Toxicity and Waste

Management Using
Bioremediation

Ashok K. Rathoure
Vardan Environet Guargaon, India

Vinod K. Dhatwalia
Uttaranchal University, India

A volume in the Advances in Environmental

Engineering and Green Technologies (AEEGT)
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Editorial Advisory Board
I. O. Agbagwa, University of Port Hartcourt, Nigeria
Idress Hamad Attitalla, Omar Al-Mukhtar University, Libya
Arun Bhatt, GBPUA&T, Ranichauri Campus, India
Manoj Bhatt, G.B. Pant Engineering College, (Uttarakhand) India
B.S. Bisht, HNBGU (A Central University) (Uttarakhand), India
S. Choi, Pusan National University, South Korea
Tejpal Dhewa, University of Delhi, India
Nelson Durán, Chemical Institute, SP, Brazil
Silas Granatovillas-Boas, University of Auckland, New Zealand
A.M. Saxena, Lucknow University Lucknow, India
N. Singh, HNBGU (A Central University) (Uttarakhand), India
P. Soni, FRI, Dehradun, India
Meena Srivastava, MP Govt. (PG) College, Hardoi-241001 (UP), India
M.K. Tripathi, APPD, CIAE, Bhopal, India
Sandip Tripathi, NIMS University Jaipur, India
Rajib Roychowdhury, Centre for Biotechnology, Visva-Bharati Santiniketan, India
G T. P. Wong, Research Centre for Environmental Changes, Academia Sinica, Taiwan


Table of Contents

Foreword.............................................................................................................................................. xvi

Preface................................................................................................................................................. xvii

Acknowledgment............................................................................................................................... xxiv

Chapter 1
Heavy Metal Pollution: A Global Pollutant of Rising Concern.............................................................. 1
Ashita Sharma, Guru Nanak Dev University, India
Mandeep Kaur, Guru Nanak Dev University, India
Jatinder Kaur Katnoria, Guru Nanak Dev University, India
Avinash Kaur Nagpal, Guru Nanak Dev University, India

Chapter 2
Heavy Metal Pollution and its Management: Bioremediation of Heavy Metal..................................... 27
Ashok K. Rathoure, Vardan Environet Guargaon, India

Chapter 3
Biosorption of Dye Molecules............................................................................................................... 51
Aisha Zaman, Jadavpur University, India
Papita Das, Jadavpur University, India
Priya Banerjee, University of Calcutta, India

Chapter 4
Microbial Response against Metal Toxicity........................................................................................... 75
Jatindra Nath Bhakta, University of Kalyani, India

Chapter 5
Application of Genomics and Proteomics in Bioremediation............................................................... 97
Amol Uttam Hivrale, Shivaji University, India
Pankaj K. Pawar, Shivaji University, India
Niraj R. Rane, Shivaji University, India
Sanjay P. Govindwar, Shivaji University, India





Chapter 6
Genetically Engineered Microorganisms for Bioremediation Processes: GEMs for 
Bioremediaton...................................................................................................................................... 113
Stephen Rathinaraj Benjamin, Universidade Federal de federal de Goiás, Brazil
Fabio de Lima, Universidade Federal de Mato Grosso do Sul, Brazil
Ashok K. Rathoure, Vardan Environet Guargaon, India

Chapter 7
Novel Bioremediation Methods in Waste Management: Novel Bioremediation Methods.................. 141
Charu Gupta, Amity University, India
Dhan Prakash, Amity University, India

Chapter 8
Effective Management of Agro-Industrial Residues as Composting in Mushroom Industry and
Utilization of Spent Mushroom Substrate for Bioremediation............................................................ 158
Rajender Singh, Directorate of Mushroom Research (ICAR) Solan, India
Mamta Chauhan, Jaypee University of Information and Technology, India

Chapter 9
Bioremediation Approaches for Recalcitrant Pollutants: Potentiality, Successes and Limitation....... 178
Bikram Basak, National Institute of Technology Durgapur, India
Apurba Dey, National Institute of Technology Durgapur, India

Chapter 10
Biodegradation of Phenol: Mechanisms and Applications.................................................................. 198
Vinod K. Dhatwalia, Uttaranchal University, India
Manisha Nanda, Dolphin (PG) Institute of Biomedical and Natural Sciences, India

Chapter 11
Phyto-Remediation: Using Plants to Clean Up Soils: Phyto-Remediation.......................................... 215
Swati Jagdale, MAEER’s Maharashtra Institute of Pharmacy, India
Aniruddha Chabukswar, MAEER’s Maharashtra Institute of Pharmacy, India

Chapter 12
A Prospective Study on Emerging Role of Phytoremediation by Endophytic Microorganisms.......... 236
Amita Verma, Sam Higginbottom Institute of Agriculture, Technology and Sciences (Deemed
University), India
Parjanya Kumar Shukla, Sam Higginbottom Institute of Agriculture, Technology and
Sciences (Deemed University), India

Chapter 13
Advances in Bioremediation for Removal of Toxic Dye from Different Streams of Wastewater....... 266
Priya Banerjee, University of Calcutta, India
Aniruddha Mukhopadhayay, University of Calcutta, India
Papita Das, Jadavpur University, India
 

Chapter 14
Decolorization of Direct Blue: 14 Dye by Thermoalkalophilic Aerobic Bacillus sp.......................... 279
Shankara S., Government College for Women, Chintamani, India
Kotresha Dupadahalli, KSPL Degree College, India
Vijayakumar M. H., Gulbarga University, India
Gaddad S. M., Gulbarga University, India

Chapter 15
Fighting Ecomafias: The Role of Biotech Networks in Achieving Sustainability............................... 295
Nadia Di Paola, University of Naples Federico II, Italy
Rosanna Spanò, University of Naples Federico II, Italy
Adele Caldarelli, University of Naples Federico II, Italy
Roberto Vona, University of Naples Federico II, Italy

Chapter 16
Effective Waste Water Treatment and its Management....................................................................... 312
Sakthivel Lakshmana Prabu, Anna University, BIT Campus, Tiruchirappalli, India
TNK Suriyaprakash, Al Shifa College of Pharmacy, India
Ruckmani Kandasamy, Anna University, BIT Campus, Tiruchirappalli, India
Thirumurugan Rathinasabapathy, International Medical University (IMU), Malaysia

Compilation of References................................................................................................................ 335

About the Contributors..................................................................................................................... 412

Index.................................................................................................................................................... 418
Detailed Table of Contents

Foreword.............................................................................................................................................. xvi

Preface................................................................................................................................................. xvii

Acknowledgment............................................................................................................................... xxiv

Chapter 1
Heavy Metal Pollution: A Global Pollutant of Rising Concern.............................................................. 1
Ashita Sharma, Guru Nanak Dev University, India
Mandeep Kaur, Guru Nanak Dev University, India
Jatinder Kaur Katnoria, Guru Nanak Dev University, India
Avinash Kaur Nagpal, Guru Nanak Dev University, India

The chapter covers various issues related to heavy metals. Here we attempt to document the possible
definitions for heavy metals. Heavy metals, the elements having density higher than 3.5 g/cm2 are being
added at high rate to our close vicinity. These metals lead to serious problems related to ecology and
mankind. Toxic effects of heavy metals are dependent on the concentration of metals, reactivity of metal
species and duration of exposure. There is a need to address the toxicological and remedial aspects of
heavy metals.

Chapter 2
Heavy Metal Pollution and its Management: Bioremediation of Heavy Metal..................................... 27
Ashok K. Rathoure, Vardan Environet Guargaon, India

Environmental degradation has become a major societal issue thanks to uncontrolled anthropogenic
activity, besides natural factors. Entry of toxic heavy metals and minerals in human system mainly
through contaminated water, food and air, leads to overt and insidious health problems. Heavy metal
pollution, a global concern today, can be managed by using bioremediation, an eco-friendly alternative.
Bioremediation is one of the most promising technological approaches to the problem of hazardous
waste. It is a technology for removing pollution from environment, restoring contaminated site and
preventing future pollution. Bioremediation can be performed in situ or ex situ. Microorganisms directly
degrade contaminants rather than merely transferring them from one medium to another, employ
metabolic degradation pathways and can be used in situ to minimize disturbance of the cleanup site.
Hence, microorganisms can be effective, economical and non-disruptive tools for eliminating hazardous
chemicals. Its advantage generally outweigh the disadvantage, therefore may be used as management tool.


 

Chapter 3
Biosorption of Dye Molecules............................................................................................................... 51
Aisha Zaman, Jadavpur University, India
Papita Das, Jadavpur University, India
Priya Banerjee, University of Calcutta, India

Water contamination due to dyes has drawn increased attention. Dyes in water bodies are greatly
perceptible and pose tremendous threat to ecosystem. Thus removal of such dye molecules is a matter
of concern. In the past various physical and chemical techniques have been employed for the removal of
colour from wastewater. However most of these methods have certain drawbacks. Biological treatment
is often efficient and economical. Many microorganisms are able to accumulate and degrade different
pollutants. Yet even the biological methods have some shortcomings such as toxicity of biodegradation
products and more.

Chapter 4
Microbial Response against Metal Toxicity........................................................................................... 75
Jatindra Nath Bhakta, University of Kalyani, India

Damage of microbial communities caused by metal toxicity in different domains of the environment is a
growing challenge worldwide. The present chapter attempted to elucidate how microorganisms tackle and
response against the metals toxicity. In response to metal toxicity, microorganisms exhibit a vast array of
acclimatization, adaptation and resistant strategies at genetic, cellular and community levels to detoxify
metals toxicity and survival. Microorganisms detoxify metals by various mechanisms (sequestration,
inhibition of influx, efflux, accumulation, precipitation and chemical modification, repair, and metabolic
by-pass) and showed resistance properties (by protein/enzyme synthesis) encoded by genes located in
chromosome, plasmid or transposon. Thus, metal toxicity hampers the microbial metabolism, growth,
activity and species diversity resulting in severe damage in environmental microbial community. Apart
from detrimental consequences of metal toxicity, the novel metal- and antibiotic- resistant microorganisms
could be used in environmental and human health benefits.

Chapter 5
Application of Genomics and Proteomics in Bioremediation............................................................... 97
Amol Uttam Hivrale, Shivaji University, India
Pankaj K. Pawar, Shivaji University, India
Niraj R. Rane, Shivaji University, India
Sanjay P. Govindwar, Shivaji University, India

Bioremediation mediated by microorganisms is proving to be cost effective, ecofriendly and sustainable

technology. Genome enable experimental and modeling techniques are of a great help in evaluating
physiology and enhancing performance of life forms to be used for bioremediation purpose. Similarly, the
application of proteomics in bioremediation research provides a global view of the protein composition
of microbial cell and offers promising approach to understand the molecular mechanism of removal of
toxic material from the environment. Combination of proteomics and genomics in bioremediation is an
insight into global metabolic and regulatory network that can enhance the understanding of gene functions.
Present chapter give a bird’s eye view of genomics and proteomics and their potential utilization in
bioremediation and for the clearer understanding of the cellular responses to environmental stimuli. An
understanding of the growth conditions governing the expression of proteome in a specific environment
is essential for developing rational strategies for successful bioremediation.


Chapter 6
Genetically Engineered Microorganisms for Bioremediation Processes: GEMs for 
Bioremediaton...................................................................................................................................... 113
Stephen Rathinaraj Benjamin, Universidade Federal de federal de Goiás, Brazil
Fabio de Lima, Universidade Federal de Mato Grosso do Sul, Brazil
Ashok K. Rathoure, Vardan Environet Guargaon, India

In the past few decades, environmental pollution is a major issue which affects biodiversity public
health and eco systems present in worldwide, nowadays, microbial potential are connected to effect the
clean-up of environmental pollutants. Conventional methods are focus on the separation, rather than the
destruction of contaminants, the use of genetically engineered microorganisms for bioremediation would
be an alternative, environmentally friendly, more effectiveness and economical clean-up technique for the
remediation of pollutants in present in contaminated sites. A combined strategies relationship between
genetic engineered microbes and bioremediation can enhance the effectiveness of contaminants sites.
Here, we have elaborated recent work on the investigation and improvement of these microbes using
genetic tools and given an outlook of what may be possible in the near future.

Chapter 7
Novel Bioremediation Methods in Waste Management: Novel Bioremediation Methods.................. 141
Charu Gupta, Amity University, India
Dhan Prakash, Amity University, India

Bioremediation technologies are one of the novel methods in the field of waste and environment
management and are presently gaining immense credibility for being eco-compatible. Bioremediation
using microbes has been well accepted as an environment friendly and economical treatment method for
disposal of hazardous petroleum hydrocarbon contaminated waste (oily waste). Besides this, earthworms
can be used to extract toxic heavy metals, including cadmium and lead, from solid waste from domestic
refuse collection and waste from vegetable and flower markets. Other novel methods used recently for
treatment of wastes are plasma incineration or plasma assisted gasification and pyrolysis technology.
The technologies applied for conditioning include ultrasonic degradation, chemical degradation, enzyme
addition, electro-coagulation and biological cell destruction. Genetic engineering is another method
for improving bioremediation of heavy metals and organic pollutants. Transgenic plants and associated
bacteria constitute a new generation of genetically modified organisms for bioremediation.

Chapter 8
Effective Management of Agro-Industrial Residues as Composting in Mushroom Industry and
Utilization of Spent Mushroom Substrate for Bioremediation............................................................ 158
Rajender Singh, Directorate of Mushroom Research (ICAR) Solan, India
Mamta Chauhan, Jaypee University of Information and Technology, India

Different types of edible mushrooms like Agaricus, bisporus, A. bitoriqus, Pleurotus spp., Volvariella
volvacea, Lentinula edodes, Calocybe indica, Flamullina, Ganoderma lucidum etc. are cultivated in
industrial scale. Majority of edible fungi secretes extracellular Ligninocellulolytic enzymes like Laccase,
lignin peroxidase, manganese peroxidase, cellulase etc. for effective conversion of ligninocellulolytic
substrate to compositing form which led to fruiting of mushrooms. Consequently, an adequate disposal
method is needed for the high quantities of spent mushroom substrate (SMS) generated in this agro-
food industrial activity. On the other side, textile industry among the largest water consuming industries
 

in the world and approximately, 10,000 different dyes and pigments are used at industrial scale. It is
estimated that nearly 40% of the total dyes used in the dyeing process may find their way in wastewater.
So, there is an attempt to utilize the ligninolytic enzymes rich SMS of different mushroom for efficiently
biodegradation of textile wastewater & polyaromatic pollutants.

Chapter 9
Bioremediation Approaches for Recalcitrant Pollutants: Potentiality, Successes and Limitation....... 178
Bikram Basak, National Institute of Technology Durgapur, India
Apurba Dey, National Institute of Technology Durgapur, India

The different chemical pollutants discharged by the industries to the environment can upset the delicate
balance of the ecosystem. Bioremediation, the use of microorganisms and plants to remediate polluted
environments, is a promising and growing area of environmental biotechnology. Bioremediation
options encompass diverse types of biotechnological mechanisms that may lead to a target pollutant’s
mineralization, partial transformation, humification, or altered redox state. The use of extra cellular and/
or cell-free enzymes has been also proposed as an innovative remediation technique. Perspectives and
limitations to evolve and use this technology are critically discussed in this chapter with respect to the
complexity of mixtures of xenobiotics often found in practice. Whereas the potential of bioremediation
is substantial, its application has important limitations that are apparent from many examples and the
authors feel that these limitations can be overcome only when adequate attention is directed to fundamental
microbiological, chemical and engineering issues.

Chapter 10
Biodegradation of Phenol: Mechanisms and Applications.................................................................. 198
Vinod K. Dhatwalia, Uttaranchal University, India
Manisha Nanda, Dolphin (PG) Institute of Biomedical and Natural Sciences, India

Aromatic compounds are widely distributed in nature. Free phenols are frequently liberated as metabolic
intermediates during the degradation of plant materials. In recent years the natural supply of phenolic
substances has been greatly increased due to the release of industrial byproducts into the environment.
Phenolic compounds are hazardous pollutants that are toxic at relatively low concentration. Effluents
from petrochemical, textile and coal industries contain phenolic compounds in very high concentration;
therefore there is a necessity to remove phenolic compounds from the environment. Microorganisms
capable of degrading phenol are common and include both aerobes and anaerobes. The use of microbial
catalysts in the biodegradation of organic compounds has advanced significantly during the past three
decades. The efficiency of biodegradation of organic compounds is influenced by the type of the organic
pollutant, the nature of the organism, the enzyme involved, the mechanism of degradation and the nature
of the influencing factors.


Chapter 11
Phyto-Remediation: Using Plants to Clean Up Soils: Phyto-Remediation.......................................... 215
Swati Jagdale, MAEER’s Maharashtra Institute of Pharmacy, India
Aniruddha Chabukswar, MAEER’s Maharashtra Institute of Pharmacy, India

In this chapter authors have discussed the role of plants to develop contaminant free environment. This
concept is also known as Phytoremediation. Phytoremediation is a word formed from the Greek prefix
“phyto” meaning plant, and the Latin suffix “remedium” meaning to clean or restore. This technology has
been receiving attention lately as an innovative, cost-effective alternative to the more established treatment
methods used at hazardous waste sites. Phytoremediation can be classified into different applications,
such as phytofiltration or rhizofiltration, phytostabilization, phytovolatilization, phytodegradation and
phyto-extraction etc. The chapter will deal with phytoremediation, its advantages, limitations and in
detail techniques of classification and application.

Chapter 12
A Prospective Study on Emerging Role of Phytoremediation by Endophytic Microorganisms.......... 236
Amita Verma, Sam Higginbottom Institute of Agriculture, Technology and Sciences (Deemed
University), India
Parjanya Kumar Shukla, Sam Higginbottom Institute of Agriculture, Technology and
Sciences (Deemed University), India

Colonies of endophytes are excellent example of beneficial association with most plants in their natural
state. Endophytic colonies and plant associations are beneficial in many ways such as supplying biologically
fixed nitrogen, regulation of phytohormone production thus enhancing the plant growth, resistance to
environmental stress etc. these associations are also important for the agriculture and industries because
they produce important medicinal, agriculture and industrial compounds as endophytic metabolites.
When we concern about the waste management, degradation and biotransformation of several toxins,
the phytoremediation by using endophytes has been developed as important tool. Current chapter
reviles, study and collect most of important knowledge, recent ongoing research, technologies, roles
and advancements in biodegradation and biotransformation of different types of toxic wastes and their
effects on environment with phytoremediation by endophytes.

Chapter 13
Advances in Bioremediation for Removal of Toxic Dye from Different Streams of Wastewater....... 266
Priya Banerjee, University of Calcutta, India
Aniruddha Mukhopadhayay, University of Calcutta, India
Papita Das, Jadavpur University, India

Azo dyes are used in abundance in several industries like textile, printing, paper, plastic, cosmetics, paints,
etc. Extensive discharge of such dyes in adjacent water bodies has raised much environmental concern.
Azo dyes are toxic to living organisms and their genotoxic and carcinogenic potentials are intensified on
being released as mixtures. In the recent years, various microorganisms have been isolated and reported
 

to possess tremendous potential for efficient dye degradation. However, the process of bioremediation
is highly controlled by experimental factors like effluent pH, temperature and concentration of dyes
in solution. Therefore, appropriate optimization of these factors is to be determined in order to ensure
maximum efficiency of this process. This review highlights application of immobilization techniques
of bacterial cells for achievement of successful biodegradation. In this study, the existing problems of
dye pollution and possible improvisations for obtaining enhanced bioremediation of dyes have also been
discussed.

Chapter 14
Decolorization of Direct Blue: 14 Dye by Thermoalkalophilic Aerobic Bacillus sp.......................... 279
Shankara S., Government College for Women, Chintamani, India
Kotresha Dupadahalli, KSPL Degree College, India
Vijayakumar M. H., Gulbarga University, India
Gaddad S. M., Gulbarga University, India

A thermo-alkalophilic bacterium isolated from textile mill effluent samples and identified as a Bacillus
sp., on the basis of biochemical tests. The selected bacterium showed high decolorization activity in
static condition as compared to shaking condition and the maximum 1000 mg l-1 Direct Blue-14 dye
decolorization was takes place in 72 h. The optimum physical parameters such as temperature 40-50 °C,
pH 8.0 with 2.5% (w/v) of nitrogen source and 4% (w/v) glucose were required for the decolorization
of Direct Blue-14 from this bacterium. UV–Visible analyses and colorless bacterial cells suggested
that Bacillus sp. exhibited decolorizing activity through biodegradation, rather than inactive surface
adsorption. The degraded dye metabolites are analyzed by TLC and diazotization, carbylamines, Ames
test for individual metabolite indicates biotransformation of Direct Blue-14 into aromatic amine and
non-toxic aromatic metabolites. These results suggest that the isolated organism Bacillus sp. as a useful
tool to treat waste water containing azo dyes at static condition.

Chapter 15
Fighting Ecomafias: The Role of Biotech Networks in Achieving Sustainability............................... 295
Nadia Di Paola, University of Naples Federico II, Italy
Rosanna Spanò, University of Naples Federico II, Italy
Adele Caldarelli, University of Naples Federico II, Italy
Roberto Vona, University of Naples Federico II, Italy

Innovation processes are becoming increasingly central, and newer industries have become already less
resource-intensive in comparison with the traditional ones. Nevertheless, this alone does not assure
sustainability, which requires a step further towards economically viable, environmentally compatible,
and socially responsible behaviours. This chapter addresses the issues relating to sustainable development
to provide a critical discussion on the potential role played by networking relationships in the biotech
field. For the purposes of the study, we employ the co-management and multi-stakeholder perspectives.
We demonstrate that the biotechnology research results may be enhanced thanks to cooperation dynamics
and interactions among heterogeneous actors, with undeniable cultural and social positive impacts. Also,
we discuss social implications and open concerns, both with regard to the relationships within innovative
networks and between institutional professional actors, allowing the identification of any grey areas and
limitations, especially relevant to policy makers.


Chapter 16
Effective Waste Water Treatment and its Management....................................................................... 312
Sakthivel Lakshmana Prabu, Anna University, BIT Campus, Tiruchirappalli, India
TNK Suriyaprakash, Al Shifa College of Pharmacy, India
Ruckmani Kandasamy, Anna University, BIT Campus, Tiruchirappalli, India
Thirumurugan Rathinasabapathy, International Medical University (IMU), Malaysia

Worldwide there is an increasing industrialization leads to increased disposal of uncontrolled waste

products into the environment which made the environment more pollute and creates hazards. Industrial
wastewater is having a major role in the environmental pollution. The major physical, chemical and biological
products of the wastewater are solid content, organic matter, in-organic compounds, detergents, soap,
cleaning products, metals, gases, volatile compounds, numerous pathogenic microorganisms, nutrients
and toxic compounds. Untreated wastewater can cause various environment pollutions problems such as
eutrophication or oxygen depletion in the environment. Hence a effective wastewater treatment process
and its management is necessary to reduce the contaminants in the permissible levels in the treated waste
streams. The final outcome of an effective wastewater treatment and its management is to ensure and
provide an appropriate environment protection to the living things and public human beings in the world.

Compilation of References................................................................................................................ 335

About the Contributors..................................................................................................................... 412

Index.................................................................................................................................................... 418
xvi

Foreword

Environmental pollution from natural source and anthropogenic sources is a major environmental con-
cern due to occurrence and persistence of many hazardous toxicants. Hence, it was deeply understand
to develop viable technologies employing microbes and plants to remediate not only metallic residues
and radionuclides, but also the xenobiotic compounds like PCBs, PAHs, PCPs, petroleum sludge and
the military wastes.
To decontaminate the soils, sediments and waters, polluted by anthropogenic activities, the scien-
tists and technologists have evolved different technologies over the years. However, naturally occurring
micro-organisms degrade the hazardous organic wastes including xenobiotic compounds, such as pesti-
cides, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in due course of
time. However, metallic residues cannot be degraded in composting, but may be converted into organic
combinations that have less bioavailability than mineral combinations of the heavy metals. In addition,
microbes can transform the oxidation states of several toxic metals and increase their bioavailability in
the rhizosphere to be taken up by metal hyper accumulating plants.
In order to give a boost to this technology, I would like to appreciate the sincere efforts of my col-
league Dr. Ashok Kumar Rathoure, Environmental Scientist to publish this volume which contains
latest information on the various aspects of bioremediation to deal with specific techniques to remove
environmental contaminations. I hope this book will serve as a ready reckoner to the new researcher and
also help the scientist working in identifying the gaps for research. I consider this book a value addition
to the scientific knowledge on bioremediation.

Sandip Triptahi
NIMS University, India

Sandip Tripathi is working as associate professor in department of Biotechnology and bioengineering, NIMS University Delhi-
Jaipur Highway, Jaipur. He has more than 10 years of teaching and research experience. He is very dynamic and founded a
national society for fluoride research at NIMS. He has also associated various research foundations and association for various
activities. He has supervised more than 10 research scholar for doctorate degree.



 xvii

Preface

Bioremediation is an emerging field of environmental research. Now a days, heavy metal pollution and
oil spillage is a major global concern. Hence, an efficient technology is needed to reduce the risk of
heavy metal pollution and oil spills. In order to avoid ambiguity with regard to the approach utilized,
the present book constitutes the recent literature on strategies and technologies of bioremediation in
different areas. The objective of a bioremediation process is to immobilize contaminants (reactants)
or to transform them to chemical products that are no longer hazardous to human health and the envi-
ronment. For certain cases in which contaminants pose no significant risk to sensitive receptors e.g.
water supply wells and surface water bodies, intrinsic bioremediation may be an appropriate strategy.
For other cases in which receptors are at risk, an enhanced (engineered) bioremediation strategy may
be necessary. Enhanced bioremediation can be performed in-situ. Bioremediation is not a panacea for
soil and groundwater contamination. A successful, cost-effective bioremediation process is dependent
on hydrogeologic conditions, contaminant signature, microbial ecology, and other spatial/temporal fac-
tors that vary widely. Biotreatability studies are necessary components of the program so that remedial
design data are collected cost-effectively. Biotreatability studies can be performed to evaluate whether
site conditions are conducive for bioremediation.
This book will aim to provide relevant theoretical and practical frameworks and the latest empirical
research findings in the area. It is written for professionals who want to improve their understanding of
the strategic role of bioremediation at different levels of the bioremediation research and knowledge, that
is, heavy metal pollution, toxicity, remediation methods and strategies to manage the waste in industries,
which is a global concern.
Bioremediation is defined by the American Academy of Microbiology as “the use of living organ-
isms to reduce or eliminate environmental hazards resulting from accumulations of toxic chemicals and
other hazardous wastes”. The sustainable development requires the development and promotion of envi-
ronmental management. Green technologies are required to treat a wide range of aquatic and terrestrial
habitats contaminated by increasing anthropogenic activities, mainly the chemical industries. Bioreme-
diation is an increasingly popular low-cost alternative to conventional methods for treating wastes and
contaminated media with the possibility to degrade these contaminants using natural microbial activity
mediated by different consortia of microbes. Over the last decade, the scientific literature has revealed
the progressive emergence of various bioremediation techniques. (Kumar et al., 2011).


 Preface

THE CHALLENGES

The quality of all life forms on Earth is linked inextricably to the overall quality of the environment.
Nature has bestowed us with unlimited abundance of land and resources; however, human beings have
shown carelessness and negligence in using them. Due to this, all the natural resources have been heavily
contaminated and causing various hazards to life. Contamination has been increased at alarming rate
due to past industrial activities when awareness of the health and environmental effects connected with
the production, use, and disposal of hazardous substances were less well recognized than today. The
problem is worldwide, and the estimated number of contaminated sites is significant (Cairney, 1993).
It is now widely recognized that contaminated land is a potential threat to human health, and remedial
action on these sites has to be carried out at a large scale so that these sites do not pose further risk of
adverse health on future generations. The conventional techniques used for remediation have been to dig
up contaminated soil and remove it to a landfill, or to cap and contain the contaminated areas of a site.
The methods have some drawbacks. A better approach than these traditional methods is to completely
destroy the pollutants if possible, or at least to transform them to innocuous substances. Some technolo-
gies that have been used are high-temperature incineration and various types of chemical decomposition
(e.g., base-catalyzed dechlorination, UV oxidation). They can be very effective at reducing levels of a
range of contaminants, but have several drawbacks, principally their technological complexity, the cost
for small-scale application, and the lack of public acceptance, especially for incineration that may increase
the exposure to contaminants for both the workers at the site and nearby residents (Vidali, 2001). Today,
biotechnology is being considered as emerging science for environmental protection. The technology
involves the use of microorganisms for biological treatment of air, water and soil pollutants. Biotech-
nological treatment is carried out at lower temperature and pressure which requires less energy than
the conventional physico-chemical treatment technology. The industries generating hazardous wastes
have found beneficial measures from the emerging trend of biotechnological treatment. Biotechnologi-
cal innovations for treatment for hazardous waste under controlled environmental conditions have been
found cost–effective means of reducing the pollution potential of waste water, leading to enhanced public
acceptance and compliance with environmental legislation (Fulekar, 2010). Environmental pollution
such as contaminated soil or surface/ground water can be solved by bioremediation by use of biological
living organisms.

SEARCHING FOR A SOLUTION

Recently, biological remediation process have also been devised to either precipitate effectively im-
mobilize inorganic pollutants such as heavy metals. Stimulation of microorganisms is achieved by
the addition of growth substances, nutrients, terminal electron acceptor/donors or some combination
thereby resulting in an increase in organic pollutant degradation and bio-transformation. The energy
and carbon are obtained through the metabolism of organic compounds by the microbes involved in
bioremediation processes (Fulekar et. al., 2009). Bioremediation is not a new concept. Microbiologists
have studied the process since the 1940s. However, bioremediation became known to a broader public
in the U.S. only in the late 1980s as a technology for cleanup of shorelines contaminated with spilled
oil. The Exxon Valdez oil spill in 1989 in Prince William Sound, Alaska was the catalyst for this atten-
tion. In the years since 1989, bioremediation has become a technology that is discussed, applied, and

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considered in many different circumstances (Hoff, 1993). There are several different bioremediation
techniques. The underlying idea is to accelerate the rates of natural hydrocarbon biodegradation by
overcoming the rate-limiting factors. Several techniques can lead to the results striven for. Indigenous
populations of microbial bacteria can be stimulated through the addition of nutrients or other materials.
Exogenous microbial populations can be introduced in the contaminated environment. The addition of
extra bacteria is known as bio augmentation. If necessary, genetically altered bacteria can be used. Once
the bacteria are chosen, the engineer must carefully meet their nutritional needs by choosing the correct
mix of fertilizer (Irwin, 1996). Furthermore, the contaminated media can be manipulated by, for example,
aeration or temperature control. The objective of a bioremediation process is to immobilize contami-
nants (reactants) or to transform them to chemical products no longer hazardous to human health and
the environment. For certain cases in which contaminants pose no significant risk to sensitive receptors
e.g. water supply wells, surface water bodies, intrinsic bioremediation may be an appropriate strategy.
For other cases in which receptors are at risk, an enhanced (engineered) bioremediation strategy may
be necessary. Enhanced bioremediation can be performed in-situ. Bioremediation is not a panacea for
soil and groundwater contamination. A successful, cost-effective bioremediation process is dependent
on hydrogeologic conditions, contaminant signature, microbial ecology, and other spatial/temporal fac-
tors that vary widely. Biotreatability studies are necessary components of the program so that remedial
design data are collected cost-effectively. Biotreatability studies can be performed to evaluate whether
site conditions are conducive for bioremediation.

ORGANIZATION OF THE BOOK

The book is organized into 15 chapters. A brief description of each of the chapters follows:
Chapter 1 identifies heavy metal pollution as a global pollutant of rising concern. The chapter covers
various issues related to heavy metals. Here author attempted to document the possible definitions for
heavy metals. Heavy metals, the elements having density higher than 3.5 g/cm2 are being added at high
rate to our close vicinity. These metals lead to serious problems related to ecology and mankind. Toxic
effects of heavy metals are dependent on the concentration of metals, reactivity of metal species and
duration of exposure. There is a need to address the toxicological and remedial aspects of heavy metals.
Chapter 2 establishes the existing challenges in the management of Heavy metal Pollution using
Bioremediation technologies. Bioremediation is one of the most promising technological approaches to
the problem of hazardous waste. It is a technology for removing pollution from environment, restoring
contaminated site and preventing future pollution. Bioremediation can be performed in situ or ex situ.
Microorganisms directly degrade contaminants rather than merely transferring them from one medium
to another, employ metabolic degradation pathways and can be used in situ to minimize disturbance
of the cleanup site. Hence, microorganisms can be effective, economical and non disruptive tools for
eliminating hazardous chemicals. Its advantage generally outweigh the disadvantage, therefore may be
used as management tool.
Chapter 3 takes philosophical orientation and debates about the biosorption techniques. The overall
aim of the chapter is to consider biosorption for removal of dye molecules. In the past various physical
and chemical techniques have been employed for the removal of colour from wastewater. However most
of these methods have certain drawbacks. Biological treatment is often efficient and economical. Many

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microorganisms are able to accumulate and degrade different pollutants. Yet even the biological methods
have some shortcomings such as toxicity of biodegradation products and more.
Chapter 4 reviews the microbial response against metal toxicity. This chapter attempted to elucidate
how microorganisms tackle and response against the metals toxicity. In response to metal toxicity,
microorganisms exhibit a vast array of acclimatization, adaptation and resistant strategies at genetic,
cellular and community levels to detoxify metals toxicity and survival. Microorganisms detoxify met-
als by various mechanisms (sequestration, inhibition of influx, efflux, accumulation, precipitation and
chemical modification, repair, and metabolic by-pass) and showed resistance properties (by protein/
enzyme synthesis) encoded by genes located in chromosome, plasmid or transposon. Thus, metal toxicity
hampers the microbial metabolism, growth, activity and species diversity resulting in severe damage in
environmental microbial community. Apart from detrimental consequences of metal toxicity, the novel
metal- and antibiotic- resistant microorganisms could be used in environmental and human health benefits.
Chapter 5 presents the application of genomics and proteomics in bioremediation. The authors of this
chapter explain the experimental and modelling techniques in genomics and proteomics in bioremedia-
tion; they have evaluated physiology and enhance the performance bioremediation process. Similarly, the
application of proteomics in bioremediation research provides a global view of the protein composition
of microbial cell and offers promising approach to understand the molecular mechanism of removal of
toxic material from the environment. Combination of proteomics and genomics in bioremediation is an
insight into global metabolic and regulatory network that can enhance the understanding of gene func-
tions. Present chapter give a bird’s eye view of genomics and proteomics and their potential utilization
in bioremediation and for the clearer understanding of the cellular responses to environmental stimuli.
An understanding of the growth conditions governing the expression of proteome in a specific environ-
ment is essential for developing rational strategies for successful bioremediation.
Chapter 6 presents an analysis of issues and concerns in using genetically engineered microorgan-
isms for bioremediation processes. The authors classify the genetically engineered microorganisms and
their potential. The microbial potential is connected to affect the clean-up of environmental pollutants.
Conventional methods are focus on the separation, rather than the destruction of contaminants, the use
of genetically engineered microorganisms for bioremediation would be an alternative, environmentally
friendly, more effectiveness and economical clean-up technique for the remediation of pollutants in pres-
ent in contaminated sites. A combined strategies relationship between genetic engineered microbes and
bioremediation can enhance the effectiveness of contaminants sites. Here, the authors have elaborated
recent work on the investigation and improvement of these microbes using genetic tools and given an
outlook of what may be possible in the near future.
Chapter 7 discusses generic concepts of novel bioremediation methods in waste management. Biore-
mediation technologies are one of the novel methods in the field of waste and environment management
and are presently gaining immense credibility for being eco-compatible. Bioremediation using microbes
has been well accepted as an environment friendly and economical treatment method for disposal of
hazardous petroleum hydrocarbon contaminated waste (oily waste). Besides this, earthworms can be
used to extract toxic heavy metals, including cadmium and lead, from solid waste from domestic refuse
collection and waste from vegetable and flower markets. Other novel methods used recently for treat-
ment of wastes are plasma incineration or plasma assisted gasification and pyrolysis technology. The
technologies applied for conditioning include ultrasonic degradation, chemical degradation, enzyme
addition, electro-coagulation and biological cell destruction. Genetic engineering is another method

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for improving bioremediation of heavy metals and organic pollutants. Transgenic plants and associated
bacteria constitute a new generation of genetically modified organisms for bioremediation.
Chapter 8 approaches the effective management of agro-industrial residues as composting in mush-
room industry and utilization of spent mushroom substrate for bioremediation. Different types of edible
mushrooms like Agaricus, bisporus, A. bitoriqus, Pleurotus spp., Volvariella volvacea, Lentinula edodes,
Calocybe indica, Flamullina, Ganoderma lucidum etc are cultivated in industrial scale. Majority of ed-
ible fungi secretes extracellular Ligninocellulolytic enzymes like Laccase, lignin peroxidase, manganese
peroxidase, cellulase etc. for effective conversion of ligninocellulolytic substrate to compositing form
which led to fruiting of mushrooms. Consequently, an adequate disposal method is needed for the high
quantities of spent mushroom substrate (SMS) generated in this agro-food industrial activity. On the
other side, textile industry among the largest water consuming industries in the world and approximately,
10,000 different dyes and pigments are used at industrial scale. It is estimated that nearly 40% of the total
dyes used in the dyeing process may find their way in wastewater. So, there is an attempt by authors to
utilize the ligninolytic enzymes rich SMS of different mushroom for efficiently biodegradation of textile
wastewater and polyaromatic pollutants.
Chapter 9 present the bioremediation approaches for recalcitrant pollutants: potentiality, successes
and limitation. The different chemical pollutants discharged by the industries to the environment can
upset the delicate balance of the ecosystem. Bioremediation, the use of microorganisms and plants to
remediate polluted environments, is a promising and growing area of environmental biotechnology.
Bioremediation options encompass diverse types of biotechnological mechanisms that may lead to a
target pollutant’s mineralization, partial transformation, humification, or altered redox state. The use of
extra cellular and/or cell-free enzymes has been also proposed as an innovative remediation technique.
Perspectives and limitations to evolve and use this technology are critically discussed in this chapter
with respect to the complexity of mixtures of xenobiotics often found in practice. Whereas the potential
of bioremediation is substantial, its application has important limitations that are apparent from many
examples and the authors feel that these limitations can be overcome only when adequate attention is
directed to fundamental microbiological, chemical and engineering issues.
Chapter 10 addresses the issue of biodegradation of phenol- mechanisms and applications. Aro-
matic compounds are widely distributed in nature. Free phenols are frequently liberated as metabolic
intermediates during the degradation of plant materials. In recent years the natural supply of phenolic
substances has been greatly increased due to the release of industrial by products into the environment.
Phenolic compounds are hazardous pollutants that are toxic at relatively low concentration. Effluents
from petrochemical, textile and coal industries contain phenolic compounds in very high concentration;
therefore there is a necessity to remove phenolic compounds from the environment. Microorganisms
capable of degrading phenol are common and include both aerobes and anaerobes. The use of micro-
bial catalysts in the biodegradation of organic compounds has advanced significantly during the past
three decades. The efficiency of biodegradation of organic compounds is influenced by the type of the
organic pollutant, the nature of the organism, the enzyme involved, the mechanism of degradation and
the nature of the influencing factors.
Chapter 11 reviews the phytoremediation. In this chapter authors have discussed the role of plants to
develop contaminant free environment. This concept is also known as Phytoremediation. Phytoremediation
is a word formed from the Greek prefix “phyto” meaning plant, and the Latin suffix “remedium” meaning
to clean or restore. This technology has been receiving attention lately as an innovative, cost-effective
alternative to the more established treatment methods used at hazardous waste sites. Phytoremediation

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can be classified into different applications, such as phytofiltration or rhizofiltration, phytostabilization,

phytovolatilization, phytodegradation and phyto-extraction etc. The chapter will deal with phytoremedia-
tion, its advantages, limitations and in detail techniques of classification and application.
Chapter 12 presents a prospective study on emerging role of phytoremediation by endophytic mi-
croorganisms. Colonies of endophytes are excellent example of beneficial association with most plants
in their natural state. Endophytic colonies and plant associations are beneficial in many ways such as
supplying biologically fixed nitrogen, regulation of phytohormone production thus enhancing the plant
growth, resistance to environmental stress etc. these associations are also important for the agriculture
and industries because they produce important medicinal, agriculture and industrial compounds as en-
dophytic metabolites. When we concern about the waste management, degradation and biotransforma-
tion of several toxins, the phytoremediation by using endophytes has been developed as important tool.
Chapter 13 analyses and compares recent approaches for removal of toxic dye from different streams
of wastewater. Azo dyes are used in abundance in several industries like textile, printing, paper, plas-
tic, cosmetics, paints, etc. Extensive discharge of such dyes in adjacent water bodies has raised much
environmental concern. Azo dyes are toxic to living organisms and their genotoxic and carcinogenic
potentials are intensified on being released as mixtures. In the recent years, various microorganisms
have been isolated and reported to possess tremendous potential for efficient dye degradation. However,
the process of bioremediation is highly controlled by experimental factors like effluent pH, temperature
and concentration of dyes in solution. Therefore, appropriate optimization of these factors is to be de-
termined in order to ensure maximum efficiency of this process. This review highlights application of
immobilization techniques of bacterial cells for achievement of successful biodegradation.
Chapter 14 discusses the decolorizition of direct blue -14 dye by thermoalkalophilic aerobic Bacillus
sp. A thermo-alkalophilic bacterium isolated from textile mill effluent samples and identified as a Bacil-
lus sp., on the basis of biochemical tests. The selected bacterium showed high decolorization activity
in static condition as compared to shaking condition and the maximum 1000 mg/L Direct Blue-14 dye
decolorization was takes place in 72 h. The optimum physical parameters such as temperature 40-50°C,
pH 8.0 with 2.5% (w/v) of nitrogen source and 4% (w/v) glucose were required for the decolorization of
Direct Blue-14 from this bacterium. UV–Vis analyses and colorless bacterial cells suggested that Bacillus
sp. exhibited decolorizing activity through biodegradation, rather than inactive surface adsorption. The
degraded dye metabolites are analyzed by TLC and diazotization, carbylamines, Ames test for individual
metabolite indicates biotransformation of Direct Blue-14 into aromatic amine and non-toxic aromatic
metabolites. These results suggest that the isolated organism Bacillus sp. as a useful tool to treat waste
water containing azo dyes at static condition.
Chapter 15 reviews issues surrounding the environment on Ecomafias. The author has discussed
various roles of biotech networks in achieving sustainability. Innovation processes are becoming increas-
ingly central, and newer industries have become already less resource-intensive in comparison with the
traditional ones. Nevertheless, this alone does not assure sustainability, which requires a step further
towards economically viable, environmentally compatible, and socially responsible behaviours. This
chapter addresses the issues relating to sustainable development to provide a critical discussion on the
potential role played by networking relationships in the biotech field. For the purposes of the study, we
employ the co-management and multi-stakeholder perspectives. The authors have demonstrated that the
biotechnology research results may be enhanced thanks to cooperation dynamics and interactions among

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heterogeneous actors, with undeniable cultural and social positive impacts. Also, it discussed the social
implications and open concerns, both with regard to the relationships within innovative networks and
between institutional professional actors, allowing the identification of any grey areas and limitations,
especially relevant to policy makers.
Chapter 16 presents about the increasing industrialization worldwide which leads to increased dis-
posal of uncontrolled waste products into the environment which made the environment more pollute
and creates hazards. Industrial wastewater is having a major role in the environmental pollution. The
major physical, chemical and biological products of the wastewater are solid content, organic matter,
in-organic compounds, detergents, soap, cleaning products, metals, gases, volatile compounds, numerous
pathogenic microorganisms, nutrients and toxic compounds. Also, untreated wastewater can cause various
environment pollutions problems such as eutrophication or oxygen depletion in the environment. Hence
an effective wastewater treatment process and its management are necessary to reduce the contaminants
in the permissible levels in the treated waste streams. The final outcome of an effective wastewater treat-
ment and its management is to ensure and provide an appropriate environment protection to the living
things and public human beings in the world.

Ashok K. Rathoure
Vardan Environet Guargaon, India

REFERENCES

Cairney, T. (1993). Contaminated Land (p. 4). London: Blackie.

Fulekar, M. H. (2010). Bioremediation Technology: Recent Advances. Springer. doi:10.1007/978-90-
481-3678-0
Fulekar, M. H., Geetha, M., & Sharma, J. (2009). Bioremediation of Trichlorpyr Butoxyethyl Ester
(TBEE) in bioreactor using adapted Pseudomonas aeruginosa in scale up process technique. Biologie
Medicale, 1(3), 1–6.
Hoff, R. Z. (1993). Bioremediation: An overview of its development and use for oil spill cleanup. Marine
Pollution Bulletin, 29(9), 476–481. doi:10.1016/0025-326X(93)90463-T
Irwin, P. (1996). To clean up environmental spill, know your medium. Electrical World 37-40.
Kumar, A.; Bisht, B. S.; Joshi, V. D. and Dhewa, T. (2011). Review on Bioremediation of Polluted
Environment: A Management Tool. International Journal of Environmental Sciences Volume 1 No.6.
Vidali, M. (2001). Bioremediation An overview. Pure and Applied Chemistry, 73(7), 1163–1172.
doi:10.1351/pac200173071163

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Acknowledgment

This book is the result of the dedicated effort of numerous individuals. We would like to acknowledge
the help of all the people involved in this project and, more specifically, to the authors and reviewers
that took part in the review process. Without their support, this book would not have become a reality.

First, we would like to thank each one of the authors for their contributions. Our sincere gratitude goes
to the chapter’s authors who contributed their time and expertise to this book.

Second, we wish to acknowledge the valuable contributions of the reviewers regarding the improvement
of quality, coherence and content presentation of chapters especially to Dr. Ajay Singh, HOD, Dr. Raja
Rasool, Dr. Waseem Ahamad, Dr. Poonam Negi Rawat, Dept. Chemistry, Uttaranchal University; Mr.
Ritesh Mishra, Assistant Professor, Dept. of Agriculture Science, IFTM, Muradabad. Most of the authors
also served as referees; we highly appreciate their double task.

Additionally, we would like to thank our Editorial Advisory Board.

Our Production Coordinator skillfully managed the production of the textbook. We wish to thank the
publisher for the sustained interest shown by him during the entire work. A sincere expression of the
thanks goes to the entire staff of IGI Global.

Generous participation of faculties and students to eliminate errors in the text and to eliminate errors in
the text and to refine the presentation will be greatly acknowledged.

Last and not least, we beg forgiveness of all those who have been with us over the course of the years
and whose names we have failed to mention.

Editors

Ashok K. Rathoure
Vardan Environet Guargaon, India

Vinod K. Dhatwalia
Uttaranchal University, India



 1

Chapter 1
Heavy Metal Pollution:
A Global Pollutant of Rising Concern

Ashita Sharma Jatinder Kaur Katnoria

Guru Nanak Dev University, India Guru Nanak Dev University, India

Mandeep Kaur Avinash Kaur Nagpal

Guru Nanak Dev University, India Guru Nanak Dev University, India

ABSTRACT
The chapter covers various issues related to heavy metals. Here we attempt to document the possible
definitions for heavy metals. Heavy metals, the elements having density higher than 3.5 g/cm2 are being
added at high rate to our close vicinity. These metals lead to serious problems related to ecology and
mankind. Toxic effects of heavy metals are dependent on the concentration of metals, reactivity of metal
species and duration of exposure. There is a need to address the toxicological and remedial aspects of
heavy metals.

INTRODUCTION

Human civilization is constantly developing and progressing and in the course of development and
progression, natural resources play a significant role. Biotic (living organisms and their products) and
abiotic resources (air, water and soil) are used to meet the requirements or demands of civilization.
In the age of rapid development, we have been exploiting resources without considering the recovery
time for the resources. Injudicious use of resources not only causes the dearth for future use, but also
leads to the addition of certain substances to our surroundings, which were not the part of biosphere or
if they were present earlier the quantity was negligible. The phenomenon of this unwanted addition of
substances to environment is referred as “Pollution” and the substances added as “pollutants”. These
pollutants added to the environment due to various developmental activities, pose a threat to ecosystem
in general and to human lives in particular. Various historical incidents including, Bhopal gas tragedy,
Minamata Bay catastrophe, London smog and many more have showcased the threat pollutants cause

DOI: 10.4018/978-1-4666-9734-8.ch001

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.
 
Heavy Metal Pollution

to mankind. Pollutants occur in gaseous, liquid or solid forms. They can be created and released due to
some human activity or they appear in biosphere due to over-exploitation of resources which leads to
unveiling of pollutants from deeper crusts of soil. Amongst different kinds of pollutants, heavy metals
have attracted attention of scientific community because of serious health hazards associated with them.
Metals were always the part of nature, but over exploitation of resources or other anthropogenic activi-
ties have added to the background concentration of these metals in the biosphere. Like, higher level of
arsenic in groundwater of many regions is result of over exploitation of underground water resources,
leading to dissolution of minerals (Nagajyoti, Lee, & Sreekanth, 2010; Hashim, Mukhopadhyay, Sahu,
& Sengupta, 2011; Bernhoft, 2012; Thomas-Mkude, 2015).
Heavy metals released from various industries and agrochemicals have proven to be a serious concern
for all types of living organisms. International bodies, like, WHO, US Environmental Protection agency
(EPA) have extensively studied and reviewed health effects related to various metals mainly cadmium,
mercury, arsenic and lead. Although the use of heavy metals have been known since more than 5000
years and their severe health effects are also known from centuries but since the middle of 19thcentury,
there is a steep rise in use of heavy metals leading to their emissions in neighboring environment and
thus triggering the cause-effect phenomenon at a faster pace. Heavy metals released through any activ-
ity remain in environment for a long time as they cannot be destroyed. Although, many of these heavy
metals are also required in traces for the physiological metabolism of both plants and animals but they
are toxic at higher concentrations (Järup, 2003; Inoue, 2013).
Extensive research is being carried out in context of heavy metals. There is need to archive basics of
heavy metals to aid in future research. This chapter aims to document most of the available definitions
of heavy metals, the sources, distribution and toxicity.

DEFINITIONS

In periodic table, metals are classified as alkali metals, transition metals, alkaline earth metals and rare
metals. The term heavy metals, which is commonly used in scientific literature does not belong to any
well-defined section in periodic table. According to Oxford dictionary, the oldest documentation of this
term was found in a book on inorganic chemistry written by Bjerrum in 1936 (Duffus, 2002; Hodson,
2004). Broadly, heavy metals can be defined on the basis of physical properties, chemical reactivity and
toxicity to living organisms.

On the Basis of Physical Properties

The term “heavy metals” is being widely used to refer toxic metals. But there is still a controversy regard-
ing the definition of these metals. In 2002, a technical report published by IUPAC (International Union of
Pure and Applied Chemistry) questions the reliability of this term and considered it to be “most loosely
defined term”. The word “heavy” signifies higher density which is usually attributed to the toxicity of
metals. The challenge here for the scientific community was to determine the threshold level of density,
above which a metal should be considered heavy metals. In past, various levels of densities were pro-
posed and metals above that limit were considered to be heavy metals. IUPAC has summarized various
studies mentioning the range of density to be considered as high. Earlier, heavy metals having density
above 7 g/cm3 were considered toxic but this definition phased out many toxic metals like arsenic from

2

Heavy Metal Pollution

the list. Thus, recent studies have reported metals having density higher than 3.5 g/cm3 as heavy metals
(Duffus, 2002; Hodson, 2004; Appenroth, 2010).
Some definitions were proposed on atomic number, atomic weight but all these definitions were
rejected in due course of time as these didn’t judiciously filter out essential metals from toxic ones.
The most widely used definition of heavy metals on the basis of physical properties is, “Metals having
density higher than 3.5 g/cm3” (Duffus, 2002).

On the Basis of Chemical Reactivity

Certain other definitions were based on the reactivity of metals, the metals which were highly reactive
were labeled to be “heavy”. Duffus (2002) pointed out that there can be certain metals which would have
low toxicity in elemental state but can prove to be highly toxic in form of compounds. The metals which
are reactive with biomolecules and form ligands with sulphur and nitrogen containing compounds are
considered to be toxic. Certain ions of toxic metals have similar affinity as that of essential metals and
thus replace essential metals in the metabolic reactions, and are considered as toxic or heavy metals.
Zn2+ is involved in various biochemical pathways and metalloenzymes but prolonged exposure to Cd2+
lead to replacement of Zn2+ with Cd2+ in biochemical reactions (Babula et al., 2008; Appenroth, 2010;
Luque-Garcia, Cabezas-Sanchez, & Camara, 2011). On the basis of reactivity we can enlist highly reac-
tive metal species as toxic heavy metals.
Appenroth (2010) has summarized metal ions on the basis of reactivity as follows:

• Toxic Metal Ions: Ti+, Ti3+, Pb4+, Bi3+, Pd2+, Pt2+, Cu+, Ag+, Au+, Hg2+
• Borderline Ions: Ga3+, In3+, Sn4+, Pb2+, As3+, Sb3+, Ti2+, V2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+,
Zn2+, Cd2+

The toxic metal ions are the ions which have high affinity to proteins or other biomolecules and
cause immediate toxicity.

On the Basis of Toxicity to Living Organisms

Many of the metals, listed in series of metals having high density are toxic to mankind only beyond a
certain concentration or after prolonged exposure (Nagajyoti et al., 2010; Saha & Panwar, 2014). Experi-
ments to monitor the toxicity and screening of heavy metals using plants and microbes are well estab-
lished. Appenronth (2010) analyzed various growth parameters of Lemna minor to measure the toxicity
of a metal and on the basis of such experiments, trends for toxicity of various metal ions are given as:
In another study in yeast it was observed that Al3+, Co3+, Mn2+, V+ produced stress responses and
thus were categorized as heavy metals (Hosiner et al., 2014).

SOURCES OF HEAVY METALS

The heavy metals existing in atmosphere, hydrosphere and lithosphere (beyond biospheric zone) come to
biosphere through natural weathering processes but anthropogenic activities involving over-exploitation
of resources and release of harmful waste increases the rate of addition of these metals to biosphere. Ac-

3
 
Heavy Metal Pollution

celeration of addition of heavy metals in ambient environment can be through natural and anthropogenic
sources. Various sources are explained as follows:

Ag + > Cd 2+ > Hg 2+ > T 1+ > Cu 2+ > Ni 2+ > Zn 2+ > Co 6+ > Cr 6+ > As 3+ > As 5+

Natural Sources

Metals exist in lithosphere, and due to natural weathering or change in pH of soil or water they get
solubilized and mobilized and are added to the biosphere. Many of these metals reach the biosphere
through natural calamities like, volcanoes and floods. Many chemical reactions (Redox reactions, acid/
base reaction, alteration of pH levels) occurring in soil and water alter the species of metals and con-
vert them to more mobile soluble forms thus increasing the concentration of toxic metals like arsenic,
chromium and mercury in our vicinity (Hashim et al., 2011; Wuana & Okieimen, 2011; Varalakshmi
& Ganeshamurthy, 2012).

Anthropogenic Sources

Natural processes do add the metals to our ambience but the rate of addition is very low and generally not
toxic or harmful for the biotic components. But the process gets accelerated due to anthropogenic activi-
ties. Certain anthropogenic activities accelerate the natural reactions and thus increase the concentration
of heavy metals. It has been observed that metals added through anthropogenic sources like mining, use
of agrochemicals, waste water discharge from industries and household are more bioavailable than the
naturally occurring ones. Various sources of heavy metals are summarized in Figure 1.

DISTRIBUTION OF HEAVY METALS IN ENVIRONMENT

Heavy metals are released to environment through various processes and reach our ambient environment
through many pathways involving abiotic components of environment viz. air, water and soil. A general
pathway for the transport of metals can be summarized as shown in Figure 2.
In our ambient air, the heavy metals are being continuously added due to emissions, which are natu-
ral or anthropogenic. Air pollutants can be inorganic, organic or the mixture of secondary pollutants
which are combination of both. Heavy metals in air become a serious concern as the spatial distribution
of pollutants through air can be high, thus effecting large population. Heavy metals get attached to the
air-borne particles of both natural and anthropogenic sources. Air-borne particles or aerosols vary from
coarse sized particles (0.5 µ) to Aitken particles (0.2µ). Natural origin of these particles can be dust
emission, evapotranspiration and volcanic eruptions (Csavina et al., 2012). Csavina et al (2011) reported
that aerosols in the vicinity of mines have high concentration of arsenic, cadmium and lead. Maximum
concentration of metals was present in particulate matter of size 0.32 µm. (Csavina et al., 2011) Also,
the pollutants can travel over a large distance and settle on soil or water, as a result of wet and dry de-
position. The distribution and deposition of pollutants through air largely depends of climatic factors.

4

Heavy Metal Pollution

Figure 1. Various sources that add heavy metals to our surroundings

Figure 2. Pathway representing transport of metals through biotic and abiotic systems

5
 
Heavy Metal Pollution

Seasonal variation in the heavy metal content in atmosphere has also been reported (Kisku, Pandey,
Negi, & Misra, 2011; Guerra, Trevizam, Muraoka, Marcante, & Caniatti-Brazaca, 2012; Kulhari et al.,
2013). According to Csavina et al (2011) during transport of heavy metals through air, the factors that
are important in spatial distribution of heavy metals include:

• Particle size: Larger the size of aerosols, lesser the spatial distribution
• Wind Velocity and direction
• Relative humidity
• Soil erodibility
• Surface friction: as it influences wind momentum

Metals present on soil crust also get transported through air. The contribution factor of metals re-
leased through anthropogenic and crustal sources is represented by Enrichment Factor (EF). It can be
represented as

(E / Al )
Ef = air

(E / Al )
soil

Enrichment factor is ratio of content of metal (E) with reference to aluminum in aerosol to soil. Here,
aluminum is considered as reference because of its significant distribution in aerosols (Wedepohl, 1995).
If value of EF is close to 1, then elements can be of crustal origin while if EF value is over 10, then
content of element is because of contribution from non-crustal sources (Waheed et al., 2011).
Hydrosphere, which covers more than 71% of our planet, also gets invaded with heavy metals. In-
dustrial waste water, agricultural run-off and municipal wastes add toxic metals to this elixir of life. As
discussed, pollutants from air also settle on water bodies. Around the globe, toxic metals like arsenic,
lead and cadmium have been found in considerably high concentrations in potable water reservoirs. At
many places, concentration of these metals is significantly high in ground water aquifers too, making
the water toxic for living organisms (Flores-Magdaleno, Mancilla-Villa, Mejia-Saenz, Olmedo-Bolanos,
& Bautista-Olivas, 2011; Harati, Varavi, Rastegar, & Foghi, 2011; Shahryari & Shehamat, 2012). Soil,
another important factor for the existence of life, is also highly contaminated by the toxic metals. Soil
has become an open dust-bin for industrial solid waste, fly ash, electronic waste and municipal waste
around the globe. Heavy metals thus added to soil not only remain in soil, but also they percolate to
water and get transported to ambient air with wind (Tomáš, Árvay, & Tóth, 2012; Aziz, Rahim, Sahid,
Idris, & Bhuiyan, 2015).
The distribution of heavy metals across various components of environment is controlled by:

• Ion exchange in soil-soil, soil-water, soil-air interactions

• Dissolution and precipitation
• Adsorption and desorption
• Aqueous reactions
• Mobilization or immobilization by biotic components
• Uptake by plants

6

Heavy Metal Pollution

Heavy metals released from underground sources are converted to methylated organic forms by the
action of bacteria and such organic forms are more bioavailable and toxic than the inorganic forms.
(Nagajyoti et al., 2010)
Example:

Bacteria 
Hg 2+ + Organic Matter  → CH 3Hg + (CH 3 )2 Hg

Similarly, other heavy metals are added to atmosphere through anthropogenic activities and are further
turned to toxic species through microbial action. If we burn pyrite containing coal, it releases oxides of
iron in the environment. This reaction is given by following chemical equation:

3FeS 2 + 8O2 → Fe3O4 + 6SO2

The oxides of metal thus produced are deposited in water bodies or soil through wet deposition.
Furthermore, during inorganic conversions in water logged soil, reducing conditions are created leading
to bioavailability of heavy metals like, manganese and iron converting them to species which are more
mobilized and more toxic to plants.

 2e → Mn 2+  +H 2O
MnO2 + 4H ++

Fe2O3 + 6H + + 2e → 2Fe 2+ + 3H 2O

Arsenic in sea water exists in the form of arsenobetaine (CH3)3As which is non-toxic form. As(III)
or arsenite, the toxic form of arsenic dominates in reducing conditions. This form of arsenic is highly
mobilized. Methylation of arsenic by microbes leads to the formation of more volatile and toxic com-
pounds like dimethyl arsine and trimethyl arsine (Dorne et al., 2011; Tangahu et al., 2011; Wuana &
Okieimen, 2011).
Transfer of metals to plants is expressed by various expressions. Here are few factors which represent
transfer of heavy metals.
Yoon et al (2006) proposed expression for Biological Concentration Factor (BCF) as:

M Root
BiologicalConcentration Factor (BCF ) =
M Soil

Also, Cui et al (2007) defined Biological Accumulation Factor (BAC) and Transfer factor (TF) as
follows:

M shoot
Biological AccumumationCoefficient (BAC ) =
M soil

7
 
Heavy Metal Pollution

M shoot
Translocation Factor (TF ) =
M root

In wastewater irrigated sites, chances of heavy metal contamination is high. Enrichment Factor (EF)
is calculated based on metal concentrations in plants collected from waste water irrigated sites to metal
concentration in plants collected from clean water irrigated soil (Singh, Sharma, Agrawal, & Marshall,
2010). Enrichment factor is represented as:

C WWI site 

 p 
 
C s WWI site 
EF =  
C CWI site 
 p 
C s CWI site 
 

CpWWI = Concentration of metal in edible parts of plant at Wastewater irrigated site;

CsWWI = Concentration of metal in soil at Wastewater irrigated site;
CpCWI = Concentration of metal in edible parts of plant at Clean water irrigated site;
CsCWI = Concentration of metal in soil at clean water irrigated site.

As discussed in Figure 2, metals travel through all components of environment, being at base of
trophic level, plants accumulate heavy metals which are transferred to higher levels of food chain. And
the biomagnification of concentration of metals from lower to higher trophic level of food chain is rep-
resented in Figure 3.

Figure 3. Inverted pyramid representing increase in concentration of metals at each trophic level

8

Heavy Metal Pollution

TOXICOLOGY OF METALS

Heavy metals have been essential accelerators in the course of development since ancient times. Use of
metals in ancient medicines is also documented. Colours derived from cadmium were used by artists
for their work. Ancient Rome was known to use lead in the fermentation process of wine, mercury to
relieve teething pain in infants and as a treatment for syphilis. The toxic form of mercury viz. methyl-
mercury was used as fungicide for food grains till 1970s. Even though the Minamata bay tragedy in
Japan (1950s) was due to toxic effects of methylmercury, several thousand people died in Iraq in early
1970s due to consumption of methylmercury treated food grain. U.S. Environmental Protection Agency
(1978) listed 24 hazardous substances which included arsenic, beryllium, cadmium, lead and mercury
that made the hazardous effects of these metals well-known (Järup, 2003; Bernhoft, 2012; Inoue, 2013).
Over-exploitation of resources and injudicious anthropogenic activities has resulted in an increase in
concentration of heavy metals in the environment much beyond the background level. This increases the
heavy metal pollution index to which a living organism is exposed. Metal pollution index (MPI) can be
evaluated as a geometric mean of content of metals present in samples of all kind of food articles and
air we breathe (Singh et al., 2010)

1
MPI = (Cf1 *Cf2 * …..Cfn )n

where Cfn = Concentration of metal in sample

Toxicity to Plants

Uptake of heavy metals by plants occurs through various routes. Metals enter plant tissue through vari-
ous sites in root and shoot zone. Toxic metals present in atmosphere in ultrafine particulate size enter
the plant tissue through cuticle, opening and closing of stomata, hydathodes and stigma. Metals present
in root zone of plants are absorbed through root tips, lateral root junctions, woundings and rhizodermis.
The level of uptake depends on the leaf area, number of stomata, rate of transpiration, branching of root,
elongation of roots and cation-exchange capacity of soil. When a metal has been absorbed through any
zone of plant, it is translocated to other parts through xylem or phloem, this translocation can happen
through ion-exchange and alteration of pH (Dietz & Herth, 2011). Figure 4 explains uptake and trans-
location of heavy metals through various points.
Toxicity of metal to plants is dependent upon:

• Size: Smaller the size, higher the penetration.

• Concentration: Higher the concentration, higher the magnitude of effect.
• Chemistry of Ions: Certain species are more reactive than others.

According to Dietz and Herth (2011) toxicity to plants can be classified as:

• Physical toxicity which is a result of clogging due to association with cellular structure.

9
 
Heavy Metal Pollution

Figure 4. Adsorption and Translocation of Metal in Plant

• Chemical toxicity which is result of binding of metal ions to biomolecules. Chemical toxicity can
further be classified as:
◦◦ Toxicity due to affinity based interactions which involve formation of covalent bonds and
binding with functional groups of proteins or lipophilic interactions.
◦◦ Catalytic activities like production of reactive oxygen species.

Heavy metal ions like, Hg2+, Cd2+ and Pb2+ have affinity towards sulphur containing amino acids,
like methionine and cysteine.

• Arsenic: Arsenic is generally non-toxic to plants in lower concentrations but higher concentra-
tion inhibit root and shoot growth. As is available to plants as As(III) or As(V) of which As(III)
is more toxic to plants (Nagajyoti et al., 2010). Uptake of As(V) is more rapid as compared to
As(III). Arsenic when translocated to sub-cellular levels, lead to the formation of As(V)-ADP,
which disrupts formation of ATP and thus affecting regular metabolism of cell (Peralta-Videa,
Lopez, Narayan, Saupe, & Gardea-Torresdey, 2009; Finnegan & Chen, 2012; Kwankua, Sengsai,
Muangphra, & Euawong, 2012).
• Cadmium: Permissible limit of cadmium in agricultural soil is 100 mg/kg. Cadmium has found
to be interfering in uptake of essential metals like calcium, potassium and phosphorus. Visible
effects of cadmium include chlorosis and growth inhibition. Guala et al (2010) reported death of
alfalfa, lettuce and raddish plants at high cadmium concentrations in soil. Increase in ROS produc-
tion due to uptake of cadmium in Pisum sativum has also been reported (Malecka et al., 2012).
In plants like Allium cepa and Eucrosia bicolor, cadmium was found to be mito-depressive and
genotoxic at a concentration as low as 0.02 mg/l (Kwankua et al., 2012).
• Chromium: Chromium is toxic to plants at a concentration of 100 µg/kg (Hayat et al., 2012).
Analysis of heavy metals in soil samples from various agricultural fields across the world has
shown levels of chromium to be much higher than toxic limit (Mwegoha & Kihampa, 2010; Hayat
et al., 2012; Varalakshmi & Ganeshamurthy, 2012; Shah et al., 2013). Zenget al (2011) studied
the translocation of chromium ions at subcellular levels and found that in root system the chro-

10

Heavy Metal Pollution

mium ions were limited to cell wall while in leaves and stems the ions were found in cell wall and
vacuoles (Zeng et al., 2011). Toxicity of chromium is associated with decrease in photosynthesis
rate as a result of decrease in number of chloroplast, increase in lipid peroxidation and superoxide
activity (Nagajyoti et al., 2010; Moloukhia & Sleem, 2011; Zeng et al., 2011; Hayat et al., 2012).
• Mercury: Mercury can be present in soil or water in elemental form, organic form or as Hg2+
ions. Presence of mercury in water bodies has been reported in many studies (Tangahu et al.,
2011; Goutte et al., 2014). Toxicity of mercury in both terrestrial and aquatic plants is well docu-
mented. Uptake of mercury in plants is dependent on the species of plant, alteration of pH and
cation-exchange capacity. In plant systems, metal is translocated through xylem/phloem system.
Toxicity symptoms due to mercury are reported in all physical, physiological and biochemical
aspects. Hg2+ ions bind with sulphur containing biomolecules including DNA base pairs leading
to mismatches in DNA base pairing and thus impairing cell metabolism. Mercury ions are also re-
sponsible for modulation of antioxidant enzymes (Azevedo & Rodriguez, 2012; Bernhoft, 2012).
• Lead: US EPA considers lead as most commonly found heavy metal. Concentration of lead in
plants above 30 µg/g of dry mass is considered to be toxic for plants (Malecka et al., 2012).
Toxicity symptoms due to uptake of lead include chromosomal damage, growth inhibition, reduc-
tion in pollen viability, increase in aberrant cells, increase in oxidative stress and reduction in seed
viability. Lead has affinity to sulphydryl groups present in enzymes and thus inhibit the activity of
enzymes (Bussche & Soares, 2011; Choudhary, Ansari, Khan & Gupta, 2012; Goswami, Thakur
& Sarma, 2010; Hamid, Bukhari & Jawaid, 2010; Kwankua et al., 2012; Nagajyoti et al., 2010).
• Copper: Copper is a micro nutrient required for physiological metabolism of plant systems. ATP
synthesis and photosynthesis require copper. But higher concentration is toxic to plants. All the
cytotoxic and genotoxic symptoms are evident in plants growing in higher concentrations of cop-
per in soil. Concentration of copper in many samples of agricultural soil has been reported in
ranges much higher than the required limits for plants (Andreazza, Bortolon, Pieniz, Camargo &
Bortolon, 2013; Gharbi, Rejeb, Ghorbal & Morel, 2005; Katnoria, Arora, Bhardwaj & Nagpal,
2011; Ping, Xingxiang, Taolin, Dongmei & Yuanqiu, 2008; Shah et al., 2013). Apoptosis was
reported in protoplasts of tomato plants at 10mM concentration of CuCl2 (Rueda et al., 2011).

