrant wastewater treatment. Process Saf Environ Prot 126:119–140

Bui X-T, Chiemchaisri C, Fujioka T, Varjani S (2019) Water and wastewater treatment technologies.

Springer

Cheremisinoff NP (2001) Handbook of water and wastewater treatment technologies. Butterworth-

Heinemann

Chin HL, Chen ZS, Chou CP (2003) Fedbatch operation using Clostridium acetobutylicum suspen-

sion culture as biocatalyst for enhancing hydrogen production. Biotechnol Prog 19(2):383–388

Collet C, Adler N, Schwitzguébel J-P, Péringer P (2004) Hydrogen production by Clostridium

thermolacticum during continuous fermentation of lactose. Int J Hydrogen Energy 29(14):1479–

1485

Deublein D, Steinhauser A (2011) Biogas from waste and renewable resources: an introduction.

Wiley

Farooq R, Ahmad Z (2017) Physico-chemical wastewater treatment and resource recovery. BoD–

Books on Demanz

178 S. I. Gadow et al.

Foteinis S, Borthwick AG, Frontistis Z, Mantzavinos D, Chatzisymeon E (2018) Environmental

sustainability of light-driven processes for wastewater treatment applications. J Clean Prod 182:8–

15

Gadow SI, Li Y-Y (2020a) Efﬁcient treatment of recalcitrant textile wastewater using two-phase

mesophilic anaerobic process: bio-hythane production and decolorization improvements. J Mater

Cycles Waste Manage 22(2):515–523

Gadow S, Li Y-Y, Liu Y (2012) Effect of temperature on continuous hydrogen production of

cellulose. Int J Hydrogen Energy 37(20):15465–15472

Gadow SI, Jiang H, Hojo T, Li Y-Y (2013a) Cellulosic hydrogen production and microbial

community characterization in hyper-thermophilic continuous bioreactor. Int J Hydrogen Energy

38(18):7259–7267

Gadow SI, Jiang H, Watanabe R, Li Y-Y (2013b) Effect of temperature and temperature shock on

the stability of continuous cellulosic-hydrogen fermentation. Biores Technol 142:304–311

Gadow SI, Jiang H, Li Y-Y (2016) Characterization and potential of three temperature ranges for

hydrogen fermentation of cellulose by means of activity test and 16s rRNA sequence analysis.

Biores Technol 209:80–89

Gadow SI, El-Shawadfy M, Abd El Zaher FH (2019a) Optimized operational parameters of anaer-

obic cellulosic-wastewater treatment for bioenergy recovery and efﬂuent quality improvements.

Curr Sci Int 8(4):789–801

Gadow SI, Ahsan HM, Li Y-Y (2019b) Continuous detoxiﬁcation of carcinogenic aromatic amines

by activated sludge treatment. Int J Environ 8(3):162–170

Gadow SI, Li Y-Y (2019) Optimization of energy recovery from cellulosic wastewater using

mesophilic single-stage bioreactor. Waste Biomass Valorizat, 1–7

Gadow SI, Li Y-Y (2020b) Development of an integrated anaerobic/aerobic bioreactor for biodegra-

dation of recalcitrant azo dye and bioenergy recovery: HRT effects and functional resilience.

Bioresour Technol Rep 100388

Habets LH, de Boerstraat T (1999). Introduction of the IC reactor in the paper industry. Technical

Report, PaquesBV, Netherlands, p 7

Hao L, Zhou X, Liu J (2020) Release of ZrO2 nanoparticles from ZrO2/Polymer nanocomposite in

wastewater treatment processes. J Environ Sci 91:85–91

Hernando M, Rodríguez A, Vaquero J, Fernández-Alba A, García E (2011) Environmental risk

assessment of emerging pollutants in water: approaches under horizontal and vertical EU

legislation. Crit Rev Environ Sci Technol 41(7):699–731

Ho Y-C, Chua S-C, Chong F-K (2020) Coagulation-ﬂocculation technology in water and wastewater

treatment. In: Handbook of research on resource management for pollution and waste treatment.

IGI Global, pp 432–457

Hou L, Ji D, Zang L (2018) Inhibition of anaerobic biological treatment: a review. In: IOP conference

series: earth and environmental science, vol 1. IOP Publishing, p 012006

Hsien C, Low JSC, Chung SY, Tan DZL (2019) Quality-based water and wastewater classiﬁcation

for waste-to-resource matching. Resour Conserv Recycl 151:104477

Hussy I, Hawkes F, Dinsdale R, Hawkes D (2003) Continuous fermentative hydrogen production

from a wheat starch co-product by mixed microﬂora. Biotechnol Bioeng 84(6):619–626

Hwang MH, Jang NJ, Hyun SH, Kim IS (2004) Anaerobic bio-hydrogen production from ethanol

fermentation: the role of pH. J Biotechnol 111(3):297–309

Jiang H, Qin Y, Gadow S, Ohnishi A, Fujimoto N, Li Y-Y (2018) Bio-hythane production from

cassava residue by two-stage fermentative process with recirculation. Biores Technol 247:769–

775

Jiang H, Qin Y, Gadow S, Li Y-Y (2017) The performance and kinetic characterization of the three

metabolic reactions in the thermophilic hydrogen and acidic fermentation of cassava residue. Int

J Hydrogen Energy 42(5):2868–2877

Kamyab S, Ataei SA, Tabatabaee M, Mirhosseinei SA (2019) Optimization of bio-hydrogen produc-

tion in dark fermentation using activated sludge and date syrup as inexpensive substrate. Int J

Green Energy 16(10):763–769

Anaerobic Biotechnology: Implementations and New Advances 179

Kaparaju P, Serrano M, Angelidaki I (2010) Optimization of biogas production from wheat straw

stillage in UASB reactor. Appl Energy 87(12):3779–3783

Khan M, Ngo HH, Guo W, Liu Y, Nghiem LD, Hai FI, Deng L, Wang J, Wu Y (2016) Optimization of

process parameters for production of volatile fatty acid, biohydrogen and methane from anaerobic

digestion. Biores Technol 219:738–748

Kim S-H, Han S-K, Shin H-S (2006) Effect of substrate concentration on hydrogen production

and 16S rDNA-based analysis of the microbial community in a continuous fermenter. Process

Biochem 41(1):199–207

Kong Z, Li L, Li Y-Y (2019) Long-term performance of UASB in treating N, N-dimethylformamide-

containing wastewater with a rapid start-up by inoculating mixed sludge. Sci Total Environ

648:1141–1150

Krishnan S, Din MFM, Taib SM, Ling YE, Puteh H, Mishra P, Nasrullah M, Sakinah M, Wahid

ZA, Rana S (2019) Process constraints in sustainable bio-hythane production from wastewater.

Bioresource Technology Reports 5:359–363

Li L, Zhou Q, Geng F, Wang Y, Jiang G (2016) Formation of nanosilver from silver sulﬁde

nanoparticles in natural waters by photoinduced Fe (II, III) redox cycling. Environ Sci Technol

50(24):13342–13350

Li Y-Y, Gadow S, Niu Q (2015) Biomass energy using methane and hydrogen from waste materials.

In: Topical themes in energy and resources. Springer, pp 131–157

Lim J, Z hou Y, Vadivelu V (2020) Enhanced volatile fatty acid production and microbial population

analysis in anaerobic treatment of high strength wastewater. J Water Process Eng 33:101058

Luo J, Zhang Q, Zhao J, Wu Y, Wu L, Li H, Tang M, Sun Y, Wen G, Feng Q (2019) Potential

inﬂuences of exogenous pollutants occurred in waste activated sludge on anaerobic digestion: a

review. J Hazard Mater 121176

Meky N, Ibrahim MG, Fujii M, Elreedy A (2020) Integrated dark-photo fermentative hydrogen

production from synthetic gelatinaceous wastewater via cost-effective hybrid reactor at ambient

temperature. Energy Convers Manage 203:112250

Miralles-Wilhelm F, Hejazi M, Kim S, Yonkofski C, Watson D, Kyle P, Liu Y, Vernon C, Delgado

A, Edmonds J (2018) Water for food and energy security: an assessment of the impacts of water

scarcity on agricultural production and electricity generation in the middle east and North Africa.

Wor ld B ank

Patel N, Khan M, Shahane S, Rai D, Chauhan D, Kant C, Chaudhary V (2020) Emerging Pollutants

in aquatic environment: source, effect, and challenges in biomonitoring and bioremediation—a

review. Pollution 6(1):99–113

Ramos LR, de Menezes CA, Soares LA, Sakamoto IK, Varesche MBA, Silva EL (2019) Controlling

methane and hydrogen production from cheese whey in an EGSB reactor by changing the HRT.

Bioprocess Biosyst Eng 1–12

Ren N, Gong M (2006) Acclimation strategy of a biohydrogen producing population in a continuous-

ﬂow reactor with carbohydrate fermentation. Eng Life Sci 6(4):403–409

Sawers R (2005) Formate and its role in hydrogen production in Escherichia coli. Portland Press

Ltd.

Shah MP (2020) Microbial bioremediation & biodegradation. Springer

Shah MP (2021) Removal of refractory pollutants from wastewater treatment plants. CRC Press

Shareef N (2020) Thermal sewage sludge disposal in stationary ﬂuidized bed combustion DN 400 by

using fuel BRAM (Fuel from Solid Waste). In: Waste management in MENA regions. Springer,

pp 259–279

Shi J, Han Y, Xu C, Han H (2019) Anaerobic bioaugmentation hydrolysis of selected nitrogen

heterocyclic compound in coal gasiﬁcation wastewater. Biores Technol 278:223–230

Shukla N, Sahoo D, Remya N (2019) Biochar from microwave pyrolysis of rice husk for tertiary

wastewater treatment and soil nourishment. J Clean Prod 235:1073–1079

Soares JF, Confortin TC, Todero I, Mayer FD, Mazutti MA (2020) Dark fermentative biohydrogen

production from lignocellulosic biomass: Technological challenges and future prospects. Renew

Sustain Energy Rev 117:109484

180 S. I. Gadow et al.

Stams AJ (1994) Metabolic interactions between anaerobic bacteria in methanogenic environments.

Antonie Van Leeuwenhoek 66(1–3):271–294

Stazi V, Tomei MC (2018) Enhancing anaerobic treatment of domestic wastewater: state of the art,

innovative technologies and future perspectives. Sci Total Environ 635:78–91

Thanos D, Maragkaki A, Venieri D, Fountoulakis M, Manios T (2020) Enhanced biogas production

in pilot digesters treating a mixture of olive mill wastewater and agro-industrial or agro-livestock

by-products in Greece. Waste Biomass Valorizat 1–9

Thauer RK, Jungermann K, Decker K (1977) Energy conservation in chemotrophic anaerobic

bacteria. Bacteriol Rev 41(1):100

Xiong W, Wang L, Zhou N, Fan A, Wang S, Su H (2020) High-strength anaerobic digestion

wastewater treatment by aerobic granular sludge in a step-by-step strategy. J Environ Manage

262:110245

Yi H, Li M, Huo X, Zeng G, Lai C, Huang D, An Z , Qin L, Liu X, Li B (2020) Recent development

of advanced biotechnology for wastewater treatment. Crit Rev Biotechnol 40(1):99–118

Z˘abav˘a B, Gh V, Ungureanu N, Dinc˘aM,FerdesM,Vl˘adut

, V (2019) Advanced technologies for

wastewater treatment by ozonation—a review. Ann Fac Eng Hunedoara-Int J Eng 17(3)

Zhang H, Bruns MA, Logan BE (2006) Biological hydrogen production by Clostridium aceto-

butylicum in an unsaturated ﬂow reactor. Water Res 40(4):728–734

Zhang B, Shan C, Hao Z, Liu J, Wu B, Pan B (2019) Transformation of dissolved organic matter

during full-scale treatment of integrated chemical wastewater: molecular composition correlated

with spectral indexes and acute toxicity. Water Res 157:472–482

Zhao W, Su X, Xia D, L i D, Guo H (2020) Contribution of microbial acclimation to lignite

biomethanization. Energy Fuels 34(3):3223–3238

Remediation of Soil Contaminated

with Heavy Metals by Immobilization

with Organic and Inorganic Amendments

Izabela Michalak and Jolanta Warchoł

Abstract Heavy metals and metalloids are hazardous chemicals that very difﬁcult

undergo microbial or chemical degradation. Their presence in natural environment

results mainly from anthropogenic sources, such as agriculture, oil and gas produc-

tion, mining industry, and military activities. Soil pollution with heavy metals is

recognized as “hot spots” posing a risk to the environment, agricultural production,

food safety, and human health. Various technologies have been developed to reduce

the potential for the release of metal ions into the environment and to scale down

changes in the land use pattern. In situ remediation of contaminated soils by supple-

menting amendments is considered as a sound alternative both environmentally and

economically. This method provides a long-term, relatively cheap remediation solu-

tion by reducing metal mobility and availability to plants. As steams from literature,

amendments’ application can improve soil biological, chemical and physical prop-

erties and consequently enhance the plant growth. The present chapter presents the

current trends (from the last decade) in the remediation of soil contaminated with

heavy metal ions by their immobilization with various by-products and low-cost

materials. The focus was put on the factors which determine the metal binding and

transformation into more stable forms. An assessment of the effectiveness of these

amendments on the soil properties and the phytoavailability to plants has been made

as well.

Keywords Heavy metals ·Contaminated soil ·(Bio)remediation techniques ·

Immobilization ·Organic and inorganic amendments

I. Michalak (B

) · J. Warchoł

Faculty of Chemistry, Department of Advanced Material Technologies, Wrocław University of

Science and Technology, Wrocław, Poland

e-mail: izabela.michalak@pwr.edu.pl

J. Warchoł

e-mail: jolanta.warchol@pwr.edu.pl

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_10

181

182 I. Michalak and J. Warchoł

1 Introduction

Heavy metals are well known toxic chemicals. Their presence in soil, resulting mainly

from the anthropogenic activities, poses a threat to the natural environment as a whole,

as well as to the production of healthy food and consequently to human health.

Agriculture, oil and gas production, burning of fossil fuels, and mining industry are

the main sources of soil pollution with heavy metals (Kamari et al. 2015; Rodríguez-

Eugenio et al. 2018; Hannan et al. 2021a; Irfan et al. 2021;Lietal. 2021; Quoc et al.

2021; Shao et al. 2021). Heavy metal contaminated soil can be a serious barrier to the

cultivation of many plant species. On the other hand, vegetation grown in polluted

areas may pose a risk to humans/animals due to bioaccumulation/biomagniﬁcation

of toxic metals in the plant biomass that may become a component of the human diet

or animal feed (Radziemska et al. 2022).

Soil washing (extraction), phytoremediation (accumulation by plants) and chem-

ical immobilization (solidiﬁcation/stabilization, vitriﬁcation, chemical treatment) are

available technologies used for cleaning up of heavy metal contaminated sites. These

conventional remediation technologies have encountered numerous obstacles, such

as long operation time, high chemicals cost, large energy consumption, secondary

pollution, soil degradation by the excessive soil nutrient loss, etc. (Hamid et al.

2018; Shah 2020; Zhou et al. 2020; Liu et al. 2021; Pei et al. 2021; Quoc et al.

2021). Therefore, low-cost and ecologically sustainable in situ remedial options are

desired to reduce the risk associated with heavy metals, to make the land resource

available for agricultural production, and to enhance food security (Mahmoud and

Abd El-Kader 2015; Shah 2021;Lwinetal. 2018; Liu et al. 2021; Shao et al. 2021).

One approach used in the remediation of contaminated soil is the application of

organic and/or inorganic soil amendments for immobilization of heavy metal ions.

These additives decrease the mobility and bioavailability of heavy metals ions to

plants (Hu et al. 2014;Lwinetal.

2018; Shan et al. 2020; Liu et al. 2021). Their use

is not aimed at eliminating heavy metals from the soil, but at lowering their activity

and blocking their entry into the food chain (Hu et al. 2014; Mahmoud and Abd

El-Kader 2015; Hamid et al. 2018; Hannan et al. 2021a). Low input, high efﬁciency

and less land disturbance are the main advantages of this cheap and environment

friendly method (Hu et al. 2014).

Certain waste biomass from forest industry (e.g., bark and wood chips, bark saw

dust), agricultural operations (e.g., straw, animal manure), kitchen waste, sewage

sludge from wastewater treatment, humic acid as well as produced from this waste

biomass compost/vermicompost or biochar can be used as inexpensive sorbents

for immobilization of heavy metal ions in the soil (Lwin et al. 2018;Xuetal.

2020; Zhou et al. 2020). These materials are available in large quantities and their

large-scale applications would ﬁt the worldwide need for circular economy. Besides

organic amendments, inorganic binders, such as natural minerals (e.g., sepiolite,

zeolite, kaolinite, bentonite, etc.), liming materials (e.g., calcium oxide, calcium

carbonate, etc.), gypsum, cement, nano-scale materials (e.g., nano-Fe, ZnO, MgO,

Remediation of Soil Contaminated with Heavy Metals … 183

Fig. 1 Soil amendments: (a) organic and (b) inorganic used in the immobilization of heavy metals

in polluted soil (on the basis of Web of Science database, access on 28 February 2022)

etc.), phosphorus-containing materials (e.g., apatite, calcium phosphate, diammo-

nium phosphate, etc.), industrial by-products (ﬂy ash, red mud, slags etc.) can also

serve as amendments in the remediation of polluted soil (Lwin et al. 2018; Zhou

et al. 2020; Pei et al. 2021). Figure 1 presents the most often used organic and

inorganic additives in the immobilization of heavy metals in polluted soil. The data

comes from the Web of Science database—in the abstracts of the publications, the

following phrases were found: “immobilization of heavy metals in soil” and “a given

soil amendment”, for example “biochar”. Nonetheless, as pointed out in some publi-

cations, amendments can negatively affect soil properties and microorganisms (Quoc

et al. 2021). Therefore, the choice of amendment should be made taking into account

the particular type of element(s), soil composition and its properties (Palansooriya

et al. 2020). Properly selected soil additive not only stabilizes heavy metals in soil,

but also reduces their uptake by plants (roots, shoots, leaves, grain) and promotes

plant growth in the contaminated soil (Wang et al. 2020).

The aim of the present chapter was to present the current trends in the remediation

of soil contaminated with heavy metal ions by immobilization with organic and inor-

ganic sorption materials. This chapter was prepared based on the recent publications

(2012–2022) from the Web of Science, Scopus, PubMed, ScienceDirect, and Google

Scholar databases.

2 Sources of Heavy Metal Ions in Soil

Soils are the major sink for heavy metals released into the environment from a

wide variety of anthropogenic sources such as: metal mine tailings, disposal of high

metal wastes, leaded gasoline and lead based paints, application of fertilizer, animal

manures, biosolids (sewage sludge), compost, pesticides, coal combustion residues,

petrochemicals, and atmospheric deposition (Dhaliwal et al. 2020). So far, there has

been no sufﬁcient data to provide an overview of the global distribution of heavy

metals in soil. In the European Union the most comprehensive s ource of information

is FOREGS data (produced by the EuroGeoSurvey), which provides continuous map

184 I. Michalak and J. Warchoł

sheet based on sampling density 1 site/5000 km2. More reliable view was provided

by Tóth et al. (2016), who analyzed the metal content in a soil sample with a density

1 site/200 km2. The analysis of heavy metals distribution by using the LUCAS

Topsoil Survey, reveals that 137,000 km2 of agricultural land in the European Union

is affected to a certain degree. About 2.6% of the samples from agricultural land

contain heavy metals in concentration qualifying for soil remediation.

Soil itself has the ability to immobilize heavy metals, which depends primarily

on the soil composition, grain size distribution, and pH. In general, acidic environ-

ment enhances heavy metals solubility and hence their mobility. Fine grained soils,

with large quantities of organic matter, silt, and clay, have higher capacity to retain

heavy metals than coarse soils, which favor the inﬁltration of water and spread of

dissolved metal ions. The inorganic colloidal fraction of soil is comprised of clay

minerals, oxides, sesquioxides and hydrous oxides. All mentioned factors inﬂuence

both the presence of certain forms of heavy metals in the water-soil environment and

immobilization properties of soil.

3 Immobilization of Heavy Metal Ions in Polluted Soil

The immobilization process of heavy metals with the use of waste biomass (metabol-

ically inactive) or other inorganic materials remains unaffected by toxicity, does not

require any growth/nutritional medium and is ﬂexible to environmental conditions

(Lwin et al. 2018). In turn, employment of living biomass for bioremediation may

not be a viable option owing to highly toxic metals which can accumulate in cells and

interrupt metabolic activities resulting in cell death (Dzionek et al. 2016). Conversion

of contaminants from their original form into a more physically and chemically stable

form due to soil amendments action, reduction in heavy metal mobility, solubility,

bioavailability, and toxicity in soil is regarded as an environmentally friendly and

economically viable remediation strategy (Kamari et al. 2015; Hannan et al. 2021a;

Malik et al. 2021; Pei et al. 2021; Quoc et al. 2021).

The effectiveness of the immobilization process depends on many parameters

such as soil pH, the content of organic matter, bulk density, cation exchange capacity

(CEC), heavy metal form, and parent material of the amendment (Hannan et al.

2021a; Malik et al. 2021; Shao et al. 2021). Because there is a direct correlation

between soil physicochemical properties and toxic metals mobility and availability

to plants, soil characteristics provides guidance for selection of the most efﬁcient

amendment (Palansooriya et al. 2020).

The immobilization process results in changing metal ions speciation from

initially highly bioavailable forms (i.e. free metals) to the much less bioavailable frac-

tions associated with organic matter, metal oxides, or carbonates (Mahmoud and Abd

El-Kader 2015; Pei et al. 2021). More speciﬁcally, toxic metal ions are taken up by the

dead waste biomass/inorganic sorbents through sorption—biosorption/adsorption or

complexation or precipitation, which results in their lower availability for plants and

lower content in cultivated crops (Lwin et al. 2018; Wang et al. 2020; Pei et al. 2021).

Remediation of Soil Contaminated with Heavy Metals … 185

Organic amendments in the form of waste biomass with a high content of organic

matter can decrease the mobility of some heavy metals due to the formation of stable

chelates (Kamari et al. 2015). This method effectively reduces the ecological risk

associated with the movement of toxic metals in soil. Simultaneously, the application

of biomass residue or products derived from it (e.g., biochar, compost) as an organic

soil amendment can improve the biological, chemical and physical properties of the

soil. An increase in the content of soil organic matter and nutrients such as N, P and

K, increases the cation exchange capacity, improves soil structure and soil microbial

and enzyme activities and ﬁnally enhances plant growth (Hu et al. 2014;Lwinetal.

2018; Hannan et al. 2021a; Irfan et al. 2021; Liu et al. 2021; Malik et al. 2021;

Pei et al. 2021). Due to improvement in soil nutrient, water retention capacity, and

porosity, soil additives establish a suitable environment for soil microbiota. Conse-

quently, immobilization of heavy metals in soil reduces their stress on microbiota

and increases enzymatic activities, which promote plant growth (Liu et al. 2021).

Chemical ﬁxation of heavy metal ions on inorganic sorption materials causes stabi-

lization rather than decontamination, which relates to transformation of metal into

an inactive form. It is claimed that there is no single mechanism responsible for

the immobilization of metal ions. Most likely, several processes are taking place

simultaneously, among which precipitation, electrostatic interaction, surface adsorp-

tion, structural sequestration and complexation are the most often mentioned. Their

share relates directly to sorption material properties as well as to the process’ condi-

tions, and can change over time inﬂuencing the degree of reduction in the bioavail-

ability of metal ions. Common features of inorganic sorption materials are porosity

and rough morphological surface with honeycomb-like anatomical or other irreg-

ular structures. The primarily properties, which characterize the material sorption

abilities are cation exchange capacity (CEC) related to the quality and quantity of

surface functional groups, and the size of sorption surface area (SSA) directly related

to material’s porosity (Palansooriya et al. 2020).

Despite many advantages, (bio)remediation of contaminated soil with

organic/inorganic amendments has some disadvantages. In case of in situ immo-

bilization, heavy metals are not removed from soil. They remain in the environment

as a potential future pollutant (Hannan et al. 2021a). It is also hypothesized that due

to degradation of organic amendments, under changing environmental conditions,

previously bound metals may be released and become bioavailable again over time

(Kabata-Pendias 2011;Huetal.

2014; Kamari et al. 2015; Palansooriya et al. 2020).

A special attention should also be paid to the chemical composition of amendments

themselves. Some of them, especially produced from waste biomass (e.g., sewage

sludge and animal manure) may contain toxic elements such as Zn, Cu, Cr, Ni, Pb,

Cd, and Hg, which may cause secondary soil contamination (Ayaz et al. 2021;Li

et al. 2021).

In the vast majority of cases, the efﬁcacy of soil amendments to immobilize

heavy metal ions is evaluated in pot experiments (Table 1). Therefore, there is a

need to perform detailed in situ experiments to check the degree of degradation of

organic amendments in soil over time, accumulation of heavy metals in cultivated

186 I. Michalak and J. Warchoł

plants, as well as in the (bio)remediated soil. Long-term ﬁeld experiments are needed

to assess the safety, biological toxicity and stability of these amendments used in

immobilization of heavy metals in soil.

3.1 Materials Used in Heavy Metal Ions Immobilization

in Soil

Table 1 presents the examples of (a) inorganic, (b) organic, as well as (c) comparison

of inorganic versus organic and (d) mixed (inorganic and organic) amendments

used in the immobilization of heavy metal ions in polluted soil and their effect on

accumulation of heavy metal ions in plants and crop growth. Most of the remediation

experiments, performed in the last decade, were carried out in pots with the use of

maize, wheat, grass, rapeseed, rice or spinach as model plants. For the immobiliza-

tion of heavy metal ions, naturally contaminated soil (multi-metal-contaminated soil

collected from polluted areas) or soil artiﬁcially polluted in the laboratory (usually

contaminated with a single heavy metal) were used. Most of the research concerned

the veriﬁcation of soil amendments effectiveness in the immobilization of cadmium

and lead. Soil amendments used in the heavy metals immobilization can be applied

at several rates (low, medium, and high) ranging usually from 0.5 to 5% (Zhou et al.

2020).

There are two main parameters used to evaluate an individual plant’s ability to

accumulate and translocate heavy metal ions. The bioconcentration factor (BCF)

refers to the ratio of metal content in plant (stem and leaf tissues) to metal content

in soil. The transfer/translocation factor (TF) is the ratio of the heavy metal content

in plant (stem and leaf tissues) to that in the root (Cakmak and Marschner 1992;

Kabata-Pendias 2011; Kamari et al. 2015; Wang et al. 2020).

Inorganic Sorbents

Clay minerals are ubiquitous phyllosilicate minerals found principally in soils,

marine sediments and argillaceous shale rocks. Their formation is a result of a

hydrothermal action, sedimentation or weathering of aluminosilicate rocks. The

basic structural unit of clays comprises tetrahedral silicate sheet(s) connected

to an octahedral aluminum hydroxide sheet by weak electrostatic interactions.

Arrangement of the tetrahedrons in layers results in a hexagonal network, while

the octahedral layer in an octahedral conﬁguration. Both networks comprise strong

covalent bonds inside their sheet. The basis for clay minerals classiﬁcation is

the ratio of tetrahedral to octahedral sheets which can be 1:1 or 2:1. The main

representatives of clay minerals are: (1) kaolinite, represented by Al2Si2O5(OH)4,is

a main component of kaoline and other clays. Its 1:1 structure consists of a silicon-

centered tetrahedral sheet and an aluminum-centered octahedral sheet; (2) bentonite

Remediation of Soil Contaminated with Heavy Metals … 187

Ta bl e 1 The examples of (a) inorganic, (b) organic, (c) comparison of inorganic versus organic and (d) mixed (inorganic and organic) amendments used in the

immobilization of heavy metal ions in polluted soil and their effect on soil/plants

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

(a) Inorganic amendments

Pb, Cd, Zn Clay mineral—bentonite, Na form Field experiment Reduction in metal ions plant uptake

(30% of Pb, 46% of Cd, and 30% of Zn),

increase in soil pH

Vrînceanu

et al. (2019)

Pb—120 mg/kg,

Cu—230 mg/kg,

Ni—270 mg/kg,

Zn—340 mg/kg

Clay mineral—montmorillonite,

25 kg per cubic meter of soil

Percolation test/7 days Increase in Zn2+ and Ni2+ adsorption

but no effect on the retention of Pb2+

and Cu2+ when compared with the

adsorption by soil alone

Correia et al.

(2020)

Pb—700 mg/kg,

Cu—430 mg/kg,

Zn—1800 mg/kg

Clay mineral—vermiculite, 0.2 g per

1.8kgofsoil

Maize (Zea mays)/greenhouse pot

experiment/4 months

Decrease in Pb accumulation in leaves,

stalks, and roots by 27.6, 22.6, and

69.7%; Cu accumulation by 29.2, 15.3,

and 19.9%; Zn accumulation by 15,

14.6, and 13.2%, respectively

Parmar et al.

(2022)

Cd—7.4 mg/kg,

Cu—229 mg/kg,

Ni—140 mg/kg,

Zn—228 mg/kg

Zeolite—clinoptilolite from 1.25 to

10% w/w in a contaminated soil

Ryegrass (Lolium

multiﬂorum)/greenhouse pot

experiment/12 weeks

Addition of 2.5% w/w of natural zeolites

caused a signiﬁcant decrease in Cu and

Zn content in soil and in plant tissue; Cd,

Cu, Ni, Zn content in roots decreased

with 10% zeolite addition rate by 17, 21,

7, and 42% while in shoots by 37, 45,

93, and 56%, respectively

Contin et al.

(2019)

Cd—5 mg/kg Zeolite obtained by

geopolymerization of metakaoline

Batch study, soil mixed with

geopolymer-supported

zeolite/20 days

Cd immobilization efﬁciency up to

58.7%

Wu et al.

(2021)

(continued)

188 I. Michalak and J. Warchoł

Tabl e 1 (continued)

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

Pb—9876 mg/kg,

Cd—224 mg/kg,

Zn—654 mg/kg

Phosphate

compounds—(NH4)H2PO4

Indian mustard (Brassica

juncea)/pot experiment/6 weeks

Variation in the solubility of P

compounds and the nature of metal

phosphate compounds formed

inﬂuenced the differential effect of

P-induced immobilization; the addition

of amendment increased supply of P and

decreased bioavailability of heavy

metals by 56.7% for Cd, 78.9% for Pb,

and 39.2% for Zn

Seshadri et al.

(2017)

Cd—1.5 mg/kg,

Pb—100 mg/kg

Flyash /30gper 3kgofsoil Rapeseed/greenhouse pot

experiment/65 days

Reduction of Pb (14.7%) and Cd (3,6%)

in contaminated soil primarily through

increased soil pH; signiﬁcant reduction

of Pb and Cd in rapeseed plant tissue

(66.1% and 48%, respectively)

Shaheen and

Rinklebe

(2014)

Cd—0.89 mg/kg,

Pb—47.35 mg/kg

Ca(OH)2Field experiments, Ca(OH)2 was

applied into alluvial soil 15 days

before nursery of rice seeds were

transplanted

Application of lime up to 3600 kg/acre,

increased soil pH which induced

changes in hydroxide and carbonate

precipitation, and caused deprotonation

of adsorption sites at soil surface; 21%

improvement of the rice grains yield and

signiﬁcant reduction in metal content in

roots, shoots, and grains

Hamid et al.

(2019)

(continued)

Remediation of Soil Contaminated with Heavy Metals … 189

Tabl e 1 (continued)

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

(b) Organic amendments

Pb—800 mg/kg Biochar from wheat straw/20,

40 g/kg; chicken manure/20, 40 g/kg;

a combination—each at 20 g/kg

Maize (Zea mays)/pot

experiment/45 days for

measurement of antioxidant

enzyme activity of leaves, 80 days

for remaining measurements

For all amendments: (1) increase in

maize plant height, biomass weight,

activity of superoxide dismutase,

peroxidase and catalase, decrease in the

malondialdehyde content; (2) signiﬁcant

decrease in Pb content in maize (roots >

stems > leaves), bioconcentration factor,

translocation factor, available Pb in soil;

biochar alone: more effective at soil

alkalinization and Pb immobilization;

chicken manure alone: more effective at

maize growth increase and antioxidant

enzymatic activity; combination: most

signiﬁcant decrease in Pb in maize

tissues (roots—53.9%, stems—75.5%,

leaves—67.5%) and soil available Pb

Liu et al.

(2021)

Cd—0.38 mg/kg Biochar from corn stalk;

polyethyleneimine (PEI)-modiﬁed

biochar/2600, 5200, 13,000 kg/ha

Wheat/ﬁeld experiments Signiﬁcant difference in the Cd content

in different parts of wheat for the same

biochar treatment: root > stem and leaf >

glumes > grains—lower than in the

control group; with the increase in the

biochar dose, decrease in Cd content in

wheat—the best results for modiﬁed

biochar at 13,000 kg/ha; enhancement of

the catalase, urease, alkaline

phosphatase and sucrase activity in soil

Tang et al.

(2022)

(continued)

190 I. Michalak and J. Warchoł

Tabl e 1 (continued)

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

Pb—20 mg/kg,

Cd—10 mg/kg,

Cr—20 mg/kg

Biochar from maize straw;

compost/0.5, 1, 2, 4%

Maize (Zea mays)/pot

experiment/60 days

Decrease in heavy metals availability in

soil treated with biochar and compost;

biochar was more effective in

immobilization of heavy metals in soil as

compared to compost; for 4% biochar,

the highest decrease in shoot Pb

content—by 71%, Cd—by 63% and

Cr—by 78%, and then 50, 50 and 71%,

respectively for 4% compost when

compared to the control

Irfan et al.

(2021)

Cd—1 mg/kg Peanut shell biochar (BC); crop straw

(CS)/5% d.w

Peanut/pot experiment/4 months Better results (statistically signiﬁcant)

for BC than for CS—enhancement of the

peanut biomass and physiological

quality; greater impact on Cd

immobilization in soil; remarkable

decrease in Cd bioavailability

Chen et al.

(2020)

Cd—6.54 mg/kg and

Pb—1344 mg/kg

Kitchen waste biochar (KWB); corn

straw biochar (CSB), peanut hulls

biochar (PHB)/20, 40, 60 g/kg

Swamp cabbage (Ipomoea

aquatica)/pot experiment/30 days

Enhancement of soil pH, reduction in the

extractable Pb and Cd in soil by all

amendments; decrease in Cd and Pb

accumulation in roots, stems, and leaves

by 45.4–97.7% for KWB, 59.3–96.6%

for CSB, and 63.9%–99.3% for PHB;

immobilization performance order:

KWB > CSB > PHB

Xu et al.

(2020)

(continued)

Remediation of Soil Contaminated with Heavy Metals … 191

Tabl e 1 (continued)

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

Brown soil polluted with Cd Peanut shell biochar (HBC);

Mg-modiﬁed peanut shell biochar

(MHBC)/1, 2%

Spinach/pot experiment/ﬁve weeks Better amendment effect for MHBC than

HBC; decrease in bioavailable Cd in soil

by 26.2% to 50.1% for MHBC; higher

reduction in Cd content in roots and

shoots for MHBC than for HBC;

decrease in Cd accumulation in roots

and shoots with increase in biochar dose

Shan et al.

(2020)

Lead smelting

slag-contaminated soil

Pb—18 300 mg/kg

Compost (C) from wild sunﬂower and

poultry liter (3:1) for 12 weeks; rice

husk biochar (RHB); cashew nut shell

(CNSB) biochar; compost-modiﬁed

biochar (RHBC and CNSBC)/0.05,

0.1, 0.2, 0.4 g/g of soil

Maize/pot experiments/6 weeks Reduction in maize Pb uptake by all

amendments, but to different extent;

better performance of compost-modiﬁed

biochars than the amendments used

singly; the least Pb uptake by maize in

soil for compost-modiﬁed biochar

RHBC followed by CNSB, RHB, CNSB

and then C; for 0.1 g/g dose of

RHBC—decrease in root content by

91% and in shoot by 86%

Ogundiran

et al. (2015)

(continued)

192 I. Michalak and J. Warchoł

Tabl e 1 (continued)

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

(c) Comparison of inorganic vs. organic amendments

Ni—50 and 100 mg/kg Biochar from bamboo; mussel shell;

zeolite; limestone/3%

Rapeseed (Brassic napus)/pot

experiment/90 days

Reduction in Ni bioavailability after soil

addition of amendments; the best results

for biochar and shells—decrease in Ni

content by 30.4% and 25.1% in roots

and by 54.5% and 50.6% in shoots,

respectively for 50 mg/kg of Ni in soil;

for 100 mg/kg of Ni in soil, the highest

decrease in Ni content for shells and

biochar—by 41.2% and 38.0% in roots,

and 48.4% and 44.2% in shoots as

compared to the control

Hannan et al.

(2021a)

Multiple-metal-contaminated

soil:

Cd—2.6 mg/kg,

Pb—1796 mg/kg,

Zn—1603 mg/kg

Pine shoots biochar (BC); cow

dung-based manure (DM),

hydroxyapatite (HAP)/0.1, 1%

Maize (Zea mays)/pot

experiment/60 days

HAP (optimal one for stabilizing heavy

metals in soil): signiﬁcant decrease in

Cd, Pb, and Zn content in shoots and

roots (better effects for the higher

dose)—for 1% reduction of the shoot

Cd, Pb, and Zn content by 67%, 85% and

84%; DM: decrease in the shoot Cd and

Pb content and root Zn content (for 0.1

and 1%), but only for 1% DM decrease

in the shoot Zn and root Pb content; BC:

decrease in the shoot Cd and Pb content

(for 0.1 and 1%), but decrease in the

shoot Zn and root Pb content only for 1%

Wang et al.

(2020)

(continued)

Remediation of Soil Contaminated with Heavy Metals … 193

Tabl e 1 (continued)

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

Multiple-metal-contaminated

soil: Zn—2273 mg/kg,

Cu—372 mg/kg,

Pb—816 mg/kg

Coconut tree sawdust (CTS);

sugarcane bagasse (SB); eggshell

(ES)/1, 3%

Water spinach (Ipomoea

aquatica)/pot experiment/8 weeks

Decrease in shoot metal content with the

increase in amendment dose; signiﬁcant

reduction in metal uptake for 3% ES;

reduced bioavailability of metals—for

example Zn was reduced by 74.5% for

3% ES, 56.2% for 3% SB and 31.0% for

3% CTS

Kamari et al.

(2015)

Cs—100 mg/kg Coconut shell biochar (BC);

incinerated sewage sludge ash (ISSA);

zeolite/10%

Napier grass (Pennisetum

purpureum)/pot

experiment/1–7 months

For all amendments—enhancement of

the grass growth (the best for ISSA

treatment); decrease in Cs content in the

leaf blade, leaf sheath and root of grass

(the best for BC treatment—decrease up

to 95% as compared to the control

group—without soil amendment)

Shao et al.

(2021)

Cd—0.86 mg/kg Maize straw biochar (BC); zeolite

(ZE); humic acid (HA)/1, 2.5, 5%;

lime (L); sodium sulﬁde (SS);

superphosphate (SP)/0.5, 1, 2%

Wheat (Triticum aestivum)/pot

experiment

5% BC, 1% ZE and 0.5% L are

recommended for Cd immobilization in

acidic or neutral soils; better

immobilization effect for high doses

than for the low doses; signiﬁcant

decrease in Cd content in the roots

within the range of 23.7–27.2% for BC,

29.5–33.0% for ZE, 16.3–37.4% for HA,

and 48.2–65.5% for L and signiﬁcant

decrease in Cd content in the straw for

2.5 and 5% ZE and 5% HA as compared

to the control

Zhou et al.

(2020)

(continued)

194 I. Michalak and J. Warchoł

Tabl e 1 (continued)

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

(d) Mixed (inorganic and organic) amendments

Multiple-metal-contaminated

soil: Pb, Cd

Corn stover biochar (BC)/coal ash

incorporated biochar (CA/BC)/1, 3,

5%

Paddy rice (Oryza sativa cv.

Nipponbare)/pot

experiment/35 days

Signiﬁcant enhancement of

immobilization efﬁciency of CA/BC for

Pb –77% and 43% for Cd as compared

to BC; for 5% CA/BC reduction in the

content of Pb (by 81%) and Cd (by

62.5%) in rice as compared to control

and signiﬁcant promotion of rice

growth—by 3.1, 2.2 and 2.0 times in

terms of root, stem length and dry mass

as compared to the control

Xiaetal.

(2021)

Multiple-metal-contaminated

soil:

As—39.5 mg/kg,

Cu—33.6 mg/kg,

Zn—108 mg/kg

Bentonite (B); talc (T); activated

carbon (AC); corn starch (CS);

composites (BAC, BCS, TAC,

TCS)/2%

Lettuce/pot experiment/45 days Small content of As in shoots after

amending soil with B, AC, BAC, T and

TAC as compared to the control; no

effect of the treatments on As

accumulation in roots; reduction in Cu

uptake by roots and shoots for tested

amendments (signiﬁcant for AC);

decrease in the Zn content in plant

tissues after amending soil (in p articular

for BCS)

Quoc et al.

(2021)

(continued)

Remediation of Soil Contaminated with Heavy Metals … 195

Tabl e 1 (continued)

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

Multiple-metal-contaminated

soil: Cu—783 mg/kg,

Cd—14.5 mg/kg,

Zn—477 mg/kg

Coal ﬂy ash/5%; straw/2%; their

combination (homogeneous mixture

of the part of two treated soils)

Rape (Brassica napus)/pot

experiment/6 weeks

The highest stabilization efﬁciency for

the combined application of

amendments—reduction in extractable

Cu, Cd and Zn by 94.6, 67.1 and 73.3%,

respectively; signiﬁcant reduction in Cu,

Cd and Zn content in plant shoot for coal

ﬂy ash and the mixture of coal ﬂy ash

and straw

Hu et al.

(2014)

Multiple-metal-contaminated

soil:

Zn—667 mg/kg,

Ni—860 mg/kg,

Pb—586 mg/kg,

Cd –122 mg/kg

Phosphogypsum (PG) used alone and

in combination with rice straw

compost (CP) (1:1)/10, 20 g d.w./kg

of soil

Canola (Brassica napus) pot

experiment/10 weeks

PG alone or in combination with CP

immobilized heavy metal ions (better

effect for PG used alone); optimal dose

was 10 g/kg; mixture of PG and CP gave

the highest canola growth at 10 g/kg

(increase by 66.8% as compared with the

control group)

Mahmoud and

Abd El-Kader

(2015)

Cd—2.5 mg/kg,

Pb—300 mg/kg,

Zn—500 mg/kg

Willow biochar; zeolite and their

combination/0.5% (15 t/ha)

Grass species: tall fescue (Fes tu ca

arundinacea) and cocksfoot

(Dactylis glomerata)

Zeolite was a more effective soil

amendment than biochar in soil

remediation; higher biomass production

for zeolite alone or mixed with the

biochar than in control treatment; zeolite

applied alone signiﬁcantly affected root

biomass and root morphology; no effect

of biochar on most root morphometric

parameters

Gł˛ab et al.

(2021)

(continued)

196 I. Michalak and J. Warchoł

Tabl e 1 (continued)

Heavy metal/content in soil Soil amendment/dose Plant species/type of

experiment/duration

Effect of sorbent on soil/plant References

Contaminated paddy ﬁeld:

Cd—0.51 mg/kg, Pb

107 mg/kg

Lime, biochar, Fe-biochar, Ca-Mg-P

fertilizer, GSA-2 (a combination of

biochar, lime, sepiolite and zeolite),

GSA-3 (a combination of organic and

inorganic additives), and GSA-4 (a

combination of manure, lime and

sepiolite)/1%

Rice (Oryza sativa)/ﬁeld

experiment—mixing of

amendments thoroughly on the

upper surface of soil, mechanical

ploughing into 0–20 cm plow layer

15 days before transplanting

General decrease in Cd and Pb content

in roots, shoots, grains and husk after

amendments application as compared to

the control; more effectivity of a

combined amendment (GSA-4) for

immobilizing Cd and Pb in ﬁeld as

compared to other tested amendment;

the best effect on reducing Cd and Pb

phytoavailability in soil and uptake by

early rice for GSA-4

treatment—reduction in Cd and Pb

uptake in shoot (42% and 44%) and in

grains (77 and 88%, respectively);

improved rice growth (56%) and grains

yield (42%) also for GSA-4 as compared

to the control

Hamid et al.

(2018)

Aged Ni contaminated

soil—100 mg/kg

Mussel shell (MS); bamboo biochar

(BC); and their combination (BC +

MS, 3:1; 1:1; 1:3)/3%

Rapeseed (Brassica napus)/pot

experiment/90 days

The combination of MS and BC was

more effective in the Ni immobilization,

reduction of metal toxicity to plants and

the improvement of soil biology; the

lowest content of Ni in shoots and roots

for BC + MS (3:1) as compared to other

experimental groups

Hannan et al.

(2021b)

Remediation of Soil Contaminated with Heavy Metals … 197

is represented by a chemical formula (Na)0.7 (Al3.3Mg0.7)Si8O20(OH)4·nH2O and is

composed of montmorillonite, hectorite, saponite, beidellite and nontronite. Its 2:1

structure comprises of an aluminum-centered octahedral layer sandwiched between

two silicon-centered tetrahedral layers; (3) montmorillonite is represented as

(Na,Ca)0.3(Al,Mg)2Si4O10 (OH)2·nH2O. Its 2:1 structure comprises of an aluminum-

centered octahedral layer sandwiched between two silicon-centered tetrahedral

layers; (4) illite, represented as (K,H3O)Al2(Si3Al)O10 (H2O,OH)2, has a basic 2:1

structure—two tetrahedral silica layers surrounding an octahedral aluminium layer in

the center but with greater silica-to-alumina ratio compared to montmorillonite; (5)

vermiculite is represented by (Mg,Fe3,Al)3(Si,Al)4O10(OH)2·4H2O, which structure

is often referred to as 2:1 phyllosilicate. Two tetrahedral silicate layers are bonded

together by one octahedral magnesium hydroxide-like layer (Shaikh et al. 2017).

Layered plate-like structure provides clay minerals with plastic properties on

wetting as well as hardness on drying. The only exception is illite, which due to

the poor hydration properties of the interlayer K+ exchangeable cations, is a non-

swelling clay. The unique swelling properties provide signiﬁcant capacity to adsorb

water. Furthermore, the empty metal centers (no occupied by Al) in the octahedral

layers act as adsorption centers for metal i ons dissolved in water. Metal ions are

attracted to siloxane faces (Si–O–Si siloxane bridges with the O atom at the surface)

by small negative charge arise from isomorphous substitution of Si4+ by Al3+ in

surface of tetrahedral sheets. Metal ions can be also adsorbed on the crystal edges on

the places resulting from bearing primary bonds Al–O and Si–O. These are so-called

permanent active sites with a pH independent charge. In the contrary, the variable

active sites are inﬂuenced by pH. The change of pH causes protonation/deprotonation

of the silanol (≡Si–OH) and aluminol (=Al–OH) hydroxyl groups. The aluminol

sites are negatively charged at pH > 9, while silanol sites at pH < 9. Both sites

interact with positively charged metal cations via H-bonding. The resulted outer-

sphere complexes (X2Me) contain water or hydroxyl ligands between the metal center

and the mineral surface. Their formation is effected by ionic strength. Inner-sphere

surface complexation is related to direct covalent bonding of metal ions to the variable

active sites on the clay mineral surface. The effect of ionic strength on inner-sphere

complexes formation is negligible. The amount of variable sites decreases along with

the decreasing pH, and at about pH 5.5 adsorption occurs exclusively at permanently

charged sites. Metals that form hydrolysis products more readily (e.g., Pb, Cu) start

to adsorb onto variable active sites, while metals with lower tendency to hydrolysis

(e.g., Zn, Cd) are more eager to be adsorbed on permanent active sites. The amount of

variable and permanent sites available for a given metal ion adsorbed from multi metal

solution can be estimated from an appropriate equilibrium model (Matłok et al. 2015).

Zeolites are crystalline aluminosilicates with three-dimensional frameworks of

SiO4 and AlO4 tetrahedral units. The aluminum ion is small enough to occupy the

position in the center of the tetrahedron of four oxygen atoms, while the isomorphous

replacement of Si4+ by Al3+ produces a negative charge in the lattice. The negative

charge is balanced by the interchangeable positive ions such as Na+,K

+,Ca

2+, and

Mg2+. These cations are exchangeable with certain cations in solutions, such as heavy

metals.

198 I. Michalak and J. Warchoł

Clinoptilolite is the most common natural zeolite, which belongs to the heulandite

family. Its chemical formula is (Na,K,Ca)4Al6Si30O72·24H2O. The characteristic

tabular morphology shows an open reticular structure of easy access, formed by

open channels of 8- to 10-membered rings. There are two channels running parallel

to each other: 10-member (tetrahedron) ring of the size of 4.4–7.2 Å and 8-member

ring with the size of 4.1–4.7 Å, and 8-member ring channel run parallel with the size of

4.0–5.5 Å. Porous structure of the clinoptilolite consists of microporosity presented

by nanotube system of aluminosilicate framework, mesoporosity formed by slot

pores determined mainly by cleavability of the zeolite crystallite, and macropores

consisted of various forms located between blocks of the zeolite crystallite and other

minerals in the zeolitic tuff. Clinoptilolite occurs in andesite, rhyolite, and basalt

rocks as veins and impregnations. But the large industrial deposits are connected with

volcanic sedimentary high-silica rocks. Zeolite tuffs often contain more than 70%

clinoptilolite. Associated minerals are usually quartz, cristobalite, calcite, aragonite,

thenardite, feldspars, chlorite, montmorillonite, and other zeolites.

Fly ash-based geopolymers (GPs) are alkali-activated aluminosilicates with tri-

dimensional network structure of –Si–O–Si(Al)– bonds. In opposite to zeolitic mate-

rials, tetrahedra of SiO4 and AlO4 are linked by sharing all the oxygen atoms. The GP

low-CO2 binders are synthesized via an alkali-activation method of a solid source

of SiO 2 and Al2O3. The most commonly used aluminosilicate raw materials are

metakaolin and, coal ﬂy ash, bottom ash, red mud, slag, and biomass ﬂy ash. They

can be used as a substrate of the geopolymers synthesis. The GPs formation mecha-

nism includes dissolution, rearrangement, condensation, and resolidiﬁcation and is

affected by the temperature, amount of water, mixing method, and physical properties

of the source material. Depending on reaction conditions and an alkaline activator

used for their manufacture, the resulting material is amorphous or semicrystalline

zeolites. The geopolymers prepared with NaOH show more crystalline morphologies

compared to the geopolymers prepared with Na2SiO3 which feature more amorphous

phases. Anyway, fast kinetics process and reaction pace do not allow for the gel or

paste to grow into a well-crystallized structure (Provis 2014). The favorable prop-

erties of geopolymers are high compressive strength and ﬁre and acid resistance

which make them an alternative material to cementitious binders. GPs application

signiﬁcantly reduce soils’ swell-shrink behavior, responsible for damage of founda-

tions, roads, or embankments. The study of Vitale et al. (2017) reveals that when

alkali activated binders contact with contaminated soil, they promote the formation

of new mineralogical phases responsible for the mechanical improvement of treated

soil. Furthermore, large total porosity, well-deﬁned pore size distribution, and low

permeability of geopolymer structures enable immobilization of heavy metal in both

cationic and an anionic forms. It opens up the possibility of GPs usage as ﬁltration

media in sand ﬁlters, permeable reactive barriers, or point-of-use water treatment

ﬁlters (Adewuyi 2021).

Phosphate compounds such as phosphate rocks, synthetic hydroxyapatite,

KH2PO4,CaH

2PO4,H

3PO4, commercial phosphate fertilizers, bone meal (from

animal bones), and slaughter-house waste products are able to immobilize heavy

metals in contaminated soils by the formation of stable minerals. The variation in

Remediation of Soil Contaminated with Heavy Metals … 199

the solubility of the P compounds inﬂuences the mechanism of metal ions immobi-

lization. In case of soluble phosphates, the process occurs with interaction of metal

ions with P-induced adsorption sites, and their precipitation in a form of insoluble

phosphates, e.g., with Pb: chloropyromorphite (Pb5(PO4)3Cl), hydroxypyromorphite

(Pb5(PO4)3OH), and ﬂuoropyromorphite (Pb5(PO4)3F); with Cu and Zn: hopeite

(Huang et al. 2016). The reaction of P with Pb proceeds through the formation of

the highly insoluble pyromorphites [Pb5(PO4)3(OH, Cl, F)]. Importantly, the formed

precipitates are not digestible for plants. The parameters that affect the process efﬁ-

ciency are pH and soluble salts content. Especially the presence of H2PO4− and

SO4 2− ions in soil enhances metal ions adsorption. pH decrease causes increase in

the negative charge on iron and aluminum oxides on soil surface. It favors formation

of metal-carbonate and metal-Fe–Mn-oxide bounds (in e.g., ferromanganese oxyhy-

droxides) (Andrunik et al. 2020). Nevertheless, the immobilization of metals was

relatively ineffective when phosphate rock was applied to calcareous soils, which

can be attributed to the poor reactivity of apatite. The possible mechanism of this

process is an ion exchange between metal cations and Ca2+ from the apatite particle.

Dissolution of apatite is rather limited in alkaline soils what diminishes lead immo-

bilization as pyromorphite. Phosphorus fertilizers, added to the contaminated soil,

release phosphoric acid, which promptly dissociates into phosphate ions and acidic

hydrogen ions. This process supports the solubilization of metal ions and their subse-

quent precipitation. Natural phosphate rocks applied as fertilizers not only release P

and Ca but can also reduce soil acidity by releasing CaCO3 (Seshadri et al. 2017).

Lime materials have been recognized as carbonates, oxides, and hydroxides of

Ca and Mg as well as a set of wastes e.g., eggshells, mussel shells, and oyster

shells, limestone, sugar beet factory lime, and cement kiln dust (Hamid et al. 2019).

Liming materials enhance soil quality by ameliorating soil acidity, thereby aiding

crop productivity and immobilization of heavy metals by precipitation as carbonates.

On the other hand, liming materials increase the content of Cd bound to mobilizable

soil fractions at the expense of the most-environmentally-inert fractions. Hence, the

combined use of liming and vegetation may increase t he long-term environmental

risk of Cd solubilization and leaching. Too higher dosages of CaO (above 10%

w/w) could negatively affect plant growth, increase metal ions phytoavailability,

and immobilization of beneﬁcial micronutrients such as Fe, Mn, Zn Cu, and B.

Therefore, determining soil qualities and its pH are important considerations prior to

lime application. Combined application of liming materials with appropriate organic

amendments (sewage sludge, manure compost) can minimize heavy metals mobility

and phytoavailability (Holland et al. 2018).

Organic Sorbents

Waste biomass from agricultural operations (e.g., straw, leaves, bagasse from

sugar cane, rice hulls from rice processing, animal manure—cattle, poultry),

forest industry (e.g., bark and wood chips, bark saw dust, wood ash), mushroom

cultivation (mushroom residue), wastewater treatment (sewage sludge), produced

200 I. Michalak and J. Warchoł

compost/vermicompost or biochar may become inexpensive sorbents of heavy metals

for soil bioremediation. These materials are characterized by large porous struc-

ture and surface area with abundant functional groups responsible for contaminants

binding. The examples of organic amendments used in the immobilization of heavy

metal ions in polluted soil and their effect on soil/plants are presented in Table 1b.

The most popular soil amendment is biochar, a carbonaceous material, being the

major product of pyrolysis or gasiﬁcation (pyrolysis combined with partial oxidation)

of biomass (EBC 2012;Lwinetal. 2018; Guo et al. 2020;Lietal. 2021). Biochar

can be obtained from a variety of feedstocks, for example wheat (Liu et al. 2021)or

maize straw (Xu et al. 2020; Zhou et al. 2020; Irfan et al. 2021), corn stalk (Tang

et al. 2022) and stover (Xia et al. 2021), rice husk (Ogundiran et al. 2015), as well as

kitchen waste (Xu et al. 2020), bamboo (Hannan et al. 2021a, b), pine shoots (Wang

et al. 2020), willow (Gł˛ab et al. 2021), pig manure digestate (Ayaz et al. 2021),

coconut shell (Shao et al. 2021), peanut hulls (Xu et al. 2020) and shells (Chen et al.

2020; Shan et al. 2020), etc. The feedstock source material determines the biochar

properties and sorption abilities as well as t he effect on morphometric parameters

of cultivated plants (Guo et al. 2020;Xuetal. 2020;Gł˛ab et al. 2021). Guo et al.

(2020) in their review provided a detailed characteristics of biochars obtained from

different organic feedstocks and under different carbonization conditions, together

with examples of the biochar-facilitated remediation of heavy-metal-contaminated

soils.

Li et al. (2021) indicated that biochar produced by co-pyrolysis of different feed-

stocks is more effective in removing heavy metals from contaminated soil than the

pristine biochar. Depending on the feedstock, biochar can vary in the content of

organic carbon and ash, plant nutrients (N, P, K), base cations (Na+,K

+,Ca

2+,Mg

2+),

as well as pH. The carbonization conditions, for example temperature, solid residence

time and the heating rate, also inﬂuence the biochar quality (e.g., Irfan et al. 2021;

Li et al. 2021; Liu et al. 2021; Tang et al. 2022). With the increase in pyrolysis

temperature, decrease in the cation exchange capacity (CEC) is observed, which is a

measure of negatively charged functional groups (e.g., –C = O, –COOH, and –OH)

on the sorbent surface binding positively charged ions (Guo et al. 2020). On the other

hand, the temperature increase favors micropore development and causes increase

in the speciﬁc surface area of sorbent (Guo et al. 2020).

The biochar properties, closely associated with heavy metal ions stabilization in

soil, are alkaline pH (increases soil pH) and high CEC (responsible for uptake and

releaseofN,P,K,Ca, Mg,Sto soil) (Lwinetal.

2018; Guo et al. 2020; Shao et al.

2021). In an alkaline environment, the majority of metals appear to be less soluble

(Hu et al. 2014). Additionally, biodegradable organic carbon can be released from

biochar to soil (Shao et al. 2021; Irfan et al. 2021). Organic matter increases soil

metal adsorption capacity by promoting exterior complexation reactions that result

in metal–organic ligand complexes (Hu et al. 2014). High content of organic matter

(in opposite to inorganic amendments) signiﬁcantly impacts the ability of metal

ions retention over the long period of time (Hannan et al. 2021a). Furthermore,

environmental recalcitrance of biochar, estimated at 90–1600 years, is a signiﬁcant

advantage over compost and other raw bio-materials (Singh et al. 2012). In this study,

Remediation of Soil Contaminated with Heavy Metals … 201

long-term (5 years) laboratory experiment revealed that between 0.5 and 8.9% of the

biochar carbon was mineralized depending on biochar feedstock (papermill sludge,

Eucalyptus saligna wood and leaves, cow manure and poultry litter) and pyrolysis

temperature. The carbon in manure-based biochars was mineralized faster than that

in plant-based biochars, and carbon in biochars produced at 400 °C mineralized faster

than that produced at 550 °C.

Other popular organic amendments used in polluted soil bioremediation are crop

straw and compost. The ﬁrst one is known to have a positive effect on soil structure,

stimulation of crops growth and development. Returning straw to the ﬁeld can result

in the production of dissolved organic carbon (DOC), which acts as an organic

ligand of heavy metals complexation (Xu et al. 2016; Chen et al. 2020). The same

concerns compost, which can also be used to stabilize heavy metals in polluted soil.

Ogundiran et al. (2015) showed that compost obtained from wild sunﬂower (Tithonia

diversifolia) and poultry liter released dissolved organic matter faster than biochar

produced from rice husk and cashew nut shell due to the action of microorganisms.

As a result, more functional groups were available for binding heavy metal ions in

soil and compost was more effective in stabilization of Pb than biochar. Different

results were obtained by Irfan et al. (2021) and Chen et al. (2020) who showed that

biochar had better immobilization properties of heavy metals than crop straw. This

indicates the complexity of the immobilization process of heavy metals in soil by

additives, which is inﬂuenced by many factors from the preparation of the additive

itself, to soil properties and others.

It is also possible to use a mixture of organic amendments for contaminated soil

treatment. Liu et al. (2021) showed that the combined use of biochar from wheat straw

and chicken manure resulted in the most signiﬁcant decrease in Pb level in maize

tissues (reduced uptake) and soil Pb bioavailability, and increase in soil enzyme

activity and maize growth when compared with single amendments treatments.

Some soil amendments (e.g., traditional biochar) have certain limitations in their

adsorption capacity, therefore more research is focusing on sorbents modiﬁcation to

improve their surface properties (Li et al. 2021; Tang et al. 2022). This can be done

through the acid or alkali, redox or magnetic modiﬁcation of biochar (Tang et al.

2022). Several modiﬁed biochars have already been tested in the bioremediation of

contaminated soil, for example biochar modiﬁed with polymer—polyethyleneimine

was used to immobilize Cd (Tang et al. 2022), coal ash incorporated biochar for the

treatment of multimetal (Pb, Cd) contaminated soil (Xia et al. 2021), Mg-modiﬁed

peanut shell biochar to immobilize Cd (Shan et al. 2020), etc. Shan et al. (2020)

showed that an increase in Mg content in biochar after modiﬁcation promoted the

exchange of Mg2+ loaded on the biochar surface with the Cd2+ in soil, thereby ﬁxing

Cd2+ on the biochar surface.

Inorganic Versus Organic Amendments

In order to select the most effective additive for the immobilization of metals in

contaminated soil, comparative studies—organic versus inorganic amendments—are

202 I. Michalak and J. Warchoł

carried out. Usually biochar is compared with inorganic amendments, like zeolite,

limestone, hydroxyapatite etc. (Table 1c). There is no clear indication which type

of amendment—organic or inorganic—exhibits higher ability to immobilize heavy

metals. Usually all tested additives reduce heavy metals mobility in soil and their

bioavailability to plants. The differences in the immobilization degree of heavy metals

resulted mainly from the physicochemical characteristics of the used additives. For

example, Shao et al. (2021) showed that biochar from coconut shell and zeolite

exhibited higher CEC and surface negative charges than the incinerated sewage

sludge ash, indicating that these two amendments had higher electrostatic adsorption

abilities for Cs+. Interesting results were presented by Zhou et al. (2020)—to achieve

a similar degree of immobilization of Cd in the acidic and neutral soil, biochar from

maize straw is recommended at a dose of 5% while zeolite at a much lower 1% dose.

Comparison of the raw biomass (unprocessed e.g., by pyrolysis) such as coconut

sawdust or sugarcane bagasse with eggshells indicated better afﬁnity for Zn, Cu and

Pb of inorganic amendment (Kamari et al. 2015).

Mixed (Organic and Inorganic) Amendments

More and more frequently the immobilization of heavy metals in contaminated soil

is done by the application of combinations of organic and inorganic amendments

(Hu et al. 2014;Lietal.

2021; Pei et al. 2021; Quoc et al. 2021). Table 1d presents

the examples of mixtures containing organic and inorganic amendments used in

soil remediation. Integrated application of adequate amendments is recommended to

maximize their efﬁciency (Palansooriya et al. 2020). It is hypothesized that blending

amendments could provide more binding sites for heavy metal(loid) stabilization in

soil (Lietal.

2021; Quoc et al. 2021). Analysis of amendments mixtures by several

morphological and physical techniques revealed that they have a higher adsorption

capacity towards metal ions than amendments applied individually (Hannan et al.

2021b). The treatment of contaminated soil with organic and inorganic additives

not only effectively immobilizes heavy metal ions, but also has a greater impact

on soil fertility parameters (e.g., higher availability of N, P, K, soil organic matter,

and microbial biomass C and N) and on microbial community composition (Gł˛ab

et al. 2021; Pei et al. 2021). Several researchers have conﬁrmed better heavy metal

immobilization efﬁciency and better plant growth stimulation for combined ﬁxes

than for s ingle treatments (e.g., Hu et al. 2014; Mahmoud and Abd El-Kader 2015;

Hamid et al. 2018; Hannan et al. 2021b; Xia et al. 2021). Nevertheless, as steams

from the work of Hu et al. (2014), the increase in the rape (Brassica napus)biomass

was observed only in the group treated with 2% straw, but not in the group with

5% coal ﬂy ash and their combination case. It probably resulted from provision of

plentiful organic matter produced during straw decomposition.

Remediation of Soil Contaminated with Heavy Metals … 203

3.2 Characteristics and Mechanism of Action of Sorbents

Used in Immobilization of Heavy Metal Ions in Soil

There are many instrumental techniques, which can be used to characterize physico-

chemical properties of amendments in order to identify their immobilization perfor-

mance towards heavy metals. As examples can serve: Brunauer–Emmett–Teller

(BET) to determine speciﬁc surface area of immobilizing agents using N2 sorp-

tion analysis (Hannan et al. 2021b; Quoc et al. 2021; Shao et al. 2021); Scanning

Electron Microscopy (SEM–EDS/EDX) to visualize the morphology of amendments

as well as their elemental composition (e.g., Kamari et al. 2015; Wang et al. 2020;

Gł˛ab et al. 2021; Hannan et al. 2021b; Quoc et al. 2021; Shao et al. 2021); Fourier

Transform Infrared Spectroscopy (FT-IR) to determine functional groups on the

surface of amendments, which participate in ion exchange (e.g., Kamari et al. 2015;

Zhou et al. 2020; Hannan et al. 2021a, b; Quoc et al. 2021); X-ray Fluorescence

(XRF) (Quoc et al. 2021) or ﬂame atomic absorption spectrometry (Quoc et al.

2021) to analyze the chemical composition of amendments; X-ray Diffraction (XRD)

to conﬁrm the formation of metal related ligands (e.g., Wang et al. 2020; Hannan

et al. 2021a; Hannan et al. 2021b). Identiﬁed characteristics of the soil amendments

enables prediction of the (bio)remediation mechanism.

Mechanism of Action of Inorganic Sorbents

An in situ ﬁeld experiment carried out on severely polluted paddy soil revealed that

alkaline clay minerals such as bentonite, sepiolite, and palygorskite immobilized

heavy metals by increasing soil pH, and consequently forming CdCO3 and Cd(OH)2

precipitates (Liang et al. 2014). Generally, where clay minerals were used as an

amendment to soil, there was an increase in the biomass of plants, whereas accu-

mulation of metals decreased. Addition of Na-bentonite and Ca-bentonite reduced

the labile fraction of heavy metal, improved wheat shoot dry matter production by

decreasing shoot metal concentration below phytotoxicity levels and decrease in

water-soluble metals in contaminated ﬂoodplain soil. It is attributed to bentonite’s

larger surface area and sorption capacity, and ability to increase soil pH which

allow forming metal ions precipitates (Vrinceanu et al. 2019). Nevertheless, raw

clay minerals have poor stabilization property and the adsorbed heavy metals can be

released in the long term through ion exchange. One of the methods to enhance their

stability is their thermal and chemical activation with e.g., magnesium carbonate. The

treated mineral particles are covered by MgO/Mg(OH)2 which results in decrease

in pore volume and surface area. An increase in the pore diameter is linked with

the formation of Mg(OH)2 clusters in the mineral interlayer space, which induces a

new phase of MgSiO3. The magnesium-montmorillonite amendment increases alka-

linity of the treated soil. This in turn causes a reduction of heavy metal solubility

in basic surrounding due to the hydrolysis reaction and electrostatic adsorption on

mineral surface as well as to precipitation with Mg(OH)2. It is also supposed that the

204 I. Michalak and J. Warchoł

reduction of heavy metal bioavailability results from the fact that montmorillonite

promotes the activity of soil bacteria to some extent and activates the expression

of functional genes (Qin et al. 2020). Clay minerals amendments can enhance soil

micronutrient availability as well. For example, vermiculite application allowed to

obtain an optimum foliar Mn concentration with a simultaneous reduction of translo-

cation factors for root-stalk and stalk-leaves (Parmar et al. 2022). This opens up the

possibility of using clay minerals as complementary fertilizers, aiming at partially

decreasing the high fertilization rates.

The mechanism associated with the successful use of natural zeolites was iden-

tiﬁed to be preliminary an ion-exchange. Beside, adsorption processes and surface

precipitation/co-precipitation controlled by insoluble/soluble products interacting

with other minerals can occur as well. The use of zeolites in acidic soils causes an

increase in pH, reduction of electrical and hydraulic conductivity and lowering pore

size of soils. As a consequence heavy metals solubility and bioavailability for plants

are reduced. However, the increase in soil pH cannot be considered as a major factor

inﬂuencing plant growth. 10% w/w zeolite addition resulted in decrease of heavy

metals uptake by plants by 20.1% for Cd, 23.4% for Cu, 29.2% for Ni, and 26% for

Zn. Additionally signiﬁcant decrease in metal ions concentration was observed in

plant tissue and shoots with efﬁciency following the order: Ni > Cu > Cd > Zn. The

decreased leachability of metals followed almost a reverse order: Cu > Cd > Ni > Zn

(Contin et al. 2019). Zeolites are unstable under acid conditions. Their presence in

soils may inﬂuence the precipitation of some metals, which in turn changes charge

density, pore distribution, and particle aggregation, subsequently affecting stabiliza-

tion and accumulation of organic C. Thus, the use of zeolite for immobilization of

soil heavy metals in urban or residential areas needs to be deeply evaluated in the

long-term perspective. The beneﬁcial effects of zeolite application as soil amendment

is a signiﬁcant increase of soil’s K and Ca exchangeability. Zeolites in homoionic K

form may serve as an effective slow-release potassium fertilizer and also as a poten-

tial soil conditioner that can improve soil inﬁltration and hydraulic conductivity.

The controlled release of potassium, ammonium or phosphates improves nutrient

use of plants and decrease runoff and sediments amount by increasing the soil water

holding capacity. Moreover, zeolites can act as carriers, stabilizers and regulators

of mineral fertilizers and macro- and micronutrients supplier. Application of zeolite

signiﬁcantly increased Zn uptake by spinach plants and favorably inﬂuenced the root

growth of Castanea sativa plants (Chatzistathis et al. 2021). The increase in plant

growth due to the addition of zeolites was observed in crop yield of potato, maize,

rice, tomato, eggplant, carrot as well as cultivation of different cereals, forage crops,

vine and fruit crops (Jakkula and Wani 2018). It could result from both alleviation

of metals toxicity stress and an improvement of plant nutrition.

The mechanism of heavy metals immobilization by ﬂy ash-based geopolymers

is complex and can combine the ion-exchange with charge-balancing cations (Na+,

K+), covalent bonding to the aluminosilicate network, the precipitation of hydroxides

and carbonates and silicates in the matrix, and the physical encapsulation in a low-

permeable geopolymeric matrix (Eleswed 2020). Siliceous composition enhances

the formation of high-surface-area three-dimensional geopolymer gel. It straight

Remediation of Soil Contaminated with Heavy Metals … 205

translates into speciﬁc surface area, which is lower than 100 m2/g. Addition of various

boron compounds to the PGs synthesis reaction increases the value beyond 200 m2/g

(Siyal et al. 2018). Synthesis of GPs from ﬂy ash precursor treated with 1 M Na2SiO3

(at 90 °C, 24 h) as alkali activator allowed to obtain the material with speciﬁc surface

area up to 2464 m2/g (Mondal et al. 2020). The increase in Ca/(Si + Al) and Al/Si

ratios in GPs material decreases efﬁciency of Pb2+ removal. While, the increase

of Na/Si ratio as well as the mass percent of the amorphous phase facilitate the

Pb2+ uptake from soil. Moreover, higher Na content enhances ion exchange of Na+

by Pb2+ in the charge balancing sites of the geopolymer framework. Nevertheless,

the microstructure composition inﬂuences the kinetics of metal ions uptake more

signiﬁcantly than the equilibrium uptake. Decrease in the Si/Al molar ratio enhances

the number of negatively charged Al tetrahedra and the pores volume but decreases

the strength of GP. The presence of borax, anhydrous borax, boric acid, amorphous

and crystalline lithium tetraborate, and colemanite limits the decrease of compressive

geopolymer strength but causes heavy metal leaching (Rozek et al. 2021). It was also

observed that the metal ions washing is lower in case of the ash-based GP synthesized

under higher alkaline conditions.

The literature review suggests that ﬂy-ash-based geopolymeric adsorbents are

generally more effective compared with raw ﬂy ash and zeolite adsorbents for the

sequestration of aqueous and air contaminants. Nevertheless, practical application of

GPs to soil stabilization is rather limited considering civil engineering requirements.

The incorporation of metal ions into geopolymer matrix reduces its compressive

strength and stability which leads to crushing and pulverization. It questions safe

disposal of the material into landﬁll. GPs application for soil amendments has not

been reported so far. Recently, Wu et al. (2021) presents in situ synthesis of zeolites

by metakaolin geopolymerization in alkaline solution. The obtained materials were

able to immobilize up to 58.7% of Cd(II) in paddy soil (Wu et al. 2021).

Mechanism of Action of Organic Sorbents

Organic sorbents possess a variety of functional groups (e.g., carboxyl, carbonyl,

hydroxyl, phenolic hydroxyl groups, etc.), which provide active sites for ion exchange

and adsorption of heavy metal ions as well as for nutrient retention (Lwin et al. 2018).

Other factors inﬂuencing immobilization process are large surface area, high porosity

and stability (Liu et al. 2021). Some of the amendments, like biochar exhibit alkaline

pH (Liu et al. 2021). Biochar’s ability to immobilize heavy metals in soil depends on

its acid neutralization ability and high cation exchange capacity (Irfan et al. 2021).

Increase soil pH transfers heavy metals into their less mobile ionic forms, controls

their bioavailability in soil and reduces their movement from soil to plant tissues (Liu

et al. 2021). Zhou et al. (2020) predicted that the increase in soil pH caused by the

addition of biochar from maize straw caused Cd immobilization by precipitation and

adsorption. Additionally, higher pH values trigger precipitation of heavy metals in a

form of hydroxide or carbonate, lowering metals content in soil (e.g., cadmium) (Bian

206 I. Michalak and J. Warchoł

et al. 2013;Xuetal. 2020). Heavy metals are immobilized by biochar amendment

primarily due to its liming effect (Bian et al. 2013).

Surface adsorption, ion-exchange, formation of stable complexes with organic

ligands, complexation and precipitation of heavy metal ions by organic soil amend-

ments are recognized as main mechanisms decreasing their content in plant tissues

(Mahmoud and Abd El-Kader 2015;Lwinetal. 2018; Liu et al. 2021; Xia et al. 2021).

In more details, the immobilization process of heavy metal ions by organic amend-

ments could be divided into following phases: (i) the bioavailable heavy metal ions in

water solution are transferred to near-surface of soil by physical attraction forces (i.e.

hydrogen bond, electrostatic attraction, and van der Waals’ force); (ii) the metal ions

are adsorbed by inner and outer active sites on the surface of organic amendment;

(iii) the adsorbed metals are immobilized by precipitation/co-precipitation (CO3 2−,

PO4 3−,CaCO

3 and MgCO3), surface complexation (Si–O bond), cation exchange

(K, Ca, Na and Mg ions), and cation-π interaction (π electrons in lignin) to form

stable metal–organic amendment complexes (Guo et al. 2020;Lietal. 2021;Xia

et al. 2021).

The dose of soil amendment is also an important factor in soil (bio)remediation

(Wang et al. 2020; Zhou et al. 2020;Gł˛ab et al. 2021). Wang et al. (2020)showeda

signiﬁcant dose-dependent effect for immobilization of Cd, Pb, and Zn in polluted

soil. More active sites for metal adsorption, precipitation, and complexation, as well

as more beneﬁcial nutrients for plants, can be provided with a higher dose of amend-

ments. In comparison to the 0.1% dose of hydroxyapatite and cow dung-based manure

applied individually, 1% dose caused lower content of Cd, Pb, and Zn in maize but

higher plant biomass. However, high-dose amendment application may result in high

costs and environmental side effects.

4 Conclusions

As steams from the literature review, immobilization is a method where relatively

cheap materials are added to (or introduced into?) contaminated soil to decrease

mobility and bioavailability of heavy metals. Various organic and inorganic by-

products or natural minerals could be used as soil amendments. The studies reveal

that there are various and complex interactions among sorption materials, heavy

metals, and soil which leads to different immobilization efﬁciencies. Thus, a practical

application of a given sorption material requires the priori analysis of soil composition

and properties as well as quantitative and qualitive analysis of metal ions mixture, in

order to identify the best soil remediation conditions.

In comparison with other sorption materials, biochar has higher porosity, larger

surface area and consequently bigger sorption capacity. This material can be obtained

from any bio-based mass resulted from agriculture or farm or food production. On the

other hand, biochars have a higher than natural sorbents energy footprint, resulting

from its production via pyrolysis.

Remediation of Soil Contaminated with Heavy Metals … 207

The review also revealed large gaps in the knowledge about soil remediation via

sorption materials amendments. First of all, most of the reported researches have been

conducted in short-term laboratory experiments, while the long-term trials regarding

mobility and bioactivity of metal ions in the ﬁeld soils are very rare. It should be

pointed out that heavy metals can never be entirely removed from soil and the immo-

bilization effect may diminish over time. Though, the sorbent-amended soils need

to be regularly monitored for heavy metal toxicity. The most suitable methods for

this process are percolation tests that take into consideration the inﬂuence of the soil’s

properties and hence reproducing the conditions of a real ﬁeld situation. Secondly,

there is a lack of research on the optimization procedures with a view to arrive at a

set of conditions that yields optimal immobilization and uptake effects. The sorption

materials discussed are more suitable for the removal of cationic forms of heavy

metals due to the negative charge of materials’ sorption surface. However, some

metalloids (e.g. As, Cr, I, U) can be present in environment in anionic f orms. Further-

more, multicomponent studies, despite being time consuming and more complex to

handle, should be done in the future to have a better understanding of the competition

between different types of pollutants on the immobilization efﬁciency. Last but not

least, practical application of low-cost amendments for heavy metals immobilization

should be preceded by cost − beneﬁt evaluations and by comparison of available

technologies, aiding progress toward large-scale applications and long-term exposure

on environmental conditions.

Acknowledgements This chapter was prepared in the framework of the project entitled “Biomass

valorization to enhance efﬁciency of toxic metals bioremediation from military and industry

areas” ﬁnanced by OPCW (No: L/ICA/ICB-105/21, The Hague, The Netherlands, 31.12.2021–

31.12.2023).

References

Adewuyi YG (2021) Recent advances in ﬂy-ash-based geopolymers: potential on the utilization for

sustainable environmental remediation. ACS Omega 6:15532–15542

Andrunik M, Wołowiec M, Wojnarski D, Zelek-Pogudz S, Bajda T (2020) Transformation of Pb,

Cd, and Zn minerals using phosphates. Minerals 10:342

Ayaz M, Stulpinaite U, Feiziene D, Tilvikiene V, Akthar K, Baltenaite-Gedien E, Striugas N,

Rehmani U, Alam S, Iqbal R, Toleikiene M, Doyeni M (2021) Pig manure digestate-derived

biochar for soil management and crop cultivation in heavy metals contaminated soil. Soil Use

Manage. https://doi.org/10.1111/sum.12773

Bian R, Chen D, Liu X, Cui L, Li L, Pan G, Xie D, Zheng J, Zhang X, Zheng J (2013) Biochar soil

amendment as a solution to prevent Cd-tainted rice from China: results from a cross-site ﬁeld

experiment. Ecol Eng 58:378–383

Cakmak I, Marschner H (1992) Magnesium deﬁciency and high light intensity enhance activi-

ties of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves.

Plant Physiol 98:1222–1227

Chatzistathis T, Papaioannou E, Giannakoula A, Papadakis IE (2021) Zeolite and vermiculite as inor-

ganic soil amendments modify shoot-root allocation, mineral nutrition, photosystem II activity

and gas exchange parameters of chestnut (Castanea sativa Mill) plants. Agronomy 11:109

208 I. Michalak and J. Warchoł

Chen X, He HZ, Chen GK, Li HS (2020) Effects of biochar and crop straws on the bioavailability

of cadmium in contaminated soil. Sci Rep 10:9528

Contin M, Miho L, Pellegrini E, Gjoka F, Shkurta E (2019) Effects of natural zeolites on ryegrass

growth and bioavailability of Cd, Ni, Pb, and Zn in an Albanian contaminated soil. J Soils

Sediments 19:4052–4062

Correia AAS, Matos MPSR, Gomes AR, Rasteiro MG (2020) Immobilization of heavy metals in

contaminated soils—performance assessment in conditions similar to a real scenario. Appl Sci

10:7950

Dhaliwal SS, Singh J, Taneja PK, Mandal A (2020) Remediation techniques for removal of heavy

metals from the soil contaminated through different sources: a review. Environ Sci Poll Res.

27:1319–1333

Dzionek A, Wojcieszy´nska D, Guzik U (2016) Natural carriers in bioremediation: a review. Electr

J Biotechnol 23:28–36

Eleswed BI (2020) Chemical evaluation of immobilization of wastes containing Pb, Cd, Cu and Zn

in alkali-activated materials: a critical review. J Environ Chem Eng 8:104–194

European Biochar Certiﬁcate, Guidelines for a Sustainable Production of Biochar (EBC, 2012)

(2012). http://www.europeanbiochar.org/en/download

Gł˛ab T, Gondek K, Mierzwa-Hersztek M (2021) Biological effects of biochar and zeolite used for

remediation of soil contaminated with toxic heavy metals. Sci Rep 11:6998

Guo M, Song W, Tian J (2020) Biochar-facilitated soil remediation. Mechanisms and efﬁcacy

variations. Front Environ Sci 8:1–23

Hamid Y, Tang L, Wang X, Hussain B, Yaseen M, Aziz MZ, Yang X (2018) Immobilization of

cadmium and lead in contaminated paddy ﬁeld using inorganic and organic additives. Sci Rep

8(1):17839

Hamid Y, Tang L, Hussain B, Usman M, Gurajal HK, Rashid MS, Yang X (2019) Efﬁciency of,

biochar, Fe containing biochar and composite amendments for Cd and Pb immobilization in a

co-contaminated alluvial soil. Environ Poll 196:113609

Hannan F, Huang Q, Farooq MA, Ayyaz A, Ma JY, Zhang N, Ali B, Deyett E, Zhou WJ, Islam F

(2021a) Organic and inorganic amendments for the remediation of nickel contaminated soil and

its improvement on Brassica napus growth and oxidative defense. J Hazard Mat 416:125921

Hannan F, Islam F, Huang Q, Farooq MA, Ayyaz A, Fang RY, Ali B, Xie XH, Zhou WJ (2021b) Inter-

active effects of biochar and mussel shell activated concoctions on immobilization of nickel and

their amelioration on the growth of rapeseed in contaminated aged soil. Chemosphere 282:130897

Holland J, Bennet A, Newton A, White P, McKenzie B, Georg T, Pakeman R, Bailey J, Fornara D,

Hayes R (2018) Liming impacts on soils, crops and biodiversity in the UK: a review. Sci Total

Environ 610:316–332

Hu XM, Yuan XS, Dong L (2014) Coal ﬂy ash and straw immobilize Cu, Cd and Zn from mining

wasteland. Environ Chem Lett 12(2):289–295

Huang GY, Su XJ, Rizwan MS, Zhu YF, Hu HQ (2016) Chemical immobilization of Pb, Cu, and

Cd by phosphatematerials and calciumcarbonate in contaminated soils. Environ Sci Pollut Res

23:16845–16856

Irfan M, Mudassir M, Khan MJ, Dawar KM, Muhammad D, Mian IA, Ali W, Fahad S, Saud S,

Hayat Z, Nawaz T, Khan SA, Alam S, Ali B, Banout J, Ahmed S, Mubeen S, Danish S, Datta

R, Elgorban AM, Dewil R (2021) Heavy metals immobilization and improvement in maize (Zea

mays L.) growth amended with biochar and compost. Sci Rep 11(1):18416

Jakkula V, Wani SP (2018) Zeolites: potential soil amendments for improving nutrient and water

use efﬁciency and agriculture productivity. Sci Rev Chem Commun 8(1):119

Kabata-Pendias A (2011) Trace elements in soils and plants, 4th edn. CRC Press, Boca Raton

Kamari A, Putra W, Yusoff S, Ishak C, Hashim N, Mohamed A, Isa I, Bakar S (2015) Immobilisation

of Cu, Pb and Zn in scrap metal yard soil using selected waste materials. Bull Environ Contamin

Toxicol 95(6):790–795

Remediation of Soil Contaminated with Heavy Metals … 209

Li YL, Yu H, Liu LN, Yu HB (2021) Application of co-pyrolysis biochar for the adsorption

and immobilization of heavy metals in contaminated environmental substrates. J Hazard Mat

420:126655

Liang X, Han J, Xu Y, Sun Y, Wang L, Tan X (2014) In situ ﬁeld-scale remediation of Cd polluted

paddy soil using sepiolite and palygorskite. Geoderma 235:9–18

Liu L, Li JW, Wu GH, Shen HT, Fu GZ, Wang YF (2021) Combined effects of biochar and chicken

manure on maize (Zea mays L.) growth, lead uptake and soil enzyme activities under lead stress.

PEERJ 9:11754

Lwin CS, Seo BH, Kim HU, Owens G, Kim KR (2018) Application of soil amendments to contam-

inated soils for heavy metal immobilization and improved soil quality—a critical review. Soil Sci

Plant Nutr 64(2):156–167

Mahmoud E, Abd E-K (2015) Heavy metal immobilization in contaminated soils using phospho-

gypsum and rice straw compost. Land Degrad Develop 26(8):819–824

Malik KM, Khan KS, Rukh S, Khan A, Akbar S, Billah M, Bashir S, Danish S, Alwahibi MS,

Elshikh MS, Al-Ghamdi AA, Mustafa AMA (2021) Immobilization of Cd, Pb and Zn through

organic amendments in wastewater irrigated soils. Sustainability 13(4):2392

Matłok M, Petrus R, Warchoł JK (2015) Equilibrium study of heavy metals adsorption on kaolin.

Ind Eng Chem Res 54:6975–6984

Mondal SK, Welz A, Rezaei F, Kumar A, Okoronkwo MU (2020) Structure–property relationship

of geopolymers for aqueous Pb removal. ACS Omega 5(34):21689–21699

Ogundiran MB, Lawal OO, Adejumo SA (2015) Stabilisation of Pb in Pb smelting slag-

contaminated soil by compost-modiﬁed biochars and their effects on maize plant growth. J

Environ Protect 6:771–780

Palansooriya KN, Shaheen SM, Chen SS, Tsang DCW, Hashimoto Y, Hou D, Bolan NS, Rinklebe J,

Ok YS (2020) Soil amendments for immobilization of potentially toxic elements in contaminated

soils: a critical review. Environ Int 134:105046

Parmar S, Singh V, Sharma VK, Yadav BK, Kharwar RN (2022) Effect of vermiculite soil amend-

ment on immobilization of selected heavy metals of rhizospheric zone of maize. Commun Soil

Sci Plant Anal 53(3):384–395

Pei PG, Xu YM, Zheng SN, Liang XF, Sun YB, Lin DS, Wang L (2021) The use of bentonite and

organic amendments for remediation of Cd contaminated ﬁelds: an environmental perspective.

Land Degrad Develop 32(13):3639–3652

Provis JL (2014) Geopolymers and other alkali activated materials: why, how, and what? Mater

Struct 47:11–25

Qin C, Yuan X, Xiong T, Zen TY, Wang H (2020) Physicochemical properties, metal availability and

bacterial community structure in heavy metal-polluted soil remediated by montmorillonite-based

amendments. Chemosphere 261:128010

Quoc TN, Nejad ZD, Jung MC (2021) Effect of commercial amendments on immobilization of

arsenic, copper, and zinc in contaminated soil: comprehensive assessing to plant uptake combined

with a microbial community approach. Minerals 11(10):1143

Radziemska M, Gusiatin ZM, Mazur Z, Hammerschmiedt T, B˛e´s A, Kintl A, Vasinova GM, Holatko

J, Blazejczyk A, Kumar V, Brtnicky M (2022) Biochar-assisted phytostabilization for potentially

toxic element immobilization. Sustainability 14:445

Rodríguez-Eugenio N, McLaughlin M, Pennock D (2018) Soil pollution: a hidden reality. FAO,

Rome, p 142

Rozek P, Florek P, Król M, Mozgawa W (2021) Immobilization of heavy metals in boroaluminosil-

icate geopolymers. Materials 14:214

Seshadri B, Bolan N, Choppala G, Kunhikrishnan A, Sanderson P, Wang H, Currie LD, Tsang D,

Ok YS, Kim K (2017) Potential value of phosphate compounds in enhancing immobilization and

reducing bioavailability of mixed heavy metal contaminants in shooting range soil. Chemosphere

184:197–206

Shah MP (2021a) Removal of emerging contaminants through microbial processes. Springer

Shah MP (2021b) Removal of refractory pollutants from wastewater treatment plants. CRC Press

210 I. Michalak and J. Warchoł

Shaheen SM, Rinklebe J (2014) Impact of emerging and low cost alternative amendments on the

(im)mobilization and phytoavailability of Cd and Pb in a contaminated ﬂoodplain soil. Ecolog

Eng 74:319–326

Shaikh SMR, Nasser MS, Hussein I, Benamor A, Onaizi SA, Qiblawey H (2017) Inﬂuence of

polyelectrolytes and other polymer complexes on the ﬂocculation and rheological behaviors of

clay minerals: a comprehensive review. Sep Pur Technol 187:137–161

Shan R, Li W, Chen Y, Sun X (2020) Effects of Mg-modiﬁed biochar on the bioavailability of

cadmium in soil. BioResources 15(4):8008–8025

Shao HJ, Wei YF, Wei CJ, Zhang FP, Li FS (2021) Insight into cesium immobilization in contam-

inated soil amended with biochar, incinerated sewage sludge ash and zeolite. Environ Technol

Innov 23:101587

Singh BP, Cowie AL, Smernik RJ (2012) Biochar carbon stability in a clayey soil as a function of

feedstock and pyrolysis temperature. Environ Sci Technol 46:11770–11778

Siyal AA, Shamsuddin MR, Khan MI, Rabat NE, Zulﬁqar M, Man Z, Siame J, Azizli KA (2018)

A review on geopolymers as emerging materials for the adsorption of heavy metals and dyes. J

Environ Manage 224:327–339

Tang B, Xu HP, Song FM, Ge HG, Chen L, Yue SY, Yang WS (2022) Effect of biochar on immobi-

lization remediation of Cd center dot contaminated soil and environmental quality. Environ Res

204:111840

Tóth G, Hermann T, Da Silva MR, Montanarella L (2016) Heavy metals in agricultural soils of the

European Union with implications for food safety. Environ Internat 88:299–309

Vitale E, Russo G, Dell’Agli G, Ferone C, Bartolomeo C (2017) Mechanical behaviour of soil

improved by alkali activated binders. Environments 4:80

Vrinceanu NO, Motelic˘a DM, Dumitru M, Calciu I, T˘anase V, Preda M (2019) Assessment of

using bentonite, dolomite, natural zeolite and manure for the immobilization of heavy metals in

a contaminated soil: the Cops

,aMic˘a case study. CATENA 176:336–342

Wang FY, Zhang SQ, Cheng P, Zhang SW, Sun YH (2020) Effects of soil amendments on heavy

metal immobilization and accumulation by maize grown in a multiple-metal-contaminated soil

and their potential for safe crop production. Toxics 8(4):102

Wu D, Huang Y, Xiao G, Li X, Yao X, Deng Z, Tan R (2021) In situ synthesis of zeolites by

geopolymerization with NaOH/KOH mixed solution and their potential application for Cd(II)

immobilization in paddy soil. Clay Miner 56:156–167

Xia Y, Li Y, Sun YT, Miao W, Liu ZG (2021) Co-pyrolysis of corn stover with industrial coal ash

for in situ efﬁcient remediation of heavy metals in multi-polluted soil. Environ Pollut 289:117840

Xu P, Sun CX, Ye XZ, Xiao WD, Zhang Q, Wang Q (2016). The effect of biochar and crop straws

on heavy metal bioavailability and plant accumulation in a Cd and Pb polluted soil. Ecotoxicol

Environ Saf 132:94–100. https://doi.org/10.1016/j.ecoenv.2016.05.031

Xu C, Zhao J, Yang W, He L, Wei W, Tan X, Wang J, Lin A (2020) Evaluation of biochar pyrolyzed

from kitchen waste, corn straw, and peanut hulls on immobilization of Pb and Cd in contaminated

soil. Environ Pollut 261:114133

Zhou C, Yuan H, Ning C, Li S, Xia Z, Zhu M, Ma Q, Yu W (2020) Evaluation of different types

and amounts of amendments on soil Cd immobilization and its uptake to wheat. Environ Manag

65:818–828

Poly-γ-Glutamic Acid and Its Application

in Bioremediation: A Critical Review

Valeria Bontà and Cinzia Calvio

Abstract Poly-gamma glutamic acid (γ-PGA) is an anionic bacterial polymer

constituted by glutamic acid residues only. It has the intrinsic ability to strongly

interact with positively charged ions and ﬂocculate them. For this reason, a large

body of literature has accumulated on its application in bioremediation, particularly

targeted to positively charged heavy metals. In this work, the most important charac-

teristics of γ-PGA and of its production are summarized, highlighting the advantages,

but also the limits, in its application i n bioremediation.

Keywords PGA ·Bacteria ·Bioremediation ·Polymer

1γ-PGA Biosynthesis

Poly-gamma glutamic acid (γ-PGA) is a natural anionic, high molecular weight

(Mw), homo-polyamide, made up of repeating units of L- and/or D-glutamic acid

residues connected by amide linkages between α-amino and γ-carboxyl groups; these

pseudopeptide bonds are resistant to classical proteases. The ribosome-independent

synthesis, that can lead to molecules most often above 106 Da, occurs via a trans-

membrane ATP-dependent γ-PGA synthase complex, which also secrets the polymer

outside the cell (Sung et al. 2005). The synthase is constituted by the products of four

genes, which are known as pgsB, pgsC and pgsA in species releasing the polymer

in the environment. A fourth small gene, pgsE, completes the pgs operon in B.

subtilis, B. amyloliquefacens, B. pumilus and B. licheniformis (Fujita et al. 2021). In

B. anthracis and other capsule forming bacteria, the pgs homologues are known as

cap genes (capBCA). In this latter group, the three biosynthetic genes are followed

by capD, which encodes a product that bears resemblance to γ-glutamyl transferases,

responsible for anchoring the polymer to the cell wall (Candela and Fouet 2005).

Microbial fermentation is the only viable way to obtain γ-PGA in signiﬁcant

amount, as the chemical synthesis of γ-PGA molecules longer than few residues

V. Bontà · C. Calvio (B

)

Department of Biology and Biotechnology, Pavia University, Pavia, Italy

e-mail: cinzia.calvio@unipv.it

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_11

211

212 V. Bontà and C. Calvio

is troublesome, inefﬁcient, expensive and non-sustainable, requiring the constant

protection of the α-carboxylic group.

Signiﬁcative amounts of γ-PGA are produced by Gram-positive bacteria, mainly

B. subtilis, B. licheniformis and B. amyloliquefaciens species. Aerobic, spore-

forming, ubiquitous in soil and plants, Bacilli are ideal workhorses for γ-PGA produc-

tion for many different reasons: they are generally non-pathogenic, fast growing,

amenable to genetic manipulation, have simple nutritional requirements, and their

products possess a long track record of safe use in human food products (Tamang

et al. 2016). B. subtilis is indeed the model organism for Gram-positive bacteria and

a large number of scholars, all over the world, have focused their studies on this

organism. In fact, scientiﬁc investigations on B. subtilis date back to 1835, although

B. subtilis got its present name only in 1872 by Ferdinand Cohn (Harwood 1989).

These has led to the accumulation of endless and thorough knowledge on its genetics

and physiology in general, and on γ-PGA production, in particular.

The polymer characteristics greatly vary among different producer strains,

different growth media and fermentation conditions. Neither the enantiomeric

composition nor the Mw are ﬁxed. Analyses of puriﬁed γ-PGA showed that it

can be made of D-glutamic acid, L-glutamic acid, or both enantiomers in different

ratios. The pathogen B. anthracis produces γ-PGA made of D-glutamate only (Jang

et al. 2011), while γ-PGA produced by the laboratory strain of B. subtilis JH642

(Srivatsan et al. 2008; Shah 2020) contains roughly 20% L-glutamate (Scoffone

et al. 2013; Morelli, personal communication). The ratio of the two stereoisomers

has been reported to vary according to the amount of Mn ions present in the growth

medium (Wu et al. 2006), although this has not been conﬁrmed by our preliminary

data (Calvio and Morelli, unpublished). γ-PGA is also highly polydisperse, but the

Mw of the molecules is on average extremely high, above 106 Da (roughly corre-

sponding to 7500 Glu residues), even though it is extremely difﬁcult to accurately

measure the Mw of a polydisperse polymer which forms inter- and intra-molecular

bonds (Park et al. 2005). The average size appears to change during bacterial growth

(Scoffone et al. 2013), according to the producing organism, in dependence to the

growth conditions, and might be reduced during puriﬁcation. For example, addition

of NaCl or (NH4)2SO4, which increase i onic strength, positively impacts on γ-PGA

molecular weight (Birrer et al. 1994;Parketal.

2005).

Being a natural secondary metabolite, γ-PGA is produced according to strictly

regulated intracellular mechanisms. In B. subtilis, the activation of the biosynthetic

operon is regulated by two different signal transduction pairs, the ComP/ComA and

the DegS/DegU two-component systems; besides these signalling systems, the SwrA

protein must be present. The most common B. subtilis laboratory strain 168, although

possessing the genes necessary for the synthesis of γ-PGA, does not transcribe the pgs

operon unless mutations that increase the intracellular level of DegU-P and produce

a functional SwrA protein are introduced (Stanley and Lazazzera 2005; Osera et al.

2009;Ohsawaetal. 2009). In a recent work, an activating mutation in degS,the

histidine kinase of the DegS/DegU two-component system, was shown to induce

elevated and prolonged γ-PGA production in domestic and wild B. subtilis strains

(Ermoli et al. 2021).

Poly-γ-Glutamic Acid and Its Application in Bioremediation: A Critical … 213

2 The Physiological Role of γ-PGA in Bacteria

The evolutionary advantage of γ-PGA biosynthesis may depend on the environ-

mental niche the producing organism inhabits. For soil organisms that release it

into the environment, γ-PGA may represent a formidable defence system against

extreme desiccation, high osmolarity or the presence of toxic metal ions thanks to

its water-holding and metal chelating capacity (Luo et al. 2016). Besides this role,

the wide diffusion of γ-PGA-hydrolases in phages infecting Bacillales indicates that

the polymer plays a fundamental role as anti-phage shield (Mamberti et al. 2015).

Conversely, the fact that the polymer is secreted at the end of the logarithmic phase of

growth, when nutrients are in short supply (Hsueh et al. 2017), cast serious doubts on

the hypothesis that γ-PGA might be produced as nutrient reserve, as reported by

several authors (Candela and Fouet 2005, Luo et al. 2016). However, once released

in the environment, the polymer represents a perfect source of carbon and nitrogen

to be exploited by both producers and non-producer strains (Mamberti et al. 2015).

Conversely, peptidoglycan-bound γ-PGA acts as virulence factor in pathogens,

by constituting a passive barrier and masking surface epitopes to the surveillance

of host immune response, protecting the capsulated bacteria from phagocytosis and

antimicrobial peptides (Kocianova et al. 2005; Scorpio et al. 2007).

3 Degradation of γ-PGA

The degradation of γ-PGA is achieved by the action of unspeciﬁc and speciﬁc γ-

PGA hydrolases, as the polymer is intrinsically resistant to common proteases. The

unspeciﬁc hydrolases are enzymes belonging to the class of γ-glutamyltransferases

(GGT, E.C. 2.3.2.2), which are ubiquitously present from bacteria to humans. GGTs

from Bacilli and other Gram-positive bacteria, but not from E. coli, can act as γ-

PGA exo-hydrolases, slowly releasing free glutamate from the N-terminal end of

the polymeric chain, independently from the stereochemical conﬁguration of the

cleaved glutamate moiety (Calvio et al. 2018; Shah 2021a, b). As for the γ-PGA-

speciﬁc enzymes, most producer strains carry pgdS, a gene encoding a secreted γ-

PGA-speciﬁc endo-hydrolase, possibly involved in the release of the nascent chain

from the synthase complex (Scoffone et al. 2013). In B. subtilis, pgdS is located just

downstream of the pgs operon, but it is independently transcribed by a promoter

regulated by SigD, the sigma factor responsible for motility genes’ expression.

Bacillus phages are endowed with other highly efﬁcient enzymes that hydrolyse γ-

PGA and are possibly released from infected cell to break down the γ-PGA shield of

surrounding target cells and ensure the reaching of cognate surface receptors for the

subsequent infection cycle (Mamberti et al. 2015; Ramaswamy et al. 2018). Genes

encoding those phage-derived enzymes are encased in several Bacilli genomes, both

in prophage regions and in their vestigial remnants. The bewildering extensive occur-

rence of these enzymes also in genomes of non-γ-PGA-producers, most typically soil

214 V. Bontà and C. Calvio

bacteria, is thought to have occurred through horizontal gene transfer events. The

selective advantage conferred by γ-PGA hydrolases in these non-Bacillales hosts

is possibly represented by the ability conferred to the organism to degrade envi-

ronmentally dispersed γ-PGA and feed on the released glutamate (Mamberti et al.

2015).

As producer strains often contain a minimum of one γ-PGA hydrolyse (GGT),

it cannot be excluded that part of the polydispersity observed in γ-PGA might be

related to post-secretion hydrolytic degradation.

4 Strategies to Improve Microbial Productivity

Although microbial production of γ-PGA is established at the industrial level, the

cost of production and puriﬁcation, which ultimately affects the market price, is

currently extremely high ($261 per 100 mg high-purity γ-PGA, sodium salt; CAS

number: 208106-41-6; Sigma Aldrich). Such prohibitive cost constitutes the major

limitation to a more widespread application of γ-PGA and, at the same time, the

driving force for research activities aimed at improving productivity.

Such strategies have followed several parallel routes, such as the identiﬁcation

of new and more efﬁcient natural producer strains, or the engineering of the genes

and pathways involved in the biosynthesis and degradation of the polymer, or the

development of better fermentation conditions (Luo et al. 2016).

γ-PGA producer strains come in two ﬂavours: those requiring an external supply of

L-glutamic acid and those that can produce it even in the absence of an external source

of the amino acid. Productivity is much higher for L-glutamic acid-dependent strains,

but polymer yield is directly correlated to the amount of L-glutamic acid added,

which impacts on the production costs. Despite major efforts have been devoted to

the isolation of new glutamic acid-independent strains, their productivity is scant in

the absence of exogenous glutamate or its metabolic precursors (Zhang et al. 2012).

To reconcile needs and costs, several efforts were dedicated to the development of

genetically engineered strains characterized by high γ-PGA productivity or simply to

improve the intracellular glutamate synthesis, which is the limiting factor for γ-PGA

synthesis (Cai et al. 2018). The genetic and metabolic optimization of all processes

responsible for γ-PGA production is relatively easy in Bacilli’s genome thanks to the

existence of well-established techniques and molecular biology tools (Appelbaum

and Schweder 2021), supporting the improvement in production yields and reduction

in fermentation costs. Over the past years, a number of genes that are involved in

γ-PGA production have been characterized, and much has been published on how

different parameters affect productivity and costs.

As one of the main costs for commercial-scale γ-PGA production is the require-

ment of large amount of L-glutamate in the fermentation media (Nair et al. 2021),

huge efforts were posed on strategies designed to reduce or replace this expen-

sive component. These approaches generally aimed at rewiring the central carbon

metabolism to enhance the carbon ﬂux toward the tricarboxylic acid (TCA) cycle,

Poly-γ-Glutamic Acid and Its Application in Bioremediation: A Critical … 215

as α-ketoglutarate is a direct precursor of γ-PGA. Metabolic models now available

can predict genes to be targeted to enhance γ-PGA synthesis (Massaiu et al. 2019).

Another successful approach was taken by Feng and collaborators’ group, who took

inspiration from a well-known glutamate producer microorganism: a 9.1% improve-

ment in γ-PGA production was obtained by introducing the NADPH-dependent

GDH pathway from Corynebacterium glutamicum into a glutamate-independent B.

amyloliquefaciens strain; the yield further improved by 66.2% by manipulating the

enzymes of the TCA cycle (Feng et al. 2017). Also, the deletion of two γ-PGA

hydrolases, GGT and PgdS, was effective in doubling productivity in the presence of

glutamate (Scoffone et al. 2013). However, the simple over-expression of the pgsBCA

operon was shown to lead to a reduction in polymer yield, possibly because the excess

of the synthase complex imbalanced other membrane-associated metabolic processes

(Feng et al. 2015). In more systemic approaches, several of the previously explored

genome engineering strategies were joined in modular way in the same strains,

leading to a substantial increase in productivity (Feng et al. 2015; Cai et al. 2018).

Bacilli are considered as “strictly aerobic” and grow poorly in oxygen-limiting

conditions (Nakano and Zuber 1998). For this reason, γ-PGA is mainly produced by

aerobic fermentation in liquid media. However, during growth, oxygen transfer from

the gas phase to the liquid phase is progressively reduced due to the high viscosity

of the medium caused by the polymer that accumulates. This phenomenon limits

cell growth and leads to a decrease in γ-PGA yield at later stages of cultivation.

To overcome this problem a successful strategy was the introduction of the gene

encoding the Vitreoscilla hemoglobin in B. subtilis chromosome, enhancing cell

growth and increasing γ-PGA yield (Su et al. 2010). Although, the efforts dedicated

to genetic improvement, the polymer is still too expensive; therefore, alternative

routes have been parallelly explored to decrease γ-PGA market price.

5 Strategies to Reduce Fermentation Costs

Besides improving strains productivity, reduction of γ-PGA production costs is

sought through the replacement of expensive components of the fermentation media

with low-cost substrates. The dominant carbon sources used in the γ-PGA biosyn-

thesis are glucose and citric acid, that are mainly derived from sources that are in

competition with human nutrition, inevitably raising social dilemmas and raising the

production costs imposed to the process. Recently, the urge to valorise agro-industrial

waste as feedstock for bacterial fermentations has encouraged investigations on solid-

state fermentation also for γ-PGA production. Indeed, a wide range of abundant and

renewable lignocellulosic biomasses or agro-industrial wastes, such as rice straw,

cane molasses, rapeseed meal, soybean residue, corncob ﬁbres, crude glycerol from

biodiesel plants, macroalgae, goose feathers and paper waste have been proposed as

cost-attractive and environmentally sustainable carbon sources (Fang et al. 2020).

Such biomasses not only represent cheap substrates for microbial growth, but their

valorisation through fermentation mitigates the environmental impact they would

216 V. Bontà and C. Calvio

have on eutrophication; moreover, the solid-state fermented substrate can be directly

used as agricultural fertilizer, exploiting the beneﬁcial effects of both Bacillus species

and γ-PGA on plant performance (Zhang et al. 2017). Among the industrial wastes,

untreated cane molasses and monosodium glutamate waste liquor, by-products of

reﬁning sugarcane and from the glutamic acid fermentation process, respectively, or

powdered fulvic acid, recovered from the wastewater of molasses fermentation by

yeast, were successfully exploited in cost-effective biosynthesis of γ-PGA (Zhang

et al. 2012;Lietal. 2020). However, most often, direct and efﬁcient fermenta-

tion of lignocellulosic feedstock is unfeasible; pretreatments are generally required

for the utilization of the sugars therein contained. For this reason, bacterial feed-

stocks based on biomass hydrolysates are preferentially used. Hydrolysates of rice

straw or corncob ﬁbers have been tested for the convenient fermentation of γ-PGA

(Hassan et al. 2014; Tang et al. 2015; Zhu et al. 2013). Unfortunately, lignocellulosic

biomass hydrolysis often implies the use of polluting agents to break the recalci-

trant matrix. An environmentally sustainable alternative for hydrolysing biomasses

is the application of microbial degradative enzymes for the pretreatment of feed-

stock biomasses. Altun (2019) employed goose feathers from poultry processing

plants as source of protein hydrolysates; in this case, feathers were ﬁrst used as

the sole carbon and nitrogen source to produce keratinolytic enzymes by Strepto-

myces pactum. The lyophilized crude enzymatic extract was applied to obtain feather

hydrolysates, which was ﬁnally used as a cheap and renewable medium for γ-PGA

production (Altun 2019).

Also, short ﬁbres produced from paper linerboard recycling could be enzymati-

cally turned into fermentable sugars and later used as carbon source for the biosyn-

thesis of γ-PGA with yields compared to the one obtained by using glucose (Scheel

et al. 2019).

Many additional industrial wastes have been suggested for production of γ-PGA,

as well as of other biocommodities. All these approaches combine the valorisation of

industrial wastes for the creation of value-added products, thus introducing circularity

in the economic process.

6 The Applications of γ-PGA

γ-PGA is basically a long chain of glutamic acid residues joined by pseudopeptide

bonds; the composition in glutamic acid mainly in the D-form makes it biocompat-

ible, non-immunogenic and even edible; in fact, it is one of the main components of

natto and chungkookjang, traditional Japanese and Korean foods, respectively, made

from soybeans fermented with Bacillus subtilis species. The slow γ-PGA degrada-

tion proﬁle prevents inﬂammatory responses. Furthermore, microbial production is

sustainable and safe also for the environment, as the polymer is not only bioproduced

but also easily biodegradable.

The conﬁguration of the γ-amide bond confers to the polymer particular chemical-

physical characteristics that make it a valid anionic biomaterial in various application

Poly-γ-Glutamic Acid and Its Application in Bioremediation: A Critical … 217

ﬁelds (Ogunleye et al. 2014). However, the extreme viscosity of the high Mw chains

makes the polymer not easily manageable. The problem can be alleviated by random

reduction of the Mw by alkaline hydrolysis, sonication, or enzymatic degradation;

on the other hand, excessive size reduction limits some properties of the polymer,

which are dependent on the length of the chains (Shih and Van 2001).

The presence of several pending and negatively charged carboxyl groups in posi-

tion α profoundly affects the solubility of the polymer: in basic conditions the −

COOH groups are deprotonated, making the polymer hydrophilic, hygroscopic and

superabsorbent, and favouring the formation of γ-PGA salts in the presence of

metal ions; conversely, in acidic environments, the carboxylic groups are proto-

nated, making the molecule water-insoluble. Besides, these pending groups repre-

sent ideal reactive centres for conjugation and crosslinking of any type of molecules.

Thanks to the above properties, high Mw γ-PGA represents a versatile candidate

for various industrial applications, as attested by the massive literature accumulated

over the last few years. Biocompatibility has prompted several potential applica-

tions in the biomedical and food sectors. The long hydrophilic moiety of γ-PGA,

which is metabolized at a slow pace in mammals, represents a perfect drug carrier

to improve the administration of poorly soluble drugs. It can also be transformed in

various γ-PGA-based composites, including hydrogels, nanoﬁbers, and nanoparti-

cles for medical applications, such as biological adhesives or scaffolding materials for

tissue engineering; in these composites, the conjugated materials provide adhesion

or mechanical strength (Park et al. 2021). γ-PGA-nanoparticles have been explored

as substitute of adenoviral vectors for the delivery of anticancer agents (Khalil et al.

2018). In addition, the ability to chelate metal ions (e.g., magnesium or calcium), and

their slow release in the digestive tract, has been exploited to create highly effective

controlled-release mineral supplements for human and farm animals health.

Another well-developed ﬁeld in which γ-PGA has already found wide application

is processed food production; it is used as an additive to improve the rheological

properties of wheat gluten by increasing the water-holding capacity (Xie et al. 2020),

as viscosity enhancer for beverages, as cryoprotectant for frozen food, aging inhibitor,

texture enhancer or bitterness relieving agent. It also acts as preservative for probiotic

bacteria during freeze drying (Bhat et al. 2015).

γ-PGA can absorb water at an amount that is several hundred times higher than

its original weight without dissolving. The high-water absorbability can be further

increased by combining the polymer with other materials or by intramolecular

crosslinking, leading to the formation of hydrogels. The super-absorbent capacity

is the key property in cosmetics and personal care products, such as γ-PGA-based

moisturizer, exfoliating and anti-wrinkle creams. γ-PGA has been shown to enhance

skin elasticity more than collagen and hyaluronic acid (Lee et al. 2014); moreover,

it has been explored also in products as diapers and napkins, as substitute of more

toxic acrylates (Castrillon et al. 2019).

Another class of γ-PGA-based products which are already on the market are

fertilizers and other agricultural adjuvants. In these types of applications, γ-PGA is

considered an environmental-friendly fertilizer synergist for improving plant uptake

of nitrogen, phosphorous, and potassium; it is often provided together with the

218 V. Bontà and C. Calvio

producing organisms, as raw fermentation broth, thanks to the numerous positive

effects that Bacillus species have on vegetable growth, making them perfect Plant

Growth Promoting bacteria (PGPB) (Zhang et al. 2017).

Moreover, γ-PGA can also positively impact on soil quality indirectly, by

promoting water retention (Guo et al. 2021) and by sustaining the ﬂourishing of

the soil microﬂora (Xu et al. 2013).

7γ-PGA in Bioremediation

Bioremediation of contaminated matrices exploiting γ-PGA has been extensively

investigated by several authors. At the basis of its application in this ﬁeld lays

the ability to efﬁciently chelate and ﬂocculate polluting metal ions such as nickel,

copper, cadmium, cobalt, chromium, aluminium, uranium, arsenic or lead, as well as

various organic compounds. The basis for these property is the presence of multiple

negatively charged carboxyl groups in the polymer structure, that readily bind to

several environmental cations, forming both intra- and inter-molecular hydrogen

bonds favouring the formation of ﬂocks (McLean et al. 1990; Inbaraj et al. 2009).

7.1 Wastewater Treatment

Detection and removal of heavy metal ions in water is of paramount threats for the

planet and for human and animal health; however, the decontamination solutions

should not impose an additional threat to already endangered sites. Aluminium- or

polyacrylamide-based coagulants, often used in water treatment plants, have indeed

been linked to the development of neurodegenerative diseases (Bondy 2010; Pennisi

et al. 2013). Being safe and biodegradable, γ-PGA represents instead the ideal solu-

tion for the removal of pollutants from wastewater, in particular of heavy metals, as it

promotes the formation of small ﬂocs entrapping the metal ions, that readily agglom-

erate into larger and sedimentable particles, without leaving toxic traces behind. The

dynamics of complexes formation with bivalent lead ions at various concentrations

of both adsorber and ligands and the characteristics of the ﬂocks at different pH

values were deeply characterized (Bodnár et al. 2008). One of the requirements

for the activity of γ-PGA as adsorber and ﬂocculant is the maintenance of a pH

that allows the ionization of the −COOH groups, which must be neither below the

pKa of γ-PGA (4.09 according to Inbaraj et al. 2009), nor to basic, so that cation

exchange reactions can occur rapidly and efﬁciently. In fact, puriﬁed acidic γ-PGA,

which is insoluble in water, was shown to adsorb cesium from radioactive wastewater

more efﬁciently than the corresponding sodium salt, even if cesium is preferentially

bound with respect to sodium and calcium. Moreover, the adsorption equilibrium is

Poly-γ-Glutamic Acid and Its Application in Bioremediation: A Critical … 219

attained in very short time (Sakamoto and Kawase 2016). Indeed, commercial bio-

adsorbers containing γ-PGA, among other components, are already on the market

(http://www.poly-glusb.jp/basic.html) and their efﬁcacy was experimentally tested,

for example on wastewater from ethanol distillation, characterized by high levels

of organic and inorganic matter. Results showed that the use of γ-PGA combined

with sodium hypochlorite and sand ﬁlters removed about 70% of the turbidity and

reduced chemical oxygen demand by 79.5% (Carvajal-Zarrabal et al. 2012).

The ﬂocculation properties of γ-PGA can also be improved by coupling with

other materials. As an example, the sequential addition of chitosan and γ-PGA was

shown to substantially reduce the chemical oxygen demand from the wastewater of

a potato starch plant, subtracting nitrogen and phosphorus and improving turbidity.

The synergistic effect is thought to be linked to the ability of chitosan to neutralize

negative charges, thereby reducing electrostatic repulsion, while γ-PGA provides a

bridging function that promotes ﬂocculation. By using biodegradable components,

the sediments, rich in organic matter, could be classiﬁed as potential soil fertilizers

(Li et al. 2020). A super Cu2+ adsorber was developed by cross-linking γ-PGA onto

Pseudomonas putida cells displaying two recombinant metal-binding proteins on its

surface. The biocomposite biosorbent allowed the quantitative recovery of copper

from liquid matrices over a sufﬁciently varied range of pH and temperatures (Hu

et al. 2017).

However, there are completely different approaches in which the multiple proper-

ties of γ-PGA can be highly valuable in wastewater treatment, e.g., as biostimulant

for other microorganisms or plants directly involved in the bioremediation processes

(Wojtowicz et al. 2022).

For the bioremediation of trichloroethylene-contaminated groundwater sites,

reductive dechlorination can be carried out by anaerobic bacteria which use H2

as electron donor to replace chlorine atoms with hydrogens. A mixture containing

γ-PGA and emulsiﬁers, vitamins and a degreaser could be successfully injected in

contaminated sites to speed up bioremediation by acting at multiple levels: (i) as

physical adsorption agent for trichloroethylene; (ii) as slow-release carbon supple-

ment for the maintenance of anaerobic dechlorinators (i.e. as biostimulant); (iii) as

pH-stabilizer, thanks to the neutralizing effect of the amine groups released by the

degrading polymer (Luo et al. 2021).