Besides above discussed heavy metals, several other heavy metals occur in high concentration in
agricultural soil and are toxic to plants. Metals like zinc, manganese, cobalt and nickel are also evalu-
ated for their toxic effects and are found to be causing severe toxicological responses in plant systems.
Alteration of enzyme activity, chromosomal aberrations, growth inhibition are common effects of these
heavy metals on various plants (Babula et al., 2008; Nagajyoti et al., 2010; Majid, Islam, & Riasmi,
2012; Srinivas, Purushotham, & Murali Krishna, 2013).
Plants exhibit many stress mediated responses in presence of heavy metals. The concentration of met-
als in plants increases in next higher trophic level of food chain due to biomagnification. The transfer of
metals from plants to animals and humans cause many toxic effects in them. Summary of some recent
literature regarding effects of heavy metals on plants is given by Table 1.

11
 
Heavy Metal Pollution

Table 1. Summary of some recent literature exhibiting effects of heavy metals on plants

Heavy Metal Plant Species Effects Reference

Eucrosis bicolor and Reduced mitotic index, spindle inhibition, increase in number of
Kwankua et al., 2012
Allium cepa micronuclei

Increase in Ca, P, Fe; Decrease in Mn, Cu; Increase in SOD, POD, Gusman, Oliveira, Farnese&
Lactuca sativa
CAT, APX, GR Cambraia, 2013

Lathyrus sativus Reduced Germination Index Talukdar, 2011

Singh, Srivastava& Prasad,

Luffaa cutangula Increase in superoxide radicals, Hydrogen Peroxide, Lipid peroxidation
2013
Arsenic
Reduced stomatal conductance; Reduction in P, Mg, Ca in shoots and
Oryza sativa Vromman et al., 2013
Mg, Ca, K in roots; Reduced grain yield.

Trigonella foenum-graecum L. Reduced Germination Index Talukdar, 2011

Increase in proline, lipid peroxidation, superoxide dismutase,

Vignamungo Srivastava & Sharma, 2013
peroxidase, ascorbic peroxidase; Decrease in catalase

Zea mays Plant growth inhibition Requejo & Tena, 2012

Zea mays Reduced growth, number of kernels and kernel weight Xiao-ke et al., 2012

Helianthus annus Stunted growth, reduced seed formation Yu, Lee, & Tsai, 2011

Decrease in stomatal conductance, transpiration rate, photosynthesis

Brassica juncea Ahmad, Nabi & Ashraf, 2011
rate and biomass; Increase in SOD, APX, CAT and GR

Martinez-Penalver, Grana,
Decrease in chlorophyll and carotenoid content and photochemical
Arabidopsis thaliana Reigosa& Sánchez-Moreiras,
operating efficiency
2012

Pena, Barcia, Azpilicueta,

Cadmium Triticum aestivum Decrease in cell cycle related proteins
Méndez& Gallego, 2012

Lactuca sativa Decrease in photosynthesis, Rubisco activity and Co2 assimilation rate Dias et al., 2013

ROS generation; Increase in Glutathione-S-Transferase activity and

Glycine max Pérez-chaca et al., 2014
NADPH-generating enzymes

Eucrosis bicolor and Reduced mitotic index, spindle inhibition, increase in number of
Kwankua et al., 2012
Allium cepa micronuclei

Pisum sativum Increase in SOD, CAT, GR Malecka et al., 2012

Oryza sativa Increase in cysteine; Decrease in GSH Qiu et al., 2013

Pisum sativum Increase in pigment content; Increase in Soluble sugars and starch Rodriguez et al., 2012

Oryza sativa Reduction in growth, dry biomass, soluble protein content Fan-rong et al., 2011

Chromium Brassica juncea Reduction in growth, chlorophyll content Ghani, 2011

Kovacik, Babula, Klejdus&

Chamomile ROS generation, Increase in SOX; Increase in soluble proteins
Hedbavny, 2013

Lactuca, Cardamine, Raphanus and

Reduced seed germination Ko, Lee & Kong, 2012
Cucumis

continued on following page

12

Heavy Metal Pollution

Table 1. Continued

Heavy Metal Plant Species Effects Reference

Israr, Jewell, Kumar& Sahi,

Sesbania drummondii Increase in SOD, APX, GR
2011

Manivasagaperumal,
Vigna radiate Reduction in growth and biomass Vijayarengan, Balamurugan&
Thiyagarajan, 2011

Prunus cerasifera Reduced fresh and dry weight, necrosis; Increase in CAT and SOD Lombardi & Sebastiani, 2005

Reduction in shoot and root length; Lipid peroxidation; Increase in

Oryza sativa (Rice) Thounaojam et al., 2012
SOD, GPX, APX, GR, ASH, GSH and Proline

Lactuca, Cardamine, Raphanus and

Reduced seed germination Ko et al., 2012
Cucumis

Raphanus sativus, Lolium perenee,

Growth inhibition, DNA damage Atha et al., 2012
Lolium rigidum

Cambrollé, Mateos-Naranjo,
Copper
Glaucium flavum Interference of Cu with Ca ions in PSII; Decrease in pigment content Redondo-Gomez, Luque&
Figueroa, 2011

Decrease in chlorophyll and carotenoid content and photochemical

Arabidopsis thaliana Martinez-Penalver et al., 2012
operating efficiency

Decrease in fresh and dry weight; Polymorphic bands in RAPD

Solanum melongena Körpe & Aras, 2011
profiles; Decrease in soluble protein content

Aydin, Gokce, Buyuk& Aras,

Cucumis sativus Growth inhibition; Polymorphic bands in RAPD profile
2012

Pisum sativum Increase in SOD, CAT, GR Malecka et al., 2012

Decrease in growth, chlorophyll, DNA and RNA content; Increase in (Vinod, Awasthi, & Chauhan,
Triticum aestivum
proline and total phenol 2012)

Lycopersicon esculentum, Lycopersicon

Mitochondrial damage Rueda et al., 2011
hirsutum

Reduction in protein, DNA, RNA, Chlorophyll and carbohydrates,

Phaseolus vulgaris Hamid et al., 2010
Increase in phenols

Eichhornia crassipes Decline in photosynthetic rate and chlorophyll Goswami et al., 2010

Rameshkumar, Baskaran,
Reduction in growth parameters, chlorophyll, protein, Amino acids and
Arachis hypogaea Rajendran & Thiyagarajan,
total sugars.
2013

Sesbania cannabina Increase in meiotic and mitotic aberrations; Decrease in pollen fertility Kumar & Srivastava, 2011

Increase in SOD, CAT, peroxidase, polymorphic bands in RAPD

Vigna sinensis Mohamed, 2011
profiles

Lead Decrease in growth parameters, decrease in photosynthetic rates; Ahmad, Ashraf, Tabassam,
Zea mays
increase in transpiration rate Hussain& Firdous, 2011

Growth inhibition; Ultrastructural changes in cell membrane; Kaur, Singh, Batish & Kohli,
Triticum aestivum
Distruption in mitochondrial structure 2013

Pisum sativum Increase in SOD, CAT, GR Malecka et al., 2012

Pluchea sagittalis Increased lipid peroxidation, SOD, ascorbic acid Rossato et al., 2012

Increased chromosomal aberrations, reduced chiasma frequency,

Trigonella foenum-graecum Choudhary et al., 2012
spindle inhibition

Increase in lipid peroxidation, SOD, CAT, APX, Glutathione-s- Lamhamdi, Bakrim, Aarab,
Triticum aestivum
transferase Lafont& Sayah, 2011

Oryza sativa Root growth inhibition; Increase in ROS, MDA, Lypoxygenase, Chen et al., 2012

Increased number of aberrant cells in meiotic division, chromosomal

Zea mays Rai & Kumar, 2010
damage
Mercury
Carrasco-Gil, Estebaranz-
Medicago sativa Increase in oxidative stress, antioxidant enzyme activity Yubero, Medel-Cuesta, Millán
& Hernández, 2012

13
 
Heavy Metal Pollution

Toxicology in Animals and Humans

Exposure of heavy metals to humans and animals can occur through inhalation, intake or penetration
through skin (Singh, Gautam, Mishra & Gupta, 2011; Tchounwou, Yedjou, Patlolla, & Sutton, 2012).
Various passages of exposure as explained as follows:

• Inhalation while breathing: Ultrafine particles or respirable particulate matter present in atmo-
sphere enter the respiratory system as we breathe in air having particulates containing heavy
metals.
• Intake of food crops or water contaminated with metals: Over exploitation of ground water or dis-
charge of wastewater from industries to water bodies increase the concentration of heavy metals
in potable water. Thus, heavy metals present in water enter alimentary canal along with the water
we drink. Food crops growing on soil contaminated with heavy metals result in transfer of these
metals to animals or humans.
• Dermal Contact: Exposure to metals also occurs through penetration through skin.
• Other factors: Apart from above mentioned factors, many other aspects which lead to the human
exposure to heavy metals include packaging of food, occupational exposure and food dyes.

USEPA analyzed the health risk associated with heavy metal intake by human beings by calculating
Hazard Quotient (Jolly, Islam, & Akbar, 2013). It is expressed as exposure concentration to reference
oral dose and if this ratio is less than 1, there is no potential risk. Hazard quotient is represented as:

(D ) * (C )
metal
HQ =
(R D ) * BO
f

where, D = daily intake of food (kg/day), Cmetal = concentration of metal (mg/kg), RfD = reference oral
dose metal (mg/kg of body weight/day) and BO = Body weight (kg).

• Arsenic: Arsenic is added to water via natural weathering of underground rocks and this can also
be deposited in soil. Above 40 million population of India is consuming arsenic contaminated
water (Jomova et al., 2011). The permissible limit for arsenic in drinking water as given by WHO
is 10 µg/l. In some parts of India Arsenic concentration has been reported as 3200 µg/l, which is
more than 300 times the permissible concentration (Jomova et al., 2011). Chromosomal aberra-
tions in human cells like, leukocytes, lymphocytes and fibroblasts are reported. Prolonged expo-
sure to arsenic leads to severe effects on cardiovascular system and nervous system which leads
to death. Arsenic induced carcinogenesis of bladder, kidney and lung cells has been reported.
Drinking water having arsenic concentration as 100 µg/l is associated with increase in cancer
patients in those areas. Arsenic is also available to human beings through inhalation of particles
laden with arsenic ions (Järup, 2003; Dorne et al., 2011; Ren et al., 2011; Csavina et al., 2014).
• Cadmium: Cadmium exposure to animals or humans can occur through the intake of food and
water and also by inhalation. Humans also get exposed to cadmium with cigarette smoking and
concentration of cadmium has been found to be much higher in blood of smokers than to non-

14

Heavy Metal Pollution

smokers. Cadmium exposure is expressed in terms of B-Cd, i.e., blood cadmium or U-Cd, urine
cadmium. B-Cd is the concentration of cadmium in blood at present time. Life time exposure to
cadmium can be higher than this. U-Cd is dependent on the concentration of cadmium on which
a person is exposed (Järup, 2003). The concentration of metal higher than permissible limit was
reported in vegetables growing in some parts of world. Also their hazard quotient was found to be
higher than as recommended by WHO. The upper limit of provisional weekly intake of cadmium
set by WHO is 7 µg/kg of body weight. The consumption of cadmium through food is reported
much higher than the recommended limits (Ata, Tayyab & Rasool, 2013; Dorne et al., 2011;
Orisakwe, Kanayochukwu, Nwadiuto, Daniel & Onyinyechi, 2012; Orisakwe, Nduka, Amadi,
Dike & Bede, 2012; Uwah, Ndahi, Abdulrahman & Ogugbuaja, 2011). Ingested cadmium is ab-
sorbed by the gastrointestinal tract and due to molecular homology with Zn2+ and Ca2+ ions, it gets
adhered to proteins and gets transported through blood to liver where it binds with mettaloproteins
and affects the liver enzyme mechanism and apoptosis for hepatocytes. When the cadmium bound
mettaloproteins reach kidney, they causes tubular dysfunction (Godt et al., 2006). Increase in con-
centration of cadmium in ambient environment lead to the occurrence of itai itai disease in Japan.
Outbreak of this disease in 1950s killed many people in the country. Toxicity associated with con-
sumption of high doses of cadmium involves renal impairment and irreversible tubular damage.
Inhalation of cadmium causes severe pulmonary disorders. International Agency for Research
in Cancer (IARC) has identified cadmium as human carcinogen (Järup, 2003; Das, Grewal, &
Banerjee, 2011). Cadmium toxicity is associated with oxidative stress and production of ROS
species and enhanced lipid peroxidation in erythrocytes, lungs, liver and brain are reported (Patra,
Rautray & Swarup, 2011). Other health effects associated with prolonged cadmium exposure in-
volves effects on bones, cardiovascular troubles, hypothalamic-pituitary gland malfunctioning and
testicular damage (Järup, 2003; Patra et al., 2011).
• Mercury: Toxicity associated with mercury was noted as “dancing cat fever” or Minamata dis-
ease. This disease came into notice in 1950s when the release of methyl mercury in bay water of
Minamata triggered bioaccumulation of mercury in entire food chain. The exposure of mercury
affected the brain and caused neurological disorders. Mercury exists in elemental form, ions or
organic form. Human exposure to mercury can occur through inhalation and consumption of
contaminated food and water. Mercury vapours when inhaled target brain directly, it reaches brain
either by getting dissolved in serum or being associated with red blood cells. Metallic mercury can
cross blood brain barrier and placental barrier. Ingested mercury affects gut and kidneys. HgCl2 is
present in many cosmetics especially skin lightening creams. It associates with sulfhydryl group
in red blood cells and metaloproteins. Only metallic mercury dissolved in blood stream cross
blood brain barrier other species of mercury gets deposited in placental, intestinal, respiratory lin-
ings (Bernhoft, 2012). Permissible limit of mercury in drinking water as given by Environmental
Protection Agency (EPA) is 2 ppb and permissible limit in seafood given by Food and Drug
Association (FDA) is 1 ppm (Das et al., 2011).
• Lead: Human exposure to lead occurs through inhalation of respirable particulate matter and in-
gestion of food and water containing lead. More than half of inhaled lead can reach to lungs and
same amount of ingested lead is absorbed through gut. It is transported to various organs while be-
ing bound to erythrocytes. In adults, it is deposited in bones, where it remains for a long time and
slowly gets released to other parts. In children, it can also cross the blood-brain barrier. Prolonged
exposure can cause acute toxicity in adults too leading to the barging of blood brain barrier and

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Heavy Metal Pollution

a disease known as lead encephalopathy (Järup, 2003; Dorne et al., 2011). The concentration of
lead in food crops of many parts of world has been found to be higher than permissible limit (0.3
mg/kg) (Nagajyoti et al., 2010; Uwah et al., 2011; Shahryari & Shehamat, 2012; Varalakshmi &
Ganeshamurthy, 2012; Shah et al., 2013). The decrease in nerve conduction and dermal sensibility
is observed in individuals having lead concentration in blood as low as 3 µmol/l. Prolonged expo-
sure can increase the concentration and can lead to severe neurological, renal and cardiovascular
disorders. Increase in reactive oxygen species leading to destruction of biomolecules like DNA
and protein. Removal of H+ ions is easier in fatty acids having more double bonds as presence of
double bond weakens the adjacent C-H bonds. Thus, saturated fatty acids are more vulnerable to
lead induced damage (Patra et al., 2011). IARC considers lead also to be probable carcinogen and
is linked with alimentary canal cancer. Shortening of telomeres was reported to be due to pro-
longed lead exposure in workers working in battery manufacturing plant (Wu et al., 2012).

Other heavy metals including, chromium, zinc, manganese and nickel cause many toxicological ef-
fects on human and animal population the transport of metals in human body is explained in Figure 5.
Throughout the world, concentration of toxic metals in ambient environment is continuously increas-
ing and exposure leads to devastating effects.

MITIGATION OF HEAVY METALS

Considering the effects of heavy metals on biota, various mitigation strategies are proposed throughout
the world. Some of the common techniques used are as mentioned in Table 2.

Figure 5. Circulation of heavy metals in human body

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Heavy Metal Pollution

Table 2. Techniques for reduction of heavy metals in environment

S. No. Component of Environment Techniques

Absorbers in chimneys, scrubbers, cyclone filters and increased height of
1. Air chimneys, biosorption, combustion filters in automobiles
Coagulation, reverse osmosis, biosorption, Adsorption, phytoremediation,
2. Water Microbial remediation
Phytoremediation, phytostabilization, microbial remediation, soil-amendments to
3. Soil sequester metals.

RECOMMENDATIONS

1. The industrial discharges releasing heavy metals should be regularly monitored and abated if they
release toxic metals beyond permissible limit.
2. Farmers should be made aware of toxicity of agricultural chemicals which have heavy metals in
composition.
3. Law enforcement at all level should be strict.
4. Public awareness programs should be implemented regularly at state, national and global levels.
5. Apart from these the individual efforts as well as focus on general strategy like following 3Rs that
is, Reduce the consumption of goods which during its life cycle release heavy metals to surround-
ing, reuse and recycle materials.

CONCLUSION

Heavy metals are adding up in our environment with each passing moment and the health and ecological
effects associated with these metals are well known. These tiny particles are culprit of mass destruction
of plants, animals and even human beings. The magnification of heavy metals at each trophic level adds
to the disastrous effects of these metals. Children are among the most affected group. Problem with
heavy metals is that they get sequestered around us thus increasing the exposure to living organisms.
Effects of heavy metals can be fatal and can cause large scale ecological destruction. There is utmost
need to establish accurate and easy diagnostic tools for diagnosis of toxicity due to metals. In research,
we need to find the alternative testing methods that should aim to reduce the ecological impacts of
research and these toxic metals are not released to the ambient environment due to research practices.
Also, it is important for the international bodies like, US EPA, WHO and FDA to clearly define the
permissible limits of metals in food, agricultural soil, water and air. Also, there is need for continuous
monitoring to locate vulnerable areas and apply suitable remediation strategies and decrease exposure
to large populations. Remediation strategies should focus on reducing the transfer through food chain
by immobilizing the metals or converting them to less mobile species. Remediation strategies should
be economical and localized.

17
 
Heavy Metal Pollution

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KEY TERMS AND DEFINITIONS

Acceptable Daily Intake: It is the minimum safe dose of heavy metals that can be consumed along
with contaminated food and water daily.
Biological Concentration Factor: It is defined as the amount of heavy metal transported from soil
to plants.
Biomagnification of Metals: The increase in concentration of heavy metals with each trophic level
due to accumulation of metal in living tissues is called biomagnification of metals.
Ecological Pyramid: The graphical representation of trophic levels signifying each level on the basis
of number, biomass or energy.
Hazard Quotient: Health risk assessment caused by consumed heavy metal with respect to body
weight is known as hazard quotient.
Heavy Metal: Metals having density higher than 3.5 g/cm3.
Transfer Factor: Amount of metal transferred from root to shoot.

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 27

Chapter 2
Heavy Metal Pollution
and its Management:
Bioremediation of Heavy Metal

Ashok K. Rathoure
Vardan Environet Guargaon, India

ABSTRACT
Environmental degradation has become a major societal issue thanks to uncontrolled anthropogenic
activity, besides natural factors. Entry of toxic heavy metals and minerals in human system mainly
through contaminated water, food and air, leads to overt and insidious health problems. Heavy metal
pollution, a global concern today, can be managed by using bioremediation, an eco-friendly alternative.
Bioremediation is one of the most promising technological approaches to the problem of hazardous waste.
It is a technology for removing pollution from environment, restoring contaminated site and preventing
future pollution. Bioremediation can be performed in situ or ex situ. Microorganisms directly degrade
contaminants rather than merely transferring them from one medium to another, employ metabolic deg-
radation pathways and can be used in situ to minimize disturbance of the cleanup site. Hence, microor-
ganisms can be effective, economical and non-disruptive tools for eliminating hazardous chemicals. Its
advantage generally outweigh the disadvantage, therefore may be used as management tool.

1. BACKGROUND

The rapid industrial developments have led to the generation of huge quantities of hazardous wastes,
which have further aggravated the environmental problems in the country by depleting and polluting
natural resources. Therefore, rational and sustainable utilization of natural resources and its protection
from toxic releases is vital for sustainable socio-economic development (Chakrabarti, 2006). Hazard-
ous waste management is a new concept for most of the Asian countries including India. The lack of
technical and financial resources and the regulatory control for the management of hazardous wastes in
the past had led to the unscientific disposal of hazardous wastes in India, which posed serious risks to
human, animal and plant life. A huge quantity of pollutants in the form of domestic and industrial ef-

DOI: 10.4018/978-1-4666-9734-8.ch002

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Heavy Metal Pollution and its Management

fluents is discharged directly or indirectly into the water bodies, which has severe impacts on its biotic
and abiotic environment. During rain, surface water with soil, mud and humus enter into the river, tanks
and other water bodies. The soil is the target of thousands of contaminants that vary in composition
and in concentration. The contaminants enter the system as a result of a wide range of actions such as
intentional applications, inadequate residue disposal, accidental wastes and inappropriate use. Some
human activities have resulted in the accumulation of metals in the environment. The inorganic miner-
als like sodium, potassium, calcium, magnesium and heavy metals like iron, manganese, lead, mercury,
chromium, cadmium, nickel, cobalt, beryllium copper etc., when reach to the river with water cause
water pollution. The use of various types of pesticides and insecticides in agriculture also cause water
pollution. The pathogenic organisms of these wastes transmit to the water and pose serious problems
(Batley, 1989; Garbarino et al., 1995; Jogdand, 1995; Agarwal, 1998; Hakeem & Bhatnagar, 2010).

2. WASTE AND ITS TYPE

Waste is an unavoidable by product of most human activity. Waste can be, in the form of solid or liquid,
posed the harmful nuisance. Most solid waste is either sent to landfills or to incinerators. Ocean dump-
ing has also been a popular way for coastal communities to dispose of their solid wastes in which large
barges carry waste out to sea and dump it into the ocean. Most municipal and non municipal waste is
sent to landfills. Landfills are popular because they are relatively easy to operate and can handle of lot
of waste material. There are two types of landfills, sanitary landfills and secure landfills. Each day after
garbage is dumped in the landfill, it is covered with clay or plastic to prevent redistribution by animals
or the wind. In natural system, there is no such thing as waste. Everything flows in a natural cycle of use
and reuse. Living organisms consume materials and eventually return them to the environment, usually
in a different form, for reuse. Solid waste refers to a variety of discarded materials, not liquid or gas that
is deemed useless or worthless. However, what is worthless to one person may be of value to someone
else and solid wastes can be considered to be misplaced resources. Solid wastes are all the waste aris-
ing from human and animal activities that are normally solid waste and that are discarded as useless
or unwanted. The term is all inclusive and it encompasses the heterogeneous mass of throwaways from
the urban community as well as the homogeneous accumulations of agricultural, industrial and mineral
wastes (USAID, 2004; Rajor & Kunal, 2011).
Electronic waste, e-waste, e-scrap or Waste Electrical and Electronic Equipment (WEEE) describe
loosely discarded, surplus, outdated, broken, electrical or electronic devices. Electronic waste may be
defined as all secondary computers, entertainment device electronics, mobile phones and other items such
as television sets and refrigerators, whether sold, donated or discarded by their original owners. The pro-
cessing of electronic waste in developing countries causes serious health and pollution problems because
electronic equipment contains some very serious contaminants such as lead, cadmium, beryllium and
brominates flame retardants. Electronic waste processing systems have matured in recent years, follow-
ing increased regulatory, public and commercial scrutiny and a proportionate increase in entrepreneurial
interest. In developed countries, electronic waste processing usually involves dismantling the equipment
into various parts like metal frames, power supplies, circuit boards, plastics, by hand. The advantages of
this process are the human’s ability to recognize and save working and repairable parts, including chips,
transistors, RAM, etc. It is a major area of concern today that the wealthy countries are dumping large
quantities of e waste into the developing world. According to BBC, currently the companies export 80

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Heavy Metal Pollution and its Management

percent of the world’s electronic trash to Asia. The problem of e-waste recycling in India involved the
urgent need for educating consumers including the potential threat to public health and the environment
posed by their products and for raising awareness for the proper waste management (Kesselman, 2007;
Dogbevi, 2007; Beary, 2008; Mcphate, 2008; Toxic Links, 2008; Pinto, 2009).
Liquid waste can be defined as such fluids as wastewater, fats, oils, grease or used oil. The disposal
of such waste, such as transmission fluid, cooking oil, spent oil, fats or grease can contaminate the
groundwater or negatively impact the wastewater system. The liquid waste basically the waste water
discharged from the industrial and domestic area. Wastewater is water that has been used for some
purpose and is deemed unfit for further use. In fact, wastewater can be used for secondary purposes in
most cases. Also, efficient use of water reduces the amount of wastewater generated. Sewage is created
by residences, institutions, hospitals and commercial and industrial establishments. Raw influent or
sewage includes household waste liquid from toilets, baths, showers, kitchens, sinks and so forth that is
disposed of via sewers. In many areas, sewage also includes liquid waste from industry and commerce.
As rainfall runs over the surface of roofs and the ground, it may pick up various contaminants including
soil particles and other sediment, heavy metals, organic compounds, animal waste, oil and grease. It
may be originated from various sites and by different uses (US EPA, 2000; Central New York Regional
Planning and Development Board, 2004; Massoud & Ahmad, 2005).
Municipal waste water is the combination of liquid or water carried wastes originating in the sanitary
conveniences of dwellings, commercial or industrial facilities and institutions, in addition to any ground-
water, surface water and storm water that may be present. Wastewater is the flow of used water from a
community. The characteristics of the wastewater discharges vary from location to location depending
upon the population and industrial sector served, land uses, groundwater levels and degree of separation
between storm water and sanitary wastes. Domestic wastewater includes typical wastes from the kitchen,
bathroom and laundry, as well as any other wastes that people may accidentally or intentionally pour
down the drain. Sanitary waste water consists of domestic wastewater as well as those discharged from
commercial, institutional and similar facilities. Industrial wastes will be as varied as the industries that
generate the wastes. Municipal waste water also contains a variety of inorganic substances from domes-
tic and industrial sources, including a number of potentially toxic elements such as arsenic, cadmium,
chromium, copper, lead, mercury, zinc etc (Xie et al., 1996, Ferrari et al., 1999).

3. INDUSTRIAL EFFLUENTS AND SOIL POLLUTION

Toxic chemicals found in wastewater pass through wastewater treatment facilities that have not been
designed to remove them and can interfere with their operation. In a biological treatment process toxic
materials can upset a treatment process or even kill the biological community and make the process
ineffective. To remove the toxic pollutants at the treatment facility can be very costly. Therefore, it is
generally advantages to remove them at the source. Source control can be achieved by the use of mu-
nicipal by laws limiting pollutant discharges to the sewerage system. The removal of toxic pollutants
at the source can be achieved by requiring treatment prior to discharge, recycling of waste by products,
manufacturing process changes and the substitution of raw material (Thorpe, 2009).
The effluent of the industries goes into the water system and changes the physico-chemical quality
of water and also makes it unfit for drinking and other uses. Since all natural waterways contain bacteria
and nutrients, almost any waste compounds introduced into such waterways will initiate biochemical

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Heavy Metal Pollution and its Management

reactions. These biochemical reactions are measured as BOD and COD in the laboratory. Both have been
widely adopted as a measure of pollution effect. Disposal of wastewaters from an industrial plant is a
difficult and costly problem. Most petroleum refineries, chemical and petrochemical plants have onsite
facilities to treat their wastewaters so that the pollutant concentrations in the treated wastewater comply
with the local or national regulations regarding the disposal of wastewaters into community treatment
plants or into rivers, lakes or oceans. The solids can be suspended 30% as well as dissolved solids which
are about 70%. The dissolved solids can be precipitated by chemical and biological processes. Organic
components may consist of carbohydrates, proteins, fats and greases, surfactants, oils, pesticides, phenols
etc. From a physical point of view the suspended solids can lead to the development of sludge deposits
and anaerobic conditions when discharged into the receiving environment. Physically, the wastewater is
usually characterized by grey colour, musty odour, 0.1% solid content and 99.9% water content (Tcho-
banoglous, 1987; Tchobanoglous et al., 2003; Clair, 2003; Maiti, 2004).
The soil is the target of thousands of contaminants that vary in composition and in concentration.
The contaminants enter the system as a result of a wide range of actions such as intentional applications,
inadequate residue disposal, accidental wastes and inappropriate use. Soil pollution is defined as the
buildup in soils of persistent toxic compounds, chemicals, salts, radioactive materials or disease causing
agents, which have adverse effects on plant growth and animal health. Soil is the thin layer of organic and
inorganic materials that covers the earth’s rocky surface. The organic portion, derived from the decayed
remains of plants and animals, is concentrated in the dark uppermost topsoil. The inorganic portion,
made up of rock fragments, was formed over thousands of years by physical and chemical weathering of
bedrock. Soil pollution is caused by the presence of manmade chemicals or other alteration in the natural
soil environment. This type of contamination typically arises from the rupture of underground storage
links, application of pesticides and percolation of contaminated surface water to subsurface strata, oil
and fuel dumping, leaching of wastes from landfills or direct discharge of industrial wastes to the soil.
The most common chemicals involved are petroleum hydrocarbons, solvents, pesticides, lead and other
heavy metals. This occurrence of this phenomenon is correlated with the degree of industrialization
and intensities of chemical usage. The waste contaminates the soil. The solid waste includes garbage,
domestic refuse and discarded solid materials such as those from commercial, industrial and agricultural
operations. They contain increasing amounts of paper, cardboards, plastics, glass, old construction ma-
terial, packaging material and toxic or otherwise hazardous substances (Knaebel et al., 1994; Franklin
Associates, 1996).
Some human activities have resulted in the accumulation of metals in the environment. Both soil
and aqueous effluents have been contaminated with heavy metals as the result of numerous industrial
activities, including mining, smelting, jewelry, automobile battery production, vehicle emission and land
filling of industrial waste and fly ash from incineration process. This contamination of the environment
poses serious health threats to humans and animals, as these heavy metals tend to persist in the environ-
ment indefinitely. This kind of contamination presents a challenge, as the presence of heavy metals in
soils and aqueous effluents leads to serious problems because they cannot be biodegraded. In this case,
the metal ion can only be converted to the base metal, methylated, precipitated, volatilized or complexed
with an organic ligand. The more common heavy metals associated with anthropogenic activities include
lead, cadmium, copper, chromium, nickel, iron, mercury and zinc. Methods of treating the contaminated
effluents currently consist of chemical precipitation, solvent extraction, dialysis, electrolytic extraction,
cementation, reverse osmosis, evaporative methods, ion exchange resins, carbon adsorption and dilution
(USEPA, 2000; European Union, 2002).

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Heavy Metal Pollution and its Management

4. HEAVY METALS

Metals play an integral role in the life processes of living organisms. Some metals like Ca, Co, Cr, Cu,
Fe, K, Mg, Mn, Na, Ni and Zn are essential, serve as micronutrients and are used for redox processes, to
stabilize molecules through electrostatic interactions; as components of various enzymes and regulation
of osmotic pressure. The essential metals must be present in a certain concentration range. Too low con-
centrations of heavy metals lead to a decrease in metabolic activity and too high concentrations, it leads
to toxicity. While other metals like Ag, Al, Cd, Au, Pb and Hg have no biological role and they are non
essential. Nonessential metals are tolerated at very low concentrations and inhibit metabolic activity at
higher concentrations. They are potentially toxic to living organism specially microorganisms. Toxicity
of non essential metals occurs through the displacement of essential metals from their native binding
sites or through ligand interactions. The many uses of heavy metals in several applications lead to wide
distribution in soil, silt, waste and waste water. Such pollution of the environment by toxic metals and
radionuclide arises as a result of many human activities, largely industrial, although such sources as
agriculture and sewage disposal also contribute. Heavy metal contamination can be a consequence of
industrial activities that eliminate residues in the soil that in long terms, promote their accumulation.
Heavy metals in wastewater come from industries and municipal sewage and they are one of the main
causes of water and soil pollution. The majority of the sources are originated by human actions like
metal manufacture and mining industries with storage, disposal and transportation problems. Among
the metals found more frequently there are Cd, Pb, Co, Cu, Hg, Ni, Si and Zn. For Cd, Pb, Cu and Zn,
their toxicity increases as follows: Pb < Zn < Cu < Cd, depending on countless abiotic and biotic factors
(Lloyd et al., 2001; Glick, 2003; Zenker et al., 2005).
Both soil and aqueous effluents have been contaminated with heavy metals as the result of numerous
industrial activities, including mining, smelting, jewelry, automobile battery production, vehicle emis-
sion and land filling of industrial waste and fly ash from incineration process. This contamination of the
environment poses serious health threats to humans and animals, as these heavy metals tend to persist
in the environment indefinitely. Metals play an integral role in the life processes of living organisms.
Some metals like Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni and Zn are essential, serve as micronutrients
and are used for redox processes, to stabilize molecules through electrostatic interactions; as components
of various enzymes and regulation of osmotic pressure. The essential metals must be present in a certain
concentration range. Too low concentrations of heavy metals lead to a decrease in metabolic activity and
too high concentrations, it leads to toxicity. While other metals like Ag, Al, Cd, Au, Pb and Hg have no
biological role and they are non essential. In order to survive in heavy metal polluted environments, many
microorganisms have developed means of resistance to toxic metal ions. These mechanisms include metal
exclusion by permeability barriers, active transport of the metal away from the cell organism, intracellular
sequestration of the metal by protein binding, extracellular sequestration, enzymatic detoxification of the
metal to a less toxic form and reduction in metal sensitivity of cellular targets. Most microorganisms are
known to have specific genes for resistance to toxic ions of heavy metals. Mostly, the resistance genes
are found on plasmids or on chromosomes. Plasmid encoded metal resistance determinants have been
reported to be inducible (Nies & Silver, 1995; Rosen, 2002).
The intake of heavy metal ions by microbial strains normally includes a redox reaction involving the
metal. Some bacteria use heavy metals for energy and their growth. Bacteria that are resistant to heavy
metals also play an important role in biogeochemical cycling of metal ions. Since, the oxidation state
of a metal ion may determine its solubility, many scientists have been attempting to use microbes that

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Heavy Metal Pollution and its Management

are capable to oxidize or reduce metals in order to remediate metal contaminated sites. Although, some
heavy metals are important and essential trace elements and others are toxic at high concentrations to
microbes, some microbes have adapted to tolerate the presence of metals or even to use them to grow. In
addition, a number of interactions between microbial strains and metals have important environmental
and health implications. Health problem due to heavy metals pollution in human being include nausea,
vomiting, bone complications, nervous system impairments and even death become a major problem
throughout many countries when metal ions concentration in the environment exceeded the admissible
limits. Due to that, various treatment technologies had been searched to reduce the concentration of
heavy metals in the environment (Nies, 1999; Lodeiro et. al., 2006).
It is needed to remove the heavy metals from the contaminated sites/waste. Several heavy metals
removal technologies including chemical precipitation, ion exchange, reverse osmosis, electrodialysis,
ultra filtration and pyhtoremediation are commonly used in industries. However, these technologies
are becoming uneconomical and unfavorable to remove heavy metals from industrial wastewaters.
The development of new treatment method such as bioremediation to remove heavy metal ions from
wastewater and solid waste which could be cost effective and more efficient has spurred to overcome
the conventional method. Bioremediation technology has received much attention as it offered low cost
technique and non hazardous biomaterials (Lovley, 1997; Ahalya et al., 2003).

5. BIOREMEDIATION

The history of bioremediation is considerably shorter and it reflects many upturns and downturns as a
result of political and economic forces. Interest in use of microorganisms to degrade specific hazard-
ous organic chemicals probably dates back to Gayle (1952), who proposed the microbial infallibility
hypothesis. Gayle postulated that for any conceivable organic compound, there exists a microorganism
that can degrade it under the right conditions. If not, evolution and adaptation would produce such a
strain. This hypothesis cannot wrong, because failure to degrade a contaminant can be attributed to the
researcher’s failure to use the right strain under the right condition. In 1970s, environmental statutes of
unprecedented scope passed, such as the Occupational Safety and Health Act (OSHA) of 1970, the Clean
Air Act (CAA) of 1970, the Clean Water Act (CWA) of 1972, the Safe Drinking Water Act (SWA) of
1974 and the Toxic Substance Control Act (TSCA) of 1976. This regulatory pressure stimulated interest
in site remediation technologies, including bioremediation. However, bioremediation failed to meet the
expectations raised by many technology salespeople, who commonly advocated the addition of special-
ized bacteria to contaminated site i.e. bioaugmentation. Early proponents of this approach generally
did not recognize that indigenous bacteria already present at a contaminated site where probably better
predisposed physiologically and genetically to mediate the degradation of the target pollutants. The first
patent for a biological remediation agent was registered in 1974, being a strain of Pseudomonas putida
that was able to degrade petroleum. In 1991, about 70 microbial genera were reported to degrade petro-
leum compounds and almost an equal number has been added to the list in the successive two decades.
Bioremediation can occur naturally or through intervention processes. Natural degradation of pollutants
relies on indigenous microflora that is effective against specific contaminants and it usually occurs at
a slow rate. With intervention processes, the rate of biodegradation is aided by encouraging growth of
microorganisms, under optimized physico-chemical conditions. Microorganisms play a vital role in
heavy metal contaminated soil and wastewater by the mechanisms of biosorption. Some microorgan-

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Heavy Metal Pollution and its Management

isms possess an astonishing catabolic versatility to degrade or transform such compounds. The need for
economical, effective and safe methods for removing heavy metals from wastewater has resulted in the
search for unconventional materials that may be useful in reducing the levels or accumulation of heavy
metals in the environment (US Congress, 1991; Agarwal, 1998; Bouwer et al., 1998; Smith et al., 1998).
Bioremediation is the use of living microorganism to reduce the environmental pollution. It is a
technology for removing pollution from the environment, restoring contaminated site and preventing
future pollution. Bioremediation can be performed in situ or ex situ. With the onset of the Industrial
Revolution, an ever increasing proportion of the earth’s surface became contaminated with natural and
xenobiotic toxic chemicals. The basic principle of bioremediation involves utilizing the activity of micro-
organisms naturally present in the soil and water or selected organisms inoculated into the environment,
to biodegrade or detoxify contaminating compounds in situ. In the majority of cases a consortium of
microorganisms will be involved in the biodegradation of the contaminant, rather than a single species.
To optimize the process, promotion of the growth of indigenous microorganisms is necessary. It can be
achieved by the addition of key nutrients such as nitrogen and phosphorus, which are normally present
in growth limiting concentrations. This enables the natural microbial flora to develop and metabolize
the contaminant (Vidali, 2001).
Alternatively, known biodegraders of the contaminant that have been identified, isolated and their
activities optimized can be used as an inoculants. For example, a recent addition to the growing list of
microorganisms able to sequester or reduce metals is Geobacter metallireducens. This bacterium can
remove uranium, a radioactive waste, from drainage waters in mining operations and from contaminated
ground waters. However, the most radiation resistant bacterium known is Deinococcus radiodurans; this
organism is also being developed to help clean up soil and water contaminated by solvents, heavy met-
als and radioactive waste. A genetically engineered strain of D. radiodurans has been produced which
can detoxify mercury (genes derived from Escherichia coli) and degrade toluene (genes derived from
Pseudomonas putida) in radioactive environments (Bouwer & Zehnder, 1993; Bernard et al., 2007).

6. REQUIREMENTS OF BIOREMEDIATION

Bioremediation is a fairly new technology and holds the promise of becoming the solution to our pol-
luted environment. This new technology gives us alternative routes to cleaning up contaminated sites
was thought to be not possible previously. Nonetheless, the technology of bioremediation still has a long
way more to go). Bioremediation faces several challenges and some of the most common ones are deficit
of knowledge, lack of integrated research, lack of revenue and inadequate tool and infrastructure (Aksu,
1998; Vidali, 2001; Ahluwalia & Goyal, 2007). A deficit of knowledge on the different fundamental
branches of sciences that are involved in the process of bioremediation hinders the progress of bioreme-
diation. Examples of the different disciplines of sciences are such as structural and molecular biology,
microbiology, genomics, geochemistry, along with hydrology and transport processes. To date, little is
known on how introduced microorganisms interact with different hydrological environment. Research
in each of the variety of fields is needed to further researchers’ comprehension on the actual activity or
rather, the chemistry that is involved and interactions between contaminants, native organisms on site
and remedial organisms. There is still much to learn and gain from research as contaminated sites are
complex systems, each composed of different types of contaminants, diverse organisms and dissimilar
environment (Doyle, 1989; Prescott, 2002). Bioremediation is a multidisciplinary field and researchers

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Heavy Metal Pollution and its Management

from various fields need to integrate their knowledge. The success and efficiency of bioremediation
requires the involvement of microbiologists, biochemists, engineers, geologists and soil scientists among
others. Knowledge on the combined factors is not enough and mostly these are the rate limiting factors
of the process of bioremediation. Interdisciplinary research of at the least two fields is unavoidable to
advance this technology. Assimilation of scientific ideas across disciplines is most needed to optimize
the potential of bioremediation (Blackburn & Hafker, 1993; Boopathy, 2000; Bernard & Jack, 2007).
There is no doubt that governmental and private sectors are investing on biotechnology companies but
not much of the revenue goes to the improvement of bioremediation. Lack of revenue is an obstruction
to advancement in bioremediation as it prevents further research and discoveries from taking place.
Thereupon, progress of bioremediation is delayed as a result of material deficiency (Gadd, 1988;
Bouwer & Zehnder, 1993). Infrastructure and materials are needed to carry out research. Accuracy in
experimental procedures is crucial to ensure successful treatment when introduced to contaminated site.
However, there is inadequacy of infrastructure to analyze the conditions of contaminated sites as well as
monitor the process of degradation that has been introduced to the site. Insufficiency of advanced tools
specifically designed to aid research on bioremediation would also hold back development of research
methodology and prevent discoveries from happening. Despite its shortcomings, its pertinence in this
world is unquestionable in the light of present day environmental hazards. Bioremediation provides a
technique for cleaning up pollution by enhancing the same biodegradation processes that occur in nature
and potential for significant advances (Bouwer et al., 1998; Boopathy, 2000; Chatterjee et al., 2008;
Shazia et al., 2009; Hakeem & Bhatnagar, 2010).

7. METAL TOXICITY AND MANAGEMENT

The pollution of the environment with toxic heavy metals is spreading throughout the world along with
industrial progress. Effluents from textile, leather, tannery, electroplating, galvanizing, dyes and pig-
ment, metallurgical industries and other metal processing and refining operations at small and large scale
sector contains considerable amounts of toxic metal ions (White et al., 1995). Cadmium, chromium,
cobalt, copper, iron, manganese, mercury, molybdenum, nickel, silver and zinc are known to be the most
commonly heavy metals used and the more widespread contaminants of the environment (FWPCA,
1998). Traces of these heavy metals are necessary as co-factors of enzymatic reactions, but high levels
of them may cause extreme toxicity to living organisms due to inhibition of metabolic reactions. Many
compounds that are legally considered to be hazardous can be transformed to harmless products. This
eliminates the chance of future liability associated with treatment and disposal of contaminated material.
Instead of transferring contaminants from one environmental medium to another from land to water or
air, the complete destruction of target pollutants is possible (Agarwal, 1998).
Arbitrary and hysterical discharge of industrial and urban wastes into the environmental sink has
become an issue of major global concern. Intensification of agriculture and manufacturing industries
has resulted in increased release of a wide range of xenobiotic compounds to the environment. Excess
loading of hazardous waste has led to scarcity of clean water and disturbances of soil, thus limiting
crop production. Although enactment of stringent regulation has led to less indiscriminate disposal of
organic and inorganic wastes, challenges remain that require other intervention (Hess et al., 1997). Waste
comprises liquid waste discharged by domestic residences, institutions, hospitals, commercial proper-
ties, industry or agriculture and can encompass a wide range of potential contaminants and concentra-

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tions. In the most common usage, it refers to the municipal wastewater that contains a broad spectrum
of contaminants resulting from the mixing of wastewater from different sources. Raw influent includes
household waste liquid from toilets, baths, showers, kitchens, sinks and so forth that is disposed to the
river via sewers. This affects the BOD, COD, turbidity and also causes the physico-chemical changes in
the river. In many areas, sewage also includes liquid waste from industry and commerce. As rainfall runs
over the surface of roofs and the ground, it may pick up various contaminants including soil particles
and other sediment, heavy metals, organic compounds, animal waste and oil and grease (APHA, 1995;
1998; FWPCA, 1998). The metal ions from metal mining pose problems to the water environment by
discharging mine water from underground and open pit mines (Knaebel, 1994). Leachate water and
runoff water from overburden/waste rock dumps also contaminate nearby water streams (Ferrari et al.,
1999). The principle pathways by which leached contaminants can enter into groundwater are leakage or
spills from storage ponds, leach pad liners, storm water run on/off, uncontrolled leaching from heaps and
dumps. These toxic metals ions not only cause potential human health hazards but also affect other life
forms (Malik, 2004). Heavy metal pollution usually results from electroplating, plastic manufacturing,
fertilizer, pigment industries, mining and metallurgical processes (Nirmal Kumar et al., 2009).
Heavy metals can be emitted into the environment by both natural and anthropogenic causes. The
major causes of emission are the anthropogenic sources specifically mining operations (Hutton & Sy-
mon, 1986; Nriagu, 1989). Much of the metal salts within the oceans, lakes, streams and estuaries are
in the form of magnesium, calcium, potassium and sodium salts. While many other constituents are
clearly available in solution, no element having a concentration less than 2 nmol in the ocean is gener-
ally considered essential for life. The metals cannot degraded to harmless products and hence persist
in environmental indefinitely (Silver, 1996). The metals leached out and in sloppy areas, are carried by
acid water downstream or run-off to the sea. Through mining activities, water bodies are most emphati-
cally polluted (INECAR, 2000; European Union, 2002). The heavy metals refer to any metallic element
that has a relatively high density than 5g/cm3, is toxic or poisonous even at low concentration and its
pollution is the major concern faced by the society today (Aksu, 1998; Nies, 1999). Heavy metals are
on the forefront of academic and regulatory concern, since millions of gallon of water containing toxic
heavy metals are generated annually from several metal processing industries and discharged into the
environment (Gardea et al., 2005).
Metals discharged into water bodies are not biodegraded but undergoes chemical or microbial
transformations, creating large impact on the environment and public health. Heavy metals influence
the microbial population by affecting their growth, morphology, biochemical activities and ultimately
resulting in decreased biomass and diversity (Roane and Pepper, 2000). The microorganisms respond to
the heavy metals by several processes; including transport across the cell membrane, biosorption to the
cell walls and entrapment in extracellular capsules, precipitation, complexation and oxidation-reduction
reactions (Veglio et al., 1997). The discharge of wastewater containing high concentrations of heavy metals
to receiving water bodies has serious adverse environmental effects. Their occurrence and accumula-
tion in the environment is a result of direct or indirect human activities, such as rapid industrialization,
urbanization and anthropogenic sources (EPA, 2000; Hussein et al., 2005; Gardea et al., 2005). Metals,
when present in our body, are capable of causing serious health problems by interfering with our normal
body functions. Some metals are useful to the body in low concentrations like arsenic, copper, iron and
nickel but are toxic at high concentrations. Other metals like aluminum, beryllium, cadmium, lead and
mercury have no biological functions and are highly toxic disrupting bodily functions to a large extent
(European Union, 2002).

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The most metal ions have to enter the microbial cell for its physiological or toxic effect. Usually,
microorganisms have solved the problem by using two types of uptake systems for metal ions. One is
fast, unspecific and driven by the chemiosmotic gradient across the cytoplasmic membrane of bacteria.
The second type of uptake system has high substrate specificity, is slower, generally uses ATP hydrolysis
as the energy source and is only produced by the cell in times of need, starvation or a special metabolic
situation (Nies & Silver, 1995; Nies, 1999). Heavy metals contamination is becoming a great concern
to the environmental awareness and government policies. Several heavy metals removal technologies
including chemical precipitation, ion exchange, reverse osmosis, electrodialysis, ultrafiltration and
phytoremediation are commonly used in industries (Ahalya et al., 2003). However, these technologies
are becoming uneconomical and unfavorable to remove heavy metals from industrial wastewaters. With
increasing environmental attention and legal constraint on discharge effluents, a need of cost effective
technologies are essential (Agarwal, 1998). Therefore, the search for efficient, eco-friendly and cost
effective remedies for wastewater treatment has been initiated. In recent years, research attention has
been focused on biological methods for the treatment of effluents, some of which are in the process of
commercialization (Prasad and Freitas, 2003). Removal of heavy metals remains a major challenge in
environmental biotechnology. The pollutants generally include organic wastes and heavy metals. Heavy
metal removal is important for survival of the environment (Lenntech, 2004; Gardea et al., 2005).
There are three principle advantages of biological technologies for the removal of pollutants; first,
biological processes can be carried out in situ at the contaminated site; second, bioprocess technologies
are usually environmentally benign, no secondary pollution and third, they are cost effective. Of the
different biological methods, bioaccumulation and biosorption have been demonstrated to possess good
potential to replace conventional methods for the removal of metals (Volesky, 1995, King et al., 1997).
Microorganisms are critical to nutrient recycling in ecosystems as they act as decomposers. Researchers
develop a feasible method to accelerate the process of decay and removal by encouraging the microbial
and associated flora and fauna within the ecosystem to accumulate, degrade and remove the pollutants
from the identified sites (Vidali, 2001; Ahalya et al., 2003; Dubey, 2004). Bioremediation is the use of
biological systems, almost invariably microorganisms to clean up a contaminated site. In bioremedia-
tion processes, microorganisms use the contaminants as nutrient or energy sources. Bioremediation uses
naturally occurring or genetically engineered microorganisms such as bacteria and fungi to transform
harmful substance into less toxic or non-toxic compounds. Compared to other methods, bioremediation
is a more promising and less expensive way for cleaning up contaminated soil and water (Hess et al.,
1997; Agarwal, 1998; USEPA, 1999; Kamaludeen et al., 2003; Tang et al., 2007).
In bioremediation, solubilization of heavy metal contaminants provides a means of removal from the
solid substance. Some microbial species subject heavy metal ions to enzymatic reduction, the reduced
form of heavy metal ions are quite insoluble and they precipitate out of solution (Nies, 1999). Most
microorganisms can reproduce rapidly and microbes such as bacteria can also freely exchange genes
by conjugation, transformation and transduction between widely divergent species. The organisms
that are utilized vary, depending on the chemical nature of the polluting agents and are to be selected
carefully as they only survive within a limited range of chemical contaminants (Shazia et al., 2009).
Contaminant compounds are transformed by living organisms through reactions that take place as a part
of their metabolic processes. Biodegradation of a compound is a result of the actions of multiple organ-
isms. When microorganisms are imported to a contaminated site to enhance degradation, the process is
known as bioaugmentation (Agarwal, 1998). Bioremediation is the process whereby organic wastes are
biologically degraded under controlled conditions to an innocuous state or to levels below concentra-

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Heavy Metal Pollution and its Management

tion limits established by regulatory authorities. Bioremediation is the elimination or reduction of toxic
pollutant from the contaminated sites with the help of biological organisms such as bacteria, fungi and
plants by degradation, assimilation or transpiration in the atmosphere. Moreover, the toxicity of heavy
metals in wastewater was shown to be dependent on factors like metal species and concentration, pH,
wastewater pollution load and solubility of the metal ions (Vidali, 2001; Strong and Burgess, 2008).
Organic contaminants may be degraded biologically and CO2 and H2O are the final products, which are
not hazardous and may be recycled. In contrast to toxic organic and inorganic substances, the metallic
species released into the environment cannot be degraded biologically, chemically or physically (Singh
and Ward, 2004). Bioaccumulation is the accumulation of materials which are not critical components
of an organism by that organism. Usually, it refers to the accumulation of metals. Heavy metal ions ac-
cumulate in microbial cells through membrane transport proteins by active transport. Various microbial
species including bacteria, fungi, algae and actinomycetes have been shown to be efficient in bioaccu-
mulation of heavy metal ions from polluted effluents (Wong et al., 1993). The bioremediation of heavy
metals using microorganisms has received a great deal of attention in recent years, not only as a scientific
novelty but also for its potential application in industry. Metal accumulative bioprocess generally falls
into one of two categories; biosorptive uptake by nonliving, non-growing biomass or biomass products
and bioaccumulation by living cells (Aksu, 1998). Microbes can remove heavy metals from contami-
nated solutions either by bioaccumulation, precipitation or biosorption. In comparison with biosorption,
bioaccumulation is a growth dependent process. Since, bioremediation is based on natural attenuation,
therefore, it is considered more acceptable than other technologies. Most bioremediation systems are
run under aerobic conditions, but running a system under anaerobic conditions may permit microbial
organisms to degrade otherwise recalcitrant molecules (Bernard & Jack, 2007, Ke et al., 2009).
The foundation of bioremediation has been the natural ability of microorganisms to degrade organic
compounds (Mostafa et al., 1992). This technology is based on the use of naturally occurring or genetically
engineered microorganisms to restore contaminated sites and protect the environment. As bioremediation
can be effective only where environmental conditions permit microbial growth and activity, its applica-
tion involves the manipulation of environmental parameters to allow microbial growth and degradation
to proceed at a faster rate. Like other technologies, bioremediation has its limitations (Teresa & Lee,
1994). Some contaminants, such as chlorinated organic or high aromatic hydrocarbons, are resistant to
microbial attack. Most frequently, in situ bioremediation is applied to the degradation of contaminants
in saturated soils and groundwater. It is a superior method to cleaning contaminated environments.
Since, it is cheaper and uses harmless microbial organisms to degrade the chemicals. Chemotaxis is
important to the study of in situ bioremediation because microbial organisms with chemotactic abili-
ties can move into an area containing contaminants. So by enhancing the cells’ chemotactic abilities,
in situ bioremediation will become a safer method in degrading harmful compounds (Takehiko, 2004).
Microbes able to degrade the contaminant increase in numbers when the contaminant is present and
when the contaminant is degraded, the biodegradative population declines (Bernard and Jack, 2007).
Theoretically, bioremediation is useful for the complete destruction of a wide variety of contaminants.

8. BIOLOGICAL TREATMENT

Recent studies in molecular biology and ecology offers numerous opportunities for more efficient bio-
logical processes. Notable accomplishments of the studies include the cleanup of polluted water and land

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Heavy Metal Pollution and its Management

areas. Bioremediation leads to changes in the solubility, sorption characteristics, transport properties and
toxicity metals. It promotes the growth of microorganisms to degrade contaminants by utilizing those
contaminants as carbon and energy sources (Teresa & Lee, 1994). The bioremediation systems in opera-
tion today rely on microorganisms native to contaminated sites, encouraging them to work by supplying
them with the optimum levels of nutrients and other chemicals essentials for their metabolism (Shazia
et al., 2009). Microorganisms can be isolated from almost any environmental conditions (Holt et al.,
1994). The search for alternative and innovative treatment techniques has focused attention on the use of
biological materials such as algae, fungi, yeast and bacteria for the removal and recovery technologies
and has gained importance during recent years because of the better performance and low cost of the
biological materials. Since, numerous types of pollutants are to be encountered in a contaminated site,
diverse types of microorganisms are likely to be required for effective mediation (Gogoi et al., 2003).
Microbes can adapt and grow at subzero temperatures, as well as extreme heat, desert conditions, in
water, with an excess of oxygen and in anaerobic conditions, with the presence of hazardous compounds
or on any waste stream. The main requirements are an energy source and a carbon source (Teresa & Lee,
1994). In situ biodegradation involves supplying oxygen and nutrients by circulating aqueous solutions
through contaminated soils to stimulate naturally occurring bacteria to degrade organic contaminants.
It can be used for soil and groundwater. In situ bioremediation means there is no need to excavate or
remove soils or water in order to accomplish remediation. Generally, this technique includes conditions
such as the infiltration of water containing nutrients and oxygen or other electron acceptors for ground-
water treatment. Bioremediation can be carried out on site without causing a major disruption of normal
activities (Thassitou & Arvanitoyannis, 2001). This also eliminates the need to transport quantities of
waste off site and the potential threats to human health and the environment that can arise during trans-
portation. Bioremediation can prove less expensive than other technologies that are used for clean-up of
hazardous waste (Singh & Ward, 2004; Ramirez et al., 2009). The complex structure of microorganisms
implies that there are many ways for the metal to be taken up by the microbial cell (Volesky & Holan,
1995). The presence and activity of microorganisms in biological wastewater treatment are vital to the
process. With regard to microbial removal of heavy metals, biosorption phenomenon is crucial (Knauer
et al., 1997). The mechanism by which microorganisms remove heavy metals can be divided into three
categories; the first mechanism is the biosorption of metals ions on the cell surface, second intracel-
lular uptake of metals ion and third chemical transformation of metal ions by microorganism (Pardo et
al., 2003). Among the different technique employed for metals removal from multi elemental system,
biosorption has been found to be highly selective. Furthermore, the metal accumulating bacteria can be
used to remove, concentrate and recover metals from industrial effluents (Loick et al., 2009).

9. BIOSORPTION

Biosorption is a metabolism independent binding of heavy metals to living cells, non living biomass or
microbial extracellular pathways resulting in bioprecipitation of heavy metals or their mechanisms of
removal (Cotoras et al., 1993). The mechanism of metal biosorption is a complicated process, mainly
ion exchange, chelations, adsorption by physical forces, entrapment in inter and intrafibrilliar capillaries
and spaces of the structural polysaccharide network as a result of the concentration gradient and diffu-
sion through cell walls and membranes. In metal biosorption process by living cells, the metal ions are
adsorbed to the surface of cells by interactions between metals and functional groups displayed on the

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Heavy Metal Pollution and its Management

surface of cells. All the metal ions before gaining access to the cell membrane and cell cytoplasm come
across the cell wall. The cell wall consists of a variety of polysaccharides and proteins and which acts as
active sites of binding metal ions. The mechanism of metal biosorption can be influenced by the status
of biomass, living or non-living, types of biomaterials, properties of metal solution chemistry, ambient/
environmental conditions such as pH (Gee & Dudeney, 1998). There are several chemical groups that
would attract and sequester the metals in biomass such as acetamido groups of chitin, structural polysac-
charides of fungi, amino and phosphate groups in nucleic acids, amido, amino, sulphhydryl and carboxyl
groups in proteins, hydroxyls in polysaccharide and mainly carboxyls and sulphates in polysaccharides
of marine algae that belong to the divisions Phaeophyta, Rhodophyta and Chlorophyta (Diels et al.,
2002). It does not necessarily mean that the presence of some functional group guarantees biosorption,
perhaps due to steric, conformational or other barriers (Gunasekaran et al., 2003).
Difference in the cell wall composition among the different groups of microorganisms, viz. algae,
bacteria, cyanobacteria and fungi, cause significant differences in the type and amount of metal ion
binding to them. The potential metal binding groups in this class of microbes are carboxylates, amines,
imidazoles, phosphates, sulfhydryls, sulfates and hydroxyls (Giller et al., 1998). The amines and imidaz-
oles groups are positively charged when protonated and may build negatively charged metal complexes.
Cell walls of bacteria are principally composed of peptidoglycans which consist of linear chains of the
disaccharide N-acetylglucosamine (Gunasekaran et al., 2003). Heavy metal binding by biopolymers hap-
pens by chance and the relative removal efficiency depends upon the metal species and concentration,
the reactivity of the available biopolymers or biomass and the composition of other wastewater compo-
nents. Moreover, surface exposure of metal binding biopolymers improves the metal binding properties
of microorganisms based not only on biosorption but also on microbial metabolic activities. Biosorption
of heavy metals by microbial cells has been studied extensively as an alternative technology for the treat-
ment of wastewaters. It is a promising process that can reduce capital costs by 20%, operational costs
by 36% and total treatment costs by 28%, compared with conventional systems (Loukidou et al., 2004).
Microbes are exploited by people in biotechnology, both in traditional food and beverage prepara-
tion and in modern technologies based on genetic engineering (Giller et al., 1999). Bacteria, like the
more complex eukaryotic organisms, have all the inner workings for cell function, cellular division and
growth, genetic transcription, translation and energy production (Silver et al., 1990). All of this activity
within a single bacterium typically takes place within a volume of 1.5-2.0 µm3. How the bacteria alter
their environment is equally important as how they are impacted by their surroundings. Much of this
interaction takes place throughout and external to the outer structures. The enveloping layers that make
up the outer structures of the bacterial cell consist of a cytoplasmic membrane, a capsule, sheaths, an
S-layer and flagella (Beveridge, 1985). The cell wall consists primarily of a cytoplasmic membrane and
a thick layer of peptidoglycan. All cells have a highly selective permeable barrier known as the cyto-
plasmic membrane, separating the life sustaining metabolic activity of the cell from its environment
and allowing all food materials, nutrients and waste products to pass (Silver, 1996). The metals can be
captured in the outer portions of the cell such that the organism can be considered to be continuously
enveloped in a brine of various ions. This phenomenon of capturing metals can act as the first step in
the bacteria’s active accumulation and assimilation of metals at various concentrations. This concentra-
tion of metal ions may have developed as a method for accumulating essential cations, if the bacteria
find itself in a more dilute environment. The outer membrane may also immobilize heavy metals, thus
preventing their penetration into the cell. Microorganisms’ ability to oxidize metals plays an important
role in the biological cycling of metals on a global scale. Microorganisms are also considered for their

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Heavy Metal Pollution and its Management

ability to accumulate metal ions within and on the membrane surface of the cell. Such interactions are
so predominant that they can be utilized for economic or ecological gain (Sattelmacher, 2001).
Bacterial and fungal survival in the presence of toxic metals mainly depends on intrinsic biochemical
and structural properties, physiological and genetically adaptation, including morphological changes
and environmental modification of metal speciation, availability and toxicity, the relative importance
of each being difficult to determine. It is probably more appropriate to define resistance by means of a
mechanism produced in direct response to the metal species concerned, e.g. synthesis of metallothio-
neins (Eccles & Hunt, 1986; Gadd, 1992). Arbitrary terms such as resistance and tolerance which are
used rather loosely and interchangeably in the literature are generally based on the ability to grow on a
certain metal concentration in laboratory media (Ehrlich, 1997). Metal tolerance may be defined as the
ability of an organism to survive metal toxicity by means of intrinsic properties and/or environmental
modification of toxicity. Intrinsic properties that can determine survival include possession of imperme-
able pigmented cell walls, extracellular polysaccharide and metabolite excretion, especially where this
leads to detoxification of the metal species by binding or perception (Aksu, 1998; Giller et al., 1999).
However, in many cases distinctions are difficult because of the involvement of several direct and indirect
physico-chemical and biological mechanisms implicated in fungal survival as distinct from environmental
modification of toxicity include extracellular precipitation, complextion and crystallization, transforma-
tion of metal species by oxidation, reduction, methylation and dealkylation, biosorption to cell walls,
pigments and extracellular polysaccharide, decreased transport or impermeability, efflux, intracellular
compartmentation and precipitation and/or sequestration. A particular organism may directly and/or
indirectly rely on several survival strategies (Peng et al., 2010).
The heavy metal removal from water and wastewater is crucial to protect the water environment from
the heavy metal pollution. The heavy metal resistant microorganisms have significant role in bioremedia-
tion (Zouboulis et al., 2004; Peng et al., 2010). The metal and organic pollutants can be removed by the
microbial flora. Bacillus sp. was very much efficient to remove the Au, Cd, Cr, Fe, Mn, Ni, Pb, U and Zn.
It was recorded that Bacillus sp. can efficiently removed the metal pollutants from the waste or industrial
effluents (Gunasekaran et al., 2003) and some aromatics, long chain alkanes, phenol, cresol (Cybulski
et al., 2003). Pseudomonas sp. were reported to Cd, Cr, Cu, Ni, Pb, U and Zn (Sar and D’Souza, 2001;
Tarangini, 2009). Ilhan et al., (2004) observed that Staphylococcus saprophyticus reduced Cr, Cu and
Pb ions. The isolate was adsorbed 100% Pb ions at different pH range of 3-5 and at 270C temperature.
Corynebacterium sp. and Flavobacterium sp. were reported for removal of organic contaminants and
halogenated hydrocarbons, phenoxyacetaes and aromatic hydrocarbons (Jogdand, 1995). Corynebac-
terium glutamicum was able to reduce the thorium and uranium (Tarangini, 2009). The biosorption of
heavy metal Cd and Cu by Flavobacterium sp. was reported by Rajbanshi, (2008) whereas, biosorp-
tion of Cu, Fe and Zn by Corynebacterium sp. was reported by Odokuma, (2009). Many investigators
(Mattuschka et al., 1993; Jogdand, 1995; Tarangini 2009) reported that Streptomyces sp. can be used to
remove the metal ions of Ag, Cd, Cu, Cr, Ni, Pb, U and Zn from the waste. Streptomyces noursei was
able to reduce the metal ions in order Pb>Ag> Cu>Cr (Mattuschka et al., 1993). Aspergillus niger was
reported to remove Ag, Au, Cd, Cu, Th, U and Zn (Townsley et al., 1986; Kuyucak & Volesky, 1988;
Gunasekaran et al., 2003). S. meliloti has huge potential to reduce the toxicity from the environment
reported by Chauhan (2015). Biosorption is being established as a useful alternative to conventional
systems for the removal of toxic metals from industrial effluents/waste. Bioremediation provides a tech-
nique for cleaning up pollution by enhancing the natural biodegradation processes. So, by developing an
understanding of microbial communities and their response to the natural environment and pollutants,

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Heavy Metal Pollution and its Management

expanding the knowledge of the genetics of the microbes to increase capabilities to degrade pollutants,
conducting field trials of new bioremediation techniques which are cost effective and dedicating sites
which are set aside for long term research purpose, these opportunities offer potential for significant
advances (Sutar & Das, 2012).
More research is needed to assess the extent to which these products affect human health. Public
awareness should be created. There should be monitoring and control over the concentration of heavy
metals in cosmetics. The existence of metals in nano form or otherwise should be determined. Toxicity of
metals bearing nano particles is a domain where systematic research needs to be carried out to establish
or negate toxic factors. Susceptibility to toxicity is influenced by age, physiological status, nutrition status
and genetic factors. More research is needed to study these interactions, particularly since malnutrition
is rampant in India. Where specific interactions are known e.g. lead and calcium, fluoride and calcium,
populations exposed to these toxic substances (factory workers, communities living near the factories)
should receive periodic health check-up and nutritional support. Health monitoring of workers engaged
in industries handling toxic metals/ minerals should be carried out regularly and nutritional support
where necessary provided. Since toxicity is insidious, mechanisms for early detection of the problem at
subclinical level through proper surveillance systems are needed. More research is needed to identify and
develop bacteria, plant, and fish-based tests. Functional consequences which may not be too obvious,
like effects on reproductive, neurological-cognitive and other functions have to be identified, through
more research on animals and humans under controlled conditions. A few recommendations are given
below to reduce the heavy metal pollution.
Anthropogenic pollution can be at the stage of fabrication or end use. Instead of pollute and clean;
mitigation strategies should receive high priority. Regulatory standards for emission and discharges
from process plants should be strictly enforced. Recycling/reprocessing of wastes containing toxic met-
als needs to be given greater emphasis not only from environmental and health considerations but also
as a resource conservation measure. Monitoring of air, water and soil in the vicinity of the toxic metal
processing units needs to be carried out more rigorously for the specific metal. Regional accredited
laboratories for analyzing pollutants in various environmental compartments should be set up to help
regulatory bodies. Guidelines for proper management of tailings and slags containing toxic metals should
be prepared taking into consideration techno- economic feasibility. Tailings dumps and process wastes
lying in locations close to the processing units need to be remediated on priority. Phytorestoration en-
hances ecological capital and provides biodiversity of choice suitable for the region where such restora-
tion measures are undertaken. Attempt should be made to replace CFL bulbs with LED (Light emitting
diode) bulbs Mercury-based medical devices and equipment should be totally phased out, since digital
options are available. Presently there is emphasis on production and use of private vehicles-two wheel-
ers, cars. This should change with emphasis on cleaner public transport systems to reduce the burden of
road run off. CNG should replace petrol and diesel. Use of diesel should be confined to public transport
and transport of goods. Manufacture of diesel cars should be stopped. Rich are taking the benefit of the
subsidy on diesel. Periodic (six monthly) examination of water quality, particularly for detection of fluo-
ride and arsenic is necessary in newer alluvium and flood plain areas in different parts of India. Water
supplied by urban municipalities and rural panchayats, should be free of (or contain within safe levels)
of biotic and abiotic toxicants including heavy metals and minerals. Inexpensive devises for purifying
water at household level have to be developed. Creation of public awareness is very important. Greater
interaction between scientists, technologists and media is needed to achieve that. School education can
be a mechanism for creating awareness.

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Heavy Metal Pollution and its Management

10. CONCLUSION

While anthropogenic activities are the major source of heavy metal pollution, natural sources contribute
significantly to the burden of arsenic and fluoride. Apart from industries, road runoff is also an important
source. The toxic elements enter the body mainly through water, food and air. Cosmetics, dental products,
some drugs, particularly Ayurvaid and Unani drugs also contribute. Major pollutants are introduced into
the aquatic systems significantly as a result of various industrial operations. The lack of technical and
financial resources and the regulatory control for the management of hazardous wastes in the past had led
to the unscientific disposal of hazardous wastes, which posed serious risks to human, animal and plant
life. A huge quantity of pollutants in the form of domestic and industrial effluents is discharged directly
or indirectly into the soil/water bodies, which has severe impacts on its biotic and abiotic environment.
During rain, surface water with soil, mud and humus enter into the river, tanks and other water bodies. The
inorganic minerals like sodium, potassium, calcium, magnesium and heavy metals like iron, manganese,
lead, mercury, chromium, cadmium, nickel, cobalt, copper etc., when reach to the soil/river water caused
pollution. Heavy metal contamination is becoming a great concern to the environmental awareness and
government policies. Several heavy metal removal technologies including chemical precipitation, ion
exchange, reverse osmosis, electrodialysis, ultrafiltration and phytoremediation are commonly used in
industries. However, these technologies are becoming uneconomical and unfavourable to remove heavy
metal from contaminated sites or wastewater. With increasing environmental attention and legal constraint
on discharge effluents, a need of cost effective technology is essential. Therefore, the search for efficient,
eco-friendly and cost effective remedies for wastewater treatment has been initiated. In recent years,
research attention has been focused on biological method, e.g. bioremediation, is in the process of com-
mercialization. Bioremediation provides a technique for cleaning up pollution by enhancing the natural
biodegradation processes. The bioremediation is one of the most promising technological approaches
to the problem of hazardous waste. This process relies on microorganisms such as bacteria or fungi to
transform hazardous chemicals into less toxic or nontoxic substances. Such biological transformation is
more attractive than direct chemical or physical treatment. Microorganisms directly degrade contami-
nants rather than merely transferring them from one medium to another, employ metabolic degradation
pathways and can be used in situ to minimize disturbance of the cleanup site. Hence, microorganisms
can be effective, economical and non disruptive tools for eliminating hazardous chemicals. There is no
doubt that bioremediation is in the process of paving a way to greater pastures. This technology offers
an efficient and cost effective way to treat contaminated soil, waste and water. Its advantage generally
outweigh the disadvantage, therefore may be used as management tool.

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 51

Chapter 3
Biosorption of Dye Molecules
Aisha Zaman
Jadavpur University, India

Papita Das
Jadavpur University, India

Priya Banerjee
University of Calcutta, India

ABSTRACT
Water contamination due to dyes has drawn increased attention. Dyes in water bodies are greatly per-
ceptible and pose tremendous threat to ecosystem. Thus removal of such dye molecules is a matter of
concern. In the past various physical and chemical techniques have been employed for the removal of
colour from wastewater. However most of these methods have certain drawbacks. Biological treatment
is often efficient and economical. Many microorganisms are able to accumulate and degrade different
pollutants. Yet even the biological methods have some shortcomings such as toxicity of biodegradation
products and more.

INTRODUCTION

The price of progress clouds the air and fouls the water across the globe. With the growth of mankind,
society, science and technology our world is attaining new heights of socio-economic development.
However this is being achieved at the huge loss of natural resources posing a great threat to future gen-
erations. As a consequence of rapid industrialization, severe environmental degradation such as water
pollution is emerging as a major obstacle in the path of sustainable development. The intake of water
by agricultural, industrial and domestic sectors is increasing tremendously whereas due to the scarcity
of water it is becoming difficult to fulfill their requirement. This has been attributed to the generation
of large amounts of wastewater containing a number of ‘pollutants’. This necessitates the proper utili-
zation of water as well as minimization of pollution so as to make the future of mankind safe. Natural
sources of water should be prohibited from pollution as they are utmost important for ecosystem and
human development. However, the quality of our water resources is deteriorating day by day due to the
continuous addition of undesirable chemicals in them. The main sources of water contamination are
DOI: 10.4018/978-1-4666-9734-8.ch003

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.
 
Biosorption of Dye Molecules

industrialization, civilization, agricultural activities and other environmental and global changes. Dyes
are known to be used by humankind for thousands of years. Till the late nineteenth century, all the dyes/
colourants were more or less natural with main sources like plants, insects and mollusks, and were
generally prepared on small scale. It was only after Perkin’s historic discovery of the first synthetic dye,
mauveine, in 1856, (Hunger, 2003; Venkataraman, 1965) that dyes were manufactured synthetically and
on a large scale. Synthetic dyestuffs are such compounds that are widely consumed by several industries
that include textile, paper, plastic, printing industries and dye houses. As a consequent they generate
substantial amount of coloured waste water. Therefore, colour removal has been a subject of increased
attention in the past few years. The process of dying textile fibres is not an efficient process. The extent
of efficiency depends on the method of dye delivery. Consequently a huge amount of coloured wastewater
is discharged by the textile industries.

SYNTHETIC DYES AND RELATED ISSUES

Synthetic dyes contribute largely to the group of hazardous compounds that are not easily biodegradable.
Their presence in water bodies is undesirable as they are difficult to eliminate due to their persistent and
recalcitrant nature. Dyes display a significant diversity in their molecular structure and can be classified
in several ways, sometimes based on their chemical structure while sometimes on the basis of their ap-
plication to the fiber type and so on. A broad classification of the dyes based on the ionic charge on the
dye molecules can be presented as follows:

• Non-Ionic: Disperse dyes

• Cationic: Basic dyes
• Anionic: Direct, acid, and reactive dyes

The properties of the dyes vary greatly with their molecular structure. The synthetic origin and complex
molecular structure of the dyes makes them more stable and difficult to be biodegraded. Therefore they
can pose tremendous threat on the environment and the natural ecosystems. Most of the dyes especially
cationic dyes are highly toxic while their degradation products can be carcinogenic as well (El-Sayed,
2007). Generally either an anthroquione or an azo group is present in non-ionic and anionic dyes Anthra-
quinone containing dyes are greatly resistant to degradation because of their complex aromatic structures
and therefore stay behind unaffected in the wastewater. Reactive dyes typically contain an azo group
associated with different types of reactive groups such as chlorotriazine or vinyl sulphone. Most of the
metal complex dyes contain chromium which is a potential carcinogenic. Disperse dyes remain undis-
sociated in aqueous medium while there some disperse dyes that have a propensity to bioaccumulate.
The toxic components of the dyes can adversely affect the ecosystem in different ways, such as:

• Dyes may significantly affect rather hinder photosynthetic activity in aquatic life as they can ab-
sorb and reflect sunlight entering the water bodies resulting in reduced light penetration and hence
interfering with the growth of microorganisms.
• Dyes can also be toxic to the aquatic life due to the presence of heavy metals, chlorides, aromatic
compounds etc., in them.

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Biosorption of Dye Molecules

• Dyes may also be problematic if they are broken down anaerobically in the sediment, as toxic
amines are often produced due to incomplete degradation by bacteria.
• Many dyes have been reported to cause, mutagenesis, chromosomal fractures, carcinogenesis,
respiratory toxicity and even teratogenecity.

In a survey conducted by the Ecological and Toxicological Association of the Dyestuffs Manufactur-
ing Industry, it was found that over 90% of some 4000 dyes tested had LD50 values higher than 2×103
mg/kg. Basic and di-azo direct dyes were noted to have the highest rates of toxicity (Shore, 1996). The
toxic effects of different dyes have been studied by many authors.
Some recent studies showed toxicity of malachite green on freshwater catfish. (Srivastava et al.,
1995) Another report presented the toxicity of acid red 73 with high LC50 values on Gambusia affinis.
(Muthukumar et al., 2005) An experimental study conducted by National Cancer Institute, 1978, showed
that rats exposed to benzidine – based dyes (for 13 week) developed hepatocellular carcinomas and
hepatic neoplastic nodules. Commercially over 100,000 dyes are available and approximately 7 × 105
tonnes are produced annually (Pearce et al., 2003; McMullan et al., 2001). Dyes have generated much
concern regarding its use, due to its toxic effects, chemical stability and low biodegradability. Besides,
dyes can be regarded as “eye catching pollutants” because of the fact that the presence of even a very
small amount of dye (less than 1 ppm) can colour large water bodies and is greatly perceptible. This
not only causes adverse effect on the environment, especially the aquatic organisms, but also affects the
aesthetic quality of the aquatic ecosystem. Hence, it is very important to treat wastewaters containing
toxic dyes before they are discharged into the water bodies.

DIFFERENT METHODS FOR WASTE WATER TREATMENT

During the past three decades, numerous colour removal methods involving physical, chemical and bio-
logical processes have been developed and implemented. However most of them have some advantages as
well as drawbacks. Pertaining to expensiveness and secondary disposal problems, many of these conven-
tional methods have not gained much attention and hence remain unsuccessful in large scale application
for treating waste waters. However, only a few have been accepted by the paper and textile industries.
Some of the existing technologies for dye removal are briefly summarized in the subsequent paragraphs.

Physical Methods

Different physical methods are widely used, such as membrane-filtration, nano filtration, reverse osmosis,
electrodialysis and adsorption techniques. The literature review reveals that the most popular method
for uptake of contaminants from wastewater is liquid-phase adsorption. This has been attributed to the
cost effectiveness and versatility of the process bundled with the availability of a wide range of cheaper
adsorbents that do not require any pretreatment prior to application.

Major Drawbacks of the Physical Methods

• Limited lifetime of membranes

• Cost of periodic replacement

53
 
Biosorption of Dye Molecules

• Technical constraints
• Economic non viability

Chemical Methods

Chemical methods include various processes such as precipitation–flocculation with Fe(II)/Ca(OH)2,

coagulation in combination with filtration and flotation,, electrokinetic coagulation, electro-flotation,
oxidation, irradiation and a few more. Recent application of the advanced oxidation processes which
involves the generation of powerful oxidizing agents such as hydroxyl radicals, have been reported for
its successful application in the field of waste water treatment.

Disadvantages of Chemical Methods

• High sludge production

• Handling and disposal problems
• Possibility of secondary pollution
• High cost, commercially unattractive

Biological Treatments

Biological treatments often prove to be an economical alternative as compared to the physical and chemi-
cal processes. Biodegradation methods involve the application of microorganisms such as fungal and
bacterial species for breaking down the contaminants into simpler and less toxic forms. Also adsorption
by (living or dead) microbial biomass and bioremediation systems are commonly applied to the treatment
of industrial effluents because many microorganisms including even yeasts, algae along with bacteria
and fungi are able to accumulate and degrade different pollutants.

Limitations of Biological Treatment

Although the biological treatments are a removal process for some organic compounds including syn-
thetic dyestuffs, but there are certain limitations such as:

• Sensitivity toward diurnal variation

• Toxicity of some products of biodegradation
• Possibility of bioaccumulation and biomagnifications of the toxic products of degradation and
hence their interference with the food chain.
• Large land area requirements & technical constraints

TECHNOLOGIES CURRENTLY IN USE FOR REMOVAL

OF COLOUR FROM EFFLUENTS

The available technologies for colour removal can be implied at two possible locations:

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Biosorption of Dye Molecules

1. At the dye house itself, to remove the colour so that the water can be re-used partially or wholly.
2. At the sewage works, to treat the coloured water prior to the available physical/chemical process-
ing, or as an ultimate brush up step. (Southern, 1995).

Microbial Degradation

Biological processes have emerged as a viable alternative for effluent treatment and have gained increasing
attention owing to their cost effectiveness, ability to produce less sludge and environmental benignity. In
recent years, a number of researchers are focusing on microbes as they have excellent ability to degrade
dyes in wastewaters. Biodegradation methods are apparently attractive from economic point of view as
several microbes such as bacteria, yeasts, algae and fungi are able to accumulate and degrade different
pollutants including a wide range of dyes. (McMullan et al., 2001; Fu & Viraraghavan, 2001)

Limitations of Microbial Degradation

However, the application of microorganisms for degradation of pollutants is often constrained because
of technical limitations. Biological treatment requires optimal favorable environment, nutrition supplies,
large land area and is often inhibited by sensitivity toward circadian deviations as well as toxic effects of
certain chemicals, and their rigidity in design and operation (Bhattacharyya & Sarma, 2003). Moreover
Biological treatment is a sluggish process and incapable of attaining adequate color elimination. (Rob-
inson et al., 2001) Although many organic compounds are degraded, but many others especially dyes are
difficult to deal with owing to their complex chemical structure and synthetic organic origin (Ravi Kumar
et al.,1998). Particularly, the azo dyes are wholly recalcitrant due to their xenobiotic nature. (Crini, 2006)

Decolorisation by Immobilized Micro-Organisms

Immobilization is a general term that is used to describe the various techniques for entrapment or at-
tachment of a cell or a particle to a substratum. It can be efficiently applied to basically all types of
biocatalysts including animal and plant cells, cellular organelles and even enzymes. Cell immobilization
has been defined as the physical confinement of viable cells within a defined localised region in such a
way that inhibits their free movement and exhibit hydrodynamic characteristic which differ from those
of the surrounding environment while retaining their catalytic activities for repeated and continuous use.

Advantages of Immobilization Technique

Immobilization of microbial cells has received increasing interest in the field of waste treatment.
Compared with conventional suspension system, the immobilized microorganism technology offers a
multitude of advantages, such as:

• High bio-mass.
• High metabolic activity.
• Strong resistance to toxic chemicals.
• Cost effective since they can be used and reused without any effective loss of activity.

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Biosorption of Dye Molecules

Therefore, the different technology of immobilization of microbes are becoming a promising tool for
wastewater treatment in the near future.

Adsorption

The process of accumulation of a substance at the interface between two phases (liquid–solid interface
or gas–solid interface) is defined by the term adsorption. The substance that collects at the interface is
called adsorbate while the solid on which adsorption occurs is termed as adsorbent (Dabrowski, 2001).
Adsorption can be classified as chemical sorption and physical sorption. Chemical adsorption involves
the formation of strong chemical associations between adsorbate molecules and adsorbent surface, usu-
ally characterized by the exchange of electrons and is generally irreversible. On the other hand Physical
adsorption or physisorption is regarded as weak van der Waals intraparticle interaction between the
adsorbate and the adsorbent and are therefore reversible in most cases (Allen S, et al., 2005).

Salient Features of Adsorption

Among various water treatment methods adsorption occupies a prominent position and is supposed to
be the best one for all the non-biodegradable organics for the removal from aqueous streams, activated
carbons being the most common adsorbent for this process due to its effectiveness and versatility. How-
ever, activated carbon is quite expensive and the cost of regeneration is also very high. On the other hand,
the alternative cheaper materials such as bagasse pith, carbonized bark, peat, soil, tree, and eucalyptus
barks, chitin, rice husk, wood, fly ash, and carbonized sewage sludge etc. have generally low adsorption
capacities so large amounts of adsorbents are needed.

Biosorption

In biosorption, which is a blend of biological and physical water treatment methods, biological materials
are used for the surface sorption of various pollutants. Biosorption refers to the accumulation and con-
centration of molecules (pollutants) from aqueous solutions by the application of biological materials,
thereby permitting the retrieval and/or environmentally suitable disposal of the pollutants. Biosorption
has been established to possess essential qualities and good potential to take over the conventional
methods for the removal of dyes/metals (Volesky & Holan, 1995; Malik, 2004) especially those that are
not easily biodegradable.

Biosorption vs Bioaccumulation

Biosorption is often confused with bioaccumulation, however this can be clarified on the basis of the
state of biomass being used. The phenomenon of bioaccumulation is always associated with living cells;
whereas, the mechanisms of biosorption are grounded on the use of dead biomass. Talking precisely,
bioaccumulation can be referred to as the active uptake of toxicants by living cells. The toxicant can
enter into the cell across the cell membrane or through the cell metabolic cycle (Malik, 2004). On the
contrary, biosorption can be considered as the passive uptake of toxicants by dead/inactive biomass
or by materials derived from biological sources. Biosorption processes have certain advantages over
bioaccumulation. Generally, the use of living organisms may not be preferable for continuous treatment

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Biosorption of Dye Molecules

of highly toxic organic/inorganic contaminants. Once the accumulated toxicant concentration reaches
the level of saturation (Eccles, 1995) an organism’s metabolism may get disturbed and broken down
resulting in death of the organism. This situation can be eluded in the case of dead biomass, which is
bendable with the environmental conditions and toxicant concentrations (Vijayaraghavan & Yun, 2008)

Biosorbents

Biosorption being effective, cheap and competitive as well, has got a novel approach and hence till date
hundreds of biosorbents have been proposed for the removal of metals and dyes. Defferent type of bio-
logical materials derived in the form of biomass from yeast, bacteria, fungi, chitin, chitosan and peat,
are used as chelating and complexing sorbents for the removal of dyes from solutions. These biosorbents
and the secondary products obtained from them possess a variety of functional groups that can form
complex with the dye molecules. The biosorbents are often more specific in terms of uptake of particular
compounds as compared to the commercially activated carbons and the traditional ion-exchange resins.
Hence biosorbents can be efficiently applied to reduce high concentration of dyes to much lower levels.
Effectiveness of biosorbent can be judged from its uptake or biosorption capacity which is mg of dye
adsorbed by one gram of biosorbent. Reported use of some of the biosorbents has been listed below in
table 1, while a few are briefly discussed in the subsequent paragraphs.

Table 1. Biosorbents used for removal of some dyes.

Biosorbent Dye Sorption Capacity Reference

(mg/g)
Crosslinked chitosan bead Reactive blue 2 2498 Chiou et al. (2004)
Chitosan bead (crab) Reactive red 222 1106 Wu et al. (2000)
Chitosan Acid orange 12 973.3 Wong et al. (2004)
Chitosan flake (lobster) Reactive red 222 398 Wu et al. (2000)
Chitosan flake (crab) Reactive red 222 293 Wu et al. (2000)
Crosslinked cyclodextrin Acid blue 25 88 Crini (2003)
Chitosan/cyclodextrin material Acid blue 25 77.4 Martel et al. (2001)
Starch-based material Acid blue 25 249 Delval et al. (2002)
Cotton waste Basic red 2 875 McKay et al. (1999)
Treated cotton Acid blue 25 589 Bouzaida and Rammah (2002)
Treated peat Basic violet 14 400 Sun and Yang (2003)
Treated peat Basic green 4 350 Sun and Yang (2003)
Peat Basic blue 69 195 Ho and McKay (1998)
Chlorella vulgaris biomass Reactive red 5 555.6 Aksu and Tezer (2005)
Activated sludge biomass Basic red 18 285.71 Gulnaz et al. (2004)
Spirodela polyrrhiza biomass Basic blue 9 144.93 Waranusantigul et al. (2003)
Living biomass Acid blue 29 6.63 Fu and Viraraghavan (2001b)
Modified fungal biomass Disperse red 1 5.59 Fu and Viraraghavan (2002b)
Dead fungus Aspergillus niger Basic blue 9 18.54 Fu and Viraraghavan (2000)
Yeasts Remazol blue 173.1 Aksu and Dönmez (2003)

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Biosorption of Dye Molecules

Chitin and Chitosan

The use of biopolymers such as chitin and chitosan has recently risen as an excellent alternative in the
field of colour removal from colour contaminated water even at low concentrations (ppm or ppb levels).
Chitin and chitosan are abundant, renewable and biodegradable resources. Chitin, a mucopolysaccharide
naturally occurs in a broad range of sources such as insects, annelids, crustaceans, fungi, and molluscs.
Chitosan has gained wide attention as a complexing agent owing to its cheapness and abundance in
availability. Moreover, the presence of a large number of amino and hydroxy functional groups in chitin
reflects its great potential to absorb of a wide range of dyes (Guibal, 2004, Varma et al., 2004; Ravi
Kumar, 2000). This biopolymer represents an attractive alternative to other biomaterials as it has been
found to show excellence in physicochemical characteristics, high reactivity as well as stability, chelation
behavior and a great deal of selectivity toward pollutants (Guibal, 2004, Varma et al., 2004 and Ravi
Kumar, 2000). Recent studies based on application of chitin and chitosan as biosorbents have revealed
their versatility and affinity towards several classes of dye compounds. Owing to their versatility chi-
tosan based biomaterials can be used in several forms such as beads, fibers, flakes and even gels. The
performance of chitosan as a biosorbent for the removal of acid dyes was investigated and the maximum
adsorption capacities of, acid orange 10, acid orange 12, acid red 73, and acid red 18 were reported
to be 922.9, 973.3, 728.2, and 693.2mg/g, respectively (Wong et al., 2004). Another study showed the
usefulness of chitosan for the removal of reactive dyes (Wu et al., 2000).

Microbial Biomass

Speaking strictly about biosorption of dyes, microbial biomass including bacteria, fungi, and microalgae,
have shown to outdo macroscopic materials such as seaweeds, crab shell, etc. The cause behind this is
possibly the nature of the cell wall constituents and functional groups involved in dye binding. Several
bacteria, fungi and microalgae have been reported to bind a variety of dye classes. (Vijayaraghavan &
Yun, 2008). Corynebacterium glutamicum was identified as a potent biosorbent of reactive red 4, which
can bind 104.6mg/g at pH 1(Won et al., 2005). Another study indicated the capability of rhizopus arrhizus
to bind 773 mg/g of Gemazol Turquise blue-G at 45 °C and pH 2. (Aksu & Dönmez, 2003; Aksu, 2003)
Moreover the biosorption capacity of fungal biomass can be increased by some physical pretreatment such
as autoclaving or by subjecting to chemical treatment prior to use. (Fu & Viraraghavan, 2001) The dye
uptake capacity of other types of biomass, such as yeasts has also been studied. Most of the large-scale
industries use yeast for their fermentation processes and the waste biomass from these processes can be
potentially used as a cheap source of biosorbent. In a study it was found that yeast acts as an efficient
bisorbent material for remazol blue and reactive black 5 with maximum adsorption capacities of173.1
and 88.5 mg/g, respectively. (Aksu & Dönmez, 2003; Aksu, 2003) Many other authors have reported
that biosorbing materials obtained from different sources of microbial biomass can be effectively em-
ployed for the removal of dyes from aqueous solutions, since most of the dyes have a particular binding
affinity for specific microbial species (Bustard et al., 1998 and Nigam et al., 1996). Moreover the use
of biomass for treating wastewater is gaining popularity day by day because of its easy availability in
abundant quantities and cost efficiency. Synthesis of valuable products such as antibiotics and enzymes
result in the formation of a huge amount of byproduct in the form of microbial biomass which can be
effectively used for removal of various pollutants from waterbodies. Besides the above mentioned facts
the physico-chemical characteristics of microbial biomass also show high potential as a sorbent material.

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Biosorption of Dye Molecules

BIOSORPTION OF DYES

A broad range of microorganisms including bacteria, fungi and yeasts are being employed for the bio-
sorption of a wide variety of dyes. Textile dyes vary greatly in their chemical composition, molecular
structure and arrangement. Therefore their interactions with microorganisms depend on the specific
characteristics and chemistry of the microbial biomass, the chemical structure of a particular dye, and
various external factors such as temperature, pH, initial dye concentration, presence of salts and heavy
meatl ions in the dye solution or wastewater (Aksu, 2005). A recent study reported that Rhizopus arrhizus
could decolorize raw water contaminated by organic humic acid via a biphasic process of adsorption. The
initial phase being fast and independent of metabolic energy, the later phase involved a slow and energy
dependent metabolic mechanism. Based on the findings of infrared spectroscopy, it was deduced that
no chemical reaction took place between cell wall and humic acid; it was merely a physical adsorption
of the contaminants onto the cell wall (Zhou & Banks, 1991).

Microorganisms and Dyes: The Machinery of Biosorption

The interface of dye molecules with live or dead biomass depends on a number of factors along with the
nature of the dyes and microbes. The decolorization of effluents by adsorption onto biological materials
may involve several complex mechanisms such as surface adsorption, ion-exchange, complexation (co-
ordination), complexation–chelation and micro-precipitation. However, the mechanisms for biosorption
have not been discussed in details in most studies; therefore, very little information is available on this.
This piece of writing presents a generalized view on the mechanism of biosorption by microbial biomass.
The extent of biosorption depends not only on the chemical nature of the dye, but also on the bacterial
genus, due to deviations in the cellular constituents, as the cell walls of all bacteria are not identical. In
fact, the cell wall composition is one of the most important factors in the analysis and differentiation
of bacterial species. It is the cell wall of bacteria which is considered to be the first constituent coming
into contact with the dye molecules where the solutes can get deposited or accumulated, either on the
surface or within the cell wall structure (Beveridge & Murray, 1976; Doyle et al., 1980)
The accumulation of dyes on dead biomass occurs through passive diffusion of the adsorbed solute
to the surface of the microbial cells. (O’Mahony et al., 2002, Aksu & Tezer, 2000; Veglio & Beolchini,
1977) Once the dye has diffused to the surface, it will bind to sites on the cell surface. The precise bind-
ing mechanisms may range from physical (i.e. electrostatic or Van der Waal forces) to chemical binding
(i.e. ionic and covalent).
The solutes i.e. the dye molecules are taken up extra-cellularly by the dead/inactive cells. Cell walls
consisting mainly of polysaccharides, proteins and lipids offer many functional groups including carboxyl,
phosphonate, amine and hydroxyl groups (Doyle et al., 1980; van der Wal et al., 1997). These functional
groups play vital roles in biosorption. The dyes can interact with these active groups on the cell surface
in a different manner. Owing to the presence of negative charge and high abundance, the carboxyl groups
can dynamically participate in the binding of metal cations. Several dye molecules, which exist in the
form of dye cations in solutions, show high affinity for carboxyl and other negatively charged groups.
The amine groups are also effectual in removing metal ions and anionic metal species by chelation and
adsorption. While the dye molecules undergo surface sorption by the amine groups via electrostatic in-
teraction or hydrogen bonding. (Vijayaraghavan & Yun, 2007). In a study it was reported that the amine
groups of C. glutamicum could bind anionic reactive dyes via electrostatic interactions (Vijayaraghavan &

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Biosorption of Dye Molecules

Yun, 2007). Generally a rise in pH favors the electrochemical attraction and adsorption of cationic dyes,
by increasing the overall negative charge on the cell surfaces until all the relevant functional groups are
deprotonated. While the anionic dyes are expected to actively bind to the cell surfaces at lower pH with
increasing positive charge due to protonation of the functional groups (Vijayaraghavan & Yun, 2007).

Structure of the Bacterial Cell Wall and Its Role in the Sorption Mechanism

The bacterial group includes a wide variety of unicellular prokaryotes and also some eukaryotes and
they are ubiquitous in soil, air and water. The presence of peptidoglycan in the bacterial cell wall dif-
ferentiates them from that of all other organism. In gram positive bacteria a thick layer of peptidoglycan
constitutes upto 90% of the cell wall. (Beveridge, 1981; Dijkstra and Keck, 1996). The peptidoglycan
layer consists of polyalcohols, mostly teichoic acids, some of which are linked to lipids and hance called
lipoteichoic acids (Sonnenfeld et al., 1985). Gram negative bacterial cell walls are composed of only
10-20% peptidoglycan. Additional outer membrane composed of phospholipids and lipopolysaccarhides
are also associated with the Gram negative cell wall (Sheu & Freese, 1973). It has been shown that
the anionic functional groupsin the peptidoglycan, teichoic acid and teichuronic acid of Gram positive
bacteria and the peptidoglycan, lipopolysaccarhides and phospholipids of Gram negative bacteria are
mostly responsible for the anionic nature and metal binding capacity of the cell wall (Sherbert, 1978).
Some extracellular polysaccharides are also found capable of binding metals (McLean et al., 1992).

PREPARATION OF BIOSORBENT

In order to achieve proficient performance in the biosorption process the sorption capacity of biomass
can be increased by some chemical or genetical modification.

Chemically Modified Biosorbents

Chemical modification can be achieved by pretreatment, binding site enhancement, binding site modi-
fication and polymerization. (Vijayaraghavan & Yun, 2008)

Pretreatment of Microorganism

It has been noted by many researchers that some physical or chemical pretreatment processes can en-
hance the adsorption capacity of biomass. Drying, autoclaving, contacting with organic chemicals, such
as formaldehyde, or inorganic chemicals, such as NaOH, H2SO4, NaHCO3, and CaCl2 are some of
the effective pretreatment methods. A comparative biosorption study using live and autoclaved Gram-
negative bacteria for the removal of reactive dyes, showed that the autolaved cells were more efficient in
accumulating the dye molecules onto their surface than the live cells. The higher uptake capacity of the
dead cells corresponds to the increased surface area caused by cell rupture during autoclaving. (Hu, 1996)

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Biosorption of Dye Molecules

Binding Site Enhancement

It has been observed by many authors that the less important functional groups can be converted to
actively binding groups by the addition of some chemical species. In one of the recent works, hydroxyl
group was replaced by carboxyl group by the introduction of chloroactic acid and then the carboxylated
biomass was further modified to form aminated biomass by the addition of enediamine and carbodiimide.
(Jeon & Holl, 2003)

Polymerization

Insertion of long polymer chain has been proven to be helpful for introducing significant functional
groups on to the surface of biomass. Direct grafting of some monomers are also formed to be efficient.
However, very little is known about this aspect of chemical modification of biosorbents (Vijayaraghavan
& Yun, 2008). In a recent study it was observed that when biomass was cross linked polyethylenimine
that contained a number of primary and secondary amine groups; the biosorption capability of the bio-
mass showed several fold enhancement particularly for some heavy metals such as chromium (VI), Cu,
Pb, Ni, and Ar (Deng & Ting, 2005a,b,c ;2007).

Genetically Modified Biosorbents

Genetical engineering and recombinant technologies provide ample scope of improvement and develop-
ment in the field of biosorption. Genetic modifications can help us to redesign microorganisms so that
they possess higher intrinsic capabilities for biosorption and enhanced specificity for metals and functional
groups and even higher resistance to ambient conditions. (Bae et al., 2000; Majare & Bulow, 2001).
Genetic modifications are more viable with microbes produced by fermentation processes. These days
several kinds of amino acids and nucleic acids are produced on the large scale by the help of genetically
engineered microorganisms (Vijayaraghavan & Yun, 2008). Several successful attempts have been made
to create recombinant bacteria with superior metals binding capacities. However, so far this technique is
mostly restricted to E.coli as it greatly assist the process and experiments involved in genetic engineering
(Chen & Wilson, 1997). In a recent experimental study, recombinant strains of Staphylococcus xylosus
and Staphylococcus carnosus were created with their surface displaying chimeric proteins that contain
polyhistidyl peptides. The insertion of H1 or H2 peptide into the surface proteins helped the strains to
achieve improved capacities for binding nickel ions (Samuelson et al., 2000). However, only a little is
known about this aspect in terms of dye molecule uptake by genetically modified organisms. But this
area has huge scope for research and implementation of this technique for separating various toxins and
other pollutants including dye molecules from contaminated solutions. This can even help in recovery
and regeneration of the important and commercially useful compounds. (Vijayaraghavan & Yun, 2008)

BATCH KINETIC STUDIES OF BIOSORPTION

It is important to attain optimum parameters and process design, operation control and sorption kinetics
for an active practical application of the process. Sorption kinetic studies are always important as they
present a clear picture of the reaction pathways and the reaction mechanism. Process design optimization

61
 
Biosorption of Dye Molecules

and kinetic values can be achieved by conducting batch experiments. In batch experiments small working
volumes (usually 100 ml) of the colored solution are used at specefic agitation speed, temperature and
other important parameters. Samples are taken before and after adding the biosorbents at predetermined
time intervals. Sample analysis is done by using UV-Visible spectrophotometer and hence the extent
of colour removal is determined which in turn helps in attaining the equilibration time. Owing to its
metabolism-independence, the process of biosorption is expected to be a rapid one. Usually it comprises
of two phases: a rapid initial uptake within 30-60 mins, and a later slower attainment of equilibrium over
a period of 2-3 hours. The uptake at a given time versus the square root of that time is plotted and the
plot reveals the rate determining step.

Effect of pH on Dye Biosorption

pH is often regarded as an important process influencing parameter, which not only determines the
colour of the dye solution and even solubility in some cases, but also effects the sorption capacity to
great extent. Hence many researchers have investigated the effect of pH on decolorisation of textile ef-
fluents using live or dead biomass. Effective removal of 11 reactive (anionic) dyes by Aeromonas sp.
Cells through biosorption was achieved at acidic range. It is noteworthy to mention that the removal
efficiency decreased as the pH of the dye solution was increased from 3.0 to 11.0. This can be possibly
explained by the association of dye anions with positively charged bacterial cell surfaces occuring at
lower pH. (Hu, 1992) Alternatively, another study on the effect of pH on biosorption of three cationic
dyes, Orlamar Red BG, Orlamar Blue G and Orlamar Red GTL, by a dead fungal species of F. carnea
showed that colour removal was directly proportional to the pH of the solution. Decrease in pH led to
a decrease in colour removal due to repulsive forces occurring between the dye cations in solution and
the positively charged biosorbent surface at pH values lower than 3.0. (Mittal and Gupta, 1996)

Effect of Temperature on Dye Biosorption

Temperature plays a vital role in bisorption process and its real application, because the coloured efflu-
ents are generally released at reasonably high temperatures (50–60 ◦) (Aksu, 2005) The biosorption of
Basic Violet 3 and Basic Yellow 24 on dried activated sludge biomass was studied and it was observed
that adsorption capacity for both the dyes decreased when the temperature was raised from 20 to 40
◦C indicating the exothermic nature of both biosorption processes (Chu & Chen, 2002). Conversely
another author reported slight or no effect on the equilibrium uptake of of six reactive dyes by three
Gram-negative bacteria (P. luteola, E. coli, and Aeromonas sp.), signifying the possibility of application
of dead biomass for the removal of dye from wastewater without decreasing its temperature (Hu, 1996).

Effect of Initial Dye Concentration on Dye Biosorption

The efficiency of colour removal varies with the initial concentration of dye present in the effluents, as
it provides the driving force required to overcome the resistances to mass transfer of the dye between the
aqueous and solid phases. Hence a higher initial concentration of dye favours the sorption mechanism.
(Aksu, 2005) The uptake of Remazol Golden Yellow dye by K. marxianus IMB3 was found to be low
at lower dye concentrations. However a considerable increase in the biosorptive capacity was noted at
higher concentrations of dye. These findings indicated some kind of cooperative interactions between

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Biosorption of Dye Molecules

the dye and the biomass at higher dye concentration (Bustard et al., 1998). Another study reported a
significant increase in the equilibrium sorption capacity of dried R. arrhizus with increasing initial
Remazol Black B concentration up to 800 mg/L, while the adsorption yield of dye showed the opposite
trend. The author suggested that the loading capacity of biomass increased from 19.3 to 500.7 mg g−1
and the adsorption yield of biomass decreased from 94.0 to 62.4%, when the initial Remazol Black B
dye concentration was increased from 20.5 to 802.4 mg/L (Aksu & Tezer, 2000).

Effect of Salts on Dye Biosorption

The process of dying involves the consumption of a large amount of salts. The concentration of salt in
dye wastewater determines its ionic strength and hence considered as one of the important factors that
influence biosorption capacity. In a study it was seen that high ionic strength due to higher concentra-
tion of NaCl led to high biosorption of humic acid by R. arrhizus. The author proposed that the effect
of ionic strength was similar to that of a colloid. At higher ionic strength formation of electrical double
layers of both R. arrhizus and humic acid occurs thereby brining the biomass and the contaminant in
close proximity. This would enhance van der Waals bonding and hence increase biosorption (Zhou &
Banks, 1993).

Effect of Heavy Metal Ions on Dye Biosorption

Beside dyes and salts, the coloured textile effluents also include some heavy metals as most of the dyes
contain metal ions. The metal ions can interfere and effect the rate and capacity of biosorption by com-
peting with dye molecules for the binding sites or conversely they can stimulate the biosorption of dye
onto the biomass. ((Aksu, 2005) The presence of high concentrations of Cd2+, Cu2+, and Al3+ caused high
biosorption of humic acid onto R. arrhizus possibly by acting as a bridge between the two. The heavy
metal ions could the negative charge on both the biosorbent and the sorbate, thereby reducing repulsive
forces between them, which in turn increased boniding. Furthermore, bi-valent and tri-valent metal
cations could interact with humic acid to form precipitates or aggregates thus reducing the solubility of
humic acid resulting in an increase in the biosorption potential (Zhou & Banks, 1993).

Effect of Particle Size on Dye Biosorption

Surface area of biosorbent is a major factor which influences biosorption kinetics. The biosorption ca-
pacity is inversely proportional to the particle size of the biomass. Biosorption of Basic Yellow 24 on
activated sludge biomass with a selected range of particle size, was found to increase with decreasing
particle size. The rise in biosrption capacity was due to the increment in the total surface area of smaller
particles for the same amount of biomass (Chu and Chen, 2002.

Effect of Shaking Rate on Dye Biosorption

Adequate rate of stirring in batch biosorption study facilitates to overcome external mass transfer re-
sistances. Biosorption of a dye Basic Yellow 24 using dried activated sludge biomass was studied at
different shaking speed or rpm (rotations per minute) ranging from 300–600 rpm. Along with increasing
rpm an increase in the dye uptake capacity was observed (Chu & Chen, 2002).

63
 
Biosorption of Dye Molecules

Effect of Size of Biosorbent on Dye Biosorption

Size of the biosorbent greatly affects the sorption process. The biosorption efficiency of any biosorbent
material varies inversely with its particle size. Smaller the size, larger is the surface area and hence
greater is the rate of biosorption. Therefore small sized biosorbing materials eventually result in short
equilibration time. But, at the same time the particles should be durable and supple enough to survive
temperature, pressure and other extreme conditions applied during the regeneration process.

Effect of Surfactants on Dye Biosorption

The dyeing process occasionally involves the use of surfactants hence they are likely to be present in
coloured waters. An author reported in his study that the binding efficiency of the cells (inactive) is re-
duced due to the presence of detergent in wastewaters. His findings also showed that high concentration
of Tween, which is a nonionic surfactant, lowers the rate of sorption. The effect of tween on different
dyes wass found to be different.

Desorption and Regeneration of Biosorbents

Biosorption is a process meant for treating polluted water and by removing the contaminants and pollut-
ants from them. However, the proficient regeneration of biosorbents is also essential. Especially when
the biomass preparation is an expensive process the regeneration of biosorbrnts becomes necessary in
order to possibly decrease the process cost and keep a continuous supply of biosorbent. Regeneration
of the biosorbent can be achieved by desorption of the contaminants/ pollutants from the biosorbents
which would result in a concentrated solution of pollutants. A successful regeneration of biosorbents
followed by desorption entails the proper choice of elutants, which strongly depends on the nature of
biosorbent and the mechanism of biosorption. Besides, the elutant must be (i) non- detrimental to the
biomass, (ii) cheap, (iii) environmentally safe and (iv) effective. Quite a few researchers have performed
comprehensive screening experiments to recognize suitable elutants for this process. (Vijayaraghavan and
Yun 2008) Several chemical agents were used to desorb Co2+ from cobalt-laden Ascophyllum nodosum,
and finally CaCl2 in the presence of HCl, and hence HCl was identified as an appropriate elutant.
(Kuyucak & Volesky, 1989)

Kinetic Models

Biosorption kinetics can be studied by using following models:

Pseudo-First-Order Kinetic Model

The rate constant of adsorption is determined from the pseudo-first-order equation given by Langergren
and Svenska as:

ln (qe − qt ) = lnqq − k1t

64

Biosorption of Dye Molecules

where qe and qt are the amounts of MB adsorbed (mg/g) at equilibrium and at time t (h), respectively
and k1 is the rate constant adsorption (h−1).

Pseudo-Second-Order Kinetic Model

The pseudo-second-order equation based on equilibrium adsorption is expressed as:

t 1 1
= 2
+ t
qt k2 qe qe

where k2 (g/mg h) is the rate constant of second-order adsorption.

Intraparticle Diffusion Model

Intraparticle diffusion model based on the theory proposed by Weber and Morris was tested to identify
the diffusion mechanism. It is an empirically found functional relationship, common to the most adsorp-
tion processes, where uptake varies almost proportionally with t1/2 rather than with the contact time t.
According to this theory:

qt = k p t1/ 2 + C

where, kp (mg/g h1/2) is the intraparticle diffusion rate constant.

The kinetic data can be fitted trying the pseudo-first-order, pseudo-second-order and intraparticle
diffusion models.
The biosorption of cationic dyes by F. carnea in the batch adsorber by was described by first-order
reaction kinetics defined in Eq. (Mittal and Gupta, 1996)

Equilibrium Modeling of Biosorption

Biosorption is a well known equilibrium separation process for wastewater treatment containing dyes.
The excellence of a biosorbent is evaluated by the amount of soorbate it can uptake from an aqueous
solution and retain in an immobilized state. Solute uptake can be calculated from the following equation:

Q = (V0C0 − VFCF ) / M

where Q0 is the solute uptake, C0 and Cf are the initial and equilibrium solute concentration (mg/L)
respectively, whileV0 and Vf are the initial and final volumes of the solution (Litre) and M is the mass
of biosorbent (g) (Vijayaraghavan and Yun, 2008). Equilibrium data, commonly known as sorption iso-
therms, are fundamental requirements for the designing of biosorption systems. The biosorption isotherm
indicates how the sorption molecules distribute between the liquid phase and the solid phase when the
biosorption process reaches an equilibrium state. The analysis of the isotherm data by fitting them to
different isotherm models is an important step to find the suitable model that can be used for design

65
 
Biosorption of Dye Molecules

purposes. Biosorption isotherm is basically important to describe how solutes interact with biosorbents,
and is critical in optimizing the use of biosorbents. The behavior of biosorbent can be studied by evalu-
ating the equilibrium isotherms and removal efficiency of dye in batch mode. Lang-muir, Freundlich,
Temkin, Redlich–Peterson, Brunauer–Emmet–Teller, Radke–Prausnitz models are commonly used for
describing the biosorption equilibrium of dyes at a constant temperature. The applicability of isotherm
equation of the study can be compared by calculating the coefficient of correlation (R2). (Aksu, 2005
and Tan et.al, 2008) Some of the commonly used models are described below.

Isotherm Models

Langmuir Isotherm Model

The Langmuir adsorption isotherm is based on the assumption that adsorption takes place on a homog-
enous surface. The Langmuir equation is given by following equation:

Ce Ce 1
= +
qe qm K L qm

where qm (mg/g) is the maximum amount of sorbate per unit weight of biosorbent for complete monolayer
coverage and KL is the Langmuir adsorption constant (L/mg). A plot of Ce/qe verses Ce should be linear
if adsorption follows Langmuir behaviour.

Freundlich Isotherm Model

Freundlich adsorption equation is given by:

1
log qe = log K F + ( ) log Ce
n

where Kf is Freundlich constant and n is heterogeneity factor.

Temkin Isotherm Model

The Temkin Isotherm model is given by the following equation.

q e = BlnA T + BlnCe

RT
B=
bT

where AT is temkin isotherm equilibrium binding constant (L/g), bT is Temkin isotherm constant, R
denotes the universal gas constant (8.314J/mol/K), T is Temperature at 298K and B is a constant related
to heat of sorption(J/mol). (Vijayaraghavan & Yun, 2008; Aksu, 2005)
The isotherm studies for Acid Blue 29 biosorption by A. nigerfungus showed that the Langmuir,
Freundlich, and BET isotherm models all fitted well with the experimental data. (Fu & Virarahavan, 2001)

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Biosorption of Dye Molecules

CONTINUOUS BIOSORPTION

Continuous biosorption studies mostly achieved by using packed bed columns are of great significance
for testing the technological and industrial feasibility of the process. Packed bed columns have emerged
as most efficient and economically convenient for continuous biosorption ((Zhao et al., 1999; Saeed &
Iqbal., 2003; Volesky et al., 2003; Chu, 2004). Other alternative for continuous biosorption are fluidized
and continuous stirred tank reactors, but these are rarely used for biosorption. Continuous stirred tank
reactors are found to be useful in case of powdered biosorbents but they maintenance requires a high
cost. (Cossich et al., 2004). The fluidized bed is needed to maintain a high flow rate so as to keep the
sorbent particles suspended (Muraleedharan et al., 1991).

Packed Bed Columns

Packed bed configuration consists of cylindrical columns that are tightly crammed with sorbent materi-
als. The waste water is allowed to pass slowly through the column under the effect of gravity. Most of
the solute particles are are sorbed at the initial layers and hence the solute concentration is expected to
be zero at the outlet (Vijayaraghavan & Yun 2008). Concentration difference is the driving force for
sorption process and it can be successfully attained by using packed bed columns and eventually results
in healthier effluent quality (Aksu & Gönen, 2004). However the initial column behavior should not be
taken into consideration as it is a time dependent process and needs some time for stabilization (Naja &
Volesky, 2006a). As time increases and the column length is traversed by the contaminated solution the
biosorbent bed becomes saturated with solute particles and the solute concentration in the biosorbent bed
gradually increases at the column outlet. The breakthrough concentration can be fixed at this point of
the column. Breakthrough concentration depends on the toxicity of the substance (solute) and is mostly
in the range of 0.01 to 1 mg/l. When the column reaches a saturation level an S-shaped breakthrough
curve is obtained. Breakthrough curves represent the equilibrium sorption isotherm relationships, mass
transfer to and all over the biosorbent in the column, and operational fluid-flow parameters, and hence
are important for the assessing the characteristics of a column (Aksu, 2005; da Silva et al., 2002).
The performance of a packed bed column can be evaluated on the basis of some important parameters
that include the following:

• Rate of uptake
• Length of the sorption zone
• Removal efficiency
• Slope of the breakthrough curve (Volesky et al., 2003; Vijayaraghavan et al., 2004)

A mass transfer zone is developed between the progressively saturated section of the column and the
clean biosorbent section (Naja & Volesky, 2006a). The length of this zone is significant for practical
applications, and is given by the following equation:

 t 
Z m = Z 1 − b 
 tc 

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Biosorption of Dye Molecules

where Z is the bed depth (cm), and tb and te are the column breakthrough and exhaustion times (h),
respectively.
Uptake can be calculated by dividing the total mass of biosorbed sorbate (mad) by that of the bio-
sorbent (M). Mass of biosorbed sorbate is obtained from the area above the breakthrough curve (C vs.
t) multiplied by the flow rate.
The removal efficiency (%) can be calculated from the given equation:

mad
Removal efficiency
 (%) = C 0Fte
X 100

where, C0 and F are the inlet solute concentration (mg/l) and flow rate (l/h), respectively. It should be
noted that the removal efficiency is independent of the biosorbent mass, but solely dependent on the
flow volume.

CONCLUSION

The use of dead/inactive microorganisms for the removal of organic contaminants such as dyes from
wastewaters and the factors influencing the biosorption process has been reviewed in detail in this chapter.
Application of untreated (live) or treated (chemical or heat treated) microorganisms as biological adsor-
bents has fascinated many researchers in the recent years due its ease of operation, less time requirement,
cost effectiveness, easy availability of biosorbents, high efficiencies in detoxification of very dilute to
concentrated effluents and no nutrient requirements. Moreover the application DNA recombinant technol-
ogy for creating genetically modified microorganisms has opened up new gates and wide opportunities
for further development and research in the field of microbial biosorption. Biosorption has been reported
by many as a cheap and convenient alternative to the traditional waste water treatment technologies.
Biosorption can be easily implemented for the cleanup of a variety of industrial effluents containing
various organic pollutants including ranging from phenolic compounds to pharmaceutical drugs and
other organic contaminants including the wide range of synthetic dyes. A wide range of microorganisms
including bacterial, fungal, and yeast strains have shown potential in the removal of dyes from coloured
effluents. However literature survey indicates that study of biosorption of organic wastes such as dyes
is restricted to a few strains of organisms for a few groups of dyes. Biosorption of dye contaminants has
been found to be strain specific and thus the type of biomass significantly affects biosorptive uptake.
Although bacterial biomass presents a wide class of biosorbents, yet some constraints such as difficulty
in reuse of biomass, limits the process from getting wide economical acceptance. The feasibility and
efficiency of the process also depends on the properties as well as composition of wastewater and envi-
ronmental conditions. Hence further investigation is needed in order to establish the best combination of
dyes, biomass types and environmental conditions. The lack of understanding of the mechanism of dye
sorption process hinders the reasonable evaluation of process performance and limitations, and thereby
the widespread application of biosorption. More information based on experimental research is required
to recognize the mechanism of dye uptake by biomass. Screening and selection of the most promising

68

Biosorption of Dye Molecules

biomass, enhancement of specificity and uptake capacity via chemical and/or genetic modifications and
analysis of biosorbent behavior with real industrial effluents and simultaneous analysis of the impact
of water quality on the uptake of specific pollutants can be helpful for successful implementation of
biosorption technology in real industrial situations on a wide scale.

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 75

Chapter 4
Microbial Response
against Metal Toxicity
Jatindra Nath Bhakta
University of Kalyani, India

ABSTRACT
Damage of microbial communities caused by metal toxicity in different domains of the environment is a
growing challenge worldwide. The present chapter attempted to elucidate how microorganisms tackle and
response against the metals toxicity. In response to metal toxicity, microorganisms exhibit a vast array of
acclimatization, adaptation and resistant strategies at genetic, cellular and community levels to detoxify
metals toxicity and survival. Microorganisms detoxify metals by various mechanisms (sequestration,
inhibition of influx, efflux, accumulation, precipitation and chemical modification, repair, and metabolic
by-pass) and showed resistance properties (by protein/enzyme synthesis) encoded by genes located in
chromosome, plasmid or transposon. Thus, metal toxicity hampers the microbial metabolism, growth,
activity and species diversity resulting in severe damage in environmental microbial community. Apart
from detrimental consequences of metal toxicity, the novel metal- and antibiotic- resistant microorgan-
isms could be used in environmental and human health benefits.

INTRODUCTION

Metals* are ubiquitous in nature, since they are an integral part of earth planet. It constitutes about 75%
of the known elements and is vital to our industry, infrastructure and daily life. Though many of them
are significantly essential in the biochemical process of organisms, nonetheless, excess concentration of
any metals causes hazardous and toxic impacts in all organisms of food chains and food webs (Jillian,
Robert, & Rajakaruna, 2015) by bioconcentration, bioaccumulation and biomagnifications phenomena.
The anthropogenic and geogenic activities are major causes for generating and releasing pollutants of
metal and its derivatives, which are undoubtedly responsible for contamination and deterioration of
different domains of global environment. Natural geologic processes continue at a very slow pace to
concentrate and disperse metals, forming large zones of elevated metal concentrations and constantly

DOI: 10.4018/978-1-4666-9734-8.ch004

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.
 
Microbial Response against Metal Toxicity

releasing metals into the environment. Besides, awful consequences of rapid and continuous economic
development and growth, industrialization, urbanization and population explosion are the prime reasons
for generation of massive amount of toxic and hazardous metal wastes in the environment in developed
and developing countries around the world. It is generally occurred in high concentration in the mining
(extraction sites) as well as industrial zones which is greatly responsible for the contamination of the
surrounding local ecosystems. Mining, manufacturing, and the use of synthetic products (e.g. pesticides,
paints, batteries, industrial waste, and land application of industrial or domestic sludge) can result the
heavy metal contamination in urban and agricultural soils. Worldwide coal burning, municipal solid
waste incineration, electronic, paper, paint, pharmaceutical industries (Biester, Muller, & Scholer, 2002;
Tack, Vanhaesebroeck, Verloo, Rompaey, & Ranst, 2005) and tailings of gold mines are identified as
the major anthropogenic origins of metals contaminating the environment.
Contamination of metals is one of the growing global problems during last few decades due to exerting
severe environmental and human health risks. The priority toxic metal (Ag, As, Be, Cd, Cr, Cu, Hg, Ni,
Pb, Sb, Se, Tl, Zn) pollutants (Sparks, 2005), their chemical derivatives and organometals significantly
pose tremendous detrimental effects in all forms of organisms in environment. Non-biodegradable and
persistent natures of metals is responsible to easily accumulate in soil (Nwachukwu, Feng, & Alinnor,
2010), sediment, plant (Ashraf, Maah, & Yusoff, 2011; Rahman, Saha, Molla, & Al-Reza, 2014) and
aquatic flora and fauna leading to biomagnifications in the food chain. It is well known that metal con-
tamination can causes various health hazardous, such as mental disorder; skin poisoning; affects kidneys,
lung, liver, heart and central nervous system; and even responsible for causing cancer in human.
Despite, the toxic metal contaminants can cause an immeasurable damage in the environmental
health especially affecting the vast array of microbial community. Microbes such as bacteria, protists,
fungi, yeast, algae, etc. are omnipresent and play key geoactive roles in the environment, particularly in
element biotransformations and biogeochemical cycling, metal, mineral and nutrients transformations,
decomposition, bioweathering, and soil and sediment formation processes (Gadd, 2010). In environ-
ment, the metal contaminants and its derivatives significantly interact with microorganisms and pose
severe adverse impacts to the significant microbial communities under certain conditions (Giller, Wit-
ter, & McGrath, 1998; Bhakta, Ohnishi, Munekage, & Iwasaki, 2010; Bhakta, Munekage, Ohnishi,
& Jana, 2012a; Bhakta, Ohnishi, Munekage, Iwasaki, & Wei, 2012b; Olaniran, Balgobind, & Pillay,
2013; Lenart-Boroń & Boroń, 2014; Bhakta, Munekage, Ohnishi, Jana, & Balcazar, 2014; Kuperman,
Siciliano, Römbke, & Oorts, 2014) that is known as metal toxicity of microbe. In general, it severely
alters the normal metabolism, growth and activity processes of microorganisms, which in turn immensely
affect the gross structure, and function of microbial communities and metabolic activities of environ-
ments (Giller et al., 1998). Extensive researches have shown that microbes exhibit response and acquire
tolerant/resistant properties against metals in contaminated environment that can play important roles
in detoxification and mobilization of metals in order to control various dangerous toxic impacts in the
environment (Gadd, 1990; Idris, Trifonova, & Puschenreiter, 2004; Bhakta et al., 2010, 2012a,b, 2014)
as well as other various toxic chemicals in the environments.
On account of above discussion, it is apparent that the comprehensive literature regarding the micro-
bial responses against the metal toxicity is insufficient and scanty. Stemming from this point of view,
the objective of present chapter has been aimed to find out answer of the question, how microbes tackle
and response against toxicity problems of metals.

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CONCEPTUAL OVERVIEW OF METAL TOXICITY

The basic concept of metal toxicity can be drawn briefly in following two processes: (a) metal transport
in microbial cell and (b) metal-microbial interaction

Metal Transport in Microbial Cell

In the process of metal toxicity, the microbial uptake of toxic metal from surrounding metal contaminated
immediate environment is the crucial event, because, the cell membrane is selective permeable (i.e.,
allows certain molecules or ions to pass through it by means of active or passive transports) in nature.
After interaction of metal with outer surface of cell membrane, it is transported though cell membrane
and reached to cytoplasm. In exceptional situation, the transportation of toxic or non-essential metals
sometimes starves cells by competitively inhibiting the transport of essential ions. Consequently, the cell
is suffered from lack of essential ions, accumulation of toxic or non-essential metals in cytoplasm and
ultimate metal poisoning. This toxic metal poisoning is the characteristic of microbial metal toxicity. The
intracellular accumulation of metals in poisonous quantities is largely governed by the protein-mediated
transport systems of metals uptake and efflux. Every microorganism has a different complement of trans-
port proteins to handle the metals uptake and efflux mechanisms. Proteins of transporter families play
definite vital role in essential metal uptake as well as in the efflux of essential and non-essential metal
ions (Nies, 2003; Ma, Jacobsen, & Giedroc, 2009). Although, up-to-date database of classification of
transporter proteins has been catalogued (Transporter Classification Database [TCDB] the knowledge
of specific proteins for transporting highly toxic non-essential metals including Pb, Al and Ag; route
and mechanism of cellular entry are unknown. The current concept regarding the transporter protein
involved in uptake of non-essential metals in microorganisms have been gained experimentally so far is
summarized in Table 1. However, there is a lack of knowledge in this respect and it is also apparent that
the uptake of non-essential metals often involves transporters those are associated with the acquisition of
essential inorganic and organic ions. Based on the current knowledge, the transportation of non-essential
metal is facilitated by two processes: (i) direct transport and (ii) co-transport with low-molecular-mass
ligands (Lemire et al., 2013).

Direct Transport

It is an energy dependent direct metal uptake process of cell. Generally, the polar solutes directly penetrate
outer cell membrane, including many essential metal ions through transporters of the general bacterial
porin (GBP) superfamily, which are β-barrel proteins in Gram-negative bacteria (Lopez, Garcia-Gimenez,
Aguilella, & Alcaraz, 2010). Role of GBP superfamily members is not experimentally demonstrated in
outer membrane transport and hence the direct uptake mechanism of non-essential metals in this respect
is unclear till date, whereas more is known about transporters in the periplasm and at the cytoplasmic
membrane. However, it has reported that non-essential metals can pass the membrane and enter to the
cytoplasm by the transporter proteins using mechanism of ionic and molecular mimicry in both bacteria
and fungi, Saccharomyces cerevisiae. The transporter proteins are found to transport some non-essential
metal cations that resemble essential metal ions and/or inorganic substrates (Table 1). Example of certain
transporters are able to transport non-essential metal cations as essential metal ions are - Zrt- and Irt-

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Microbial Response against Metal Toxicity

like protein (ZIP) family (Grass et al., 2005; Lin, Chai, Love, & Fu, 2010), natural resistance-associated
macrophage protein (NRAMP) family (Makui et al., 2000; Cohen, Nelson, & Nelson, 2000), ATP-binding
cassette (ABC)-type protein family (Anderson, Adhikari, Nowalk, Chen, & Mietzner, 2004) and P-type
ATPase transporter family (Hao, Chen, & Wilson, 1999; Lemire et al., 2013). Additionally, examples of
transporters from the major intrinsic protein (MIP) (Sanders, Rensing, Kuroda, Mitra, & Rosen, 1997;
Meng, Liu, & Rosen, 2004; Wysocki et al., 2001), sulphate permease (SulP) (Pereira et al., 2008), solute
Na symporter (SSS) (Borghese & Zannoni, 2010) and inorganic-phosphate transporter (PiT) (Elias et
al., 2012) families that facilitate the transport of non-essential metal oxyanions owing to their similarity
to other inorganic substrates (Lemire et al., 2013).

Table 1. Transporter proteins and their experimentally defined roles for uptaking non-essential metals
in microorganism (Lemire et al., 2013)

Superfamily or Family* Examples Organism Substrates References

α-Type Channels

Major intrinsic protein (MIP) GlpF Escherichia coli Glycerol, Sb(III)‡ and As(III)‡ Sanders et al., 1997;
superfamily Meng et al., 2004

Fps1 Saccharomyces cerevisiae Glycerol, Sb(III)‡ and As(III)‡ Wysocki et al. 2001

Mercuric ion transporter (Mer) MerH Mycobacterium marinum Hg(II)‡ Schue, Dover, Besra,
superfamily§ Parkhill, & Brown, (2009)
β-Barrel Porins

General bacterial porin (GBP) FpvA Pseudomonas aeruginosa Mn(II), Fe(III), Co(II), Zn(II), Hannauer et al., 2012;
superfamily|| Ni(II), Cu(II), Cd(II)‡ and Braud et al., 2009
Ga(III)‡

FptA P. aeruginosa Fe(III), Co(II), Ni(II) and Pereira et al., 2008

Ga(III)‡
Porters (Uniporters, Symporters and Antiporters)

Zrt- and Irt-like protein (ZIP) family ZipB Bordetella bronchiseptica Zn(II) and Cd(II)‡ Lin et al, 2010

ZupT E. coli Mn(II), Fe(III), Co(II), Zn(II) Grass et al., 2005

and Cd(II)‡

Inorganic-phosphate transporter (PiT) PitA E. coli Phosphate, Zn(II), As(III)‡ and Elias et al., 2012
family Te(IV)‡

Solute Na symporter (SSS) family ActP Rhodobacter capsulatus Acetate and Te(IV)‡ Borghese et al., 2010

Sulphate permease (SulP) family Sul1 S. cerevisiae Sulphate and Cr(VI) ‡

Pereira et al., 2008

Natural resistance-associated Smf1 S. cerevisiae Mn(II), Fe(II), Co(II), Ni(II), Cohen et al., 2000
macrophage protein (NRAMP) family Cu(II), Zn(II) and Cd(II)‡

MntH E. coli Mn(II), Fe(II), Co(II), Zn(II) Makui et al., 2000

and Cd(II)‡
Transporters Driven by Hydrolysis of Phosphate–Phosphate Bonds

ATP-binding cassette (ABC)-type FbpA, FbpB, Haemophilus influenzae Fe(III) and Ga(III)‡ Anderson et al., 2004
ATPase superfamily FbpC

P-type ATPase superfamily MntA Lactobacillus plantarum Mn(II) and Cd(II)‡ Hao et al., 1999

Note: ActP, acetate permease; Fbp, Fe -binding protein; Fps1, glycerol uptake and efflux facilitator; FptA, Fe –pyochelin receptor A; FpvA, ferripyoverdine
3+ 3+

receptor A; GlpF, glycerol uptake facilitator; MntA, Cd- and Mn-transporting P-type ATPase; Sul1, sulphate permease 1; ZupT, Zn uptake transporter.
*Taxonomy based on the Transporter Classification system of the International Union of Biochemistry and Molecular Biology (IUBMB) (Saier, 2000). ‡Non-
essential metal. §The Mer superfamily consists of five permease families (MerF, MerH, MerTP, MerC and MerE) known to mediate Hg(II) uptake. These
transporters are functionally coupled with resistance determinants in various microorganisms and are encoded on chromosomes, plasmids and transposons
(Schue et al., 2009). ||Although FpvA and FptA are grouped taxonomically with the GBP superfamily, these transporters are distinguished from other porins
because they use active mechanisms of transport.

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Microbial Response against Metal Toxicity

Co-Transport with Ligands

It refers to the ligands (such as, phosphate, amino acids, peptides and organic acids) mediated metal
transport mechanism of cell where ligands act as a vehicle for carrying metals. The metals can be co-
transported through the membrane of cell by binding to ligands. Reduction potential property of metals
helps to bind to functional group of ligands and to pass through membrane in this transport mechanism.
Likewise, the specificity of functional group of ligand has the potential to bind to specific metal. Example
of ligands are - citrate-dependent transport of Fe(III) by the FecA system of Escherichia coli (Hussein,
Hantke, & Braun, 1981); phosphate transporters of S. cerevisiae Pho84 (Jensen, Ajua-Alemanji, & Culotta,
2003) and E. coli PitA (Beard et al., 2000), can transport essential divalent metals as part of phosphate
complexes. Similarly, the non-essential metals are also transported in cell through these transporters
using the same mechanism. For example, Hg(II) is entered to the cell by the formation of Hg–cysteine
complexes in Geobacter sulfurreducens (Schaefer & Morel, 2009).
Additionally, another well known example of low molecular weight ligand is the siderophore. It is
a high-affinity metal (such as V, Mo and Fe) (Schalk, Hannauer, & Braud, 2011) chelating molecules
secreted by microorganisms such as bacteria, fungi to bind, transport or shuttle these metals in cell.
These low-molecular-mass coordination molecules are excreted by a wide variety of fungi and bacteria
basically to aid Fe assimilation; probably because Fe is needed in larger amounts by cells than other
poorly soluble metals. Organisms have most likely evolved mechanisms to ensure that Fe demand can
met through the production of species-specific siderophores, or by attachment to a solid Fe mineral, e.g.
Fe oxides, to shorten the pathway between the Fe substrate and cellular site of uptake. It forms complexes
with oxides, hydroxides and other molecules for sequestration. Although siderophores are found to
chelate metals other than Fe(III), the non-essential metals such as Cd(II) and Ga(III) (Braud, Hannauer,
Mislin, & Schalk, 2009; Hannauer et al., 2012) have also been shown to accumulate in cells through an
Fe siderophore-mediated transport. For instance, Pseudomonas aeruginosa acquires Ga(III) through the
siderophores pyochelin and pyoverdine, but at a rate that is approximately 20-fold slower than that of
Fe(III)–siderophore complexes (Braud et al., 2009; Braud, Hoegy, Jezequel, Lebeau, & Schalk, 2009).

Metal-Microbial Interaction

Microbial metal toxicity is a complex effect resulted from metal geochemical process of environment and
cellular biochemical process. Metals are directly and/or indirectly involved in all aspects of microbial
growth, metabolism and differentiation (Gadd, 1992). Metal–microbe interactions, therefore, are of key
importance within the framework of metal toxicity, geomicrobiology, and also fundamental to microbial
biomineralization processes. Microbes interact with metals and minerals in natural and synthetic environ-
ments, altering their physical and chemical states which lead to toxicity growth, activity and survival.
Metals interact throughout the cell, especially essential cellular components, from the cell wall, outer
membrane, periplasm, and inner membrane (in Gram-negative bacteria) through to the cytoplasm through
covalent and ionic bonding and are responsible for changing the biochemical reactions in the metabolic
process. When microorganisms are exposed to metals containing surrounding immediate environment,
the metals are come in contact with the bacterial cell and various biochemical process of metal-microbe
interaction is initiated. This process can generally be differentiated in three following sequential phases
on the basis of interaction site of cell: (i) Outer membrane interaction – it is initial interaction of metals
with the extracellular polymers, proteins and various metabolites of microbes at outer surface of cell wall

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Microbial Response against Metal Toxicity

or membrane involves (ii) Intra membrane interaction – it covers the interaction of metals with porins,
ligand, siderophores and membrane-bound importers during the period of metal transportation across
the membrane and (iii) Intracellular or cytoplasmic interaction – it comprises the interaction of metals
with various cytoplasmic components through complex cytoplasmic biochemical reactions.
However, the entire mechanism of metal toxicity includes different aspects of metal-microbial in-
teractions through a series of complex biochemical reactions to damage the microbial cell as follows:
reactive oxygen, species (ROS) stress and antioxidant depletion, lipid peroxidation, protein dysfunction
and loss of enzyme activity, membrane damage, competitive inhibition of nutrient assimilation and metal
genotoxicity (Figure 1).

Figure 1. The overview of gross damages caused by metal toxicity in cell.

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Microbial Response against Metal Toxicity

Table 2. Metal specific resistance mechanisms experimentally observed in microorganism (Modified

form of Rajendran et al, 2003)

Resistance Mechanisms Metals

Extracellular complexation Cd , Ga , Ni , U , Th and Cu2+
2+ 2+ 2+ 2+ 2+

Suppression of influx Cd2+, Cu2+, and Zn2+

Efflux Cd2+, Cu2+, Co2+, Mn2+, Zn2+, Mg2+, Ni2+, AsO2-, AsO43-, Sb3+and
K+
Accumulation Cd2+, Cu2+, Zn2+, Au+ and Ag2+
Precipitation Co2+, Mn2+, Zn2+, Mg2+ and K+
Redox transformations and chemical modification As5+ and Hg2+
Metabolic by-pass As5+
Volatilization Hg2+

MICROBIAL RESPONSE: ACCLIMATIZATION, ADAPTATION AND RESISTANCE

The microbe responses in different ways by exerting the self-defense and detoxifying mechanisms in order
to combat and protect the deadly metal toxicity. Microbial cellular responses to metals are heterogeneous
in both their biochemical and genetic bases, which are ultimately reflected in the alteration of microbial
growth, activity and community diversity. In response to metal toxicity, however, microorganisms develop
various acclimatization, adaptation and resistance strategies (Jarosławiecka & Piotrowska-Seget, 2014)
(Table 2), which are characterized by cellular (biochemical and genetic) (Jarosławiecka & Piotrowska-
Seget, 2014) as well as growth, activity and community levels.

Cellular Response

Biochemical Basis of Response

Microorganisms have the biochemical nature of acclimatization, adaptation and resistance characteristics
to render appropriate responses against the metal toxicity. To withstand metal toxicity, microorganisms
acclimatize and adapt the adverse situation and/or exhibit resistance ability by adopting different bio-
chemical and genetic mechanisms. For metal ion homeostasis system in bacteria to work properly, the
bacterial cell must balance the uptake, efflux and sequestration of metal ions, and if they cannot prevent
non-essential or toxic metal ions from entering the cell, they must remove or detoxify them. Therefore,
microorganisms are well equipped with uptake, efflux and sequestration systems those work uniquely
to deal the metal toxicity. However, it is noticeable that no single strategy provides universal acclima-
tization, adaptation and resistance capabilities to all toxic metals; it is probably due to the distinctive
physiochemical properties of different metal atoms. Although microorganisms exerted the mechanisms
of complex and diverse biochemical natures for controlling metal toxicity, the acclimatization, adaptation
and resistance processes can be categorized functionally as follows from the exterior of the cell through
to the interior of the cell (Figure 2):

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Microbial Response against Metal Toxicity

Figure 2. Various acclimatization, adaptation and resistance mechanisms of microorganisms exhibited

in response to deadly metal toxicity. The corresponding numbers of upper panel and lower panel (at
bottom) of the figure depict the response strategies of microorganisms.