The complexation with γ-PGA was also shown to improve the performances of

a catalytic dechlorination method based on the used palladium-doped zero-valent

iron nanoparticles. γ-PGA complexation stabilized and provided electrostatic and

steric repulsion to prevent particle aggregation due to attractive magnetic forces. The

positive role of γ-PGA was demonstrated by comparing the dechlorination activity

of naked and γ-PGA-complexed nanoparticles on chlorophenol as model pollutant

(Zhang et al. 2018).

In marine sediments treatments, a new role for γ-PGA was devised as biological

glue and protective agent to wrap oil-degrading bacteria adsorbed on solid zeolite

particles, thus preventing marine currents dispersion of the microbial species directly

responsible for the bioremediation of sediments contaminated by crude oil (Zhao et al.

2018).

220 V. Bontà and C. Calvio

Moreover, due to its highly efﬁcient cation-binding character, it can also be used

as biosensor of metal contamination. γ-PGA-stabilized gold nanoparticles have been

synthetized and used to sense trivalent chromium levels in aqueous solution, which

could be conveniently assessed via a colorimetric change occurring upon chromium

binding. The reported sensitivity is very high (up to 0.2 ppb) and the biosensor was

successfully tested in different liquid matrices (Yuan et al. 2020).

7.2 Soil Bioremediation

The suitability of γ-PGA properties in soil washing simulations has been poorly

explored. This is probably linked to the existence of several alternative soil remedia-

tion methods and to the technical problems in envisaging the scale up of soil-washing

procedures based on an expensive bio-adsorber as γ-PGA. The only real advantage

that the polymer offers with respect to similar conventional techniques relies on the

fact that upon γ-PGA washing, soil characteristics do not change, and the soil micro-

biota appears unaffected (Peng et al. 2020), which is a fundamental asset for future

restoration of degraded soil (Coban et al. 2022).

However, the metal-chelation properties of γ-PGA can be indirectly exploited

in contaminated soils. γ-PGA, applied to soil spiked with Cd and Pb, reduced the

growth inhibitory effects of those metal ions on cucumber seedlings by reducing

their bioavailability, as demonstrated by the lower metal ions content in the plant

tissue (Pang et al. 2018).

Moreover, in phytoremediation of hypersaline soil, the growth of two different

halophytes was shown to increase upon addition of γ-PGA solutions to the soil

(Mu et al. 2021). This effect is supposedly linked to the γ-PGA-mediated reduced

bioavailability of Ca2+ ,Mg

2+ and NO3−, the ions analysed in this study, which miti-

gates the salt stress thereby promoting plant growth. The increase in plant biomass,

in turn, led to a larger amount of ions adsorbed by the plants and, ultimately, to a

more efﬁcient phytoremediation of the hypersaline site (Mu et al. 2021).

Analogously to what described for wastewater treatments, γ-PGA can also be

used as biostimulant for the activity of microbial consortia able to degrade speciﬁc

pollutants. This strategy has been applied for the bioremediation of soil samples

contaminated by petroleum hydrocarbons by Wojtowicz and collaborators ( 2022).

An autochthonous degrading microbial consortium was introduced in soil derived

from a contaminated site either alone or together with γ-PGA, in a combined bioaug-

mentation and biostimulation approach. The rate of pollutants degradation over a

6-month period improved signiﬁcantly in the presence of the polymer with respect

to the non-biostimulated consortium (Wojtowicz et al. 2022). This approach has the

advantage of being potentially applicable in situ even to urbanized polluted sites.

Poly-γ-Glutamic Acid and Its Application in Bioremediation: A Critical … 221

8 Open Challenges and Perspectives

A large body of literature explored the propensity of γ-PGA to ﬂocculate several

cationic compounds rapidly and efﬁciently, a feature that has prompted a wide array

of research studies on its applicability in bioremediation. The efﬁciency of ion binding

is pH dependent, and the selectivity mainly depends on compounds’ relative concen-

tration and charge. However, the ﬁeld implementation of the lab-conceived applica-

tions is still far from being realized. γ-PGA, as a commercial product, is currently

too expensive for massive use in large wastewater and soil treatment plants.

γ-PGA appears more valuable as a slow-release carbon and nitrogen source for

the biostimulation of microbes degrading organic compounds, (Luo et al. 2021;

Zhang et al. 2018; Zhao et al. 2018). It has also shown to be applicable as

metals-immobilization agent to support phytoremediation techniques. However, its

biodegradability in this respect is a double-edged weapon: it makes it a perfectly

biologically safe immobilization agent, able to preserve and even improve soil quality,

but it does not guarantee the long-term stability of the immobilized metals, because of

the ubiquitous presence of viral, bacterial, and eukaryotic γ-PGA hydrolases. Experi-

mental proof of its actual survival in real natural environments is still missing, as most

of the data have been collected in simulated natural conditions (Pang et al. 2018).

The unstable nature of γ-PGA in an uncontrolled environment is also a major short-

coming when considering t he option of recycling it after desorption of the payload

through mild acidic conditions.

A still unexplored strategy to decrease γ-PGA-based bioremediation costs and,

concurrently, overcome its degradation might be represented by the use of γ-PGA-

producing bacteria grown in situ, alone or in combination with other suitable micro-

bial species to ensure a constant γ-PGA-based bioremediating action. Alterna-

tively, in order to stabilize γ-PGA molecules in the natural environment, conju-

gated molecules can be explored, decreasing the degradation rate and prolonging its

survival.

The high production costs may be overcome by exploring novel fermentation

strategies, for instance, bioﬁlm fermentation. This technique has been applied by

Moni and colleagues (2022) for the convenient production of proteases by Bacillus

subtilis (Moni et al. 2022). However, the suitability of this technique largely depends

on the speciﬁc nature of the substrate for bacterial growth, the aeration conditions

and the stability of the external conditions, therefore further investigations should be

done for its application in γ-PGA production.

In conclusion, γ-PGA is an attractive biopolymer with high potential in several

application ﬁelds. However, ﬁrst and foremost, the hopes placed in new overproducer

engineered strains and ideal fermentation conditions of low-cost recycled organic

wastes, must be fulﬁlled. Once these two conditions are met, solving the other issues,

as the one correlated to the stability of the polymer, would be downhill.

222 V. Bontà and C. Calvio

References

Altun M (2019) Bioproduction of γ-Poly (glutamic acid) using feather hydrolysate as a fermentation

substrate. Trak Univ J Nat Sci 20(1):27–34. https://doi.org/10.23902/trkjnat.448851

Appelbaum M, Schweder T (2021) Metabolic engineering of bacillus—new tools, strains, and

concepts. In: Nielsen J, Stephanopoulos G, Lee SY (eds) Metabolic engineering. https://doi.org/

10.1002/9783527823468.ch13

Bhat AR et al (2015) Improving survival of probiotic bacteria using bacterial poly-γ-glutamic acid.

Int J Food Microbiol 196:24–31. https://doi.org/10.1016/j.ijfoodmicro.2014.11.031

Birrer GA, Cromwick A, Gross RA (1994) γ-Poly (glutamic acid) formation by Bacillus licheni-

formis 9945a: physiological and biochemical studies. Int J Biol Macromol 16(5):265–275. https://

doi.org/10.1016/0141-8130(94)90032-9

Bodnár M et al (2008) Nanoparticles formed by complexation of poly-gamma-glutamic acid with

lead ions. J Hazard Mater 153(3):1185–1192. https://doi.org/10.1016/j.jhazmat.2007.09.080

Bondy SC (2010) The neurotoxicity of environmental aluminium is still an issue. Neurotoxicology

31(5):575–581. https://doi.org/10.1016/j.neuro.2010.05.009

Cai D et al (2018) Enhanced production of poly-γ-glutamic acid by improving ATP supply in

metabolically engineered Bacillus licheniformis. Biotechnol Bioeng 115(10):2541–2553. https://

doi.org/10.1002/bit.26774

Calvio C, Romagnuolo F, Vulcano F, Speranza G, Morelli CF (2018) Evidences on the role of

the lid loop of γ-glutamyltransferases (GGT) in substrate selection. Enzyme Microb Technol

114:55–62. https://doi.org/10.1016/j.enzmictec.2018.04.001

Candela T, Fouet A (2005) Bacillus anthracis CapD, belonging to the gamma-

glutamyltranspeptidase family, is required for the covalent anchoring of capsule to peptidoglycan.

Mol Microbiol 57(3):717–726. https://doi.org/10.1111/j.1365-2958.2005.04718.x

Carvajal-Zarrabal O, Nolasco-Hipólito C, Barradas-Dermitz DM, Hayward-Jones PM, Aguilar-

Uscanga MG, Bujang K (2012) Treatment of vinasse from tequila production using polyglutamic

acid. J Environ Manage 95. https://doi.org/10.1016/j.jenvman.2011.05.001

Castrillon N, Molina EM, Fu H, Roy A, Toombs J (2019) Super absorbent polymer replacement

for disposable baby diapers. https://doi.org/10.13140/RG.2.2.15095.98720

Coban O, De Deyn GB, Van der Ploeg M (2022) Soil microbiota as game-changers in restoration

of degraded lands. J Sci 375:6584. https://doi.org/10.1126/science.abe072

Ermoli F, Bontà V, Vitali G, Calvio C (2021) SwrA as global modulator of the two-component

system DegSU i n Bacillus subtilis. Res Microbiol 172(6):103877. https://doi.org/10.1016/j.res

mic.2021.103877

Fang J, Liu Y, Huan C, Xu L, Ji G, Yan Z (2020) Comparison of poly-γ-glutamic acid produc-

tion between sterilized and non-sterilized solid-state fermentation using agricultural waste as

substrates. J Clean Prod 255:120248. https://doi.org/10.1016/j.jclepro.2020.120248

Feng J et al (2015) Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by

modular pathway engineering. Metab Eng 32:106–115. https://doi.org/10.1016/j.ymben.2015.

09.011

Feng J et al (2017) Enhancing poly-γ-glutamic acid production in Bacillus amyloliquefaciens by

introducing the glutamate synthesis features from Corynebacterium glutamicum. Microb Cell

Fact 16(1):88. https://doi.org/10.1186/s12934-017-0704-y

Fujita KI et al (2021) Effect of pgsE expression on the molecular weight of poly (γ-glutamic acid)

in fermentative production. Polym J 53:409–414. https://doi.org/10.1038/s41428-020-00413-7

Guo J, Shi W, Li J, Zhai Z (2021) Effects of poly-γ-glutamic acid and poly-γ-glutamic acid super

absorbent polymer on the sandy loam soil hydro-physical properties. PLoS One 16(1):e0245365.

https://doi.org/10.1371/journal.pone.0245365

Harwood CR (1989) Introduction to the Biotechnology of Bacillus. In: Harwood CR (eds) Bacillus.

Biotechnology handbooks, vol 2. Springer, Boston, MA. https://doi.org/10.1007/978-1-4899-350

2-1_1

Poly-γ-Glutamic Acid and Its Application in Bioremediation: A Critical … 223

Hassan SHA et al (2014) Electricity generation from rice straw using a microbial fuel cell. Int J

Hydrog Energy 39(17):9490–9496. https://doi.org/10.1016/j.ijhydene.2014.03.259

Hsueh YH, Huang KY, Kunene SC, Lee TY (2017) Poly-γ-glutamic acid synthesis, gene regulation,

phylogenetic relationships, and role in fermentation. Int J Mol Sci 18(12):2644. https://doi.org/

10.3390/ijms18122644

Hu P et al (2017) Poly-γ-glutamic acid coupled Pseudomonas putida cells surface-displaying metal-

lothioneins: composited copper(ii) biosorption and inducible ﬂocculation in aqueous solution.

RSC Adv 7(30):18578–18587. https://doi.org/10.1039/C7RA01546A

Inbaraj BS, Wang JS, Lu JF, Siao FY, Chen BH (2009) Adsorption of toxic mercury (II) by an extra-

cellular biopolymer poly (gamma-glutamic acid). Bioresour Technol 100(1):200–207. https://

doi.org/10.1016/j.biortech.2008.05.014

Jang J, Cho M, Chun JH et al (2011) The poly-γ-D-glutamic acid capsule of Bacillus anthracis

enhances lethal toxin activity. Infect Immun 79(9):3846–3854. https://doi.org/10.1128/IAI.011

45-10

Khalil IR et al (2018) Poly-Gamma-Glutamic Acid (γ-PGA)-based encapsulation of adenovirus

to evade neutralizing antibodies. Molecules 23(10):2565. https://doi.org/10.3390/molecules231

02565

Kocianova S et al (2005) Key role of poly-γ-dl-glutamic acid in immune evasion and virulence of

Staphylococcus epidermidis. J Clin Invest 115(3):688–694. https://doi.org/10.1172/JCI23523

Lee NR et al (2014) In vitro evaluation of new functional properties of poly-γ-glutamic acid

produced by Bacillus subtilis D7. Saudi J Biol Sci 21(2):153–158. https://doi.org/10.1016/j.sjbs.

2013.09.004

Li M et al (2020) Treatment of potato starch wastewater by dual natural ﬂocculants of chitosan and

poly-glutamic acid. J Clean Prod 264. https://doi.org/10.1016/j.jclepro.2020.121641

Luo Z, Guo Y, Liu J, Qiu H, Zhao M, Zou W, Li S (2016) Microbial synthesis of poly-γ-glutamic

acid: current progress, challenges, and future perspectives. Biotechnol Biofuels 9:134. https://

doi.org/10.1186/s13068-016-0537-7

Luo SG, Chien CC, Sheu YT, Verpoort F, Chen SC, Kao CM (2021) Enhanced bioremediation

of trichloroethene-contaminated groundwater using modiﬁed γ-PGA for continuous substrate

supplement and pH control: batch and pilot-scale studies. J Clean Prod 278. https://doi.org/10.

1016/j.jclepro.2020.123736

Mamberti S et al (2015) γ-PGA hydrolases of phage origin in bacillus subtilis and other microbial

genomes. PLoS One 10(7):e0130810. https://doi.org/10.1371/journal.pone.0130810

Massaiu I et al (2019) Integration of enzymatic data in Bacillus subtilis genome-scale metabolic

model improves phenotype predictions and enables in silico design of poly-γ-glutamic acid

production strains. Microb Cell Fact 18:3. https://doi.org/10.1186/s12934-018-1052-2

McLean RJ, Beauchemin D, Clapham L, Beveridge TJ (1990) Metal-binding characteristics of the

gamma-glutamyl capsular polymer of bacillus licheniformis ATCC 9945. Appl Environ Microbiol

56(12):3671–3677. https://doi.org/10.1128/aem.56.12.3671-3677.1990

Moni R, Khan AAN, Islam Z, Zohora US, Rahman MS (2022) Bioﬁlm fermentation: a propitious

method for the production of protease enzyme by bacillus subtilis RB14. Ind Biotechnol 18(1).

https://doi.org/10.1089/ind.2021.0016

Mu Y et al (2021) Phytoremediation of secondary saline soil by halophytes with the enhancement

of γ-polyglutamic acid. Chemosphere 285. https://doi.org/10.1016/j.chemosphere.2021.131450

Nair P, Navale GR, Dharne MS (2021) Poly-gamma-glutamic acid biopolymer: a sleeping giant

with diverse applications and unique opportunities for commercialization. Biomass Conv Bioref.

https://doi.org/10.1007/s13399-021-01467-0

Nakano MM, Zuber P (1998) Anaerobic growth of a “strict aerobe” (Bacillus subtilis). Ann Rev

Microbiol 52(1):165–190. https://doi.org/10.1146/annurev.micro.52.1.165

Ogunleye A, Bhat A, Irorere VU, Hill D, Williams C, Radecka I (2014) Poly-γ-glutamic acid—

production, properties and applications. Microbiology 6. https://doi.org/10.1099/mic.0.081448-0

224 V. Bontà and C. Calvio

Ohsawa T, Tsukahara K, Ogura M (2009) Bacillus subtilis response regulator DegU is a direct acti-

vator of pgsB transcription involved in gamma-poly-glutamic acid synthesis. Biosci Biotechnol

Biochem 73(9):2096–2102. https://doi.org/10.1271/bbb.90341

Osera C, Amati G, Calvio C, Galizzi A (2009) SwrAA activates poly-gamma-glutamate synthesis

in addition to swarming in Bacillus subtilis. Microbiology (reading) 155(7):2282–2287. https://

doi.org/10.1099/mic.0.026435-0

Pang X, Lei P, Feng X, Xu Z, Xu H, Liu K (2018) Poly-γ-glutamic acid, a bio-chelator, alleviates

the toxicity of Cd and Pb in the soil and promotes the establishment of healthy Cucumis sativus

L. seedling. Environ. Sci. Pollut Res Int 25(20):19975–19988. https://doi.org/10.1007/s11356-

018-1890-9

Park SB, Sung MH, Uyama H, Han DK (2021) Poly (glutamic acid): production, composites, and

medical applications of the next-generation biopolymer. Prog Polym Sci 113:101341. https://doi.

org/10.1016/j.progpolymsci.2020.101341

Park C et al (2005) Synthesis of super-high-molecular-weight poly-γ-glutamic acid by Bacillus

subtilis subsp. chungkookjang. J Mol Catal B Enzym 35(4–6):128–133. https://doi.org/10.1016/

j.molcatb.2005.06.007

Peng YP, Chang YC, Chen KF, Wang CH (2020) A ﬁeld pilot-scale study on heavy metal-

contaminated soil washing by using an environmentally friendly agent-poly-γ-glutamic acid

(γ-PGA). Environ Sci Pollut Res Int 27(28):34760–34769. https://doi.org/10.1007/s11356-019-

07444-5

Pennisi M, Malaguarnera G, Puglisi V, Vinciguerra L, Vacante M, Malaguarnera M (2013) Neuro-

toxicity of acrylamide in exposed workers. Int J Environ Res Public Health 10(9):3843–3854.

https://doi.org/10.3390/ijerph10093843

Ramaswamy S, Rasheed M, Morelli CF, Calvio C, Sutton BJ, Pastore A (2018) The structure of

PghL hydrolase bound to its substrate poly-γ-glutamate. FEBS J 285:4575–4589. https://doi.org/

10.1111/febs.14688

Sakamoto S, Kawase Y (2016) Adsorption capacities of poly-γ-glutamic acid and its sodium salt

for cesium removal from radioactive wastewaters. J Environ Radioact 165:151–158. https://doi.

org/10.1016/j.jenvrad.2016.10.004

Scheel RA, Fusi AD, Min BC, Thomas CM, Ramarao BV, Nomura CT (2019) Increased production

of the value-added biopolymers poly(R-3-Hydroxyalkanoate) and poly (γ-Glutamic acid) from

hydrolyzed paper recycling waste ﬁnes. Front Bioeng Biotechnol 7. https://doi.org/10.3389/fbioe.

2019.00409

Scoffone V, Dond D, Biino G, Borghese G, Pasini D, Galizzi A, Calvio C. Knockout of pgdS and ggt

genes improves γ-PGA yield in B. subtilis. Biotechnol. Bioeng., 110: 2006–2012. 2013. https://

doi.org/10.1002/bit.24846.

Scorpio A et al (2007) Poly-γ-glutamate capsule-degrading enzyme treatment enhances phagocy-

tosis and killing of encapsulated bacillus anthracis. Antimicrob Agents Chemother 51(1):215–

222. https://doi.org/10.1128/AAC.00706-06

Shah MP (2020) Microbial Bioremediation & Biodegradation. Springer

Shah MP (2021a) Removal of refractory pollutants from wastewater treatment plants. CRC Press

Shah MP (2021b) Removal of emerging contaminants through microbial processes. Springer

Shih IL, Van YT (2001) The production of poly- (gamma-glutamic acid) from microorganisms

and its various applications. Bioresour Technol 79:207–225. https://doi.org/10.1016/s0960-852

4(01)00074-8

Srivatsan A et al (2008) High-precision, whole-genome sequencing of laboratory strains facilitates

genetic studies. PLoS Genet 4(8):e1000139. https://doi.org/10.1371/journal.pgen.1000139

Stanley NR, Lazazzera BA (2005) Deﬁning the genetic differences between wild and domestic

strains of Bacillus subtilis that affect poly-γ-dl-glutamic acid production and bioﬁlm formation.

Mol Microbiol 57:1143–1158. https://doi.org/10.1111/j.1365-2958.2005.04746.x

Su Y et al (2010) Improved poly-gamma-glutamic acid production by chromosomal integration of

the Vitreoscilla hemoglobin gene (vgb) in Bacillus subtilis. Bioresour Technol 101(12):4733–

4736. https://doi.org/10.1016/j.biortech.2010.01.128

Poly-γ-Glutamic Acid and Its Application in Bioremediation: A Critical … 225

Sung MH, Park C, Kim CJ, Poo H, Soda K, Ashiuchi M (2005) Natural and edible biopolymer

poly-gamma-glutamic acid: synthesis, production, and applications. Chem Rec 5(6):352–366.

https://doi.org/10.1002/tcr.20061. PMID: 16278834

Tamang JP, Watanabe K, Holzapfel WH (2016) Review: diversity of microorganisms in global

fermented foods and beverages. Front Microbiol 7. https://doi.org/10.3389/fmicb.2016.00377

Tang B et al (2015) Highly efﬁcient rice straw utilization for poly-(γ-glutamic acid) production by

Bacillus subtilis NX-2. Bioresour Technol 193:370–376. https://doi.org/10.1016/j.biortech.2015.

05.110

Wojtowicz K, Steliga T, Kapusta P, Brzeszcz J, Skalski T (2022) Evaluation of the effectiveness of

the biopreparation in combination with the polymer γ-PGA for the biodegradation of petroleum

contaminants in soil. Mater Lett 15(2):400. https://doi.org/10.3390/ma15020400

Wu Q, Xu H, Xu L, Ouyang P (2006) Biosynthesis of poly (gamma-glutamic acid) in Bacillus

subtilis NX-2: regulation of stereochemical composition of poly (gamma-glutamic acid). Process

Biochem 41:1650–1655. https://doi.org/10.1016/j.procbio.2006.03.034

Xie X et al (2020) Effect of poly-γ-glutamic acid on hydration and structure of wheat gluten. J Food

Sci 85(10):3214–3219. https://doi.org/10.1111/1750-3841.15400

Xu ZQ et al. Effect of poly (glutamic acid) on microbial community and nitrogen pools of soil. Acta

Agric. Scand. B Soil Plant Sci. 63:657–668. https://doi.org/10.1080/09064710.2013.849752

Yuan X, Zhou B, Li M Shen M, Shi X (2020) Colorimetric detection of Cr3+ ions in aqueous solution

using poly (γ-glutamic acid)-stabilized gold nanoparticles. Anal Methods 12:3145–3150. https://

doi.org/10.1039/D0AY00842G

Zhang et al (2012) High-level exogenous glutamic acid-independent production of poly-(γ-glutamic

acid) with organic acid addition in a new isolated Bacillus subtilis C10. Bioresour Technol

116:241–246. https://doi.org/10.1016/j.biortech.2011.11.085

Zhang L et al (2017) Effects of poly-γ-glutamic acid (γ-PGA) on plant growth and its distribution

in a controlled plant-soil system. Sci Rep 7:6090. https://doi.org/10.1038/s41598-017-06248-2

Zhang et al (2018) Poly (γ-Glutamic acid) promotes enhanced dechlorination of p-Chlorophenol

by Fe-Pd nanoparticles. Nanoscale Res Lett 13:219. https://doi.org/10.1186/s11671-018-2634-y

Zhao G, Sheng Y, Wang C, Yang J, Wang Q, Chen L (2018) In situ microbial remediation of

crude oil-soaked marine sediments using zeolite carrier with a polymer coating. Mar Pollut Bull

129(1):172–178. https://doi.org/10.1016/j.marpolbul.2018.02.030

Zhu F, Cai J, Zheng Q, Zhu X, Cen P, Xu Z (2013) A novel approach for poly-γ-glutamic acid

production using xylose and corncob ﬁbres hydrolysate in Bacillus subtillis HB-1. J Chem Technol

Biotechnol 89:616–622. https://doi.org/10.1002/jctb.4169

Metagenomics Analysis of Extremophiles

and Its Potential Use in Industrial Waste

Water Treatment

Ashok Kumar Shettihalli, Saisha Vinjamuri, S. Divijendra Natha Reddy,

Renu Pai, and Prathibha Narayanan

Abstract Industrial waste water treatment is signiﬁcant in managing the water crisis

being faced globally. Though number of physical and chemical technologies have

been developed for waste water treatment, there are challenges being faced with

respect to the cost of implementation and removal of pollutants. Use of enzymes from

mesophilic organisms for the treatment of toxic pollutants is cost-effective but limited

due to their narrow range of stability. Alternatively, enzymes from extremophiles

show a broader range of stability under extreme environmental conditions and can

aid in effective biodegradation of heavy metals. The recent advances in metagenomics

and meta transcriptomics analysis gives information on operational responses of these

extremophiles to environmental disturbances which is crucial in optimizing waste

water treatment processes on a large scale in bioreactors. This chapter gives insight

on how these new and advanced approaches can result in design of novel enzymes

for potential application in waste water treatment.

Keywords Waste water treatment ·Enzymatic treatment ·Metagenomics ·

Extremophiles ·Meta transcriptomics

1 Introduction

The remarkable ability of certain organisms to thrive under extreme environmental

conditions make them very attractive prospect of their usage for various biotechnolog-

ical applications. These are classiﬁed as extremophiles having the ability to survive

at extreme conditions such as high or low temperature (Elleuche et al. 2015), acidic

or basic pH (Aguilera 2013;Luísetal. 2022; Qiu et al. 2021; Cavicchioli et al. 2002;

Shah 2021a, b; Struvay and Feller 2012) high or low pressure (Ichiye 2018; Jin et al.

2019; Canganella and Wiegel 2011; Qiu et al. 2021). Predominantly, microbes espe-

cially various types of bacteria have this ability to survive under extreme conditions

A. K. Shettihalli · S. Vinjamuri · S. Divijendra Natha Reddy · R. Pai · P. Narayanan (B

)

Department of Biotechnology, B.M.S College of Engineering, Bull Temple Road, Basavanagudi,

Bengaluru 560019, India

e-mail: prathibhan.bt@bmsce.ac.in

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_12

227

228 A. K. Shettihalli et al.

(Qiu et al. 2021; Fenice et al. 2021; Shah 2020; Van den Burg 2003; Elleuche et al.

2015). These extremophiles provide unique opportunity of exploiting their proteins

or enzymes for various applications (Elleuche et al. 2015) as these products can func-

tion in suboptimal conditions such as extremes of temperatures, pH and toxicity of

the reaction conditions. Unfortunately, most of the extremophiles are not culturable

and very few of them actually grow into viable organisms in the lab. This makes

possible loss of certain important enzymes or proteins that may have potential utility

for biotechnological applications. Metagenomics is an approach of identifying the

genome features of organisms especially microbes that are not culturable (Cowan

et al. 2015; Escuder-Rodríguez et al. 2018; Verma et al. 2021; Liu et al. 2021).

The traditional metagenomic approach involves isolation of the DNA from a given

sample that is representative of all organisms in that sample, fragmentation of DNA,

adapters ligation and cloning into suitable vectors for creation of genomic library

and sequencing of the library (clones) and annotation or identifying the genomes

by assembling them to reference genomes or homologous searching against genome

databases. This process is tedious and lengthy in terms of time, labour and cost.

However, with the advent of next generation sequencing, the DNA sequencing is

straightforward after adapter ligation to DNA fragments, it is fast and less expensive

(Fig. 1). After high throughput sequencing, obtained sequences can be assembled

to many genomes as there is availability of many reference genomes and very efﬁ-

cient sequences assembling bioinformatics tools. From this metagenomic approach,

it is possible to identify enzymes or protein products that are capable of functioning

normally under sub optimal conditions.

Water is the universal solvent and many large-scale industries require constant

supply of water for various reaction conditions and treatment. Unfortunately, the by-

product of the industrial process is the release of large amount of spent water with

enormous amount of toxic chemicals and other undesirable entities. As the release of

Fig. 1 Metagenomics approach for using next generation sequencing

Metagenomics Analysis of Extremophiles and Its Potential Use … 229

untreated industrial waste water into the surrounding environment is prohibited by

industrial regulations that are in place, various methods of treating the industrial waste

water already are under use. However, many of these methods suffer from inherent

issues and not cost effective. Usage of biological agents is actively being considered

and are being utilized or under consideration for use (Sharma et al. 2021). As the

industrial water is stuffed with lot of toxic impurities, usually at high temperature

with pH extremes, most of the biological agents or protein products such as enzymes

may not actively function and the process could be slow and expensive. In this

context, as extremophiles thrive under extremes of temperature, pH and salinity,

either themselves or their products such as proteins and enzymes make an attractive

prospect for treating industrial waste water. In this chapter, we provide the utility of

extremophiles and their products, identiﬁcation of them by metagenomic approach

and their usage in the treatment of industrial waste water.

2 Extremophiles and Their Characteristic Features

Extremophiles are mostly prokaryotic microorganisms that thrive in extreme condi-

tions. The conditions in which the existence of life seems impossible are the places

where extremophiles lives. These organisms can sustain and are viable in places

that impose high pressure, temperature and pH. Some of them survive using the

nutrients that are considered toxic and some can withstand high amounts of radia-

tion and salinity. There are few more that can survive vacuum and extremely xeric

conditions. As contrast to extremotophiles, extremotolerants can tolerate extreme

condition for certain period of time but they grow optimally only at normal condi-

tions. They are sometimes called moderate extremophiles. Sometimes extremophiles

have shown to utilize more than one extreme condition to survive. These are called

as poly extremophiles. For instance, the microorganisms that dwell on deep sea

ﬂoor and trenches live under extreme hydro pressure and cold temperature. The

organisms have successfully adapted their structure and metabolism to these harsh

conditions to their advantage. These characters of adaptation can be harvested for

working in areas which have potentially a high risk involved like radioactive sites,

industrial waste management sites, mines, etc. Extremophiles has been elaborately

reviewed with reference to their physiology and genetics in the past (Seckbach and

Stan-Lotter 2013).

2.1 Organisms

Extremophiles mostly consist of Archaean microorganisms. They also include

eukaryotes such as protozoa, algae, fungi, bacteria and even some multicellular

organisms.

230 A. K. Shettihalli et al.

Archean

Archaean’s being obligate anaerobes can live in oxygen free environment. Their

adaptability makes them ﬂourish in almost all of the extreme conditions that are

known. They grow in the most acidic, basic, thermophilic and halophilic environ-

ments. Methanopyrus kandleri strain 116 (Takai et al. 2008) is a methanogen, that

grows in deep sea can be cultured at high temperature of about 122 °C and high

pressure of 20 MPa.

Protozoa

These being mostly parasitic, are found with algae and bacteria that are present in

hypersaline conditions. For example, Trachelocerca conifer, Metacystis truncata &

Chilophrya utahensis are found in the brackish lagoons. Algae-Microalgae sustain

and live in most of the extreme conditions. Oscillatoria terebriformis is the blue-

green algae found in hot springs, at about 70 °C. The temperature tolerance of these

algae is due to the presence of homo polar bonds in their proteins.

Fungi

Eutypella sp. D-1 (Lu et al. 2014) have been isolated from soil of high altitude Arctic

region. Cladosporium sphaeospermum showed increased viability in the Chernobyl

Nuclear Power plant (Dadachova et al. 2007). Cryomyces antarcticus is the toughest

eukaryotic organism, known as black fungi, is known to survive solar radiation,

desiccation, extreme polar coldness and dryness (Onofri et al. 2007).

Bacteria

These are the most versatile among all the other extremophiles. Serpentinomonas sp.

B1 can grow in highly basic pH of 11–12.5 (Suzuki et al. 2014) While Halarsenat-

ibacter silvermanii SLAS-1 uses extreme salinity of 35% of Soda Lake in USA and

grows, it is Planococcus halocryophilus Or1 that survive a freezing temperature of

−15 °C. Pyrococcus yayanosii (Birrien et al. 2011), an obligate barophile, grows on

ocean ﬂoor, at a pressure of 50–52 MPa. Deinococcus radiodurans can survive grave

radiation exposure of 5000 Gy without losing viability. It is known as the world’s

toughest bacterium as it can live even after exposure to extreme cold, radioactive

rays, desiccation, acidic condition and also in vacuum.

Metagenomics Analysis of Extremophiles and Its Potential Use … 231

Multicellular Organisms

Tardigrades, one of the most interesting species of multicellular organisms, are actu-

ally extremophiles. Their presence is reported in hot springs, deep sea, polar regions

and also in outer space.

2.2 Characteristics and Adaptations

Acidophiles

Acidophiles are those that thrive in environment with extremely acidic pH. Some of

the most acidic environments found on earth are at the mining sites and geothermal

springs. For the extremophiles to survive in the extremely acidic pH (nearing 0), the

cytoplasmic pH at around 6.0 should be maintained. This is achieved by increasing

the activity of the pathway that pushes the excess cytoplasmic protons out of the

cell or by consuming it. The pathways that express the proteins for outward proton

pumping is active in these organisms (Krulwich and Pandan 2011). They also keep

the organic acid from moving out of the cells by degrading them within the cytoplasm.

Alkaliphiles

Alkaliphiles are the extremophiles that live in extremely basic pH. The Soda Lake

in USA and Lake Turkana in Kenya are major alkali lakes in the world. With pH

range of 8.5–10, these lakes have a unique ecological condition. The organisms that

thrive in these conditions, the alkaline challenges are faced by enhancing the Na+/H+

antiporter activity. These antiporter proteins also hold a pH sensor domain that results

in considerably large fold increase in ion exchange. They can also secrete the organic

acids like acetic acid and lactic acid to change the immediate surrounding pH (Zhang

et al. 2016).

Halophiles

Halophiles are those extremophiles that survive high salt concentration. Salinibacter

ruber and Halobacterium salinarum are among the extreme halophiles that grow

around 20–23% salinity ranges of The Great Salt Lake. To escape the loss of water due

to high salt concentration, these organisms have developed two approaches. Firstly,

they can equilibrate their salt concentration to reach the same level as that of external

environment by salt accumulation. These involves the action of Cl−/K+ pumps,

rhodopsins (Adamiak et al. 2015). The organisms accumulate K+ or Cl− in their

cytoplasm. Secondly, they produce organic solutes known as osmolytes from external

environment that protects the proteins from denaturation (Saum and Müller 2008).

232 A. K. Shettihalli et al.

Thermophiles

Thermophiles are those that ﬂourish in very high temperatures. Being one of the

most important physical attributes for an organism to sustain, thermophiles breach

the human boundaries and grow in some of the most hostile temperature condi-

tions. The minimum temperature at which the microbes can live is reported by

bacteria Deinococcus geothermalis DSM1130 at −25 °C (Frösler et al. 2017) and

the maximum by archaea Geogemma barossii at 130 °C (Kasheﬁ and Loveley

2003). The proteins necessary for their survival have shown to be adapted by two

means. The archaean’s show a structure-based stability as they have evolved from

a hyperthermophilic habitat. Whereas the bacteria show sequence-based stability

which is achieved by less compaction and additional salt bridges (Berezovsky and

Shakhnovich 2005).

Psychrophiles

Psychrophiles are those that are opposed to thermophiles and survive in very low

temperature. They have adapted membrane fatty acids with LC-PUFAs in them

which serves as antioxidant. They have polar carotenoids as membrane pigments that

increases ﬂuidity of the membrane. The anti-freeze proteins (also called ice struc-

turing or thermal hysteresis proteins) are non-colligative biological antifreezes. These

can bind to ice and inhibit ice growth and recrystallization, thereby decreasing the

freezing point resulting in reduced ice growth (Voets 2017). They prevent ice recrys-

tallization as they keep a liquid environment around the cell (Raymond). They are also

equipped with anti-nucleating proteins, a large membrane bound protein, which initi-

ates heterogeneous ice formation (Lorv et al. 2014). Biosurfactant like glycolipids

and phospholipids produced in these organisms helps in movement across the cells.

They have very effective and efﬁcient DNA/RNA chaperons that prevent misfolding

and stabilize the DNA and RNA secondary structure (Collins and Margesin 2019).

Radiophiles

Radiophiles, as the name suggests, grows well in radioactive contaminated soils.

The two types of radiation that affect the functioning of the cell are ionizing and

non-ionizing radiations. The ionizing radiations, like gamma rays, causes double

stranded breaks in the DNA and also damage proteins and lipids. To overcome

these adversaries, the microorganism has developed precise and fast DNA repair

mechanism (Pavlopoulou et al. 2016) and condensed nucleoid. They prevent protein

damage by suppressing ROS production cell cleaning functions and selective protein

protection. In case of non-ionizing radiations like UV rays, the DNA damage is subtle,

by pyrimidine dimer formation causing photolesions. They have photoreactivation

genes and numerous other repair strategies that help to overcome the damage (Jones

Metagenomics Analysis of Extremophiles and Its Potential Use … 233

and Baxter 2017). They also employ gene duplication and carotenoids and superoxide

dismutases formation for their protection from continuous exposure to radiation.

Barophiles

Barophiles are the organisms that live under high pressure. They are also called

as pzeiophiles. They colonize and grow under high hydrostatic pressure (HHP),

usually in great depths of the ocean. The cytoplasmic membrane in these organism

gets compacted and becomes impervious to water. To increase the ﬂuidity of the

membrane, unsaturated fatty acids are packed more. The porins like OmpH/L and

proteins ToxR/S act as pressure sensors in these organisms. Their ribosomes are

stabilized by extended loops at regions (Oger and Jebbar 2010). Barophiles also

includes organisms that survive in low pressures present on mountains and outer

space, with a pressure range of 0.0033–10–13 MPa. They develop bioﬁlms around

them or undergo spore formation to protect itself (Horneck et al. 2010).

Metallophiles

Metallophiles which can survive in high concentration of heavy metals. Most of these

are chemolithoautotrophs. They are found in acidic environments especially in the

mines, industrially polluted sites, volcanic eruption sites and the hydrothermal vents.

They use heavy metals like cadmium, cobalt, copper, lead, mercury, nickel, zinc and

so on for their survival. Their ability to solubilize metals has been successfully used in

bio mining applications (Rohwerder et al. 2003). These organisms produce bioﬁlms

around their consortium which protects the organism layer beneath and also helps in

horizontal gene transfer among them. They have a capsule that protects cell and an

efﬁcient proton efﬂux system along biomolecules repair mechanisms allows these

organisms survive the harsh environment (Hu et al. 2020).

But not all the extremophiles that have been isolated from these harshest envi-

ronments can be cultured. Isolation and culturing of extremophiles have completely

different challenges. Isolating and identifying an extremophile is easier. But when it

comes to culturing, the organisms that survive at the extreme ends of the nutrition or

physical condition spectra, have been hard to culture.

3 Bioprospecting of Extremozymes in Industrial

Wastewater treatment

Industrial waste water has high percentage of heavy metals and other toxic

compounds which has to be treated before discharging to landﬁlls and water bodies

or reusing for agriculture and industrial processes. Conventionally, a series of phys-

ical and chemical techniques are followed in industries for waste water treatment

234 A. K. Shettihalli et al.

to meet the standards required before its discharge or reuse. But these methods

have several limitations which includes high expenditure, production of by-products

during the treatment process which could be harmful and not so adequate heavy

metals removal efﬁciency. The alternative approach which minimises these disad-

vantages is enzymatic treatment. Enzymes have unique properties such as high speci-

ﬁcity and stability, faster reaction by reducing the activation energy and relatively low

process costs. Enzymes obtained from extremophiles are known as extremozymes

and they have the potential to contribute signiﬁcantly to the global enzyme market in

the near future. The beneﬁts of using extremozymes is that they can catalyse r eactions

even in harsh environment. However, the challenge is to create such harsh conditions

in the industrial laboratories to grow extremophiles for production of extremozymes.

Additionally, the biomass yield of extremophiles is very low relative to mesophylls

(Sysoev et al. 2021). Due to all these hurdles, there is very little research with respect

to structure, function, properties and applications of extremozymes.

3.1 Extremozymes Having Potential in Industrial Waste

Water Treatment

Some of the extremozymes known to have capacity to degrade and remove heavy

metals and toxic compounds from waste water are discussed below.

Nitrilase

Nitrilases have wide applications concerning compound exterior surface changes,

sewage treatment and formation of carboxylic acids. Nitrilase converts nitrile

compounds to carboxylic acid and releasing ammonia by acting on carbon–nitrogen

triple bond. Extremozyme Nitrilases have been isolated from wide variety of bacteria,

fungi, yeast and plants. Extremozyme Nitrilases relatively have faster biotransfor-

mation, high substrate solubility, and reduced viscosity and contamination (Gunjal

Aparna et al. 2021).

Peroxidases

These enzymes degrade by oxidizing phenolic compounds, polychlorinated

biphenyls (PCBs) compounds, hydrocarbons and dyes. Lignin peroxidases and

manganese peroxidases are commonly used enzymes which act by breakup of alpha

and beta carbon bonds, oxidation of phenolic groups and benzyl alcohols and hydrox-

ylation of benzylic methylene groups. Lignin peroxidases requires hydrogen peroxide

for its catalytic reactions. Manganese peroxidases accelerates the conversion process

of Mn2+ to Mn3+ (Gunjal Aparna et al. 2021).

Metagenomics Analysis of Extremophiles and Its Potential Use … 235

Lipases

Lipases are utilized in the biodegradation of oil, proteins and salts present in waste

water from dairy and tannery industries (Gunjal Aparna et al. 2021).

Laccases

Laccase belongs to class of oxidoreductase enzymes. Extremozyme laccase are

very stable in extracellular ﬂuids. Some investigations observed the use of laccase

in degradation of polymers esp. polycyclic aromatic hydrocarbons (PAHs). PAHs

accumulate as a result of incomplete combustion of organic matter and automo-

bile exhausts. They are extremely detrimental due to their carcinogenic properties.

Laccase catalyse by oxidation or decarboxylation of substrates which can be phenols,

phenolic compounds or lignin and reducing oxygen to water and carbon dioxide. The

main source of laccases are white rot fungi and molds. Furthermore, laccases can

degrade xenobiotic and non-phenolic compounds. Another important application of

extremozyme laccase from alkali-halotolerant bacteria is in degradation of azo dyes

in textile industry waste waters which is a threat to aquatic life and human beings

(Gunjal Aparna et al. 2021).

Pectinases

Pectin is abundantly present in fruits. Apparently, waste water f rom fruit processing

industries has high concentration of pectin as by-product. This in turn can affect

treatment as pectin can produce methane which can interfere in activated sludge

process. Studies have shown Alkalophilic Bacillus spp. produces pectinase enzyme

which is used for pre-treatment of waste water to remove pectin before water enters

the activated sludge tank (Gunjal Aparna et al. 2021).

Catalase

Hydrogen peroxide is used often in textile and semiconductor industries. It can

cause severe damage to the existence of marine life if waste water containing

hydrogen peroxide is discharged without treatment to water bodies. Catalase enzymes

effectively removes hydrogen peroxide from efﬂuents (Gunjal Aparna et al. 2021).

Cellulases

Cellulases from alkaliphilic organism Bacillus sp. ﬁnds applications in textile

industry where indigo dyes are used as colouring agent. The waste dye gets released

into efﬂuents. Cellulase enzyme help in removal of stains and dyes from waste water.

236 A. K. Shettihalli et al.

There are three groups of cellulases which acts in different ways. Endoglucanases

converts cellulose ﬁber into free chain ends, exoglucanase separates cellobiose units

and β-glucosidase forms glucose (Gunjal Aparna et al. 2021).

Esterases

The esterases belong to the hydrolase group and hydrolases ester containing

compounds such as carbamates, organophosphates etc. and used in degradation

of xenobiotic and various other toxic compounds like cypermethrin, sulfosul-

furon and ﬁpronil. The esterases extremozyme are isolated from the thermophilic

microorganisms (Gunjal Aparna et al. 2021).

Tyrosinases

Efﬂuents from coal, plastic, resin manufacturing, and petroleum reﬁning industries

has huge content of harmful phenolic compounds which can hinder the activated

sludge process. These enzymes catalyse oxidization of phenols and convert to useful

products like amino acids (Gunjal Aparna et al. 2021).

Dioxygenases

Dioxygenases oxidizes aromatic compounds into aliphatic products. They ﬁnd appli-

cations in detoxiﬁcation in response to oil spills in oceans (Gunjal Aparna et al. 2021).

Monooxygenases

Monooxygenases are also used for biotransformation of aromatic compounds

and hydrocarbons. They act by stereo selectivity on different types of substrates

which are applied as reducing agents. One such commercial enzyme is methane

monooxygenases. (Gunjal Aparna et al. 2021).

4 Metagenomics Analysis

The extremity of survival conditions of organisms has been expanding ever since

man began to isolate and culture them. The microorganisms were isolated from

every possible habitat on Earth and from space. They were cultured on different

growth enrichment media to get pure cultures and ﬁnally identiﬁed them by various

genomic studies. This led to ﬂow of information regarding a speciﬁc organism and

its genomic content. The cultured organisms were also used for proteome analysis

Metagenomics Analysis of Extremophiles and Its Potential Use … 237

and thereby its structural and functional uniqueness. But the organisms that survive

at the extreme end of the physicochemical spectra were still left out as they were very

hard to culture. There were communities of organisms that grew in close proximities

forming a complex consortium that could adapt instantly to changing environments.

Studying these microorganisms was a distant vision until genome sequencing was

developed and used in the study of genomes.

The idea of “metagenomics” was ﬁrst conceptualized by Handelsman et al. (1998),

where they envisioned the idea of sequencing of collection of genes from a milieu

and study them in a way that was similar to that of study of a single genome. In Chen

and Pachter (2005), Unviersity of California researchers, deﬁned metagenomics as

“the application of modern genomics technique without the need for isolation and lab

cultivation of individual species”. Metagenomics uses shot gun or PCR based genome

sequencing techniques to analyze a collection of genes of an entire community of

organisms that dwells in a particular environment.

Metagenomics involves two primary studies. It begins with isolation of metage-

nomic DNA followed by PCR ampliﬁcation of molecular markers with taxonomic

values. If it is a bacterial genome 16S rRNA gene ampliﬁcation is done and if

fungi then 18S rRNA gene ampliﬁcation is accomplished. The results are carefully

analyzed to construct library. This involves reading short metagenomic sequences

and join them to form contigs which in turn are clustered in a process called binning to

form classes. The sequences thus formed are analyzed by either simple or multiple

alignment method. The relationship between the protein and the source family is

built. And ﬁnally, protein structure prediction and phylogenetic analysis is carried

out. The metagenomics analysis is elaborately studied in the recent past with new and

sophisticated software which can be read in papers by Prayogo et al. 2020, Garlapati

et al. 2019, Lorenz and Eck (2005).

4.1 Structural Metagenomics

Structural metagenomics involves the study of the characters of the microbial

community. The relationship between components in the community, helps in

understanding about the ecology and biological functioning of the community.

The methods consist of assembly, binning and taxonomic proﬁling via community

analysis (Jimenez et al. 2012).

4.2 Functional Metagenomics

Functional metagenomics concentrate on the genes that code for a particular protein

and ﬁnding its application in the industry. The protein structure predicted after

metagenomic analysis is used for the characterization of the protein like its activity,

optimum temperature and pH, etc.

238 A. K. Shettihalli et al.

4.3 DNA Extraction

Metagenomic analysis needs entire genetic compliment to be cloned to construct

libraries. For this process the need for intact DNA is essential. Therefore, DNA extrac-

tion and puriﬁcation is a primary and crucial step which involves careful sampling

and minimum transportation time (Felczykowska et al. 2015).

Water Habitat

Seas and oceans that covers majority of earth are also the source of some extreme

microbes that can be beneﬁcial for man. There have metagenomics studies mostly on

the surface water inhabiting micro-organisms as underground water with oligotrophic

environment shows less diversity. Most of the projects are dedicated to marine water

environment, but there is also focus on the lagoons, lakes, river beds, hot water

springs and also frozen water that is in the form of ice.

Concentration of the sample is important to get desirable amount of DNA. The

large volumes of water should be centrifuged to acquire sufﬁcient biomass. The

water can or/and should be ﬁltered using different ﬂow ﬁlter system equipped with

ﬁlters of appropriate pore size to r etain the target microbes. The cell lysis can be

accomplished using enzymatic, detergent, high temperature or mechanical treatment.

The pre-requisite of sequencing is that the sample should be as pure as possible.

Thus, after extracting DNA from water, it is important to remove all the chemical

and enzymatic contaminants. C-TAB has shown good puriﬁcation effects on the

isolated DNA (Ranjan et al. 2005). Sometimes even after concentrating large volumes

of water, amount of DNA extracted will be much smaller than expected. In such a

scenario, DNA can be ampliﬁed using linker ampliﬁed shotgun library (LASL) (Kim

and Bae 2011) or by multiple displacement ampliﬁcation using random hexamers

and ϕ29 polymerase, a bacteriophage DNA polymerase.

Soil Habitat

One of the most challenging habitats, soil, has highest concentrations of microbes.

There are commercially available DNA isolation kits for the process. But it has been

reported that manual isolation gives substantial quality DNA (Tanveer et al. 2016).

The extraction from soil can be direct wherein the DNA is isolated in situ (Robe et al.

2003) or indirect where the microbes are segregated from the soil ﬁrst and then the

DNA is isolated (Hu et al. 2010).

Both in direct and indirect extraction, the access to the nucleic acid come after

the cell lysis. The means by which the cell lysis takes place may differ from source

to source. Some of them require physical disruption like bead beating sonication,

vortexing, homogenization, thermal treatments, etc. In case of direct extraction, the

cell lysis buffer is added to the soil sample and physical treatments are placed to

Metagenomics Analysis of Extremophiles and Its Potential Use … 239

act after which the soil aggregates are separated. In case of indirect extraction, the

microbes are ﬁrst separated from the soil and then the cell lysis is done. Soil is

separated from the sample using centrifugation or density gradient centrifugation.

Centrifugation is employed to separate the heavier soil impurities at low speed

ﬁrst and then to sediment the microbial community from the supernatant at higher

speed. Density gradient centrifugation is a less efﬁcient alternate to separate soil

from the community. It is used in the case of very challenging high clay soil. The

lysis buffer used for extraction contains chemicals like SDS, PEG, sodium deoxy-

cholate, etc. Puriﬁcation involves complete removal of soil and humic acid. The

charge characteristics of humic acids are similar to DNA, as a result gets precipitated

along with it. Humic acid can be quantiﬁed with UV absorbance at 230 nm as DNA

shows absorbance at 260 nm. Puriﬁcation can be accomplished using precipitation

with potassium acetate, isopropanol and ethanol. To get high quality puriﬁed nucleic

acid sample, the puriﬁcation can be continued using Sephadex gel ﬁltration or ion

exchange chromatography. Sometimes Cesium chloride gradient centrifugation is

done to isolate a relatively pure product.

Sludge Habitat

Waste water milieu hosts diverse microbes that are of much interest and signiﬁ-

cance. The ﬁrst reason being the health of humans depends on safe drinking water

and secondly to understand function of the microbes in foaming, nitriﬁcation, etc.

(Yadav and Kapley 2019). The waste water contains different pollutants like less toxic

domestic sewage and toxic wastes from the industries. To carry out a successful

DNA extraction these pollutants should be removed. Larger insoluble impurities

are removed by mechanical separation. In the second step the dissolved organic

compounds are removed by biological treatments with the help of microorganisms.

In the third step the inorganic waste, mainly nitrates and phosphates produced during

second step, are removed. Alternately, the commercial kits for extraction of DNA

from soil can also be used in DNA preparation (Arumugam et al. 2021).

Human Body Microbiome

The most common human microbiome samples used in metagenomic analysis is

the human faeces. To extract DNA, modiﬁed cetyltrimethylammonium bromide–

polyethylene glycol (CTAB) phenol:chloroform extraction protocol is mostly used.

The CTAB is used along with phenol:chloroform to lyse the cells and separate protein

from the sample in a bead beating process. Chloroform, linear acrylamide and PEG

are used for puriﬁcation of the DNA (Sui et al. 2020).

240 A. K. Shettihalli et al.

4.4 16S rRNA Sequencing

Sanger sequencing is the conclusive sequencing approaches that can be used for

the analysis. But because of its labour-intensive process and high cost, newer and

much cheaper Next-Generation Sequencing is being used. The Illumina/Solexa and

454/Roche are the most predominantly used systems (Shuikan et al. 2019).

454/Roche clonally ampliﬁes the DNA fragments attached to beads by emulsion

polymerase chain reaction. This is then placed in picotitre plate and sequenced in

parallel pyrosequencing that is, all the four deoxy ribonucleotides which gets incor-

porated by DNA polymerase when complementary. The polymerization releases

pyrophosohates resulting in light beam which is measured. A charged coupled device

camera converts the light to the sequence of the template. In the Illumina/Solexa

NGS (Pérez-Cobas et al. 2020), random DNA fragments are immobilized on a solid

surface. Then PCR ampliﬁcation is done and the sequences are r ead. This is a highly

efﬁcient, fast system with yields as high as 60Gbp.

4.5 Assembly

In this step, based on the desired outcome like need for functional information or

determining genome, the short sequence reads are assembled to develop longer

contigs. Assembly of sequences can be either reference based or de novo. The

reference-based assembly can used when the sequences in the contigs are similar

to the datasets that are already available. De novo assembly is put to use when the

conserved sequences do not match to the existing datasets. The de novo approach

works with either OLC graphs or de-bruijn graph. With respect to input reads, OLC

graph provides more information than de-bruijn, but they also require more compu-

tational resources. In case of de-bruijn, the graphs are simpler and more arguments

can be added to analyze the contigs more efﬁciently (Rizzi et al. 2019). It is to be

noted that de novo type of assembly requires complex computational strategies along

with expertise in the ﬁeld.

4.6 Binning

The organization of the contigs into different bins depending on its composition and

gene present is termed as binning. Taxonomic dependent binning relies on the fact that

the genomes contain conserved set of nucleotides that can be exploited to segregate

the sequences. But in case of unknown DNA fragments the similarity is searched for

the genes encoded by the genome independently, without any reference, to classify

them. This is called as taxonomic independent binning or genome binning. Genome

binning can be either based on the assumption that features like essential single copy

Metagenomics Analysis of Extremophiles and Its Potential Use … 241

genes, %G + C, and nucleotide frequency are similar in same genome (Sangwan

et al. 2016) or on the hypothesis that abundance of sequences of same genome have

parallel abundance in the sample taken and sequences of same species have similar

abundance in multiple samples (Sedlar et al. 2017), or a hybrid of both the strategies

(Alneberg et al. 2014). There are numerous algorithms that are available to cluster

the sequences into respective genomes that are discussed by Yue et al. (2020).

4.7 Annotation

The identiﬁcation of the genes, which is feature prediction, is structural annota-

tion, and it’s supposed function done to predict the taxonomic neighbour comprises

functional annotation. Structural annotations need the identiﬁcation of the reading

frames, coding and non-coding regions, start/stop codons, regulatory motifs and

splicing sites. Depending on the outcome of the structural annotation we can predict

the functional annotation of a gene like its biochemical and biological functions, its

expression and the pathways that are regulated by the products. The tools required for

annotation can be studied in the reviews given by Dong and Strous (2019), Tamames

et al. (2019).

Every stage in the metagenomic analysis requires efﬁciency and precision. The

loss of DNA due to fragmentation or the process itself may result in inaccuracy of

the sequence that is read in 16 s rRNA sequencing. Shorter reads and contigs are not

entertained during the binning process. Most of the work after DNA extraction and

ampliﬁcation are completed with the computational algorithm and software. But the

in-depth familiarity about the sequences, genomes and thereby its lineage is very

much essential for the successful completion of the analysis. Although these anal-

yses show optimistic results, it is worth the hardship only if there is any productive

outcome like industrial application. The sequenced genes upon use should yield a

product that can be used in industries to increase yield or decrease the environmental

damage caused by the industrial activity. Research in waste water management from

commercial, industrial as well as domestic areas are need of the hour. The metage-

nomic analysis of the extremophiles and/or their gene products that can clean the

waste water is necessary to stop the already depleting fresh water reservoir from

being unable to use.

5 Optimization of Waste Water Treatment Using

Metagenomics and Meta Transcriptomic Analysis

Waste water treatment plants (WWTP) are essential for efﬁcient management of

water resources. The efﬁciency of these plants depends on the type of treatment

being carried out (Ma et al. 2011; Saikaly et al. 2005; Tchobanoglous et al. 2003).

242 A. K. Shettihalli et al.

The activated sludge aeration basin is the heart of WWTP‘s and its functionality is

dependent on a wide range of conditions. One of the major reasons which effect the

overall waste water treatment process is the change in microbial population in the

activated sludge basin during different stages of treatment & operational parameters.

The variation in the population of the microorganisms is also due to the varying

environmental/climatic conditions (Saikaly et al. 2005). Hence, an understanding

of the varied activities of the microbiome present during the different processes of

waste water treatment would aid in developing an efﬁcient treatment strategy.

Traditionally, culture-based assays are used to detect the microorganisms,

however, they are not highly efﬁcient and fail to identify the pathogenic organisms

at low concentrations and are time consuming. Second generation sequencing (SGS)

technologies help in detecting, identifying and quantifying population of microbes

including those which are un-culturable by traditional methods. SGS like 16S r DNA

sequencing and whole metagenome sequencing techniques are widely being used for

determination of microbial populations in waste water treatment plants. Ilumina plat-

forms and Pyrosequencing techniques (high throughput) are widely adapted for SGS

due to their low error rates and greater data output compared to other platforms. Sana-

pareedy et al. (2009), Zhang et al. (2012) have reported the use of pyro sequencing

techniques to determine the microbial population in the activated sludge of waste

water treatment plants.

Buettner and Noll (2018) reported the various microbial communities present in

anaerobic digesters of biogas plants and sewage treatment plants (STP’s) using 16 S

rDNA sequencing. They reported a greater diversity of microbial population in STP

than in the anaerobic digesters. El Chakhtoura et al. (2015) reported the variation

in microbial population within the water distribution network by using 16S r DNA

sequencing methods. They observed an abundance of rare taxa Viz, Nitrospirae,

Acidobacteria and Gemmatimonadetes.

16S rDNA encodes for the highly conserved 16S RNA subunit of bacterial and

archaeal ribosomes and the phylogeny of the organism can be established based

on the degree of similarities of these sequences. The hyper variable regions (HVR)

ﬂanked by conserved sequences present in the 16S rDNA can be easily ampliﬁed

and identiﬁed by PCR. The different groups of microorganisms can be identiﬁed

based on the composition of the HVR and the number of reads per group gives the

relative abundance of those microbes in a given sample. However, the accuracy of

identiﬁcation and classiﬁcation by this method decreases at species level. Taxonomic

assignment at species level is therefore recommended for those HVR sequences

that match exactly with a reference data base. Identiﬁcation at species level with

conserved gene markers and indels can also be also done with species speciﬁc PCR

after 16S rDNA sequencing.

An alternative to 16S rDNA sequencing is whole-metagenome s equencing

(WMS). The WMS sequences do not require primers and are taken from the frag-

mented genomes of the entire population. Hence can be used to identify unchar-

acterized sequences unlike in 16S rDNA sequencing. WMS can also be used for

identiﬁcation of metabolic pathways of functional signiﬁcance.

Metagenomics Analysis of Extremophiles and Its Potential Use … 243

Metagenomic analysis enumerates the entire population of the organism’s present

and can aid in identifying pathogenic microbial species, antibiotic resistance genes,

microbial species which hinder the treatment process like blooms of mycolic acid

producing bacteria which result in increased foaming. Further comparative analysis

of metagenomic and meta-transcriptomic datasets could help in determining the rela-

tive metabolic activities of the different microbial populations. Several researchers

have used the metagenomic approaches to study the changes in microbial populations

in anaerobic sludge digesters. They have identiﬁed the changes in microbial commu-

nity at functional level. WMS analysis can give insight into the overall population of

microbes at species level as well as the metabolic and functional activities involving

the various interactions of the microbial population at a given time. WMS does not

involve any ampliﬁcation bias and can be used for detection and quantiﬁcation of

pathogens and other microorganisms of low abundance.

6 Design of Novel Enzymes from Extreme Environments

Using Metagenomic Analysis for Effective

Bioremediation

To survive in extreme ecological niches contaminated with pesticides, aromatic

hydrocarbons, heavy metals and nuclear wastes, microorganisms have developed

several physiological adaptations. These microorganisms are potential source of

enzymes for environmental bioremediation. The scanty information on the factors

inﬂuencing the microbial growth under stressed environments is t he major bottle-

neck for the isolation of these novel biomolecules from these extreme habitats. For

the effective utilization of novel biomolecules for bioremediation depends on the

deeper understanding on the microbial physiology, metabolic capabilities, factors

inﬂuencing the composition and role of indigenous microorganism. Metagenomics

is an emerging approach used to study in detail structure, physiology and metabolic

processes of these novel microorganisms. The metagenomics studies in conjunc-

tion with high-throughput sequencing technologies are used for the isolation and

characterization of novel biomolecules used in environmental bioremediation.

As more than 99% of microorganisms in the environment are uncultivable,

substantial portion of microbial community has remained unexplored for their poten-

tial applications. The new method of study has been used for the identiﬁcation of

the novel microbial communities based on the 5S and 16S rRNA gene without

culturing the microorganism. The information required for culturing of the uncul-

tivable microorganisms is obtained from the sequencing of 16S rRNA gene. The

metagenomics approach whereby the extracted whole DNA from the sample is

sequenced using the various next generation sequencing platforms such as SOLiD,

Oxford NanoPore, Illumina and PacBio has revolutionized the studies in search of

novel biomolecules (Handelsman et al. 1998). The insights given by the metagenome

analysis of indigenous microbes can be used for designing the optimum conditions

244 A. K. Shettihalli et al.

required for the isolation of uncultured potential microbes with novel biomolecules

(Lorenz et al. 2002; Prosser 2015). Both gene centric and genome centric metage-

nomics approach is used to ﬁnd the presence of new taxa with metabolic and func-

tional genes and to reconstruct the complete genome respectively from the sequence

data obtained from the environmental DNA.

In addition to acceleration of in situ bioremediation using metagenomics, rate of

decontamination is enhanced either by bioaugmentation with the addition of whole

cells or by biostimulation with the addition of rate limiting nutrients (Nikolopoulou

and Kalogerakis 2009). The molecular tools based phylogenetic studies revealed

that representation of cultured bacteria is only <1% of the bacterial diversity and

the great majority of the microorganisms are unculturables or yet to be cultured

which can be harnessed for the isolation of novel enzymes with the right culture

conditions. To tap the substantial reservoir of unseen natural diversity containing

microbial community with potential novel enzymes, collective genomes of all the

organisms of that particular habitat is used for the metagenomics approach where

the analysis of the 16S rRNA gene reveals the diversity, physiology and metabolic

requirements of microbial communities of a given habitat. Currently the hypervari-

able regions (V1–V9) reﬂecting the signiﬁcant sequence variation among bacteria

are used for sequencing and identiﬁcation by the researchers. Moreover, the struc-

tural and physiological information obtained from the metagenomics analysis of the

indigenous microbes can be used for designing the culture conditions including the

media f or the isolation of microorganisms with novel enzymes (Singh and Gabani

2011; Jeon et al. 2009).

Metagenomic approach involves the extraction of DNA from the environmental

sample, restriction digestion, cloning of DNA fragments in a suitable vector (plasmid,

cosmid, fosmid or BAC vectors), transformation of the engineered vector into a suit-

able host followed by the screening of the clones in a DNA library for a particular

function using either “function based” or “sequence based” approach. The function-

based screening involves selection of clones expressing desired traits followed by

the characterization of genes and encoded products. The sequence-based screening

is the widely accepted method which involves the generation of metagenomics DNA

library, sequencing, assembly of the raw data into longer contigs and identiﬁcation

of the protein sequences by aligning database against reference databases such as

NCBI-NR or NCBIRefseq. Further, this database can be used for gene synthesis,

codon optimization and biochemical characterization of identiﬁed enzymes. With

the advent of next generation sequencing (NGS) in conjunction with bioinfor-

matics tools, sequence-based screening has facilitated the isolation, identiﬁcation

and characterization of several novel enzymes of industrial importance (Knietsch

et al. 2003).

Using function-based approach, the screening of metagenomic libraries enabled

in the identiﬁcation of genes encoding novel lipases, proteases, novel phosphodi-

esterase, alcohol oxidoreductase, cellulase, chitinase, dehydratase, β-lactamase and

amylases.

Metagenomics Analysis of Extremophiles and Its Potential Use … 245

As the environmental pollution is a serious global concern, several microorgan-

isms especially bacteria which produce novel catabolic enzymes are used for biore-

mediation to transform or degrade the pollutants. The metagenomics is a promising

ﬁeld which can be effectively used to access the untapped microbial resources for

effective bioremediation. Whereas the earlier microbiological processes for the iden-

tiﬁcation of the novel molecule was labour and cost intensive, metagenomics is time

saving and cost-effective technology for the same. The high-throughput sequencing

technology and functional screening of metagenomics libraries immensely help the

mankind for the detection of novel biocatalysts for effective bioremediation. The

huge data set obtained from the metagenomic analysis can aid in discovering the

potentially novel biomolecules.

7 Conclusion

Biological waste water treatment plants host a diverse population of microorganisms

which is dynamic in nature. The variations in the microbial consortia are dependent

on the environmental and process conditions occurring during the different stages of

waste water treatment. Integrated omics technologies comprising of metagenomic

analysis, transcriptomic analysis and metabolomic analysis has an immense appli-

cation in the identiﬁcation and characterization of these microbial communities.

In recent years, these technologies have been used to understand the diversity in

microbial population during waste water treatments. The knowledge of this varia-

tion in the microbial consortia at different stages of waste water treatment can help

in designing effective treatment strategies as well as help in identifying pathogenic

microorganisms and antimicrobial resistance over a broad range.

Acknowledgements We thank Ram N. Manas, currently third year student of BE Biotech-

nology, BMS College of Engineering, Bangalore, India, for his efforts in creating Fig. 1, depicting

metagenomics approach for using next generation sequencing, for this chapter.

References

Adamiak J, Otlewska A, Gutarowska B (2015) Halophilic microbial communities in deteriorated

buildings. World J Microbiol Biotechnol 31(10):1489–1499. https://doi.org/10.1007/s11274-015-

1913-3

Aguilera A (2013) Eukaryotic organisms in extreme acidic environments, the Río Tinto case. Life

3:363–374. https://doi.org/10.3390/life3030363

Alneberg J, Bjarnason BS, De Bruijn I, Schirmer M, Quick J, Ijaz UZ, Lahti L, Loman NJ, Andersson

AF, Quince C (2014) Binning metagenomic contigs by coverage and composition. Nat Methods

11(11):1144–1146. https://doi.org/10.1038/nmeth.3103

246 A. K. Shettihalli et al.

Arumugam K, Bessarab I, Haryono MA, Liu X, Zuniga–Montanez RE, Roy S, Qiu G, Drautz–

Moses DI, Law YY, Wuertz, S. and Lauro, F.M. (2021) Recovery of complete genomes and

non-chromosomal replicons from activated sludge enrichment microbial communities with long

read metagenome sequencing. NPJ bioﬁlms and microbiomes, 7(1):1–13. https://doi.org/10.1038/

s41522-021-00196-6

Berezovsky IN, Shakhnovich EI (2005) Physics and evolution of thermophilic adaptation. Biophys

Comput Biol 102(36):12742–12747. https://doi.org/10.1073/pnas.0503890102

Birrien JL, Zeng X, Jebbar M, Cambon-Bonavita MA, Quérellou J, Oger P, Bienvenu N, Xiao

X, Prieur D (2011) Pyrococcus yayanosii sp. nov., an obligate piezophilic hyperthermophilic

archaeon isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 61(12):2827–

2881. https://doi.org/10.1099/ijs.0.024653-0

Buettner C, Noll M (2018) Differences in microbial key players in anaerobic degradation between

biogas and sewage treatment plants. Int Biodeterior Biodegr 133:124–132. https://doi.org/10.

1016/j.ibiod.2018.06.012

Canganella F, Wiegel J (2011) Extremophiles: from abyssal to terrestrial ecosystems and possibly

beyond. Naturwissenschaften 98:253–279. https://doi.org/10.1007/S00114-011-0775-2

Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR (2002) Low-temperature extremophiles

and their applications. Curr Opin Biotechnol 13:253–261. https://doi.org/10.1016/S0958-166

9(02)00317-8

Chen K, Pachter L (2005) Bioinformatics for whole-genome shotgun sequencing of microbial

communities. PLoS Comput Biol 1(2):e24. https://doi.org/10.1371/journal.pcbi.0010024

Collins T, Margesin R (2019) Psychrophilic lifestyles: mechanisms of adaptation and biotech-

nological tools. Appl Microbiol Biotechnol 103(7):2857–2871. https://doi.org/10.1007/s00253-

019-09659-5

Cowan DA, Ramond JB, Makhalanyane TP, de Maayer P (2015) Metagenomics of extreme

environments. Curr Opin Microbiol 25:97–102. https://doi.org/10.1016/j.mib.2015.05.005

Dadachova E, Bryan RA, Huang X, Moadel T, Schweitzer AD, Aisen P, Nosanchuk JD, Casadevall

A (2007) Ionizing radiation changes the electronic properties of melanin and enhances the growth

of melanized fungi. PLoS One 2(5):e457. https://doi.org/10.1371/journal.pone.0000457

Dong X, Strous M (2019) An integrated pipeline for annotation and visualization of metagenomic

contigs. Front Genet 10:999. https://doi.org/10.3389/fgene.2019.00999

El-Chakhtoura J, Prest E, Saikaly P et al (2015) Dynamics of bacterial communities before and

after distribution in a full-scale drinking water network. Water Res 74:180–190. https://doi.org/

10.1016/j.watres.2015.02.015

Elleuche S, Schäfers C, Blank S et al (2015) Exploration of extremophiles for high tempera-

ture biotechnological processes. Curr Opin Microbiol 25:113–119. https://doi.org/10.1016/j.mib.

2015.05.011

Escuder-Rodríguez JJ, Decastro ME, Cerdán ME et al (2018) Cellulases from thermophiles found

by metagenomics. Microorganisms 6(3):66. https://doi.org/10.3390/microorganisms6030066

Felczykowska A, Krajewska A, Zieli ´nska S, Ło´s JM (2015) Sampling, metadata and DNA

extraction-important steps in metagenomic studies. Acta Biochimica Polonica 62(1):151–160.

https://doi.org/10.18388/abp.2014_916

Fenice M, Khare SK, Gorrasi S (2021) Editorial: mining, designing, mechanisms and applications of

extremophilic enzymes. Front Microbiol 12:709377. https://doi.org/10.3389/fmicb.2021.709377

Frösler J, Panitz C, Wingender J, Flemming HC, Rettberg P (2017) Survival of Deinococcus geother-

malis in bioﬁlms under desiccation and simulated space and Martian conditions. Astrobiology

17(5):431–447. https://doi.org/10.1089/ast.2015.1431

Garlapati D, Charankumar B, Ramu K, Madeswaran P, Murthy R (2019) A review on the applications

and recent advances in environmental DNA (eDNA) metagenomics. Rev Environ Sci Bio/technol

18(3):389–411. https://doi.org/10.1007/s11157-019-09501-4

Gunjal Aparna B, Waghmode Meghmala S, Patil Neha N (2021) Role of extremozymes in bioreme-

diation. Res J Biotechnol 16(3):240–252. https://www.researchgate.net/publication/342821710_

Role_of_Extremozymes_in_Bioremediation_A_Review

Metagenomics Analysis of Extremophiles and Its Potential Use … 247

Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM (1998) Molecular biological access

to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol

5(10):245–249. https://doi.org/10.1016/s1074-5521(98)90108-9

Horneck G, Klaus DM, Mancinelli RL (2010) Space microbiology. Microbiol Mol Biol Rev

74(1):121–156. https://doi.org/10.1128/MMBR.00016-09

Hu W, Feng S, Tong Y, Zhang H, Yang H (2020) Adaptive defensive mechanism of bioleaching

microorganisms under extremely environmental acid stress: advances and perspectives.

Biotechnol Adv 42:107580. https://doi.org/10.1016/j.biotechadv.2020.107580

Hu Y, Liu Z, Yan J, Qi X, Li J, Zhong S, Yu J, Liu Q (2010) A developed DNA extraction method

for different soil samples. J Basic Microbiol 50(4):401–407. https://doi.org/10.1002/jobm.200

900373

Ichiye T (2018) Enzymes from piezophiles. Semin Cell Dev Biol 84:138–146. https://doi.org/10.

1016/J.SEMCDB.2018.01.004

Jeon JH, Kim JT, Kim YJ, Kim HK, Lee HS, Kang SG, Kim SJ, Lee JH (2009) Cloning and char-

acterization of a new cold-active lipase from a deep-sea sediment metagenome. Appl Microbiol

Biotechnol 81:865–874. https://doi.org/10.1007/s00253-008-1656-2

Jimenez DJ, Andreote FD, Chaves D, Montaña JS, Osorio-Forero C, Junca H, Zambrano MM,

Baena S (2012) Structural and functional insights from the metagenome of an acidic hot spring

microbial planktonic community in the Colombian Andes. PLoS One 7(12):e52069. https://doi.

org/10.1371/journal.pone.0052069

Jin M, Gai Y, Guo X et al (2019) Properties and applications of extremozymes from deep-sea

extremophilic microorganisms: a mini review. Mar Drugs 17(12):656. https://doi.org/10.3390/

md17120656

Jones DL, Baxter BK (2017) DNA repair and photoprotection: mechanisms of overcoming envi-

ronmental ultraviolet radiation exposure in halophilic archaea. Front Microbiol 8:1882. https://

doi.org/10.3389/fmicb.2017.01882

Kasheﬁ K, Lovley DR (2003) Extending the upper temperature limit for life. Science 301(5635):934.

https://doi.org/10.1126/science.1086823

Kim KH, Bae JW (2011) Ampliﬁcation methods bias metagenomic libraries of uncultured single-

stranded and double-stranded DNA viruses. Appl Environ Microbiol 77(21):7663–7668. https://

doi.org/10.1128/AEM.00289-11

Knietsch A, Bowien S, Whited G, Gottschalk G, Daniel R (2003) Identiﬁcation and characterization

of coenzyme B12-dependent glycerol dehydratase—and diol dehydratase—encoding genes from

metagenomic DNA libraries derived from enrichment cultures. Appl Environ Microbiol 69:3048–

3060. https://doi.org/10.1128/AEM.69.6.3048-3060.2003

Krulwich TA, Sachs G, Padan E (2011) Molecular aspects of bacterial pH sensing and homeostasis.

Nat Rev Microbiol 9(5):330–343. https://doi.org/10.1038/nrmicro2549

Liu YX, Qin Y, Chen T et al (2021) A practical guide to amplicon and metagenomic analysis of

microbiome data. Protein Cell 12:315–330. https://doi.org/10.1007/S13238-020-00724-8

Lorenz P, Eck J (2005) Metagenomics and industrial applications. Nat Rev Microbiol 3(6):510–516.

https://doi.org/10.1038/nrmicro1161

Lorenz P, Liebeton K, Niehaus F, Eck J (2002) Screening for novel enzymes for biocatalytic

processes: accessing the metagenome as a resource of novel functional sequence space. Curr

Opin Biotechnol 13:572–577. https://doi.org/10.1016/s0958-1669(02)00345-2

Lorv JS, Rose DR, Glick BR (2014) Bacterial ice crystal controlling proteins. Scientiﬁca. Article

ID 976895. https://doi.org/10.1155/2014/976895

Lu XL, Liu JT, Liu XY, Gao Y, Zhang J, Jiao BH, Zheng H (2014) Pimarane diterpenes from the

Arctic fungus Eutypella sp. D-1. J Antibiot 67(2):171–174. https://doi.org/10.1038/ja.2013.104

Luís AT, Córdoba F, Antunes C et al (2022) Extremely acidic eukaryotic (Micro) organisms: life

in acid mine drainage polluted environments—mini-review. Int J Environ Res Public Health

19(1):376. https://doi.org/10.3390/ijerph19010376

248 A. K. Shettihalli et al.

Ma Y, Wilson CA, Novak JT, Riffat R, Aynur S, Murthy S, Pruden A (2011) Effect of various

sludge digestion conditions on sulfonamide, macrolide, and tetracycline resistance genes and

class I integrons. Environ Sci Technol 45(18):7855–7861. https://doi.org/10.1021/es200827t

Nikolopoulou M, Kalogerakis N (2009) Biostimulation strategies for fresh and chronically polluted

marine environments with petroleum hydrocarbons. J Chem Technol Biotechnol 84:802–807.

https://doi.org/10.1002/jctb.2182

Oger PM, Jebbar M (2010) The many ways of coping with pressure. Res Microbiol 161(10):799–

809. https://doi.org/10.1016/j.resmic.2010.09.017

Onofri S, Selbmann L, De Hoog GS, Grube M, Barreca D, Ruisi S, Zucconi L (2007) Evolution

and adaptation of fungi at boundaries of life. Adv Space Res 40(11):1657–1664. https://doi.org/

10.1016/j.asr.2007.06.004

Pavlopoulou A, Savva GD, Louka M, Bagos PG, Vorgias CE, Michalopoulos I, Georgakilas AG

(2016) Unraveling the mechanisms of extreme radioresistance in prokaryotes: lessons from nature.

Mutation Res/Rev Mutation Res 767:92–107. https://doi.org/10.1016/j.mrrev.2015.10.001

Pérez-Cobas AE, Gomez-Valero L, Buchrieser C (2020) Metagenomic approaches in microbial

ecology: an update on whole-genome and marker gene sequencing analyses. Microbial Genom

6(8). https://doi.org/10.1099/mgen.0.000409

Prayogo FA, Budiharjo A, Kusumaningrum HP, Wijanarka W, Suprihadi A, Nurhayati N (2020)

Metagenomic applications in exploration and development of novel enzymes from nature: a

review. J Gen Eng Biotechnol 18(1):39. https://doi.org/10.1186/s43141-020-00043-9

Prosser JI (2015) Dispersing misconceptions and identifying opportunities for the use of ‘omics’

in soil microbial ecology. Nat Rev Microbiol 13:439–446. https://doi.org/10.1038/nrmicro3468

Qiu J, Han R, Wang C (2021) Microbial halophilic lipases: a review. J Basic Microbiol 61:594–602.

https://doi.org/10.1002/jobm.202100107

Ranjan R, Grover A, Kapardar RK, Sharma R (2005) Isolation of novel lipolytic genes from uncul-

tured bacteria of pond water. Biochem Biophys Res Commun 335(1):57–65. https://doi.org/10.

1016/j.bbrc.2005.07.046

Rizzi R, Beretta S, Patterson M, Pirola Y, Previtali M, Della Vedova G, Bonizzoni P (2019) Overlap

graphs and de Bruijn graphs: data structures for de novo genome assembly in the big data era.

Quant Biol 7(4):278–292. https://doi.org/10.1007/s40484-019-0181-x

Robe P, Nalin R, Capellano C, Vogel TM, Simonet P (2003) Extraction of DNA from soil. Eur J

Soil Biol 39(4):183–190. https://doi.org/10.1016/S1164-5563(03)00033-5

Rohwerder T, Gehrke T, Kinzler K, Sand W (2003) Bioleaching review part A. Appl Microbiol

Biotechnol 63(3):239–248. https://doi.org/10.1007/s00253-003-1448-7

Shah Maulin P (2020) Microbial bioremediation & biodegradation. Springer

Shah Maulin P (2021a) Removal of refractory pollutants from wastewater treatment plants. CRC

Press

Shah Maulin P (2021b) Removal of emerging contaminants through microbial processes. Springer

Saikaly PE, Stroot PG, Oerther DB (2005) Use of 16S rRNA gene terminal restriction fragment anal-

ysis to assess the impact of solids retention time on the bacterial diversity of activated sludge. Appl

Environ Microbiol 71(10):5814–5822. https://doi.org/10.1128/AEM.71.10.5814-5822.2005

Sanapareddy N, Hamp TJ, Gonzalez LC, Hilger HA, Fodor AA et al (2009) Molecular diversity

of a North Carolina wastewater treatment plant as revealed by pyrosequencing. Appl Environ

Microbiol 75:1688–1696. https://doi.org/10.1128/AEM.01210-08

Sangwan N, Xia F, Gilbert JA (2016) Recovering complete and draft population genomes from

metagenome datasets. Microbiome 4(8):1–11. https://doi.org/10.1186/s40168-016-0154-5

Saum SH, Müller V (2008) Regulation of osmoadaptation in the moderate halophile Halobacillus

halophilus: chloride, glutamate and switching osmolyte strategies. Saline Syst 4(4):1–15. https://

doi.org/10.1186/1746-1448-4-4

Metagenomics Analysis of Extremophiles and Its Potential Use … 249

Seckbach J, Oren A, Stan-Lotter H (eds) (2013) Polyextremophiles: life under multiple forms of

stress. In: Cellular origin, life in extreme habitats and astrobiology, vol 27. Springer Science &

Business Media. https://doi.org/10.1007/978-94-007-6488-0

Sedlar K, Kupkova K, Provaznik I (2017) Bioinformatics strategies for taxonomy independent

binning and visualization of sequences in shotgun metagenomics. Comput Struct Biotechnol J

15:48–55. https://doi.org/10.1016/j.csbj.2016.11.005

Sharma J, Goutam J, Dhuriya YK, Sharma D (2021) Bioremediation of Industrial Pollutants. In:

Panpatte DG, Jhala YK (eds) Microbial rejuvenation of polluted environment. microorganisms

for sustainability, vol 26. Springer, Singapore, pp 1–31. https://doi.org/10.1007/978-981-15-745

5-9_1

Shuikan A, Alharbi SA, Alkhalifah DHM, Hozzein WN (2019) High-throughput sequencing and

metagenomic data analysis. In: Metagenomics-basics, methods and applications. IntechOpen.

https://doi.org/10.5772/intechopen.89944

Singh OV, Gabani P (2011) Extremophiles: radiation resistance microbial reserves and therapeutic

implications. J Appl Microbiol 110:851–861. https://doi.org/10.1111/j.1365-2672.2011.04971.x

Struvay C, Feller G (2012) Optimization to low temperature activity in psychrophilic enzymes. Int

J Mol Sci 13:11643–11665. https://doi.org/10.3390/ijms130911643

Sui HY, Weil AA, Nuwagira E, Qadri F, Ryan ET, Mezzari MP, Phipatanakul W, Lai PS (2020)

Impact of DNA extraction method on variation in human and built environment microbial commu-

nity and functional proﬁles assessed by shotgun metagenomics sequencing. Front Microbiol

11:953. https://doi.org/10.3389/fmicb.2020.00953

Suzuki S, Kuenen JG, Schipper K, Van Der Velde S, Ishii SI, Wu A, Sorokin DY, Tenney

A, Meng X, Morrill PL, Kamagata Y (2014) Physiological and genomic features of highly

alkaliphilic hydrogen-utilizing Betaproteobacteria from a continental serpentinizing site. Nat

Commun 5(1):3900. https://doi.org/10.1038/ncomms4900

Sysoev M, Grötzinger SW, Renn D, Eppinger J, Rueping M, Karan R (2021) Bioprospecting of novel

extremozymes from prokaryotes—the advent of culture-independent methods. Front Microbiol

12:630013. https://doi.org/10.3389/fmicb.2021.630013

Takai K, Nakamura K, Toki T, Tsunogai U, Miyazaki M, Miyazaki J, Hirayama H, Nakagawa S,

Nunoura T, Horikoshi K (2008) Cell proliferation at 122 C and isotopically heavy CH4 produc-

tion by a hyperthermophilic methanogen under high-pressure cultivation. Proc Natl Acad Sci

105(31):10949–10954. https://doi.org/10.1073/pnas.0712334105

Tamames J, Cobo-Simón M, Puente-Sánchez F (2019) Assessing the performance of different

approaches for functional and taxonomic annotation of metagenomes. BMC Genom 20(1):960.

https://doi.org/10.1186/s12864-019-6289-6

Tanveer A, Yadav S, Yadav D (2016) Comparative assessment of methods for metagenomic DNA

isolation from soils of different crop growing ﬁelds. 3 Biotech 6(2):220. https://doi.org/10.1007/

s13205-016-0543-2

Tchobanoglous G, Burton FL, Stensel HD (2003) Solution manual for use with wastewater

engineering: treatment and reuse. McGraw-Hill

Van den Burg B (2003) Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6:213–

218. https://doi.org/10.1016/S1369-5274(03)00060-2

Verma S, Meghwanshi GK, Kumar R (2021) Current perspectives for microbial lipases from

extremophiles and metagenomics. Biochimie 182:23–36. https://doi.org/10.1016/J.BIOCHI.

2020.12.027

Voets IK (2017) From ice-binding proteins to bio-inspired antifreeze materials. Soft Matter

13(28):4808–4823. https://doi.org/10.1039/C6SM02867E

Yadav S, Kapley A (2019) Exploration of activated sludge resistome using metagenomics. Sci Total

Environ 692:1155–1164. https://doi.org/10.1016/j.scitotenv.2019.07.267

Yue Y, Huang H, Qi Z, Dou HM, Liu XY, Han TF, Chen Y, Song XJ, Zhang YH, Tu J (2020)

Evaluating metagenomics tools for genome binning with real metagenomic datasets and CAMI

datasets. BMC Bioinformatics 21(1):334. https://doi.org/10.1186/s12859-020-03667-3

250 A. K. Shettihalli et al.

Zhang T, Shao M-F, Ye L (2012) 454 pyrosequencing reveals bacterial diversity of activated sludge

from 14 s ewage treatment plants. ISME J 6:1137–1147. https://doi.org/10.1038/ismej.2011.188

Zhang L, Su F, Kong X, Lee F, Day K, Gao W, Vecera ME, Sohr JM, Buizer S, Tian Y, Meldrum

DR (2016) Ratiometric ﬂuorescent pH-sensitive polymers for high-throughput monitoring of

extracellular pH. RSC Adv 6(52):46134–46142. https://doi.org/10.1039/C6RA06468J

Prospects of Nanobioremediation

as a Sustainable and Eco-Friendly

Technology in Separation of Heavy

Metals From Industrial Wastewater

Prathibha Narayanan, S. Divijendra Natha Reddy, and Praphulla Rao

Abstract Rapid industrial development and discharge of efﬂuents into rivers has

polluted water with heavy metals and other toxic matter. Most of the conventional

techniques for waste water treatment are known to cause harmful impact on envi-

ronment by releasing hazardous components. Some industries use bioremediation to

remove heavy metals which is eco-friendly but ineffective where the environment

itself is toxic to microorganisms due to presence of chemicals and non-biodegradable

metals. Advanced nanotechnology such as nano-adsorption and nanomembrane, is an

emerging ﬁeld to treat efﬂuents with high efﬁciency though it’s not cost effective and

eco-friendly. The solution to these problems can be integrating bioremediation with

nanotechnology. In this chapter, the structure and characteristics of nanomaterials are

discussed. Additionally, the chapter also highlights the integration of microbiology

and nanotechnology in two ways: Firstly, in green synthesis of nanoparticles which

is less expensive and sustainable and secondly, coating nanoparticles in microbes for

effective remediation.

Keywords Nanotechnology ·Bioremediation ·Nanobioremediation ·Industrial

waste water treatment ·Heavy metals ·Green synthesis

1 Introduction

The sustainability of human civilization depends on the judicious usage of natural

resources such as water. With the advent of rapid industrialization during last century

and usage of water as the universal solvent for many industrial processes, it is

becoming apparent to deal with the waste water being generated by many indus-

tries. As the discharge of large amounts of untreated water in to the environment

from industry is not desirable and governed by many regulations, there are various

process/methods that are being used for treating the waste water (Rajeshwari et al.

P. Narayanan (B

) · S. Divijendra Natha Reddy · P. Rao

Department of Biotechnology, B.M.S College of Engineering, Bull Temple Road, Basavanagudi,

Bengaluru 560019, India

e-mail: prathibhan.bt@bmsce.ac.in

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_13

251

252 P. Narayanan et al.

2000;Ali 2012; Zheng et al. 2015; Edwards 2019; Shah 2021a, b; Singh et al.

2019; Ajiboye et al. 2021; Nazaripour et al. 2021). Most of industrial waste water

contains toxic chemicals and metals that could pose problems for the environment

and can cause serious health issues to the surrounding population. If left untreated

and released into the surroundings, this water could contaminate and pollute the

ground water and could cause chronic health issues ranging from skin diseases to

cancer. Hence the removal of metals and other toxicants from the waste water is

an urgent priority. There are many technological processes that utilize chemical or

biological agents for industrial water remediation. Conventional physicochemical

methods are being used for treating the water to remove toxic metals and chemicals

(Shahedi et al.2020; Shah 2020; Gunatilake 2015; Zawierucha et al. 2016; Edwards

2019; Crini and Lichtfouse 2019; Mao et al. 2021). However, these methods suffer

from being cost intensive and cannot be adapted universally for all kinds of industrial

waste water treatment (Crini and Lichtfouse 2019). During last few decades, with

the better understanding of microorganisms spanning all life forms, their utility as a

bioremediation agent has been actively explored as the process of bioremediations

seems to be natural and cost effective. There are many microorganisms or their prod-

ucts that are being used or exploited for removing organic and non-organic compo-

nents such as metals from the industrial waste water (Rajeshwari et al. 2000;Crini

and Lichtfouse 2019; Sharma et al. 2021). Although bioremediation is successful

in many instances, its use for large scale or complete remediation or control over

microbes is still debatable. Nanotechnology is relatively modern ﬁeld having impli-

cations across various spectrum of sciences, engineering and medicine (Rajeshwari

et al. 2000; Sawhney et al. 2008; Rashidi et al. 2011; Hussain et al. 2017; Ramos

et al. 2017; Crini and Lichtfouse 2019). This ﬁeld has seen an explosion in develop-

ment over the last couple of decades and its applications virtually touch all the ﬁelds.

Recently, nanomaterials are being conceived as remediations agents for treatment of

polluted water not only from industry but from other sources of human consumption

(Sawhney et al. 2008; Rashidi et al. 2011;Lietal. 2015; Hussain et al. 2017; Ramos

et al. 2017; Nasrollahzadeh et al. 2021).

In this chapter, we will review the nature of nanomaterials and their usage in the

treatment of waste water and future perspectives of use of nanomaterials in waste

water treatment.

2 Structure and Characteristic Properties of Nanomaterials

Nanotechnology, during last few decades burst in to attention due to its poten-

tial applications to all the ﬁelds of technology and engineering. Nanomaterials are

expected to perform or to be used in endless potential applications due to their size

ranging in nanoscale. Nano scale is deﬁned as the range where at least one dimen-

sion of the material falls under 100 nm (nM) (Emil Roduner 2006). Because of their

size, the physical and chemical properties of nanomaterials are different from the

bulk materials of the same element with which they are made (Wu et al. 2020;Juh

Prospects of Nanobioremediation as a Sustainable and Eco-Friendly Technology … 253

Tzeng Lue 2007; Murty et al. 2013). One important aspect of being at nanoscale is

the surface to volume ratio is more and this is being exploited for better utility or for

many applications as the availability of more atoms on the surface in comparison to

the bulk material. Nanomaterials comes in various shapes and forms. Based on the

number of dimensions nanomaterials can be classiﬁed as zero-, one-, two- and three-

dimensional nanomaterials. In case of zero all dimensions are at nano scale (nano

spheres), in one dimension, two dimensions are at nanoscale and the other is outside

nano scale and in two-dimensional (nanotubes, nanoﬁbres, nanorods, nano wires),

one dimension is at nano scale and the other two outside the nanoscale (nano ﬁlms,

nano layers). Three dimensional nanomaterials are not at nanoscale but are made

up of repeating units of nanomaterials (dendrimers). Based on the composition of

elements, nanomaterials can be classiﬁed as carbon based (nanoﬁbers, nano tubes,)

and metal based such as gold and silver nanoparticles and TiO2 oxide nanoparti-

cles. The physical and chemical properties of nanomaterials are different from the

corresponding bulk material. Catalytic activity of some nanomaterials is better as

the surface to volume ratio is more(ref). They possess better electrical conductivity

in case of ceramics and magnetic composite nanomaterials while metal nanomate-

rials may have increased resistance. Nanomatrials generally have increased magnetic

activity and also behave as super paramagnetic material. Some metal nanomaterials

show marked toughness, ductility and plasticity (Wu et al. 2020; nanotechnology

and 2007 2007). Some nanomaterials are characterized by shift in optical absorp-

tion, ﬂuorescence and increased quantum efﬁciency. When it comes to biological

applications or in medicine, nanomaterials are proved to be better in overcoming

biological barriers such as cell membranes and also shown to be more biocompat-

ible (Hu et al. 2006, 2017; Kyriakides et al. 2021). Because of changes in physical

and chemical properties at nano scale, nanomaterials are being exploited for their

usage in puriﬁcation of water either from natural sources or from the sources of

contamination such as an industry.

3 The Science Behind Nanobioremediation

Industrial harmful contaminants have distinct physical and chemical properties and

each contaminant interacts in a different manner with environmental parameters. Due

to this the conventional techniques to treat waste water is not so effective. Bioreme-

diation is used in many industries for their low cost and varied applications Recently

in several investigations, nanomaterials have been used in association with bioreme-

diation technologies which prevents formation of dangerous by-products as well as

accelerates the process of degradation or removal of contaminants (Vázquez-Núñez

et al. 2020). Using nanomaterials for bioremediation has many beneﬁts. Because of

its nanosize, the material will have high surface area which increases reactivity with

surrounding components. They require less activation energy for reactions. Nano-

materials have the advantage of exhibiting surface plasmon resonance which can

be applied in detecting toxic substances. Due to small size, nanomaterials have the

254 P. Narayanan et al.

potential to reach deep into contamination zones. Also, oxide coating with nanomate-

rials can increase reactivity to a large extent. Some metal nanoparticles can function

as biocatalysts. For example, few studies revealed Palladium nanoparticles which

are coated on the cell walls of Shewanella oneidensis gets charged with radicals in

the presence of substrates which act as electron donors such as acetate, hydrogen

etc. When these cells coated with nanoparticles and charged with substrates come

in contact with compounds containing chlorine, the radicals present in palladium

removes chlorine. Immobilization of microorganisms is possible by using metal

nanoparticles which are magnetic in nature. These cells have been efﬁciently applied

in degradation or recovery of components. In one of the observations, the microor-

ganisms treated or coated with magnetic nanoparticle effectively separated organic

sulfur from fossil fuels on large scale in reactors (Rizwan et al. 2014). Interaction of

nanoparticles with living organisms can lead to any outcomes like sorption, biotrans-

formation, dissolution etc. which leads to degradation of toxic compounds (Vázquez-

Núñez et al. 2020). Nanoparticles can either inhibit or activate the metabolism of

living organisms participating in bioremediation. Therefore, it is very essential to

conﬁrm the effects of nanoparticles with living organisms before its application in

remediation processes. Some of the parameters that needs to be studied are nanopar-

ticles size and shape, surface area, chemical properties of both toxic components

and nanoparticles, nature and type of organism, growth media, pH, temperature etc.

(Tan et al. 2018). Due to involvement of so many factors, it is very difﬁcult to study

their individual effects. There are not many research studies done which establish

the r elationship between these parameters. Sorption studies are one of the signiﬁcant

methods in nanobioremediation. Contaminant removal either can be through adsorp-

tion which is surface phenomena or through absorption where the toxic compound

penetrates the nanoparticle and gets separated as a solution. Additionally, either

the sorption can be by chemical means or by physical methods. Several research

has been performed on adsorption isotherms, thermodynamics, and kinetic studies

to understand the type of adsorption processes and interaction between nanoparti-

cles and contaminants. Nanoparticles can degrade contaminants by photocatalytic

processes. In some cases, enzymes produced by living organisms degrade pollutants

(Vázquez-Núñez et al. 2020).

4 Formation of Nanoparticles Using Natural Sources

Synthesis of nanoparticles naturally using microorganisms and plants is cost effec-

tive, safe and eco-friendly. Not all organisms can produce metal nanoparticles.

Formation of nanoparticles from organisms is natural and can be in any two ways:

First method is by bio-reduction where metal nanoparticles are produced by reduc-

tion using biological tools. In this method, metal ions are reduced and enzymes are

oxidized. Nanoparticles can be then recovered from the contaminated samples. The

Prospects of Nanobioremediation as a Sustainable and Eco-Friendly Technology … 255

second method is bio-sorption where metal ions from polluted locations are bound

to the cell walls of organisms or the organisms synthesize peptides which assemble

into suitable nanoparticles (Zhang et al. 2020).

4.1 Sources for Synthesis of Nanoparticles

Bacteria

Several species of bacteria are widely studied to have the capacity to produce nanopar-

ticles by reducing metal ions. It is relatively easy to engineer bacteria as per the

requirements for the synthesis of nanoparticles (Singh et al. 2018). Some bacteria

known for production of silver nanoparticles are Escherichia coli, Lactobacillus

casei, Bacillus sp., Pseudomonas sp., Enterobacter cloacae, Corynebacterium sp.

and Shewanella oneidensis. Gold nanoparticles production from bacteria are compar-

atively complex and few bacterial species recognised includes Shewanella alga,

Escherichia coli, Bacillus sp., Desulfovibrio desulfuricans, and Rhodopseudomonas

capsulate.

Synthesis of palladium has been studied for Escherichia coli and Psedomonas

cells. Reports claim that copper nanoparticles from Morganella morganii need to be

stabilized immediately after their formation since copper metal is usually unstable

and readily gets oxidized to form cupric oxide. Copper nanomaterials accumulates

extracellularly as a result of intracellular uptake of copper ions which is then enzy-

matically reduced (Zhang et al. 2020). Nanomaterials formed from different strains

of bacteria vary in size, shape, morphology and time duration for their synthesis.

Fungi

Fungi are found to be better sources relative to bacteria for production of metal and

metal oxide nanoparticles in view of the fact that many intracellular enzymes, proteins

and reducing agents are present on their cell surfaces which aid in the mechanism.

The process is cost effective and efﬁcient resulting in large amount of nanoparticles

of well-deﬁned morphologies (Singh et al. 2018). Fusarium oxysporum, Aspergillus

fumigatus and Trichoderma reesei have been identiﬁed to produce silver nanopar-

ticles extracellularly. Since Trichoderma reesei morphology is extensively studied,

there is advantage of manipulating them for the nanoparticle synthesis. Aspergillus

fumigatus produce silver nanoparticles within minutes of exposure. White rot fungi

form silver nanoparticles intracellularly. Although not much work is done on produc-

tion of gold nanoparticles f orm fungi, but there are studies reported on the forma-

tion of gold nanoparticles from Verticillium species by reducing Tetrachloroaurate

(AuCl4) ions externally on the mycelium (Zhang et al. 2020).

256 P. Narayanan et al.

Few research work reveals synthesis of single platinum nanoparticles (PtNPs)

intracellularly by Neurospora crassa. Fusarium oxysporum also synthesizes PtNPs

both extra and intracellularly. Some fungi such as Fusarium oxysporum and Verticil-

lium sp. produce metal oxides like magnetite nanoparticles (MaNPs) intracellularly

(Zhang et al. 2020).

Fungal mycelia have relatively larger surface area. In addition, for large scale

production and puriﬁcation process use of fungal species has been proven efﬁ-

cient and less expensive through investigations. Furthermore, enormous number of

proteins and enzymes are formed in fungi, which are required for high production

of nanoparticles. However, most of the fungi are pathogenic and might be a concern

(Zhang et al. 2020).

Yeasts

Like fungi, yeasts also have large surface area to their advantage. They synthe-

size metal nanoparticles through various methods such as precipitation, sorption,

sequestration etc. Most of the nanoparticles are produced intracellularly by yeasts.

For example, Candida glabrata produces CdS quantum dots intracellularly in the

presence of cadmium salts. Also, Torulopsis sp. in the presence of lead ions forms

PbS quantum dots and Pichia jadinii is known to produce gold nanoparticles

intracellularly (Zhang et al. 2020).

Plants

Plants have the tendency to store heavy metals. In addition, plants have varied

biomolecules such as carbohydrates, proteins, fats, enzymes etc. and phytochemicals

like sugars, ketones, aldehydes, carboxylic acids, ﬂavonoids and terpenoids, which

play a key role in reduction of metal ions to nanoparticles and makes them one of

the best sources with regards to ease of process, faster synthesis, stability and cost.

Many plants have been studied over the past few years for production of nanopar-

ticles which includes Aloe barbadensis (Aloe vera), Azadirachta indica (Neem),

Avena sativa (Oats), Osimum sanctum (Tulsi), Coriandrum sativum (coriander) and

Cymbopogon ﬂexuosus (lemon grass) for synthesis of silver and gold nanoparti-

cles to name a few. Other metal nanoparticles like zinc, cobalt, nickel and copper

are found to be synthesized from Brassica juncea (mustard), Helianthus annuus

(sunﬂower), Coriandrum sativum (coriander), Hibiscus rosa-sinensis (China rose),

Camellia sinensis (green tea), Acalypha indica (copper leaf) and Aloe barbadensis

(Aloe vera). Most of the metal and metal oxides nanoparticles are produced by reduc-

tion of metal salt ions accumulated in plants. For example, ﬂavonoids reduce metal

ions into metal nanoparticles by transformation of enol to the keto forms. Studies

show that conversion of enol- to keto is the key mechanism in the formation of silver

nanoparticles from Ocimum basilicum (sweet basil) extracts. Sugars like glucose

also participate in metal nanoparticles synthesis with different size and shapes. Also,

Prospects of Nanobioremediation as a Sustainable and Eco-Friendly Technology … 257

proteins comprising of amino acids reduce the metal ions to form nanoparticles.

Some investigations reveal that few amino acids are capable of binding with silver

ions and hence produce silver nanoparticles. Furthermore all the naturally occurring

amino acids were examined and tested to study their reduction process of gold ions

(Singh et al. 2018).

Plants used for metal nanoparticle synthesis differ in their process times and

form nanoparticles of various shapes and sizes. Jatroa curcas extracts synthesize

homogenous silver nanoparticles of small sizes from silver nitrate (AgNO3)saltin

about 4 h. On contrary, Acalypha indica leaves form homogeneous silver nanopar-

ticles of slightly bigger sizes (Singh et al. 2018). Medicago sativa seeds were found

very effective in the synthesis of silver nanoparticles as the process was faster and

took less than an hour and more than 90% of silver ions reduced. The tempera-

ture requirement is also low to enable action of enzymes. The nanoparticles were

triangular or spherical with a rather heterogeneous size range (Zhang et al. 2020).

In another study, silver and gold nanoparticles were formed from a single compo-

nent such as phyllanthin isolated from the plant Phyllanthus amarus, rather than

using whole plant or plant parts. Concentrations of phyllanthin highly inﬂuenced the

shapes of nanoparticles. For instance, phyllanthin added in large amounts resulted

in spherical nanoparticles (Singh et al. 2018).

Some extracts from plants like Anacardium occidentale (cashew nut), Azadirachta

indica (neem), Swieteni amahagony (mahogany) and vegetable extracts (Zhang et al.

2020) have been reported to synthesize bimetallic silver and gold nanoparticles.

Algae

Cyanobacteria or blue green algae, such as C. vulgaris, and S. Platensis L. majus-

cule, and C. prolifera, are yet another cost-effective sources for green synthesis

of nanoparticles. Recently, synthesis of gold metal nanoparticles by reduction of

Au3+ ions to gold oxide (AuO) using cyanobacteria S. platensis proteins, have been

reported (Aman Gour et al. 2019).

Viruses

The capsid proteins on the surface of virus make them highly reactive towards metals

leading to formation of nanoparticles. For example, Tobacco mosaic virus (TMV) has

close to 2000 capsid proteins on their surface which reacts with silver or gold ions in

addition to plant extracts of Nicotiana benthamiana (Round-leaved native tobacco)

or Hordeum vulgare (Barley) (Zhang et al. 2020). The synthesized nanoparticles

were relatively smaller in size.

258 P. Narayanan et al.

4.2 Solvents

Water is the most commonly used solvent for nanoparticle synthesis as it is rela-

tively cheaper and readily available (Shanker et al. 2016). For example, gold and

silver nanoparticles are produced from gallic acid prepared in an aqueous medium.

Furthermore, oxidation of gold nanoparticles by the oxygen available in the water,

increases its efﬁciency and reactivity and enhances its role in bioremediation (Singh

et al. 2018).

4.3 Factors Modulating Formation of Metal Nanoparticles

The size and shape of nanoparticles depends on various parameters such as reac-

tion time, reactant concentrations, pH, and temperature. These factors need to be

optimized for the synthesis of nanoparticles of desired morphology.

pH

pH of the reaction medium is very important factor and alters the size and shape of

nanoparticles. For example, rod-shaped gold nanoparticles of size in the range 25-

85 nm were formed from biomass of Avena sativa (Oat) at acidic pH of 2. These gold

nanoparticles were relatively smaller (5–20 nm) and nucleation was comparatively

good when pH was maintained at 3 or 4. Slight alkaline conditions (pH > 5) are

required for synthesis of silver nanoparticles from Cinnamon zeylanicum extracts

which are homogenous and spherical in nature (Zhang et al. 2020).

Reactant Concentration

The presence and concentration of phytochemicals and other bio-components in

the extract determine the size and shape of the nanoparticles. For example, the

growth of gold and silver nanomaterials formed from extracts of Cinnamomum

camphora (camphor) were reported to be inﬂuenced by the quantity of biomass

in the reaction mixture. Also, addition of chloroauric acid to Aloe vera leaf extract

led to spherical nanoparticles rather than triangular plates. It was also reported that

the size of the nanoparticles can be manipulated by varying the concentrations of the

substrates. In another similar study, varied shapes of silver nanoparticles including

the desired spherical proﬁles, were synthesized by altering the concentration

of Plectranthu samboinicus extract in the process mixture (Zhang et al. 2020).

Prospects of Nanobioremediation as a Sustainable and Eco-Friendly Technology … 259

Reaction Time

Yet another factor inﬂuencing morphological characteristics of the nanoparticles is

the reaction time. A research investigation observed formation of spherical silver

nanoparticles of average size 12 nm from Anana scomosus (Pineapple) extract and

AgNO3, within 2 min of time showing a rapid color change. Chenopodium album leaf

extract produced silver and gold nanoparticles within 15–20 min of the reaction and

the process persisted for more than 2 h. In another study, it was revealed that different

sizes of nanoparticles formed from Azadirachta indica leaf extract and silver nitrate

(ranging 10–35 nm) resulted in an increase in reaction time from half an hour to

more than 4 h (Zhang et al. 2020).

Reaction Temperature

The temperature modulates the structure, size, and productivity of nanoparticles

synthesized from different plants. One of the experimental studies reported that

temperature had reciprocal effects on the size of nanoparticles synthesized from the

Citrus sinensis (sweet orange) extracts from its rind. The nanoparticle size decreased

with an increase in temperature. Also, in few other studies it was noted that the

temperature altered shape of gold nanoparticles produced using Avena sativa (oat).

The gold nanoparticles formed at lower temperature were spherical compared to

nanoparticles at higher temperatures which exhibited non-spherical patterns. Process

temperature conditions also affects productivity like it was noticed in few observa-

tions that the yield of platinum nanoparticles synthesized extracellularly, was around

5.66 mg l−1 which varied with temperature (Zhang et al. 2020).

4.4 Identiﬁcation and Removal of Heavy Metal Ions

from Waste Water

Industrial development has led to accumulation of contaminants and pollutants in

soil, air and water. Industrial efﬂuents especially are discharged into water bodies.

Most of the efﬂuents include harmful heavy metals like copper, lead and cadmium

which are detrimental even at low concentrations. There are several methods for

identifying these pollutants but these conventional techniques are not cost effective,

consume lot of time and also require skilled and experienced personnel. Use of

metal nanoparticles has many advantages due to its size and optical properties like

the process is simple, relatively cheaper and has high sensitivity to detect toxic

heavy metals even at low concentrations. In one of the investigations reported, silver

nanoparticles were synthesized from different plant sources which were sensitive

in identiﬁcation of heavy metals based on their plant source. For example, silver

nanoparticles prepared from mango leaves displayed calorimetric sensing selectively

260 P. Narayanan et al.

for lead and mercury ions. Another signiﬁcant pollutant from industries like textile,

plastic, leather, paper and food are coloured dyes. Dyes are important component used

in manufacturing process in textile industries. About 10–15% of dyes are discharged

as wastes into water bodies. These are very dangerous as they increase turbidity

in water thereby preventing penetration of sunlight which can be fatal for marine

life. For removal of dyes from water, usually metal oxide nanoparticles like zinc

oxide (ZnO), titanium dioxide (TiO2), and copper oxide (CuO) with photocatalytic

properties are used for their high surface area. High surface area increases absorption

of reactive dyes at low concentrations as nanoparticles have large number of reactive

sites available due to high surface energies. Furthermore, very less quantity of these

nanoparticles is required for large mass of efﬂuents (Singh et al. 2018).

5 Coating of Nanomaterials Inside Microbes for Effective

Bioremediation

Nanomaterials can be potentially used for treating industrial wastewater and ground

water. Nanomaterials can treat the wastewater by means of adsorption, photocatal-

ysis, disinfection and membrane application. Adsorption is a common mechanism

used with nanoparticles to treat the wastewater. Properties such as high surface area,

selective adsorption sites, short diffusion distance between particles, alterable surface

chemistry, reusability and so on gives the advantage of nanoparticles in wastewater

treatment. Most commonly, heavy metals, arsenic, chromium, mercury phosphates,

organic compounds, PAHs, DDT are adsorbed by nanoparticles. Organic compounds,

volatile organic carbon, azo dye, congo red dye and so forth are removed from

wastewater by means of photocatalytic oxidation. Nanoparticles with high photocat-

alytic activity such as TiO2, ZnO, iron oxide nanoparticles are used in this technique.

Nanoscale zero-valent iron (nZVI), nanoscale calcium peroxide nanoparticles are

used to treat wastewater with halogenated organic compounds, metals, nitrate, arse-

nate, oil, PAH, PCB by means of redox reactions. Some of the nanoparticles have

strong antimicrobial properties like silver and TiO2 nanoparticies that can be used

to disinfect the waste water. These nanoparticles have low toxicity, high chemical

stability and reusability. Several research studies have conﬁrmed use of nanoparti-

cles of metal and metal oxides such as silver, TiO2, Zeolites and CNTs that exhibit

strong hydrophilicity, high mechanical and chemical stability, high permeability and

selectivity, can be embedded to the membranes to increase their effectiveness in

puriﬁcation of the wastewater (Yadav et al. 2017).

Few investigations observed that Palladium, Pd(0) nanoparticles can be used for

removal of chlorine. The cell wall and inside the cytoplasm of Shewanella oneidensis

were coated with Pd(0) nanoparticles which gets charged on reaction with suitable

substrates The protons present in these charged Pd(0) coated cells when exposed

to chlorine rich mixtures, react with phencyclidine, leading to the separation of the

chlorine molecule.

Prospects of Nanobioremediation as a Sustainable and Eco-Friendly Technology … 261

Sarioglu et al. (2017) studied the dye removal capacity of bacterial cells encapsu-

lated within electrospun nanoﬁbrous webs. Commercially available strain of Pseu-

domonas aeruginosa was immobilized using polymers like polyvinyl alcohol (PVA)

and polyethylene oxide (PEO). The extent of encapsulation and the viability of

bacteria were examined using advanced and modern tools and techniques. It was

reported that bacteria encapsulated in PEO web showed higher methylene blue dye

removal efﬁciency which could be due to better accessibility of bacteria trapped in

the nanoﬁbrous webs. Studies reported that the encapsulated bacterial cells remained

alive for about a couple of months when the webs were stored at 4 °C and thus could

be preferred to lyophilized cells which selective culture media and space for storage.

In addition, some research reveal that the electrospun nanoﬁbrous webs containing

viable cells ﬁnd scope in bioremediation of water systems.

The electrospun cyclodextrin ﬁbers (CD-F) manufactured by electrospinning tech-

nique has been used in few studies as carrier matrix and feed for encapsulation of

bacteria which can be effectively applied for bioremediation purposes. N.O San

Keskin et al. (2018) reported in his ﬁndings that electrospinning process is simple

and easy for immobilization of bacteria into CD-F matrix and is widely applied

in wastewater treatment. CD-F are not toxic and hence display cell viability for

more than a weeks of time when stored at 4 °C. The observations were promising

for nanobioremediation as the bacteria encapsulated with CD-F showed high heavy

metals and dye removal efﬁciencies showing results as 70 ± 0.2%, 58 ± 1.4% and 82

± 0.8% for Nickel, chromium and reactive dye, RB5, respectively. Added advantage

is bacteria can consume CD as an extra carbon source when primary carbon source

depletes thereby increasing their growth rate.

6 Conclusion

Over the last few years research studies on green synthesis of metal and metal oxide

nanoparticles has increased due to its various advantages. Among the numerous

sources, plant extracts have been observed to have signiﬁcant roles as stabilizing and

reducing agents which under optimized reaction conditions can lead to formation

of nanoparticles with desired sizes, shapes, and other morphological characteris-

tics. Nanoparticles play a vital role in degradation of heavy metals in waste water.

Nanoparticles on one hand degrades waste that might be toxic to microorganisms and

on the contrary, improves performance of microorganisms in remediation of harmful

substances. Though studies have investigated the inter-relation between nanoparti-

cles and microorganisms in degradation of harmful substances in batch experiments,

but the ﬁndings are not sufﬁcient to comprehend the synergetic effect of nanoparticles

and microorganisms during a nanobioremediation process and its applications. Also,

there is no safety data available that indicates the long-term effects on use of nanopar-

ticles with microorganisms. Hence, there is lot of scope in this ﬁeld to explore and

research and apply nanobioremediation technologies in varied applications following

the regulatory framework and safety guidelines.

262 P. Narayanan et al.

References

Ajiboye TO, Oyewo OA, Onwudiwe DC (2021) Simultaneous removal of organics and heavy metals

from industrial wastewater: a review. Chemosphere 262.https://doi.org/10.1016/j.chemosphere.

2020.128379

Ali I (2012) New generation adsorbents for water treatment. Chem Rev 112:5073–5091. https://

doi.org/10.1021/cr300133d

Crini G, Lichtfouse E (2019) Advantages and disadvantages of techniques used for wastewater

treatment. Environ Chem Lett 17:145–155. https://doi.org/10.1007/S10311-018-0785-9

Edwards JD (2019) Industrial wastewater treatment CRC Press. https://doi.org/10.1201/978135107

3509

Gour A, Jain NK (2019) Advances in green synthesis of nanoparticles. Artif Cells Nanomed

Biotechnol 47(1):844-851.https://doi.org/10.1080/21691401.2019.1577878

Gunatilake SK (2015) Methods of removing heavy metals from industrial wastewater. J Multidis-

ciplinary Eng Sci Studies 1:2912–1309

Hu ZG, Zhang J, Chan WL, Szeto YS (2006) The sorption of acid dye onto chitosan nanoparticles.

Polymer 47:5338–5842. https://doi.org/10.1016/j.polymer.2006.05.071

Hu X, Sun A, Kang W et al (2017) Strategies and knowledge gaps for improving nanomaterial

biocompatibility. Environ Int Elsevier 102:177–189. https://doi.org/10.1016/j.envint.2017.03.001

Hussain P, Teknologi U, Kakooei S et al (2017) Engineering applications of nanotechnology, 334

Juh Tzeng L (2007) Physical properties of nanomaterials. Encyclopaedia of nanoscience and

nanotechnology, X:1–46

Keskin NOS, Celebioglu A, Sarioglu OF, Uyar T, Tekinay T (2018) Encapsulation of living bacteria

in electrospun cyclodextrin ultrathin ﬁbers for bioremediation of heavy metals and reactive dye

from wastewater. Colloids Surf B 161:169–176. https://doi.org/10.1016/j.colsurfb.2017.10.047

Kyriakides T, Raj A, Tseng T et al (2021) Biocompatibility of nanomaterials and their immunolog-

ical properties. Biomed Mater, 16(4)

Li R, Zhang L, Wang P (2015) Rational design of nanomaterials for water treatment. Nanoscale

7:17167–17194. https://doi.org/10.1039/C5NR04870B

Mao M, Yan T, Shen J et al (2021) Capacitive removal of heavy metal ions from wastewater via an

electro-Adsorption and electro-reaction coupling process. Environ Sci Technol 55:3333–3340.

https://doi.org/10.1021/ACS.EST.0C07849

Murty BS, Shankar P, Raj B et al (2013) Unique properties of nanomaterials. Textbook Nanosci

Nanotechnol, 29–65.https://doi.org/10.1007/978-3-642-28030-6_2

Nasrollahzadeh M, Sajjadi M, Iravani S, Varma RS (2021) Carbon-based sustainable nanomaterials

for water treatment: state-of-art and future perspectives. Chemosphere 263:128005. https://doi.

org/10.1016/j.chemosphere.2020.128005

Nazaripour M, Reshadi MAM, Mirbagheri SA et al (2021) Research trends of heavy metal removal

from aqueous environments. J Environ Manage 287:112322. https://doi.org/10.1016/j.jenvman.