(1) Extracellular sequestration: Microbial responses against metal toxicity is initiated with metals
sequestration at the outer surface of the cell membrane/wall exhibiting various mechanisms such as, bind-
ing, crystallization, precipitation, extracellular metal and mineral nanoparticle deposition, complexation
and biomineralization, redox transformation, volatilization, etc. (Figure 2).
Microorganisms possess a characteristic extracellular polymeric substances (EPS) (Jarosławiecka
and Piotrowska-Seget, 2014), a complex high-molecular structure constituted with carbohydrates, lipid,
proteins, humic substances, nucleic acids etc., are associated with the functions of microbial aggregates,
mass transfer, sequestration of nutrients and metals, surface characteristics, adsorption ability, stability,
biodegradability etc. (Sheng, Yu, & Li, 2010) (Figure 2). Many microorganisms upregulate the expres-

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Microbial Response against Metal Toxicity

sion of EPS in response to metal exposure, and these molecules contain functional groups capable of
coordinating metal ions as efficient metal binding agents. EPS and siderophores can trap metal ions in
the extracellular environment (Harrison, Ceri, & Turner, 2007). Siderophores are also able to bind other
metals, e.g. Ga, Ni, U, Th and Cu (Gadd, 1988; Gadd & White, 1989) by forming metal complexations
(Figure 2). Metals interact with proteins or cell-associated polysaccharides (such as, lipopolysaccharide)
of membrane and bind to bacterial cell surfaces (Langley & Beveridge, 1999).
The trapped metal is also found to precipitate on cell surfaces of microorganisms by EPS and sid-
erophores (Langley & Beveridge, 1999) (Figure 2). It is well known that some hydrogen sulphide pro-
ducing bacteria can precipitate metals as metal sulphides. For instance, Cd-grown Klebsiella aerogenes
contained 2-4% of the dry weight as cadmium and large numbers of electron-dense granules of CdS
occurred on outer cell surfaces (Aiking, Stijnman, Garderen, Heerikhuizen, & Van’t Riet, 1984). Such,
metal sulphide precipitation can also occur in algae, yeasts and fungi as well as metal precipitation as
phosphates or oxalates (Gadd, 1988).
Microorganism can crystallize the metal and deposit as nanoparticles on membrane surface. Microbes
are implicated in ferromanganese nodule formation on ocean floors and a variety of bacteria, algae and
fungi can become encrusted with manganic oxides (Kelly, Norris, & Brierley, 1979) (Figure 2). Crystal-
line deposits of many other elements, e.g. Au and U, have been observed in a variety of microbial types
(Gadd, 1988).
Microbial redox transformation is also another mechanism to prevent metal binding to and entry
through the cell membrane (Picard et al., 2011) (Figure 2). Recently, formation of nanopaticles of met-
als and deposition on the outer surface of the cell membrane has also been observed in the extracellular
sequestration of microbes. Some bacteria have the ability to transform the metals into gases form, for
instance, volatilization of Hg by some Lactobacillus sp., Beijerinckia sp (KDr2), etc. (Ruiz, Alvarez,
Gonzalez-Ruiz, & Cesar, 2011; Ray, Gachhui, Pahan, Chaudhury, & Mandal, 1989).
(2) Suppression of influx: It is kind of active mechanism for preventing the metals from entering the
cell by reduced permeability. Microorganisms have evolved complex transporter systems in the mem-
brane which regulates the passage of ions or molecules through it. These transporting systems exhibit
the impermeability for entering some toxic and nonessential metal ions under certain conditions which
refers to suppression of metal influx (Figure 2). The mechanisms involved in this concern are regulated
by means of expression and activities of proteins associated with metal influx/efflux are crucial for
metal resistance, and different bacterial species have distinct complements of these systems. EPS and
siderophores those are bound to toxic metals may also act as suppressor to decrease uptake or increase
efflux by membrane transporters. In bacteria, the complex and tight regulatory mechanisms are found
to control the activity of membrane transporters that take up metals, and regulators that can bind metal
ions with femtomolar affinities (Ma, Jacobsen, & Giedroc, 2009) control some of these transporters.
(3) Efflux: Efflux is the active transport phenomena of the non-essential, toxic and/or excess es-
sential metals away from the cell. Unlike other molecules, metals are neither synthesized nor degraded
in accordance with requirements of the cell. Therefore, the intracellular concentrations of metal ions
are maintained by influx (influx pump) and efflux (efflux pump) systems of the cell membrane. The
presence of relatively higher concentrations of non-essential, toxic and/or excess essential metals within
cells is responsible for toxicity effects. To avoid this adverse situation and to reach a homeostasis condi-
tion of metals, the cell adopts the efflux pump to transport these metals from intracellular space to the
outside (Figure 2). For example, Cd2+-efflux pumps of bacteria. In microorganisms, many resistance
determinants on chromosomes and mobile genetic elements encode a range of membrane transporters

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Microbial Response against Metal Toxicity

that efflux specific toxic metals out of the cell. The expression of these systems is controlled by ultra-
sensitive regulators that bind metals with zeptomolar affinities, and the activity of these transporters
may be driven by ATP hydrolysis or chemiosmotic potential (Ma et al., 2009; Nies, 2003).
(4) Intracellular sequestration: After metal uptake, the microbial cell exhibits several responses of
metals sequestration in intracellular regions as well as periplasmic space in order to minimize or reduce
metal toxicity. The intracellular sequestration of metals basically includes the following accumulation,
precipitation, redox transformations and chemical modification, repair, and metabolic by-pass processes
using the intracellular binding proteins, enzymes and some other important biomolecules:

• Accumulation: The accumulation of metals in nontoxic forms within the cell is the most common
phenomena of microorganisms to overcome the metal toxicity. In toxic condition, the genetic sys-
tems are unregulated and synthesized the intracellular metal-binding proteins in different microor-
ganisms, such as- bacteria, cyanobacteria, algae, fungi and yeasts. These proteins, bacterioferritin
and metallothioneins (MTs) (Carrondo, 2003) and phytochelatins (PCs) can effectively bind the
metal such as, Cu, Zn, Cd, Au, Ag etc.
• Precipitation: Another common microbial response is metal precipitation by compartmentalizing
and/or converting to more innocuous forms (Figure 2). Many microorganisms precipitate metals
as metal oxides, metal sulphides, metal–protein aggregates or elemental metal crystals, which
form particulates that are closely associated with the cytoplasmic membrane (Zannoni, Borsetti,
& Turner, 2008). Dense intracellular deposits of uranium were observed in Pseudomonas aerugi-
nosa 32, while other electron-dense bodies, including polyphosphate, have been associated with
intracellular metal accumulation in several bacteria, algae and fungi (Gadd, 1988). In eukaryotic
microbes, e.g. yeasts, a majority of intracellular Co2+, Mn2+, Zn2+, Mg2+ and K+ is located in the
vacuole where there may be binding to low molecular weight polyphosphates (Gadd & White,
1989). Besides, metals are precipitated in the form of oxalates, carbonates, phosphates, oxides,
hydroxides, sulfides and nanoparticulate biominerals.
• Redox Transformations and Chemical Modification: In addition, it seems that most survival
mechanisms depend on redox transformations of some metal species from higher to lower toxic
forms leading to reduction of metal toxicity (Figure 2). Metals can undergo specific redox and
covalent reactions in cells, and these reactions alter the chemical reactivity of the metal atoms and
convert toxic metal species to less toxic or less available forms (Silver & Phung, 1996). This modi-
fication process can alter the redox state of the metal, create metal crystal precipitates or generate
organometallic small-molecule compounds.
• Repair: This process repairs the cellular molecules oxidized by ROS (Figure 2). Some molecules
having redox-sensitive functional groups are oxidized by the primary reactions with metals or by
highly reactive, catalytic by-products of metals (such as ROS). These oxidized molecules can be
repaired by cellular chaperones, enzymes or antioxidants (Harrison et al., 2009).
• Metabolic By-Pass: It is a kind of enzyme mediated metal detoxification response (such as, oxi-
dation, reduction, methylation, and demethylation) of microorganism (Figure 2). Microbial cell
synthesize the metal-disrupted enzymes by producing alternative proteins with catalytic cores
that do not bind to the toxic metal ligand or by shunting metabolites towards alternative pathways
(Lemire, Mailloux, Auger, Whalen, & Appanna, 2010). For example, arsenate reductase converts
the arsenate (As(V)) to arsenite (As(III)) which is then exported from the cell by energy-depen-
dent efflux pump.

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Microbial Response against Metal Toxicity

Genetic Basis of Response: Genomics and Metal Resistance

Microbial resistance to metal toxicity is a significant phenomenon. Despite apparent toxicity, however,
many microbes grow and even flourish in apparently metal-polluted habitats developing variety of active
and/or incidental mechanisms, which contribute to resist against metal toxicity (Mowll & Gadd, 1984;
Holden & Adams, 2003). The metal resistance is varied with the variations of microorganisms as well
as qualitative and quantitative properties of non-essential and toxic metals in the environment. Besides,
it is greatly influenced by physico-chemical nature of the environment and chemical behaviour of metal
species as discussed elsewhere. Microbial resistance to toxic metals is widespread, with frequencies
ranging from a few per cent in pristine environments to nearly 100% in heavily polluted environments
(Silver & Phung, 2009).
Both biochemical and genetic basis of cellular response and resistance are complex and interlinked
mechanisms widely varied in different microorganisms. The genetic basis and mechanisms of resistance
are substantial and well-documented in microbes. The interaction of, and selection for resistance to, toxic
substances in addition to metals, such as antibiotics and toxic analogues, involve similar principles to
those concerning metals. However, it is well known that metal resistance is chromosomally-, plasmid- or
transposon-encoded, and one or more genes may be involved at the biochemical levels which are respon-
sible for resistance. The mechanisms of action involve the sensing respective genetic systems for protein/
enzyme synthesis and distribution of proteins as improvised defending systems for metal-detoxification
process. Various types of resistance mechanisms can occur singly or in combination and for a particular
metal different mechanism of resistance can occur in the same species.
The mechanism of an organism for sensing to a change in the local environment or within the cell is
in changes in mRNA transcription. In response to regulation process, bacteria use the initiation of RNA-
transcription as the key step for sensing (Browning & Busby, 2004), which is controlled by the RNA
polymerase and different σ factor subunits of RNA polymerase. These effectively control the expression
of different subsets of genes responsible for translation of sense specific proteins (i.e., protein synthesis).
Due to specific regulons associated with different transcription factors and conserved DNA binding
sites, the transcription factors recognize and bind to specific nucleotide sequence of DNA strands (or
promoter site). In response to specific signals (i.e., stimulons; such as specific metals), the transcription
factors can alter the gene expression by expression or activating the transcription. To rapidly overcome
the adverse situation caused by metal ions, an appropriate response system is improvised in response to
alarm signal of metal through transcription and protein synthesis of gene involved in the microorganisms.
However, all microorganisms essentially have several specific genes for toxic metal ion resistances.
These genes are located in chromosomes, plasmids, or transposons encode specific resistance to a vari-
ety of metal ions and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+,
Pb2+, TeO3(2-), Ti+, Zn2+ etc. The functional mechanism involved in gene mediated resistance systems
are (i) energy-dependent active efflux pumping of toxic ions, (ii) enzymatic detoxification by redox
transformations (example of enzyme, oxidation, reduction, methylation, and demethylation) and/or (iii)
metal-binding proteins
Different types of transporter genes belonging to various superfamily or family are identified ex-
perimentally in different microorganisms (Table 1). Generally, following types of transporter genes are
supposed to be important for synthesizing the proteins in this respect, such as, ATPases/ABC transporters
cassette, CDF (cation diffusion facilitator) and resistance/nodulation (RND).

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Microbial Response against Metal Toxicity

The energy-dependent efflux of toxic ions has been reported as the largest group of resistance sys-
tems in bacteria (Silver & Phung, 2005). Most of the microbes have energy dependent ATPases (P-type
ATPases; ATPases forming a phosphorylated intermediate during their catalytic cycle) as the efflux
resistance systems. The newly-coined family name ABC transporters (for ATP cassette) is a change
from ‘periplasmic protein-requiring’ transport systems (Fath & Kolter, 1993). For example, Cd2+-efflux
pumps of bacteria are either inner membrane P-type ATPases. It is first known from the Ca2+ ATPase of
animal muscles and the Na+ K+ ATPase, but is now being found in bacteria as well. Another instance is
Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is
present in most bacterial genomes and many plasmids. The arsenic resistance efflux system transports
arsenite [As(III)], alternatively using either a double-polypeptide (ArsA and ArsB) ATPase or a single-
polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene in the arsenic resistance
system, ArsC, encodes an enzyme that converts intracellular arsenate [As(V)] to arsenite [As(III)], the
substrate of the efflux system.
In addition, Another new protein family, named CDF for ‘cation diffusion facilitator’ has as proto-
type the protein Czc which is a regulatory component of a Cd2+-Zn2+-Co2 resistance determinant in the
Gram-negative bacterium Alcaligenes eutrophus and are known that efflux Ag+, Cu+, Ni2+, and Zn2+.
The triple-polypeptide Czc (Cd2+, Zn2+ and Co2+) chemiosmotic efflux pump consists of inner membrane
(CzcA), outer membrane (CzcC) and membrane-spanning (CzcB) proteins that together transport cations
from the cytoplasm across the periplasmic space to the outside of the cell. (Silver, 1996).
In contrast, resistance to Zn2+, Ni2+, Co2+ and Cd2+ in Gram-negative bacteria is based on the action
of proton-cation antiporters, members of a newly-recognized protein family that has been implicated in
diverse functions such as metal resistance/nodulation of legumes/cell division (therefore, the family is
called RND). Three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump,
a periplasmic-bridging protein and an outer membrane channel.
In spite of these, resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+
and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the
enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic
forms. More recently recognized arr genes for the periplasmic arsenate reductase that functions in an-
aerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase
that functions as an initial electron donor in aerobic resistance to arsenite.
In response to metal toxicity, the respective gene is upregulated and metal binding protein is synthe-
sized in microbial genetic system. The prevailing mechanism is the synthesis of metal-binding peptides
and formation of protein-metal complexes. The metallothioneins (MTs), phytochelatins (PCs), chaperone,
periplasmic silver binding proteins are recognized for formation of complexes with heavy metals. MTs,
chaperone and periplasmic silver binding proteins are encoded by the SmtA, CopZ and SilE genes, re-
spectively (Silver & Phung, 2005). MTs, first discovered in Synechococcus PCC 7942, are encoded by
the Smt locus, which consists of two divergently transcribed genes smtA and smtB. The smtA encodes
a class II MT, and the product of smtB represses the transcription of smtA. The MTs, low molecular
weight (6-7 kDa), cystine-rich proteins essentially play key roles in metal detoxification process in many
microorganisms such as, Escherichi coli, Cyanobacterium, Syneococcus spp., Pseudomonus putida,
Saccharomyces cerevisiae etc. SmtA is also associated with zinc and lead homeostasis in Synechococ-
cus PCC 7942 (Blindauer, 2011), Bacillus cereus, Streptomyces sp., Salmonella choleraesuis 4A and

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Microbial Response against Metal Toxicity

Proteus penneri GM-10 (Murthy, Geetha, & Sarangi, 2011; Naik, Pandey, & Dubey, 2012). However,
genes encoding PCs syntheses have not yet been identified. The basic structure of PCs peptides is γ-Glu-
Cys)nX, (in which X is Gly, γ-Ala, Ser or Glu and n = 2–11) and varied depending on the organism.
The biosynthesis of MTs and PCs is induced by many metals including Cd, Hg Ag, Cu, Ni, Au, Pb and
Zn; however, Cd is by far the strongest inducer.

Microbial Growth, Activity, and Community Diversity

Microbial activity includes the microbial metabolism and growth processes using the available nutri-
ents, metals and minerals of surrounding environment which is generally the potential indicator of soil
and water qualities, as autotrophic organisms (plants, algae, fungi etc.) directly and indirectly rely on
microorganisms for growth. Therefore, the metals are directly or indirectly involved in all aspects of
microbial metabolism, growth, activity, differentiation and diversity (Gadd, 1992). Such, metal toxicity
can interrupt the normal microbial metabolism, growth and activity.
Fungi and bacteria constitute the main components of the soil microbial biomass. The study has re-
ported that metal toxicity effects differently in bacterial and fungal activities. Fungi are less sensitive to
heavy metals pollution than bacteria. The concentration and species varieties of metals are responsible
for shifting the growth, activity and diversity of microbes especially fungi and bacteria. It is commonly
accepted that toxic metals, their chemical derivatives and organometals can have significant effects on
both the quantitative (numerical population) and qualitative (species composition) microbial diversities.
Toxic effects of metals on soil microorganisms have been extensively studied by phospholipid fatty acid
(PLFA) analysis and indicated that bacteria and fungi were affected differently by metal toxicity.
The metal toxicity has significant impacts on both the quantitative (numerical population) and qualitative
(species) microbial diversities (Koechler, Farasin, Cleiss-Arnold, & Arsène-Ploetze, in press). Different
assays of biomass measurements, plate counting and minimum inhibitory concentration (MIC) have also
indicated that heavy metals affect mirobial diversity in soil differently (Gadd, 2010; Bhakta et al., 2012,
2014). The microbial genetic materials are muted accordingly in response to certain condition of metal
toxicity resulting in microbial death or development of new microbial strains. Studies of soil bacterial
community using metagenomic microbial DNA by polymerage chain reaction and denaturing gradient
gel electrophoresis (PCR-DGGE) based DNA fingerprinting revealed that heavy metal contamination
in agricultural soils close to copper and zinc smelters may provoke changes in the composition of soil
bacterial community and a decrease of the bacterial diversity (Li, Xu, Tang, Wu, Muhammad, & Wang,
2006; Wang et al. 2007; Altimira et al., 2012, Bhakta et al., 2012, 2014) (Figure 3). In some cases, mi-
crobial activity can result in remobilization of metals from non-bioavailability to bioavailability form
(Gadd, 2009; Violante, Huang, & Gadd, 2008). However, changes in the soil bacterial community exposed
to heavy metal may vary depending of soil properties, heavy metal bioavailability and the indigenous
microbial groups in soil (Ranjard et al. 2006; Das, Patnaik, Sahu, Chakraborty, Sudarshan, & Thatoi,
2013). Both bacterial growth rate and community composition are affected approximately at the same
contamination level of metal. At this level, heavy metal-sensitive bacteria are probably responsible for the
decrease in bacterial activity and the competitive advantage of more tolerant ones resulted in a change in
community composition. This phenomenon parallels the coupling between changes in pollution-induced
community tolerance and changes in species composition.

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Microbial Response against Metal Toxicity

Figure 3. Metagenomic–DNA fingerprinting pattern of As–, Cd– and Hg–resistant bacteria in four
sludge/sediment samples (S1 – S4) using PCR–DGGE. The different bands (A – H) of metagenomic–DNA
fingerprint indicate the different bacterial strains identified by 16S rDNA sequencing [(Lysinibacillus
sp. (A), uncultured Lactobacillaceae bacterium (B), an uncultured soil bacterium clone (C), Staphy-
lococcus sciuri (D), Bacillus fastidiosus (E), Bacillus niacin (F), Clostridium sp. (G) and Bacillus sp.
(H)] (unpublished).

ENVIRONMENTAL IMPACT AND FUTURE PROSPECTS

In order to detoxify the metals toxicity and survival, microorganisms response against metal toxicity by
means of acclimatization, adaptation and resistant strategies at genetic, cellular and community levels
using various detoxifying mechanisms (sequestration, inhibition of influx, efflux, accumulation, precipi-
tation, redox transformations and chemical modification, repair, and metabolic by-pass) and developed
resistance properties (by protein/enzyme synthesis) encoded by genes located in chromosome, plasmid
or transposon.
However, it has been well known that the toxic impact of metals contamination invites an immeasur-
able damage in environmental microbial community, since; metal toxicity is responsible for damaging
normal structural profile of microbial community and developing the microbial metal-resistant properties
along with the acquisition of antibiotic-resistant properties as described herein which are severe alarming
threat for environmental and human health.
Apart from detrimental consequences, the novel metal- and antibiotic- resistant microorganisms with
beneficial characteristics could be explored from metagenomic environmental samples using DGGE tech-

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Microbial Response against Metal Toxicity

nology for future biotechnological prospective applications in environmental and human health benefits
as follows: (i) important natural biosphere processes, metal and mineral transformations, mobilization,
bioleaching and biomining (Gadd, 2010), (ii) reclamation and bioremediation of metal contaminated
environment (Bhakta et al., 2012a,b, 2014) and (iii) novel antibiotic production from metal-resistant
microbes, because most of the metal resistant microbes are generally of multiantibiotic tolerant (Tuckfield
& McArthur, 2008). Besides, some biominerals or metallic elements deposited by microbes may have
catalytic and other properties of nanoparticle, crystalline or colloidal forms, and these are potentially
relevant to develop the novel biomaterials for structural, technological, environmental and antimicrobial
purposes (Lloyd et al., 2008; Theng & Yuan, 2008; Petkov et al., 2009; Hennebel, Gusseme, & Vers-
traete, 2009). On account of above comprehensive discussion, it can be apprehended that mechanism of
microbial response against metal toxicity and acquisition of metal-resistant properties of microorganisms
would open a new vista in developing clean and sustainable environment as well as in pharmaceutical
and medicinal perspectives for human health benefits in future.

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KEY TERMS AND DEFINITIONS

Ames Test: A microbiological assay that is used to assess the mutagenic potential of chemical
compounds.
ATPases Metal-Transporter: Microbe transport metals trough the membrane using the energy
dependent transporter ATPases. It is also known as ABC transporter.
Cation Diffusion Facilitator (CDF): It is an integral membrane protein consists of three polypeptides,
inner membrane (CzcA), outer membrane (CzcC) and membrane-spanning (CzcB) that increase tolerance
to divalent metals by facilitating the transportation of divalent metal ions, cadmium, zinc and cobalt.
Denaturing Gradient Gel Electrophoresis (DGGE): It is a kind of electrophoresis method for
molecular fingerprinting that separates polymerase chain reaction (PCR)-generated DNA products on
the basis of sequence differences that results in differential denaturing characteristics of the DNA.
Metallothioneins (MTs): It is a low molecular weight (6-7 kDa), cystine-rich proteins encoded by
the Smt locus and essentially play key roles in metal detoxification process in many microorganisms,
first discovered in Synechococcus PCC 7942.
Metal-Resistance: Microbes exert various self-defense and detoxifying mechanisms in order to
protect the deadly metal toxicity which is known as metal resistance.
Microbial Metal-Sequestration: Microbe sequesters metals by various mechanisms such as, bind-
ing, crystallization, precipitation, metal and mineral nanoparticle deposition, complexation, chemical
transformation, volatilization, etc., to tackle metal toxicity, which is referred as metal sequestration in
this chapter.
Redox Transformation: It refers to redox transformations of some metals species from higher to
lower toxic forms leading to reduce metal toxicity which is used as survival mechanisms of microorgan-
isms to save from metal toxicity.

ENDNOTE
*
Transition metals, other metals, and metalloids are collectively used as the term “metals” in this
chapter.

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Chapter 5
Application of Genomics and
Proteomics in Bioremediation
Amol Uttam Hivrale Niraj R. Rane
Shivaji University, India Shivaji University, India

Pankaj K. Pawar Sanjay P. Govindwar

Shivaji University, India Shivaji University, India

ABSTRACT
Bioremediation mediated by microorganisms is proving to be cost effective, ecofriendly and sustainable
technology. Genome enable experimental and modeling techniques are of a great help in evaluating
physiology and enhancing performance of life forms to be used for bioremediation purpose. Similarly, the
application of proteomics in bioremediation research provides a global view of the protein composition
of microbial cell and offers promising approach to understand the molecular mechanism of removal of
toxic material from the environment. Combination of proteomics and genomics in bioremediation is an
insight into global metabolic and regulatory network that can enhance the understanding of gene func-
tions. Present chapter give a bird’s eye view of genomics and proteomics and their potential utilization
in bioremediation and for the clearer understanding of the cellular responses to environmental stimuli.
An understanding of the growth conditions governing the expression of proteome in a specific environ-
ment is essential for developing rational strategies for successful bioremediation.

1. INTRODUCTION

Bioremediation is a process in which naturally occurring organisms are used for rapid degradation /
removal of hazardous pollutants from environment in order to obtain healthy soil, sediments, substances
and ground water (Kumar et al., 2011). In natural way biodegradation is the recycling of waste or break-
ing down organic matter in to nutrients for the other organisms (Alexander, 1994). Bioremediation is
carried out with the help of life forms, including bacteria, fungi, insects, worms, plants, etc. by taking
nutrients such as C, N and P from the contaminant ultimately transforming xenobiotics in to environ-
ment friendly products (Vidali, 2001). Bioremediation approach becomes important when it comes to

DOI: 10.4018/978-1-4666-9734-8.ch005

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.
 
Application of Genomics and Proteomics in Bioremediation

remediation of water reserves. Industrial effluents especially textile industry waste are responsible for
contamination of water bodies which result in limiting the water availability for drinking and agriculture
purpose (King et al., 1997).

Microbes and Bioremediation

Dynamic behavior, flexibility in nutritional requirements and ability to adopt under extreme stress condi-
tions makes the microbe the most eligible life forms for survival. This virtue of the microbe is proving
to be beneficial to human kind especially when it comes to removal of contaminants / toxic entities
from environment. A large number of microorganisms have been reported for degradation of different
industrial wastes such as dyes (Sartale et al., 2011, Keharia & Madamwar, 2003); hydrocarbons, specially
related to petrochemiscal waste (Chhatre et al., 1996; Kapley et al., 2009; Mishra et al., 2001); tannery
effluent (Shrivastava et al., 2003); chlorinated aromatics (Banta and Kahlon, 2007); distillery spentwash
(Kumar et al., 2007); pesticides (Malhotra et al., 2007) heavy metals (Tripati & Shrivastava, 2007) and
so on. Similarly, a phenomenon such as chemotaxis and its relevance in bioremediation using the pure
culture system in model study has also been reported (Paul et al., 2006). The Energy Research Institute,
New Delhi, demonstrated an application of carrier based hydrocarbon- degrading bacterial consortium
for bioremediation of crude oil contaminated agricultural land in northeastern and western part of India
(Mishra et al., 2001). Utilization of genomic tools in the identification of microbial community has led
to the discovery of unique bacteria that were not accessible by traditional techniques. DNA extraction
from target niches and amplification of the DNA bar-coding region by polymerase chain reaction (PCR)
has proved extremely useful in meaningful characterization of microbial community (Malik et al., 2008).
Real time microbial community analysis is also possible with the help of sequencing based approach
where microbial population dynamics can be studied at different time interval and as function of carbon
sources utilized by microbes.

Cellular Building Blocks: Role in Microbial Analysis

Recent advantages in molecular biology techniques have enable to researcher to overcome the drawback
of the culture based analysis resulting in increased to understand of microbial diversity and functionality
in the environment. However, these mew method rely on characterization of cellular constituents such as
fatty acid, proteins and nucleic acid that can be extracted directly from environmental sample without the
need for culturing and their analysis can be used to elucidate the of the microbial community (Rossello
& Amann, 2001). Phospholipids is an important fraction of the cell biomass, and PLFA (Phospholipids
fatty acids analysis) have been used previously to identify microorganism especially bacteria. Invention
of Gas- Liquid chromatography (GLC) have changed the dimension of chromatography technology and
considered as revolution in the analysis of fatty acid and lipid based microbial fingerprinting is sly de-
pendent on GLC techniques now days. Since every microorganism has unique FAME (fatty acid methyl
ester) profiles which can be used as a tool for microbial source tracking. Fatty acid composition can be
influenced by temperature and nutrition and individual fatty acid cannot be used to represent specific
species (Kirk et al., 2004). Though, protein profile is used to understand functional families in vast
diversity, but certain drawbacks are associated with this (Jansson et al., 2000). The most popular cell
constituent used in microbial analysis is nucleic acid. DNA sequences provide the basis for our current

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Application of Genomics and Proteomics in Bioremediation

classification of microbial species and most tools focus on the use of DNA for analyzing the microbial
secret (Muyzer, 1999).
Molecular biology approaches such as genetic engineering, transcriptomics, proteomics and interac-
tomics are now routinely been used in bioremediation process to study exact mechanism involved. The
efficiency of bioremediation system definitely increased if precise molecular approaches are used and
systematically followed. Even combination of genomics and proteomics data may be utilized to under-
stand / study the metabolism of organism involved in the bioremediation process. This kind of study
will help in developing efficient stains of microbes to enhance the metabolism of xenobiotics (Desai
et al., 2010). Currently wide array of molecular biology techniques are used to identify and analyze
various aspects of microorganism such as gene and protein function, their interactions, metabolic and
regulatory pathways. These techniques are facility quick analysis at molecular level to understand the
cellular metabolism of microorganism (Desai et al., 2010). Biodegradation and bioremediation area
open a opportunity for interesting and unexplored possibilities in the field of proteomics, genomics and
bioinformatics, which provides the details structure and reactivity of the organic compound, sequence,
structure and function of protein (enzymes), comparative genomics and so on. Similarly, bioremediation
can be studies with respect to degradation pathway, prediction of genes in degradation pathways and
degradation of compounds (Ellis et al., 2002).
This chapter is an attempt to assist the researchers working in the area of bioremediation by provid-
ing the linkage of bioremediation with cutting edge sciences like genomics, transcriptomics, proteomics
and bioinformatics (Fleming et al., 1993; Schena et al., 1998; Sikkema et al., 1995; Kuhner et al., 2005;
Ellis et al., 2000).

2. GENOMICS IN BIOREMEDIATION

Genomics is a related to the genetics that applies recombinant DNA, DNA sequencing methods, and
bioinformatics to sequence, assemble, and analyze the function and structure of genomes (the complete
set of DNA within a single cell of an organism). In traditional bioremediation, contaminated samples
from the environment are incubated in the laboratory and rates of contaminant degradation or biotrans-
formation are monitored and lead to estimation of the potential of metabolic activity of the microbes
responsible for bioremediation. A large magnitude of research in the area of bioremediation, initially,
was focused on isolation and characterization of efficient microbes for pure culture and characterization
of biotransformed/ biodegraded metabolites at molecular level using a sophisticated instrumentation in
chromatography and spectroscopy (Fulekar, 2005a). As the research progressed, evolutionary approaches
are proving to be extremely useful for the correct identification and isolation of an efficient strain of
potential bioremediator (may be a novel one) from a microbial community present in the complex
mixture of pollutants (Wilfred, 2005). Improved technologies in genomics, especially those which are
useful for correct identification of microorganisms led to increased interest in the use of isolation and
establishment of pure culture of the candidate organisms for bioremediation of organic and inorganic
environmental pollutants (Nierman & Nelson, 2002). Currently, whole genome sequences of most of the
known microorganisms useful in bioremediation are available in public domain. Sequencing technol-
ogy, especially next generation sequencing (NGS) technology is proving to be vital in the enrichment
of sequence database and gene sequence information thus obtained is important in understanding the

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physiological and genomic features of microorganisms involved in bioremediation (Bihari, 2013). Ex-
pression analysis of genes under various environmental conditions using DNA microarray technology
is also possible because of availability of whole genome sequence (Nierman & Nelson, 2002). Such ge-
nome wide expression analysis provides a global picture of genetic interplay during environmental stress
which can be useful in identifying regulatory circuits in these organisms (Baldi & Hatfield, 2002). This
becomes significant as currently, the mechanism that controls the regulation of catalytic and respiratory
genes that are most important in bioremediation are unknown (Lovely D. 2003). Availability of detailed
information of genetic system of environmentally significant organisms makes the elucidation of many
genes of previously unknown function and understanding bioremediation pathway possible (Fulekar,
2005). This information, further lead to isolation of key genes in the bioremediation pathway for their
future use in the development of genetically modified organisms with enhanced bioremediation ability.

Use of GMOs in Bioremediation

Recent development in the field of rDNA technology allows the incorporation of novel genes coding for
specific proteins which may play a vital role in the degradation or biotransformation of toxic contami-
nants in the environment. Now it is known fact that genetically engineered microbes are more efficient
in removing the organic pollutants and ultimately improves the rate of degradation of these chemicals
and their transformation into less toxic to non-toxic products at least at laboratory level (Sayler & Ripp,
2000). A lot of genetic systems are currently being and will be used as potential bioremediation agents
(Menn et al., 2008; Sayler et al., 1998). A large number of opportunities that can be utilized for enhanc-
ing degradation performances using recombinant DNA technology approaches have been described by
Timms and Pieper (1999). Degradation rate can be increased by manipulation at the rate limiting steps
of metabolic pathways of microbes or entire novel pathway can also be incorporated into previously
non performing bacteria (Mann et al., 2008). One of the pioneering examples of utilization of genetic
modifications in bioremediation is of Pseudomonas fluroescens which was isolated from manufactured
gas plant facility which is heavily contaminated with polyaromatic hydrocarbons (PAHs) and utilized for
genetic modification and designated as P. fluroescens HK44. Wherein, a naphthalene catabolic plasmid
pUTK21 was introduced and in addition to this, transposon based lux gene responsible for production of
bioluminescence was fused with a promoter for naphthalene catabolic genes. Increased catabolic gene
expression along with bioluminescent response was observed after exposure of HK44 strain to naphthalene
or the intermediate metabolite salicylate (King et al., 1990). Genetic engineering approaches appear to
be fascinating, but are also associated with technical and ethical difficulties. The lack of information on
the key genes involved in the degradation / bioremediation pathway of a wide range of pollutants poses
a hurdle in the process of identifying a candidate gene for developing genetically engineered organism.
There are ethical issues like horizontal gene transfer, which currently is the most debated issue world-
wide. Isolation and characterization of an efficient microbe from the natural sources using traditional
techniques, though, appears to be cumbersome, but often preferred by researchers because once screened
and isolated, the microbe is free from all such debates (Cha et al., 1999). Complementation of traditional
isolation techniques with modern molecular tools for correct identification of potential organism still
remains an approach of choice.

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2.1 16S rRNA Approach in Bioremediation

The advances in the field of microbial ecology was the discovery of 16S r RNA sequence with highly
conserved gene and involved similar phylogenic characteristics of microorganism with their communities
(Pace et al. 1986; Amann et al. 1995). The advantages of 16S rRNA sequence analysis in bioremediation
is to analyzed the sequence in contaminated environment and determine phylogenic characteristics of
microorganism with bioremediation processes (Rogers & McClure, 2003; Watanabe & Baker, 2000).
Besides this, using this techniques researcher can observe dominant microorganisms during bioremediation
which are obtained from environmental sample (Lovley, 2001). In general, it is very difficult to isolate
the environmentally useful organisms in culture (Amann et al, 1995) but using the 16S rRNA approach
researcher can able to analyzed the closely related organism which may be helpful for biodegradation.
For example, in aquifers, most microorganisms oxidize the contaminants with reduction of Fe having
closely similar phylogeny with the Geobactor species (Rooney-varga et al. l999; Snoeyenbos-West et
al. 2000; Roling et al. 2001). This Geobacter species effectively removes the uranium from the pol-
luted water (Lovley et al. 1991). The major drawback of 16S rRNA technique is that information of the
phylogeny of organisms that are linked with bioremediation unable to predict important aspect in their
phylogenetic characteristics (Pace, l997, Achenbach and Coates, 2000). The prediction of similarity in
phylogeny is more difficult when absence of no closely related organisms.

2.2 DNA Microarray in Bioremediation

Though, the complete genome sequence of microorganism having potential in bioremediation studies
are not yet accelerated in quick way (Golyshin et al., 2003; Tiedje, 2002; Heidelberg et al.,2002; Ses-
hadri et al., 2005; Rabus et al., 2005). Using the complete genome sequence researchers can analyzed
the expression of all genes in each genome under different environmental condition with the help of
DNA microarray technique (Gao et al., 2004; Muffler et al., 2002; Schut et al., 2003). Such type of
genome expression analysis provides important information for identification of regulatory pathway in
the microorganisms (Lovley, 2003; Rabus et al., 2005; Muffler et al., 2002). Now days, DNA microar-
ray are routinely used to evaluate the structure and gene expression profile of microorganism involved
in bioremediation (Schut et al., 2003; Dennis et al., 2003). For example, using DNA microarray mRNA
expression in Bacillus subtilis was achieved in anaerobic conditions (Ye et al., 2000). DNA microarray
is effectively used to monitor microbial communities which are helpful in bioremediation by knowing
genes and pathways involved in biodegradation (Rhee et al., 2004). Such type of DNA microarray use-
ful to analyze the naphthalene enriched soil microorganism by changing the some parameter (Cho &
Tiedje, 2002). DNA microarrays now routinely used to determine bacterial species identification, gene
analysis of microbial genomes and genome-wide transcriptional profiles (Muffler et al.2002; Greene
& Voordouw, 2003).

2.3 Next-Generation Sequencing in Bioremediation

Next- Generation sequencing (NGS) technology lead to start real revolution in environmental biotechnology
and bioremediation, it extends its novelty with the other edge of science such as genomics, proteomics,

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metagenomics and transcriptomics (Ma & Zhai, 2012). Bioremediation and biodegradation is the now
developed with standard molecular technologies and play important role in degradation process. NGS
technology is capable to produced new monoclonal and digital DNA data in huge amount with negli-
gible prices. Because of this, it may be more convenient tools and techniques for researcher working
in the field of environmental science (Eiler et al., 2012). NGS approach is helpful for the identification
and quantification of microbes which are uncultivable in laboratory condition and very important in
the bioremediation studies. This type of long read sequence contains two hyper variable region which
enables specific taxonomic classification of bacteria, archaea, fungi, protozoa and algae (Sims, 2013).
NGS technology enhanced the functional genomics for cultivated microorganism laboratory and with
the help of daily routing molecular techniques such as fragmentation of DNA, restriction digestion and
sequencing it leads to standardize the organisms and this will be helpful in development of bioremedia-
tion area (Bihari, 2013). This technology may help to investigate the novel catabolic pathways, muta-
tions, peculiar genetic arrangements in chromosomes or in cryptic plasmids. NGS technique is strong,
straightforward and cost effective technology with high accuracy but it required proper instrumental and
infrastructure. Now days this technology is much closer among the scientists, technologists, biologists
expert to build order in bioremediation and biodegradation.

2.4 Stable Isotope Probing (SIP) Technique in Bioremediation

The new technology, who gives information to study about complex nature of microorganism in lab
scale, is the application of Stable Isotope Probing (SIP) technique. Here, known labeled isotopes are
added in the microbial community and important fraction of gDNA was isolated and analog study was
conducted (Radajewski et al., 2000). SIP technology gives entire genome data of microbes which are
involved in biodegradation. In this whole genome amplification can be achieved by shotgun sequencing
and assembly and annotation of catalytic enzymes can be achieved. The SIP technology now routinely
applied to absorbed the number of substances such as naphthalene (Yu and Chu, 2005); phenol (DeRito
et al., 2005); methanol (Lueders et al., 2004); methane (Morris et al., 2002); propionate (Lueders et al.,
2004); methyl bromide, methyl chloride (Miller et al., 2004), pentachlorophenol (Mahmood et al., 2005),
ammonium (Cupples et al., 2007), and 2,4-D (Cupples et al., 2006).

3. PROTEOMICS IN BIOREMEDIATION

Proteomics is the large-scale study of proteins, particularly their structures and functions. The terms
‘proteomics’ was coined by Wasinger and co-workers in 1995 (Wasinger et al., 1995); which involved
post genomic characteristic of organisms that emerge from the growth of large and complex genome
sequencing datasets. Proteomics analysis of microorganism is very important because researcher can
directly observed phenotypes of microorganism that may not be possible in the genome sequence (Fulekar
& Sharma, 2008; Kumavath & Devarapalli, 2013). Proteomics is totally based on method of separation
of protein using modem techniques such as two-dimensional polyacrylamide gel electrophoresis (2-DE)
and mass spectrometry (MS) (Hochstrasser, 1995). The advent of proteomics has allowed an extensive
examination of global changes in the composition or abundance of proteins as well as identification of
key proteins involved in the response of microorganisms in a given physiological state. A number of
reports have described sets of proteins that are up- or down regulated in response to the presence of

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specific pollutants. In bioremediation membrane protein is the high interest for proteomics analysis For
example’ PAHs ubiquitous environmental pollutants are extremely important to remove from the environ-
ment In situ and ex situ bioremediation of pAHs has been partially achieved using natural and genetically
engineered microorganisms. Using a proteomics approach, the physiological changes in an organism
during bioremediation provide further insight into bioremediation-rerated genes and their regulation.
In pAH biodegradation, alterations occurred in bacterium affects cell-surface proteins and receptors
(Sikkema et al. 1995). While here 2-DE is very useful and alternative approach for multidimensional
protein identification technology (MudpIT) (Santos, 2004). The other 81-kDa proteinto catalase –per-
oxidase that expressed in response to pyrene exposure was recovered using 2-DE from mycobacterium
sp. Strain PYR-1. Later, six major proteins were significantly induced and over expressed on 2-DE
when Mycobacterium sp. Starin PYR-1 was exposed to phenantherene, dibenzothiophene and pyrene.
Several pyrene- specific polypeptides were identified by N-terminal and internal peptide sequencing as
putative enzyme. Furthermore, the induction of two ring hydroxylating dioygenses, i.e. Pdo1 and Pdo2.
In response to pyrene was proposed during pyrene catabolism by mycobacterium sp. Stain 6PY1. A
composite profile for 20 PAH induced protein was presented when organism mycobacterium sp. Stain
PY1 was grown in the presence of high molecular weight PAHs.
Mass spectroscopy has modernized techniques for the environmental proteomics here researcher can
analyses the small molecules to peptides and proteins (Aebersold & Mann. 2003). The MS techniques
coupled with standard database and played a crucial role in proteomics for protein identification. Matrix
associated laser desorption/ionization time-offlight MS (MALDI-TOF-MS) is the most commonly used
approach to identifying proteins of interest with the help of 2-D gel electrophoresis by mass peptide
fingerprinting (Aebersold & Mann. 2003.; Aitken and Learmonth.2002., Landry et al. 2000). Another
technique surface-enhanced laser-desorption-ionization MS (SELDI-TOF-MS) is the combination of
direct sample fractions on a chip integrated with MALDI-TOF-MS analysis (Merchant and Weinberger,
2000; Seibert et al., 2005). The different expressed proteins were analyzed using SELDITOF- MS in
blue exposed to PAHs and heavy metals (Knigge et al., 2004). The liquid chromatography MS (LC-MS)
technique has been now routinely used for direct detection and identification of potential contaminants
in water (Joo and Kim. 2005).

3.1 Environmental Proteomics in Bioremediation

In nature, microorganism always faced many expeditious and harsh changes of environmental parameter
such as temperature, humidity, nutrients and predators. The main approach of microbes to overcome
these problems is the modification of their protein expression profile. Transcriptomics and proteomics
are playing important role to investigate the physiology of complex microbial consortium at molecular
level while here, individual genes are not enough to understand the microbial adaptation. Environmen-
tal proteomics involved of new technologies ranging simple protein purification to the quantitative and
comparative proteomics of a new identified proteins. It means that mapping of proteins in ecosystem
to the protein expression at different environmental condition. The advances in proteomics are post
translational modification which involves protein functionality, protein-protein interaction and protein
sequence analysis. Hence, prospective applications of modern proteomics techniques in microbial ecology
are identification of novel functional genes, identification of new enzymatic and metabolic pathways,
to identify the novel proteomes in the biodegrading organisms. Besides this, it is helpful to monitor
dynamic and sustainability of other environmental factor (Maron et al., 2007).

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3.2 Insight of Bioremediation Using Proteomics

The protein expression in the organisms varies with their environmental condition, which change their
physiological state as well as changes may occurs in the adaptive response to different external stimuli.
The proteomics technology allows an extensive examination of composition of proteins, also helpful
for identification of key proteins involved physiological state of microorganism (Vasseur et a1., 1999;
Wilkins et al., 200l). These key proteins regulated the response that occurs in the presence of specific
pollutants (Kim et al., 2002). The proteomics approach is helpful to analyze the physiological changes
in an organism during bioremediation or researcher can observed related genes and their regulation us-
ing proteomics. The advents of proteomics technology led to identify the unknown genes and proteins
during the anaerobic biodegradation of toluene and ethyl-benzene (Kuhner et al., 2005). In anaerobic
biodegradation of toluene, various genes and related proteins are expressed besides this, two toluene
related operon (bss and bbs) was specially induced ion the adapted cells. In expression study of ethyl-
benzene pathway, Ebd protein was formed in ethyl-benzene-adapted cells but not in acetophenon-adapted
cells, while Apc proteins were formed in both conditions (Kuhner et al., 2005). The proteomics approach
exposed new pathway for aerobic and anaerobic biodegradation of toxic wastes which certainly gives an
idea for identification of new proteins. The most of the regulated proteins involved in different category
such as oxidative stress response, general stress response, energy metabolism, transcription regulation and
transport molecule besides this, it involved nucleotide biosynthesis and cell mobility (Santos et al., 2004).
The standard proteomics experiment having basically in four steps such as sample preparation,
protein denaturation, Protein separation by MS analysis and last one is protein identification based on
obtained MS/ MS data. The proteomics analysis can be further increased if data is validated by other
complementary method such as transcriptome analysis, phenotypical analysis. This is the state of art of
proteomics analysis of environmental sample or environmental proteomics.

3.3 Stress Response Study Using Proteomics in Bioremediation

In the prokaryotes, variety of changes may occurs in the cellular constituents due to heat shock, cold shock
and presence of heavy metals which results into change in the existing proteomes of an organism. The
resolution of 2D gel proteomics analysis having important to cellular protein contents in bioremediation.
The proteomics analysis exposes the microorganism to stress inducing conditions or toxic pollutants. This
can help to identify the stress proteins that were expressed in environmental pollutant. The combination
of flow cytometry and proteomics now recently used to show the cell physiology of microorganisms
involved in the bioremediation (Wiacek et al., 2006). For example, P. alcaligenes degrade gentisate at
420C and proteomic analysis shows the over expression of regulatory protein or catabolic enzymes and
it is possible to facilitate the degradation of aromatic hydrocarbon with change in the proteomes with
high temperature during bioremediation. The role of different proteins in the resistance of P. fluorescens
against heavy metals such as lead, copper and cobalt was analyzed by using the proteomics approaches
(Sharma et al., 2006). The proteomics techniques helpful to understand the cellular mechanism of un-
identified mixed cultured of variable environmental conditions. The proteomics techniques such as 2-D,
MALDI-TOF/TOF MS and de novo sequencing are helpful for the identification of ATPases synthesis,
oxido-reductases and several transport proteins associated with several efflux pumps. The 2-DE is very
useful to study the stress response proteomes to solve the post-translational modification of proteins.
This modified protein have very important role in bioremediation and it will become an interesting new

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area of research in the field of bioremediation. The other application of proteomics in bioremediation is
to understand the stress response such as acid and base stress response in useful microorganism.

4. CONCLUSION

The application of modern technologies such as proteomics and genomics to study the bioremediation/
biodegradation is an excellent approach to study at molecular level and become a novel approach in
environmental biotechnology. The proteomics approach for protein analysis in environmental biotech-
nology is still fancy. Proteomics analysis of degradative bacteria gives novel information of proteins/
genes involved in degradation pathways. While, the genomics approach used to understand function of
genes, genes pathways but for this pure culture of environmentally friendly organisms is required. The
other techniques of molecular biology such as genetic engineering, microarray, and transcriptomics are
used to study the details mechanism in the degradation pathways. Application of these technologies
provides large amount of data in environmental biotechnology field and there is need to organized data
in stepwise manner within database. The other molecular technique such as genetic engineering shows
more prominent progress towards the bioremediation. All this modern techniques gives the knowledge
about biodegradation pathways, structure and functions of key proteins and molecular characterization
of microorganism.

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Chapter 6
Genetically Engineered
Microorganisms for
Bioremediation Processes:
GEMs for Bioremediaton

Stephen Rathinaraj Benjamin

Universidade Federal de federal de Goiás, Brazil

Fabio de Lima
Universidade Federal de Mato Grosso do Sul, Brazil

Ashok K. Rathoure
Vardan Environet Guargaon, India

ABSTRACT
In the past few decades, environmental pollution is a major issue which affects biodiversity public health
and eco systems present in worldwide, nowadays, microbial potential are connected to effect the clean-up
of environmental pollutants. Conventional methods are focus on the separation, rather than the destruc-
tion of contaminants, the use of genetically engineered microorganisms for bioremediation would be
an alternative, environmentally friendly, more effectiveness and economical clean-up technique for the
remediation of pollutants in present in contaminated sites. A combined strategies relationship between
genetic engineered microbes and bioremediation can enhance the effectiveness of contaminants sites.
Here, we have elaborated recent work on the investigation and improvement of these microbes using
genetic tools and given an outlook of what may be possible in the near future.

1. INTRODUCTION

The introduction of synthetic compounds into the environment, composed with the massive removal of
natural materials to distinctive environmental compartments. In some cases this accumulation can con-
stitute a severe hazard. During the past several decades as a result of human activities, has resulted in the
various elements of the chemical structures of many of these pollutants are beyond the biodegradation

DOI: 10.4018/978-1-4666-9734-8.ch006

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.
 
Genetically Engineered Microorganisms for Bioremediation Processes

capabilities of microorganisms by existing pathways (Pieper & Reineke, 2000; Dejoinghe et al., 2000;
Bruins et al., 2000). A vast number of pollutants and waste materials containing heavy metals are disposed
into the environment per annum. The contaminants causing ecological problems leading to imbalance
between nature and global concern. Thus, control and treatment strategies to reduce the hazardous ef-
fects of pollutants are needed. Instead, conventional physical, chemical and thermal processes have high
costs; require high energy demand and consumption of many chemical reagents. The search for alterna-
tive methods for traditional methods resulted in evolution of bioremediation techniques. It approaches
is cost effective, economical and alternative to conventional treatments, which generally incinerations,
immobilization of the pollutants. Generally used approach to bioremediation involves multidisciplinary
approach include biostimulation where organisms selected for high degradation abilities are used to in-
oculate the contaminated site, bioaugmentation adding microorganisms that naturally contain catabolic
genes, bioaccumulation this process requires live cells, biosorption where metal sorption to cell surface
by physicochemical mechanisms and phytoremediation use of plants to concentrate and metabolize
toxic compounds in contaminated sites. During bioremediation, bacteria utilized for metal removal from
contaminated sites is also a promising technology. However, the bacterial or plant-based processes may
include the large volumes of production based on pollutant-loaded biomass, which is problematic dis-
posal. Accordingly, in the current scenario, biological methods, i.e. bacterial mediated bioremediation,
have the upper hand in terms of sustainability and easy applicability in-situ. The development of genetic
engineering has given us the substantial knowledge of molecular biology and biochemistry has also led
to the development of efficient techniques to monitor the fate of genetically engineered microorganisms
(GEMs) upon release into the environment. Genetically modification technique has resulted often in
a wide variety of current and promising applications for use in the process of bioremediation. Today,
genetically modified microorganisms have found applications in human health, agriculture, and biore-
mediation and in industries such as food, paper, and textiles. Genetic engineering offers the advantages
over traditional methods of increasing molecular diversity and improving chemical selectivity. GEMs
have the possible to be a persuasive tool for cleaning up certain kinds of environmental contamination.
This present discussion delineates several molecular tools and strategies to engineer microorganisms;
the advantages and limitations of the methods are addressed. The final part of this chapter reviews and
evaluates several applications of GEMs currently employed in commercial ventures.

2. BACTERIAL-RECOMBINANT DNA USED IN GEMS

2.1 Natural Horizontal Transference of DNA in Bacteria

The importance of genetic factors pivotal steps in deliberating biodegradation potentials on microorganisms.
The role of plasmid DNA plays on important in genetic adaptation and bacterial genomes of microbial
populations to distinguishing environmental changes are generally accepted nowadays. It represents a
highly mobile form of DNA which can be transferred via conjugation or transformation. Besides, the
most useful to consider horizontal transfer of recombinant DNA in the overall context of horizontal gene
transfer among bacteria, for many genes (antibiotic resistance, heavy metal resistance, symbiotic and

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degradative), and in many environmental situations (Davison, 1999). It has been suggested that it is an
important key role in contributing to the development of novel biodegradation capacities of microbial
communities when they are exposed to organic pollutants (Rittmann et. al., 1990; Dennis, 2005). It oc-
curs via conjugative transfer of self-transmissible or mobilizable catabolic plasmids and transposons,
integrons, bacteriophages (Dejonghe et al., 2000). The transfers of genes encoding biodegradation func-
tions are associated with conjugative plasmids, transposable elements, and integrative and conjugative
transposons (Springael & Top, 2004).
Transformation has been observed in a number of gram-positive or gram-negative, pathogenic or
environmental bacteria such as Acinetobacter sp. ADP1, Bacillus subtilis, Streptococcus pneumoniae,
Neisseria gonorrhoeae, Pseudomonas stutzeri, Ralstonia solanacearum and H. influenzae take up free
DNA from their under surrounding environment (Smith et al., 1995; Hamoen et al., 2001; Barbe et
al., 2004; Meier & Wackernagel, 2005; Fall et al., 2007). Recently, a novel area of T4SS function has
been favored in N. gonorrhoeae that secretes chromosomal DNA in the surrounding environment in a
noncontact-dependent manner (Hamilton et al., 2005). This T4SS is localized in the large, horizontally
acquired gonococcal genetic island (GGI) present in the chromosome of N. gonorrhoeae; thus by fa-
cilitating chromosomal DNA secretion, this genomic island (GEI) also encodes the mechanism of its
own dissemination. On the biological point of view, the SOS response to antibiotic stress-induced DNA
damage has been shown to induce genetic transformability of bacteria and hence to promote horizontal
dissemination of antibiotic resistance genes (Beaber et al., 2004; Prudhomme et al., 2006).
Conjugation is a mechanism of the development of DNA transfer from donor to recipient through a
composed specialized apparatus which consists of a cell-envelope spanning translocation channel joined
in gram-negative bacteria or to the gram-positive bacteria present in surface-associated adhesins (Chen et
al., 2005). It constitutes part of a large and versatile family of T4SS-dependent transport systems (Chen et
al., 2005; Christie et al., 2005). Generally, T4SSs are encoded by multiple genes organized into a single
operon. Due to genetic determinant organizations, shared homologies and evolutionary relationships,
T4SSs have been classified into several types (Juhas et al., 2008). Type F and P T4SSs, often referred
to as type IVA systems, resemble the archetypal VirB/VirD4 system of Agrobacterium tumefaciens and
are considered to be the paradigm of type IV secretion. The most recently described T4SSs that are
evolutionarily distant from all previously described GI T4SSs, play a key role in the horizontal trans-
fer of a wide eriety of GEIs derived from a broad spectrum of bacteria, including Haemophilus spp,
Pseudomonas spp, Erwinia carotovora, Salmonella enterica serovar Typhi, L. pneumophila and others
(Juhas et al., 2007a, b, 2008).
Transduction is the process of DNA transfer from one bacterium to another via bacterial viruses
(bacteriophages). Many bacteriophages are able to transfer bacterial genes, including genomic islands
(GEIs), as passengers in their genomes. Transduction occurs in a variety of bacteria, including the genera
Desulfovibrio, Escherichia, Pseudomonas, Rhodococcus, Rhodobacter, Salmonella, Staphylococcus, and
Xanthobacter, as well as Methanothermobacter thermautotrophicus, a species of Archaea. One good ex-
ample is the SaPI family of Staphylococcus aureus islands (Maiques et al., 2007). Interestingly, SaPIbov2,
a member of the SaPI family of genomic islands, has been shown to be induced to replicate by different
staphylococcal phages, encapsidated and transferred to a variety of recipient bacteria, including differ-
ent Staphylococcus strains (Maiques et al., 2007). Certain genomic regions of staphylococci resemble

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slightly deteriorated prophages that could be mobilized by other phages. Generalized transduction may
mechanistically resemble certain aspects indicative of an existence of phage- and plasmid-like ances-
tors: the transfer of such islands may include self-replicating plasmid-like states (Ubeda et al., 2007).

3. TRANSGENICS AND GENETIC ENGINEERING

Transgenic and genetic engineering are new techniques in molecular biology particularly environmental
benefit typically containing; plants and either reduce the input of agrochemicals into the environment
or make the biological remediation of contaminated areas more efficient (Macek et al., 2008). Many
genes are involved in metal uptake, translocation and sequestration; the transfer of any of these genes
into candidate plants is a possible strategy for genetic engineering of plants to improve phytoremediation
traits. Depending on the strategy, transgenic plants can be engineered to accumulate high concentrations
of metals in harvestable parts. Transfer or overexpression of genes will lead to enhanced metal uptake,
translocation, sequestration chemical modification, and tolerance. Genetic engineering of plants for
synthesis of metal chelators will improve the capability of plant for metal uptake (Karenlampi et al.,
2000; Pilon-Smits & Pilon, 2002; Clemens et al., 2002; Eapen & D´Souza, 2005).
Molecular genetic techniques have been applies mainly identify a range of gene families that are likely
to be involved in transition metal transport. Considerable progress has been made recently in identifying
plant genes encoding metal ion transporters and their homologous in hyper accumulator plants. Bennett
et al., (2003) conducted a greenhouse experiment using transgenic Indian mustard plants overexpressing
adenosine triphosphate sulfurylase (APS) or J-glutamylcysteine synthetase (J-ECS) or GSH synthetase
(GS). The ECS and GS transgenic plants accumulated 1.5-fold more Cd and 1.5- to 2-fold more Zn
compared to control while APS plants did not. J-ECS transgenics also accumulated 2.4- to 3-fold more
Cu, Cr and Pb compared to wild plants. Transgenic Indian mustard plants overproducing PC accumulated
significantly high level of Zn and Cd in contaminated soil from Leadville, Colorado. Through on the
strategy, transgenic plants can be engineered to collect high concentrations of metals in harvestable parts.
Transfer or overexpression of genes will lead to enhanced metal uptake, translocation, sequestration or
intracellular targeting. Besides, genetic engineering of plants for synthesis of metal chelators will improve
the capability of plant for metal uptake (Karenlampi et al., 2000; Pilon-Smits & Pilon, 2002; Clemens et
al., 2002; Eapen & D´Souza, 2005). Therefore, it is hoped that genetic engineering may offer a powerful
new means by which to improve the capacity of plants to remediate environmental pollutants (Yang et
al., 2005, Mello-Farias & Chaves, 2008). A genetic system has been recently developed (Coppi et al.,
2001) in which Geobacter species predominated, followed by a sulfate-reducing phase during which
Fe(III) and U(VI) reduction ceased and acetate-oxidizing sulfate reducers related to Desulfobacter were
more abundant (Anderson et al., 2003; Miletto et al., 2011). With regard to Geobacter, the extracellular
reduction of uranium by conductive Geobacter pili is one such focus. Findings suggest that Geobacter pili
have an essential function as protective and catalytic cellular mechanisms for bioremediation of ground
water contaminated by uranium (Cologgi et al., 2011). The genetic engineering of the producer strain
influences biosurfactant yields and is critical for industrial applications. The biosurfactant structural
information consist of regulatory genes and secretion mechanisms is valuable information to have and
can direct genetic modifications. Pseudomonas aeruginosa and Bacillus subtilis are often used known
their genetics (Sullivan, 1998).

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4. GENETICALLY MODIFIED MICROBIAL STRAIN FOR REMEDIATION

4.1 Recombinant Bacterial Strain

Conventional approaches to the remediation of sites having a mix of waste by physico-chemical or

thermal methods may present a complex situation and may also lead to the formation of unknown toxic
intermediates (Dua et al., 2002). Thus, decontamination of such sites by GE bacteria may be preferred
over the conventional approaches because of the special attributes of microorganisms, involving designed
metabolic pathways, to bioremediate the numerous mix environmental pollutants without producing toxic
intermediates (Pieper & Reineke, 2000; Furukawa, 2003) (Table 1). GE bacteria have also contributed
to the detoxification of heavy metals and other recalcitrant compounds, the metallothioneins were also
tested in microcosm field study (Valls et al., 2000b). Another approach attempted to enhance the metal
accumulation by combining the specific metal transporter with MTs in the cytoplasm (Wolfram & Bau-
erfeind et al., 2009). Heavy metals like mercury (Hg), arsenic (As), cadmium (Cd), nickel (Ni), cobalt
(Co), and lead (Pb) highly reactive and toxic to the vital organs in living organisms (Singh et al., (2011)
. Escherichia coli strain was found to transport organomercurial compounds into the cytoplasm in the
presence of only merA and merB genes due to the reduction of toxicity by chelation between polyphos-
phate and Hg2+ (Pan-Hou et al., 2002). Owing to their involvement in resistance against mercury, mer
operon usually consists of structural genes encoding functional proteins associated with various functions
such as transport (mer T and mer P), regulation (mer R and mer D) and reduction (mer A and mer B)
(Ruiz & Daniell et al., 2009; Mathema et al., 2011). Recently, two other mer genes, mer E and mer H
(membrane bound) assisting in the membrane transport of mercury has been reported in bacteria (Kiyono
et al., 2009; Schue et al., 2009). Recently, a new mercuric ion transporter genet Mer B present on the
genes coding IncP-1β plasmid with table bacterial strain useful for mercury bioremediation. Cupriavidus
metallidurans strain with MSR33 was genetically and biochemically characterized maintained stabled
with plasmid pTP6 over 70 generations under non-selective conditions (Rojas et al., 2011).
The microorganism Deinococcus radiodurans has been studied to detoxify Cr (VI), U(VI) and
Tc(VII) from soil (Fredrickson et al., 2000). A genetically engineered D. radiodurans strain was created
by cloning the E. coli gene (merA) that provides the ability to utilize carbon and energy from catabolism
of toluene and mercury (radioactive contaminants) (Brim et al., 2000). The microbial family Geobac-
teriaceae has shown potential for radioactive metal reduction (Lloyd et al., 2003). The gene dcuB from
G. sulfurreducens, which encodes a fumarate transporter, was engineered in G. metallireducens to grow
with fumarate as a terminal electron acceptor, revealing the ruling approach of GE with expanded respira-
tory capabilities (Butler et al., 2006). Recently Hasin et al., (2010) reported a well-characterized model
methanotroph Methylococcus capsulatus (Bath), able to bioremediate chromium(VI) pollution over a
wide range of concentrations (1.4 – 1000 mg L− 1 of Cr6+), thus extending the bioremediation potential
via successful genetic manipulation of this major group of microorganisms. Leebana et al (2012) studied
P. putida was used as an indicator strain to study the biodecolorization performance to decolorize Direct
golden yellow dye using a bacterial strain effectively under static conditions (desalination and water
treatment). It has been reported that the metal regulatory genes are involved in encoding the proteins
that help in the conversion of more toxic form of heavy metals to lesser toxic forms (Bondarenko et
al., 2008; Jan et al., 2009; Ng et al., 2009; Hasin et al., 2010). Sometimes, complexity of the problems,
arising due to presence of mixed contaminants, presents a situation where survival of GE microbe is in

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Table 1. Microorganisms capable of degrading organic compounds GEMs

Organisms Genus/Species Contaminant Reference

Bacteria Achromobacter sp Hg Ng et al., (2009)
AO22
Bacillus Idriensis As Shuang Li et al., (2011)
strains and
Sphingomonas
desiccabilis
Citrobacter As, NO3- Baohua Li et al., (2015)
Cupriavidus Hg Luis, A et al., (2011)
metallidurans CH34
Cyanobacteria Cr Codd, G.A et al., (2005)
Enterobacter aerogenes Cr Jigisha, P et al., (2012)
and Cu Marcela, B et al., (2014)
cloacae
Pseudomonas Cd, Pb, Cu, U, Ra, Ni, Ag, Jayashree R, et al.,(2012)
aeruginosa Zn, Th
Pseudomonas K-62 Hg Kiyono and Pan-Hou (2006)
Pseudomonas Cd, Zn, Hg and Pb Bondarenko et al., (2008)
fluorescens OS8;
Pseudomonas strain Hg Kiyono et al., (2009)
K-62
Streptomyces sp Cu, Zn, Cd, Pb, Fe, Ni, Selatnia A, et al., (2004)
Ag, Th,
Ra and U
Zoogloea ramigera Cu Gupta et. al., (2000)
P. putida strain Cr, 1,2,3-Trichloropropane Samin, G et al., (2014)
P. putida 06909 Cd Lee, SW. et al., (2001)
E. coli SE5000 Ni Deng, X. et al., (2005)
Ralstonia eutropha As Mondal P. et al., (2008)
MTCC 2487
Deinococcus Hg Brim et al., (2000)
radiodurans strains
Pseudomonas Cd,Zn,Hg,Pb Bondarenko et al., (2008)
fluorescens OS8;
Pseudomonas strain Hg Kiyono et al., (2009)
K-62
Mesorhizobium huakuii Cd Sriprang et al., (2003)
B3
Caulobacter crescentus Cd II Patel et al., (2010)
JS4022/p723-
6H
Sphingomonas As Shuang et al., (2011)
desiccabilis and
Bacillus idriensis
strains
B. subtilis BR151 Cd Ivask et al., (2011)
(pTOO24)

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danger (Shukla et al., 2010). Thus, a combination of microbiological and ecological knowledge of the
site would be a precondition for application of bioremediation technology by using the GE bacteria.
Besides the competitive situation and adverse environmental conditions existing in the field conditions
hampers the survival of GE bacteria.

4.2 Recombinant Fungal Strain

Fungi are of special interest for this study as they play a potential role in bioremediation, and there is
distinct lack of appreciation of by bacteria. Though, despite clear evidence of metabolic and morpho-
logical versatility, filamentous nature of mycelium, including mycorrhizas this can reach the majority
of applications of pollutant transformations. The past 40 years use of algae to remove pollutants from
water, algal bioremediation, has been well studied Ryther (1972), Kuyucak (1988), RomeroGonzalez
(2001) and Wang (2011). The considerable research has been conducted on the development of algal
biosorbents to remediate pollutants, particularly heavy metals (Hubbe et al., 2011). The application of
fungal bioremediation at these sites can reduce nutrient loads in effluents prior to discharge, thereby
providing an opportunity to increase feed inputs, consequently farm productivity, an easier control of
cellular response, the avoidance of arable land use, and the ability to extract micro- and macronutrients
from wastewaters or industrial flue gasses (Anemaet et al., 2010; McGinn et al., 2011; Pittman, 2011).
They are capable to binding various heavy metals and related eukaryotic photosynthetic organisms, and
some fungi have preferentially developed the production of peptides. However, Microalgae are superior
in remediation processes as a wide range of toxic and other wastes materials; Some algae which are
generally used for the waste water treatment are Chlorella, Scenedesmus, Synechoccystis, Gloeocapsa,
Chroococcus, Anabaena, Lyngbya, Oscillatoria, Spirulina etc. Pollution of soil by heavy metals affects
the functioning of microorganisms and induces alteration in their population structure. Filamentous fungi
Aspergillus flavus strain KRP1 were reported to carried out by 18S rRNA analysis exhibit considerable
potential use in bioremediation of aqueous substrates containing mercury(II) through a biosorption
mechanism (Kurniati, et al., 2014). The use of wild fungi for the bio reclamation of polluted soils had
become the focus of considerable attention due to their high detoxification potential of a great variety
of toxicants, possess a suite of extracellular enzymes that oxidize several and the considerable cost sav-
ings, compared with other technologies (Coccia et al., 2009; Romero et al., 2010; Sannino et al., 2010).

4.3 Recombinant Algal Strain

Algae are one of the proposed platforms for renewable energy production that is not likely to impact
agriculture (Pittman et al., 2011). The algae have several advantages such as rapid growth rate, high
photosynthetic efficiency and high biomass production. Lately, a great deal of interest has been centered
on algae as potential candidates for bioremediation of polluted water bodies (Hassett et al., 1981). The
groups of algae with such potential are Cyanobacteria (blue green algae), Microalgae (generally green)
and Macroalgae. The algae have many features that make them ideal for the selective removal and
reducing the concentration of heavy metals, which include high bio sorption capacity, high tolerance
to heavy metals, ability to grow both autotrophically and heterotrophically, large surface area/ volume
ratios, phototaxy, phytochelatin production and its potential for genetic manipulation. The combination of
microbiological and ecological knowledge, biochemical mechanisms, and field engineering designs are
essential elements for successful in situ bioremediation using genetically modified bacteria and microal-

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gae (Das & Chandran, 2011, Subashchandrabose et al., 2013). Recently the genetic engineering has also
been used to improve the bioremediation of organic pollutants. The transgenic approach for engineering
the plants either with bacterial or animal xenobiotic degrading genes has been successfully tried for in
situ bioremediation and accumulation of heavy metal as well as degradation of various xenobiotics like
explosives and hydrocarbons (French et al., 1999, Suresh and Ravishankar et al., 2004). Genetically
modified organisms have been developed to degrade or modify many different compounds including
carbozole, a petroleum component that inhibits catalysts used in refining (Riddle et al., 2003), pesticides
(Qiao et al.,2003), explosives (Duque et al., 1993), aromatic compounds (Lorenzo et al., 2003; Watanabe
et al., 2003). Microalgae can provide many competitive advantages over bacteria and fungi in degrading
organic pollutants and molecular methods and metabolic and genomic information will help not only in
identification and selection of mixotrophic species of cyanobacteria and microalgae with capabilities to
degrade organic pollutants but also in monitoring the efficiency of remediation efforts under the field
condition (Subashchandrabose et al., 2013). Recently, Sivakumar et al., (2012) reported that microalgae
are capable of producing lipids and hydrocarbons quickly and their photosynthetic abilities make them
a promising candidate for wastewater treatment (bioremediation) and can be used as an alternative en-
ergy source (Biodiesel). Some microorganisms could be used in biomonitoring of organic pollution by
constructing these organisms using bioluminescence genes (Horsburgh et al., 2002). The expression of
the catabolic genes of PCB-degrading microorganisms is a key factor for PCB biodegradation in con-
taminated soils (Seeger et al., 2010). Transgenic plants expressing the bacterial xenobiotic degradation
genes combine the advantages of both the systems, i.e. the greater ability of biodegradation by bacteria
and the high biomass and stability of the plants to have an ideal system for in situ bioremediation of
contaminants (French et al., 1999). Walker et al., (1975) isolated an alga, Prototheca zopfi which was
capable of utilizing crude oil and a mixed hydrocarbon substrate and exhibited extensive degradation of
n-alkanes and isoalkanes as well as aromatic hydrocarbons. Some research has confirmed that certain
fresh algae (e.g. Chlorella vulgaris, Scenedesmus platydiscus, S. quadricauda and S. capricornutum)
are capable of uptaking and degrading PAHs (Atlas et al., 2009).

5. POTENTIAL EFFECTS OF GEMS IN BIO-REMEDIATION

5.1 Bioremediation Activity

Bioremediation has shown a great potential of several bacterial strains. A large number of bacteria which
have the potential of degrading the toxic metals. Heavy metals such as Pb, As, Cd and Hg are abundant
in nature and cause a destructive significance on the surroundings mainly at high concentrations. This
was also the evidence of microbial resistance in heavy metal stress. The quantity of cadmium degrada-
tion by these strains was observed favorably in heavy metal resistance assay. Pseudomonas aeruginosa,
RSA-4 strain was ability to tolerate cadmium up to the concentration of 80 ppm while PB-5 strain only
upto 60 ppm. In various strains such as; Proteus vulgaris (BC1), Pseudomonas aeruginosa (BC2),
Acinetobacter radioresistens (BC3) and Pseudomonas aeruginosa (BC5) grown in pH 7.0, 30°C which
is based on high level of heavy metal and antibiotic resistances from sewage water sample (Sinha and
Mukherjee, 2009). However, Pseudomonas aeruginosa strain KUCd1 was observed more efficient to
remove 40 ppm of cadmium, the active growth of bacterium reduced 83.5% while in 60 ppm only 39%
(Raja et al., 2009). The characterization of cadmium resistant bacteria from mangrove ecosystem was

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analyzed phylogenetically and the active strain was identified as Bacillus safensis and similarly, Cheung
and Gu, (2002) proved that reduction in blue colour pigment production occurs with Vogesella indigofera
occurs on exposure of higher concentrations of chromium nearly 150 µg of chromium/ml. Bio reduction
of Cr (VI) by actinobacteria and their potential for bioremediation processes in soil (Desjardin et al.,
2003; Polti et al., 2009). Polti et al., (2009) showed that a Streptomyces strain, previously isolated from
sugarcane, was able to reduce 90% of Cr (VI) bioavailability in soil samples supplemented with 50 mg
kg 1 after 7 days of incubation without the addition of any substrate or pretreatment. Also, they showed
that the reduction of Cr (VI) by Streptomyces sp. MC1 is a useful mechanism for the bioremediation
of soils and this could improve biotechnological processes. Streptomyces sp. MC1 not only could be
applied to semi-liquid or liquid systems, but also solid systems such as soil. Moreover, Benimeli et al.
(2008) studied the lindane bioremediation ability of Streptomyces sp. M7 in soil samples and the pesticide
effects on maize plants seeded in lindane-contaminated soil previously inoculated with Streptomyces
sp. M7. Their results showed that Streptomyces sp. M7 increased the biomass and concomitantly de-
creased residual lindane. Bacterial strains of Pseudomonas, Burkholderia, Comamonas, Cupriavidus,
Sphingo-monas, Acidovorax, Rhodococcus, Corneybacterium and Bacillus genera have been character-
ized the diverse aerobic bacteria capable of oxidizing PCBs have been reported (Pieper and Seeger,
2008, Furukawa and Fujihara, 2008; Seeger and Pieper, 2009). Burkolderia xenovorans LB400 is able
to degrade a broad range of PCBs (Haddock et al, 1995; Seeger et al., 1995) and is a model bacterium
for PCB degradation. Rhodococcus jostii RHA1 is another potent PCB-degrading soil bacterium (Seto
et al., 1995; Warren et al., 2004; McLeod et al., 2006). Most efficient strains for bioremediation of
effluents contaminated by copper were identified by mass spectrometry as Pseudomonas aeruginosa
and Enterobacter cloacae presenting a high score for species identification. P.aeruginosa showed high
tolerance to concentrations up to 160ppm of copper and E.cloacae up to 320ppm of copper. Consider-
ing, P.aeruginosa and E.cloacae could be very efficient in the reusing process of copper, moreover, the
strains of P.aeruginosa was able to remove 30% of copper from a medium containing 160ppm of copper
compared with 23% of removal, and E.cloacae adsorbed 50% of 320 ppm of copper, compared to 20%
in the same study (Sethuraman & Kumar et al., 2011). Assessment of heavy metal accumulation in the
microbial cells can be done by transmission electron microscopy (TEM). In a study, TEM analysis of P.
putida 62BN demonstrated intracellular and periplasmic accumulation of cadmium (Rani et al; 2009).
Similarly, heavy metal transport through bioaccumulation has been reported in many bacterial genera
like, Citrobacter sp. (Pb,Cd), Thiobacillus ferrooxidans (Ag), Bacillus cereus (cd), Bacillus subtilis
(Cr), Pseudomonas aeruginosa (U) Micrococcus luteus (Sr) Rhizopus arrhizus (Hg), Aspergillus niger
(Th), Saccharomyces cerevisiae (U) respectively (Rani et al; 2009, Ahemad et al; 2011). Above results
Acidithiobacillus ferrooxidans has been shown to have a high tolerance to many metal ions like nickel,
copper, cadmium, zinc etc. (Modak & Natarajan 1995; Novo et al., 2000). Survival of Acidithiobacillus
ferrooxidans in arsenic–rich mine waters and the ability of this organism to oxidize ferrous to ferric of-
fers a promising alternative to the remediation of arsenic from contaminated water. In addition, Vinas
et al., (2005) found that soil moisture is a key factor modulating biodegradation rates. Though, highest
biodegradation rate were observed with 40% and 60% WHC soils. An account of this result shows that
soil moisture content impacts PAH biodegradation, and that both too little and too much soil moisture
can reduce PAH biodegradation rates. Dariusz Dziga et al., (2014), employed transformed cells to hy-
drolyse Microcystins (MCs), performed in immobilized E.coli BL21_MlrA cells and Sphingomonas
sp. ACM 3962 efficiently eliminate from contaminated freshwater. Furthermore, a laccase-producing
white-rot fungus Trametes sp. has been tested for bioremediation. The maximum effluent decolourization

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of 73.3% and chemical oxygen demand (COD) reduction of 61.7% was achieved after 7 days of fungal
treatment to 20% v/v of distillery waste in culture medium. Under these conditions, a 35-fold increase
in laccase production by this fungus was observed (Gonzalez, et al., 2000). Rkodococcus erytkropolis
to lower the interfacial tension between the oil and aqueous phases. All of the bacterial colony types
isolated produced trehalose tetra esters similar to those of R. erythropolis. In a later study (Tuleva et al.,
2008) using the same model system, purified glycolipids were added, and all types added decreased the
adaptation times for the model oil degradation and increased the hydrocarbon degradation efficiency.
The white-rot fungi Bjerkandera fumosa, Phlebia radiata, and Trametes versicolor and the brown-
rot fungus Fomitopsis pinicola produce oxalate crystals in high levels on ZnO, Co3(PO4)2, and CaCO3
(Jarosz-Wilkolazka and Gadd, 2003). In brown-rot fungi, induction of oxalic acid is related to copper
tolerance (Green & Clausen, 2003). Brown-rot fungi can maintain oxalic acid concentrations as high as
600μM/g. Oxalic acid is also produced by brown-rot fungi during leaching of metals from the treated
wood (Humar et al., 2004). One-third of the isolates of soil fungi are able to solubilize at least one toxic
metal compound, ZnO, CO3(PO4)2, and Zn3(PO4)2, and 10% solubilize all three (Sayer et al., 1995). In
Penicillium simplicissimum, adsorption of zinc is accompanied by the production of citric acid (Franz
et al., 1991). The cultural filtrate of Aspergillus niger can render the solubility of 18% Cu, 7% Ni, and
4% Co, and these amounts are enhanced by the addition of HCl (Sukla et al., 1992). Fe(III) can be
solubilized by a low-molecular-weight chelating compound known as the ferrichrome (Crichton, 1991).
In Rhodopseudomonas palustris, an arsM gene, encoding bacterial and archaeal homologues of the
mammalian Cyt19 As(III) S-adenosyl methionine methytransferase, was regulated by arsenicals. An
expression of arsM was introduced into strains for the methylation of arsenic. When arsM was expressed
in Sphingomonas desiccabilis and Bacillus idriensis, it had 10 folds increase of methyled arsenic gas
compared to wild type in aqueous system. In soil system, about 2.2%–4.5% of arsenic was removed by
biovolatilization during 30 days (Shuang et al., 2011).

6. SUICIDAL GENETICALLY ENGINEERED MICROORGANISM

Assuming that the environment safety is a thing, the construction of transposition vectors is required
which do not harbor antibiotic resistance genes as antibiotic vectors are unacceptable for environmental
safety. The most promising strategy is designing suicidal genetically engineered microorganisms with
bacterial containment systems as far as mitigation of the risks combined with environmental release
of recombinant microorganisms is concerned. Genetically engineered microorganisms have potential
risks affecting the biological environment, recent progress in generating suicidal genetically engineered
microorganisms will make it possible to apply GEMs expressing suitable P450 for the bioremediation
of soil contaminated with PCDDs and PCDFs in the near future. Although the concept of containment
has been conceptualized by the design of suicidal genetically engineered microorganisms the resolution
for successful containment involves programming the death of genetically engineered microorganisms
after the reduction of the concerning pollutant. Paul et al., proposed a genetic design model that repre-
sents for the uncertainty of the chance of genetically engineered microorganisms after they have fulfilled
their task. This reviews proposing the killer–anti-killer genes, which are induced by the pollutant. When
the pollutant is depleted the killer genes are expressed, which exterminate the genetically engineered
microorganism. Indeed, the horizontal gene transfer was mitigate by the use of killer genes on plasmid
eliminates the microbial recipient if a transfer occurs. Such a design removes the risks, and associated

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concerns of use, generally associated with introducing genetically engineered microorganisms into the
environment. This is a platform by which the uncontrolled proliferation of genetically engineered mi-
croorganisms could be mitigated and the risks associated with developing these microorganisms for suc-
cessful bioremediation minimized. Unfortunately, many of these containment technologies have evolved
but not been taken into account in the regulatory framework or applied by scientists in the design of
genetically engineered microbes for bioremediation. There can be two fates of the genetically engineered
microorganisms: the preferred one is the completion of the prescribed work by the organism and then
its complete elimination from the environment. The alternative way, which is generally not supported,
is that the organism will not be eliminated but will survive and proliferate. The first option is the best
suited as if the recombinant microorganisms will remain in the environment, there may arise undesirable
effects in the ecosystems (Paul et al., 2005). So, to minimize the undesirable affects in the environment,
the survival ability of the strains should be limited by the construction of special containment systems.

7. PLASMID ADDICTION SYSTEM

A demanding concern with microbial systems is that self-replicating re-engineered cells may produce
undesired consequences if they escape or overwhelm their intended environment. To address this bio-
safety issue, various mechanisms for limiting microbial replication and horizontal gene transfer have
been proposed. These include the use of host–construct dependencies such as toxin–antitoxin pairs,
poison-antidote, post-segregation killing, or programmed cell death conditional plasmid replication or
the requirement for a specific metabolite to be present for cellular function. While refactoring of the
existing genetic code or tailoring of orthogonal systems.
As antibiotic-resistance genes are commonly used as markers during plasmid construction, there is
therefore major concern that their presence in environmentally released GEMs could contribute to the
generation of antibiotic-resistant superbugs (Mulvey & Simor, 2009). Generally, this system contains at
least two genes, specified a stable toxin which is certainly a protein and another gene is for the produc-
tion of a factor (protein or antisense RNA) that provides resistance to the cell against this toxin (Paul et
al., 2005). Functioning of this system is based on the differential decay rate of toxins and the antidotes
underlie molecular mechanisms of toxin activation in plasmid-free cells. The degradation of the antidote
PemI/Kis is due to the Lon protease (Tsuchimoto et al., 1992). The presence of the antidote factor is de-
pends upon the survival ability of the cell. Since, the cell bears its plasmid, the cell death is prevented. In
plasmid free cells, the concentration of the antidote quickly declines, and the toxic action of lethal protein
causes cell death (Pieper and Reineke, 2000). The killing mechanism of toxin is not well explored; in
fact only a few techniques are characterized completely. Liu et al., (2008) determined the mode of action
of the Doc toxin of the Phd (prevent host death) – doc (death on cure) toxin-antitoxin system. Instead,
the Doc induction mechanism showed interaction with 30S ribosomal subunits, stabilized polysomes,
and resulted in a significant increase in mRNA half-life. In this sense, Doc was mimicking the action
of hygromycin B. In another report, killing mechanism of action of Ccd, PemK, and Kid toxins was
revealed, which are encoded by ccd and pem/par D located on plasmids F and R100/R1, respectively.
CcdB toxin inhibits the gyrase by forming a complex with the free Gyr A subunit. Nevertheless, CcdA
antidote prevents the formation of this complex and also releases CcdB by forming inactive complexes,
that is, CcdA-CcdB (Mario et al., 2009). The PemK and Kid toxins act on DnaB helicase and inactivate
replication. The replication is restored when cognate antitoxins are present in the cell (Jensen et al., 1995).

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The killer gene and the regulatory circuit are the two key parts of a bacterial containment system. The
regulatory circuits contain inducible promoters and regulators that control the activity of the promoters
in the presence of suitable effector molecules. The expression of the killer gene is controlled by the
regulatory circuit in response to the environmental signals.
Pseudomonas putida carrying a deletion of the asd gene was provided with an external asd gene under
the positive control of XylS activator, the same activator which also negatively controls gef expressions,
Hence, a strain deprived of 3-methylbenzoate would die due to killing by Gef protein and also due to
diaminopimelic acid deprivation (Ronchel and Ramos, 2001). Alternatively few more approaches have
been developed based upon the protein colicin E3 encoded by col E3 gene which cleaves 16S rRNA
and kills the bacterial cell. The killing effect of imm E3 gene has a killing effect was neutralized by the
antidote effect by an immunity function, In this approach, imm E3 was placed in the chromosome of
E.coli and col E3 gene was clone in the plasmid observed the transfer of the plasmid to other cells in
the death of the recipient (Torres et al., 2004).Two lethal genes,colE3 for colE3 toxin and ecoRIR for
EcoRI, were cloned in different plasmid, and the gene coding for antidotes (immE3 and ecoRM) against
the toxins was placed on the bacterial genome.

8. TECHNIQUES

A number of new recombinant DNA techniques have been developed for genetically engineered micro-
organisms for biodegradation of environmental contaminants and improve the degradation of hazardous.
These techniques include new expression vectors to carry the heterologous genes into the host organ-
ism, new mechanisms to control gene expression, containment mechanisms to control persistence of
genetically-engineered microorganisms, application of site-directed and random mutagenesis to increase
the substrate range or activity of biodegradative enzymes, and methods to track genetically-engineered
microorganisms. The recombinant DNA technology explores PCR, anti-sense RNA technique, site di-
rected mutagenesis, electroporation and particle bombardment techniques. Other fingerprinting technique
(mainly DGGE/TGGE), ARDRA, TRFLP, FISH, RISA and gene reporter technique have been intensively
applied to gain further insight into the mechanism of degradation of pesticides. The biotechnology armed
with recombinant DNA technology is now fine tuning the bioremediation technology by improving
pollutant-degrading microbes through strain improvement and genetic modification of specific regula-
tory and metabolic genes that are crucial in developing effective, safe and economical techniques for
bioremediation. Different strategies have been developed using recombinant DNA technology to produce
genetically improved strains for use in the biosorption process, and many of these strategies equip the
bacterial cell wall with metal ion-binding polypeptides to act as anchors. One of these studies was on
the fusion protein: the β-domain of IgA protease of N. gonorrhoeae with metallothionein (MT) from rats
(Valls et. al., 2000), and lpp-ompA-various sizes of peptides (EC20) (Bae et al., 2000). The following
items are the main biosorption mechanisms, which can also be considered mechanisms of resistance or
tolerance to heavy metals developed by microorganisms. Genetic engineering techniques such as DNA
shuffling, site-directed mutagenesis or error-prone PCR have been applied to improve the catalytic
activity and substrate specificity with the help of enzyme called organophosphate hydrolase (OPH).
Mutants with 25-fold and 725- fold higher hydrolytic activity for two poorly hydrolysable OPs, methyl
parathion, and chlorpyrifos, respectively, were obtained using DNA shuffling (Cho et al., 2002, Cho et

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al., 2004). Genetic fingerprinting techniques provide a specific profile of a given microbial community
and based on the separation of amplicons after PCR amplification of phylogenetic such as 16S rRNA.
Some of these fingerprinting techniques such as terminal restriction fragment length polymorphism
(T-RFLP), length heterogeneity analysis by PCR (LH-PCR), single-strand conformation polymorphism
(SSCP), denaturing gradient gel electrophoresis (DGGE), or ribosomal intergenic spacer analysis (RISA)
applied in bioremediation. In addition, the identification of T-RFLP peaks can be directly obtained by
comparing them to databases (Marsh et al., 2000). However, the fingerprints of bacteria of the genera
Enterobacter and Ochrobactrum were detected by T-RFLP particularly, provides the genes encoding
catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery land
formed soil (Katsivelal et al., 2005). RISA sequence variability may be too great for environmental
applications. Its level of taxonomic resolution is greater than 16S rRNA and hence may lead to very
complex community profiles. An automated version of this technique (ARISA) is also available which
detects the abundance and size of PCR amplicons by measuring the fluorescence emission of labeled
primers (Ranjard et al., 2001). Moreover, RISA which allows distinguish between different strains and
closely related species of Staphylococcus (Spiegelman D et al., 2005; Mendoza M et al., 1998), Bacillus
(Bes et al., 2002., Bourque et al., 1995), Vibrio (Daffonchio et al., 2003, Chun et al., 2002), and other
medically important microorganisms. Real-time quantitative PCR (also referred to as qPCR) has emerged
as a promising tool for rapid, reproducible and accurate estimations of microbial community dynamics
or monitoring their catabolic activity during active bioremediation processes. Recently, (Desai et al.,
2009b) determined abundance of active bacterial populations of an enriched bacterial consortium-AIE2
during the steady-state condition within continuous bioreactors treating Cr(VI) and azo dye mixtures by
calculating 16S rRNA gene copy numbers using qPCR assays. In amplified ribosomal DNA restriction
analysis (ADRDA), PCR amplified 16S rRNA fragments are digested or ut at specific sites with restric-
tion enzymes and the resulting digest separated by gel electrophoresis.

9. FUTURE RESEARCH DIRECTIONS

With the advantages of low cost raw material, big adsorbing capacity, and no secondary pollution etc.
microorganisms are promising for purification of degrading pollution. However, the future application
of GE bacteria for pollution remediation will not be free from the risks associated with their release in
the environment. The future risk regarding use of other engineered bacteria is still unclear. Therefore,
the future perspectives of engineered bacterial strains in the field conditions which is at present limited
to laboratory experiments only. Some limitations on the use of bioremediation of sites contaminated
with certain hazardous compounds are the slow rate of degradation. A slow development in the field of
application of GE bacteria is mainly ascribed to the possible risks, low public acceptability. Perhaps the
selection of indigenous microflora in the recombinant technology will shed the public apprehension also.
Nevertheless, it is not exclusive to biological materials and which may not always meet analytical needs
perfectly. In contrast, innovative engineering technique such as molecular genetics, genetic engineering,
transcriptomics, proteomics and interactomics; can best capitalize on the inherent properties of biological
cells, including overproduction of proteins and the detoxification system, for analytical purposes. Phage-
displayed peptide-library technology offers a suitable platform for screening for heavy-metal-binding
peptides; peptides themselves, radiolabeled peptides, peptides conjugated with chemotherapeutic agents.

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10. CONCLUSION

The use of genetically modified (GM) bacteria represents a research boundary with extensive implica-
tions. The promising benefits of using genetically modified bacteria are significant. But the requirement
for GM bacteria may be uncertain for many cases, considering that domestic species often accomplish
adequately but we do not tap the full potential of wild species due to our limited understanding of key
component in the cleanup strategy mechanisms, including the regulation of enzyme systems that degrade
pollutants. Moreover, uncontrolled growth of the GEMs and their high potential to spread new genetic
information to recipients is a major obstacle in the validation of a recombinant under field conditions.
Therefore, designing of novel bacteria suicidal systems in order to control the growth and prevention
of horizontal transfer of genes and therefore to avoid the appearance of undesirable genetic traits can
provide the further direction in developing novel recombinants. In order to restore environmental balance
the bioremediation technique evidently does indicate several benefits and is one of the most preferred
methods to deal with restoration of environment. Though polluted environments by genetic engineering
opens up new possibilities for bioremediation, it is still in its research and development phase, with many
technical issues needing to be addressed.

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Chapter 7
Novel Bioremediation Methods
in Waste Management:
Novel Bioremediation Methods

Charu Gupta
Amity University, India

Dhan Prakash
Amity University, India

ABSTRACT
Bioremediation technologies are one of the novel methods in the field of waste and environment man-
agement and are presently gaining immense credibility for being eco-compatible. Bioremediation using
microbes has been well accepted as an environment friendly and economical treatment method for dis-
posal of hazardous petroleum hydrocarbon contaminated waste (oily waste). Besides this, earthworms
can be used to extract toxic heavy metals, including cadmium and lead, from solid waste from domestic
refuse collection and waste from vegetable and flower markets. Other novel methods used recently for
treatment of wastes are plasma incineration or plasma assisted gasification and pyrolysis technology.
The technologies applied for conditioning include ultrasonic degradation, chemical degradation, enzyme
addition, electro-coagulation and biological cell destruction. Genetic engineering is another method
for improving bioremediation of heavy metals and organic pollutants. Transgenic plants and associated
bacteria constitute a new generation of genetically modified organisms for bioremediation.

1. INTRODUCTION

It is believed now that wastes are no longer treated as waste and they can be used as a valuable resource.
Biomass can interact and confront with water and soil pollutants in both active (live) as well as passive
(dead) way, thereby offering numerous opportunities of exploring them for environmental clean-up. With
rapid increase in urban populations particularly in the developing world, there is a growing problem of
how to manage organic waste and to find alternatives to landfill disposal particularly for domestic food
waste and that from vegetable markets. According to the research team, it is an unfortunate fact of life
DOI: 10.4018/978-1-4666-9734-8.ch007

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.
 
Novel Bioremediation Methods in Waste Management

that much of this waste is currently dumped on the outskirts of many towns and cities and is causing
serious pollution, disease risk and general ecological harm. It also represents a considerable wasted
resource, whereas the organic matter might be exploited usefully in growing food crops (Glazer & Ni-
kaido, 2007). Besides this, vast number of pollutants and waste materials containing heavy metals are
also disposed into the environment per annum. Approximately 6 x 106 chemical compounds have been
synthesized, with 1,000 new chemicals being synthesized annually. Almost 60,000 to 95,000 chemicals
are in commercial use. According to Third World Network reports, more than one billion pounds (450
million kilograms) of toxins are released globally in air and water. The contaminants causing ecological
problems leading to imbalance in nature is of global concern. Bioremediation is an option that offers the
possibility to destroy or render harmless various contaminants using natural biological activity. As such,
it uses relatively low-cost, low-technology techniques, which generally have a high public acceptance
and can often be carried out on site (Vidali et al., 2001). Compared to other methods, bioremediation is
a more promising and less expensive way for cleaning up contaminated soil and water (Kamaludeen et
al., 2003). Bioremediation uses biological agents, mainly microorganisms, e.g. yeast, fungi or bacteria
to clean up contaminated soil and water (Strong et al., 2008). It results in the elimination, attenuation
and/or transformation of polluting or contaminating substances by the use of biological processes.

1.1 Principles of Bioremediation

Bioremediation is defined as the process whereby organic wastes are biologically degraded under con-
trolled conditions to an innocuous state, or to levels below concentration limits established by regulatory
authorities (Mueller et al., 1996). Most bioremediation systems are run under aerobic conditions, but
running a system under anaerobic conditions may permit microbial organisms to degrade otherwise recal-
citrant molecules. The microorganisms may be indigenous to a contaminated area or they may be isolated
from elsewhere and brought to the contaminated site. Contaminant compounds are transformed by living
organisms through reactions that take place as a part of their metabolic processes. Biodegradation of a
compound is often a result of the actions of multiple organisms. When microorganisms are imported to
a contaminated site to enhance degradation, the process is known as bio-augmentation. Since bioreme-
diation mainly utilizes living organisms (plants) including microbes (bacteria and fungi) that degrade or
detoxify substances hazardous to human health and environment. Microorganisms secrete extracellular
enzymes that attack the pollutants and convert them into harmless products. Therefore bioremediation can
be effective only where environmental conditions permit microbial growth and activity; its application
often involves the manipulation of environmental parameters to allow microbial growth and degradation
to proceed at a faster rate. For degradation it is necessary that bacteria and the contaminants should be
in contact. This is not easily achieved, as neither the microbes nor contaminants are uniformly spread
in the soil. Some bacteria are mobile and exhibit a chemotactic response, sensing the contaminant and
moving towards it. Other microbes such as fungi grow in a filamentous form toward the contaminant.
It is possible to enhance the mobilization of the contaminant utilizing some surfactants such as sodium
dodecyl sulphate (SDS) (Hetherington et al., 2006). However, contaminants like chlorinated organic or
high aromatic hydrocarbons are resistant to microbial attack and they are degraded either slowly or not
at all. The main advantage of using bioremediation techniques is that they are more economical than the
traditional methods of incineration and some pollutants can be treated on site thereby reducing exposure
risks for clean-up personnel and potentially wider exposure as a result of transportation accidents. Since

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bioremediation is based on natural attenuation it is considered more acceptable than other technologies
(Glazer & Nikaido, 2007).
Besides this, earthworms can also be used to extract toxic heavy metals, including cadmium and lead,
from solid waste from domestic refuse collection and waste from vegetable and flower markets. Three
species of earthworm, Eudrilus eugeniae, Eisenia fetida and Perionyx excavates can be used to assist in
the composting of urban waste and to extract heavy metals, cadmium, copper, lead, manganese, zinc,
prior to subsequent processing. The process of vermicomposting in this way allows such waste materials
to be remediated and the compost can be used subsequently in growing human food without the risk of
accumulating heavy metals in crops (Kamarudheen et al., 2014).

2. FACTORS AFFECTING BIOREMEDIATION

The factors affecting bioremediation process include existence of a microbial population capable of
degrading the pollutants; availability of contaminants to the microbial population; environment factors
(soil type, temperature, pH and the presence of oxygen or other electron acceptors and nutrients).

2.1 Microbial Populations for Bioremediation Processes

Microbes are diverse in their habitat, they will adapt and grow at subzero temperatures, as well as ex-
treme heat, desert conditions, in water, with an excess of oxygen, and in anaerobic conditions, with the
presence of hazardous compounds or on any waste stream. Their main requirements are an energy source
and a carbon source. Because of the adaptability of microbes they can be used to degrade or remediate
environmental hazards. Aerobic bacteria recognized for their degradative abilities are Pseudomonas,
Alcaligenes, Sphingomonas, Rhodococcus and Mycobacterium. These microbes degrade pesticides and
hydrocarbons, both alkanes and polyaromatic compounds as they use the contaminant as sole source
of carbon and energy (Vidali, 2001; Prescott, 2002). Anaerobic bacteria are not as frequently used for
degradation as aerobic bacteria but some of them are used for bioremediation of polychlorinated biphe-
nyls (PCBs) in river sediments, dechlorination of the solvent trichloroethylene (TCE) and chloroform.
Ligninolytic fungi such as the white rot fungus Phanaerochaete chrysosporium have the ability to degrade
an extremely diverse range of persistent or toxic environmental pollutants. They utilize the substrates
such as straw, saw dust, or corn cobs.
Methylotrophs grow by utilizing methane for carbon and energy. The initial enzyme in the pathway
for aerobic degradation, methane monooxygenase, has a broad substrate range and is active against a
wide range of compounds, including the chlorinated aliphatic trichloroethylene and 1,2-dichloroethane
(Glazer & Nikaido, 2007).

2.2 Environmental Factors for Bioremediation Processes

The growth and activity of the microorganisms must be stimulated to increase their population for bio-
remediation. It is achieved through ‘bio-stimulation’ that involves the addition of nutrients and oxygen
to help indigenous microorganisms. These nutrients are the basic building blocks of life and allow mi-
crobes to create the necessary enzymes to break down the contaminants. All of them will need nitrogen,

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phosphorous and carbon for their growth (Prescott, 2002). Microbial growth and activity is affected by
pH, temperature and moisture. Although microorganisms have also been isolated in extreme conditions,
most of them grow optimally over a narrow range, so that it is important to achieve optimal conditions.
Hydrocarbons are readily degraded under aerobic conditions, whereas chlorinated compounds are de-
graded only in anaerobic ones. To increase the oxygen amount in the soil it is possible to till or spurge
air. In some cases, hydrogen peroxide or magnesium peroxide can be introduced in the environment.
Soil structure can be improved by adding gypsum and organic matter (Prescott, 2002). Nowadays, there
are four main biological techniques used for treating soil and groundwater for stimulation of activity of
indigenous microorganisms by the addition of nutrients, regulation of redox conditions, optimizing pH
conditions; inoculation of the site by microorganisms with specific biotransforming abilities; application
of immobilized enzymes; and use of plants (phytoremediation) to remove or transform pollutants (Vidali,
2001; Gupta, 2012). In the specific methods used for bioremediating contaminated soil and water, land-
farming, composting, intrinsic bioremediation and slurry bioreactor are included.

3. BIOREMEDIATION STRATEGIES

3.1 In-Situ Bioremediation

Bioventing encourages the in-situ biodegradation of POLs (petroleum-oil-lubricants) by providing oxy-

gen to microorganisms in the soil. The system supplies oxygen by injecting air directly into the residual
contamination. Bioventing uses low airflow rates to provide only enough oxygen to keep up microbial
activity. Optimal flow rates maximize biodegradation as vapors move slowly through biologically ac-
tive soil while minimizing volatilization of contaminants. A basic bioventing system includes a well
and a blower, which pumps air through the well and into the soil (Lee et al., 2006). Biopile treatment
is a full-scale technology in which excavated soils are mixed with soil amendments, placed on a treat-
ment area, and bioremediated using forced aeration. The contaminants are reduced to carbon dioxide
and water. The basic biopile system includes a treatment bed, an aeration system, an irrigation/nutrient
system and a leachate collection system. Moisture, heat, nutrients, oxygen, and pH are controlled to
enhance biodegradation. The irrigation/nutrient system is buried under the soil to pass air and nutrients
either by vacuum or positive pressure. The treatment time is typically 3 to 6 months (Wu et al., 2009).

3.2 Ex-Situ Bioremediation

These techniques mainly involve the excavation or removal of contaminated soil from ground. Land
farming is a simple technique in which contaminated soil is excavated and spread over a prepared bed
and periodically tilled until pollutants are degraded. The goal is to stimulate indigenous bio-degradative
microorganisms and facilitate their aerobic degradation of contaminants. In general, the practice is limited
to the treatment of superficial 10–35cm of soil. Composting is a process by which organic wastes are
degraded by microorganisms, typically at elevated temperatures in the range of 55° to 65°C. The increased
temperatures result from heat produced by microorganisms during the degradation of the organic material
in the waste. In windrow composting, firstly, contaminated soils are excavated and screened to remove
large rocks and debris (Blanca et al., 2007; 2008).The soil is transported to a composting pad with a
temporary structure to provide containment and protection from weather extremes. Amendments (straw,

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alfalfa, manure, agricultural wastes and wood chips) are used for bulking agents and as a supplemental
carbon source. Soil and amendments are layered into long piles, known as windrows. The windrow is
thoroughly mixed by turning with a commercially available windrow turning machine. Moisture, pH,
temperature and explosives concentration are monitored. At the completion of the composting period,
the windrows are disassembled and the compost is taken to the final disposal area. Biopiles are a hy-
brid of land-farming and composting. These are typically used for treatment of surface contamination
with petroleum hydrocarbons. They are a refined version of land farming that tends to control physical
losses of the contaminants by leaching and volatilization. Biopiles provide a favorable environment for
indigenous aerobic and anaerobic microorganisms. Here engineered cells are used for construction of
aerated compost piles (Fahnestock et al., 1998). Bioreactors/ slurry reactors/ aqueous reactors are used
for ex situ treatment of contaminated soil and water. It involves the processing of contaminated solid
material (soil, sediment, sludge) or water through an engineered containment system. Slurry bioreac-
tor creates a three-phase (solid, liquid and gas) mixing condition to increase the bioremediation rate of
soil bound and water-soluble pollutants as a water slurry of the contaminated soil and biomass (usually
indigenous microorganisms) capable of degrading target contaminants. In general, the rate and extent
of biodegradation are greater in a bioreactor system than in situ or in solid-phase systems because the
contained environment is more manageable and hence more controllable and predictable.
The first patent for a biological remediation agent was registered in 1974, being a strain of Pseudo-
monas putida (Prescott et al., 2002) that was able to degrade petroleum. In 1991, about 70 microbial
genera were reported to degrade petroleum compounds and almost an equal number has been added to
the list in the successive two decades. These organisms belong to at least 11 different prokaryotic divi-
sions (Glazer et al., 2007).

4. NOVEL BIOREMEDIATION METHODS

4.1 Plasma Incineration

A novel method developed and used recently for treatment of wastes is plasma incineration or plasma
assisted gasification. It uses plasma that causes the volatilization of solid waste materials under anaero-
bic conditions. The plasma generates a huge amount of energy which otherwise cannot be produced by
nuclear fission/fusion. Under such extreme conditions, waste materials get decomposed and oxidized
partially to harmless innocuous products like carbon monoxide, hydrogen and water. During this pro-
cess the organic matter of waste is converted into synthetic gas called SynGas while inorganic fraction
is converted into an inert vitrified glass and thus no ash is left to be land-filled. The major advantages
of this technology are that it reduces the production of exhaust gas and thus lessens the air pollution;
toxic materials produced are entrapped and encapsulated thereby making easier and safer to handle; and
finally equipment is compact so it takes a minimum space for large disposal wastes (Amin et al., 2012).

4.2 Pyrolysis Technology

Another recently developed method for bioremediation is Pyrolysis technology (developed by Energystics
Technologies, Ltd). This method involves the use of electromagnetic waves (EM). Direct and concentrated
electromagnetic waves are allowed to fall on solid, liquid or gaseous waste. This technology directly

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couples electromagnetic energy with a target material to produce heat. When EM waves fall on the target,
it absorbs energy producing temperature which is above the melting or vaporization temperature of target
waste material. Such a high temperature causes the disassociation of strong molecular bonds in waste
materials. This technology requires only a relatively small energy input because the technology does
not utilize conductive medium. In order to demonstrate the effect of this technology on animal tissues,
pyrolysis of beef carcass was done which resulted in complete dematerialization of the tissue leaving no
residues or smoke. The major advantages of Pyrolysis technology are that it produces instantaneous but
controllable heating and releases no harmful emissions (Shepera, 2004; Jones et al., 2004).

4.3 Conditioning

Conditioning is done to enhance the characteristics of bio-solids for their further processing. There are
many conditioning technologies like ultrasonic degradation, chemical degradation, enzyme addition,
electro-coagulation and biological cell destruction. These processes modify the inorganic and organic
characteristics of waste which is critical to other processes (Hetherington et al., 2006).

4.3.1 Ultrasonic Degradation

This is the first step of the conditioning process. Solid wastes are subjected to acoustic waves to attain
extremely high temperatures and pressures within biosolids. This result in the complete destruction of
microbial cells due to shear stresses produced as a result of explosion of gas bubbles. This process is
adjustable to high frequency and low frequency waves. The major advantages of this technique is that
it causes an increase in rate of cell disruption, decrease in anaerobic digestion time, thereby reducing
sludge quantity and increases the biogas production (Hetherington et al., 2006).

4.3.2 Chemical Cell Destruction

This process ensures disruption of the cell membrane of microorganisms in waste activated sludge process
for the improved efficiency of anaerobic digestion and amount of biogas. The waste material is treated
with caustic soda for about one hour. This causes the weakening of cell membrane of microorganisms
and also decreases the viscosity of solution. For industrial scale treatment, MicrosludgeTM (Biocell)
is used for this purpose. It uses industrial-scale high pressure homogenizer which causes a sudden and
enormous pressure drop responsible for causing lysis of microbial cell in the sludge. This sludge is then
liquefied, mixed with primary sludge followed by anaerobic digestion to produce biosolids and biogas
(Hetherington et al., 2006).

4.3.3 Enzyme Conditioning

Enzyme conditioning is done with the help of microbes (both aerobic and anaerobic) that produces
specialized enzymes (usually an enzyme mixture with specific nutrients like humic acids, amino acids)
which degrade organic materials converting them into carbon dioxide and water. This method increases the
dewaterability of biosolids and also reduces odours during digestion process (Hetherington et al., 2006).

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4.3.4 Electro-Coagulation

This is the recently developed method where electric current is used to increase the rate of anaerobic
digestion of waste material. The electric current so produced dissolves an anode (sacrificial anode) and
releases chemically reactive aluminum into the wastewater stream. The positively charged aluminum
ions attract very fine negatively charged ions and particles towards them resulting in agglomeration.
As a result, the agglomerated particles increase in size and settle down due to their increased weight
(Hetherington et al., 2006; Amin et al., 2012).

4.3.5 Biological Cell Destruction

This process involves the use of a chemical agent that possesses strong oxidation properties. The agent
weakens the cell wall of bacteria. These processed bacteria are then returned back to the activated sludge
process where they decompose into carbon dioxide and water.

4.3.6 Thermophilic Fermentation

This method utilizes the microorganisms to increase the rate of digestion process at high temperatures.
Thus sewage sludge and other waste residuals are converted into a fertilizer grade product. Another
technology called ThermoMaster™ process has been developed in which auto-heated aerobic digestion
is operated at a relatively short residence time (30 hours) to maximize the production of single-cell
protein using an influent waste material as a substrate. The solids thus obtained are dried and pelletized
(Hetherington et al., 2006; Amin et al., 2012).

4.4 Anaerobic Digestion

The first phase consists of volatile fatty acid digester where digestion takes place at around 35°C. In the
next phase, the temperature is further increased in a thermophilic range of 50-56°C where further digestion
takes place under anaerobic conditions. In the final phase, the heating is slowed down till temperature
reaches around 35°C. Recent advancements in technology have led to the use of ozone treatment for
anaerobic digestion. The biosolids are first subjected to anaerobic digestion and then are passed into
a reaction tank where they are exposed to low levels of ozone. Studies have shown that only 0.06g of
ozone per gram of dissolved solids is sufficient to destroy the biological activity of digested biosolids.
The ozone treated waste material is then sent to thickening tank followed and back to digester in which
both ozone treated and non-ozone treated waste materials are mixed. After this, waste material is either
sent for dewatering or for ozone treatment (Vranitsky & Lahnsteiner, 2002).

4.5 Electro Dewatering of Waste Material

Conventional dewatering technologies, such as centrifuges, belt filter presses, and rotary vacuum filters,
are not effective methods for treating sewage sludge with high water content. Alternatively, waste material
is subjected to direct current (DC) which in the initial stage causes particles to migrate to the oppositely
charged electrode (electrophoresis). Due to electro-osmosis, cake formation occurs as ions migrate to

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the oppositely charged electrode. This technology is suitable for use in combination with conventional
filtration (Kim et al., 2014).

4.6 Electroacoustic Dewatering

This is another method of dewatering waste where both electric field and ultrasound waves are used
to increase the efficiency of dewatering. Electric field is used for electrophoresis and electro-osmosis
whereas acoustic waves (sound waves) are used in maintaining electrical continuity throughout the waste
material. The main advantage of using acoustic waves is that they decrease specific energy consumption,
increases the filtration rate and helps to keep cathode clean (Hetherington et al., 2006).

4.7 Mechanical Freeze Thaw

In this process, the sludge is frozen followed by crushing and thawing under natural conditions. During
freezing, the chemical bonds in the sludge are altered thus making removal of water easier.
The treated sludge is then subjected to conventional sludge dewatering equipment (Amin et al., 2012).

4.8 Hydrothermal Oxidation

This technology is also known as ‘Supercritical water oxidation’. In this process, the water is heated and
pressurized above the critical point at 374°C and 3,191 pounds per square inch (psi). Consequently, the
solubility of organic substances and oxygen into water is significantly increased. The main advantage
of this technology is that it decomposes organic matter completely and produces a high quality effluent
(Amin et al., 2012).

4.9 Drying of Waste Material

Drying is done to remove the water from the waste residues collected from both domestic, industrial and
agriculture waste. This reduces weight and volume with high solid percentage. Drying can be through
direct or indirect heating. The different methods of drying are discussed below:

4.9.1 Belt Drying

It involves the use of two or more slow moving belts that are arranged in series. The air is supplied
around the belts. The dewatered sludge is spread in the form of a thin layer on a belt to increase the
surface area (Amin et al., 2012).

4.9.2 Direct Microwave Heating

This process involves the use of high-efficiency multi-mode microwave system which is specifically
designed to remove moisture. It removes water and pathogens from dewatered sludge. This process is
highly automated and can dry waste materials having an initial moisture content of 85% to a final moisture
content of 10%. This technology has an added advantage that it has the capacity of about 100% pathogen
without any change in its nutrient content (Amin et al., 2012).

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4.9.3 Flash Drying

This method can dry waste material to up to 90% or more solids. Sludge containing water is fed into a
centrifuge machine where dewatering takes place and its solid content reach up to 25% concentration.
This material is then discharged into thermal stage as a fine-grained spray. Once the particles of solid
waste enter thermal cyclone chamber, they get dried up instantly. These dried particles are then taken
out of the chamber in a fraction of second, with the help of sweep gas. The sweep gas, drawn through
ventilator fan is reheated in hot gas generator before re-entering into dryer loop (Amin et al., 2012).

4.9.4 Fluidized Bed Dryer

After the wet cake is formed, it is passed through the fluidized bed dryer where it comes into contact
with already dried granules. Indirect heating is then applied by means of tubular heat exchangers that
are immersed in fluidized layer of solids. The major advantages of this technology are that it can dry
the sewage material up to 90% solid content (Amin et al., 2012).

4.9.5 Chemical Drying

This process involves the drying of solids through chemical reaction. The dewatered waste material is
allowed to react with ammonium salts or anhydrous ammonia and concentrated organic acids like sul-
phuric acid and phosphoric acid. The reaction of ammonia with organic acids generates heat and during
this reaction sulphates and phosphates are produced. The heat produced from the reaction is utilized for
drying of waste materials. During this process, hard granular materials are produced that can be mixed
with plant nutrients to raise their nutritive value (Amin et al., 2012).

5. BIOREMEDIATION BY GENETIC ENGINEERING

Biotechnological technique like genetic engineering can be used for improving bioremediation of heavy
metals and organic pollutants. Transgenic plants and associated bacteria can be used to create genetically
modified organisms for bioremediation by over-expressing the genes responsible for their metal-binding
proteins or peptides that in turn enhance their heavy metal accumulation and/or tolerance. Furthermore
transgenic algae and microorganisms mutated with bioluminescence genes can be used in bio-monitoring
of organic and inorganic pollution. There are several approaches for the construction of GEMs for bio-
remediation application. First approach is the identification of organisms suitable for modification with
the relevant genes, e.g. aquatic microbes can be used to develop GEMs for bioremediation of aquatic
sources. The use of such organisms would avoid the supplementation of nutrients to the inoculated
environment, thereby reducing the costs incurred and maintenance required. Scientists have developed
Anabaena sp. and Nostoc ellipsosporum by the insertion of linA (from P. paucimobilus) and fcbABC
(from Arthrobacter globiformis) respectively (Kulshreshtha, 2013). The second approach is the pathway
construction, extension and regulation. GEMs have developed by improving existing catabolic path-
ways or to extend these pathways to degrade some more compounds which are not possible to degrade
by using wild strain. The complete catabolic pathway may be encoded by a single microorganism, or
by a consortium of microorganisms, each performing one or more of the stages of bioremediation of

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xenobiotics. In this way, constructed GEMs possess the degradation capabilities of different microbial
communities due to the alteration of gene sequences which further improve the efficiency and efficacy
of the catabolic pathways (Kulshreshtha, 2013). Third approach is modification of enzyme specificity
and affinity, e.g. E. coli strain is genetically modified to express a hybrid gene cluster for the degrada-
tion of trichloroethylene (TCE). Fourth approach is bioprocess development, monitoring, and control,
and bioaffinity, bioreporter, sensor applications for chemical sensing, toxicity reduction, and end point
analysis. Bioluminescence can be easily detected and do not require expensive devices, exogenous ad-
dition of chemicals or co-factors. Further, GEMs possess chemical sensors that allow the monitoring of
contaminant bioavailability rather than just contaminant presence. Bioluminescence producing GEMs
also help us to understand the spread of microbes in the polluted area and end point of the bioremedia-
tion (Kulshreshtha, 2013).
In a recent study, a Pseudomonas putida strain was genetically engineered for 1,2,3-trichloropropane
bioremediation. 1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in
the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed.
Finally, the dehalogenase gene (dhaA31) was cloned and was introduced into the genome of strain MC4
using a transposon delivery system. Growth of the resulting engineered bacterium, P. putida MC4-5222,
on TCP was observed, and all organic chlorine was released as chloride. The results demonstrated the
successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective,
plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated
hydrocarbon (Samin et al., 2014). Transgenic Chlamydomonas cells express metallothionin, a metal
binding protein. These cells grow at normal rates in the presence of lethal concentrations of cadmium
accumulating five-fold more cadmium compared to wild type cells. Mixotrophy in cyanobacteria and
microalgae can provide many competitive advantages over bacteria and fungi in degrading persistent
organic pollutants (POPs) (Subashchandrabose et al., 2013).

6. BIOREMEDIATION USING EARTHWORMS (VERMICULTURE)

Earthworms could be used to extract toxic heavy metals, including cadmium and lead, from solid waste
from domestic refuse collection and waste from vegetable and flower markets. Three species of earth-
worm, Eudrilus eugeniae, Eisenia fetida and Perionyx excavates are used to assist in the composting of
urban waste and to extract heavy metals, cadmium, copper, lead, manganese, zinc, prior to subsequent
processing. The process of vermicomposting in this way allows such waste materials to be remediated
and the compost can then be used in growing human food without the risk of accumulating heavy metals
in crops. Studies have shown that up to three-quarters of the various heavy metals can be removed by
the worms from solid waste. The E. eugeniae species is the most effective worm at remediating solid
waste and producing rich compost. Research has shown that the heavy metal content of such waste can
be reduced to levels significantly below the permissible safe limits. The mechanism involved is because
of worm’s digestive system that is capable of detaching heavy metal ions from the complex aggregates
between the ions and humic substances in the waste during the process of rotting. Various enzyme-driven
processes leads to assimilation of the metal ions by the worms so that they are locked up in the organism’s
tissues rather than being released back into the compost as worm casts. The separation of dead worms
from compost is a relatively straightforward process allowing the heavy metal to be removed from the
organic waste (Science Daily, 2012). In a recent research, phenol bioremediation potential of Earthworms

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was studied. Earthworms were maintained under proper growth conditions. Different concentrations of
phenol ranging from 10ppm to 50ppm were taken for the assays. All the bioremediation assays were
done in the soil with phenol dispersed in water and remediation rates were observed for 24 hours, 48
hours, 72 hours, 96 hours and 120 hours. The initial concentration of phenol was increased eventually
and the rate of bioremediation was estimated using 4-amino anti-pyrine method. The phenol degradation
activity of the gut microflora of the earthworm along with the soil microflora was also observed on a
formulated media. The amount of phenol bioremediated was found higher in case of earthworm and not
the microflora solely. It was found to be 30 ppm in 48 hours when the initial concentration was 50 ppm
(Kamarudheen et al., 2014). In another study, the earthworms’ capacity to remediate the quality of soils
polluted with heavy metals (Zn, Cu, Mn, Pb) as a result of using the swine residual water in agriculture
(50 m3 ha-1) was distinguished. The study has been conducted in a Chernozem (FAO System), in a culture
of Zea mays L. The collected data consisted of soil analyses and tissue analyses to establish the heavy
metal uptake by earthworm species. Determination of the heavy metal contents of soil has been realized
on two depth intervals: 0-10 cm and respectively 10-20 cm, through the method of flame atomic absorp-
tion spectrometry. Earthworm extraction was done using formaldehyde according to the methodology
in force at six months since the residual water has been applied on soil. Both for earthworm number and
earthworm weight there were significant differences (<0.05) between the values recorded in controls
and treatments, i.e. the earthworm number considerably increased in the variants fertilized with swine
residual water and also the earthworm weight, which confirm the earthworm preference for the organic
matter provided by the residual water. Also there were significant differences (<0.05) between the three
analyzed earthworm species regarding the accumulation process of the investigated microelements and,
among them, Lumbricus rubellus showed in its tissues the lowest concentration of microelements and
Allolobophora rosea the highest concentrations. There were also much lower concentrations of the studied
microelements in the earthworm tissues as compared with the adjacent soil in all analyzed species, and
among them there was recorded the lowest content of heavy metals in Lumbricus castaneus. This aspect
and also the fact that this species has been the best represented as numeric abundance in the experimental
soil confirm the good capacity of this species to tolerate the presence of the heavy metals in soil and
to metabolize them with the soil ingestion within the feeding process. The results of this study support
the idea claiming the earthworms’ bioremediator potential of the soils polluted with heavy metals, as
a possibility to restore their quality by maintaining in appropriate levels the earthworm populations in
the agricultural lands. These findings also come to sustain the specialization of this role on earthworm
species (Iordache & Borza, 2012).

7. DUCKWEEDS FOR PHYTOREMEDIATION

Effective wastewater treatments through conventional methods that rely on heavy aeration are expensive
to install and operate. Duckweeds are capable of recovering or extracting nutrients or pollutants and are an
excellent candidate for bio-remediation of wastewaters. Such plants grow very fast, utilizing wastewater
nutrients and also yield cost effective protein-rich biomass as a by-product. Duckweeds being tiny surface
floating plants are easy to harvest and have an appreciable amount of protein (15%–45% dry mass basis)
and a lower fiber (7%–14% dry mass basis) content. Besides nutrient extraction, duckweeds have been
found to reduce total suspended solids, biochemical oxygen demand (BOD), and chemical oxygen de-
mand in wastewater significantly. Depending on the initial concentrations of nutrients, duckweed-covered

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systems can remove nitrate at daily rates of 120–590 mg NO3- m-2 (73%–97% of initial concentration)
and phosphate at 14–74 mg PO4m-2 (63%–99% of initial concentration). Removal efficiencies within 3
days of 96% and 99% have been reported for BOD and ammonia. Besides several genera of duckweeds
(Spirodela, Lemna, Wolffia), other surface-floating aquatic plants like water hyacinth (Eichhornia) are
well known for their phyto-remediation qualities (Gupta & Prakash, 2014). Duckweed culture is an eco-
friendly approach of phyto-remediation which will not only help in free-of-cost extraction of nutrients
in the form of protein-rich biomass but also bio-remediate the ponds, lakes, and other water bodies and
make them a more suitable water resource for aquaculture. Wastewater-duckweed-carp poly-culture
makes the perfect integrated package for pollution control and re-use of recovered nutrients. Moreover,
in view of increased pressure on land over the years for production of food and fodder (due to ever
increasing population, urbanization, industrialization, etc.), utilization of an alternate resource for the
purpose makes sense. In this direction, duckweed culture holds ample scope not only for high quality
food production through aquaculture but also releasing pressure on the underground water resources
(Gupta & Prakash, 2014).

8. MUSHROOMS AND BIOREMEDIATION

Spent mushroom compost is generated as waste after the harvesting of mushroom crop. Recently, it is
proposed to call it as “post mushroom substrate” because it is not really spent and has many uses. The
composition of spent mushroom substrate varies depending on the type of mushroom to be cultivated
and substrate used. It is proved as an excellent source of humus, although much of its nitrogen content
has been used up by the growing mushrooms. It remains a good source of general nutrients (N, P, K
along with trace elements) which makes it well suited for supporting plant growth (Svoboda et al., 2002).
Mushrooms are found to possess the two basic abilities of biodegradation and bioaccumulation for the
bioremediation of waste. Biodegradation ability of mushrooms is due to the presence of many enzymes
involved in the degradation of pollutants and solid waste residues though the extent and safe level is
unknown. Moreover, mushrooms are known for the bioaccumulation of pollutants or heavy metals from
the habitat in which they are growing. Mushroom possesses a very effective mechanism that enables
them to take up some trace elements from the substrate. Heavy metal accumulation ability of mushroom
has been studied by the number of authors (Kala & Svoboda, 2000; Svoboda et al., 2002; Kulshreshtha
& Sharma, 2014).

9. BIOREMEDIATION OF PLASTIC AND RUBBER BY ENDOPHYTIC FUNGI

The massive and rapid accumulation of plastic and rubber waste is a problem that cannot be ignored.
While there has been an enormous boom in synthetic polymer production, methods of treating these
materials after disposal have not been able to keep up. Since several types of plastic and rubber contain
the same chemical bonds that are found in natural plant polymers, specific attention is given to the ability
of endophytes, organisms that grow symbiotically inside plants, to degrade synthetic plastic and rubber.
The endophytic fungus Pestalotiopsis microspore has previously been identified in the degradation of
the plastic polyester polyurethane. This fungus secretes a small enzyme that is responsible for plastic
degradation (Legaspi & Elaine, 2014).

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10. BIOREMEDIATION THROUGH MICROBES: GROWTH PARAMETERS

Most bioremediation systems work under aerobic conditions, but recalcitrant molecules are degraded by
microbial organisms only under anaerobic conditions. Bioremediation depends on various parameters:
nature of pollutants, soil structure, pH, moisture content and hydrogeology, nutritional state, microbial
diversity of the site, temperature and oxidation-reduction (redox-potential) (Dua et al., 2002). In biore-
mediation processes, microorganisms use the contaminants as nutrient or energy sources (Tang et al.,
2007). Bioremediation activity through microbes is stimulated by supplementing nutrients (nitrogen
and phosphorus), electron acceptors (oxygen), and substrates (methane, phenol, and toluene), or by
introducing microorganisms with desired catalytic capabilities (Ma et al., 2007; Baldwin et al., 2008).
Plant and soil microbes develop a rhizospheric zone which is also used as a tool for accelerating the
rate of degradation or to remove contaminants. In addition to this, aromatic compounds are persistent
environmental pollutants that are widely distributed in the biosphere due to anthropogenic activities.
These compounds are highly toxic to living beings; therefore, many of them have been listed as prior-
ity pollutants by Environmental Protection Agency. Microbial degradation has emerged as an effective
technology to remove these compounds from environment. Many microbial enzymes such as oxygenases,
dehalogenases, reductases, hydroxylases and dehydrogenases are involved in the degradation of these
environmental pollutants. Among them, oxygenases are the key enzymes for aerobic biodegradation of
aromatic compounds because they are involved in the initial reaction of degradation and also catalyze
the ring cleavage of the aromatic compounds which is the essential step for the complete mineralization
of these compounds (Arora et al., 2009). Oxygenases catalyze insertion of one or two oxygen atoms
into the substrates. Two classes of oxygenases have been identified based on number of oxygen atom
used in the oxidation: mono-oxygenases and dioxygenases. Monooxygenases incorporate one atom of
the oxygen into the substrate whereas dioxygenases add both atoms of the oxygen (Arora et al., 2009).

11. PROS AND CONS OF BIOREMEDIATION

Bioremediation is a natural process and is therefore perceived by the public as an acceptable waste treat-
ment process for contaminated material such as soil. The residues for the treatment are usually harmless
products and include carbon dioxide, water and cell biomass. Theoretically, bioremediation is useful for
the complete destruction of a wide variety of contaminants. Many compounds that are legally considered
to be hazardous can be transformed to harmless products. This eliminates the chance of future liability
associated with treatment and disposal of contaminated material. In bioremediation, there is no need
of transferring contaminants from one environmental medium to another and the complete destruction
of target pollutants is possible. Bioremediation can also be carried out on site, without causing a major
disruption of normal activities. This also eliminates the need to transport quantities of waste off site
and the potential threats to human health and the environment that can arise during transportation. Bio-
remediation can prove less expensive than other technologies that are used for clean-up of hazardous
waste (Dua et al., 2002).
The major con of bioremediation is that the process is limited to those compounds that are biodegrad-
able. Not all compounds are susceptible to rapid and complete degradation. There are some concerns
that the products of biodegradation may be more persistent or toxic than the parent compound. Biodeg-
radation process is a natural phenomenon and is highly specific. There are major site factors required

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for complete biodegradation that include the presence of metabolically capable microbial populations,
suitable environmental growth conditions, and appropriate levels of nutrients and contaminants. Biodeg-
radation is often difficult to extrapolate from bench and pilot-scale studies to full-scale field operations.
Research is needed to develop and engineer bioremediation technologies that are appropriate for sites
with complex mixtures of contaminants that are not evenly dispersed in the environment. Contaminants
may be present as solids, liquids, and gases. Bioremediation often takes longer than other treatment op-
tions, such as excavation and removal of soil or incineration. Regulatory uncertainty remains regarding
acceptable performance criteria for bioremediation. There is no accepted definition of “clean”, evaluating
performance of bioremediation is difficult, and there are no acceptable endpoints for bioremediation
treatments (Dua et al., 2002).

12. CONCLUSION

Environmental pollution affects quality of life and environmental ecosystem. Since decades, many at-
tempts are being done to reduce environmental pollution. Among these attempts bioremediation is one
of the good remedial techniques. Bioremediation technique involves the use of microorganisms to reduce
the toxicity of harmful wastes and heavy metals from contaminated wastes water. Some other methods
of bioremediation are by using earthworms (vermiculture), duckweeds and mushrooms, thermophilic
fermentation or by genetically engineered microbes including endophytic fungi for biodegrading plas-
tic wastes. Recently bioremediation is used in combination with other physical and chemical methods
such as plasma incineration, pyrolysis, conditioning, electro-coagulation, electroacoustic dewatering,
mechanical freeze thaw, hydrothermal oxidation, flash drying, microwave heating, fluidized bed dryer,
and chemical drying. Bioremediation can thus be proved as a problem solving approach in the field of
solid waste by detoxifying wastes. Due to its cost effectiveness and lesser environmental impact, it offers
an attractive and more conventional clean-up technology.

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Chapter 8
Effective Management of
Agro-Industrial Residues as
Composting in Mushroom
Industry and Utilization of
Spent Mushroom Substrate
for Bioremediation
Rajender Singh
Directorate of Mushroom Research (ICAR) Solan, India

Mamta Chauhan
Jaypee University of Information and Technology, India
ABSTRACT
Different types of edible mushrooms like Agaricus, bisporus, A. bitoriqus, Pleurotus spp., Volvariella
volvacea, Lentinula edodes, Calocybe indica, Flamullina, Ganoderma lucidum etc. are cultivated in
industrial scale. Majority of edible fungi secretes extracellular Ligninocellulolytic enzymes like Laccase,
lignin peroxidase, manganese peroxidase, cellulase etc. for effective conversion of ligninocellulolytic
substrate to compositing form which led to fruiting of mushrooms. Consequently, an adequate disposal
method is needed for the high quantities of spent mushroom substrate (SMS) generated in this agro-food
industrial activity. On the other side, textile industry among the largest water consuming industries in
the world and approximately, 10,000 different dyes and pigments are used at industrial scale. It is es-
timated that nearly 40% of the total dyes used in the dyeing process may find their way in wastewater.
So, there is an attempt to utilize the ligninolytic enzymes rich SMS of different mushroom for efficiently
biodegradation of textile wastewater & polyaromatic pollutants.

1. INTRODUCTION

Mushroom cultivation is a common practice all over the world and is a major income source in China
and other developing countries which also suffer from serious pollution. Total edible species of mush-
rooms are approximately 2000 and total edible species under cultivation are nearly 20 at commercial
DOI: 10.4018/978-1-4666-9734-8.ch008

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.

Effective Management of Agro-Industrial Residues

level. World mushrooms production annually is greater than 25 million ton (Li, 2012). Total annual
mushroom production in India is 1, 20, 000 tons (DMR, 2014). Annually, world production of major
lignocellulolytic biomass waste is around 1088.258 million tons (FAO). Mushroom cultivation offers a
highly efficient method capable of not only biodegradation and bioremediation of agro-industrial waste
but also biotransformation into proteinaceous food that can sustain food security in the developing coun-
tries (Ingale & Ramteke 2010; Narain et al. 2011; Philippoussis & Diamantopoulou 2011; Kulshreshtha
et al. 2010). Many basidiomycetes fungi are edible mushrooms whose industrial production generates
significant amount of spent mushroom substrate (SMS) with residual high levels of lignin-degrading
extracellular enzymatic activities. Annual spent mushroom substrate required to be disposed of in India
is around 6,00,000 tons (Table 1) . Spent mushroom substrate (SMS), has recently gained importance
because of its unique physical, chemical and biological properties. Spent mushroom substrate (SMS) is
a biomass waste generated from mushroom production. About 5 kg of SMS is generated for every kg of
mushroom produced (Williams et al., 2001). Spent mushroom substrate (SMS) is the substrate left over
after mushroom harvesting. The rapid growth in mushroom production worldwide has resulted in large
quantities of SMS (about 13.6 million tons per year) (Williams et al., 2001; Uzun, 2004). These massive
amounts of waste can cause environmental problems, which has led to increased research to develop
technologies for treating these type of waste substrate. SMS mainly contains lignocellulosic materials,
such as sawdust, wheat straw, paddy straw, wheat bran, chicken manure and cotton seed hulls, which
have been decomposed and permeated by mycelium.
The composting of spent mushroom substrate yields a product that is valuable for agricultural uti-
lization and soil reclamation. Mushroom produces don’t reuse this spent mushroom substrate (SMS),
because disease incidence, nutrient depletion and growth factor depletion. SMS has rich heterogeneous
microbial populations with remains of mushrooms mycelia, actinomycetes and bacteria. It has a high
organic content and low concentration of essential plant nutrient contents.
Activities in mushroom industry is steadily growing, the quantity of mushroom waste (SMS) gener-
ated annually is increasing. In recent years, the mushroom industry has faced challenges in storing and
disposing these waste substrates. The obvious solution is to explore new applications and development
of new technologies for the management and effective utilization of SMS. In last decades, there has been
considerable discussion recently about the potential of using SMS for bioremediation of environmental
pollutants.
The increasing industrialization has specially put pressure on natural resources like water bodies,
agricultural land and the physical area by adding unwanted chemicals in the environment. The manu-
facturing and use of dyes and pigments is a multibillion-dollar industry. The use of these chemicals is
an integral part of almost all manufacturing processes. Dyes are synthetic chemical compounds having
complex aromatic structures, which are extensively used in the textile, cosmetics, plastic, food, paper
printing, colour photography and pharmaceuticals industries. Approximately 10,000 different dyes and
pigments are used at industrial scale and over 0.7 million tonnes of synthetic dyes are produced annually,
worldwide (see Figure 1 and Table 1).
When such wastewater effluent is discharged onto the land adjoining the agriculture crops, it causes the
gradual deterioration of land quality and ultimate reduction in crop yields. Colour is the first recognised
contaminant in textile wastewater and has to be removed before discharging it into receiving water body.
Among various industrial effluents involved in an increase of chemical load (COD) of water systems,
effluents from textile dyeing industries are the major source, resulting discharge of coloured water caus-
ing toxicities to aquatic life. The presence of a variety of dyes in the wastewater emanating from textile

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Effective Management of Agro-Industrial Residues

Table 1. Worldwide annual mushrooms production and spent mushroom substrate (SMS) waste generation

S.No. Country Crop Annual mushrooms Approximate spent mushroom substrate

production (tonnes) production (tonnes)
(Annual mushrooms productions × Five times
SMS)(tonnes)
1. Australia Mushrooms and truffles 49954.00 249,770.00
2. India Mushrooms and truffles 40000.00 200,000.00
3 Belgium Mushrooms and truffles 42000.00 21,0000.00
4 China Mushrooms and truffles 7076842.00 35384210.00
5 Taiwan Mushrooms and truffles 8700.00 43,500.00
6 South Korea Mushrooms and truffles 5700.00 28,500.00
7 Denmark Mushrooms and truffles 10936.00 54680.00
8 France Mushrooms and truffles 104621.00 523105.00
9 Germany Mushrooms and truffles 59884.00 299420.00
10 Hungary Mushrooms and truffles 19000.00 95,000.00
11 Indonesia Mushrooms and truffles 39682.00 198410.00
12 Iran Mushrooms and truffles 87675.00 438375.00
13 Ireland Mushrooms and truffles 63600.00 295,000 (Barry et al. 2012)
14 Israel Mushrooms and truffles 10000.00 50,000.00
15 Italy Mushrooms and truffles 792000.00 39,96000.00
16 Japan Mushrooms and truffles 61500.00
17 Netherlands Mushrooms and truffles 323000.00 800,000 (Oei and Albert 2012).
18 New Zealand Mushrooms and truffles 10116.00 307500.00
19 Poland Mushrooms and truffles 220000.00 11,00000.00
20 Republic of Korea Mushrooms and truffles 25502.00 127510.00
21 South Africa Mushrooms and truffles 14284.00 71420.00
22 Spain Mushrooms and truffles 149700.00 825,000.00 FAO, 2007
23 United Kingdom Mushrooms and truffles 79500.00 397500.00
24 United States of Mushrooms and truffles 406198.00 2030990.00
America
25 others Mushrooms and truffles Nearly 400000.00 2000000.00
Source= FAOSTAT 2013

dyeing industries is a serious concern because of their resistance to environmental conditions like light,
temperature and pH, resulting in low biodegradability. The presence of high level of heavy metals viz.,
copper, zinc, cadmium, chromium in wastewater further aggravates the problem. Available physical
and chemical methods used for wastewater treatment have limitation due to high cost, low efficiency
and in-applicability to a wide variety of dyes. Hence feasible and environmental friendly approach that
is Bioremediationis used. The use of microorganisms to detoxify environmental pollutants is generally
referred as bioremediation. Environmental issues are now of increasing concern and biological meth-
ods are being more directed towards technologies to minimize pollution or to remediate it if it occurs.
Mushroom species like, Pleurotus, Agaricus, Volvariella, Lentinula, Ganoderma spp. along with white
rot fungi and their ligninolytic enzymes appear to have wide industrial applications (Figure 2).

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Effective Management of Agro-Industrial Residues

Figure 1. Location of major textile industries in different parts of India

The biotechnology based processes involving dead or live microbial cells or their components, as
biota agents are more user friendly under such conditions, as these restrict the introduction of new forms
of chemicals in the environment. Mushroom cultivation is an eco-friendly method of converting a wide
range of agro-residues and agro-industrial wastes in to a protein rich food in the form of mushrooms.
After several cycles of mushroom cultivation, the mushroom productivity decreases and the residual
by-product, generated in the form of spent mushroom substrate (SMS) required to be properly disposed
of to maintain hygiene and avoid environmental problems. If not properly disposed of, it leads to envi-
ronmental problems, such as soil and water contamination, due to high load of organic carbon, nitrogen
and salt in SMSs of different mushrooms. The biological component of SMS is also very important,
as it is constituted of mushroom mycelial biomass along with high population of heterotrophic bacte-
ria, actinomycetes and the fungi having potential to produce full range of ligninolytic enzymes (Singh
et al., 2013a, 2013b). The past studies have also supported the role of laccase, lignin peroxidase and
manganese dependent peroxidase along with some more enzymes in decolourisation of colouring dyes.

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Effective Management of Agro-Industrial Residues

Figure 2. Different species of edible mushrooms cultivated in India and worldwide

Highly immobilized microbial cultures in the form of SMS tend to have a higher level of bioremedia-
tive activity and are more resilient to environmental perturbations such as pH and toxic chemicals than
the suspended cultures. Besides this, the immobilized cultures offer other advantages like reusability
of the same biocatalyst, control of reactions, and the non-contamination of products. Immobilized form
of living microorganisms have also been described more effective in biological wastewater treatment
and treatment of polyaromatic hydrocarbons than the suspended form of microorganisms. The overall
objective of this book chapter is to introduce with the effective management of agriculture waste residue
for mushroom composting and waste generated from mushroom can be efficiently used for treatment of
textile wastewater and polyaromatic hydrocarbons or others pollutants which led to bioremediation of
environmental pollutants and generation of green technology.

2. CURRENT STATUS OF MUSHROOM CULTIVATION

Worldwide, the top three mushroom species cultivated are the white button mushroom (Agaricus bispo-
rus), shiitake (Lentinula edodes) and oyster mushroom (Pleurotus spp.) (Suguimoto et al. 2001; Li 2012).
Cultivation of these mushrooms represents a major industry in the countries of Southeast Asia (Mehta et
al. 1990; Ragunathan & Swaminathan 2003). Amongst the different cultivated species, genus Pleurotus

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is an industrially important and occupying a third place in worldwide production of edible mushrooms,
i.e., 16.3% of the total production (Chang, 1996). Pleurotus spp. can grow and produce fruit bodies
in temperate and tropical zones and does not require any pretreatment of substrate. The production of
Pleurotus spp. results in a large amount of protein (as extra-cellular enzymes) on substrates consisting
primarily of wide agro-industrial and forestry waste materials (Poppe, 2000). In South Korea, the area
of mushroom cultivation has increased to 1,102 km2 and 117,600 t of mushroom was produced in 2000
(Ko et al., 2005). It has been reported that approximately five times of SMS is produced in South Korea
annually, of which 58% is from Pleurotus ostreatus cultivation.
In the United States, the annual production of Pleurotus spp. has reached 880 t in 1996 and it has
increased by 120-fold in 2002 (Royse et al., 2004). In 2010/2011, the oyster mushroom production has
exceeded 3,700 tonnes in the United States (United States Department of Agriculture 2012). In Ireland,
mushroom production is an appreciable industry and has been estimated to contribute approximately
£90 million to the economies of both Northern Ireland and the Republic of Ireland. In Northern Ireland,
24,000 t of mushrooms are produced annually, offering 3,000 jobs to the nation. (Russell et al. 2005;
Williams et al. 2001).