2021.112322

Rashidi L, Khosravi-Darani K (2011) The applications of nanotechnology in food industry. Critical

reviewsin food science and nutrition. Taylor & Francis 51:723–730.https://doi.org/10.1080/104

08391003785417

Rajeshwari KV, Balakrishnan M, Kansal Kusum Lata A, Kishore VVN (2000) State-of-the-art of

anaerobic digestion technology for industrial wastewater treatment. Renew Sustain Energy Rev

Elsevier 4(2):135–156.https://doi.org/10.1016/S1364-0321(99)00014-3

Ramos AP, Cruz MAE, Tovani CB, Ciancaglini P (2017) Biomedical applications of nanotech-

nology. Biophys Rev 9:79–89. https://doi.org/10.1007/S12551-016-0246-2

Rizwan Md, Singh M, Mitra CK, Morve RK (2014) ecofriendly application of nanomaterials:

Nanobioremediation. J Nanoparticles. Article ID 431787. https://doi.org/10.1155/2014/431787

Roduner E (2006) Size matters: why nanomaterials are different. Chem Soc Rev 35:583–592. https://

doi.org/10.1039/B502142C

Prospects of Nanobioremediation as a Sustainable and Eco-Friendly Technology … 263

Sarioglu OF, Keskin NOS, Celebioglu A, Tekinay T, Uyar T (2017) Bacteria encapsulated elec-

trospun nanoﬁbrous webs for remediation of methylene blue dye in water. Colloids Surf B

152:245–251. https://doi.org/10.1016/j.colsurfb.2017.01.034

Sawhney APS, Condon B, Singh KV et al (2008) Modern applications of nanotechnology in textiles.

Text Res J 78:731–739. https://doi.org/10.1177/0040517508091066

Shah MP (2020) Microbial bioremediation & biodegradation. Springer

Shah MP (2021a) Removal of refractory pollutants from wastewater treatment plants. CRC Press

Shah MP (2021b) Removal of emerging contaminants through microbial processes. Springer

Shahedi A, Darban AK, Taghipour F, Jamshidi-Zanjani A (2020) A review on industrial wastewater

treatment via electrocoagulation processes. Elsevier. Current Opinion Electrochemistry 22:154–

169. https://doi.org/10.1016/j.coelec.2020.05.009

Shanker U, Jassal V, Rani M, Kaith BS (2016) Towards green synthesis of nanoparticles: from

bio-assisted sources to benign solvents. A review. Int J Environ Anal Chem 96:801–835. https://

doi.org/10.1080/03067319.2016.1209663

Sharma R, Jasrotia T, Sharma S et al (2021) Sustainable removal of Ni(II) from waste water by

freshly isolated fungal strains. Chemosphere 282:130871. https://doi.org/10.1016/j.chemosphere.

2021.130871

Singh J, Dutta T, Kim KH et al (2018) Green synthesis of metals and their oxide nanoparticles:

applications for environmental remediation. J Nanobiotechnology 16:84. https://doi.org/10.1186/

s12951-018-0408-4

Singh R, Singh R, Gupta R, Singh R (2019) Treatment and recycling of wastewater from textile

industry. Advances in biological treatment of industrial waste water and their recycling for a

sustainable future. Appl Environ Sci Eng Sustain Future. Springer. https://doi.org/10.1007/978-

981-13-1468-1_8

Tan W, Peralta-Videa JR, Gardea-Torresdey JL (2018) Interaction of titanium dioxide nanoparticles

with soil components and plants: current knowledge and future research needs-a critical review.

Environ Sci Nano 5:257–278.https://doi.org/10.1039/c7en00985b

Vázquez-Núñez E, Molina-Guerrero CE, Peña-Castro JM, Fernández-Luqueño F, de la Rosa-

Álvarez MG (2020) Use of nanotechnology for the bioremediation of contaminants: a review.

Processes 8(7):826.https://doi.org/10.3390/pr8070826

Wu Q, Miao W, Zhang Y, Gao H, Hui D (2020) Mechanical properties of nanomaterials: a review.

Nanotechnol Rev 9(1):259–273. https://doi.org/10.1515/ntrev-2020-0021

Yadav KK, Singh JK, Gupta N, Kumar V (2017) A Review of Nanobioremediation technologies

for environmental cleanup: a novel biological approach. J Mate Environ Sci 8(2):740–757

Zawierucha I, Kozlowski C, Malina G (2016) Immobilized materials for removal of toxic metal ions

from surface/groundwaters and aqueous waste streams. Environ Sci Process Impacts 18:429–444.

https://doi.org/10.1039/C5EM00670H

Zhang D, Ma X-l, Gu Y, Huang H, Zhang G-w (2020) Green synthesis of metallic nanoparticles

and their potential applications to treat cancer. Front Chem 8:799. https://doi.org/10.3389/fchem.

2020.00799

Zheng T, Wang J, Wang Q et al (2015) A bibliometric analysis of industrial wastewater research:

current trends and future prospects. Scientometrics 105:863–882. https://doi.org/10.1007/S11

192-015-1736-X

Nanotechnology for Bioremediation

of Industrial Wastewater Treatment

Harshala S. Naik, Parvindar M. Sah, Swapnali B. Dhage, Smita G. Gite,

and Rajesh W. Raut

Abstract At an ongoing pace, the population of the world will increase mani-

fold. The population explosion and global warming will impact the available useful

water for drinking. Simultaneously to fulﬁl the demand of other sectors like indus-

tries and agriculture there will be pressure on water availability on earth. Due to

human activity, various pollutants are released from the industrial sector. Quick

attention is needed towards the treatment of the wastewater before it gets released

into the environment to maintain the quality and purity of water. For the treatment

of wastewater, various methodologies have developed in the last few years. One

of the most interesting ways to treat wastewater is the use of nano bioremedia-

tion. The present book chapter enlightens the use of nanotechnology-based biore-

mediation for wastewater treatment. Nanobioremediation is a method or process

in which a combination of biology and nanotechnology is used to remove heavy

metal pollutants from wastewater and make water clean. The removal of environ-

mental contaminants (such as heavy metals, organic and inorganic pollutants) from

contaminated sites using nanoparticles/nanomaterials formed by plants, fungi, and

bacteria with the help of nanotechnology is called Nano bioremediation. In tradi-

tional ways using either nanotechnology-based or biological-based methods, these

H. S. Naik · P. M . Sah · R. W. Raut (B

)

Department of Botany, The Institute of Science, 15 Madam Cama Road, Mumbai 400032, MS,

India

e-mail: rajesh.w.raut@gmail.com

H. S. Naik

e-mail: harshalasandipnaik14@gmail.com

P. M. S a h

e-mail: sahparvindar701@gmail.com

S. B. Dhage · S. G. Gite

Department of Chemistry, The Institute of Science, 15 Madam Cama Road, Mumbai 400032, MS,

India

e-mail: swapnali.dhage23@gmail.com

S. G. Gite

e-mail: smitagite5@gmail.com

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_14

265

266 H. S. Naik et al.

both are capable of removal of toxic metals from the environment. Using nanopar-

ticles along with microorganisms acts synergistically for the removal of pollutants.

Sometimes a single enzyme is also effective for bioremediation but the enzyme is

not stable. Using functionalized nanoparticles will provide stability and improve

bioremediation speed.

Keywords Nanotechnology ·Nano bioremediation ·Industrial wastewater

treatment ·Environmental science

1 Introduction of Nano Bioremediation for Industrial

Wastewater Treatment

The need for water is just as important as those for food, shelter, and clothes for human

beings. Water is indispensable to our existence and life originated here without it, we

cannot exist. While water is a natural resource, its availability is limited. According

to reports, one of the six individuals has difﬁculty getting fresh water. It is estimated

that until 2025, around 50% of the global population will suffer from water-related

issues (Aguilar-Pérez et al. 2021). Clean water is essential for all creatures on the

planet. Water access can impact the environment and climate change if there is no

proper access. Lack of water will affect biodiversity and destroy habitats in addition

to causing landscape erosion (Hashem 2014). With the increase in population, rapid

commercialization, and less precipitation, hazardous chemicals have spread across

the earth’s surface and are affecting the groundwater system. As a result, ecosystems

have undergone drastic changes (Zekic et al. 2018). The scarcity of pure water is the

primary problem throughout the world, so there is a need to explore new methods

and technologies for cleaning water. One method is to use nanotechnology to clean

wastewater. During the past few years, water pollution has received a lot of attention.

By providing quality water, people will be able to satisfy their current needs for water

(Pandey 2018; Shah 2020a, b).

Nanoscience and nanotechnology are experiencing a new era of opportunities

due to technological developments. Nanoscience is a branch of science that deals

with molecules that have at least one dimension smaller than 100 nm. Even though

nanoparticles are made from metal ions, they behave differently from metal ions

(Rizwan and Ahmed 2018).

This chapter mainly addressed nanotechnology-based solutions for bioremedi-

ating wastewater. The goal of this chapter is to provide an introduction to bioremedi-

ation, different methods of bioremediation, nanobioremediation, and different kinds

of nanoparticles used in bioremediation, their role, drawbacks, and conclusion.

Before we look at nanotechnology-based wastewater treatment, we should get a

better understanding of water pollutants, their sources, types, and effects, as well as

the methods used to remove contaminants from water.

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 267

1.1 The Main Source of Water Pollutants

Chemical and biotechnological industries produce a variety of useful products. There

is no doubt that industries produce inevitable products, but they are also producing

products which are ultimately responsible for a source of pollution. Due to various

industrial activities, toxic chemicals are released into water.

Here are a few examples of pollutants released by different industries. They can

be classiﬁed as organic or inorganic. Chlorinated phenols, phthalic esters, persistent

organic pollutants (POPs), azo dyes, petroleum hydrocarbons, pesticides, etc., are

among the most common organic pollutants. In contrast, inorganic pollutants include

heavy metals such as aluminum (Al), cadmium (Cd), nickel (Ni), chromium (Cr),

lead (Pb), and mercury (Hg) (Hussain et al. 2016; Bharagava et al. 2020).

Organic Pollutant: Their Sources, and Effects

Chlorinated Phenols

Chlorinated phenols is a major constituent released by coking plants, oil reﬁneries,

resin manufacturing, paper and pulp industries. They are recalcitrant and toxic, and

the level of chlorination determines their toxicity (Annachhatre and Gheewala 1996;

Shah 2021a, b).

Phthalic Esters

They are a class of organic refractory plasticizers used in various products such

as plastic rooﬁng, piping, gaskets, medical equipment, rainwear, plastic ﬁlm. The

phthalic ester in plastics is responsible for its ﬂexibility. More than one million tons

of phthalic ester is used in the manufacture of plastic in China. It has a serious

effect on human health as these compounds are mutagenic and affect the embryonic

development of the foetus (He et al. 2015).

Persistent organic pollutants (POPs)

Persistent organic pollutants (POPs) consist of different chemical groups that require

attention. Polychlorinated dibenzo-p-dioxins, dibenzo-furans (PCDD/Fs), polychlo-

rinated biphenyls (PCBs), and heptachloride present in pesticides require special

attention. Forest ﬁre and volcanic activity are main natural sources of POPs such

as dibenzofurans and dioxins. POPs can also be produced by furnaces, incinerators,

power plants, and heating systems used in industrial sites. Various countries have

banned this type of chemical in recent years. These are organic compounds that are

harmful to the environment. POPs are mainly non-biodegradable and lipophilic.

It affects the respiratory, circulatory, and digestive systems of the human body

(Alshemmari 2021).

268 H. S. Naik et al.

Azo Dyes

In many industries, dyes are used as colouring compounds. Colour-producing

compounds are classiﬁed according to their use and chromophore group. Phytocya-

nine dyes, anthraquinone dyes, azo dyes and phthalocyanine dyes are chromophore-

containing dyes. An Azo dye has a special azo bond responsible for absorbing light

in visible wavelength regions.

More than 3000 types of dyes are available on the market. Each year, more than 2.8

tons of dyes are added to the water system. The reason why so many dyes are available

is that they are chemically stable and easy to synthesize. A number of industries

use azo dyes, including food, paper, l eather, cosmetics, textiles, pharmaceuticals.

According to the survey, the dye which is used in the dyeing process is not completely

utilized, about 10% of the dye is not utilized and unutilized dye discharges into water

bodies. These azo dyes released from efﬂuent have many side effects. It damages the

cells of the organism, the rate of photosynthesis is reduced, it leads to a decrease in

oxygen content which affects the ﬂora and fauna of water bodies. Mainly azo dye

consists of a nitro group which is responsible for mutation. When dye is released from

industrial water, it breaks down and forms a new kind of toxic product. Such products

are mutagenic and carcinogenic. Several derivatives of azo dye after breakdown

are o-toluidine, 1,4-phenylenediamine, and 3-methoxy-4-aminoazobenzene which

are carcinogenic. According to a study, unsulfonated azo dyes are more toxic and

mutagenic than sulfonated azo dyes (Singh and Singh 2017).

Petroleum Hydrocarbons

The oil industry releases wastewater that contains a lot of hydrocarbons, ethylben-

zene, benzene, xylene, toluene, small amounts of oxygen, sulfur, nitrogen, and heavy

metals. The presence of these hydrocarbons in terrestrial systems or on land changes

the original composition of soil and affects the growth of plants. Through different

transport processes occurring in nature, petroleum hydrocarbons eventually end up

in the sea and pollute marine ecosystems. As a result of fuel spills, large environ-

mental losses occurred in cold regions and threatened the health of organisms. These

large numbers of hydrocarbons are responsible for damage to the central nervous

system and also lead to suppression of bone marrow activity. In order to remove this

kind of contamination from contaminated sites, nanoparticle-based tools can be used

(Mohammadi et al. 2020).

Inorganic Pollutants

Industrial wastewater from different sources contain different kinds of heavy metals,

such as copper, cobalt, cadmium, chromium, lead, iron, zinc, nickel, and magnesium.

Metals such as manganese, copper, zinc, nickel, cadmium, mercury, lead, aluminium,

chromium, iron, and cobalt require immediate attention, according to the WHO.

Many countries are concerned about the release of metals or heavy metals into

industrial water because these metals or heavy metals persist in the environment and

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 269

lead to bioaccumulation. Their non-biodegradable nature makes them responsible

for continuous transfer into the next generation if they bioaccumulate in organisms.

Radioactive metals can cause harm to the human body if they accumulate in the body

(Bernard 2008; Assi et al. 2016; Hussain et al. 2016; Koul and Taak 2018a; Magrì

et al. 2018).

Here, we will discuss different types of metals, their sources, and their effects on

the environment.

Zinc

Industries like coal mining, steel processing, and waste combustion emit zinc. A direct

accumulation of zinc causes diarrhoea and gastrointestinal distress, while inhalation

of zinc fumes causes fever.

Nickel

Fossil fuel combustion, metal plating industries, nickel mining, electroplating fumes

from alloys used in welding and brazing, and nickel-reﬁning industries release nickel.

Nickel acts as a carcinogen and causes respiratory tract disease.

Mercury

In paper and pulp manufacturing, gold extraction, chloro-alkali operations, smelting

operations, and amalgam tooth ﬁllings, mercury is released. It leads to kidney

damage. As a result of chronic poisoning, the blood’s ability to carry oxygen is

reduced. Mercury can also cause nutritional disturbances, excessive irritation of

tissues, loss of appetite, gingivitis, and excessive salivation. Exposure to mercury

fumes may cause respiratory problems.

Manganese

Steel and welding industries release large amounts of manganese. Consequently,

neurotological disease is caused by this metal. Poisoning can lead to irritability and

speech problems.

Lead

Rubber production, glass polishing, jewellery making, lead-glazed plastic manufac-

turing, stained glass crafting, pottery, and painting make up this industry. Lead affects

many different organs of the body. The toxins not only damage organs, but also affect

cellular mechanisms and enzymes.

Iron

It includes Hematite mining, metal industries, and welding. Lung silicosis is caused

by iron fume accumulation in heavy fumes.

Copper

Copper emissions are mainly caused by welding, copper mining, and metal fumes

from smelting operations. Copper causes Wilson’s disease and anaemia as well as

higher accumulation of copper in the body. Aside from kidney and liver damage,

270 H. S. Naik et al.

other vital organs can also be harmed and it affects digestion also. At high levels,

sulfate and copper induce necrosis and kill the organism.

Cobalt

Paints and cement are the main industries that use cobalt. At low concentrations, it

causes respiratory problems such as irritation of the respiratory tract, while at high

concentrations, it may cause pneumoconiosis.

Chromium

Chromium is mainly used in paint and colouring companies. Moreover, it is released

in the manufacturing of chemical and refractory materials, cement manufacturing,

chrome plating, ferrochrome manufacturing, metal ﬁnishing industries, combus-

tion of fossil fuels, tanneries, and textile plants. It damages kidneys when at low

concentrations, while causing respiratory chromogenic substances when at high

concentrations, which are mostly carcinogenic and also cause allergies.

Cadmium

Cadmium is released by several industries, including the automotive and aircraft

industries, alloys’ production, metallurgy processing, nickel–cadmium battery manu-

facturing industries, paint industries, plastic industries mining, and textile printing

industries. Inhalation and ingestion can cause acute and chronic toxicity. A number

of metabolic pathways are affected by Cd, including alcohol dehydrogenase, pyru-

vate dehydrogenase, pyruvate decarboxylase, delta-aminolevulinic acid dehydratase,

arylsulfatase, and lipoamide dehydrogenase.

Aluminium

The production of aluminum alloys, the pharmaceutical industry, and packaging

units release aluminium. Humans can develop lung ﬁbrosis from the fumes from

these sources. Fumes from these sources can cause lung ﬁbrosis and osteomalacia.

Large accumulations in bones can cause lung ﬁbrosis.

Contaminated water also contains microorganisms in addition to organic and inor-

ganic pollutants. These microorganisms are present in wastewater from the healthcare

industry because of improperly sterilized medical swabs, needles, and bandages. In

normal water bodies, unsterilized tools make the water contaminated.

1.2 The Science Behind the Use of Nanomaterials

in Bioremediation

The goal of remediation is to remove toxic, heavy metal, and harmful waste from the

environment. The three main methods of environmental remediation are physical,

chemical, and biological (Singh et al. 2020).

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 271

Remediation of the environment usually involves chemical methods such as reduc-

tion, chemical washing, chelating ﬂushing, ion exchange, electrowinning, adsorp-

tion, membrane ﬁltration, and concentration. There are a number of biotechnology

methods that remove pollutants from the environment, including rhizoﬁltration,

bioslurping, windrows, biosorption, land farming, bioplies, bioventing, bioleaching

bioreactors, biosparging, composting, bioaugmentation, biostimulation phytoreme-

diation, and mycoremediation. Among these three major processes, biological

processes are the most commonly used because they do not involve toxic chem-

icals or energy consumption. In most cases, bioremediation does not require the

use of external chemicals, as the process relies on naturally occurring plants and

microorganisms (Yadav et al. 2017).

Bioremediation can be classiﬁed into two major categories: in-situ and ex-situ.

In situ bioremediation occurs at the original site where contaminated and toxic

substances were excavated before being treated. As a result, it is faster, easier, and

more effective than ex-situ bioremediation (Singh et al. 2020).

In soil, there are bacteria capable of degrading soil chemicals. These bacteria

are called “bugs”. These tiny bugs are often found in oil spills and gasoline.

Microbes can digest chemicals and then convert them into harmless forms, such

as water and carbon dioxide. The process occurs in two different ways. As an initial

step, it is necessary to manipulate parameters such as nutrient levels, temperature,

and oxygen ions in order to increase the ability of microorganisms to digest harmful

metals. Adding microorganisms to contaminated sites is the second method, which

is not the most common. The two methods serve the same purpose, however (Yadav

et al. 2017).

In bioremediation, pollutants are removed from the environment. Nanobioreme-

diation involves removing pollutants from the environment using nanoparticle-based

solutions made from plants, fungi, or bacteria.

In their talk, Richard Feynman introduced the concept of nanotechnology. In a talk

titled ‘There’s Plenty of Room at the Bottom,’ he did not use t he term nanotechnology

directly. Nanotechnology is a milestone in the development of new branches of

science. Nanotechnology creates new materials by manipulating materials at very

small scales. Reduced metal ions have completely different properties than their

original forms. Nanoparticles or nanomaterials differ in two important ways; the ﬁrst

is the surface area ratio, and the second is the quantum effect. In order to effectively

remove contaminants, both of these factors are necessary. Quantum effects accelerate

response time, and when there is a large surface area, the interaction is greater

(Mourdikoudis et al. 2018; Rizwan and Ahmed 2018). Based on its applications,

nanotechnology can be classiﬁed into many branches. In wastewater treatment, a

variety of nanomaterials are used. In order to remove pollutant size dependence,

nanoparticles play a vital role. Nanoparticles possess special properties that make

them suitable for use in different methods of removing contaminants.

Another sub-branch of nanotechnology is green nanotechnology. Eco-friendly

nanoparticles and nanoproducts can be made with green nanotechnology. Nanopar-

ticles are most efﬁcient at removing contaminants, which requires fewer resources, in

272 H. S. Naik et al.

wastewater treatment. These simple, cost-effective processes can be used to remove

contaminants from wastewater (Alshemmari 2021).

Nanotechnology-based solutions are widely used in bioremediation because they

can be easily synthesized and are safe. In addition, nanoparticles increase the

efﬁciency and speed of bioremediation.

The following examples illustrate how bioremediation works. Phytoremediation

involves using plants to eliminate toxic pollutants. Nanoparticles functionalized with

speciﬁc enzymes will produce the greatest productivity. The procedure is essentially

very straightforward. Plants cannot degrade large molecules such as oligosaccharides

and large chain hydrocarbons. Plants degrade these complex molecules into simple

molecules, which are then degraded by nanoparticles. With this method, bioremedia-

tion will be more effective if it is combined with nanoparticles. Shewanella oneidensis

introduced into a contaminated site with Pd(0) deposition on the cell wall is another

good example of nano bioremediation. As a result of their smaller size particles and

semipermeable nature of the plasma membrane, these particles also enter the cyto-

plasm. As well as showing t he presence of electron donors such as hydrogen acetate,

it will also charge and show the presence of chlorine in water bodies. In addition to

acting as indicators, they degrade chlorine (Singh et al. 2020).

1.3 Outline of the Nano Biosynthesis Mechanism Used

in Bioremediation

Heavy metals can be removed from a variety of sources using nanoparticles. Nanopar-

ticles are ﬁrst synthesized using different synthesis methods such as physical, chem-

ical, and biological, and then they are used in bioremediation. Alternatively, nanopar-

ticles can be engineered to combine with microbes, and then these engineered

nanoparticles can be used in bioremediation (Koul and Taak 2018a).

There are variety of methods and techniques that can be employed to

remove toxic heavy metals from the environment. Bioremediation using nanopar-

ticles/nanomaterials created by plants, fungi, and bacteria with nanotechnology

is called nanobioremediation (NBR). There are differences between normal and

nano-bioremediation. In remediation, biological agents are typically used, but when

combined with nanoparticles, their efﬁcacy is increased. NBR is the most suitable

and environmentally friendly method for removing pollutants. As of now, remedia-

tion techniques are either in-situ or ex-situ. Plants, microbes, and enzymes present

in organisms that use these techniques for remediation degrade heavy metals. Heavy

metals are naturally absorbed by some plants through their roots; this process is

known as phytoremediation (Rizwan et al. 2014). When nanoparticles made from

plants, microbes, and enzymes were applied, the plant’s phytoremediation efﬁciency

was enhanced. Phytoremediation uses enzyme-functionalized nanoparticles in order

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 273

to degrade long-chain hydrocarbons. Plants and microbes cannot degrade these

long-chain hydrocarbons (Shah 2020a, b).

In addition to wastewater treatment, nanoparticles are also used to treat contami-

nated soil and air pollution (Ibrahim et al. 2016) (Fig. 1).

Fig. 1 Outline of bioremediation process for industrial waste water

274 H. S. Naik et al.

2 The Method Used in Bioremediation of Wastewater

Several methods are available for wastewater treatment, including adsorption, photo-

catalytic degradation, redox reaction, disinfection and membrane-based techniques.

Due to the unique properties of nanoparticles, such as strong absorption, high reac-

tivity, and quantum size effect, these techniques are widely used (Yadav et al. 2017)

(Fig. 2).

2.1 Adsorption

This is a surface-based method for removing pollutants. An adsorption process occurs

when adsorbable solutes interact with solid adsorbents. Nanoparticles serve as solid

adsorbents, whereas heavy metals serve as adsorbable solutes. Due to the interaction

force between the adsorbent and adsorbable solute, adsorbable solute deposits on

adsorbable surfaces. Adsorption stands out for wastewater treatment because of its

versatility, unique properties, superior efﬁcacy, large surface area, multiple adsorp-

tion sites, temperature-dependent modiﬁcations, speciﬁc pore sizes, unique surface

chemistry, ease of handling, low cost, and the fact that it does not require pretreat-

ment. Shape and morphology also play an important role in nanobioremediation.

Because of their high absorption capacity, modiﬁed nanosorbents can sometimes

become toxic (Ali and Gupta 2007).

In the removal of heavy metals, adsorbents play an important role. Nano adsor-

bents like activated carbon, clay minerals, and natural zeolites can also remove heavy

metals, but their adsorption capacities are low, resulting in poor results. Nanoparticles

with sizes of 1-100 nm are often used in bioremediation instead of traditional sorbents.

Chemical, physical, and biological methods can be used to synthesize nanoadsor-

bents. Biosynthesis materials are widely used because they do not require toxic

chemicals or high energy. Chemistry and physical synthesis nanoparticles have a

well-deﬁned shape and size, while biological synthesis nanoparticles require special

Fig. 2 Method used in

Bioremediation

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 275

attention to their shape and size. In the biological synthesis of nanoadsorbents, plants

such as algae, bryophytes, angiosperms, microbes, and enzymes secreted by living

organisms are used as reducing agents. Functionalized nanoparticles can be synthe-

sized intracellularly or extracellularly by secreting enzymes from the organism where

they function to reduce metal ions and stabilize them (Mourdikoudis et al. 2018).

Nanoadsorbents such as nanotubes, nanowires, and quantum dots are used to treat

wastewater. Nanoadsorption is currently performed with nanoparticles of zero-valent

metals, metal oxides, and nanocomposites. The most commonly used nanoparti-

cles in wastewater treatment are titanium oxides, cerium oxides, manganese oxides,

nanosized zero-valent metals, ferric oxides, aluminium oxides, and magnesium

oxides. Phosphate, an organic pollutant, can be selectively absorbed by cerium oxide.

Metallic nanoparticles are known for their simplicity, super magnetic properties, and

high adsorption capacities, which make them ideal for remediation. Besides adsorp-

tion, chemical and photodegradation methods are also used for wastewater remedi-

ation. However, metallic nanoparticles also have some disadvantages, such as their

small size, which makes them less stable, causing agglomeration, and reducing their

properties. Further, separation from water bodies is difﬁcult, and higher deposition

causes toxic effects on water ecosystems (Aguilar-Pérez et al. 2021).

The pharmaceutical industry uses a variety of drugs that are released into the

environment. Pharmaceutical drugs can disrupt the body’s homeostasis as well as the

endocrine system. In addition, these products produce a large amount of free radicals,

which are toxic. If these medications are used excessively, they can cause genotoxicity

and immunosuppression. For instance, Ibuprofen, paracetamol, and diclofenac do not

require a prescription. Paraben, which is found in many skincare products, is another

pharmaceutical drug that causes environmental problems. Medically, diclofenac is

used to disinfect wounds, but it produces antibiotic-resistant bacteria that cannot be

killed by any antibiotic. The drug is also responsible for the emergence of drug-

resistant bacteria (Sherry Davis et al. 2017).

The ﬁrst report on carbon nanotubes was published by Iijima in (1991). As

cylinder-shaped macromolecules, CNTs have a radius as small as a few nanome-

tres and varying lengths. CNTS are hexagonal lattices of fullerenes with grooves.

These highly hydrophobic nanoparticles are used in wastewater treatment because of

their ability to entrap organic molecules due to the hydrophobic aggregation between

them (Roy 2014a).

In a recent study, green-synthesised Cu nanoparticles were effective at removing

three selected pharmaceutical drugs from aquatic media (Husein et al. 2019).

Nanoparticles of different types are used in nanobioremediation. Metal nanopar-

ticles of Ag, Au have good degradation properties for organic dyes (Sherry Davis

et al. 2017). Bimetallic Fe/Ni nanoparticles of size 60–85 NM produced by green

synthesis can remove 85.8% of triclosan from wastewater. By using biologically

produced platinum and palladium nanoparticles from D. vulgaris, other pharmaceu-

tical drugs, such as sulfamethoxazole, have been successfully removed. It is capable

of removing 85% of the contaminants (Sherry Davis et al. 2017).

Nanoparticles can be produced using plants such as Noaea mucronata, Euphorbia

maroclada, which can remove Pb, Cd, Zn, Cd, and Ni. Nanoparticles can also be

produced by bacteria. For the removal of toxic metals, iron nanoparticles are effective.

276 H. S. Naik et al.

Maghemite is a type of iron nanoparticle produced by Actinobacter. A study found

that bacteria produce iron nanoparticles aerobically and participate in bioremediation.

Additionally, thermophilic nanoparticles can be used to create magnetic nanoparticles

that degrade heavy metals. nZVI nanoparticles produced from black tea, grape marcs,

and vine leaves remove ibuprofen from an aqueous medium (Koul and Taak 2018a).

The orange peel powder has also been used to synthesize Fe3O4 nanoparticles

with good adsorption capacity of cadmium from an aqueous solution. According to

a comparative study of orange peel powder, chemically produced magnetic nanopar-

ticles and orange peel-mediated magnetic nanoparticles, they have surface areas of

47.03, 76.32 and 65.19 m2g−1 BET respectively (Ali 2017).

Nano clay is also used in the absorption process. In addition to removing contami-

nated water, these nano clays are used in soil bioremediation. Nano clay is used for the

successful reduction of phosphorus, according to a report by Allophane. According

to this study, phosphorus concentration reduces from 14.2 to 4.2 mg/L, a reduction

of nearly 70% from the original medium (Koul and Taak 2018a).

2.2 Photocatalysis

In this process, UV light is used to oxidize organic pollutants and convert them

into water and carbon dioxide as by-products. Photocatalysis can also be used to

disinfect certain bacteria. During photolysis, the photocatalyst area accelerates the

reaction rate without consuming it. It is an advanced oxidation process (Ibrahim et al.

2016) where nanoparticles act as photocatalysts. By absorbing UV light, photocat-

alysts get charged and can convert hazardous, non-biodegradable contaminants into

non-hazardous, biodegradable compounds. Using light to convert contaminants has

advantages and disadvantages. Reaction rates may increase or decrease as a result

of light. In case of low light intensity, reaction rate and photocatalytic activity are

affected. As photocatalysts, different nanoparticles are used, such as TiO2,Si, Cd,

ZnS, SnO2,WSe

2,Fe

2O3,WO

3,TiO

3,ZnO,etc.(Roy 2014b) (Fig. 3).

A photocatalytic material is typically illuminated with light that balances the

valence and conduction bands during the photolysis process. Exposure of light

photons to irradiation is more powerful than their band gap energy. As a result

of this process, electronic holes are created, which combine with organic pollutants

and produce reactive oxygen species. In photocatalysis, the size of the nanoparticles

plays a crucial role (Aguilar-Pérez et al. 2021). Water molecules capture the hole (hh)

generated by electron–hole pairs in photocatalysis. By releasing hydroxyl radicals

(OH-) from water molecules, a wide range of microbial cells and organic pollu-

tants can be degraded. There has been a great deal of interest in the role of titanium

nanoparticles and silver in environmental bioremediation, especially in wastewater

treatment (Ojha 2020).

Fujishima and Honda discovered the photocatalytic thin-ﬁlm mechanism while

studying the electrochemical photolysis of water on TiO2 semiconductor electrodes.

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 277

Fig. 3 Degradation of Methylene blue through photocatalysis

The titanium nano-oxide nanocomposite inactivates microorganisms by UV irradia-

tion (Berekaa 2016). To enhance the photocatalytic activity of nanoparticles, titanium

quantum dots (QDs), titanium nanotubes, titanium nanosheets, titanium nanowires,

and titanium mesoporous hollow shells are used. Different morphological forms of

Tio2 increase the number of photoreactive sites exposed. TiO2 nanoparticles with

metal or non-metal doping should enhance charge separation or board the absorption

spectrum. In addition to difﬁcult recovery, agglomeration, and short activity, titanium

oxide nanoparticles have several disadvantages (Ibrahim et al. 2016).

Copper nanoparticles are synthesized from Escherichia sp SINT7, which is copper

resistant. The nanoparticles successfully degrade azo dye at a very low concentration

of 25 mg g−1. A blank Cango-T shows a reduction of 83.61%, while Direct Blue-1

reduces up to 88.42% (Mandeep and Shukla 2020). In addition to their function as

a semiconductor, Zn NPs are also used as a photocatalyst to degrade organic dyes,

phenols, and pharmaceuticals (Sherry Davis et al. 2017).

2.3 Redox Reaction

During wastewater treatment, an oxidation and reduction reaction takes place because

of the redox reaction conversion of hazardous contaminants or toxic metals into non-

hazardous contaminated or nontoxic metals that become more stable, less mobile,

or inert. To enhance degradation and extraction capacity, redox reactions are used in

remediation to change the water chemistry and microbiology by introducing speciﬁc

substances into wastewater containing contaminants.

Water contaminated with organic or inorganic contaminants can be treated with

this redox process technique. It is not only laboratory practice used for bioremedia-

tion, but all processes on earth use energy generated from redox reactions (Tandon

and Singh 2016).

278 H. S. Naik et al.

On earth, basic transformations such as the nitrogen cycle, carbon cycle, sulphur

cycle, magnesium cycle, iron cycle, and nitrous cycle take place. Redox-sensitive

elements such as chromium (Cr), arsenic (As), uranium (U) and copper (Cu) may be

present in the environment (Tandon and Singh 2016). Redox-active CeO2 nanopar-

ticles are used for the treatment of wastewater in a variety of industries. Redox

nanoparticles react with Cr (VI) (aq) and Fe2+ species, leading to the change in

surface morphology (Division 2019). As part of traditional methods, chlorine and

ozone are used to disinfect; this product produces carcinogenic substances. By using

nanoparticles, these problems are solved. Ferrate (VI) is used as a disinfectant for

algae, bacteria, microcystins, viruses. Many natural and advanced nanomaterials

exhibit disinfectant properties. Nanoparticles such as silver nanoparticles, chitosan,

photocatalytic TiO2, aqueous fullerene nanoparticles and carbon nanotubes, fullerol

interact with the cell membrane and cause the membrane to break down and release

reactive oxygen species. Nickel, selenium, chromium, cobalt, arsenic, lead, and

copper are easily controlled by redox reactions in order to determine their toxicity

and mobility (Tandon and Singh 2016).

2.4 Disinfection

Water emitted from various sources contains many pathogens that may cause

serious health problems in humans. Bacteria (Escherichia coli, Pseudomonas aerug-

inosa, Burkholderia pseudomallei, Salmonella typhi, Plesiomonas, Yersinia ente-

rocolitica, Vibrio cholera, Campylobacter spp., Shigella spp., etc.),cyanobacteria

(Anabaena, Nostoc, Microcystis, Planktothrix), viruses (astroviruses, hepatitis A

viruses, hepatitis E viruses, noroviruses enteroviruses, sapoviruses, rotavirus, aden-

oviruses), prions, protozoa (Cyclospora cayetanensis, Isospora belli, Entamoeba

histolytica, Giardia intestinalis, Cryptosporidium, Balantidium coli, Naegleria

fowleri, Toxoplasma Gondii, Acanthamoeba spp.), helminths (Schistosoma spp.,

Dracunculus medinensis), cysts, fungi, Rickettsia, etc., are various kind of microbes

that pose a serious threat to people and ecosystems alike (Ojha 2020). Water treatment

must be done properly. It is one of the s imple processes in which microorganisms are

killed by suitable compounds and water becomes free of microorganisms. The use

of disinfectants is not limited to eliminating microbial contamination; they also help

eliminate other parameters like colour, taste, oxidize iron and manganese in water

bodies, and enhance ﬁltration as well as coagulation activity (Berekaa 2016).

These disinfection practices are mostly used in the pharmaceutical industry,

medical ﬁeld, research labs, and diagnosis centers. In traditional disinfection

methods, chlorine, ozone is used to kill compound toxics and cancerous byproducts,

like halo acetonitrile, dibutyl phthalate, halo acetic acid, chlorite, and chloral hydrate.

According to the report, there are about 600 byproducts of disinfection that are very

harmful to living things. Nanoparticles are a safer alternative. The silver nanoparti-

cles, zinc oxide, iron oxide, manganese oxide nanoparticles (Mn2O3, MnO2, MnO

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 279

and Mn3O4) NPs, titanium oxide nanoparticles, cerium oxide nanoparticles, magne-

sium oxide nanoparticles, silica-based nanocomposites, and carbon-based nanoma-

terials play an important role as disinfectants either by generating reactive oxygen

species or by disrupting cell membranes. Several metals play an important role

in the disinfection of E. coli bacteria. A superior ability to kill bacteria has been

demonstrated by silver nanoparticles (Berekaathe 2016;Ojha 2020).

A Double-layered superparamagnetic Fe3O4@SiO2@Ag@porousSiO2

nanoparticle shows good disinfectant properties against E. coli, as well as good recy-

cling properties. It is a two-layer system, with Fe 3 O 4 @SiO 2 nanoparticles on the

inner layers and Ag@porous SiO2 nanoparticles on the outer layers. It is believed that

the inner layer of Fe 3 O 4 @SiO 2 was protected by a dense s ilica matrix and allowed

Ag to be loaded on the outer surface. The outer mesoporous silica helps to dissolve

and oxidize Ag, giving Ag+ ions. Combinational double-layered nanoparticles can

meet the MCL for drinking water after treatment (Wang et al. 2019).

The use of titanium nanoparticles led to fast killing of bacteria E coli in the pres-

ence of solar irradiation followed by a ﬁrst order kinetic mechanism. Iron oxide

nanoparticles also show great properties, and they are easy to synthesize, cost-

effective, and recyclable, which shows great disinfectant properties (Ojha 2020). Tita-

nium nano-oxide and thin-ﬁlm nanocomposite demonstrated the ability to inactivate

microorganisms (Berekaa 2016).

According to a review article, iron granules used on a commercial scale success-

fully inactivated viruses and nanoscale zero-valent iron (nZVI) particles completely

destroyed bacteria like Pseudomonas ﬂuroscens and B. subtilis. Hybrid copper and

ZVI nanoparticle clusters were used in a case study in Tirupur, Tamil Nadu, India to

treat industrial efﬂuents (textile, tannery, pharmaceutical) and sewage. The hybrid

cluster uses more copper, ZVI, and less silver to reduce treatment costs. These clus-

ters remove excess salts, ﬂuorides, heavy metals, excess salt, dyes from the efﬂuent

(Kiruba Daniel et al. 2014). Nanoparticles may act as an antimicrobial agent for

wastewater treatment, along with a combination of UV disinfection to improve water

quality (Berekaa 2016).

2.5 Membrane

Using Membrane in wastewater treatment is one of the most effective strategies

for removing water pollutants s ince it has a great separation capacity and does not

require the addition of chemicals during operation. The efﬁciency of membranes is

determined by the permeability of the membrane material. Membranes are generally

made of cellulose acetate, polyamides, or polyacrylonitrile. In accordance with the

size of membranes, there are different categories of membranes. Bacteria, suspended

solids, and protozoa are removed by micro membranes. Viruses can be removed

with Ultraﬁltration, whereas heavy metals and organic matter can be removed with

Nanoﬁltration (Abdelbasir and Shalan 2019).

280 H. S. Naik et al.

Fouling of the membrane is a disadvantage of membrane ﬁltration. There are two

types of fouling: organic fouling and biological fouling. Organic fouling is a depo-

sition of organic molecules inside the membrane pore, whereas biological fouling

occurs when microorganisms present in the water attach to membranes and reduce

ﬂux capacity. The limitations of the membrane can be overcome by modifying the

membrane with hydrophobicity parameters which are capable of enhancing organic

antifouling (Ibrahim et al. 2016).

The use of nanoﬁltration membranes is a good practice for removing pathogens

such as Cryptosporidium oocysts (Ojha 2020). Nanoﬁltration membranes (NF) not

only remove toxic metals, but are also important for recovering nutrients from

industrial wastewater. NF with 90 mm pore size is commonly used in paper and

pulp industry and has the advantage of rejecting more than 70% of phosphorus.

However, the disadvantage of these NF is high phosphorus, which causes fouling of

the membrane. Gold nanoparticles embedded with a polymer blend of NF membrane

exhibit better recovery, rejection of trivalent phosphate at 96.1%, and increased

fouling resistance. Graphene oxide (GO) or attapulgite (ATP) supported on ceramic

shows nearly 100% rejection of heavy metals like nickel, lead, cadmium, and copper

(Mandeep and Shukla 2020).

As nanotechnology advances, new membranes are developed for water treatment.

Grafting, bending, and other methods are used to modify membranes. The modiﬁ-

cations are necessary to make the membrane highly permeable, increase catalytic

properties, and degrade contaminants (Ibrahim et al. 2016).

The membranes used in bioremediation processes are produced by the electrospin-

ning process, the purpose of using the electrospinning process is to generate polymer

or composite nanoﬁbrous membranes from different materials between 20–200 m

diameter. Electrospinning provides a ﬁne porous size with a large speciﬁc surface

area and gives the membranes high water ﬂux. A study revealed that nanomem-

branes can effectively remove a wide range of heavy metals and after this process,

the membrane can be recovered again and reused after cleaning it (Abdelbasir and

Shalan 2019).

The afﬁnity of the membrane can be increased by adding a functional group to

it. Through covalent interaction, ligands are attached to the membrane for function-

alization. Cibacron blue is attached to the cellulose nanoﬁber membrane for func-

tionalization during aluminium puriﬁcation. Attaching ceramic nanoparticles such as

alumina hydroxide or iron nanoparticles to polymer nanoﬁber membranes removes

heavy metals from the nanoﬁbers. As a ligand molecule, cyclodextrin can also be

added to polymethyl methacrylate nanoﬁber membranes to improve the removal of

organic waste (Ibrahim et al. 2016). Nanoparticles can be used individually or in

combination with appropriate matrices to remove pollutants f rom industrial waste

water. In a review on Microbial Nanotechnology for Bioremediation of Industrial

Wastewater, it was found that porous magnesium oxide can absorb 1000 mg. g −1

toxic dye from the iron industry. The grafted iron oxide with hyperbranched polyg-

lycerol had the ability to remove copper, nickel, and aluminium from wastewater in

just 35 s (Mandeep and Shukla 2020).

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 281

Nanoparticles are supported on suitable supporting materials during the membrane

process. For example, cellulose acetate, alginate, and polyvinyl alcohol embedded

with silver nanoparticles. Different fabrication processes have been developed based

on the separation process. Silver-coated Mn Zn ferrite, silver-containing thermo-

plastic hydrogels, silver-ion-exchanged titanium phosphate ﬁlms, and silver nanopar-

ticles within third-generation dendritic poly (amidoamine) (PAMAM) grafted onto

multiwalled carbon nanotubes are used in membrane ﬁltration. Many transport mech-

anisms in bacteria are inhibited by silver ions, which bind to bacterial DNA (Mandeep

and Shukla 2020).

Nano-zeolites in Thin Film Nanocomposite Membranes (TFN) increase

membrane permeability. 80% of the salt is rejected by these TFNs. Nano-TiO2 in

TFCs increases rejection rates, reduces biofouling, reduces biological contaminants,

and inactivates microorganisms using UV light (Kiruba Daniel et al. 2014).

3 Nanoparticles Used in Bioremediation

A signiﬁcant increase has occurred in the biological synthesis of nanoparticles to

create new materials that are cost-effective and stable with important applications in

electronics, medicine and agriculture. It is possible to synthesize nanoparticles using

an array of conventional methods, but biological synthesis is superior because of its

ease of rapid synthesis, control, ease of processing, control of size characteristics,

low cost, and eco-friendly approach. Biological contaminants (such as bacteria) and

chemical contaminants including organic pollutants, are extensively removed with

the help of nanoparticles. During the last decade, nanoparticles and nanomaterials

have attracted considerable attention because of their unique size-dependent physical

and chemical properties (Okhovat et al.2015).

Biological systems can synthesize molecules with highly selective properties and

are self-organizing. Nanoparticles produced traditionally by physical and chemical

methods are costly. As a way to synthesize nanoparticles at a lower cost, microorgan-

isms and plant extracts were used. Using a bottom-up approach, nanoparticles are

biosynthesized by reduction and oxidation reactions. It is usually microbial enzymes

or plant phytochemicals with antioxidant or reducing properties that are responsible

for reducing metal ions into nanoparticles (Sastry et al. 2003).

3.1 Nanoparticle Synthesized by Plants

The green synthesis of nanoparticles by plants has become very popular in recent

years due to a single-step biosynthesis process, lack of toxicants, and presence of

282 H. S. Naik et al.

natural capping agents. It is advantageous to use plants for the synthesis of nanoparti-

cles since they are readily available, safe to handle, and have a broad range of metabo-

lites. Water-soluble phytochemicals reduce metal ions in a much shorter period than

fungi and bacteria (Yadav et al. 2017).

The synthesis of nanoparticles by plants is therefore superior to that of bacteria

and fungi. Compiled information indicates that the effects of nanoparticles vary from

plant to plant and depend on their mode of application, size, and concentration (Yadav

et al. 2017).

Silver Nanoparticles

To remove toxicity from wastewater, silver nanoparticles can be used in simu-

lated wastewater treatment. The plants that have been reported for the synthesis

of silver nanoparticle are Elettaria cardamomum, Parthenium hysterophorus

Ocimum sp, Euphorbia hirta, Nerium indicum Azadirachta indica, Brassica juncea,

Pongamia pinnata, Clerodendrum inerme, Gliricidia sepium, Desmodium triﬂorum,

Opuntia,ﬁcus indica, Coriandrum sativum, Carica papaya, (fruit) Pelargonium

graveolens, Aloe vera extract, Capsicum annuum, Avicennia marina, Rhizophora

mucronata, Ceriops tagal, Rumex hymenosepalu,s Pterocarpus santalinus, Sonchus

asper. Most commonly, silver nanoparticles are used in disinfection techniques due

to their toxic effect on microorganisms at certain levels.

Gold Nanoparticles

Heavy metals, fertilizers, detergents, and pesticides seriously reduce the avail-

ability of pure drinking water and potable water. Research on several fronts is

advancing the idea of nano based solution using gold for cost-effective solution

in water treatment, which has intriguing potential to deal with the water pollution

problem. The plants responsible for synthesizing the gold nanoparticles are Te rmi -

nalia catappa, Banana peel, Mucuna pruriens, Cinnamomum zeylanicum, Medicago

sativa, Magnolia kobus, Dyopiros kaki, Allium cepa L., Azadirachta indica A. Juss.,

Camellia sinensis L., Chenopodium album L., Justicia gendarussa L., Macroty-

loma uniﬂorum, Mentha piperita L. Mirabilis jalapa L., Syzygium aromaticum (L),

Terminalia catappa L., Amaranthus spinosus (Lietal.

2011).

The degradation of methylene blue is achieved by using more than 20 plant-

mediated gold nanoparticles. Oil of leaves of Myristica fragrans, Fagonia indica,

Persea americana seed extract, Nigella arvensis leaf extract, Lagerstroemia

speciosa, Sterculia acuminate, Mimosa tenuifolia, Salmalia malabarica, Sesbania

grandiﬂora L., Edible coconut oil, Parkia roxburghii, leaf extract, Costus Pictus leaf

extract, Actinidia deliciosa fruit, Costus speciosus rhizome, Citrifolia fruit extract,

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 283

Hydrocotyle asiatica leaf extract can produce gold nanoparticles and degrade methy-

lene blue under different conditions (Dabhane et al. 2021). Although Gold nanopar-

ticles have been used to degrade dyes in several reports, this process is not commer-

cially viable because the gold salt is expensive and the nanoparticle cannot be recov-

ered after degradation. A commercially viable process could be created by using

nanoparticles supported by matrices.

The catalytic activities of gold and silver nanoparticles were determined by

catalytic degradation of pollutants with excess amounts of NaBH4 at room tempera-

ture. A photocatalytic mechanism can be used to bioremediate industrial water using

such nanoparticles (Vo et al. 2019).

CuO Nanoparticle

Plant-mediated copper oxide nanoparticles are the easiest, simple and most efﬁcient

way for the production of nanoparticles. Madhuca longifolia extract mediated CuO

nanoparticles exhibit great characterization aspects like great crystalline nature on

XRD and on change in different sizes it shows an increase in photoluminescence

property. This nanoparticle shows photocatalytic degradation of methylene blue (Das

et al. 2018).

3.2 Nanoparticles P roduced by Bacteria

Bacteria produce enzymes that catalyze speciﬁc reactions which are responsible for

the synthesis of nanoparticles. This provides a new rational biosynthetic strategy

that involves the use of enzymes, microbial enzymes, vitamins, polysaccharides and

biodegradable polymers. The extracellular secretion of enzymes has the advantage

of producing highly pure nanoparticles of size 100–200 nm that are free of other

cellular proteins. The properties of nanoparticles are determined by optimizing the

parameters controlling organism growth, cellular activities, and enzymatic processes.

A large-scale synthesis of nanoparticles using bacteria is attractive because it does not

require hazardous, toxic, and expensive chemical materials (Iravani 2014). Bacteria

are considered potential sources of nanoparticle synthesis, like gold, silver, platinum,

palladium, titanium, titanium dioxide, magnetite, cadmium sulphide, and so on.

Silver Nanoparticles

Silver nanoparticles (Ag NPs) are highly toxic to microorganisms and thus have

strong antibacterial effects against a wide range of microorganisms, including viruses,

bacteria, and fungi. Silver nanoparticles attached to ﬁlter materials have been consid-

ered promising for water disinfection due to their high antibacterial activity and cost-

effectiveness. The bacteria responsible for the synthesis of silver nanoparticles are

284 H. S. Naik et al.

Bacillus cereus, Oscillatory willie, Escherichia coli, Pseudomonas stuzeri, Bacillus

subtilis, Bacillus cereus, Bacillus thuringiensis, Lactobacillus strains, Corynebac-

terium, Staphylococcus aureus, Ureibacillus thermosphaericus (Lietal.

2011). Pseu-

domonas aeruginosa JP-11 was extracted from marine water produced 40 nm, spher-

ical shape cadmium sulphide nanoparticle removes cadmium pollutant from aqueous

solution. Other bacteria like Klebsiella pneumoniae, Escherichia coli, Pseudomonas

jessinii isolated from tiger nuts, carrot juice and faces are responsible for silver

nanoparticles. Shewanella loihica PV-4, produced spherical shape silver nanoparticle

responsible for degradation of methyl orange dye (Gahlawat and Choudhury 2019).

3.3 Nanoparticles P roduced by Yeast and Fungi

The fungi are an excellent source of extracellular enzymes that inﬂuence nanopar-

ticle synthesis. Many of them have been used for the biosynthesis of nanoparti-

cles. As compared to bacteria, fungi can produce larger amounts of nanoparticles

(Munisamy).

It is equally important to understand the nature of the biogenic nanoparticles.

As a result of microbiological methods, nanoparticles are formed more slowly than

those derived from plant extracts. Enzymes produced by fungi are responsible for

synthesis of nanoparticles. Due to the wide diversity, easy culture methods, reduced

time, and cost-effectiveness of fungi, they offer an advantage over other methods.

Thus, nanoparticles can be synthesized in an eco-friendly manner (Ali et al. 2011).

CuO Nanoparticles

CuO nanoparticles can be produced using fungus-like Penicillium chrysogenum.

In this study, copper sulfate with fungal extract was exposed to gamma

dose radiation. The nanoparticles form were found to be effective against six

different kinds of fungi Ralstonia solanacearum, Fusarium oxysporum, Ralstonia

solanacearum, Aspergillus niger, Penicillium citrinum, Erysiphe cichoracearum,

Erwinia amylovora, and Alternaria solani.

Maximum antifungal activity of penicillium-mediated copper oxide nanoparticles

was observed in Fusarium oxysporum and it was least effective in Erwinia amylovora.

This nanoparticle is best for disinfection of agricultural packaging tools because these

six fungi infects agricultural crops and when it comes to the packaging industry there

might be a chance of contamination of water. It is necessary to take action against

these fungi for future contamination (El-Batal et al. 2020).

Gold nanoparticles synthesized using fungus Streptomyces griseoruber extracted

from soil samples of Mercara region are responsible for degradation of methylene

blue (Gahlawat and Choudhury 2019).

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 285

4 Target Speciﬁc Removal of Pollutants Using Nano

Bioremediation

Studies are being conducted on removing contaminants from industrial wastewater,

which contains various types of contamination. A few examples of nanoparticles

that have been used for metal or nonmetal removal from industrial wastewater are

shown in Table 1. Industrial wastewater contains major metals and nonmetals such

as Cr (VI), Pb (II), Mercury, Cu II, Cd (II) / Pb (II), Ar (III), Nickel, Anionic chromat

(Ibrahim et al. 2016) (Puthukkara et al. 2021a).

4.1 Metal or Non-Metal

Chromium VI

1 g/L of guar gum zinc oxide nanoparticles can remove almost 98.63% Cr (VI) within

50 min at pH 7. Green tea leaf extract, Syzygium jambos (l.), Eucalyptus globulus

leaf, Citrus limetta fruit peel mediated iron nanoparticles also remove chromium VI

from industrial wastewater (Ibrahim et al. 2016).

Pb (II)

A graphene oxide/zinc oxide nanocomposite (GO-ZnO), iron phosphate-mediated

magnetite nanoparticle (Fe3O4), and a Syzygium aromaticum ﬂower extract-

mediated zerovalent iron nanoparticle (ZIF) have been shown to remove Pb II. The

adsorption capacities of CeO2, Fe3O4, and TiO2 are 189 mg Pb/g, 83 mg Pb/g, and

159 mg Pb/g, respectively. The TiO2, Fe3O4 and CeO2 nanoparticles do not show

any toxicity, but CeO2 exhibits high phytotoxicity.

Mercury (Hg2+)

Mercury ions are removed by iron sulﬁde (FeS) nanoparticles synthesised using

carboxymethylcellulose (CMC) as a stabilizing agent.

Cadmium (II) / Lead (II)

Cd (II) and Pb (II) were removed from wastewater using NiO nanoparticles. Both

heavy metals can be absorbed by the NiO nanoparticles. NiO nanoparticles had

maximum adsorption capacities f or Pb (II) and Cd (II) ions of 625 mg/g and 909 mg/g,

286 H. S. Naik et al.

respectively. Contaminants are removed spontaneously and endothermically during

this process.

Arsenic (III)

Fe3O4 nanoparticles with ascorbic acid show 46.06 mg/g absorption of arsenic (III)

and arsenic (V) from contaminated sites. The advantage of these nanoparticles is that

they prevent metals from leaching from water.

Copper (II)

The copper (II) adsorption capacities of ZnO and MgO are 593 and 226 mg/g, respec-

tively, at 3 to 4 pH. Iron phosphate nanoparticles coated with sodium carboxymethyl

cellulose can absorb copper (II) from soil and water (Table 1).

4.2 Organic or Inorganic Molecules

Various types of nanoparticles are used for removing organic and inorganic pollu-

tants. They are mostly carbon-based nanoparticles that are used to remove toxic

compounds from the environment. Dye, pesticides, benzene/phenol derivatives and

pharmaceuticals are among the organic and inorganic pollutants released into indus-

trial water. Table 2 provides data regarding different types of organic/inorganic pollu-

tants, speciﬁc contaminants released by different sectors and nanoparticle-based

solutions for removing contaminants.

Organic Dyes

Different dyes are eluted in the textile industry. Methylene blue, methylene orange,

bromophenol blue, malachite green, Reactive Blue 4 (RB4), Alizarin red S (ARS) and

Morin are some of the most common dyes. Due to electrostatic interaction, methy-

lene blue can interact with magnetite carbon nanotubes and exhibit high absorp-

tion capacity. Carbon nanotubes (CNT) prepared by acid washing and oxidation of

nanoparticles in puriﬁed air are used to degrade methyl orange. A high CNT dosage

is responsible for inhibiting the surface methyl orange dye molecules. Dye degra-

dation is affected by temperature and pH. Dye-like reactive blue degrades at high

temperatures, due to the high temperature, particle diffusion between dye molecules

increases, leading to the degradation of the dye. The adsorption capacity of Alizarin

red S (ARS) and Morin is increased with low pH.

Additionally, plant extract synthesized nanoparticles are capable of degrading dye

molecules. Mango peel, Cupressus sempervirens, Camellia sinensis, Green and black

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 287

Tabl e 1 Types of contaminant and nanoparticles used in removal of contaminantes

Types of

Contaminants

Target speciﬁc

contaminant

Nanoparticles used in

bioremediation

References

Metals /Non-metal Heavy metals Thiol-functionalized

superparamagnetic

nanoparticles, ZnO, FeO3

Ibrahim et al. (2016)

Cr (VI) Guar gum–nano zinc oxide

(GG/nZnO)

Green tea leaf mediated

zerovalent iron nanoparticle

Syzygium jambos (l.)

Alston leaf extract-based

ZVI nanoparticle

Puthukkara et al.

(2021a)

Eucalyptus globulus leaf

Citrus limeta fruit peel

Pb (II) Nanocomposite

GO–ZnO(OH)2, Magnetite

(Fe3O4)

Ibrahim et al. (2016)

Iron phosphate mediated

nanoparticle

Nano ZnO nanoparticle

CeO2, TiO2 nanoparticles

Syzygium aromaticum

ﬂower mediated zero-valent

iron nanoparticles

Puthukkara et al.

(2021a)

Mercury Iron sulphide (FeS)

nanoparticle

Ibrahim et al. (2016)

Copper II Iron phosphate nanoparticle

Magnesium and zinc oxide

(MgO and ZnO)

carbon nanotube modiﬁed

with silver nanoparticle

Cd (II) / Pb (II) Nanocrystalline

hydroxyapatite (nHA)

NiO nanoparticles

Ar (III) Fe3O4 nanoparticles

Coated with ascorbic acid

Nickel Carbon nanotube modiﬁed

with sodium hypochlorite

Anionic chromate Oxidation on carbon

nanotube

288 H. S. Naik et al.

Tabl e 2 Nanoparticle used in removal of organic/inorganic pollutant

Organic /inorganic

Contaminants

Target speciﬁc

contaminant

Nanoparticles used in

bioremediation

References

Organic dyes Methylene blue (MB) Carbon nanotubes

loaded with magnetite

Ibrahim et al.

(2016)

Psidium guajava leaf

extract-based ZVI

nanoparticle

Puthukkara et al.

(2021a)

Methyl orange (MO) Carbon nanotube Ibrahim et al.

(2016)

Mango Peel mediated

ZVI nanoparticle

Puthukkara et al.

(2021a)

Cupressus sempervirens

leaf mediated ZVI

nanoparticle

Bromophenol blue Camellia sinensis leaf

extract mediated

zero-valent valent iron

nanoparticle synthesis

Malachite green Green, oolong and black

tea leaf extract mediated

ZVI nanoparticles

Ibrahim et al.

(2016)

Reactive blue 4 (RB4) Carbon nanotube

Alizarin red S (ARS)

and Morin

Pesticides Dubinin Carbon nanotube

Diuron and dichlobenil

Atrazine

Benzene

Derivatives

Phenolic

Compounds

2,4,6-Trichlorophenol

(TCP)

Carbon nanotubes with

treatment of HNO3

Benzene, toluene,

ethylbenzene, and

p-xylene

Carbon nanotube with

treatment of sodium

hypochlorite (NaOCl)

1,2-Dichlorobenzene

(1,2 DCP)

Graphitized carbon

nanotube

Chlorophenol Graphitized carbon

incorporated by Ni/Fe

Nanoparticles

Zhuang et al.

(2021)

Pharmaceuticals Ciproﬂoxacin (CPI) Graphitized (MG),

hydroxylated (MH) and

carboxylized (MC)

Ibrahim et al.

(2016)

Ibuprofen (IBU) HNO3 on carbon

nanotube (OMWCNTs)

(continued)

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 289

Tabl e 2 (continued)

Organic /inorganic

Contaminants

Target speciﬁc

contaminant

Nanoparticles used in

bioremediation

References

Tetracycline (TC) sodium hypochlorite on

carbon nanotube

Epirubicin (EPI) Modiﬁcation of carbon

nanotube with HNO3

Natural organic

maters (NOMs)

Humic acid (HA) Carbon nanotube with

modiﬁcation of HNO3

and H2SO4

Fulvic acid (FA)

tea extracts assisted synthesis of zero-valent iron nanoparticles are responsible for

degradation of methylene blue, methylene orange, bromophenol blue and malachite

green dyes (Ibrahim et al. 2016; Puthukkara et al. 2021b).

Pesticides

Carbon nanotubes are responsible for degradation of diuron, dichlobenil, atrazine

and dubinin. Diuron degradation is inﬂuenced by hydrogen bonding. Addition of an

oxygen-containing group increases the adsorption of pesticides on carbon nanotubes

(Ibrahim et al. 2016).

Benzene Derivatives, Phenolic Compounds

Benzene-containing compounds like 1,2-Dichlorobenzene (1,2 DCP) are degraded

at high pH with the help of graphitized carbon nanotubes. Dye degradation requires

a low pH, but compounds like benzene require a high pH for degradation. A decrease

in adsorption capacity results from more water molecules being formed due to photo-

catalytic degradation. With the help of HNO3, carbon nanotubes can also be used

for the degradation of 2,4,6-Trichlorophenol (TCP). Carbon nanotubes oxidized

with sodium hypochlorite (NaOCl) generally degrade compounds such as p-xylene,

ethylbenzene, toluene and benzene (Ibrahim et al. 2016).

Pharmaceuticals

The wastewater released by pharmaceutical industries contains several traces of drugs

which adversely affect both humans and the environment. A number of drugs in phar-

maceutical industry wastewater are epirubicin (EPI), ciproﬂoxacin (CFX), tetracy-

cline (TC), ibuprofen (IBU). Carbon nanotubes are used to degrade pharmaceutical

drugs (Ibrahim et al. 2016) (Table 2).

290 H. S. Naik et al.

Tabl e 3 Bioremediation of microbial contamination through nanoparticles

Microbial

Contaminants

Target speciﬁc

contaminant

Nanoparticles used in

bioremediation

References

E. coli Silver nanoparticles, Ag

Nanowire

Yadav and Kumar (2017)

AgNPs/PU, Ag/CNT/PU

sponges, AgNPs

polypropylene ﬁlters,

Polysulfone/AgNPs,

AgNW/CNT

Ojha (2020)

MgO nanoparticles Ojha (2020)

Bacillus subtillus Magnesium nanoparticles Yadav and Kumar (2017)

P. aeruginosa, Magnetic Fe3O4/SiO2, Dimapilis et al. (2018),

Al-Issai et al. (2019),

Wang et al. (2019), Ojha

(2020)

Staphylococcus

aureus

Silver nanoparticles, ZnO,

CuO, Tio2

Salmonella typhi Silver nanoparticle, ZnO,

CuO, Tio2

Natural Organic Matter

For absorption of natural organic matter like Fulvic acid (FA), Humic acid (HA)

carbon nanotubes are used with a combination of H2SO4 and HNO3.

4.3 Microorganism

Microorganisms play a great role in toxic metal reduction but sometimes microor-

ganisms may lead to contamination of water. Nanoparticles from various syntheses

can remove microorganisms’ contamination. In most cases, silver nanoparticles

are used to kill microorganisms. The fungi mediated silver nanoparticle is found

to be effective against bacteria like S. aureus and C. violaceum (Durán et al. 2007),

Table 3 shows Microbial contamination and nanoparticles for removal of it.

5 Recent Research Trends and Advances in Bioremediation

Various industries emit wastewater that contains Microplastics (MPs), which are

plastic particles with a size of about 5 mm, and nano plastics, which are plastic

particles with a s ize less than 100 nm. Plastics of this type are either formed by

the degradation of larger plastic materials or directly from products in which they

are used. In both cases, such plastics have serious health risks. Studies on drinking

water treatment plants show more than 400 mg MPs per litre, which is dangerous

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 291

to human health. Currently, no treatment is available for the complete removal of

MPs. Due to its hydrophobic surface and ability to absorb pollutants, MPs may cause

problems in other wastewater treatment systems. As a result, it is difﬁcult to evaluate

the effectiveness of particular wastewater treatment methods. MPs can be removed

at a certain level using activated carbon (AC) inﬁltration (Jjagwe et al. 2021).

Recently, one interesting strategy for wastewater treatment has been the use

of hydrogel-mediated composites for the removal of organic and inorganic pollu-

tants. Hydrogel based solutions have a good adsorbent capacity as compared to

conventional adsorbents.

The polymer networks in hydrogel have a large absorption capacity, and they have

several better properties like durability, porosity, photostability, and odourlessness.

Yi et al. demonstrated the ﬁve-cycle reusability of hydrogels formed from graphene

oxide, polyvinyl alcohol, and alginate on the methyl orange dye in 20 min. Using

two different kinds of polymer of alginate, i.e., alloy site nano-composite beads and

calcium alginate show reusability after ten cycles of the cycle (Thakur et al. 2018).

A novel kind of titanate nanocomposite sheet immobilized from silk ﬁbroin has

been reported by Magri et al., approximately 75% of Pb2+ can be absorbed on this

sheet. During the whole process, even after several rounds of treatment, it does not

lose its capacity or show any degradation of the composite material. The selectivity of

these immobilized titanate sheets on solid silk ﬁbroin nanocomposite was enhanced

by the addition of sodium ions (Magrì et al. 2018).

Today, researchers are making use of surface functionalized silica nanoparticles

for water puriﬁcation. Silanol group on silica increases surface activity of nanopar-

ticles and protects them from leaching. Wang et al. describe the development of

silica-based adsorbent models for heavy metals. Study by Di Natalea et al. showed

that carbon nanoparticles supported by silica are capable of absorbing heavy metals

from wastewater, such as Pb2+, Ni2+ and Cd2+. In certain conditions, such as

when the temperature is around 100 0C and the pH is neutral, Ni2+ is removed

faster than Cd2+. Other investigations of heavy metal removal by Sheeta et al. using

different nanostructures such as silica/graphite oxide composites, graphite oxide,

and silica nanoparticles. Composites of silica/graphite oxide in a ratio of 2:3 exhibit

the highest efﬁciency of heavy metals among these three materials. In a recent study,

Li et al. developed a new silica nano adsorbent whose surface has a functionalizing

component designed speciﬁcally to eliminate heavy metals from wastewater. The

novel kind of surface modiﬁcation is made with ﬁve kinds of functional groups; these

modiﬁcations are unique and were initially used for the removal of heavy metals. The

silica modiﬁed with EDTA as a functionalizing group was able to absorb more Pb

than the other four groups. As a method of removing trihalomethanes (THMs) from

water by using the sintered process, Ulucan et al. reported that Fe2O3 nanoparticles

sintered in zeolite showed a superior level of absorption (Janani et al. 2022).

For r emoval of Pb2+ from aqueous medium two kinds of nano-zero-valent iron

were used. One type is iron produced by a reduction method and the other is commer-

cially available iron. This comparative study shows zero valent iron showed more

absorption of Pb2 + and in less than 15 min Pb2+ was removed from the aqueous

system. Elliott et al. synthesized nano-zero-valent iron nanoparticles using sodium

292 H. S. Naik et al.

borohydride and ferrous sulphate and successfully removed Hexachlorocyclohexane

from contaminated water. This nanoparticle formulation can remove almost 95%

contaminated Hexachlorocyclohexane with help of a little amount of zero-valent

iron nanoparticle (2.2 to 27 g.L−1) within 2-day (Puthukkara et al. 2021a).

There are many applications of nanotechnology in bioremediation. It is known

that enzyme-based nanoparticles can be used for bioremediation since enzymes act

as biocatalysts. The main disadvantage of enzymes is that they are less stable than

synthetic catalysts. Since enzymes have a short lifetime, they are not applicable

commercially. The stability of enzymes can be increased in many ways. Enzymes

play a signiﬁcant role when attached to the nanoparticle (Rizwan and Ahmed 2018).

The advantage of nanoparticles is that they extend the half-life of enzymes by

providing extra stability, protecting the enzymes from biotic and mechanical degra-

dation. Enzymes encapsulated within nanoparticles are more stable and can be reused

several times, the nanoparticles provide support for the enzymes and protect them

from protease attack and therefore biodegradation of enzymes is prevented, ensuring

that enzymes last longer. The properties of nanoparticles were tested using complex

nanoﬁber esterase enzymes over a 100-day period. Yadav and Kumar (2017) proved

that enzymes continue to function after 100 days.

An enzyme can be separated easily from a product or reactant by combining with

iron nanoparticles, which have great magnetic properties. An enzyme can also play an

important role in bioremediation, but a combination of enzymes with nanoparticles

can also be helpful. A study of this type was conducted by Qiang et al. These exper-

iments used two different enzymes, peroxides and trypsin. As a result of combining

these enzymes in core–shell nanoparticles, the activity efﬁciency, stability, and life-

time of the enzymes was enhanced from an hour to a week. It helps to protect enzymes

from oxidation (Rizwan and Ahmed 2018).

It is necessary to recycle waste materials produced by industry into useful products

in order to reduce pollution. Due to advances in technology, it is now possible to turn

this waste into useful products thanks to excellent technology. In the industry, this

concept is more easily applied to the production of biomolecules, adsorbents, biogas,

clinker, and biohydrogen from waste. In 2019, Kumar described fermentative bacteria

producing biohydrogen in a dark reaction in the presence of nanoparticles (Mandeep

and Shukla 2020).

Several researchers are interested in converting industrial wastewater into biohy-

drogen. There is evidence that Elreedy et al. used mixed kinds of bacteria cultures to

produce monometallic, bimetallic and multimetallic nanoparticles for biohydrogen

production. The study found that biohydrogen is produced by nanoparticles with

multiple combinations of metal ions. As a result, various types of nanoparticles,

hydrogenase and dehydrogenase activities increased (Preethi et al. 2019). Gadhe

et al., found that biohydrogen production increased in addition to nickel oxide and

hematite nanoparticles. The production of biohydrogen is lower in monometallic

nanoparticles than in combinations of different metals. Ferredoxin oxidoreductase

and hydrogenase enzymes, a mixed metal nanoparticle exhibits 8.83 mmol/g COD

(Mandeep and Shuklathe 2020).

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 293

In the environment, hydrophobic chemicals such as polycyclic aromatic hydrocar-

bons (PAHs) tend to persist for long periods of time and affect environmental condi-

tions. Tungittiplakorn et al. develop nanoparticles with modiﬁed polyethylene glycol

urethane acrylate to deal with this problem. Since polycyclic aromatic hydrocarbons

are not soluble in water, they cannot be removed easily by the normal biosorption

process; therefore, modiﬁed nanoparticles are used. Polymer embedded nanoparti-

cles increase the solubility of hydrophobic chemicals or contaminants (Rizwan and

Ahmed 2018).

ZVI can degrade a variety of chlorinated aromatics and polychlorinated biphenyls,

as well as aliphatic compounds. During these degradation processes, chlorinated by-

products are formed, and these by-products affect the reactivity of iron nanoparticles.

This problem will also be solved with advances in nanotechnology. Combination

metal therapy plays an important role in chemical degradation. When zero-valent

iron nanoparticles combine with metals like Ni, Cu, Zn, and Pd, they show a greater

catalytic response than iron nanoparticles alone.

Most commonly, palladium is used as a catalyst for dehalogenation in bioremedi-

ation processes using nano-zero-valent iron. By using bimetallic nanoparticles such

as Ni/Fe, Xie et al. report on the degradation of polybrominated diphenyl ethers

(PBDEs) in the environment. Different types of organic coatings are applied in order

to increase the productivity of nanoparticles. These coatings play a variety of roles

in activating ZVI molecules. It plays an important role in the modiﬁcation of the

ZVI surface resulting in an increase in the adsorption rate of contaminants on the

surface of nanoparticles. Due to redox reactions, electrons get shut down and are

responsible for increasing the speed of reaction, so these coatings are also essen-

tial to redox reactions. Hydrophobic compounds become more mobile when they

are coated with electrolyte organic compounds. Hydrophobic compounds become

more mobile when they are coated with electrolyte organic compounds. In the Fe-Pb

bimetallic system, contaminants are adsorbent to a greater extent and there are no

harmful by-products formed at the end. As a result of the reaction, reactive oxygen

species are produced, which are again helpful to dechlorinate the reaction (Koul and

Taak 2018b).

The Texas Rice University developed a new kind of nanomaterial called a nano

mat, which resembles paper and is used to clean oil spills from ground surfaces as

well as water bodies. Numerous petroleum hydrocarbon releases from the oil industry

have toxic effects on aquatic environments. The researchers found that thin metal

and carbon can trap oil droplets. Multifunctional nanowire structures with a coating

of zirconium particles, which have multiple faces, can be used to separate oil from

water. A new kind of structure can absorb particles with molecular weights 10 times

greater than their original weight (Mohammadi et al. 2020).

294 H. S. Naik et al.

6 The Drawback of Nano Bioremediation

In addition to being beneﬁcial, nanoparticle-based solutions are also toxic. A limited

amount of literature exists on nanotechnology’s drawbacks in bioremediation. Below

are some disadvantages of nanotechnology in wastewater treatment. In terms of

wastewater treatment, hydrogel has many advantages, but one major disadvantage is

the difﬁculty in recovering it after treatment. After treatment, the substance tends to

dissolve and form a solution, so it is difﬁcult to recover it (Thakur et al. 2018).

Advanced nanoparticles such as metallic nanoparticles MNPs accumulate in the

environment and are toxic to various ecosystems, including aquatic, terrestrial, and

air. Nanoparticles may be accumulated in the environment in different ways; they

may enter the environment through the site where they are produced or during their

transportation process at the time of their actual application or ﬁnal disposal. As

Nano-engineered materials pass through the aqueous medium, they affect natural

processes such as chemical, physical, and also biological processes such as reac-

tions, aggregation, transformation, sedimentation, dissolution, transformation, and

sorption (Aguilar-Pérez et al. 2021).

Sharifabadi et al. studied the disinfection of bacteria using modiﬁed sodium

dodecyl sulphate when it was combined with an ag/cation resin ﬁlter system. They

found that after treatment there were no bacteria present, but when using a different

ﬁlter like Ag/ ﬁbre, Ag/sand, Ag/zeolite, and Ag/anion resin show low bacteria

removal. This is one of the drawbacks of nanotechnology (Manikandan et al. 2021).

Nanoparticles are rapidly incorporated into animal cells as compared to plant

cells because plant cells have a maximal level of external barrier. A nanoparticle

present in the soil accumulates in plants through their roots, and in an aqueous

medium, heteroaggregation and settlement of the particles may contribute to toxi-

city. The interaction of nanoparticles with natural organic matter affects the aquatic

environment. Various processes are affected by these interactions, including surface

transformation, adsorption, dissolution (oxidation and sulﬁdation), and stabiliza-

tion/aggregation. According to a study carried out by Cáceres-Vélez et al. on the toxic

effect of AgNPs on zebraﬁsh in the presence of humic acid (HA). The result from

the experiment shows that if there is more amount of humic acid then AgNPs uptake

by zebraﬁsh will reduce. But it doesn’t change the effect of AgNPs on zebraﬁsh.

The presence of AgNPs in zebraﬁsh bodies leads to toxicity in their body. The main

advantage of this mechanism is that it will lower the toxic effects by reducing the

uptake of AgNPs in the presence of humic acid. Another experiment was conducted

by Selmani et al. to determine the effect of coated selenium nanoparticles on aquatic

organisms Vibrio ﬁscheri and Daphnia magna. The stability of nanoparticles in an

aqueous environment depends on the type of medium and surface coating. A compar-

ative study between selenium and selenium coated polyacrylic acid (PAA) shows a

toxic effect on 24 and 48 h of exposure. There is another kind of nanoparticle that is

most commonly used in bioremediation i.e., iron oxide nanoparticles. Besides their

use in bioremediation, it causes toxic effects on the nitrogen cycle (Aguilar-Pérez

et al. 2021).

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 295

7 Conclusion

We conclude from these chapters that nanotechnology-based solutions for indus-

trial waste are a promising technology for removing pollutants from water bodies.

Throughout the last few decades, due to rapid industrialization, new industries have

developed day by day. These industries offer immense comfort products for the

beneﬁt of humans. Various industries release toxic chemicals that have many side

effects. The conventional methods remove toxic chemicals from the environment

and provide a toxin-free environment, however if nanotechnology-based solutions

are added to conventional methods, speed and effectiveness of the process will be

enhanced. In today’s world, nanotechnology-based bioremediation solutions can

remove various contaminants including organic, inorganic, and biological contam-

inants. Nanoparticles make this possible due to their unique properties. Because of

their easy synthesis, cost-effectiveness, and great functionality, they are widely used.

Environmental toxic pollutants can be removed using methods such as adsorption,

membrane-based, photocatalysis, disinfection, and redox reaction. Each of these

methods has advantages and disadvantages.

References

Abdelbasir SM, Shalan AE (2019) An overview of nanomaterials for industrial wastewater

treatment. Korean J Chem Eng 36(8):1209–1225. https://doi.org/10.1007/s11814-019-0306-y

Aguilar-Pérez KM et al (2021) Nanoadsorbents in focus for the remediation of environmentally-

related contaminants with rising toxicity concerns. Sci the Total Environ. Elsevier B.V. https://

doi.org/10.1016/j.scitotenv.2021b.146465

Ali AEI et al (2011) Pharmacologyonline 1:710–720

Ali I, Gupta VK (2007) Advances in water treatment by adsorption technology. Nat Protoc

1(6):2661–2667. https://doi.org/10.1038/nprot.2006.370

Al-Issai L et al (2019) Use of nanoparticles for the disinfection of desalinated water. Water

(switzerland) 11(3):1–20. https://doi.org/10.3390/w11030559

Alshemmari H (2021) An overview of persistent organic pollutants along with the coastal

environment of Kuwait. Open Chem 19(1):149–156. https://doi.org/10.1515/chem-2021-0198

Annachhatre AP, Gheewala SH (1996) Biodegradation of chlorinated phenolic compounds.

Biotechnol Adv 14(1):35–56. https://doi.org/10.1016/0734-9750(96)00002-X

Assi MA et al (2016) The detrimental effects of lead on human and animal health. Veterinary World

9(6). https://doi.org/10.14202/vetworld.2016.660-671

Berekaa MM (2016) Nanotechnology in Wastewater Treatment; Inﬂuence of Nanomaterials on

Microbial Systems. Int J Curr Microbiol App Sci 5(1):713–726. https://doi.org/10.20546/ijcmas.

2016.501.072

Bernard A (2008) Cadmium & its adverse effects on human health. Indian J Med Res

Bharagava RN, Saxena G, Mulla SI (2020) Introduction to industrial wastes containing organic and

inorganic pollutants and bioremediation approaches for environmental management. In: Biore-

mediation of industrial waste for environmental safety. Springer Singapore, pp 1–18. https://doi.

org/10.1007/978-981-13-1891-7_1

Dabhane H et al (2021) Phytogenic synthesis of gold nanoparticles and applications for removal of

methylene blue dye: A review. Environ Chem Ecotoxicol 3:160–171. https://doi.org/10.1016/j.

enceco.2021.04.002

296 H. S. Naik et al.

Das P et al (2018) Madhuca longifolia plant-mediated green synthesis of cupric oxide nanoparti-

cles: A promising environmentally sustainable material for wastewater treatment and efﬁcient

antibacterial agent. J Photochem Photobiol B 189:66–73. https://doi.org/10.1016/j.jphotobiol.

2018.09.023

Dimapilis EAS et al (2018) Zinc oxide nanoparticles for water disinfection. Sustain Environ Res

28(2):47–56. https://doi.org/10.1016/j.serj.2017.10.001

Division S (2019) Environmental science: nano redox chemistry of CeO 2 Nanoparticles in aquatic

systems containing Cr (VI )( aq ) and Fe 2+ ions environmental science: nano environmental

signiﬁcance : redox-active CeO 2 NPs are used in many industrial applications. O. Environ Sci:

Nano [Preprint], (Vi)

Durán N et al (2007) Antibacterial effect of silver nanoparticles produced by fungal process on

textile fabrics and their efﬂuent treatment. J Biomed Nanotechnol 3(2):203–208. https://doi.org/

10.1166/jbn.2007.022

El-Batal AI et al (2020) Penicillium chrysogenum-Mediated Mycogenic synthesis of copper oxide

nanoparticles using gamma rays for in vitro antimicrobial activity against some plant pathogens.

J Cluster Sci 31(1):79–90. https://doi.org/10.1007/s10876-019-01619-3

Gahlawat G, Choudhury AR (2019 A review on the biosynthesis of metal and metal salt nanoparticles

by microbes. RSC advances. Royal Soc Chem, pp 12944–12967. https://doi.org/10.1039/c8ra10

483b

Hashem EA (2014) Nanotechnology in water treatment, case study: Egypt. J Econ Dev Studies

2(3):244–259. https://doi.org/10.15640/jeds.v2n3a18

He L et al (2015) Contamination and remediation of phthalic acid esters in agricultural soils in

China: a review. Agronomy Sustain Dev, 519–534. https://doi.org/10.1007/s13593-014-0270-1

Husein DZ, Hassanien R, Al-Hakkani MF (2019) Green-synthesized copper nano-adsorbent for

the removal of pharmaceutical pollutants from real wastewater samples. Heliyon 5(8):e02339.

https://doi.org/10.1016/j.heliyon.2019.e02339

Hussain A et al (2016) Exploited application of sulfate-reducing bacteria for concomitant treatment

of metallic and non-metallic wastes: a mini review. 3 Biotech 6(2). https://doi.org/10.1007/s13

205-016-0437-3

Ibrahim RK et al (2016) Environmental application of nanotechnology: air, soil, and water. Environ

Sci Pollut Res. Springer, pp 13754–13788. https://doi.org/10.1007/s11356-016-6457-z

Iravani S (2014) Bacteria in nanoparticle synthesis: current status and future prospects. Int Scholarly

Res Notices 2014:1–18. https://doi.org/10.1155/2014/359316

Janani R et al (2022) Advancements in heavy metals removal from efﬂuents employing nano-

adsorbents: way towards cleaner production. Environ Res, 203.https://doi.org/10.1016/j.envres.

2021.111815

Jjagwe J et al (2021) Synthesis and application of granular activated carbon from biomass waste

materials for water treatment: a review. J Bioresources Bioproducts. KeAi Communications Co.,

pp 292–322. https://doi.org/10.1016/j.jobab.2021.03.003

Kiruba Daniel SCG et al (2014) Multifunctional silver, copper and zero valent iron metallic nanopar-

ticles for wastewater treatment. In: Application of nanotechnology in water research, pp 435–457.

https://doi.org/10.1002/9781118939314.ch15

Koul B, Taak P (2018a) Nanobioremediation. In: Biotechnological strategies for effective remedia-

tion of polluted soils. Springer Singapore, pp 197–220. https://doi.org/10.1007/978-981-13-242

0-8_8

Koul B, Taak P (2018b) Nanobioremediation. In: Biotechnological strategies for effective remedia-

tion of polluted soils. Springer Singapore, pp 197–220. https://doi.org/10.1007/978-981-13-242

0-8_8

Li X et al (2011) Biosynthesis of nanoparticles by microorganisms and their applications. J

Nanomater. https://doi.org/10.1155/2011/270974

Manikandan S et al (2021) Emerging nano-structured innovative materials as adsorbents in wastew-

ater treatment. Bioresource Technol 320. Elsevier Ltd, Jan. 01, 2021. https://doi.org/10.1016/j.

biortech.2020.124394

Nanotechnology for Bioremediation of Industrial Wastewater Treatment 297

Magrì D et al (2018) Titanate Fibroin nanocomposites: a novel approach for the removal of heavy-

metal Ions from water. ACS Appl Mater Interfaces 10(1):651–659. https://doi.org/10.1021/acs

ami.7b15440

Mandeep, Shukla P (2020) Microbial nanotechnology for bioremediation of industrial wastewater.

Front Microbiol. Frontiers Media S.A. https://doi.org/10.3389/fmicb.2020.590631

Mohammadi L et al (2020) Petroleum hydrocarbon removal fromwastewaters: A reviewMoham-

madi, Leili, Abbas Rahdar, Edris Bazrafshan, Hamid Dahmardeh, Md Abu Bin Hasan Susan,

and George Z. Kyzas. 2020. ‘Petroleum Hydrocarbon Removal Fromwastewaters: A Review.’

Processes, 1–34.,” Processes, 8, p 447

Mourdikoudis S, Pallares RM, Thanh NTK (2018) Characterization techniques for nanoparticles:

comparison and complementarity upon studying nanoparticle properties, Nanoscale. Royal Soc

Chem, pp 12871–12934. https://doi.org/10.1039/c8nr02278j

Munisamy P (no date) Phytochemical analysis and green synthesis of silver nanoparticles by

Kariveppillai fruits extract-biological activity study phytochemistry view project. Available at

www.tnsroindia.org.in.

Shah MP (2020a) Microbial bioremediation & biodegradation. Springer

Ojha A (2020) Nanomaterials for removal of waterborne pathogens. Elsevier, Waterborne

Pathogens. https://doi.org/10.1016/b978-0-12-818783-8.00019-0

Okhovat N, Hashemi M, Golpayegani AA (2015) Photocatalytic decomposition of Metronidazolein

aqueous solutions using titanium dioxide nanoparticles. J Mater Environ Sci 6(3):792–799

Pandey G (2018) Prospects of Nanobioremediation in environmental cleanup. Orient J Chem

34(6):2838–2850. https://doi.org/10.13005/ojc/340622

Shah MP (2021a) Removal of refractory pollutants from wastewater treatment plants. CRC Press

Preethi et al (2019) Biohydrogen production from industrial wastewater: an overview. Bioresource

Technol Reports, p 100287. https://doi.org/10.1016/j.biteb.2019.100287

Puthukkara PAR, Jose TS, lal SD (2021a) Plant mediated synthesis of zero-valent iron nanoparticles

and its application in water treatment. J Environ Chem Eng. Elsevier, p 104569. https://doi.org/

10.1016/j.jece.2020.104569

Puthukkara PAR, Jose TS, lal SD (2021b) Plant mediated synthesis of zero valent iron nanoparticles

and its application in water treatment. J Environ Chem Eng. Elsevier Ltd. https://doi.org/10.1016/

j.jece.2020.104569

Shah MP (2021b) Removal of emerging contaminants through microbial processes. Springer

R Ali H (2017) Applications of bio-waste materials as green synthesis of nanoparticles and water

puriﬁcation. Adv Mater 6(5):85.https://doi.org/10.11648/j.am.20170605.16

Rizwan M, Ahmed MU (2018) Nanobioremediation: Ecofriendly application of nanomaterials. In:

Martínez LMT et al. (eds), Springer International Publishing AG, part of S.N. 2018 and, H. of E.

(eds) handbook of ecomaterials. Springer International Publishing, pp 1–14. https://doi.org/10.

1007/978-3-319-48281-1_97-2

Rizwan Md et al (2014) Eco Friendly application of nanomaterials: Nanobioremediation. J

Nanoparticles, pp 1–7. https://doi.org/10.1155/2014/431787

Roy A (2014a) Nanotechnology in industrial wastewater treatment. Water Intell Online 13:220.

https://doi.org/10.2166/9781780406886

Roy A (2014b) Nanotechnology in industrial wastewater treatment. Water Intell Online. https://doi.

org/10.2166/9781780406886

Rumschlag SL et al (2020) Consistent effects of pesticides on community structure and ecosystem

function in freshwater systems. Nat Commun 11(1):1–9. https://doi.org/10.1038/s41467-020-201

92-2

Sastry M et al (2003) Biosynthesis of metal nanoparticles using fungi and actinomycete. CurrentSci,

pp 162–170. Available at https://www.researchgate.net/publication/228550063

Shah MP (2020b) Microbial bioremediation & biodegradation, microbial bioremediation &

biodegradation. Springer Singaporehttps://doi.org/10.1007/978-981-15-1812-6

298 H. S. Naik et al.

Sherry Davis A, Prakash P, Thamaraiselvi K (2017) Nanobioremediation technologies for sustain-

able environment. Environmental science and engineering (Subseries: Environmental Science),

(9783319484389), pp 13–33. https://doi.org/10.1007/978-3-319-48439-6_2

Singh E, Osmani RAM, Banerjee R (2020) Nanobioremediation: an emerging approach for a cleaner

environment. In Microbial bioremediation & biodegradation, pp 309–363. https://doi.org/10.

1007/978-981-15-1812-6_12

Singh PK, Singh RL (2017) Bio-removal of azo dyes: a review. Int J Appl Sci Biotechnol 5(2):108–

126. https://doi.org/10.3126/ijasbt.v5i2.16881

Tandon PK, Singh SB (2016) Redox processes in water remediation. Environ Chem Lett 14(1):15–

25. https://doi.org/10.1007/s10311-015-0540-4

Thakur S et al (2018) Recent progress in sodium alginate based sustainable hydrogels for

environmental applications. J Clean Prod 198:143–159. https://doi.org/10.1016/j.jclepro.2018.

06.259

Vo TT et al (2019) Biosynthesis of silver and gold nanoparticles using aqueous extract from crinum

latifolium leaf and their applications forward antibacterial effect and wastewater treatment. J

Nanomaterials.https://doi.org/10.1155/2019/8385935

Wang X et al (2019) Mesoporous silica-protected silver nanoparticle disinfectant with controlled

Ag+ ion release, efﬁcient magnetic separation, and effective antibacterial activity. Nanoscale Adv

1(2):840–848. https://doi.org/10.1039/c8na00275d

Yadav KK et al (2017) A review of nanobioremediation technologies for environmental cleanup: a

novel biological approach. J Mater Environ Sci 8(2):740–757

Yadav KK, Kumar V (2017) A review of Nanobioremediation technologies for environmental

cleanup: a novel biological approach micro plastic in aquatic biota view project testbook for

students View project. JMES. Available at http://www.jmaterenvironsci.com/

Zeki´cE,Vukovi´c Ž, Halkijevi´c I (2018) Application of nanotechnology in wastewater treatment.

Gradjevinar 70(4):315–323. https://doi.org/10.14256/JCE.2165.2017

Zhuang M et al (2021) Carbothermal synthesis of ni/fe bimetallic nanoparticles embedded into

graphitized carbon for efﬁcient removal of chlorophenol. Nanomaterials 11(6). https://doi.org/

10.3390/nano11061417

Plant Mediated Nanomaterials:

An Overview on Preparation Strategies,

Characterisation, and Their Potential

Application in Remediation

of Wastewater

Neha Kumari, Lakhan Kumar, and Navneeta Bharadvaja

Abstract Waste water containing inorganic/organic pollutants and heavy metals

has become a major problem nowadays. Some heavy metals are toxic even if they

are present in trace amount. So, removal through adsorption of these heavy metals

is necessary as they are non-biodegradable and can easily enter in the soil and

consequently harm plants and other life-forms. Nanotechnology provides a potential

approach for environmental remediation as it helps in photocatalytic degradation

of dyes and heavy metal adsorption from aqueous environment. Nanoparticles can

be synthesized from various techniques including chemical, physical and biological.

Synthesis of nanoparticles from plants is an eco-friendly, inexpensive and simple way

to remediate environmental pollutants. Here, we reviewed the synthesis of nanopar-

ticles using a wide range of plants. Plant extracts, contains a number of bioactive

compounds, which serve as reducing and capping agent for the metal precursor. Plant

based nanomaterials have shown ability to selectively sense the toxic hazardous heavy

metals like Pb, As, Ni, Hg, Cr, Zi, Co, Fe, Mn, Pd and Se in various environmental

niche. The chapter commences with the introduction to environmental degradation

and discusses the phytonanoparticles synthesis approaches and their characterization.

Besides this, it illustrates on role of various plant bioactive compounds in synthesis

of nanomaterials. Further, it discusses role of various plant mediated nanomaterials

in remediation of dyes and heavy metals. The article concludes by highlighting chal-

lenges and prospects of this emerging green technology for environmental pollution

remediation.

Keywords Wastewater ·Inorganic/organic pollutants ·Adsorption ·

Photocatalytic degradation ·Nanotechnology ·Nanoparticles

N. Kumari · L. Kumar · N. Bharadvaja (B

)

Department of Biotechnology, Delhi Technological University, New Delhi 110042, India

e-mail: navneetab@dce.ac.in

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_15

299

300 N. Kumari et al.

1 Introduction

Phyto-nanotechnology is the emerging branch of nanomaterials synthesis from plant

biomolecules that are used for a number of purposes including biomedical, microfab-

rication, energy storage devices, agriculture, health care, and remediation purpose

as it has large surface area and high reactivity. Phyto-nanotechnology is known to be

successful to reduce the environmental pollution which involves water treatment and

puriﬁcation of gases. Treatment of ground water is also somewhere related to inor-

ganic and organic pollutants present in the soil. According to United Nations report,

globally 0.5% of fresh water is present on earth and 80% of freshwater goes back

to the ecosystem without being treated. Contaminated water and soil have become

a great issue for us and the environment as well. Water can be contaminated by the

presence of different chemicals and heavy metals like lead, arsenic, nickel, mercury,

chromium, zinc, cobalt, and selenium. They are present in trace amount but still are

highly toxic. Metallic elements are considered to be toxic due to its high density,

speciﬁc gravity and atomic weight. There are 50 heavy metal presents out of which

17 are highly toxic and. normally, most of the heavy metals are found on the earth

crust but due to excessive industrialization, deforestation and various other human

activities these metals transfer to ground water (Chowdhury et al. 2016). Not all the

heavy metals are hazardous, some are essential to humans like cobalt, copper, zinc

and mercury but they can be harmful if present in excessive amount. Toxicity of a

metal depends upon its dosage, chemical reaction and means of exposure. Sources

of heavy metals like mercury, cadmium, arsenic, lead include coal mining, contact

with acid rain, TDS reﬁning procedure of other metals, manufacturing of chemi-

cals and corrosion of pipes (Rosenberg 2015). Exposure of contaminated water for

drinking purpose can affect human health in many ways. It can lead to several types

of cancer, organ damage, neurotoxicity, nephrotoxicity, malfunction of kidney, lungs,

liver, circulatory system, learning difﬁculty, rheumatoid arthritis and even death in

extreme cases (Lellis et al. 2019). Also, in agriculture, the toxic heavy metal present

in the soil can be taken up by plants leading to ROS strengthening, crops damage

and adverse effect on human and animal health.

On the other hand, dyes are known to be carcinogenic and pollute water bodies.

Exposure of dye in water bodies result in reduction in availability of light to the life

forms living under water which leads to the reduction in the rate of photosynthesis,

affecting the growth of the plant. 15–50% of azo dye is found in water bodies because

it does not bind to fabric. This wastewater incorporated with dye when used in irri-

gation affects the soil microbial communities, enzyme activity and growth of plant.

Azo dyes are chemically stable, non-biodegradable, durable coloured compounds

that are carcinogenic and mutagenic in nature (Ismail et al. 2019) Congo red dye,

also known as carcinogen. Benzedrine exhibit optimal, thermal and physiochem-

ical stability which contributes to their non-biodegradable nature (Rai et al. 2014).

Triphenylmethane dye used in manufacturing process such as textile dyeing, food,

and cosmetics etc., causes reproductive diseases in rabbit and marine animals. O-

phenylenediamine used a substrate in dyeing composition acts as a xenobiotic and

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 301

recalcitrant. It causes breathing problem, ingestion and eye irritation in humans.

Brilliant Cresyl Blue is used in scientiﬁc labs and industries. Rhodamine 6 G is a

ﬂuorescent dye widely used for staining purpose but has toxic effects on microor-

ganisms and humans. Rhodamine B dye is a water soluble red dye that causes eye

and skin diseases (Glossman-Mitnik 2013). Methyl blue is a type of cationic dye

extensively used in dyeing paper, clothes leather etc. Jaundice, cyanosis, high blood

pressure, vomiting are some of the common diseases associated with soluble methyl

blue dye (Kushwaha et al. 2014). There are many other water soluble dyes like mala-

chite green dye, crystal violet dye, and phenol red dye that reduce the transparency

of water, affect photosynthesis of aquatic ecosystem and are hazardous to humans.

Recent studies show that nanoparticles successfully remediate organic, inorganic

pollutants, and heavy metals from the aqueous solution. There are several methods

to synthesize nanoparticles, but biogenic synthesis of nanoparticles is chosen over

other chemical and physical methods since they are simple, eco-friendly, cost effec-

tive and clean. Biological synthesis can be done from various sources like bacteria,

fungi, algae and plants. In this chapter, we are focusing on phytonanotechnology

which utilise plant extracts which act as reducing and capping agent in the synthesis

of nanoparticles and provide stability. Generally, the plant extract is prepared as a

stock and certain amount of metal precursors are allowed to react with plant extract

under optimized reaction conditions. After a particular time, visual observation like

colour change indicates the formation of nanoparticles without the use of any toxic,

expensive chemical or the formation of any harmful by product. Metals and oxides

of metals act as adsorbents following the adsorption isotherm, adsorption thermody-

namics and adsorption kinetic modelling (Guerra et al. 2018). The Nano Zero Valent

Iron particle is best for the reduction process as it makes use of phyto-compounds

present in the plant extract. Zero valent form of iron is efﬁcient in adsorption of

hexavalent chromium (Madhavi et al. 2013a). Similarly zero valent form of silver

nanoparticles removes cadmium from aqueous solution (Al-Qahtani 2017). To keep

the particle stable and prevent its aggregation certain stabilizing agents are used

like carboxyl methyl cellulose, sodium borohydride etc. Maghemite nanoparticles is

effective against lead and cadmium from water instead of electro exploding wire tech-

niques (Yadav and Fulekar 2018a). The chapter aims to cover several aspects of nano-

materials synthesis, their characterization and potential application in environmental

remediation.

2 Synthesis of Nanoparticles

Nanoparticles are generally produced by top down and bottom-up approaches that

require heavy machines, chemicals and a very high maintenance cost. So, instead

of using these physical and chemical methods, biological method which is less time

consuming, non-toxic and economically suitable can be preferred. The biological

method involves microorganism assisted biogenesis using yeast, fungi, bacteria and

algae, bio template and plant extract assisted biogenesis. In this chapter, we focused

302 N. Kumari et al.

on studying various approaches of synthesis of nanoparticles using plant extracts

(Fig. 1). The biomolecules present in the plant extracts act as reducing and capping

agents which help in bio-reduction of metal ions under optimized conditions. Silver

nanoparticles formed from the Gardenia jasminoides extract help in reduction of

silver ions to zero oxidation state without using any toxic chemical to form silver

nanoparticles (Lü et al. 2014). Hence, bio-compounds like polyphenol, ﬂavonoid,

alkaloids, proteins, enzymes and co-enzymes help in reduction to form nanoparticles

whose size depends upon the phytochemicals compounds present in the plant extract.

Synthesis of metal nanoparticles with different plant extract is given in Table 1.

Prepared extract kept at magnetic stirrer for reaaction to take place

Adding suitable concentration of metal ions in plant extract

Filter to get pure aqueous plant extract

Heating the solution at hot plate

Plant powder added in distilled water to form solution

Cut into small peices or formed powered

Non diseased healthy plant

Nanoparticle

formed

•Factors affecting

nanoparticles

formation

•Temperature

•pH

•Reaction time

•Concentration of

plant extract and

metal precursor

Characterization

of nanoparticles

•Uv-Vis spectroscopy

•FTIR

•SEM/TEM

•XRD etc.

Environmental

applications

•Removal of organic

and inorganic

pollutants

•Removal of heavy

metal

•Degradation of dyes

etc.

Fig. 1 Schematic representation of plant mediated synthesis of nanoparticles, their characterisation,

and applications

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 303

Ta bl e 1 Nanoparticle synthesis from various plants, their characterization, and potential applications

Metal precursor Plant Characterization techniques Observation Applications References

UV–vis FT-IR SEM TEM EDX Initial Final

Copper

nanoparticle

Copper sulphate

pentahydrate

Piper

Retrofractum Vahl

234–255 nm 550–570 cm Spherical shape

high Cu

content-70.3%

2–10 nm Crystallinity phase −26.4% Yellow Greenish

black

Inhibit E.coli and

Staphylococcus aureus

Amaliyah et al.

2020)

Copper

nanoparticle

Copper sulphate

pentahydrate

Cedrus deodara 205 nm 607 cm Agglomerated

and form large

particle

Spherical No impurities Greenish Dark

brown

Inhibit against

pathogenic strains

Ramzan et al.

(2021)

Copper

nanoparticle

Copper acetate

monohydrate

Punica granatum

peel extrac

–Broad spectrum at 3400 for

OH present at surfaces of

CuO

Spherical shape,

diameter =

12.5 mm

–XRD-crystalline size =

35.80 nm monoclinic phase

Blue Brown Antibacterial activity

against E.coli

Siddiqui et al.

(2020)

Copper

nanoparticle

Copper sulphate

pentahydrate

(5 mM)

Celastrus

paniculatus leaf

extract

269 nm Spherical

diameter = 5nm

2–10 nm DLS- zeta potential =−

22.2 mV

Zeta deviation = 3.61 mV

EDS-purity (79.87%)

Yellow Green Antifungal property

Degradation efﬁciency

on organic dye

Mali et al.

(2020)

Copper

nanoparticle

Copper sulphate

pentahydrate

Neem ﬂower

extract

550–560 nm Tightly packed

nanocrystal

Spherical (5 nm) –Light blue

= light

green

Dark

yellow =

brown ppt

Antibacterial study

Max. efﬁciency against

Proteus mirabilis =

40 mg/ml

Gopalakrishnan

and Muniraj

(2021)

Copper

nanoparticle

Copper sulphate

pentahydrate

Red extract

cabbage

(Brassica oleracea

var. capitata f.

rubra extract)

255 nm 3100–3600 cm

Band energy gap = 2.75 eV

Spherical Particle size =

77.5 nm

XRD-crystalline nature, Fcc

structure, crystalline size =

78 nm

–White ppt Antibacterial agent

against E.coli and S.

aureus

Fernandez and

Rajagopal

(2020)

Copper

nanoparticle

Copper sulphate

pentahydrate

Seedless dates 576 nm 624.57 cm –Spherical XRD

DLS

Particle size distribution =

mean diameter 78 nm

Zeta potential =+41 mV

Pale yellow Red brown

color

More feasible than

chemical method

Mohamed

(2020)

Copper

nanoparticle

Copper chloride

Tinospora

cardifolia

248 nm 3329 cm − OH stretching

1620-C=O stretching

1395-OH bending

1319-C-O bending

1049- C-O–H stretching

Spherical XRD- crystalline

phase -metallic

copper and copper

oxide

PSD = 63.5 nm

Polydispersity index = 0.26

Zeta potential =−33.98 mV

AFM = 178 nm height

Sky blue Dark green Antimicrobial activity

Efﬁcacy of coated

cotton against

gram-positive is 65% &

gram-negative is 50.5%

Sharma et al.

(2019)

(continued)

304 N. Kumari et al.

Tabl e 1 (continued)

Metal precursor Plant Characterization techniques Observation Applications References

UV–vis FT-IR SEM TEM EDX Initial Final

Zinc oxide

nanoparticle

Zinc nitrate

hexahydrate

Cassia ﬁstula plant

extract

370 nm Photolytic degradation-

(batch reactor) = 5 ppm

dye conc. Gives 90%

degradation; ph-2 & 4

96.26% & 98.71%

Photodegradation efﬁciency

Antioxidant (DPPH) Ic50

= 54 Mg/ml

Hexagonal

wurtzite

structure

5–15 nm XRD- 100,002,101

plane—increases purity of

particles

– – Exhibit Antioxidant,

Bactericidal property

and catalytic property

against methyl blue

Suresh et al.

(2015)

Silver

nanoparticle

Silver nitrate

Morinda tinctoria

Leaf extract

420 nm 3296 cm—carboxylic group

3436–3220 cm—O–H

stretching, H bonded

phenol and alcohol group

1634 cm—N–H bending

primary amines

1672 cm—C = O stretching

Spherical rod

shaped

79–96 nm 4 peaks of 2 theta

38.26 degree = (111)

44.44 = (220)

64.58 = (220)

77.67 = (311)

Fcc and crystalline in nature

Pale yellow Dark

brown

Degradation of methyl

blue dye

Vanaja et al.

(2014)

Silver

nanoparticle

Silver nitrate

Neem leaf

Neem bark

Mango leaf

Green tea

Pepper seeds

420 -Spherical – – Pale yellow Brownish

yellow

Calorimetric sensing of

toxic metal (Hg2+,

Pb2+, Zn2+, Cr 3+,

Cd2+, Ca2+. Cu2+,

Mg2+, Ni2+, Fe2+)

Karthiga and

Anthony (2013)

Silver

nanoparticle

Silver nitrate

Amaranthus

gangeticus Linn

leaf extract

416 nm 1635 cm—C = O stretching

3441 cm—OH & NH2

Globular shaped 11–15 nm – – Brown Antifungal and

antibacterial activity

inhibitory activity

towards gram +ve & −

ve

Degrade congo red dye

Kolya et al.

(2015)

Silver

nanoparticle

Silver nitrate

Nigella Sativa seed

extract

426 nm 3423- 2880 cm

Indicates CH, −OH, −NH,

C=C acting as reducing &

stabilizing/capping agent

Spherical 10.88 nm XRD-

2theta = 38.14 degree- (111);

44.36 degree (200); 64.71

(220)

77.40 (311)

White Light

brown

Congo Dye

degradation

Chand et al.

(2021)

(continued)

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 305

Tabl e 1 (continued)

Metal precursor Plant Characterization techniques Observation Applications References

UV–vis FT-IR SEM TEM EDX Initial Final

Silver

nanoparticle

Silver nitrate

Zanthoxylum

armatum leaves

419 nm 3431 cm- N–H stretching

OH hydroxyl grp

2922 cm- C-H stretching

1744 cm—carbonyl

stretching

1630 cm- N–H bond

1375 & 1238 cm—N = O

symmetry

1045 cm- C-N amines

Spherical shape Diameter =

15–20 nm

Zeta potential =−21.2 mV

Average size −36 nm

XRD

Fcc

2theta = 38.23 degree (111);

46.45

(200); 66.65 (220);

77.55 (311)

Avg crystallize size—22 nm

Colourless Brownish Dye

degradation—safranin

O

Methyl red

Orange and methyl

blue

Jyoti and Singh

(2016)

Silver

nanoparticle

Silver nitrate

Viburnum opulus

fruit extract

513 nm; 415 nm

(350–450 nm)

3398 cm- stretching of O–H

1733 cm- C=O stretching

2931 cm—C-H stretching

1380 cm-C-O

stretching

1030 cm- C-O bending

Spherical 7–26 nm

Avg size- 16 nm

XRD-

2theta

38.33 degree (111); 44.56

(200); 64.62 (220);

77.44(311);82.41 (222); 32.2-

organic compound in sample

(Fcc)

TGA- 35.14% bioactive

compounds present at AgNP

surface

Faint red Yellowish

brown

Dye degradation

Brilliant blue

Tartrazine

Carmoisine

David and

Moldovan

(2020)

Silver

nanoparticle

Silver nitrate

Calendula

ofﬁcinalis

436 nm 2theta –

665 cm; 3438 cm; 1635 cm-

stretching vibration of

carbonyl grp; 3338 cm-

O–H stretching

Uniform and

spherical

50–60 nm

140–150 nm

XRD-

Fcc, crystal size = 14.37 nm

Yellow Brown Methyl blue and

methyl orange

degradation

Chandra Paul

et al. (2020)

Silver

nanoparticle

Silver nitrate

Dahlia pinnata

leaf extract

460 nm 1064 cm- C-N bond

3265 cm- O–H stretching

2916 cm- C-H stretching

1423 cm- C-H bending

673- stretching vibration of

halo alkene

1595 cm- C-H present

Almost spherical Diameter- 15 nm XRD-

Face centre cubic structure,

27.5- (220); 32.75- (122);

46.25(111); 54.65(331);

57.25(241); 76.68(311)

Colourless Dark

yellow

Rapid colorimetric

detection of Hg2 +

Roy et al. (2015)

Silver

nanoparticle

Silver nitrate

Panax Ginseng

root extract

404 nm 3640 cm- O–H stretching

1740- carbonyl stretching

vibration of aldehyde

1640- symmetry-COO

stretching

1407- asymmetry-COO

stretching 1065- bending

vibration of C–C-O &

C–C-OH

spherical 4–20 nm ––Dark

brown

Detection of Hg2+ Tag ad et a l.

(2017)

(continued)

306 N. Kumari et al.

Tabl e 1 (continued)

Metal precursor Plant Characterization techniques Observation Applications References

UV–vis FT-IR SEM TEM EDX Initial Final

Gold nanoparticle

Gold(lll)chloride

trihydrate

Capsicum annum 335.26 nm 3324- 3329 cm- =−OH

grp

2100 cm- -CN grp

1636 cm- C = Ogrp

Triangular –XRD-

111, 200, 220, 311 degree

Crystal size = 13.71 nm

Yellow Dark red Water pollution

removal and

Show strong

antimicrobial activity

at low concentration

Baran et al.

(2020a)

SnO2

nanoparticle

(SnCl2)

Vitex agnus-castus

fruit extract

373.45 nm 3434.27

1633.02

1027.50

647.45

Anti-symmetric Sn–O-Sn;

Sn–O symmetric

Spherical 4–13 nm

Avg si ze = 8nm

XRD

26.9 degree- (110);34- (101);

38-(200); 52-(211); 58-(002);

62.1-(310); 65.2-(301);

71.4(202)

–Light grey Photocatalytic

degradation of organic

dye RhB, under and

removal of heavy metal

Co+2

Ebrahimian et al.

(2020)

Iron nanoparticle

(FeSO4.7H2O)

Eucalyptus

globules leaf

‘graph given =

above 420 nm

3346.25 cm; 1635.58;

524.68 cm (polyphenolic

content present

Spherical 50–80 nm XRD

Peak at 2 theta 46.40 indicates

zero valent iron

Brown Black Adsorption of Cr(VI) Madhavi et al.

(2013a)

Maghemite

nanoparticle

2MFeCl3+1M

FeSO4.7H2O

Tridax plant 210 nm 454 cm,

569 cm,

632 cm, attributed to

Fe–O;

563 cm—gamma attributed

to FeO

1443–1600 attributed to

C=C/C=N bond

326, 402, 500, 706 are

peaks of Maghemite

Cubic spinal

structure

(spherical and

cuboidal shape)

9.59–15.42 nm XRD

18.22,

29.3,30.02,32.08,43.14,57.48

degree with miller indices

011,210,220,310,330

–Blackish

brown ppt

Removal of heavy

metal Pb and Cd

Yadav and

Fulekar (2018a)

Zinc Nanoparticle

Zinc acetate

(0.1 M)

Sphagneticola

trilobata

(leaf, stem and

root extract)

-3144 cm-C=O stretching

1665 cm −N–H stretch

1640 cm-N–H bending in

amine & amide grp

Irregular and

complex

65–80 nm EDS- zinc 41.4% synthesized

in

Nanomaterial

XRD-

200,111,221 planes

correspond to 2 theta angle

Face center cubic structure

–Yellow

colour

Removal of Chromium

by DPC method

Shaik et al.

(2020a)

Cupric Oxide

nanoparticle

Calotropis procera 3444 cm −OH grp;

1636 cm;

1231 cm;1084– protein

latex

519; 598 cm −Cu–O band

Spherical 10–15 nm;

diameter =

15.06 nm standard

deviation- 5.17

XRD

Peak on range 20 < 2 theta

<80 degree

Monoclinic symmetry

Green ppt Black ppt Removal of Cr(VI)

from aqueous solution

Dubey and

Sharma (2017)

(continued)

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 307

Tabl e 1 (continued)

Metal precursor Plant Characterization techniques Observation Applications References

UV–vis FT-IR SEM TEM EDX Initial Final

Iron based NP

FeSO4.7H2O

Eucalyptus leaf –3388 cm −O–H stretching;

2932 &2840 −C-H

stretching;1720—carbonyl

grp; 1605& 1515 −C=C

aromatic skeletal vib.;1330

−CH3 asymmetric

vib.;1048- C-N stretching

NP rxn with Cr(VI) &

Cu(II) show band at 460 &

546 cm -Fe–O stretch

Polydispersed 20–80 nm EDS-

Element % of Fe = 16.3;

C = 47.4

O = 36.3

XRD

2 theta at 44.8;

24 degree show biomolecule

capped with Fe NP

– – Removal efﬁciency of

Cr (VI) and Cu (II)

when existed together

or separated

Weng et al.

(2016a)

Zero valent silver

NP

Silver nitrate

Ficus Benjamina 420 nm 3461- O–H stretching;

1632 cm—amide grp

Dendritic

structure

60–105 nm ––Brown Cadmium removal

from aqueous solution

Al-Qahtani

(2017)

Zero valent Iron

NP

Rose damascene,

Thymus vulgaris,

Utrica dioica leaf

extract

–3400 & 3430 cm for

polyphenols;

1126–1190 cm for carbonyl

grp; 1628 &1640 cm

corresponds to C=C in

alkene grp; 615–617

indicating aromatic

compounds of alkanes

Non uniform

exhibit differed

space & void

space

100 nm

BET

Surface area =

1.63(TV);

2.42(UD); 1.42

(RD

)m3/g total pore vol

= 4.52*10–2(TV)

2.97*10–2 (UD);

2.08*10–2 (RD)

XRD

Perfectly index crystalline Fe

– – Removal of Cr(VI)

from aqueous solution

Fazlzadeh et al.

(2017)

Silver

nanoparticle

Silver nitrate

Perilla frutescens 469 nm 615 & 627- C-H group;

1048 & 1124—stretching

vibration; 1381 &1374

-C-N stretching vibrations;

1608 & 1601- secondary

amide groups

Spherical,

rhombic,

triangular and

rod shaped

25.71 nm Zeta potential-

−23.33 mV

FCC cubic

Yellow Brown Exhibits antioxidant,

anticancer and

antibacterial properties

Reddy et al.

(2021)

Copper

nanoparticle

Cu(OAc)2

Stachys

lavanaulifolia

400 nm 3100–3385-hydroxyl

group; 2922, 1618, 1398

represent saturated

hydrocarbon; C=C and

C=O—aromatic stretching

frequency; 3400- O–H

stretching; 1473-bonding

vibration of sp2 bond

1627- carbonyl stretching

Monodispersed 20–35 nm XRD

2theta = 32.59, 35.61, 38.78,

48.82, 53.24, 58.37, 61.60,

66.31, 68.15, 72.46, 75.30

assigned to (110), (−111),

(111), (−202), (020), (202),

(−113), (220), (311), and

(-222) planes

Pale yellow Dark

brown

Represents catalysed

C-heteroatom coupling

reaction

Veisietal.

(2021)

(continued)

308 N. Kumari et al.

Tabl e 1 (continued)

Metal precursor Plant Characterization techniques Observation Applications References

UV–vis FT-IR SEM TEM EDX Initial Final

Iron based

nanoparticle

FeSO4.7H2O

Green tea extract –1611- C = C stretching

vibrations; 1362- C-N

bond; 1039- C–O–C

bonding

–50–80 nm GC–MS

Phenol, 1,1-biphenyl, 2-ethyl,

1,2,3- benzenetriol, caffeine

and bis (2-ethylnexyl)

phthalate involve in FeNP

synthesis as reducing and

capping agent

Removal of hexavalent

chromium

Hao et al. (2021)

Nickel oxide

nanoparticle

Ni(NO3)2.6H2O

Abutilon indicum

leaf extract

200–385 nm 3410 (O–H); 2990 (C-H);

1702 (C = O) 1650 (amine

I and amide II); 1259

(O–H) 1140 (C-O)

Agglomerated

form

452.49 nm XRD

Highly crystalline attributed to

(111), (200), (220), (311),

(222) plane with diffraction

angle 37.5, 43.26, 63.11,

75.46, 49.46 degree

respectively

Yellow Green Exhibit anticancer,

antioxidant, and

antibacterial property

Khan et al.

(2021)

Zinc oxide- based

nanoparticle

Zn(NO3)2.7H2O

Saponaria

ofﬁcinalis extract

380 nm -Agglomerated

form

100–200 nm

diameter

XRD

Hexagonal wurtzite phase

attributed to (100), (002),

(101), (102), (210), (103),

(200), (212), (201) planes

Dark

brown

Dye degradation

efﬁciency of methyl

blue and show

antibacterial activity

T˘anase et al.

(2021)

Silver

nanoparticle

Silver nitrate

Plant gum of-

Araucaria

heterophylla

Azadirachta indica

Prosopis chilensis

(bark of tree)

– – Spherical 30 nm

DLS =

50 nm

35 nm; DLS =

50 nm

DLS

Diameter = 75 nm

Zeta potential

(EDX)

−20.87 mV

−23.65 mV

16.41 mV

(1) Effective against

gram positive and

gram-negative bacteria

(2) not much effective

against cell lines, but

still exhibit anticancer

anti-cancer activity

against human breast

cancer cell line MCF 7

at higher concentration

(3) Chromium removal

efﬁcacy was not

efﬁcient

Samrot et al.

(2019)

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 309

3 Role of Phytocompounds in Nanoparticles Synthesis

Phytocompounds play a major role in the formation of nanoparticles. Due to exten-

sive study on phytochemicals analysis, our understanding has increased about them.

It has facilitated in identiﬁcation of particular biochemical compound present in

the plant which acts as a reducing, capping and stabilising agent in nanoparticles

synthesis. The phytochemicals analysis, if carried out by performing phytochemi-

cals tests can give the exact phyto component participating in reduction, which can

be further determined by FTIR (Fourier Transform Infrared Spectroscopy) analysis.

FTIR studies reveal that amino acids help in reduction of metal ions. For example,

alpha amino acids reduces silver ion (Tan et al. 2010). Flavonoids are secondary

metabolites produced by plants that have the ability to transform enol-form to keto

form by releasing one hydrogen atom. Functional group of phenolic acids comprises

the phenolic ring which is responsible for chelating metal and carboxylic acid. In

synthesis of silver nanoparticle, using plant extracts of three plants namely Schinus

molle, Equisetum giganteum, and Ilex paraguariensis, removal of electrons occur

so that Gallic acid is oxidised to quinine (Barberia-Roque et al. 2019). Phytocom-

pounds polyols and polysaccharides actively present in Cinnamomum verum help

in reducing Ag + ion in silver nanoparticle synthesis (Sathishkumar et al. 2009).

Its functional group containing carbohydrate and hydroxyl group is known to be

soluble in water while methyl and isopentyl are lipophilic in nature. It also has

potential to donate hydrogen atom, thus helping in reduction process. Flavonoids

are divided into major s ubclasses which included chalcones, ﬂavones, ﬂavanols,

isoﬂavones and anthocyanidin. Quercetin, which comes under the class of ﬂavanol,

chelates metal ion at three different positions namely the catechol group, carbonyl

group and hydroxyl group. Quercetin and plant pigmentation helps in bioreduc-

tion of silver nanoparticle synthesized from Acalypha indica leaf extract (Krishnaraj

et al. 2010). Flavonoids have potential to tolerate heavy metals like cadmium and

zinc in Arabidopsis thaliana. Terpenoids are the derivative of essential oil and have

diverse structure containing 5 carbon isoprene units. They show strong antioxidant

activity. They also help in bio-reduction by deprotonating the OH− group to form

conjugate base structure and preventing it from further oxidation. Terpenes, which

are converted into terpenoids upon oxidation, help in metal reduction. This active

redox reaction leads to the formation of nanoparticles. Lantana camara leaf extract

contains terpenoids as their main reducing and capping agent in the synthesis of silver

nanoparticles (Ajitha et al. 2015). Excessive thermal heating can inactivate essential

phytocompounds. Monosaccharide sugar like glucose and fructose also take part in

formation of metallic nanoparticles through the conversion of ketone to aldehydes.

Disaccharides and polysaccharides form an open chain with the help of monosac-

charides up to 7–8 units and provide the metal ion to aldehydes group and facilitate

reduction process.

310 N. Kumari et al.

4 Characterization of Nanoparticles

To detect the reduction of metal precursor, the optical absorbance of synthesised

nanoparticles is performed by a UV–Vis spectrophotometer. The spectra are recorded

within the range of 200–700 nm. UV–Vis spectrophotometer is used to determine

the concentration of different molecules present in a solution utilising the charac-

teristics wavelength of the molecule at which it maximally absorbs the light. After

the plant extract and metal precursor are mixed, a change in colour of the solution

is observed as the time progress. One concern is there that with time aggregation

of nanoparticles proceeds, which ultimately alter the peak of absorbance. At partic-

ular wavelength of localized surface plasmon resonance, we get different maximum

absorbance of nanoparticles as compared to literature values of those particular metal

precursors. A study reported t hat the gold nanoparticles synthesized from Garcinia

mangostana fruit peels turned purple instead of traditional yellow (Xin Lee et al.

2016). Maximum absorbance of silver and gold nanoparticles synthesized from

Rumex roseus leaf extract was recorded at 429 nm and 549 nm respectively by

UV–Vis spectrophotometer (Chelly et al. 2021). SEM/TEM is used to determine the

morphology of the synthesized nanoparticles. In TEM analysis, once electron hit

the sample it gets absorbed, and gives much higher resolution than light microscopy

whereas in SEM analysis of nanoparticles, the electron scans for different region

of sample. In some regions, more scattered electrons are present due to which the

absorbed atom is less and in other region less scattered electron found give a clear indi-

cation of more absorbed atom. This scattering of electron in different region results

in more contrasting three- dimensional image as obtained from SEM micrograph.

SEM/TEM studies are done to observe the shape and size of the synthesised nanoma-

terials. Garcinia magostana mediated magnetite nanoparticles are of 13.42 nm and

displayed diffraction rings of Fe3O4 phase (Yuseﬁ et al. 2021). FTIR spectra deter-

mine the functional group present in the plant extract which are held responsible

for the synthesis of nanoparticles. The principle behind FTIR tell us that the bond

between two atoms or molecules are not ﬁxed and are involved in different types

of motions called bond vibrations. Every compound has its signature vibration and

stretching between the bonds by which the functional group can be determined. For

example, formaldehyde has carbonyl carbon attached with oxygen where stretching

and wagging goes on simultaneously. Therefore, absorbance determines the type of

functional group present in the sample. The peaks in the spectrum show the bending

and stretching vibrations of the biomolecules. Zinc oxide nanoparticles synthesized

from Cayratia pedata leaf extract exhibit zinc bonding at 400 cm−1 and oxygen

bonding at 600 cm−1 (Jayachandran and Nair 2021). Crystal structure of nanomate-

rials are analysed by XRD. We use X-ray crystallography because X-ray has shorter

wavelength. Its working involves projecting a high energy electron beam falls on

a rotating target that throws out the X-ray generated, which are then measured by

a detector containing photomultiplier tube of X-ray diffractometer. The position of

the peaks is determined by the planes which diffract coherently at an angle where

Bragg’s law holds good.

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 311

5 Different Types of Plant Mediated Nanoparticles Helping

in Dye Degradation and Heavy Metal Removal

from Aqueous Solution

Chemical compounds which are released from industries, households, oil pollution,

acid rain, sewage and agriculture waste makes remediation process of water more

complicated and difﬁcult. There are many techniques available for puriﬁcation of

water but the nanotechnological approach has been termed as potential method due

to the small size and high reactivity of nanoparticles. Several types of plant-based

nanomaterials have been explored for remediation of environmental pollutants as

illustrated in Fig. 2.

5.1 Nanoscale Zero Valent Iron (nZVI) Nanoparticle

Nanoscale zero valent iron (nZVI) is preferred because it can be separated easily

under the inﬂuence of external magnetic ﬁeld. nZVI cannot remove contaminants

on its own because of high probability of aggregation which can easily alter the

surface chemistry to make it unreactive (Pasinszki and Krebsz 2020). To prevent these

problems, nZVI nanoparticles come into use without damaging the active catalytic

sites of nZVI. Sodium borohydride is used for stabilizing these nanoparticles as it

increases surface reduction and prevents oxidation. Polyvinyl-pyrrolidone (PVP),

polyethylene glycol (PEG), and carboxymethyl cellulose (CMC) used in coating of

nZVI increase the stability by catalytic reduction of ketones to alcohol (Parimala

Fig. 2 Types of plant

derived nanoparticles

Plant derived

nanoparticle

Nanoscale

Zero Valent

Iron

(nZVI)

Titanium

diodide

(TiO2)

Metal

Based

Oxides of

metal

Bimetallic/

Trimetalic

NPs

312 N. Kumari et al.

and Santhanalakshmi 2014). Fenton’s reagent is also used in remediating wastew-

ater along with nZVI nanoparticle as it produces hydroxyl radicle at low pH with

higher concentration of H2O2 and Fe2+ . Reduction of oxygen by nZVI and subse-

quent formation of hydrogen peroxide leads to hydroxyl radical formation and an

oxidized organic compound (Babuponnusami and Muthukumar 2014). Nanoscale

zero valent iron nanoparticle synthesis from green tea extract using bentonite for

stabilization successfully degraded 96.2% RB 238 dye within 60 min followed by

Fenton like oxidation (Hassan et al. 2020). Catalysis of dye resulted in the formation

of hydrogen peroxide in which the OH radicle and dye degraded to form carbon

dioxide and water molecule. Nanoscale zero valent iron used along with magnetite

nanoparticle followed Fenton’s reaction to remediate wastewater by degrading chlo-

rinated compound like 2, 4-Dichlorophenoxyacetic Acid at pH 3–6.5 within 90 min

(Nanoparticles 2020).

5.2 Titanium Dioxide (TiO2) Nanoparticle

Titanium dioxide obtained from the three different minerals namely anatase, brookite

and rutile. Anatase grade of TiO2 exhibits similar properties as of rutile but brookite

form is very rarely formed and is highly unstable. TiO2 nanoparticle synthesized from

Cinnamon powder are of spherical anatase phase and shows enhanced photocatalytic

property for solar cells with band gap of 3.2 eV which is determined by UV–Vis

spectroscopy (Nabi et al. 2020a). Titanium dioxide is a potent photocatalyst which

gets activated by UV light such as sunlight for photocatalytic reaction to take place.

Titania exhibits unique physical properties due to which TiO2 is insoluble in water

and show white colouration or precipitate, that’s why it is extensively used as food

additive. Chemical and physical methods have several limitations like small scale

production, not environment friendly, maintenance of temperature and pressure leads

to high cost, use of surfactants result in toxicity and complexity. TiO2 nanoparticles

can be successfully synthesized from biological methods like Aloe vera extract,

Annona squamosa peel extract, and Jatropha curcas leaf extract, Nycetanthes Arbor-

Tristis leaf extract, Psidium guajava and various other plant extracts given in Table 2.

TiO2 is another way to generate hydroxyl radical which oxidizes contaminants

present in water under the inﬂuence of light. Under the exposure of UV light, the

band energy gap of TiO2 is 3–3.5 eV. The transfer of negative charged electron from

valence band to conduction band exhibited by the forming positive charged holes.

The photogenerated positive charge carriers (holes) from valence band diffuse to

photocatalyst surface and react with water molecule to form free radical which is

further oxidizes (Nakata and Fujishima 2012). The negatively charged electron from

conduction band helps to promote reduction followed by reacting with atmospheric

air to produce non-hazardous compound like water molecule, carbon dioxide etc. To

enhance the photocatalytic efﬁciency, TiO2 is trapped within nanoparticles without

damaging the active sites followed by surface modiﬁcation via doping with carbon,

nitrogen and metal (Zahra et al. 2020).

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 313

Ta bl e 2 Types of phyto-nanoparticles and their application in treatment of wastewater

Type of nanoparticles Plant source Absorption capacity/percentage

degradation

Applications References

nZVI Rosa damascene (RD)

Thymus vulgaris (TV)

Utrica dioica (UT ) leaf extract

94.87%

83.45%

86.8%

Removal of Cr(VI)from aqueous

solution

Fazlzadeh et al. (2017)

Kaolin-supported nZVI Ruellia tuberosa 99.8% Degradation of reactive black 5

azo dye

Khunjan and Kasikamphaiboon

(2021)

nZVI Spinacia oleracea COD- 73.82%

BOD- 60.31%

Removal of chemical and

biological oxygen demand from

municipal waste water

Turakhia et al. 2018)

nZVI Cupressus sempervirens 95.4% Decolourization efﬁciency of

methyl orange

Ebrahiminezhad et al. 2018)

nZVI Oak

Mulberry

Cherry

Leaf extract

– Removal of As(III) and Cr(VI)

from aqueous solution

Poguberovi´c et al. (2016)

nZVI Mentha piperita PO4 3−85.01%

NH3 −99.51%

NO3 −86.33%

Pb2+ −83.4%

Cl −79.33%

Removal of agricultural

contaminants like phosphate,

ammonia, nitrate, lead, and

chloride from aqueous solution

Shad et al. (2020)

nZVI Shorea robusta leaf extract 96% Degradation of congo red dye

from aqueous solution

Jha and Chakraborty (2020)

nZVI Grape seed extract + Fe3+ 82.8–96.1% Removal of Cr(VI) from aqueous

solution

Guo et al. (2020)

(continued)

314 N. Kumari et al.

Tabl e 2 (continued)

Type of nanoparticles Plant source Absorption capacity/percentage

degradation

Applications References

nZVI Ficus Benjamina leaf extract 75.5–85% Cadmium removal from aqueous

solution

Al-Qahtani (2017)

TiO2

Au-TiO2

Azadiracta indica –Degrade methyl red dye Sankar et al. (2015)

Cinnamomum tamala leaves 64% Degradation of methyl orange Naik et al. (2013)

Jatropha curcas leaf 82.26%

76.48%

Removal of chemical oxygen

demand(COD)

Removal of Cr from tannery

wastewater

Goutam et al. (2018)

Piper betel (PS)

Ocimum tenuiﬂorum (OT)

Moringa oleifera (MO)

Cariandrum sativum (CS)

MO degrades faster among all

within 30 min

Degradation of malachite green

dye

Pushpamalini et al. (2020)

Lemon peel extract 70% Degradation of rhodamine B dye Nabi et al. (2020b)

Syzygium cumini leaf extract 75.5%

82.53%

Photocatalytic removal of

chemical oxygen demand (COD)

Removal of lead (Pb)

Sethy et al. (2020)

TiO2Caltropis gigantea leaf extract 96.7% Catalytic degradation of

metformin by solar

photocatalysis

Prashanth et al. 2021)

Metal -FeNP Green tea extract 81%

31%

Removal of methyl

chlorobenzene (MCB) from

wastewater

Removal of chemical oxygen

demand(COD)

Kuang et al. (2013)

(continued)

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 315

Tabl e 2 (continued)

Type of nanoparticles Plant source Absorption capacity/percentage

degradation

Applications References

FeNP Eucalyptus leaf extract N-71.7%

COD-84.5%

Removal of total nitrogen and

COD from wastewater

Wang et al. (2014)

Nickel NP Camellia sinensis leaf extract 94.5% Degradation of CV dye Bibi et al. (2017)

AgNP Morinda tinctoria leaf extract 95.3% Degradation of methyl blue dye Vanaja et al. (2014)

FeNP Camellia sinensis leaf extract

Vitis vinifera extract

RR-52%

RY-64%

RB-78%

RRB-76%

RR-49%

RY-61%

RB-71%

RRB-68%

Degradation of Reactive Red 195

(RR), Reactive Yellow 145 (RY),

Reactive Blue (RB) and Reactive

Black 5 dyes

Raman et al. (2021)

AgNP Nigella sativa seed extract 98.5% Decolouration of Congo red dye Chand et al. (2021)

AgNP Zanthoxylum armatum leaves SO-1.02 × 10–3

MR-1.03 × 10–3

MO-1.86 × 10–3

MB-1.44 × 10–3

Dye Degradation of safranin O

(SO), Methyl red (MR), Methyl

orange (MO), Methyl blue(MB)

Jyoti and Singh (2016)

AgNP Calendula ofﬁcinalis MB- 27.12%

MO-69.79%

Degrade methyl blue (MB) and

methyl orange(MO) dye in

aqueous solution

Chandra Paul et al. (2020)

AuNP Capsicum annum Pb-63.46%

Cd-60.20%

Cu-51.50%

Fe-68.20%

Ni- 42.18%

Co- 23.47%

Mn-21.62%

Zn-35.37%

Pb-75.75%

Removal of contaminant present

in water including Pb, Cd, Cu,

Fe, Ni, Co, Mn and Zn

Baran et al. (2020a)

(continued)

316 N. Kumari et al.

Tabl e 2 (continued)

Type of nanoparticles Plant source Absorption capacity/percentage

degradation

Applications References

SnO2 NP Vitex agnus castus fruit extract RhB- 91.7%

Co+2–93.6%

Photocatalytic degradation of

Rhodamine B dye and removal

of heavy metal Co+2

Ebrahimian et al. (2020)

FeNP Green tea extract 19.9 mg/g Removal of As (V) at 298 K Wu et al. (2021)

Oxide of metal—iron Pomegranate seed extract 95.08% Degradation of reactive blue 4

dye from aqueous solution

Bibi et al. (2019)

iron Cynometra ramiﬂora fruit

extract

94%-91% Degradation of methylene blue

dye under sunlight irradiation

Bishnoi et al. (2018)

iron Piper betle leaves MG-93%

MO-73.29%

Degradation of malachite green

(MG) and methyl orange (MO)

dyes

Badmapriya and Asharani (2016)

Copper Carica papaya leaf extract –Degradation of Coomassie

brilliant bye R-250 dye

Sankar et al. (2014)

Copper Pridium guajava leaf extract NB- 93%

RY160-81%

Degradation of Nile blue (NB)

dye and reactive yellow (RY160)

dye

Singh et al. (2019)

Alpha- manganese oxide Ficus Retusa MO- 116.1 mg/g

MR- 74.02 mg/g

Determining adsorption

efﬁciency of azo dye like methyl

red and methyl orange

Srivastava and Choubey (2021)

Zinc Carissa edulis extract 97% Degradation of Congo red dye Fowsiya et al. (2016)

Zinc Trianthema Portulaca strum’s

extract

91% Show antimicrobial activity

against Staphylococcus aureus

and E.coli,

Antifungal activity against

Aspergillus niger and Aspergillus

ﬂavus and degradation of

Synozol Navy blue-KBF textile

dye under solar irradiation

Khan et al. (2019)

(continued)

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 317

Tabl e 2 (continued)

Type of nanoparticles Plant source Absorption capacity/percentage

degradation

Applications References

Zinc Calliandra haematocephala

leaf extract

88% Catalytic degradation of

methylene blue dye under solar

radiation

Vinayagam et al. (2020)

Zinc Saponaria ofﬁcinalis 42% Degradation of methyl blue and

exhibit antibacterial property

Sharma et al. (2019)

Bimetallic—Fe/Pd NP Grape leaf aqueous extract 98% Removal of orange (II) dye Luo et al. (2016b)

nZVI/Ni Green tea extract 100% Removal of Cr(VI) for

groundwater

Zhu et al. (2018)

Ni/Fe NP Punica granatum peel extract 76–78%- groundwater

68–72%- lake water

92–94%- DW

Removal of tetracycline from

ground water, lake water and

deionized distilled water

Ravikumar et al. (2019)

nZVI-Cu NP bentonite

supported nZVI-Cu NP

Pomegranate rind extract 72%

95%

Removal of antibiotic

tetracycline from natural water

Gopal et al. (2020)

Ag/AgCl NP Azadirachta indica 92.5% Degradation of methyl blue dye

from aqueous solution and

exhibit antibacterial property

Panchal et al. (2021)

Mn:CeO2Cassia angustifolia

Seed extract

–Photo degradation of malachite

green dye

Antony and Yadav (2021)

NiFe2O4 NP Juglans regia 85% Photocatalytic degradation of

congo red dye and ciproﬂoxacin

from water

Tajetal. (2021)

318 N. Kumari et al.

5.3 Metal and Metal Oxide Nanoparticle

Metals and oxides of metal nanoparticles are governed by physical and chemical

properties of metals which play a major role in providing stability. Also, catalytic

property enhances the degradation of contaminants like toxic metals, organochlori-

nated pesticides, polychlorinated biphenyl (PCB) (Nguyen et al. 2018). Metal oxide

nanoparticles are very speciﬁc to size, shape as well as nanostructure. They have

high density which results in small size of nanoparticles. So much of concern with

the size is related to reactivity, magnetic and electric property of nanomaterials.

Rather than remediating wastewater, iron oxide and magnetite are also considered

as a potential approach in MRI and MSD. Similarly, silicon dioxide, manganese

oxide, copper oxide, zirconium oxide, acts as catalysts in oxidation process and also

possess electrolytic property. CeO2 nanoparticle synthesised from Oleo Europaea,

Rubia cordifolia leaf show various catalytic properties, used in medical sciences and

optical sensor technology (Nadeem et al. 2020). Iron oxide nanoparticle conﬂicts our

interest, used in removal of contaminants from water. iron oxide nanoparticle poten-

tially removes lead, cadmium and chromium from aqueous solution (Ehrampoush

et al. 2015). Bio-compounds such as coumarin and oleﬁns facilitates reduction of

metal ion by donating electron followed by hydroxyl and methyl group containing

compound. polyphenolic compounds chelate the metal ion therefore nanoparticle

can be reused up to ﬁve cycles without losing stability (Groiss et al. 2017). Several

examples of metallic nanoparticles and metal oxide nanoparticles are given in Table 2.

5.4 Bimetallic/Trimetallic Nanoparticle

Combination of metals by optimizing their energies and reaction conditions refers

to bimetallic or trimetallic nanoparticles based upon the number of metals partici-

pating in the formation and enhance their remediation strategy. They can be used

as multipurpose tool. Bimetallic increases the efﬁcacy of reduction of metal by

altering the individual component, or geometrical structure to achieve better func-

tionality and application. Phoenix dactylifera synthesised bimetallic copper-silver

nanoparticle exhibit catalytic property to degrade methyl blue from aqueous solution

and antibacterial activity against Bacillus subtilis and Escherichia coli (Al-Haddad

et al. 2020). Some monometallic nanoparticle aggregates easily and loss their reac-

tivity therefore addition of catalytic metal is preferred which result in formation of

bimetallic nanoparticle in order to increases the reactivity, catalytic selectivity and

great efﬁciency useful in multiple applications like exhibiting antimicrobial property,

anticancer property and potent nano catalyst. Green synthesis of Au–Ag bimetallic

nanoparticle by using Pulicaria undulata extract shows catalytic activity in reduction

of 4-nitrophenol to 4-aminophenol under the inﬂuence of sodium borohydride (Khan

et al. 2020). Silver nanoparticle which exhibits excellent antibacterial property used

in pharmaceutical sector and antimicrobial property used in cosmetics, biosensor,

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 319

food processing etc,. On the other hand, gold nanoparticle used in medical ﬁeld like

cancer therapy. The idea is to combine the dual nature of silver and gold nanoparticle

to improve the application and efﬁciency in bimetallic nanoparticle. Fe/Pd nanopar-

ticle synthesis from aqueous extract of grape leaf in order to determine the reactivity

of bimetallic nanoparticle in comparison with monometallic iron nanoparticle (Luo

et al. 2016a).

Green synthesis of Au/Pt/Ag trimetallic nanoparticle using Lamis albi ﬂos extract

for determination of antimicrobial activity (Dlugaszewska and Dobrucka 2019). Vitex

angus-castus synthesized Au-ZnO-Ag trimetallic nanoparticle has ability to degrade

methylene blue dye within 36 min as well as 97% degradation of crystal violet

dye (Dobrucka 2019). Nanomaterial along with nanoparticles is more speciﬁc in

treatment of water as it increases the degradation speed and reactivity, preventing

any kind of by product formation. Nanomaterials when reacts with pollutants (inor-

ganic/organic) or heavy metals, result in photocatalytic reaction, chemical reaction,

absorption and adsorption. Au–Ag-Sr nanoparticle synthesize from root extract of

three different plants namely Coriandrum sativum, Aloe i ndica and Plectranthus

amboinics by using gold chloride, silver nitrate and strontium chloride as a metal

precursor (Binod et al. 2018). Trimetallic Fe-Ag-Pt synthesize from Platycodon

grandiﬂorum shows excellent catalytic efﬁciency in reducing 4- nitroaniline to p-

phenylenediamine within 25 min and complete decolourization of rhodamine B dye

within 15 min (Basavegowda et al. 2017).

6 Remediation Potential of Phyto-Nanoparticles

in Wastewater Treatment

6.1 Dye Degradation from Wastewater

Heterogeneous photocatalysis is technique used for puriﬁcation of wastewater. The

mechanism can be classiﬁed based upon the type of catalyst. Photo-decolouration

involves photo-oxidation and photo-reduction in which dye converted to its orig-

inal form. Photo-degradation converted the dye into some non-toxic stable product.

Photo-mineralization gives the potential to decompose the dye into carbon dioxide,

water, nitrate, etc.. Photo-decomposition involves photo-degradation and photo-

mineralization. In photocatalytic degradation of dye, excited electron moves from

valence band to conductance band, generating electron hole pair which result in

oxidative photo-degradation by generating the hydroxyl radical. thus, atmospheric

oxygen comes in contact with electron resulting in complete degradation of dye to

non-toxic by products such as carbon dioxide, water molecule, etc. (Marimuthu et al.

2020). TiO2 can be used as potent oxidizing agent because of its high energy gap

between valance band and conductance band. A suitable metal which is stable, act

as good conductor as well as absorbs light easily can be doped with TiO2 to reduce

the energy gap. Electron captured by oxygen in water forming a free radical. Hole

320 N. Kumari et al.

created by due to excitation of electron ﬁnally accepts the electron from absorbed

dye resulting in reduction of dye. Determination of dye degradation through UV-

Vis spectroscopy carried out by evaluating the optical density of nanoparticles. The

electron transfer from donor to acceptor held on the surface of nanoparticle thus, it

acts as a catalyst for the reaction. The dye degradation is dependent upon the size

and shape of synthesized nanoparticle and the target dye chemical structure. Silver

and palladium nanoparticle synthesized from Daucus carota leaf extract shows high

efﬁciency of removing rhodamine 6-G dye. Catalytic property was evaluated that

came to be 98% and 89.4% of rhodamine dye get decolourized within 2 min and

30 min under the treatment of palladium nanoparticle and silver nanoparticle respec-

tively (Joseph Kirubaharan et al. 2020). Silver nanoparticle synthesized from Albizia

procera shows promising results in removing methyl blue dye. Optimized pH at

11.5 to get removal efﬁciency of 99.6% of methyl blue dye. Similarly, temperature

optimized at 30 degree Celsius and contact time of around 70 min to get removal efﬁ-

ciency of 93.65% and 51.54% respectively (Raﬁque et al. 2019). Green synthesis of

copper nanoparticle is successful in degrading 96% of methyl blue dye from aqueous

solution under optimize conditions (Sinha and Ahmaruzzaman 2015). Several studies

conducted on applicability of plant-based nanomaterials for dyes and heavy metal

pollution, UV–Vis range for dye degradation analysis, and additives etc., have been

presented in Table 3.

6.2 Heavy Metal Removal from Aqueous Solution

Adsorption mechanism assisted by electrostatic interaction, complexation and adsor-

bent nature. electrostatic interaction between metal ion and adsorbent are driving

forces for adsorption process (Sarma et al. 2019). plant mediated nanoparticles

provide a function group which increases in binding site and form surface complex by

electrostatic attraction. Adsorbate interaction with adsorbent determines with the help

of different isotherm include Freundlich isotherm and Langmuir isotherm. Kinetic

model include pseudo ﬁrst order reaction and pseudo second order reaction helps

in determining the type of adsorption on adsorbent along with reaction pathways.

combination of theoretical and experimental calculation obtained from adsorption

isotherms and kinetic model explain the efﬁcient removal of heavy metal from the

water (Al-Senani and Al-Fawzan 2018). Adsorption capacity of silicon nanoparticle

synthesized from plant extracted Saccharum ravannae, Saccharum ofﬁcinarum and

Oryza sativa found to be above 95% for Pb2+ and Ca2+. The adsorption studies

of Silica nanoparticle is done by optimizing various parameters like pH, metal ion

concentration, temperature, adsorbent dose and contact (Sachan et al. 2021). The

adsorption mechanism is understood by various isotherm models like Freundlich

isotherm and Langmuir isotherm and thermodynamic studies. Iron nanoparticle

synthesized by using tea extract was irradiated with 60Co gamma radiation. The

adsorption capacity of Cu2+ ions in aqueous solution before and after radiation was

observed 81.67% and 97% respectively under optimize condition (Amin et al. 2021).

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 321

Ta bl e 3 Phytonanoparticles showing removal efﬁciency of heavy metals and dye degradation from aqueous solution

Nanoparticle Heavy metal/dye UV- Vis of dye/NP

(obs.)

Reaction time Additives pH Percentage

removal/observation

References

Silver nanoparticle Methylene blue dye 660 nm/420 nm 0–72 h –5.6

6.6

7.6

8.6

95.3% at 72 h at 8.6

pH

Vanaja et al.

(2014)

Silver nanoparticle Congo red dye SPR band—498

(pie-pie)

338(n-pie)

At interval of

1.5 min upto

15 min

Sodium

borohydride

–Not given Kolya et al.

(2015)

Silver nanoparticle Congo red dye 496 nm dye/426

AgNP

0–13 mintues Sodium

borohydride

–98.5 upto 5 cycles Chand et al.

(2021)

Silver nanoparticle Safranin O

Methyl red,

Methyl Orange,

Methyl blue

(10 mg/l)

519 nm

415 nm

460 nm

664 nm

(AgNP- 419 nm)

0–24 h – – Degradation rate

constant

1.02 × 10–3/min

1.03 × 10–3

1.86 × 10–3

1.44 × 10–3

Jyoti and

Singh (2016)

Silver nanoparticle Brilliant blue

Tartrazine

Carmoisine

629 nm

420 nm

504 nm

Reaction rate

constant-k

0.2097

0.0076

0.0496

Sodium

borohydride

– – David and

Moldovan

(2020)

(continued)

322 N. Kumari et al.

Tabl e 3 (continued)

Nanoparticle Heavy metal/dye UV- Vis of dye/NP

(obs.)

Reaction time Additives pH Percentage

removal/observation

References

Silver nanoparticle Methyl blue

Methyl orange

500–700

350–550 nm

1mM

5min

2 mM–5 min

1mM

5min

2mM

5min

Sodium

borohydride

–27.12%

18.08%

69.79%

42.11%

Chandra Paul

et al. (2020)

Silver nanoparticle Hg2 + detection Not mentioned

(given a graph)

– – pH- 3–8

(no effect

of pH)

Dark yellow changes

to colourless

Roy et al.

(2015)

Silver nanoparticle Detection of Hg2+ Not mention (graph

given)

– – – Dark brown changes

to colourless

Tag ad et al .

(2017)

Gold nanoparticle Toxic metal removal

Pd

Cd

Cu

Fe

Ni

Co

Mn

Zn

Pb

Absorption capacity

of adsorbent used in

aqueous soln. of

toxic metal was

determined by batch

method

120 min – 5

6

6.5

Removal%

63.46

60.20

51.50

68.20

42.18

23.47

21.62

35.37

75.76

Baran et al.

(2020b)

(continued)

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 323

Tabl e 3 (continued)

Nanoparticle Heavy metal/dye UV- Vis of dye/NP

(obs.)

Reaction time Additives pH Percentage

removal/observation

References

SnO2 nanoparticle Photodegradation of

RhB

Removal of heavy

metal Co + 2

553.5/370–375 nm 190 min

60 min

– 7

>7

>8

91.7%

Adsorption capacity

93.6%

Formation of soluble

hydroxyl complex in

excess of OH

Cobalt hydroxide

started to ppt from aq.

Sol. & adsorption was

impossible

Ebrahimian

et al. (2020)

Zero valent iron

nanoparticle

Adsorption of Cr(VI) –30 min

90 min

Oenothein B – Adsorption efﬁciency

98.1%

71.9%

Madhavi

et al. (2013a)

Maghemite

nanoparticle

Removal of heavy

metal in ﬂy ash

Pb

Cd

–1h

24 h

1h

24 h

– 7 85.56%

90.85%

67.8%

Conc. Reached below

the detection level of

ICP-OES (not

deﬁned)

Yadav and

Fulekar

(2018a)

(continued)

324 N. Kumari et al.

Tabl e 3 (continued)

Nanoparticle Heavy metal/dye UV- Vis of dye/NP

(obs.)

Reaction time Additives pH Percentage

removal/observation

References

Iron based

nanoparticle

Removal of Cr (VI) and

Cu (II) when co-existed

together

Cr (VI) and Cu (II)

present separately

Removal efﬁciency of

Cr

Cu (II)

Pb (II)

Zn (II)

–

1h

T = 288 K

T = 308 K

– 5 Cr (VI)- 58.9%

Cu (II)-33%

Cr (VI) = 74.2%; Cu

(II) = 45.2%

Cu-26.8%

Cr-50.7%

Cu-40.8%

Cr-62.6%

Cr = from 75.1% to

50.8%

Cu 28.3% to 64.2%

44.8

21.5

31.4

10.8

Ajitha et al.

(2015)

Zero valent Silver

nanoparticle

Cadmium removal from

aqueous solution

–40 min – 6 85%

75.5%

Al-Qahtani

(2017)

Zero valent iron

nanoparticles

Removal of Cr (VI)

from aqueous solution

TV-Fe (higher%

removal)

UD-Fe

RD-Fe

Adsorption

capacities

466

462

453.7

1–10

30 min

–2–9 91.75%

60.95%

97.5%

97.1%

96.94%

Fazlzadeh

et al. (2017)

Silver nanoparticles Chromium removal 350 nm –Activated

carbon

–Absorbing pattern was

irregular

Samrot et al.

(2019)

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 325

Iron oxide nanoparticles synthesized from Ramalina sinesis extract was successfully

able to remove lead and cadmium by following Langmuir adsorption isotherm and

Freundlich adsorption isotherm respectively also both removal follows second order

kinetic model with removal capacity of 82% for lead and 77% for cadmium under the

pH ranges between 4 to 5 and initial ion concentration was 50 mg/l with 70-degree

temperature in 1 h (Arjaghi et al. 2021).

Factor Affecting Removal of Heavy Metal

Biomass concentration

Increase in biomass concentration results in increases in number of metal binding sites

hence increase adsorption efﬁciency. Lead, zinc and chromium removal efﬁciency

increases from 94.35% to 100%, 44% to 36.9% and 55% to 81.9% respectively,

when concentration of biomass changes from 0.2 g to2g(Chandra Sekhar et al.

2003). At very high concentration of biomass, metal removal efﬁciency decreases

because of the reduction average distance available for absorption sites due to the

aggregation of biomass. Copper uptake efﬁciency decreases from 85 to 58%. With

increased biosorption concentration from 0.5 g/l to 2 g/l (Chandra Sekhar et al.

2003). From comparative studies, we can conclude that low biomass concentration

results in decreased biosorption efﬁciency. With increase in dosage of zerovalent

iron nanoparticle from 0.5 g/l to 2.0 g/l, the removal efﬁcacy of Cu, Zn, Cr and Pb

was increased from 76%, 14% 51% and 78% to more than 80% in both Zn and Pb

followed by complete removal of Cu and Cr within 30 min. higher pH was obtained

at higher biomass concentration for the removal of heavy metal like Zn, Pb, Cr and

Cu (Chen et al. 2008). Since metal removal occurs at the surface of the nanoparticle,

the maximum removal of heavy metal become unchanged. Also, removal efﬁciency

of heavy metal is inversely proportional to the initial metal ion concentration under

a constant biomass concentration. Magnetite concentration was increased from 1 g/l

to 4 g/l in order to increases the removal efﬁciency of hexavalent chromium from

29.1% to complete removal (Ataabadi et al. 2015).

pH

The important factor is pH, affecting the chemistry of metal ions and biosorbent by

inﬂuencing their solubility as well as toxicity. Metal uptake related to complexation

chemistry of metal ion and behaviour of function group present at the surface of

the plant. The carbonyl group of biomolecule provide a negative charge at acidic

condition due to which an electrostatic interaction occurs between two cationic which

results in biosorption of metal. At low pH, H +ions are occupied in the active sites of

adsorption, with increases in pH these sites become free and available for the heavy

metal on the surface of adsorbent. In the pH range of 3–7 there is slight increase in

removal process of heavy metal like Cr(VI) removal favoured at low pH due to the

positive charged surface of nanoparticle at low pH attract the negative charge anions

as a r esult an electrostatic attraction occurred (Weng et al. 2016b). Also, at some point

326 N. Kumari et al.

adsorption sites become independent of pH change indicating saturation point which

is attributed to formation of hydroxyl complex in excess of OH ion, depends upon the

type of heavy metal that is to be removed and the surface of nanoparticle. Adsorption

capacity increases with increases in pH until it attains a maximum biosorption at

optimum pH because further increase in pH will result in precipitation of metal.

Addition of NaOH and HNO3 leads to increment and reduction of pH respectively.

Studies have been be conducted over silver nanoparticle synthesis from different

plant extract like neem leaf, sun dried leaf of neem, neem bark, mango leaf, sun

dried leaf of mango, green tea and pepper seed extract selectively sense heavy metal

like mercury, lead, zinc, cobalt and zinc over a wide range of pH from initial pH and

highest pH that maintains acidic and basic environment respectively. At initial pH

4, mercury was detected by neem bark synthesized silver nanoparticle but it was not

detected when increasing the pH up to 9 rest other metal like cobalt and nickel was

detected (Karthiga and Anthony 2013).

Temperature

Higher temperature increases the solubility of metal ion in water, therefore biosorp-

tion of these metal ions become difﬁcult. Temperature depends upon the type of chem-

ical reaction occurring between biosorbent and metal ion. Metal removal efﬁciency

decreases with increase temperature for exothermic biosorption as the absorption of

the molecule becomes easier and when temperature rise, desorption of the molecule

take place. Sorption of lead and cadmium by Caladium bicolor biomass is affected

between 30 to 80 degrees Celsius. Increase in temperature results in weak attractive

forces between bisorbent and bisorbate, thinning of the outer boundary layer which

help the metal ion which lead to decreases in sorption. Lower temperature helps in

enhancing the activation energy and solubility of chemical to increase the rate of

the reaction. Uptake of lead ion t o biosorbent by peanut shell effectively remove

66% of lead at 20 degrees Celsius. Removal efﬁciency decreases with increase in

temperature from 20 degree Celsius to 40 degree Celsius showed exothermic biosorp-

tion (Ta¸sar et al. 2014). In zinc oxide nanoparticle, increase in temperature from 30

to 70-degree result in increase in adsorption capacity of lead from 16.19 mg/g to

19.96 mg/g (Azizi et al. 2017) whereas increase in temperature from 288 to 308 K

results in higher adsorption of Cr (VI) and Cu (II) on the surface of iron nanopar-

ticle (Weng et al. 2016b). Interaction between available sites of the absorbent with

absorbate efﬁciently increases the removal rate from 73.8% to 100% of the toxic

hexavalent chromium with increase in temperature from 25 degrees Celsius to 40

degrees Celsius respectively (Ataabadi et al. 2015). Therefore, temperature depends

upon the nature of the process.

Initial metal ion concentration

Adsorption capacity increases with initial metal ion concentration. When all binding

sites occupied with metal result in increase in concentration slope. There would be

decrease in adsorption rate with further increasing the concentration of metal ion

as all the obtainable nanoparticle sites are ﬁlled. Removal efﬁciency of chromium

is 86% with initial iron concentration 30 mg/l. increase in iron concentration up to

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 327

150 mg/l result in deduce the adsorption efﬁciency from 86 to 70% (Al-Qahtani

2017). 100 mg/ml is the concentration of metal ion where the removal efﬁciency of

chromium is highest that is 42.37%. Further increasing the metal ion concentration up

to 200 mg/ml, the removal efﬁciency of chromium decreased from 42.37% to 33.75%

as all available site occupied which result in closing of the pores and hence metal ion

preventing penetrating deep into adsorbent pore (Shaik et al. 2020b). Zerovalent iron

nanoparticle effectively remove hexavalent chromium with adsorption efﬁciency of

98.1% observed within 30 min when initial metal ion concentration is 200 mg/ml.

adsorption efﬁciency decreases to 71.9% with increase in metal ion concentration

up to 400 mg/ml (Madhavi et al. 2013b).

Contact time

Contact time indirectly effects the rate of adsorption and the data was analysed by

kinetic models namely pseudo ﬁrst order and pseudo second order model. It has

similar affect as of initial metal concentration, biosorption increases with increase

in contact time. When all the binding sites become fully saturated, the reaction

become independent of time. Cadmium removal from aqueous solution carried

out at optimum contact time of 40 min. over that time, no further increase in

cadmium removal efﬁciency (Al-Qahtani 2017). Absorption capacity of hexavalent

chromium decreases from 98.1% to 72.9% with increase in contact time of 30 min

to 90 min (Madhavi et al. 2013b). Maghemite nanoparticle effectively remove lead

and cadmium from ﬂy ash with respect to time. The removal process observed upto

0 to 24 h. It has been observed that initially, 85.56% of lead is removed and cadmium

removal was not deﬁned by ICP-OES. After 2 h, 67.8% removal of cadmium was

detected followed by detection of 90.85% lead at 24 h (Yadav and Fulekar 2018b).

Most of the adsorption site are occupied initially that’s why it attains an equilibrium

and removal percentage does not increase rapidly.

6.3 Desorption Analysis

Desorption studies refer to the removal of absorbed metal from the surface of

absorbent. There are various types of desorbing agents including tap water, sodium

hydroxide, hydrochloric acid, deionized water, sulphuric acid, ammonium hydroxide,

potassium hydroxide. For example, the desorption efﬁciency of HCl and H2SO4 is

very high in removing the Cr (VI) and Pb (II) ions respectively from the adsorbent. It

was found that adsorbent exhibited good removal efﬁciency of chromium and lead up

to ﬁve consecutive cycles (Bayuo et al. 2020). Sodium hydroxide is considered best

for the removal of chromium up to ﬁve consecutive cycles as its desorption efﬁcacy

reduces from 98 to 89% after the ﬁfth cycle (Al-Haddad et al. 2020). Chromium

desorption from red peanut skin synthesized iron nanoparticle was done by using

16 M hydrochloric acid and distilled water as desorbents. Iron nanoparticle dried by

vacuum under 40 degrees Celsius and 60 degrees Celsius as well as air dried at room

temperature. Maximum chromium efﬁciency that is 100% within 1 min was achieved

328 N. Kumari et al.

by vacuum dried at 60 degrees Celsius then followed by vacuum dried iron nanopar-

ticle at 40 degrees Celsius. It has been hypothesized that with increase in temperature

of vacuum drying, reduction of radius of iron nanoparticle occurred due to which

at higher temperature chromium removal occurred more rapidly. Minimum removal

efﬁciency was 90% in 4.5 h given by air dried iron nanoparticle due to formation of

ferrosoferric oxide (Pan et al. 2019). Bimetallic silver- copper nanoparticle at zinc

oxide surface efﬁciently able to degrade rhodamine B to leuco rhodamine B dye and

Congo red dye within 12 s and 9 s respectively up to ﬁve consecutive cycles because

after that leaching of metal started which can be determined by ICP-AES (Manjari

et al. 2020).

7 Challenges and Future Prospects

Nanoparticles have been successfully found active in remediating the waste water but

at the same time, they also face some critical challenges in their synthesis. Since we

discussed the green synthesis of nanoparticle, ﬁrst of all there is need for the selection

of appropriate plants which are rich in phytocompounds like polyphenol, ﬂavonoids,

terpenoids, alkaloids etc. Identiﬁcation of particular biochemical compound present

in the plant that acts as a reducing capping and stabilising agent of the nanoparticle

is also necessary. After selection of suitable plant with exact targeted active phyto-

chemical compound, the selection of suitable metal precursor is needed. Nanopar-

ticles have small size and larger surface area due to which they have more inter-

action sites available on their surface with cells. This gives several toxic biological

responses. Smaller the size of the nanoparticle, greater the toxicity attributed with

it. Toxicity of nanoparticle depends upon various parameters including structure,

shape, hydrophilicity, composition, concentration, reaction temperature and surface

chemistry. The toxicological effects of nanoparticle affect humans and environment.

Acute and chronic toxicity occur upon oral exposure of several metal nanoparticles.

Due to their small size, some nanoparticles pass the blood brain barrier which may

lead to dangerous neural diseases. Reuse of nanoparticles is another challenge.

Nanotechnology is establishing in every ﬁeld, be it agricultural engineering, drug

delivery, X-ray imaging, dentistry, cosmetics or other environmental aspects. From

past studies, we can conclude that nanoparticle synthesis by traditional methods

requires heavy machine equipment and toxic chemicals with complex handling

which is a very costly procedure. To overcome these disadvantages, we moved to

green synthesis of nanoparticles from plant, algae, bacteria and fungi. This chapter

presents an insight into nanoparticle synthesis from plant extract. Exact mechanism

of the targeted biochemical process occurs at cellular level for increased production

of nanoparticle, which can be discovered in further studies. More studies can

be carried out on capping agents which prevent the further reaction aggregation

and result in providing stability for a longer period of time. We have to expand

the area of the sources of nanoparticle synthesis so that they can be synthesized

from waste materials like algae and several other plants which are cheap, easily

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 329

available and suitable. Very limited studies have been carried out on the toxicity

of phyto-nanoparticles. We need to develop several strategies for detoxiﬁcation of

green nanoparticles. Research cannot be restricted to water pollutant removal only.

We can explore new opportunities in the environmental air puriﬁcation also, to

reduce the amount of particulate matter (PM), ozone, sulphur, etc.

8 Conclusion

Pollutants such as heavy metal and various organic or synthetic dyes contaminate the

water bodies leading to water pollution thus creating many problems related to human

and animal health and affecting the environment. Therefore, there is an immediate

requirement of water treatment. Phyto-nanotechnology holds an enormous potential

to remediate wastewater as it offers inexpensive, eco-friendly and efﬁcient way. In

this chapter, we have discussed the various types of plant synthesized nanoparticles

which can be used to remediate wastewater. The exploration of biotechnological

prospects for nanoparticles synthesis at industrial scale remediation of wastewater is

needed. The article concludes that phytonanoparticles can be a potential, inexpensive

and ecofriendly agents for environmental pollution remediation.

Declarations Funding: Not Applicable.

Conﬂicts of Interest: The authors declare that they have no conﬂict of interest.

Availabity of data and material: Not Applicable.

Code Availability: Not Applicable.

References

Ajitha B, Ashok Kumar Reddy Y, Sreedhara Reddy P (2015) Green synthesis and characterization

of silver nanoparticles using Lantana camara leaf extract. Mater Sci Eng C 49:373–381. https://

doi.org/10.1016/j.msec.2015.01.035

Al-Haddad J, Alzaabi F, Pal P et al (2020) Green synthesis of bimetallic copper–silver nanoparti-

cles and their application in catalytic and antibacterial activities. Clean Technol Environ Policy

22:269–277. https://doi.org/10.1007/s10098-019-01765-2

Al-Qahtani KM (2017) Cadmium removal from aqueous solution by green synthesis zero valent

silver nanoparticles with Benjamina leaves extract. Egypt J Aquat Res 43:269–274. https://doi.

org/10.1016/j.ejar.2017.10.003

Al-Senani GM, Al-Fawzan FF (2018) Adsorption study of heavy metal ions from aqueous solution

by nanoparticle of wild herbs. Egypt J Aquat Res 44:187–194. https://doi.org/10.1016/j.ejar.2018.

07.006

Amaliyah S, Pangesti DP, Masruri M, Sabarudin A, Sumitro SB (2020) Green synthesis and char-

acterization of copper nanoparticles using Piper retrofractum Vahl extract as bioreductor and

capping agent. Heliyon 6:e04636. https://doi.org/10.1016/j.heliyon.2020.e04636

330 N. Kumari et al.

Amin RM, Mahmoud RK, Gadelhak Y, El-Ela FIA (2021) Gamma irradiated green synthesized

zero valent iron nanoparticles as promising antibacterial agents and heavy metal nano-adsorbents.

Environ Nanotechnol Monit Manag, 100461.https://doi.org/10.1016/j.enmm.2021.100461

Antony D, Yadav R (2021) Facile fabrication of green nano pure CeO2 and Mn-decorated CeO2

with Cassia angustifolia seed extract in water reﬁnement by optimal photodegradation kinetics of

malachite green. Environ Sci Pollut Res 28:18589–18603. https://doi.org/10.1007/s11356-020-

11153-9

Arjaghi SK, Alasl MK, Sajjadi N et al (2021) Green synthesis of iron oxide nanoparticles by RS

lichen extract and its application in removing heavy metals of lead and cadmium. Biol Trace

Elem Res 199:763–768. https://doi.org/10.1007/s12011-020-02170-3

Ataabadi M, Hoodaji M, Tahmourespour A, et al (2015) Optimization of factors affecting hexava-

lent chromium removal from simulated electroplating wastewater by synthesized magnetite

nanoparticles. Environ Monit Assess, 187.https://doi.org/10.1007/s10661-014-4165-z

Azizi S, Shahri MM, Mohamad R (2017) Green synthesis of zinc oxide nanoparticles for enhanced

adsorption of lead Ions from aqueous solutions: equilibrium, kinetic and thermodynamic studies.

Molecules, 22https://doi.org/10.3390/molecules22060831

Babuponnusami A, Muthukumar K (2014) A review on Fenton and improvements to the Fenton

process for wastewater treatment. J Environ Chem Eng 2:557–572. https://doi.org/10.1016/j.jece.

2013.10.011

Badmapriya D, Asharani IV (2016) Dye degradation studies catalysed by green synthesized iron

oxide nanoparticles. Int J ChemTech Res 9:409–416

Baran MF, Acay H, Keskin C (2020a) Saponaria ofﬁcinalis determination of antimicrobial and toxic

metal removal activities of plant-based synthesized (Capsicum annuum L. Leaves), ecofriendly,

gold nanomaterials, Glob. Challenges 4:1900104. https://doi.org/10.1002/gch2.201900104

Baran MF, Acay H, Keskin C (2020b) Determination of antimicrobial and toxic metal removal activ-

ities of plant-based synthesized (Capsicum annuum L. Leaves), ecofriendly, gold nanomaterials.

Glob Challenges 4:1900104. https://doi.org/10.1002/gch2.201900104

Barberia-Roque L, Gámez-Espinosa E, Viera M, Bellotti N (2019) Assessment of three plant extracts

to obtain silver nanoparticles as alternative additives to control biodeterioration of coatings. Int

Biodeterior Biodegrad 141:52–61. https://doi.org/10.1016/j.ibiod.2018.06.011

Basavegowda N, Mishra K, Lee YR (2017) Trimetallic FeAgPt alloy as a nanocatalyst for the

reduction of 4-nitroaniline and decolorization of rhodamine B: a comparative study. J Alloys

Compd 701:456–464. https://doi.org/10.1016/j.jallcom.2017.01.122

Bayuo J, Abukari MA, Pelig-Ba KB (2020) Desorption of chromium (VI) and lead (II) ions and

regeneration of the exhausted adsorbent. Appl Water Sci 10:1–6. https://doi.org/10.1007/s13201-

020-01250-y

Bibi I, Kamal S, Ahmed A, Iqbal M, Nouren S, Jilani K, Nazar N, Amir M, Abbas A, Ata S, Majid

F (2017) Nickel nanoparticle synthesis using Camellia Sinensis as reducing and capping agent:

Growth mechanism and photo-catalytic activity evaluation. Int J Biol Macromol 103:783–790.

https://doi.org/10.1016/j.ijbiomac.2017.05.023

Bibi I, Nazar N, Ata S, Sultan M, Ali A, Abbas A, Jilani K, Kamal S, Sarim FM, Khan MI, Jalal

F, Iqbal M (2019) Green synthesis of iron oxide nanoparticles using pomegranate seeds extract

and photocatalytic activity evaluation for the degradation of textile dye. J Mater Res Technol

8:6115–6124. https://doi.org/10.1016/j.jmrt.2019.10.006

Binod A, Ganachari S V, Yaradoddi JS et al (2018) Biological synthesis and characterization of tri-

metallic alloy (Au Ag, Sr) nanoparticles and its sensing studies. IOP Conf Ser Mater Sci Eng

376.https://doi.org/10.1088/1757-899X/376/1/012054

Bishnoi S, Kumar A, Selvaraj R (2018) Facile synthesis of magnetic iron oxide nanoparticles using

inedible Cynometra ramiﬂora fruit extract waste and their photocatalytic degradation of methylene

blue dye. Mater Res Bull 97:121–127. https://doi.org/10.1016/j.materresbull.2017.08.040

Chand K, Jiao C, Lakhan MN, Shah AH, Kumar V, Fouad DE, Chandio MB, Ali Maitlo A, Ahmed M,

Cao D (2021) Green synthesis, characterization and photocatalytic activity of silver nanoparticles

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 331

synthesized with Nigella Sativa seed extract. Chem Phys Lett 763:138218. https://doi.org/10.

1016/j.cplett.2020.138218

Chandra Paul S, Bhowmik S, Rani Nath M, Islam MS, Kanti Paul S, Neazi J, Sabnam Binta Monir

T, Dewanjee S, Abdus Salam M (2020) Silver nanoparticles synthesis in a green approach: size

dependent catalytic degradation of cationic and anionic dyes, orient. J Chem 36:353–360. https://

doi.org/10.13005/ojc/360301

Chandra Sekhar K, Kamala CT, Chary NS, Anjaneyulu Y (2003) Removal of heavy metals using

a plant biomass with reference to environmental control. Int J Miner Process 68:37–45. https://

doi.org/10.1016/S0301-7516(02)00047-9

Chelly M, Chelly S, Zribi R, Bouaziz-ketata H, Gdoura R, Lavanya N, Veerapandi G, Sekar C, Neri

G (2021) Synthesis of silver and gold nanoparticles from rumex roseus plant extract and their

application in electrochemical sensors. Nanomaterials 11:1–18. https://doi.org/10.3390/nano11

030739

Chen SY, Chen WH, Shih CJ (2008) Heavy metal removal from wastewater using zero-valent iron

nanoparticles. Water Sci Technol 58:1947–1954. https://doi.org/10.2166/wst.2008.556

Chowdhury S, Mazumder MAJ, Al-Attas O, Husain T (2016) Heavy metals in drinking water:

occurrences, implications, and future needs in developing countries. Sci Total Environ 569–

570:476–488. https://doi.org/10.1016/j.scitotenv.2016.06.166

David L, Moldovan B (2020) Green synthesis of biogenic silver nanoparticles for efﬁcient catalytic

removal of harmful organic dyes. Nanomaterials 10. https://doi.org/10.3390/nano10020202

Dlugaszewska J, Dobrucka R (2019) Effectiveness of biosynthesized Trimetallic Au/Pt/Ag nanopar-

ticles on planktonic and bioﬁlm enterococcus faecalis and enterococcus faecium Forms. J Clust

Sci 30:1091–1101. https://doi.org/10.1007/s10876-019-01570-3

Dobrucka R (2019) Facile synthesis of trimetallic nanoparticles Au/CuO/ZnO using Vitex agnus-

castus extract and their activity in degradation of organic dyes. Int J Environ Anal Chem 00:1–12.

https://doi.org/10.1080/03067319.2019.1691543

Dubey S, Sharma YC (2017) Calotropis procera mediated one pot green synthesis of Cupric

oxide nanoparticles (CuO-NPs) for adsorptive removal of Cr(VI) from aqueous solutions. Appl

Organomet Chem 31:1–15. https://doi.org/10.1002/aoc.3849

Ebrahimian J, Mohsennia M, Khayatkashani M (2020) Photocatalytic-degradation of organic dye

and removal of heavy metal ions using synthesized SnO2 nanoparticles by Vitex agnus-castus

fruit via a green route. Mater Lett 263:127255. https://doi.org/10.1016/j.matlet.2019.127255

Ebrahiminezhad A, Taghizadeh S, Ghasemi Y, Berenjian A (2018) Green synthesized nanoclusters

of ultra-small zero valent iron nanoparticles as a novel dye removing material. Sci Total Environ

621:1527–1532. https://doi.org/10.1016/j.scitotenv.2017.10.076

Ehrampoush MH, Miria M, Salmani MH, Mahvi AH (2015) Cadmium removal from aqueous

solution by green synthesis iron oxide nanoparticles with tangerine peel extract. J Environ Heal

Sci Eng 13:1–7. https://doi.org/10.1186/s40201-015-0237-4

Fazlzadeh M, Rahmani K, Zarei A, Abdoallahzadeh H, Nasiri F, Khosravi R (2017) A novel green

synthesis of zero valent iron nanoparticles (NZVI) using three plant extracts and their efﬁcient

application for removal of Cr(VI) from aqueous solutions. Adv Powder Technol 28:122–130.

https://doi.org/10.1016/j.apt.2016.09.003

Fernandez AC, KM A, Rajagopal R (2020) Green synthesis, characterization, catalytic and antibac-

terial studies of copper iodide nanoparticles synthesized using Brassica oleracea var. capitata f.

rubra extract. Chem Data Collect 29:100538. https://doi.org/10.1016/j.cdc.2020.100538

Fowsiya J, Madhumitha G, Al-Dhabi NA, Arasu MV (2016) Photocatalytic degradation of Congo

red using Carissa edulis extract capped zinc oxide nanoparticles. J Photochem Photobiol B Biol

162:395–401. https://doi.org/10.1016/j.jphotobiol.2016.07.011

Glossman-Mitnik D (2013) Computational study of the chemical reactivity properties of the

Rhodamine B molecule. Procedia Comput Sci 18:816–825. https://doi.org/10.1016/j.procs.2013.

05.246

332 N. Kumari et al.

Gopal G, Sankar H, Natarajan C, Mukherjee A (2020) Tetracycline removal using green synthesized

bimetallic nZVI-Cu and bentonite supported green nZVI-Cu nanocomposite: a comparative study.

J Environ Manage 254:109812. https://doi.org/10.1016/j.jenvman.2019.109812

Gopalakrishnan V, Muniraj S (2021) Neem ﬂower extract assisted green synthesis of copper nanopar-

ticles—optimisation, characterisation and anti-bacterial study. Mater Today Proc 36:832–836.

https://doi.org/10.1016/j.matpr.2020.07.013

Goutam SP, Saxena G, Singh V, Yadav AK, Bharagava RN, Thapa KB (2018) Green synthesis

of TiO2 nanoparticles using leaf extract of Jatropha curcas L. for photocatalytic degradation of

tannery wastewater. Chem Eng J 336:386–396. https://doi.org/10.1016/j.cej.2017.12.029

Groiss S, Selvaraj R, Varadavenkatesan T, Vinayagam R (2017) Structural characterization,

antibacterial and catalytic effect of iron oxide nanoparticles synthesised using the leaf extract

of Cynometra ramiﬂora. J Mol Struct 1128:572–578. https://doi.org/10.1016/j.molstruc.2016.

09.031

Guerra FD, Attia MF, Whitehead DC, Alexis F (2018) Nanotechnology for environmental reme-

diation: materials and applications. Molecules 23:1–23. https://doi.org/10.3390/molecules230

71760

Guo B, Li M, Li S (2020) The comparative study of a homogeneous and a heterogeneous system

with green synthesized iron nanoparticles for removal of Cr(VI). Sci Rep 10:1–11. https://doi.

org/10.1038/s41598-020-64476-5

Hao R, Li D, Zhang J, Jiao T (2021) Green synthesis of iron nanoparticles using green tea and

its removal of hexavalent chromium. Nanomaterials 11:1–13. https://doi.org/10.3390/nano11

030650

Hassan AK, Al-Kindi GY, Ghanim D (2020) Green synthesis of bentonite-supported iron nanopar-

ticles as a heterogeneous Fenton-like catalyst: kinetics of decolorization of reactive blue 238 dye.

Water Sci Eng 13:286–298. https://doi.org/10.1016/j.wse.2020.12.001

Ismail M, Akhtar K, Khan MI, Kamal T, Khan MA, Asiri AM, Seo J, Khan SB (2019) Pollution,

toxicity and carcinogenicity of organic dyes and their catalytic bio-remediation. Curr Pharm Des

25:3645–3663. https://doi.org/10.2174/1381612825666191021142026

Jayachandran A, ATR, Nair AS (2021) Green synthesis and characterization of zinc oxide nanopar-

ticles using Cayratia pedata leaf extract. Biochem Biophys Reports 26:100995. https://doi.org/

10.1016/j.bbrep.2021.100995

Jha AK, Chakraborty S (2020) Photocatalytic degradation of Congo Red under UV irradiation by

zero valent iron nano particles (nZVI) synthesized using Shorea robusta (Sal) leaf extract. Water

Sci Technol 82:2491–2502. https://doi.org/10.2166/wst.2020.517

Joseph Kirubaharan C, Fang Z, Sha C, Yong YC (2020) Green synthesis of Ag and Pd nanoparticles

for water pollutants treatment. Water Sci Technol 82:2344–2352. https://doi.org/10.2166/wst.202

0.498

Jyoti K, Singh A (2016) Green synthesis of nanostructured silver particles and their catalytic appli-

cation in dye degradation. J Genet Eng Biotechnol 14:311–317. https://doi.org/10.1016/j.jgeb.

2016.09.005

Karthiga D, Anthony SP (2013) Selective colorimetric sensing of toxic metal cations by green

synthesized silver nanoparticles over a wide pH range. RSC Adv 3:16765–16774. https://doi.org/

10.1039/c3ra42308e

Khan M, Al-Hamoud K, Liaqat Z et al (2020) Synthesis of au, ag, and au–ag bimetallic nanoparticles

using pulicaria undulata extract and their catalytic activity for the reduction of 4-nitrophenol.

Nanomaterials 10:1–14. https://doi.org/10.3390/nano10091885

Khan SA, Shahid S, Ayaz A, Alkahtani J, Elshikh MS, Riaz T (2021) Phytomolecules-coated

NiO nanoparticles synthesis using abutilon indicum leaf extract: antioxidant, antibacterial, and

anticancer activities. Int J Nanomedicine 16:1757–1773. https://doi.org/10.2147/IJN.S294012

Khan ZUH, Sadiq HM, Shah NS, Khan AU, Muhammad N, Hassan SU, Tahir K, saﬁ SZ, Khan

FU, Imran M, Ahmad N, Ullah F, Ahmad A, Sayed M, Khalid MS, Qaisrani SA, Ali M, Zakir

M (2019) Greener synthesis of zinc oxide nanoparticles using Trianthema portulacastrum extract

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 333

and evaluation of its photocatalytic and biological applications. J Photochem Photobiol B Biol

192:147–157. https://doi.org/10.1016/j.jphotobiol.2019.01.013

Khunjan U, Kasikamphaiboon P (2021) Green synthesis of kaolin-supported nanoscale zero-valent

iron Using Ruellia tuberosa leaf extract for effective decolorization of azo dye reactive black 5.

Arab J Sci Eng 46:383–394. https://doi.org/10.1007/s13369-020-04831-w

Kolya H, Maiti P, Pandey A, Tripathy T (2015) Green synthesis of silver nanoparticles with antimi-

crobial and azo dye (Congo red) degradation properties using Amaranthus gangeticus Linn leaf

extract. J Anal Sci Technol 6:4–10. https://doi.org/10.1186/s40543-015-0074-1

Krishnaraj C, Jagan EG, Rajasekar S, Selvakumar P, Kalaichelvan PT, Mohan N (2010) Synthesis

of silver nanoparticles using Acalypha indica leaf extracts and its antibacterial activity against

water borne pathogens. Colloids Surfaces B Biointerfaces 76:50–56. https://doi.org/10.1016/j.

colsurfb.2009.10.008

Kuang Y, Wang Q, Chen Z, Megharaj M, Naidu R (2013) Heterogeneous Fenton-like oxidation of

monochlorobenzene using green synthesis of iron nanoparticles. J Colloid Interface Sci 410:67–

73. https://doi.org/10.1016/j.jcis.2013.08.020

Kushwaha AK, Gupta N, Chattopadhyaya MC (2014) Removal of cationic methylene blue and

malachite green dyes from aqueous solution by waste materials of Daucus carota. J Saudi Chem

Soc 18:200–207. https://doi.org/10.1016/j.jscs.2011.06.011

Lellis B, Fávaro-Polonio CZ, Pamphile JA, Polonio JC (2019) Effects of textile dyes on health

and the environment and bioremediation potential of living organisms. Biotechnol Res Innov

3:275–290. https://doi.org/10.1016/j.biori.2019.09.001

Lü F, Gao Y, Huang J, Sun D, Li Q (2014) Roles of biomolecules in the biosynthesis of silver

nanoparticles: case of gardenia jasminoides extract, Chinese. J Chem Eng 22:706–712. https://

doi.org/10.1016/S1004-9541(14)60086-0

Luo F, Yang D, Chen Z et al (2016a) Characterization of bimetallic Fe/Pd nanoparticles by grape

leaf aqueous extract and identiﬁcation of active biomolecules involved in the synthesis. Sci Total

Environ 562:526–532. https://doi.org/10.1016/j.scitotenv.2016.04.060

Luo F, Yang D, Chen Z, Megharaj M, Naidu R (2016b) One-step green synthesis of bimetallic Fe/Pd

nanoparticles used to degrade Orange II. J Hazard Mater 303:145–153. https://doi.org/10.1016/

j.jhazmat.2015.10.034

Madhavi V, Prasad TNVKV, Reddy AVB, Ravindra Reddy B, Madhavi G (2013a) Application of

phytogenic zerovalent iron nanoparticles in the adsorption of hexavalent chromium, Spectrochim.

Acta—Part A Mol. Biomol. Spectrosc 116:17–25. https://doi.org/10.1016/j.saa.2013a.06.045

Madhavi V, Prasad TNVKV, Reddy AVB et al (2013b) Application of phytogenic zerovalent iron

nanoparticles in the adsorption of hexavalent chromium. Spectrochim Acta—Part A Mol Biomol

Spectrosc 116:17–25. https://doi.org/10.1016/j.saa.2013b.06.045

Mali SC, Dhaka A, Githala CK, Trivedi R (2020) Green synthesis of copper nanoparticles using

Celastrus paniculatus Willd. leaf extract and their photocatalytic and antifungal properties.

Biotechnol Reports 27:e00518. https://doi.org/10.1016/j.btre.2020.e00518

Manjari G, Saran S, Radhakrishanan S et al (2020) Facile green synthesis of Ag–Cu decorated ZnO

nanocomposite for effective removal of toxic organic compounds and an efﬁcient detection of

nitrite ions. J Environ Manage 262:110282. https://doi.org/10.1016/j.jenvman.2020.110282

Marimuthu S, Antonisamy AJ, Malayandi S et al (2020) Silver nanoparticles in dye efﬂuent treat-

ment: a review on synthesis, treatment methods, mechanisms, photocatalytic degradation, toxic

effects and mitigation of toxicity. J Photochem Photobiol B Biol 205:111823. https://doi.org/10.

1016/j.jphotobiol.2020.111823

Mohamed EA (2020) Green synthesis of copper & copper oxide nanoparticles using the extract of

seedless dates. Heliyon 6:e03123. https://doi.org/10.1016/j.heliyon.2019.e03123

Nabi G, Raza W, Tahir MB (2020a) Green synthesis of TiO2 nanoparticle using cinnamon powder

extract and the study of optical properties. J Inorg Organomet Polym Mater 30:1425–1429. https://

doi.org/10.1007/s10904-019-01248-3

Nabi G, Ain QU, Tahir MB, Nadeem Riaz K, Iqbal T, Raﬁque M, Hussain S, Raza W, Aslam I,

Rizwan M (2020b) Green synthesis of TiO2 nanoparticles using lemon peel extract: their optical

334 N. Kumari et al.

and photocatalytic properties. Int J Environ Anal Chem, 1–9. https://doi.org/10.1080/03067319.

2020b.1722816

Nadeem M, Khan R, Afridi K et al (2020) Green synthesis of cerium oxide nanoparticles (Ceo2

nps) and their antimicrobial applications: a review. Int J Nanomedicine 15:5951–5961

Naik GK, Mishra PM, Parida K (2013) Green synthesis of Au/TiO2 for effective dye degradation

in aqueous system. Chem Eng J 229:492–497. https://doi.org/10.1016/j.cej.2013.06.053

Nakata K, Fujishima A (2012) TiO2 photocatalysis: design and applications. J Photochem Photobiol

C Photochem Rev 13:169–189. https://doi.org/10.1016/j.jphotochemrev.2012.06.001

Nanoparticles M (2020) Heterogeneous fenton-like catalytic degradation of

Nguyen NHA, Padil VVT, Slaveykova VI, ˇ

Cerník M, Ševc˚u A (2018) Green synthesis of metal and

metal oxide nanoparticles and their effect on the unicellular alga Chlamydomonas reinhardtii.

Nanoscale Res Lett 13. https://doi.org/10.1186/s11671-018-2575-5

Pan Z, Lin Y, Sarkar B et al (2019) Green synthesis of iron nanoparticles using red peanut skin

extract: synthesis mechanism, characterization and effect of conditions on chromium removal. J

Colloid Interface Sci 558:106–114. https://doi.org/10.1016/j.jcis.2019.09.106

Panchal P, Meena P, Nehra SP (2021) A rapid green synthesis of Ag/AgCl-NC photocatalyst for

environmental applications. Environ Sci Pollut Res 28:3972–3982. https://doi.org/10.1007/s11

356-020-11834-5

Parimala L, Santhanalakshmi J (2014) Studies on the iron nanoparticles catalyzed reduction of

substituted aromatic ketones to alcohols. J Nanoparticles 2014:1–10. https://doi.org/10.1155/

2014/156868

Pasinszki T, Krebsz M (2020) Synthesis and application of zero-valent iron nanoparticles in water

treatment, environmental remediation, catalysis, and their biological effects. Nanomaterials 10.

https://doi.org/10.3390/nano10050917

Poguberovi´c SS, Krˇcmar DM, Maleti´c SP, Kónya Z, Pilipovi´c DDT, Kerkez DV, Ronˇcevi´cSD

(2016) Removal of As(III) and Cr(VI) from aqueous solutions using “green” zero-valent iron

nanoparticles produced by oak, mulberry and cherry leaf extracts. Ecol Eng 90:42–49. https://

doi.org/10.1016/j.ecoleng.2016.01.083

Prashanth V, Priyanka K, Remya N (2021) Solar photocatalytic degradation of metformin by TiO2

synthesized using Calotropis gigantea leaf extract. Water Sci Technol 83:1072–1084. https://doi.

org/10.2166/wst.2021.040

Pushpamalini T, Keerthana M, Sangavi R, Nagaraj A, Kamaraj P (2020) Comparative analysis

of green synthesis of TiO2 nanoparticles using four different leaf extract. Mater Today Proc

40:S180–S184. https://doi.org/10.1016/j.matpr.2020.08.438

Raﬁque M, Sadaf I, Tahir MB et al (2019) Novel and facile synthesis of silver nanoparticles using

Albizia procera leaf extract for dye degradation and antibacterial applications. Mater Sci Eng C

99:1313–1324. https://doi.org/10.1016/j.msec.2019.02.059

Rai MS, Bhat PR, Prajna PS, Jayadev K, Rao PSV (2014) Original research article degradation of

malachite green and congo red using aloe barabadensis mill. Extract 3:330–340

Raman CD, Sellappa K, Mkandawire M (2021) Facile one step green synthesis of iron nanoparticles

using grape leaves extract: textile dye decolorization and wastewater treatment. Water Sci Technol

83. https://doi.org/10.2166/wst.2021.140

Ramzan M, Obodo RM, Mukhtar S, Ilyas SZ, Aziz F, Thovhogi N (2021) Green synthesis of copper

oxide nanoparticles using Cedrus deodara aqueous extract for antibacterial activity. Mater Today

Proc 36:576–581. https://doi.org/10.1016/j.matpr.2020.05.472

Ravikumar KVG, Sudakaran SV, Ravichandran K, Pulimi M, Natarajan C, Mukherjee A (2019)

Green synthesis of NiFe nano particles using Punica granatum peel extract for tetracycline

removal. J Clean Prod 210:767–776. https://doi.org/10.1016/j.jclepro.2018.11.108

Reddy NV, Li H, Hou T, Bethu MS, Ren Z, Zhang Z (2021) Phytosynthesis of silver nanoparticles

using perilla frutescens leaf extract: characterization and evaluation of antibacterial, antioxidant,

and anticancer activities. Int J Nanomedicine 16:15–29. https://doi.org/10.2147/IJN.S265003

Rosenberg E (2015) Heavy metals in water: presence, removal and safety. Johnson Matthey Technol

Rev 59:293–297. https://doi.org/10.1595/205651315x689009

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 335

Roy K, Sarkar CK, Ghosh CK (2015) Rapid colorimetric detection of Hg2+ ion by green silver

nanoparticles synthesized using Dahlia pinnata leaf extract. Green Process Synth 4:455–461.

https://doi.org/10.1515/gps-2015-0052

Sachan D, Ramesh A, Das G (2021) Green synthesis of silica nanoparticles from leaf biomass

and its application to remove heavy metals from synthetic wastewater: a comparative analysis.

Environ Nanotechnol Monit Manag 16:100467. https://doi.org/10.1016/j.enmm.2021.100467

Samrot AV, Angalene JLA, Roshini SM, Raji P, Steﬁ SM, Preethi R, Selvarani AJ, Madankumar A

(2019) Bioactivity and heavy metal removal using plant gum mediated green synthesized silver

nanoparticles. J Clust Sci 30:1599–1610. https://doi.org/10.1007/s10876-019-01602-y

Sankar R, Rizwana K, Shivashangari KS, Ravikumar V (2015) Ultra-rapid photocatalytic activity of

Azadirachta indica engineered colloidal titanium dioxide nanoparticles. Appl Nanosci 5:731–736.

https://doi.org/10.1007/s13204-014-0369-3

Sankar R, Manikandan P, Malarvizhi V, Fathima T, Shivashangari KS, Ravikumar V (2014) Green

synthesis of colloidal copper oxide nanoparticles using Carica papaya and its application in

photocatalytic dye degradation. Spectrochim. Acta—Part A Mol Biomol Spectrosc 121:746–750.

https://doi.org/10.1016/j.saa.2013.12.020

Sarma GK, Sen Gupta S, Bhattacharyya KG (2019) Nanomaterials as versatile adsorbents for heavy

metal ions in water: a review. Environ Sci Pollut Res 26:6245–6278

Sathishkumar M, Sneha K, Won SW, Cho CW, Kim S, Yun YS (2009) Cinnamon zeylanicum

bark extract and powder mediated green synthesis of nano-crystalline silver particles and its

bactericidal activity. Colloids Surfaces B Biointerfaces 73:332–338. https://doi.org/10.1016/j.col

surfb.2009.06.005

Sethy NK, Arif Z, Mishra PK, Kumar P (2020) Green synthesis of TiO2 nanoparticles from Syzy-

gium cumini extract for photo-catalytic removal of lead (Pb) in explosive industrial wastewater.

Green Process Synth 9:171–181. https://doi.org/10.1515/gps-2020-0018

Shad S, Belinga-Desaunay-Nault MFA, Sohail, Bashir N, Lynch I (2020) Removal of contaminants

from canal water using microwave synthesized zero valent iron nanoparticles. Environ Sci Water

Res Technol 6:3057–3065. https://doi.org/10.1039/d0ew00157k

Shaik AM, David Raju M, Rama Sekhara Reddy D (2020b) Green synthesis of zinc oxide nanoparti-

cles using aqueous root extract of Sphagneticola trilobata Lin and investigate its role in toxic metal

removal, sowing germination and fostering of plant growth. Inorg Nano-Metal Chem 50:569–579.

https://doi.org/10.1080/24701556.2020.1722694

Shaik AM, David Raju M, Rama Sekhara Reddy D (2020a) Green synthesis of zinc oxide nanopar-

ticles using aqueous root extract of Sphagneticola trilobata Lin and investigate its role in toxic

metal removal, sowing germination and fostering of plant growth. Inorg. Nano-Metal Chem

50:569–579. https://doi.org/10.1080/24701556.2020a.1722694

Sharma P, Pant S, Dave V, Tak K, Sadhu V, Reddy KR (2019) Green synthesis and characterization

of copper nanoparticles by Tinospora cardifolia to produce nature-friendly copper nano-coated

fabric and their antimicrobial evaluation. J Microbiol Methods 160:107–116. https://doi.org/10.

1016/j.mimet.2019.03.007

Siddiqui VU, Ansari A, Chauhan R, Siddiqi WA (2020) Green synthesis of copper oxide (CuO)

nanoparticles by Punica granatum peel extract. Mater Today Proc. https://doi.org/10.1016/j.matpr.

2020.05.504

Singh J, Kumar V, Kim KH, Rawat M (2019) Biogenic synthesis of copper oxide nanoparticles

using plant extract and its prodigious potential for photocatalytic degradation of dyes. Environ

Res 177:108569. https://doi.org/10.1016/j.envres.2019.108569

Sinha T, Ahmaruzzaman M (2015) Green synthesis of copper nanoparticles for the efﬁcient removal

(degradation) of dye from aqueous phase. Environ Sci Pollut Res 22:20092–20100. https://doi.

org/10.1007/s11356-015-5223-y

Srivastava V, Choubey AK (2021) Study of adsorption of anionic dyes over biofabricated crystalline

α-MnO2 nanoparticles. Environ Sci Pollut Res 28:15504–15518. https://doi.org/10.1007/s11356-

020-11622-1

336 N. Kumari et al.

Suresh D, Nethravathi PC, Udayabhanu, Rajanaika H, Nagabhushana H, Sharma SC (2015) Green

synthesis of multifunctional zinc oxide (ZnO) nanoparticles using Cassia ﬁstula plant extract

and their photodegradative, antioxidant and antibacterial activities. Mater Sci Semicond Process

31:446–454. https://doi.org/10.1016/j.mssp.2014.12.023

Tagad C, Seo HH, Tongaonkar R, Yu YW, Lee JH, Dingre M, Kulkarni A, Fouad H, Ansari SA,

Moh SH (2017) Green synthesis of silver nanoparticles using Panax ginseng root extract for the

detection of Hg2+. Sensors Mater 29:205–215. https://doi.org/10.18494/SAM.2017.1475

Taj MB, Alkahtani MDF, Raheel A, Shabbir S, Fatima R, Aroob S, Yahya R, Alelwani W, Alahmadi

N, Abualnaja M, Noor S, Ahmad RH, Alshater H (2021) Bioconjugate synthesis, phytochemical

analysis, and optical activity of NiFe2O4 nanoparticles for the removal of ciproﬂoxacin and

Congo red from water. Sci Rep 11. https://doi.org/10.1038/s41598-021-84983-3

Tan YN, Lee JY, Wang DIC (2010) Uncovering the design rules for peptide synthesis of metal

nanoparticles. J Am Chem Soc 132:5677–5686. https://doi.org/10.1021/ja907454f

T˘anase MA, Marinescu M, Oancea P, R˘aducan A, Mihaescu CI, Alexandrescu E, Nistor CL, Jinga

L-I, Di¸tu LM, Petcu C, Cinteza LO (2021) Antibacterial and photocatalytic properties of ZnO

nanoparticles obtained from chemical versus Saponaria ofﬁcinalis extract-mediated synthesis.

Molecules 26:2072. https://doi.org/10.3390/molecules26072072

Ta¸sar ¸S, Kaya F, Özer A (2014) Biosorption of lead(II) ions from aqueous solution by peanut shells:

equilibrium, thermodynamic and kinetic studies. J Environ Chem Eng 2:1018–1026. https://doi.

org/10.1016/j.jece.2014.03.015

Turakhia B, Turakhia P, Shah S (2018) Green synthesis of zero valent iron nanoparticles from

Spinacia oleracea (spinach ) and its application in waste water treatment, Iaetsd J Adv Res. Appl

Sci 5:46–51

Vanaja M, Paulkumar K, Baburaja M, Rajeshkumar S, Gnanajobitha G, Malarkodi C, Sivakavinesan

M, Annadurai G (2014) Degradation of methylene blue using biologically synthesized silver

nanoparticles. Bioinorg Chem Appl 2014. https://doi.org/10.1155/2014/742346

Veisi H, Karmakar B, Tamoradi T, Hemmati S, Hekmati M, Hamelian M (2021) Biosynthesis

of CuO nanoparticles using aqueous extract of herbal tea (Stachys Lavandulifolia) ﬂowers and

evaluation of its catalytic activity. Sci Rep 11. https://doi.org/10.1038/s41598-021-81320-6

Vinayagam R, Selvaraj R, Arivalagan P, Varadavenkatesan T (2020) Synthesis, characterization and

photocatalytic dye degradation capability of Calliandra haematocephala-mediated zinc oxide

nanoﬂowers. J Photochem Photobiol B Biol 203:111760. https://doi.org/10.1016/j.jphotobiol.

2019.111760

Wang T, Jin X, Chen Z, Megharaj M, Naidu R (2014) Green synthesis of Fe nanoparticles using

eucalyptus leaf extracts for treatment of eutrophic wastewater. Sci Total Environ 466–467:210–

213. https://doi.org/10.1016/j.scitotenv.2013.07.022

Weng X, Jin X, Lin J, Naidu R, Chen Z (2016a) Removal of mixed contaminants Cr(VI) and Cu(II)

by green synthesized iron based nanoparticles. Ecol Eng 97:32–39. https://doi.org/10.1016/j.eco

leng.2016.08.003

Weng X, Jin X, Lin J et al (2016b) Removal of mixed contaminants Cr(VI) and Cu(II) by green

synthesized iron based nanoparticles. Ecol Eng 97:32–39. https://doi.org/10.1016/j.ecoleng.2016.

08.003

Wu Z, Su X, Lin X, Khan NI, Owens G, Chen Z (2021) Removal of As(V) by iron-based nanopar-

ticles synthesized via the complexation of biomolecules in green tea extracts and an iron salt. Sci

Total Environ 764. https://doi.org/10.1016/j.scitotenv.2020.142883

Xin Lee K, Shameli K, Miyake M, Kuwano N, Bt Ahmad Khairudin NB, Bt Mohamad SE, Yew

YP (2016) Green synthesis of gold nanoparticles using aqueous extract of Garcinia mangostana

fruit peels. J Nanomater 2016. https://doi.org/10.1155/2016/8489094

Yadav VK, Fulekar MH (2018b) Biogenic synthesis of maghemite nanoparticles (γ-Fe2O3)using

Tridax leaf extract and its application for removal of ﬂy ash heavy metals (Pb, Cd). In: Materials

today: proceedings. Elsevier Ltd, pp 20704–20710

Plant Mediated Nanomaterials: An Overview on Preparation Strategies … 337

Yadav VK, Fulekar MH (2018a) Biogenic synthesis of maghemite nanoparticles (γ-Fe2O3) using

Tridax leaf extract and its application for removal of ﬂy ash heavy metals (Pb, Cd). Mater Today

Proc 5:20704–20710. https://doi.org/10.1016/j.matpr.2018.06.454

Yuseﬁ M, Shameli K, Yee OS, Teow SY, Hedayatnasab Z, Jahangirian H, Webster TJ, Kuˇca K

(2021) Green synthesis of fe3o4 nanoparticles stabilized by a garcinia mangostana fruit peel

extract for hyperthermia and anticancer activities. Int J Nanomedicine 16:2515–2532. https://doi.

org/10.2147/IJN.S284134

Zahra Z, Habib Z, Chung S, Badshah MA (2020) Exposure route of TiO2 nps from industrial

applications to wastewater treatment and their impacts on the agro-environment. Nanomaterials

10:1–22. https://doi.org/10.3390/nano10081469

Zhu F, He S, Liu T (2018) Effect of pH, temperature and co-existing anions on the Removal of

Cr(VI) in groundwater by green synthesized nZVI/Ni. Ecotoxicol Environ Saf 163:544–550.

https://doi.org/10.1016/j.ecoenv.2018.07.082

Bacteria and Pollutants

Sonia Kaura, Akansha Mathur, and Aakanksha Kalra

Abstract With the immense technological advances in the twenty-ﬁrst century,

regarding the twenty-ﬁrst century as the technological era would not be an exag-

geration. These advances have revolutionized almost all the arenas be it in terms

of automobiles, electronics, medical and healthcare, agriculture and food crops, etc.

However, recent decades have focussed on the sustainability of the environment to a

similar extent as to human advancements and beneﬁts. In this regard, waste manage-

ment in terms of both solid as well as liquid waste, is one of the most important sectors

which needs and has gained immense value. Compared to traditional physical and

chemical treatment methods, bioremediation technology utilising microorganisms

and their aggregates is acknowledged as an efﬁcient and economic green treatment

(biological origin) method. These microbes have been used in various ways some

of which include direct mixing, immobilisation or encapsulation. Analysis of these

microbes in different set ups including wastewater treatment plants or solid waste

removals have been studied extensively and plethora of knowledge has been gained in

this regard suggesting that bacterial populations differ greatly depending on the oper-

ating circumstances of the waste disposal and the properties of the waste. It has also

been observed that variations in the organic or nutritional composition of the waste

might potentially impact the bacterial community. Emerging contaminants (ECs),

which include endocrine-disrupting compounds, pharmaceuticals (lipid regulators,

antibiotics, diuretics, non-steroid anti-inﬂammatory drugs, stimulant drugs, antisep-

tics, analgesics, beta blockers), detergents, disinfectants, and personal care products,

have become a grave concern owing to their bioactive presence on environmental

matrices. It is therefore vital to undermine the potential of microbes in the proper

disposal of both solid and liquid waste. The current chapter, in this regard, is about

the microbes that have been identiﬁed as potential waste disposal mechanisms. In

addition, the chapter also focuses on the novel technologies that have been employed

using microbes for waste disposal.

Keywords Pollutants ·Bacteria ·Emerging contaminants ·Green treatment ·

Novel technologies ·Solid waste

S. Kaura · A. Mathur · A. Kalra (B

)

Dr. B. Lal Institute of Biotechnology, Jaipur, India

e-mail: aakankshakalra.07@gmail.com

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_16

339

340 S. Kaura et al.

1 Introduction

Since time immemorial we have observed that every discovery brings with itself a

new set of advantages and challenges opening even more scope for research and

development and breaking the boundaries that were once created. Needless to say,

the inventions and discoveries in the twenty-ﬁrst century have been immense and in

this regard the challenges too have been extensive. One of those challenges include

the generation of large amounts of waste products.

The acknowledgement of the excessive rise in the waste generated has been done

via almost all sections of the society and affected the economy of the world altogether.

Some of the instances from the same are shared here. “Poor waste management is

contaminating the world’s oceans, clogging drains and causing ﬂooding, transmit-

ting diseases, increasing respiratory problems from burning, harming animals who

unknowingly consume waste, and affecting economic development, such as through

tourism,” said Sameh Wahba, World Bank Director for Urban and Territorial Devel-

opment, Disaster Risk Management, and Resilience (Ayilara et al. 2020). According

to the World Bank’s What a Waste 2.0 report, the world produces 2.01 billion tonnes

of municipal solid waste per year, with at least 33% not being managed in an envi-

ronmentally sound manner. Take, for example, plastic garbage, which is choking our

oceans and accounts for 90% of all marine debris. In 2016, the globe produced 242

million tonnes of plastic waste, which is roughly equivalent to 24 trillion 500-mm,

10-g plastic bottles (Ayilara et al. 2020).

The recent years have shed light on the essentiality of environmental sustainability

for the survival of humans and other life forms on earth leading to the generation of

Sustainable Development Goals commonly called as SDGs in 2015 by the United

Nations of which SDG No. 6 majorly focuses on waste disposal and treatment. A

decade back these were considered as the sources of infection spread in addition to

creating sub-standard conditions for living. However, research in the last decade has

shed light on the role of microbes particularly bacteria in the removal or disposal of

these waste products referred to as bioremediation. This chapter is therefore focused

on the waste generated owing to the plethora of advancements and industrialization

in the twenty-ﬁrst century and the traditional strategies employed for the disposal of

this waste. Also, the chapter described the role of microbes employed in the disposal

of solid as well as liquid waste emphasizing on the novel strategies employed for the

same.

2 Novel Waste Generated in the Twenty-First Century

Waste is usually referred to as the materials or substances that are to be discarded

after primary use. However, there is no accurate deﬁnition of waste products since

waste for one can be the raw material for the other and thus a huge amount of focus

is on the reuse and recycling of the same. Similarly the classiﬁcation of the waste

Bacteria and Pollutants 341

can also be done on various parameters such as on the basis of physical state (solid

and liquid), the toxicity (hazardous and non-hazardous) and the source (municipal or

industrial waste). All these waste types have been described brieﬂy in the upcoming

section.

3 Solid and Liquid Waste

As per the physical state, waste is categorized as liquid or solid waste wherein

liquid waste include dirty water, organic liquids, wash water, waste detergents,

industry efﬂuents and even rainwater. Thus, this waste can be sourced via house-

holds or industries both and depending on the components present can be hazardous

or non-hazardous. However, it is quite clear that the household waste is usually

non-hazardous. On the other hand, solid waste includes the waste from household

such as municipal waste, plastic waste etc. or industrial waste such as chemicals,

construction or demolition waste. The solid waste is usually categorized as:

•Plastic Waste: This includes bags, containers, jars, bottles and a variety of other

items found in any household. Though plastic is non-biodegradable, it can be

recycled in various forms.

•Paper Waste: Packaging materials, newspapers, cardboards and other items are

included in this category which can be readily recycled and reused.

•Tins and Metals: This includes sheet metal, siding, rooﬁng, rebar, ﬂashings, pipes,

window frames, doors, furnaces, duct work, wire, cable, bathtubs, fencing, bicycle

frames, automotive body components, machines, garbage cans, metal furniture,

tyre rims, propane cylinders and other ferrous and nonferrous metallic items.

•Ceramics and Glass Waste: Ceramic waste powder (CWP) is produced during

the polishing of ceramic tiles and is thrown in landﬁlls, where it can pollute the

soil, air, and groundwater, causing major environmental issues. Another waste

product that is produced in great amounts and is difﬁcult to eliminate is waste

glass. It is well known that the majority of waste glass, particularly container

glass, is collected, remelted, and utilized to make new glass. However, not all

waste glass can be used in the manufacture of new glass and thus becomes an

accumulated waste.

4 Hazardous and Non-Hazardous Waste

As per toxicity of the generated waste, it can be segregated into hazardous and

non hazardous waste. As per conventional deﬁnition, hazardous wastes are the ones

which are potentially harmful to the humans and the environment and thus needs to

be controlled with utmost care. These are described as the ones which are ignitable,

corrosive or reactive and meet one of the following criteria: ignitability, corrosivity,

reactivity, or toxicity along with ecological, geological, and environmental calamities

342 S. Kaura et al.

that last a long time well. These are usually produced owing to manufacturing opera-

tions. These include radioactive materials, medical wastes, combustion waste, waste

from mining etc. These categories are described brieﬂy in the upcoming section.

•Medical Waste

According to US EPA 2022 (United States Environmental Protection Agency) this

includes any solid waste generated during diagnosis, treatment or biological research

and is produced by hospitals, private clinics, nursing homes for elderly people, blood

banks, autopsy or mortuary facilities, research institutes or laboratories and usually

contains blood, body ﬂuids or other infectious elements. As per WHO reports about

85% of the waste generated by health care activities are non-hazardous but the

remaining 15% is infectious, chemically reactive or radioactive (Pujara et al. 2019).

It is further segregated in the following categories:

•Infectious waste including waste contaminated with blood and other bodily ﬂuids

(e.g. discarded diagnostic samples), infectious agent cultures and stocks from

laboratory work (e.g. waste from autopsies and infected animals from labora-

tories), and waste from infected patients (e.g. swabs, bandages, and disposable

medical devices)

•Expired, unused and infected medications and vaccines are among the common

pharmaceutical waste.

•Waste containing genotoxic compounds (i.e. substances that are mutagenic, terato-

genic or carcinogenic), such as cytotoxic medications used in cancer treatment

and their metabolites

•Products polluted by radionuclides, such as radioactive diagnostic or radiothera-

peutic materials, are examples of radioactive waste.

•Human tissues, organs or ﬂuids, body parts, and contaminated animal carcasses

constitute pathological waste

•Syringes, needles, disposable scalpels and blades, and other sharp objects

•Solvents and reagents used in laboratory preparations, disinfectants, sterilants and

heavy metals found in medical devices (e.g. mercury in broken thermometers) and

batteries are examples of chemical waste.

•Radioactive Waste

Radioactive waste is the waste consisting of ionizing radiation-emitting chemicals.

As per the regulations of US EPA, these are categorized as high-level, low level

and transuranic wastes on the basis of their source and content of the radioactive

materials.

•High Level Radioactive Waste (HLRW): This includes the one obtained from

production of nuclear materials especially for defense purposes and is held as

sludge, liquid or pellets which needs to be solidiﬁed before being disposed of.

The other sources of this waste include the laboratories or diagnostic and therapy

labs which use the radioactive materials in surplus.

Bacteria and Pollutants 343

•Low Level Radioactive Waste (LLRW): The sources for these wastes include

nuclear power sector, medical and academic institutions where small amounts of

radionuclides are being used in the respective processes such as medical tracers.

•Transuranic Waste (TRU): These include the waste consisting of radioactive

elements with atomic numbers higher than uranium such as plutonium and any

items contaminated with such elements are included in the category (Gupta et al.

2021). These are further divided into remote handled (RH TRU) which emit pene-

trating radiation and must be shielded and contact handled transuranic waste (CH

TRU) in which the radiations are not penetrating and need not be shielded. 96%

of the TRU is composed of CH TRU (Lee et al. 2013). This type of waste is

particularly harmful over long periods of time.

Though the waste removal strategies are discussed in great detail in the upcom-

ings sections, radioactive waste disposal requires extra attention particularly the

HLRW. To dispose of HLRW, various methods have been proposed, including outer

space disposal, ocean disposal, and subsurface burial. For the prospective storage

and disposal of HLRW, deep geological disposal in subterranean repositories/tunnels

500–1000 m deep is usually considered. For deep geological HLRW disposal, multi-

barrier methods have been proposed, with three consecutive barriers: waste canisters

(made of carbon steel, copper, copper steel, and titanium), an engineered struc-

ture (made of cement or clay), and a third barrier (known as buffer and/or back-

ﬁll). Deep geological HLRW disposal’s ultimate purpose is to isolate radioactive

waste from humans and the environment until residual/leaked radioactive waste (also

known as radwaste) has no meaningful impact on humans or the environment. When

researchers are attempting to safely dispose of HLRW, they must consider a number

of factors.

•Combustion Waste

Coal is one the most commonly used fuel in the present scenario and is respon-

sible for innumerable facilities such as electricity and power production, heating,

transportation and thus is the major source of air pollution. It can be used alone or

in combination with other fuels such as diesel, gasoline oil, natural gas, etc. but is

particularly signiﬁcant in the cases where coal constitutes about 50% of the total

fuel (Gupta et al. 2021). This not only is a source of air pollution but also affects the

health of the individuals particularly children during and after birth.

•Extraction and Mining Waste

Intensive mining activities have resulted in a massive amount of hazardous wastes all

over the world which are commonly associated with high levels of acid-generating

sulﬁde minerals as well as potentially toxic metals and metalloids (PTMs) such as

As, Sb, Cu, Pb, Cd, Zn, Hg, Ag, Sn, Fe, Al, Mn, Tl, U, Th, and W (Anawar 2015).

The extraction and mining waste majorly includes the water used for inﬁltration of

the mine during the extraction process and also the soil and rock created during

the process. The entire mining process is a combination of multiple steps such as

crushing, grinding, dissolution, calcining etc. that may either lead to an intermediate

344 S. Kaura et al.

or a ﬁnal product (Gupta et al. 2021). The toxic components produced during the

process are distributed in the aquatic and terrestrial systems via means of erosion,

dispersal, leaching or air. In spite of multiple strategies such as burial, pedogenesis,

chemical weathering etc., the potentially toxic metals and metalloids (PTMs) and

acid mine drainage (AMD) fractions cause signiﬁcant damage to the aquatic and

terrestrial systems ultimately posing a serious health concern (Anawar 2015).

•E Waste

E waste also known as Waste Electrical and Electronic Equipment (WEEE) includes

any electrical or electronic device that has been thrown, surplused, obsoleted or

broken. Needless to say, with the advancements in technology and industrializa-

tion, this is the fastest growing waste throughout the world and thus has become a

major public health concern. Besides, the inadequate information regarding its correct

disposal has led to its accumulation in households, especially in developing coun-

tries like India. This ever-increasing trash is extremely complicated in nature, and it’s

also a rich source of metals like gold, silver, and copper which may be recovered and

reintroduced into the manufacturing process. As a result, e waste trade and recycling

coalitions employ a diverse range of people (Mishra et al. 2019). The composition

analysis of e waste suggests that metals account for about 60% of the total waste

while plastics account for about 30% with about 3% hazardous pollutants (Kishore

2010). Many toxic metallic pollutants, such as lead, cadmium, and beryllium, as well

as brominated ﬂame-retardants, can also be found in electronic equipment. Lead is

the most extensively utilized dangerous heavy metal in electronic devices for a range

of reasons, resulting in a variety of health risks due to environmental contamination.

•Industrial Waste

The expansion of industrial sectors such as agro-, food-, paper-, and pulp industries

is the need of the hour to meet the demands of the growing population. These indus-

tries produce hazardous waste, the majority of which is organic in nature and so is

deposited or processed in the environment. These wastes cause increased contamina-

tion resulting in high mortality in addition to the physical and morphological changes

in the organisms/animals that come in contact. Although the waste generated is toxic,

it is primarily composed of macromolecules and bioactive chemicals, making it suit-

able for extraction and manufacturing of value-added goods. The creation of value-

added products such as bioplastics, biofuels, and biosurfactants at the same time

improves the process’ economics and contributes to environmental sustainability

(Gaur et al. 2020).

5 Non-Hazardous Waste

After a detailed description of the hazardous waste, in the upcoming section, the

chapter focuses on the non-hazardous waste generated in the twenty-ﬁrst century.

Though it does not appear to be a concern by deﬁnition, this form of garbage can

Bacteria and Pollutants 345

cause considerable environmental damage. Non-hazardous waste can be generated

during the manufacturing of goods and products, especially in the industrial sector.

Electric power generation and the production of materials such as pulp and paper, iron

and steel, glass and concrete are other examples. Ash, sludges, antifreeze, grinding

dusts, and liquids polluted with non-hazardous chemicals are examples of common

industrial pollutants that are deemed non-hazardous in most jurisdictions. This can

further be categorized broadly in three types including municipal waste, agricultural

waste and construction and demolition waste which are brieﬂy described below:

•Municipal waste: It is the most common and socially signiﬁcant source of the

waste and an enormous source of waste particularly in urban areas owing to high

population density. Depending on the physical state of the waste, it can be both

solid or liquid in nature.

– Solid waste: A recent study has suggested East Asia and Paciﬁc region to be

the source of major amounts of municipal solid waste with China and India

accounting for 15% and 11.94% of the global waste respectively (Kishore

2010). Besides, owing to expanding populations and ongoing migration from

rural to urban regions, and other factors, many cities in lower-income nations

in Africa and Asia are expected to double their MSW generation within 15–

20 years (Vikaspedia). According to a study, only 6–7% of MSW gets converted

to compost in India, with the rest disposed of through landﬁlling with almost

50% content as the organic matter thereby further complicating the disposal

process (Sharma and Jain 2019).

– Liquid waste: This includes human waste, kitchen and food processing trash,

pathogens, parasite eggs, inorganic materials, sand and grit, and other organic

waste components. All hazardous components must be removed from waste,

leaving an inert residue that may be properly disposed of. Sewage and sludge

are the most common types of liquid waste. Sewage is wastewater that contains

human waste, whereas sludge is wastewater that is generated by everyday activ-

ities, such as washing and cooking residue, but does not contain human waste.

Though it is considered to be non-hazardous, it may contain toxic elements

which in the case of developing countries are frequently dumped in natural

ecosystems while puriﬁed by certain processes in developed countries before

disposal.

•Agricultural waste: Waste generated during the production and harvesting of

crops or trees, as well as animal raising belongs to this category. Animal waste

is a subcategory of agricultural waste that includes waste f rom cattle, dairy and

other animal-related agricultural and farming practices (e.g., feed waste, bedding

and litter, and feedlot and paddock runoff) (Gupta et al. 2021). This waste is

speciﬁcally harmful owing to the accumulation of N2O, SO2, CH4, and smoke

owing to improper disposal of agricultural waste. Every year, India produces over

350 million tonnes of agro-industrial waste (Katare et al. 2020) which is tradi-

tionally burned/incinerated or left to rot in the ﬁelds, resulting in the production

346 S. Kaura et al.

of smoke, toxic gasses, carcinogens (for example, polycyclic aromatic hydrocar-

bons, furans, and dioxins), and greenhouse gasses, all of which have negative

health and environmental consequences (Sharma et al. 2020). This traditional

disposal particularly of pesticide waste results in degradation of natural resources

in addition to posing a threat on human health (Ghosh et al. 2018). To combat

this waste disposal, research in 4 major categories have been focused including

low-carbon and energy utilization of agricultural waste explored by conversion

of straw, wood, and other waste into bioethanol to develop the biomass industry,

reducing the discharge of agricultural waste contributing to the mitigation of

greenhouse gas emissions, the material and energy ﬂow of agricultural waste and

the prevention and control of agricultural waste pollution (Ghosh et al. 2018).

•Construction and demolition waste: Commonly called the “C&D waste”, it is

referred to as the waste generated during construction, renovation and demolition

activities including land excavation or formation, civil and building construction,

site clearance, demolition activities, roadwork and building renovation. Multiple

adverse impacts of C&D waste generation are observed including usage of a

large amount of land resources for waste landﬁlling, harming the surroundings

by hazardous pollution, and wasting natural resources (Kornberg et al. 2019). It

is worrisome to note that India alone produces about 530 million tonnes of this

waste being the second largest in the world (According to a report by Earth5R

Organization in 2020) and thus must build a comprehensive system to monitor

and utilize the same including government-led popular awareness campaigns.

6 Traditional Strategies of Waste Disposal (Solid

andLiquidWaste)

The previous sections have described the waste generated in the twenty-ﬁrst century

in great detail therefore this section focuses on the disposal strategies of the same.

Multiple strategies have been employed for the same since long ago and these have

been varied in terms of the country’s economic and technical developments as well

as the kind of waste generated. Besides, over the course of time, innovations have

been made in the same strategies. This section describes the traditional strategies

of waste disposal and treatment focusing separately on the methods for solid and

liquid waste disposal. Certain major strategies for the disposal of waste have been

described below:

•Disposal of solid waste:

Although a majority of the solid waste produced in India is dumped in landﬁlls

without appropriate sorting or pretreatment ultimately leading to greenhouse gas

emissions endangering both human health and environment (Mohanty et al. 2020),

several other strategies have been developed. The major factors to be kept in mind

for developing integrated solid waste management include environmental friendli-

ness, cost-effectiveness and social acceptability. Besides, “zero-waste production”

Bacteria and Pollutants 347

and “waste prevention” attempts are the highlights for the same in order to reduce

gaseous emissions, solid leftovers and pollution, also contributing to climate and envi-

ronmental protection (Mohanty et al. 2020). The common methods included in this

section include composting, incineration, landﬁlls, mechanical biological treatment

and pyrolysis and gasiﬁcation which are brieﬂy described below:

•Composting: It is a way of transforming garbage into a useful product rather

than a ﬁnal disposal method thus ultimately reducing waste. Compost has been

used as a soil conditioner in both rural and semi-urban regions for a long time

for growth of vegetables and crops. The technologies involve simple processes

such as windrow composting involving shredded plant material to automated

enclosed vessel digestion of mixed home trash. The aerobic process is termed

as composting while the anaerobic is termed as digestion and their hybrids have

also been attempted. It is one of the safest ways to dispose off the trash leading

to the conversion of organic waste under aerobic conditions and production of

useful products like biogas, biofertilizers, etc. The prepared compost is useful

for a variety of purposes including boosting agricultural productivity, bioreme-

diation, plant disease control, weed control etc. It also boosts soil biodiversity

while lowering the environmental dangers of synthetic fertilizers. Rather than

being a natural and uncontrollable process, composting is started and managed

in a controlled atmosphere and this is how it is different from decomposition.

Though the process is coupled with all these advantages, it also possesses certain

disadvantages such as long time duration, disagreeable odor and may contain

infections that can tolerate high temperatures to some extent, i.e. thermotolerant

pathogens, as well as having insufﬁcient nutrient content (Ayilara et al. 2020). The

detailed description and the role of microorganisms in composting is discussed

in upcoming sections.

•Incineration: Waste destruction via burning and energy generation is referred to

as incineration which is also known as “energy-from-waste” (EfW) or “waste-to-

energy” though the terms are misleading. This process has been used many times

for getting rid of medical waste and recovery of metals and energy in addition

to reduction in the volume of waste. Though it is still one of the most widely

used methods, release of toxic elements such as dioxins and furans limit its use.

Besides, it has also been a technical challenge in developed countries owing to

the high amount of organic matter and construction wastes (Kornberg 2019).

•Landﬁll: It is one of the most traditional ways of trash disposal which began with

building landﬁlls in abandoned quarries, mining voids and borrow pits. Though

the method minimizes environmental problems and was a relatively economical

strategy of waste disposal, the increase in generated waste and reduction in avail-

able land over the time has forced it to shift to other strategies. Additionally,

wind-blown litter, vermin attraction and pollutants like leachate that drain into

ground water and the landﬁll gases, including methane and carbon dioxide are

negative impacts of the process. The major anions observed in the leachate include

chlorides, nitrates and sulphates owing to sewage, agricultural and animal waste

deposits which ultimately contaminates nearby water bodies, underground and

surface waters.

348 S. Kaura et al.

•Mechanical Biological Treatment: It is a combinatorial method particularly used

for organic waste via both mechanical sorting and biological treatment. Depending

on the processing order, it can be referred t o as MBT or BMT. This is particularly

helpful in developing countries where the source-point waste classiﬁcation cannot

always be guaranteed (Fei et al. 2018). The mechanical stage involves the separa-

tion of the recyclable materials such as metals, plastic or glass for fuel generation

referred to as refuse derived fuels (RDF). The biological method includes diges-

tion or composting depending on whether the method is anaerobic or aerobic,

leading to the production of biogas or green energy (optional, only with diges-

tion). Studies have also shown that coupling MBT to a biogas puriﬁcation system

gives about 40% energy efﬁciency with the lowest amount of pollutants (Fei et al.

2018). Many metro and urban cities in India including Delhi, Mumbai, Ahmed-

abad, Hyderabad, Indore, and Rajkot, have RDF plants that create ﬂuffy solid fuels

and pallets that can be used as an alternative fuel in the industry. This approach is

a technically validated MSW disposal technology that minimizes environmental

and land consequences. RDF has a caloriﬁc value of 8 to 14 MJ/kg, and the gener-

ated energy/electricity can be used for district heating and industrial purposes. As

a result, RDF is a viable alternative to fossil fuels in the manufacturing of steel

and cement kilns. In India, 36 RDF plants are operational, with many more in

the development stages to generate segregated combustible fraction (SCF), which

accounts for around 10% of total waste generation. The Indian government has

mandated that industries use at least 5%–15% RDF replacement fuel. SCF/RDF

applications are environmentally friendly since they divert trash from landﬁlls

and reduce greenhouse gas emissions (Pujara et al. 2019). High economic costs

is one of the limitations of the method.

•Pyrolysis and gasiﬁcation: These are two types of thermal treatment involving

heating materials at high temperatures with little oxygen in a sealed vessel. This

method has a higher efﬁciency than direct incineration. Pyrolysis, carried out

between 673 to 973 K, is the conversion of solid waste to solid, liquid and gaseous

products of which liquid and gaseous counterparts are burned to generate energy

while solid residue is processed to form activated carbon and other goods. An

example of the same is a model unit created by TERI and located in Gwal Pahari,

Gurugram, Haryana. However, owing to being an energy-intensive and a non-self-

sustaining process, little investments have been made in this technology. Another

reason for its failure to conquer the market is the unpleasant odor evolution that

occurs during the procedure. Gasiﬁcation, on the other hand, converts organic

molecules into synthetic gas (syngas) containing carbon monoxide and hydrogen

which is further burned to generate power and steam as a renewable source of

energy. The aforementioned plants primarily used biomass as a feedstock. There

are two types of gasiﬁer designs available in India: the NERIFIER model and

the TERI model. Navreet Energy Research and Information installed the NERI-

FIER model in Nohar, Rajasthan (NERI) and Sawdust, agricultural debris, and

forest trash are used in the NERI model. TERI has deployed over 650 thermal

biomass gasiﬁer units in various micro, small, and medium businesses (MSME)

and rural electricity access across the country. The TERI unit, on the other hand,

Bacteria and Pollutants 349

is a two-stage unit that also seeds on biomass. So far, no successful MSW gasi-

ﬁcation projects have been reported, owing to the greater moisture content of

Indian OFMSW. The OFMSW gasiﬁcation technique, when used as RDF pellets,

is predicted to be successful in the near future. The good news is that, following

a successful experiment in Dubai, an Indian business is attempting to develop an

MSW gasiﬁcation plant (Ghosh et al. 2018).

•Disposal of liquid waste:

Speciﬁc care for liquid waste disposal has to be taken focusing majorly on envi-

ronmental protection, human health protection and aesthetic concerns. The envi-

ronmental concerns include destabilization of aquatic ecosystems and destruction

of marine animals. Improper liquid waste disposal leads to contamination of both

ground and surface water ultimately affecting human health via numerous diseases.

Similarly, aesthetic concerns include unpleasant odor making life uncomfortable. As

already mentioned in the previous section multiple types of liquid wastes are obtained

such as municipal sewage, industry efﬂuents, storm sewage, etc. thereby requiring

different strategies for the same. Some of the most commonly used strategies are

described in the upcoming section.

•Dewatering: Elimination of water from the waste leading to its condensation and

further landﬁlling is one of the attractive approaches for non-hazardous waste. The

eliminated water can then be ﬁltered and treated for secondary uses. The process

initiated with transfer of waste in strong plastic bags for water elimination and

has moved up to centrifugation in a cylindrical tank for the same.

•Sedimentation: Similar to dewatering the purpose of this method is to eliminate

water from the waste via gravity instead of centrifugation in dewatering. The

liquid waste is deposited in a sedimentation basin designed in such a way to reduce

the velocity of water thereby helping sedimentation of the suspended particles.

Eventually, the liquid and the solid components can then be separately treated via

appropriate strategies.

•Composting: This is another strategy that can be employed to get rid of non-

hazardous waste wherein organic materials with nutrients such as nitrate, potas-

sium etc. can be separated and used for agricultural purposes. The strategy is

eco-friendly and relatively economical as compared to other strategies of liquid

waste disposal.

•Incineration: A strategy employed for the disposal of hazardous waste wherein the

liquids consisting of acids, chemicals, oils, etc. are removed via heat in specialized

furnaces leaving just the water behind. For this process, two types of specialized

furnaces are used including ﬂuidised bed furnace and multiple hearth furnaces.

In a ﬂuidised bed furnace, waste coupled with a bed of solid particle matter or

solid–ﬂuid mixes made up of sand, ash or limestone under pressure are heated in

the presence of oxygen for complete and efﬁcient burning of the waste. On the

other hand, a multiple-hearth furnace incinerates massive volumes of garbage at

350 S. Kaura et al.

different stages at a uniform rate using multiple stacked chambers. The chambers

are layered and thus compact and easy to ﬁt into tight spaces and are reasonably

inexpensive to produce and install. However, this method also has a negative

impact on the environment via release of hazardous pollutants and greenhouse

gases affecting both human health and climate change.

•Root Zone Treatment: This strategy is particularly useful for domestic wastewa-

ters which are relatively clean and involves a multistep procedure consisting of

sedimentation followed by ﬁltration process ultimately leading the water to the

roots of growing plants. The series of steps employed in this process include

pretreatment sedimentation, anaerobic reactor, anaerobic ﬁlter and plant ﬁlled

gravel ﬁlter. Pretreatment sedimentation involves removal of solid particles in

the sedimentation basis which is followed by pumping the water through bafﬂed

design in an anaerobic reactor. The numerous internal compartments of the reactor

possess accumulated microorganisms on the surface which decompose most of

the suspended particles in water. This is followed by further treatment of the

wastewater by microorganism colonies growing in the media present in the anaer-

obic ﬁlter. It is at this stage that the majority of the suspended solids are being

consumed by the bacteria and the water is ready to be transported to the roots

of the plants present in the gravel in a plant ﬁlled gravel ﬁlter. The plants are

usually tough reeds that provide resistance to the passage of the water. Plants

respire, supplying oxygen to the efﬂuent and assisting in the removal of any left-

over pollutants. The method has multiple advantages. It usually relies on gravity

because the water ﬂows downhill from stage to stage, reducing the need for pumps

and valves. It’s also extremely eco-friendly, as root-zone technology requires only

20% of the energy that a standard sewage treatment facility does. In addition, a

well-established plant bed usually requires relatively little maintenance. Root-

zone therapy, on the other hand, can be costly to conduct since it comprises

multiple parts, and its complicated installation means may not be possible in

some places.

•Solidiﬁcation: Adding binding chemicals to wastewater in order to solidify the

waste to a compact, stiff, and easily disposable solid is the process of liquid waste

solidiﬁcation. Solidiﬁcation changes the physical qualities of garbage, making it

harder, stronger, or less permeable, as well as enclosing any dangerous materials.

The most common solidifying agents used in the process are lime ash, sawdust,

cement kiln dust, lime kiln dust, gypsum, ﬂy dust, asphalt or cement. The follow

up strategies after this conversion include landﬁlls or incineration. Solidiﬁcation

is a reasonably inexpensive and simple process, however the extra solid material

produces a lot of waste. The extra weight and mass can result in increased shipping

and disposal expenses, as well as a disproportionate amount of landﬁll space.

Bacteria and Pollutants 351

7 Soil Microbes Associated with Municipal Solid Waste

The soil is home to a sizable colony of bacteria involved in the bioconversion of

waste. In the form of single cell proteins, enzymes, antibiotics, and other precious

molecules, microorganisms provide a wide range of beneﬁts. Municipal trash is made

up of a variety of components that serve as a substrate for a wide range of microorgan-

isms to ﬂourish. A variety of microorganism species, including cellulolytic fungal

strains, which have been employed to transform cellulosic materials into signiﬁcant

components like alcohol and organic acid, thrive in the biologically rich environment

provided by municipal solid trash. These fungi come in both mesophilic and ther-

mophilic types. Typically, compared to their mesophile counterparts, the enzymes

produced by thermophiles are more dynamic at high temperatures and more thermally

stable (Lo et al. 2009; Gautam 2011).

By using waste materials as a carbon source for themselves, bacteria are able

to make a number of straightforward and useful compounds that are essential for

the health of the soil, plant growth, and the maintenance of the natural ecosystem

(Barman et al. 2011) An essential biological component of excellent farming practises

and the bioconversion of wastes from the kitchen includes bacteria and some fungus.

According to the ﬁndings of their study, W.M.F. WanIshak et al. (2011) concluded that

municipal solid waste should be sanitised before being released into the environment

in order to decrease microbe activity and thereby prevent or delay the release of

hazardous chemicals into the environment as well as reduce odour production.

8 Role of Bacteria in Treatment Systems

The fundamental biological components of aerobic waste treatment systems are

bacteria. The majority, if not all, of the organic chemicals included in industrial wastes

can be metabolised by bacteria thanks to their complex biochemical makeup. In all

aerobic waste treatment systems, obligate aerobes and facultative bacteria are present.

Any species’ capacity to compete for a portion of the system’s available organic

material determines how quickly it can reproduce. Typically, bacterial predominance

will separate into two main groups: those that use the organic waste compounds and

those that use the lysed byproducts of the ﬁrst group of bacteria. The most signiﬁcant

group of bacteria will inﬂuence the characteristics of the treatment by utilising the

organic components in the waste.

The majority of the bacteria eventually die and lyse after the organic substrate is

depleted. Other bacteria can thrive because the bacteria’s biological components are

released. Secondary predomination will happen because biological treatment systems

are typically over designed for safety. The ability of the bacteria to ﬂocculate is by far

the most signiﬁcant trait, after their metabolic traits. For full stabilisation, all aerobic

biological waste treatment systems rely on the separation of the microorganisms

from the liquid phase through ﬂocculation.

352 S. Kaura et al.

Initially, it was believed that Zoogloea ramigeria, a single bacterial species, was

responsible for ﬂocculation, but more recent research has revealed that numerous

other bacterial species are also capable of ﬂocculation. All bacteria may be able to

ﬂocculate in speciﬁc environmental conditions, according to a theory. The primary

inﬂuences on ﬂocculation are the energy level and surface charges of the bacterium.

It has been demonstrated that the electrical surface charge on bacteria cultured in

diluted organic waste systems is less than the 0.020 V threshold charge for auto-

agglutination. This means that when two bacteria approach one another, Brownian

movement generates enough energy to overcome the electrical forces that repel them,

allowing the Van der Waal forces of attraction t o prevail and hold the two together.

9 Novel Strategies of Waste Treatment by Using Bacteria

1. Aerobic Composting

The process of composting is a natural decomposition of solid organic waste by the

local microbial community under the ideal environmental conditions of hot, humid

air. It is a pathogen-free procedure that signiﬁcantly reduces waste by up to 85%. The

process produces CO2, water, mineral ions, and stabilised organic matter, commonly

known as compost or “humus.“ Compost is a stable nutrient-enrichment material

that is helpful to plant growth (Riddech et al. 2002).

Compost undergoes in the following stages:

(i) An initial rapid stage of decomposition

(ii) Stabilization stage

(iii) Incomplete humiﬁcation stage

The bacterium acts as an energy transducer in this process, which is entirely microbe-

driven. A signiﬁcant quantity of energy is produced during this process, some of

which is utilised by bacteria. The leftover energy is released as heat, which raises

the temperature of the pile and speeds up the regular composting process. Here, a

rise in temperature acts as a sanitizer, while microbial activity promotes the miner-

alization of organic materials and lowers the C: N ratio. The ﬁnished item was reli-

able and secure for gardening and farming. It boosts plant development, conserves

water, minimises soil erosion, soil acidity, pathogen assault, and dependence on

agrochemicals, to name just a few of its numerous advantages (Rastogi et al. 2020).

Commonly occurring bacteria in composting mixture are Alcaligenes faecalis,

Arthrobacter sp., Brevibacillus brevis, Bacillus circulans, Bacillus licheniformis,

Bacillus megaterium, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis,

Clostridium thermocellum, Flavobacterium sp., Pseudomonas sp., Thermus sp., and

Vib rio sp. whereas, fungi involved in composting are Aspergillus fumigatus, Basid-

iomycetes sp., Humicola grisea, H. insolens, H. lanuginosa, Malbranchea pulchella,

Myriococcum thermophilum, Paecilomyces variotii, Papulaspora thermophilia,

Bacteria and Pollutants 353

Penicillium sp., Scytalidium thermophilum, Termitomyces sp., and Trichoderma sp.,

and actinobacteria are Streptomyces sp., Frankia sp., and Micromonospora sp.