3. PRESENT WORLDWIDE SCENARIO OF SPENT MUSHROOM SUBSTRATE

Wheat straw, the above-ground biomass remaining after wheat harvest, was estimated to be around
70.9 million tons only in the U.S (Johnson et al., 2006). Individually, yard trimmings and wheat straw
have exhibited low degradability due to their lignocellulosic recalcitrance (Cui et al., 2011; Zhao et al.,
2014a).Although paddy straw, wheat straw, soybean, wheat bran, chicken manure are the traditional
substrates forAgaricus spp.Pleurotus spp., different biological efficiencies (BE) have been reported by
various authors (Ragunathan et al., 1996; Zhang et al., 2002). Other non-traditional substrates used alone
or in combination with the traditional substrates include cotton stalks (BE, 32.69–41.42%) maize stover
(BE, 25.18–35.39%), coir pith (BE, 26.11–27.33%), sorghum stover (BE, 32.17–36.84%), saw dust (BE,
73.5%), banana leaves (BE, 10.25%), mango leaves (BE, 5.96%), bagasse (BE, 34.29–41.31%), peanut
shell, corn cobs, coffee pulp, Eupatorium adenophorum, Populus deltoids etc. (Ingale & Ramteke 2010;
Moonmoon et al. 2010; Patrabansh & Madan 1997; Ragunathan & Swaminathan 2003). Pleurotus sa-
jor caju is grown commercially on supplemented rubber-wood sawdust. For every 200 g of mushroom
produced, about 800 g of spent mushroom substrate (SMS) is available. An average farm discards about
24 tons of SMS per month. The SMS consists of mycelium, extracellular enzymes produced by fungus
during growth and unutilized lignocellulosic substrate. The disposal of SMS is a major problem to farm-
ers. Of the total spent compost produced in Malaysia, 69% is dumped in landfill sites, 28% is applied
to agricultural land and remaining 3% is incinerated (Vigneswaran et al., 1997). However, most spent
compost is produced in large urban areas and it can be extremely expensive to transport it to farming
areas where it is given free to farmers. A further difficulty is that farmers only require compost at certain
times of the year whereas spent compost is produced all year round. On the other hand, the incineration
of compost is an alternative method of disposal whereby it offers a large reduction in the volume of the
solid waste to be disposed of, along with the elimination of nuisance from biological decomposition. The

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method of incineration requires smaller space, relatively to land application but has the disadvantage of
generation of ash (25 - 40% of the incoming load on a dry basis) which can have high polluting qualities
(Vigneswaran et al., 1997). As both the main methods currently employed for the disposal of compost
has some drawbacks, it becomes necessary to explore new applications of the spent mushroom compost.
SMS is commonly made from renewable agricultural residues such as sawdust, sugarcane bagasse,
oil palm empty fruit bunch, wheat straw-bedded horse manure, hay, poultry manure, ground corncobs,
cottonseed meal, cocoa shells, gypsum and other substances (Jordan et al., 2008). One of the main dif-
ficulties in the management of SMS is the lack of waste management supervision (Magette et al., 1998).
The current largest mushroom producer of mushrooms, China has attained more than 20 mt and ac-
counted for over 80% of the world’s mushroom production (Li, 2012). An average farm discards about
24 t of SMS per month (Singh et al. 2011). In Ireland, approximately 254,000 t of SMS is generated
each year (Barry et al. 2012) and in The Netherlands, more than 800,000 t of SMS is produced per year
(Oei & Albert 2012). In some countries, waste management of SMS is a major problem faced by farm-
ers. Apparently, the obvious solution is to increase the demand for SMS through exploration of new
applications for utilization (Figure 3).

4. PHYSICO-CHEMICAL PROPERTIES OF SPENT MUSHROOM

SUBSTRATE (SMS) OF DIFFERENT MUSHROOMS

The major physico-chemical properties of fresh spent mushroom substrate of different mushrooms were
determined and the pH of spent substrates varied between 7.7 in Agaricus bisporus to 6.8 in Pleurotus
sajor-caju SMS. Moisture content was higher (64%) in SMS of Agaricus bisporus, followed by spent
substrate of Pleurotus sajor-caju (55%). Moisture content (64%), Nitrogen (1.8%), Sodium (105 ppm),
Potassium (275 ppm) and Calcium (1045 ppm) were higher in spent substrate of A. bisporus (Table 2)
compared to SMS of P. sajou-caju with an exception of Carbon (33.2%). The values of Carbon, Nitro-

Figure 3. Schematic diagram for development of mushroom culture, spawn, compost, cultivation and
spent mushroom substrate generation

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gen, Sodium, Potassium and Calcium were 42%, 1.7%, 48 ppm, 134 ppm and 896 ppm, respectively
in spent substrate of Pleurotus sajor-caju. These results indicate that spent substrate of both Agaricus
bisporus and Pleurotus sajor-caju provides good nutrient source for the growth of different bacteria
and fungi (Table 2).

5. MICROFLORA OF SPENT MUSHROOM SUBSTRATE

SMS is the left over substrate after harvesting mushrooms; it is harboring both bacteria and fungi. The
microorganisms from SMS were characterised as Enterobacter sp, Bacillus polymxa, Micrococcus roseus,
Citrobacter fruedi, Bacillus subtilis, Clostridium perfringens, Pseudomonas aeruginosa, Bacillus cereus,
Bacillus licheniformis, Escherichia coli (Table 3)and the fungi fromSMS were characterized as Tricho-
derma hazianum, Trichoderma polysporum, Trichoderma viride, Pencillium janthinellum, Mycogene
perniciosa and Aspergillus bisporus (Viji et al., 2003). All the three SMS namely, Agaricus bisporus,
Pleurotus spp., and Volvariella volvacea have fungal isolates namely Penicillum spp, Rhizopus sppand
Aspergillus niger with potential for dye decolourization (Table 4). Among these,Aspergillus niger showed
greaterdecolourization potential during 16 days incubation. Decolourization may be due biosorption,which
is dependent on functional groups in the dye molecule in fungal biomass. The SMSisolates are able to
decolourize and detoxify highly concentrated effluent (Manikandan et al., 2012). The catalytic stabil-
ity is often improved by immobilization, microorganisms may degrade higher concentration of toxic
components than their free cell counterparts.

6. BIOREMEDIATION OF POLYAROMATIC HYDROCARBONS

BY SPENT MUSHROOM SUBSTRATE

Bioremediation of PAH-contaminated soils is based either on the biostimulation of the indigenous

microbiota (Sayara et al., 2010) or on the addition of exogenous microbes, the latter approach being
referred to as bioaugmentation (Haritash & Kaushik, 2009). Bioaugmentation with lignin-degrading
fungi (LDF) to perform the clean-up of PAH-contaminated soils has received increasing attention in

Table 2. Physico-chemical properties of spent mushroom substrate (SMS) of different mushrooms

Physico-Chemical Properties Spent Mushroom Substrate

Agaricus bisporus Pleurotussajor-caju
Moisture (%) 64.00 55.00
pH 7.70 6.80
Total Nitrogen (%) 1.8 1.70
Total Carbon (%) 33.2 42
Sodium (ppm) 105.00 48.00
Potassium (ppm) 275.00 134.00
Calcium (ppm) 1045.00 896.00

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Table 3. Bacterial population in spent substrate of different mushrooms

Spent Substrate Bacteria Isolates Isolates Identified by Gene 16S rRNA Gene
Sequencing
A. bisporus DMRB-3 Bacillus subtilis
CFU[(16.1×107)/ml]
     A. DMRB-1 Bacillus pumilus
     A. DMRB-6 Sphingobacterium multivorum-I
     A. DMRB-7 Sphingobacterium multivorum-II
P. sajor-caju DMRB-2 Bacillus licheniformis
CFU[(35.74×107)/ml]
DMRB-4 Bacillus subtilis
DMRB-9 Rummelibacillus stabekisii
DMRB-10 Pseudomonas fluorescens-I
DMRB-11 Pseudomonas fluorescens-II
V. Volvacea DMRB-3 Bacillus pumilus
CFU[(29.6×107)/ml]

Table 4. Fungal population in spent substrate of different mushrooms

Spent Substrate Fungal Isolates Isolates Identified by Gene 5.8S rRNA Gene
Sequencing
Agaricus bisporus DMRF-1 Aspergillus fumigatus
CFU[(5x104)/ml]
Pleurotus sajor-caju DMRF-2 Aspergillus fumigatus
CFU[(14.5x104)/ml]
DMRF-7 Schizophyllum commune
DMRF-8 Pezizomycotina sp.
Volvariella volvacea DMRF-6 Aspergillus fumigatus
CFU[(4x104)/ml]
DMRF-4 Paecilomyces variotii
DMRF-5 Pichia guilliermondii

recent years due to its reported efficacy (Covino et al., 2010b; Federici et al., 2012a). LDF are known
to produce extracellular lignin-modifying enzymes (LME) with low substrate specificity which enables
them to degrade a wide range of organic pollutants, including PAH (Covino et al., 2010c). The main
LME enzymes include multi-copper oxidases, such as laccase, and heme-peroxidases (Mn-peroxidase
(MnP), versatile peroxidase and lignin peroxidase).Contaminant degradation by Lignin degrading fungi
(LDF) in soil requires the addition of lignocellulosicmaterials either as amendments, or inoculum car-
riers (Covino et al., 2010a). The use of these additives has been found to have a favorable impact on
the resident microbiota, including specialized populations (Federici et al., 2012a). PAH degradation
via bioaugmentation with LDF has been shown to involve either synergistic or antagonist interactions
between the fungi added and the autochthonous microflora.
The use of spentmushroom substrate of some ligninolytic fungi as a source of viable inocula for soil
clean up applications has been proposed (Li et al., 2012). However spent Agaricus bisporus substrate (SAS),
without previous treatment, has never been used for bioremediation of PAH polluted soil. Spent substrate
of Agaricus bisporus, has been shown to enhance the ability of the fungi to endure the toxic effects of

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both cadmium and lead in a PAH-contaminated substrate (García-Delgado et al., 2013a). Marín-Benito
et al., (2014) reported the ability of pesticide degradation by spent substrate of Agaricus bisporusused
as composted with spent substrate of P. ostreatus(75:25) and its adsorption capacity for fungicides with
low polarity that reduce their mobility in the environment. In addition, spent substrate of A. bisporus
has been reported to be an excellent source of LME, mainly laccase that were able to biodegrade PAH in
aqueous solutions (Mayolo-Deloisa et al., 2011). The annual production of this organic waste in Europe
has been estimated at 3.5 × 106 t (Pardo-Giménez & Pardo-González, 2008) consequently its potential
use in bioaugmentation applications would certainly help to reduce this amount (Table 5).
This needs a variety of manipulations of the waste prior to its application, to gain more insight into
its ability to act as an organic amendment, fungal carrier or a supplier of exogenous complex microbiota
or microflora.The addition of the sterilized spent A. bisporus substrate to polluted site was effective in

Table 5. Bioremediation of pollutants by spent mushroom substrate

S.No. Type of Spent Mushroom Substrate Type of Pollutants References

1. Pleurotus sajor-caju Textile dyes Singh et al., 2010, 2011
2. Pleurotus spp. Phenolic and non-phenolic aromatic Rodriguez et al., 2004
Agaricus bisporus compounds Trejo-Hernandez et al. (2001)
3. Pleurotus pulmonarius Removal of petroleum from industrial soil Chiu et al., 2009
4. Pleurotus pulmonarius Biocide pentachlorophenol Law et al., 2003
5. Mixed spent mushroom substrate Polycyclic aromatic hydrocarbons Garcia Delgado et al., 2013
6. Pleurotus ostreatus Creosote contaminated Eggen, 1999
soil
7. Mixed spent mushroom compost Detoxification Russo et al., 2012
and benzo(a)pyrene
8. Lentinus polychrous Remazol Khammuang et al., 2007
brilliant blue R
9. Pleurotus ostreatus Polycyclic aromatic hydrocarbons Ayotamuno et al., 2010
10. Agaricus bisporus Fungicides Ahlawat et al., 2010
Agaricus bisporus (75%) and Biocide and fungicides Kadian et al. (2008)
Pleurotus sp. (25%) Fungicides Chiu et al. (1998)
Pleurotus pulmonarius Law et al. (2003)
Córdova Juarez et al. (2011)
11. Pleurotus Remazol brilliant blue R and Congo red Lim et al., 2013
ostreatus, P. eryngii, and P.
cornucopiae
12. Agaricus bisporus Fungicides (metalaxyl, benalaxyl, penconazole, Marín-Benito et al., 2012
tebuconazole, pyrimethanil, cyprodinil,
iprovalicarb and azoxystrobin)
13. Pleurotus sajor-caju Methyl violet 2B Singh et al., 2013
14. Pleurotus ostreatus, Pleurotus sajor- Rhodamine B, Chicago sky blue, Methyl violet Singh et al., 2013
caju, Agaricus bisporus 2B
15. Lentinula edodes, Agaricus bisporus, Textile azo dyes Ahlawat et al., 2009
Volvariella volvacea Synthetic textile dyes Annuar et al. (2009)
Pycnoporus sanguineus Dyes Khammuang and Sarnthima
Lentinus polychrous (2007)
16. Lentinula edodes Heavy metals Chen et al. (2005)

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stimulating the resident soil bacteriawhich resulted in higher levels of 3-ring PAH being removed. Bio-
augmentation treatments with spent substrate of A. bisporus, were more effective in removing 5, 6-ring
PAH. The wide availability of this agro-waste combined with its proved efficacy in PAH biodegradation
make its use technically feasible for environmental remediation purposes. Another field of the use of spent
mushroom substrate from commercial production of edible fungi is bioremediation of soil contaminated
with different organopullutants. Spent substrate of A. bisporus was successfully used in remediation
of soil contaminated with chlorophenols, hydrocarbons and volatile organic compounds (Semple et al.,
2001). The ability to degrade chemically different compounds was attributed to the extracellular enzyme
systems (lignin peroxidase, manganese-dependent peroxidase and laccase) and production of reactive
free radicals (Field et al., 1993). Several species of cultivated and medicinal mushrooms (e.g. Pleurotus
ostreatus, Agrocybe aegerita, Kuehneromyces mutabilis, Trametes versicolor, Stropharia rugosoannulata
were also found to be able to degrade different organopollutants (Sack & Fritsche, 1997; Song, 1999).
Many factors influence the effectiveness of fungal soil bioremediation and one such include the con-
centration of respective pollutants in the soil. Eggen (1999) successfully applied spent oyster mushroom
substrate to remediation of soil with initial concentration of poly -aromatic hydrocarbon of 1900 mg/
kg. In this study we used a similar substrate for remediation of soil in which PAH concentration was
three times as high. Polycyclic aromatic hydrocarbons (PAHs) belong to a class of organic compounds
that are persistent and recalcitrant in the environment. Biodegradation of PAHs especially by white-rot
fungus Pycnoporus sanguineus is considered environmentally friendly when compared to the conven-
tional physical–chemical remediation which might lead to secondary contamination and the need for
additional post-treatment (Annuar et al. 2009). Similarly, Vikineswary et al., (2006) have shown that a
higher laccase productivity (7.60 U/g substrate) during solid substrate fermentation of sago hampas was
achieved as compared to oil palm frond parenchyma tissue (7.52 U/g substrate) and sawdust (5.68 U/g).
The capacity of P. sanguineus to remove PAHs like anthracene, phenanthrene and pyrene was attributed
to the enzyme laccase secreted (Munusamy et al., 2008).
Now, the potential of SMS in bioremediation of PAH has attracted more attention. Bioremediation
by employing crude extracts containing PAH-degrading enzymes from SMS are supposed to be more
economical compared to pure enzyme/s. Purification of pure enzyme is often expensive and has limited
application in bioremediation. However, it could not be a one-to-one relationship between PAH degra-
dation rate and a particular enzyme activity, since crude extracts contained more than one enzyme. An
intergraded waste management approach by combining ozone oxidation pre-treatment and SMS-mediated
aerobic biological treatment of benzo(α)pyrene was granted successful (Russo et al. 2012). The matrix of
Pleurotus pulmonarius-degraded paddy straw consist predominantly –OH groups which hypothetically
contributed by the cellulosic cell wall of plant matter and they facilitated the biosorption of PAHs (Lau
et al., 2003). Laccase was found in the SMS of P. pulmonarius and its activity was 1.5-fold higher than
that of manganese peroxidase (MnP). More recently, pilotscale PAH removal capacity of spent substrate
of P. ostreatus in Nigerian oil-based drill cuttings has been investigated and after 56 days, the degradation
of total PAHs was as high as 92.38% (Ayotamuno et al. 2010). Besides that, the highest total degradation
rate of 15 PAHs were achieved by adding crude extracts from spent substrate of Pleurotus eryngii, fol-
lowed by A. bisporus, P. ostreatusand Coprinus comatus (Li et al. 2010). Interestingly, laccase activity
from the crude extracts of spent substrate from P. eryngii was not the highest amongst all. This showed
that the laccase activity was not positively correlated to the PAH degradation rate, implying that natural

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mediators present in different crude extracts might contribute to the degradation rate, rather than lac-
case alone. Oxidation of phenol by crude extracts of spent substrate from A. bisporus was reported and
laccase was identified as the main enzyme responsible for the oxidation (Trejo- Hernandez et al. 2001).

7. COCKTAIL OF LIGNINOLYTIC ENZYMES GENERATED

FROM SPENT MUSHROOM SUBSTARTE

Mushroom enzymes reported in the literature are derived from mycelia of macro-fungi grown in submerged
fermentation. Enzymes can also be extracted from solid substrate fermentation technology which is a
process mimicking nature (Pandey et al. 2000). After mushroom crop harvesting, the spent mushroom
compost will have plenty of extra-cellular enzymes. Of all the enzymes, laccase (EC 1.10.3.2) is the
most reserved and common in spent substrate from Agaricus bisporus (Mayolo- Deloisa et al. 2009),
Pleurotus sajor-caju (Kumaran et al. 1997; Singh et al. 2003), P. ostreatus, Lentinula edodes, Flam-
mulina velutipes and Hericium erinaceum (Ko et al. 2005). However, lignin peroxidase productivity
(per microgram of SMS) was found to be the highest from the spent substrate of P. sajor-caju (Singh et
al. 2003) and it was two-, 22-, 30-, 86-fold higher than that of β-glucosidase, laccase, xylanase and cel-
lulase, respectively. It is believed that the type of enzymes produced by mushroom during cultivation is
directly affected by the ingredient of growing substrates and the species of mushrooms (Ball and Jackson
1995).A total of eight dyes from the triphenylmethane, azo and polymeric/heterocyclic dye group were
decolourized by enzyme cocktail extracted from five month old spent compost of Pleurotus sajor-caju
with lignin peroxidase as the main enzyme (Singh et al., 2010).So far spent compost of Agaricus bisporus
has been employed for the recovery of laccase (Ball & Jackson, 1995; Mayolo-Deloisa et al., 2009). The
decolourisation of Remazol brilliant blue R by laccase from the spent compost of Lentinus polychrous
Lev. has also been reported (Khammuang & Sarnthima, 2007). Spent mushroom compost of Pleurotus
spp. which represent the second largest group of cultivated basidiomycetes, has only been implicated
in the bioremediation of polyaromatic hydrocarbons (Chiu et al., 1998; Eggen, 1999; Lau et al., 2003).
However, the potential of enzymes from the spent compost of Pleurotus spp. for the decolourisation
of dyes has not been explored. However, there has been an earlier report on the extraction of high titers
of lignin peroxidase (LiP) from the spent compost of P. sajor-caju and its potential in thedecolourisation
of selected synthetic dyes (Avneesh et al., 2002). Decolourisation of azo dyes by the cultures of P. sajor
caju has already been reported (Chagas & Durrant, 2001). Since azo dyes make the largest group of
synthetic colourants used in textile industries which were released into the environment, decolourization
of azo dyes by the enzyme cocktail from the spent compost offers economical bioremediation alternative.
Simona Di Gregorio et al., 2010 also demonstrated that the enzyme showed also a significant stabil-
ity with respect to the temperature of incubation, and all the characteristics previously de-scribed make
the laccase from spent substrate of P. ostreatus showed extremely attractive for industrial application,
suggesting the exploitation of the SMS not only as a low cost substrate with oxidative capacity, but also
as a source of robust laccase. In this context, the induction of laccase production could be advantageous
for enzyme-purifying purpose, even though, in the experimental condition here explored, an effect of
induction of the chromo baths on the ligninolytic battery of enzymes eventually associated to the SMS
was absent.

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8. ROLE OF SPENT MUSHROOM SUBSTRATE IN DYES DECOLOURISATION

Mushrooms are ligninolytic fungi and represent an important organism in natural recycling events and
in bioremediation (Gadd 2001). The enzyme systems of mushrooms contain lignin peroxidase, laccase
and manganese-dependent peroxidase which catalyses metabolisation of many ligninlike structures, for
example, PAHs and phenols. However, only in this decade, greater attention has been drawn to reuse
SMS and the enzymes recovered from it as an economical approach in bioremediation of such pollutants
(Eggen & Sasek 2002).
Textile industry is one of the largest water consuming industries in the world. The manufacturing
and use of dyes and pigments is a multibillion-dollar industry. The use of these chemicals is an integral
part of almost all manufacturing processes. Dyes are synthetic chemical compounds having complex
aromatic structures, which are extensively used in the textile, cosmetics, plastic, food, paper printing,
colour photography and pharmaceuticals industries (Forgacs et al., 2004). Approximately 10,000 different
dyes and pigments are used at industrial scale and over 0.7 million tonnes of synthetic dyes are produced
annually, worldwide (Knapp et al., 1997). United States Department of Commerce has predicted a 3.5 fold
increase in textile manufacturing by 2020 (Ganesh et al., 1994; Walsh et al., 1980). In India, an average
textile mill producing 60 x 104 m of fabric per day is likely to discharge approximately 1.5 Megalitres
per day (MLD) of effluents (COINDS, Comprehensive Industry Document Series, 1999-2000). Dur-
ing industrial dyeing process a substantial amount of dye is lost in the waste water. It is estimated that
nearly 40% of the total dyes used in the dyeing process may find their way in wastewater (Vaidya et al.,
1982).Evenin modern days of industrialization, an industrial waste effluent is being often discharged
into water bodies without any adequate pre treatment. When such wastewater effluent is discharged onto
the land adjoining the agriculture crops, it causes the gradual deterioration of land quality and ultimate
reduction in crop yields. This has led to an enhanced concern and awareness, forcing Government of
India to strict legislations for controlling the discharge of untreated effluents into the environment, may
it be land, water or air.
Major classes of synthetic dyes are azo, anthraquinone and triaryl methane, and majority of them
are toxic and even carcinogenic with long turnover times. With the increased use of a wide variety of
dyes, pollution due to dye wastewater is becoming increasingly alarming. Colour is the first recognised
contaminant in textile wastewater and has to be removed before discharging it into receiving water body
(Padamavathy et al., 2003). The presence of high level of heavy metals viz., copper, zinc, cadmium,
chromium in wastewater further aggravates the problem (Pagga & Taeger, 1994). Textile industry is
also one of the water intensive industries, which consumes large quantity of water for various processes
and discharges equally large volume of wastewater containing a variety of pollutants. According to an
estimate, a mill having production capacity of 35,000 m cloth per day consumes 1,000 kl water and
produces effluents of 800 kl day-1. Textile industry in India is one of the oldest industries. Large textile
mills are (Fig. 1) mainly situated in Ahmadabad, Surat, Mumbai, Coimbatore, Kanpur and Delhi.The
exact data on the number of processing houses operating in the cottage and household sector is not avail-
able and it has remained by and large unorganized. The textile units are scattered all across the country
and out of 21,076 units, Tamilnadu tops with 5,285, whereas Maharashtra has the next highest number
of units (ISPCH, 1995).
Physical processes if followed by chemical or biological process have shown good results. However,
wide variation in BOD and other parameters necessitates the requirement of equalization of various
physio-chemical processes viz., chemical coagulation / flocculation, adsorption, ion exchange, etc.

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Chemical coagulation employing various chemical coagulants followed by setting, have proven to be
highly efficient in removing solids, BOD and COD from textile effluents and attained its reliability as
physical and chemical unit (Mohan et al., 1997; Robinson et al., 2001 a). Removal of colour from the
effluents is difficult by using conventional physico-chemical treatment system. However, biological
treatment may be the best alternative, as the operational costs involved in it are relatively low compared
to conventional technologies (Arutchelvan et al., 2003).
Mushroom cultivation is an eco-friendly method of converting a wide range of agro-residues and
agro-industrial wastes in to a protein rich food in the form of mushrooms. After several cycles of mush-
room cultivation, the mushroom productivity decreases and the residual by-product considered as spent
mushroom substrate which required to be properly disposed off to maintain hygiene and avoid environ-
mental problems. If not properly disposed off, it leads to environmental problems, such as soil and water
contamination, due to high load of organic carbon, nitrogen and salt in SMSs of different mushrooms.
Syntheticdyes find application in different industrial divisions including textile industry. An annual
consumption of about 0.7 million tons of synthetic dyes has been reported (Deveci et al., 2004).Textile
industry alone ac-counts for two-thirds of the total dyestuff market. Accordingly, discoloration of textile
wastewaters is one of the major environmental concerns since last decades. However, it is worth to mention
that P. ostreatus and many other basidiomycetes are edible mushrooms and their industrial cultivation
produces a significant amount of spent mushroom substrate (SMS), reported as harboring high levels
of residual oxidative enzymatic activity (Ball et al, 1995). Thus, SMS would be a low cost source of
ligninolytic enzyme and their use for bioremediation for environmental pollutants (Trejo Hernandez et
al., 2001; Karla et al., 2009). Gregorio et al., 2010 also studied that the low cost organic substrate, the
SMS deriving from the cultivation of the basidiomycetes Pleurotus ostreatus, is able to discolor anthra-
quinonic, diazo and monoazo-dyes when incubated in dying chromo-reactive and chromo-acid baths
containing surfactants and anti-foams, where the concentrations of the different dyes are exceeding the
one recovered in the corresponding wastewaters.

9. BIOREMEDIATION OF FUNGICIDE/INSECTICIDES

Disposal of Spent Mushroom Substrate (SMS) generated after button mushroom cultivation is a matter
of great concern as it is produced nearly five times to the quantity of mushrooms produced. The bulk
of SMS makes its disposal more arduous besides the involvement of finance for its disposal. Moreover,
unplanned disposal causes considerable land and water pollution along with nuisance in the surround-
ings. SMS have potential to bioremediate several agricultural grade fungicides and pesticides (Ahlawat
et al., 2010, 2011).

10. DISCUSSION

Many fungal species are cultivated either for culinary or medicinal purposes, and some of the fungi are
produced on large scales. The spent substrate left after fruit body collection is partly recycled in open
field agriculture, but partly it causes environmental problems as bulk waste material. However, the spent
substrates might represent a potential tool to cure polluted environment especially soils. Up to now spent
mushroom substrate from only from few species i.e. Agaricus bisporus, Lentinula edodes, Pleurotus

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spp., Trametes versicolor have been studied either under lab conditions or in field scale. In the research
work performed, spent oyster mushroom substrate demonstrate higher degradation capability to degrade
3-ring PAH compounds even in soil with initial concentrations.Of many different spent substrates from
edible and medicinal mushroom cultivation. Only a few have been tried in soil remediation up to now.
Results obtained are variable; on one side, encouraging data on depletion of PCP and PAHs from con-
taminated soil using spent substrate of Lentinula edodes, Pleurotus pulmonarium and P. ostreatus have
been published (Okeke et al., 1993, 1996; Chiu et al., 1996; Eggen, 1999). The advantage of the use of
spent mushroom substrate in soil restoration is rgar it is a cheap inoculums source and its application
can to some extent reduce the cost of its disposal.

11. CONCLUSION

It could be concluded that the enzymes extracted from the spent compost of P. sajor-caju offers an
economical advantage of obtaining industrially important enzymes, which have potential in the biore-
mediation of synthetic dyes. Furthermore, the utilization of spent compost for the extraction of enzymes
can also offer a possible solution for the problem posed due to the disposal of large amounts of spent
mushroom compost. It has being shown that enzymes extracted from the spent compost of P. sajor-
caju have potential in the decolourisation of chemically different dyes. In addition, the effect of certain
physical parameters on the rate of decolourisation of each dye was also shown. The study also provided
insight on how the knowledge of these parameters can help in the optimization of processes employing
enzymes for bioremediation on an industrial scale. Moreover, the in vitro treatment of environmental
pollutants with crude ligninolytic enzymes of white rot fungi represents a simpler and effective method
as compared to the direct application of the fungi by eliminating the need to grow biomass and absorp-
tion effects of the pollutants on the mycelia. The utilization of SMS for the extraction of enzymes offers
an economical advantage of obtaining industrially important enzymes without long incubation periods
and additional cost of specialized fermentations.

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Chapter 9
Bioremediation Approaches
for Recalcitrant Pollutants:
Potentiality, Successes and Limitation

Bikram Basak
National Institute of Technology Durgapur, India

Apurba Dey
National Institute of Technology Durgapur, India

ABSTRACT
The different chemical pollutants discharged by the industries to the environment can upset the delicate
balance of the ecosystem. Bioremediation, the use of microorganisms and plants to remediate polluted
environments, is a promising and growing area of environmental biotechnology. Bioremediation options
encompass diverse types of biotechnological mechanisms that may lead to a target pollutant’s miner-
alization, partial transformation, humification, or altered redox state. The use of extra cellular and/
or cell-free enzymes has been also proposed as an innovative remediation technique. Perspectives and
limitations to evolve and use this technology are critically discussed in this chapter with respect to the
complexity of mixtures of xenobiotics often found in practice. Whereas the potential of bioremediation
is substantial, its application has important limitations that are apparent from many examples and the
authors feel that these limitations can be overcome only when adequate attention is directed to funda-
mental microbiological, chemical and engineering issues.

1. INTRODUCTION

Environmental contamination due to anthropogenic and natural sources is increasing day by day because
of increase in human population, industrialization and urbanization. The paradox for the public, scien-
tists, academicians and politicians is how to tackle the contaminants that jeopardize the environment.
Human activities to a greater extent and natural processes to some extent cause serious issues of pol-
luting soil and aquatic environments by releasing a large number of organic chemical substances such
as petroleum hydrocarbons, phenolic compounds, halogenated and nitroaromatic compounds, phthalate
esters, solvents and pesticides, endocrine disrupting chemicals, toxic heavy metals etc. Organic pollutants

DOI: 10.4018/978-1-4666-9734-8.ch009

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.

Bioremediation Approaches for Recalcitrant Pollutants

comprise a potential group of chemicals which can be dreadfully hazardous to human health. Many of
these are recalcitrant. As they persist in the environment, they are capable of long range transportation,
bioaccumulation, in human and animals, and biomagnifications in food chain (Nair et al., 2008).Most
of the compounds belong to groups that are widespread and are generally persistent and/or toxic. The
term “bioremediation” has been used to describe the process of using microorganisms to degrade or
remove hazardous components of the wastes from the environment. Biodegradation and its application
in bioremediation of organic pollutants have benefited from the biochemical and molecular studies of
microbial processes. Biodegradation is defined as the biologically catalyzed reduction in complexity of
chemical compounds. It is based on two processes: growth and co-metabolism. In growth, an organic
pollutant is used as sole source of carbon and energy and it results in the complete degradation of the
pollutant molecules. Co-metabolism can be defined as the transformation of a pollutant by a microorgan-
ism incapable of using the pollutant as a sole source of energy or of one of its constituent elements in
the presence of a growth substrate that is used as primary carbon and energy source (Basak et al, 2014).
Biotransformation of organic contaminants in the natural environment, which is defined as transforma-
tion of recalcitrant toxic organic pollutant into the lesser one, has been extensively studied to understand
microbial ecology, physiology and evolution for their potential in bioremediation. Bioremediation has
considerable strength and certain limitations too. Remediation, achieved whether by biological, chemical
or a combination of both means, is the only option as the problem of pollution is to be solved without
transferring to the future. As the knowledge mandate and complexities vary for different bioremediation
treatments, a better understanding of the principles together with the limitations of bioremediation aids
in maximizing the benefits and minimizing the cost of treatments. In this chapter we shall be critically
reviewing (i) the potentiality, (ii) successes, the advances made thus far and the requisite foci of research
on maximizing the biodegradation of pollutants, and (iii) limitations of bioremediation techniques in
removing toxic pollutants from the environments.

2. BACKGROUND

Relative to the pre-industrialization era, industrialization and intensive use of chemical substances such
as petroleum oil, hydrocarbons (e.g., aliphatic, aromatic, polycyclic aromatic hydrocarbons (PAHs),
BTEX(benzene, toluene, ethylbenzene, and xylenes), chlorinated hydrocarbons like polychlorinated
biphenyls (PCBs), trichloroethylene(TCE), and perchloroethylene, nitroaromatic compounds, organophos-
phorus compounds) solvents, pesticides, and heavy metals are contributing to environmental pollution
(Megharaj et al., 2011). Large-scale pollution due to man-made chemical substances and to some extent
by natural substances is of global concern now. Seepage and run-offs due to the mobility, and continuous
cycling of volatilization and condensation of many organic chemicals such as pesticides have even led
to their presence in rain, fog and snow (Dubus et al., 2000). Remediation of these polluted sites follow-
ing the conventional engineering approaches based on physicochemical methods is both technically and
economically challenging. Bioremediation employs the capabilities of microorganisms in the removal
of pollutants. With advances in biotechnology, bioremediation has become one of the most rapidly de-
veloping fields of environmental restoration, utilizing microorganisms to reduce the concentration and
toxicity of various chemical pollutants. Although, this novel technology has a multidisciplinary approach,
its central drive depends on microbiology. This technology includes biostimulation (stimulating viable
native microbial population), bioaugmentation (artificial introduction of viable Microbial population),

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Bioremediation Approaches for Recalcitrant Pollutants

bioaccumulation (live cells), biosorption (by dead microbial biomass), phytoremediation (bioremediation
by plants) and rhizoremediation (bioremediation through plant and microbe interaction). Compared to
other methods, bioremediation is a more promising, more efficient and cost effective way for cleaning
up contaminated soil and water. A number of bioremediation strategies have been developed to treat
contaminated wastes and sites. Selecting the most appropriate strategy to treat a specific site can be
guided by considering three basic principles: the amenability of the pollutant to biological transformation
to less toxic products (biochemistry), the accessibility of the contaminant to microorganisms (bioavail-
ability) and the opportunity for optimization of biological activity (bioactivity). However, the current
bioremediation approaches suffer from a number of limitations which include the poor capabilities of
microbial communities in the field, lesser bioavailability of contaminants on spatial and temporal scales,
and absence of bench-mark values for efficacy testing of bioremediation for their widespread applica-
tion in the field. The restoration of all natural functions of some polluted soils remains impractical and,
hence, the application of the principle of function-directed remediation may be sufficient to minimize
the risks of persistence and spreading of pollutants.

3. POTENTIALITY, SUCCESSES AND LIMITATION

OF BIOREMEDIATION AT A GLANCE

Approximately 6x106 chemical compounds have been synthesized, with 1,000 new chemicals being
synthesized annually. Significant amounts of a wide variety of industrial organic chemicals are released
into the environment deliberately, to function as pesticides or to preserve wood or insulate electric trans-
formers. Others are released accidentally or disposed of as waste. Deleterious effects or damages by
these contaminants lead to ‘pollution’, a process by which a resource (natural or man-made) is rendered
unfit for use, more often than not, by humans. Incomplete combustion of organic substances leads to
generation of about 100 different PAHs which are the ubiquitous pollutants. Except for a few PAHs used
in medicines, dyes, plastics and pesticides, they are rarely of industrial use. Some PAHs and their epox-
ides are highly toxic, and mutagenic even to microorganisms. About six specific PAHs are listed among
the top 126 priority pollutants by the US Environmental Protection Agency. Polychlorinated biphenyls
(PCBs), used in hydraulic fluids, plasticizers, adhesives, lubricants, flame retardants and dielectric fluids
in transformers are toxic, carcinogenic, and persistent. Polychlorinated dibenzodioxins and dibenzofurans
are recalcitrant chemicals and some of the congeners with lateral chlorine substitutions at positions 2,
3, 7 and 8 are carcinogenic to humans. Many solvents such as TCE and carbon tetrachloride pollute the
environments due to large-scale industrial production and anthropogenic uses. Pesticides are regularly
used in agricultural- and public health-programs worldwide. In many cases, the environmental effects
of these chemical substances outweigh the benefits they accrue to humans and necessitate the need of
their degradation after the intended uses.

Issues, Controversies, and Problems

Several processes have been used to remove xenobiotic compounds like physical and chemical meth-
ods. Recently applied physical method includes quick sorption on the activated sludge, adsorption by
low-cost adsorbents such as carbon blacks, powdered activated carbon (PAC) and pyrolysed rice husk
(PRH) etc. and subsequent biodegradation. Pulsed high-voltage discharge system is also a good physi-

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Bioremediation Approaches for Recalcitrant Pollutants

cal method to remove organic contaminants, use of granular or biological activated carbon. Chemical
method includes Photo-Fenton reaction for the removal of natural or synthetic aromatic compounds
and solvent-impregnated resin (SIR) system for the removal of phenols and thiophenols from water by
complex formation by hydrogen bonding of phosphine oxides and phosphates. Electrochemical detoxifi-
cation of waste water can be achieved by ion exchange membranes as solid polymer electrolytes (SPE);
other methods include H2O2/UV processes, O3/UV processes, Fenton processes (Fe2+/H2O2) solvent
extraction, membrane processes. Practice shows that the application of physicochemical technologies
is quite expensive and not completely effective. One of the main disadvantages of these processes is the
formation of toxic secondary intermediate compounds. Biological treatments are preferred for large-
scale removal of this type of pollutants. It is one of the reasons why activated sludge reactors have been
widely used for phenol removal from industrial wastewater. Biological treatment is a practical and not
very expensive solution to treat this kind of effluents compared to chemical methods; because various
population of microorganisms in the activated sludge are able to degrade organic compounds and most
of aromatic pollutants can be biologically degraded (Agarry et al., 2008).

4. WHY MICROBES?

The microbial remediation of these pollutants may be driven by energy needs, or a need to detoxify the
pollutants, or may be fortuitous in nature (co-metabolism). Because of the ubiquitous nature of microor-
ganisms, their numbers and large biomass relative to other living organisms in the earth, wider diversity
and capabilities in their catalytic mechanisms, and their ability to function even in the absence of oxygen
and other extreme conditions, the search for pollutant-degrading microorganisms, understanding their
genetics and biochemistry, and developing methods for their application in the field have become an
important human endeavor. During the last few decades, extensive research has led to isolation of unusual
microbes, capable of degrading a vast array of toxic organic compounds even under extreme conditions
and environments. Though these pollutants are relatively alien for the microbes, they have evolved novel
pathway(s) for their metabolism. However, development of novel pathway(s) in the evolutionary time
scale is an extremely slow process, during which the microbes modify an existing pathway useful for the
naturally occurring metabolites to structurally related recalcitrant analogous substrates. When a microbial
species is exposed to a nitro-aromatic compound, it may be subjected to either (i) mineralization leading
to formation of inorganic end products (viz. CO2 and water) through catabolism (where the nitro-aromatic
compound serves as a sole source of C, N and energy) or (ii) co-metabolism, anon-specific transforma-
tion by enzymes specific for other substrates (where the nitroaromatic compound can be transformed
only in the presence of another substrate and may or may not serve as a C and N source).

5. PHYTOREMEDIATION

Phytoremediation is a promising new technology that uses plants to degrade, assimilate, metabolize, or
detoxify metals, hydrocarbons, pesticides, and chlorinated solvents. Phytoremediation is an emerging
technology that utilizes plants and then the associated rhizosphere microorganisms to remove, trans-
form, or contain toxic chemicals located in soils, sediments, ground water, surface water, and even the
atmosphere. Currently, phytoremediation is used for treating many classes of contaminants including

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petroleum hydrocarbons, chlorinated solvents, pesticides, explosives, heavy metals and radionuclides, and
landfill leachates. in situ, in vivo and in vitro methods of application has been described for remediation
of these compounds. Phytoremediation prescriptions have recommended being site-specific; however,
these applications have the potential for providing the most cost-effective and resource-conservative ap-
proach for remediating sites contaminated with a variety of hazardous chemicals. Phytoremediation is
seen as a final polishing step following the initial treatment of the high-level contamination. However,
when contaminants are in low concentration, phytoremediation alone may be the most economical and
effective remediation strategy. Many sites with less toxic contaminants are suitable for phytoremediation
as a long-term solution to the problem.

6. DEVELOPMENTS AND SUCCESSES IN BIOREMEDIATION TECHNOLOGIES

Bioremediation of toxic wastes can be categorized as in situ and ex situ and ex situ slurry bioremediation.
Their ultimate objective is to degrade organic pollutants to concentrations below the permissible limits
established by regulatory authorities and preferably to undetectable levels. Table 1 represents the dif-
ferent strategies of in situ and ex situ bioremediation technologies. With in situ techniques, the soil and
associated ground water is treated in place without excavation, whereas it is excavated prior to treatment
with ex situ applications. Selection of appropriate technology among the wide range of bioremediation
strategies developed to treat contaminants depends upon three basic principles i.e., the amenability of
the pollutant to biological transformation (Biochemistry), the accessibility of the contaminant to mi-
croorganisms (Bioavailability) and the opportunity for optimization of biological activity (Bioactivity)
(Dua et al., 2002; Shukla et al., 2010).

In Situ Bioremediation

An in situ process is applied at the contaminated site, without removal of contaminated material from
its original location. in situ processes include:

1. Land farming, Bioventing: In land farming, contaminated soil is mixed with nutrients and moisture
and periodically aerated. Bioventing encourages the in situ biodegradation of POLs (petroleum-oil-
lubricants) by providing oxygen to microorganisms in the contaminated soil. The system supplies
oxygen by injecting air directly into the residual contamination. Bioventing uses low airflow rates
to provide only enough oxygen to keep up optimal microbial activity. Optimal flow rates maximize
biodegradation as vapors move slowly through biologically active soil while minimizing volatiliza-
tion of contaminants.
2. Soil biopiling: Biopiling treatment is a full-scale technology in which excavated soils are mixed
with soil amendments, placed on a treatment area, and bioremediated using forced aeration. The
contaminants are reduced to carbon dioxide and water. The basic biopile system includes a treatment
bed, an aeration system, an irrigation/nutrient system and a leach ate collection system. Moisture,
heat, nutrients, oxygen, and pH are controlled to enhance biodegradation. The irrigation/nutrient
system is buried under the soil to pass air and nutrients either by vacuum or positive pressure.
3. Composting: Composting is a process by which organic wastes are degraded by microorganisms,
typically at elevated temperatures. Typical compost temperatures are in the range of 55° to 65° C.

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Table 1. Different bioremediation technologies

Bioremediation Methods Benefits Limitations Applications References

(Kulkarni &
Most cost efficient, Biodegradative abilities
Biosparging Environmental constraints Chaudhari,
noninvasive ofindigenous microorganisms
2007)
Bioremediation of
(Kulkarni &
Environmental constraints, TNT contaminated soil by the
Simple, efficient and cost Chaudhari,
Land farming time consuming, lack of application of molasses,Surface
effective 2007; Shukla, et
monitoring system application,
al., 2010)
aerobic process,
Relatively passive, less Extended treatment time Presence of metals and other (Shukla, et al.,
Bioventing
effective Monitoring difficulties inorganic 2010)
Presence of metals and other
Monitoring difficulties,Need inorganic Environmental (Kulkarni &
in situ More efficient than
to control abiotic loss, parameters Biodegradability of Chaudhari,
Soil biopiling Biosparging, Land
Mass transfer problem, pollutants Chemical solubility 2007; Shukla, et
farming and Bioventing
Bioavailabilitylimitation Geologicalfactors Distribution al., 2010)
of pollutants
Extended treatment time,
Requires
nitrogensupplementation,
(Kulkarni &
Low cost Rapid reaction incubation periodsmonths
Surface application, Chaudhari,
Composting rate, Inexpensive, self- to years, lack of full-scale
agricultural tomunicipal waste 2007; Megharaj,
heating information about biological
et al., 2011)
systems, poor chemical
characterization of the
outcome of theprocess
Rapid degradation
kinetic, Optimized (Behkish,
Soil requires excavation, Bioaugmentation,
Bioreactors environmental Lemoine,
Relatively high cost capital, Toxicity of amendments,
(Slurry reactors Parameters, Sehabiague,
Relatively high operating Toxic concentrations of
Aqueous reactors) Enhances mass transfer, Oukaci, &
cost contaminants
Effective use of Morsi, 2007)
inoculantsand surfactant
Precipitation
or Flocculation
(Non-directed
physico-chemical
complexation
Cost-effective, simple Yet to be exploited Removal of heavy
reaction between (Natrajan, 2008)
operations commercially metals
ex situ dissolved
contaminants and
charged cellular
components (dead
biomass)
Microfiltration
(Microfiltration Waste water treatment;
Remove dissolved Yet to be exploited (Shukla, et al.,
membranes are recovery and reuse of morethan
solids rapidly commercially 2010)
used at a constant 90% of original wastewater
pressure)
Present a novel method Studied for applications as
Microbial fuel cells forsimultaneous Yet to be exploited biosensors such as sensors (Sevda, et al.,
(MFC) bioelectricitygeneration commercially for biological oxygen demand 2013)
and wastewater treatment monitoring

The increased temperatures result from heat produced by microorganisms during the degradation
of the organic material in the waste. The main drawbacks of the composting method are: (a) lack
of full-scale information about biological systems, (b) long incubation period, (c) lack of effective

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control on various parameters that may affect the outcome, and (d) poor chemical characterization
of the outcome of the process. The merit of the composting method is its moderate cost by virtue
of in situ application (Kulkarni & Chaudhari, 2007).Addition of compost to contaminated soil for
bioremediation makes it a sustainable technology since the biodegradable organic waste in the
compost is being utilized for beneficial activity. Also, composting improves the soil structure, nu-
trient status and microbial activity. During composting the contaminant can disappear via different
mechanisms such as mineralization by microbial activity, transformation to products, volatilization,
and formation of nonextractable bound residues with organic matter.

Ex Situ Bioremediation

ex situ bioremediation refers to removal of contaminated material from the source (soil/water) and pro-
cessing it in bioreactors under controlled operating parameters (temperature, pH, and aeration).Therefore,
this process is more expensive than thein situ approach. In this process, heavily contaminated soil in a
soil: water ratio of 1:1 (w/w) is stirred in a reactor to form slurry and treated under aerobic and anaerobic
conditions (Esteve-Nunez et al., 2007). ex situ technologies include use of Bioreactors (Slurry reac-
tors, Aqueous reactors), Precipitation or Flocculation, Microfiltration, Electrodialysis. There are many
benefits of ex-situ which can be enlisted as follows: Cost efficient and simple procedure, inexpensive,
self-heating, low cost rapid reaction rate, inexpensive, self-heating, can be done on site, rapid degrada-
tion kinetic optimized environmental parameters, enhances mass transfer, effective use of inoculant
and surfactant, removes dissolved solids rapidly, withstand high temperature and can be reused. The
ex situ bioremediation treatment could use free and immobilized cell systems. In free cell systems, live
bacteria/fungi or their consortia are used as an inoculum to degrade the organics. However, it suffers
from drawbacks: (i) survival of inoculum gets difficult at threshold concentration of toxic chemicals,
(ii) reduction in chemical load is limited, and (iii) presence of heavy metals inhibits treatment. To avoid
wash out of biomass at low concentrations of toxic chemicals and increase its rate of degradation, im-
mobilization of degradative bacteria has been proposed by several researchers. Whole cells immobilized
using adhesion, crosslinking, entrapment to various matrices (peat, agarose, clay, alginate, diatomaceous
earth, k-carragenan, etc.) or self-immobilization on granular sludge provide an alternative to overcome
the existing limitations experienced with the free cell system of bioremediation. The ex situ bioremedia-
tion of aromatic compounds using enzymatic treatment is also proving to be greatly useful. Enzymatic
treatment is simply the better biological way to remove aromatic compounds from industrial wastewater.
Treatment of aqueous phenols using oxidoreductive enzymes is an efficient and cost effective method,
Peroxidasesorpolyphenol oxidases (PPO) can be better utilized for the removal of phenolic contaminants.
Apart from peroxidases or polyphenol oxidases (PPO), there are numbers of enzymes like phenol oxidase,
phenol hydroxylase, phenolase, cresolase, cytochromeP450etc, and these enzymes are very efficient in
the biodegradation of phenols from industrial waste water and effluents. Enzymatic water treatment has
been carried out employing both free and immobilized PPO; in fact better efficiency has been obtained
from the later in terms of reusability, stability and longer viability. Enzymeactivity may be hampered
due to some peculiar properties of the enzymatic proteins such as their non-reusability, high sensitivity
towards several denaturating agents and presence of adverse sensory or toxicological effects. Many of
these undesirable constraints may be removed by the use of immobilized enzymes. This approach has
proven to be more advantageous for enzyme catalysis than the use of free enzymes. Enzyme bioreactor
technology is another approach which has been developed for obtaining better performance and high

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degree of removal of phenolic contaminants. Enzymatic reactor allows enzyme and substrate to come
into contact for a sufficient period of time for reaction to take place; and product may then easily be
separated. Various kinds of enzymatic reactors like batch, stirred tank, packed bed, fluidized bed and
membrane reactor has been utilized for removal of phenols from industrial waste water on large scale
basis (Ibrahim et al., 2001).

Bioavailability

The process of bioremediation depends on the metabolic potential of microorganisms to detoxify or trans-
form the pollutant molecule, which is dependent on both accessibility and bioavailability. Bioavailablity
of is the fraction of pollutants which is available to microorganisms for degradation and is a critical limit-
ing factor. Following entry into the soil environment, pollutants rapidly bind to the mineral and organic
matter (solid phases) via a combination of physical and chemical processes. Sorption, complexation and
precipitation constitute the pollutant–soil interaction. The ability of soils to release (desorb) pollutants
determines its susceptibility to microbial degradation, thereby influencing effectiveness of the bioreme-
diation process (Avio et al, 2015). There are two types of bioavailability considered and the consequent
biodegradation of organic contaminants: (i) the pre-requisite release of contaminant from sorbed phase
to aqueous phase for its degradation by microorganisms and (ii) biodegradation of the contaminant in
the sorbed phase, without being desorbed, by the enzymes (Figure 2). Aqueous solubility, volatility
or reactivity of organic pollutants varies greatly, and all of them may influence their bioavailability in
water and soils. Moreover, the sequestration of pollutants over time may occur due to the contact and
interaction of soil with pollutant molecules. Several factors like organic matter, cation exchange capacity,
micropore volume, soil texture and surface area affect the pollutant sequestration (Chung & Alexan-
der, 2002). Even a weakly sorbed and easily degraded pollutant can be effectively sequestrated in soil
with aging, thereby rendering it partly inaccessible to microorganisms and affecting the bioavailability.
Hence, the generalizations about the effects of aging on the sorption-desorption behavior of different
organic chemicals are difficult to achieve. Some pertinent issues that need to be considered include: (a)
bioavailability and toxicity of parent molecules and their residues in soils,(b) standardized protocols for

Figure 2. Schematic representation of bioavailability and degradation of a bioavailable pollutant

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different pollutants and their use across the sites, (c) assessment on remobilization of pollutants during
the post-remediation period, and (d) determination of environmentally acceptable pollutant end-points
in the bioremediated soils (Megharaj et al., 2011).

Biosurfactants: Bioavailability Enhancers

Biosurfactants are surface-active microbial products that have numerous industrial applications. Many
microorganisms, especially bacteria, produce biosurfactants when grown on water-immiscible substrates.
Most common biosurfactants are glycolipids in which carbohydrates are attached to a long-chain ali-
phatic acid, while others, like lipopeptides, lipoproteins and heteropolysaccharides, are more complex.
The genes involved in biosurfactant synthesis have been characterized and the molecular genetics of
regulatory mechanisms of biosurfactant production have been studied in detail The most promising
applications of biosurfactants are in the cleaning of oil-contaminated tankers, oil-spill management,
transportation of heavy crude, enhanced oil recovery, recovery of oil from sludge and bioremediation of
sites contaminated with hydrocarbons, heavy metals and other pollutants. Application of surfactants to
polluted soils has been used as one of the treatment strategies for increasing the mass transfer of hydro-
phobic organic contaminants. Biosurfactants are surface-active microbial products that have numerous
industrial applications. The surfactants are amphiphilic molecules that contain hydrophilic and hydro-
phobic moieties; hydrophilic groups can be anionic, cationic, zwitter ionic, and nonionic. The synthetic
surfactants contain sulfate, sulfonate or carboxylate group (anionic); quaternary ammonium group
(cationic); polyoxyethylene, sucrose, or polypeptide (nonionic)and the hydrophobic parts of paraffins,
olefins, alkylbenzenes, alkylphenols, or alcohols. The common chemical surfactants such as Triton X-100,
Tween 80 and sodium dodecyl sulphate tested as surfactants for bioremediation techniques are petroleum
derived products. The zwitter ionic surfactants (e.g., N-dodecylbetaine) which contain both anionic and
cationic groups have low critical micelle concentration (CMC) values, more surface active, and high
solubilization capacity. Increased desorption rates of sorbed pollutants from soils by the application of
surfactants make the pollutants available for remediation. The food-grade surfactants, the plant-based
surfactants (e.g., fruit pericarp from Sapindus mukurossi) or the natural surfactants such as humic acids
may be preferred to the synthetic surfactants due to high biodegradability, low toxicity, and higher public
acceptance. Microorganisms also produce surfactants (surface-active amphiphilic metabolites such as
glycolipids, phospholipids, lipopeptides, lipoproteins, and lipopolysaccharides).The in situ application
of surfactants to improve bioavailability of recalcitrant organic pollutants requires careful planning and
selection based on the previous information about the fate and behavior of the surfactant and the target
pollutant. Caution is needed to prevent groundwater contamination via leaching and consequent toxic-
ity to microorganisms. Hence, a good strategy would be to choose bacteria that are capable of not only
metabolizing the target pollutant but also producing biosurfactant.

Biosurfactant-Producing and Pollutant-Degrading

Microbial Inoculants: Dual Role

Microorganisms also produce surfactants (surface-active amphiphilic metabolites such asglycolipids,

phospholipids, lipopeptides, lipoproteins, and lipopolysaccharides). These low- and high-molecular
weight biosurfactants find their uses in food processing, cosmetic and pharmaceutical industries, in
addition to bioremediation efforts. The application of biosurfactant-producing and pollutant-degrading

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microorganisms offers dual advantages of a continuous supply of biodegradable surfactant and the abil-
ity to degrade pollutant(s) (Megharaj et al., 2011). Using genes encoding the biosynthetic pathway of
biosurfactants can enhance biodegradation rates by improving the bioavailability of the substrates; and
genes encoding resistance to critical stress factors may enhance both the survival and the performance
of designed catalysts (Dua et al., 2002).

Exploitation of Microbial Natural Attenuation, Cooperation and Networking

The biodegradative capability of microorganisms as the principles of microbial infallibility expressing

the empirical observation that no naturally occurring organic compound is totally resistant to microbial
biodegradation provided that the environmental conditions are favorable. – Alexander (1965)

During bioattenuation (natural attenuation), the pollutants are transformed to less harmful forms or
immobilized. Such transformation and immobilization processes are largely due to biodegradation by
microorganisms. The chemical properties and quantities of xenobiotic pollutants determine their toxic-
ity and persistence in the environment. Their interaction with targeted and non-targeted organisms has
extensively damaged the ecosystem through entry into the food chains. The net result has been bioac-
cumulation and biomagnification of these xenobiotic pollutants in aquatic and terrestrial organisms.
Such ecological stress, imposed by massive amounts of xenobiotics, is partially surmounted through
microbial attenuation. While microbial attenuation may be almost infallible, it is not adequate to protect
the biosphere from indiscriminate agricultural and industrial activities. This may be because (i) xenobiot-
ics are relatively new to the biosphere, (ii) enhanced resistance to attenuation is inherent due to unusual
chemical bonds or functional groups in their structure, (iii) microbes have not had enough time to evolve
suitable metabolic apparatus to deal with incorporated xenobiotics, and (iv) only a few microbes have
evolved with the ability to sustain in adverse ecological states. The natural attenuation processes are
contaminant-specific, accepted as methods for treating fuel components (e.g., BTEX) (Atteia & Guil-
lot, 2007), but not for many other classes. The time required for natural attenuation varies considerably
with site conditions. Many polluted sites may not require an aggressive approach to remediation, and
bioattenuation is efficient and cost-effective (Atteia & Guillot, 2007; Megharaj et al., 2011).
If a natural compound is available in larger quantities, biomass and catabolic activity increases by an
autocatalytic process until an essential nutrient e.g. the carbon and energy source becomes limiting. In
contrast, the transformation rate of a xenobiotic compound and the number of active cells in the popula-
tion remain constant if degradation is incomplete and does not yield energy for growth. Thus, under these
conditions the fate of xenobiotic compounds is essentially determined by gratuitous processes that are
named co-metabolism. As much as the diversity in sources and chemical complexities in organic pollut-
ants exists, there is probably more diversity in microbial members and their capabilities to synthesize or
degrade organic compounds. The microbial populations of soil or aquatic environments are composed of
varied, synergistic or antagonistic communities rather than a single strain. In the natural environments,
biodegradation involves transferring the substrates and products within a well-coordinated microbial
community, a process referred to as metabolic cooperation. The potential to degrade organic pollutants
varies among microbial groups or different guilds (group of species that exploit the same class of envi-
ronmental resources in a similar way) and is dose-dependent. It is very challenging to introduce all the
genes required for degradation for many organic pollutants or stable maintenance of even a single gene

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single organism. Hence, the microbial consortia of ecologically relevant candidate taxa which are known
to degrade xenobiotic pollutants and respond to different environmental stimuli are desired, rather than
the singlei solate for augmentation.
The application of molecular ecological techniques for community profiling, soil metagenomics
using isotope distribution analysis, and functional genomics and proteomics can help in identifying the
partners and the patterns of responses to external stimuli within the network and the ‘system complexi-
ties’ of contaminated sites. Stable isotope probing (SIP) analyses, either DNA-SIP or RNA-SIP, provide
opportunities to link microbial diversity with function and identify those culturable as well as uncultured
organisms which are involved in biodegradation in the field. Likewise, the high-throughput approaches
such as DNA microarrays, metagenomics, metatranscriptomics, metaproteomics, metabolomics, and
whole cell-based biosensors are useful to characterize the contaminated sites, identify new degradative
activities and monitor bioremediation efficiency (Desai et al., 2010; Megharaj, et al., 2011).

Biostimulation and Bioaugmentation

The acceleration of microbial degradation of xenobiotic pollutants generally depends on the supply of
carbon, nutrients such as N and P, temperature, available oxygen, soil pH, redox potential, and the type
and concentration of organic pollutant itself. Biostimulation is the addition of right ratio of different
nutrients and other possible stimuli for the optimal bioremediation by native microbial community at a
cleanup site. To stimulate microbial degradation, nutrients in the form of fertilizers (water soluble (e.g.,
KNO3, NaNO3, NH4NO3,K2HPO4 and MgNH4PO4), slow release (e.g., customblen, IBDU, max-bac),
and oleophilic (e.g., Inipol EAP22, F1, MM80, S200)) are added (Megharaj et al., 2011). These addi-
tions may be insufficient or inaccurate for polluted sites with different types of pollutants. Formulation
of nutrient-treatment strategies and maintenance of control on the degradation rates and the outcomes
of degradation need to be tailored to specific site/pollutant combinations.
Bioaugmentation is the defined as the practice of enhancing the performance of indigenous micro-
bial population especially when they are not up to the task through addition of commercially prepared
microbial strain with specific catabolic activities. Pre-adaptation of catabolic bacteria to the target
environment,prior to inoculation, improves survival, persistence and degradative activities, leading to
enhanced remediation of the polluted soil (Megharaj et al., 2011). However, bioaugmentation efforts
are often met with failures more often due to lesser efficiency, competitiveness and adaptability, rela-
tive to the indigenous members of natural communities. For example, the well-known bacteria capable
of degrading PCBs in laboratory culture media survived poorly in natural soils, and when these strains
were inoculated to remediate PCB-contaminated soils, the resultant was the failure of bioaugmentation.
Indeed, bioaugmentation itself is undesirable in all the environmentally sensitive locations, especially
those protected from the introduction of exotic flora or fauna (Megharaj et al., 2011).Scott et al. (2010)
proposed a new strategy of using a free enzyme-based product to remediate waterbodies contaminated
with atrazine(Scott et al., 2010).The ecological or environmental issues associated with degrading or-
ganisms can be circumvented by this strategy. The soils do have exoenzymes (cell-free enzymes)which
include proteases, and the presence of proteases along with other inhibitors may limit the longevity of
free enzymes applied for bioremediation.

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Potential of Extra Cellular Enzymes in in Situ Remediation of Polluted Soils

The use of extra cellular and/or cell-free enzymes has been also proposed as an innovative remediation
technique. They can offer some advantages over the use of microbial cells. In order to be biodegraded,
contaminants must interact with enzymatic systems in the degrading organisms. If soluble, they can
easily enter cells, if insoluble; they must be transformed into soluble or easily cell-available products
(Figure 1).The first effective step for cell-transformation of insoluble substances, including xenobiotics
and even plastic materials, is usually the reaction catalyzed by ecto- and extra cellular enzymes, which
are deliberately released by the cells into their nearby environment. The process can be quite rapid for
some natural compounds like cellulose or very slow for many xenobiotic compounds. Extra cellular
enzymes include a large range of oxidoreductases and hydrolases (Table 2). Both these enzymes may
explicate a degradative function and transform polymeric substances into partially degraded or oxidized
products that can be easily taken up by cells. Several extra cellular enzymes, either as cell-associated or
cell-free enzymes, may behave as powerful catalysts in the biodegradation of harmful pollutants. How-
ever, their large-scale application for remediation of polluted soils is still limited. This may derive from
several drawbacks and disadvantages depending on both the pollutants and the enzymes. For instance,
the simultaneous presence of several polluting substances in a contaminated site with synergistic, often
negative, effects on the enzyme efficiency, the high costs associated with the isolation and purification of
free enzymes, the low stability of enzymes to the harsh conditions of soil all concur to restrict the wide
use of enzymes as remediating agents of polluted soils. Although immobilized enzymes may present
a high stability under soil conditions, they are not widely applied in the remediation of polluted soils.

Figure 1. Roles of extracellular enzymes in biodegradation of toxic pollutants

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Table 2. Biodegradation of various pollutants by microbial cell-free enzymes

Pollutant Enzyme Source

Azo dyes Laccase Pycnoporus sanguineis
Biopolymers (kraft, lignin), Lignin Peroxidases (LiP), White-rot fungi
Mn-dependent Peroxidases (MnP)
Anthracene, pyrene Lignin Peroxidases, Nematoloma forwardii
Mn-dependent Peroxidases, Laccase
Asphaltenes Chloroperoxidase Cladariomyces fumago
Phenols, PAH Polyphenol oxidase, Lignin Peroxidases, Phenol degrading microorganisms, white rot fungi
Mn-dependent Peroxidases
PCP, DDT, PCBs Lindane Dehalogenases, Laccase Several microorganisms
Estrogenic chemicals MnP, laccase Trametes versicolor
Nitrile compounds Nitrilase, nitrile hydratase, Nocardiasp, Rhodococcus sp.,
amidase Fusariumsolani
PCBs (1-6 Cl substitutions) LiP, MnP Coriolopsis polyzona, Pleurotusostreatus, T. versicolor
PCP lignin-degrading enzyme system P. chrysosporium, T. versicolor, Inonatus dryophilus
TNT, RDX LiP, MnP, cellobiose dehydrogenase White-rot fungi
Bleach plant effluents Laccase P. sanguineis

Application of Genetically Engineered Microorganisms

Advances in genetic and protein engineering techniques have opened up new avenues to move towards the
goal of genetically engineered microorganisms (GEMs) to function as “designer biocatalysts”, in which
certain desirable biodegradation pathways or enzymes from different organisms are brought together
in a single host with the aim of performing specific reactions. Microorganisms respond differently to
various kinds of stresses and gain fitness in the polluted environment. This process can be accelerated
by applying genetic engineering techniques. The recombinant DNA and other molecular biological tech-
niques have enabled (i) amplification, disruption, and/or modification of the targeted genes that encode
the enzymes in the metabolic pathways, (ii) minimization of pathway bottlenecks, (iii) enhancement of
redox and energy generation, and (iv)recruiting heterologous genes to give new characteristics. Various
genetic approaches have been developed and used to optimize the enzymes, metabolic pathways and
organisms relevant for biodegradation. Through the genetic engineering of metabolic pathways, it is
possible to extend the range of substrates that an organism can utilize. Hybrid dioxygenases that have
acquired enhanced degradation capabilities for PCBs, TCE and some other aromatic hydrocarbons.
Genetic engineering also permits the combination of several degradative activities within a single
host organism. If a single strain is constructed to perform several related or unrelated metabolic activi-
ties, the efficiency and predictability of the process may be significantly enhanced. Such recombinant
strains may be useful for the bioremediation of recalcitrant compounds. Requirements for the design
of bacteria with multiple pathways for use in bioremediation have been described (Dua et al., 2002).
Timmis and Piper (1999) suggested a strategy for designing organisms with novel pathways and the
creation of a bank of genetic modules encoding broad specificity enzymes or pathway segments that can
be combined at will to generate new or improved activities. The use of appropriate regulatory circuits
can enhance substrate flux through these designed pathways; and rationally engineering the pathway

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branch-points can avoid or reduce substrate misrouting. Nevertheless, the introduced genes or enzymes,
even in a single modified organism, need to be integrated within the regulatory and metabolic network
for proper expression (Megharaj et al., 2011).

7. HEAVY METAL TOXICITY REMOVAL BY MICROBES

Unlike many other pollutants, metals can undergo biodegradation and produce less toxic, less mobile and/
or less bioavailable products, but heavy metals are difficult to be removed from contaminated environment.
These metals cannot be degraded biologically, and are ultimately everlasting, though the speciation and
bioavailability of metals may change with variation in the environmental factors. At high concentrations,
metal ions can either completely inhibit the microbial population by inhibiting their various metabolic
activities or organisms can develop resistance or tolerance to the elevated levels of metals. Some metals
such as, zinc, copper, nickel and chromium are essential or beneficial micronutrients for plants, animals
and microorganisms while others (e.g., cadmium, mercury and lead) have no known biological and/
or physiological functions. However, the higher concentration of these metals has great effects on the
microbial communities in soils in several ways- (1) it may lead to a reduction of total microbial biomass
(2) it decreases numbers of specific populations or (3) it may change microbial community structure
(Shukla et al., 2010). The removal of different kinds of heavy metals including Cu, Zn, Ni and Cr by free
and immobilized microalgae has been well demonstrated. An adsorption–desorption cycle was developed
for repeated uses of the algal beads (microalgal beads) for the removal and recovery of heavy metals.
Microorganisms have evolved several mechanisms by which they can immobilize, mobilize or transform
metals rendering them inactive to tolerate the uptake of heavy metal ions. These mechanisms include (1)
exclusion-the metal ions are kept away from the target sites (2) extrusion-the metals are pushed out of the
cell through chromosomal/plasmid mediated events (3) accommodation metals form complex with the
metal binding proteins (e.g. metallothienins, a low molecular weight proteins) or other cell components
(4) bio-transformation—toxic metal is reduced to less toxic forms and (5) methylation and demethylation.

8. DEVELOPMENTS IN PHYTOREMEDIATION

Plant-assisted bioremediation, or phytoremediation, is commonly defined as the use of green or higher

terrestrial plants for treating chemically or radioactively polluted soils. Herbicides are economically
important, but the non-point pollution that they cause may disrupt the surrounding environment. Phy-
toremediation of herbicides has been well studied using conventional plants. Transgenic plants produced
for metabolizing herbicides and long-persisting pollutants can be used for phytoremediation of foreign
chemicals in contaminated soil and water. The major advantages of phytoremediation are as follows:

1. The cost of the phytoremediation is lower than that of traditional processes both in situ and ex situ.
2. The plants can be easily monitored.
3. The possibility of the recovery and re-use of valuable products.
4. It uses naturally occurring organisms and preserves the natural state of the environment.
5. The low cost of phytoremediation (up to 1000 times cheaper than excavation and reburial) is the
main advantage of phytoremediation.

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Phytoextraction

Phytoextraction (or phytoaccumulation) uses plants or algae to remove contaminants from soils, sedi-
ments or water into harvestable plant biomass. Generally, this process has been tried more often for
extracting heavy metals. At the time of disposal, contaminants are typically concentrated in the much
smaller volume of the plant matter than in the initially contaminated soil or sediment. ‘Mining with
plants’, or phytomining, is also being experimented with. The plants absorb contaminants through the
root system and store them in the root biomass and/or transport them up into the stems and/or leaves. A
living plant may continue to absorb pollutant still its death or until it is harvested. After harvest, a lower
level of the contaminant may remain in the soil, so the growth/harvest cycle must usually be repeated
through several crops to achieve a significant cleanup. After the process, the cleaned soil can support
other vegetation. The fungal treated plants grown in Cd–Ni combination contaminated soils showed
higher phytoextraction efficiency than those in Cd or Ni contaminated soils.

Phytotransformation

Certain plants, such as can as, render organic pollutants, such as pesticides, explosives, solvents, indus-
trial chemicals, and other xenobiotic substances non-toxic by their metabolism. Microorganisms living
in association with plant roots may metabolize these substances in soil or water. These recalcitrant
pollutants cannot be broken down to basic molecules (water, carbon dioxide etc.) by plant molecules,
and hence the term phytotransformation represents a change into less toxic chemical structure without
complete breakdown of the compound. However, microorganisms living as symbiont can utilize these
transformed pollutants further and metabolize and mineralize completely.

Rhizoremediation

Rhizodegradation or rhizoremediation (also called enhanced rhizosphere biodegradation, phytostimu-

lation, and plant assisted bioremediation) is the breakdown of organic contaminants in the soil by soil
dwelling microbes which is enhanced by the rhizosphere’s presence. Certain soil dwelling microbes
digest organic pollutants such as fuels and solvents, producing harmless products. Rhizoremediation, an
integral component of phytoremediation can occur naturally or can be triggered by introducing specific
pollutant degrading microbes or plant growth promoting microorganisms. Plants produce many second-
ary plant metabolites (SPMEs)which include allelopathic chemicals, root exudates, phytohormones/
phytoalexins, phytosiderophores, andphytoanticipins and are derived from isoprenoid, phenylpropanoid,
alkaloid or fatty acid/polyketidepathways(Megharaj et al., 2011). Singer et al. (2004) stated that SPMEs
are pollutant analogues within the network of suprametabolism, having implications for predicting the
fate of pollutants.
Since the root depth of herbaceous plants varies from plant to plant, from soil to soil, and season
to season, the presence of contaminants in soils which is deeper than the root zone of plants requires
excavation, other agronomic practices or selection of trees with deeper roots. Nevertheless, most of the
recalcitrant organic contaminants are typically found in the top few cm of the soil.