Multiple variants of composting that are used in the current scenario are

described as:

•Static Pile Composting

The aerated static pile composting method uses either positive or negative ambient

air. Along with organic wastes and bulking agents, air is circulated through the

compost pile. Layers of bulking agent are layered into the pile to improve air ﬂow

and add porosity. The aerated static pile method is nearly identical to the windrow

composting method in terms of structure, with the exception that the aerated static pile

method does not require rotating to produce aeration. Aerated static pile composting

can produce compost in as little as three to six months and is suited for a wide

range of organic wastes, including yard trimmings, papers, and food scraps. Further-

more, when compared to the windrow composting method, it requires less land. This

composting method is expensive and ineffective at decomposing animal manures

and fats (Palaniveloo et al. 2020).

•Windrow Composting

Windrow composting is a popular approach because it can handle a signiﬁcant

amount of organic waste. This form of composting entails piling organic waste

into long, narrow heaps known as “windrows,” which have a triangular or circular

cross-sectional area. The piles are then either manually or mechanically turned. The

temperature of raw materials is used in the turning process, as a turning signal, the

windrows will be rotated if the temperature reaches a certain level. As a result, the pile

is aerated. This method can break down large amounts of organic wastes like grease,

liquids, and animal manures. Because the pile is large enough to generate adequate

heat and maintain the temperature, windrow composting is ideal for restaurants,

cafeterias, and markets that produce enormous amounts of food waste. However, to

accommodate the enormous equipment, this composting method necessitates a large

amount of land and is time-consuming ( Palaniveloo et al. 2020).

•In-vessel Composting

In-vessel composting is often utilized in the industry as well since it can also treat a

large amount of organic wastes. This approach encloses all organic wastes in various

containers or vessels, which are then manually or automatically spun to ensure that the

wastes are aerated. When compared to windrow composting, in-vessel composting

requires a smaller amount of land and manual labor. This procedure has disadvantages

in that it is pricey and may necessitate equipment knowledge (Palaniveloo et al. 2020).

•Vermiculture Technology

Vermiculture technology is a system that uses earthworms to convert organic waste

into vermicompost. It has a wide range of applications in waste management and

sustainable organic farming, and has proven to be one of the most efﬁcient and

cost-effective ways to handle organic waste. For the biodung and vermicomposting

354 S. Kaura et al.

processes, a mixture of grass clippings, water hyacinth, and cattle dung was used

as organic waste. The results shows the organic wastes were successfully treated

over a 60-day period using partial bio-dung composting and vermicomposting. It

contains growth-promoting chemicals including auxins and cytokinins and adds to

the delivery of important micronutrients.

2. Anaerobic Digestion

By simultaneously creating biogas as a source of energy, Anaerobic digestion (AD)

demonstrates a promising sustainable method for treating MSW (McCarty 2001). Its

potential for expansion is constrained by its sluggish digestion rate and the production

of chemicals that hinder methanogenesis (Stams and Plugge 2009; Shah 2021a, b).

However, due to its capacity to effectively reduce the chemical oxygen demand

(COD) and biological oxygen demand (BOD) from the waste streams and convert

them into biogas/methane, the AD process has recently been used to treat diverse

agricultural wastes, food residues, and wastewater (Kwietniewska and Tys 2014).

AD is a promising method for treating MSW and generating CO2 and CH4, which,

coupled with the manure produced after decomposition and the ability to be utilised

as a biofertilizer, can satisfy the energy demand.

AD process can be further subdivided into four distinct steps:

1. Enzymatic hydrolysis: Extracellular enzymes convert complicated organic stuff

(protein, lipid, and carbohydrates) into more easily soluble molecules (amino

acids, fatty acids, and sugars).

2. Fermentation: By the presence of fermentative bacteria, reduced end products

from hydrolysis are transformed into a combination of short-chain volatile fatty

acids (VFAs) and other products such CO2, hydrogen, and acetic acid during

fermentation.

3. Acetogenesis and Methanogenesis: Using acetogenic bacteria, organic acids

are changed in this stage into acetate, CO2, and hydrogen. Clostridium,

Ruminococcus, and Eubacterium genera contain both acetogenic and non-

acetogenic bacteria that are crucial for acetogenesis. Acetobacterium and

Sporomusa are two exclusively acetogenic bacteria.

3. Nitrogen Fixation

A vital natural supply of reactive nitrogen for the wetland environment is the nitrogen

ﬁxation by bacteria. The rhizosphere’s enrichment of nitrogen-metabolizing bacteria

is facilitated by the roots’ supply of oxygen and organic matter (Lamers et al. 2012).

Bacteria change nitrogen in the rhizosphere of wetland plants through nitriﬁca-

tion, denitriﬁcation, absorption, and anaerobic oxidation of ammonia by nitrate and

nitrogen ﬁxation (Bañeras et al. 2012). This process’ metabolic energy comes from

the oxidation of organic materials and lithotrophy. Microbes perform an N-ﬁxation of

non-reactive N2, and nitrogen is produced. The heterotroph and autotroph prokary-

otes contribute toward the production of a large amount of reactive nitrogen by

nitrogen ﬁxation.

Bacteria and Pollutants 355

Cyanobacteria in wetlands must have access to light in order to ﬁx nitrogen. In

wetlands, the signiﬁcant genera of N-ﬁxing bacteria include Enterobacter, Azospir-

illum, Pseudomonas, Klebsiella, and Vib ri o. Typically, the roots and the heterotrophic

nitrogen ﬁxer form a mutually beneﬁcial symbiosis, and the roots’ carbohydrates are

traded for the ammonia the bacteria create.

4. Degradation of Organic Pollutants

Because they can degrade almost all kinds of organic contaminants, microbes are

referred to as bioremediators (Fenchel et al. 2012; Shah 2020). The organic contami-

nants are broken down by microbes through a co-metabolism process. In this process,

the complex carbon-based compounds are broken down by microorganisms in the

rhizosphere of aquatic and terrestrial plants to produce organic carbon and electron

acceptors. The microbial population and amount of xenobiotics in natural water deter-

mine the biodegradation rate, and the macrophyte species has a signiﬁcant impact

on the microbe population. Microbes in the rhizosphere receive organic carbon from

plants, which helps them break down complex organic molecules like hydrocarbons

and aromatic hydrocarbons. In order to promote plant growth, bacteria also emit

indole acetic acid (IAA).

Many bacteria that were isolated from aquatic plants also exhibited activities

that promote plant growth and pollution breakdown. Organic substances including

phenolics, amines, and aliphatic aldehydes can be broken down by the bioﬁlms that

are connected to aquatic plants. These bioﬁlms can also break down dissolved organic

materials, including polychlorinated biphenyls (PCBs) and atrazine.

5. Metal Biosorption and Bioaccumulation

In general, bacteria execute passive and active processes for metal ion biosorption

within their cell walls. The cell walls of both active and dormant bacteria facilitate

passive biosorption through a variety of metabolic activities. Metal ions adhere to

the cell surface as a result of their reactivity with functional groups (such as amine,

amide, carbonyl, hydroxyl, and sulfonate) in the cell wall. Different mechanisms,

such as ion exchange, sorption, complexation, chelation, and micro-precipitation,

may each play a separate or complementary role in the metal ion binding process.

On the other hand, live cells take up metal ions during the active biosorption

process. Metals that enter live cells have different outcomes depending on the species

and particular elements. The elements may be delivered to a particular structure and

may be bound, stored, precipitated, and sequestered in certain particular intracellular

organelles. The endophytic bacteria displayed exceptional capacities for heavy metal

bioaccumulation and detoxiﬁcation.

Additionally, bacteria create biosurfactants and exude them as root exudates. By

interacting and complexing with insoluble metals, these biosurfactants increase the

bioavailability of metals in the soil and aquatic media. A crucial role in the complex-

ation of metals is played by extracellular polymeric molecules, which are mostly

made up of proteins, polysaccharides, nucleic acids, and lipids. This decreases the

bioavailability of the metals. For instance, Azobacter sp. reduced the uptake of metals

by Triticum aestivum by producing extracellular polymeric substances (EPS) that

356 S. Kaura et al.

formed complexes with chromium and cadmium. In plants, the production of several

metabolites, including siderophores and organic acids (such as citric acids, oxalic

acids, and acetic acids), affects the bioavailability and translocation of heavy metals

(Visioli et al. 2014).

6. Bioventing

Any substance that is aerobically degradable can be broken down using this method.

In bioventing, nitrogen and phosphorus as well as oxygen are injected into the

contaminated area (Rockne and Reddy 2003). The texture of the soil affects how

these nutrients and oxygen are distributed in the soil. In bioventing, a modest air

ﬂow rate provides ample oxygen for bacteria. Bioventing is nothing more than the

process of pushing air into a well that had previously drawn air into contaminated

soil above the water table. When the water table is far below the surface and the loca-

tion is hot, bioventing is more effective. It is primarily used to remove petroleum,

oil, and other hazardous materials. From one place to another, various chemicals are

removed at varying rates.

7. Biosparging

In biosparging, air is injected under pressure beneath the groundwater to raise the

oxygen concentration. For the microbial breakdown of the contaminant, oxygen is

injected. The aerobic decomposition and volatilization are increased by biosparging.

To stop volatile material from being transferred into the environment while injecting

oxygen at the polluted site, pressure must be under control. By decreasing the diam-

eter of the injection point, the cost can be decreased. It is important to understand

the permeability and texture of the soil before infusing oxygen. This technology

was used to measure the extent of remediation accomplished in terms of both mass

removal and reduction in mass discharge into groundwater at a known source of gaso-

line contamination. Underground storage tank (UST) locations can reduce petroleum

products by using biosparging. Mid-weight petroleum products, such as diesel and

jet fuel, are most frequently used at sites where biosparging is employed; lighter

petroleum products, such as gasoline, have a tendency to volatilize more easily and

be removed more quickly by air sparging.

8. Bioaugmentation

To speed up waste decomposition, microorganisms with appropriate metabolic capa-

bilities are introduced to the contaminated location. Bioaugmentation is used to

ensure that in situ microorganisms can completely break down chlorinated ethenes,

such as tetrachloroethylene and trichloroethylene, to ethylene and chloride, which

are non-toxic, at areas where soil and groundwater are contaminated.

9. Biopiling

It is a synthesis of farming and composting. A treatment bed, an aeration system,

an irrigation/nutrient system, and a leachate collection system make up the funda-

mental biopile system. Moisture, heat, nutrition, oxygen, and pH should all be under

control for effective breakdown. Underground irrigation equipment uses vacuum

Bacteria and Pollutants 357

to deliver nutrients and air. The soil is coated with plastic to avoid runoff, which

also prevents evaporation and volatilization and encourages sun heating. The biopile

therapy method takes 20 to 3 months to ﬁnish (Niu et al. 2009).

10. Landforming

Make a layer of excavated earth sandwiched between clean soil, clay, and concrete

while constructing land. The two uppermost layers should be the concrete layer and

the clear soil at the bottom. After that, let nature take its course. Additionally, add

oxygen, nutrients, and moisture, and use lime to keep the pH level close to 7. Land

formation is primarily beneﬁcial for pesticides.

10 Bacteria and Liquid Waste Management—Novel

Strategies

The primary goal of wastewater treatment is to prevent water sources from being

contaminated and to protect the general public’s health by preventing the spread of

illness. This is accomplished using a number of treatment technologies, including

onsite and offsite treatment systems. Therefore, the purpose of this part is to describe

the off-site wastewater treatment technologies (activated sludge, trickling ﬁlters,

stabilisation ponds, built wetlands, and membrane bioreactors) (USEPA 2005).

1. Activated sludge

The goal of the activated sludge process is to remove organic materials from wastew-

ater by using a high concentration of microorganisms, primarily bacteria, protozoa,

and fungi, which are present as a loose clumped mass of tiny particles that are kept

in suspension by stirring (Templeton and Butler 2011). Sewage that contains active

microorganisms that aid in the decomposition of organic waste is known as acti-

vated sludge. Of all the wastewater treatment technologies, it is the most ﬂexible and

efﬁcient.

The microorganisms in the aeration tank of an activated sludge system can break

down the organic stuff in the wastewater. 70–90% of the microbes’ weight is made

up of organic material, and 10–30% is inorganic. The various cell types change

according to the chemistry present and the unique traits of the organisms that make

up the biological mass. A clariﬁer, also known as a settling or sedimentation tank,

separates the suspended solids from the treated wastewater by gravity after the mixed

liquor is emptied from the tank. In order to maintain a concentrated population of

microorganisms to treat the wastewater, the concentrated biological solids are subse-

quently returned back to the aeration tank. Because the system is constantly producing

microbes, a means of removing the extra biological solids must be supplied. A bigger

volume of sludge must be handled since the waste solids from the aeration t ank are

less concentrated than those from the clariﬁer. It’s feasible to maximise or reduce

the creation of solids depending on how the process is s et up and run.

358 S. Kaura et al.

The oxidation of sewage organic matter into carbon dioxide and water is facilitated

by the action of the microorganisms. Prior to allowing the sewage to travel through

the settling chamber, which aids in the removal of sand and other materials, while

the ﬂoating debris is shred and ground, during the early stage of treatment, the large

ﬂoating materials in the wastewater are ﬁrst screened out. The wastewater is ﬁrst

treated by passing air through it in the system’s aeration tank. According to research,

an activated sludge system is effective in removing 75–90% of the biological oxygen

demand (BOD) from sewage (Tortora et al. 2010).

2. Trickling ﬁlter

A trickling ﬁlter is a commonly used method of secondary wastewater treatment. It

is made up of a ﬁlter bed that contains a highly permeable media (gravel or plastic

material etc.), which has a layer of microorganisms on the surface that leads to

the formation of a slime layer. In a trickling ﬁlter system, the microorganisms are

attached to the media in the bed and form a bioﬁlm over it. As the wastewater passes

through the media, the microorganisms consume and remove contaminants from the

wastewater.

One typical technique for secondary wastewater treatment is a trickling ﬁlter. It

is constructed of a ﬁlter bed with a highly porous media (gravel, plastic, etc.) that

has a layer of microorganisms on the surface that causes a slime layer to form. The

microorganisms in a trickling ﬁlter system cling to the medium in the bed and create

a bioﬁlm on top of it. The microorganisms consume and eliminate pollutants from

the wastewater as it moves through the media.

A septic tank, a clariﬁer, and an application system make up trickling ﬁlters. The

application system aids in distributing the treated wastewater to the correct location,

the clariﬁer helps the biological materials settle out of the wastewater, and the septic

tank aids in the removal of wastewater solids. To prevent them from covering the thin

layer of microorganisms present and from killing them, solid and oily debris must

ﬁrst be removed from wastewater before it is processed and transferred to a trickling

ﬁlter.

Depending on the amount of hydraulic or organic loading, trickling ﬁlters can

be categorised as high rate or low rate. Low rate ﬁlters involve straightforward

processing that results in consistently high-quality efﬂuent. 80–85% of the applied

BOD should be able to be removed by the low rate trickling system. Higher organic

and hydraulic loadings than low rate ﬁlters are often characteristics of high rate

ﬁlters. Recirculation happens with high rate ﬁlters but not with low rate ﬁlters. Filter

efﬂuent is returned and put to the ﬁlter once more through a process called recircu-

lation. This wastewater recycling increases the amount of trash that is applied with

microorganisms, causing the efﬂuent to undergo adequate treatment. (Van Haandel

and van der Lubbe 2007).

3. Membrane bioreactor

In a membrane bioreactor, direct solid–liquid separation by membrane ﬁltration

using micro or ultraﬁltration membrane technology is combined with the biological

degrading process of activated sludge. The technique enables total physical retention

Bacteria and Pollutants 359

of all suspended particles and bacterial ﬂocs inside the bioreactor. A membrane biore-

actor has advantages over other treatment systems, including high efﬂuent quality,

effective disinfection, increased volumetric loading, and less sludge generation.

In order to separate the solid from the liquid components of the sludge suspen-

sion, a membrane bioreactor (MBR) uses membrane technology instead of the

gravity settling used in the traditional activated sludge process. Biologically active

wastewater inputs from municipal or industrial sources are treated in membrane

bioreactors. There are two possible MBR conﬁgurations: internal/submerged and

external/side stream. While in the external/side stream, the membranes are a sepa-

rate unit process requiring intermediate pumping stages, in the submerged, the

membranes are immersed in and integral to the biological reactor. (Lofrano et al.

2013).

Polymers or inorganic materials are used to create the membranes in membrane

bioreactor systems. They are composed of numerous tiny pores that can only be

seen under a microscope. Only minuscule particles and water can ﬂow across the

membrane due to its microscopic hole size. Although there are other membrane

forms, the hollow ﬁbre, ﬂat sheet, and tubular membranes are t he most often used

ones in membrane bioreactors. While the tubular membrane is often put outside the

bioreactor, the hollow ﬁbre and ﬂat sheet membranes are typically submerged in

water.

The conventional activated sludge system and other biological wastewater treat-

ment methods are competitors of the MBR process. Conventional biological methods

can struggle to achieve treatment standards for discharge into sensitive settings, even

when they perform well in meeting typical discharge norms and are economical.

Additionally, it has been noted that using conventional methods to reuse wastewater

is not cost-effective unless ultraﬁltration or microﬁltration membranes are utilised

as a post treatment (United Nations Environmental Programme, Wastewater and

stormwater Treatment (2012).

11 Roles and Dynamics of Microorganisms in Wastewater

Treatment Systems

Bacteria, protozoa, viruses, fungi, algae, and helminthes are the main microbiological

species identiﬁed in wastewater treatment systems. The majority of these organisms

are found in water, which promotes the spread of diseases.

11.1 Bacteria

Bacteria are crucial to the conversion of organic materials contained in wastewater

treatment systems to less complicated molecules. Bacteria that range in size from

360 S. Kaura et al.

0.2 to 2.0 mm in diameter are in charge of the majority of the wastewater treat-

ment in septic tanks. Even though not all bacteria are hazardous, some of them

do cause illnesses in people and animals that are tied to water. Cholera, dysentery,

typhoid fever, salmonellosis, and gastroenteritis are a few of these ailments (Akpor

and Muchie 2010).

As in the case of activated sludge, bacteria can be discovered entangled in ﬂocs.

While some bacteria, such as ﬁlamentous bacteria, are essential in biological treat-

ment, others, such as these, can seriously interfere with settling and foaming. There

are several reports of waterborne gastroenteritis with no identiﬁed cause, and bacteria

are the vulnerable agent. This illness may be caused by speciﬁc bacteria of Pseu-

domonas and Escherichia coli that can harm newborns. These microorganism strains

have also been linked to epidemics of gastrointestinal diseases. The most signiﬁ-

cant number of bacteria in wastewater treatment systems. The majority of organ-

isms are facultative, meaning they can survive with or without oxygen. Although

heterotrophic and autotrophic bacteria can both be found in wastewater treatment

systems, heterotrophic bacteria are more common.

The carbonaceous organic materials in wastewater discharge is often where

heterotrophic bacteria get their energy. The energy produced is used to create new

cells as well as to release energy by converting organic material and water. Achro-

mobacter, Alcaligenes, Arthrobacter, Citromonas, Flavobacterium, Pseudomonas,

Zoogloe and Acinetobacter are a few notable bacterial species that are present in

wastewater treatment systems (Oehmen et al. 2007).

11.2 Protozoa

Protozoa, which are tiny, unicellular creatures, are also present in wastewater treat-

ment facilities. They carry out a variety of helpful tasks during the treatment

process, including clarifying the secondary efﬂuent by removing bacteria, ﬂoccu-

lating suspended debris, and acting as bioindicators of the sludge’s health. In at least

one stage of development, the protozoa that live in wastewater treatment systems can

move. They are unicellular creatures with organelles that are enveloped in membranes

and are 10 times larger than bacteria.

Protozoa have an advantage in wastewater because they feed on pathogenic

bacteria. Depending on how they move, they can be divided into ﬁve groups: free

swimming ciliates, crawling ciliates, stalked and sessile ciliates, ﬂagellates, and

amoeboid. Protozoa are helpful biological markers of the health of wastewater treat-

ment systems. Although some protozoa may survive for up to 12 h without oxygen,

they are typically classiﬁed as obligate aerobes, making them ideal markers of an

aerobic environment.

They can also display more sensitivity to toxins than bacteria and act as markers

of a toxic environment. The absence or immobility of protozoa in a treatment system

is a sign of potential harm. It is suggested that a sign of a well-run and stable system

Bacteria and Pollutants 361

is the presence of signiﬁcant numbers of highly developed protozoa in the biolog-

ical mass in a wastewater treatment system. Depending on how they move, they

can be divided into ﬁve categories. The free-swimming ciliates, crawling ciliates,

stalked/sessile ciliates, ﬂagellates, and amoebae are the members of these categories.

Free-swimming ciliates, crawling ciliates, and stalked ciliates are the three different

types of ciliates. These three all have cilia, which resemble small hairs and beat in

unison.

Aspidiscacostata, Carchesiumpolypinum, Chilodonellauncinata, Opercularia-

coarcta, Operculariamicrodiscum, Trachelophyllumpusillum, Vorticella convallaria,

and Vorticella microstoma are the ciliated protozoa species that are most frequently

seen in wastewater treatment operations. There is evidence that the free-swimming

ciliates, which contain cilia on every surface of their bodies, are often found

suspended or swimming freely in the bulk solution, including Litonotus sp. and

Paramecium sp. In contrast, the crawling ciliates, like Aspidisca sp. and Euplotes

sp., only have cilia on the surface of their belly, or ventral, where the mouth opening is

situated. The stalked ciliates, including Carchesium sp. and Vorticella sp., have their

cilia only around the mouth opening and are attached to ﬂoc particles, as opposed

to the crawling ciliates, which are typically found on ﬂoc particles. Their anterior

portion is larger, while their posterior portion is thin. Dispersed bacteria are drawn

into the mouth opening by a water vortex created by the beating of the cilia and the

springing motion of the stalk.

There are two main forms of amoebae that are found in wastewater systems:

naked amoebae like Actinophyrs sp., Mayorella sp., and Thecamoeba sp., and shelled

amoebae or testate amoebae like Cyclopyxis sp. The shelled amoeba has a protective

covering made of calciﬁed material, whereas the naked amoeba has no protective

covering at all. Protozoa that have ﬂagella have an oval form and one or more whip-

like ﬂagella. The ﬂagella of ﬂagellated protozoa help drive them through wastewater

treatment systems in a corkscrew pattern of movement.

11.3 Viruses

In particular, human viruses that are heavily discharged in faeces can be discovered in

wastewaters. Although bacterial viruses may also be present, native animal and plant

viruses can be found in wastewater in lesser amounts. They are the responsible parties

for a number of water-related illnesses in people, including conjunctivitis, meningitis,

and gastrointestinal and respiratory infections. According to reports, enteric viruses

were the primary cause of the majority of aquatic illnesses with unknown sources.

When present in wastewater, they are very notorious and persistent and can continue

to be an active source of infection for months.

362 S. Kaura et al.

11.4 Fungi

The microorganisms present in wastewater treatment systems include fungi as well.

Multicellular organisms called fungi are also found in activated sludge. They may

effectively compete with bacteria in a mixed culture under speciﬁc environmental

conditions and metabolise organic molecules. Additionally, only a few fungi have

the ability to oxidise ammonia to nitrite and even fewer to nitrate. The sewage fungus

species Sphaerotilus natans and Zoogloea sp. are the most prevalent. Although they

can also metabolise organic materials, a variety of ﬁlamentous fungi are naturally

found in wastewater treatment systems as spores or vegetative cells.

11.5 Algae

Algae are a type of biological plant that contributes to the overall stabilization of

organic wastes. Because algae get their energy for synthesis from sunlight, they

don’t need to digest organic substances like bacteria and fungi do. The inorganic

components of wastes, including ammonia, carbon dioxide, phosphate, magnesium,

potassium, iron, calcium, sulphate, sodium, and other ions, are predominantly used

by algae to make protoplasm. Because algae and bacteria do not need the same waste

components, it is possible for algae and bacteria to coexist. The bacteria break down

the waste’s organic components and release some of the inorganic components that

the algae need. The bacteria use the oxygen released by the algae during protoplasm

synthesis to complete aerobic stabilization of the organic matter. In the absence of

sunlight, algae, like bacteria and fungus, must rely on the metabolism of organic

materials to receive the energy they require to stay alive. This organic substance is

generally derived from stored food within the cell, but it can also be derived from

organic waste in some algae species (Adebayo and Obiekezie 2018).

References

Adebayo FO, Obiekezie SO (2018) Microorganisms in waste management. Res J Sci Technol

10(1):28–39

Akpor OB, Muchie M (2010) Remediation of heavy metals in drinking water and wastewater

treatment systems: processes and application. Int J Phys Sci 5(12):1807–1817

Anawar HM (2015) Sustainable rehabilitation of mining waste and acid mine drainage using

geochemistry, mine type, mineralogy, texture, ore extraction and climate knowledge. J Environ

Manage 158:111–121

Ayilara MS, Olanrewaju OS, Babalola OO, Odeyemi O (2020) Waste management through

composting: challenges and potentials. Sustainability 12(11):4456

Bañeras L, Ruiz-Rueda O, López-Flores R, Quintana XD, Hallin S (2012) The role of plant type and

salinity in the selection for the denitrifying community structure in the rhizosphere of wetland

vegetation. Int Microbiol 15:89–99

Bacteria and Pollutants 363

Barman D et al (2011) Isolation of cellulytic bacterial strains from soil for effective and efﬁcient

bioconversion of solid waste. Life Sci Med Res 10:1–7

Fei F, Wen Z, Huang S, De Clercq D (2018) Mechanical biological treatment of municipal solid

waste: Energy efﬁciency, environmental impact and economic feasibility analysis. J Clean Prod

178:731–739

Fenchel T, Blackburn H, King GM, Blackburn TH (2012) Bacterial biogeochemistry: the

ecophysiology of mineral cycling. Academic Press, Cambridge, MA, USA (2012)

Gaur VK, Sharma P, Sirohi R, Awasthi MK, Dussap CG, Pandey A (2020) Assessing the impact

of industrial waste on environment and mitigation strategies: a comprehensive review. J Hazard

Mater 398:123019

Gautam SP (2011) Diversity of cellulolytic microbes and the biodegradation of municipal solid

waste by a potential strain. Int J Microbiol 1:1–12

Ghosh A, Debnath B, Ghosh SK, Das B, Sarkar JP (2018) Sustainability analysis of organic fraction

of municipal solid waste conversion techniques for efﬁcient resource recovery in India through

case studies. J Mater Cycles Waste Manage 20(4):1969–1985

Gupta G, Datta M, Ramana GV, Alappat BJ, Bishnoi S (2021) Contaminants of concern (CoCs)

pivotal in assessing the fate of MSW incineration bottom ash (MIBA): First results from India

and analogy between several countries. Waste Manage 135:167–181

https://earth5r.org/sustainable-construction-waste-management-india/

https://vikaspedia.in/energy/environment/waste-management/solid-and-liquid-wastemanagement-

in-rural-areas

https://www.epa.gov/rcra/medical-waste

Katare VD, Madurwar MV, Raut S (2020) Agro-industrial waste as a cementitious binder for

sustainable concrete: an overview. Sustainable waste management: policies and case studies, pp

683–702

Shah MP (2020) Microbial bioremediation & biodegradation. Springer

Kishore J (2010) E-waste management: as a challenge to public health in India. Indian J Commun

Med Ofﬁcial Publication Indian Assoc Preventive Soc Med 35(3):382

Kornberg D (2019) Garbage as fuel: pursuing incineration to counter stigma in postcolonial urban

India. Local Environ 24(1):1–17

Kwietniewska E, Tys J (2014) Process characteristics, inhibition factors and methane yields of

anaerobic digestion process, with particular focus on microalgal biomass fermentation. Renew

Sustain Energy Rev 34:491

Shah MP (2021a) Removal of refractory pollutants from wastewater treatment plants. CRC Press

Lamers LP, Van Diggelen JM, Op Den Camp HJ, Visser EJ, Lucassen EC, Vile MA, Jetten MS,

Smolders AJ, Roelofs JG (2012) Microbial transformations of nitrogen, sulfur, and iron dictate

vegetation composition in wetlands: a review. Front Microbiol 3:156

Lee WE, Ojovan MI, Jantzen CM (Eds) (2013) Radioactive waste management and contaminated

site clean-up: processes, technologies and international experience. Elsevier

Lo YC, Saratale GD, Chen WM, Bai MD, Chang JS (2009) Isolation of cellulose hydrolytic bacteria

and applications of the cellulolytic enzymes for cellulosic biohydrogen production. Enzyme

Microb Technol 44:417

Shah MP (2021b) Removal of emerging contaminants through microbial processes. Springer

Lofrano G, Sureyya M, Gülsüm EZ, Derin O (2013) Chemical and biological treatment technologies

for leather tannery chemicals and wastewaters: a review. Sci Total Environ 461–462:265–281

McCarty P (2001) The development of anaerobic treatment and its future. Water Sci Technol 44:149

Mishra S, Tiwary D, Ohri A, Agnihotri AK (2019) Impact of municipal solid waste landﬁll leachate

on groundwater quality in Varanasi. India. Groundwater Sustain Dev 9:100230

Niu GL, Zhang JJ, Zhao S, Liu H, Boon N (2009) Bioaugmentation of a 4-chloronitrobenzene

contaminated soil with Pseudomonas putida ZWL73. Environ Pollut 157:763–771

Oehmen A, Lemos PC, Carvalho G, Yuan Z, Keler J, Blackall LL, Reis AM (2007) Advances in

enhanced biological phosphorus: from micro to macro scale. Water Res 41:2271–2300

364 S. Kaura et al.

Palaniveloo K, Amran MA, Norhashim NA, Mohamad-Fauzi N, Peng-Hui F, Hui-Wen L, ... Razak

SA (2020) Food waste composting and microbial community structure proﬁling. Processes

8(6):723

Pujara Y, Pathak P, Sharma A, Govani J (2019) Review on Indian municipal solid waste management

practices for reduction of environmental impacts to achieve sustainable development goals. J

Environ Manage 248:109238

Rastogi M, Nandal M, Khosla B (2020) Microbes as vital additives for solid waste composting.

Heliyon 6(e03343):2020. https://doi.org/10.1016/j.heliyon.2020.e03343

Riddech N, Klammer S, Insam H (2002) Characterisation of microbial communities during

composting of organic wastes. In: Insam H, Riddech N, Klammer S (eds) Microbiology of

composting. Springer, Berlin, Heidelberg, pp 43–51

Rockne K, Reddy K (2003) Bioremediation of contaminated sites. University of Illinois at Chicago

Sharma KD, Jain S (2019) Overview of municipal solid waste generation, composition, and

management in India. J Environ Eng 145(3):04018143

Sharma S, Basu S, Shetti NP, Kamali M, Walvekar P, Aminabhavi TM (2020) Waste-to-energy

nexus: a sustainable development. Environ Pollut 267:115501

Stams AJ, Plugge CM (2009) Electron transfer in syntrophic communities of anaerobic bacteria

and archaea. Nat Rev Microbiol 7:568

Templeton MR, Butler D (2011) An introduction to wastewater treatment. Ventus Publishing ISBN:

978-87-7681-843-2

Tortora GJ, Berdell RF, Christine LC (2010) Microbiology: an introduction (10th edition)

United Nations Environmental Programme, Wastewater and stormwater Treatment (2012)

US Environmental Protection Agency (USEPA) (2005) Pollution Prevention (P2) Framework. EPA-

748-B04–001.Ofﬁce of Pollution Prevention and Toxics

Van Haandel A, van der Lubbe J (2007) Handbook biological wastewater treatment. Quist

Publishing, Leidschendam

Visioli G, D’Egidio S, Vamerali T, Mattarozzi M, Sanangelantoni AM (2014) Culturable endophytic

bacteria enhance Ni translocation in the hyperaccumulator Noccaea caerulescens. Chemosphere

117:538–544

WanIshak WMF et al (2011) Isolation and identiﬁcation of bacteria from activated sludge and

compost for municipal solid waste treatment system. (24):450–454

Bioﬁlms in Porous Media

Esha Garg, Ajit Varma, and M. S. Smitha

Abstract Bioﬁlms are microbial communities that are attached to a surface in three

dimensions. Bioﬁlms formed when microorganisms like bacteria adhere, prolif-

erate and enclose themselves in extracellular polymeric polymers (EPS) which is

self-produced. It is formed in natural ecosystem or engineered systems and plays

remarkable role in hydrodynamics in porous media. Microbial bioﬁlms are resis-

tant to environmental factors like temperature, pH, and water activity, mechanical

stress. Microbial bioﬁlms can impact the hydrodynamics of porous medium in both

natural and artiﬁcial systems. Porosity, permeability, dispersion, diffusion, and mass

transfer of reactive and nonreactive solutes are all inﬂuenced by bioﬁlm develop-

ment in porous media. Understanding and regulating bioﬁlm development in porous

medium will maximize the potential value of porous media bioﬁlms while mini-

mizing the negative consequences. Beneﬁcial porous media bioﬁlm uses include

subsurface cleanup, improved oil recovery, and carbon sequestration, to name a few.

The objective of the chapter is to focus on the various aspects of bioﬁlm development

in porous media.

Keywords Bacteria ·EPS ·Hydrodynamics ·Microbial bioﬁlms ·Permeability ·

Porous media

1 Introduction

Bioﬁlms are microbial communities that are adhered to a surface in three dimen-

sions. A bioﬁlm is mainly produced by microorganisms like fungi, bacteria, protozoa,

algae that are attach to a surface and encapsulated in an EPS array that they have

self-produced. Bioﬁlms can be found on a variety of surfaces as well as in a variety

of industrial, environmental, and medical systems. Depending on the culture condi-

tions, bioﬁlm communities are found to form continuous ﬁlms and distinct colonies

(Halan et al. 2012). The huge amount of EPS associated with bioﬁlms provides

E. Garg · A. Varma · M. S. Smitha (B

)

Amity Institute of Microbial Technology, Amity University, Uttar Pradesh, Sector-125, Noida,

U.P. 201313, India

e-mail: smsreedharan@amity.edu

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_17

365

366 E. Garg et al.

EPS

Maintains symbiotic

relatio nships among

organis ms

Keeps biofilm

communities spatially

organized

Reduces cell movement

which leads to stable

community

Fig. 1 Functions of EPS

structural support and is distinguish feature between bioﬁlms and suspended cells.

The presence of EPS helps to keep bioﬁlm communities organized spatially. EPS

immobilize or drastically reduces microbial cell movement, leading to the creation

of stable microbial community which is less rigid (Fig. 1). Its presence provides an

edge because of the symbiotic or mutualistic relationships among organisms.

A porous medium is a solid matrix (Flemming and Wingendet 2010) whose pores

are of several micrometers in size and provides an amiable environment to bioﬁlm

forming microbes because of its high speciﬁc surface. The formation of bioﬁlms in

porous media is of immense application in bioremediation of soil (Shah Maulin 2020)

and aquifers (Meckenstock et al. 2015), sequestration of carbon dioxide, bio-assisted

oil recovery etc.

Bioﬁlms in porous media can have a wide range of morphologies, including

continuous ﬁlms of varying thickness (Ye et al. 2015) or patchy, colony-like bioﬁlms.

Bioﬁlm structure formation is greatly inﬂuenced by following factors like growth

regulating factors, existence of inhibitors and the hydrodynamics (Hobley 2015).

The comprehensiveness of formation of bioﬁlm depends on the ability of growth

of adhered microorganism and the rate of reproduction. The subsequent factors that

inﬂuence bioﬁlm formation are described (Fig. 2).

(1) Availability of nutrients and energy

(2) Conditions that are appropriate in terms of geochemistry

(3) Presence of inhibitors

(4) Several bioﬁlm-eating microorganisms, and

(5) Inﬂuence of Hydrodynamics on solute mass transport which causes mechanical

stress on bioﬁlms.

2 Morphological Features of Bioﬁlms in Porous Media

Bioﬁlms appear opaque in natural and synthetic porous media. Planar pore networks

etched in glass and ﬂow chambers jammed with beads of glass have been employed

Bioﬁlms in Porous Media 367

Appropriate

Geochemical

Conditions

Absence of

inhibitors

Biofilm-

Eating

Organisms

Nutritional

and

Energetic

Availability

Hydrodynamics

Fig. 2 Major factors that inﬂuence bioﬁlm formation

as porous media model to review the expansion of bioﬁlms, allowing direct optical

examination of core and pore scale development (Wildenschild and Sheppard 2013).

According to some studies, methods like magnetic resonance (Vogt et al. 2013) and

confocal microscopy fully supported the investigation of bioﬁlm growth in porous

media. Paulsen et al. discovered the three different bioﬁlm growth stages when he

photolithographed the sandstone having a porous structure on glass plates- (a) forma-

tion of smooth bioﬁlms on the pore walls, (b) Non-uniformity in bioﬁlms over time,

and (c) strands of bioﬁlm eventually spanned the pores (web-like structure). Patchy

bioﬁlms, smooth bioﬁlms continuous bioﬁlms and irregular aggregates were discov-

ered by McCarty and Dupin. By using a chamber-and-throat pore network etched

on glass, Vayenas et al. investigated the biodegradation of a combination of organic

toxicants at the time of growth of bioﬁlm. According to one of the study’s intriguing

ﬁndings, the average width of bioﬁlms had a concrete interaction with the pore size.

3 Bacterial Interactions with Porous Media

The deposition of planktonic cells to the substratum is the ﬁrst step in bioﬁlm forma-

tion and growth. Deposition is commonly thought of as a series of transport and adhe-

sion steps. The adhesion step is controlled by cell substratum interaction, whereas

transport is determined by hydrodynamics.

Between the cell and the substratum is a Gibbs energy barrier. Bacterial cell surface

macromolecules can break through this energy barrier, allowing them to reach the

substratum with high ionic strength. As a result, interactions between cell surface

macromolecules can break through this energy barrier and reach the substratum with

high ionic strength. At high ionic strength, adhesion is determined by interactions

368 E. Garg et al.

between cell surface macromolecules and the substratum, which are referred to as

steric interactions. Bridging, which promotes adhesion and lowers the activation

energy of adhesion, and, steric hindrance, which inhibits adhesion and thus are the

two types of steric interactions that commonly occur.

Modeling bioﬁlm growth is one of the main tools used to improve current under-

standing of the correlation between the hydrodynamics of porous media and bacterial

bioﬁlms. Over geological time scales, this approach can provide predictions that are

difﬁcult to observe experimentally.

4 Bacterial Transportation in Porous Media

Convective movement in the aqueous phase, slowed by attachment to surfaces and

straining or trapping into interstitial pores are administered by bacterial transport

on porous media (Xiong et al. 2016). Several factors inﬂuence bacterial transport

through porous media, including bacterial cell properties, solution chemistry, porous

media characteristics, and interspatial ﬂuid acceleration (Kone et al. 2014). The

presence of molecules such as proteins or polysaccharides on the cell surface, as

well as motility and chemotaxis, can inﬂuence bacterial transport, retardation, and

adhesion to surfaces.

Cell size and shape, as well as cell surface charge and cell by drophobicity, have

all been linked to transport through porous media. Bacterial transport and adhesion

to surfaces have been shown to be inﬂuenced by solute characteristics which includes

ionic strength, pH, temperature, concentrations of dissolved organic matter, surfac-

tants, and nutrients. Attachment of a bacterial will increase with increase in iconic

strength according to many studies.

In the presence of high ion concentrations, this effect is usually attributed to

compression of the electrostatic double layer. Porous media properties such as pore

water velocity, typore size, the presence of Fe minerals, the organic matter content,

and grain and pore size distribution have been reported to i nﬂuence bacterial transport

and adhesion. Some combination of the aforementioned parameters may inﬂuence

bacterial transport through porous media.

To predict bacterial transport through porous media, experiments should be

conducted with the aquifer material of interest and under conditions as close to those

expected in the ﬁeld as possible. The convection–dispersion equation for bacteria

has been used in many basic bacterial transport models. To account for the extent of

bacterial attachment to surfaces, empirically determined collision efﬁciency factors

are typically used.

The bacterial transport can be explained by the convection–dispersion transport

model. Due to the numerous interactions occurs within porous media, transporta-

tion of bacteria in the subsurface (Sams et al. 2016) notably in the vadose zone, is

a complicated task. Depending on the prevailing interactions of the bacteria in the

Bioﬁlms in Porous Media 369

pore system, bacteria may be captured at the media-air–water three-phase interface,

at the air–water interface or on the media surface, when transported within the vadose

zone. By using column experiments, transportation of Bacillus subtilis (Wilking et al.

2013), Pseudomonas ﬂuorescens and E.coli in silica sand under water-unsaturated

conditions was reviewed in one of the research. To ascertain media surface ther-

modynamic properties and bacteria, interfacial tension measurements were used to

interpret bacterial interactions within the system.

5 Effect of Growth of Bioﬁlm on Porous Media

Hydrodynamics

Hydrodynamics in porous media is affected by EPS and microbial cells. Formation

of discrete clusters marked as an initial phase of bioﬁlm accumulation. The local

hydrodynamics, effective porous media particle size and availability of nutrients all

inﬂuence bioﬁlm distribution in porous media. Carbon availability also inﬂuence the

formation of bioﬁlm in porous media.

The method of measurement and the pore size of media inﬂuence the porosity in

media. The permeability of porous media is thought to be reduced as biomass and

polysaccharides accumulate.

With increased biomass production, permeability decreases are usually not

uniform, but rather vary spatially, and temporally. In bioﬁlm-affected porous media,

dispersivity increases over time, if the bioﬁlm reaches to a pseudo steady phase, it

often reaches to semi-stable values.

Nondestructive, spatially and temporally resolved measurements of hydrody-

namic dispersion and velocity in porous media (Holzner et al. 2015)aswellas

limited bioﬁlm imaging, are well-suited to magnetic resonance microscopy (MRM)

techniques. Growth of bioﬁlm in porous media signiﬁcantly reduces space in pores

for advective ﬂow.

Carbon availability is known to impact the formation of bioﬁlm in porous media

(Hand et al. 2008). However, increased bioﬁlm growth has been reported in untreated

sewage areas with trace of carbon (Dixon et al. 2018; Godzieba et al. 2022). Another

limiting growth factor, such as oxygen, may have become more available, which

could explain this. Microbial activity growth takes place when oxygen is introduced

into column systems that are repeatedly tried to open or attached to gas-permeable

piping system, for example silicon piping system.

Bioﬁlm-induced mineral formation, such as divalent or trivalent (e.g. Ca,Fe,Mg)

sulphate, phosphate minerals, carbonate and sulphide frequently causes transition in

hydrodynamics in bioﬁlm-affected porous media (Li et al. 2015). In one of the inves-

tigation, it was found that debris substance majorly was calcium carbonate which

has an impact on growth of bioﬁlms in porous media (Zhang and Klapper 2014).

370 E. Garg et al.

6 Visualization of Bioﬁlms in Porous Media

Counterproductive and non-invasive measurements is used to evaluate the circula-

tion, presence and framework of bioﬁlms in porous media. Examples of such tech-

niques include microscopy and high-resolution photography, transmission electron

microscopy or scanning electron microscopy (TEM or SEM), nuclear magnetic reso-

nance (NMR) spectroscopy, ultrasound-based imaging techniques, X-ray tomog-

raphy. Microscopes and image analysis software can be used to measure the

dimensions of stained or unstained bioﬁlms.

Optical techniques for monitoring bioﬁlms in porous media are limited by the

fact that porous media particles are not smooth (e.g. glass beads, sand grains). Back-

ground signal and image chromatic aberration are increased on porous media surfaces

because they are typically irregularly shaped (Chen 2002). Porous media are also

opaque, and because high-resolution microscopy objectives have a deﬁned working

distance, thus the detailed analysis is restricted.

The thickness of surface bioﬁlms is measured by using electron microscopy

methodologies such as TEM and SEM. Microscale imaging of bioﬁlms in porous

media (Neu and Lawrence 2014) is continuing, despite the emergence of X-ray

tomography techniques for imaging porous media (Beltran et al. 2011). NMR, ultra-

sound, and complex conductivity imaging are non-optical techniques for visualising

bioﬁlms in opaque media that have a lot of upside.

7 Bioﬁlm Systems in Porous Media as Models

The model used for understanding the process of bioﬁlms in porous media should take

into account the impact of classiﬁcations and microscale heterogeneities. Admittedly,

modelling an outsized scale system on the microscale are often computationally

intensive.

Bulk-scale models based on logical or fast analytical simulation are computation-

ally intensive (Peszynska et al. 2015) and perform well in situations where generous

bulk is adequate and it is, when microscopic mechanisms are minimal in compar-

ison to total system conduct. As a function of formation of bioﬁlm in porous media,

these models can imitate bulk changes in speciﬁcations such as speciﬁc surface area,

porous structure, dispersivity and permeability. Microscale models, one on either

hand, treat porous media affected with bioﬁlms as multi–faceted on the pore scale,

permitting them to analyze regionalized obstruction, which impacts the total device

hydrodynamics.

Multiscale models have recently been described that numerically overcome

the Brinkman and Navier–Stokes equations which incorporate the approach with

Lagrangian-type simulations or cellular automaton of detached segment pathways

(Kapellos et al. 2007a, b).

Bioﬁlms in Porous Media 371

8 Nature and Innovation of Bioﬁlms in Porous Media

Bioﬁlms have been found in a wide range of manufacturing, ecologic, and medical

conﬁgurations. At ﬁrst, study focused on eliminating bioﬁlms by employing antimi-

crobials, but several characteristics of bioﬁlms have recently been recognized as

potentially beneﬁcial for engineered applications.

Due to their elevated degree of environmental pressure and toxicants, bioﬁlm reac-

tors are quite often suggested f or the intervention of recalcitrant compounds. Such

compounds involve dyes, surfactants, organic solvents and herbicides (Mondragón-

Parada et al. 2008). Bioﬁlm hinders colloidal transport (Majumdar et al. 2014).

Healthcare system depends on micropollutants (colloid-mediated contaminant)

transport and infectious agent (biological colloids) through porous media. Forma-

tion of established bioﬁlm communities is proved to be advantageous despite the fact

that its development and organization is still unexplained. Bioﬁlms are enticing to

innovators because of their near vicinity to one another, which enables for cell–cell

interaction, genetic component transfer (DNA, RNA), colocation of physiologically

various organisms, which can enhance metabolite exchange, and enhanced tolerance

to ecological stresses.

Because of their increased resilience to toxic compounds and environmental

stress, bioﬁlm reactors are oftenl postulated for the cure of recalcitrant compounds.

Subsurface bioﬁlm barriers prefer maximum porosity, permeability reduction and

groundwater ﬂow control. Adequate thickness is preferred in bio trickling ﬁlters

(used in sewage treatment) having an impact on removal of excess of solute with

high consistent permeability. Grid of ﬂow channels is regarded as a special type of

porous medium, bioﬁlm growth (bio-fouling) in such processes can have a signif-

icant impact on ﬂow, as evidenced by increased back-pressure in (bio-) fouled

membrane processes. Membranes including reverse osmosis ﬁltration cartridges are

also precluded from evaluation.

Bioﬁlms builds up solutes and potentially vast quantities of minerals in environ-

ment and manufacturing in industries. Calcium carbonate, sulfur-containing minerals

and iron oxides are most prominent inorganic components of bioﬁlms in porous

media. Mineral precipitation results in semi-permanent to permanent bioﬁlm cell

encrustation, along with clogging of key locations in a porous medium (Thullner

2010).

8.1 Profound Subsurface Bioﬁlms for Improved Oil Recovery

and Carbon Capture

Selective plugging of high-permeability networks in reservoir rock to strengthen

sweep efﬁciency, manufacturing of biosurfactants or gases to improve mobility of

oil and in situ biocracking, that incorporates microorganisms that breaks down long

alkane chains and produces higher-solubility alkane with short chains, all these are

372 E. Garg et al.

instances of microbially improved oil recuperation (Armstrong and Wildenchild

2012).

Bioﬁlms are beneﬁcial at preferentially plugging high-permeability water-ﬁlled

residential areas as crook zones. In high-permeability areas, lowered permeability

grant ﬂuids such as water or supercritical CO2 (scCO2) used for improved secondary

or tertiary oil recovery to construct remaining oil in areas with low-permeability and

this marks for establishment which is quite successful.

8.2 Bioﬁlm Reactors with Porous Media in Industry

and Waste Treatment

In sewage water treatment and water treatment, bioﬁlms in porous media plays vital

role (Wingender and Flemming 2011). Microorganisms adhered to water ﬁltration

can have signiﬁcant effect on water treatment processes as they either grow inside

just at infﬂuent or sewage waters of ﬁltration apparatus.

Microorganisms, fairly immobilized in porous media, have for quite some time

been utilized in wastewater expulsion of toxicants from wastewater (Shah Maulin

2021) in trickling ﬁlters and inﬁltration systems. Role of bioﬁlms in large-scale

applications and primary analysis are currently in progress (Qureshi et al. 2005;

Iliuta and Larachi 2004; Shah 2020, 2021; Mauclaire et al. 2006).

Porous media bioﬁlms, on the other hand, have been witnessed to aid in the oxida-

tive precipitation of metals like reduced manganese and iron, sorption or degradation

of organic particulates and solutes, in water.

8.3 Bioﬁlm Barriers in the Subsurface for Contaminated

Groundwater Control and Remediation

Growth and expansion of microbial activity helps in advancement of subsurface

bioﬁlm barriers. In order to monitor and govern the groundwater and environ-

ment soil, semipermeable, permeable and impermeable bioﬁlm barriers have been

suggested (Hiebert et al. 2001; Komlos et al. 2006; Cunningham et al. 2003).

By stimulating dense growth of bioﬁlms, these barriers are able to limit the perme-

ability and thus it allows to reduce or direct groundwater ﬂow through speciﬁc

subsurface areas. These subsurface bioﬁlm barriers are quite competent in removing

solutes.

Bioﬁlms in Porous Media 373

9 Conclusion

Bioﬁlm development in porous media involves a complex set of biological, phys-

ical and chemical interactions. Microbial bioﬁlm produces Extracellular Polymeric

Substance (EPS) when it adheres to the porous media and starts proliferating. Its

growth affects porous structure, absorption, diffusion, dispersion, and transport

systems of reactive and nonreactive solutes in porous media. Many latest techniques

and technologies have enabled unveiling the bioﬁlms formation in porous media.

The application of microbial bioﬁlm in the areas of soil improvement, pollutant

restoration, waste treatment etc. without the complete elucidation of bioﬁlm forma-

tion displays its eco-social impact. The difﬁculties associated with bioﬁlm in porous

media need to be analysed in detail using sophisticated instruments and improvised

work plan. In future, we anticipate that new knowledge would be generated describing

the interactions between bioﬁlms and their adaptation in porous habitats.

References

Armstrong RT, Wildenschild D (2012) Investigating the pore-scale mechanisms of microbial

enhanced oil recovery. J Pet Sci Eng 94–95:155–164

Battin TJ, Besemer K, Bengtsson MM, Romani AM, Packmann AI (2016) The ecology and

biogeochemistry of stream bioﬁlms. Nat Rev Microbiol 14:251–263

Beltran MA, Paganin DM, Siu KKW, Fouras A, Hooper SB, Reser DH, Kitchen MJ (2011) Interface-

speciﬁc x-ray phase retrieval tomography of complex biological organs. Phys Med Biol. 56:7353–

7369. pmid:22048612

Billings N, Birjiniuk A, Samad TS, Doyle PS, Ribbeck K (2015) Material properties of bioﬁlms—a

review of methods for understanding permeability and mechanics. Rep Prog Phys 78:036601

Chen X, Schauder S, Potier N, Van Dorsselaer A, Pelczer I, Bassler BL, Hughson FM (2002)

Structural identiﬁcation of a bacterial quorum-sensing signal containing boron. Nature 415:545–

549

Cunningham AB, Sharp RR, Hiebert R, James G (2003) Subsurface bioﬁlm barriers for the

containment and remediation of contaminated groundwater. Bioremediat J 7(3–4):151–164

De Vos WM (2015) Microbial bioﬁlms and the human intestinal microbiome. NPJ Bioﬁlms

Microbiomes 1:15005

Dixon MJ, Flint SH, Palmer JS, Love R, Chabas C, Beuger AL (2018) The effect of calcium on

bioﬁlm formation in dairy wastewater. Water Pract Technol 13(2):400–409

Flemming H-C (2011) In: Flemming H-C, Wingender J, Szewzyk U (eds). Springer, pp 81–109

Flemming H-C, Wingender J (2010) The bioﬁlm matrix. Nat Rev Microbiol 8:623–633

Godzieba M, Zubrowska-Sudol M, Walczak J, Ciesielski S (2022) Development of microbial

communities in bioﬁlm and activated sludge in a hybrid reactor

Halan B, Bühler K, Schmid A (2012) Bioﬁlms as living catalysts in continuous chemical syntheses.

Trends Biotechnol 30:453–465

Hand VL, L loyd JR, Vaughan DJ, Wilkins MJ, Boult S (2008) Experimental studies of the inﬂuence

of grain size, oxygen availability and organic carbon availability on bioclogging in porous media.

Environ Sci Technol 42:1485–1491

Hiebert R, Sharp RR, Cunningham AB, James G (2001, August) Development and demonstration

of subsurface bioﬁlm barriers using starved bacterial cultures. Contaminated Soil Sediment Water

45–47

374 E. Garg et al.

Hobley L, Harkins C, MacPhee CE, Stanley-Wall NR (2015) Giving structure to the bioﬁlm matrix:

an overview of individual strategies and emerging common themes. FEMS Microbiol Rev 39:649–

669

Holzner M, Morales VL, Willmann M, Dentz M (2015) Intermittent Lagrangian velocities and

accelerations in three-dimensional porous medium ﬂow. Phys Rev E 92:013015

Iliuta I, Larachi F (2004) Biomass accumulation and clogging in tricklebed bioreactors. Aiche J

50:2541–2551

Kapellos GE, Alexiou TS, Payatakes AC (2007a) Hierarchical simulator of bioﬁlm growth and

dynamics in granular porous materials. Adv Water Resour 30:1648–1667

Kapellos GE, Alexiou TS, Payatakes AC (2007b) A multiscale theoretical model for diffusive mass

transfer in cellular biological media. Math Biosci. https://doi.org/10.1016/j.mbs.2007.04.008

Kaplan JB (2014). In: Donelli G (ed) Microbial bioﬁlms: methods and protocols. Springer, pp

203–213

Komlos J, Cunningham AB, Camper AK, Sharp RR (2006) Effect of substrate concentration on

dual-species bioﬁlm population densities of Klebsiella oxytoca and Burkholderia cepacia in

porous media. Biotechnol Bioeng 93:434–442

Kone T, Golﬁer F, Orgogozo L, Oltéan C, Lefèvre E, Block JC et al (2014) Impact of bioﬁlm-induced

heterogeneities on solute transport in porous media. Water Resour Res 50:9103–9119

Majumdar U, Alexander T, Waskar M, Dagaonkar MV (2014) Effect of bioﬁlm on colloid attachment

in saturated porous media. Water Sci Technol 70(2):241–248

Meckenstock R et al (2015) Biodegradation: updating the concepts of control for microbial cleanup

in contaminated aquifers. Environ Sci Technol 49:7073–7081

Mondragón-Parada ME, Ruiz-Ordaz N, Tafoya-Garnica A, Juarez-Ramirez C, Curiel-Quesada E,

Galindez-Mayer J (2008) Chemostat selection of a bacterial community able to degrade s-triazinic

compounds: continuous simazine biodegradation in a multi-stage packed bed bioﬁlm reactor. J

Ind Microbiol Biotechnol 35(7):767–776

Neu TR, Lawrence JR (2014) Innovative techniques, sensors, and approaches for imaging bioﬁlms

at different scales. Trends Microbiol 23:233–242

Peszynska M, Trykozko A, Iltis G, Schlueter S, Wildenschild D (2015) Bioﬁlm growth in porous

media: Experiments, computational modeling at the porescale, and upscaling. Adv Water Resour

95:288–301

Qureshi N, Annous BA, Ezeji TC, Karcher P, Maddox IS (2005) Bioﬁlm reactors for indus-

trial bioconversion processes: employing potential of enhanced reaction rates. Microb Cell Fact

4(1):1–21

Rosenzweig R, Furman A, Dosoretz C, Shavit U (2014) Modeling bioﬁlm dynamics and hydraulic

properties in variably saturated soils using a channel network model. Water Resour Res

50(7):5678–5697

Sams R, Garca J, Molle P, Forquet N (2016) Modelling bioclogging in variably saturated porous

media and the interactions between surface/subsurface ﬂows: application

Shah Maulin P (2021) Removal of refractory pollutants from wastewater treatment plants. CRC

Press

Shah Maulin P (2020) Microbial bioremediation & biodegradation. Springer

Thullner M (2010) Comparison of bioclogging effects in saturated porous media within one-and

two-dimensional ﬂow systems. Ecol Eng 36(2):176–196

Vogt SJ, Sanderlin AB, Seymour JD, Codd SL (2013) Permeability of a growing bioﬁlm in a

porous media ﬂuid ﬂow analyzed by magnetic resonance displacement-relaxation correlations.

Biotechnol Bioeng 110:1366–1375. pmid:23239390

Wildenschild D, Sheppard AP (2013) X-ray imaging and analysis techniques for quantifying pore-

scale structure and processes in subsurface porous medium systems. Adv Water Resour 51:217–

246

Wilking JN et al (2013) Liquid transport facilitated by channels in Bacillus subtilis. Proc Natl Acad

Sci USA 110:848–852

Bioﬁlms in Porous Media 375

Wingender J, Flemming H-C (2011) Bioﬁlms in drinking water and their role as reservoir for

pathogens. Int J Hyg Environ Health 214:417–423

Xiong Q, Baychev TG, Jivkov AP (2016) Review of pore network modelling of porous media:

experimental characterisations, network constructions and applications to reactive transport. J

Contam Hydrol 192:101–117

Ye S, Zhang Y, Sleep BE (2015) Distribution of bioﬁlm thickness in porous media and implications

for permeability models. Hydrogeol J 23(8):1695–1702

Zhang T, Klapper I (2014) Critical occlusion via bioﬁlm induced calcite precipitation in porous

media. New J Phys 16(5):055009

Removal of Heavy Metals

from Industrial Wastewater Using

Bioremediation Approach

Pooja M. Patil, Abhijeet R. Matkar, Vitthal B. Patil, Ranjit Gurav,

and Maruti J. Dhanavade

Abstract Heavy metal contamination has developed great attention throughout the

globe as an effect of their persistent and recalcitrant nature which results in toxic

effects on the environment, shelf lives of animals and plants, estimating chronic

diseases in individuals. Heavy metals exist a wider scope for science and inno-

vation, with pressure on cost-effectiveness technology and to minimize the effect

of anthropogenic accomplishments on the environment, and exploration of innova-

tive, eco-friendly methods for ecological restoration. Bioremediation is known to

be promising and eco-friendly technology for the remediation of contaminated sites

and for a huge range of pollutant removals this treatment has been applied. Biore-

mediation technology has the potential to treat various types of waste and it is one

method through which contaminant or toxic metals are treated by using living cells

like bacteria, fungus, and algae, and this bioremediation method is better over than

conventional methods. In this book chapter heavy metals and their types, effects of

industrial discharge heavy metals on the water body and human beings are explained.

This chapter also reviews the conventional treatment for the treatment of heavy metals

that existed in wastewater. Then further bioremediation and its mechanism, types of

bioremediations, the interaction between microorganisms and metals, and advantages

of bioremediation methods for industrial wastewater are also discussed.

P. M. P atil · V. B . Pa t i l

Department of Environment Management, Chhatrapati Shahu Institute of Business Education and

Research, Kolhapur, India

A. R. Matkar

Department of Mechanical Engineering, D.Y. Patil College of Engineering and Technology,

Kolhapur, India

R. Gurav (B

)

Ingram School of Engineering, Texas State University, San Marcos, TX 78666, United States

e-mail: ranjit.g@txstate.edu

M. J. Dhanavade (B

)

Department of Microbiology, Bharati Vidyapeeth’s Dr. Patangrao Kadam Mahavidyalaya, Sangli,

India

e-mail: maruti.dhanavade@bharatividyapeeth.edu.in

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_18

377

378 P. M. Patil et al.

Keywords Bioremediation ·Heavy metals ·Anthropogenic activities ·

Ecofriendly ·Toxicity

1 Introduction

The presence of heavy metals in industrial emission leads to solid deposits, apoptosis

along wastewater body streams and in faunas that live near dumping sites. In daily

life exposure to contaminated wastewater is extensive, speciﬁcally in populated areas

and where for agriculture purposes wastewater is used (Elgallal et al. 2016). Earlier

research revealed that effectual metal waste management is a signiﬁcant worldwide

concern. Several industrial activities, like paper pulp, tanneries industries, batteries,

electrical gadgets cause a severe impact on marine life. Heavy metal pollution is a

worldwide issue and its level changes from source to source. Sewage and industrial

wastewater released into waterways are a substantial source of heavy metal pollution

in the water bodies (Van Oosten and Maggio 2015). The metals substantially bind

to stripy materials, accumulate in the bottom of a river, and then are disposed into

the water surrounding, pretending a secondary contamination source, and putting

ecosystems at threat. Anthropogenic sources of metallic compounds in the natural

ecosystem include acid rain leaking from the soil, lead in gasoline, runoff from non-

point sources, atmospheric and industrial pollution, landﬁll, mining, manufacturing

operations, and processing of nuclear fuel (Ahmed et al. 2017).

Industrial wastewater affects the fauna and ﬂora of the water body with ground-

water eminence. Metals like zinc (Zn), cobalt (Co), copper (Cu), selenium (Se),

nickel (Ni), cadmium (Cd), vanadium (V), arsenic (As), mercury (Hg), lead (Pb),

and chromium (Cr) are dangerous to humans and other living organisms. The

hazardous chemicals should be eliminated from the industrial water to restore valu-

able compounds and to avoid negative signiﬁcances. In their elemental forms, several

metals like Cr, Cd, Pb, Ni, and Hg are severely toxic. (Patil and Bohara 2020). Thus,

the heavy metals leached quickly into water and it gets accumulated into the human

cell or tissues. Heavy metals are found in the gills, tissues, liver, and muscle of various

species of ﬁsh in contaminated marine bodies. Metals get accumulated in various parts

of the human body organs when they enter the food chain. Though most heavy metals

are frequently used in industries, people and employees living close by facilities are

possibly exposed and get contaminated. Heavy metals above the permissible limit,

may have negative signs on people, other organisms, and the whole ecosystem. In

food, the acceptable, permissible level of heavy metals has been allied to fewer human

health threats. Heavy metal pharmaceutical contaminants, phosphorus, bisphenol-A,

hydrocarbons, cations, textile dyes, detergents, nitrogenous elements, and pesticides

are chemical impurities that commonly exist in wastewater (Koedrith et al. 2013;

Gurav et al. 2019, 2021a; Choi et al. 2020a, b; Gurav et al. 2022).

With the growing number of contaminated sites worldwide, it is necessary to use

an effective approach to control the spread of pollutants and reduce toxin concentra-

tions. Innovative technologies should develop to harness the microbe’s capacity to

Removal of Heavy Metals from Industrial Wastewater Using … 379

extract pollutants from contaminated sites. Biological treatment includes the utiliza-

tion of indigenous microbes for the removal of organic matter in wastewater (Patil

et al. 2019a, b; Shah Maulin 2020; Liu et al. 2015). Bioremediation microbes-

based technologies are a substitute treatment and are ﬂexible, safe, economically

feasible, low-risk, and environmental friendly (Gurav et al. 2017). Microorganisms

mostly fungi, and bacteria, which degrade environmental contaminants into fewer

damaging types are identiﬁed as bioremediation. In bioremediation radioactive pollu-

tants are enhanced for remediation by altering them into less poisonous forms to

eliminate or immobilize the contaminants. The treatment needs naturally existing

microbes that decompose and utilize dangerous pollutants as a source of food for

their progress (Patil et al. 2019a, b). The process of bioremediation can be efﬁca-

cious in environments that consent to the activity of microorganisms. Depending on

its working, bioremediation is stated to be in-situ and ex-situ bioremediation. In-

situ bioremediation treatment of wastewater is carried out on spot, with negligible

excavation. In ex-situ bioremediation, the pollutant clearing and removing process

are done after it has been shifted to a suitable location by excavation or pumping.

In ex-situ the wastewater is collected from the contaminated site and in a distinct

location it is treated using physicochemical and biological treatment (Ali et al. 2013,

Shah 2021a, b). This book chapter presents heavy metals and their types, effects

of the industrial discharge of heavy metals on the water body and human beings,

conventional methods for heavy metals wastewater treatment. Bioremediation, types

of bioremediations, and advantages of bioremediation are also discussed in this book

chapter.

2 Industrial Wastewater and its Current Status

The universe is transitioning to a sustainable age. The wastewater which is produced

not only disturbs the environment but also reduces the pure water concentration that

will harm to the upcoming generations. To recover and reuse the wastewater there

is a need for eco-friendly and innovative wastewater treatments. In this universe,

there is a persistent need to overcome the water pollution problems. The concerns of

industrial discharges are far-extensive, and they are affecting living creatures. For the

working purpose, almost all industries require a huge amount of water (Sharma et al.

2021a, b). Water gains contact with toxic pollutants, hazardous waste, toxic metals,

and biotic sludge through several processes. This polluted water contaminates the soil

and negatively impacts the cropland, and also destroys the diversity of species that

existed in that area. Individuals who comes in contact with contaminated soil or water

also suffer prolonged health issues. Due to air pollution, there is a prominent growth

in numerous diseases, and it seems to affect us frequently. Air and water pollution

is affecting the atmosphere and health of individuals due to the arrival of medium,

small, and huge industries. Pollution due to industries damages the normal patterns

and cycles, signifying severe biodiversity consequences. Industries emit greenhouse

gases and smoke into the environment, which subsidizes the hazardous products,

380 P. M. Patil et al.

global warming, chemical waste, and other pollutants that continue to destruct the

universe and its inhabitants. Industrial emission also enriches the concentration of

heavy metals present in soil like cadmium, iron, etc. A comprehensive range of

heavy metals has been shown by polluted topsoil. The pre-treated industrial water

is generally discharged to the surface water body. The concentration of metals in

the bottom sediments and riverbanks is usually high. Unintended industrial develop-

ment, deﬁciency of pollution control guidelines, a huge number of enterprises, use

of outmoded machinery, and improper waste disposal are the signiﬁcant causes of

industrial pollution (Ali et al. 2020; Gurav et al. 2021b, c).

2.1 Distillery Wastewater

In distillery wastewater sugarcane juice, di-butyl phthalate, benzenepropanoic acid,

and 2-hydroxy caproic acid are being exist. Few of these contaminants like 2-hydroxy

caproic acid, di-n-octyl phthalate, and butanedioic acid dibutyl are responsible for

endocrine disruptors in living creatures. The several contaminants that existed in

distillery wastewater are genotoxic, cytotoxic, and carcinogenic and they should be

removed before disposing it into the environment. Generally, untreated disposal of

these contaminants into aquatic ecosystems results in high chemical oxygen demand,

biochemical oxygen demand, sulfate, high nitrogen, and phosphate content that

causes eutrophication. If these contaminants are disposed on the terrestrial ecosystem

results in inhibition in seed germination, soil acidiﬁcation, and less crop growth

(Zhang et al. 2021).

Advancement in technology has resulted in the development of organic treatment

methods these treatments signiﬁcantly reduce numerous problems of the environment

but are ineffectual in treating inorganic waste existing in wastewater. To cope with the

hazardous pollutants numerous physical, biological, and organic approaches can be

applied individually or in a mixture. Flocculation, coagulation treatment will reduce

biological load, color, and suspended solids but it is not solely effective f or inorganic

materials. Organic treatments are known to be eco-friendly, and they can be applied

aerobically and anaerobic for the treatment. The anaerobic treatment is effective for

reducing the organic load from wastewater, but an appropriate sample and microor-

ganism’s requirement is essential. The microbe bio-transfers the contaminants that

are existed in wastewater into comparatively less hazardous elements. Anaerobic

digestion can be done with conventional reaction methods like up-ﬂow anaerobic

sludge blankets or with continuously stirred tank reactors, anaerobic batch reactors,

single and biphasic systems, anaerobic ﬁlters, etc. (Singh et al. 2021).

Removal of Heavy Metals from Industrial Wastewater Using … 381

2.2 Pulp and Paper Industry

Wastewater from the paper and pulp industry is a huge source of heavy metals adulter-

ation in the environment all-round the universe. The extensive quantity of wastewater

has been produced from the paper and pulp industry due to the elaborate processing

of material at a diverse stage prominent to difﬁculty maintaining the contaminant

release levels laid by numerous environmental protection policies. Subsequently, the

discharge of wastewater into and environment have adverse impacts like scum forma-

tion, increase in temperature, slime production, odor, and decrease in aesthetic beauty.

In recent years due to advance technology the amount of wastewater discharge has

been reduced. The production scale also impacts wastewater quantity as compared

to bigger mills medium and small-scale mills generate less concentration of wastew-

ater. Thus, the pulp and paper industry wastewater lead to ecotoxicity so there is a

need to improve the manufacturing process and treatment amenities to overwhelm

the problem. Approximately a ton of paper production discharges 190–200 m3 of

wastewater with huge suspended elements and dissolved solids. In 2016–2017, the

Central Pulp and Paper Research Institute has reported that there are 850 pulp and

paper industries, comprising the minor paper industry. It characterizes the signiﬁcant

quantity of lignocellulosic waste and toxic metal contamination produced by these

productions in India (Zhang et al. 2021).

These treatments are known to be better in terms of performance and expense

in addition to being environment companionable, nevertheless, organic treatments

are not predominantly effective for reducing recalcitrant pollutants and color of

wastewater. The efﬁciency of these treatments is based upon the approaches like

biodegradability index, nitrogen to carbon ratio of generated wastewater. Hence,

more research is needed to express the norms for approving any treatment system

and attain current outcomes from the designated approach. Therefore, research is

also needed to implement strategies to eliminate recalcitrant contaminants from the

wastewater with the concurrent generation of energy like the bioelectricity, biogas by

sustainable technique (Gurav et al. 2020; Gurav et al. 2021d). Heavy metal contam-

ination in surroundings poses risk to zooplankton, microbes, phytoplankton, human

health, and wildlife due to recycling in the food chain. The paper factories generate

kraft paper, writing paper, and hardboard produces numerous metals, principally Mn,

Cu, Fe, Pb, Cd, Cr, and Zn as well as a huge concentration of unwanted waste. These

toxic metals have a robust binding propensity with lignocellulosic waste due to their

cations, causing an organometallic complex. Due to their composite composition,

these chemicals perform as insistent biological contaminants. As a result of their

lasting persistence and damage caused by continuing bio-concentration in animals

and plants tissue, these toxic metals and persistent organic contaminants create a

negative impact on the ecosystem, disturbing the food chain, food web by bioaccu-

mulation and biomagniﬁcation. Mostly in industrial sludge, these heavy metals are

being existed, which are enormously harmful to living creatures, and the environment

(Singh et al. 2021).

382 P. M. Patil et al.

2.3 Tannery Industry

In-universe, yearly tannery industry produced approximately 300 million tonnes of

wastewater. China individually produces around 1.2 hundred million tons of liquid

waste from approximately 1.4 hundred million tons of water disbursed in the tannery

processes yearly. Currently, liquid waste reduction and the development of a sustain-

able tannery industry are the main signiﬁcant factors to be pursued. Because of

its huge pollution, tanneries trades are levied upon severe guidelines concerning

the discharge of the contaminants. Countries like Southeast Asia, the USA, Europe,

China, and Brazil are more conscious and effective in following rules and regulations

about pollution. These nations have implemented and placed standards regarding the

limits of biological oxygen demand (BOD), chemical oxygen demand (COD), pH,

chromium, sulphide, etc. There are numerous processes in the tannery industry, which

contribute to generating a huge amount of wastewater generated like production,

processing, and manufacturing of leather each single step generates a speciﬁc quan-

tity of wastewater with a diverse concentration of pollutants. The generated water

from the tannery industry is extremely alkaline, with high COD, BOD, suspended

solids level, saline and contain a pungent odor, with dark color (Gurav and Jadhav

2013). In the tannery industry from the individual process, the wastewater is treated

distinctly before merging with the integrated liquid waste (Singh et al. 2021).

The primary treatment eliminates suspended solids, scarves, hairs, and other

biological matter intending to decrease the biological load for the secondary treat-

ment. By several methods, the primary treatment is performed like neutralization,

electro-dialysis, air ﬂoatation, adsorption, coagulation, micro-electrolysis, etc. Every

treatment system conﬁrms the toxic element removal that is existed in the liquid

waste and is ready to be progressed to the biological treatment. Secondary treatment

methods involve anaerobic and aerobic treatment systems applied to tannery liquid

waste followed by primary treatment methods. The numerous treatments systems are

included in secondary treatment methods like sequencing batch reactor, oxidation,

oxidation ditch, activated sludge, moving bed bioﬁlm reactor, microbial remedia-

tion, constructed wetland, and anaerobic treatment method with biogas production.

Almost all these treatments are eco-friendly and stable even in medium, small, and big

scale tannery industries but they can handle the low organic load. Secondary treatment

methods do not efﬁciently eliminate phosphorus and nitrogen from wastewater, and

numerous other contaminants require strong and advanced innovative technology. In

tertiary treatment, by applying membrane ﬁltration and oxidation the contaminants

are removed. Above both treatments, effectively eliminate the lasting pollutants to

comply with the set level of standards. Still, they are expensive, suffer drawbacks like

membrane fouling, and need further expansion to develop their performance (Ayele

and Godeto 2021).

To decrease wastewater, various tanneries throughout the world have applied

procedures to make appropriate resources use like liquid waste recycling or applying

it for washing ground. Few recognized uses of tannery-treated liquid waste are irri-

gation to sunﬂower ﬁelds and tannery liquid waste in microbial fuel cells. To ensure

Removal of Heavy Metals from Industrial Wastewater Using … 383

the lower discharge of tannery liquid waste, to reduce the pollutants, and for effective

consumption of tannery waste, research and measures are still needed to be executed

throughout the complete tannery industry process. Approaches to ensure cleaner

manufacture are required, including hair preservation, salt reduction, low chrome

tanning, and salt reduction (Gurav et al. 2016). Lessening in COD, BOD and concur-

rent mitigation of phosphorus and nitrogen must be safeguarded while treating the

environmental balance of the receiving aquatic body or land (Meruvu 2021).

3 Types of Heavy Metals

Pollutants exits in the industrial efﬂuent are classiﬁed as organic and inorganic

contaminants that exist a huge range of toxic levels in it. Chemical, physical, and

biological treatments are commonly applied in treating organic pollutants (Lyu et al.

2016; Zhang et al. 2016). But the above methods are not appropriate for treating

inorganic contaminants like heavy metals. Since they have qualities like oxidation,

reduction, complex formation, and solubility, the decomposition of heavy metals

plays a primary concern (Lu et al. 2020). The element which has an atomic mass

between 63 and 200 and speciﬁc gravity of more than 5.0 speciﬁes that the compound

is heavy metal. In the environment, they exist as a natural compound. The heavy metal

word refers to the compound whose density is higher and toxic or harmful even at

less concentration. In current years, heavy metal pollution has become a major threat

to the environment, because even at a very minute level high risk relates to human

health and the ecosystem. Because of its accumulation, endurance, magniﬁcation,

and ﬂexibility, it is a huge burden to the environment. Toxic metals either exist in a

mixed or chemical form that is challenging to eliminate from the wastewater (Zhang

et al. 2021). The presence of heavy metals in the open aquatic bodies tends to algal

blooms, oxygen deﬁciency, and the end of ﬂora and fauna. The discharge of heavy

metals into rivers converts into hydrated ions which have an extremely toxic property

than metal elements. The hydrated compounds interrupt the enzymatic process and

immersion is more rapid (Soliman and Moustafa 2020). So heavy metal removal

is obligatory to lower the risks of living organisms. To limit the pollution levels of

water, World Health Organization (WHO) and Environmental Protection Agency

(EPA) have recognized the most allowable discharge of heavy metal levels into the

surrounding. So far if the discharged efﬂuent comprises heavy metals with high

concentrations, then the allowable limits will cause environmental and human health

problems (Sharma et al. 2021a, b).

3.1 Cadmium

Cadmium exists in natural deposits, industrial efﬂuent and is known to be the most

toxic metal. It has a key role in plating, nickel–cadmium batteries, alloys, stabilizers,

384 P. M. Patil et al.

and phosphate fertilizers industries. In the eco-system, even at less concentration,

the compound of cadmium is harmful. Due to cadmium “Itai-Itai” disease is induced

by accumulation of cadmium in the aquatic bodies and occurs bones softening and

fractures to individuals (Sharma et al. 2021a, b). Furthermore, they bring out special

effects like lung cancer, hepatic toxicity, and produce harm to the liver, respiratory

system, reproductive organs, and kidney. So efﬁcient and economically consistent

treatment is necessary for cadmium removal from the wastewater (Ali et al. 2020).

3.2 Chromium

On earth the utmost existing seventh element is chromium. It occurs in the ore

form, which is composed of crocoite (PbCrO4), chrome ochre (Cr2O3), and ferric

chromite (FeCr2O4). In tanning industries, textile industries, electroplating indus-

tries, and leather industries are the major sources of chromium. The above manufac-

turers generate hexavalent chromium Cr (VI), and trivalent chromium Cr (III) but for

animals, plants, and other organisms the hexavalent chromium is more injurious than

trivalent chromium. Cr (VI) exists mostly in salt chromate production and Cr (III)

is beneﬁcial in fat metabolism and shows a chief part in sugar (Ayele and Godeto

2021). The above-mentioned forms are used in the chrome plating, glass industry,

steel production, wood conservation, plating and electroplating, pigment fabrication.

Chromium metal functions as a titrating agent, cleaning agent, and additive in-mold

construction and fabrication process in magnetic tape. When humans are exposed

to chromium it causes kidney and liver damage, skin inﬂammation, ulcer creation,

vomiting, and pulmonary congestion. So, from wastewater chromium should be

signiﬁcantly removed before it reaches the environment or it should be modiﬁed into

a low toxic form (Singh et al. 2021).

3.3 Nickel

Nickel is a hard silver metal with 28 atomic numbers. It is non-biodegradable and

persistent heavy metals mostly exist in industrial wastewater. The industrial sources

of wastewater are silver reﬁneries, printing, electroplating industries, alloy industries,

and battery manufacturing industries. Nickel is also used in several applications

like jewelry, catalysis, alloys, batteries, coins, machinery parts, and resistance wires

(Goswami et al. 2021). The utilization of nickel in various appliances creates a threat

to the environment and humans. The human effects of nickel are chest pain, nausea,

dry cough, breathing problem, skin eruption, renal edema, gastrointestinal ache, and

pulmonary ﬁbrosis. To avoid environmental and health risks, a striking and innovative

technology is necessary to recuperate the nickel metal (Meruvu 2021).

Removal of Heavy Metals from Industrial Wastewater Using … 385

3.4 Lead

Lead is a soft and heavy metal that occurs in the cerussite, galena sulﬁte form. The

chief source of the lead association in the industrial efﬂuent is primarily due to

lead-acid batteries. Lead exists often in wastewater from electroplating industries,

steel industries, electrical industries, and explosive manufacturers (Aibeche et al.

2021). It affects DNA and protein synthesis and cell replication. It is known to be

hazardous and in the body of humans, it gets readily collected. It generates illnesses

like damaging the nervous system, kidney damage, cancer, and mental retardation. To

animals and plants, exposure to lead is dangerous and causes environmental pollution.

So, numerous researchers around the globe are focusing their ideas on treatment for

lead removal from wastewater (Sharma et al. 2020).

3.5 Copper

Copper is considered toxic when it is at a high concentration. It is the vital element

needed for living organisms and plays a necessary role in the synthesis of an enzyme,

in bone and tissue development. There are different forms of copper like cuprous

ions, metal, and cupric ions from these cupric ions are more hazardous and toxic

for humans than that for others (Zvab et al. 2021). The chief contributors of copper

are metallurgy, mining industries, electroplating industries, chemical manufacturing,

printing circuit, steel industries, fertilizers, and paints. The human effects of copper

are anemia, hair loss, headache, kidney damage, brain, and liver damage, and even

death. So, for copper recovery from wastewater satisfactory treatment technology is

necessary (Wang et al. 2019).

3.6 Zinc

Zinc controls the physiological mechanisms and biochemical operations of human

tissues. For other metals, zinc functions as a decorative and protective layer for steel

zinc acts as an anti-corrosive agent. In various coal combustion, steel processing,

and mining industries zinc is exploited. Although it is necessary for trace levels in

organisms if it is more than limited health disputes such as vomiting, pain, anemia,

skin inﬂammation, and fever are detected. The zinc industrial sources, accountable

are pulp and paper, steel making industries, electroplating industries, and brass work

industries. The above-stated things were collected with the desirable for zinc removal

active treatment (Areco et al. 2018).

386 P. M. Patil et al.

3.7 Mercury

Global awareness regarding mercury pollution has enhanced due to the hazardous

incidents that occurred in Japan. The extremely toxic and hazardous heavy metal

contamination in the water stream is mercury. In various forms, mercury exists

in the environment like mercurous ion, elemental mercury, and mercuric ion. The

transportation of mercury metal will contaminate, damage, disturb the entire water

stream (Zhao et al. 2021). As it is available from industrial sources that pollute the

environment so to preserve the environment and human health in 2013 the Mina-

mata convention is weighed up. This convention has synchronized the materials

comprising mercury fetch out stricter standards emission. Methyl mercury disturbs

the synthesis of protein and harms the sites of enzymes. Mostly, from plastic indus-

tries, pulp and paper, oil reﬁneries, pharmaceutical and chloro-alkali industries more

concentration of mercury is discharged into the environment. The probable mercury

consequences to humans are damage to the brain, kidney, respiratory and reproduc-

tive systems. Hence, the researchers have gained more devotion to removing mercury

from industrial wastewater (Matsuyama et al. 2021).

3.8 Arsenic

Arsenic exists in rock, topsoil, midair, and water. Organic arsenic composites are

chieﬂy exited in ﬁsh. Inorganic arsenic exits in groundwater further consumed for

domestic and drinking purposes. Arsenic human exposure is signiﬁcant via food

consumption and potable water. Foodstuff is known to be the imperative arsenic

source, inorganic arsenic is departures in potable water and through potable water, and

individuals get wide-open to arsenic. Arsenic disclosure occurs through polluted soil

like mine tailings (Ostermeyer et al. 2021). Contingent upon the solubility and particle

size of arsenic the inhale in airborne absorption is dependent. Arsenic is the soluble

form that is effortlessly immersed by the gastrointestinal tract and arsenic is ethylated

and metabolites through urine are evacuated. Arsenic concentrations in nails, blood,

hair, and urine, are used as exposure biomarkers. Great absorption of arsenic in

the human body damages the liver, kidney, rises cardiovascular, immunological,

metabolic disease (Sher et al. 2020).

4 The Effects of Heavy Metals Released from Industries

on Natural Water Bodies and Living Organisms

Heavy metals like nickel, arsenic, antimony, zinc, cadmium, chromium, etc. are

hazardous at greater concentrations and induce toxic sound effects on the biotic

components. Ionic forms of metals react with biological molecules in the living

Removal of Heavy Metals from Industrial Wastewater Using … 387

body and form more toxic compounds. The toxic characteristics of these elements

depend on the bioavailability, and critical concentration which is elevated through

ecological progressions like bioaccumulation, and biomagniﬁcation. In many cases,

biomagniﬁcation affects ecosystem composition and services. Oxidized forms and

the ligand state of heavy metals play a vital role in the accessibility of toxic metals.

When the heavy metal levels are beyond the threshold level, the heavy metal becomes

toxic and disrupts metabolic reactions at the cellular level which affects the organ

and organism. The injuriousness of metals touches the delicate organs and disturbs

the nervous role, harms the blood content, affects kidneys, lungs functions, and other

organs (Ahmed et al. 2012). This consequently results in a softness, loss of memory,

an upsurge of allergies, raise blood pressure. Death of cells takes place due to the

free radicals formation which is signiﬁcant for the oxidative mechanisms in the body.

Thus, heavy metals can change the biological composition of ecosystems and how

living organisms interact with one another and their surroundings. Subsequently, it

may result in the threatening of ecosystem services. Several regulatory bodies have

adopted the permissible limits for the discharge of heavy metals. The researchers have

concentrated on the development of innovative treatment techniques. If not properly

treated, the fate of heavy metal-loaded wastewater will be entered the ecological food

chains from the polluted water and soil resources (Damtie et al. 2018).

The entry of these elements in the biotic communities and ecological food chains

is determined by various environmental factors. Uptake of these elements depends on

the chemical property, solubility, bioavailability, chelation, properties of the medium,

etc. The problem associated with heavy metals is that they may accumulate in the

creature’s bodies (microbes, ﬂoras, and animals) without killing them. If this living

organism receives small quantities of toxic metals in food, then they will not elimi-

nate, their concentration within the body of the organism increases. This process of

accumulating higher and higher amounts of material within the body of the organism

is called bioaccumulation. The small quantity of ﬂoras is called hyperaccumulators

that effortlessly absorb the great levels of heavy metals from the surrounding. If

the above ﬂoras are harvested the much more concentration of toxic metals will get

reduced. Soil acidity (pH) is responsible for plants metal uptake. If the acidity is high

the mobility and solubility of metals are more, ultimately, they are more accumulated

and uptake by plants (Carlos et al. 2018).

The unusual changes in the coloring and growth patterns are signs that indicate

the plants are polluted and adulterated with metals. In the body of carnivorous and

herbivorous organisms, the metals enter through eating food, ﬁsh, meat, leafy vegeta-

bles, and by drinking milk, water with an eminent level of metals. From the creature

body, the toxic metals are not eliminated, they get accumulated in the organisms

organs, bones, skin, and hair. Lowest feeders predominantly accumulate these toxic

metals and with sediments, the metals are ingested. The seaweed from sediments,

and aquatic surroundings accumulate the metals. Thus, when they enter the food

chain, this phenomenon of increasing levels of metals in the bodies of higher trophic

level creatures is recognized as biological ampliﬁcation. As humans are at the end

of food chains, they are more exposed to metal adulteration from soil and aquatic

388 P. M. Patil et al.

water bodies. Due to their persistence, their effects on organisms at higher trophic

levels, concerns about long-term human health problems (Liu et al. 2019).

4.1 Effect of Heavy Metals on Human

The Environmental Protection Act (EPA) has revealed that cadmium and its

compounds, mercuric chloride, lead, methyl mercury are probably suspected to be

carcinogens. To all the mercury forms the individual’s nervous system is sensi-

tive. Great concentration exposure permanently harms the brain, developing fetus

is damaged, and kidney failure. The brain effect may result in tremors, irritability,

memory problems, vision change, hearing loss, shyness. Metallic mercury high-level

exposure in short term causes diarrhea, lung damage, high blood pressure, eye irri-

tation, increase in heart rate, nausea, skin rashes, diarrhea. Disclosure to cadmium

diarrhea, vomiting, breathing difﬁculties, abdominal cramps, high or low blood pres-

sure, weakness in the muscle, and face numbness. Large amounts of barium intake

can cause, high blood pressure, changes in heart rhythm or paralysis, and life loss (Wu

et al. 2016). The arsenic is odorless and tasteless. Minor level exposure decreases

the production of red and white blood cells, irregular heart rhythm, nausea, blood

vessels injury, vomiting, and deadness of ‘pins and needles in hands and feet. Lead

can affect every body organ. Lasting lead exposure results in decreased performance

of a nervous system, weakness in ﬁngers, wrists, or ankles, small increases in blood

pressure, and anemia (Kondzior and Butarewicz 2018). Disclosure to high lead levels

can severely damage the kidney and injure the brain and ultimately cause death. In

pregnant women, great levels of exposure to lead may cause miscarriage. High-

level exposé in men can damage the organs responsible for loss in sperm genera-

tion. Ingesting high concentration and long-term acquaintance even to low concen-

tration severely leads to and build-up possible kidney disease, lung damage, and

fragile bones. Table 1 and Fig. 1 represents impact of heavy metals on humans and

ecosystems is represented (Singh et al. 2017).

5 Conventional Treatment and Technologies for Heavy

Metal Removal

The discharge of contaminants in water can be reduced by following adequate proce-

dures. Due to its inhibitory characteristics, to remove the contaminant from wastew-

ater high elimination enforcement treatment is required. The industries are facing

more difﬁculties in reducing the contaminants from discharge. Therefore, to conserve

and protect the surrounding, various technological methods have been innovated by

researchers like ion exchange, dissolved air ﬂotation, membrane bioreactors, reverse

osmosis, etc. Depending upon the efﬂuent type, and heavy metal concentration, the

treatments is applied to the wastewater (Chai et al. 2021).

Removal of Heavy Metals from Industrial Wastewater Using … 389

Tabl e 1 Impact of heavy metals on humans and the ecosystem (Jia et al. 2018; Damtie et al. 2018;

Kondzior and Butarewicz 2018)

Heavy metals Impact on humans Impact on ecosystem

Cu Damage liver and kidney, headaches,

dizziness, nausea, stomach cramps,

vomiting

Affects the water purity, root growth

Cr Hyperaemia, renal failure, cancers,

necrosis, acute tubular necrosis,

histiocytic inﬁltration, and lymphocytic

Damage the nutritional cycle of the

ecosystem

Hg Behavioral and neurological illnesses

like tremors,

memory loss, and insomnia

Seed germination in the plant is

reduced, chlorosis; reduces the plant

height

Co Effect on pulmonary functions, and eye Declined the starch, amino acids sugar,

and protein content

Cd Damage the bones and kidney Affect the rate of germination and

declined the quality of water

As Pregnancy damage, cancer, and

skin problems

Reduced the plant and leaf health,

bio-accumulate, and biomagniﬁes in

organisms

Hg Behavioral and neurological illnesses

such as tremors, insomnia, and

memory loss

Seed germination in the plants is

reduced, chlorosis, improper in

development plant

Ni Reduced the function of the lung,

respiratory ache Syndrome, lung and

nasal sinus cancer

Enzyme activity is decreased which

affects the CO2 ﬁxation and Calvin

cycle

Mn Parkinson’s diseases affect the rate of

mortality

The concentration of chlorophyll is

decreased

Zn Hair loss, necrosis, etc Sugar concentration is declined,

carotenoid, decrease in starch, amino

acid, and chlorophyll content

Pb Abdominal pain, hypertension, birth

ﬂaws fatigue, mental retardation,

sleeplessness, hallucinations, paralysis,

weight loss, vertigo, alteration in the

function of the nervous system, and

renal dysfunction

Effect on the enzyme action of

organisms; biomagniﬁes in living

organisms, declines the quality of water

5.1 Ion Exchange

Ion exchange is the separation treatment, where the ions are replaced with other ions,

and metal ions are removed from the industrial efﬂuent. The deposition of sludge

is low in the process of ion exchange as compared to coagulation treatment. Ion

exchange resin is used to eliminate the material from water. The resin is designed

in stress-free and strain form to avoid natural decomposition. Through cross allied

polymer, matrix resins are designed which are attached with functional groups and

covalent bonding, and space in resin structures permits the ions to shift applicably.

390 P. M. Patil et al.

Fig. 1 Effects of heavy metal on humans and the environment

The resins are categorized into two types natural and synthetic resins from these

two in the treatment process one resin has been utilized to interchange the metal

ion with cation (Bashir et al. 2019). For the separation of metals from efﬂuent than

natural resins, synthetic resins are extensively preferred. The synthetic resins fore

mostly remove the arsenic metals from a waste liquid. The cationic exchanger is

widely preferred which includes weak basic and strongly acidic resins. In acidic

resins, the sulfonic acid is an exit and in basic resins carboxylic acid is present. The

metal cations and transmutable ions are delivered by hydrogen ions. Natural zeolites

have great cation exchange capability for heavy metal removal from the waste liquid.

The structure of zeolite is in crystalline form which includes silicate and aluminum

atoms linked with oxygen bridges. Zeolites remove a great amount of chromium

metal from wastewater, and it is also applicable for Pb, Cu, Zn, Cd. From the efﬂuent

98% of heavy metals are eliminated by zeolites. Hence the ion exchange technology

has been applied in various industries and an abundant quantity of efﬂuent is treated

for efﬁcient heavy metal exclusion (Pan and An 2019).

5.2 Dissolved Air Flotation (DAF)

In dissolved air ﬂotation the accumulation of suspended particles has raised by tran-

siting the air bubbles in the liquid that can be effortlessly separable from the shallow

of the water. Surfactants are supplemented in the process to surge the performance

and enhanced the accumulation between negatively charged ﬂocks with positively

charged air bubbles. In DAF organic polymers are extensively utilized and on the

particle surface during the agglomeration phase, the monolayer is formed which is

Removal of Heavy Metals from Industrial Wastewater Using … 391

signiﬁed by the polymer chain length and molecular weight. Various types of poly-

mers are utilized in DAF like polyvinyl alcohol (PVA), chitosan, and polyethylene

glycol in the DAF, modiﬁed PVA for Cd, Mn, Zn, Ni, Pb (Pooja et al. 2021). By

applying chitosan to waste liquid in DAF 29% of Cd, 29% Pb, 31% Mn, and 27%

Ni, were removed. In modiﬁed PVA treatment extensively Ni and Zn are removed

and this treatment help in the elimination of other heavy metals. In DAF treatments

the micro and nanobubbles are utilized from heavy metals exclusion. With the help

of DAF treatment exclusive concentrations of heavy metals are eliminated from

the industries so, this treatment has the prospective to conﬁscate the heavy metal

overloaded with wastewaters (Azevedo et al. 2018).

5.3 Membrane Filtration

In membrane ﬁltration, pressure-driven departure treatment is applied to waste liquid.

Rather than heavy metal elimination, disinfection also occurs in this method. In

membrane ﬁltration, the particles are separated based on their concentration, pres-

sure, size, and pH. By merging the membrane with chemical solutions, the mechanism

of ﬁltration is enhanced. The membrane consists of a precise porous surface which

has a crucial role in eliminating heavy metals from polluted water. The material of the

membrane is categorized into two types namely, polymer and ceramic. The ceramic

membrane is preferred mostly in treating industrial efﬂuent than polymer membrane

because it is expansively resistant to a chemical due to its hydrophobic capacity.

In polymeric membrane polypropylene, polyethylene, and polyvinylidene ﬂuoride

materials are mostly used due to their hydrophobic nature and they can foul effort-

lessly (Almasian et al. 2018). The inter collaboration between the heavy metal and

polymeric membrane is great. Dependent upon the membrane pore size, the perme-

ability of the membrane is achieved. The solute is on one cross and a pure solution

is on the other side. This treatment is considered an optimistic technology due to

its ease of operation, efﬁciency, and it requires less space. Supplementary organic

elements and suspended solids are also eliminated in this treatment. The organic

matter (OM) and dissolved organic matter (DOM) are major exits in the membranes.

To enhance the membrane performance and to remove the OM, DOM efﬂuent should

be pretreated. So, to separate non-polluting material some focused membrane ﬁlters

are exploited like nanoﬁltration, microﬁltration, reverse osmosis, and ultraﬁltration

to segregate the heavy metal from efﬂuent depending upon its size. These methods

can grip the extent volume of aqueous liquid for heavy metal elimination (Efome

et al. 2018).

392 P. M. Patil et al.

5.4 Reverse Osmosis (RO)

In reverse osmosis, the charge exclusion and size exclusion principal work. The

semi-permeable membrane is used for dissolved species removal and waste liquid is

passed through the membrane. The range of pore size in RO is from 0.1 to 1.0 nm.

For the process of desalination, it is greatly used. In treating industrial waste solu-

tions for heavy metal removal reverse osmosis has extensively been used. In RO

membrane various modiﬁcations have been carried out to treat the electroplating of

an aqueous solution. The treatment efﬁciency of RO membrane is reliable on pres-

sure, pH, membrane material, temperature, and clogging properties of the membrane

(Thaçi and Gashi 2019). To prevent membrane fouling, wastewater should be pre-

treated to remove the colloidal and surface particles from the waste solution. For

the last ten years, this method has been applied for treated wastewater. Before RO

treatment ultraﬁltration is applied to the waste solution to eradicate the clogging

problem. This RO technology removes 92.3–99.8% of the organic pollutants, heavy

metals, and inorganic materials from waste solutions. The RO removes most of the

contaminants from the waste solutions hence it is preferred in extensive industries

and for commercial purposes (Wang et al. 2016).

6 Bioremediation and its Mechanism

Bioremediation is a biotechnological intervention for the cleanup of the polluted

sites, which can be done in situ as well as in ex-situ form. It relies on the integral

characteristics and abilities of indigenous plants, fungi, and bacterial species. It has

applications on the global, regional and local scale for preventing future pollution.

For groundwater protection and soil conservation in current years bioremediation

has been used. Bioremediation is a grouping of technologies that utilizes microbiota

mostly, fungi, and heterotrophic bacteria to decompose or to modify the hazardous

contaminants into other substances which are fewer hazardous byproducts than the

parent substance. The beginning of bioremediation is the massive natural ability of

microbes and plants to degrade organic forms and to accumulate inorganic forms of

pollutants (Abatenh et al. 2017). The removal mechanism called bioaccumulation

is the accumulation of substances or chemicals in an organism. There are a larger

number of plants called hyperaccumulators that signiﬁcantly absorb high concentra-

tions of heavy metals from the soil environment. If such plants species are grown

the living organisms will get more explored to these harmful metals, so most caution

should take while utilizing these plants for animal and human use. The treatment

site should be protected from the entry of wild animals. Thus, the entry of these

materials into the food chain and unintended environmental consequences can be

avoided (Bhatt et al. 2020).

The mechanisms used for microbial bioremediation are toxic metals sequestra-

tion by components of the cell wall or by intracellular metal-binding peptides and

Removal of Heavy Metals from Industrial Wastewater Using … 393

proteins like phytochelatins and metallothioneins (MT) along with elements like

bacterial siderophores which are generally catecholate, related to fungi that generate

hydroxamate siderophores. Altering biochemical pathways to block metal uptake

may be a second mechanism. Transformation of metals by enzymes and reduction of

intracellular concentration of metals using precise efﬂux systems is the consequent

mechanism (Bhatt et al. 2020).

The capacity can be improved by applying genetically modiﬁed microbes and

plants. As the toxic waste material remains in the vapor, liquid, or solid-state, the

bioremediation technique differs depending upon the type of waste, the concentration

of waste, and the site. When site conditions are not suitable, bioremediation requires

the construction of engineered systems to supply materials that stimulate microbial

as well as plant growth. Physiochemical conditions are optimized in engineered

bioremediation (Kumar and Dwivedi 2021).

Various advantages are offered by applying the bioremediation process. Bioreme-

diation solely relies on natural processes, and the threat to the ecosystems is reduced.

In bioremediation to clean up the pollutants or impurities, the microbes are emended

in ex-situ and in situ way. In the bioremediation process, the organisms or habitat is

not disturbed and it is known to be a cheaper clean method because in this method

there is no need for substantial labor or equipment. This process does not create any

pollution or hazardous substance. Mostly the contaminant and hazardous products

are converted into a sustainable f orm. In the case of inorganic bioremediation inor-

ganic metal elements are collected, after being concentrated by the accumulators,

and can be reused, or properly disposed of (Azubuike et al. 2016).

7 Interaction Between the Metals and Microorganisms

Microbes are involved in the surrounding fate of heavy metals, inducing transitions

between insoluble and soluble phage through a range of biological and physico-

chemical mechanisms. These methods are signiﬁcant components of normal biogeo-

chemical cycles for metalloids and metals, and a few of them may be applied to treat

contaminated material. Through reduction, methylation, dealkylation, and oxidation

microbes can alter metalloid and metal species. Metalloids undertake two major alter-

ations: (i) decrease of metalloid oxyanions to chemical form like SeO2−3toSeO,

and SeO2−4 (ii) metalloid methylation, organometallics to methyl products, metalloid

oxyanions, like AsO−2, methylarsonic acid to (trimethylarsine) (CH3)3As, AsO−2.

The surface phenomena, bio-sorption retaining dead biomass, and application by

alive biomass are examples of microbe metal interaction. The existence of dissimilar

resistance mechanisms in microbes is a decent survival strategy for microbes to alter

metal in the existence is even a great application in the soil and aqueous system

(Azubuike et al. 2016). Economically, innovative practical biomass restoration and

transformation of recovered metal into functional form are the outstanding selec-

tions for biosorption treatment. Additionally, remediation of metal from polluted

394 P. M. Patil et al.

soil demanded a great assimilated approach combining in situ and ex-situ tech-

niques. To cope with these complex environmental issues, advanced development

and supreme practicable innovative technologies are needed. The genetic and physi-

ological foundation of microbe metal interactions, the microbial treatment seems to

be the solution. Methods are often biogeochemically crucial because they alter the

toxicity and stability of metals and contain biotechnological probable in bioremedi-

ation. Due to early toxic metal exposure, numerous microorganisms are presumed

to have advanced metal resistance. The resistance of metal in the last 50 years is the

outcome of increased exposure to metal contamination. Due to human activities, the

heavy metal concentration has enhanced in the environment, but active microbes have

developed the metal tolerant capacity and detoxifying approaches. A greater number

of metal resistant and tolerant microbes were found in contaminated water, and soil.

In recent years for the removal of excess chemical, physical parameters, and harmful

toxic metal, the application of metal resistant and tolerant bacteria has attracted

great interest (Kumar and Dwivedi 2021). Metal resistance elements in microbes

are found on plasmids, which are certainly transmissible and extent by horizontal

transfer. The essential resistance genes can be introduced into the appropriate area by

genetic transfer method for manufacturing commercial inoculants, or use of enzymes

for soil and water bioremediation. Bacterial adaptive responses to ecological stress

and molecular processes of variation have been researched using molecular (PCR-

based) and phenotypic methods. In the above section recent advances in microbe

metal interaction, with special reference to metal toxicological effects on living

creatures, responses of microorganisms in developing metal resistance and tolerance

mechanisms, and applications of microorganisms in metal-contaminated environ-

ment bioremediation is explained (Verma and Kuila 2019). In Table 2, possible

microbes for the great tolerance of heavy metals are described.

Tabl e 2 Probable microbes for the great tolerance of heavy metals (Sharma et al. 2021a, b)

Sr. no Heavy metals Microbes

1Cu Kocu ria sp. CRB15

2 Pb, As, Cu Microbacterium sp. CE3R2, Curtobacterium sp. NM1R1,

3Cd Flavobacterium sp., Rhodococcus sp., Klebsiella pneumoniae

4Hg, Cd, Ag Pseudomonas putida

5Cd Enterobacter, Klebsiella, Leifsonia, Bacillus

6Ni Bacillus licheniformis

7Cr, Co, Mn, Pb Pseudomonas moraviensis, Bacillus cereus

8Fe,Ni, Cr,Zn Bacillus sp. PS-6

9Zn, Cd Chryseobacterium humi, Ralstonia eutropha

10 Pb Bacillus sp. MN3-4

11 Pb, Cd Azospirillum

12 As, Cd Mesorhizobium huakuii

Removal of Heavy Metals from Industrial Wastewater Using … 395

8 Types of Bioremediations

Various types existed in the bioremediation process. Bioremediation can be done

by applying bacteria, fungi and in the phytoremediation process, numerous plants

species have been used for the treatment of contaminated sites or to purify the pollu-

tant material. In treating the hazardous substance or site the above methods are

combined and the researchers have modiﬁed the process and treatment is imple-

mented (Sharma 2021). In Fig. 2 types of bioremediations are represented and the

details of the methods are deliberated under the subsequent heads.

8.1 Microbial Bioremediation

The heavy metal-microbe interactions are the basis of bioremediation. Microorgan-

isms are ubiquitous and play a key role in the environmental fate of heavy metals. All

types of microorganisms like bacteria, protozoa, fungi, algae, and lichens can mediate

some types of interactions with heavy metals which can be explored in bioremedia-

tion. During their contact, heavy metals strongly affect the activities and survival of

these organisms. High concentrations of many heavy metals produce harmful effects

on many microbial species, and heavy metals are toxic to many microorganisms even

Fig. 2 Types of Bioremediations

396 P. M. Patil et al.

at very lower concentrations. On the other hand, heavy metal-resistant microorgan-

isms with their varying phenotypic expressions also inﬂuence the heavy metals in

the environment (Verma and Kuila 2019).

Unlike bioremediation of oil, solvents, and pesticides which works as a source of

food, energy, and hydrolyze contaminants which relies on stimulating the growth of

certain microbes, in the case of heavy metals these are absorbed and accumulated

inside the microbial body. Bioremediation requires a combination of the factors like

nutrients, moisture, pH, osmotic balance, aeration, and the right temperature. The

absence of these elements may prolong the process. Conditions that are unfavorable

for bioremediation may be improved by adding amendments to the treatment site,

such as molasses, vegetable oil, or simply air. These amendments optimize conditions

for microbes to ﬂourish, thereby accelerating the completion of the said bioremedia-

tion process. The presence of heavy metals at elevated concentrations of toxic chem-

icals, high salt concentration adversely affect bioremediation. So, the technology is

feasible only for diluted metal contaminants (Ojuederie and Babalola 2017).

Bacterial diversity, ubiquitous nature, augmentation, and easy handling of bacteria

have a bright future in heavy metal remediation of contaminated sites and wastewater.

Acidic wastewater containing heavy metals, rich in sulphates is fed to the bioreactors

which are speciﬁcally designed to facilitate microbial bioremediation. Bacteria in the

bioreactors produce hydrogen sulﬁde, which is circulated to the metal precipitation

tank where metal ions bound to sulfur forms metal sulﬁdes which are least soluble and

precipitated out from the water. These sulphides are collected and processed further to

harvest minerals of economic value. The treated water after bioremediation becomes

pure enough to meet safety standards so safely discharged and clean enough to use

for agriculture and other related activities (Bharagava et al. 2019).

Biosurfactants are compounds produced by microbes like bacteria and fungi,

which act as biological complexing agents for various heavy metals. Both biosurfac-

tants and biosurfactant-producing microorganisms can be explored at heavy metal-

contaminated sites for their removal by washing and ﬂushing for ex-situ bioremedia-

tion. Bio-volatilization is a process that converts heavy metals into volatile derivatives

using the catalytic activity of microorganisms. This process offers treatment for As

and Hg contamination. Moreover, bio-volatilization has also been reported for other

heavy metals like Bi, Sb, and Tc. Biosorption is a passive uptake process to bind

heavy metals on the cellular structure of the biological mass. It involves physical and

chemical attachments with some selected bio-functional groups, where ion exchange,

covalent bonding, complexation, electrostatic attraction, Van der Waal’s force, and

microprecipitation play important roles in their interactions. For this process, a great

variety of inactive and nonliving microbial biomass has been explored with a diverse

range of biosorption capabilities. But pH, temperature and ionic strength of the solu-

tion, porosity, prehistory, and pretreatment of biosorbents, concentration and speci-

ation of heavy metals strongly inﬂuence the capacity, and adsorption rate. Chemical

modiﬁcations of biomass with accelerated binding capacity and afﬁnity for heavy

metals have a promising future. Table 3 represents the bacteria and fungus used in

the bioremediation process (Wang et al. 2020).

Removal of Heavy Metals from Industrial Wastewater Using … 397

Tabl e 3 Microorganisms used in bioremediation process (Wang et al. 2020; Bharagava et al. 2019;

Verm a a nd K uila 2019)

Heavy Metal Microorganisms Fungus

Pb Bacillus subtilis, Micrococcus luteus,

B. ﬁrmus, Aspergillus niger, B.

megaterium, Brevibacterium iodinium,

Penicillium species, Streptomyces spp.,

Staphylococcus spp., Pseudomonas spp.

Candida sphaerica

Cd Alcaligenes faecalis, B. megaterium,

Pseudomonas aeruginosa, Bacillus

subtilis

Coprinosis atramentaria

Cu Streptomyces sp., Staphylococcus sp.,

Enterobactercloacae,

Methylobacterium organophilum,

Desulfovibrio desulfuricans, A. faecalis

(GP06), Enterobactercloaceae,

Flavobacterium spp., Arthrobacter

strain, Micrococcus sp., Gemella spp.,

Pseudomonas aeruginosa Micrococcus

spp., Pseudomonas sp., Flavobacterium

spp.

Aspergillus versicolor, Sphaerotilus

natans, Aspergillus niger (pre-treated

with Na2CO3), Candida spp.

Aspergillus niger

Ni Acinetobacter sp., Micrococcus sp.,

Desulfovibrio desulfuricans

Pseudomonas spp.

Aspergillus spp., Aspergillus niger,

Aspergillus versicolor,

Aspergillusniger (0.2 N) pre-treated

with Na2CO3), Candida spp.

Hg Pseudomonas aeruginosa, Klebsiella

pneumoniae, Vibrio parahaemolyticus

(PG02), Vibrio ﬂuvialis, Bacillus

licheniformis

Candida parapsilosis

Cr Acinetobacter spp., Bacillus cereus,

and Arthrobacter sp.

Aspergillus niger, Saccharomyces

cerevisiae, Rhizopus oryzae,

Aspergillus versicolor, Penicillium

chrysogenum, Sphaerotilus natans,

Phanerochaete chrysosporium,

Saccharomyces cerevisiae, Hansenula

polymorpha, Yarrowiali polytica, S.

cerevisiae, Rhodotorula pilimanae,

Rhodotorula mucilage, and

Pichiaguillier mondii

Zn Pseudomonas spp., Bacillus ﬁrmus

Co Enterobacter cloacae

8.2 Algal Bioremediation (Phycoremediation)

Algal bioremediation the known to be an innovative treatment for heavy metal elim-

ination from wastewater applying chieﬂy inactive and non-living biomass and algae.

Live algae have limited sorption capacity as the heavy metal ions often adversely

398 P. M. Patil et al.

affect the living cells and numerous factors of the environment which directly impact

the absorption capacity of ions. Additionally, the sorption method shows high differ-

ences based on the development phase of algae. Absorption mechanisms in living

algae are more complex than in non-living algae. The non-living algae cells absorb

metal ions from the cell membrane surface, and it is a kind of extracellular process

where metal recovery becomes easy. Non-living algal biomass is an assemblage of

polymers (such as complex carbohydrates, cellulose, pectins, and other associated

glycoproteins, etc.) that is capable of binding to heavy metal cations as adsorbents

with the potential for cost-effective wastewater treatment (Zeraatkar et al. 2016).

8.3 Phytoremediation (Plant bioremediation)

Phytoremediation is a process that uses various types of vegetation (green plants)

for situ treatments to remove, transfer, and stabilize, the metal contaminants. In

this innovative treatment, the natural properties of phytoplanktons are utilized in

engineered methods to remediate toxic waste. The advantage of the plant nutrient

utilization process is to absorb nutrients and water through roots, emerge water by

leaves; and performs as a transformation structure to metabolize to biological element

(Patil et al. 2022). The plant bio-accumulate and absorb heavy metals including

trace elements. For plants, the eagerly bioavailable heavy metals are nickel, sele-

nium, cadmium, copper, arsenic, and zinc. Temperately bioavailable heavy metals

are cobalt, chromium, manganese, lead, and iron. Uranium is mostly not available

metal. By applying chelating agents much more lead can be accessible. Similarly, to

enhance the accessibility of radio-cesium 137 and uranium ammonium nitrate and

citric acid should be used.

Factors affecting phytoremediation other than bioavailability are the selection of

proper accumulator which is responsive to agricultural practices that allow repeated

planting and harvesting. Production of sufﬁcient biomass and accumulation in shoots

are also essential criteria. More than four hundred species of plants are considered

suitable for use in phytoremediation (Gerhardt et al. 2017).

Phytoaccumulation ( phytoextraction) is the process, where plant roots of selected

hyperaccumulator plant sorb (absorb/adsorb) store the metal contaminants along

with other nutrients and water. The mass which is contaminated is not devastated

but ends up with the leaves and plant shoots. This treatment is primarily used for

wastewater containing a lower concentration of metals. The metals are stored in the

plant in aerial shoots, which are harvested and either smelted for potential metal

recycling/recovery or are eventually disposed of as hazardous waste (Pandey and

Bajpai 2019).

Phytoextraction includes the absorption and uptake of polluted metals which is

existed in the soil through roots and underground elements of plants, by hyperac-

cumulation mechanism. From the polluted soil, the hyperaccumulator plants uptake

huge concentrations of heavy metals, and accumulate and transfer them in other body

organs beyond the ground at from 100 to 1000 times greater concentrations than those

Removal of Heavy Metals from Industrial Wastewater Using … 399

existing in non-hyperaccumulating plant species. These hyperaccumulators will not

have any phytotoxic effect or disease hence they are very suitable for phytoremedia-

tion. The hyperaccumulators are usually grown in metal and toxic contaminated soil

areas and also generate plentiful biomass. These plants are unique because of the

characteristics like the much greater capacity to take up heavy metals from the soil;

enhanced root-to-shoot translocation of metal ions; a much greater ability to detoxify

and sequester extremely large amounts of heavy metals in the shoots, and the ability to

grow fast with the profuse root system (Suman et al. 2018). Rhizoﬁltration is related

to phyto-accumulation, but the phytoplankton applied for cleaning are elevated in

greenhouses with their origins in water. This system can be used for ex-situ treat-

ment. That is, wastewater is impelled to the plant’s surface irrigation. For hydroponic

systems, the artiﬁcial medium soil like sand is submerged with perlite or vermiculite

are used. As the roots become s aturated with contaminants, they are harvested for

valuable metal recovery and disposed off if they are useless (Pajevi´cetal.

2016).

8.4 Nano-Bioremediation

The nanomaterials are categorized as substances having sizes 1–100 nm. The size of

nanoparticles (NPs) is precisely tiny that has quantum special effects by restraining

their electrons. Due to the NP’s size, they hold numerous exclusive and special proper-

ties. NP’s have been applied in several ﬁelds like electronics, biomedicine, catalysis,

and photonics. NP’s signify changeable properties with the help of bulk counterparts,

so there are various opportunities to develop innovative materials that can be utilized

in several industrial applications. By utilizing the single-step treatment method the

NP’s can treat the wastewater efﬁciently and remove the contaminants that existed

in wastewater. In industrial wastewater treatment (WWT) the NP’s are applied as

adsorbents. Due to its unique structure and properties like adsorption capacity and

high selectivity, it is signiﬁcant to remove heavy metal exited in wastewater. Their

high volume and surface ratio permits them to absorb heavy metals and other contam-

inants. Nanomaterials can increase reactivity, penetrate deeper, and eliminate heavy

metals efﬁciently. Current research highlights the possible use of materials such

as nanocomposite, nanowires, nanotubes, and nano-spheres merged with conven-

tional WWT which is beneﬁcial to eliminate several inorganic and organic contam-

inants, including heavy metals. Depending upon the existence of heavy metals and

the external factors dissemination potential of nanomaterials is inﬂuenced. External

factors affect the nano-adsorbents properties like sorbents size, shape, surface chem-

istry, chemical composition, fractal dimension, solubility, crystal structure, agglom-

eration state. Nanomaterials bargain atomic level modiﬁcation, unlike bulk materials

that have several innovative characteristics and properties which bulk materials are

not offered (Tripathi et al. 2022).

400 P. M. Patil et al.

9 Advantages of Bioremediation for Industrial Wastewater

Treatment

Bioremediation using indigenous microbial and plants species is one of the waste

treatment technologies that utilizes the natural state of the environment. It is an

engineering and scientiﬁc application to the processing materials through biolog-

ical agents which are renewable. It is an eco-friendly method that needs very low

energy consumption. As biotic factors are explored in the treatment process it is an

aesthetically pleasing and cheaper option. In the ex-situ process, easy monitoring of

microbes and plants can be done. Periodically harvested material can be used for

the recovery of valuable precious metal species for further use. Toxic and hazardous

metals can be properly disposed of at the disposal site (Pajevi´cetal.

2016).

Thus, the use of plants for the bioremediation of water offers a wide range of

advantages. In phytoremediation, solar energy has been utilized and in an undisturbed

condition, without excavation, the huge concentrations of contaminants are removed.

The numerous metals are treated and removed with the phytoremediation method.

For the polluted sites, phytoremediation delivers a valuable tool, mostly these sites are

not remediated by other treatments like huge extension sites at shallow depths with

fewer impurities concentrations. The plant species utilized in the phytoremediation

process ﬁts studied crop plants hence there is extensive knowledge available for the

management, harvesting, and cultivation, of these plant species (Gerhardt el at. 2017).

In phytoremediation, the ground area is covered by the plants, so the waste and air

pollutants are restricted by the plants, which decreases water and wind-based erosion

and steadies the soil. The wind erosion exposes the direct pathway of air contami-

nation inhalation and contaminated food ingestion by suspended particle deposition

onto the leafy vegetables and plants. If we used hyperaccumulators then its biomass

can be incinerated which declines the waste volume and mass. As compared with

other remediation treatments phytoremediation is a cost-effective method. In contam-

inated site removal, many steps are included like removal, excavation, isolation, and

returning of the residue to the speciﬁc site. The human–plants relationship in the

evolutionary past is rooted deeply. With the help of remediation, the contaminated

site or the barren land can get converted into an aesthetic green ﬁeld. The social and

public acceptance of phytoremediation is also high. Huge research has still to be done

to increase the efﬁciency of bioremediation treatment, to enhance the production and

number of organisms for the treatment. Another limitation long time duration of the

site clean-up (Pandey and Bajpai 2019; Suman et al. 2018) (Fig. 3).

Removal of Heavy Metals from Industrial Wastewater Using … 401

Fig. 3 Advantages of Bioremediation

10 Perspectives, Challenges, and Opportunities

Bioremediation is a low maintenance cost, highly selective, less contaminated, and

low water generated, microorganism-based promising and remediated technology.

This process is mostly employed to exclude toxins from the surroundings and the

adsorption of harmful pollutants from wastewaters, pesticides, or fertilizers. The

several advantages are offered by biosorption, microbes the probable potentials of

enormous scale application seem encouraging when limited costs are considered.

In bioremediation selection and screening of microbes, strain is very important.

Constant mechanisms study of sustaining heavy metal biosorption, and bioaccu-

mulation by microbes, development in equilibrium and kinetic model to upgrade

the process of bioremediation. Also, the commendation for encouraging toxic metal

elimination, utilization of biomass including chemical modiﬁcation treatment, and

combining or incorporating this method with conventional for heavy metal removal is

needed. Microorganism role, identiﬁcation, catabolic and metabolic pathways have

been promoted from advanced molecular treatments like proteomics, genomics, tran-

scriptomics, and metabolomics (Pandey and Bajpai 2019). Contaminant degrading

microorganisms often existed in contaminated sites; the types and pollutants concen-

tration may stimulate their growth, development, and metabolic activities, but it

can be applied for agro-industrial waste, which encompasses high content of phos-

phorus, nitrogen, and potassium, and act as a nutrient source for a most extremely

contaminated site. Then the pure isolates microbial consortium degrades contami-

nated material more effectively. Optimizing the application of genetically engineered

402 P. M. Patil et al.

microorganisms (GEM) to enhance the capability of bioremediation is a promising

approach. This is outstanding to generate biocatalyst target pollutants, which contain

resistant elements by combining efﬁcient and novel metabolic pathways, spreading

the substrate level of prevailing pathways, and increasing the stability of catabolic

activity. GEM multiplication and parallel gene transferrin a bioremediation applica-

tion has become a successful strategy. Additionally, applying biological, organic, and

eco-friendly methods, smearing GEM to a target polluted material could enhance the

efﬁciency of bioremediation (Pajevi´cetal.

2016).

11 Conclusion

Bioremediation of metals from wastewater uses indigenous as well as genetically

modiﬁed strains of microbes as well as plants. It is ecological, economical, and

socially acceptable technology where renewable biological resources are explored.

Phytoremediation enhances photosynthesis through cultivated plants for phytoreme-

diation. The activity can be intervened by modifying hyperaccumulators for their

luxurious growth, disease resistance, other environmental stress resistance as well

as herbicide resistance features as per the requirements. These Genetically Modiﬁed

Organisms (GMOs) contain foreign genes implanted into the genome of another

creature of the different or same species by applying recombinant DNA technology.

These genetically engineered microbes and plants have been applied to acquire

capable strains for bioremediation of polluted environments by having the enriched

capability to break-down abundant contaminants. Considering the importance of

transgenic microbes in heavy metal contaminants removal, more research must be

done to enrich the survival rate of microbes when released into the ecosystem for

bioremediation, because their survivability is poor in the current situation. Many

environmental factors like temperature and nutrient concentrations and other related

factors, can hamper their utilization and need to be controlled for the effectiveness of

the bioremediation process. Anticipation of horizontal gene transmission from engi-

neered microorganisms to indigenous microbes, use of anti-sense RNA and suicidal

genes, antibiotic genes markers should be avoided and can be replaced with other

selectable markers to avoid unintentional transfer of antibiotic resistance genes.

However, research is essential to appreciate the metabolic pathways of microbes

and transgenic plants applied in bioremediation to discover their efﬁciency. High

biomass hyper-accumulator plants should be identiﬁed, and they should be improved

through genetic engineering. The capacity of the microbes applied in bioremediation

to compete with the indigenous microbial population is essential for the success of

bioremediation.

Removal of Heavy Metals from Industrial Wastewater Using … 403

References

Abatenh E, Gizaw B, Tsegaye Z, Wassie M (2017) The role of microorganisms in bioremediation-a

review. Open J Environ Biol 2(1):038–046

Ahmed J, Thakur A, Goyal A (2021). Industrial Wastewater and Its Toxic Effects. https://doi.org/

10.1039/9781839165399-00001

Ahmed MB, Zhou JL, Ngo HH, Guo W, Thomaidis NS, Xu J (2017) Progress in the biological and

chemical treatment technologies for emerging contaminant removal from wastewater: a critical

review. J Hazard Mater 323:274–298. https://doi.org/10.1016/j.jhazmat.2016.04.045

Aibeche C, Selami N, Zitouni-Haouar FEH, Oeunzar K, Addou A, Kaid-Harche M, Djabeur A

(2021) Bioremediation potential and lead removal capacity of heavy metal-tolerant yeasts isolated

from Dayet Oum Ghellaz Lake water (northwest of Algeria). Int Microbiol 1–13. https://doi.org/

10.1007/s10123-021-00191-z

Ali H, Khan E, Sajad MA (2013) Phytoremediation of heavy metals—concepts and applications.

Chemosphere 91(7):869–881. https://doi.org/10.1016/j.chemosphere.2013.01.075

AliS,AbbasZ,RizwanM,Zaheer IE,Yava¸s ˙

I, Ünay A, Kalderis D (2020) Application of ﬂoating

aquatic plants in phytoremediation of heavy metals polluted water: a review. Sustainability

12(5):1927. https://doi.org/10.3390/su12051927

Almasian A, Giahi M, Fard GC, Dehdast SA, Maleknia L (2018) Removal of heavy metal

ions by modiﬁed PAN/PANI-nylon core-shell nanoﬁbers membrane: ﬁltration performance,

antifouling and regeneration behavior. Chem Eng J 351:1166–1178. https://doi.org/10.1016/j.

cej.2018.06.127

Areco MM, Haug E, Curutchet G (2018) Studies on bioremediation of Zn and acid waters using

Botryococcus braunii. J Environ Chem Eng 6(4):3849–3859. https://doi.org/10.1016/j.jece.2018.

05.041

Ayele A, Godeto YG (2021) Bioremediation of chromium by microorganisms and its mechanisms

related to functional groups. J Chem 2021. https://doi.org/10.1155/2021/7694157

Azevedo A, Oliveira HA, Rubio J (2018) Treatment and water reuse of lead-zinc sulphide ore mill

wastewaters by high rate dissolved air ﬂotation. Miner Eng 127:114–121. https://doi.org/10.1016/

j.mineng.2018.07.011

Azubuike CC, Chikere CB, Okpokwasili GC (2016) Bioremediation techniques–classiﬁcation based

on site of application: principles, advantages, limitations and prospects. World J Microbiol

Biotechnol 32(11):1–18. https://doi.org/10.1007/s11274-016-2137-x

Bashir A, Malik LA, Ahad S, Manzoor T, Bhat MA, Dar GN, Pandith AH (2019) Removal of heavy

metal ions from aqueous system by ion-exchange and biosorption methods. Environ Chem Lett

17(2):729–754. https://doi.org/10.1007/s10311-018-00828-y

Bharagava, R. N., Purchase, D., Saxena, G., Mulla, S. I. 2019. Applications of metagenomics in

microbial bioremediation of pollutants: from genomics to environmental cleanup. In: Microbial

diversity in the genomic era. Academic Press, pp 459–477. https://doi.org/10.1016/B978-0-12-

814849-5.00026-5

Bhatt P, Rene ER, Kumar AJ, Zhang W, Chen S (2020) Binding interaction of allethrin with esterase:

bioremediation potential and mechanism. Biores Technol 315:123845. https://doi.org/10.1016/j.

biortech.2020.123845

Carlos FS, Schaffer N, Andreazza R, Morris LA, Tedesco MJ, Boechat CL, Camargo FADO (2018)

Treated industrial wastewater effects on chemical constitution maize biomass, physicochemical

soil properties, and economic balance. Commun Soil Sci Plant Anal 49(3):319–333. https://doi.

org/10.1080/00103624.2018.1427257

Chai WS, Cheun JY, Kumar PS, Mubashir M, Majeed Z, Banat F, Show PL (2021) A review

on conventional and novel materials towards heavy metal adsorption in wastewater treatment

application. J Clean Prod 126589. https://doi.org/10.1016/j.jclepro.2021.126589

Choi Y-K, Choi T-R, Gurav R, Bhatia SK, Park Y-L, Kim HJ, Kan E, Yang Y-H (2020a) Adsorption

behavior of tetracycline onto Spirulina sp. (microalgae)-derived biochars produced at different

temperatures. Sci Total Environ 710:136282

404 P. M. Patil et al.

Choi Y-K, Gurav R, Kim HJ, Yang Y-H, Bhatia SK (2020b) Evaluation for simultaneous removal of

anionic and cationic dyes onto maple leaf-derived biochar using response surface methodology.

Appl Sci 10(9)

Damtie MM, Kim B, Woo YC, Choi JS (2018) Membrane distillation for industrial wastewater treat-

ment: studying the effects of membrane parameters on the wetting performance. Chemosphere

206:793–801. https://doi.org/10.1016/j.chemosphere.2018.05.070

Efome JE, Rana D, Matsuura T, Lan CQ (2018) Experiment and modeling for ﬂux and permeate

concentration of heavy metal ion in adsorptive membrane ﬁltration using a metal-organic frame-

work incorporated nanoﬁbrous membrane. Chem Eng J 352:737–744. https://doi.org/10.1016/j.

cej.2018.07.077

Elgallal M, Fletcher L, Evans B (2016) Assessment of potential risks associated with chemicals in

wastewater used for irrigation in arid and semiarid zones: a review. Agric Water Manag 177:419–

431. https://doi.org/10.1016/j.agwat.2016.08.027

Gerhardt KE, Gerwing PD, Greenberg BM (2017) Opinion: Taking phytoremediation from proven

technology to accepted practice. Plant Sci 256:170–185. https://doi.org/10.1016/j.plantsci.2016.

11.016

Goswami RK, Agrawal K, Shah MP, Verma P (2021) Bioremediation of heavy metals from wastew-

ater: a current perspective on microalgae-based future. Lett Appl Microbiol. https://doi.org/10.

1111/lam.13564

Gurav RG, Bhatia SK, Jagtap UB, Yang Y-H, Choi Y-K, Tang J, Bhatnagar A (2021a) Utilization of

invasive weed biomass for biochar production and its application in agriculture and environmental

clean-up. In: Pant D, Bhatia SK, Patel AK, Giri A (eds) Bioremediation using weeds. Springer,

Singapore. Singapore, pp 207–224

Gurav R, Bhatia SK, Choi T-R, Choi Y-K, Kim HJ, Song H-S, Lee SM, Lee Park S, Lee HS, Koh

J, Jeon J-M, Yoon J-J, Yang Y-H (2021b) Application of macroalgal biomass derived biochar

and bioelectrochemical system with Shewanella for the adsorptive removal and biodegradation

of toxic azo dye. Chemosphere 264:128539

Gurav R, Bhatia SK, Choi T-R, Choi Y-K, Kim HJ, Song H-S, Park SL, Lee HS, Lee SM, Choi

K-Y, Yang Y-H (2021c) Adsorptive removal of crude petroleum oil from freshwater using ﬂoating

pinewood biochar decorated with coconut oil-derived fatty acids. Sci Total Environ 146636

Gurav R, Bhatia SK, Choi T-R, Kim H-J, Lee H-J, Cho J, Ham S, Suh M-J, Kim S, Kim S-K, Yoo

D, Yang Y-H (2021d) Seafood processing chitin waste for electricity generation in a microbial

fuel cell using halotolerant catalyst Oceanisphaera arctica YHY1. Sustainability 13(15)

Gurav R, Bhatia SK, Choi T-R, Kim HJ, Choi Y-K, Lee H-J, Ham S, Cho JY, Kim SH, Lee SH, Yun

J, Yang Y-H (2022) Adsorptive removal of synthetic plastic components bisphenol-A and solvent

black-3 dye from single and binary solutions using pristine pinecone biochar. Chemosphere

296:134034

Gurav R, Bhatia SK, Choi T-R, Kim HJ, Song H-S, Park S-L, Lee S-M, Lee H-S, Kim S-H, Yoon

J-J, Yang Y-H (2020) Utilization of different lignocellulosic hydrolysates as carbon source for

electricity generation using novel Shewanella marisﬂavi BBL25. J Clean Prod 124084

Gurav R, Bhatia SK, Choi T-R, Park Y-L, Park JY, Han Y-H, Vyavahare G, Jadhav J, Song H-S,

Yang P, Yoon J-J, Bhatnagar A, Choi Y-K, Yang Y-H (2019) Treatment of furazolidone contam-

inated water using banana pseudostem biochar engineered with facile synthesized magnetic

nanocomposites. Bioresour Technol 122472

Gurav R, Lyu H, Ma J, Tang J, Liu Q, Zhang H (2017) Degradation of n-alkanes and PAHs from

the heavy crude oil using salt-tolerant bacterial consortia and analysis of their catabolic genes.

Environ Sci Pollut Res 24(12):11392–11403

Gurav RG, Jadhav JP (2013) Biodegradation of keratinous waste by Chryseobacterium sp. RBT

isolated from soil contaminated with poultry waste. J Basic Microbiol 53(2):128–135

Gurav RG, Tang J, Jadhav JP (2016) Sulﬁtolytic and keratinolytic potential of Chryseobacterium

sp. RBT revealed hydrolysis of melanin containing feathers. 3 Biotech 6(2):145

Removal of Heavy Metals from Industrial Wastewater Using … 405

Jia Z, Li S, Wang L (2018) Assessment of soil heavy metals for eco-environment and human health

in a rapidly urbanization area of the upper Yangtze Basin. Sci Rep 8(1):1–14. https://doi.org/10.

1038/s41598-018-21569-6

Koedrith P, Kim H, Weon JI, Seo YR (2013) Toxicogenomic approaches for understanding molec-

ular mechanisms of heavy metal mutagenicity and carcinogenicity. Int J Hyg Environ Health

216(5):587–598. https://doi.org/10.1016/j.ijheh.2013.02.010

Kondzior P, Butarewicz A (2018) Effect of heavy metals (Cu and Zn) on the content of photosynthetic

pigments in the cells of algae Chlorella vulgaris. J Ecol Eng 19(3). https://doi.org/10.12911/229

98993/85375

Kumar V, Dwivedi SK (2021) Bioremediation mechanism a nd potential of copper by actively

growing fungus Trichoderma lixii CR700 isolated from electroplating wastewater. J Environ

Manag 277:111370. https://doi.org/10.1016/j.jenvman.2020.111370

Liu Q, Tang J, Bai Z, Hecker M, Giesy JP (2015) Distribution of petroleum degrading genes and

factor analysis of petroleum contaminated soil from the Dagang Oilﬁeld, China. Sci Rep 5:11068

Liu Z, Lin H, Cai T, Chen K, Lin Y, Xi Y, Chhuond K (2019) Effects of Phytoremediation on

industrial wastewater. In: IOP conference series: earth and environmental science, vol 371, no 3.

IOP Publishing, pp 032011. https://doi.org/10.1088/1755-1315/371/3/032011

Lu Q, Bai J, Zhang G, Wu J (2020) Effects of coastal reclamation history on heavy metals in different

types of wetland soils in the Pearl River Delta: levels, sources and ecological risks. J Clean Prod

272:122668. https://doi.org/10.1016/j.jclepro.2020.122668

Lyu H, Gong Y, Gurav R, Tang J (2016) Chapter 9-Potential application of biochar for bioremedia-

tion of contaminated systems. In: Ralebitso-Senior TK (ed) Biochar application. Elsevier, C. H.

Orr, pp 221–246

Matsuyama A, Yano S, Taniguchi Y, Kindaichi M, Tada A, Wada M (2021) Trends in mercury

concentrations and methylation in Minamata Bay, Japan, between 2014 and 2018. Mar Pollut

Bull 173:112886. https://doi.org/10.1016/j.marpolbul.2021.112886

Meruvu H (2021) Bacterial bioremediation of heavy metals from polluted wastewaters. In: New

trends in removal of heavy metals from industrial wastewater, pp 105–114. https://doi.org/10.

1016/B978-0-12-822965-1.00005-2

Ojuederie OB, Babalola OO (2017) Microbial and plant-assisted bioremediation of heavy metal

polluted environments: a review. Int J Environ Res Public Health 14(12):1504. https://doi.org/10.

3390/ijerph14121504

Ostermeyer P, Bonin L, Folens K, Verbruggen F, García-Timermans C, Verbeken K, Hennebel T

(2021) Effect of speciation and composition on the kinetics and precipitation of arsenic sulﬁde

from industrial metallurgical wastewater. J Hazard Mater 409, 124418. https://doi.org/10.1016/

j.jhazmat.2020.124418

Pajevi´c S, Borišev M, Nikoli´c N, Arsenov DD, Orlovi´c S, Župunski M (2016) Phytoextraction of

heavy metals by fast-growing trees: a review. Phytoremediation 29–64. https://doi.org/10.1007/

978-3-319-40148-5_2

Pan ZF, An L (2019) Removal of heavy metal from wastewater using ion exchange membranes. In:

Applications of ion exchange materials in the environment. Cham, pp. 25–46. https://doi.org/10.

1007/978-3-030-10430-6_2

Pandey VC, Bajpai O (2019) Phytoremediation: from theory toward practice. In: Phytomanagement

of polluted sites, pp 1–49. https://doi.org/10.1016/B978-0-12-813912-7.00001-6

Patil PM, Bohara RA (2020) Nanoparticles impact in biomedical waste management. Waste Manag

Res 38(11):1189–1203. https://doi.org/10.1177/0734242X20936761

Patil PM, Mahamuni PP, Abdel-Daim MM, Aleya L, Chougule RA, Shadija PG, Bohara RA (2019a)

Conversion of organic biomedical waste into potential fertilizer using isolated organisms from

cow dung for a cleaner environment. Environ Sci Pollut Res 26(27):27897–27904. https://doi.

org/10.1007/s11356-019-05795-7

Patil PM, Mahamuni PP, Shadija PG, Bohara RA (2019b) Conversion of organic biomedical waste

into value added product using green approach. Environ Sci Pollut Res 26(7):6696–6705. https://

doi.org/10.1007/s11356-018-4001-z

406 P. M. Patil et al.

Patil R, Zahid M, Govindwar S, Khandare R, Vyavahare G, Gurav R, Desai N, Pandit S, Jadhav J

(2022) Chapter 8-Constructed wetland: a promising technology for the treatment of hazardous

textile dyes and efﬂuent. In: Shah M, Rodriguez-Couto S (eds) Development in wastewater

treatment research and processes. Elsevier, J. Biswas, pp 173–198

Pooja G, Kumar PS, Prasannamedha G, Varjani S, Vo DVN (2021) Sustainable approach on removal

of toxic metals from electroplating industrial wastewater using dissolved air ﬂotation. J Environ

Manag 295:113147. https://doi.org/10.1016/j.jenvman.2021.113147

Shah Maulin P (2021a) Removal of Emerging Contaminants through Microbial Processes. Springer

Shah Maulin P (2021b) Removal of refractory pollutants from wastewater treatment plants. CRC

Press

Sharma P (2021) Efﬁciency of bacteria and bacterial assisted phytoremediation of heavy metals:

an update. Bioresour Technol 124835. https://doi.org/10.1016/j.biortech.2021.124835

Sharma P, Pandey AK, Kim SH, Singh SP, Chaturvedi P, Varjani S (2021a) Critical review on

microbial community during in-situ bioremediation of heavy metals from industrial wastewater.

Environ Technol Innov 24:101826. https://doi.org/10.1016/j.eti.2021.101826

Sharma, P., Pandey, A. K., Udayan, A., Kumar, S. 2021b. Role of microbial community and metal-

binding proteins in phytoremediation of heavy metals from industrial wastewater. Bioresour

Technol 124750. https://doi.org/10.1016/j.biortech.2021.124750

Shah Maulin P (2020) Microbial bioremediation & biodegradation. Springer

Sharma R, Talukdar D, Bhardwaj S, Jaglan S, Kumar R, Kumar R, Umar A (2020) Bioremediation

potential of novel fungal species isolated from wastewater for the removal of lead from liquid

medium. Environ Technol Innov 18:100757. https://doi.org/10.1016/j.eti.2020.100757

Sher S, Hussain SZ, Rehman A (2020) Phenotypic and genomic analysis of multiple heavy metal–

resistant Micrococcus luteus strain AS2 isolated from industrial waste water and its potential

use in arsenic bioremediation. Appl Microbiol Biotechnol 104(5):2243–2254. https://doi.org/10.

1007/s00253-020-10351-2

Singh N, Gupta VK, Kumar A, Sharma B (2017) Synergistic effects of heavy metals and pesticides

in living systems. Front Chem 5:70. https://doi.org/10.3389/fchem.2017.00070

Singh P, Itankar N, Patil Y (2021) Biomanagement of hexavalent chromium: current trends and

promising perspectives. J Environ Manag 279:111547. https://doi.org/10.1016/j.jenvman.2020.

111547

Soliman NK, Moustafa AF (2020) Industrial solid waste for heavy metals adsorption features and

challenges; a review. J Market Res 9(5):10235–10253. https://doi.org/10.1016/j.jmrt.2020.07.045

Suman J, Uhlik O, Viktorova J, Macek T (2018) Phytoextraction of heavy metals: a promising tool

for clean-up of polluted environment? Front Plant Sci 9:1476. https://doi.org/10.3389/fpls.2018.

01476

Thaçi BS, Gashi ST (2019) Reverse osmosis removal of heavy metals from wastewater efﬂuents

using biowaste materials pretreatment. Pol J Environ Stud 28(1):337–341. https://doi.org/10.

15244/pjoes/81268

Tripathi S, Sanjeevi R, Anuradha J, Chauhan DS, Rathoure AK (2022) Nano-bioremediation:

nanotechnology and bioremediation. In: Research anthology on emerging techniques in envi-

ronmental remediation. IGI Global, pp 135–149. https://doi.org/10.4018/978-1-6684-3714-8.

ch007

Van Oosten MJ, Maggio A (2015) Functional biology of halophytes in the phytoremediation of

heavy metal contaminated soils. Environ Exp Bot 111:135–146. https://doi.org/10.1016/j.envexp

bot.2014.11.010

Verma S, Kuila A (2019) Bioremediation of heavy metals by microbial process. Environ Technol

Innov 14:100369. https://doi.org/10.1016/j.eti.2019.100369

Wang W, Jiang F, Wu F, Li J, Ge R, Li J, Lyu J (2019) Biodetection and bioremediation of copper

ions in environmental water samples using a temperature-controlled, dual-functional Escherichia

coli cell. Appl Microbiol Biotechnol 103(16):6797–6807. https://doi.org/10.1007/s00253-019-

09984-9

Removal of Heavy Metals from Industrial Wastewater Using … 407

Wang XX, Wu YH, Zhang TY, Xu XQ, Dao GH, Hu HY (2016) Simultaneous nitrogen, phospho-

rous, and hardness removal from reverse osmosis concentrate by microalgae cultivation. Water

Res 94:215–224. https://doi.org/10.1016/j.watres.2016.02.062

Wang X, Aulenta F, Puig S, Esteve-Núnez A, He Y, Mu Y, Rabaey K (2020) Microbial

electrochemistry for bioremediation. Environ Sci Ecotechnol 1:100013

Wu X, Cobbina SJ, Mao G, Xu H, Zhang Z, Yang L (2016) A review of toxicity and mechanisms of

individual and mixtures of heavy metals in the environment. Environ Sci Pollut Res 23(9):8244–

8259. https://doi.org/10.1007/s11356-016-6333-x

Zeraatkar AK, Ahmadzadeh H, Talebi AF, Moheimani NR, McHenry MP (2016) Potential use of

algae for heavy metal bioremediation, a critical review. J Environ Manag 181:817–831. https://

doi.org/10.1016/j.jenvman.2016.06.059

Zhang H, Tang J, Wang L, Liu J, Gurav RG, Sun K (2016) A novel bioremediation strategy

for petroleum hydrocarbon pollutants using salt tolerant Corynebacterium variabile HRJ4 and

biochar. J Environ Sci 47:7–13

Zhang Y, Zhao M, Cheng Q, Wang C, Li H, Han X, Li Z (2021) Research progress of adsorption

and removal of heavy metals by chitosan and its derivatives: a review. Chemosphere 279:130927.

https://doi.org/10.1016/j.chemosphere.2021.130927

Zhao MM, Kou JB, Chen YP, Xue LG, Fan TT, Wang SM (2021) Bioremediation of wastewater

containing mercury using three newly isolated bacterial strains. J Clean Prod 299:126869. https://

doi.org/10.1016/j.jclepro.2021.126869

Zvab U, Kukulin DS, Fanetti M, Valant M (2021) Bioremediation of copper polluted wastewater-

like nutrient media and simultaneous synthesis of stable copper nanoparticles by a viable green

alga. J Water Process Eng 42:102123. https://doi.org/10.1016/j.jwpe.2021.102123

Membrane Reactor and Moving Bed

Bioﬁlm Reactor for Tannery Wastewater

Treatment

C. Raja, J. Anandkumar, and B. P. Sahariah

Abstract Tannery efﬂuent is one of most complex industrial efﬂuent, huge amount

of wastewater rich in organic and inorganic pollutants including heavy metals and

toxic elements. High COD along with presence of chromium and sulphides is basic

characteristics of tannery wastewater (TWW). Research community emphasizes

on tannery efﬂuent treatment using individually or in various hybrid processes

owe to complexity and requirement of non-harmful dischargeable limit of this

wastewater. For preserving water in its natural state, researchers all over the world

develop numerous new tools and technologies such as membrane systems, advanced

oxidation system, sorption systems and moving bed bioﬁlm reactor etc., along

with conventional bioremediation/physicochemical treatment processes. Membrane

reactor (MBR) follows principle of separation of pollutants using organic and inor-

ganic membranes developed from kaolin, clay, polyvinylidene ﬂuoride, poly sulfone,

poly ether sulfone material etc., also has drawn attention for tannery wastewater

treatment. Moving bed bioﬁlm reactor (MBBR) uses microbe’s pollutants remedia-

tion potential providing a substratum for its settlement, high density, speedy growth

and shock tolerance. This chapter discusses fundamentals of MBR and MBBR, their

applicability in individual and hybrid system conﬁguration for achieving successful

treatment of wastewater from tanneries.

Keywords Tannery wastewater treatment ·Membrane reactor (MBR) ·Moving

bed bioﬁlm reactor (MBBR) ·Hybrid process ·Chromium

C. Raja · J. Anandkumar

Department of Chemical Engineering, National Institute of Technology Raipur (C.G),

Raipur 492010, India

B. P. Sahariah (B

)

University Teaching Department, Chhattisgarh Swami Vivekanand Technical University, Bhilai

(C.G) 491107, India

e-mail: biju.sahariah@gmail.com

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_19

409

410 C. Raja et al.

1 Introduction

Tannery industries are one of the most important commercial successful industries

in the world. In leather processing, tanning is the major and essential operation for

producing various leather products. Leather processing is divided into a few basic

operations such as pre-tanning, tanning, post tanning, and ﬁnishing. The average

consumption of water for raw material processing is from 25,000 to 80,000 L per

unit operations (Mannucci et al. 2010). In pre-tanning operation includes soaking,

unhairing, liming, bating, and deliming. Degreasing, pickling, tanning, pressing,

and drying are the major operations in the tanning process. Chrome and vegetable

tanning processes are important steps in tannery process and produce a huge volume

of wastewater and responsible to cause pollution in the aqueous system (Lofrano

et al. 2013). Reports states that for more than 2,500 tanneries in India, the waste

bio-sludge generation in TWW is as high as 165,200 tons/year and the scenario for

treatment becoming ﬁnancially heavy (Bharagava et al. 2017; Sodhi et al. 2021).

1.1 Characteristics of Tannery Wastewater

TWW is diverged in characteristics from tannery to tannery due to the raw mate-

rial used and the processing. Type of tanning process, organic loading, and size of

the tannery highly inﬂuences the characteristics. TWW contains organic & inor-

ganic biomolecules such as blood, fats, hair; processing chemicals such as calcium

sulﬁde, trivalent chromium, tannins, sulfonated oil, spent dyes, resulting in chlo-

rides, sulfates, and sulﬁde, etc., in signiﬁcant concentrations; and a high quantity

of total solids, BOD, and COD (Korpe and Rao 2021; Lofrano et al. 2013;Chowd-

hury and Mostafa 2015; Sawalha et al. 2019). Re-tanning and dyeing operations can

produce wastewater with 71,040 mg BOD/l and 2400 mg COD/l.

General composition of TWW observed in the different studies is summarized in

Table 1.

Pickling and chrome tanning operations produce a huge quantity of solids (TS

and TDS). The discharged wastewater from the liming and unhairing operations has

high pH conditions. Post tanning process consumes as high as 8.6 m3 of water on

averagely which has high conductivity due to the presence of chloride and sulphate

ions. High sulphide is characteristic of liming process with a discharge of as high as

0.12 m3 WW/ton. Dye, metal complex, fat liquoring agents, and tannins are important

chemicals present in the TWW that can cause toxicity to the environment (Hansen

et al. 2021). Signiﬁcant amounts of heavy metals such as Cr, Ni, Cd, Pb, Cu, Zn

(Abdel-shafy et al. 1997) are regular in the TWW. Sivagami et al. (2018), Shah (2020)

reported that after primary and secondary treatment failed to meet the discharge limit

of sulfate and total dissolved solids from tannery wastewater containing 3980 mg/l

of COD and 4000 sulfates.

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery … 411

Tabl e 1 Characteristics of tannery wastewater reported in different literature

pH BOD (mg/l) COD (mg/l) Chloride

(mg/l)

Chromium

(mg/l)

Sulphate

(mg/l)

Dissolved

solids (mg/l)

Suspended

solids

Total solids References

7.5 1000 ±7200 ± 20 – – – 8100 ± 50 1240 ± 40 9340 ± 70– Chowdhury

and Mostafa

(2015)

7.8–8.8 150–250 1500–2500 1360–1740 2100–2760 3000–5000 200–1000 Sivagami

et al. (2018)

7.5 920 ± 15.81 3980 ± 29.66 4000 ± 14.32 14,000 ± 50.99 6800 ± 35.35 20,800 ± 55.01 Chowdhurty

et al. (2013)

7.15 – 35,200 43,100 – – 52,000 16,000 – Sawalha

et al. (2019)

4.12–4.68 2099 ± 1185 4767 ± 2422 1302 ± 420 2671 ± 1582 7677 ± 4084 – – Boopathy

et al. (2013)

4.62–8.13 900–1200 1100–3000 – 0.360- < 0.001 – 58–200 –Rangel

et al. (2007)

7.5 2000 > 10,000 – – – – 4000 45,000 Song et al.

(2000)

8–12 1243–1984 6593–7267 14–26 –3250–7489 1217–2847 –Aregu et al.

(2021)

10.2 – 10,500 4620 8.2 2560 7420 – – Pal et al.

(2020a)

(continued)

412 C. Raja et al.

Tabl e 1 (continued)

pH BOD (mg/l) COD (mg/l) Chloride

(mg/l)

Chromium

(mg/l)

Sulphate

(mg/l)

Dissolved

solids (mg/l)

Suspended

solids

Total solids References

– – 55,000 – 198 0.14 –985 –Stoller et al.

(2013)

5.11 – 21.305 ± 150.58 – – – – 4831 ± 131.16 Yang et al.

(2021)

7.2–8.5 –541–6309 – – – 9900–14,090 – – Vo et al.

(2021)

5.65 ± 0.86 –992 ± 21.5 – – 1.165 ± 14 – – – Serkaran

et al. (2013)

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery … 413

1.2 Conventional Treatment Process

The presence of large molecular organic and inorganic compounds, diverse heavy

metals at high concentration in TWW are prone to cause serious pollution issues

to the environment if released without any proper treatment. Conventional treat-

ment methods used for the TWW include bioremediation in activated sludge process

(ASP), up-ﬂow anaerobic sludge blanket (UASB), chemical precipitation, adsorp-

tion, coagulation-ﬂocculation, ﬂoatation, ion exchange and wetland systems etc. The

following section brieﬂy describe a few conventional processes in view of TWW.

Bioremediation

Bioremediation is the process of removal /reduction /transformation of pollutants into

less harmful or elemental component with the help of biological species. Generally,

bioremediation involves biodegradation, biosorption, bioleaching and bioaccumu-

lations where microbial species and numerous plant species and live/dead biomass

ﬁx the pollutants; and the same are applicable for the treatment of TWW (Ghumra

et al. 2021). In microbial -based bioremediation process of TWW, microbes degrade

organic compounds originated from animal skin/ leathers; colouring agents like dye

material and process biosorption of heavy metals present in the TWW. Efﬁciency of

microorganisms such as bacteria, fungi, yeast, and algae are well documented for

TWW bioremediation provided suitable operating and environmental conditions.

Similarly, the phytoremediation process where plants are used to treat contami-

nates, the treatment plants follow mechanisms of phytostabilization (ﬁxed state/ loca-

tion of pollutants), phytoaccumulation (absorb and accumulate in plant body parts)

phytovolatilization (evaporation), phytodegradation (breakdown) and phytoextrac-

tion (extract and absorb) of the pollutants. Speciﬁc organic acids, enzymes and root

exudates consists of phytosiderophores (metal solubilizing substances) present in

plant species play the major role in phytoremediation.

The bioremediation is governed by the cellular and molecular components present

in the microbial cells that releases enzymes and other functional proteins suitable for

remediation mechanisms. Numerous conventional reactors with various environment

such as activated sludge process (ASPs), up ﬂow anaerobic sludge blanket (UASBs)

and Constructed wetland system are frequently employed for TWW treatment. ASP

is one of the prominent processes aerobic biological processes used in the secondary

treatment of TWW efﬁcient for removal of BOD and COD in the continuous treat-

ment process along with sludge separation. It is an easy to operate and econom-

ical viable method. Excess activated sludge is the major issue of this process. Bera

et al. (2012) evaluated the efﬁciency of ASP and achieved 95.58% of chromium

removal from the tannery wastewater. Ballén-segura et al. (2016) achieved removal

of chromium, nitrate, phosphate, and BOD using. Scenedesmus and observed that

growth of the algae species is directly proportional to pollutant removal efﬁciency.

Sekaran et al. (2013) reported efﬁciency of immobilised Bacillus sp. and algae species

414 C. Raja et al.

Synechocystis to remove BOD, COD, VFA in a chemo autotrophic activated carbon

oxidation reactor with complete sulphide removal. Serrata marcescens, Bacillus

aryabhattai HU-39, Wickerhamomyces anomalus M10, Saccharomyces cerevisiae,

Aspergillus ﬂavus CR500 are a few important microorganisms f requently recog-

nised for heavy metal removal in the aqueous solutions (Kumar and Dwivedi 2021).

Removal of 4-n-nonylphenol, mono- and di-ethoxylated nonylphenols from conven-

tional tannery efﬂuents using plants are well recognised (Gregorio and Rufﬁni 2015).

Kassaye et al. (2017) observed chromium removal using Polygonum coccineum,

Brachiara mutica and Cyprus papyrus.

Constructed wetland system is another conventional method highly considered for

treatment of tannery wastewater. Rangel et al. (2007) applied Stenotaphrum secun-

datum, Canna indica, Typha latifolia , Iris pseudacorus and Phragmites australis for

treatment of the tannery wastewater in construct wetland system where Typha lati-

folia, and Phragmites australis, exhibited satisfactory performance. (Younas et al.

2022; Shah 2021a, b) reviewed the importance of plant species and microorganism

in constructed wetland system for the treatment of chromium rich tannery treatment

wastewater and wet land plants and growth media are the important factors for high

removal efﬁciency of chromium. However, the different wetland methods are used

in the lab scale level only and further development is needed. (Aregu et al. 2021)

reported Chrysopogon zizanioides and pumice as a species in wetland system that can

signiﬁcantly remove more than 98% of chromium concentration at HRT 5 h. Adsorp-

tion, ﬁltration, microbial activities, and plant uptake are the prime mechanism of the

process.

The presence of perilous organics, various tannins and several inorganic elements

upshots high strength and complexity of the TWW which are many a times respon-

sible for inhibition of microbial activities (Bharagava et al. 2017; Sodhi et al. 2021).

Therefore, advanced and combine/ hybrid treatment options are recommended for

remediation of complex wastewaters before releasing to receiving environment.

Physicochemical Methods

The conventional physicochemical methods used for wastewater treatment are

precipitation, coagulation-ﬂocculation, Ion exchange, and Floatation etc. to name

a few. Precipitation method require addition of precipitating agents to form precip-

itates with the pollutants is generally used to reduce COD, chromium, sulfate from

the tannery industries (Fettig et al. 2017). Reyes-Serrano et al. (2020) observed more

than 99% chromium removal along with COD while using calcium hydroxide as

precipitant for chromium removal from TWW. Precipitation is also employed for the

removal of suspended solids present in the tannery efﬂuent. The major advantages

of the system are its efﬁciency to reduce the settling period of particles present in

the wastewater (Ghumra et al. 2021). Aluminum sulphate, ferric chloride, ferrous

sulphate are the few important coagulants can be used as precipitant in the TWW

treatment process (Lofrano et al. 2013). Chowdhury et al. (2013) observed 150 mg/l

ferric chloride coagulant dosage can provide removal of COD, and Cr removal as 92

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery … 415

and 96% respectively. Time, pH, and coagulant dosage were inﬂuencing the treatment

process. Elsheikh (2009) reported the 98.8% chromium and 31% removal of COD

using calcium hydroxide for precipitation of chromium in the coagulation method.

Ion-exchange process, resins are used to remove pollutants from solutions which

are insoluble substances and exchange their own radicals from the solutions (Fu and

Wang 2011). Low consumption of chemicals, less sludge production, and low cost

are the few advantages of this process. A macroporous carboxylic resin exhibited

90% removal of chromium from other metals and organics (Tiravanti et al. 1991).

Flotation is based on imparting the ionic metal species in wastewaters hydrophobic

by use of surfactants (Fu and Wang 2011). Flotation is an easy and faster method

comparatively than coagulation and sedimentation used for heavy metal removal.

Bubble attachment, dissolved air ﬂoatation, ion ﬂoatation and precipitation ﬂoatation

are the few important ﬂoatation methods used in the mineral processing. (Fu and

Wang 2011). Medina et al. (2005) reported, precipitate ﬂoatation method for the

removal of Cr (III) and achieved 96.2% at pH 8.0. The advantages and limitations

drawbacks of different conventional system are listed in Table 2.

Tabl e 2 Limitations and advantages of conventional treatment methods

Methods Advantages Disadvantages

Activated sludge method Low capital cost

Easy to operate

Sludge formation

Phytoremediation Low cost eco friendly Slow process, not suitable for

higher concentration

Constructed wetland Eco friendly

Cost-effective

Controlled environmental

condition is required

Precipitation Ease to operate

Suitable for high pollutant load

Integrated physiochemical

process

High sludge formation

High chemical consumption

Handling and disposal issue

Coagulation Simplicity in operation

Low capital cost

Huge sludge generation

High chemical consumption

Floatation Low retention time

Suitable for low-density

particles

High over ﬂow rate

High metal selectivity

High capital cost

High maintenance and

operating cost

Ion exchange High removal efﬁciency High

regeneration

Rapid and efﬁcient process

Highly sensitive to Ph

Limited commercial use

416 C. Raja et al.

1.3 Advanced Treatment Process

The conventional treatment methods frequently turn inefﬁcient in the treatment

process due to the complex nature of efﬂuent and are not achieve a high removal

rate. High chemicals consumption, expensive, and sludge formation are the few

limitations of the methods. Hence, the improvement of efﬁciency, advanced treat-

ment methods such as oxidation and membrane system are focused and followed for

the removal of pollutants from the aqueous solution.

Oxidation Process

In the oxidation process, the organic compounds are oxidised by the hydroxy ions

that are powerful for degradation. A few different types of oxidation processes such

as Fenton, ozone, and photocatalytic are employed for the waste treatment process

(Lofrano et al. 2013). The amount of free radical generation and reaction time is the

most important factor for the degradation process and this method is often applicable

in pre-treatment of efﬂuent (Korpe and Rao 2021). Sivagami et al. (2018) reported,

ozonation method to be more suitable than the Fenton oxidation for COD and TOC

removal; COD removal is inﬂuenced by pH, hydrogen peroxide and ferrous ions

concentration.

Electrocoagulation and Adsorption

Electrocoagulation process is advanced coagulation mechanisms that facilitates

reduction of the settling period comparatively than the conventional coagulation

process. Metal electrodes, such as aluminum and iron are in general employed for

ﬂocculation and precipitation in electrocoagulation. (Ghumra et al. 2021; Züleyha

et al. 2021; Züleyha et al. 2021) reported the removal of COD at different Ph 5 and

3 as 83.3 and 83.5% respectively using the electrocoagulation method from initial

concentration of 6000–9000 mg/l.

Adsorption is a promising, cost-effective, eco-friendly, and easily applicable tech-

niques. The adsorbents used for chromium removals are activated carbon, graphite,

serpentine and zeolite etc. Farahat and Sanad (2021) reported adsorption capacity of

138 mg/g for chromium removal at low pH, 25 °C, and 60 min of contact time.

2 Membrane System

The membrane process is gradually receiving importance as promising tool for treat-

ment of industrial wastewater/ pollutants in an efﬁcient way. The process necessitates

moderate cost, low energy, and there is no need for any additives compared to the

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery … 417

Tabl e 3 Classiﬁcation of

Pressure driven membrane

process

Types of

membranes

Pressure (Bar) Pore size Mechanism

Microﬁltration 2 0.05–10 µmSieving

Ultraﬁltration 1–10 2–100 nm Sieving

Nanoﬁltration 10–25 2nm Solution

diffusion

Reverse

Osmosis

10–100 <2 nm Solution

diffusion

other treatment technologies. Several types of membranes made of ceramic, poly-

meric, and composite elements are in usage for the treatment process in wastewater

industries.

2.1 Principle of Membrane System

Membrane separation is a physical phenomenon that separates a solute from the

aqueous mixture on the basis of size of molecules. Pressure-driven membrane

process removes different sizes of molecules and is classiﬁed in Table 3 according

to the pore size of the membrane into microﬁltration (MF), ultraﬁltration (UF),

reverse osmosis (RO), and nanoﬁltration (NF) membranes respectively (Hakami

et al. 2020). Membrane pore size and distribution, surface charge, ﬂuid ﬂow, and

an active layer are the most important inﬂuencing factors (Mestre et al. 2019). The

separation mechanism of the membrane is generally governed by size exclusion,

Donnan exclusion effect, and adsorption. In size exclusion, the pore size of the

membrane is less than molecule size, which will retain the molecule, and for large

pore size passes through the membrane (Mulder 1996). The adsorption mechanism

is similar to the typical adsorption process via physical or chemical interactions.

Donnan exclusion is mostly employed for dense membranes. The feed solution pH

is above the iso electric point, membrane repulse cation and vice versa for lower pH

(Abdullah et al. 2019; Mulder 1996).

2.2 MBR Conﬁguration for TWW Treatment

The selection of membrane module is important for the commercial treatment of

TWW and the different factors such as ﬂow rate, cleaning ability, module replacement

are considered during the module selection. There are four types of commercially

available membrane modules, namely, plate and frame module, tubular module, spiral

wound module, and hollow ﬁber modules (Ezugbe and Rathilal 2020).

418 C. Raja et al.

Plate and frame module is one of the earliest modules consists of membrane

and spacers interconnected with a metallic frame. The module is mostly suitable

for wastewater containing high level of suspended solids and it is easy to clean

(Ezugbe and Rathilal 2020). Yang et al. (2021) studied the efﬁciency of the PVDF

and PES ultraﬁltration membrane with permeation efﬁciency and fouling behaviour.

Spiral Wound Module consists of more membranes and spacers around the perforated

collection tube and widely applied in RO and NF operations. The conﬁguration offers

a high packing density leading to high membrane surface area. It is suitable for large-

scale operations due to its easy replacement (Ezugbe and Rathilal 2020). Cassano

et al. (2001) used commercial polysulphone membrane with 20KDa of Molecular

weight cut-off and achieved 55% removal of fat. Stoller et al. (2013) used commercial

NF spiral wound module and achieved COD as 102 mg/l and removed total suspended

solids. Similarly, the removal rate of chromium and COD for RO membrane was

86 mg/l and 0.04 mg/l respectively. The threshold ﬂux of the NF membrane was

also reported. Tubular Module consists of an outer shell and an inner tube. Tubular

membranes are adapted to treating feed with high solid contents (Ezugbe and Rathilal

2020). Mohammed and Sahu (2015) reported, that chromium removal using three

different commercial membranes and RO membrane achieved 99.9% removal efﬁ-

ciency. Amine functionalized tubular composite membrane employed for the heavy

metal removal. The removal percentage of chromium and arsenic was reported as

97.22% and 96.75%. Hollow Fiber Module houses a bundle of hollow ﬁbers, whether

closed or open-end, in a pressure vessel. A very notable advantage of this module type

is its ability to house large membrane areas in a single module (Ezugbe and Rathilal,

2020). Mukherjee et al. (2019) conduct a scale up study of chromium removal on

mixed matrix ultraﬁltration membrane using modelling. Vo et al. (2021) reported the

COD and TDS of equalization tank wastewater using PVDF hollow ﬁber membrane

and achieved 87 ± 14% of COD where the fouling level was reduced by the sponge

material.

2.3 Factors Inﬂuencing Performance of MBRs

Different types of membranes are utilised for separation processes in TWW treatment

depending on steps of the TWW generated. Membrane type, and material, pore size,

pre-treatment of feed water, and fouling control methods are responsible to inﬂuence

the membrane performance (Rosman et al. 2018). The Beam house process efﬂuent

contains high suspended solids with a signiﬁcant quantity of colloidal particles. In

this situation, a microﬁltration membrane is mostly suitable for separation. Simi-

larly, the ﬁnal stage operation is mostly organic and some metals are present in the

feed. However, ultraﬁltration and nanoﬁltration membrane is suitable for the oper-

ation to get high efﬁciency. Hakami et al. (2020). Foulant signiﬁcantly affects the

membrane material because of the interaction between the membrane surface and the

foulants. The cost of the membrane material is another important consideration for

membrane selection. Du et al. (2020). The selection of membrane material depends

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery … 419

on the sources of wastewater. Both organic and inorganic membranes are used for

the treatment of beam house efﬂuent and attain better efﬁciency. Pore size is another

important parameter to be considered for membrane selection. The membrane pore

is important for ﬂux and removal efﬁciency. Membrane pore size is also one of

the factors considered for better efﬁciency as well as membrane fouling. Micro-

ﬁltration membrane typically used for elimination of turbidity, suspended solids,

and pathogens. Nanoﬁltration membranes can affect the removal of organic matter,

sulphates, turbidity, suspended solids and other pathogens. Common application is

for removal of monovalent salts in reverse osmosis membrane (Ghumra et al., 2021).

Paletal. (

2020b) investigated NF and graphene oxide membrane for COD, TDS,

and chromium removal. Both membranes revealed varied ﬂux and efﬁciency and

indicated that membrane hydrophilicity and formation of nanochannel inﬂuences

the performance. Stoller et al. (2013) suggested polyamide RO membrane provides

better removal in terms of chromium than NF. Roy (2020) studied heavy metal

rejection by different polymeric membranes with pore radii about 1 nm where the

membrane offered better rejection for Cu (II), As(V), Cr (97.22) and sustained better

stability for continuous operation. Arif et al. (2020) applied ultraﬁltration membrane

for removal and reduction of chromium. Karunanidhi et al. (2020) observed removal

of BOD, COD, and dye removed from tannery efﬂuent using keratin-polysulfone

membrane. The keratin modiﬁed polysulfone membrane has less pore size than the

polysulfone membrane with high ﬂux. Kaplan-Bekaroglu and Gode (2016) reported

90% COD removal by ceramic ultraﬁltration membrane with membrane pore size

of 10 nm. Whereas, membranes with high pore sizes have high ﬂux with low efﬁ-

ciency than other membranes. Bhattacharya et al. (2013) reported that a microﬁltra-

tion membrane can reduce the fouling tendency of the membrane due to the pres-

ence of dissolved solids during pre-treatment providing suitable inﬂuent to reverse

osmosis membrane in a combination of ceramic microﬁltration and reverse osmosis

membrane system. Roy Choudhury et al. (2018) found hydroxyethyl cellulose and

CuO blend composite membrane showed better metal removal from the wastew-

ater. The membrane showed better rejection in chromium due to strong adhesion

between support and nanoparticle which enhance the pore size reduction. Elomari

et al. (2016) reported clay membrane with different pore sizes showed good reten-

tion in turbidity and conductivity. Romero-Dondiz et al. (2015) reported the perfor-

mance of different molecular weight cut-off polysulfone ultraﬁltration membranes

in vegetable tanning wastewater. Low molecular weight cutoff membrane provides

high rejection than other compared membranes due to its size exclusion mecha-

nism. Kiril Mert and Kestioglu (2014) reported removal of pollutant from tanning

process by three different types of membrane with different molecular weight cut-

off. Membrane showed better efﬁciency in sulfate and suspended solids removal

when 200 MWCO membrane shows better performance than other membranes in

the chromium recovery. Rambabu and Velu (2016) studied modiﬁed PES membrane

for BOD, COD and TDS removal. CaCl2 modiﬁcation improved the performance

than pure PES membrane. Similarly, chitosan modiﬁed PES membrane showed the

signiﬁcant removal in chromium separation from the wastewater. The membrane

shows better rejection in salts.

420 C. Raja et al.

2.4 Advantages and Challenges of Membrane Reactors

Membrane technology has several advantages over other separation processes which

include no requirement of chemicals, low energy consumption, no phase change, and

is suitable for continuous processes and easy adaptation with other systems (Mulder

1996). However, fouling is a major challenge due to the relatively higher amounts of

organic matter and particulates in the feed wastewater. Membrane fouling is reduced

ﬂux, membrane life, increases energy like pressure, and also process cost (Ezugbe

and Rathilal 2020). Suspended solids, microbes, and organic materials are a few

components that are the reasons for fouling due to deposition on the membrane

surface (Hakami et al. 2020). Fouling is characterized by colloidal, organic, inor-

ganic, and biofouling. Membrane fouling depends on feed characteristics like pH and

ionic strength, membrane characteristics like roughness, hydrophobicity, etc., and

process conditions like cross-ﬂow velocity, trans-membrane pressure, and tempera-

ture. The different techniques are used to avoid or reduce the fouling in the membrane.

Turbulence promotors, ultra-sonication, backwashing are the techniques reduces the

membrane fouling (Ezugbe and Rathilal 2020; Hakami et al. 2020).

3 Moving Bed Bioﬁlm Reactor (MBBR)

Moving bed bioﬁlm reactor (MBBR) is a bioﬁlm reactor system, currently with wide

attraction from researchers and environment scientists for treatment of complex and

municipal wastewater due to its efﬁciency, cost effectiveness and easy operating

facilities. Microbes are most suitable agents selected for bioremediation; however

indigenous microbes due to its poor populations many times fail to attain the treat-

ment level. In MBBR, microbes are provided with inert porous carrier media as

substratum in suspension for microbial adhesion and growth (Odegaard et al. 1994).

High microbial density, tolerance toward toxic/ hazardous chemicals, environmental

stress and parameters are major attributes for high performance efﬁciency of MBBR.

3.1 Conﬁguration of MBBR

Bioﬁlms are intricate surface architecture made of well-structured communities of

adherent microorganisms attached to inert/abiotic or living/biotic surface (Huang

and Li 2014; Muhammad et al. 2020). The aggregated microorganisms may belong

to same species or diverse species of bacteria, protozoa, archaea, algae, ﬁlamen-

tous fungi, and yeast that strongly attach to each other and/or to surfaces and are

held together by self-produced polymer matrix known as extracellular polysac-

charide matrix (EPS). Production of EPS, interaction between microbial cells as

well as surrounding media, and their intermolecular forces such as electrostatic/

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery … 421

van der Walls interaction, hydrophobicity etc. predominantly regulates adherence

of microbes to the substratum (Carniello et al. 2018). Surface charge of microbes

is another prime factor that affects bioﬁlm production through adhesion of nega-

tively charged microbes (due to presence of considerable amount of carboxyl, amino,

and phosphate groups in cell wall) with positively charged substratum. EPS, the

very signiﬁcant component responsible for bioﬁlm formation and performance is

mainly composed of polysaccharides, secreted proteins, and extracellular DNAs

(Costa et al. 2020). EPS inﬂuences protection of cells, transportation of nutrients

and electron acceptors, solubilization of hydrophobic/recalcitrant elements and cell

to cell signalling by Quorum Sensing Interference (QS). Bestowing the physicochem-

ical and molecular properties of the microbes, bioﬁlms get maturation followed by

dispersal or detachment. The microbial position at substratum in two-dimensional

Brownian motion facilitates easy separation of bioﬁlms triggered by any shear effects

or bacterial movement (Carniello et al. 2018) (Fig. 1).

The growth of microbes in the biocarriers in the MBBR creates several zones

such as aerobic (in the biocarrier surface) and micro anoxic (interior bioﬁlms) zones.

These stratiﬁcations highly facilitate slow growth nitriﬁers in the bioﬁlm surface and

anoxic denitriﬁes providing nitriﬁcation and partial denitriﬁcation of accumulated

nitrite and nitrates in the inﬂuent (di Biase et al. 2019). The ﬂexibility of MBBRs

with its suspended carrier bed make it feasible to operate in batch mode, sequencing

as well as sequential conﬁguration for achieving high treatment efﬁciency (Di Iaconi

et al. 2003; Odegaard et al. 1994; Sahariah and Chakraborty 2013). For a commercial

laundry with daily ﬂow of 0.6–1.0 m3/d wastewater, a two staged MBBR treatment

with Kaldane K5 carrier media provided more than 95% of organics and anionic/

nonionic surfactants removals with attainment to its limit for discharged to receiving

environment (Bering et al. 2018). Treatment of high organic strength wastewater,

Fig. 1 Lab scale MBBRs with Polyurethane cubes as bioﬁlm carrier media

422 C. Raja et al.

for example, Cheese industries, MBBR stands suitable treatment process for carbon,

phosphorous and ammonia removals Tsitouras et al. (2021). Similarly, MBBRs are

continuously employed for treatment of metals, dyes from various industries with

higher rate of success (Leyva-Díaz et al. 2020;Lietal. 2015;Suetal. 2019). MBBR

with modiﬁed low-density polyethylene–polypropylene carriers provided maximum

elimination capacity of Congo red as high as 214.4 mg/L day (Sonwani et al. 2021).

In a study for mixed azo dye (1200 mg/l) and chromium (300 mg/l) treatment from

textile wastewater, MBBR with bacterial consortium of Bacillus circulans, Bacillus

circulans, Bacillus subtilis and Terribacillus gorriensis accomplished more than 80%

decolorization simultaneously with 56% degradation of chromium from the initial

stages of treatment (Biju et al. 2022).

3.2 Inﬂuencing Factors in MBBRs

Being a bioreactor, MBBRs are also responsive to environmental parameters (pH,

temperature etc.) and operational parameters (HRT, loadings etc.) to some extents.

However, it is signiﬁcantly resistance to minor changes to these parameters compared

to other biological reactors. Major inﬂuencing parameter for MBBR performance

involves its carrier media for bioﬁlm growth and ﬁlling density of the carrier element

in the reactor. Carrier media with higher surface space for bioﬁlm growth with

minimum weight and 30–40% ﬁll of the reactor volume is recommended for better

performance of MBBRs.

3.3 Advantages and Limitations of MBBRs

The advantages of bioﬁlm reactor are more enhanced in case of MBBR due to of

MBBR conﬁguration with carrier material of high surface area for the growth of the

microbes, that keep moving inside the reactor due to movement of external force or

reactor ﬂuid. Requirement of less land area, high sludge retention time, reduction of

head loss in MBBR, no sludge recirculation, lower sensitivity to shock loading, no

sludge bulking, and appropriateness for slow-growing microbes, are another a few

positive attributes of MBBR recognised for its high utility for industrial wastewater

treatment (Bachmann Pinto et al. 2018;Lietal.

2015, 2019a, b; Sahariah et al. 2018;

Sodhi et al. 2021; Yadu et al. 2018).

Along with so much more advantages there are few limitations come across appli-

cation of MBBRs such as necessity of manual monitoring as its functional attribute is

microorganisms. Therefore, there is need of skilled technician and operators having

rich knowledge in biological wastewater treatment to monitor reactor performance

and avoid unwanted situations such as entry of insects that spoil carrier element in

the reactor and direct escaping of the same.

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery … 423

4 Conclusion

High efﬁciency for pollutant removal is utmost association with MBRs and MBBRs.

Pollutants of various category, organics, r ecalcitrant, heavy metals, inorganics are

well manged in these reactors. Tannery wastewater is a complex wastewater with

huge load of organics, large biomolecules responsible for high COD, dyes, heavy

metals such as chromium, along with inorganics sulphides, nitrogen etc. The fact

of these pollutant removals in MBR and MBBR are well established. Considering

requirement and high consumption of water resources in tannery industry, these

advanced but easy operation MBR and MBBR are recommended to achieve high

efﬂuent quality.

References

Abdel-Shafy HI, Hegemann W, Genschow E (1997) The elimination of chromium in the treatment

of tannery industrial wastewater. Environ Manag Health 8(2):73–79. https://doi.org/10.1108/095

66169710166566

Abdullah N, Yusof N, Abu Shah MH, Wan Ikhsan SN, Ng ZC, Maji S, Lau WJ, Jaafar J, Ismail AF,

Ariga K (2019) Hydrous ferric oxide nanoparticles hosted porous polyethersulfone adsorptive

membrane: chromium (VI) adsorptive studies and its applicability for water/wastewater treatment.

Environ Sci Pollut Res 26:20386–20399. https://doi.org/10.1007/s11356-019-05208-9

Aregu MB, Asfaw SL, Khan MM (2021) Developing horizontal subsurface ﬂow constructed wetland

using pumice and Chrysopogon zizanioides for tannery wastewater treatment. Environ Syst Res

https://doi.org/10.1186/s40068-021-00238-0

Arif Z, Sethy NK, Mishra PK, Verma B (2020) Green approach for the synthesis of ultraﬁltration

photocatalytic membrane for tannery wastewater: modeling and optimization. Int J Environ Sci

Technol 17:3397–3410. https://doi.org/10.1007/s13762-020-02719-8

Bachmann Pinto H, Miguel de Souza B, Dezotti M (2018) Treatment of a pesticide industry wastew-

ater mixture in a moving bed bioﬁlm reactor followed by conventional and membrane processes

for water reuse. J Clean Prod 201:1061–1070. https://doi.org/10.1016/J.JCLEPRO.2018.08.113

Ballén-segura M, Rodríguez LH, Ospina DP, Bolaños AV, Pérez K (2016) Using Scenedesmus sp.

for the phycoremediation of tannery wastewater, vol 12, pp 69–75

Bera D, Chattopadhyay P, Ray L (2012) Continuous removal of chromium from tannery wastewater

using activated sludge process—Determination of kinetic parameters, vol 19, pp 32–36

Bering S, Mazur J, Tarnowski K, Janus M, Mozia S, Morawski AW (2018) The application of

moving bed bio-reactor (MBBR) in commercial laundry wastewater treatment. Sci Total Environ

627:1638–1643. https://doi.org/10.1016/J.SCITOTENV.2018.02.029

Bharagava RN, Saxena G, Mulla SI, Patel DK (2017) Characterization and identiﬁcation of recal-

citrant organic pollutants (ROPs) in tannery wastewater and its phytotoxicity evaluation for envi-

ronmental safety. Arch Environ Contam Toxicol 752(75):259–272. https://doi.org/10.1007/S00

244-017-0490-X

Bhattacharya P, Roy A, Sarkar S, Ghosh S, Majumdar S, Chakraborty S, Mandal S, Mukhopadhyay

A, Bandyopadhyay S (2013) Combination technology of ceramic microﬁltration and reverse

osmosis for tannery wastewater recovery. Water Resour Ind 3:48–62. https://doi.org/10.1016/j.

wri.2013.09.002

424 C. Raja et al.

Biju LM, Pooshana V, Kumar PS, Gayathri KV, Ansar S, Govindaraju S (2022) Treatment of

textile wastewater containing mixed toxic azo dye and chromium (VI) BY haloalkaliphilic bacte-

rial consortium. Chemosphere 287:132280. https://doi.org/10.1016/J.CHEMOSPHERE.2021.

132280

Boopathy R, Karthikeyan S, Mandal AB, Sekaran G (2013) Characterisation a nd recovery of sodium

chloride from salt-laden solid waste generated from leather industry, pp 117–124. https://doi.org/

10.1007/s10098-012-0489-y

Carniello V, Peterson BW, van der Mei HC, Busscher HJ (2018) Physico-chemistry from initial

bacterial adhesion to surface-programmed bioﬁlm growth. Adv Colloid Interface Sci 261:1–14.

https://doi.org/10.1016/J.CIS.2018.10.005

Cassano A, Molinari R, Romano M, Drioli E (2001) Treatment of aqueous efﬂuents of the leather

industry by membrane processes: a review. J Memb Sci 181:111–126. https://doi.org/10.1016/

S0376-7388(00)00399-9

Chowdhury M, Mostafa MG (2015) Characterization of the efﬂuents from leather processing

industries, pp 173–187. https://doi.org/10.1007/s40710-015-0065-7

Chowdhury M, Mostafa MG, Biswas TK, Saha AK (2013) Treatment of leather industrial efﬂuents

by ﬁltration and coagulation processes. Water Resour Ind 3:11–22. https://doi.org/10.1016/j.wri.

2013.05.002

Costa RC, Souza JGS, Bertolini M, Retamal-Valdes B, Feres M, Barão VAR (2020) Extracellular

bioﬁlm matrix leads to microbial dysbiosis and reduces bioﬁlm susceptibility to antimicrobials

on titanium biomaterial: an in vitro and in situ study. Clin Oral Implants Res 31:1173–1186.

https://doi.org/10.1111/CLR.13663

di Biase A, Kowalski MS, Devlin TR, Oleszkiewicz JA (2019) Moving bed bioﬁlm reactor tech-

nology in municipal wastewater treatment: a review. J Environ Manag 247:849–866. https://doi.

org/10.1016/J.JENVMAN.2019.06.053

Di Iaconi C, Lopez A, Ramadori R, Passino R (2003) Tannery wastewater treatment by sequencing

batch bioﬁlm reactor. Environ Sci Technol 37:3199–3205. https://doi.org/10.1021/ES030002U

Du X, Shi Y, Jegatheesan V, Ul Haq I (2020) A review on the mechanism, impacts and control

methods of membrane fouling in MBR system. Membranes. https://doi.org/10.3390/membranes

10020024

Elomari H, Achiou B, Ouammou M, Albizane A, Bennazha J, Alami Younssi S, Elamrani I (2016)

Elaboration and characterization of ﬂat membrane supports from Moroccan clays. Application

for the treatment of wastewater. Desalin Water Treat 57:20298–20306. https://doi.org/10.1080/

19443994.2015.1110722

Elsheikh MA (2009) Tannery wastewater pre-treatment, pp 433–440. https://doi.org/10.2166/wst.

2009.351

Ezugbe EO, Rathilal S (2020) Membrane technologies in wastewater treatment: a review.

Membranes (basel) 10. https://doi.org/10.3390/membranes10050089

Farahat MM, Sanad MMS (2021) Decoration of serpentine with iron ore as an ef ﬁ cient low-cost

magnetic adsorbent for Cr (VI) removal from tannery wastewater, vol 388, pp 51–62

Fettig J, Pick V, Oldenburg M, Phuoc NV (2017) Treatment of tannery wastewater for reuse by

physico- chemical processes and a membrane bioreactor, pp 420–428. https://doi.org/10.2166/

wrd.2016.036

Fu F, Wang Q (2011) Removal of heavy metal ions from wastewaters: a review. J Environ Manag

92:407–418. https://doi.org/10.1016/j.jenvman.2010.11.011

Ghumra DP, Agarkoti C, Gogate PR (2021) Improvements in efﬂuent treatment technologies in

common efﬂuent treatment plants (CETPs): review and recent advances. Process Saf Environ

Prot 147:1018–1051. https://doi.org/10.1016/j.psep.2021.01.021

Gregorio SD, Rufﬁni LGM (2015) Phytoremediation for improving the quality of efﬂuents from

a conventional tannery wastewater treatment plant, pp 1387–1400. https://doi.org/10.1007/s13

762-014-0522-2

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery … 425

Hakami MW, Alkhudhiri A, Al-Batty S, Zacharof MP, Maddy J, Hilal N (2020) Ceramic microﬁltra-

tion membranes in wastewater treatment: ﬁltration behavior, fouling and prevention. Membranes

(Basel) 10:1–34. https://doi.org/10.3390/membranes10090248

Hansen E, de Aquim PM, Gutterres M (2021) Environmental assessment of water, chemicals and

efﬂuents in leather post-tanning process: A review. Environ Impact Assess Rev 89(1):106597.

https://doi.org/10.1016/j.eiar.2021.106597

Huang T, Li D (2014) Presentation on mechanisms and applications of chalcopyrite and pyrite

bioleaching in biohydrometallurgy-a presentation. Biotechnol Rep. https://doi.org/10.1016/j.btre.

2014.09.003

Kaplan-Bekaroglu SS, Gode S (2016) Investigation of ceramic membranes performance for tannery

wastewater treatment. Desalin Water Treat 57:17300–17307. https://doi.org/10.1080/19443994.

2015.1084595

Karunanidhi A, David PS, Fathima NN (2020) Electrospun keratin-polysulfone blend membranes

for treatment of tannery efﬂuents. Water Air Soil Pollut. 231. https://doi.org/10.1007/s11270-

020-04682-z

Kassaye G, Gabbiye N, Alemu A (2017) Phytoremediation of chromium from tannery wastewater

using local plant species, vol 12, pp 894–901. https://doi.org/10.2166/wpt.2017.094

Kiril Mert B, Kestioglu K (2014) Recovery of Cr(III) from tanning process using membrane sepa-

ration processes. Clean Technol Environ Policy 16:1615–1624. https://doi.org/10.1007/s10098-

014-0737-4

Korpe S, Rao PV (2021) Application of advanced oxidation processes and cavitation techniques

for treatment of tannery wastewater-a review. J Environ Chem Eng 9:105234. https://doi.org/10.

1016/j.jece.2021.105234

Kumar V, Dwivedi SK (2021) A review on accessible techniques for removal of hexavalent

Chromium and divalent Nickel from industrial wastewater: Recent research and future outlook,

vol 295

Leyva-Díaz JC, Monteoliva-García A, Martín-Pascual J, Munio MM, García-Mesa JJ, Poyatos JM

(2020) Moving bed bioﬁlm reactor as an alternative wastewater treatment process for nutrient

removal and recovery in the circular economy model. Bioresour Technol 299:122631. https://doi.

org/10.1016/J.BIORTECH.2019.122631

Li C, Zhang Z, Li Y, Cao J (2015) Study on dyeing wastewater treatment at high temperature by

MBBR and the thermotolerant mechanism based on its microbial analysis. Process Biochem

50:1934–1941. https://doi.org/10.1016/J.PROCBIO.2015.08.007

Li C, Liang J, Lin X, Xu H, Tadda MA, Lan L, Liu D (2019a) Fast start-up strategies of MBBR for

mariculture wastewater treatment. J Environ Manag 248:109267. https://doi.org/10.1016/J.JEN

VMAN.2019.109267

Li J, Peng Y, Zhang L, Liu J, Wang X, Gao R, Pang L, Zhou Y (2019b) Quantify the contribution

of anammox for enhanced nitrogen removal through metagenomic analysis and mass balance in

an anoxic moving bed bioﬁlm reactor. Water Res 160:178–187. https://doi.org/10.1016/J.WAT

RES.2019.05.070

Lofrano G, Meriç S, Zengin GE, Orhon D (2013) Chemical and biological treatment technologies

for leather tannery chemicals and wastewaters: a review. Sci Total Environ 461–462:265–281.

https://doi.org/10.1016/j.scitotenv.2013.05.004

Mannucci A, Munz G, Mori G, Lubello C (2010) Anaerobic treatment of vegetable tannery

wastewaters: a review. Desalination 264:1–8. https://doi.org/10.1016/j.desal.2010.07.021

Medina BY, Torem ML, De Mesquita LMS (2005) On the kinetics of precipitate ﬂotation of Cr III

using sodium dodecylsulfate and ethanol. Miner Eng 18:225–231. https://doi.org/10.1016/j.min

eng.2004.08.018

Mestre S, Gozalbo A, Lorente-ayza MM, Sánchez E (2019) Low-cost ceramic membranes: a

research opportunity for industrial application. J Eur Ceram Soc 39:3392–3407. https://doi.org/

10.1016/j.jeurceramsoc.2019.03.054

426 C. Raja et al.

Mohammed K, Sahu O (2015) Bioadsorption and membrane technology for reduction and recovery

of chromium from tannery industry wastewater. Environ Technol Innov 4:150–158. https://doi.

org/10.1016/j.eti.2015.06.003

Muhammad MH, Idris AL, Fan X, Guo Y, Yu Y, Jin X, Qiu J, Guan X, Huang T (2020) Beyond

risk: bacterial bioﬁlms and their regulating approaches. Front Microbiol 0:928. https://doi.org/

10.3389/FMICB.2020.00928

Mukherjee R, Bhunia P, De S (2019) Long term ﬁltration modelling and scaling up of mixed matrix

ultraﬁltration hollow ﬁber membrane: a case study of chromium ( VI ) removal, vol 571, pp

204–214

Mulder M (1996) Basic principles of membrane technology, 2E.pdf

Odegaard H, Rusten B, Westrum T (1994) A new moving bed bioﬁlm reactor-applications and

results. Water Sci Technol 29:157–165. https://doi.org/10.2166/WST.1994.0757

Pal M, Malhotra M, Mandal MK, Paine TK, Pal P (2020a) Recycling of wastewater from

tannery industry through membrane-integrated hybrid treatment using a novel graphene oxide

nanocomposite. J Water Process Eng 36:101324. https://doi.org/10.1016/j.jwpe.2020.101324

Pal M, Malhotra M, Mandal MK, Paine TK, Pal P (2020b) Recycling of wastewater from

tannery industry through membrane- integrated hybrid treatment using a novel graphene oxide

nanocomposite. J Water Process Eng 36

Rambabu K, Velu S (2016) Modiﬁed polyethersulfone ultraﬁltration membrane for the treatment

of tannery wastewater. Int J Environ Stud 73:819–826. https://doi.org/10.1080/00207233.2016.

1153900

Rangel OSSÃ, Castro PML, Calheiros CSC (2007) Constructed wetland systems vegetated with

different plants a pplied to the treatment of tannery wastewater, vol 41, pp 1790–1798. https://doi.

org/10.1016/j.watres.2007.01.012

Reyes-Serrano A, López-Alejo JE, Hernández-Cortázar MA, Elizalde I (2020) Removing contam-

inants from tannery wastewater by chemical precipitation using CaO and Ca(OH)2. J Chinese

Chem Eng 28:1107–1111. https://doi.org/10.1016/j.cjche.2019.12.023

Romero-Dondiz EM, Almazán JE, Rajal VB, Castro-Vidaurre EF (2015) Removal of vegetable

tannins to recover water in the leather industry by ultraﬁltration polymeric membranes. Chem

Eng Res Des 93:727–735. https://doi.org/10.1016/j.cherd.2014.06.022

Rosman N, Salleh WNW, Mohamed MA, Jaafar J, Ismail AF, Harun Z (2018) Hybrid membrane

ﬁltration-advanced oxidation processes for removal of pharmaceutical residue. J Colloid Interface

Sci 532:236–260. https://doi.org/10.1016/j.jcis.2018.07.118

Roy Choudhury P, Majumdar S, Sahoo GC, Saha S, Mondal P (2018) High pressure ultraﬁltration

CuO/hydroxyethyl cellulose composite ceramic membrane for separation of Cr (VI) and Pb (II)

from contaminated water. Chem Eng J 336:570–578. https://doi.org/10.1016/j.cej.2017.12.062

Roy S (2020) Removal of As(V), Cr(VI) and Cu(II) using novel amine functionalized composite

nanoﬁltration membranes fabricated on ceramic tubular substrate

Sahariah BP, Anandkumar J, Chakraborty S (2018) Stability of continuous and fed batch sequen-

tial anaerobic–anoxic–aerobic moving bed bioreactor systems at phenol shock load application.

Environ Technol (United Kingdom) 39. https://doi.org/10.1080/09593330.2017.1343388

Sahariah BP, Chakraborty S (2013) Effects of cycle time and ﬁll time on the performance of an

anaerobic-anoxic-aerobic-fed batch moving-bed reactor. Environ Technol (United Kingdom) 34.

https://doi.org/10.1080/09593330.2012.692712

Sawalha H, Alsharabaty R, Sarsour S, Al-jabari M (2019) Wastewater from leather tanning and

processing in Palestine: characterization and management aspects. J Environ Manag 251:109596.

https://doi.org/10.1016/j.jenvman.2019.109596

Shah MP (2020) Microbial bioremediation & biodegradation. Springer

Sekaran G, Karthikeyan S, Nagalakshmi C (2013) Integrated Bacillus sp. immobilized cell reactor

and Synechocystis sp. algal reactor for the treatment of tannery wastewater, pp 281–291. https://

doi.org/10.1007/s11356-012-0891-3

Sivagami K, Sakthivel KP, Nambi IM (2018) Advanced Oxidation Processes for the Treatment of

Tannery Wastewater. J Environ Chem Eng 6:3656–3663

Membrane Reactor and Moving Bed Bioﬁlm Reactor for Tannery … 427

Shah MP (2021a) Removal of refractory pollutants from wastewater treatment plants. CRC Press

Shah MP (2021b) Removal of emerging contaminants through microbial processes. Springer

Sodhi V, Singh C, Pal Singh Cheema P, Sharma R, Bansal A, Kumar Jha M (2021) Simulta-

neous sludge minimization, pollutant and nitrogen removal using integrated MBBR conﬁguration

for tannery wastewater treatment. Bioresour Technol 341:125748. https://doi.org/10.1016/J.BIO

RTECH.2021.125748

Song Z, Williams CJ, Edyvean RGJ (2000) Sedimentation of tannery wastewater. Water Res

34:2171–2176. https://doi.org/10.1016/S0043-1354(99)00358-9

Sonwani RK, Swain G, Jaiswal RP, Singh RS, Rai BN (2021) Moving bed bioﬁlm reactor with immo-

bilized low-density polyethylene–polypropylene for Congo red dye removal. Environ Technol

Innov 23:101558. https://doi.org/10.1016/J.ETI.2021.101558

Stoller M, Sacco O, Sannino D, Chianese A (2013) Successful integration of membrane technologies

in a conventional puriﬁcation process of tannery wastewater streams. Membranes (basel) 3:126–

135. https://doi.org/10.3390/membranes3030126

feng Su J, Xue L, lin Huang T, Wei L, yu Gao C, Wen Q (2019) Performance and microbial

community of simultaneous removal of NO3−-N, Cd2+ and Ca2+ in MBBR. J Environ Manag

250:109548. https://doi.org/10.1016/J.JENVMAN.2019.109548

Tiravanti G, Petruzzelli D, Passino R (1991) Pretreatment of Tannery. Water Sci Technol 36:197–

207. https://doi.org/10.1016/S0273-1223(97)00388-0

Tsitouras A, Al-Ghussain N, Delatolla R (2021) Two moving bed bioﬁlm reactors in series for

carbon, nitrogen, and phosphorous removal from high organic wastewaters. J Water Process Eng

41:102088. https://doi.org/10.1016/J.JWPE.2021.102088

Vo T, Bui X, Dang B (2021) Environmental Technology & Innovation Inﬂuence of organic loading

rates on treatment performance of membrane bioreactor treating tannery wastewater. Environ

Technol Innov 24:101810. https://doi.org/10.1016/j.eti.2021.101810

Yadu A, Sahariah BP, Anandkumar J (2018) Inﬂuence of COD/ammonia ratio on simultaneous

removal of NH<inf>4</inf>+-N and COD in surface water using moving bed batch reactor. J

Water Process Eng 22. https://doi.org/10.1016/j.jwpe.2018.01.007

Yang F, Huang Z, Huang J, Wu C, Zhou R, Jin Y (2021) Tanning wastewater treatment by

ultraﬁltration: process efﬁciency and fouling behavior, pp 1–17

Younas F, Khan N, Bibi I, Afzal M, Hussain K, Shahid M, Aslam Z, Bashir S, Mahroz M (2022)

Constructed wetlands as a sustainable technology for wastewater treatment with emphasis on

chromium-rich tannery wastewater, vol 422

Züleyha B, ¸Sahset I, Nuhi D (2021) Effect of controlled and uncontrolled pH on tannery wastewater

treatment by the electrocoagulation process. Int J Environ Anal Chem 00:1–16. https://doi.org/

10.1080/03067319.2021.1925261

Nanobioremediation: A Sustainable

Approach for Wastewater Treatment

Sougata Ghosh, Bishwarup Sarkar, and Sirikanjana Thongmee

Abstract Urbanization and industrial revolution has led to the pollution of the

existing water bodies at an alarming rate. Heavy metals, pesticides, dye, oil spills and

other hazardous chemicals are among the key refractory pollutants that cannot be

removed effectively by the conventional wastewater treatment processes which are

often expensive and have high energy requirements. Nanotechnology is employed

in various ﬁelds more recently that include textiles, food, pharmaceutics, agriculture

and even environment. Small dimension of the nanoparticles offers larger surface

area for adsorption of the toxic pollutants that can be eventually removed. Such

nanoparticles have exotic physicochemical and optoelectronic properties that deter-

mine their appropriate ﬁeld of applications. Nanotechnology driven solutions for

wastewater treatment is not only rapid and efﬁcient but also economical. In view of

the background, this chapter discusses in detail, the recent advances in the ﬁeld of

wastewater nanobioremediation through application of various surface active nanos-

tructures. Photocatalytic degradation of pollutants in wastewater by nanostructured

catalyst in presence of appropriate source of illumination has been reported in several

studies. Some of the common nanocatalysts covered here include titanium dioxide

(TiO2), zinc oxide (ZnO), ferric oxide (Fe2O3), zinc sulﬁde (ZnS), magnetic nanopar-

ticles (MNPs). Further, pressure-driven nanoﬁltration using different nanomaterials

such as carbon, metal oxides are also elaborated. Eventually, nanosorbents mediated

removal of several organic and inorganic pollutants from wastewater samples are also

covered. Hence, further optimization and scale up of nanomaterial mediated wastew-

ater treatment can help to implement the process for treating industrial efﬂuents to

ensure a safe environment.

S. Ghosh (B

) · S. Thongmee

Faculty of Science, Department of Physics, Kasetsart University, Bangkok, Thailand

e-mail: ghoshsibb@gmail.com

S. Ghosh

Department of Microbiology, School of Science, RK. University, Rajkot, Gujarat, India

B. Sarkar

College of Science, Northeastern University, Boston, MA, USA

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_20

429

430 S. Ghosh et al.

Keywords Wastewater treatment ·Refractory pollutants ·Nanotechnology ·

Photocatalysis ·Nanoﬁltration ·Nanosorbents

1 Introduction

Water is a vital part of the planet which is naturally recycled to maintain an adequate

supply of clean water for human consumption (Bora and Dutta 2014). However,

uncontrolled growth rate of the human population along with excessive industrializa-

tion has resulted in shortage of potable water in various parts of the world (Luikham

et al. 2018). In addition, majority of water supply processes require a large amount

of resources that are primarily involved in puriﬁcation and treatment of wastewater

to control the organic and inorganic content in such contaminated water samples

(Chorawala and Mehta 2015).

Conventional wastewater treatment methods are cumbersome and typically

consists of three stages namely, preliminary, primary and secondary (Ghosh 2020).

Moreover, the treated samples are also subjected to ultraviolet (UV) disinfection

before they could be discharged into the water bodies. In some instances, tertiary

methods are also employed to remove trace amounts of contaminants which are

extremely costly (Ghosh et al. 2021a, b). Hence, novel, cost-effective and improved

wastewater treatment methods are required that are rapid and environment friendly

as well (Qu et al. 2013). Nanotechnology has recently been demonstrated to provide

such solutions wherein different nanomaterials such as titanium dioxide (TiO2), zinc

oxide (ZnO), polymer membranes, carbon nanotubes (CNTs), and magnetic nanopar-

ticles (MNPs) were used for effective wastewater treatment (Ghosh and Webster

2021a, b; Ghosh et al. 2021c, d, e). Therefore, this chapter discusses in detail the

use of nanomaterials for photocatalysis, nanoﬁltration as well as nanosorption of

organic and inorganic pollutants from wastewater. Different kinds of nanomaterials

are summarized in Table 1 that have effectively displayed removal of various pollu-

tants such as dyes, heavy metals, organic and inorganic pollutants from wastew-

ater samples. Further studies on improvements, optimization and risk evaluation of

these nanomaterials for their application in the environment can provide an effective

nano-based wastewater treatment method that can be useful on a large scale.

2 Photocatalysis

Metal and metal oxide nanoparticles with attractive physicochemical and

optoelectronic properties are reported to show promising catalytic activity that

can be exploited for degradation and removal of refractory pollutants (Ghosh et al.

2016a, b, c; Shende et al. 2017, 2018; Karmakar et al. 2020). In a recent study, Abd

Elkodous et al. (2021) loaded carbon nanomaterials onto nanocomposite matrix

that was utilized for photocatalytic treatment of wastewater. The matrix was made

Nanobioremediation: A Sustainable Approach for Wastewater Treatment 431

Tabl e 1 Nanomaterials used for removal of refractory pollutants for treatment of wastewater

Type of nanomaterial Pollutant degraded Maximum

removal

References

Photocatalysis

CNFST/C-dots 10%,

CNFST/rGO 10%, and

CNFST/SWCNTs 10%

nanocomposites

Chloramine-T 65% Abd Elkodous et al.

(2021)

SeSnNPs Methylene blue and

malachite green

98% and >

95%

Saray et al. (2019)

TiO2NPs Cyanide –Ijadpanah-Saravy et al.

(2014)

ZnS QDs Naphthalene 95% Rajabi et al. (2016)

ZnSe/PANI

nanocomposite

Methylene blue, Cr(VI) >50% Shirmardi et al. (2018)

Nanoﬁltration

Cellulose acetate NF

membrane

Sulphate, sodium and

calcium

– Choi et al. (2002)

Commercial nanoﬁlter Potassium, chloride –Nataraj et al. (2006)

ES10, ES10C, LES90,

LF10, NTR729HF, and

NTR-7410

Various organic and

inorganic pollutants

– Thanuttamavong et al.

(2002)

MPF34, NF90, and NF270 Arsenite, sulphate, and

chloride

68%, 98%,

and 84%

Andrade et al. (2017)

Polyacrylonitrile (PAN)

membrane with

polydopamine

(PDA)/polyethyleneimine

(PEI) layer and β-FeOOH

nanorods

Methyl blue, Congo red,

methyl orange and

rhodamine B

100%, 100%,

69.9% and

77.3%

Lv et al. (2017)

Nanosorption

GEPCD-MNPs Congo red and Cr(VI) –Cai et al. (2017a)

GO/Chitosan–PVA

nanopolymer composite

Congo red 88.17% Das et al. (2020)

NiONPs Rhodamine B dye 76% Motahari et al. (2015)

Polyindole nanoﬁber Cu(II) 121.95 mg Cai et al. (2017b)

PVA coated PES-TiO2

nanohybrid membrane

Organics, salts and

ammonia–nitrogen

95.83%,

72.40% and

95.66%

Kusworo et al. (2021)

432 S. Ghosh et al.

up of Co0.5Ni0.5Fe2O4/SiO2/TiO2 (CNFST) that was conjugated with C-dots, single

walled-carbon nanotubes (SWCNTs), and graphene oxide (rGO) nanomaterials.

X-ray diffraction (XRD) patterns of the nanocomposites then displayed anatase

phase of the titanium dioxide (TiO2) that was one of the primary components of

the matrix along with presence of the three different carbon nanomaterials loaded

onto the CNFST. Further, scanning electron microscopy (SEM) displayed uniform

distribution of C-dots and rGO nanomaterials over the surface of CNFST while

SWCNTs demonstrated sheet-like soots as well as uniform cross-linking with

CNFST. Thereafter, transmission electron microscopy (TEM) results demonstrated

spherical shaped particles with an average diameter of 90 ± 14 nm. Brunauer–

Emmett–Teller (BET) surface area of the synthesized nanocomposites loaded with

rGO and C-dots was smaller when compared to standard TiO2 photocatalyst (P25)

samples which was a result of increase in size of the nanoformulations as well as

poor loading or increased agglomeration of the conjugated carbon nanomaterials. On

the other hand, SWCNTs conjugation led to signiﬁcant increase in the surface area.

The pores in all the conjugated samples were multimodal and broad-shaped wherein

the mesopores had a diameter ranging from 2 to 50 nm while the macropores had

a diameter greater than 50 nm. Raman analysis further showed anatase TiO2 as the

dominant phase in the nanomaterial coated nanomatrix.

A comparative study of the three different carbon-loaded nanocomposites showed

28, 32.7, and 41.6% chloramine-T dye degradation by the CNFST/C-dots 10%,

CNFST/rGO 10%, and CNFST/SWCNTs 10% nanocomposites, respectively after

40 min of ultraviolet (UV) irradiation. Moreover, subsequent increase in contact time

to 90 min resulted in further increase in dye degradation ability with best removal

activity observed using CNFST/SWCNTs 10% nanocomposites. In addition, the

increase in the dosage concentration of the prepared nanoformulations resulted in

subsequent increase i n photocatalytic degradation of dye. Likewise, dye degrada-

tion ability of the CNFST/SWCNTs 10% nanocomposites was increased from 35

to 65% with concomitant decrease in the pH value of the system from 10.0 to 3.0,

respectively. The zeta potential of the SWCNTs loaded nanocomposite was +15 mV

which facilitated effective binding of the dye through electrostatic attraction for

improved photocatalytic activity. The primary reactive oxygen species (ROS) medi-

ated chloramine-T dye degradation was mainly attributed to the hydroxyl radical that

was further scavenged on addition of isopropanol resulting in 40% decrease in dye

degradation.

Organic pollutants were also reported to be degraded by tin selenide nanoparti-

cles (SnSeNPs) that were prepared using co-precipitation method (Saray et al. 2019).

XRD pattern analysis demonstrated orthorhombic phase of the particles. However,

the crystallinity of the particles was dependent upon the Se/Sn ratio wherein a ratio

of 1.0 resulted in formation of crystalline particles while particles made with a Se/Sn

ratio of 1.3 did not exhibited a crystalline phase. Moreover, ﬁeld emission scanning

electron microscopy (FESEM) and TEM results demonstrated complete agglomera-

tion of the particles when the Se/Sn ratio was 0.8 with presence of oxygen as impurity

as conﬁrmed by energy dispersive X-ray (EDX) spectroscopy results. Meanwhile,

particles with Se/Sn ratio of 1.2 were pure and smaller in size. The photocatalytic

Nanobioremediation: A Sustainable Approach for Wastewater Treatment 433

activity of the prepared SnSeNPs was then evaluated in which methylene blue degra-

dation was analysed in presence of visible light. NPs prepared using a Se/Sn ratio

of 1.2 was able to effectively degrade 98% of methylene blue dye within 25 min

of incubation. Furthermore, the reusability of the nanomaterials was also evaluated

wherein similar dye reduction ability of the SnSeNPs was observed after four succes-

sive cycles. The phase of the NPs prepared using Se/Sn ratio of 1.2 was stable as

observed in XRD patterns even after four cycles of dye degradation while particles

with Se/Sn ratio of 0.8 completely changed its phase after four successive photocat-

alytic processes. Likewise, malachite green degradation ability was also monitored

in presence of the SnSeNPs with varying ratios of Se and Sn, respectively. More

than 95% of the dye was effectively degraded by NPs with Se/Sn ratio of 1.0 within

45 min of incubation. The textural properties of the NPs were also evaluated wherein

a maximum speciﬁc surface area equivalent to 77.441 m2/g was observed with Se/Sn

ratio of 1.2 along with a pore diameter and volume of 2.43 nm and 0.124 cm3/g,

respectively. The energy of the valence bond of SnSeNPs with Se/Sn ratio of 1.2

was calculated to be 1.21 eV that was further used for investigating the electronic

structure of the particles. Therefore on the basis of electronic structure, formation of

hydroxyl radical using SeSnNPs was proposed to facilitate efﬁcient dye degradation

which could be applicable in wastewater treatment as well.

In another study, Ijadpanah-Saravy et al. (2014) reported fabrication of titanium

dioxide nanoparticles (TiO2NPs) by controlled hydrolysis of 3 M titanium tetra-

chloride. The nanomaterial demonstrated photocatalytic degradation of cyanide in

wastewater samples. X-ray diffraction (XRD) patterns then displayed characteristic

peaks of anatase and rutile form of TiO2NPs with a crystalline size of 18 and 22 nm,

respectively while scanning electron microscopic (SEM) images demonstrated spher-

ical morphology of the particles with an average size of 20 nm. Thereafter, the photo-

catalytic properties of the NPs were determined wherein a 4:1 ratio of anatase to rutile

form of the particles was optimal for maximum photocatalytic activity to degrade

cyanide. Interestingly, pH 11 was optimal for cyanide degradation with maximum

photocatalytic efﬁciency as well as lowest electrical energy consumption as compared

to other catalysts.

Rajabi et al. (2016) also demonstrated effective photocatalytic removal of indus-

trial pollutants using zinc sulphide quantum dots (ZnS QDs). Fast and efﬁcient chem-

ical precipitation technique was followed for preparation of ZnS QDs which exhibited

a blue shift in UV–vis absorption peak at 235 nm that was attributed to the particle

size decrease, band-gap energy increase as well as due to quantum size conﬁnement

effect. Additionally, the direct optical band gap value of the prepared nanomate-

rial was 4.09 eV. XRD patterns then demonstrated a cubic zinc blend crystalline

structure of the ZnS QDs without presence of any impurities. TEM image analysis

further revealed spherical shape of the QDs with an average dimension of around

1 nm. Thereafter, naphthalene was used for demonstrating pollutant degradation

activity of the prepared ZnS QDs wherein only 25–31% degradation was obtained in

absence of light. An optimal pH of 11 resulted in maximum degradation efﬁciency of

naphthalene which was decreased with concomitant increase in initial concentration

of the pollutant. Moreover, a rapid increase in degradation rate was observed for

434 S. Ghosh et al.

90 min that was reduced for the next 45 min followed by equilibrium. A low initial

concentration of 10 mg of ZnS QDs then demonstrated up to 95% naphthalene

degradation efﬁciency that remained fairly constant up to four cycles highlighting its

reusability. Further, a maximum degradation rate constant of 6.90 × 10–5 min−1 was

attained when the initial concentration of naphthalene was 20 ppm. The mechanism

of degradation was then proposed to involve photoexcitation that may have resulted

in electron–hole pair formation on the surface of ZnS QDs semiconductor that may

have then oxidized naphthalene to 2-formylcinnamaldehyde.

Shirmardi et al. (2018) also demonstrated improved photocatalytic activity of zinc

selenide nanoparticles (ZnSeNPs) after addition of polyaniline (PANI) that acted as

an organic semiconductor. The ZnSe/PANI nanocomposite was prepared using co-

precipitation technique whose heterostructure form was conﬁrmed by XRD patterns.

In addition, TEM images showed a distinct ZnSeNPs core and PANI shell structure

whereas high resolution transmission electron microscopy (HRTEM) images demon-

strated interplanar distance of 0.33 nm between the zinc blend structure as evident

from Fig. 1. Raman spectroscopy of the nanocomposite indicated a weaker ZnSe

peak at 500 cm−1 as compared to pristine ZnSeNPs that was attributed to the pres-

ence of PANI shell in the composite. Raman spectrum of the nanocomposite also

demonstrated two other peaks at 1105 and 420 cm−1 that corresponded with C-H

vibration of the semi-quinonoid rings and out-of-plane ring-deformation of PANI,

respectively. X-ray photon spectroscopy (XPS) based valence band (VB) spectral

analysis then demonstrated a nanocomposite VB potential of 0.48 ± 0.05 eV while

the pristine ZnSeNPs had a VB potential of approximately 1.13 eV. Further, methy-

lene blue dye degradation ability of the prepared nanocomposite was analysed under

visible light irradiation. More than 50% of the initial dye concentration was reduced

by the ZnSe/PANI nanocomposite within 30 min whereas it took 120 min for the

pristine ZnSeNPs for completing similar levels of reduction. Additionally, presence

of PANI displayed an increase in photocurrent intensity along with a decrease in

the photoconductivity resistance. In addition, ZnSe/PANI nanocomposite was also

investigated for its inorganic pollutant removal activity wherein absorption peak of

K2Cr2O7 at 375 nm was decreased in presence of the nanocomposite with gradual

increase in visible light irradiation time. Hence, Cr(VI) ions were reduced to Cr(III)

efﬁciently by the nanocomposite as well.

3 Nanoﬁltration

Nanoparticles impregnated polymeric ﬁlms are often used as superior membranes

for nanoﬁltration of water in order to remove certain hazardous pollutants which

are recalcitrant in nature (Ghosh et al. 2022a, b; Ghosh and Webster 2022). Nataraj

et al. (2006) removed colour and contaminants from distillery efﬂuent using a hybrid

system of nanoﬁltration (NF) combined with reverse osmosis (RO). A commercial

nanoﬁlter as well as thin-ﬁlm composite (TFC) polyamide RO membrane was used

in this study wherein effective colour removal ability of the NF was attained when

Nanobioremediation: A Sustainable Approach for Wastewater Treatment 435

Fig. 1 TEM image of a pristine ZnSe NPs and b ZnSe/PANI nanocomposites. The inset shows an

HRTEM image of ZnSe/PANI nanocomposites. Reprinted from Shirmardi A, Teridi MA, Azimi HR,

Basirun WJ, Jamali-Sheini F, Youseﬁ R, 2018. Enhanced photocatalytic performance of ZnSe/PANI

nanocomposites for degradation of organic and inorganic pollutants. Applied Surface Science

the size of the particles was in the colloidal range. Thus, in at an optimal pressure of

30–50 bar, the total dissolved solids (TDS) was reduced from 51,500 to 9050 ppm

while conductivity and chloride concentration was reduced from 346 mS/cm and

4900 ppm to 15.06 mS/cm and 2650 ppm, respectively. High rejection efﬁciency

against bivalent and trivalent ions was also exhibited by the NF membrane. There-

after, evaluation of RO based pollutant removal was performed. The impact of feed

pressure on the permeate properties was analysed in which the pressure increased

linearly from 20 to 70 bar when distillery spent wash was used as feed. TDS and

chemical oxygen demand (COD) rejection was altered from 83 to 99.06% and 95.6 to

98.96%, respectively when 50 bar pressure was applied. Additionally, high rejection

percentages for potassium and chloride ions were observed.

Choi et al. (2002) used a cellulose acetate NF membrane for treatment of wastew-

ater. The effective surface area of the hollow NF membrane used in this study was 11.7

m2 along with 55% salt rejection property. A membrane bioreactor (MBR) system

was set up to evaluate the efﬁcacy of treatment which was operated for 71 days. The

transmembrane pressure along with the relative productivity permeate after 20 days

of incubation were 36 kPa and 1.0–1.2, respectively after which a drastic increase

in relative productivity was observed. The change in sulphate, sodium and calcium

ions concentration were similar to that of conductivity wherein no electrolytes were

accumulated in the bioreactor due to charge effect of the membrane. It was hence

proposed that the bioreactor could be operated under low suction pressure as the

effect of osmotic pressure was insigniﬁcant along with high rejection of organic

matter. The biodegradable membrane remained stable for 60 days in the bioreactor

wherein nitriﬁcation and denitriﬁcation occurred simultaneously. In addition, atomic

436 S. Ghosh et al.

force microscope (AFM) images revealed a larger surface roughness on the cellulose

acetate membrane after 40 days of bioreactor operation which was further increased

till 71 days thus highlighting biodegradation of the membrane.

Thanuttamavong et al. (2002) also characterized NF rejection efﬁciency of organic

and inorganic pollutants for treatment of wastewater. Six different commercial NF

membranes namely ES10, ES10C, LES90, LF10, NTR729HF, and NTR-7410 were

used in this study that were composed of aromatic polyamides, polyvinyl alcohols,

and sulfonated polysulfones. The total dissolved organic and inorganic content of

the polluted water sample were 1.8 mg/L and 3 mM, respectively. The pH and the

turbidity of the water samples were in the range of 7.2–7.5 and 2.5–5.5 NTU, respec-

tively. Long-term operations for a time period of 120 and 20 days were carried out

with and without microﬁltration pre-treatment, respectively. A transmembrane pres-

sure of 0.15 MPa was maintained in both cases along with a standardized permeate

ﬂux temperature of 25 °C. A signiﬁcant decline in permeate ﬂux was observed for

all the membranes during the ﬁrst 10 days of operation. The permeate ﬂux of the

untreated loose membrane NTR7410 continuously declined and reached a steady

state value of 0.28 m/d in 15 days of operation whereas the treated membranes

showed a faster steady state attainment. Further, a stable rejection of all the compo-

nents was observed throughout the NF operation which highlighted that membrane

fouling does not interfere with the rejection mechanisms. Additionally, signiﬁcant

change in zeta potential values of the membrane surface was observed after long-term

operation. The MWCO value for ES-10, ES-10C, LES-90 and LF-10 was 100 Da

while it was 200 Da and more than 350 Da for NTR-729HF and NTR-7410, respec-

tively. Moreover, organic matter with a size range of 300–1800 Da was found in

the permeate of NTR7410 NF membrane. With regards to inorganic matter rejec-

tion, divalent ions such as Ca2+ and Mg2+ exhibited higher rejection as compared to

monovalent ions such as Na+ and Cl− which was attributed to the charge effect of the

membrane. Thereafter, the effective charge density of the NTR-729HF membrane

was changed from −2to −0.5 mol/L after long-term operation which suggested a

decrease in t he electrostatic property of the membrane after its utilization. The parti-

tioning coefﬁcient of the membrane for nitrate ion was also decreased from 5.0 to

2.6 after operation.

Likewise, Andrade et al. (2017) demonstrated gold mining efﬂuent treatment

using NF and compared with RO. Five different membranes were used in this study

namely, TFC-HR and BW30 that were RO membranes while MPF34, NF90, and

NF270 were the three NF membranes. The permeate ﬂux of RO membranes were

7–12 folds lower than NF membranes that was attributed to higher resistance in the

RO membrane. Moreover, a more intense membrane fouling was also observed in

case of TFC-HR whereas MPF34 exhibited minimum fouling. The initial pollutant

retention efﬁciency of all the membranes was considerably high. However, a high

sulphate concentration in the mining efﬂuent was proposed to interfere with the reuse

of water as it could cause metal precipitation which could further result in membrane

fouling. Among the RO membranes, TFC-HR demonstrated maximum pollutant

retention efﬁciency with 75%, 99%, and 78% retention of arsenic, sulphate, and

chloride ions, respectively. Similarly, NF90 membrane exhibited excellent arsenic,

Nanobioremediation: A Sustainable Approach for Wastewater Treatment 437

sulphate, and chloride retention of 68%, 98%, and 84%, respectively. Moreover, the

efﬂuent was pre-treated to increase its pH which resulted in decrease in conductivity

as well as concentrations of calcium and magnesium. Concentration of arsenic was

also reduced with subsequent increase in pH of the efﬂuent from 2.2 to 5.0 that

subsequently enhanced recovery rate from 27 to 70% along with higher resistance to

membrane fouling. The permeate recovery rate (RR) was also studied in which the

quality of the permeate remained almost similar when the RR value was up to 40%

above which the removal efﬁciency started to gradually decrease. The concentration

of polarization of NF membranes was then increased to 1.45 using a synthetic solution

of MgSO4 which resulted in increase of RR to 70% while decreasing the removal

efﬁciency. Hence, it was concluded that such NF based efﬂuent treatment could be

optimal as well as feasible for obtaining industrial water samples from the same.

Lv et al. (2017) reported fabrication of a photocatalytic nanoﬁltration (NF)

membrane that had self-cleaning property and thus, could be used for wastew-

ater treatment as represented schematically in Fig. 2. Co-deposition method was

carried out on an ultraﬁltration polyacrylonitrile (PAN) membrane with poly-

dopamine (PDA)/polyethyleneimine (PEI) to form an intermediate layer after which

β-FeOOH nanorods were mineralized to further create a photocatalytic layer on

the NF system. X-ray photoelectron spectrometer (XPS) results of the mineral-

ized membrane then displayed additional binding energy peaks at Fe 2p along with

a change in oxygen/carbon ratio from 0.32 to 1.22 which conﬁrmed presence of

FeOOH group on the membrane surface. SEM images also demonstrated uniform

distribution of β-FeOOH nanorods over the surface of the membrane with a vertical

orientation and a thickness of around 0.45 μm. The enhanced wettability of the

membrane was analysed through calculation of the dynamic water contact angle

that was decreased from 60° to 20° within 100 s. Such improved permeability of the

mineralized membrane was attributed to the hydrophilicity of the β-FeOOH nanorods

that were attached on the surface of the membrane. The surface charge properties

of the membrane were then analysed wherein the isoelectric point of the membrane

surface decreased from 6.3 to 5.6 after mineralization. In addition, the molecular

weight cut-off (MWCO) value showed a steady decrease from 20,000 to 2000 Da

with subsequent increase in deposition time from 1 to 6 h, respectively. Thereafter,

the dye rejection performance of the stable mineralized NF membrane was studied

in which 100% rejection of methyl blue and Congo red was attained at pH 3 and 7,

respectively with a deposition time of 2 h. Likewise, 60.8 and 69.9% of methyl orange

rejections were observed at pH 7 and 3, respectively. On the other hand, rhodamine B

dye rejection was increased up to 77.3% when a positive surface charge was attained

on the membrane. Thereafter, a continuous cross-ﬂow membrane reactor was set

up for observing the photocatalytic performance of the prepared membranes. The

colour of the feed solution containing 20 mg/L of methyl blue and 30% H2O2 became

colourless after 6 h of incubation. The self-cleaning property of the membrane was

also evaluated by immersing the fouling membrane into an acidic H2O2 solution

and in presence of visible light wherein the water ﬂux was recovered to its original

value. A high NF performance of 97.3% was achieved even after ﬁve successful

photocatalytic cycles indicating its reusability and recyclability.

438 S. Ghosh et al.

Fig. 2 Schematic representation of preparation process for the β–FeOOH mineralized NFM and

its application in photocatalytic nanoﬁltration. Reprinted with permission from Lv Y, Zhang C, He

A, Yang SJ, Wu GP, Darling SB, Xu ZK, 2017. Photocatalytic nanoﬁltration membranes with

self-cleaning property for wastewater treatment. Advanced Functional Materials 27:1,700,251.

4 Nanosorption

Nanocomposite based adsorbents are advantageous compared to conventional

sorbents due to their superior ability for puriﬁcation and convenience in manipu-

lation of their activity by rational physico-chemical modiﬁcation. Cai et al. (2017a)

also reported formulation of a novel multi-layer magnetic adsorbent (GEPCD-

MNPs) using ring opening polymerization process wherein a multi-layer cationic

polymer was coated on the surface of magnetic nanoparticles (MNPs). These nano-

adsorbents were then utilized for dyeing wastewater treatment. SEM images of the

nano-adsorbents showed spherical morphology of the MNPs that had a smaller diam-

eter as compared to pristine MNPs which suggested presence of the polymer on the

active sites of MNPs. The contact angle of the nano-adsorbent was less than 90°

when immersed in Congo red and Cr(VI) containing solutions thus indicating its

hydrophilicity. The magnetic properties of the nano-adsorbent were also evaluated

which provided a saturation magnetization value of 54.7 emu/g. The removal efﬁ-

ciency of Congo red increased with subsequent increase in the pH value of the system

from 7.25 to 9.25 that was attributed to protonation of the MNPs and the functional

groups of the polymer which may result in improved coordinate afﬁnity with the dye.

On the contrary, removal rate of Cr(VI) from the solution decreased with concomitant

increase in pH value from 2.98 to 11.94 which was proposed to transform Cr2O7 2−

into CrO2− that has lower electrostatic interaction with the nano-adsorbent. The

adsorption kinetics analyses for Congo red and Cr(VI) adsorption followed a pseudo-

second order reaction while both the Langmuir and Freundlich adsorption isotherm

models were properly ﬁtted for the adsorption processes. Therefore, GEPCD-MNPs

Nanobioremediation: A Sustainable Approach for Wastewater Treatment 439

were proposed to be economically viable and effective nano-adsorbents that could

be potentially be used in large-scale wastewater treatment processes with further

optimization.

A hybrid hydrogel nanopolymer was prepared by Das et al. (2020) using graphene

oxide (GO), chitosan and polyvinyl alcohol (PVA) that was investigated for its

wastewater treatment efﬁcacy. SEM images of the prepared GO reinforced chitosan-

PVA hydrogel nanopolymer displayed homogenous distribution of GO over the

matrix without any agglomeration. Fourier transform infrared (FTIR) spectroscopy

analysis then displayed peaks at 3268.06 and 1377.33 cm−1 which corresponded

with –NH2 stretching vibrations of chitosan and –CH2 deformation vibrations of

PVA due to cross-linking, respectively. FTIR spectra of the prepared nanopolymer

then displayed characteristic peaks corresponding with surface functional groups

of chitosan, PVA and GO. A maximum positive surface charge on the nanopolymer

membrane was observed at a pH value of 3.3 that was considered ideal for anionic dye

adsorption. The swelling behaviour of the nanopolymer membrane was also evalu-

ated wherein maximum swelling of 17.24 g/g was achieved after 90 min of immersion

in water that further increased to 25.89 g/g at a pH value of 2.0. This change was

attributed to ionization of the functional groups present on the membrane. Thereafter,

dye adsorption studies were conducted using a biological oxygen demand (BOD)

shaker at 140 rotations per minute (rpm) under room temperature. A maximum Congo

red dye removal efﬁciency of 84.31% was obtained at an initial dye concentration

of 10 mg/L. Additionally, the adsorption efﬁciency increased from 76.62 to 81.1%

with subsequent increase in dosage of adsorbent from 1 to 6 g/L, respectively. An

acidic pH of 2.0 was further demonstrated to provide maximum adsorption efﬁ-

ciency of 88.17% because of effective electrostatic interaction between the cationic

nanopolymer membrane and anionic dye molecules. Adsorption isotherm studies

predicted that Langmuir model was best ﬁtted for explaining the dye adsorption

while adsorption kinetics revealed pseudo second order reaction of the adsorption

process.

In another study, Motahari et al. (2015) cost-effectively synthesized nickel oxide

nanoparticles (NiONPs) for rhodamine B dye removal from wastewater samples.

H2acacen ligand was used for hydrothermal formation of nano-scale Ni(OH)2

followed by calcination to obtain NiONPs. XRD patterns of the prepared NPs corre-

sponded with the face-centered cubic as well as crystalline structure of nickel oxide.

FESEM images then displayed monodispersed NiONPs while TEM micrographs

showed spherical morphology of the particles with an average size of 10 nm. The

BET surface area of the NiONPs was 176.56 m2/g that were porous in nature with

an average pore size of around 9.7 nm. Later on, dye adsorption studies were carried

out in which 76% of rhodamine B dye at a concentration of 10 mg/L was efﬁciently

removed within the initial 30 min of reaction. The reusability of the particles was

also highlighted wherein no change in dye adsorption activity was observed after

reusing the same particles. Adsorption isotherm studies then provided maximum dye

adsorption capacity of 111 mg/g using Langmuir isotherm model whereas kinetic

studies showed suitability of pseudo second order reaction for explaining the NiONPs

440 S. Ghosh et al.

mediated rhodamine B dye adsorption reaction. Optimal pH 7.0 then demonstrated

efﬁcient dye adsorption that was exothermic in nature as the dye adsorption capacity

decreased with subsequent increase in the temperature of the system.

Cai et al. (2017b) also fabricated polyindole nanoﬁbers using electrospinning

that could act as nano-adsorbent for removal of heavy metal ions from wastewater.

SEM and TEM images displayed spherical morphology of the nanoﬁbers that had a

smooth surface along with an average diameter range of 140–300 nm. The ranges of

pore size, diameter and speciﬁc surface area were 0.217–0.250 cm3 g−1, 55–68 nm

and 64.43–86.41 m2 g−1, respectively. Moreover, the speciﬁc surface area and pore

volume decreased with concomitant decrease in the ﬁbre diameter while the opposite

was observed for the pore diameter. Thereafter, effect of pH on the metal adsorption

capacity of the nanoﬁbers was investigated wherein maximum Cu(II) adsorption

was observed at an acidic pH value of 6.0 that was used for further experiments

in this study. The maximum adsorption capacity of the nanoﬁbers having a polyin-

dole concentration of 1.6% was 121.95 mg/g within 15 min of incubation that was

the most effective contact time as well. In addition, the Cu(II) adsorption capacity

of the nanoﬁber was insigniﬁcantly affected in presence of low concentrations of

other metal ions such as Na+,K

+,Mg

2+ and Ca2+ that highlighted the speciﬁc Cu(II)

adsorption capacity of the nanoﬁbers. Moreover, the adsorption isotherm studies

then demonstrated Langmuir model to be a best ﬁt for the Cu(II) adsorption using

polyindole nanoﬁbers. Additionally, the pseudo second order reaction was followed

by the adsorption process. Moreover, the reusability of the nanoﬁber was also inves-

tigated wherein the adsorption efﬁciencies were 95%, 88.7% and 82% of the original

adsorption capacity of the nanoﬁbers after the third, ﬁfth and seventh cycle, respec-

tively. In addition, Cu(II) adsorption by the nanoﬁbers was proposed to be mediated

by chemisorption which consists of coordination between the metal ion and nitrogen

containing functional groups of the membrane may result in chelation.

In another similar study, a nanohybrid membrane that was cross-linked with PVA

and coated with polyether sulfone (PES)-TiO2NPs was reported as an effective adsor-

bent in wastewater treatment (Kusworo et al. 2021). SEM images of the prepared

membranes showed a smooth surface with minimal nodules while the sub-layer struc-

tures of the membrane were asymmetric with a dense skin layer along with a porous

sub-layer made up of ﬁnger-like structures as evident from Fig. 3. XRD patterns of

PVA coated PES-TiO2 nanohybrid membrane further showed a wide band at 19–20°

that corresponded with orthorhombic lattice along with the polycrystalline behaviour

of PVA along with weak bands that highlighted the amorphous PES and TiO2 crys-

talline peaks. In addition, the water contact angle of the 3.0 wt.% PVA complexed

nanohybrid membrane was 27.67° that highlighted its hydrophilicity. Mechanical

properties of the 3.0 wt.% PVA linked membrane was also studied that demonstrated

a thickness of 90 μm with a tensile strength and elongation break of 6.4 ± 0.15 MPa

and 1.6 ± 0.10%, respectively. Thereafter, addition of TiO2NPs on the membrane

was attributed to the increase and stabilization of the permeate ﬂux at 80 L/m2/h as it

could degrade the attached foulants. Hence, gradual increase in TiO2 concentration

resulted in improved COD, total dissolved solids (TDS), surface wettability as well

as rejection percentages. Likewise, addition of PVA resulted in considerable increase

Nanobioremediation: A Sustainable Approach for Wastewater Treatment 441

in COD, TDS, and NH3-N rejection by 244.80%, 56.70%, and 5.29%, respectively.

Furthermore, organics, salts and ammonia–nitrogen rejection were also enhanced up

to 75.00%, 51.61%, and 90.47% that was further improved by 95.83%, 72.40% and

95.66%, respectively when integrated with ozonation.

Fig. 3 Membrane surface morphology of a PES-TiO2 1wt-%, b PES-TiO2/PVA 0.5 wt-%, c PES-

TiO2/PVA 3.0 wt-% and cross-section images of d PES-TiO2 1wt-%, e PES-TiO2/PVA 0.5 wt-%,

f PES-TiO2/PVA 3.0 wt-%. Reprinted with permission from Kusworo TD, Kumoro AC, Utomo

DP, Kusumah FM, Pratiwi MD, 2021. Performance of the crosslinked PVA coated PES-TiO2 nano

hybrid membrane for the treatment of pretreated natural rubber wastewater involving sequential

adsorption–ozonation processes. Journal of Environmental Chemical Engineering 9:104,855.

442 S. Ghosh et al.

5 Conclusion and Future Perspectives

Surface active nanoparticles exhibit notable physical and chemical properties that

are exploited in various biomedical applications (Ghosh et al. 2018). The attractive

features of the nanoparticles have extended their applications for infection control,

agriculture, textiles, and even environment (Adersh et al. 2015; Rokade et al. 2017;

Jamdade et al. 2019; Bhagwat et al. 2018; Ghosh et al. 2016d). The nanostruc-

tures used so far for the treatment of wastewater for photocatalysis, nanoﬁltration

or nanosorption are either fabricated by physical or chemical methods which often

use hazardous chemicals and reaction conditions (Bloch et al. 2021; Ranpariya et al.

2021). However, biological route for synthesis of metal and metal oxide nanoparti-

cles are environmentally benign, rapid and efﬁcient (Shinde et al. 2018). Microbe

synthesized nanoparticles are reported to have efﬁcient photocatalytic activity that

can be further integrated in the membranes for effective dye degradation and removal

(Ghosh 2018). Similarly, various medicinal plants like Gloriosa superba, Dioscorea

bulbifera, Gnidia glauca, Plumbago zeylanica, and others are reported to synthesize

exotic gold, silver, copper, platinum, and palladium nanoparticles that can be further

explored for wastewater treatment (Rokade et al. 2018; Ghosh et al. 2015a, b, c;

Jamdade et al. 2019; Salunke et al. 2014). Composite nanoparticles either bimetallic

or functionalized with metal removing or dye degrading enzymes, bioactive princi-

ples can be employed for multimodal wastewater treatment (Robkhob et al. 2020;

Ghosh et al. 2015d; Kitture et al. 2015). In view of the background it can be concluded

that development of nanotechnology assisted strategies for wastewater treatment can

serve as a powerful tool to ensure clean environment.

Acknowledgements Dr. Sougata Ghosh acknowledges Kasetsart University, Bangkok,

Thailand for Post Doctoral Fellowship and funding under Reinventing University Program (Ref. No.

6501.0207/10870 dated 9th November, 2021 and Ref. No. 6501.0207/9219 dated 14th September,

2022).

References

Abd Elkodous M, El-Sayyad GS, Maksoud MA, Kumar R, Maegawa K, Kawamura G, Tan

WK, Matsuda A (2021) Nanocomposite matrix conjugated with carbon nanomaterials for

photocatalytic wastewater treatment. J Hazard Mater 410:124657

Adersh A, Ghosh S, More PA, Chopade BA, Gandhi MN, Kulkarni AR (2015) Surface defect

rich ZnO quantum dots as antioxidants inhibiting α-amylase and α-glucosidase: a potential anti-

diabetic nanomedicine. J Mater Chem B 3:4597–4606

Andrade LH, Aguiar AO, Pires WL, Miranda GA, Teixeira LP, Almeida GC, Amaral MC (2017)

Nanoﬁltration and reverse osmosis applied to gold mining efﬂuent treatment and reuse. Braz J

Chem Eng 34(1):93–107

Bhagwat TR, Joshi KA, Parihar VS, Asok A, Bellare J, Ghosh S (2018) Biogenic copper

nanoparticles from medicinal plants as novel antidiabetic nanomedicine. World J Pharm Res

7(4):183–196

Nanobioremediation: A Sustainable Approach for Wastewater Treatment 443

Bloch K, Pardesi K, Satriano C, Ghosh S (2021) Bacteriogenic platinum nanoparticles for

application in nanomedicine. Front Chem 9:624344

Bora T, Dutta J (2014) Applications of nanotechnology in wastewater treatment—a review. J Nanosci

Nanotechnol 14:613–626

Cai D, Zhang T, Zhang F, Luo X (2017a) Quaternary ammonium β-cyclodextrin-conjugated

magnetic nanoparticles as nano-adsorbents for the treatment of dyeing wastewater: synthesis

and adsorption studies. J Environ Chem Eng 5:2869–2878

Cai Z, Song X, Zhang Q, Zhai T (2017b) Electrospun polyindole nanoﬁbers as a nano-adsorbent

for heavy metal ions adsorption for wastewater treatment. Fibers Polym 18(3):502–513

Choi JH, Dockko S, Fukushi K, Yamamoto K (2002) A novel application of a submerged nanoﬁl-

tration membrane bioreactor (NF MBR) for wastewater treatment. Desalination 146:413–420

Chorawala KK, Mehta MJ (2015) Applications of nanotechnology in wastewater treatment. Int J

Innov E merg Res Eng 2(1):21–26

Das L, Das P, Bhowal A, Bhattachariee C (2020) Synthesis of hybrid hydrogel nano-polymer

composite using graphene oxide, chitosan and PVA and its application in waste water treatment.