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9. WASTEWATERS CAN BE BIOREMEDIATED IN MICROBIAL FUEL CELLS

Microbial fuel cells (MFCs) present a novel method for simultaneous bioelectricity generation and
wastewater treatment. A microbial fuel cell (MFC) is a bioreactor that converts chemical energy in the
chemical bonds in organic compounds to electrical energy through catalytic reactions of microorganisms
under anaerobic conditions. It has been known for many years that it is possible to generate electricity
directly by using bacteria to break down organic substrates. The recent energy crisis has reinvigorated
interests in MFCs among academic researchers as a way to generate electric power or hydrogen from
biomass without a net carbon emission into the ecosystem. MFCs can also be used in wastewater treat-
ment facilities to break down organic matters (Sevda et al., 2013). They have also been studied for
applications as biosensors such as sensors for biological oxygen demand monitoring. The use of an
anode as a final electron acceptor by bacteria has led to the possibility of a wide range of applications.
It should be noted that many of these envisaged applications are not currently feasible and require sig-
nificant improvements if they are to become viable technologies (Franks & Nevin, 2010). One of the
most active areas of MFC research is the production of power from wastewaters combined with the
oxidation of organic or inorganic compounds. Studies are demonstrating that any compound degradable
by bacteria can be converted into electricity. The range of compounds include, but by no means limited
to, acetate, glucose, starch, cellulose, wheat straw, pyridine, phenol, p-nitrophenol and complex solu-
tions such as domestic waste water, brewery waste, land file leachate, chocolate industry waste, mixed
fatty acids and petroleum contaminates. Within these systems less biomass is also generally produced
than their equivalent aerobic processes and without the need for energy intensive aeration process less
energy is required. However, MFCs for the large scale treatment of wastewaters still face problems of
scale up from laboratory experiments and slow rates of substrate degradation. The ability of the MFC
microbial communities to degrade a wide range of environmental pollutants may be more valuable than
production of electricity itself in certain settings, especially when the MFC technology can be used for
environmental cleanup in situ. Geobacter sp. have been known to be important in the anaerobic degrada-
tion of petroleum components and landfill leachate contaminants in ground water. The oxidation of the
contaminant is linked to the reduction of Fe (III). The oxidation and reduction process can be increased
through the addition of Fe (III) chelators or electron shuttles to promote increased transfer of electrons
between cells and insoluble Fe (III) oxides. Contaminants often persist in the environment due to absence
of suitable electrons acceptors and the addition of chelators, electron shuttles or other electron acceptors
in the subsurface environment is not feasible.

10. LIMITATIONS OF BIOREMEDIATION: A GRIM FACE

Although a wide range of new microorganisms have been discovered that are able to degrade highly
stable, toxic organic xenobiotics, still many pollutants persist in the environment. A number of reasons
have been identified as challenges posed to the microorganisms working in contaminated sites. Such
potential limitations to biological treatments include: poor bioavailability of chemicals, presence of other
toxic compounds, inadequate supply of nutrients and insufficient biochemical potential for effective bio-
degradation. There are some drawbacks with the field release of genetically engineered microorganisms
(GEMs), which include the decreased levels of fitness and the extra energy demands imposed by the
presence of foreign genetic material in the cells. More importantly, there remains a great risk of mobile

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genetic elements entering the environment and being acquired by undesirable organisms. One of the criti-
cal knowledge gaps of composting is lack of sufficient knowledge about microorganisms involved during
various stages of composting, the thermophilic stage in particular, which is almost like a black-box. In
fact, there are conflicting views about the role of the thermophilic stage of composting in bioremediation
of contaminants. Added to this complexity is the fate of bound residues and whether or not they pose a
risk in the future. Knowledge about (a) the nature and activity of microorganisms involved in various
stages of composting, and (b) the degree of stability of compost and its humic matter content will greatly
assist in better designing of composting as a bioremediation strategy for contaminated soils (Megharaj
et al., 2011).Although co-metabolism can constitute an integral part of successful bioremediation ap-
proach to degradation of xenobiotic compounds, it still has an important limitation which concerns the
researchers. As exemplified by the naphthalene sulfonic acids complete mineralization and utilization
of xenobiotic compounds may be accomplished through complex interactions between single species
of mixed populations. This kind of syntrophic degradation is, however, susceptible to disturbing influ-
ences such as loss and misrouting of metabolites. Thus, under conditions of biological waste treatment
a considerable portion of xenobiotic compounds is co-metabolized by the indigenous microorganisms
to undefined secondary products. This metabolic and chemical misrouting of metabolites constitutes a
major part of the residual organic carbon of the effluent of industrial treatment plants. Surely, bacterial
cells that harbor complete catabolic pathways and thus can degrade xenobiotic compounds completely
and productively should have a clear growth advantage. Nevertheless, an important hurdle of using
specifically adapted microorganisms for remediation of industrial waste streams or contaminated soils
is the material complexity of mixtures of pollutants.
Disadvantages to the in situ application of extra cellular, cell-associated or cell-free enzymes may
also arise from the enzymes. Enzymes may lose their activity upon pollutant transformation. In the soil,
enzymes are present in complex, three dimensional assemblages of mineral and organic particles that
will restrict their mobility and affect their activity. In activation or degradation of enzymatic molecules
may also take place. As a consequence, changes in their kinetics, stability and mobility will occur. These
will determine the operating range of enzymes in soil around microorganisms and enzymes. As regards
to isolated enzymes, a drawback that greatly hampers their practical application is the cost of enzymes
isolation and purification (Mukherjee et al. 2013). Given the best producer of the selected enzyme, the
production of a purified enzyme requires long and expensive isolation and purification procedures.
Furthermore, very low amounts of the purified protein are usually obtained thus rendering the whole
process too costly for practical applications (Gianfreda & Rao, 2004). Immobilized enzymes have usu-
ally a long-term and operational stability, being very stable toward physical, chemical, and biological
denaturing agents. Furthermore, they may be reused and recovered at the end of the process. However,
their large-scale application in the bioremediation of polluted soils is not reported yet, largely due to the
process is not cost effective.

11. FUTURE RESEARCH TOWARDS SOLUTION TO THE LIMITATIONS

1. The biological response to environmental pollutants varies within a microbial guild, and the pres-
ence of co-contaminants can elicit variable responses. The choice of methods in each technology
requires careful consideration. What is now important is to gain a better understanding on the
metabolic cooperation among the microbial communities.

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2. The existing biological waste treatment processes work at an exceedingly slow pace. The term
slow pace refers to the rate of degradation (complete mineralization) of pollutants in the effluent
within a given period, before new effluent arrives to place an additional load of pollutants. This
limitation can be alleviated by pinpointing a rate-limiting step and elevating the level of the rate-
limiting enzyme or its regulatory protein by increasing either transcription/translation of encoding
gene or its stability.
3. Some pollutants are not completely mineralized by microbes. Instead, they are transformed to dead
end products, which can serve as substrate for other microorganisms. By combining the genes
from two parent strains, an effective organism can be engineered to achieve the ultimate goal of
mineralization.
4. Since industrial effluents are mixtures of toxic pollutants (instead of a single toxic pollutants),
strains could be constructed which concomitantly destroy target pollutant along with other toxic
pollutants.
5. The environmental application of genetically engineered microbes needs an appropriate cloning
system, which can (a) perform under particular environmental conditions, (b) be non-transmissible,
(c) be cost-effective, and (d) environment friendly, while not fostering the spread of resistance to
chemotherapeutic antibiotics.
6. The potential of enzymes for bioremediation purposes can greatly increase by the use of micro-
organisms and their enzymes from extreme environments. Enzymes from both thermophilic and
psycrophilic microorganisms usually display some unusual and particular features that may render
them potential, powerful catalysts for the degradation of polluting chemicals even under extreme
environmental conditions.

12. CONCLUSION

Increase in the application of agro-chemicals and contamination with toxic industrial effluents are
resulting in a continued contamination of our environment and food. More and more options are be-
ing researched for environmentally safe solutions like use of biopesticides including biofungicides and
bioherbicides in place of synthetic pesticides. Though biopesticides are expected to become important
tools in future pest management, conventional chemicals will remain to be a significant part of pest
management both in developed and developing countries. Hence it is important to concentrate efforts on
biological transformation of chemical agricultural inputs and on-site remediation strategies be developed
with particular reference to agriculture soils of which bioremediation can play a very important role.
The potential of microorganisms in the remediation of some of the compounds previously known to be
undegradable has been widely acknowledged globally. With advances in biotechnology, bioremediation
has become a rapidly growing area and has been commercially applied for the treatment of hazardous
wastes and contaminated sites. A wide range of bioremediation strategies have been developed for the
treatment of contaminated soils using natural and modified microorganisms. Rapid progress in various
pathways, operative in microbes for the degradation of xenobiotic pollutants have shed more light on its
mechanism. Recent advances in the molecular genetics of degradation and studies on enzyme-tailoring
and DNA-shuffling are on the way to be a robust bioremediation technique. Selecting the most appropri-

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ate strategy to treat a specific site can be guided by considering three basic principles: the amenability of
the pollutant to biological transformation to less toxic products, the bioavailability of the contaminant to
microorganisms and the opportunity for bioprocess optimization. With the help of advances in bioinfor-
matics, biotechnology holds a bright future for developing bioprocesses for environmental applications
towards cleanup of toxic wastes.

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Chapter 10
Biodegradation of Phenol:
Mechanisms and Applications

Vinod K. Dhatwalia
Uttaranchal University, India

Manisha Nanda
Dolphin (PG) Institute of Biomedical and Natural Sciences, India

ABSTRACT
Aromatic compounds are widely distributed in nature. Free phenols are frequently liberated as metabolic
intermediates during the degradation of plant materials. In recent years the natural supply of phenolic
substances has been greatly increased due to the release of industrial byproducts into the environment.
Phenolic compounds are hazardous pollutants that are toxic at relatively low concentration. Effluents
from petrochemical, textile and coal industries contain phenolic compounds in very high concentration;
therefore there is a necessity to remove phenolic compounds from the environment. Microorganisms
capable of degrading phenol are common and include both aerobes and anaerobes. The use of micro-
bial catalysts in the biodegradation of organic compounds has advanced significantly during the past
three decades. The efficiency of biodegradation of organic compounds is influenced by the type of the
organic pollutant, the nature of the organism, the enzyme involved, the mechanism of degradation and
the nature of the influencing factors.

INTRODUCTION

Phenol (hydroxy benzene) is an organic aromatic compound (C6H5OH) consisting of a phenyl group
(-C6H5) bonded to a hydroxyl group (-OH) Figure 1. It is a volatile, white crystalline solid with acidic
nature. Phenol naturally occurs in decaying dead organic matters (rotting vegetables) and coal. At room
temperature phenol is a translucent, colorless, crystalline mass, white powder or syrupy liquid when
mixed with water. The crystals are hygroscopic and turn pink to red in air. Phenol has a sweet tar like
odour and is soluble in alcohol, glycerol, petroleum and water to a lesser extent. Phenol nowadays is
produced from petroleum on a large scale (about 7 billion kg/year). Generally phenol is synthesized from
1-methylethylbenzene (cumene), which can be used as an indication of the levels of phenol production.

DOI: 10.4018/978-1-4666-9734-8.ch010

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.

Biodegradation of Phenol

Figure 1.

(Basha et al., 2010). A germen chemist, Runge in 1834, first isolated phenol from coal tar and named
it as karbolsaure (coal-oil acid or carbolic acid), but its composition was not known until 1841. Phenol
can be produced by synthetically and naturally. By fractional distillation of coal tar phenol is obtained
naturally. Phenol was first used in the raw state, as creosote, to prevent the weathering of railway ties and
ships timber. It was also used to reduce the odour of decomposition in sewage. It is frequently used in
pharma Industries, synthetic resin, dyes, pesticides, etc. Phenol is very toxic and its increasing presence
displays a significant environmental toxicity hazard. Acute exposure of phenol can result in myocardial
depression and central nervous system disorders. A large number of microorganisms are capable of
degrading phenol with bacteria as the major player. Characterization of bacteria that are capable of de-
grading phenol has resulted in bringing out the possible biological mechanism to remediate the phenol
contaminant in the environment.

Uses of Phenol

Phenol has been produced since 1860s. By the end of the 19th century, industrial scientists revealed
many applications of phenol. It is widely used in the synthesis of dyes, aspirin, and one of the first high
explosives, picric acid. As early as in 1872, it was found that phenol could be condensed with aldehydes
(for example methanal) to make resinous compounds, a process still in use today. Phenolmethanal
(formaldehyde) resins are the basis of the oldest plastics. It is still used to make low cost thermosetting
plastics such as melamine and bakelite used in electrical equipment. Phenol is widely used to make
pharmaceuticals, perfumes, pesticides, synthetic tanning agents, lubricating oils and solvents. Due to
its wide application phenolic compounds are frequently found in effluent from coke-oven batteries, coal
gasification refinery, petrochemical plants and other industries, such as herbicides, synthetic chemicals,
pesticides, pulp-and-paper, photo developing chemicals, antioxidants, tannery and foundries (washing
of the gas effluents) etc. (Basha et al., 2010) (see Figure 1).

Global Phenol Production

There was a decline in the global phenol during the recession, but they returned to normal in 2010. The
world phenol production from 2010 to 2012, showed a positive annual growth (nearly 2.5%) Figure 2
(Global phenol production broken down by country, 2012) and figure 3 (Phenol capacity broken down
by region, 2012). It increased from 8.34 million tonnes in 2010 to more than 8.9 million tonnes in 2012.
APAC accounted for over 41% (above 3.7 million tonnes) of the overall output volume and became the

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Biodegradation of Phenol

Figure 2.

world’s unrivalled leader in phenol production. With a combined production of more than 5.5 million
tonnes in volume China, the USA, Japan, Taiwan and South Korea were the top five phenol manufactur-
ing countries in 2012 (Merchant Research & Consulting, Ltd 2014).

Phenol Production through Micro Reactor Technology

In the near future, comparatively small reactors (Figure 4) may be used for the production of phenol and
many other chemicals. One such potential micro-reactor for the production of phenol involves the use of
a small diameter (2 mm), porous tube of alumina coated with a layer of palladium metal. A mixture of
benzene and oxygen is fed through the tube and hydrogen gas is passed over the tube. The temperature of
the tube is then maintained to 150 - 250°C. Further, the palladium catalyst coverts Hydrogen to atomic

Figure 3.

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Biodegradation of Phenol

Figure 4.

hydrogen as it permeates though the alumina tube. Oxygen atoms released by the reaction of hydrogen
atoms with oxygen gas. These oxygen atoms react with benzene forming benzene epoxide which finally
isomerises to phenol. Easy separation of phenol is favoured by the differences in boiling points of phenol
(182°C) and benzene (80°C). Thus, finally liquid phenol is obtained in a highly pure form. Researchers
claim that this method saves on capital cost and also reduces the energy and waste. This method can be
easily scaled up by the addition of more tubes effectively using a modular approach. One single micro-
reactor could produce up to 100,000 tonnes of phenol per year. This technology can also be applied to
the manufacture of other materials (Basha et al 2010).

Toxicity of Phenol

Phenol and its derivatives are toxic and classified as hazardous materials (Zumriye & Gultac, 1999).Several
central nervous system disorders are a consequence of excessive exposure to phenol including collapse,
coma and muscular convulsions. Muscle weakness and tremors are also seen sometimes. It also leads to
a reduction in body temperature, this is known as hypothermia. Mucus membrane is also very sensitive
to the action of phenol. Acute exposure of phenol can cause myocardial depression. Phenol exposure
causes a burning effect on skin ultimately leading to whitening and erosion of the. Usually <10 mg/L
urine of the person is normal level of phenol. Once absorbed, phenol is widely distributed throughout
the body and the liver and kidneys generally have the greatest amount of phenol-derived products. Renal
damage and salivation are also the consequences of continuous exposure to phenol. Excessive exposure
to phenol can also cause irritation of the eye, conjunctional swelling, corneal whitening and finally
blindness. Other effects include frothing from nose, mouth and headache. Hepatic damage can also be
induced by phenol. Chronic exposure may result in anorexia, dermal rash, dysphasia, gastrointestinal
disturbance, vomiting, weakness, weightlessness, muscle pain, hepatic tenderness and nervous disorder. It
is also suspected that exposure to phenol may cause paralysis, cancer and gene to fibre striation. Phenolic
compounds display varying degrees of toxicity. Thus, their fate in the environment is important (Bollag
et al., 1988). In recent years, most of the research work has been focused towards the development of
enzymatic processes to remove phenolic contaminants (Ghioureliotis & Nicell, 1999). Phenol is also
an antiseptic agent and is used in surgery, which indicates that it is also toxic to many microorganisms
(EPA, 1979), and may also have antiseptic properties when gargled as a mouthwash.

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Biodegradation of Phenol

Regulations of Phenol

Phenol is listed as a hazardous waste under the Resource Conservation and Recovery Act (RCRA). Vari-
ous regulatory authorities have imposed strict limits to phenol concentration in industrial discharges,
drinking waters and workroom. According to the guidelines prescribed by WHO (1994) a concentra-
tion of 1 μg/l phenol is allowed for drinking waters. The Environmental Protection Agency (EPA) has
declared that the concentration of phenol in lakes and streams should be limited to 0.3 milligrams per
liter of water (0.3 mg/l). This would protect human health from the possible harmful effects of phenol
contaminated drinking water, plants and animals (ATSDR, 2008).

Mechanism of Phenol Biodegradation

Biological treatments convert the wastes into simple end products (Pradeep et al., 2011). Thus, the use
of biological methods is preferred over chemical methods (Lika & Papadakis, 2009). The rate and extent
of biodegradation of a chemical compound largely depends upon its structure and the environment in
its vicinity (Annachatre & Gheewala, 1996). Biological removal can be classified into microbial and
enzymatic methods.

Microbial Removal of Phenol

Soil microflora appears to be an important means by which phenolic substances are removed from the
environment. Phenol can be degraded by the activity of a large number of microorganisms including
bacteria, fungi and actinomycetes. Bacterial species that can effectively remove phenol include Bacillus
sp., Pseudomonas sp., Acinetobacter sp., Achromobacter sp.etc (Table 1). Phenol can also be biodegraded
by fungal species like Fusarium sp, Phanerocheate chrysosporium, Corious versicolor, Ralstonia sp,
Streptomyces sp. etc. However, the growth of these microorganisms is inhibited at higher concentrations
of phenol (Prieto et al., 2002).
Phenol is metabolised by dihydroxylating the benzene ring into a catechol derivative which is further
converted to an open ring through ortho- or metaoxidation (Figure 5). The enzyme catechol 1,2-dioxy-
genase oxidizes catechol to acetaldehyde and pyruvate via ortho-cleavage pathway (Figure 6). Catechol
can also be oxidized by the metapathway to 2-hydroxymuconic semialdehyde by the enzyme catechol
2,3-dioxygenase. The final products of both the pathways can enter the tricarboxylic acid (TCA) cycle.
Catechols are cleaved either by ortho-fission (intradiol, i.e., carbon bond between two hydroxyl groups)
or by a metafission (extra diol, i.e., between one of the hydroxyl groups and a nonhydroxylated carbon)
as given in Figure 5 and Figure 6. Finally the ring is opened and subsequently degraded.

Aerobic Biodegradation of Phenol

Aerobic biodegradation of phenol results in its complete mineralization. This method is generally pre-
ferred in wastewater cleanup. Phenol is recognized as an inhibitory substrate at relatively low concentra-
tions (100 mg/L) and is a preferred model for studying the kinetics of aromatic molecule degradation

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Table 1. Microorganisms in the biodegradation of phenolic compounds.

S/N Types of Phenol Microorganisms (Bacteria) Reference

1 Phenol Pseudomonas putida Allsop et al. (1993)
2 2- cholrophenol Pseudomanas putida Overmeyar and Rehm (1995)
3 Phenol Pseudomonas sp. Bodzek et al. (1996)
4 Phenol Pseudomonas sp. Gotz and Reuss(1997)
5 Phenol Bacillus sp. Ali et al. (1998)
6 Phenol Rhizoctonia praticola Bollag et al. (1988)
7 Phenol Trametes trogii Garzillo et al. (1998)
8 Phenol Pseudomonas putida Loh and Wang (1998)
9 Phenol Pseudomonas flurorescens Torres et al. (1998)
10 Phenol Pseudomonas putida Mordocco et al. (1999)
11 Phenol Ralstonia eutropha Leonard et al. (1999 a,b)
12 Phenol Pseudomonas putida Wang and Loh (1999)
13 Phenol Pseudomonas putida Zumriye and Gultac (1999)
14 Phenol Pseudomonas pictorium Annadurai et al. (2000)
15 Phenol, Nitrophenol Nocardioides Cho et al. (2000)
16 Phenol Acinetobacter calcoaceticus Nakamura and Sawada (2000)
17 Phenol Streptomyces setonii An et al. (2001)
18 Phenol Alcaligenes sp. Baek et al. (2001)
19 Phenol Pseudomonas sp. Gonzalez et al. (2001)
20 Phenol Pseudomonas putida Loh and Jun (2001)
21 Phenol Acinetobacter sp. Hao et al. (2002)
22 Phenol Rhodococcus erythropolis Prieto et al. (2002)
23 Chloro phenol Achromobacter sp. Xiangchun et al. (2003)
24 Phenol Alcaligenes sp. Nair and Shashidhar (2004)
25 Pentachlorophenol Sphingomonas chlorophenolica Bielefeldt and Cort (2005)
26 Phenol Bacillus brevis Arutchelvan et al. (2006)
S/N Types of Phenol Microorganisms (Fungus) Reference
1 Phenol Aspergillus terreus Garcia Garcia et al., 1997
2 Phenol Coriolus versicolor Kadhim et al. (1999)
3 Phenol Coprinus cinereus Schneider et al. (1999)
4 Phenol Phanerocheate chrysosporium Garcia et al. (2000)
5 Phenol Pleurotus ostreatus Hublik and Schinner (2000)
6 Phenol Chalara paradoxa Robles et al. (2000)
7 Phenol Aspergillus niger Garcia et al. (2000)
8 Phenol Trichosporon cutaneum Godjevargova et al. (2003)
9 Phenol Fusarium sp. Santos and Linardi (2004)
10 Nonyl phenol Clavariopsis aquatic Moeder et al. (2006)

continued on following page

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Table 1. Continued

S/N Types of Phenol Microorganisms (Yeast) Reference

1 Phenol Candida maltosa Ariana Fialova et al. (2004)
2 Candida tropicalis Salmeron- Alcocer et al. (2007)
S/N Types of Phenol Microorganism (Algae) Reference
1 Phenol Ochromonas danica Semple and Cain (1995)
2 Phenol Ankistrodesmus braunii Gabriele Pinto et al. (2002)
3 Phenol Scenedesmus quadricauda Gabriele Pinto et al. (2002)

(Christen et al., 2012). Microbial growth is inhibited by higher concentrations of phenol. Therefore,
various approaches were used to overcome this substrate inhibition. Aerobic biodegradation was first
studied in the early 19th centuries. In the first step of the aerobic pathway (Figure 6) for the biodegrada-
tion of phenol, molecular oxygen is used by the enzyme phenol hydroxylase to add a second hydroxyl
group in ortho-position to the one already present to form catechol(1, 2- dihydroxybenzene). This step
requires a reduced pyridine nucleotide (NADH2). Catechol can be further degraded via two alternative
pathways. In the ortho- or β-ketoadipate pathway, the enzyme catechol 1, 2-dioxygenase cleaves the
aromatic ring between the catechol hydroxyls (intradiol fission) (Parales, 1996; Stanier and Ornston,
1973). Evans and Kilby (1948) first reported the production of β-ketoadipate during the degradation of
phenol by strain ‘Vibrio 01’. The resulting cis, cis muconate is further metabolized via β-ketoadipate to
Krebs cycle intermediates. In the meta-pathway, extradiol fission occurs where the ring fission occurs
adjacent to the two hydroxyl groups of catechol .The enzyme catechol 2, 3-dioxygenase transforms
catechol to 2-hydroxymuconic semialdehyde. This compound is metabolized further to intermediates
of the Krebs cycle (Figure 6).

Anaerobic Biodegradation of Phenol

Phenol can also be degraded anaerobically (in the absence of oxygen). This technology is less advanced
than the aerobic process. It is based on the analogy with the anaerobic benzoate pathway proposed by
Williams and Evans (1975) for Paracoccus denitrificans. In the first step in this anaerobic pathway
phenol is carboxylated in the para position to 4 hydroxybenzoate by the enzyme 4-hydroxy benezoate
carboxylase. The anaerobic degradation of several other aromatic compounds has been shown to include
a carboxylation reaction. For denitrifying Paracoccus like organisms, carboxylation of the aromatic ring
in para position to the hydroxyl group of o-cresol resulting in 3-methyl 4-hydroxybenzoate has been
reported. Methogenic consortium was later shown to travel a varity of phenolic compounds including
o-cresol, catechol and ortho halogenated phenols via para carboxylation followed by dehydroxylation.
Energy rich biogas and a nutrient-rich digestate are produced during anaerobic digestion of organic
waste. If the levels of hazardous compounds and pathogens are low in the digestate it can be used as a
fertilizer in agricultural soils. Chemical analysis of digestate from bioreactors operating at thermophilic
temperature has detected higher content of phenols compared to mesophilic bioreactors, verifying the
degradation results. Digestate with the highest phenol content has the greatest negative impact on soil
microbial activity (Leven et al., 2012) (see Table 1).

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Biodegradation of Phenol

Figure 5. Figure 6.

Enzymatic Polymerization of Phenol

Enzymes are proteins. Hundreds of enzymes are found in all types of living cells. Enzymes are bio- cata-
lysts that increase the rate of chemical reactions taking place within living cells. An enzyme increases
the rate of reaction, but it does not affect the reaction equilibrium. The acceleration achieved by enzymes
is often tremendous, with some reaction rates increased to one million times faster than the rate in the
absence of enzymes (Bailey & Ollis, 1986). Enzymes catalyze only specific reactions. They generally

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Biodegradation of Phenol

act under moderate conditions of temperature, pH, solvents and ionic strength. Enzymes appear to be a
promising tool for the selective removal of pollutants from waste streams (Demarche et al., 2011). The
mechanism for degradation of aromatic compounds by the enzyme peroxidases is as following:

E + H2O2 →Ei + H2O (1)

Ei + AH2→Eii + AH. (2)

Eii + AH2→E + AH. + Eii (3)

Eii + H2O2→ Eiii + H2O. (4)

Hydrogen peroxide (H2O2) oxidizes the native enzyme (E) to an active intermediate enzymatic form
called compound I (Ei). The compound I accepts an aromatic compound (AH2) into its active site and
carries out its oxidation (Pradeep et al., 2011). A free radical (AH) is produced and released into solu-
tion. This leaves the enzyme in the compound II (Eii) state. Compound II oxidizes a second aromatic
molecule, generating another free radical product. The enzyme then returns to its native state, thereby
completing the cycle. The overall peroxidase reaction are described by Eqs. (1)–(3). In the presence of
excess hydrogen peroxide, the reaction of Eq. (4) becomes important because compound III (Eiii) is a
reversibly inactivated form of the enzyme. This describes that the enzyme is inhibited by H2O2 in excess.
On the other side, the rate of reaction is limited by the lack of hydrogen peroxide during the reaction
step. To suppress this inhibition semi-batch addition of H2O2 can be done. This maintains an optimized
ratio between hydrogen peroxide and enzyme concentrations (Kulkarni & Kaware 2013; Wilberg et al.,
2000) (see Table 2).

Factors Affecting Biodegradation of Phenol

Phenol can be degraded both aerobically and anaerobically (Shashidhar et al., 2008; Nuhoglu & Yalcin,
2005; Jin et al., 2009). The process of Biodegradation is regulated by many factors like temperature,
pH, chemical structure and substrate concentration (Trigo et al., 2009; Shashidhar et al., 2008; Agarry
et al., 2008). In order to achieve the maximum biodegradation of the desired organic compound each of
these factors needs to be optimized.

Effect of Substrate Concentration

Phenol biodegradation by microbes is inhibited by substrate itself at higher concentrations. Thus, the
optimization of the substrate concentration for biodegradation of phenols is important.

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Biodegradation of Phenol

Table 2. Enzymes involved in the biodegradation of phenolic compounds.

S/N Types of Phenol Enzyme Reference

1 Phenol Polyphenol Oxidase Burton et al. (1993)
2 Phenol Polyphenol Oxidase Okeke Cano et al. (1997)
3 Phenol Polyphenol Oxidase Shashirekha et al. (1997)
4 Phenol Phenol Oxidase Okeke et al. (1997)
5 Phenol Catechol 2,3 dioxygenase Ali et al.(1998)
6 Phenol Laccase Bollag et al. (1998)
7 Phenol Polyphenol oxidase Garzillo et al. (1998)
8 Phenol Peroxidase Ghioureliotis and Icell (1998)
9 Phenol Horse radish peroxidase Wu et al. (1998)
10 Phenol Horse radish peroxidase Zahida et al. (1998)
11 Phenol Polyphenol oxidase Edwards et al. (1999)
12 Phenol Laccase Kadhim et al. (1998)
13 Phenol Laccase Schneider et al. (1999)
14 Methoxyphenol Laccase Setti et al. (1999)
15 Phenol Laccase Hublik and Schinner (2000)
16 Phenol Laccase Robles et al. (2000)
17 Phenol Catechol 1,2oxygenase An et al. (2001)
18 Phenol Polyphenol oxidase Luke and Burton (2001)
19 Bis phenol Peroxidase Sakurai et al. (2001)
20 Phenol Polyphenol oxidase Steffens (2002)
21 Phenol Phenol oxidase Johjima et al. (2003)
22 Phenol Tyrosinase Xiangchun (2003)
23 Lignophenols Peroxidase Xia et al. (2003)

Effect of pH

Extreme pH values of the wastewater (less than 3 or greater than 9) inhibit the growth of microorganisms.
Most of soils have values of pH between 5.0 and 9.0 which is optimal for microbial enhanced biodegra-
dation of waste contamination. Most fertile native soils maintain this pH by a natural buffering capacity
due to the presence of carbonates and other minerals. However, this buffering capacity can be depleted
over time as a result of acidic by products of degradation. Although microbes can adapt to a broader
range of pH values, there typically is an accompanying decrease in growth/metabolic rates. Generally,
laboratory studies on phenol biodegradation are carried out near neutral pH (pH = 7.0).

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Biodegradation of Phenol

Effect of Temperature

Each microorganism has an optimum temperature range for growth. Thus, temperature plays an important
role in the degradation of organic pollutants by microbes. Generally, sudden exposure to temperatures
higher than 350 oC displays detrimental effect on the bacterial enzymes responsible for the benzene
ring cleavage. Whereas, exposure to temperatures lower than 300 oC slows down the bacterial activity.
P. putida has been reported to degrade phenol at a lower temperature (i.e., 100oC) than Bacillus stearo-
thermophiles that effectively degrades phenol at a temperature of about 500 oC (Williams et al., 1975).

Effect of Chemical Structure (Substituents Present on Ring)

Chemical structure is an important factor that can affect the biodegradation of phenols. It is determined
by the number of substituents, type of substituents, position of substituents and degree of branching. More
the number of substituents in the structure, the less biodegradable it becomes. It has been reported that
substituted phenols such as mono, di-, tri-, and pentachlorophenol are less degradable than unsubstituted
phenol. Also, o- and p-substituted phenols are more degradable than m-substituted phenols (Agarry et
al., 2008).

CONCLUSION

Phenol has become one of the major components of the industrial effluent. Thus, the degradation of
phenol has become one of the major environmental issues. For the degradation of phenol many physical
and chemical methods have been in use at present. An alternative method for degradation is biological
process or “Bioremediation”. It is a relatively cheaper and effective process that releases lesser harmful
products. Phenol can biodegraded both by aerobic and anaerobic processes. In the aerobic process the
end product is carbon dioxide and in the anaerobic process the end product is methane or carbon dioxide.
Biodegradation of phenol is regulated by many factors like temperature, pH, chemical structure and sub-
strate concentration. These factors must be optimized in order to achieve the maximum biodegradation
of the desired organic compound.

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Chapter 11
Phyto-Remediation: Using
Plants to Clean Up Soils:
Phyto-Remediation

Swati Jagdale
MAEER’s Maharashtra Institute of Pharmacy, India

Aniruddha Chabukswar
MAEER’s Maharashtra Institute of Pharmacy, India

ABSTRACT
In this chapter authors have discussed the role of plants to develop contaminant free environment. This
concept is also known as Phytoremediation. Phytoremediation is a word formed from the Greek prefix
“phyto” meaning plant, and the Latin suffix “remedium” meaning to clean or restore. This technology
has been receiving attention lately as an innovative, cost-effective alternative to the more established
treatment methods used at hazardous waste sites. Phytoremediation can be classified into different applica-
tions, such as phytofiltration or rhizofiltration, phytostabilization, phytovolatilization, phytodegradation
and phyto-extraction etc. The chapter will deal with phytoremediation, its advantages, limitations and
in detail techniques of classification and application.

1. INTRODUCTION

Soil is the fundamental foundation of our agricultural resources, food security, global economy and
environmental quality. Increasing demand for agricultural products has led to extensive cultivation in
agricultural lands (Oh et al., 2013). Applying fertilizers, pesticides and herbicides is necessary to protect
the quality and quantities of these products. However, the excessive use of these agro-chemicals creates
environmental problems, such as accumulation of these chemical substances in the soil and plant uptake
(Sahibin et al., 2002). With the development of urbanization and industrialization, soils have become
increasingly polluted by heavy metals and organic pollutants, which threaten ecosystems, surface and
ground waters, food safety and human health (Li et al., 2009). The main factors contributing to soil
pollution are the increased growth of industry; nearly 1000 new chemicals are being synthesized every
year (Shukla et al., 2010). According to Third World Network reports, more than one billion pounds
DOI: 10.4018/978-1-4666-9734-8.ch011

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Phyto-Remediation: Using Plants to Clean Up Soils

(450 million kilograms) of toxins are released globally in air and water. Therefore environmental pollu-
tion with organic xenobiotics (pesticides, pharmaceuticals, petroleum compounds, polychlorobiphenyls
(PCBs) and polycyclic aromatic hydrocarbons (PAHs) etc.) is a global problem (Woolhouse, 1983).
Excessive metal concentration in soils pose significant hazard to human, animal and plant health, and
to the environment in general. Contamination of soils with toxic metals has often resulted from human
activities, especially those related to mining, industrial emissions, disposal or leakage of industrial
wastes, application of sewage sludge to agricultural soils, manure, fertilizer and pesticide use. Due to
the potential toxicity and high persistence of metals, soils polluted with these elements are environmen-
tal problem that requires an effective and affordable solution. Although a number of techniques have
been developed to remove metals from contaminated soils, many sites remain contaminated because of
economic and environmental costs to clean up those sites with the available technologies are too high.
According to Ensley (2000), the estimated expenses incurred in the remediation of a site contaminated
with Pb using the conventional excavation-landfill approach most commonly practiced in the United
States are approximately $150-$350 t-1. Taking into account such a high demand of economic resources,
methods of environmental restoration of metal-polluted soils using a plant-based technology have at-
tracted increasing interest in the last two decades. In this context, phytoremediation has been developed
as a cost effective and environmentally friendly remediation method of contaminated soils.
The idea of using metal-accumulating plants to remove heavy metals and other compounds was first
introduced in 1983, but the concept has actually been implemented for the past 300 years on wastewater
discharges. The generic term phytoremediation consists of the Greek prefix phyto (plant) attached to the
Latin root remedium (to correct or remove an evil) (Cunningham et al., 1996).Phytoremediation tech-
nology is an in innovative field of science and technology for cleaning up contaminated soil, water and
air (Pulford and Watson,2003). Certain plants have endogenous, genetic biochemical and physiological
qualities to combat against the soil, water and air pollution (Meagher, 2000). Phytoremediation, also
called green remediation, botano-remediation, agroremediation, or vegetative remediation is considered
a publicly appealing (green) remediation technology that uses vegetation and associated microbiota, soil
amendments and agronomic techniques to remove, contain, or render the heavy metals harmless in the
soil (Cunningham et al., 1996). Phytoremediation for metal-contaminated soils represents a market op-
portunity of approximately US$1 billion per year in the USA alone; the U.S. phytoremediation market
currently comprises only 0.5% of the total remediation market, equivalent to circa US$ 100-150 million
per year (Glass, 2000) (see Table 1).

2. SIGNIFICANCE OF PHYTOREMEDIATION (VISHNOI ET AL., 2008 )

1. Plants control 80% of the energy in most ecosystems and do not need external energy sources.
a. Photosystem I make NADPH.
b. Reduce CO2 and make large biomass.
c. Can reduce toxic metal ions.
2. Plants grow extensive root systems (100 million/acre/yr).
a. Plants mines 16 metals for normal growth.
b. Some plants hyper accumulate heavy metals.
c. Some plants degrade toxic organic chemicals.
3. Phytoremediation is a sound support to bacterial remediation methods.

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Table 1. Advantages and limitations of phytoremediation (Paulo et al., 2014)

S. No Advantages Limitations
1. In situ and passive technique Limited to shallow soils or where contamination is localized to the surface (< 5 m)

2. Uses solar energy and is low cost Still under development and therefore not accepted by many regulatory agencies

3. Has reduced environmental impact and contributes to the There is little knowledge of farming, genetics,
landscape improvement reproduction and diseases of phytoremediating plants

4. High acceptance by the public Metal concentrations in the soil can be toxic and lethal to plants

5. Provides habitat for animal life Generally, plants are selective in metal remediation

6. Reduction in dispersal of dust and contaminants by wind Treatment slower than the traditional physico-chemical techniques

7. Reduction of surface runoff Contamination may spread through the food chain if accumulator plants are ingested
by animals

8. Reduction of leaching and mobilization of contaminants Efficient phytoremediating plants may not adapt to climatic and environmental
in soil conditions at contaminated sites

9. Harvesting of the plants or organs that have accumulated If the plants release compounds to increase the mobility of the metals, these can be
metals is easy to accomplish with existing technology leached into groundwater

10. The harvested biomass can be economically valuable The area to be decontaminated must be large enough to allow application of
cultivation techniques

11. Plant process more easily controlled than those of Toxicity and bioavailability of degradation products remain largely unknown
microorganisms

4. Phytoremediation is affordable on grand scale needed for marginal land reclamation and cleaning
the water in lakes, streams and marshes.
5. Phytoremediation is a rapid gaining support by the public as “Green” solution to our environmental
problems.

Plants that are able to decontaminate soils do one or more of the following:

1. Plant uptake of contaminant from soil particles or soil liquid into their roots;
2. Bind the contaminant into their root tissue, physically or chemically; and
3. Transport the contaminant from their roots into growing shoots and prevent or inhibit the contami-
nant from leaching out of the soil.

The remediation of a site using phytoremediation depends on many factors. Some major factors are
type of contaminant, type of plant, contamination levels, size and depth of the contaminated area, site
conditions such as nutrient availability, soil organic matter content, soil water, soil aeration, and other
desirable soil quality parameters conducive to initiation and survival of plant species, type and number
of plants needed to remediate the site.

3. GENETIC ENGINEERING OF PLANTS

To create suitable plants for phytoremediation, one possibility is the selection of high biomass plants
like poplar, Brassica juncea, or tobacco with the highest tolerance and accumulation capacities, provided
that the species displays sufficient genetic variation for those characteristics. Another avenue of recent

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Phyto-Remediation: Using Plants to Clean Up Soils

research is the association of metal tolerant micro-organisms like bacteria or fungi to enhance tolerance.
In this alternative, the genetic engineering of plant-associated bacteria seems promising. Improvement
of hyperaccumulator biomass is also envisaged by the genetic manipulation of plant hormone levels . A
hyper accumulator is a plant capable of growing in soils with very high concentrations of metals, absorb-
ing these metals through their roots, and concentrating extremely high levels of metals in their tissues.
The metals are concentrated at levels that are toxic to closely related species not adapted to growing on
the metalliferous soils. Compared to non-hyperaccumulating species, hyperaccumulator roots extract
the metal from the soil at a higher rate, transfer it more quickly to their shoots, and store large amounts
in leaves and roots. The ability to hyperaccumulate toxic metals compared to related species has been
shown to be due to differential gene expression and regulation of the same genes in both plants. Genetic
engineering of plants can substantially improve phytoremediation efficiency of plants. Genetic ma-
nipulation has aimed to improve accumulation, tolerance and detoxification capacities of high biomass
and rapid growing plants in order to optimise the phytoextraction process. The choice of genes to be
transferred is difficult. Many genes are involved in metal uptake, translocation, and sequestration. The
genes to be transferred depend on the heavy metal to be extracted from the soil. At least three catego-
ries of genes are generally considered: transporters, proteins involved in metal chelation, and metabolic
enzymes involved in detoxification. Transgenic plants with improved metal uptake and/or sequestration
have been developed for cadmium, zinc, lead, mercury, arsenic and selenium. The genes that have been
transferred, thus far, are mainly cloned from micro-organisms for which the tolerance or detoxification
mechanisms were known or from the non-tolerant, non-accumulator model plant species, Arabidopsis
thaliana but usually not from hyperaccumulators. A common strategy to identify genes necessary to
tolerate heavy metals or non-metals is to study plants that are naturally adapted to those toxic trace ele-
ments. Hyperaccumulators constitute an exceptional biological material and gene reservoir to understand
adaptation to extreme metallic environments. Recently transcriptomic studies on hyperaccumulators have
provided novel insights into the molecular mechanisms underlying metal tolerance and accumulation
as well as access to the identification of a large array of genes which are constitutively (in the absence
of excess of metallic ions) over expressed and are thought to be involved in the hyperaccumulation trait
(Verbruggen et al., 2009) (see Figure 1).

4. TECHNIQUES OF PHYTOREMEDIATION

For removal of different hazardous compounds from contaminated soil and water, plant potentials have
been exploited that resulted in several technological subsets. Schwitzguebel (2000) has gave different
techniques of phytoremediation as:

1. Phytoextraction: The use of pollutant accumulating plants to remove pollutants like metal organics
from soil by concentrating them in harvestable plant parts.
2. Phytotransformation: The degradation of complex organic to simple molecules or the incorpora-
tion of these molecules into plants tissues.
3. Phytostimulation: Plant-assisted bioremediation or the stimulation of microbial and fungal deg-
radation by release of exudates / enzymes into the root zone (rhizosphere).
4. Phytovolatilization: The use of plants to volatilize pollutants or metabolites.

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Phyto-Remediation: Using Plants to Clean Up Soils

Figure 1. Transition mechanism in plants for metal accumulation (Singh et al, 2011)

5. Phytodegradation: Enzymatic breakdown of organic pollutants such as trichloroethylene (TCE)

and herbicides, both internally and externally and through secreted plant enzymes.
6. Phytorhizofiltration: The use of plant roots to ab/adsorb pollutants, mainly metals, but also organic
pollutants, from water and aqueous waste streams, Pump and tree (Dendroremediation): the use of
trees to evaporate water and thus to extract pollutants from the soil.
7. Phytostabilization: The use of plants to reduce the mobility and bioavailability of pollutants in
the environment, thus preventing their migration to groundwater or their entry into the food chain.
8. Hydraulic Control: The control of the water and the soil field capacity by plant canopies.

5. PHYTOEXTRACTION/PHYTOACCUMULATION/PHYTOABSORPTION

Phytoextraction involves removal of toxins, especially heavy metals and metalloids, by the roots of the
plants with subsequent transport to aerial plant organs (Salt et al., 1998). Pollutants accumulated in stems
and leaves are harvested with accumulating plants and removed from the site. The aim of phytoextraction
is reducing the concentration of metals in contaminated soils to regulatory levels within a reasonable
time frame. This extraction process depends on the ability of selected plants to grow and accumulate
metals under the specific climatic and soil conditions of the site being remediated. Two approaches have
currently been used to reach this goal: the use of plants with exceptional, natural metal-accumulating
capacity, the so-called hyperaccumulators, and the utilization of high-biomass crop plants, such as corn,
barley, peas, oats, rice, and Indian mustard with a chemically enhanced method of phytoextraction. The
main characteristics of these two phytoextraction systems are summarized in Table 2 (Nascimento et
al., 2006).
Phytoextraction can also be divided into two categories i.e. continuous and induced. Continuous
phytoextraction requires use of plants that accumulate particularly high levels of the toxic contaminants

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Phyto-Remediation: Using Plants to Clean Up Soils

Table 2. Main characteristics of the two strategies of phytoextraction of metals from soils

Chemically Assisted Phytoextraction Natural Phytoextraction

Plants are normally metal excluders Plants naturally hyperaccumulate metals
Fast growing, high biomass plants Slow growing, low biomass production
Synthetic chelators and organic acids are used to enhance metal uptake Natural ability to extract high amount of metals from soils
Chemical amendments increase the metal transfer from roots to shoots Efficient translocation of metals from roots to shoots
Low tolerance to metals; the increase in absorption leads to plant death High tolerance; survival with high concentrations of metals in
tissues
Risk of leaching of metal chelates to groundwater No environmental drawback regarding leaching of metals

throughout their lifetime. The roots of the established plants absorb metal elements from the soil and
translocate them to the above-ground shoots where they accumulate (hyperaccumulators), while induced
phytoextraction take place if metal availability in the soil is not adequate for sufficient plant uptake,
chelates or acidifying agents may be used to liberate them into the soil solution. This technique employs
metal chelators, such as ethylenediaminetetraacetic acid (EDTA), N-hydroxyethyl-EDTA (HEDTA) or
citric acid in an attempt to increase the metal uptake capacity and the translocation of metals inside plants
from high biomass species (Huang et al., 1997; Lasat et al., 1998). Taking into account the negative
side-effects of applied synthetic chelators, the use of root-produced agents which are naturally degrad-
able by microorganisms is preferable.
More benefits are derived through phytoextraction. It enables scientists to reclaim and recycle usable
materials, including a wide variety of precious metals from the soil. Also, its potential benefits are ex-
tremely high and extremely attractive to scientists and businessmen alike. Furthermore, phytoextraction
is economical because only solar energy must be present to maintain the system . Finally, the greatest
advantage of this technology is that it utilizes the inherent agronomic benefits of plants. These benefits
include high biomass, extensive root systems that both stabilize the ecosystem by preventing contaminant
to spread through leaching as well as reaching a large volume of contaminated soil and a greater ability
to withstand adverse environmental conditions and interspecies competition than bacteria . Salt et al.
(1995) reported that the costs involved in phytoextraction would be more than ten times less per hectare
compared to conventional soil remediation techniques. It can be applied in mineral industry to commer-
cially produce metals by cropping (Sheoran et al., 2009). As extensive as these benefits are, the possible
costs of using plants for bioremediation should not be ignored (Annie et al., 2013). Phytoextraction also
has environmental benefits because it is considered a low impact technology. Furthermore, during the
phytoextraction procedure, plants cover the soil and erosion and leaching will thus be reduced. With
successive cropping and harvesting, the levels of contaminants in the soil can be reduced (Vandenhove et
al., 2001). However, potential limitations and risks should be considered when employing phytoextrac-
tion and these concerns should be considered during the design of any operation. A principle concern
is the slow growth rates of plants and therefore, the low rate of production compared to mechanical
methods. Phytoextraction has also been referred to as phytomining or biomining. A narrower defini-
tion of phytomining is the use of plants to obtain an economic return from metals extracted by a plant,
whether from contaminated soils or from soils having naturally high concentrations of metals (Brooks,
1998). Plants may use two strategies to deal with high metal concentrations adjacent to their roots: 1)

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Phyto-Remediation: Using Plants to Clean Up Soils

exclusion (avoidance) mechanisms by which the uptake and/ or root-to-shoot transport of metals are
restricted; and 2) internal tolerance mechanisms that immobilize, compartmentalize or detoxify metals
in the symplasm through production of metal binding compounds (Kupper et al., 1999). The goal of
phytoextraction is to maximize metal accumulation in plant tissues; mechanisms of internal tolerance
are likely to be important. Phytoextraction is mainly applied to metals (Cd, Ni, Cu, Zn, Pb) but can also
be used for other elements (Se, As) and organic compounds. This technique preferentially uses hyperac-
cumulator plants that have the ability to store high concentrations of specific metals in their aerial parts
(0.01% to 1% dry weight, depending on the metal). Elsholtzia splendens, Alyssum bertolonii, Thlaspi
caerulescens and Pteris vittata are known examples of hyperaccumulator plants for Cu, Ni, Zn/Cd and
As, respectively (Paulo et al, 2014). Jiang et al. (2004) determined the growth performance and ability
for Cu phytoextraction of Elsholtzia splendens. Zhang et al. (2009) expressed that as Cd phytoextraction
is observed by maize, the percentage of exchangeable form of Cd decreased in the planted soil. Similar
finding of decrease in Cd level in soil planted with maize have also been reported by Mojiri (2011).
T. Venkateswara et al., 2014 have reported some of the finding as follows:

• Phytoextraction of Lead: In natural conditions, lead hyper accumulation has not been docu-
mented. Plants belong to families: Brassicaceae, Euphorbiaceae, Asteraceae, Lamiaceae, and
Scrophulariaceae have the potential to uptake lead.
• Phytoextraction of Arsenic: Researchers in Florida discovered that ferns are growing in soil
contaminated with arsenic at an abandoned lumber yard. The ferns had been soaking up arsenic
from the soil through their roots and storing it in their fronds. Two arsenic hyper accumulators,
Pteris cretica cv Mayii (Moonlight fern) and Pteris vittata (Chinese brake fern) are identified by
applying the above process.
• Phytoextraction of Cadmium: Solanum nigrum (Solanaceae) has a potential application for phy-
toextraction of Cd from contaminated soils. A fast growing weed plant Physalis minima Linn. is
used for the removal of Cadmium from the contaminated soils.
• Phytoextraction of Chromium: The findings indicated that family Cruciferae (Brassica camp-
estri L.- raya) was most tolerant to Cr toxicity, followed by Chenopodiacea (Spinacia oleracea
L.- spinach) and Leguminosae (Trigonella foenumgraecum L.- fenugreek).
• Rhenium (Re): is one of the rarest elements in the Earth’s crust, and is one of the ten most
expensive metals on the world market. Re phytomining requires a plant hyperaccumulator of
Re that should be simple to cultivate and grow fast. Four plant species Mountain-spinach (atri-
plex hortensis), Buckwheat (polygonum fagopyrum), Alfalfa (medicago), White clover (trifolium
repens) were used (Ognyan et. al., 2012). Two approaches for the development of commercial
phytoextraction technologies are believed to have significant promise: (i) domesticate natural ele-
ment hyperaccumulators; (ii) clone all genes needed for hyperaccumulation and hypertolerance
and express them in a high-biomass yielding transgenic hyperaccumulator. A recent examination
of genes expressed in Thlaspi caerulescens vs. A. thaliana using the microarray devices based
on genes expressed in A. thaliana provides important advances in understanding of the unusual
characteristics of T. caerulescens Genomic, transcriptomic, and proteomic approaches can each
contribute to improved understanding of phytoextraction biochemistry and agronomy (Chaney et
al, 2007).

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6. BIOMEDICAL APPLICATIONS

The application of plant-synthesized nanoparticles to environmentally-relevant catalytic reactions

could be an exciting, value-added application for “phytomined” metals. In this field, the unique proper-
ties of nanoparticles enable reactions that were not possible or significantly low yielding when using
conventional catalysts. For example, Sharma et al.(2012) carried out a study into the uptake of Au by
Sesbania drummondii seedlings. The Au nanoparticles were shown to be effective catalysts for the re-
duction of the pollutant 4-nitrophenol without extraction of the nanoparticles from the plant. The use of
nanoparticles for biomedical applications is a heavily researched area. This is especially true in the case
of Au nanoparticles, where the ever increasing diversity of published applications includes biosensors,
genomics, clinical chemistry, photothermolysis of cancer cells and tumors, targeted drug delivery and
optical bioimaging of cells and tissues Whilst no examples of plant-synthesized nanoparticles being used
in biomedical applications are currently reported, a greener route for their synthesis could potentially
increase the safety of nanoparticle manufacture. Increased safety could be afforded through the elimina-
tion of toxic chemicals often used in conventional nanoparticle synthesis techniques (Hunt et al, 2014).

7. RHIZOFILTRATION/ PHYTOFILTRATION/ RHIZODEGRADATION

Rhizofiltration is applicable for the treatment of surface water and groundwater, industrial and residential
effluents, downwashes from power lines, storm waters, acid mine drainage, agricultural runoffs, diluted
sludges, and radionuclide-contaminated solutions or all kinds of contaminated water can be treated with
rhizofiltration (Rawat et al, 2012; Tiyasha et al.,2013). This is primarily used to remediate extracted
groundwater, surface water, and wastewater with low contaminant concentrations. It is the adsorption
or precipitation onto plant roots or absorption of contaminants in the solution surrounding the root zone
(USEPA, 2000). An illustration of this method is shown in Fig. 2. Plants suitable for rhizofiltration ap-
plications can efficiently remove toxic metals from a solution using rapid growth root systems. Initially
most of aquatic plants were considered for rhizofiltration but now, Various terrestrial plant species have

Figure 2. Engineered rhizofiltration system.

Source: Phytoremediation: using plant to remove pollutants from the environment. http//www.aspp.org/pubaff/phytorem.htm

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Phyto-Remediation: Using Plants to Clean Up Soils

been found to effectively remove toxic metals such as Cu, Cd, Cr, Ni, Pb, and Zn from aqueous solu-
tions. It was also found that low level radioactive contaminants can successfully be removed from liquid
streams (Rawat et al, 2012). The mechanism of rhizofiltration lies in physical and biochemical impacts of
plant roots in waste water treatment. Efficiency of mechanism of rhizofiltration lies in the efficiency of
roots to synthesis certain chemicals which causes heavy metals to rise in plant body. Root exudates and
changes in rhizosphere, pH may cause metals to precipitate onto root surfaces. The root environment or
root exudates may produce biogeochemical conditions that result in precipitation of contaminants onto
the roots or into the water body. As they become saturated with the metal contaminants, roots or whole
plants are harvested for disposal. Plants with high root biomass, or high absorption surface, with more
accumulation capacity (aquatic hyperaccumulators) and tolerance to contaminants achieve the best re-
sults. Promising examples include Helianthus annus, Brassica juncea, Phragmites australis, Fontinalis
antipyretica and several species of Salix, Populus, Lemna and Callitriche (Paulo et al., 2014)
Constructed wetlands have been used for a wide range of inorganics including metals, Se, perchlo-
rate, cyanide, nitrate and phosphate. In constructed wetlands water hyacinth, Azolla and duckweed are
popular because they are good metal accumulators and can be harvested easily. Recently two bacterial
strains, Bacillus mycoides and Stenotrophomonas maltophilia have shown potential to detoxify Se and a
model system for Se rhizofiltration based on Astragalus bisulcatus – rhizobacteria interactions has also
been proposed(Hooda, 2007). Rhizofiltration and phytoextraction are similar in that they each result in
accumulation of the contaminant in or on the plant. However, in rhizofiltration this accumulation can
occur in the roots or in the portion of the plant above water, whereas for effective phytoextraction the
accumulation occurs aboveground, not in the roots. In addition, rhizofiltration differs from phytoextrac-
tion in that the contaminant is initially in water, rather than in soil (Pivetz, 2001). The plants to be used
in rhizofiltration for cleanup are raised in greenhouses with their roots in water rather than in soil. To
acclimatize the plants, once a large root system has been developed, contaminated water is collected from
a waste site and brought to the plants where it is substituted for their water source. The plants are then
planted in the contaminated area where the roots take up the water and the contaminants along with it. As
the roots become saturated with contaminants, they are harvested. Sunflower, Indian mustard, tobacco,
rye, spinach, and corn have been studied for their ability to remove lead from water, with sunflower
having the greatest ability (Etim, 2012). The advantages associated with rhizofiltration are the ability to
use both terrestrial and aquatic plants for either in situ or ex situ applications. Another advantage is that
contaminants do not have to be translocated to the shoots. Thus, species other than hyperaccumulators
may be used. Terrestrial plants are preferred because they have a fibrous and much longer root system,
increasing the amount of root area (Raskin et al., 2000). Disadvantages and limitations include the con-
stant need to adjust pH, plants may first need to be grown in a greenhouse or nursery; there is periodic
harvesting and plant disposal; tank design must be well engineered; and a good understanding of the
chemical speciation/interactions is needed. The cost of remediation by rhizofiltration has been estimated
to be $2-$6 per 1000 gallons of water (USEPA, 2000) (see Figure 2).

8. PHYTOSTABILIZATION/ PHYTORESTORATION/PHYTOSEQUESTRATION

Phytostabilization referred to as in-place inactivation or phytoimmobilization, is primarily used for the

remediation of soil, sediment, and sludges (USEPA, 2000). Phytostabilization is the use of vegetation
to contain soil contaminants in situ, through modification of the chemical, biological, and physical

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conditions in the soil. Contaminant transport in soil, sediments, or sludges can be reduced through
absorption and accumulation by roots; adsorption onto roots; precipitation, complexation, or metal va-
lence reduction in soil within the root zone; or binding into humic (organic) matter through the process
of humification. In addition, vegetation can reduce wind and water erosion of the soil, thus preventing
dispersal of the contaminant in runoff or fugitive dust emissions, and may reduce or prevent leachate
generation. Phytostabilization research to date has generally focused on metals contamination, with
lead, chromium, and mercury being identified as the top potential candidates for phytostabilization. It
is the use of plant roots to limit contaminant mobility and bioavailability in the soil (Pivetz, 2001). In
this remedial technique, plant stabilizes wastes and prevents exposure pathway through wind and water
erosion, enables hydraulic control that restricts the vertical migration of pollutants into ground water,
and immobilizes the pollutants physically and chemically by root sorption and chemical fixation with
different soil amendments. Selected plant for this technique should be poor translocators for metal con-
tamination towards aerial parts likely to be consumed by humans or animals, easy to establish, quick to
grow, having well developed canopies and root systems, and tolerant to metal pollution and other climatic
and site stresses that could limit plant growth (Sarwat,2012). The main objective is to avoid mobiliza-
tion of contaminants and limit their diffusion in the soil. Species of genera Haumaniastrum, Eragrostis,
Ascolepis,Gladiolus and Alyssum are examples of plants cultivated for this purpose (Paulo et al.,2014).
The research of Smith and Bradshaw led to the development of two cultivars of Agrosists tenuis Sibth
and one of Festuca rubra L which are now commercially available for the Phytostabilization of Pb, Zn,
Cu contaminated soils (Sarwat, 2012).
In phytostabilization, plants are responsible for reducing the percolation of water within the soil matrix,
which may create a hazardous leachate, inhibiting direct contact with polluted soil by acting as barrier
and interfering with soil erosion, which results in the spread of toxic metals to the other sites. The plants
primary purposes are to (1) decrease the amount of water percolating through the soil matrix, which may
result in the formation of a hazardous leachate, (2) act as a barrier to prevent direct contact with the con-
taminated soil and (3) prevent soil erosion and the distribution of the toxic metal to other areas (Raskin
et al., 2000). It is useful for the treatment of lead (Pb) as well as arsenic (As), cadmium (Cd), chromium
(Cr), copper (Cu) and zinc (Zn). Vigorously growing plants are necessary to exert hydraulic control and
immobilization at the site; plants cannot die or be removed during the phytostabilization design period.
Low-level radionuclide contaminants can also be held in place by phytostabilization, and this alterna-
tive can result in significant risk reduction if their half-lives are not too long. Soil amendments such
as phosphate, lime, and organic matter are sometimes needed to immobilize toxic metals such as lead,
cadmium, zinc, and arsenic. Cadmium is readily translocated to leaves in many plants, which represents a
risk to the food chain, and this pathway may be the limiting consideration in applying phytostabilization
at some metals contaminated sites (Schnoor,1997). For phytostabilization of metals a combination of
trees and grasses work best. Fast-transpiring trees such as ‘Poplar’ maintain an upward flow to prevent
downward leaching, while grasses prevent wind erosion and lateral runoff with thin dense root system.
Further, grasses do not accumulate as much metals in their shoots as dicot species, minimizing exposure
of wildlife to toxic elements (Hooda, 2007). Some of the advantages associated with this technology
are that the disposal of hazardous material/biomass is not required (USEPA, 2000) and it is very effec-
tive when rapid immobilization is needed to preserve ground and surface waters (Jadia et al., 2009).
However, this clean-up technology has several major disadvantages including: contaminant remaining
in soil, application of extensive fertilization or soil amendments, mandatory monitoring is required, and

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the stabilization of the contaminants may be primarily due to the soil amendments. Disadvantages of
phytostabilization include the necessity for long-term maintenance of the vegetation or verification that
the vegetation will be self-sustaining (Pivetz et al., 2001).

9. PHYTOVOLATILIZATION

Toxic metals such as Se (Selenium), As and Hg (Mercury) may exist as gaseous species in environment.
Recently it is discovered that plants that absorb elemental form of metals from soil, could convert them
biologically into gaseous species inside the plant, i.e., biomethylated to form volatile molecules and
finally release them to the atmosphere. Phytovolatilization is the use of green plants to extract volatile
contaminants, such as Hg and Se, from polluted soils and to ascend them into the air from their foliage
(Karami & Shamsuddin, 2010). In other word, Phytovolatilization involves the use of plants to take
up contaminants from the soil, transforming them into volatile forms and transpiring them into the at-
mosphere (United States Protection Agency, 2000). There are some naturally occurring or genetically
modified plants that are capable of absorbing elemental forms of these metals from the soil, biologi-
cally converting them to gaseous species within the plant and volatized into the atmosphere through the
stomata. The mentioned process is controversial of all techniques due to its dubious nature that whether
release of these volatilized elements in atmosphere is safe. Phytovolatilization has mainly been applied
to groundwater, but it can be applied to soil, sediments, and sludges. The advantage of this method is
that the contaminant, mercuric ion, may be transformed into a less toxic substance (that is, elemental
Hg). The disadvantage to this is that the mercury released into the atmosphere is likely to be recycled by
precipitation and then redeposited back into lakes and oceans, repeating the production of methyl-mercury
by anaerobic bacteria. The disadvantage is the volatilized element could be recycled by precipitation
and then redeposit back into ecosystem.
Volatile Se compounds, such as dimethylselenide, are 1/600 to 1/500 as toxic as inorganic forms of Se
found in the soil. After genetic modification of Arabidopsis thaliana L. and Nicotiana tobacum L. with
bacterial organomercurial lyase (Mer B) and mercuric reductase (Mer A) genes plants have developed
abilities to absorb elemental Hg(11) and methyl mercury (Mer Hg) from the soil and release volatile Hg
(0) from leaves to atmosphere. This technology does not require much management after plant seeding.
In addition, it has advantage of minimum site disturbance, low erosion rate and there is no need for
disposal of hazardous plant material (Sarwat Ismail, 2012) . Banuelos perceived that some plants were
able to transform Se in the form of dimethylselenide and dimethyldiselenide in high-selenium media.
Unlike other remediation techniques, once the contaminants have been removed via volatilization, one
has no control over their migration to other areas. A similar case of volatilization based soil remediation
has also been reported in many recently published reports (Tangahu et al., 2011). Similarly transformed
yellow poplar (Liriodendron tulipifera) plantlets had resistance to, and grew well in, normally toxic
concentrations of ionic mercury. The transformed plantlets volatilized about ten times more elemental
mercury than did untransformed plantlets. Indian mustard and canola (Brassica napus) may be effec-
tive for phytovolatilization of selenium, and, in addition, accumulate the selenium (Jadia et al., 2009).
Phytovolatalization of Mercury: - There is some evidence that certain plant species have the ability
to extract and accumulate mercury both from the atmospheric and soil sources. No plant species with
mercury hyper accumulating properties has been identified. Due to this, scientists have been researching

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the use of genetically engineered plants by inserting bacterial genes specific for detoxifying toxic forms
of mercury. Transgenic plants like Arabidopsis thaliana L. and tobacco (Nicotiana tobacum) containing
both the merA and merB bacterial genes have the ability to transform methyl-mercury into elemental
mercury, releasing it into the atmosphere through a process termed phytovolatilization (Venkateswara
Rao et al, 2014).

10. PHYTODEGRADATION / PHYTOTRANSFORMATION

This is also referred to as phytotransformation. It involves the degradation of complex organic mol-
ecules to simple molecules or the incorporation of these molecules into plant tissues (Trap et al., 2005).
Phyto-degradation is the metabolism of contaminants within plant tissues. Plants produce enzymes,
such as dehalogenase and oxygenase that help catalyze degradation. Phytodegradation is the uptake,
metabolizing, and degradation of contaminants within the plant, or the degradation of contaminants in
the soil, sediments, sludges, ground water, or surface water by enzymes produced and released by the
plant (Vishnoi et al, 2008). When the phytodegradation mechanism is at work, contaminants are broken
down after they have been taken up by the plant. As with phytoextraction and phytovolatilization, plant
uptake generally occurs only when the contaminants’ solubility and hydrophobicity fall into a certain
acceptable range. Phytodegradation has been observed to remediate some organic contaminants, such
as chlorinated solvents, herbicides, and munitions, and it can address contaminants in soil, sediment, or
groundwater (EPA, 2000). Phytodegradation is not dependent on microorganisms associated with the
rhizosphere. Contaminants subject to phytodegradation include organic compounds such as chlorinated
solvents, herbicides, and insecticides, and inorganic nutrients. For phytodegradation, the plant must be
able to take up the compound.

11. RHIZODEGRADATION/ PHYTOSTIMULATION

Rhizodegradation refers to the breakdown of contaminants within the plant root zone, or rhizosphere.
It is believed to be carried out by bacteria or other microorganisms whose numbers typically flourish
in the rhizosphere. This activity is due to the presence of proteins and enzymes produced by the plants
or soil organism such as bacteria yeast and fungi. Rhizodegradation is a symbiotic relationship that has
evolved between plants and microbes. Plants provide nutrients necessary for the microbes to thrive, while
microbes provide a healthier soil environment . Plant enzymes have been identified that break down am-
munition wastes, chlorinated solvents such as TCE (Trichloraethane) and others which degrade organic
herbicides. The localized nature of rhizodegradation means that it is primarily useful in contaminated
soil, and it has been investigated and found to have at least some successes in treating a wide variety of
mostly organic chemicals, including petroleum hydrocarbons, polycyclic aromatic hydrocarbons (PAHs),
chlorinated solvents, pesticides, polychlorinated biphenyls (PCBs), benzene, toluene, ethylbenzene, and
xylenes (EPA, 2000). It can also be seen as plant-assisted bioremediation, the stimulation of microbial and
fungal degradation by release of exudates/enzymes into the root zone (rhizosphere) (Zhang et al., 2005).
Some examples are where Rhizodigandation is used (Singh et al, 2012).

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1. Petroleum & hydrocarbons, using, Juniper, fescue in ground water.

2. Explosive waste, using duck weed, Parrot feather in ground water.
3. Nitrates wastes using hybrid popular in ground water.

Phytoremediation of Petroleum Compounds

The rhizosphere degradation of chlorinated solvents under new plantations is limited, but work by
Godsy et al. indicates that as the trees age the microbial populations might change and foster anaerobic
degradation. With compounds such as the polycyclic aromatic hydrocarbons (PAHs), petroleum com-
pounds and polychlorinated biphenyls (PCBs), the role of the rhizosphere is much more critical. Efforts
to demonstrate plant or rhizosphere effects on petroleum degradation, particularly PAHs, continue. In
a greenhouse study, Banks et al. observed generally greater remediation efficiency in soil planted with
four genotypes of Sorghum bicolor (L.) compared with unvegetated controls (Newman et al., 2004).
There are other strategies, which are considered categories of phytoremediation by some authors, but
actually, they are mixed techniques or variations of the above mentioned strategies (Paulo et al, 2014;
Pivetz, 2012; USEPA 2001).
These include:

1. Hydraulic Barriers: Some large trees, particularly those with deep roots (e.g., Populus sp.), re-
move large quantities of groundwater during transpiration. Hydraulic control (or hydraulic plume
control) is the use of vegetation to influence the movement of ground water and soil water, through
the uptake and consumption of large volumes of water. Contaminants in this water are metabo-
lized by plant enzymes, and vaporized together with water or simply sequestered in plant tissues .
Vegetation water uptake and transpiration rates are important for hydraulic control and remediation
of ground water. Water uptake and the transpiration rate depend on the species, age, mass, size, leaf
surface area, and growth stage of the vegetation. They also are affected by climatic factors, such
as temperature, precipitation, humidity, insolation, and wind velocity, and will vary seasonally.
2. Vegetation Covers/ Caps: Herbs (usually grasses), eventually shrubs or trees, establish on landfills
or tailings, are used to minimize the infiltration of rain water, and contain the spread of pollutants.
The roots increase soil aeration thus, promoting biodegradation, evaporation and transpiration. A
vegetated cap (or cover) is a long-term, self-sustaining cap of plants growing in and/or over con-
taminated materials, designed to minimize exposure pathways and risk. The primary purpose of
the vegetation is to provide hydraulic control and prevent or minimize infiltration of precipitation
and snowmelt into the contaminated subsurface, thus preventing or minimizing leachate formation.
This is done by maximizing evapotranspiration and maximizing the storage capacity of the soil.
A cap designed for this purpose is called an evapotranspiration cap or waterbalance cover. The
difficulty of this technique is that tailings generally are not suitable for the development of plant
roots. However, various investigations have been undertaken with the aim of developing processes
of cultivation in tailings. For example a technique in which an organic soil composed of sawdust,
plant remains, and some NPK-fertilizers is deposited on the surface was utilized by Hungarian
agronomists (Biological Reclamation Process, BRP). The workers were able to obtain, at the end
of a single biological cycle, 76 different plant species including cereals, shrubs, fruit trees and even
large trees like oaks and pines.

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3. Constructed Wetlands: These are ecosystems consisting of organic soils, microorganisms, algae
and vascular aquatic plants in areas where the water level is at/near the surface, at least part of the
year. Constructed wetlands or treatment wetlands are artificial wetlands that are used for treating
organic, inorganic, and nutrient contaminants in contaminated surface water, municipal waste
water, domestic sewage, refinery effluents, acid mine drainage, or landfill leachate. All the com-
ponents work together in the treatment of effluents, through the combined actions of filtration, ion
exchange, adsorption and precipitation. It is the oldest method of wastewater treatment and is not
regarded as proper phytoremediation, since it is based on the contributions of the entire system.
Good cleaning efficiency, low cost of construction along with easy operation and maintenance are
the main advantages. It is widely applied in the treatment of domestic, agricultural and industrial
waste water, but has proved to be suitable also for treating acid mine drainages.
4. Phytodesalination: It is a recently reported emerging technique that utilizes halophytes to remove
excess salts from saline soils. The potential of Suaeda maritima and Sesuvium portulacastrum in
removal and accumulation of NaCl, from highly saline soils, has been demonstrated. Although it
has its peculiarities, this technique is a modality of phytoextraction.