Environ Technol Innov 18:100664

Ghosh S (2018) Copper and palladium nanostructures: A bacteriogenic approach. Appl Microbiol

Biotechnol 101(18):7693–7701

Ghosh S, Nitnavare R, Dewle A, Tomar GB, Chippalkatti R, More P, Kitture R, Kale S, Bellare

J, Chopade BA (2015) Novel platinum-palladium bimetallic nanoparticles synthesized by

Dioscorea bulbifera: Anticancer and antioxidant activities. Int J Nanomed 10(1):7477–7490

Ghosh S, Jagtap S, More P, Shete UJ, Maheshwari NO, Rao SJ, Kitture R, Kale S, Bellare J, Patil S,

Pal JK (2015) Dioscorea bulbifera mediated synthesis of novel Aucore Agshell nanoparticles with

potent antibioﬁlm and antileishmanial activity. J Nanomater 2015:562938

Ghosh S, More P, Nitnavare R, Jagtap S, Chippalkatti R, Derle A, Kitture R, Asok A, Kale S,

Singh S, Shaikh ML, Ramanamurthy B, Bellare J, Chopade BA (2015) Antidiabetic and antiox-

idant properties of copper nanoparticles synthesized by medicinal plant Dioscorea bulbifera.J

Nanomed Nanotechnol S6:007

Ghosh S, More P, Derle A, Kitture R, Kale T, Gorain M, Avasthi A, Markad P, Kundu GC, Kale S,

Dhavale DD, Bellare J, Chopade BA (2015) Diosgenin functionalized iron oxide nanoparticles

as novel nanomaterial against breast cancer. J Nanosci Nanotechnol 15(12):9464–9472

Ghosh S, Chacko MJ, Harke AN, Gurav SP, Joshi KA, Dhepe A, Kulkarni AS, Shinde VS, Parihar

VS, Asok A, Banerjee K, Bellare J, Chopade BA (2016a) Barleria prionitis leaf mediated

synthesis of silver and gold nanocatalysts. J Nanomed Nanotechnol 7:4

Ghosh S, Gurav SP, Harke AN, Chacko MJ, Joshi KA, Dhepe A, Charolkar C, Shinde VS, Kitture R,

Parihar VS, Banerjee K, Kamble N, Bellare J, Chopade BA (2016b) Dioscorea oppositifolia medi-

ated synthesis of gold and silver nanoparticles with catalytic activity. J Nanomed Nanotechnol

7:5

Ghosh S, Patil S, Chopade NB, Luikham S, Kitture R, Gurav DD, Patil AB, Phadatare SD, Sontakke

V, Kale S, Shinde V, Bellare J, Chopade BA (2016c) Gnidia glauca leaf and stem extract mediated

synthesis of gold nanocatalysts with free radical scavenging potential. J Nanomed Nanotechnol

7:358

Ghosh S, Harke AN, Chacko MJ, Gurav SP, Joshi KA, Dhepe A, Dewle A, Tomar GB, Kitture

R, Parihar VS, Banerjee K (2016d) Gloriosa superba mediated synthesis of silver and gold

nanoparticles for anticancer applications. J Nanomed Nanotechnol 7(4)

Ghosh S, Webster TJ (2021a) Nanotechnology for water processing. In: Shah MP, Rodriguez-Couto

S, Mehta K (eds) The future of efﬂuent treatment plants-biological treatment systems. Elsevier,

pp 335–360. Paperback ISBN: 9780128229569

Ghosh S, Webster TJ, 2021b. Biologically synthesized nanoparticles for dye removal. In: Shah

M, Couto SR, Biswas JK (eds) Removal of emerging contaminants from wastewater through

bio-nanotechnology. Elsevier, pp 573–603. Paperback ISBN: 9780323855839

Ghosh S, Webster TJ (2022) Nanotechnological advances for oil spill management: removal,

recovery and remediation. In: Das P, Manna S, Pandey JK (eds) Advances in oil-water separation:

444 S. Ghosh et al.

a complete guide for physical, chemical, and biochemical processes. Elsevier, pp 175–194. ISBN:

978-0-323-89978-9

Ghosh S, Sanghavi S, Sancheti P (2018) Metallic biomaterial for bone support and replacement. In:

Balakrishnan, P, Sreekala MS, Thomas S (eds), Fundamental biomaterials: metals, vol 2. Wood-

head Publishing Series in Biomaterials, Woodhead Publishing, pp 139–165. ISBN: 0081022069

(print); ISBN: 978-0-08-102206-1 (online)

Ghosh S, Selvakumar G, Ajilda AAK, Webster TJ (2021a) Microbial biosorbents for heavy metal

removal. In: Shah MP, Couto SR, Rudra VK (eds) New trends in removal of heavy metals

from industrial wastewater. Elsevier B.V., Amsterdam, Netherlands, pp 213–262. eBook ISBN:

978-0-12-823108-1; Paperback ISBN: 978-0-12-822965-1

Ghosh S, Sharma I, Nath S, Webster TJ (2021b) Bioremediation-the natural solution. In: Shah MP,

Couto SR (eds) Microbial ecology of waste water treatment plants (WWTPs). Elsevier, pp 11–40.

eBook ISBN: 978-0-12-822504-2; Paperback ISBN: 978-0-12-822503-5

Ghosh S, Joshi K, Webster TJ (2021c) Removal of heavy metals by microbial communities. In: Shah

MP, Couto SR (eds) Waste water treatment reactors: microbial community structure. Elsevier, pp

537–566. eBook ISBN: 978-0-12-824244-5; Paperback ISBN: 978-0-12-823991-9

Ghosh S, Bhagwat T, Webster TJ (2021d) Arsenic removal using nanotechnology. In: Kalamdhad A,

Haq I (eds) Emerging treatment technologies for waste management. Springer Nature, Singapore,

pp 73–102. eBook ISBN: 9789811620157; Paperback ISBN: 9789811620140

Ghosh S, Khunt N, Webster TJ (2021e) Arsenic removing prokaryotes as potential bioﬁlters. In:

Shah M, Couto SR, Biswas JK (eds) An innovative role of bioﬁltration in waste water treat-

ment plants (WWTPs). Elsevier, pp 65–111. Paperback ISBN: 9780128239469; eBook ISBN:

9780128239476

Ghosh S, Bhattacharya J, Nitnavare R, Webster TJ (2022a) Heavy metal removal by Bacillus for

sustainable agriculture. In: Islam MT, Rahman M, Pandey P, (eds) Bacilli in Agrobiotechnology:

plant stress tolerance, bioremediation, and bioprospecting. Springer Nature, Switzerland, pp 1–30.

eBook ISBN 978-3-030-85465-2; Print ISBN 978-3-030-85464-5.

Ghosh S, Bhattacharya J, Nitnavare R, Webster TJ (2022b) Microbial remediation of metals by

marine bacteria. In: Shah MP, Couto SR (eds) Development in wastewater treatment research and

processes: microbial degradation of xenobiotics through bacterial and fungal approach. Elsevier,

USA, pp 131–158. ISBN: 978-0-323-85839-7

Ghosh S (2020) Toxic metal removal using microbial nanotechnology. In: Rai M, Golinska P (eds)

Microbial nanotechnology. CRC Press, Boca Raton. eBook ISBN: 9780429276330

Ijadpanah-Saravy H, Safari M, Khodadadi-Darban A, Rezaei A (2014) Synthesis of titanium dioxide

nanoparticles for photocatalytic degradation of cyanide in wastewater. Anal Lett 47(10):1772–

1782

Jamdade DA, Rajpali D, Joshi KA, Kitture R, Kulkarni AS, Shinde VS, Bellare J, Babiya KR,

Ghosh S (2019) Gnidia glauca-and Plumbago zeylanica-mediated synthesis of novel copper

nanoparticles as promising antidiabetic agents. Adv Pharmacol Sci 2019:9080279

Karmakar S, Ghosh S, Kumbhakar P (2020) Enhanced sunlight-driven photocatalytic and

antibacterial activities of ﬂower-like ZnO@ MoS2 nanocomposite. J Nanopart Res 22:11

Kitture R, Chordiya K, Gaware S, Ghosh S, More PA, Kulkarni P, Chopade BA, Kale SN (2015) ZnO

nanoparticles-red sandalwood conjugate: a promising anti-diabetic agent. J Nanosci Nanotechnol

15:4046–4051

Kusworo TD, Kumoro AC, Utomo DP, Kusumah FM, Pratiwi MD (2021) Performance of the

crosslinked PVA coated PES-TiO2 nano hybrid membrane for the treatment of pretreated natural

rubber wastewater involving sequential adsorption–ozonation processes. J Environ Chem Eng

9:104855

Luikham S, Malve S, Gawali P, Ghosh S (2018) A novel strategy towards agro-waste mediated dye

biosorption for water treatment. World J Pharm Res 7(4):197–208

Lv Y, Zhang C, He A, Yang SJ, Wu GP, Darling SB, Xu ZK (2017) Photocatalytic nanoﬁltration

membranes with self-cleaning property for wastewater treatment. Adv Func Mater 27:1700251

Nanobioremediation: A Sustainable Approach for Wastewater Treatment 445

Motahari F, Mozdianfard MR, Salavati-Niasari M (2015) Synthesis and adsorption studies of NiO

nanoparticles in the presence of H2acacen ligand, for removing Rhodamine B in wastewater

treatment. Process Saf Environ Prot 93:282–292

Nataraj SK, Hosamani KM, Aminabhavi TM (2006) Distillery wastewater treatment by the

membrane-based nanoﬁltration and reverse osmosis processes. Water Res 40(12):2349–2356

Qu X, Alvarez PJ, Li Q (2013) Applications of nanotechnology in water and wastewater treatment.

Water Res 47:3931–3946

Rajabi HR, Shahrezaei F, Farsi M (2016) Zinc sulﬁde quantum dots as powerful and efﬁcient

nanophotocatalysts for the removal of industrial pollutant. J Mater Sci: Mater Electron 27:9297–

9305

Ranpariya B, Salunke G, Karmakar S, Babiya K, Sutar S, Kadoo N, Kumbhakar P, Ghosh S

(2021) Antimicrobial synergy of silver-platinum nanohybrids with antibiotics. Front Microbiol

11:610968

Robkhob P, Ghosh S, Bellare J, Jamdade D, Tang IM, Thongmee S (2020) Effect of silver doping

on antidiabetic and antioxidant potential of ZnO nanorods. J Trace Elem Med Biol 58:126448

Rokade SS, Joshi KA, Mahajan K, Tomar G, Dubal DS, Singh V, Kitture R , Bellare J, Ghosh S

(2017) Novel anticancer platinum and palladium nanoparticles from Barleria prionitis. Global J

Nanomed 2(5):555600

Rokade S, Joshi K, Mahajan K, Patil S, Tomar G, Dubal DS, Parihar VS, Kitture R, Bellare JR,

Ghosh S (2018) Gloriosa superba mediated synthesis of platinum and palladium nanoparticles

for induction of apoptosis in breast cancer. Bioinorg Chem Appl 2018:4924186

Salunke GR, Ghosh S, Kumar RS, Khade S, Vashisth P, Kale T, Chopade S, Pruthi V, Kundu G,

Bellare JR, Chopade BA (2014) Rapid efﬁcient synthesis and characterization of silver, gold, and

bimetallic nanoparticles from the medicinal plant Plumbago zeylanica and their application in

bioﬁlm control. Int J Nanomed 9:2635–2653

Saray AM, Youseﬁ R, Zare-Dehnavi N, Jamali-Sheini F (2019) Improvement visible-light photocat-

alytic performance of single-crystalline SnSe1±x NPs toward degradation of organic pollutants.

Solid State Sci 98:106044

Shende S, Joshi KA, Kulkarni AS, Shinde VS, Parihar VS, Kitture R, Banerjee K, Kamble N,

Bellare J, Ghosh S (2017) Litchi chinensis peel: a novel source for synthesis of gold and silver

nanocatalysts. Global J Nanomed 3(1):555603

Shende S, Joshi KA, Kulkarni AS, C harolkar C, Shinde VS, Parihar VS, Kitture R, Banerjee K,

Kamble N, Bellare J, Ghosh S (2018) Platanus orientalis leaf mediated rapid synthesis of catalytic

gold and silver nanoparticles. J Nanomed Nanotechnol 9:2

Shinde SS, Joshi KA, Patil S, Singh S, Kitture R, Bellare J, Ghosh S (2018) Green synthesis of

silver nanoparticles using Gnidia glauca and computational evaluation of synergistic potential

with antimicrobial drugs. World J Pharm Res 7(4):156–171

Shirmardi A, Teridi MA, Azimi HR, Basirun WJ, Jamali-Sheini F, Youseﬁ R (2018) Enhanced photo-

catalytic performance of ZnSe/PANI nanocomposites for degradation of organic and inorganic

pollutants. Appl Surf Sci 462:730–738

Thanuttamavong M, Yamamoto K, Oh JI, Choo KH, Choi SJ (2002) Rejection characteristics of

organic and inorganic pollutants by ultra low-pressure nanoﬁltration of surface water for drinking

water treatment. Desalination 145:257–264

Bioremediation of Textile Dyes

for Sustainable Environment—A Review

Rajalakshmi Sridharan and Veena Gayathri Krishnaswamy

Abstract Development of science has taken over our lives and made it mandatory

to live with science. The synthetic technology takes more than it has given for our

welfare. The process of meeting the demand of the consumers, industries supported

the synthetic products to meet the same. Textile industry is one among them which

uses synthetic dyes to dye fabrics. The end result of the process is the production of

fabrics resistant to physical, chemical and biological stresses. The process not only

meets the consumers demands it also consumes customers health by polluting the

environment. The pre-treated or non-treated textile dyes released into the environ-

ment requires effective treatment process. Bioremediation, an eco-friendly method

is a broad spectrum which includes—microbial remediation, enzymatic remedia-

tion, phyto-remediation, phyco-remediation and bioreactors -. The current review

focuses on the microbial, enzymatic and bioreactor remediation of textile dye contam-

inated efﬂuents. This is the need of the hour to restore the healthy and sustainable

environment.

Keywords Textile dyes ·Bioremediation ·Enzymatic process ·Bioreactors

1 Introduction

Industrialization, a signiﬁcant factor in the growth of textile industries met the

demands of the consumer. The handmade cloths have become mechanized and sold

for lesser rate to reach wider consumers. The contribution of textile industries to

the country’s economy was nearly 1 trillion dollars which involved approximately

35 million workers. Textile industries started consuming fuel, electricity, water, and

chemicals for production. Nearly three trillion galloons of fresh water were consumed

to produce 60 billion Kg of fabrics (Desore and Narula 2018). Handling of pollu-

tants released from the industries requires special attention. The mutagenic and the

carcinogenic effects of the textile dye pollutants/efﬂuents were reported as a case

R. Sridharan · V. G. Krishnaswamy (B

)

Department of Biotechnology, Stella Maris College (Autonomous), University of Madras,

Chennai, India

e-mail: veenagayathri@stellamariscollege.edu.in

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_21

447

448 R. Sridharan and V. G. Krishnaswamy

Tabl e 1 Types of dyes and its applications

Dyes Applications References

Direct dyes Cotton, paper, leather, nylon and blends Cavaco-Paulo (1998)

Vat dyes Cotton, linen and rayon Liang et al. (2008)

Reactive dyes Cellulosic, fabrics and ﬁbre Tanthapanichakoon et al. (2005)

Acid dyes Silk, wool, leather, nylon, paper, food and

cosmetics

Daneshvar et al. (2004)

study. The direct discharge of industrial efﬂuents into the Amani Shah Ka Nallah

drainage in Rajasthan polluted the surface and ground water making it unﬁt for

agriculture and other purposes (Mathur et al. 2005).

Dyes are colouring agents that is majorly used in textile, pharmaceutical, paper,

printing industries etc. In dyes the lights are absorbed in visible spectrum and they

contain minimum a single chromophore group and has a structure of single and

double bonds in alternate manner. The stabilizing force for the organic structure of

the dye is given by electron resonance (Gowri et al. 2014). There are more than

10,000 different dyes were used in textile industries (Gupta et al. 2015). Table 1

gives the types of dyes and its applications.

Synthetic dyes were discovered by Willian Henry Perkin in 1856. These synthetic

dyes are resistant to sweat, sunlight, chemicals etc. and are applied on fabrics for

permanent colour. Textile industries uses the major quantity of synthetic dyes and

which are 70% composed of azo dyes (Saratale et al. 2011; Shah 2020). Reactive azo

dyes are dyes that have heterocyclic rings containing halogens substituent and forms

covalent link between the functional groups in the dye and the cellulose (Xie et al.

2008).These reactive dyes undergo nucleophilic substitution as it is electronegative.

This attack causes the ﬁxation on fabric after r eactive dye undergoes hydrolysis and

are resistant to chemical, physical and environmental agents (Al-degs et al. 2000).

The general structural formula for reactive dyes

S...F...T...X

S- Solubilizing group, F- chromophore, T- binding group, X- reactive system

(Gowri et al. 2014).The major group of dyes are composed of azo dyes containing

(–N = N-) azo group which is a chromophore (Lavanya et al. 2014) (Fig. 1).

Different types are azo dyes are formed when there is substitution of azo group by

benzene or naphthalene containing –Cl, –CH3,–NO

2, –NH2 and –OH and –COOH

Fig. 1 Structure of azo dye

Bioremediation of Textile Dyes for Sustainable Environment—A Review 449

(Saratale et al. 2011).Based on the number of azo groups, they are classiﬁed as

monoazo, di azo, triazo, tetrazo and polyazo dyes. Azo dyes are produced when

primary amine is deazotised by HCl and NaNO2 at very low temperature forms

diazonium salt which is conjugated with aromatic compounds, the azo dye are

formed (Lavanya et al. 2014). More than ten thousands of dyes are used in which ﬁfty

percentage are azo dyes (Leena and Selva Raj 2008). Since the azo dye comprises

maximum percentage in the wastewater from tanneries, treatment of this water is

required to reduce environmental impact. International Agency for Research on

Cancer (IARC) has classiﬁed many of these amines as carcinogens to humans (Sax

1975). Since azo dyes have poor exhaustion properties, the applied initial dyes

remains unbound to the fabric and could be found in the efﬂuents. Removal of these

dyes from the efﬂuents is a difﬁcult process because of the stability of dyes towards

light, oxidizing agents and heat. These properties make them non-biodegradable

(Rani et al. 2014).

Exposure to the textile pollution might cause ailments such as—headaches, lung

infection, skin infection, nausea, congenital malformation, carcinogenetic, and muta-

genic illness (cancer)–. The discharge of the efﬂuent containing azo dyes into the

ecosystem causes the formation of unpleasant smell. As dyes are exposed to sunlight,

they are reduced and the reduced metabolites affects photosynthesis, concentration

of dissolved oxygen, decreases the water quality and also affects aquatic organisms,

fauna, ﬂora and human beings. This discharge of azo dyes also adversely affects the

Total Carbon Concentration (TOC), Biological Oxygen Demand (BOD) and Chem-

ical Oxygen Demand (COD) (Saratale et al. 2009).The review provides a glimpse of

different biological methods employed in the removal/treatment of textile efﬂuents

released from the textile industries.

2 Treatment of Wastewater

Conventional methods for treatment of azo dye contaminated efﬂuents are difﬁ-

cult and less effective. Azo dyes are now widely treated by physical, chemical,

photo catalytic and biological methods (Lade et al. 2015). Dye treatment produces

more toxic metabolites which pollutes the land and water. To avoid further prob-

lems, it is necessary to ﬁnd an effective, less time consuming and cost effective

treatment process. Physical and chemical methods include ﬂocculation, ozonation,

bleaching, membrane ﬁltration, adsorption by activated carbon and irradiation are

the most commonly used methods for the treatment of dyes in wastewater. Since the

dye contains a complicated molecular structure these conventional process such as

oxidation by ozonation, activated carbon, coagulation, ultra ﬁltration t reatment can

be done. Though these treatments are expensive they aren’t efﬁcient and also they

produce abundant secondary waste products which require further treatment to be

eradicated (Robinson et al. 2001) (Table 2).

450 R. Sridharan and V. G. Krishnaswamy

Tabl e 2 Merits and demerits of physical and chemical methods of dye degradation

Physical and chemical

methods

Merits Demerits

Membrane seperation Decolorization of chemical

class dyes

Sludge generation

Coagulation/ﬂocculation Simple, cost effective Sludge production, handling

and disposal problem

Oxidation Quick process and effective Expensive

Ion exchange Effective Economic constraints

Ozonation COD and color removal is

complete, there are no toxic

metabolites produced

Expensive, half life is short

Fenton reagent Decolourises both soluble and

insoluble dyes

Sludge formation

Photo chemical Absence of sludge production

and no odours are produced

Production of secondary

pollutants

Electrochemical destruction Absence of sludge production

and Chemical consumption is

less

Electricity requirement is high

Electro kinetic coagulation Economical, dye removal is

high

Unsuitable for acid dyes,

generation of sludge

Adsorption by Activated

carbon

Effective process Unsuitable for disperse and vat

dyes

Irradiation Effective for lab scale and less

volume of efﬂuent

DO requirement is high

Bioremediation methods have drawn many scientists as they degrade the azo

dyes in an efﬁcient and effective manner which is cost effective and eco-friendly.

The microorganisms adapt to the toxic environment and they become resistant to

the toxicity of the dyes and transform the toxic dyes to non toxic or less toxic

compounds (Saratale et al. 2011; Shah 2021). Decolourisation by microorganisms

are carried either by aerobic or anaerobic methods. In aerobic conditions, the microor-

ganism takes long time for chemostatic growth in the presence of oxygen and then

decolousises the azo dyes. But in case of anaerobic microorganisms, they are not

speciﬁc for the azo compound and hence, this method is used universally (Pearce

et al. 2003). Wide varieties of microorganisms exist that are capable of decolorizing

dyes of various classes. Usage of single isolate isn’t effective because single isolates

can only decolorize particular class of dyes. Therefore in the biological treatment

consortium is used to decolorize the dyes present in the efﬂuent. Mixed cultures were

effective when compared to individual strain. Since the process is inexpensive and the

end products are mineralized with less toxicity, there are numerous studies done on

biological treatment of industrial textile wastewater (Mohana et al. 2008; Chang et al.

2001). Microorganisms also biosorb the azo dyes present in the efﬂuent. Biosorption

is the characteristics of certain biomolecule produced by microorganisms that bind

Bioremediation of Textile Dyes for Sustainable Environment—A Review 451

speciﬁcally to the ions or molecules present in the textile efﬂuent. Microorganisms

produces enzymes that degrades the azo dyes and forms metabolites that are less

toxic (Revankar et al. 2007).

Biological degradation is usually done by using fungi, bacteria, algae and yeast

and some are listed below in Table 3.

The below Table 4 shows the advantages and disadvantages of biological

treatment.

Tabl e 3 Microorganisms used in degradation of dyes

Organisms Dyes References

Bacteria

Bacillus sps

Methyl red

Methyl orange

Congo red

Erichrome black T

Kalyanietal. (2008)

E.coli

P.ﬂuorescence

Reactive orange –MER

Reactive blue-M58

Reactive yellow-M46

Reactive black-B

Saranraj et al. (2014)

B.odyssey

B.thuringiensis

B.subtilis

Alcaligenes sps

Nocardiopsis alba

Acid red Saranraj et al. (2014)

P.extremorientalis Congo red Neifar et al. (2016)

P.aeruginosa Acid blue 113 Rani et al. (2016)

Fungi

Trametes versicolor

Acid orange 7

Acid blue 74

Reactive red 2

Reactive black 5

Ramírez-Montoya et al. (2015)

Tabl e 4 Advantages and disadvantages of biological treatments (Robinson et al. 2001)

Biological methods Advantages Disadvantages

Biodegradation Economical, dependent on

metabolism of the microbes

Nutrition requirement is continuous,

slow, continuous monitoring

Biosorption Inexpensive Large scale treatment has not been

studied yet, disposal is trickier

Enzymatic Effective Expensive

Phytoremediation Eco-friendly High maintenance and slow

452 R. Sridharan and V. G. Krishnaswamy

3 Aerobic Treatment

Depending on the requirement of oxygen bacteria can be divided into aerobic bacteria,

anaerobic bacteria and facultative bacteria. In the aerobic biological treatment,

aerobic and facultative bacteria are used for the treatment at an aerobic environ-

ment (Wang et al. 2011). The process is classiﬁed into Activated sludge process and

Bioﬁlm formation.

4 Activated Sludge Process

In activated sludge process a ﬂoc is used which comprises of numerous organisms

which are capable of degrading and absorbing the organics and therefore it is called as

Activated sludge. Activated sludge process is an effective method for the degradation

and this process is used in oxidation ditch and sequential batch reactor.

In this Sequential batch reactor process, there are ﬁve steps involved they are

inﬂow, reaction, sedimentation, outﬂow and standby. This method of sequential batch

reactor activated sludge process is commonly used because it shows high COD

removal and decolorization. In the bioﬁlm process, numerous organisms are allowed

to grow and attach to an object surface and the wastewater is let to ﬂow on the surface.

The contact between the wastewater and the object surface with the bioﬁlm attached

will result in degradation and puriﬁcation. The main bioﬁlm processes are biological

contact oxidation, rotating biological contactor and biological ﬂuidized bed.

5 Anaerobic Treatment

In anaerobic treatment anaerobic microorganisms are used. There is high

concentration and low concentration treatment in anaerobic treatment process.

There are many studies done on anaerobic treatment of textile wastewater but a

combination of aerobic-anaerobic treatment gives better results for dye degradation

(Wang et al. 2011).

6 Enzymatic Methods

Microorganisms degrade azo dyes by the enzymes, so the decolourisation and miner-

alization of the dyes occurs in speciﬁc conditions. The enzymes that degrade the dyes

have wide substrate speciﬁcity (Pandey et al. 2007; Esposito and Durán 2000;Mester

Bioremediation of Textile Dyes for Sustainable Environment—A Review 453

and Tien 2000). The major enzymes involved in the azo dye degradation are azore-

ductase, laccase, lignin peroxidise, manganese peroxidise and hydroxylases (Rani

et al. 2016).

7 Azoreductase

The reaction carried by azoreducatse is catalysed in presence of NADH and FADH

(Robinson et al. 2001). It carries reductive azo dye degradation. In aerobic reaction,

oxygen inhibits reduction of azo bond and NADH consumption is dominated and

they interfere with the transfer of electrons from NADH to azo dyes (Sandhya et al.

2008; Elisangela et al. 2009) and azo dye reduction in extracellular environment. It

has also been studied that redox mediator compounds with low molecular weight

which acts as electron shuttles for NADH dependent azoreductase. Microorganism

that produces azo reducatses are mostly Bacteria and Fungi. Lactobacillus casei

(Seesuriyachan et al. 2007), Bacillus odyssey, B.thuringiensis, B.subtilis, B.cereus,

Alcaligenes sp, Nocardia alba (Saranraj et al. 2014).

Laccases are enzymes containing four atoms of copper, type 1 paramagnetic

copper are responsible for blue colour and the substrate oxidation. Type 2 copper and

two 3rd type copper reduces the oxygen molecule to two water molecules. Phenolic

compounds are oxidised by laccases and forms non toxic aromatic amines (Ramírez-

Montoya et al. 2015).The azo dye containing phenolic group is oxidised by laccase

enzyme which generates phenoxy radical and oxidises to carbonium ion (Camarero

et al. 2005). Bacteria that produces Laccase enzymes are Pseudomonas desmolyticum

(Kalme et al. 2007), Bacillus sps (Dawkar et al. 2008), Coriolus versicolor,

Paraconiothyrium variabile, and Tremetes versicolor (Asadgol et al. 2014).

Peroxidises are enzyme groups with hemoproteins groups that carries the reaction

in Hydrogen peroxide presence. The lignin and manganese peroxidise have similar

mechanism which works in hydrogen peroxide presence. Lignin peroxidise were

ﬁrst isolated from P.chrysosporium which oxidises the non- phenolic compounds.

Whereas Mn2+ to Mn3+ by Manganese peroxidise because Mn3+ is responsible for

oxidation of phenolic compounds (Glenn et al. 1983).

8 Bioreactors in Textile Wastewater Treatment

Bioreactors are ex situ bioremediation process to treat the industrial wastewater.

To treat the industrial efﬂuents there are many types of bioreactor conﬁguration to

achieve maximum degradation. The process usually is aerobic or anaerobic and some-

times combination of both. Decolorization and degradation of the textile efﬂuents are

done using biological source in the bioreactors. There are numerous research papers

has been published on sequential, combined and even integrated aerobic-anaerobic

treatment in bioreactors to degrade the dyes. Different conﬁguration of bioreactors

454 R. Sridharan and V. G. Krishnaswamy

Fig. 2 Packed bed

bioreactor

PRODUCT OUTLET

ENZYME PACKED

COLUMN

HEATING JACKET

for the dye degradation has been used such as packed bed bioreactor, Sequential

Batch reactor, ﬂuidized bed reactor, Trickiling ﬁlter and Moving bed bioﬁlm reactor.

Bioreactors are mainly used as to retain the biomass for a longer period of time for

effective degradation of dyes in the efﬂuent (Ramachandran et al. 2013). Packed bed

bioreactors are mainly used when fungi are used in the treatment of dye degradation

in reactors. To degrade synthetic dyes, Irpex lacteus, a white rot fungi has been used

in a packed bed reactor which had a solid support of polyurethane foam (PUF) and

pine wood. The synthetic dye, Remezol Brilliant Blue R was degraded by the white

rot fungi in the packed bed reactor containing the support (Kasinath et al. 2003).

In some packed bed bioreactors activated carbon used for the treatment also gave

effective results in degradation of synthetic dyes (Mesquita et al. 2012). Packed bed

bioreactors can be continuous ﬂow or fed-batch. Most of the studies on degradation

of textile dyes were done using a continuous ﬂow packed bed bioreactor. Congo red

dye has been decolorized in a continuous ﬂow packed bed bioreactor that was packed

with rice hull, Schizophullum sp. F17 was used in the process (Li and Jia 2008). The

Fig. 2 shows packed bed bioreactor.

Fluidized bed bioreactors have been used in the treatment of wastewater. In a

ﬂuidized bed reactor, a ﬂuid (or gas) is passed through a solid that is suspended

by the velocity of the ﬂuid ﬂow. The velocity of the ﬂuid should be as such that it

suspends the solid but doesn’t wash off the solid from the reactor. For the wastewater

treatment, to increase the degradation capacity, fenton process is combined with the

ﬂuidized bed and is called as ﬂuidized bed fenton process. There are many such

researches published on the usage of FBF process for the treatment of industrial

efﬂuent. Effective results were reported on the degradation of dyes using Fluidized

bed fenton process in which SiO2 were used as carriers which gave a complete

removal of dye in a s hort period of time (Wang et al. 2011). The sequential batch

reactor contains a cycle of reaction such as ﬁll, react, settle and draw. The time period

of these cycles will differ with the type of set up and the type of reaction in the reactor.

Sandhya et al. (2005) designed a microaerophilic-aerobic sequential batch reactor

to treat simulated wastewater containing mixture of dyes and the ﬁnal concentra-

tion of dye in the synthetic wastewater was 56 mg/L. There were two reactors R1

Bioremediation of Textile Dyes for Sustainable Environment—A Review 455

and R2 which was microaerophilic and aerobic reactor respectively. There was no

decolourization observed under shaking condition, only COD removal was observed

but under static condition, degradation of the dye was observed. Therefore the biore-

actor was switched between static and shaking conditions to achieve COD, BOD

removal and decolourization of the dyes. During static condition 78.9% of decolour-

izationwas obtained and during static-shaking condition 31.8% of decolourization

was reported. Baskar and Sukumaran (2015) used a sequencing batch reactor for

treating wastewater produced by meat processing industry. Initially the BOD was

330 mg/L. The SBR contained an air diffuser and had a volume of 1200 L. There

was a drastic decrease in the COD and BOD levels in 9 days. From 50 mg/L the

BOD reduced to 10 mg/L and from 80 mg/L the COD reduced to 30 mg/L in nine

days. Therefore SBR was proved to reduce the BOD and COD and also the turbidity

of the wastewater was decreased to 90%, making the wastewater harmless to the

environment.

There are mainly two technologies used for the treatment of wastewater they are

activated sludge process and trickling ﬁlters. Compilation of these two technolo-

gies, a new type of treatment called Moving Bed Bioﬁlm Reactor was developed in

Norway by Norwegian University of Science and Technology in co-operation with

Anox Kaldnes AS, a Norwegian Company. The biomass present in MBBR is usually

in two forms, one is suspended and the other is attached to carrier material. In the

year of 1989, the ﬁrst MBBR was installed. In a MBBR, carrier materials are used

which supports the growth of bioﬁlm. Since the carrier is of lighter density than

water, they have a movement along with the stream of water inside the reactor. For

aerobic MBBR, the movement of the carrier element is given by air diffuser from

the bottom of the reactor. In case of anaerobic treatment, a mechanical stirrer is used

for the movement of the carrier element in the reactor. Francis and Sosamony (2016)

studied on Moving bed bio-ﬁlm reactor to treat textile wastewater that was pre-treated

in a ﬂuidized bed fenton process. The moving bed bio-ﬁlm reactor had a height of

50 cm, internal size of 15 X 15, a total volume of 11.25 L. They used Poly Vinyl Chlo-

ride (PVC), the inlet pipe of a washing machine as carrier material for formation of

bioﬁlm. The sludge was collected from Augastan textiles and Micobacterium marni-

lacus was isolated from the sludge. The synthetic wastewater contained Chemistar

turq blue (100 mg/L) as the dye, which was treated in the fenton process to bring

the COD from 780 mg/L to 336 mg/L. The pre-treated textile wastewater was then

treated in the moving bed, the maximum COD removal was seen in 2.5 days, at a pH

of 7.33 and a carrier ﬁlling ratio of 67%. COD removal was estimated to be 87.22%

and BOD removal of 80% was achieved using moving bed bioﬁlm reactor. Koupaie

et al. (2011) treated synthetic wastewater containing Acid Red18 (AR18) 40 mg/L

in a conventional Sequential batch reactor and three Moving bed bio-ﬁlm reactor

containing three different carrier materials in each. The ﬁlling ratio was 50%. All the

reactors measured 50 cm of height, 14 cm inner diameter, 9.8 L total volume and 5

L working volume. The sludge was collected from Zargandeh municipal wastewater

treatment plant, Tehran (Iran). The HRT was set for 2 days. The dye concentration

was gradually increased from 40 mg/L to 1000 mg/L. COD removal in sequential

batch reactor, moving bed bio-ﬁlm reactor 1, moving bed bio-ﬁlm reactor 2 and

456 R. Sridharan and V. G. Krishnaswamy

moving bed bio-ﬁlm reactor 3 were 96.1%, 97.7%, 97.6% and 97.5% respectively.

Usage of different carrier materials didn’t show any inﬂuence on the COD removal.

The decolorization of acid red 18 dye wasn’t observed and was concluded that the

bio-ﬁlm attachment wasn’t enough for the dye removal. Delnavaz et al. (2008)used

three moving bed bio-ﬁlm reactors measuring 70 cm of height, 10 cm of inner diam-

eter, 0.4 cm wall thickness and 60 cm of working volume with Light Expanded Clay

Aggregate (LECA) as carriers of 50% ﬁlling ratio. They set a HRT of 8, 24, 48, 72 h

to degrade the amine compounds such as aniline, para-diaminobenzene, and para-

amino phenol. Efﬁcient results were obtained after three days of operation. 90% of

COD removal was seen for inﬂuent COD that was 750 mg/L, 87% of COD removal in

inﬂuent COD of 100 mg/L and 90% for 2000 mg/L for para-diaminobenzene, para-

aminophenol and aniline respectively. The Fig. 8 below shows typical moving bed

bioﬁlm reactor design. Koupaie et al. (2011) used anaerobic sequencing batch reactor/

moving bed sequencing batch bioﬁlm reactor to study the kinetics of decolourization

and biodegradation of Acid Red 18 azo dye. The reactors measured a diameter of

14 cm, height of 50 cm and effective volume of 5.5 L. Polyethylene bioﬁlm carriers

at a ﬁlling ratio of 50% were used as support in the moving bed bioﬁlm reactor.

There were three anaerobic sequential batch reactor to which different concentration

of dyes were added, 100, 500 and 1000 mg/L. in the operation period of 90 days,

83% of COD removal was seen. Therefore anaerobic sequencing batch reactor along

with moving bed gave an effective result for wastewater treatment. Jafari et al. (2013)

built an anaerobic ﬂuidized bed reactor and aerobic moving bed bioreactor to treat

synthetic wastewater using Bee-Cell 2000 as carriers in the moving bed which had

a height of 90 cm, inner diameter of 10 cm, 60 cm of working volume and 1.5 L of

the carriers. The operation period was 255 days at a HRT of 48, 40, 32, 24, 18 h.

The sludge was collected from aerobic digesters of municipal wastewater treatment

plant. The efﬂuent COD of the ﬂuidized bed was 935 mg/L and the moving bed

bioﬁlm reactor reduced the COD level from 935 mg/L to 350 mg/L. The removal of

COD was 97.5% in the moving bed bioﬁlm reactor.

Synthetic wastewater containing different concentrations of Reactive orange 16

dye in 25, 50, 75 and 100 mg/L was treated in a combination of ozonation and

moving bed bioﬁlm reactor. Two moving bed bioreactors were used in which one had

ozonated synthetic wastewater and the other with non ozonated synthetic wastewater

in a 200 mL cylindrical glass. Kaldnes K1 was used as support material in the reactors,

the ﬁlling ratio was 40%. The activated sludge was collected from municipal sewage

treatment plant and the HRT was set for 6 h. The operation continued for 60 days.

About 94% of the inﬂuent COD was achieved in the moving bed. It was concluded

that the moving bed treating non ozonated synthetic wastewater didn’t show much

degradation of dye and COD. Therefore moving bed bioﬁlm reactor can only enhance

the removal of COD and dye in a pre-treated wastewater.

A pilot scale moving bed bioreactors which contained three 15 L reactors with

mechanical stirrer and air diffuser was designed by Park et al. (2007). All the reactors

contained Polyurethane-Activated carbon (PU-AC) foam as carrier material with a

20% ﬁlling ratio in each reactor. The ﬁrst two reactors were anaerobic and the last

reactor was aerobic reactor. The pilot scale reactor was initially operated without

Bioremediation of Textile Dyes for Sustainable Environment—A Review 457

the carriers and then compared the performance with the carrier material in it. The

carriers were incubated with the microorganisms for seven days for better attachment

of the microorganisms to the foam. Removal of COD was seen in the ﬁrst day of

operation, HRT was set as 44 h in all the reactors. The COD removal was high with

high packing material. They concluded that A20 moving bed bioreactor was a better

treatment method than other conventional process. Treatment of synthetic wastewater

in a moving bed bioﬁlm reactor followed by a membrane separation process. The

moving bed bioﬁlm reactor contained aerobic and anaerobic section. The total volume

was 70 L in which 60 L was the effective volume. The length was 70 cm, width was

25 cm and depth was 40 cm. The cylindrical carriers were used and the ﬁlling ratio

was 35%. The synthetic wastewater contained an average of 500 mg/L of COD,

after the treatment 85% of the COD was removed. The synthetic wastewater was

added with Brilliant Red X-3B dye which was reduced in the anaerobic moving bed

bioreactor. Highest of COD removal was seen in the aerobic moving bed bioreactor

(Dong et al. 2014).

9 Treatment of Textile Azo Dyes Using Combination

of Bioreactors

Moving bed bioﬁlm reactor plays an important role to enhance the pre-treated water

and to result in high quality ﬁnal efﬂuent. Therefore, the combination of reactors is

used for the maximum degradation. A system of anaerobic ﬂuidized bed reactor and

aerobic moving bed bioﬁlm reactor was used by Jafari et al. (2013). The efﬂuent of

anaerobic ﬂuidized bed reactor was fed to an aerobic moving bed bioﬁlm reactor

was operated at different hydraulic retention time. Maximum COD removal was

obtained in anaerobic ﬂuidized bed reactor, aerobic moving bioﬁlm reactor just

polished the results of the pre-treated synthetic wastewater. Francis and Sosamony

(2016) worked on a combination of ﬂuidized bed fenton process and moving bed

bioﬁlm reactor. Comparison study of moving bed bioﬁlm reactor and conventional

sequential batch reactor was studied by Koupaie et al. (2011).They have concluded

COD, dye concentration and turbidity results of both the reactors did not show much

difference.

10 Conclusion

The treatment of the textile efﬂuent is the need of the hour. The efﬁcient and eco-

friendly process (Bioremediation) converts the complex dye compounds into simpler

structure which could be lesser toxic than parent compound. The current review

summarizes the methods of bioremediation used in the treatment of textile dye

contaminated efﬂuents. The method of selection of bioremediation depends on the

requirement and the site of treatment.

458 R. Sridharan and V. G. Krishnaswamy

References

Al-degs Y, Khraisheh MAM, Allen SJ, Ahmad MN (2000) Effect of carbon surface chemistry on

the removal of reactive dyes from textile efﬂuent. Wat Res 34(3):927–935. https://doi.org/10.

1111/j.1748-1716.1958.tb01584.x

Asadgol Z, Forootanfar H, Rezaei S, Mahvi AH, Faramarzi MA (2014) Removal of phenol and

bisphenol-a catalyzed by laccase in aqueous solution. J Environ Health Sci Eng 12(1):1–5. https://

doi.org/10.1186/2052-336X-12-93

Baskar M, Sukumaran B (2015) Effective method of treating wastewater from meat processing

industry using sequencing batch reactor, pp 27–31

Camarero S, Ibarra D, Martínez MJ, Martínez ÁT (2005) Lignin-derived compounds as efﬁ-

cient laccase mediators for decolorization of different types of recalcitrant dyes. Appl Environ

Microbiol 71(4):1775–1784. https://doi.org/10.1128/AEM.71.4.1775-1784.2005

Cavaco-Paulo A (1998) Mechanism of cellulase action in textile processes. Carbohydr Polym

37(3):273–77. https://doi.org/10.1016/S0144-8617(98)00070-8

Chang JS, Chou C, Lin YC, Lin PJ, Ho JY, Hu TL (2001) Kinetic characteristics of bacterial

azo-dye decolorization by Pseudomonas Luteola. Water Res 35(12):2841–50. https://doi.org/10.

1016/S0043-1354(00)00581-9

Daneshvar N, Salari D, Khataee AR (2004) Photocatalytic degradation of azo dye acid red 14 in

water on ZnO as an alternative catalyst to TiO2. J Photochem Photobiol, A 162(2–3):317–322.

https://doi.org/10.1016/S1010-6030(03)00378-2

Dawkar VV, Jadhav UU, Jadhav SU, Govindwar SP (2008) Biodegradation of disperse textile dye

brown 3REL by newly isolated Bacillus Sp. VUS. J Appl Microbiol 105(1):14–24. https://doi.

org/10.1111/j.1365-2672.2008.03738.x

Delnavaz M, Ayati B, Ganjidoust H (2008) Biodegradation of aromatic amine compounds using

moving bed bioﬁlm reactors. Iranian J Environ Health Sci Eng 5(4):243–250

Desore A, Narula SA (2018) An overview on corporate response towards sustainability issues

in textile industry. Environ Dev Sustain 20(4):1439–1459. https://doi.org/10.1007/s10668-017-

9949-1

Dong B, Chen H, Yang Y, He Q, Dai X (2014) Treatment of printing and dyeing wastewater using

MBBR followed by membrane separation process. Desalin Water Treat 52(22–24):4562–4567.

https://doi.org/10.1080/19443994.2013.803780

Elisangela F, Andrea Z, Fabio DG, de Menezes Cristiano R, Regina DL, Artur CP (2009) Biodegra-

dation of textile azo dyes by a Facultative Staphylococcus Arlettae Strain VN-11 using a sequen-

tial microaerophilic/aerobic process. Int Biodeterior Biodegrad 63(3):280–88. https://doi.org/10.

1016/j.ibiod.2008.10.003

Esposito E, Durán N (2000) Potential applications of oxidative enzymes and phenoloxidase-like

compounds in wastewater and soil treatment: a review. Appl Catal B 28(2):83–99

Francis A, Sosamony KJ (2016) Treatment of pre-treated textile wastewater using moving bed

bio-ﬁlm reactor. Proc Technol. https://doi.org/10.1016/j.protcy.2016.05.033

Glenn JK, Morgan MA, Mayﬁeld MB, Kuwahara M, Gold MH (1983) An extracellular H2O2-

requiring enzyme preparation involved in lignin biodegradation by the white rot basidiomycete

phanerochaete chrysosporium. Biochem Biophys Res Commun 114(3):1077–1083. https://doi.

org/10.1016/0006-291X(83)90672-1

Gowri RS, Vijayaraghavan R, Meenambigai P (2014) Microbial degradation of reactive dyes- a

review. Int J Current Microbiol Appl Sci 3(3):421–436

Gupta VK, Khamparia S, Tyagi I, Jaspal D, Malviya A (2015) Decolorization of mixture of dyes:

a critical review. Glob J Environ Sci Manage 1(1):71–94. https://doi.org/10.7508/gjesm.2015.

01.007

Hosseini Koupaie E, Alavi Moghaddam MR, Hashemi H (2011) Comparison of overall performance

between moving-bed and conventional sequencing batch reactor. Iranian J Environ Health Sci

Eng 8(3):235–44

Bioremediation of Textile Dyes for Sustainable Environment—A Review 459

Jafari J, Mesdaghinia A, Nabizadeh R, Farrokhi M, Mahvi AH (2013) Investigation of anaerobic

ﬂuidized bed reactor/ aerobic moving bed bio reactor (AFBR/MMBR) system for treatment of

currant wastewater. Iran J Public Health 42(8):860–867

Kalme SD, Parshetti GK, Jadhav SU, Govindwar SP (2007) Biodegradation of Benzidine based

dye direct blue-6 by Pseudomonas Desmolyticum NCIM 2112. Biores Technol 98(7):1405–1410.

https://doi.org/10.1016/j.biortech.2006.05.023

Kalyani DC, Patil PS, Jadhav JP, Govindwar SP (2008) Biodegradation of reactive textile dye

red BLI by an isolated Bacterium Pseudomonas Sp. SUK1. Biores Technol 99(11):4635–4641.

https://doi.org/10.1016/j.biortech.2007.06.058

Kasinath A, Novotný ˇ

C, Svobodová K, Patel KC, Šašek V (2003) Decolorization of synthetic

dyes by Irpex Lacteus in liquid cultures and packed-bed bioreactor. Enzyme Microb Technol

32(1):167–173. https://doi.org/10.1016/S0141-0229(02)00279-X

Lade H, Govindwar S, Paul D (2015) Mineralization and detoxiﬁcation of the carcinogenic azo dye

congo red and real textile efﬂuent by a polyurethane foam immobilized microbial consortium in

an upﬂow column bioreactor. Int J Environ Res Public Health 12(6):6894–6918. https://doi.org/

10.3390/ijerph120606894

Lavanya C, Dhankar R, Chhikara S, Sheoran S (2014) Review article degradation of toxic dyes: a

review. Int J Curr Microbiol App Sci 3(6):189–199

Leena R, Selva Raj D (2008) Bio-decolourization of textile efﬂuent containing reactive black-b by

efﬂuent-adapted and non-adapted Bacteria. African J Biotechnol 7(18):3309–3313. https://doi.

org/10.4314/ajb.v7i18.59276

Li X, Jia R (2008) Decolorization and biosorption for congo red by system rice hull- Schizophyllum

Sp. F17 under solid-state condition in a continuous ﬂow packed-bed bioreactor. Biores Technol

99(15):6885–6892. https://doi.org/10.1016/j.biortech.2008.01.049

Liang C, Huang CF, Chen YJ (2008) Potential for activated persulfate degradation of BTEX

contamination. Water Res 42(15):4091–4100. https://doi.org/10.1016/j.watres.2008.06.022

Mathur N, Bhatnagar P, Nagar P, Bijarnia MK (2005) Mutagenicity assessment of efﬂuents

from textile/dye Industries of Sanganer, Jaipur (India): A Case Study. Ecotoxicol Environ Saf

61(1):105–113. https://doi.org/10.1016/j.ecoenv.2004.08.003

Mesquita I, Matos LC, Filipa Duarte FJ, Maldonado-Hódar AM, Madeira LM (2012) Treatment of

azo dye-containing wastewater by a fenton-like process in a continuous packed-bed reactor ﬁlled

with activated carbon. J Hazard Mater 237–238:30–37. https://doi.org/10.1016/j.jhazmat.2012.

07.066

Mester T, Tien M (2000) Oxidation mechanism of ligninolytic enzymes involved in the degrada-

tion of environmental pollutants. Int Biodeterior Biodegrad 46(1):51–59. https://doi.org/10.1016/

S0964-8305(00)00071-8

Mohana S, Shrivastava S, Divecha J, Madamwar D (2008) Response surface methodology for

optimization of medium for decolorization of textile dye direct black 22 by a novel Bacterial

consortium. Biores Technol 99(3):562–569. https://doi.org/10.1016/j.biortech.2006.12.033

Neifar M, Chouchane H, Mahjoubi M, Jaouani A, Cherif A (2016) Pseudomonas extremorientalis

BU118: a new salt-tolerant laccase-secreting bacterium with biotechnological potential in textile

azo dye decolourization. 3 Biotech 6(1):1–9. https://doi.org/10.1007/s13205-016-0425-7

Pandey A, Singh P, Iyengar L (2007) Bacterial decolorization and degradation of azo dyes. Int

Biodeterior Biodegrad 59(2):73–84. https://doi.org/10.1016/j.ibiod.2006.08.006

Park C, Lee M, Lee B, Kim SW, Chase HA, Lee J, Kim S (2007) Biodegradation and biosorption

for decolorization of synthetic dyes by Funalia Trogii. Biochem Eng J 36(1):59–65. https://doi.

org/10.1016/j.bej.2006.06.007

Pearce CI, Lloyd JR, Guthrie JT (2003) The removal of colour from textile wastewater using whole

bacterial cells: a review. Dyes Pigm 58(3):179–196. https://doi.org/10.1016/S0143-7208(03)000

64-0

Ramachandran P, Sundharam R, Palaniyappan J, Munusamy AP (2013) Potential process implicated

in bioremediation of textile efﬂuents: a review. Pelagia Res Library Adv Appl Sci Res 4(1):131–45.

www.pelagiaresearchlibrary.com

460 R. Sridharan and V. G. Krishnaswamy

Ramírez-Montoya LA, Hernández-Montoya V, Montes-Morán MA, Jáuregui-Rincón J, Cervantes

FJ (2015) Decolorization of dyes with different molecular properties using free and immobilized

laccases from trametes versicolor. J Molecul Liquids 212:30–37. https://doi.org/10.1016/j.mol

liq.2015.08.040

Rani B, Kumar V, Singh J, Bisht S, Teotia P, Sharma S, Kela R (2014) Bioremediation of dyes by

fungi isolated from contaminated dye efﬂuent sites for bio-usability. Braz J Microbiol 45(3):1055–

1063. https://doi.org/10.1590/S1517-83822014000300039

Rani VP, Priya KS, Nancy AA, Kumari GM (2016) Investigation of enzymatic degradation of acid

blue 113 by Halotolerant Strain Pseudomonas Aeruginosa.” World Journal of Pharmaceutical

Research 5 (3): 1673–80

Revankar MS, Desai KM, Lele SS (2007) Solid-state fermentation for enhanced production of

laccase using indigenously isolated Ganoderma Sp. Appl Biochem Biotechnol 143(1):16–26.

https://doi.org/10.1007/s12010-007-0029-0

Robinson T, McMullan G, Marchant R, Nigam P (2001) Remediation of dyes in textile efﬂuent:

a critical review on current treatment technologies with a proposed alternative. Biores Technol.

https://doi.org/10.1016/S0960-8524(00)00080-8

Sandhya S, Padmavathy S, Swaminathan K, Subrahmanyam YV, Kaul SN (2005) Microaerophilic-

aerobic sequential batch reactor for treatment of azo dyes containing simulated wastewater.

Process Biochem 40(2):885–890. https://doi.org/10.1016/j.procbio.2004.02.015

Sandhya S, Sarayu K, Uma B, Swaminathan K (2008) Decolorizing kinetics of a Recombinant

Escherichia Coli SS125 strain harboring Azoreductase gene from Bacillus Latrosporus RRK1.

Biores Technol 99(7):2187–2191. https://doi.org/10.1016/j.biortech.2007.05.027

Saranraj P, Stella D, Sivasakthivelan P (2014) Scholars research library separation, puriﬁcation and

characterization of dye degrading enzyme Azoreductase from Bacterial isolates. Central Eur J

Experim Biol 3(2):19–25. http://scholarsresearchlibrary.com/archive.html

Saratale RG, Saratale GD, Kalyani DC, Chang JS, Govindwar SP (2009) Enhanced decolorization

and biodegradation of textile azo dye scarlet R by using developed microbial consortium-GR.

Biores Technol 100(9):2493–2500. https://doi.org/10.1016/j.biortech.2008.12.013

Saratale RG, Saratale GD, Chang JS, Govindwar SP (2011) Bacterial decolorization and degradation

of azo dyes: a review. J Taiwan Inst Chem Eng 42(1):138–157. https://doi.org/10.1016/j.jtice.

2010.06.006

Sax NI (1975) Dangerous properties of industrial materials.” Van Nostrand Reinhold £21.25. https://

doi.org/10.2105/ajph.54.5.866-b

Seesuriyachan P, Takenaka S, Kuntiya A, Klayraung S, Murakami S, Aoki K (2007) Metabolism

of azo dyes by Lactobacillus Casei TISTR 1500 and effects of various factors on decolorization.

Water Res 41(5):985–992. https://doi.org/10.1016/j.watres.2006.12.001

Shah Maulin P (2020) Microbial bioremediation and biodegradation. Springer

Shah Maulin P (2021a) Removal of refractory pollutants from wastewater treatment plants. CRC

Press

Shah Maulin P (2021b) Removal of emerging contaminants through microbial processes. Springer

Tanthapanichakoon W, Ariyadejwanich P, Japthong P, Nakagawa K, Mukai SR, Tamon H (2005)

Adsorption-desorption characteristics of phenol and reactive dyes from aqueous solution on

mesoporous activated carbon prepared from waste tires. Water Res 39(7):1347–1353. https://doi.

org/10.1016/j.watres.2004.12.044

Wang Z, Xue M, Huang K, Liu Z (2011) Textile dyeing wastewater treatment. Advances in Treating

Textile Efﬂuent. https://doi.org/10.5772/22670

Xie K, Hou A, Sun Y (2008) Chemical graft of cellulose with the ion-pair emulsion containing the

reactive groups and its dyeing properties. J Dispersion Sci Technol 29(10):1385–1390. https://

doi.org/10.1080/01932690802313105

Zee FP, Der V, Villaverde S (2005) Combined anaerobic-aerobic treatment of azo dyes - a short

review of bioreactor studies. Water Res 39(8):1425–1440. https://doi.org/10.1016/j.watres.2005.

03.007

Microbial Contamination

of Environmental Waters

and Wastewater: Detection Methods

and Treatment Technologies

José Gonçalves, Israel Díaz, Andrés Torres-Franco, Elisa Rodríguez,

Priscilla Gomes da Silva, João R. Mesquita, Raúl Muñoz,

and Pedro A. Garcia-Encina

Abstract The majority of waterborne pathogens of public health concern do not

originate in natural water reservoirs but are instead introduced by point sources of

pollution and non-point sources of pollution. Pathogens introduced in the environ-

ment by anthropogenic activities are diverse and even if present in low concentrations

have serious public health implications. Due to their diversity and low concentrations,

there are technical challenges to their accurate quantiﬁcation, which is particularly

true for pathogenic viruses. To overcome the limitations, or as a result of the limita-

tions, water monitoring programs use indicators of fecal contamination as a surrogate

for pathogen occurrence. Most methods to detect human viruses in aquatic environ-

ments follow these steps: Concentration, nucleic acid extraction, ampliﬁcation of the

genomic segment (or segments) chosen, and detection/quantiﬁcation of the ampli-

ﬁed genomic segment. Wastewater treatment technologies are the most important

step to prevent public health burden and to safely reuse waste. Due to the difference

in health conditions of people living in different countries, the pathogen content is

notably different and therefore the appropriate treatment options are also different

and should be diverse. There are warnings that climate change is likely to exacerbate

health risks related to deﬁciencies in water supply, sanitation and hygiene in many

regions of the world. These risks can be minimized though a continuum surveillance

system using the One Health Approach. This book chapter discusses the pathways

J. Gonçalves (B

) · I. Díaz · A. Torres-Franco · E. Rodríguez · R. Muñoz · P. A. Garcia-Encina

Institute of Sustainable Processes, University of Valladolid, Dr. Mergelina s/n, 47011 Valladolid,

Spain

e-mail: zemcg5@gmail.com

P. A. Garcia-Encina

e-mail: pedroantonio.garcia@uva.es

Department of Chemical Engineering and Environmental Technology, University of Valladolid,

Dr. Mergelina s/n, 47011 Valladolid, Spain

P. G. d a S ilv a · J. R. Mesquita

ICBAS – Abel Salazar Institute of Biomedical Sciences, University of Porto, Porto, Portugal

LEPABE – Laboratory for Process Engineering, Environment, Biotechnology and Energy,

Faculty of Engineering, University of Porto, Porto, Portugal

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_22

461

462 J. Gonçalves et al.

and routes of transmission of waterborne pathogens in water bodies, water treat-

ment technologies and their role to control the spread of pathogens, as well as the

challenges to detect waterborne pathogens in the environment.

Keywords Emerging diseases ·Emerging pathogens ·Fecal indicators ·Human

viruses ·Pathogen removal ·Wastewater treatment ·Waterborne pathogens

1 Introduction

Sewage pollution is a major human health concern due to the known risk of

exposure to human waste and the public and regulatory will to reduce sewage

pollution. Pathogens are microorganisms that cause diseases. Pathogens that are

transmitted through water are called waterborne pathogens. When present in water

bodies, pathogens are capable of infecting humans by skin contact during bathing

and swimming, consumption of contaminated ﬁsh and shellﬁsh, and ingestion of

contaminated water (Gonçalves et al. 2018). The nature and abundance of waterborne

microbial communities and their composition are highly dependent on location and

time, as well as various environmental and anthropogenic factors. Most waterborne

pathogens of public health concern do not originate in natural water reservoirs but

are instead introduced by point sources of pollution such as sewage discharges and

non-point sources of pollution such as agricultural runoff, precipitation, and direct

pollution by livestock and wildlife efﬂuents (Fig. 1). Stormwater runoff generated

during rainfall and snow events contains pollutants that include pathogens. In the

USA, polluted stormwater runoff is a leading cause of contamination of water

bodies, both chemical and biological (USEPA 2002). The issue is not present only in

the USA, but is instead a global problem as surface water is polluted with pathogens

in most countries. A study in China analyzed samples from 16 surface waters and in

all of them waterborne pathogens were detected (Jin et al. 2018). A study conducted

in Finland analyzed 139 surface waters from 7 lakes and 15 rivers, and 51 of them

were contaminated with pathogens (Hörman et al. 2004). Similar studies with a

wide range of pathogens have been made worldwide and all report contamination

of pathogens in surface waters (Baker et al. 2021; Kongprajug et al. 2021).

The main source of pathogens entering waters that can be used for recreation and

ﬁshing is fecal contamination. Diseases caused by waterborne pathogens are a major

global burden, estimated to cause more than 2.2 million deaths each year. The most

common diseases include diarrhea, gastrointestinal diseases, and systemic diseases.

Of the 2.2 million deaths per year, about 1.4 million are children. It is estimated

that waterborne diseases have a global economic cost of nearly 12 billion US dollars

per year. Interestingly, the number of outbreaks underestimates the true incidence of

waterborne infections. Nonetheless, the number of waterborne diseases has declined

dramatically since the 1900s as a result of the widespread implementation of waste

treatment systems. Water treatment systems, both for drinking water and wastewater,

Microbial Contamination of Environmental Waters … 463

Fig. 1 Illustration of the main routes of contamination of water bodies with waterborne pathogens,

such as sewage discharges and non-point sources of pollution such as agricultural runoff, precipi-

tation, and direct pollution of streams and rivers by livestock and wildlife. Examples of activities

that pose risk to humans and animals by using contaminated waters are also illustrated (recreational

activities and consumption of ﬁsh)

have been and continue to be key players in the reduction of waterborne infections

(Mbanga et al. 2020; Shah 2020).

Over time, bacteria, viruses, protozoa, and helminths have evolved various mech-

anisms that facilitate their rapid responses to environmental changes. These rapid

responses may be key factors in their infectivity and pathogenicity under climate vari-

ability. The major groups of waterborne pathogens present in water bodies include

bacteria, viruses, protozoa, and helminths. In this book chapter, we will focus mainly

on two groups: Bacteria and Viruses.

Climate change and climate variability has the potential to increase even further the

burden of climate-sensitive diseases, in particular waterborne and foodborne, through

direct impacts such as in the case of extreme events (ﬂood and sea level rise). Indirect

impacts, such as temperature ad humidity, inﬂuence the process of pathogen growth

and survival. Other indirect impacts include agriculture, water resource management,

conﬂicts, displacements, among others. There are warnings that climate change is

likely to exacerbate health risks related to deﬁciencies in water supply, sanitation

and hygiene in many regions of the world. (Cissé 2019; Shah 2021).

The risks of climate change can be minimized though a continuum surveillance

system using the One Health Approach. This approach connects human, animal, and

environmental health by implementing programs, policies, legislation, and research

in which multiple sectors communicate and cooperate to achieve better public health.

464 J. Gonçalves et al.

Future actions to control the emergence and re-emergence of infectious diseases need

to consider the impacts of climate change and integrated interventions. The present

book chapter will discuss the pathways and routes of transmission of waterborne

pathogens in water bodies, water treatment technologies and their r ole to control the

spread of pathogens, as well as the challenges to detect waterborne viruses in the

environment.

2 Fecal Indicator Bacteria in Water Bodies and Emerging

Waterborne Pathogens

The detection of pathogens in contaminated water by fecal sources presents some

challenges, including the high cost of pathogen detection and the non-continuous

transport of pathogens in water bodies. In addition, the pathogens present in water are

diverse and usually present in low concentrations, and thus, their detection poses some

technical difﬁculties. To overcome the limitations, or as a result of the limitations,

water monitoring programs use indicators of f ecal contamination as a surrogate for

pathogen occurrence (Gonçalves 2018; Korajkic et al. 2018).

Fecal indicator bacteria (FIB), particularly E. coli and enterococci, are considered

good indicators of risk to human health in freshwater and untreated sewage. An impor-

tant reason for their wide acceptance as fecal indicators is because they are present in

high concentrations in the feces of mammals. Nevertheless, concentrations of fecal

indicator bacteria might not hold a good correlation with other pathogens once they

enter the aquatic environment due to a range of factors, such as dilution, water ﬂow

and survival rates in different environments (Ahmed et al. 2018; Balasubramanian

et al. 2016; Gonçalves 2018; Gonçalves et al. 2018). Results regarding the use of

faecal indicator bacteria and microbial source tracking (MST) markers to determine

the presence of pathogens in aquatic environments are not unanimous. Korajkic et al.

(2018) reviewed 73 publications that were generated over a 40-year period studying

the relationship between one or more faecal indicator bacteria with one or a group of

pathogens. Nearly half of the review publications did not include statistical analysis

and the rest were evenly split into those who observed statistically signiﬁcant corre-

lations and those who did not. The study concluded that FIB and MST markers might

be suitable as indicators of faecal pollution, but their relationship with waterborne

pathogens is not clear. Several factors might inﬂuence the relationship, such as the

frequency of detection, variable shedding rates, differential fate and transport charac-

teristics. In the case of waterborne viruses, the relationships are even less understood

(Gonçalves et al. 2018a). The efﬁciency of FIB is even less optimal where the domi-

nant sources of faecal pollution in water are low levels of diffuse pollution from a range

of non-human and human sources (Evans et al. 2019), including leaking from sewer

pipes and land runoff from wildlife and agricultural s ources. On the other hand, point

sources originate from municipal wastewater treatment plants (WWTPs) and gener-

ally result in high levels of FIB that are identiﬁed more quickly than with non-point

Microbial Contamination of Environmental Waters … 465

sources. The FIB originated from non-point sources exhibit lower concentrations but

might be persistent and include a mixture of recent and aged faecal inputs, making

difﬁcult to track the source of pollution (Teixeira et al. 2020).

The detection of fecal coliforms was one of the ﬁrst methods proposed to assess

water quality in 1966, although in this period, it was though that fecal coliforms can

only survive and replicate while in the homeostatic intestinal environment of an host

(Geldreich and Clarke 1966). Nowadays, it is known that fecal coliforms are not

exclusively found in the intestinal environment of hosts and research has been done

suggesting that they can growth or persist in water bodies (Teixeira et al. 2020).

3 Antimicrobial Resistance Genes as Emerging

Environmental Contaminants

Antibiotics are small molecules that can either inhibit or kill bacteria and are an essen-

tial therapy for bacterial infections. However, some bacteria grow and survive despite

antibiotic administration. This property, known as antimicrobial resistance, decreases

available treatment options in clinical settings, resulting in increased morbidity and

mortality (Boolchandani et al. 2019).

Antibioticresistancehasbeen historicallyregardedasa clinicalconcernandconsid-

ered to be exclusively related to the excessive use and misuse of antibiotics (Cacace

et al. 2019). In recent years, the fate of antimicrobial resistance genes (ARGs) released

to wastewaters has received increasing interest and there is a worldwide consensus

that raw municipal wastewater, treated efﬂuent and wastewater sludge are reservoirs

of ARGs and crucial hotspots for the evolution and spread of antibiotic resistance (Liu

et al. 2018). Antibiotics entering water and wastewater are insufﬁciently removed

and/or inactivated in treatment plants, causing a signiﬁcant fraction being released

directly into the environment in efﬂuent waters. A part of these are retained in the

sludge, which accumulates these compounds (Núñez-Delgado et al. 2019).

Direct contact between pathogenic bacteria and environmental ARG carriers,

as well as, the continuous selective pressure enforced by traces of antibiotics in

wastewaters make WWTPs an ideal hub for horizontal transfer of ARGs between

microorganisms. There are two main mechanisms that render wastewaters critical

hotspots for the evolution and spread of antimicrobial resistance: Selective pressure

and horizontal gene transfer (Fig. 2). Thus, the presence of antibiotics in wastewater

affects the natural selection of bacteria present by favouring one group of organisms

over another. Antibiotics cause a selective pressure by killing susceptible bacteria,

while allowing antibiotic-resistant bacteria to survive and multiply and proliferate

(Zhang et al. 2021).

In addition to the selective pressure, wastewaters are also hotspots for horizontal

gene transfer, which enable the spread of ARGs between different bacterial species.

Horizontal gene transfer is the movement of genetic information between organisms.

Several resistance genes evolved long ago in natural environments without inﬂuence,

466 J. Gonçalves et al.

Fig. 2 Mechanisms that aid the spread and evolution of ARGs in wastewaters. There are two main

mechanisms: Selective pressure and horizontal gene transfer. Horizontal gene transfer can occur by

transduction, conjugation, and transformation

but these genes are now rapidly spreading to and among human pathogens. As seen in

Fig. 2, there are three genetic mechanisms for horizontal gene transfer: transduction,

conjugation and transformation. Transduction of ARGs occurs when a bacteriophage

accidentally packages host ARG gene(s) within it capsid, either in place of the viral

genome or with the viral genome. The ARG or ARGs are then incorporated into

the genome of the next bacteria being infected by the bacteriophage (Harper et al.

2021). As opposed to transduction and transformation, conjugation requires direct

cell-to-cell contact via cell surface pilus or adhesions. Conjugation offers a better

protection from the environment and a more efﬁcient way to transfer genes from

a donor to a recipient bacterium. Once connected, the two bacteria will directly

contact and form a coupling bridge by which DNA is transferred from the donor

to the recipient. Transformation is the process in which extracellular free fragments

of DNA are incorporated by certain bacteria. For this to occur, several conditions

need to be in place, such as the availability of free DNA fragments and competent

recipient bacteria. In addition, the translocated DNA must be integrated into the

recipient genome or encircled as plasmid DNA (Thomas and Nielsen 2005).

With the rapid spread of ARGs among pathogenic bacteria and the consequent

increase in human and animal disease caused by these pathogens, ARGs have been

considered as emerging environmental contaminants by the World Health Organi-

zation (World Health Organization 2014). Due to the global occurrence of environ-

mental ARGs (Xia et al. 2017) and their potential acquisition by clinical pathogens

in environmental settings, they are an ongoing global concern (Hatosy and Martiny

2015). Studies have demonstrated that raw wastewater contains signiﬁcantly higher

amounts of ARGs than treated wastewater. However, its discharge to the receiving

Microbial Contamination of Environmental Waters … 467

water bodies signiﬁcantly increases ARG quantities in the environment (Jäger et al.

2018). ARG-carrying mobile genetic elements, such as conjugative plasmids, inte-

grative and conjugative elements, transposons and integrons, were also reported to

be present downstream of WWTPs (Marano and Cytryn 2017).

Bacterial communities found in wastewater are complex but show high resem-

blance at high taxonomic ranks. Raw wastewater is dominated by members of

the phyla Proteobacteria, Actinobacteria and Firmicutes, and classes such as

Bacilli, Clostridia, Bacteroides, Alpha-, Beta- and Gammaproteobacteria. Human

commensal microorganisms are the main bacterial representatives in WWTP inﬂuent

water. These groups include bacteria frequently reported as potential ARG carriers,

such as Enterobacteria, Enterococci, Staphylococci and Pseudomonas (Narciso-da-

Rocha and Manaia 2017). Among the screened ARGs and genetic elements, intI1 is

the most abundant in both efﬂuents and water environments and it has been proposed

as a potential marker of anthropogenic pollution (Gillings et al. 2015). The Infectious

Disease Society of America (IDSA) has identiﬁed six bacterial species, the ESKAPE

pathogens, as especially dangerous due to their patterns of antibiotic resistance:

Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinobacter

baumannii, Pseudomonas aeruginosa, and Enterobacter spp (Boucher et al. 2009).

Several different approaches have been used to describe the genetic backgrounds

and hosts of ARGs with different levels of success, including cultivation and isola-

tion (Paiva et al. 2017), high-throughput sequencing (Guo et al. 2017) and Emulsion,

Paired Isolation and Concatenation PCR (epicPCR) (Hultman et al. 2018). The isola-

tion of pure cultures is still considered the most important method to study antimi-

crobial resistance. Antibiotic susceptibility testing of pure isolates is relatively inex-

pensive and provides important information on resistant patterns needed for clinical

microbiology. Harmonized databases of clinical breakpoints, such as EUCAST, are

used worldwide to monitor antibiotic resistance with common epidemiological cut-

off values for resistance (ECOFFs). Despite its importance for clinical breakpoints,

ECOFFs cannot be directly applied to wastewaters since it requires a large number of

environmental isolates. Only a fraction of environmental bacteria can be grown under

laboratory conditions. Thus, culturing, and antimicrobial susceptibility testing has

limitations for a comprehensive study of resistance in wastewaters. The combination

of culture methods, susceptibility testing and molecular biology tools allows the iden-

tiﬁcation of previously unknown resistance determinants that may be either intrinsic

or acquired by mutations and horizontal gene transfer. Whole-genome sequencing is

a powerful tool to study the genetic environment of ARGs and enables the detection

of genes located on mobile genetic elements, capable of horizontal gene transfer.

Metagenomic analyses of urban wastewater have been suggested as an ethically

acceptable and feasible approach for continuous global surveillance and prediction

of antimicrobial resistance (Hendriksen et al. 2019). Metagenomics overcomes the

need for prior knowledge of resistance gene diversity, and it is not restricted to a

few chosen genes. Through the sequencing of the total DNA extracted from the

microbial community, the whole resistome can be predicted (Lakin et al. 2017).

On the other hand, the annotation of ARGs relies on known genes in antibiotic

resistance databases, such as Resfams, MEGARes and CARD. The big advantage is

468 J. Gonçalves et al.

that ARG databases are regularly updated with novel gene variants and sequences

can be re-analysed with the updated databases (Wallace et al. 2017).

Metagenomic analyses have been used in several environments to study antibi-

otic resistance (Fresia et al. 2019; Hendriksen et al. 2019). However, due to the

low number of ARGs in comparison to other functional genes, deep sequencing is

needed, which signiﬁcantly increases sequencing costs. The produced short-reads

provide only limited information and, by assembling short reads to longer overlap-

ping regions (contigs), information about phylogeny and genetic location of genes can

be inferred. Long-read sequencing technologies (also known as third-generation tech-

nologies), such as Paciﬁc Biosciences and Oxford Nanopore, were recently devel-

oped and differ from second-generation sequencing (NGS) (e.g. Illumina) in their

ability to generate long reads that can span long genomic regions, thus providing

an opportunity to link the ARGs with their ﬂanking regions at a much lower cost.

The higher per-read error rate of long-read sequencing, when compared with the

well-established NGS technologies, such as Illumina, is a limitation that has been

recently addressed by specially designed bioinformatic algorithms for error correc-

tion and high accuracy de novo assembly (van der Helm et al. 2017). Recent studies

on the resistome from wastewaters have shown that these bioinformatic tools are reli-

able and that Nanopore sequencing is largely consistent with that based on Illumina

sequencing platforms (Che et al. 2019).Nevertheless, the study and understanding of

environmental and public health implications of the resistome in wastewaters needs

to be further explored.

4 Human Enteric Viruses in Receiving Waters. Challenges

for Detection and Quantiﬁcation

Monitoring of human enteric viruses in the aquatic environment began in the 1940s

with the goal of monitoring microbial water quality and identifying major sources

of water pollution. Wastewater treatment technologies have improved signiﬁcantly

in the last decade, however, waterborne diseases continue to have public health and

socioeconomic implications and viruses are the main cause of waterborne infections.

A wide range of enteric viruses were reported to be the major agents of waterborne

outbreaks. Illness can occur upon exposure to few viral particles, therefore methods

to detect viruses from environmental samples should be sensitive enough to detect

very low concentrations (Zhang et al. 2019). The available diagnostic techniques have

limited the detection of enteric viruses from environmental samples and consequently

these pathogens have not been frequently identiﬁed as agents of waterborne diseases.

Most methods to detect human viruses in aquatic environments follow these steps:

(1) Concentration of the targeted virus from the water sample into a suitable volume;

(2) RNA or DNA extraction from the targeted organism; (3) Ampliﬁcation of the

genomic segment (or segments) chosen; (4) Detection and/or quantiﬁcation of the

ampliﬁed genomic segment.

Microbial Contamination of Environmental Waters … 469

4.1 Concentration Methods for Viruses in Aquatic

Environments

Due to the low concentrations of human viruses in natural waters, the implementation

of a concentration step is crucial for a successful detection (Gonçalves et al. 2018b).

This procedure should be simple and fast, have a high recovery rate, be suitable

for a wide range of enteric viruses, produce a small amount of concentrate, and be

inexpensive. The efﬁciency of the concentration method is highly dependent on the

quality of the water sampled. A wide range of methods for concentrating viruses from

environmental water samples have been tested with varying degrees of success. Most

methods exploit the physicochemical properties of viruses. Concentration techniques

include ultracentrifugation, ultraﬁltration, adsorption and elution-based methods, and

viral precipitation (Symonds and Breitbart 2015). Each of the available methods has

important advantages and limitations.

Ultracentrifugationusesforcesrangingfrom 100,000 ×g to 235,000×gtoconcen-

trateviruses from a givensample.It ismostlyusedasa secondaryconcentrationmethod

to adsorption-elution because it is difﬁcult to process large volumes of water samples.

However, it has been successfully used as a direct concentration method for viruses

from wastewater and heavily polluted recreational waters (Zheng et al. 2022).

Methods based on ultraﬁltration, such as vortex ﬁltration or tangential ﬂow ﬁltra-

tion, use size exclusion to concentrate viruses. Water and ions smaller than the pores of

the ﬁlter pass through the membrane, while microorganisms that are larger are concen-

trated in the retentate. Due to the generic size exclusion principle of ultraﬁltration, this

ﬁltrationtechniquecanbe used toconcentrateseveralwaterbornepathogens,including

viruses, bacteria and protozoa (Liu et al. 2012; Zheng et al. 2022).

Precipitation methods are typically used as a secondary step. In organic ﬂoccula-

tion, buffered beef extract is used to precipitate viruses from concentrated samples by

lowering the pH to 3.5. The precipitate formed is centrifuged and the pellet suspended

in sodium phosphate. A well validated technique using polyethylene glycol (PEG) has

been used on several viruses and various environmental waters (Hmaïed et al. 2016).

PEG precipitation consists of precipitation of viral particles by adding 0.5 M NaCl and

7% PEG to beef extract with constant stirring for 2 h at 4 ºC, followed by a centrifu-

gation step and resuspension of the pellet in Tris-buffered saline. Despite its use, beef

extracthasbeen reported tohaveinhibitory effectsinPCRassays and widelydiscarded.

Skimmedmilkﬂocculationwassuccessfully used as a one-stepprotocoltoconcentrate

viruses from coastal waters. The viruses adsorb to the pre-ﬂocculated milk proteins.

The ﬂocs sediment by gravity and the precipitate is dissolved in PBS and centrifuged.

Skimmed milk ﬂocculation was tested for human adenovirus (HAdV) in seawater

samples, with recoveries ranging from 42 to 52%. The protocol requires agitation of

samples for 8–10 h to successfully ensure adsorption of viruses by skimmed milk ﬂoc-

cules, followed by another 8–10 h of sedimentation (Calgua et al. 2008). Recently,

skimmed milk ﬂocculation has also been efﬁciently used for simultaneous concen-

tration of Human Adenovirus (HAdV), Bacteriophage MS2 (MS2), Rotavirus (RoV)

and Bovine Viral Diarrhea Virus (BVDV) as well as various bacteria and protozoa

470 J. Gonçalves et al.

(Gonzales-Gustavson et al. 2017). The method is reproducible, reliable, and inexpen-

sive, but has the disadvantage of requiring more than 16 h to process the samples. A

combination of ﬁltration through nitrocellulose membranes with skimmed milk ﬂoc-

culation was successfully used to concentrate HAdV and NoV from seawater samples.

In the same study, glass wool ﬁlters were used in combination with skimmed milk

ﬂocculation for freshwater samples. The processing times were signiﬁcantly reduced

(Wyn-Jones et al. 2011).

A wide range of ﬁlters and ﬁltration methods are traditionally used to concentrate

large volumes of water (up to thousands of liters), such as electropositive and elec-

tronegative cartridge ﬁlters, glass ﬁber ﬁlters, glass wool ﬁlters, vortex ﬂow ﬁltration,

tangential ﬂow ﬁltration and acid ﬂocculation (Wyn-Jones et al. 2011).

Adsorption-elution based methods are the most common procedure in environ-

mental virology and are based on adsorption of the viral particle to a surface followed

by elution. This approach is effective to recover viruses from water samples, but it is

highly dependent on water quality conditions such as pH, ionic strength and organic

content. There are two main ﬁlter types to adsorb virus: negatively charged ﬁlters and

positively charged ﬁlters. Electronegative ﬁlters rely on the manipulation of the water

sample to cause a positive surface charge of a viral particle. Enteric viruses are nega-

tively charged in waters and will only adsorb to a negatively charged membrane under

acid conditions or in the presence of Mg2+, or other multivalent cations. Compared to

electropositive ﬁlters, electronegative ﬁlters show higher virus recoveries in marine

waters and waters of high turbidity. The major weakness of electronegative ﬁlters

is the need for preconditioning the water sample or ﬁlter, prior to ﬁltration, as the

sample must be pH adjusted. It is also a complex and time-consuming procedure

(Haramoto et al. 2018).

Electropositive ﬁlters are based on the innate negative charge of virus particles

in environmental waters, and consequently they do not require preconditioning of

the water before ﬁltration. Viruses that are more electronegative adsorb better to

positively charged surfaces, which has an impact on the elution efﬁciency. To elute

viruses from the ﬁlters, solutions with various amino acids and complex proteina-

ceous solutions are commonly used. The most used solution consists of beef extract

at a pH of 9.0–9.5 and a concentration of 1.5–3.0% of 0.05–0.1 M glycine. 1MDS

Zetapor Virosporb (CUNO, Meriden, CN) is the most used electropositive ﬁlter and it

was designed by the U.S. Environmental Protection Agency to recover viruses from

drinking water (U.S. Environmental Protection Agency 1996). NanoCeram ﬁlters

(Argonide, Sanford, FL) are a good and cheaper alternative, as they have been efﬁ-

ciently used to concentrate various enteric viruses from tap and river water samples.

Most electropositive ﬁlters are easy t o use and are suitable for ﬁeld use. However

they are easily clogged and have low recovery rates for viruses in marine water due

to the presence of salt and the alkalinity of seawater, which cause low adsorption

of the viruses to the ﬁlter. Glass wool coated with mineral oil has hydrophobic and

electropositive sites on its surface and have been used as an adsorption material for

viral concentration. The suspension of viruses ﬂows through the pore space of the

glass wool membrane and the ﬁber surface attracts and retains negatively charged

Microbial Contamination of Environmental Waters … 471

viruses at a neutral pH and without addition of cations. The efﬁciency of concen-

tration by glass wool depends on the type of viruses, water pH and water matrix.

Glass powder, such as borosilicate glass beads (with a diameter of 100 ± 200 µm),

are a good adsorbent for viruses. Due to the formation of a ﬂuidized bed, the ﬁlter

matrix cannot be clogged as with glass wool materials. In both glass wool materials

and glass powder, the recoveries widely depend on the sample type. Magnetic beads

coated with antibodies or other molecules can be used to concentrate viruses. Viruses

bind to the beads and are magnetically separated from the sample. Viruses are eluted

using an appropriate buffer (Haramoto et al. 2018).

4.2 Quantiﬁcation Methods for Viruses in Water

Environments

Available detection techniques have limited the detection of enteric viruses from

environmental samples and, consequently, these pathogens have frequently not been

identiﬁed as agents of waterborne diseases. Culture based methods can determine the

concentration of infectious viruses, however, these methods have a high associated

costs, require intensive laboratory work, give late results and fail to detect many

waterborne viruses, as for example NoV that cannot yet be cultivated (Symonds and

Breitbart 2015).

Transmission electron microscopy (TEM) is widely used to detect enteric viruses

in public health laboratories, but this method is subjective, requires specialized

personnel, is laborious and time consuming, and exhibits a low sensitivity. For

these reasons it has been largely dismissed for water samples. Immunological tests,

such as enzyme immunoassay (EIA), radioimmunoassay (RIA) and enzyme-linked

immunosorbent assay (ELISA), have been used to detect enteric viruses and many

have commercially available kits tested for the main enteric viruses. Immunological

tests are widely used in clinical samples, but due to their poor analytical sensitivity,

these tests are not good enough for environmental samples. The detection of enteric

viruses in surface waters is most frequently based on molecular technologies, due

to their highest sensitivity, speciﬁcity, and reduced processing times. However, they

have the disadvantage that infectivity cannot be conﬁrmed.

Molecular detection of viruses requires the extraction of genomic nucleic acids

(either DNA or RNA) followed by virus-speciﬁc DNA or RNA target detection using

several different approaches. Extraction of viral DNA or RNA can be done by a wide

range of commercially available kits that are reliable, offer high reproducibility and

are easy to use. Proteinase K treatment followed by phenol chloroform extraction and

ethanol precipitation, sonication and heat treatment are also widely used. Automated

methods for nucleic acids extraction have been developed in recent years and have

been successfully applied to analyze viruses in water samples (Wang et al. 2020).

472 J. Gonçalves et al.

The ﬁrst molecular technique used to detect viruses was PCR, invented by Kary

Mullis et al. in 1986 (Mullis et al. 1986). PCR detection is based on the ampli-

ﬁcation of a virus-speciﬁc section of viral genome using a pair of short oligonu-

cleotides (primers) that guide the PCR polymerase to amplify the target sequence

of the viral genome. In the case of RNA viruses, reverse transcription (RT) of viral

RNA to complementary DNA (cDNA) is required before the PCR. PCR products

are most commonly visualized by agarose gel electrophoresis and results can be

further conﬁrmed by hybridization of the PCR product using an internal oligonu-

cleotide probe or by sequencing the DNA product (Kojima et al. 2002). Semi-nested

or nested PCR use a second internal primer or primer set, which allows to improve

the sensitivity and speciﬁcity. It can also be used as a conﬁrmation step.

In the nineties, real-time PCR (qPCR) was developed by Heid and collaborators

(Heid et al. 1996) and it is nowadays widely used in research and diagnostics. qPCR

shares the same principle of target ampliﬁcation as PCR, but it combines primer

ampliﬁcation with detection of the ampliﬁed product in a single reaction mix in real-

time. By using ﬂuorescent labelled primers or an oligonucleotide probe, in addition

to the primer set, the signal is detected via intercalating ﬂuorescent dyes that bind to

the ampliﬁed PCR products. In the same way as PCR, only DNA can be ampliﬁed.

In order to amplify RNA, a reverse transcription of the RNA to cDNA needs to

be performed ﬁrst, and the process is called RT-qPCR. Recent qPCR approaches

allow very speciﬁc detection of one or more targets in one reaction. One of its

best properties is that qPCR allows the quantiﬁcation of the target template initially

present in the sample. qPCR is the most widely used molecular biology technique due

to its robustness, speed, miniaturization of the reactions, and excellent automation

possibilities. It is well established in scientiﬁc research, industrial development and

it has become an essential tool of service companies, quality control applications

and diagnostics. qPCR, as well as PCR and many enzymatic reactions, is prone

to inhibition. Environmental samples often contain inhibitory compounds that in

extreme situations can lead to false negative results. By using low sample volumes

or by diluting the samples this effect can be minimized.

Another important variant of PCR is digital PCR (dPCR). In dPCR, a single bulk

PCR reaction is partitioned into thousands or in some cases millions of nanoliter or

picolitre reactions. Partitioning of the reaction was only possible by recent advances

in nanoﬂuidics and emulsion chemistries (droplet dPCR or ddPCR). The partitioning

of the sample struggles to assure that each individual partition only contains one target

molecule or none. Quantiﬁcation is obtained by counting partitions with ampliﬁed

products and those without ampliﬁed products. However, since some partitions may

have multiple ampliﬁed products, a Poisson correction coefﬁcient is applied to the

positive partitions for compensation. The advantages of dPCR in relation to qPCR are

the precision of nucleic acids quantiﬁcation, no need for standard curve, high repro-

ducibility, and lower susceptibility for environmental PCR inhibition (very small

reaction volume). The costs of dPCR are high but the trend is to decrease. More and

more equipment are available in the market, which indicates that dPCR can become

popular as a tool to monitor water quality.

Microbial Contamination of Environmental Waters … 473

Isothermal methods, which require a single constant temperature for ampliﬁcation,

can also be used for sensitive detection of nucleic acids. While in PCR reactions,

temperature changes are required, isothermal methods, such as nucleic acid sequence-

based ampliﬁcation (NASBA) and loop mediated isothermal ampliﬁcation (LAMP)

perform ampliﬁcation without temperature changes and thus require less complicated

and cheaper instruments. Some of these methods involve different enzymes than DNA

polymerase or in some cases several enzymes. The popularity of NASBA and LAMP

has been increasing as alternatives to PCR method (Becherer et al. 2020).

Next Generation Sequencing (NGS) offers the sequencing of a massive quantity

of short DNA fragments in a single sequencing reaction that produces base pair

reads covering an enormous amount of information in just a few days. NGS can be

used to conﬁrm positive PCR results or as a powerful detection tool. This technique

overcomes the limitation of target-based methods, such as PCR, qPCR and isothermal

methods, which require prior knowledge of the sequences of at least part of the viral

genomes. Additionally, NGS provides an exceptional insight into viral diversity by

viral metagenomics studies. Most NGS platforms are still very costly, but there

are a lot of service providers available. The processing and interpretation of the

results obtained by NGS rely heavily on bioinformatic tools, which requires skilled

personnel (Vinkšel et al. 2021). The development of sensitive detection methods for

enteric viruses is a challenging task. The standardization and validation of protocols

is an imperative requirement to implement molecular techniques in either clinical or

environmental ﬁelds (Cassedy et al. 2021).

5 Wastewater Treatment Technologies to Minimize

the Spread of Waterborne Pathogens

In wastewater treatment Plants (WWTPs) pathogens are removed from wastew-

ater and concentrated in the form of sewage sludge during the primary treatment,

common in most WWTP of any size. Preliminary and primary treatment are aimed at

removing larger materials and solids and includes sorting units and primary settlers

where coagulants or polymers may be applied to enhance sedimentation. In medium

and large-size WWTP, the biological process during secondary treatment converts

approximately 40% of the available organic matter into CO2 and H2O and the rest

accumulates and is used for the growth of biological sludge, where viable forms of

pathogens can accumulate. The removal of soluble and particulate organic matter

occurs during secondary treatment, typically in Activated Sludge (AS) reactors and

their variants, including bioﬁlm and membrane systems. Several variants of AS tech-

nology can achieve high removal efﬁciencies of nitrogen and phosphorus, which are

the main focus of tertiary units. Additional treatment may be applied at the WWTP

(tertiary treatment) before water is discharged; depending on the ﬁnal use of water,

disinfection and membrane processes are sometimes applied to further reduce the

concentration of pathogens. Bio trickling ﬁlters, pond and membrane bioreactors

474 J. Gonçalves et al.

Fig. 3 Main wastewater treatment steps commonly found in wastewater treatment plants

(MBRs) are widely applied tertiary treatment technologies. In facilities in which

the removal of pathogens or speciﬁc pollutants are required, advanced treatment

processes are applied and include UV radiation, ozonisation, activated carbon, chlo-

rination among others. Further details of pathogens an virus removal reported in

recent literature are provide for each treatment step, as well as the removal of antimi-

crobial resistant bacteria (ARB) and ARG, for which detection methods are currently

being developed and standardized (Miłobedzka et al. 2022). Figure 3 summarizes

the main treatment steps in common WWTPs.

5.1 Preliminary Treatment

Preliminary treatment is used to remove ﬂoating organic material (called scum) and

coarse solids dragged in the sewer system that would interfere with mechanical equip-

ment, reducing the suspended solids load for subsequent treatment processes. This

step comprises gravity sedimentation of settleable solids in the screened wastewater

and scums, and may involve the use of coagulants to enhance the process. Prelimi-

nary treatment has a limited effect on the removal of pathogen from the liquid stream,

as removal by settling is not expected. However, pathogens may be removed when

attached to particles, typically reaching removals from 0 to 1 Log10 units of coliforms,

norovirus and other fecal indicators. Chemically enhanced primary treatment and

advanced primary treatment may increase removals during primary sedimentation

from 1 to 2 Log10 for virus, bacteria, and protozoa and 1–3 Log10 for helminth eggs.

Screenings, grit, and sludge will contain high concentrations of pathogens and must

be safely treated and/or disposed to protect public health.

Microbial Contamination of Environmental Waters … 475

5.2 Secondary Treatment

Secondary treatment typically comprises aerobic or anaerobic processes f or biolog-

ical removal of carbon and nitrogen. Other technologies include systems usually

applied for decentralized solutions, such as septic tanks, pond systems, wetlands,

and sand ﬁlters.

Activated sludge/MBR: The most common technology is Activated Sludge

System, which in its most basic conﬁguration comprises an aerated reactor followed

by a secondary settler conventional Activated Sludge (CAS). Advanced conﬁgura-

tions include the substitution of the secondary settler by MBRs for the enhancement of

biomass separation or process modiﬁcations to achieve nitriﬁcation–denitriﬁcation

and enhanced biological phosphorus removal (EBPR). Together, primary settling

followed by CAS or MBR have a certain disinfection capacity due to the natural

predation, decay or adsorption onto the biological ﬂocs of pathogens, subsequently

removed by the solid’s separation processes. Secondary treatment usually increases

in about 1 Log10 unit the reduction achieved during primary sedimentation, with

removals of coliforms and indicators in CAS ranging from 1.0 to 1.6 Log10 units

(Lucena et al. 2004). Similarly, Alcalde et al. (2012) reported removals ranging

1.6 to 1.8 Log10 units for E. coli, Fecal enterococci, somatic bacteriophages, and

spores of sulphite-reducing clostridia. Furthermore, MBR systems can substantially

increase pathogen removal during secondary treatment (Alcalde et al. 2012; Lucena

et al. 2004). In this sense, De Luca et al. (2013) reported 2.7 and 1.7 Log10 higher

reductions of somatic coliphages and F-RNA speciﬁc bacteriophages compared with

conventional activated sludge process (De Luca et al. 2013), whereas Zhang and

Farahbakhsh (2007) reported a complete removal of fecal coliforms and up to 5.8 logs

removal of coliphages was observed in a pilot MBR system (Zhang and Farahbakhsh

2007). The use of aerobic granular sludge also showed higher removals than CAS

systems, with increases from 1.3 to 2.1 Log10 removals for F-speciﬁc RNA bacte-

riophages and 1.1–2.3 Log10 removals for E. coli, Enterococci, and Thermotolerant

coliforms (Barrios-Hernández et al. 2020).

Other technologies: Other technologies for secondary treatment include the use

of anaerobic processes or trickling ﬁlters (TF). Although their total removal is not

achieved, a reduction of 2.34 and 1.36 Log10 in the concentration of E. coli was

produced along the water lines, respectively, in a systems applying trickling ﬁlters

(TF) followed by secondary settlers (Marín et al. 2015).

For anaerobic technologies, although signiﬁcant contributions to coliforms

removal are not expected, Tandukar et al. (2007) reported total and fecal coliform

removals of 4 and 3.7 Log10 units, respectively, in a upﬂow anerobic sludge blanket

reactor followed by down-ﬂow hanging sponge unit (Tandukar et al. 2007). In addi-

tion, decentralized systems such as septic tanks, sand ﬁlters, wetlands and pond

systems have shown potential to remove substantial quantities of pathogens, with

HRT being the most signiﬁcant factor that can be optimized. Particularly, high

removals in ﬁltration units were favoured by ﬁne grain sizes of ﬁlter media, uniform

water distribution methods and low hydraulic loading rates (Wang et al. 2021a, b).

476 J. Gonçalves et al.

5.3 Tertiary and Advanced Systems

In a CAS systems followed by nitrifying rotating biological contactors (RBCs), sand

ﬁltration and chlorination/dechlorination, Zang and Farahbakhsh (2007) recorded

up to 5.7 Log10 removal of coliforms and 5.5 Log10 of coliphages were observed

in the conventional treatment process with advanced tertiary treatment (Zhang and

Farahbakhsh 2007). The addition of chemical coagulants appeared to improve the

efﬁcacy of primary and secondary treatment for microorganism removal. Other

technologies of tertiary/advanced treatment may include electro-Fenton (EF) with

continuous hydroxyl radical generation, which can achieve up to 4 Log10 units in

coliforms/E. coli inactivation (Wang et al. 2021a, b). Similarly, a water reclamation

system treating efﬂuent of a CAS WWTP in Spain, and comprising coagulation-

ﬂocculation, settling, sand ﬁltration and chlorination achieved additional reductions

of 3.4 2.5, 0.9 and 0.6 Log10 units, respectively (Alcalde et al. 2012).

ARG and ARB, antibiotic resistant E. coli and several ARG providing resistance

can be removed from discharged water in WWTP with primary and secondary treat-

ment or secondary treatment followed by disinfection; 2 Log10 removal was found

for E. coli and 1.5–2 Log10 for other resistant bacteria (Proia et al. 2018b). However,

most of these resistant bacteria and resistance genes are concentrated in sewage

sludge and can potentially re-enter water bodies if reused for land application or not

properly managed.

Techniques of virus surveillance in wastewater are expensive and due to limita-

tions in their cultivation methods, only a few enteric viruses are regulated by standards

for virus control. During the COVID-19 pandemic, environmental risks of viruses

gained more attention and several studies focused on the development of indicators

of enteric viruses in wastewater samples as their relative abundance is expected to

be higher than in other aquatic environments. In this regard, the concentration of

norovirus and coliphages was determined in reclaimed water from two WWTP in

Spain, revealing a correlation between the concentration of each of them only at high

concentrations (Truchado et al. 2021). On the contrary, JC and BK polyomaviruses

were good indicators of total enteric viruses and the Log10 reduction of their concen-

tration during wastewater treatment was lower than that of faecal indicator bacteria

(Tandukar et al. 2021) indicating the necessity of further application of advanced

and tertiary processes.

5.4 Sewage Sludge Treatment and Disposal

The use of sludge for land application is common throughout the world. Some coun-

tries prohibit its use for this purpose and some others require sludge to be treated and

converted into a biosolid, which depending on its physic and chemical characteristics

and pathogen content, that could be applied to soil. In this regard, the Standards for

Microbial Contamination of Environmental Waters … 477

the Use or Disposal of Sewage Sludge (2003) published by the United States Envi-

ronmental Protection Agency (40 CFR Part 503) (Environmental Protection Agency

2003) and the Proposal for a Directive of the European parliament and of the Council

on the spreading of sludge on land (2003) are a reference and limits the concentra-

tions of faecal coliforms, Salmonella sp, enteric viruses, Clostridium perfringens

and viable helminth ova in biosolids to be used for land application. Depending

on the concentration of those, biosolids are classiﬁed and the lowest concentration

is required for allowing its use for fertilization. Those biosolids that do not meet

requirements cannot be used for land application and are commonly incinerated.

In addition, the presence of regulated indicators does not correlate with the pres-

ence of other pathogens (Fijalkowski et al. 2017), such as those carrying ARGs and

thus antibiotic resistant. Despite regulated concentrations of some pathogen, detailed

studies on the effect of pathogens transmissibility by land application are scarce as

well as its release to water bodies by drainage. In this regard, long-term studies

on trial ﬁelds revealed no association between the relative abundance of potentially

pathogenic bacteria and the seasonal application of stabilized (under thermophilic

conditions) biosolids; however, an increased relative abundance of sul1 and tetW was

signiﬁcantly related to biosolid application (Stiborova et al. 2021).

Sludge treatment in medium and large WWTP is often performed by anaer-

obic digestion of the sewage sludge to recover energy in the form of biogas. Many

pathogens are inactivated during anaerobic digestion, particularly at the thermophilic

range (~3 Log10 reduction) and at long retention times (Zhao and Liu 2019)but the

mechanisms of pathogenic inactivation are not well reported (Li et al. 2022). Ther-

mophilic AD can achieve t he requirements for land application; however, recent

studies on pathogens not covered by regulations revealed the presence of several

parasites such as Cryptosporidium spp. Entamoeba spp. Giardia duodenalis (Benito

et al. 2020) and some controversy is found regarding ARG removal at different

temperatures during AD; because some pathogens are heat-resistant and the abun-

dance of total ARG was higher at 55ºC than al lower temperatures (Huang et al.

2019).

Virus infectivity has been reported to decrease by more than 90% during anaerobic

digestion of sludge. However, biosolids obtained from AD contain higher levels of

somatic coliphages than traditional bacterial indicators which are more persistent in

solids and may be a complementary indicator of pathogenic viruses (Martín-Díaz

et al. 2020) since at high concentration of coliphages and enteric viruses a positive

correlation could be established (Truchado et al. 2021).

Thermal pre-treatments of sludge are popular for increasing pathogenic reduction

when AD does not meet quality criteria, due to the high temperatures employed (up

to 160–180 ºC) and the fact that some of the energy required can be recovered as

additional biogas. Although the process has been traditionally employed as a pre-

treatment to increase energy recovery, recent studies show an increased potential of

pathogens, ARB and ARG inactivation when performed as a post-treatment because

bacterial regrowth during AD of solid fraction is prevented by digesting only the

liquid hydrolysate (Cai et al. 2021). Anyhow, temperature during AD and thermal

treatments is the dominant factor for the inactivation of pathogens in sewage sludge

478 J. Gonçalves et al.

management systems (Li et al. 2022). Thermal hydrolysis achieved a very high

removal of E. coli and coliphages, in contrast to the sonication process and also a

positive correlation between coliphages and enteric viruses; according to the reported

ratios between them, a tentative concentration of < 104 PFU/g DW was proposed as

quality limit for land application (Levantesi et al. 2015).

6 Conclusion

The World Health Organization (WHO) estimates that, in 2012, 12.6 million deaths

in the world were attributable to environmental issues, which represents 23% of

all deaths. Among the environmental factors, food and water contaminations are

of particular relevance in the transmission of diseases (WHO 2018). Waterborne

diseases include many different types of infections that are transmitted through

water. These include pathogens from a range of taxa (viruses, bacteria, protozoa

and helminths).

Waterborne pathogens are a public health problem worldwide and are still a major

cause of serious illness and mortality. Pathogen indicators need to be constantly

improved as many emerging pathogens cause waterborne diseases and outbreaks.

Increasing demands are being placed on the water treatment industry to reduce

the risk of disease from both chemicals and microorganisms. To meet these demands,

we need a better understanding of microbial resistance at the molecular level, and

we need to develop faster methods to evaluate treatment efﬁcacy.

Climate change and climate variability has the potential to increase even further the

burden of climate-sensitive diseases. The risks can be minimized though a continuum

surveillance system using the One Health Approach. This approach connects human,

animal, and environmental health by implementing programs, policies, legislation,

and research in which multiple sectors communicate and cooperate to achieve better

public health.

References

Ahmed W, Hamilton KA, Lobos A, Hughes B, Staley C, Sadowsky MJ, Harwood VJ (2018) Quan-

titative microbial risk assessment of microbial source tracking markers in recreational water

contaminated with fresh untreated and secondary treated sewage. Environ Int 117:243–249.

https://doi.org/10.1016/j.envint.2018.05.012

Alcalde L, Folch M, Tapias JC (2012) Removal and relationships of microbial indicators in a water

treatment and reclamation facility. J Water Health 10:549–556. https://doi.org/10.2166/wh.201

2.213

Baker CA, Almeida G, Lee JA, Gibson KE (2021) Pathogen and surrogate survival in relation

to fecal indicator bacteria in freshwater mesocosms. Appl Environ Microbiol 87:e00558-e621.

https://doi.org/10.1128/AEM.00558-21

Microbial Contamination of Environmental Waters … 479

Balasubramanian MN, Raˇcki N, Gonçalves J, Kovaˇc K, Žnidariˇc MT, Turk V, Ravnikar M, Gutiérrez-

Aguirre I (2016) Enhanced detection of pathogenic enteric viruses in coastal marine environ-

ment by concentration using methacrylate monolithic chromatographic supports paired with

quantitative PCR. Water Res 106:405–414. https://doi.org/10.1016/j.watres.2016.10.020

Barrios-Hernández ML, Pronk M, Garcia H, Boersma A, Brdjanovic D, van Loosdrecht MCM,

Hooijmans CM (2020) Removal of bacterial and viral indicator organisms in full-scale aerobic

granular sludge and conventional activated sludge systems. Water Res. X 6:100040. https://doi.

org/10.1016/j.wroa.2019.100040

Becherer L, Borst N, Bakheit M, Frischmann S, Zengerle R, von Stetten F (2020) Loop-mediated

isothermal ampliﬁcation (LAMP) – review and classiﬁcation of methods for sequence-speciﬁc

detection. Anal Methods 12:717–746. https://doi.org/10.1039/C9AY02246E

Benito M, Menacho C, Chueca P, Ormad MP, Goñi P (2020) Seeking the reuse of efﬂuents and sludge

from conventional wastewater treatment plants: analysis of the presence of intestinal protozoa and

nematode eggs. J Environ Manage 261:110268. https://doi.org/10.1016/j.jenvman.2020.110268

Boolchandani M, D’Souza AW, Dantas G (2019) Sequencing-based methods and resources to study

antimicrobial resistance. Nat Rev Genet 20:356–370. https://doi.org/10.1038/s41576-019-0108-4

Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, Scheld M, Spellberg B,

Bartlett J (2009) Bad bugs, no drugs: no ESKAPE! an update from the infectious diseases society

of America. Clin. Infect Dis off Publ Infect Dis Soc Am 48:1–12. https://doi.org/10.1086/595011

Cacace D, Fatta-Kassinos D, Manaia CM, Cytryn E, Kreuzinger N, Rizzo L, Karaolia P, Schwartz

T, Alexander J, Merlin C, Garelick H, Schmitt H, de Vries D, Schwermer CU, Meric S, Ozkal CB,

Pons M-N, Kneis D, Berendonk TU (2019) Antibiotic resistance genes in treated wastewater and

in the receiving water bodies: a pan-European survey of urban settings. Water Res 162:320–330.

https://doi.org/10.1016/j.watres.2019.06.039

Cai C, Hu C, Yang W, Hua Y, Li L, Yang D, Dai X (2021) Sustainable disposal of excess sludge:

post-thermal hydrolysis for anaerobically digested sludge. J Clean Prod 321:128893. https://doi.

org/10.1016/j.jclepro.2021.128893

Calgua B, Mengewein A, Grunert A, Boﬁll-Mas S, Clemente-Casares P, Hundesa A, Wyn-Jones AP,

López-Pila JM, Girones R (2008) Development and application of a one-step low cost procedure

to concentrate viruses from seawater samples. J Virol Methods 153:79–83. https://doi.org/10.

1016/j.jviromet.2008.08.003

Cassedy A, Parle-McDermott A, O’Kennedy R (2021) Virus detection: a review of the current and

emerging molecular and immunological methods. Front Mol Biosci 8:637559. https://doi.org/10.

3389/fmolb.2021.637559

Che Y, Xia Y, Liu L, Li A-D, Yang Y, Zhang T (2019) Mobile antibiotic resistome in wastewater

treatment plants revealed by Nanopore metagenomic sequencing. Microbiome 7:44. https://doi.

org/10.1186/s40168-019-0663-0

Cissé G (2019) Food-borne and water-borne diseases under climate change in low- and middle-

income countries: further efforts needed for reducing environmental health exposure risks. Acta

Trop 194:181–188. https://doi.org/10.1016/j.actatropica.2019.03.012

De Luca G, Sacchetti R, Leoni E, Zanetti F (2013) Removal of indicator bacteriophages from

municipal wastewater by a full-scale membrane bioreactor and a conventional activated sludge

process: Implications to water reuse. Bioresour Technol 129:526–531. https://doi.org/10.1016/j.

biortech.2012.11.113

Environmental Protection Agency (2003) Standards for the use or disposal of sewage sludge; ﬁnal

agency response to the national research council report on biosolids applied to land and the results

of EPA’s review of existing sewage sludge regulations

Evans AE, Mateo-Sagasta J, Qadir M, Boelee E, Ippolito A (2019) Agricultural water pollution:

key knowledge gaps and research needs. Curr Opin Environ Sustain. Environ Change Assess

36:20–27. https://doi.org/10.1016/j.cosust.2018.10.003

Fijalkowski K, Rorat A, Grobelak A, Kacprzak MJ (2017) The presence of contaminations in sewage

sludge – the current situation. J Environ Manage 203:1126–1136. https://doi.org/10.1016/j.jen

vman.2017.05.068

480 J. Gonçalves et al.

Fresia P, Antelo V, Salazar C, Giménez M, D’Alessandro B, Afshinnekoo E, Mason C, Gonnet GH,

Iraola G (2019) Urban metagenomics uncover antibiotic resistance reservoirs in coastal beach

and sewage waters. Microbiome 7:35. https://doi.org/10.1186/s40168-019-0648-z

Geldreich EE, Clarke NA (1966) Bacterial pollution indicators in the intestinal tract of freshwater

ﬁsh. Appl Microbiol 14:429–437. https://doi.org/10.1128/am.14.3.429-437.1966

Gillings MR, Gaze WH, Pruden A, Smalla K, Tiedje JM, Zhu Y-G (2015) Using the class 1 integron-

integrase gene as a proxy for anthropogenic pollution. ISME J 9:1269–1279. https://doi.org/10.

1038/ismej.2014.226

Gonçalves J, Gutiérrez-Aguirre I, Balasubramanian MN, Zagorš ˇcak M, Ravnikar M, Turk V (2018)

Surveillance of human enteric viruses in coastal waters using concentration with methacrylate

monolithic supports prior to detection by RT-qPCR. Mar Pollut Bull 128:307–317. https://doi.

org/10.1016/j.marpolbul.2018.01.040

Gonçalves J (2018) Distribution of enteric viruses in the Gulf of Trieste and their interactions with

environmental and biological parameters dissertation. https://doi.org/10.13140/RG.2.2.31906.

76482

Gonzales-Gustavson E, Cárdenas-Youngs Y, Calvo M, da Silva MFM, Hundesa A, Amorós I,

Moreno Y, Moreno-Mesonero L, Rosell R, Ganges L, Araujo R, Girones R (2017) Characteriza-

tion of the efﬁciency and uncertainty of skimmed milk ﬂocculation for the simultaneous concen-

tration and quantiﬁcation of water-borne viruses, bacteria and protozoa. J Microbiol Methods

134:46–53. https://doi.org/10.1016/j.mimet.2017.01.006

Guo J, Li J, Chen H, Bond PL, Yuan Z (2017) Metagenomic analysis reveals wastewater treatment

plants as hotspots of antibiotic resistance genes and mobile genetic elements. Water Res 123:468–

478. https://doi.org/10.1016/j.watres.2017.07.002

Haramoto E, Kitajima M, Hata A, Torrey JR, Masago Y, Sano D, Katayama H (2018) A review on

recent progress in the detection methods and prevalence of human enteric viruses in water. Water

Res 135:168–186. https://doi.org/10.1016/j.watres.2018.02.004

Harper DR, Abedon ST, Burrowes BH, McConville ML (eds) (2021) Bacteriophages: biology,

technology, therapy, 1st edn. Springer (2021)

Hatosy SM, Martiny AC (2015) The ocean as a global reservoir of antibiotic resistance genes. Appl

Environ Microbiol 81:7593–7599. https://doi.org/10.1128/AEM.00736-15

Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res

6:986–994. https://doi.org/10.1101/gr.6.10.986

Hendriksen RS, Munk P, Njage P, van Bunnik B, McNally L, Lukjancenko O, Röder T, Nieuwen-

huijse D, Pedersen SK, Kjeldgaard J, Kaas RS, Clausen PTLC, Vogt JK, Leekitcharoenphon P,

van de Schans MGM, Zuidema T, de Roda Husman AM, Rasmussen S, Petersen B, Surveil-

lance GS, project consortium, Amid, C, Cochrane, G, Sicheritz-Ponten, T, Schmitt, H, Alvarez,

J.R.M, Aidara-Kane, A, Pamp, S.J, Lund, O, Hald, T, Woolhouse, M, Koopmans, M.P, Vigre,

H, Petersen, T.N, Aarestrup, F.M. (2019) Global monitoring of antimicrobial resistance based

on metagenomics analyses of urban sewage. Nat Commun 10:1124. https://doi.org/10.1038/s41

467-019-08853-3

Hmaïed F, Jebri S, Saavedra MER, Yahya M, Amri I, Lucena F, Hamdi M (2016) Comparison of two

concentration methods for the molecular detection of enteroviruses in raw and treated sewage.

Curr Microbiol 72:12–18. https://doi.org/10.1007/s00284-015-0909-4

Hörman A, Rimhanen-Finne R, Maunula L, Bonsdorff C-H, von Torvela N, Heikinheimo A,

Hänninen M-L (2004) Campylobacter spp, Giardia spp, Cryptosporidium spp, noroviruses,

and indicator organisms in surface water in Southwestern Finland 2000–2001. Appl Environ

Microbiol. https://doi.org/10.1128/AEM.70.1.87-95.2004

Huang X, Zheng J, Tian S, Liu C, Liu L, Wei L, Fan H, Zhang T, Wang L, Zhu G, Xu K (2019)

Higher temperatures do not always achieve better antibiotic resistance gene removal in anaerobic

digestion of swine manure. Appl Environ Microbiol 85:e02878-e2918. https://doi.org/10.1128/

AEM.02878-18

Microbial Contamination of Environmental Waters … 481

Hultman J, Tamminen M, Pärnänen K, Cairns J, Karkman A, Virta M (2018) Host range of antibiotic

resistance genes in wastewater treatment plant inﬂuent and efﬂuent. FEMS Microbiol Ecol 94.

https://doi.org/10.1093/femsec/ﬁy038

Jäger T, Hembach N, Elpers C, Wieland A, Alexander J, Hiller C, Krauter G, Schwartz T (2018)

Reduction of antibiotic resistant bacteria during conventional and advanced wastewater treatment,

and the disseminated loads released to the environment. Front Microbiol 9. https://doi.org/10.

3389/fmicb.2018.02599

Jin D, Kong X, Cui B, Jin S, Xie Y, Wang X, Deng Y (2018) Bacterial communities and potential

waterborne pathogens within the typical urban surface waters. Sci Rep 8:13368. https://doi.org/

10.1038/s41598-018-31706-w

Kojima S, Kageyama T, Fukushi S, Hoshino F, Shinohara M, Uchida K, Natori K, Takeda N,

Katayama K (2002) Genogroup-speciﬁc PCR primers for detection of Norwalk-like viruses. J

Virol Methods. https://doi.org/10.1016/S0166-0934(01)00404-9

Kongprajug A, Chyerochana N, Rattanakul S, Denpetkul T, Sangkaew W, Somnark P, Patara-

pongsant Y, Tomyim K, Sresung M, Mongkolsuk S, Sirikanchana K (2021) Integrated analyses

of fecal indicator bacteria, microbial source tracking markers, and pathogens for Southeast Asian

beach water quality assessment. Water Res 203:117479. https://doi.org/10.1016/j.watres.2021.

117479

Korajkic A, McMinn BR, Harwood VJ (2018) Relationships between microbial indicators and

pathogens in recreational water settings. Int J Environ Res Public Health 15:2842. https://doi.org/

10.3390/ijerph15122842

Lakin SM, Dean C, Noyes NR, Dettenwanger A, Ross AS, Doster E, Rovira P, Abdo Z, Jones KL,

Ruiz J, Belk KE, Morley PS, Boucher C (2017) MEGARes: an antimicrobial resistance database

for high throughput sequencing. Nucleic Acids Res 45:D574–D580. https://doi.org/10.1093/nar/

gkw1009

Levantesi C, Beimfohr C, Blanch AR, Carducci A, Gianico A, Lucena F, Tomei MC, Mininni G

(2015) Hygienization performances of innovative sludge treatment solutions to assure safe land

spreading. Environ Sci Pollut Res Int 22:7237–7247. https://doi.org/10.1007/s11356-014-3572-6

Li M, Song G, Liu R, Huang X, Liu H (2022) Inactivation and risk control of pathogenic microor-

ganisms in municipal sludge treatment: a review. Front Environ Sci Eng. https://doi.org/10.1007/

s11783-021-1504-5

Liu P, Hill VR, Hahn D, Johnson TB, Pan Y, Jothikumar N, Moe CL (2012) Hollow-ﬁber ultra-

ﬁltration for simultaneous recovery of viruses, bacteria and parasites from reclaimed water. J

Microbiol Methods 88:155–161. https://doi.org/10.1016/j.mimet.2011.11.007

Liu L, Su J-Q, Guo Y, Wilkinson DM, Liu Z, Zhu Y-G, Yang J (2018) Large-scale biogeographical

patterns of bacterial antibiotic resistome in the waterbodies of China. Environ Int 117:292–299.

https://doi.org/10.1016/j.envint.2018.05.023

Lucena F, Duran A, e, Morón, A, Calderón, E, Campos, C, Gantzer, C, Skraber, S, Jofre, J. (2004)

Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and

secondary wastewater treatments. J Appl Microbiol 97:1069–1076. https://doi.org/10.1111/j.

1365-2672.2004.02397.x

Marano RBM, Cytryn E (2017) The mobile resistome in wastewater treatment facilities and down-

stream environments. In: Antimicrobial resistance in wastewater treatment processes. Wiley, Ltd,

pp 129–155. https://doi.org/10.1002/9781119192428.ch8

Marín I, Goñi P, Lasheras AM, Ormad MP (2015) Efﬁciency of a Spanish wastewater treatment

plant for removal potentially pathogens: Characterization of bacteria and protozoa along water

and sludge treatment lines. Ecol Eng 74:28–32. https://doi.org/10.1016/j.ecoleng.2014.09.027

Martín-Díaz J, Lucena F, Blanch AR, Jofre J (2020) Review: Indicator bacteriophages in sludge,

biosolids, sediments and soils. Environ Res 182:109133. https://doi.org/10.1016/j.envres.2020.

109133

Mbanga J, Abia ALK, Amoako DG, Essack, Sabiha.Y. (2020) Quantitative microbial risk assessment

for waterborne pathogens in a wastewater treatment plant and its receiving surface water body.

BMC Microbiol 20:346. https://doi.org/10.1186/s12866-020-02036-7

482 J. Gonçalves et al.

Miłobedzka A, Ferreira C, Vaz-Moreira I, Calderón-Franco D, Gorecki A, Purkrtova S, Bartacek J,

Dziewit L, Singleton CM, Nielsen PH, Weissbrodt DG, Manaia CM (2022) Monitoring antibiotic

resistance genes in wastewater environments: the challenges of ﬁlling a gap in the one-health cycle.

J Hazard Mater 424:127407. https://doi.org/10.1016/J.JHAZMAT.2021.127407

Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H (1986) Speciﬁc enzymatic ampliﬁcation

of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51(Pt

1):263–273. https://doi.org/10.1101/sqb.1986.051.01.032

Narciso-da-Rocha C, Manaia CM (2017) The inﬂuence of the autochthonous wastewater microbiota

and gene host on the fate of invasive antibiotic resistance genes. Sci Total Environ 575:932–940.

https://doi.org/10.1016/j.scitotenv.2016.09.157

Núñez-Delgado A, Pousada-Ferradás Y, Álvarez-Rodríguez E, Fernández-Sanjurjo MJ, Conde-Cid

M, Nóvoa-Muñoz JC, Arias-Estévez M (2019) Chapter 1 - effects of microbiological and non-

microbiological treatments of sewage sludge on antibiotics as emerging pollutants present in

wastewater: a review. In: Shah MP, Rodriguez-Couto S (eds) Microbial wastewater treatment.

Elsevier, pp 1–17. https://doi.org/10.1016/B978-0-12-816809-7.00001-4

Paiva MC, Reis MP, Costa PS, Dias MF, Bleicher L, Scholte LLS, Nardi RMD, Nascimento AMA

(2017) Identiﬁcation of new bacteria harboring qnrS and aac(6')-Ib/cr and mutations possibly

involved in ﬂuoroquinolone resistance in raw sewage and activated sludge samples from a full-

scale WWTP. Water R es 110:27–37. https://doi.org/10.1016/j.watres.2016.11.056

Proia L, Adriana A, Jessica S, Carles B, Marinella F, Marta L, Luis BJ, Pierre S (2018) Antibiotic

resistance in urban and hospital wastewaters and their impact on a receiving freshwater ecosystem.

Chemosphere 206:70–82. 1016/j.chemosphere.2018.04.163

Shah MP (2021) Removal of emerging contaminants through microbial processes. Springer

Shah Maulin P (2020) Microbial bioremediation and biodegradation. Springer

Shah Maulin P (2021) Removal of refractory pollutants from wastewater treatment plants. CRC

Press

Stiborova H, Kracmarova M, Vesela T, Biesiekierska M, Cerny J, Balik J, Demnerova K (2021)

Impact of long-term manure and sewage sludge application to soil as organic fertilizer on the

incidence of pathogenic microorganisms and antibiotic resistance genes. Agronomy 11:1423.

https://doi.org/10.3390/agronomy11071423

Symonds EM, Breitbart M (2015) Affordable enteric virus detection techniques are needed to

support changing paradigms in water quality management. CLEAN – Soil Air Water 43:8–12.

https://doi.org/10.1002/clen.201400235

Tandukar M, Ohashi A, Harada H (2007) Performance comparison of a pilot-scale UASB and

DHS system and activated sludge process for the treatment of municipal wastewater. Water Res

41:2697–2705. https://doi.org/10.1016/j.watres.2007.02.027

Tandukar S, Ghaju Shrestha R, Malla B, Sthapit N, Sherchand JB, Sherchan SP, Haramoto E (2021)

Virus reduction at wastewater treatment plants in Nepal. Environ. Chall. 5:100281. https://doi.

org/10.1016/j.envc.2021.100281

Teixeira P, Salvador D, Brandão J, Ahmed W, Sadowsky MJ, Valério E (2020) Environmental and

adaptive changes necessitate a paradigm shift for indicators of fecal contamination. Microbiol

Spectr 8:8.2.1. https://doi.org/10.1128/microbiolspec.ERV-0001-2019

Thomas CM, Nielsen KM (2005) Mechanisms of, and barriers to, horizontal gene transfer between

Bacteria. Nat Rev Microbiol 3:711–721. https://doi.org/10.1038/nrmicro1234

Truchado P, Garre A, Gil MI, Simón-Andreu PJ, Sánchez G, Allende A (2021) Monitoring of human

enteric virus and coliphages throughout water reuse system of wastewater treatment plants to

irrigation endpoint of leafy greens. Sci Total Environ 782:146837. https://doi.org/10.1016/j.sci

totenv.2021.146837

USEPA (2002) Implementation guidance for ambient water quality criteria for Bacteria, EPA Fact

Sheet.

van der Helm E, Imamovic L, Hashim Ellabaan MM, van Schaik W, Koza A, Sommer MOA (2017)

Rapid resistome mapping using nanopore sequencing. Nucleic Acids Res 45:e61. https://doi.org/

10.1093/nar/gkw1328

Microbial Contamination of Environmental Waters … 483

Vinkšel M, Writzl K, Maver A, Peterlin B (2021) Improving diagnostics of rare genetic diseases

with NGS approaches. J Community Genet 12:247–256. https://doi.org/10.1007/s12687-020-005

00-5

Wallace JC, Port JA, Smith MN, Faustman EM (2017) FARME DB: a functional antibiotic resistance

element database. Database. https://doi.org/10.1093/database/baw165

Wang S, Ai Z, Zhang Z, Tang M, Zhang N, Liu F, Han G, Hong S-L, Liu K (2020) Simultaneous

and automated detection of inﬂuenza A virus hemagglutinin H7 and H9 based on magnetism and

size mediated microﬂuidic chip. Sens Actuat B Chem 308:127675. https://doi.org/10.1016/j.snb.

2020.127675

Wang M, Chen H, Liu S, Xiao L (2021a) Removal of pathogen and antibiotic resistance genes from

waste activated sludge by different pre-treatment approaches. Sci Total Environ 763:143014.

https://doi.org/10.1016/j.scitotenv.2020.143014

Wang M, Zhu J, Mao X (2021b) Removal of pathogens in onsite wastewater treatment systems: a

review of design considerations and inﬂuencing factors. Water 13:1190. https://doi.org/10.3390/

w13091190

WHO (2018) Preventing disease through healthy environments: a global assessment of the burden

of disease from environmental risks [WWW Document]. https://www.who.int/publications-det

ail-redirect/9789241565196 (accessed 2.19.22)

World Health Organization (2014) Antimicrobial resistance: 2014 Global report on surveillance

Wyn-Jones AP, Carducci A, Cook N, D’Agostino M, Divizia M, Fleischer J, Gantzer C, Gawler

A, Girones R, Höller C, de Roda Husman AM, Kay D, Kozyra I, López-Pila J, Muscillo M,

Nascimento MSJ, Papageorgiou G, Rutjes S, Sellwood J, Szewzyk R, Wyer M (2011) Surveillance

of adenoviruses and noroviruses in European recreational waters. Water Res 45:1025–1038.

https://doi.org/10.1016/j.watres.2010.10.015

Xia Y, Li A-D, Deng Y, Jiang X-T, Li L-G, Zhang T (2017) MinION nanopore sequencing enables

correlation between resistome phenotype and genotype of coliform bacteria in municipal sewage.

Front Microbiol 8. https://doi.org/10.3389/fmicb.2017.02105

Zhang K, Farahbakhsh K (2007) Removal of native coliphages and coliform bacteria from municipal

wastewater by various wastewater treatment processes: Implications to water reuse. Water Res

41:2816–2824. https://doi.org/10.1016/j.watres.2007.03.010

Zhang Q, Gallard J, Wu B, Harwood VJ, Sadowsky MJ, Hamilton KA, Ahmed W (2019) Synergy

between quantitative microbial source tracking (qMST) and quantitative microbial risk assess-

ment (QMRA): a review and prospectus. Environ Int 130:104703. https://doi.org/10.1016/j.env

int.2019.03.051

Zhang Y, Pei M, Zhang B, He Y, Zhong Y (2021) Changes of antibiotic resistance genes and bacterial

communities in the advanced biological wastewater treatment system under low selective pressure

of tetracycline. Water Res 207:117834. https://doi.org/10.1016/j.watres.2021.117834

Zhao Q, Liu Y (2019) Is anaerobic digestion a reliable barrier for deactivation of pathogens in

biosludge? Sci Total Environ 668:893–902. https://doi.org/10.1016/J.SCITOTENV.2019.03.063

Zheng X, Deng Y, Xu X, Li S, Zhang Y, Ding J, On HY, Lai JCC, In Yau C, Chin AWH, Poon

LLM, Tun HM, Zhang T (2022) Comparison of virus concentration methods and RNA extraction

methods for SARS-CoV-2 wastewater surveillance. Sci Total Environ 153687. https://doi.org/10.

1016/j.scitotenv.2022.153687

Use of Genetic Engineering Approach

in Bioremediation of Wastewater

Jutishna Bora, Saqueib Imam, Vardan Vaibhav, and Sumira Malik

Abstract The principles of genetic engineering led to unlocking the door to many

possibilities, expanding its application into several ﬁelds of life sciences. Recently

scientists have attempted to combine the disciplines of genetic engineering and biore-

mediation as a possible solution to free the environment of possible organic contam-

inants such as oil, solvents, and herbicides, as well as cycling out toxic heavy chem-

icals, which are further used by the microbes for carrying out their metabolic cycles

and as their source of food and energy. This paper addresses the various microbes

and genetic tools involved in bioremediation. Potential novel ways are highlighted

that are being developed using gene editing tools to better the waste management

system. Further, the key metabolic pathways are discussed, by which microbes cycle

the harmful toxic pollutants from the environment into their system. The various

actively used Gene editing tools for bioremediation of wastewater to get speciﬁc

microbes included are TALEN, ZFNs, and CRISPR Cas9.

Keywords Bioremediation ·Genetic engineering ·Genetically Modiﬁed

Microorganisms (GMO) ·CRISPR/Cas 9 ·ZFN ·TALEN ·Biosensor ·

Bioaccumulation

1 Introduction

With the increase in industrialisation over the years, the rate of pollution has increased

drastically worldwide. Industrial instruments and batch processes have led to the

release of toxic and carcinogenic substances in the air, land and water, such as

pharmaceutical waste, by-products from petroleum reﬁning, and dyes—polymers

J. Bora · S. Malik (B

)

Amity Institute of Biotechnology, Amity University Jharkhand, Ranchi, Jharkhand, India

e-mail: smalik@rnc.amity.edu

S. Imam

Amity Institute of Biotechnology, Amity University Kolkata, Kolkata, West Bengal, India

V. Vaibhav

Amity Institute of Biotechnology, Amity University, Uttar Pradesh, Noida, India

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_23

485

486 J. Bora et al.

and even explosives. Extensive use of pesticides and herbicides by farmers to curb the

growth of weeds and unwanted microorganisms on their crops has also contributed

to polluting a more signiﬁcant part of the environment since these pesticides and

herbicides use various nitrogen-containing chemicals and other compounds which

cannot be degraded or removed easily. WHO has reported that by the year 2021,

84% of the population all over India will have been living in areas with polluted air,

which has exceeded India’s overall own quality of air, with an average presence of 63

microns of particulate matter (PM) per cubic meter of air. Another survey has shown

that, in the year 2020, India alone reported an average of 51.90% of land ﬁlled with

various pollutants, damaging crops and human life altogether.

Further, reports by WHO have revealed that the average life expectancy of Indian

life has also decreased by 5.2 years, indicating signiﬁcant endangerment to human

life in the upcoming years if the rate of pollution keeps increasing at this steady

rate. Such an increased accumulation in the environment can render the soil, air and

water bodies unﬁt for further use and cause health hazards to humans and animals.

Hence, the demand for recycling and degradation/removal of these pollutants from

the environment has gained a higher ground. Scientists have attempted to combine

various life sciences disciplines with different technological advances, to develop

an efﬁcient and suitable technique for curing the environment from such harmful

pollutants.

Conventional remediation techniques for the degradation/removal of pollutants

were costly since they involved using speciﬁc instruments and chemicals, which

not only contributed to causing additional environmental damage but were also

not economically viable. Hence an alternative technique was needed for remedi-

ation of pollutants, which was inexpensive, caused minimum damage, was less

time consuming, required minimum labour and was a long-term investment overall.

That is when biological microorganisms were considered a possible replacement for

the conventional techniques for bioremediation. These microorganisms were exten-

sively studied under simulated environmental conditions with speciﬁc pollutants,

which served as the substrate for the growth of microorganisms. It was observed

that these microorganisms could degrade/bioaccumulate these pollutants via their

various metabolic pathways, which was also found to be different for different strains

of microorganisms. Microorganisms offer the remediation of pollutants via a natural

process. Hence it became not only the ideal choice for farmers and industrialists,

but also further research and experiments were conducted to modify their genome

genetically, enzymes they produced, their structure or inserted with genes from other

species to produce speciﬁc proteins, enhancing their productivity, meanwhile main-

taining their biomass to a minimum concentration. The by-products released by them

were also less harmful (Fig. 1).

Thus, when genetic engineering principles were applied to the ﬁeld of microbi-

ology, along with the use of nanotechnology to produce genetically modiﬁed organ-

isms (GMO), it was observed that they provided enhanced efﬁciency with which

remediation could be carried out compared to the conventional techniques. Microbes

were naturally found to degrade certain compounds, regarded as pollutants in the

environment, but the main problem was that these naturally occurring microbes

Use of Genetic Engineering Approach in Bioremediation of Wastewater 487

Fig. 1 The above ﬁgure

depicts the various in-situ

and ex-situ bioremediation

techniques carried out by

microbes and conventional

instruments (Dixit and

Malaviya 2015;Chibuike

and Obiora 2014;

Bhattacharya et al. 2015;

Burgess et al. 2001)

provided a prolonged rate of degradation, which meant a very time-consuming

process. Some microbes could not survive in a variety of extreme environmental

conditions. That is when genetic engineering came into play, where we could manipu-

late an organism’s genome to I mpart additional abilities into it. This included manip-

ulating the organism’s critical genes involved in bioremediation or modifying the

metabolic pathways. Such developed GMOs were successfully operated in the ﬁeld

to achieve the destruction of organic and inorganic waste to some extent. Genetic

engineering helps unravel an organism’s genome through which we can identify the

physical and chemical mechanisms involved in transforming pathways for speciﬁc

compounds. This knowledge can help us design a synthetic organism of our own

from scratch.

Some of the principles of genetic engineering involved in the production of

genetically modiﬁed organisms include.

•Bacterial engineering- Includes the optimisation of biocatalyst involved in the

metabolic processes in a bacterial strain or the involvement of protein engineering

to enhance particular enzyme activity.

•Genetically engineered fungi for Mycoremediation- Recent advances in molecular

biology have made it possible to engineer a genetically advanced and improved

488 J. Bora et al.

version of the original fungal strain for enhanced mycoremediation. Mycoreme-

diation simply means the employment of fungal strains for remediation of toxic

products from the environment. They were successful in the removal of harmful

PCBs (Polychlorinated biphenyls) from the environment.

•Genetically engineered plants for phytoremediation- Even though the geneti-

cally modiﬁed microorganism offers enhanced activity over their naturally occur-

ring counterparts, they might not perform as efﬁciently as under lab conditions.

Biodegradation might not even happen since some of these engineered microor-

ganisms have a poor survival rate in the contaminated soil. Hence, phytoreme-

diation was considered the next best option since it was easier to manage as the

autotrophs require very little nutrient input to work. Plants also offered protec-

tion against water and wind erosion; hence contaminants did not spread enough.

They are renewable and hence an ideal option after microorganisms. Genetic engi-

neering is used to overexpress the genes involved in metabolism, uptake or trans-

port of speciﬁc pollutants since usual; unmodiﬁed plants are very slow towards

biodegradation.

In this paper, we will discuss the coupling of the simple natural processes carried

out by the microorganism with the modern-day principles of genetic engineering

to develop a synthetic genetically modiﬁed microorganism for bioremediation of

the environment, occurring safely without contributing additionally to polluting the

environment.

2 Type of Contaminants Found in Everyday Waste

Products

There are numerous amounts of contaminants and waste material being dumped

into the environment by large-scale industries every day. Some of these contami-

nants include the widely used herbicides and pesticides to control crop damage by

farmers and the agriculture industries (Dixit and Malaviya 2015). In doing so, the use

of harmful chemicals has increased environmental pollution, leading to disastrous

effects such as loss of soil viability, poor crop production and quality, and loss of

ﬂora and fauna (Dixit and Malaviya 2015; Chibuike and Obiora 2014). The different

types of contaminants found in the environment include.

•Heavy metals- These are the most harmful contaminants found in the environ-

ment. Heavy metals accumulate in the environment, primarily because of burning

fossil fuels, everyday mining, and extensive chemicals in the form of pesticides

for crop growth. A microbial process cannot degrade heavy metals since they

are not part of the microbial metabolism, so instead, these metals are trans-

formed from their current oxygen state to the other. Mechanisms followed by

microbes to cycle metals are biosorption, bioleaching, biomineralisation and

enzyme-catalysed transformation. The most common mechanism involves the

Use of Genetic Engineering Approach in Bioremediation of Wastewater 489

methylation of the heavy metals, making them volatile and easy to oxidise now.

Heavy metals can accumulate in our body via the food we eat, initially grown

in the soil with toxic metal ions. Such accumulation can lead to health problems

such as destroying vital organs and glands such as the heart, brain, kidney and

even liver (Chibuike and Obiora 2014).

•PVC- PVC and its related products, generated during the regularly conducted

industrial processes, have contributed immensely to polluting the environment.

Such a massive surge has been since there has been an increase in the demand

for plastics for daily usage, where PVC ﬁnds its practical use in making drainage

pipes, water pipes, window frames and even medical devices. Chlorine is the basic

building block of PVC, which is a very toxic gas when exposed. The burning of

PVC and its related products releases chlorine and dioxins into the environment.

Chlorine is said to destroy the ozone layer of the atmosphere. It is the most

concerning the reason for global warming, whereas dioxins are created wher-

ever chlorine or chlorine-based compounds are formed, and it is one of the most

toxic chemicals to be produced, contributing to health hazards such as cancer,

mental health problems (is a neurotoxin), damage to the immune system and even

reproductive problems (Chibuike and Obiora 2014; Avrilescu 2005).

•Radioactive metals- These are metals with an unstable nucleus emitting harmful

radiation in the environment, which can even penetrate the human skin, damage the

underlying organs, and cause-speciﬁc life-threatening mutations. Such emission

includes alpha, beta and gamma rays. Hence, such metals are highly toxic to

human health, causing various health hazards (Karaouzas et al. 2021). One of

the prime Examples of radioactive poisoning in human history is the bombing

of Hiroshima in 1945, where an estimated 90,000 to 120,000 people were killed,

and many are still suffering the effects to date! (Singh 2017).

•Herbicides- They primarily contaminate the groundwater and are toxic to the

plants. Damage to the plant species by herbicides is very selective; for example, it

has been found that some herbicides are said to seep into the soil and inhibit photo-

synthesis; 2,4-d herbicide is said to kill the broadleaved plants but has minimal

effect on the grasses. Increased use of 2,4-D herbicides can make broadleaved

plants extinct and increase grasses. The most lethal herbicide now banned or

being phased out in the European Union is Paraquat, which has increased the risk

of neurological diseases in the human body, such as Alzheimer’s and Parkinson’s

disease. Other harmful herbicides include Methomyl, 1,3-Dichloropropene, and

Pyrethroid insecticides which are either banned or used and have been limited

(Singh 2017).

•Petroleum Hydrocarbons- It is the most toxic contaminant for the existing aquatic

life. Industries dumping their wastes, transportation or pipeline failure can lead

to the accumulation of crude petroleum and its related products in the water,

damaging the water quality and making it unﬁt for further use. Consumption

of such untreated water can lead to health problems such as headaches, dizzi-

ness, nausea, nerve disorders such as the development of seizures, irregular heart

rhythms, and the immune system is also drastically affected (Singh 2017;Guo

et al. 2019).

490 J. Bora et al.

3 Role of Microbes in Bioremediation

Microbes, as the name suggests, are microscopic living organisms with a unique

metabolic system to carry out their day-to-day activities and play an essential role

as part of nature’s food chain. Their habitats range from soil on the earth to the air

we breathe and are even present in the water we drink (Singh 2017). Some microbes

also habitat the human body, such as Staphylococcus epidermidis (which lives on the

skin and inside the nose) and obligate anaerobes (found in the abdomen and colon),

to name a few. Some microbes can be single-celled such as bacteria (Rigét et al.

2010;Yietal. 2011;Skerker 2008).

Previously used conventional techniques for bioremediation were soon becoming

obsolete for the sole reason of being inefﬁcient and uneconomic (Yi et al. 2011;

Skerker 2008). One of these methods included the addition of a reagent to an aqueous

solution to remove heavy metals. Such reagents increase the pH of that solution,

which further converts the heavy metals into insoluble ones, which have to be next

removed via precipitation (Skerker 2008; Gavrilescu et al. 2019). The problem with

such methods was that the aggregates formed in the end were difﬁcult to remove and

expose. In a way, heavy metals removed from the aqueous solution were now disposed

into the land as solid waste, so such methods proved unethical and uneconomical.

On the other hand, after years of rigorous research and recent breakthroughs in

the ﬁeld of life sciences, particularly the combined ﬁeld of microbiology and genetic

engineering, scientists found a possible and efﬁcient way of manipulating some of the

naturally found microbes to save the environment. In the simple process of bioremedi-

ation, microbes cycle the environmental polluting toxic organic and chemical waste,

through their metabolic pathways, as their source of food, for generating energy to

carry out daily tasks for their survival (Yi et al. 2011;Skerker 2008; Gavrilescu et al.

2019; Smaranda et al. 2016). In this way, with the employment of microbes in the

areas of possible organic contamination, we can degrade these wastes into less or

no toxic forms, potentially cleaning the environment of such harmful contaminants

posing a threat to the life of humans as well as the environment.

Microorganisms have come up as a critical alternative to cleaning those polluted

areas, which are out of bounds for regular human interaction for that very same

purpose because their most important feature includes their survival in a variety of

extreme conditions of the environment, which makes them suitable for cleaning the

environment (Skerker 2008).

There are two factors in the ecosystem that work together to control the rate of

degradation by microbes, which are-

•Abiotic components- This includes all the non-living components of the biosphere

that affect the growth and survival of living things, such as temperature, pH,

humidity, sunlight, oxygen and minerals (including the heavy metal waste in the

soil) (Smaranda et al. 2016).

•Biotic components- These include all the living being present in the ecosystem,

which is carefully sub-divided to show their signiﬁcance and survival of the

Use of Genetic Engineering Approach in Bioremediation of Wastewater 491

Fig. 2 Above ﬁgure shows

the simple cyclic relation

between the pollutant, the

environment and the

microorganism for

bioremediation (Karaouzas

et al. 2021; Singh 2017)

ENVIRONMEN

(POLLUTANT

ACCUMULATI

ON POOL)

MICROORGANISM(

DEGRADATION)

SUBSTRATE(

POLLUTANT)

ecosystem as a whole. These components include- producers, consumers, decom-

posers and detritivores (which include our microbes) (Smaranda et al. 2016)

(Fig. 2).

4 Genetic Engineering of Microorganisms

The advantage it gives over the conventional methods is the ability to manipulate

and edit the genome of a microorganism in such a way to produce a modiﬁed version

of the original species, with much better metabolic rates, controlled growth, and

showing resistance to extreme temperatures (Kaniecki et al. 2018). Microorganisms

are engineered and designed according to the need to show maximum catalytic ability

towards bioremediation, with a minimum cell mass production, so they do not hamper

the working conditions of any other plant or its related biological process (Kaniecki

et al. 2018; El Fantroussi and Agathos 2005). They are thus supplemented with

additional genetic properties by insertion of parts of the genome from various other

organisms showing capabilities for the biodegradation of speciﬁc pollutants, which

the naturally occurring microorganisms cannot degrade or, even if they can, is not

efﬁcient enough to be applied on a large scale (Kaniecki et al. 2018; El Fantroussi and

Agathos 2005; Madhavi and Mohini 2012). The combination of different life sciences

disciplines to create a hybrid microorganism using various genes of different species

owing to different metabolic abilities, inﬂuencing the biodegradation and biopro-

cessing of pollutants produced daily, which cannot be easily degraded or removed

by the conventional methods (Boopathy 2000).

492 J. Bora et al.

5 Steps Followed During Gene Editing of Microorganisms

Creating a genetically modiﬁed organism to clean up the environment depends

on many factors critical to its development (Paranthaman and Karthikeyan 2015).

Selecting strains which show speciﬁc responses towards the presence of a particular

pollutant depends on the-

•Pollutant type and its composition.

•Species to which the microorganism belongs, showing response to the particular

pollutant.

•Gene editing is done keeping in mind the physiochemical conditions of the envi-

ronment harbouring the harmful pollutants to ensure the species’ survival in

bioremediation.

Now, keeping the above factors in mind, we design our microbes based on four

basic approaches, which ensure not only a safe, efﬁcient bioremediation process

but also maintain a minimum microbial growth altogether, thereby minimising any

further counter pollution by our own employed Genetically modiﬁed microorganism

(GMM) (Paranthaman and Karthikeyan 2015; Jaysankar et al. 2008; Shah et al.

2013; Kukumar and Nirmala 2016). These three basic approaches which modify the

organism’s genome and capabilities include.

(1) Modiﬁcation of enzyme speciﬁcity and afﬁnity- The enzymes present in some

of the speciﬁc strains of microorganisms are said to show biodegradable prop-

erties towards a wide variety of pollutants. These pollutants are recognised as

the substrate by the different microbial enzymes, which at optimum temper-

ature, pH and ionic concentration, help transform these harmful pollutants

into harmless by-products of their metabolism. Examples of the enzymes

include- cytochrome P450s, laccases (benzenediol oxidoreductases), hydro-

lases, dehalogenases, proteases and lipases extracted from microbial strains,

which are naturally involved in bioremediation (Jaysankar et al. 2008; Shah

et al. 2013; Kukumar and Nirmala 2016). These microbial strains include

Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium.

Some pollutants degraded by the enzymes produced by above mentioned micro-

bial strains include- polymers, PAH, dyes, detergents and pesticides (Kehinde

and Isaac 2016). Now such enzymes offer biodegradable properties towards

selected pollutants but have a relatively slow conversion rate, coupled with an

increased microbial biomass production (Kehinde and Isaac 2016; Phulpoto

et al. 2016). Hence, to tackle this problem, genetic engineering principles come

in handy. Gene editing tools are applied for the modiﬁcation of enzymes. For

example, we can chemically modify lipases by attaching aldehydes, polyethene

glycols and imido-esters, thereby altering their conformation and hydropho-

bicity (Kehinde and Isaac 2016; Phulpoto et al. 2016). These alterations bring

with them enhanced microbial activity, stability and selectivity. Hence, we can

easily manipulate to produce a synthetic enzyme from a suitable microbial strain

Use of Genetic Engineering Approach in Bioremediation of Wastewater 493

to impart capabilities of efﬁcient degradation of different types of pollutants that

are engineered depending on their operational environmental conditions.

(2) Metabolic Pathway construction and regulation- Microorganisms use their

complex metabolic pathways to cycle the elements from the external environ-

ment into their system to obtain energy and carry out their everyday activities.

Now, with the advancement of science and technology, we can design, manipu-

late and regulate the metabolic pathway of a suitable microorganism, such that it

can use the pollutants from the external environment as its nitrogen and carbon

source for obtaining energy assisting in bioremediation (Kukumar and Nirmala

2016; Phulpoto et al. 2016; Hesham et al. 2012; Aranda et al. 2010). The main

aim for the development of a genetically modiﬁed organism was to introduce

such modiﬁcations so that the modiﬁed organism can exhibit a desired catabolic

pathway, depending on selectivity and speciﬁcity, derived from a combina-

tion of determinants having a similar pathway, complementary to the pathway

segments required to be integrated into the GMM. For example- the degradation

of 2-nitrobenzoate is done by a new metabolic pathway by the bacterial strain

Arthrobacter sp. SPG. 2-nitrobenzoate is a nitro-aromatic compound considered

a harmful pollutant capable of causing high environmental toxicity and a signif-

icant threat since they harbour mutagenic activity (Aranda et al. 2010; Sarang

et al. 2013). Hence, when strains of Arthrobacter sp. SPG was isolated and simu-

lated in sterile and non-sterile environments, and they showed promising results

of degrading more than 90% of 2-nitrobenzoate within 10–12 days (Kadirvelu

et al. 2002). The metabolites produced on degradation were Salicylate and cate-

chol produced from the native oxidative pathway, which is a ﬁrst and only

followed by the SPG strain. The native pathway followed for the degradation

of nitroaromatic compounds was reductive, and the metabolites produced at the

end of the reaction were also different from those produced by the SPG strain,

i.e., the reductive pathway produced pyruvate and acetaldehyde as end-products

(Riggle and Kumamoto 2000). Henceforth, due to the discovery of a more efﬁ-

cient pathway for degradation of nitroaromatic compounds, scientists started

taking a keen interest in extracting the genes responsible for taking this new

metabolic pathway for manipulation and designing of a genetically modiﬁed

organism showing the combined capabilities of efﬁcient degradation provided

by the SPG strain of Arthrobacter. The surviving capabilities are derived from a

different strain to create an organism capable of surviving in extreme environ-

mental conditions, showing minimum unwanted microbial biomass enlargement

and efﬁcient working capacity (Riggle and Kumamoto 2000; Fomina and Gadd

2014; Mulligan et al. 2001). Another example of a modiﬁed pathway for degra-

dation of 2-nitrobenzoate (2-NBA) was followed by the bacterial strain KU-7

belonging to Pseudomonas ﬂuorescens. It was discovered that these strains

could efﬁciently accumulate 2-NBA from polluted areas, serving as the sole

source of carbon, nitrogen and energy sources for this bacterial strain (Texier

et al. 1993; Perpetuo et al. 2011). KU-7 followed the reductive pathway for the

degradation of 2-NBA, producing intermediate 3-hydroxyanthranalite, which

was different from the intermediates anthranilate or catechol produced from

494 J. Bora et al.

Tabl e 1 Above table shows the list of microorganisms whose genes are modiﬁed by gene editing

tools for bioremediation

Pollutant Degradation by

microorganism

Modiﬁcation References

Toluene/benzoate P. putida KT2442 Metabolic Pathway Panke et al. (1998)

Polychlorinated biphenyl

(PCB)

Pseudomonas sp.

LB400

Speciﬁcity for the

substrate

Panke et al. (1998)

Mono/dichlorobenzoats Pseudomonas sp.

B13

Metabolic Pathway Panke et al. (1998)

Trichloroethylene, toluene E. coli

FM5/Pky287

Regulating Pathway Panke et al. (1998)

4-ethyl benzoate P. putida Metabolic Pathway Panke et al. (1998)

PCB, benzene, toluene E. coli JM109

(Pshf1003)

Speciﬁcity for the

substrate

Panke et al. (1998)

Chloro-, methyl benzoate Pseudomonas sp.

FR1

Metabolic Pathway Panke et al. (1998)

the reductive-degradative pathway followed by other bacteria. Hence, this indi-

cated that an alternate pathway was followed by the KU-7 cells, which was

different and more efﬁcient, requiring less energy expenditure, i.e., oxidation

of only 2 NADPH was required for completing the reaction. The intermediate

3-hydroxyanthranalite produced upon degradation was also less harmful and

less toxic and thus could be easily removed via physical methods (Jaysankar

et al. 2008; Shah et al. 2013; Texier et al. 1993; Perpetuo et al. 2011).

(3) Bio-afﬁnity biosensor- Applications of such genetically modiﬁed biosensors

include chemical sensing for detecting the speciﬁc chemical in a suspected

environment and reducing or degrading the toxic products on its detection.

Biosensors are also used to analyse various samples’ chemicals by modifying the

biosensors showing different selectivity traits. Some of the widely used geneti-

cally modiﬁed biosensors used for identifying and analysing speciﬁc pollutants

from the environment include- R. metropolis used for successfully identifying

2,4-Dinitrophenol and Staphylococcus plasmid pI258 for the identiﬁcation of

arsenic in the polluted pool of water (Gavrilescu et al. 2019; Madhavi and

Mohini 2012; Jaysankar et al. 2008) (Table 1).

6 Gene Editing Tools Employed for Waste Management

With the growing advances in biological science and engineering principles, it has

become possible to modify and produce our microbes suited for safely disposing

of everyday harmful waste via bioremediation. Various gene editing tools have

been employed and are widely used for generating microorganisms speciﬁcally for

managing waste, with the added advantage of displaying maximum catalytic ability

Use of Genetic Engineering Approach in Bioremediation of Wastewater 495

with minimum cell mass, thereby not hampering the working of any other plant

or its related biological process (Boopathy 2000; Jaysankar et al. 2008; Shah et al.

2013). Each gene editing tool below includes a nuclease that can be engineered and

customised according to our need to recognise, bind and cleave the speciﬁc sequences

in the foreign genome.

6.1 CRISPR/Cas 9

CRISPR/Cas 9 protein, also known as the ( CLUSTERED REGULARLY INTER-

SPACED PALINDROMIC REPEATS), is a specialised molecular scissor discov-

ered and developed by Nobel prize-winning scientists Emmanuelle Charpentier

and Jennifer Doudna. This protein has a precise targeting mechanism, which has

been shown to edit any type of genome of any given size. Due to its efﬁcient and

versatile gene editing capabilities, it has shown promising results in the ﬁeld of life

sciences, helping in producing drugs, thereby providing a much better therapeutic

approach towards the treatment of many neurological and immunological diseases

such as SCID (Severe combined immunodeﬁciency disorder), Alzheimer’s disease

(Jaysankar et al. 2008; Shah et al. 2013; Kukumar and Nirmala 2016).

Cas9 is a DNA endonuclease, usually found in bacteria Streptococcus pyogenes,

which is guided by a particular type of RNA molecule called the gRNA. CRISPR, on

the other hand, is a base repeat of about 30–40 bp, separated by a spacer sequence that

is said to complement the sequence of the genome being edited, i.e., the foreign DNA

sequences (Jaysankar et al. 2008; Shah et al. 2013; Kukumar and Nirmala 2016).

Nowadays, CRISPR is being applied to increase and modify the genetic metabolic

pathway of that microorganism already known for degrading environmental wastes

such as hydrocarbons, plastics, Pesticides, heavy metals etc. The edited microor-

ganisms formed are called Genetically Modiﬁed organisms (GMO) (Perpetuo et al.

2011; Roane et al. 2001).

The CRISPR/Cas 9 protein consists of two components-

•a single guide RNA (gRNA).

•Cas 9 protein.

Both the components work in close sync to edit the target genome. Before

CRISPR/Cas 9 protein is delivered to the patient’s body via a suitably designed

vector, the gene sequences targeted by the protein must be identiﬁed ﬁrst. CRISPR is

highly suited for editing Archeal and bacterial genomes (El Fantroussi and Agathos

2005; Madhavi and Mohini 2012; Boopathy 2000).

496 J. Bora et al.

Mechanism of Action of CRISPR/Cas 9

We ﬁrst identify and select the gene of interest by DNA sequencing methods. For

this, we use high-end gene sequencing tools such as microarray and High throughput

sequencing (HTS) to get the gene’s correct, accurate base sequence to be edited. Next,

we transcribe the CRISPR repeats giving us the crRNA (Fantroussi and Agathos

2005; Madhavi and Mohini 2012; Boopathy 2000). The gRNA (or the guide RNA)

is next obtained by combining the transcribed crRNA and Cas9 protein, giving us

the Ribonucleoprotein called the crRNP or the gRNA. The RNA controls the target

speciﬁcity since it contains a 20-base long protospacer complementary to the target

sequence of DNA.

The gRNA works by identifying the speciﬁcally targeted sequence of DNA in the

foreign genome and thus guiding the Cas9 protein to come and bind to the targeted

site on the DNA, introducing double-stranded breaks (DSB) (Boopathy 2000). Now,

these breaks are repaired naturally by the host’s system in two ways-

•error-prone nonhomologous end joining (NHEJ)- causes the Knockout of gene

function.

•High-ﬁdelity homology-directed repair (HDR)- causes the Knockdown of gene

expression.

Before these repairs are done, we can efﬁciently insert another gene or even

delete our target sequence according to the required speciﬁc modiﬁcation. Hence,

after insertion/deletion, the nick in the ds DNA ﬁnally gets sealed through

repairing mentioned above pathways. Prokaryotes usually apply the HDR method to

repair their dsDNA breaks, thereby creating precise modiﬁcations in their genome

(Boopathy 2000).

Let us, for example, there is a bacterial strain Bacillus cereus A, which has been

found to have great application for the bioremediation of diesel oil. Its optimum pH

range was 6.5, and the optimum working temperature was found to be 45 °C. Hence,

this bacterial strain having a very narrow working range of pH and temperature made

it difﬁcult to apply to areas of environment with extremely low pH or low-temperature

ranges. Hence, to make this strain suitable to work in various pH and temperature

ranges, we would use the CRISPR/Cas9 technology to edit its genome. We do this by

identifying the gene we have to edit with DNA sequencing tools; after that, we design

our gRNA having the protospacer sequence complementary to the gene of interest.

Then we will insert another DNA sequence at the targeted site, which was obtained

from a bacterial strain of its related family member, such that the new genetically

modiﬁed strain of Bacillus cereus A obtained can work in a wide range of pH and

temperature changes.

Use of Genetic Engineering Approach in Bioremediation of Wastewater 497

6.2 TALEN (Find an Advantage Over CRISPR)

TALENs is a naturally occurring protein which has been found to hold powerful

gene editing capabilities. It is originally derived from the plant pathogenic bacteria,

Xanthomonas spp, which can efﬁciently alter the gene expression in the host plant

cell it attaches. TALEN is an acronym which stands for Transcription activator-

like effector nucleases. Unlike CRISPR/Cas9, which has protein and RNA compo-

nents, TALENS are protein based (Kadirvelu et al. 2002); Riggle and Kumamoto

2000). They are chimeric protein molecules consisting of DNA-binding modules

called TAL. TAL comprises 33–35 amino acids, differing only at the 12 and 13

positions, respectively, representing the DNA contact sites called Repeat variable

residues (DVDs) (Riggle and Kumamoto 2000; Fomina and Gadd 2014). DVDs

dictate the DNA binding speciﬁcity of each TALEN molecule; hence engineering

the TAL repeats harbouring the DVDs enable us to introduce the necessary modiﬁ-

cation required for gene alteration, thereby giving us the ﬂexibility to target different

sequence. It has the sensitivity of recognising a single base pair of the DNA, at a

time, in the host cell. Hence, TALENs is a ﬁve times more efﬁcient gene editing tool

than CRISPR/Cas9. It is made up of two functional domains-

•A DNA-recognition Transcription activator-like effector (TAL) domain

•A nuclease domain

TALENs are created by the fusion between DNA-recognition Transcription

activator-like effector (TAL) with DNA cleaving domains, i.e., the nuclease domain.

TAL effectors bind to the DNA sequences, activate its gene expression, and even

alter it. TAL effectors having the DVDs are engineered in such a way to show high

afﬁnity to a predetermined target DNA sequence in the host (Riggle and Kumamoto

2000; Fomina and Gadd 2014) (Table 2).

Tabl e 2 The table, as mentioned earlier, describes the different combinations of DVDs in the TAL

of TALENS and its target nucleotide in a DNA sequence

Target nucleotide RVD (Repeat variable Di-residues) References

Adenine Asparagine-isoleucine Boopathy (2000), Jaysankar et al.

(2008), Fomina and Gadd (2014)

Guanine Asparagine-Asparagine Boopathy (2000), Jaysankar et al.

(2008), Fomina and Gadd (2014)

Cytosine Histidine-aspartate Boopathy (2000), Jaysankar et al.

(2008), Fomina and Gadd (2014)

Thymine Asparagine-glycine Boopathy (2000), Jaysankar et al.

(2008), Fomina and Gadd (2014)

498 J. Bora et al.

Mechanism of Action of TALENs

The mechanism of action of TALENs for editing a genome is similar to the

CRISPR/cas9 method, where a DBS (Double-stranded break) is introduced in the

genome, and the cells respond to the break by repairing it via the two previously

mentioned mechanisms of DNA repair, i.e., error-prone nonhomologous end joining

(NHEJ) and high-ﬁdelity homology-directed repair (HDR). When the TALENs intro-

duced the break at the targeted site, the engineered TAL part having the right combi-

nation of DVDs targets the nucleotide and modiﬁes the host cell’s genome. Now, the

DBS breaks are ligated via any of the two repair mechanisms, and we ﬁnally get our

genetically modiﬁed organism (GMO) (Riggle and Kumamoto 2000; Fomina and

Gadd 2014).

6.3 ZFNs

ZFNs are a much more commonly used endonuclease for gene editing. It is a synthetic

protein, which means they are genetically engineered in a lab. Hence they can only

target and manipulate the speciﬁc targeted part of the organism’s genome, which

needs editing, thereby giving us accurate results and minimum chance of error. Hence,

ZFNs are “Highly Speciﬁc Genomic Scissors” (Smaranda et al. 2016; Kaniecki et al.

2018; Mulligan et al. 2001); Mu. The construction of ZFNs depends on the species of

the organism whose genome is being edited and the type of modiﬁcation that needs

to be done in its genome, i.e., insertion or deletion (Smaranda et al. 2016; Kaniecki

et al. 2018).

ZFNs stand for Zinc Finger Nucleases and are also classiﬁed as DNA binding

proteins, similar to TALENs. They are made up of two components which are-

•DNA binding domain is made of two modules (Zinc Finger Protein).

•DNA cleaving endonuclease domain, obtained from a Bacteria.

The DNA binding domain or the Zinc ﬁnger domains are the part of ZFNs

engineered in the lab to specify the DNA sequence it needs to bind in the host

organism. The zinc ﬁnger domain is a minimal protein motif with multiple ﬁnger-like

projections in their 3-D structure, making tandem contacts with their targeted DNA

sequences and binding zinc in its structure. This domain consists of two modules,

where each module recognises a unique hexamer of a 6 bp long DNA sequence in

the host cell. Both these modules, when fused as a single domain in the ZFNs, form

a ﬁnger-like structure called the Zinc Finger Protein (ZFP), and each ZFP shows a

unique speciﬁcity of up to 24 bp long DNA sequences in the genome (Smaranda

et al. 2016; Kaniecki et al. 2018).

The DNA cleaving endonuclease usually fuses with the ZFP while constructing

ZFNs is the Folk endonuclease enzyme. Folk endonuclease enzyme is obtained via

recombinant DNA technology, where the restriction endonuclease gene from the

bacterial strain Flavobacterium okeanokoites IFO 12,536 is ﬁrst introduced into the

Use of Genetic Engineering Approach in Bioremediation of Wastewater 499

host cell, where the gene expresses itself completely under suitable conditions. Then

the endonuclease is isolated from the host cell, puriﬁed and ﬁnally fused with the

ZFP t o give the ZFNs gene editing synthetic protein molecule (Kaniecki et al. 2018).

The Fo lk domain needs to dimerise to effectively introduce a Double Stranded

Break (DBS) in the host genome while gene editing. The dimerisation of the Fo lk

domain helps increase the speciﬁcity factor of the ZFNs, thereby reducing off-target

attacks on the host’s genome.

Mechanism of Action of ZFNs

ZFNs also work via the exact general mechanism as all the other gene editing tools,

i.e., by the introduction of the Double-Stranded Breaks (DBS) in the structure of the

host’s genome, which enables us to either insert our gene of interest at that speciﬁc

position or delete the speciﬁc targeted site, in order to get the modiﬁed version of the

original genome with enhanced capabilities and survival rates. The introduction of

a DBS break in the genomic DNA stimulates the natural DNA repair mechanism of

the cell, which includes- the nonhomologous end joining (NHEJ) followed in all the

phases of the cell cycle or the high-ﬁdelity homology-directed repair (HDR) followed

exclusively during the G2 and the S phase of the cell cycle (Boopathy 2000; Mulligan

et al. 2001). Both of these repair-mechanism work efﬁciently to accurately seal the

single-stranded overhangs at the end of DBS breaks, thereby completing the process

of gene editing. We must ensure that the repair mechanism is completed accurately

because an imprecise repair can also lead to the loss of nucleotides, including our

gene of interest (Fig. 3).

7 Types of Genetically Modiﬁed Microorganisms (GMM)

and Techniques they Apply for Bioremediation

With the previously mentioned gene editing tools, scientists and researchers have

created a wide array of genetically modiﬁed organisms and developed novel tech-

niques targeted explicitly for bioremediation and effective waste management, with

minimum side effects or addition of any unwanted pollutant in the environment

(Kadirvelu et al. 2002; Mulligan et al. 2001). The primary reason for a shift

towards adaptation of microorganism and biotechnology principles to carry out waste

management was expensive alternative and conventional strategies for remediation.

They showed destructive side-effects since these techniques involved the use of many

harmful chemicals which, when accumulated in the environment, contributed addi-

tionally to polluting the environment, i.e., conventional methods were less efﬁcient

and not ideal (Mulligan et al. 2001). Hence, with the discovery of the possibility for

quickly editing any genome, more complex models on the metabolism and working

of microorganisms were being created, with certain modiﬁcations. These changes

500 J. Bora et al.

Fig. 3 Above ﬁgure depicts the three genome editing nucleases (ZFNs, TALENs and

CRISPR/Cas9) that modify the genome by inducing DSBs (Double-stranded breaks) at the targeted

speciﬁc site requiring modiﬁcation. Now, these DSBs are repaired by the two cell repair pathways:

NHEJ and HDR. DSB repair by the NHEJ pathway leads to indels (Insertion or Deletion) in the

genome. Hence, when two DSBs in the genome carry out an insertion, deletion of the intervening

sequences is created, leading to the repair by the NHEJ pathway. In the HDR pathway, a donor DNA

template is required. When there is a donor repaired HDR DNA template, the gene responsible for

the HDR pathway induces a DSB at the desired locus (Kaniecki et al. 2018)

were introduced to enhance the uptake rate of contaminants, increase the degradation

rate of certain pollutants, the survival rate in non-ideal conditions, and reduce the

oxygen demand of microorganisms, which made them an ideal choice for treatment

of the daily generated waste products. Contaminated soil, water and even air could be

puriﬁed using genetically modiﬁed organisms (GMOs). There are different types of

genetically modiﬁed organisms belonging to different species of organisms, used for

detecting, degrading and cyclising pollutants (Dixit and Malaviya 2015; Avrilescu

2005; Gavrilescu et al. 2019).

7.1 Genetically Modiﬁed Biosensors for Pollutant Detection

Biosensors have been used by biologists for a very long time for tagging and identi-

fying certain chemicals, biological molecules and even a cell. Their application has

expanded to a wide array of industrial ﬁelds, ﬁnding their main application in the

food industry, where they help in quantifying the presence of alcohol, carbohydrates

and acids during the ongoing fermentation processes, as well as the pharmaceutical

Use of Genetic Engineering Approach in Bioremediation of Wastewater 501

industry, where they help in indicating drug production or presence of any unwanted

molecule of chemical or biological origin (Kaniecki et al. 2018; El Fantroussi and

Agathos 2005; Madhavi and Mohini 2012). Recent studies have expanded their use

in marine applications, where the level of eutrophication of the water bodies could

be detected by applying nitrite and nitrate sensors.

A biosensor is a device applied f or measurement of any reaction of either biolog-

ical or chemical origin, which works by the generation of signals, having the intensity

proportional to the concentration of the analyte in the reaction, where the analyte in

the reaction mixture is the substance which we are interested in for detection (Texier

et al. 1993; Roane et al. 2001). There are three different types of biosensors, based

on their target and mechanism-

•A biocatalytic group comprises enzymes.

•Bio-afﬁnity group, including antibodies and nucleic acids.

•Microbe-based containing microorganisms.

We will be more focused on the Microbe-based biosensors, which are exclusively

used for detecting certain pollutants for bioremediation (Texier et al. 1993; Roane

et al. 2001). Using biosensors, an environmentalist can analyse and assess the type

of pollutant present in the given environment and design and engineer any suitable

microorganism to degrade that pollutant (Mejáre and Bülow 2001; Wasilkowski

et al. 2012).

Usually, the original microbes currently in use as biosensors show some limi-

tations when working under certain environmental conditions. They either do not

remain viable long enough to show accurate signals or degrade due to unfavourable

environmental conditions. Hence, this is where genetic engineering comes in handy

(Mejáre and Bülow 2001; Wasilkowski et al. 2012). We can use any of the genes

mentioned above editing tools to modify the genome of the microbes, which are

already being used as biosensors, to impart capabilities to them, such that they can

not only survive but also perform efﬁciently under a wide variety of environmental

conditions, i.e., in extreme pH, temperature, acidic or essential environment.

In order to create a genetically modiﬁed organism, we usually use the

CRISPR/Cas9 technology due to its advantage over other gene editing tools being

cheap, simple and easy to apply by researchers as it can evaluate genetic interaction

and their related genotypic and phenotypic expression when replaced by another gene

of interest. Gene modiﬁcation for creating a biosensor is done by inserting an “indi-

cator gene” into the organism’s genome (Mulligan et al. 2001; Mejáre and Bülow

2001; Wasilkowski et al. 2012; Roane et al. 2001). The organisms usually selected

to serve as biosensors are bacterium since it has sensitivity detection of <0.2 mM.

Although, some strains of yeast, such as Yeast SPT1 and SPT2, can also be used

as biosensors. The presence of a pollutant and the environmental conditions make it

suitable for the indicator gene to be expressed, and the resulting protein represents

the detectible signal analysed and assessed.

502 J. Bora et al.

Indicator Genes Involved in Genetic Modiﬁcation of Biosensors

Luciferase is the most frequently used indicator gene for creating a genetically modi-

ﬁed microorganism. It belongs to the class of oxidative enzymes isolated from those

species of animal which produce bioluminescence. Hence, when luciferase is inserted

in the genome of a selected microorganism, it works with the promoter gene that

activates it in the presence of a speciﬁc pollutant, thereby generating a signal and

indicating its presence.

The second indicator gene for pollutant detection is a green ﬂuorescent protein

(GFP) (Paranthaman and Karthikeyan 2015; Jaysankar et al. 2008). This protein

is naturally extracted from jellyﬁsh, a marine life animal. GFP expresses a green

ﬂuorescence signal when exposed to the ray of U-V light, which ionises it. It is

made of 238 amino acids, out of which amino acids present at positions 65–67

(i.e. Ser–dehydroTyr–Gly) are responsible for reacting and producing visible green

ﬂuorescence under the U-V light. The gene expressing this protein can be inserted

in several microorganisms, alongside many different promoter sequences, which

activates this gene in the presence of different speciﬁc contaminants (Paranthaman

and Karthikeyan 2015; Jaysankar et al. 2008).

The third type of indicator gene inserted to modify the microorganism’s genome

for detection genetically is the lac-z gene. This is the most widely used indi-

cator/reporter gene for microbial and animal modelling systems (Paranthaman and

Karthikeyan 2015; Jaysankar et al. 2008). This gene is also inserted alongside

different 5’ regulatory elements of many genes, which are known to express when

exposed to different types of pollutants, speciﬁc to their activation (Paranthaman and

Karthikeyan 2015; Jaysankar et al. 2008).

Example of Biosensors

Some of the widely used biosensors for pollutant detection include.

(1) Thauera butanivorans- This bacterium is used as a biosensor for detecting 1-

butanol which poses a signiﬁcant threat to aquatic life. 1-butanol is said to be

easily biodegraded and can lead to oxygen depletion in the water bodies, ulti-

mately harming aquatic life. This biosensor is created in E coli using the σ

factor and promoter sequence present in this species. The promoter sequence

(PBMO) was attached at the 5’ end of the tetracycline resistance fusion gene

called TETA-GFP, a fusion of GFP protein and tetr gene. The σ factor (BMOR),

on the other hand, was expressed under the inﬂuence of the BMOR promoter

(PBMOR) (Jaysankar et al. 2008). Now, both the PBMO and PBMOR promoters

are activated upon the binding of 1-butanol. Under increased concentrations of

butanol, the σ factor (BMOR) caused an increase in the expression of TETA-

GFP, causing an ampliﬁcation in the signal generated by the GFP. This ampli-

ﬁed signal is recorded to sense the quantity and presence of butanol in the

environment (Jaysankar et al. 2008).

Use of Genetic Engineering Approach in Bioremediation of Wastewater 503

(2) R. erthropolis- This strain belongs to the class of gram-positive bacteria

belonging to the genus Rhodococcus. Environmentalists and industrialists are

widely using this strain to successfully identify 2,4-Dinitrophenol, which is

said to pose a harmful contaminant. R. erthropolis is a whole-cell biosensor

which feeds on the substrate 2,4-Dinitrophenol, as its sole nitrogen source.

The microorganism response to the presence of that pollutant is in the form of

increased biomass production (Shah et al. 2013). However, this strain for use

as a biosensor has become almost obsolete. However, the genes responsible

for identifying and detecting 2,4-Dinitrophenol, i.e., npdG and npdI genes, are

genetically inserted into another host to create a genetically modiﬁed organism

to attain a much greater sensitivity and efﬁciency towards detection and its

degradation. An example of such GEM includes B (Shah et al. 2013). cepacia

BRI6001L. 2,4-Dinitrophenol is a harmful air pollutant, which can lead to skin

lesions on human skin, Cardiovascular disorders, and even cause chronic effects

on the Central Nervous System (CNS) (Shah et al. 2013) (Table 3).

Tabl e 3 An overview of all the microorganisms potentially found as useful biosensors with target

detection in the environment

Target for detection Microorganism Limit of detection References

Bioavailable Copper P. ﬂuorescens DF57 with

a Tn5 lux AB promoter

probe transposon

0.3 ppm Park et al. (2013)

Bioavailable

Naphthalene

P. putida with NAH7m

plasmid and an

insertional gene fusion

between sal promoter

and the l ux AB genes

50–500 nM Park et al. (2013)

Ni+ and Co+2 Ralstonia eutropha

AE2515

0.1 μMfor Ni + 2 and

9 μMfor Co

+2

Park et al. (2013)

Bioavailable

Phosphorus

Synechococcus PCC

7942 reporter strain

0.03 μMPark et al. (2013)

Halogenated organic

acids

Recombinant E.coli had

DL-2-haloacid

dehalogenase encoding

thegeneand luxCDABE

genes

> 100 mg/l Park et al. (2013)

Tributyltin Bioluminescent

recombinant E.colir::

lux AB strain

0.02 μM i n synthetic

glucose medium and

1.5 μM

Park et al. (2013)

Hg+2 E.coli HMS174 was

harbouring mer-lux

plasmid pRB27 or

pRB28

0.2 ng/g Park et al. (2013)

504 J. Bora et al.

7.2 Bioaccumulation for Heavy Metal Removal Using

Genetically Modiﬁed Organisms

In essential words, Bioaccumulation is the simple process of accumulating foreign

substances by metabolically active living cells. These foreign substances include

harmful environmental pollutants such as pesticides, heavy metals, PCBs, PAH (poly-

cyclic aromatic hydrocarbons), and herbicides; which are absorbed directly by the

cells of microorganisms at a rate faster than the rate at which the substance is lost

or eliminated from its system by catabolism and excretion (Kehinde and Isaac 2016;

Phulpoto et al. 2016).

Heavy metals are the most toxic and harmful contaminants found in the environ-

ment. It is the cause of various chronic neurological diseases in the human body, as

well as the cause of death of many organisms (plants, land and water included) which

thrive on the water and soil contaminated by heavy metals. Heavy metal removal by

conventional technique is complicated, expensive, and a slow process; hence it is not

deemed economically viable. The alternative and the best option for remediation of

heavy metals was Bioaccumulation by genetically engineered microbes.

Bioaccumulation by the microorganisms comes in very useful for the removal of

heavy metal ions from the water bodies and land, which, if left unchecked, would

harm not only the aquatic life but also the humans who consume the crops growing on

these ﬁelds or the water and the ﬁshes ﬂoating in that water (Phulpoto et al. 2016). In

this essential process, microbial biomass makes use of certain speciﬁc proteins that

helps in creating a differential gradient, allowing the uptake of the metallic ions from

the water into its intercellular space. Now, these metal ions are utilised in various

cellular processes such as enzyme catalysis, signalling processes and stabilisation of

charges in many biomolecules. With the increasing living standards, and the need to

reduce the reliance on carbon emission products, protecting the environment from

various pollutants has become a priority worldwide. The demand for metal resources

increased by an average of 32% in 2021. Hence an efﬁcient system for curbing the

effects of this growing demand on the environment was needed. Hence, researchers

innovated with the idea of using the strains of microorganisms that actively uptake

metal ions as part of their natural metabolic system and genetically engineering it to

increase the efﬁciency and selectivity of uptake while maintaining a lower microbial

biomass growth (Phulpoto et al. 2016). Bioaccumulation has been thought to change

the dynamic of industries disposing of waste by providing an alternative, such that

this genetically engineered microorganism can not only remove but also recover

those heavy metal ions from the waste products (Kehinde and Isaac 2016; Phulpoto

et al. 2016; Aranda et al. 2010). Hence, these metal ions can then be reduced, puriﬁed

and reﬁned in the various downstream processing to obtain the metal in its pure form,

thereby diminishing the need for disposal (Phulpoto et al. 2016; Aranda et al. 2010).

First of all, we should know that the processes of biosorption and Bioaccumu-

lation are very different. Biosorption is the process of adsorption of the molecules

on the surface of the microorganism using physical interactions such as electrostatic

forces or chemical interactions such as Van der Waal’s forces, whereas, Bioaccumu-

lation is the metabolically active process of uptake of molecules around it into their

Use of Genetic Engineering Approach in Bioremediation of Wastewater 505

intercellular space by creation of a translocation pathway from the external to the

internal environment by the microorganism (Hesham et al. 2012; Aranda et al. 2010;

Sarang et al. 2013). Once accumulated in the intercellular space, these molecules are

sequestered or broken down by different proteins and peptide ligands.

Advantage of GEM (Genetically Engineered Microorganisms) Over

the Wild-Type Strains

Using GEM (Genetically engineered microorganisms) for Bioaccumulation over the

wild-type strains provides the advantage of being more robust than the latter. GEM

help bioaccumulates heavy metal ions, pesticides, herbicides, and PAH, beyond the

minimum inhibitory concentrations of wild-type strains. Since we know the wild

type or the edited strain of the microorganism applied for Bioaccumulation uses the

protein machinery which is controlled via the regulatory elements of the genome

which has evolved to show the capability to bioaccumulate, i.e., this capability was

not present in the primitive or the traditional form of this strain (Hesham et al. 2012;

Aranda et al. 2010; Sarang et al. 2013). Hence, when their genome is genetically

edited, we can insert additionally vital regulatory elements from different organisms

to better the uptake and sequestering system. These regulatory elements inserted can

be the promoter, ribosome binding sites, terminator or even a new protein-producing

gene. The choice of insertion is a researcher’s choice system to target the speciﬁcity

of the chemical that needs to be absorbed into the system. Thus, GEM provides easy

manipulation over the wild-type strain, offering superior control over the import-

storage system of the pollutants surrounding the GEM (Aranda et al. 2010; Sarang

et al. 2013). GEM is created by transferring the gene, mainly encoding this import-

storage system from the wild-type strain to a different host microorganism. This is

then simulated to test its growth and efﬁciency in the wastewater having single or

multiple efﬂuents with various pollutants for targeted and speciﬁc Bioaccumulation

(Hesham et al. 2012; Aranda et al. 2010; Sarang et al. 2013).

Working of a Genetically Engineered Microorganism

for Bioaccumulation (Import-Storage System)

Genetic engineering focuses on improving the uptake rate of pollutants by microor-

ganisms into their cytoplasm. Such microorganisms are prepared via recombinant

DNA technology, where a recombinant microorganism is prepared by genetically

engineering a synthetic import-storage system for Bioaccumulation and cycling of

the heavy metal ions and other chemicals from the environment. For creating such

a system, genes are selected from different organisms based on their speciﬁcity for

importing and storing certain chemicals, studied in simulations, and then combined

in a different host to give a Genetically modiﬁed microorganism (Phulpoto et al.

2016; Hesham et al. 2012; Aranda et al. 2010; Sarang et al. 2013).

506 J. Bora et al.

We start by selecting the importers for the import-storage system. Selection is

based on the efﬁciency with which those microorganisms having the appropriate

importers can uptake the chemical substances around them (Hesham et al. 2012;

Aranda et al. 2010; Sarang et al. 2013). For example, for creating a GEM involved

in the Bioaccumulation of Heavy metals from a polluted waterbody, we developed

our import-storage system speciﬁc to uptake such harmful heavy metals.

Importers

Importers fall under three classes: the channels, secondary carrier protein-producing

genes and active transporters.

•Channels- These are described as α-helical proteins that help in the passive diffu-

sion of heavy metal ions from its surrounding via the creation of a concentra-

tion gradient across the inner membrane. These are energy-independent proteins,

helping reduce the overall energy requirement by the microorganism for Bioac-

cumulation. Example-to increases the bioaccumulation rate of As3+ and Hg, and

we use channels such as the homotetramer glycerol facilitators (GlpF) obtained

from Escherichia coli, Corynebacterium diphtheria or Streptomyces coelicolor

(Kadirvelu et al. 2002). These channels have zero-energy requirements and are

the ideal choice for use as Bioaccumulation. However, in many cases, when the

concentration of heavy metal surrounding the GEM is very high, Bioaccumu-

lation must be carried out against the equilibrium concentration. To tackle this

problem, porins are used, which have β-barrels that form translocation pathways

working across the outer membrane of the GEM (Sarang et al. 2013; Kadirvelu

et al. 2002). One such porin widely used for the uptake of Ni ion is the FrpB4

channel obtained from Helicobacter pylori. We overexpress the divalent cation,

selecting porins to increase Ni’s uptake concentration.

•Secondary carriers- These carrier proteins also facilitate the active transport of

ions across the membrane. These carriers are again divided into three classes of-

uniporters, symporters and antiporters. Out of these three, symporters are used for

performing Bioaccumulation. Example- For uptake of Ni, Hg and Co, Nixa from

H. pylori and its homologs from Staphylococcus, Novosphingobium aromati-

civorans, and Rhodopseudomonas palustris have been used. These symporters

belong to the NiCoT family, which falls under the receptor superfamily called the

transporter-opsin-G-protein Receptor (Jaysankar et al. 2008; Sarang et al. 2013;

Kadirvelu et al. 2002). Symporters are driven by the PMF (Proton Motive Force),

where protons that generate the charge difference are used as co-substrates during

the targeted metal ion uptake. Hence, it can perform efﬁcient uptake of metal ions

from wastewater.

•Primary Active Transporters-These transporters belong to a group of multicompo-

nent protein complexes consisting of: the transmembrane component for allowing

the translocation across the membrane, an ATPase component (for phospho-

anhydride bond hydrolysis) which is an energy coupling component required in the

Use of Genetic Engineering Approach in Bioremediation of Wastewater 507

cytoplasm to thrust the translocation of substrates from the external environment,

and a periplasmic solute binding component (Sarang et al. 2013; Kadirvelu et al.

2002). These importers can also carry the ions against the equilibrium concen-

tration gradient using the energy derived from the hydrolysis of high-energy

compounds such as ATP and GTP. These transporters primarily help bioaccu-

mulate ions such as Cd and Cu. Primary active transporters such as MntA, and

cdtB found in Lactobacillus Plantarum and TcHMA3 found in the plant Thlaspi

caerulescens. are selected for the uptake of Cd, whereas for uptake of Cu involves

the transporter Copa from Enterobacter hirae. Both of the mentioned primary

active transporters belong to the P-type ATPase superfamily and hence utilise the

ATP reserves of the cells to carry out the import of these ions (Jaysankar et al.

2008; Kadirvelu et al. 2002).

Now, after the appropriate selection of the importer for the uptake of the heavy

metal ion from the wastewater, we have to design the proper storage system for the

GEM to store and sequester the heavy metal ions inside its system.

Genetically Engineered Metal-Binding Entities as Storage System

For designing a storage system, scientists have stressed developing synthetic cyto-

plasmic metal-binding entities for the proper and efﬁcient sequestering of the Heavy

metal ions. These metal-binding entities can be either the protein ligands or the

enzymes that produce peptides and other protein polymers for binding to the heavy

metal ions (Smaranda et al. 2016; Kaniecki et al. 2018). These entities provide efﬁ-

cient binding sites for heavy metal ions, which importers earlier transported into the

cytoplasm.

•Genetically encoded metal-binding proteins- The genes usually selected for genet-

ically coding these metal-binding proteins belong to the largest protein-producing

group. The protein is produced in the MT, a polyphyletic superfamily of the MBPs.

The MT protein used for the storage system is found in every species, be it the

prokaryotes, archaea or eukaryotes. They have a very high content of Cysteine

residue in their amino acid chains, which is a necessary component for properly

coordinated sequestration of the heavy metal ion by multiple MT proteins. MT

protein can provide a binding site to metal ions such as Zinc (Zn), Cadmium

(Cd), Copper (Cu), Mercury (Hg), Arsenic (As), and Nickel (Ni) and cobalt (Co)

(Kaniecki et al. 2018; El Fantroussi and Agathos 2005). Usually, the overexpres-

sion of MT can reduce its storage capacity; to overcome this limitation, MT protein

is fused with a soluble fusion partner such as the maltose-binding protein or the

glutathione-S-transferase. To avoid fusion or Cysteine-rich MTs to prevent over-

expression, histidine-rich Metal-Binding proteins are used. Such histidine-rich

proteins function as a natural storage system for heavy metal ions (Kaniecki et al.

2018; El Fantroussi and Agathos 2005). An example of histidine-rich MT protein-

Hpn obtained from H.pylori- is a characteristic Heavy metal storage protein that

allows binding sites for Nickel.

508 J. Bora et al.

Tabl e 4 Above the table is a list of the microbes and their gene modiﬁcation to uptake heavy metals

Heavy metals Microbes Modiﬁed gene expression References

Mercury Achromobacter sp

AO22

Mercury reductase expressing mer gene Ng et al.

(2009)

Cadmium,

Lead, Mercury,

Zinc

Pseudomonas

ﬂuorescens OS8;

Escherichia

coliMC1061;

Bacillus

subtilisBR151;

Staphylococcus

aureus RN4220

MerR/CadC/ZntR/Pmer/PcadA/PzntA Ng et al.

(2009)

Chromium (VI) Methylococcus

capsulatus (Bath)

CrR genes for Cr (VI) reductase activity Ng et al.

(2009)

Cadmium B. subtilis BR151

(pTOO24)

Luminescent Cadmium sensors Ng et al.

(2009)

Arsenic Sphingomonas

desiccabilis and

Bacillus Idriensis

strain

Overexpression of arsM gene Ng et al.

(2009)

•The enzyme produced Metal-Binding peptides and polymers- In this type of

storage system, a gene selected from a speciﬁc species is said to produce an

enzyme that is involved in mediating the production of small polymers and

peptides from raw materials already present in the cytosol of the microorganism’s

cells (Kaniecki et al. 2018; El Fantroussi and Agathos 2005). Such polymers

include phytochelatin, an oligomer of glutathione (GSH) (El Fantroussi and

Agathos 2005). This polymer is produced in two steps: in the ﬁrst step, there is a

ligation between the L-forms of cysteine and glutamate to give γ-glutamylcysteine

(γEC), which is ﬁnally followed by another ligation between the L-form of

glycine and γEC in the second step. These steps are mediated by ATP-Dependent

enzymes- ligase GshI and ligase GshII, respectively (Kaniecki et al. 2018;El

Fantroussi and Agathos 2005). A total of eleven such fused γECs can be added in

a sequential order to the GSH chain using the enzyme phytochelatin synthase (or

PCS), commonly found in plants showing resistance to heavy metals (Kaniecki

et al. 2018; El Fantroussi and Agathos 2005; Aranda et al. 2010). Although PCS

is not very efﬁcient in carrying out Bioaccumulation alone, its binding capacity

is increased by the overexpression of the enzymes cysE, GshI and GshII, respec-

tively, via genetic engineering principles. Another notable example is the produc-

tion of polyphosphate (polyP) ester by the enzyme polyphosphate kinase found

in Klebsiella pneumonia. PolyP is responsible for bioaccumulating Hg, As3+ ,Cu,

and Ni (Jaysankar et al. 2008; El Fantroussi and Agathos 2005; Aranda et al.

2010) (Table 4).

Use of Genetic Engineering Approach in Bioremediation of Wastewater 509

8 Challenges Faced During Gene Editing of a Microbe

Even though the concept of applying genetic engineering for the development of

genetically modiﬁed plants (GMP) and microorganisms (GMM) sounds easy, it does

bring many challenges. These challenges include the problems faced while the isola-

tion and selection of proper species for gene editing and the political challenges

faced after the formation and application of these GMM. Some of these prominent

challenges include-

•Bioethical issues- Sometimes, the changes introduced during genome manipula-

tion are not always advantageous and effective. Due to the insertion or deletion of

a particular gene or genes, the germline or the species might mutate, or an unde-

sirable change has been introduced unintentionally or even die and might lead

to extinction. Furthermore, in order for a successful and broad application of the

various gene editing tools for the creation and safe use of GMM and GMP in all

areas, the worldwide legislation needs the ﬁrst-hand opinion of the social and life

workers, the policymakers of each state, as well as, the stakeholders of the sectors

that whether the creation of GMM/GMP is an economically viable and safe option

or not (Perpetuo et al. 2011; Rajendran et al. 2003; Mejáre and Bülow 2001).

•Genetic contamination- The introduction of GMM/GMP in the environment also

leads to the genetic contamination of the naturally occurring species of organism

in the environment. This may include the plants growing or the microorganisms

involved in making the soil fertile (Mejáre and Bülow 2001; Wasilkowski et al.

2012; Demnerova et al. 2005). If a species in the environment that is sexually

compatible with the GMM/GMP is introduced, it might lead to interbreeding

among them. This will result in the disappearance of the novel traits introduced in

the GMM, and the genes might get transferred into the native occurring species.

This could be advantageous because speciﬁc tolerance abilities might develop

in the native species, and efﬁciency for carrying out their natural day-to-day

processes might also increase. However, introducing new genes might disturb

their relationship with their current habitat and eventually destroy the ecosystem

(Wasilkowski et al. 2012; Roane et al. 2001).

•Horizontal Transfer of Recombinant genes (HGT) to Native species- This is the

transfer of foreign genes introduced in the ecosystem from the newly introduced

GMM/GMP to the naturally occurring native species. The main concern for the

chance of occurrence of HGT is that it becomes impossible to eliminate these

interbred species if the phenotypic traits are harming the ecosystem (Wasilkowski

et al. 2012; Roane et al. 2001; Congeevaram et al. 2007). Such HGT can occur via

various methods such as transformation, conjugation or transduction. An example

of the harmful effects of the HGT on the ecosystem can be the transfer of antibiotic

resistance genes to a pathogen, potentially causing health hazards to humans

(Roane et al. 2001; Congeevaram, et al. 2007). This transfer results in novel trait

resistance to the antibiotic through pathogen, which could have been otherwise

destroyed by an appropriate antibiotic treatment (Roane et al. 2001; Congeevaram

et al. 2007; Chojnacka 2010).

510 J. Bora et al.

•Impact on the non-targeted organism- Selective pressure can increase between the

targeted species needing modiﬁcation and non-targeted species not to be disturbed.

The environment around the non-targeted species changes drastically due to the

foreign genes entering the ecosystem via the GMM/GMP, causing a shift in the

ecology, thereby increasing the survival pressure drastically on the non-targeted

species. They need to adapt to the new ecological changes, or else they might

not evolve and can become a distinct population (Blackmore and Reddish 1996;

Bharagava and Mishra 2018; Singh et al. 2014; Wasilkowski et al. 2012).

9 Conclusion and Future Perspective

According to various studies and surveys, bioremediation has been considered the

best alternative to other conventional techniques. Biotechnology application towards

improving living standards and controlling environmental pollution has been recog-

nised as one of our generation’s growing and future technology, opening new oppor-

tunities and providing the future groundwork for innovation and application in

various other ﬁelds. Furthermore, with bioinformatics and metagenomic studies,

it has become much easier to generate valuable data from soil and sea, to iden-

tify and isolate the source of new genes, which show different unique capabili-

ties, to be applied in developing transgenic organisms and microorganisms. Various

new approaches are being applied to make bioremediation more efﬁcient and more

straightforward, such as genome shufﬂing for generating cross-species organisms,

enzyme modiﬁcation to alter its activity and modiﬁcation in the metabolic pathway to

provide a minor energy spending route for bioremediation. Advancement for devel-

oping new ways for handling the genome of any organism is still going on in combi-

nation with the different ﬁelds of sciences aiding in this venture to ﬁnd common

ground.

Presently bioremediation using bacteria and other microorganisms is still in devel-

opment and is not widely used. Such limited application is the sole reason for the

difﬁculty for these engineered microorganisms to survive in an environment outside

the ideal lab conditions since no matter how many lab simulations these GMM can

survive, the natural environment always holds a different condition altogether. Lesser

survival rate could be due to the higher level of toxicity in the ground soil or the

constant change of season, which also changes the pH, temperature and soil fertility.

Hence, for these reasons, phytoremediation using transgenic plants has been pushed

as the current choice for bioremediation, owing to their ability to require less nutrient

input and protect water and soil from erosion. Genetic risk validation methods have

also made it possible to assess, analyse and proceed with caution to ensure the trans-

genes inserted in plants or microorganisms do not escape or alter the environment,

further contributing to pollution. The potential of phytoremediation by transgenic

plants exceeds the limitation of typical plants involved in remediation. Experiments

are also being conducted, giving suitable groundwork for the future development of

a technique capable of using transgenic plants and transgenic organisms to carry out

Use of Genetic Engineering Approach in Bioremediation of Wastewater 511

bioremediation at an efﬁciency level that has never been observed before. The combi-

nation being tested involved the plants for degrading the toxic contaminants from

the environment and the rhizospheric microorganisms working towards enhancing

the availability of hydrophobic compounds that can degrade a wide variety of toxic

chemicals mixed with pesticides, herbicides and dyes.

References

Aranda E, Ullrich R, Hofrichter M (2010) Conversion of polycyclic aromatic hydrocarbons, methyl

naphthalenes and dibenzofuran by two fungal peroxygenases. Biodegradation 21:267–281

Avrilescu M (2005) Fate of pesticides in the environment and its bioremediation. Eng Life Sci

5:497–526

Bharagava RN, Mishra S (2018) Hexavalent chromium reduction potential of Cellulosimicrobium

sp. isolated from common efﬂuent treatment plant of tannery industries. Ecotoxicol Environ Saf

147:102–109

Bhattacharya M, Guchhait S, Biswas D, Datta S (2015) Waste lubricating oil removal in a batch

reactor by mixed bacterial consortium: a kinetic study. Bioprocess Biosyst Eng 38:2095–2106

Blackmore R, Reddish A (1996) Global environmental issues Hodder and Stoughton, United

Kingdom

Boopathy R (2000) Factors limiting bioremediation technologies. Biores Technol 74:63–67

Burgess JE, Parsons SA, Stuetz RM (2001) Developments in odour control and waste gas treatment

biotechnology: a review. Biotechnol Adv 19:35–63

Chibuike G, Obiora S (2014) Heavy metal polluted soils: Effect on plants and bioremediation

methods. Appl Environ Soil Sci

Chojnacka K (2010) Biosorption and Bioaccumulation–the prospects for practical applications.

Environ Int 36(3):299–307

Congeevaram S, Dhanarani S et al (2007) Biosorption of chromium and Nickel by heavy metal

resistant fungal and bacterial isolates. J Hazardous Mater 146(1–2):270–277

Demnerova K, Mackova M, Spevakova V, Beranova K, Kochankova L et al (2005) Two approaches

to biological decontamination of groundwater and soil polluted by aromatics characterisation of

microbial populations. Int Microbiol 8:205–211

Dixit R, Malaviya D et al (2015) bioremediation of heavy metals from soil and aquatic environment:

an overview of principles and criteria of fundamental processes. Sustainability 7:2189–2212

El Fantroussi S, Agathos SN (2005) Is bioaugmentation a feasible strategy for pollutant removal

and site remediation? Curr Opin Microbiol 8:268–275

Fomina M, Gadd GM (2014) Biosorption: current perspectives on concept, deﬁnition and

application. Bioresour Technol 160:3–14

Gavrilescu M, Diaconu M, Bulgaria L et al (2019) Exploring and exploiting the abilities of microor-

ganisms and plants and their interactions for environmental bioremediation (in Romanian).

Performantica Publishing House, Iasi, Romania

Guo W, Pan B, Sakkiah S, Yavas G, Ge W, Zou W, Tong W, Hong H (2019) Persistent organic

pollutants in food: contamination sources, health effects and detection methods. Int J Environ

Res Public Health 16:4361

Hesham A, Khan S, Tao Y, Li D, Zhang Y et al (2012) Biodegradation of high molecular weight

PAHs using isolated yeast mixtures: application of metagenomic methods for community structure

analyses

Jaysankar D, Ramaiah N, Vardanyan L (2008) Detoxiﬁcation of toxic heavy metals by marine

bacteria highly resistant to mercury. Mar Biotechnol 10:471–477

Kadirvelu K, Senthilkumar P, Thamaraiselvi K, Subburam V (2002) Activated carbon prepared from

biomass as adsorbent: elimination of Ni (II) from aqueous solution. Bioresour Technol 81:87–90

512 J. Bora et al.

Kaniecki K, De Tullio L, Greene EC (2018) A change of view: homologous recombination at

single-molecule resolution. Nat Rev Genet

Karaouzas I, Kapetanaki N, Mentzafou A, Kanellopoulos TD, Skoulikidis N (2021) Heavy metal

contamination status in Greek surface waters: a review with application and evaluation of pollution

indices. Chemosphere 263:128192

Kehinde FO, Isaac SA (2016) Effectiveness of augmented consortia of Bacillus coagulans,

Citrobacter koseri and Serratia ﬁcaria in the degradation of diesel polluted soil supplemented

with pig dung. Afr J Microbiol Res 10:1637–1644

Kukumar S, Nirmala P (2016) Screening of diesel oil-degrading bacteria from petroleum

hydrocarbon contaminated soil. Int J Adv Res Biol Sci 3:18–22

Madhavi GN, Mohini DD (2012) Review paper on–parameters affecting bioremediation. Int J Life

Sci Pharma Res 2:77–80

Mejáre M, Bülow L (2001) Metal-binding proteins and peptides in bioremediation and phytoreme-

diation of heavy metals. Trends Biotechnol 19(2):67–73

Mulligan CN, Yong R, Gibbs BF (2001) Remediation technologies for metal contaminated soils

and groundwater: an evaluation. Eng Geol 60(1–4):193–207

Ng et al (2009), Bondarenko et al (2008), Hasin et al (2010), Ivask et al (2011), Liu et al (2011)

Panke et al (1998), Kumammru et al (1998), Reineke and Knackmuss (1979, 1980), Winter et al

(1989), Ramos et al (1987), Kumammru et al (1998), Rojo et al. (1987)

Paranthaman SR, Karthikeyan B (2015) Bioremediation of heavy metal in paper mill efﬂuent using

Pseudomonasspp. Int J Microbiol 1:1–5

Park M, Tsai SL, Chen W (2013) Microbial biosensors: engineered microorganisms as the sensing

machinery. Sensors 13(5):5777–5795

Perpetuo EA, Souza CB, Nascimento CAO (2011) Engineering bacteria for bioremediation. In:

Carpi A (ed) Progress in molecular and environmental bioengineering—from analysis and

modeling to technology applications. InTech, Rijeka, pp 605–632

Phulpoto H, Qazi MA, Mangi S, Ahmed S, Kanhar NA (2016) Biodegradation of oil-based paint

by Bacillus species monocultures isolated from the paint warehouses

Rajendran P, Muthukrishnan J, Gunasekaran P (2003) Microbes in heavy metal remediation. Indian

J Exp Biol 41:935–944

Rigét F, Biggert A, Braune B, Stow J, Wilson S (2010) Temporal trends of legacy POPs in Arctic

biota, an update. Sci Total Environ 408:2874–2884

Riggle PJ, Kumamoto CA (2000) Role of a Candida albicans P1-type ATPase in resistance to copper

and silver ion toxicity. J Bacteriol 182:4899–4905

Roane TM, Josephson KL, Pepper IL (2001) Dual-bioaugmentation strategy to enhance remediation

of contaminated soil. Appl Environ Microbiol 67(7):3208–3215

Sarang B, Richa K, Ram C (2013) Comparative study of bioremediation of hydrocarbon fuel. Int J

Biotechnol Bioeng Res 4:677–686

Shah MP, Patel KA, Nair SS, Darji AM (2013) Microbial degradation of Textile Dye (Remazol

Black B) by Bacillus spp. ETL-2012. J Biomed Biodeg 4:1–5

Singh RL (ed) (2017) Principles and Applications of Environmental Biotechnology for a Sustainable

Future. Springer, Singapore

Singh R, Singh P, Sharma R (2014) Microorganism as a tool of bioremediation technology for

cleaning environment: a review. Proc Int Acad Ecol Environ Sci 4(1):1–6

Skerker JM (2008) Rewiring the speciﬁcity of two-component signal transduction systems. Cell

Smaranda C, Hlihor R-M, Apostol LC, Bulgaria, et al (2016) Chapter 2. Biosorption and bioaccu-

mulation: Principles and potential applications in bioremediation. In: Biosorption and bioaccu-

mulation: principles and applications in environmental bioremediation. Politehnium Publishing

House, Iasi, Romania

Texier AC, Andres Y, le Cloirec P (1993) Selective biosorption of lanthanide (La, Eu, Yb) ions

by Pseudomonas aeruginosa. Environ Sci Technol 33:489–495

Use of Genetic Engineering Approach in Bioremediation of Wastewater 513

Wasilkowski D, Swedziol ˙

Z, Mrozik A (2012) The applicability of genetically modiﬁed microor-

ganisms in bioremediation of contaminated environments. Chemik 66(8):822–826

Yi Y, Yang Z, Zhang S (2011) Ecological risk assessment of heavy metals in sediment and human

health risk assessment of heavy metals in ﬁshes in the middle and lower reaches of the Yangtze

River basin. Environ Pollut 159:2575–2585

Nanotechnology for Bioremediation

of Heavy Metals

Anu Kumar, Bhanu Krishan, Shivani, Sunny Dhiman, and Akshita Sharma

Abstract Heavy metals do exist as an important source for industrial processes

such as textiles, power plants, and pesticide production plants but their discharge

in the environment is a serious concern because of their high toxicity and non-

biodegradable properties which can cause serious illness and dreadful effects to

humans and animals including aquatic habitats. Due to their adverse effect, removal

of heavy metals from any contaminated source through bioremediation with the

inclusion of Nanotechnology has been an eloquent method in recent times. Nanoma-

terials exhibit unique properties such as selectivity; relative small size due to which

they have been reported to act as an effective absorbent of heavy metals. Nanoma-

terials synthesized from microbes and fungi have been revealed to reduce the level

of heavy metals contamination through enzyme-based routes or through absorption.

Thus, this chapter will focus on different strategies employed using nanomaterials

for heavy metal degradation.

Keywords Biogenic nanoparticles ·Adsorption ·Heavy metals ·

Nanocomposites ·Nanoﬁltration ·Nano-phytoremediation

1 Introduction

Starting from the era of industrialisation, numerous problems have been associated

with the release of toxic contaminants into the environment. The main ﬁeld where

these contaminants are associated involves the natural water and soil sources which

are further directly associated with the risks involved to human health as well as

producing economic damages to the environment (Marcovecchio and Freije 2007).

One of the most concerning contaminants in the environment is the release of heavy

A. Kumar (B

) · B. Krishan · Shivani · S. Dhiman

University Institute of Biotechnology, Chandigarh University, Mohali-140 413, Gharuan, Punjab,

India

e-mail: anu.uibt@cumail.in

B. Krishan · Shivani · A. Sharma

GGDSD College, Sector 32-C, Chandigarh, India

M. P. Shah (ed.), Modern Approaches in Waste Bioremediation,

https://doi.org/10.1007/978-3-031-24086-7_24

515

516 A. Kumar et al.

metals in high concentrations. The elements possessing metallic properties with a

density greater than 5 g per cubic centimetre are deﬁned as heavy metals; due to

their toxicity these elements are also termed carcinogenic and well-known water-

soluble toxins (Gunatilake 2015). Heavy metals include Arsenic (As), Lead (Pb),

Selenium (Se), Tin (Sn), Copper (Cu), Silver (Ag), Zinc (Zn), Cadmium (Cd), Gold

(Au), Mercury (Hg), Molybdenum (Mb), Nickel (Ni), Iron (Fe), Chromium (Cr),

Manganese (Mn), Aluminium (Al), Thallium (Tl), Titanium (T), Thorium (Th) and

Cobalt (Co). According to US-EPA, As, Cd, Cr, Cu, Pb, Ni and Zn are characterized

as hazardous as well as toxic metals.

The release of such toxic metals into the environment is based upon two different

sources of origin (a) Natural origin which may include mineralisation, leaching of

ore and volcanism, (b) which includes the anthropogenic sources such as disposal of

waste, industrial waste efﬂuents, municipal waste and agricultural waste (Marcov-

ecchio and Freije 2007; Gunatilake 2015). The largest recognised release of heavy

metals resides near the urban and industrial areas. The industries associated with

the release of heavy metals include tannery, semiconductor, mining, and reﬁnement

industry. The utilisation of these toxins in the industrial process is associated with

shaping, forming, coating, and dyeing of products, metal surface treatment, installa-

tion of atomic energy and weaving of ﬁnished material into a ﬁnalised manufactured

product (Ayres 1992). The toxicity and effects of metals are dependent upon the

concentration and type of contaminant. Chromium is considered the dominant heavy

metal in efﬂuents,the use of Cr in industries is mainly associated with dyeing, paints,

fabrication of steel and textiles.

The ineffective treatment and release of untreated efﬂuent into water sources

containing a higher concentration of chromium can signiﬁcant changes in the aquatic

ecosystem and life form, resulting in accumulation in aquatic plants, algae, inverte-

brates and ﬁshes. Cr(VI) also has an adverse on humans which when present in the

bloodstream can vandalise the blood cells, ingestion of Cr(VI) in abundance causes

organ failure due to ruptured blood cells. Also, the accumulation of Cr in tannery

workers are reported to cause altered iron metabolism. Other adverse effects include

severe burns, systemic poisoning, eye damage, and respiratory problems (Singh et al.

2016). The presence of Zinc in water bodies is toxic to aquatic animals and upon

ingestion, by humans,it is responsible to cause fatigue and dizziness (Awofolu 2005).

Copper has the potential of forming a compound with sulphide ions which is directly

harmful to humans. Copper sulphide can lead to kidney dysfunction, disruption of

the capillaries, gastrointestinal, renal and respiratory anaemia (Gupta and Lutsenko

2009; SenthilKumar et al. 2011). A study was conducted on F344/N rats and B6C3F1

mice by the US National Toxicology program in order to determine the effect of

extreme concentration of CuSO4 present in drinking water. The highest concentra-

tion 45 mg Cu/kg BW/day for male rats and 31 mg Cu/kg BW/day for females

was found to be highly toxic with death and organ weight loss of the subjected

animals (Lum et al. 2021). The main sources for exposure to Nickel are drinking

water and food. Nickel, on the other hand, when ingested can cause an allergic reac-

tion such as dermatitis, lung ﬁbrosis, cardiovascular disease and cancer in the body

Nanotechnology for Bioremediation of Heavy Metals 517

(Duda-Chodak and Błaszczyk 2008). The standard conventional methods chosen

for the removal of these toxic heavy metals from wastewater includes bioremedi-

ation, solvent extraction, evaporation, ion-exchange, biosorption, advanced oxida-

tion processes, photocatalysis and reverse osmosis (Gode and Atalay 2008). These

processes are considered less efﬁcient for t he removal of such metals and create

a challenging issue of inadequate removal with a cost-effective process. Thus, to

resolve these challenges, principles of nanotechnology are applied in wastewater

treatment, ﬁltration to remove these metals from the environment even under minute

concentration.

2 Nanotechnology in Heavy Metal Removal

Nanotechnology is an offshoot that involves the principles of nano-sciences, i.e., the

study of structures and materials on a diminutive level. The study is based upon the

manipulation and application of nanomaterials, a wide class of matter that encom-

passes particulate solidity, having atmost 100 nm as one of its dimensions (Balaji

2019). The demand for nanomaterials has become evident in many empirical appli-

cations. To deﬁne nanomaterials, it can be said a substance that possesses at least one

dimension of lesser than 100 nm. The synthesis of such nanomaterials can be from

bulk i.e. etching or mechanical milling; the process is referred to as the top-down

approach. Another way is the bottom-up approach where nuclei are grown from

atoms or molecules that can further be synthesized into nanomaterials by employing

methods like green synthesis, laser pyrolysis, sol–gel process, spinning, etc. (Baig

et al. 2021). There are various unique properties of NMs which makes them feasible

for research studies. It is seen that nanomaterials may ﬂatter magnetic properties even

if their parent element is non-magnetic (Baig et al. 2021). The external magnetic ﬁeld

may help in exploiting these magnetic components. Magnetic nanoparticles have

divergent characteristics, for instance, super magnetism, high-ﬁeld irreversibility

and shifting loop after ﬁeld cooling (Sajid 2022). Nanomaterials acquire a high

surface area ratio in contrast to their bulk counterparts. Due to the mentioned prop-

erty, these NMs are highly beneﬁcial in adsorption, catalysis as well as thermal and

electrochemical sensing applications (Baig et al. 2021; Sajid 2022). Acting as a cata-

lyst these NMs in 2D conﬁguration provides an avenue for the catalyst to disperse

atomically on it, inﬂating the catalyst activity (Baig et al. 2021). Based upon the

size and shape these materials can be classiﬁed into carbon-based nanomaterials,

metallic nanoparticles, ceramic-based nanoparticles, polymeric nanomaterials and

biomolecules derived nanomaterials (Sajid 2022).

518 A. Kumar et al.

2.1 Metal and Metal Oxide Nanoparticles in Heavy Metals

Removal

Metal and metal oxide nanoparticles (NPs) are of great study these days, this is

because of the fact that the synthesis of such NPs is cost-effective, easy regeneration

and exhibit high adsorption capacity, some of the metal NPs are illustrated in Fig. 1.

Metal oxides such as Iron Oxide (Fe2O3) NP, Zinc oxide (ZnO) NPs, Nickel Oxide

(NiO) NPs, Copper oxide (CuO) NPs and Titanium Oxide (TiO2) are the extensively

applied nano-adsorbents for the removal of toxic heavy metals. The surfactant func-

tionalization of metal oxide NPs enhances the absorption capacity and is reported

to increase with the decrease in the size of NPs. The studies related to the use of

CuO, NiO and ZnO are found to efﬁciently remove heavy metals from the water

(Parvin and Rikta 2019) but the use of bare metallic NPs is a less common process

as the separation of NPs from the water is a deliberately difﬁcult task. Therefore, to

overcome this problem, capping and functionalization of nano-adsorbents are opted

to enhance the structural stability and enables the removal of NMs from water easily.

CuO NPs synthesized from natural sources as well as using precipitation method are

reported for an excellent potential for absorption of heavy metals. A study regarding

plant-mediated synthesis of CuO from Catharanthus leaf extract showed 2.11% and

2.91% removal efﬁciency for chromium and cadmium respectively when incubated

with stock solutions of respected metals for up to 24 h (Verma and Bharadvaja 2021).

Another study related to the green synthesis of CuO NPs from mint leaves and

orange peels showed the shreds of evidences for the removal of Pb(III), Ni(II) and

Cd(II) from contaminated water. The optimum uptake capacity was calculated after

the treatment of the metal oxide NPs with metal-contaminated water to determine

the adsorption capacity of the NPs. The optimum uptake capacity for Pb(III), Ni(II)

and Cd(II) was found to be 88.80, 54.90 and 15.60 mg/g with a sorbent dose of

0.33 g/L at pH 6 (Mahmoud et al. 2021). Iron oxide NPs with excellent adsorption

Fig. 1 Illustration of various metal and metal oxide NPs

Nanotechnology for Bioremediation of Heavy Metals 519

Fig. 2 Surface adsorption

phenomenon of NZVI

against toxic heavy metals

efﬁciency has been reported to adsorb Cr, Pb and Zn with an efﬁciency of 92.26%,

75.57% and 89.36% (Venkatraman and Priya 2021). Iron oxide NPs synthesized

from Taranjabin have been analysed to remove lead from wastewater with an

efﬁciency of 96.73% at 50 mg/L of the highest concentration (Miri et al. 2021).

Bimetal oxide NPs tends to show higher adsorption capacity than single metal

oxide NPs from wastewater. These NPs absorb the heavy metals by sites binding,

electrostatic interaction, and selective adsorption. Bimetal NPs such as Fe-magnetic

NPs has been found eloquent for the removal of Arsenic (As) from wastewater in

a short contact period by the interaction of the hydroxyl group of the NPs with As

(Parvin and Rikta 2019; Deliyanni et al. 2006).

Metal NPs such as nano-size zero-valent ions (NZVI) with improved stability are

found effective for the elimination of heavy metals. In a study, NZVI were entrapped

in a non-toxic biodegradable stabilizer composed of chitosan carboxymethyl β-

cyclodextrin complex was found to remove Cr6+ and Cu2+ , following the principle of

physisorption reduction of Cr6+ to Cr3+ , oxidising Fe0 to Fe3+ (illustrated in Fig. 2)

(Kanel et al. 2006). Other metal and metal oxide NPs which have been studied for

their extensive r emoval properties against toxic metals includes dendrimer nano-

absorbents, chitosan NPs, inorganic molecules functionalized NPs and polymer

functionalized NPs (Wadhawan et al. 2020).

3 Nanocomposites in Heavy Metal Removal

Nanocomposites are the inorganic or organic composite materials having one dimen-

sion less than a nanometre, these nano-scale composites can be classiﬁed into ﬁve

major groups which are (i) sol–gel composites, (ii) intercalation-type nanocompos-

ites, (iii) entrapment type, (iv) electroceramics and (v) structural ceramics. These

types of nanocomposites differ in production strategies and overall structural integrity

(Komarneni 1992). The approach that makes nanocomposites feasible for their

application in the removal of heavy metals is surface functionalization through the

introduction of functional groups such as hydroxyl, sulphates and animo groups.

These functional groups, therefore, supports the adsorption and chelation of the

520 A. Kumar et al.

heavy metals, thereby, increasing the adsorption capacity of the nanocomposites

(Wang et al. 2018). These NCs are incorporated with different solid substrates which

enhance the overall physical and chemical properties of the NCs. Solid substrates

such as carbon nanotubes, zeolites, graphene oxide, bentonite, magnesium oxide and

silica are studied for size control of magnetic iron oxide NPs (Hayati et al. 2018).

Because of the easy solid–liquid separation under magnetic ﬁeld and reusability,

various studies have been reported for nanocomposites in heavy metal removal from

wastewater. Magnetic carbon nanocomposites such as Zr-Magnetic organic frame-

works (MOFs) composites, these composited prepared in a study consisted of a

nano-sized Fe3O4@SiO2 core coated with Zr-MOFs, to study the efﬁciency of these

adsorbents for the heavy metals, Fe3O4@SiO2@UiO-66 and its amino derivatives

(Fe3O4@SiO2@UiO-66-NH2 and Fe3O4@SiO2@UiO-66-Urea) were also synthe-

sized. The obtained Zr-MOFs were reported to exhibit high adsorption capacity and

fast adsorption kinetics for metals ions (Huang et al. 2018).

Novel carbon nanotubes coated with poly-aminodoamine dendrimer have also

been reported to absorb As(III) and Co2+ and Zn2+ from aqueous solutions. The

reaction was carried out in a ﬁxed-bed column under controlled pH and ﬂow rate.

The maximum absorption observed was 432 mg/g for As(III) (Hayati et al. 2018).

Hydrothermal fabrications of TiO2-MoO

3 nanocomposites synthesized by precipi-

tation method were studied to remove selected heavy metals from standard solution.

The nanocomposites showed the highest adsorption of 59 mg/g for Cr(VI under

elevated room temperature, therefore, making it a considerable good choice for the

removal of toxic heavy metals (Zhao et al. 2018).

4 Nanoﬁltration and Nanoﬁlter Membranes

A recent advancement involving well-developed pressure-driven membrane ﬁltration

has been utilised for the separation of molecules from the liquid phase. The property

of NFs which makes it a special methodology for treatment and removal of heavy

metals is ﬁxed charge possessed due to the surface charge dissociation, the charge

groups can be either sulphonated or carboxyl groups. This property enables this

technique to be involved in the treatment of pharmaceutical waste, metal recovery

and removal of organic and inorganic pollutants present in surface water (Bowen and

Welfoot 2002; Abdel-Fatah 2018). Another property of NFs which makes them an

efﬁcient choice is of smaller pore size of membranes. With a high recovery rate and

low cost these NFs are studied for removal of heavy metals, studies have reported

92% of copper removal from the high volume of wastewater, with 150–35 mg/L of

COD removal and 80% salt rejection (Khedr 2008; Liu et al. 2008). The separation

mechanism involved for NF rejection is deﬁned by the following steps by Macoum

(Macoun 1997).

(a) Wetted Surface: The transport of molecules occurs through the membrane due

to the formation of hydrogen bonding between the water and the NF membrane.

Nanotechnology for Bioremediation of Heavy Metals 521

(b) Capillary rejection: This occurs due to the electrostatic repulsions between the

solution and membrane.

(c) Diffusion of solution: The solute and solvent gets dissolved in the active layer

of the membrane and diffusion of solution through the NF layer

(d) Charged capillary: Same charged ion gets attracted into the membrane and the

counter ions are rejected due to streaming.

(e) Finely porous: The dense material of NF is punctured by pores. Therefore,

partitioning between bulk and pore ﬂuid determines the transport.

A novel type of NF membrane synthesized using chitosan and 1,3,5-triglycidyl

isocyanurate gradient cross-link on polyethersulfone ultraﬁltration membrane

showed a high rejection performance to MgCl2, Na2SO4, MgSO4 and NaCl. This

positively charged membrane manifested superior permeability and mechanical

strength as compared to the earlier reported membranes (Yang et al. 2021). A cost-

effective NF composed of thin-ﬁlm composites were studied to separate selected

heavy metals from the solution. These optimized NF membranes successfully

rejected 93.9%, 97.9% and 87.7% of Cu2+ ,Mn

2+ and Cd2+ (Cheng et al. 2021).

Fe3O4 NP dispersed uniformly on the surface of MXene sheets was studied to exhibit

greater removal efﬁciency of heavy metals as compared to virgin MXene sheets. The

prepared NM membrane were found to achieve removal of 63.2%, 64.1% and 70.2%

removal of Cu2+ ,Cd

2+ and Cr6+ from wastewater. These membranes were reusable

after washing with HCl solution (Yang et al. 2021).

Application of NF in heavy metal treatment is an effective strategy because of its

operation at low pressure, high salt concentration tolerance and cost-effectiveness.

The only limitation to this method is membrane fouling due to colloidal substances

and high molecular weight components (Abdel-Fatah 2018).

5 Nanobioremediation and Nanophytoremediation

An emerging concept of Nanobioremediation (NBT) has been widely used for soil,

water and air pollutants removal and treatment. This technique involves the integra-

tion of nanotechnology and bioremediation for an efﬁcient treatment process under

minimum time as bioremediation alone is a time-consuming process. Thus, this

method has served potential beneﬁts in the removal of pollutants, organic compounds

and toxic metals (Carata et al. 2017). Metal-microbe interaction has excelled the

fabrication of various NPs with the involvement of microbial and plant extracts.

Such biogenic NPs are the hybrid strategy for the sustainable removal of toxic heavy

metals. The synthesis of Biogenic NPs (BNP) is accomplished through two different

techniques—biologically controlled mineralization, where the microbes control the

nucleation of intracellular BNPs and biologically induced mineralization, where the

synthesis of NPs takes place when metal ions are precipitated or reduced after inter-

acting with cell wall or membrane ﬁltrate (Misra and Ghosh Sachan 2021; Yue et al.

2016;Suetal.

2014). The synthesis of BNPs including their shape and size is greatly

522 A. Kumar et al.

Tabl e 1 The biogenic NPs summarized for the removal of toxic heavy metals

NPs type Microorganism/

Plant used

Heavy metal

removed

Removal efﬁciency Findings

CuS Shewanella

oneidensis MR-10

Cr6+ biosorption 94.1% Xiao et al. (2017)

CdS Pseudomonas

aeruginosa JP-11

Cd removal 88.7% Rajetal. (2016)

Pd Enterococcus

faecalis

Cr6+ reduction 100% Ha et al. (2016)

CuO Mint leaves and

orange leaves

extract

Pb2+ removal 88.80 mg/g uptake

capacity

Mahmoud et al.

(2021)

Ag Convolvulus

arvensis

Pb and Cd removal 82%, 77% Arjaghi et al.

(2021)

inﬂuenced by variables such as aeration, pH, temperature and metal ion concentra-

tion (Misra and Ghosh Sachan 2021). Several NPs synthesised biogenically from

microbes and plants have been studied for an eloquent eviction of heavy metals by

biosorption or reduction mechanism summarised in the table 1 (Misra and Ghosh

Sachan 2021).

Nano-phytoremediation (NPT) is a greater potential study against pollutants that

are poorly degraded by phytoremediation. This method is effectively been utilized

in the ﬁelds of textiles, paints and cosmetics for the treatment of contaminants that

includes heavy metals, organic and inorganic pollutants such as atrazine, PCBs,

PAHs and organic solvents. This novel strategy is based upon the percipience of

NPs by the plant roots, which upon entering the plant root transports in two ways-

Apoplastic transport (outside the plasma membrane and xylem vessels), Symplastic

transport (the water movement between cytoplasm and sieve tubes) (Srivastav et al.

2018; Roberts and Oparka 2003). The interaction between NPs and plants is studied

to stimulate propitious effects to the plant,AgNPs can increase the ABA and GA

phytohormones, whereas magnetic NPs can enhance the nitrogen assimilation and

improvement in plant metabolism. Also, these NPs lead to an increase in the chloro-

phyll amount and the seed germination ability of the plant (Srivastav et al. 2018).

Certain NPs assisted plant-based remediation studies are mentioned in the Table 2.

Although these biogenic NPs are eloquent in the control and degradation of heavy

metals, certain challenges that limit their usage. BNPs form reactive oxygen species

inside the microorganism that can lead to defective cellular structure and inhibition

of growth. In the case of nano-phytoremediation, an efﬁcacious degradation requires

long experimentation studies; also, these studies are suitable only for a moderate

level of toxic metal contamination.

Nanotechnology for Bioremediation of Heavy Metals 523

Tabl e 2 NMs utilisation with the plant in order to facilitate removal of heavy metal by combined

Nano-phytoremediation

Nanomaterial Plant species

involved

Heavy metal Removal efﬁciency Finding

Nano-scaled

zero-valent ions

Oak Cu immobilization 76%, 73% Slijepˇcevi´c

et al. (2021)

Salicylic acid

NPs

Isatiscappadocica Arsenic 705 ppm and

1188 ppm

accumulation of

metal in roots and

shoots of plant

Souri et al.

(2017)

Nano-scaled

zero-valent ions

Tradescantia

spathacea

Pb, Cd 73.7% Jesitha and

Harikumar

(2018)

Nano-scaled

zero-valent ions

Alternanthera

dentata

Pd, Cd 71.3% Jesitha and

Harikumar

(2018)

6 Conclusion

The most concerning contaminant in the environment is the release of heavy metals

in high concentrations. These heavy metals are toxic to the environment as well as

human health causing severe disease and long term health problems. The conven-

tional strategies exploited for the treatment and removal of these toxic metals are

less eloquent and found to release secondary toxic compounds. The employment

of nanotechnology in the ﬁelds of treatment and degradation is an effective process

that includes the exploitation of nanomaterials because of their unique properties.

Due to their small size and high adsorption capacity, these NMs targets speciﬁcally

these toxic metals and therefore, serve as an alternative for the removal of these

heavy metals. Metal and metal oxide nanoparticles such as CuO, Fe2O3, ZnO and

TiO2 synthesized from plants and chemically are studied for their high adsorption

capacity. Metal Oxide NPs are found to be highly eloquent for the removal of Cu,

Cr and Pb from wastewater samples. Also, NZVI which are effective metal nanopar-

ticles with unique magnetic properties is found to stabilize toxic metals from water

sources. Novel carbon nanotubes coated with poly-aminodoamine dendrimer and

nanocomposites composed of metal oxides are studied for removal of metals such

as, Co and Zn through adsorption under controlled pH and temperature with high

kinetic properties. The other effective and cost-effective process is the exploitation

of NF membranes which are highly porous with efﬁcient rejection capability. These

membranes are coated with NPs or equivalent particles which increase their efﬁ-

ciency in order to separate heavy metals from the solution. Nanobioremediation and

nanophytoremediation on the other hand is an emerging ﬁeld under the principles of

nanotechnology which aims to use the biogenic NPs synthesized through microbial

routes or exploited with their aid. Such NPs are non-toxic to the environment and

524 A. Kumar et al.

facilitate the complete removal of toxic heavy metals under controlled experimen-

tation. The exploitation of nanotechnology is not only focused on the removal but

recently these principles are used for sensing and detection of the toxic l evels of

contaminants which includes inorganic pollutants, organic pollutants, heavy metals

and xenobiotics. Thus, the ﬁeld of nanotechnology is a promising concept for the

pollutant-free environment.

References

Abdel-Fatah MA (2018) Nanoﬁltration systems and applications in wastewater treatment: review

article. Ain Shams Eng J 9:3077–92. https://www.sciencedirect.com/science/article/pii/S20904

47918300534

Arjaghi SK, Alasl MK, Sajjadi N, Fataei E, Rajaei GE (2021) Retracted article: green synthesis of

iron oxide nanoparticles by RS lichen extract and its application in removing heavy metals of

lead and cadmium. Biol Trace Elem Res 199:763–768. https://doi.org/10.1007/s12011-020-021

70-3

Awofolu OR (2005) A survey of trace metals in vegetation, soil and lower animal along some

selected major roads in metropolitan city of Lagos. Environ Monitor Assess 105:431–47. https://

doi.org/10.1007/s10661-005-4440-0

Ayres RU (1992) Toxic heavy metals: materials cycle optimization. Proc Natl Acad Sci 89:815–20.

https://doi.org/10.1073/pnas.89.3.815

Baig N, Kammakakam I, Falath W (2021) Nanomaterials: a review of synthesis methods, properties,

recent progress, and challenges. Mater Adv 2:1821–1871. http://xlink.rsc.org/?DOI=D0MA00

807A

Balaji S (2019) Nanobiotechnology: an overview. MJP Publisher

Bowen WR, Welfoot JS (2002) Modelling the performance of membrane nanoﬁltration—critical

assessment and model development. Chem Eng Sci 57:1121–37. https://www.sciencedirect.com/

science/article/pii/S0009250901004134

Carata E, Panzarini E, Dini L (2017) Environmental nanoremediation and electron microscopies.

Nanotechnologies for environmental remediation. Springer International Publishing, Cham, pp

115–136. https://doi.org/10.1007/978-3-319-53162-5_4

Cheng X, Lai C, Li J, Zhou W, Zhu X, Wang Z, et al. (2021) Toward enhancing desalination and

heavy metal removal of TFC nanoﬁltration membranes: a cost-effective interface temperature-

regulated interfacial polymerization. ACS Appl Mater Interfaces 13:57998–8010. https://doi.org/

10.1021/acsami.1c17783

Deliyanni EA, Nalbandian L, Matis KA (2006) Adsorptive removal of arsenites by a nanocrystalline

hybrid surfactant–akaganeite sorbent. J Colloid Interface Sci 302:458–66. https://linkinghub.els

evier.com/retrieve/pii/S0021979706006011

Duda-Chodak A, Błaszczyk U (2008) The impact of nickel on human health. J Elementol 13:685–

693

Gode F, Atalay ED, Pehlivan E (2008) Removal of Cr(VI) from aqueous solutions using modiﬁed

red pine sawdust. J Hazardous Mater 152:1201–1207. https://linkinghub.elsevier.com/retrieve/

pii/S030438940701151X

Gunatilake SK (2015) Methods of removing heavy metals from. J Multidiscip Eng Sci Stud Indust

Wastewater 1:13–8. www.jmess.org

Gupta A, Lutsenko S (2009) Human copper transporters: mechanism, role in human diseases and

therapeutic potential. Future Med Chem 1:1125–42.https://doi.org/10.4155/fmc.09.84

Nanotechnology for Bioremediation of Heavy Metals 525

Ha C, Zhu N, Shang R, Shi C, Cui J, Sohoo I, et al. (2016) Biorecovery of palladium as nanoparticles

by Enterococcus faecalis and its catalysis for chromate reduction. Chem Eng J 288:246–254.

https://www.sciencedirect.com/science/article/pii/S1385894715016782

Hayati B, Maleki A, Najaﬁ F, Gharibi F, McKay G, Gupta VK, et al. (2018) Heavy metal adsorption

using PAMAM/CNT nanocomposite from aqueous solution in batch and continuous ﬁxed bed

systems. Chem Eng J 346:258–70. https://www.sciencedirect.com/science/article/pii/S13858947

18305308

Huang L, He M, Chen B, Hu B (2018) Magnetic Zr-MOFs nanocomposites for rapid removal of

heavy metal ions and dyes from water. Chemosphere 199:435–44. https://www.sciencedirect.

com/science/article/pii/S0045653518302194

Jesitha K, Harikumar PS (2018) Application of nano-phytoremediation technology for soil polluted

with pesticide residues and heavy metals. Phytoremediation. Springer International Publishing,

Cham, pp 415–439. https://doi.org/10.1007/978-3-319-99651-6_18

Kanel SR, Grenèche J-M, Choi H (2006) Arsenic(V) Removal from groundwater using nano scale

zero-valent iron as a colloidal reactive barrier material. Environ Sci Technol 40:2045–50. https://

doi.org/10.1021/es0520924

Khedr MG (2008) Membrane methods in tailoring simpler, more efﬁcient, and cost effective wastew-

ater treatment alternatives. Desalination 222:135–145. https://www.sciencedirect.com/science/

article/pii/S0011916407007631

Komarneni S (1992) Feature article. Nanocomposites. J Mater Chem 2:1219. http://xlink.rsc.org/?

DOI=jm9920201219

Liu F, Zhang G, Meng Q, Zhang H (2008) Performance of nanoﬁltration and reverse osmosis

membranes in metal efﬂuent treatment* *Supported by the National Natural Science Founda-

tion of China (20476096, 20776133), Zhejiang Provincial Bureau of Science & Technology

(2005C33040) and Bureau of Edu. Chinese J Chem Eng 16:441–5. https://www.sciencedirect.

com/science/article/pii/S1004954108601020

Lum JS, Brown ML, Farrawell NE, McAlary L, Ly D, Chisholm CG, et al. (2021) CuATSM improves

motor function and extends survival but is not tolerated at a high dose in SOD1G93A mice with

a C57BL/6 background. Sci Rep 11:19392. https://doi.org/10.1038/s41598-021-98317-w

Macoun RG (1997) The mechanisms of ionic rejection in nanoﬁltration 1–408. http://www.uns

works.unsw.edu.au/primo_library/libweb/action/dlDisplay.do?vid=UNSWORKS&docId=uns

works_43106&fromSitemap=1&afterPDS=true

Mahmoud AED, Al-Qahtani KM, Alﬂaij SO, Al-Qahtani SF, Alsamhan FA (2021) Green copper

oxide nanoparticles for lead, nickel, and cadmium removal from contaminated water. Sci Rep

11:12547. https://doi.org/10.1038/s41598-021-91093-7

Marcovecchio J, Freije H (2007) Heavy metals, major metals, trace elements

Miri A, Najafzadeh H, Darroudi M, Miri MJ, Kouhbanani MAJ, Sarani M (2021) Iron oxide nanopar-

ticles: biosynthesis, magnetic behavior, cytotoxic effect. ChemistryOpen 10:327–33. https://doi.

org/10.1002/open.202000186

Misra M, Ghosh Sachan S (2021) Nanobioremediation of heavy metals: perspectives and challenges.

J Basic Microbiol https://doi.org/10.1002/jobm.202100384

Parvin F, Rikta SY (2019) Tareq SM. 8 - application of nanomaterials for the removal of heavy metal

from wastewater. In: Ahsan A, Ismail AFBT-N in W and WT (eds) Micro and nano technologies.

Elsevier, pp 137–57. https://www.sciencedirect.com/science/article/pii/B9780128139028000083

Raj R, Dalei K, Chakraborty J, Das S (2016) Extracellular polymeric substances of a marine

bacterium mediated synthesis of CdS nanoparticles for removal of cadmium from aqueous solu-

tion. J Colloid Interface Sci 462:166–175. https://www.sciencedirect.com/science/article/pii/S00

21979715302496

Roberts AG, Oparka KJ (2003) Plasmodesmata and the control of symplastic transport. Plant, Cell

Environ 26:103–24. https://doi.org/10.1046/j.1365-3040.2003.00950.x

Sajid M (2022) Nanomaterials: types, properties, recent advances, and toxicity concerns. Current

Opinion Environ Sci Health 25:100319. https://www.sciencedirect.com/science/article/pii/S24

6858442100091X

526 A. Kumar et al.

SenthilKumar P, Ramalingam S, Sathyaselvabala V, Kirupha SD, Sivanesan S (2011) Removal

of copper(II) ions from aqueous solution by adsorption using cashew nut shell. Desalination

266:63–71. https://linkinghub.elsevier.com/retrieve/pii/S0011916410005680

Singh R, Kumar M, Bishnoi NR (2016) Development of biomaterial for chromium (VI) detoxiﬁca-

tion using Aspergillus ﬂavus system supported with iron. Ecol Eng 91:31–40. https://linkinghub.

elsevier.com/retrieve/pii/S092585741630060X

Slijepˇcevi´c N, Pilipovi´c DT, Kerkez Ð, Krˇcmar D, Beˇceli ´c-Tomin M, Beljin J, et al. (2021) A cost

effective method for immobilization of Cu and Ni polluted river sediment with nZVI synthesized

from leaf extract. Chemosphere 263:127816. https://www.sciencedirect.com/science/article/pii/

S0045653520320117

Souri Z, Karimi N, Sarmadi M, Rostami E (2017) Salicylic acid nanoparticles (SANPs) improve

growth and phytoremediation efﬁciency of Isatis cappadocica Desv., under As stress. IET

Nanobiotechnol 11:650–655. https://doi.org/10.1049/iet-nbt.2016.0202

Srivastav A, Yadav KK, Yadav S, Gupta N, Singh JK, Katiyar R, et al. (2018) Nano-phytoremediation

of pollutants from contaminated soil environment: current scenario and future prospects. Phytore-

mediation. Springer International Publishing, Cham, pp 383–401. https://doi.org/10.1007/978-3-

319-99651-6_16

Su J, Deng L, Huang L, Guo S, Liu F, He J (2014) Catalytic oxidation of manganese(II) by multi-

copper oxidase CueO and characterization of the biogenic Mn oxide. Water Res 56:304–13.

https://www.sciencedirect.com/science/article/pii/S0043135414002036

Venkatraman Y, Priya AK (2021) Removal of heavy metal ion concentrations from the wastewater

using tobacco leaves coated with iron oxide nanoparticles. Int J Environ Sci Technol. https://doi.

org/10.1007/s13762-021-03202-8

Verma A, Bharadvaja N (2021) Plant-mediated synthesis and characterization of silver and copper

oxide nanoparticles: antibacterial and heavy metal removal activity. J Cluster Sci 2021. https://

doi.org/10.1007/s10876-021-02091-8

Wadhawan S, Jain A, Nayyar J, Mehta SK (2020) Role of nanomaterials as adsorbents in heavy

metal ion removal from waste water: a review. J Water Process Eng 33:101038. , https://www.sci

encedirect.com/science/article/pii/S221471441930529X

Wang B, Wu T, Angaiah S, Murugadoss V, Ryu J-E, Wujcik EK, et al. (2018) Development of

nanocomposite adsorbents for heavy metal removal from wastewater . ES Mater Manufact 35–44.

https://doi.org/10.30919/esmm5f17524

Xiao X, Liu Q-Y, Lu X-R, Li T-T, Feng X-L, Li Q, et al. (2017) Self-assembly of complex hollow

CuS nano/micro shell by an electrochemically active bacterium Shewanella oneidensis MR-1.

Int Biodeterior Biodegrad 116:10–6. https://www.sciencedirect.com/science/article/pii/S09648

30516304243

Yang B, Gu K, Wang S, Yi Z, Zhou Y, Gao C (2021) Chitosan nanoﬁltration membranes with

gradient cross-linking and improved mechanical performance for the removal of divalent salts

and heavy metal ions. Desalination 516:115200. https://www.sciencedirect.com/science/article/

pii/S001191642100271X

Yang X, Liu Y, Hu S, Yu F, He Z, Zeng G, et al. (2021) Construction of <scp> Fe 3 O 4</scp>

@ <scp>MXene composite</scp> nanoﬁltration membrane for heavy metal ions removal from

wastewater. Polym Adv Technol 32:1000–1010. https://doi.org/10.1002/pat.5148

Yue L, Wang J, Zhang Y, Qi S, Xin B (2016) Controllable biosynthesis of high-purity lead-sulﬁde

(PbS) nanocrystals by regulating the concentration of polyethylene glycol in microbial system.

Bioprocess Biosyst Eng 39:1839–1846. https://doi.org/10.1007/s00449-016-1658-x

Zhao Y, Sun Q, Luo J, Chen H, Cai W, Su X (2018) Hydrothermal fabrication of TiO2-MoO3

nanocomposites with superior performance for water treatment. Nano-Struct Nano-Objects

13:93–9. https://www.sciencedirect.com/science/article/pii/S2352507X17303505

Use of Genetic Engineering Approach in Bioremediation of Wastewater

Abstract

Recommended publications

Bioremediation: The Remedy to Expanding Pollution

Microbial Contamination of Environmental Waters and Wastewater: Detection Methods and Treatment Tech...

Removal of Heavy Metals from Industrial Wastewater Using Bioremediation Approach

Removal of Heavy Metals Using Bio-remedial Techniques