Buffer Strips and Riparian Corridors (USEPA 2001)

Buffer strips are areas of vegetation placed downgradient of a contaminant source or plume, or along a
waterway (i.e., riparian corridor). The vegetation contains, extracts, and/or destroys contaminants in soil,
surface water, and ground water passing underneath the buffer through hydraulic control, phytodegra-
dation, phytostabilization, rhizodegradation, phytovolatilization, and perhaps phytoextraction. The use
of buffer strips might be limited to easily assimilated and metabolized compounds. Relatively soluble
contaminants, such as nutrients and some organics (especially pesticides), have been addressed using
buffer strips and riparian corridors.

Combinations of Phytoremediation Processes

At a phytoremediation site, combinations of the phytoremediation processes discussed above may occur
simultaneously or in sequence for a particular contaminant, or different processes may act on different
contaminants or at different exposure concentrations. For example, TCE in soil can be subject to bio-
degradation in the root zone (rhizodegradation) and metabolism within the plant (phytodegradation),
with loss of some contaminant or metabolite through volatilization from the plant (phytovolatilization).
Some metals or radionuclides in water can be accumulated on or within roots (rhizofiltration) while other
metals or radionuclides are simultaneously taken up into the aerial portion of the plant (phytoextraction).

12. CONCLUSION

Phytoremediation is well suited for applications in low-permeability soils, where most currently used
technologies have a low degree of feasibility or success, as well as in combination with more conventional
clean up technologies (electromigration, foam migration, etc.). Phytoremediation actually benefits the
soil, leaving an improved, functional, soil ecosystem at costs estimated at approximately one-tenth of
those currently adopted technologies

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Table 3. Overview of different mechanisms of Phytoremediation (Pandey et al., 2013)

Mechanism Process Media Contaminants Plants

Phytoextraction Hyper- Soil, sediment, Metals: Cd,Cu, Ni, Pb, Zn Indian mustard, sunflowers, pennycress,
accumulation brown fields with EDTA addition of Pb, Crusifers, Rape seed plants, barley,
selenium alyssum
Hyper- Soil, sediment lead (Pb), cadmium (Cd), Brassica juncea (Indian mustard) and
accumulation chromium (Cr), copper (Cu), Helianthus anuus (sunflower)
nickel (Ni), and zinc (Zn)
with EDTA addition
Hyper- Soil, sediment Zn, Co, Cu Se, Pb and Cd Brassicaceae, Fabaceae,
accumulation Euphorbiaceae, Asteraceae,
and Contaminant Lamiaceae, and
extraction Scrophulariaceae
Contaminant Soil, sediment, Metals:Ag, Cd, Co, Cr, Cu, Indian mustard, sunflowers, hybrid
extraction and sludges Hg, Mn, Mo, Ni, Pb, Zn;
capture Radionuclides:90Sr, 137Cs,
239Pu, 234U,238U
Contaminant Soil Soil Perennial ryegrass (Lolium perenne)
extraction
Rhizofiltration Rizosphere Ground water, Metals: Cd,Cu, Ni, Pb, Zn; Aquatic plants-emergents
accumulation waste water Radionuclides: 90Sr, 137Cs, (Bullrush,cattail, pondwed, arrow root,
logoons or created 238U duckweed) Sebmergents (algae,,hydrilla,
weetlands stonewort,parrotfeather)
Contaminant Ground water and Metals, radionuclides Sunflowers, Indian mustard, water
extraction and surface water hyacinth
capture
Phytostabilization Contaminant Soil, sediment As, Cd, Cr, Cu, Hs, Pb, Zn Indian mustard, hybrid poplars, grasses
containment sludges
Complexa-tion Soil, sediment Metals: Cd,Cu, Ni, Grasses with fibrous root
Pb, Zn,Cr,As,Se,U;
Hydrophobic
Organism
Phytodegradation Contaminant Soil, sediment Organic compounds, Algae, stonewort, hybrid poplar, black
destruction sludges chlorinated solvents, willow, bald cypress
phenols, herbicides,
munitions
Degradation in Soil, ground water, Herbisides (atrazine, Phreatophyte trees (Popular willow,
plants land fill leachate alachlor) Aromatics cotton wood, grasses rye, Bermuda
land application of (BTEX) Choriated alipatics sorghum fescue)
waste water (TCE), Nutrients (NO3-, Legumes clover alfalfa, cowpeas
NH4+,PO3-), Alnmunition
waste TNT, RDX
Phytovolatilization Contaminant Soil, sediment Chlorinated solvents, some Poplars, alfalfa black locust, Indian
extraction from sludges inorganics (Se, Hg, and As) mustard
media and release
to air
Volatilization by Soil, sediment, Se, Hg, and Ti Poplars, Indian mustard, Canola,
leaves ground water Tobacco plant
volatilization to Soil Inorganic pollutant Soil plants
the atmosphere Ni,Zn, Cd, As, Se,
Cu,Co,Pb,Hg, and
Radionuclides

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Table 4. United States Department of Agriculture National Conservation Practice Standards (Hetti-
arachchi, 2012)

Phytoremediation Mechanism Facilitating USDA NRCS Conservation Practices

Phytoextraction/ 327 – Conservation Cover
Phytoaccumulation 340 – Cover Crop
342 – Critical Area Planting
612 – Tree/shrub Planting
Phytodegradation 311 – Alley Cropping
(Phytotransformation) 327 – Conservation Cover
390 – Riparian Herbaceous Cover
391 – Riparian Forest Buffer
393 – Filter Strip
Rhizofiltration/ 332 – Contour Buffer Strips
Rhizodegradation 340 – Cover Crop
342 – Critical Area Planting
390 – Riparian Herbaceous Cover
391 – Riparian Forest Buffer
585 – Stripcropping
601 – Vegetative Barrier
Phytovolatilization 327 – Conservation Cover
340 – Cover Crop
342 – Critical Area Planting
512 – Forage and Biomass Planting
612 – Tree Planting
Phytosequestration/ 327 – Conservation Cover
Phytostabilization 340 – Cover Crop
391 – Riparian Forest Buffer
612 – Tree Planting
Phytohydraulics 340 – Cover Crop
391 – Riparian Forest Buffer
393 – Filter Strip
512 – Forage and Biomass Planting
612 – Tree Planting
635 – Vegetated Treatment Area
Phytostabilization 327 – Conservation Cover
329 – Residue and Tillage Management –
No-till/Strip-till/Direct Seed
330 – Contour Farming
332 – Contour Buffer Strips
342 – Critical Area Planting
380 – Windbreak/Shelterbelt Establishment
585 – Stripcropping
589C – Cross Wind Trap Strips
601 – Vegetative Barrier
603 – Herbaceous Wind Barriers

• Plants act as solar-driven pumping and filtering systems as they take up contaminants (mainly wa-
ter soluble) through their roots and transport/translocate them through various plant tissues where
they can be metabolized, sequestered, or volatilized.
• Approximately 400 plant species from at least 45 plant families have been so far, reported to
hyperaccumulate metals. Some of the families are Brassicaceae, Fabaceae, Euphorbiaceae,
Asterraceae, Lamiaceae and Scrophulariaceae. Crops like alpine pennycress (Thlaspi caerule-
scens), Ipomea alpine, Haumaniastrum robertii, Astragalus racemosus, Sebertia acuminate have

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Phyto-Remediation: Using Plants to Clean Up Soils

very high bioaccumulation potential for Cd/Zn, Cu, Co, Se and Ni, respectively (Lasat, 2000).
Willow (Salix viminalis L.), maize (Zea mays L.), Indian mustard (Brassica juncea L.), and sun-
flower (Helianthus annuus L.) have reportedly shown high uptake and tolerance to heavy metals
(Moosavi et al., 2013).
• Among the plants of the Brassica species, the Brassica juneca deserve special attention because
its relevance to the process of phytoexctration of heavy metals from soil was confirmed in many
experiments. It has been found that B. juncea exhibits a high capacity to accumulate Cd- mainly in
the shoots, where Cd level was recorded at level of 1450 μg Cd/g dry wt.This is three times more
than reported in Brassica napus (555 μ g/g dry wt). In addition, this plant exhibit a high removal
efficiency of other metals such as Pb (28% reduction) and Se (reduced between 13–48%) (Salt et
al., 1998) and this plant is more effective at removing Zn from soil than Thlaspi caerulescens,
a known hyperaccumulator of zinc. This is due to the fact, that B. juneca produces ten-times
more biomass than T. cearullescens.Some species, such as cabbage (Brassica oleracea L.), lettuce
(Latuca sativa L.) and tobacco (Nicotiana tabacum L.),accumulate high levels of Cd in leaves
rather than in roots and increases or decreases the bioavailability of metal ions. The root of Indian
mustard are found to be effective in the removal of Cd, Cr, Cu, Ni, Pb, and Zn, and sunflower can
remove Pb, U, Cs and Sr from hydroponic solutions. Tang et al. reported the increase in uptake
of copper by Indian mustard and sunflower plant. Nehnevajova et al., investigated that the high-
est metal concentration was found in leaves (shoot) of commercial cultivars of sunflower plants
grown on metals-contaminated soil (Moosavi et al., 2013).
• Among the cultivated crops rape and sunflower revealed higher cadmium concentrations in their
shoots than in the roots. Nagaraju and Karimulla described that some species, including Jatropha
curcas (from Euphorbiaceae), Dodonaea viscose (from Sapindaceae) and Cassia auriculata (from
Fabaceae), had potential for remediation of soils polluted with different kinds of trace and major
elements. Also, high heavy metal accumulating ability has been reported for cereal crops such as
maize (Zea mays L.), sorghum (Sorghum bicolor) and alfalfa (Medicago sativa L.). Such plants
can be used successfully to clean up heavy metal polluted soils if their biomass and metal content
are large enough to complete remediation within a reasonable period (Moosavi et al., 2013).

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Chapter 12
A Prospective Study
on Emerging Role of
Phytoremediation by
Endophytic Microorganisms
Amita Verma
Sam Higginbottom Institute of Agriculture, Technology and Sciences (Deemed University), India

Parjanya Kumar Shukla

Sam Higginbottom Institute of Agriculture, Technology and Sciences (Deemed University), India

ABSTRACT
Colonies of endophytes are excellent example of beneficial association with most plants in their natu-
ral state. Endophytic colonies and plant associations are beneficial in many ways such as supplying
biologically fixed nitrogen, regulation of phytohormone production thus enhancing the plant growth,
resistance to environmental stress etc. these associations are also important for the agriculture and in-
dustries because they produce important medicinal, agriculture and industrial compounds as endophytic
metabolites. When we concern about the waste management, degradation and biotransformation of
several toxins, the phytoremediation by using endophytes has been developed as important tool. Current
chapter reviles, study and collect most of important knowledge, recent ongoing research, technologies,
roles and advancements in biodegradation and biotransformation of different types of toxic wastes and
their effects on environment with phytoremediation by endophytes.

INTRODUCTION

Bioremediation is a method of removal of pollutants and wastes from the environment by the use of
micro-organisms. According to Ministry of environment and forest, Govt. of India, Bioremediation
is the use of biological interventions of biodiversity for mitigation (and wherever possible, complete
elimination) of the noxious effects caused by environmental pollutants in a given site. Bioremediation
is generally considered to include natural attenuation (little or no human action), bio-stimulation or bio-
augmentation, the deliberate addition of natural or engineered micro-organisms to accelerate the desired

DOI: 10.4018/978-1-4666-9734-8.ch012

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A Prospective Study on Emerging Role of Phytoremediation

catalytic capabilities Thus bioremediation, phytoremediation and rhizoremediation contribute signifi-

cantly to the fate of hazardous waste and can be used to remove these unwanted compounds from the
biosphere. It relies on the biological processes in microbes to breakdown these wastes. Phytoremediation
is a technology that is based on the combined action of plants and their associated microbial communi-
ties to degrade, remove, transform, or immobilize toxic compounds located in soils, sediments, ground
water and surface water. Phytoremediation has been used to treat many classes of contaminants including
petroleum hydrocarbons, chlorinated solvents, pesticides, explosives, heavy metals and radionuclides
in soil and polluted water. There are several advantages of phytoremediation compared to conventional
techniques, such as low cost, low disruptiveness to the environment, public acceptance, and potentiality
to remediate various pollutants. In addition, plants as autotrophic systems with large biomass require
only a modest nutrient input, and they also prevent the spread of contaminants through water and wind
erosion (Cherian, 2005). Candidate plant for phytoremediation should have the characteristics such as
high biomass production, extensive root system, and ability to tolerate high concentration of pollutants
and withstand environmental stress. Like other treatment technologies, phytoremediation has its disad-
vantages e.g. climatic and geological limitations, potential phytotoxicity of the contaminant, potential
for the contaminant or its metabolites to enter the food chain, and potentially longer timescale compared
to other technologies (Macek, 2000). Phytoremediation, the use of plants to degrade toxic contaminants
in the environment involves a number of processes including phytoextraction, phytotransformation,
phytostabilization, phytovolatilization and rhizofiltration (Prasad, 2011). The uptake and concentration
of pollutants into harvestable biomass for sequestration or incineration is known as Phytoextraction (or
phytoaccumulation). Phytotransformation involves enzymatic modification resulting in inactivation,
degradation (phytodegradation), or immobilization (phytostabilization) of pollutants. Phytovolatiliza-
tion involves the removal of pollutants from soil and their release through leaves via evapotranspiration
processes and rhizofiltration involves the filtering of water through a mass of roots to remove pollut-
ants. While some success has been reported using plants alone in bioremediation, the use of plants in
conjunction with plant associated bacteria offers much potential for rhizoremediation (Mejare, 2001).
Plants and their associated microorganisms are characterized by varied and complex interactions
which has been the subject of extensive research and diverse applications (Mishra, 2014). Endophytes
lives symbiotically within the plants. The interaction between Endophytic bacteria and host plants has
not been fully understood, it is well established that some of these interactions are beneficial to the
plant. The endophytes close association with internal tissues of host plant has increasingly gained them
scientific and commercial interest due to their potential to improve plant quality and growth. A promis-
ing field to exploit plant-endophyte partnerships is the remediation of contaminated soils and (ground)
water. Like for Treatment wetlands (also known as constructed wetlands) are effective and low-cost
operational alternatives to conventional technologies for the elimination of a wide range of contaminants
from wastewaters and polluted groundwater (Braeckevelt, 2007; Calheiros, 2007). Many plant growth
promoting endophytes can assist their host plant to overcome contaminant-induced stress responses,
thus providing improved plant growth and also affects the environment.
Endophytes are defined as microorganisms (fungi, bacteria) that colonize living, internal tissues of
plants without causing any immediate, negative effects. The term endophyte was first introduced in 1886
by De Bary for microorganisms (fungi, yeast, and bacteria) colonizing internal plant tissues (De Bary,
1884). One of the latest definitions of endophytes was proposed by Posada and Vega (2005) who used

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this term to describe all organisms inhabiting different internal parts of plants, including seeds. The huge
variety of the metabolic pathways employed by endophytes makes them valuable tools for bioremedia-
tion, which can be used for assimilation of methane, fixation of nitrogen, bioremediation of pollutants
(e.g., pesticides, herbicides, insecticides, petrochemicals, polychlorobiphenyls, phenols/chlorophenols),
and biotransformation of organic substances, for example propylene to epoxypropane and production of
chiral alcohols (Gai, 2009; Kim, 2012) as shown in figure 1.

Categories

According to Tan (2001) on the basis of different classification method, endophytes can be divided into
different categories. But the popular classification method is according to the microbes to differentiate,
mainly including endophytic fungi, endophytic bacteria, endophytic actinomyces.

Endophytic Fungi

Strobel (1993) first isolated endophytic fungi (Taxomyces andreanae) from a medicinal plant Taxus
brevifolia. Huang (2007) also got a total 42 endophytic fungi strains from Nerium oleander L. In tropi-
cal host, the kinds and number are very abundant because of high biodiversity, which of them are new
species unfound and unnamed (Azevedo, 2000). Besides, endopytic fungi also exist in the marine plants.
Osterhage (2000) isolated the endophytic and obligate marine fungus Ascochyta salicorniae from
the green alga Ulva sp. Endophytic fungi is the earliest and most isolated which is also most deepening
studied about endophyte. Endopytic fungi, which has endophytic fungi in more than 80 genera of the
hundreds of grass species, is of widely applications in promoting plant growth and protecting plant to
reduce diseases and insect pests.

Figure 1.

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Endophytic Bacteria

Endophytic bacteria usually exist in intercellular space and vascular tissue of the plant. There are, So far,
lots of research of insolating endophytic becteria from various plants, such as cotton, sweet corn culti-
vars and pea cultivars (Cho, 2007). Li (2008) isolated a total of 98 non-symbiotic endophytic bacterial
strains from 150 soybean root nodules. More than 129 kinds of the endophytic bacteria were isolated
so far from different crop plants, including both Gram-negative and Gram-positive species representing
over 54 genera. The major bacterial taxa belong to the former Pseudomonas group (Pseudomonas, Burk-
holderia, Phyllobacterium) and Enterobacteriaceae (Enterobacter, Erwinia, Klebsiella) (Pullen, 2002).
Additionally, some plants grown in natural abominable condition also were colonized by endophytic
bacteria. For example, Kan (2007) isolated 61 endophytic bacterium from Vicia, Oxytropis, Medicago,
Melilotus and Onobrychis species grown in Qinghai-Tibet plateau and loess plateau, some of which can
resistant to high alkaline (pH 11) and high concentration of NaCl (3-5%, w/v).

Endophytic Actinomyces

Streptomyces is the most important endophytic actinomyces isolated, usually colonized highest in the
roots and also in stems and leaves of plant (Wang, 2008). Castillo (2000) isolated Streptomyces NRRL
3052 from the native medicinal plant snakevine (Kennedia nigriscans) of Australia, this is a good example
to show that Medicinal plants are the important resource of isolating endophytic actinomyces which
can induce secondary metabolite of very important value. Streptomyces NRRL 3052 strain can release
munumbicins B which can be used against a methicillin-resistant strain of Staphylococcus aureus, and
it’s also show activity against Bacillus anthracis and multidrug-resistant Mycobacterium tuberculosis.
The research about endophytic actinomyces is relative less, but its play important role in application in
medicine.
Several endophytic metabolites were evaluated till now, among which we identified few compounds
with their structure and their source of origin given in Table 1.

Isolation and Identification

Pirttil (2003) studied that endophytic organisms have been isolated from different parts of plant. They
were isolated from scale primordia, meristem and resin ducts. Endophytic organisms are also isolated from
leaf segments with midrib and roots (Hata, 2002) and from stem, bark, leaf blade, petiole (Hata, 2008),
and buds (Pirttil, 2008). Sequence-based approach was used for investigating the transmission of diverse
fungal endophytes in seed and needles of Pinusmonticola, westernwhite pine (Ganley and Newcombe
2006).They isolated 2003 fungal endophytes from 750 surface-sterilized needles. In contrast, only 16
endophytic isolates were obtained from 800 surface-sterilized seeds. There are endophytic bacteria, fungi,
and/or actinomycetes whose isolation from the plant tissues has been a challenge since the studies on
endophytes started. Several researchers have reviewed extensively different methods of the isolation of
bacterial endophytes (Hallmann, 1997). Endophytes are isolated by initial surface sterilization followed
by culturing from ground tissue extract (Rai, 2007) or by direct culturing of plant tissues (Hata, 2002) on
media suitable for bacteria or fungi or actinomycetes. The impact of different culture media on isolation
of endophytic fungal flora from root and fruits of Azadirachta indica A. was studied by Verma (2011).
According to them, mycological agar (MCA) medium yielded the highest number of isolates, with the

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A Prospective Study on Emerging Role of Phytoremediation

Table 1. Several endophytic metabolites with their origin

Endophytic Source Endophyte Cytotoxicity Test Reference

Metabolite
3-ketotauranin Platycladus orientalis Phyllosticta spinarum Kithsiri Wijeratne,
2008

Tyrosol V. arenaria Glomerella cingulata Huang, 2008

Xylariaquinone A Xylaria sp. Plasmodium falciparum Verma

K1 strain

Penicillenols A1 Penicillium sp. GQ-7, Aegiceras corniculatum Tansuwan, 2007

Nigerasperone A Colpomenia sinuosa Aspergillus niger EN-13 Huang, 2007

Nigerasperone B Colpomenia sinuosa Aspergillus niger EN-13 Huang, 2007

continued on following page

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A Prospective Study on Emerging Role of Phytoremediation

Table 1. Continued
Endophytic Source Endophyte Cytotoxicity Test Reference
Metabolite
Nigerasperone C Colpomenia sinuosa Aspergillus niger EN-13 Huang, 2007

Excelsione Knightia excelsa unidentified fungus Zhang, 2007

Preussomerin EG1 Callicarpa acuminata Edenia gomezpompae Lang, 2007

Leptosphaerone C Aegiceras corniculatum. Penicillium sp. JP-1 Macías-Rubalcava,

2008

continued on following page

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A Prospective Study on Emerging Role of Phytoremediation

Table 1. Continued
Endophytic Source Endophyte Cytotoxicity Test Reference
Metabolite
Nectriapyrone V. arenaria Glomerella cingulata Lin Z, 2008

Phyllospinarone Platycladus orientalis Phyllosticta spinarum, Huang, 2008

greatest species richness. Madmony (2005) obtained an obligatory endophyte, Enterobacter cloacae,
was found to be associated with the pollen of several Mediterranean pines. Rungjindamai (2008) found
that most fungal endophytes isolated from plants and algae are members of the Ascomycota or their
anamorphs, with only a few numbers of basidiomycetous endophytes, these often being orchid mycor-
rhizas. Basidiomycetous morphotypes were isolated from healthy leaves, rachis, and petioles of the oil
palm Elaeis guineensis in a Thai plantation which were further characterized by molecular analysis using
ribosomal DNA sequences. For the first time ever, the microorganism species Acremonium terricola,
Monodictys castaneae, Penicillium glandicola, Phoma tropica and Tetraploa aristata were reported as
endophytic fungi by Bezerra (1995).
On the basis of phylogeny and life history endophytic fungi have been classified into two broad
groups known as clavicipitaceous (C-endophytes) which infect some grasses and the nonclavicipitaceous
endophytes (NC-endophytes) which can be recovered from asymptomatic tissues of nonvascular plants,
ferns and allies, conifers, and angiosperms (Rodriguez, 2009). NC-endophytes represent three distinct
functional classes based on host colonization and transmission, in planta biodiversity and fitness benefits
conferred to hosts while the C group has just one class. Conventionally, identification of endophytes
was done based on morphological characteristics for bacteria, fungi, and actinomycetes and with the
help of biochemical tests for bacteria and actinomycetes.With the development of molecular biology,
ribosomal DNA Internal Transcribed Spacer (ITS) sequence analysis is widely used for the identifica-
tion of microorganisms. RibosomalDNA (rDNA) ITS was proved to be a valuable source of evidence to

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A Prospective Study on Emerging Role of Phytoremediation

resolve phylogenetic relationships at lower levels, such as among genera or species (Youngbae, 1997). It
was also reported that ITS sequences analysis was especially effective in nonsporulating fungi identifica-
tion which reduced the impact of biased judgement (Chen, 2008) and the Large Subunit (LSU) and ITS
data are powerful tools to resolve the taxonomy of basidiomycetous endophytes (Rungjindamai, 2008).
Pleurostoma, Chaetomium, Coniochaeta (Lecythophora), Daldinia, Xylaria, Hypoxylon, Nodulisporium,
Cazia, and Phellinus isolated as endophytes from Huperzia serrata were confirmed for the first time by
rDNA ITS analysis (Chen, 2011).

Phytoremediation and Endophytes: Basic Relationship and Recent Research

Due to its “green” approach phytoremediation is a rapidly growing field in which remediation of envi-
ronmentally toxic compounds done by the use of plants. White (2003) describes a division of phytore-
mediation, rhizodegradation, is the use of plants to stimulate the microbial community near the root soil
interface to enhance the degradation of recalcitrant compounds in the soil. The production of bioactive
substances by endophytes is directly related to the independent evolution of these microorganisms,
which might have incorporated genetic information from higher plants, allowing them to better adapt
to plant host and carry out some functions such as protection from pathogens, insects, and grazing
animals (Souza, 2004). One of the major limitations of phytoremidation which is, even plants that are
tolerant to the presence of contaminants often remain relatively small, due to toxicity of the pollutants
that they are accumulating or the toxic end products of their degradation. Along with the production of
novel chemicals, many endophytes have shown a natural capacity for xenobiotic degradation or may act
as vectors to introduce degradative traits. The ability of some endophytes to show resistance to heavy
metals/antimicrobials and degrade organic compounds probably stems from their exposure to diverse
compounds in the plant/soil niche. This natural ability to degrade these xenobiotics is being investigated
with regard to improving phytoremediation (Siciliano, 2001; Barac, 2004; Germaine, 2006). Numer-
ous plant-derived chemicals, counting those generated from root turnover, stimulate microorganisms to
biodegrade xenobiotics (Isidorov & Jdanova, 2002; Miya & Firestone, 2001) In particular, salicylate,
which induces systemic acquired resistance in plants (Meyer, 1999), has been linked to the microbial
degradation of naphthalene, a polycyclic aromatic hydrocarbon (PAH) (Yen & Gunsalus, 1982). Chen
& Aitken (1999) demonstrated the ability of salicylate to greatly enhance the rate of removal of other
PAHs, such as fluoranthene, pyrene, benzanthracene, chrysene, and benzpyrene. Master (2001) studied
that salicylate elevated expression of bphA, which encodes biphenyl dioxygenase, in the PCB-degrader
Pseudomonas sp. Cam-1, whereas several terpenes, (s)-(þ)- carvone, p-cymene, and pinene) inhibited
bphA activity at similar concentrations (1mM). Recognizing the plethora of plant-derived PAH ana-
logues, it is no surprise that the rhizosphere has been repeatedly shown to contain a diverse population
of PAH-degrading microorganisms (Daane, 2001). Researchers have begun to investigate the efficacy of
amending PAH-contaminated soil with salicylate in an effort to induce PAH degradation by indigenous
soil microorganisms (Ogunseitan, 1991). Presumably, the exogenous source of salicylate enhances the
survival of PAH-degrading microorganisms and induces the genes encoding enzymes involved in PAH
degradation. Recent evidence also suggests a link between salicylate and PCB degradation (Singer, 2001).
Potential endophytic microorganisms to improve phytoremediation Xenobiotics degrading ability
founds in many endophytes of that host plants growing in soil contaminated with xenobiotics these
organisms have contaminant degrading genes (Siciliano, 2001). Contamination of field with nitro aro-

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A Prospective Study on Emerging Role of Phytoremediation

matic compounds known for presence of prevalent endophytic strains than of plant rhizospheric or soil
microbial microbes. Phyto-symbiotic strain of Methylbacterium which was isolated from hybrid poplar
trees (Populus deltoidsx nigra) able to biodegrade many nitro-aromatic compounds such as 2,4,6-trini-
trotoluene (Van, 2004). Endophytic bacteria and their prospective application of biotechnology was
described by Barac (2004) .They engineered Burkholderia cepacia G4 studied to increase plant tolerance
to toluene and decrease the transpiration of toluene to the atmosphere. Toluene is one of the components
four for BTEX contamination, and this has the potential to improve phytoremediation by decreasing
toxicity and increasing degradation of the xenobiotic component Barac 2004 .Endophytic bacteria plays
a hopeful role in the remediation of contaminants as well as their degradation. The capability of several
endophytes to be evidence for resistance to heavy metals and degrade organic compounds in the plant/
soil. This ability to degrade these xenobiotics is being investigated with respect to improving phytoreme-
diation (Barac, 2004; Porteous-Moore, 2006; Ryan, 2007) .Bacteria degrading recalcitrant compounds
are more abundant among endophytic populations than in the rhizosphere of the plants in contaminated
sites (Siciliano, 2001), which could mean that endophytes have a role in metabolizing these substances.
Engineered endophytic Burkholderia cepacia strain improved phytoremediation and promoted plant
tolerance to toluene (Barac, 2006). Study by Porteous Moore (2006) describes the range of endophytes
found in poplar trees, growing at a phytoremediation field site contaminated with toluene (Taghavi, 2005),
with mean of identifying potential candidates for enhancing phytoremediation of toluene, ethylbenzene
and xylene (BTEX)compounds (Germaine, 2004). The isolated endophytic bacteria were characterized
by comparative sequences analysis of partial 16SRNA genes, Box-PCR profiling of genomic DNA and
physiological categorization, with respect to substrate utilization, antibiotics and heavy metals sensi-
tivities. Germaine, (2006) confirmed that inside the large bacterial communities found in poplar tree
several endophytic strains were present that had the potential to get better phytoremediation of volatile
organics and herbicides. Possible advantages of using endophytes to improve xenobiotic remediation
were summarized by Newman and Reynolds, 2005. These research areas need further investigation to
establish the development of endophytic niche for applications related with environmental biotechnology.
Doty (2008) claims a major benefit of using endophytic bacteria over rhizospheric bacteria in phy-
toremediation is that while a rhizospheric bacterial population is difficult to control, and competition
between rhizospheric bacterial strains often reduces the number of the desired strains (unless metabolism
of the pollutant is selective), the use of endophytes that naturally inhabit the internal tissues of plants
reduces the problem of competition between bacterial strains Plant-associated endophytes with potential
for bioremediation identified include endophytes of poplar trees as shown in Table 2. Van Aken (2004)
describes a methylotrophic endophytic bacterium isolated from hybrid poplar trees (Populus deltoides
X Populus nigra DN34) that was capable of degrading the explosives TNT, RDX and HMX, to carbon
dioxide evocative of that these endophytes are imminent for remediation of environmental soil containing
these explosive nitro aromatic compounds. Germaine (2006) studied that when pea (Pisum sativum) used
as host plants and inoculated with Pseudomonas endophytes, isolated from hybrid poplars (P. trichocarpa
X P. deltoides cv. Hoogvorst) and capable of degrading 2,4-D, the pea plants showed no accumulation
of 2,4-D in their tissues and showed little or no signs of phytotoxicity when compared to uninoculated
controls portentous that these endophytes have possible ability for bioremediation of environmental soil
contaminated with 2,4-D . Pseudomonas putida VM1441P was able to degrade Naphthalene (Germaine,
2009).Degradation of chlorobenzoic acids by Pseudomonas aeruginosa R75 and Pseudomonas savas-
tanoi CB35 isolated from Elymus dauricus was studies Siciliano (1998). Herbaspirillum sp.K1 isolated
from Triticum spp bioremediate compounds like PCB and TCP (Mannisto, 2001).

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Table 2. Plant-associated endophytes with potential for bioremediation

Enzyme Target Pollutant

Aromatic dehalogenase     Chlorinated aromatics (DDT, PCBs etc.)
Carboxyl esterases     Xenobiotics
Cytochrome P450     Xenobiotics (PCBs)
Dehalogenase     Chlorinated solvents and Ethylene
Glutathione s-transferase     Xenobiotics
Peroxygenases     Xenobiotics
Peroxidases     Xenobiotics
Laccase     Oxidative step in degradation of explosives
N-glucosyl transferases     Xenobiotics
Nitrilase     Herbicides
Nitroreductase     Explosives (RDX and TNT)
N-malonyl transferases     Xenobiotics
O-demethylase     Alachlor, metalachor
O-glucosyl transferases     Xenobiotics
O-malonyl transferases     Xenobiotics
Peroxdase     Phenols
Phosphatase     Organophosphates

Application of Endophyte Mediated Phytoremediation

Endophytes in Bioremediation of Metals

Endophytic bacteria have been shown to possess several characters that can alter heavy metals bioavail-
ability (Lasat, 2002) through the release of chelating substances, acidification of the microenvironment,
and by influencing changes in redox potential (Smith & Read, 1997). For example, Abou-Shanab (2003)
reported that the addition of Sphingomonas macrogoltabidus, Microbacterium liquefaciens, and Micro-
bacterium arabinogalactanolyticum to Alyssum murale grown in serpentine soil significantly increased
the plant uptake of Ni when compared with the un-inoculated controls as a result of soil pH reduction.
However, heavy metals are known to be toxic to plants and most organisms when present in soils in
excessive concentrations. Giller (1998) reported that there was a detrimental effect to soil microbial di-
versity and microbial activities (indexes of microbial metabolism and of soil fertility) in metal-polluted
environments. Burd (2000) states that soil microorganisms are known to affect the metal mobility and
availability to the plant, through acidification, and redox changes or by producing iron chelators and
siderophores for ensuring the iron availability, and/or mobilizing the metal phosphates. A large propor-
tion of metal contaminants are unavailable for the root uptake by plants, because heavy metals in soils
are generally bound to organic and inorganic soil constituents, or alternatively, present as insoluble pre-
cipitates. Siderophore producing plant growth-promoting rhizobacteria (PGPR) solubilize unavailable
forms of heavy metal-bearing Fe and also form complexes with bivalent heavy metal ions that can be
assimilated by root mediated processes (Braud, 2009). The PGPR may also contribute in reducing the

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metal phytotoxicity by biosorption mechanism (Zaidi, 2006). The bacterial cells could adsorb a greater
amount of heavy metals either by a metabolism in dependent passive, or by a metabolism-dependent
active process (Khan, 2007). Bacterial biosorption/bioaccumulation mechanism, together with other
plant growth promoting features (e.g. production of ACC deaminase and phytohormones) accounted for
improved plant growth in metal-contaminated soils (Zaidi, 2006). The efficiency of phytoremediation
is also influenced by the bioavailability of metals to plants in soil. Bacteria may transform toxic heavy
metals to forms that are more readily taken up into roots. For example, bacteria could enhance Se accu-
mulation in plants by reducing selenate to organic Se, and organoselenium forms like SeMet are known
to be taken up at faster rates into roots than inorganic forms (Zayed, 1998). Huang (2005) further depicts
the relative changes as the percentages of the speciation concentration difference between bulked soil
and rhizosphere to the concentration of bulked soil. Results showed that the relative changes of organic
bound Cu, Zn and Pb were, respectively, +5%, +23%, +3% in the infected rhizosphere, and 0.8%, −3%,
−2% in the noninfected rhizosphere. Thus, significant amounts of Cu, Zn and Pb were bounded by or-
ganic matter in the infected rhizosphere.
Because of the broad host range, the endophytic nature and resistant to multiple metals, endophytic
fungi from plants accumulating multiple metals should be valuable microorganism resources for bio-
remediation of water and soils contaminated by multiple heavy metals. Zhang (2002) found that Pteris
vittata (Chinese brake fern) efficiently hyperaccumulates arsenic in its fronds which can be effectively
harvested. Plants that are especially good at concentrating the pollutants are termed hyperaccumulators.
The hyperaccumulator-associated endophytes could be metal resistant, due to long-term adaptation to
the high concentration of metals accumulated in the plants (Idris, 2004). Baker (2000) defined a metal
hyperaccumulator as a plant that can concentrate the metals to a level of 0.1% for nickel, cobalt, copper,
and lead, 1% for zinc, and 0.01% for cadmium. Pteris vittata can effectively remove this metalloid from
soil. Vido (2001) studied another hyperaccumulator is Thlaspi caerulescens, which can concentrate
cadmium, a highly toxic and probably carcinogenic metal. The mechanism of uptake of cadmium and
zinc by this member of the Brassicaceae family has been well studied by Pence (2000) this involves a
highly expressed metal transporter. The zinc/cadmium pumping ATPase was recently purified directly
from T. caerulescens and was shown to transport both zinc and cadmium (Parameswaran, 2007). Bras-
sica napus, Nicotiana tabacum, Thlapsi caerulescens, Alyssum bertoloni, Alnus firma, T. goesingense
and Solanum nigrum are some of the hyperaccumulating plants from which isolation of metal resistant
endophytes is done in recent years. On the other hand many metal resistant endophytes have also been
isolated from non – hyperaccumulating plants such as Arabis hirusta, Acacia decurrens, and Symplocos
paniculata (Li, 2012). The examples of metal resistant endophytic fungi are Peyronellaea, Stegano-
sporium, Microspaeropsis, Aspergillus. Mucor, Phoma and Alternaria. Mastretta, (2009) and his team
studied the role of endophytes in bioremediation in Nicotiana tabaccum plants. Inoculation of Nicotiana
tabaccum with endophytes resulted in improved biomass production under conditions of Cadmium(Cd)
stress, and the total plant Cd concentration was higher compared to noninoculated plants. These results
demonstrated the beneficial effects of seed endophytes on metal toxicity and accumulation. Ahmad (2006)
studied promising phytoremidation capacity of Ni, Cr and Cd by Aspergillus niger and Pencillium sp from
single and multi-metal solutions and this study also highlighted possible exploitation of the filamentous
fungi of metal polluted habitat. Endophytic fungus Microsphaeropsissp LSE10 isolated from cadmium
hyperaccumulator Solanum nigrum L was utilized as a bioabsorbent for the detoxification of cadmium
(Luo, 2010). In another research Kaoushik (2013) isolates two arsenic tolerant fungal strains Aspergil-
lus flavus and Aspergillus.niger. These two strains are capable of removing 50%-76% of arsenic from

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different arsenic enriched medium, simultaneously also tolerant to different other heavy metals like Cd,
Pb, Hg, Zn and Cr. In near future these two fungal strains will be effective in arsenic removal planning
from arsenic contaminated sites. According to Deng (2014) Lasiodiplodia sp. MXSF31 the Cd, Pb, Zn
resistant endophytic fungi was isolated from metal accumulating Portulaca oleracea and re-isolated
from the shoots and roots of inoculated rape . Lasiodiplodia sp. MXSF31 showed high biosorption and
bioaccumulation capacities of Cd, Pb and Zn from the metal contaminated solutions and enhanced the
metal extraction efficacy of rape in soils contaminated by multiple metals. Another interesting study
which study indicates that isolated fungi of contaminated water can be used as bioremediation agents
done by Chandrakar (2014). A comparative level of metal resistance was also shown by filamentous
fungi originated from unpolluted sites. The tolerance and resistance of the isolates depended much more
on the fungus tested than on the site of its isolation. This variation may be explained by the development
of tolerance and adaptation of the fungi to heavy metals. Aspergillus sp. and Fusarium sp. was the most
resistance to all the metals tested, which make them promising candidates for further investigations
regarding their ability to remove metals from contaminated environment. The main conclusion of this
study states that fungal population isolated from heavy metal contaminated sites has the ability to resist
higher concentration of metals, some of plants with their endophytes in bioremediation of metals given
in table 3.

Endophytes in Hydrocarbon Bioremediation

The importance of hydrocarbon remediation is due mainly to the environmental impacts caused by petro-
leum and its derivatives. Hydrocarbon degradation is, undoubtedly, the most important bioremediation
process and, although not yet totally clear, is directly linked to the recruiting ability of microorganisms
and provides the conditions for these to perform their degrading activity (Muratova, 2003). Hydrocarbon
chains present light fractions forming gases and heavy fractions forming oil. The distribution of these
chains is differentiated according to the place of origin (Ramos, 2006). The source of the environmen-

Table 3. Endophytes in bioremediation of metals

Endophyte Microorganism Plant Metal Reference

Pseudomona sp. Ps29C, Bacillus megaterium Bm4C Brassica Juncea Nickel Rajkumar & Freitas (2008)
Mesorhizobium sp. Rengei B3 Astragalus sinicus Cadmium Sriprang (2003)
Azotobacter chroococcum HKN-5, Bacillus megaterium Brassica Juncea Lead, Zinc Wu (2006)
HKP-1, Bacillus mucilaginosus HKK-1
Pseudomonas chlororaphis SZY6, Azotobacter Brassica napus Copper He (2010)
vinelandii GZC24, Microbacterium lactium YJ7
Kluyvera ascorbata SUD165 Indian mustard Nickel, Lead & Zinc Burd (2000)
Achromobacter xylosoxidans Ax10 Brassica Juncea Copper Ma (2009)
Bacillus subtilis SJ-101 Brassica Juncea Nickel Zaidi (2006)
Microbacterium oxydans AY509223 Alyssum murale Nickel Abou-Shanab (2006)
Pseudomonas fluorescens G10, Microbacterium sp. Brassica napus Lead Sheng (2008)
G16
Pseudomonas sp. PsA, Bacillus sp. Ba32 Brassica Juncea Chromium Rajkumar (2008)
Microsphaeropsissp LSE10 Solanum nigrum L Cadmium Luo (2010)

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tal impact caused by the oil industry is in the processes involved in the discovery of new oil wells, oil
refinement and transportation, where roughly 10% of the product’s use is lost in spills with catastrophic
consequences to coastlines (Van Hamme, 2003). A large amount of crude oil is also lost in storage causing
highly contaminating sludge. Soil contamination by hydrocarbons, as well as underground and surface
water, seriously affects the ecosystem, especially through the accumulation within animals and plants
resulting in death and mutation (Ilyina, 2003). The activity of microorganisms in the cycle and remedia-
tion of hydrocarbons in the environment is undeniable. They are often found in contaminated environ-
ments (Labud, 2007), although, their action depends on the contaminant present and its availability as a
carbon source. A number of bacteria have, therefore, been described as responsible for petroleum and its
derivatives bioremediation processes, among these are Pseudomonas, Mycobacterium (Solano-Serena,
2000), Acinetobacter sp. (Gallego, 2001) and Serratia marcescens (Wongsa, 2004). Most of the bacteria
involved in these bioremediation processes present aerobic metabolisms, some of which are capable of
degrading both crude oil and more refined fractions. The main purpose of this study was the isolation of
endophytic bacteria present in vegetables growing in locations impacted by asphalt paving waste, their
identification and the determination of their degrading capacity for petroleum and its derivatives, with
a view to investigating new forms of hydrocarbon bioremediation.
Bacteria of Pseudomonas genus are known for their bioremediation potential, particularly in hydro-
carbons (Evans, 2004; Vetrova, 2007). Bacteria of the Bacillus genus are recognized as great enzyme
producers (Heck, 2002; Anto, 2006), and may also present bioremediation potential. Cubitto (2004)
describe a sample of Bacillus subtilis as a biosurfactant producer contributing to the bioremediation
process by the autochthonous microbiota in sites contaminated by petroleum. Johnsen (2005) found
that bacteria initiate PAHs degradation by the action of intracellular dioxygenases, oxygenase, dehy-
drogenase, phosphatases, dehalogenases, nitrilases, nitroreductases and lignolytic enzymes. Chadhain
(2006) states that aromatic ring dioxygenases are multicomponent enzymes which consist of an electron
transport chain containing a ferredoxin and a reductase and a terminal dioxygenase. The best studied
PAHs dioxygenase is naphthalene dioxygenase from Pseudomonas putida encoded by the NAH plasmid
pDTG1 (Dennis & Zylstra 2004).

Endophytes in Plastic Bioremediation

Plastic is a macromolecule comprising of smaller repetitive units made up of either natural or synthetic
substances that enabled most of the industrial, technological revolutions of the 20th century and are
composed of petroleum based materials called resin which are widely used for packaging applications
like Polyethylene (LDPE, MDPE, HDPE, LLDE), Polypropylene (PP), Polystyrene (PS), Polybutylene
terephthalate (PBT) materials that are resistant to biodegradation (Raaman, 2012). Polyurethane is a
xenobiotic substance that offers to a kind of polymers derived from the condensation of polyisocyanate
and polyols having intra molecular urethane bonds (-NHCOO-) connected with a series of urethane link-
ages (Shah, 2008). Microorganisms are known to survive in environment where intractable materials
like polyurethane are present, so it is possible that these microorganisms can use this substance and can
be useful tools in biodegradation. Isolation of nineteen medium chain length (mcl) poly (3-hydroxy-
alkanoate) PHA-degrading microorganisms was characterized for its extracellular depolymerase from
natural sources (Gangoiti, 2008). These microorganisms hydrolysed biodegradable plastics such as short
chain-length (scl) PHA, poly (ɛ-caprolactene) (PCL), poly ethylene succinate (PES) and poly (L-lactide)

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(PLA). Howard (2001) gives a report on the degradation of polyester PUR by microorganisms especially
by fungi deals mainly by the hydrolysis of ester bonds by esterases. Fungi colonize the wastes as substrate
and secrete extracellular enzymes which play a major role in breaking down the polymers into simple
dimer and monomers. Studies from fungal colonization and biodeterioration of polyvinyl chloride in in
situ and ex situ conditions recommended that microbial progression may occur for the longer period of
exposure in in situ conditions6. Aureobasidium pullulans, Rhodotorula aurantica and Kluyveromyces
spp. which were the principle colonizing fungi identified by Webb (2000) and Oda (1998). Putative
polyurethanases have been isolated and characterized from protein extracts of several organisms, in-
cluding the bacteria Pseudomonas chlororaphis (smith, 2008) and Comamonas acidovorans (Akutsu,
1998, Allen, 1999), as well as the fungus Candida rugosa (Gautam, 2007). The active enzymes have
been classified as esterases (Crabbe, 1994), lipases (Stern, 2008), and proteases and ureases (Pathirana,
1984), suggesting degradation of the PUR substrate by cleavage of the ester bond.
Enzymatic degradation of PUR by both fungi (Cosgrove, 2007; Crabbe, 1994) and bacteria (Howard,
1998; Howard, 2001; Kay, 1991) has been demonstrated. Soil fungi comprise the majority of organisms
screened for PUR degradation activity (Cosgrove, 2007; Crabbe, 1994). Fungi of the genera Alternaria,
Aspergillus, Phoma, Pennicilium, Plectosphaerella, Geomyces, Nectria, and Neonectria were isolated
with access to mixed nutrient sources from buried PUR samples. Geomyces pannorum was the most
commonly isolated PUR-degrading organism with this method (Cosgrove, 2007). Few organisms have
been shown to degrade PUR as a sole carbon source. Aspergillus niger has some reported degradation
activity; however, it was observed to be quite slow, with visible signs of degradation occurring only
after 30 days.

Endophytes in Pesticide Bioremediation

Pesticides can be used to control or to manage pest populations at a tolerable level. The suffix “-cide”
literally means “kill”, therefore, the term pesticide refers to a chemical substance that kills pests. Koo-
kana (1998) states that major environmental concern of used pesticides is their capacity to leach down
to subsoil and contaminate the ground water and if immobile, they would persist on the top soil where
it could accumulate to toxic level in the soil and become harmful to microorganisms, plants, animals
and man (Amakiri, 1982). Mervat (2009) demonstrated the use of microorganisms for the degradation
and detoxification of numerous toxic xenobiotics, especially pesticides, proved to be an efficient tool to
decontaminate the polluted sites in the prevailing environment. Complete biodegradation of a pesticide
involves the oxidation of parent compound to form carbon dioxide and water. This process provides both
carbon and energy for the growth and reproduction of microbes. A diverse group of bacteria, includ-
ing members of the genera Alcaligenes, Flavobacterium, Pseudomonas and Rhodococcus, metabolize
pesticides (Aislabie & Lloyd-Jones, 1995; Richin, 1997; Mulchandani, 1999). Actinomycetes have con-
siderable potential for the biotransformation and biodegradation of pesticides. Members of this group of
Gram-positive bacteria have been found to degrade pesticides with widely different chemical structures,
including organochlorines, striazines, triazinones, carbamates, organophosphates, organophosphonates,
acetanilides, and sulfonylureas. In nature, plants for example, a hydrophyte Typha latifolia metabolize
organic pollutants (Ops), remove malathion and dimethoate pollutants from soil. Germaine reported that
when pea (Pisum sativum) plants were inoculated with Pseudomonas endophytes, isolated from hybrid
poplars (P. trichocarpa X P. deltoides cv. Hoogvorst) and capable of degrading 2,4-D, the pea plants

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showed no accumulation of 2,4-D in their tissues and showed little or no signs of phytotoxicity when
compared to uninoculated controls suggesting that these endophytes have potential for bioremediation
of environmental soil contaminated with 2,4-D .

Endophytes in Organic Waste Bioremediation

Though phytodegradation is still a relatively new area of green research, there are many research groups
engaged in studying the underlying science necessary for a wide range of applications for plant-based
remediation system against organic contaminants (Newman and Reynolds, 2004). Much work remains to
be done in carrying out field studies based on laboratory-scale results/experiments using plant-associated
endophytic and rhizospheric bacteria to degrade a wide range of toxic organic compounds of concern in
environmental soil before commercially viable systems. Using biotechnology tools, bacterial strains can
be engineered expressing specific enzymes to degrade toxic organic substances in an effective way. Ad-
ditionally bacteria (rhizospheric and/or endophytic) can be engineered, through gene transfer to degrade
toxic organic pollutants present in prevailing environment. However, genetic engineering of endophytic
as well as rhizospheric bacteria along with transgenic plants is seems to be promising approach for
remediation of contaminated environmental sites.

Effect on Climate of Endophytic Population and Vice Versa

Endophytic population varies from plants to plants and from species to species. Within the same species
it not only varies from region to region but also differs with change in climatic conditions of the same
region. Temporal changes in relative frequency of total endophytic fungi were studied by Chareprasert,
(2006). They found that matured leaves of teak (Tectona grandis L.) and rain tree (Samanea saman Merr.)
had greater number of genera and species, with higher colonization frequency, than those in the young
leaves and their occurrence in leaves increased during rainy season. The endophytic population and fre-
quency tended to differ among sampling dates for all the organs studied, namely, young leaves, petiole,
and twigs of Gingko biloba L.They proved that the occurrence of Phyllosticta sp. in both leaves and
petioles was first detected in August and peaked in October with none in the month of May. Phomopsis
sp. was detected in twigs throughout the growing season. These results suggest that the distribution of
the two dominant endophytic fungi was organ-specific and differed within seasons.
Greenhouse gas emissions (particularly methane and carbon dioxide) depending on the composition
of vegetation is also a area of research besides bioremediation of volatile organics. (Goraj, 2013; López,
2013). The main greenhouse gases are methane and carbon dioxide. Raghoebarsing (2005) has been
demonstrated that methanotrophs inhabiting Sphagnum spp., e.g., Methylocella palustris and Methy-
locapsa acidiphila, oxidize methane to carbon dioxide, which is later used by Sphagnum plants in the
process of photosynthesis. This discovery substantially changed the description of the carbon cycle in
peat ecosystems and at the same time the global carbon cycle. In this way, methanotrophic endophytes
inhabiting Sphagnum spp. Can act as a natural methane filter that can reduce CH4 and CO2 emission
from peat lands by up to 50% (Kip, 2012). Other field studies of Goraj, (2013) have shown the potential
ability of the plant–methanotrophic bacteria systems to reduce methane emission up to 77%, depending
on the season and the host plant. Furthermore, isolated endophytes from Sphagnum spp. could colonize
crops and promote their growth. Molecular genetic analysis has shown that the dominant endophytic

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groups belong to the genera Burkholderia, Pseudomonas, Flavobacterium, Serratia, and Collimonas.
Shcherbakov, (2013) have suggested that the isolated strain can be a promising object for the develop-
ment of effective growth-promoting and protective microbiological preparations to be used in agricul-
ture. Furthermore, the endophytes inhabiting Sphagnum spp. can be used for the inoculation of plants
inhabiting an artificial wetland system used to treat mixed contaminations.
Recent studies have indicated the big potential of plants in the remediation of polluted sites. The
excellence of adaptation abilities and promising remediation efficiencies strongly imply the superiority
of endophytes in the bioremediation of mixed contamination at their low concentrations. It could be
useful for developing an efficient metal removal system (Li, 2012). On the other hand, the adaptation
abilities and the remediation efficiencies of endophytic microorganisms still need further understanding
and recognition for practical applications. Environmental water pollution is a serious problem in China
and other developing countries because of the discharge of organic pollutants such as pesticides and
nutrients including phosphorus (Zhao, 2006; Wang, 2007; Perelo, 2010). Because organic pollutants in
aquatic environments are generally present in low concentrations, bioremediation and/or phytoremedia-
tion may be the most economic and reliable approach to address the problem. In addition, phosphorus,
which can stimulate eutro phication, usually precipitates in sediments. This makes phosphorus largely
unavailable to plants. Furthermore, the phosphorus precipitate can persist in a lake for a considerable
length of time and continue to periodically cause algal blooms. The release of in soluble phosphorus and
its removal from the environment using aquatic plants is a so called ecoengineering approach (Weyens,
2009) in which released phosphorus is taken up by plants and then removed from the aquatic environ-
ment by harvesting the plants. Apart from Toyama (2009) who showed that an aquatic plant−bacterial
collaboration could accelerate contaminant degradation in the aquatic environment, there has been little
research into endophytic bacteria associated with aquatic plants. Heavy metal contamination to water
and soil poses a major environmental and human health problem. In addition, excessive metal concentra-
tions in contaminated soils result in decreased soil microbial activity and soil fertility, and yield losses
(McGrath, 1995). Phytoremediation, the use of plants to extract, sequester, and/or detoxify pollutants
through physical, chemical, and biological processes (Wenzel, 1999) has been reported to be an effective,
in situ, non-intrusive, low-cost, aesthetically pleasing, ecologically benign, socially accepted technol-
ogy to remediate polluted soils (Alkorta & Garbisu, 2001). It also helps prevent landscape destruction
and enhances activity and diversity of soil microorganisms to maintain healthy ecosystems, which is
consequently considered to be a more attractive alternative than traditional methods to the approaches
that are currently in use for dealing with heavy metal contamination (Cunningham & Ow, 1996; Bogardt
& Hemmingsen, 1992).

Advantages of Bioremediation by Endophytes

Bioremediation by endophytes can often be carried out on site, often without causing a major disrup-
tion of normal activities. This also eliminates the need to transport quantities of waste off site and the
potential threats to human health and the environment that can arise during transportation. This technique
of bioremediation can prove less expensive than other technologies that are used for cleanup of hazard-
ous waste. Ryan (2007) listed some of the advantages associated with the use of endophytic bacteria
in phytoremediation of contaminated environmental soil when compared with the use of plants alone.
They include

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• Quantitative gene expression of bacterial pollutant catabolic genes can be used to assess the ef-
ficiency of the remediation process,
• Genetic engineering of a bacterial catabolic pathway is easier to manipulate than a plant catabolic
pathway, and
• Toxic pollutants taken up by the plant may be degraded in planta by endophytic degraders reduc-
ing the toxic effects of contaminants in environmental soil on flora and fauna.

Disadvantages of Bioremediation by Endophytes

Biological processes are often highly specific. Important site factors required for success include the
presence of metabolically capable microbial populations, suitable environmental growth conditions,
and appropriate levels of nutrients and contaminants and bioremediation is limited to those compounds
that are biodegradable. Not all compounds are susceptible to rapid and complete degradation. There
are some concerns that the products of biodegradation may be more persistent or toxic than the parent
compound. Some disadvantages associated with the use of bacteria in plant-associated bioremediation
of contaminated environmental soil. Weyens, 2009 reviewed the benefits of using plant-associated endo-
phytes in bioremediation and emphasized that although successfully applied in several laboratory-scale
experiments, the large-scale field application of this technology is limited by a number of issues including

• The levels of contaminants tolerated by plants,

• Limited bioavailability of organic contaminants, and
• Unacceptable levels of vapor transpiration of VOCs into the atmosphere.

Some other disadvantages were also given by Ryan, 2007. They include,

• This technology is limited to shallow contaminants in environmental soil,

• It is slower than traditional remediation technologies,
• The choice of plant can mean that it is only seasonally effective,
• It is associated with phytotoxic effects of contaminants,
• There is potential for the environmental contaminants or their metabolites to enter the food chain
if contaminants are not completely detoxified and if the plants are consumed by local fauna.

CONCLUSION

Bioremediation by endophytes and with plant relation is useful for the complete destruction of a wide
variety of contaminants like organics, plastics, metals, etc. Bioremediation by endophytes is a natural
process and is therefore perceived by the public as an acceptable waste treatment process for contaminated
material such as soil. These microbes in association with plants are able to degrade the contaminant
increase in numbers when the contaminant is present, the contaminant is degraded, the biodegradative
population declines. The residues by these remediation methods treatments are usually harmless products
and include carbon dioxide, water, and cell biomass. Many compounds that are legally considered to be
hazardous can be transformed to harmless products. This eliminates the chance of future liability associ-
ated with treatment and disposal of contaminated material. Despite the disadvantages associated with

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the use of plant-associated endophytic bacteria to degrade toxic organic compounds in environmental
soil, it is clear that there is potential for these bacteria to make a significant contribution to sustainable
bioremediation. Bioremediation by endophytes although considered a boon in the midst of present day
environmental situations, can also be considered problematic because, while additives are added to enhance
the functioning of one particular bacterium, fungi or any other microorganisms and their association with
plants. it may be disruptive to other organisms inhabiting that same environment when done in situ. Even
if genetically modified microorganisms are released into the environment after a certain point of time it
becomes difficult to remove them. Bioremediation is generally very costly, is labor intensive, and It is
difficult to extrapolate from bench and pilot scale studies to full scale field operations. But Endophytes
mediated phytoremediation and endophytic metabolites have a great promise for providing excellent,
cheaper, and safer bioremediation process, which deserve an extensive investigation in near future.

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Chapter 13
Advances in Bioremediation
for Removal of Toxic Dye from
Different Streams of Wastewater
Priya Banerjee
University of Calcutta, India

Aniruddha Mukhopadhayay
University of Calcutta, India

Papita Das
Jadavpur University, India

ABSTRACT
Azo dyes are used in abundance in several industries like textile, printing, paper, plastic, cosmetics, paints,
etc. Extensive discharge of such dyes in adjacent water bodies has raised much environmental concern.
Azo dyes are toxic to living organisms and their genotoxic and carcinogenic potentials are intensified on
being released as mixtures. In the recent years, various microorganisms have been isolated and reported
to possess tremendous potential for efficient dye degradation. However, the process of bioremediation is
highly controlled by experimental factors like effluent pH, temperature and concentration of dyes in solu-
tion. Therefore, appropriate optimization of these factors is to be determined in order to ensure maximum
efficiency of this process. This review highlights application of immobilization techniques of bacterial
cells for achievement of successful biodegradation. In this study, the existing problems of dye pollution
and possible improvisations for obtaining enhanced bioremediation of dyes have also been discussed.

1. INTRODUCTION

Unplanned and unmonitored industrialization and urbanization in developing countries necessitate sus-
tainable utilization of water via reclamation and recycling of industrial wastewater. Of different types of
industries, those associated with textile processing and manufacturing reportedly consume a huge volume
of water primarily for the dyeing and finishing procedures. This in turn is an area of prime concern for
environmental audit. Besides, inept dyeing procedures often end up discharging large amounts of the
potentially hazardous dyes as dispersion or true solution in wastewater to adjacent water bodies thereby

DOI: 10.4018/978-1-4666-9734-8.ch013

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.

Advances in Bioremediation for Removal

severely contaminating the environment (Stolz, 2001). Owing to the quantity and constituents of the ef-
fluents produced, the textile industry has been considered as the most polluting industrial sector (Sen &
Demirer, 2003). Anthroquinoid, indigoid, and azo aromatic compounds are the primary classes of dyes
in application. Azo dyes are made up of conjugated structures comprised of double bonds and aromatic
rings which participate in strong π- π interactions. These azo dyes are of immense environmental con-
cern, especially in case of reactive dyeing of cellulosic fibres, as huge quantities of unbound dyes are
released through the effluent (Khan & Banerjee, 2010). During the process of utilization, loss of dyes
incurred may range from up to 2% (basic dyes) to 50% (reactive azo dyes) depending on the type of dye
being used (Robinson et al., 2001). Azo, nitro or sulfo groups present in the chemical structure of the
dyes, often render them recalcitrant to microbial degradation thereby leading to accumulation of their
residues in the biotic community (Pourbabaee et al. 2006). Textile dyes present in effluents have been
reported to exhibit mutagenic and genotoxic potentials as well (Sharma et al. 2007).
In recent years, significant attention is being allotted for preventing direct discharge of untreated textile
effluents into neighboring water bodies. The conventional physical and chemical methods developed and
implemented so far for the treatment of dye containing effluents are costly, involve operational glitches,
and in nearly all cases result in CO2 formation due to incomplete degradation of dyes (Karapinar et al.
2000). Complete degradation of textile effluents has been achieved only through biological oxidation
(Karapinar et al., 2000). Nevertheless, challenges encountered by the conventional activated sludge
process (CASP) include maintenance of special nutritional requirements and survival conditions of the
functional microorganisms. Due to an increasing global scarcity of potable water sources, laws regard-
ing uses of water resources have become more stringent making it imperative to design new, efficient,
cost effective and time saving methodologies for the treatment and reuse of wastewater. It stresses upon
the application of biodegradation in combination with adsorption processes for ensuring faster effluent
treatment and maintenance of better survival conditions for the participating microorganisms. Immobi-
lized cells are also widely used for yielding important biological compounds, wastewater reclamation
and soil bioremediation (Khan & Banerjee, 2010). Besides simplifying separation and enhancing the
recovery of immobilized bacteria, other advantages of this process include reusability, cost reduction,
comparatively longer effective lifetime and protection of immobilized strains from adverse experimen-
tal conditions. Immobilization also protects the bacterial cells from high concentrations of recalcitrant
compounds and provides a longer contact time for better degradation of the same. Immobilized cells
have been efficiently applied as biocatalysts for treating large amounts of liquid or soil samples in a
simple and cost effective procedure.
This study summarizes the recent investigations in the removal of azo dyes (using both simulated
and real textile effluents) using immobilized bacterial species which is an emerging procedure for the
biological treatment of dye-rich industrial effluents. This review also analyses the process parameters
of concern and their interaction that affect dye removal. It also describes two models for optimization
namely Response Surface Methodology and Artificial Neural Network used to ensure maximum ef-
ficiency of the designed process.

2. BACKGROUND

Of all types of synthetic dyes being utilized for commercial applications, azo dyes are most extensively
used. This type of dyes are aromatic compounds having one or more –N=N– groups in their chemi-

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Advances in Bioremediation for Removal

cal structure (Pandey et al., 2007). They are widely used in many industries like textile dyeing, food,
cosmetics, paper printing, etc. with the textile industry being its largest consumer (Pandey et al., 2007).
Inefficient utilization procedures lead to the discharge of large quantities of residual dyes into the sur-
roundings along with the industrial effluents that are generated. Presence of several dyes are evident in
water even at concentrations nearing 1 mg/L. Textile effluents with dye concentrations ranging between
10–200 mg/L are considered as highly colored (Pandey et al., 2007). Such types of colored effluents
require proper treatment methodologies prior to discharge. Many reports are available which illustrate
the use of physicochemical methods for treatment of dye-rich effluents. Even the two extensively used
process namely coagulation/ flocculation produced large amounts of sludge, safe disposal of which in
turn was a concern. Processes like adsorption and membrane filtration were effective but yielded second-
ary waste streams which required further treatment (Pandey et al., 2007). Due to such drawbacks in the
conventional technologies, in recent studies, biological methods have provided attractive options for the
efficient treatment of dye-bearing wastewaters. Biological methods are also energy and cost effective. Such
methods are generally considered environmentally benign, as they can achieve complete mineralization
of organic pollutants in a cost effective manner. Azo dyes are reported xenobiotics. Hence, they can be
anticipated to exhibit recalcitrance to biodegradation. Studies have proved that dyes resist biodegradation
when treated in conventional activated sludge treatment units (Stolz, 2001). However recent studies have
shown that several microorganisms, including fungi, bacteria, yeasts and algae, possess the potential to
decolorize and even completely mineralize various azo dyes under optimized growth conditions.
In recent times, immobilized microbial cell technology has been established as the most appropri-
ate treatment method for treatment of effluents rich in recalcitrant reactive azo dyes along with their
metabolites. Cell immobilization facilitates increased biomass concentration, higher process stability,
reusability of biocatalysts, increased hydraulic loading rates, and improved stable microbial activities in
the immobilized environment as compared to CASP and suspended cell cultures (Guo et al., 2007; Chen
et al., 2009a). Immobilized cell cultures (ICC) exhibit higher activity, more resilience to fluctuations in
environmental conditions like temperature, pH, variations in concentrations of toxic chemicals, etc. ICC
can be easily applied for solid-liquid separation within settling tanks, and can also eradicate difficulties
related to bulking amount in comparison to suspension culture (Ramsay et al., 2005). Natural (agro and
organic wastes) or synthetic (polymers and carbon compounds) matrices are specifically suitable for
entrapment of cells as they create a local anaerobic environment which in turn promotes oxygen-sensitive
decolorization (Steffan et al., 2005). It is also essential to design a suitable bioreactor in order to achieve
maximum efficiency of ICC. Implementation of the ICC technology significantly raises the cell count
of the selected bacterial specie (azo dye-degrading bacteria), thereby bringing about a reduction in the
bioreactor volume, which is a critical criterion in designing a wastewater treatment unit (Pandey et al.,
2007). This review is expected to serve as a useful reference for the further development of effective
bioprocesses for treatment of dye-rich wastewater utilizing immobilized bacterial cells as the biocatalyst.

3. BACTERIAL DECOLOURISATION AND DEGRADATION MECHANISMS

Bacterial treatment of dye-rich wastewater may proceed along two principal mechanisms: biosorption
and enzymatic degradation. It may also occur via a combination of both of the above (Phugare et al.,
2010; Wu et al., 2012).

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Biosorption

The biosorption capacity of selected bacterial specie is accredited to the property of the heteropolysaccha-
ride and lipid components of the cell wall, which bear several functional groups (such as –NH2, -COOH,
-OH, -PO4, etc.) and other charged groups, that help in development of strong attractive forces between
the azo dye molecules and the cell wall (Srinivasan & Viraraghavan, 2010; Charumathi & Das, 2012).
Various types of pre-treatment processes like autoclaving (Lim et al., 2010), treatment with acid (Srini-
vasan & Viraraghavan, 2010), formaldehyde (Ambrosio et al., 2012), alkali or CaCl2 (Vijayaraghavan
& Yun, 2007), may bring about changes on the cell surface and alter the capacity of the binding sites.
The non requirement of nutrients, unhampered storage and usage for prolonged periods and convenient
regeneration using organic solvents and detergents render the dead cells as more suitable for biosorp-
tion in comparison to their living forms (Solis et al., 2012). The process of biosorption is guided by
experimental conditions like pH, temperature and ionic strength of the solution, contact time, adsorbent
dose and adsorbate concentration, chemical structure of dye and the type of microorganism being used
(Vijayaraghavan & Yun, 2007; Ambrosio et al., 2012; Solis et al., 2012).

Enzymatic Degradation

Azo dyes are electron-deficient in nature owing to the presence of azo linkage (-N=N) which in many
cases are associated with other electron-withdrawing moieties like the sulphonic (SO3 −) group. These
moieties create an electron deficiency and render the dye less susceptible to bacterial degradation (Ku-
beran et al., 2011; Kurade et al., 2011). Under optimum conditions, these dyes may be degraded by
the various enzymes like reductases, oxidases, laccases, etc (Solis et al., 2012). Of these the laccase
producing species have higher potential in bioremediation owing to their non-specific oxidation capac-
ity, non requirement for cofactors and use of ambient oxygen as an electron acceptor (Wu et al., 2009).

4. IMMOBILIZATION OF BACTERIAL CELLS

Immobilization of microorganism for biological treatment of wastewater has been widely reported. Of
the various methods of immobilization of bacterial cells, four main procedures have been described as
follows.

Methods of Immobilization

Methods of immobilization can be classified as ‘‘passive” when microorganisms naturally grow on natural
or synthetic surfaces and ‘‘active” when external agents like flocculants, gels and other chemicals are
used to facilitate attachment (Moreno-Garrido, 2008). Immobilization of microbial cells can occur in
either of the following methods (Martins et al., 2013).

• Cross linking: The method involves formation of covalent bonds between activated inorganic
support and cell when aided by a binding (cross-linking) agent. For covalent linking, it is essential
to pre-treat the matrix substrate with chemicals.

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• Entrapment: In case of entrapment, cells are encapsulated in a support matrix or embedded in

fibers. It is an irreversible mechanism that protects the immobilized microbes by creating a bar-
rier around them. This technique ensures the prolonged viability of the immobilized cells during
processing as well as in storage conditions (Górecka & Jastrzębska, 2011). Entrapment is the
most extensively applied method for cell immobilization. The matrices used are discussed in the
following section. In this method, the cells are entrapped within a rigid network to prevent the
cells from diffusing into surrounding medium while simultaneously permitting the penetration of
substrate. Entrapment provides high mechanical strength, but possesses certain disadvantages like
cell leakage, preparation costs, limitations in diffusion, and probable abrasion of support mate-
rial as a result of usage. It can only support low loading capacity as biocatalysts are incorporated
within the support matrix (Gao et al., 2010; Stolarzewicz et al., 2011).
• Encapsulation: Encapsulation is another irreversible process of immobilization in which biocata-
lysts are encapsulated by the membrane and remain free-floating within the core space (Górecka
& Jastrzębska, 2011). The membrane being semi-permeable, allows free flow of substrates and
nutrients when being used. The factor influencing this phenomenon is the appropriate pore size of
the membrane in addition to the size of core material. The capsule protects the biocatalyst from
adverse environmental conditions. This method prevents biocatalyst leakage thereby raising the
process efficiency (Martins et al., 2013). However, inspite of acheiving high cell loading the cap-
sules are still weak (Martins et al., 2013). The diffusion limitation is only disadvantage associated
with this method (Martins et al., 2013).
• Adsorption: Adsorption is the “passive” route of immobilization of microorganisms onto porous
and inert support materials (Araujo et al., 2010). Apparently, it is the simplest method of revers-
ible immobilization (Martins et al., 2013). Firstly, transportation of the cells occurs from the bulk
phase to the surface of support. This is followed by adhesion of cells on the support and succeed-
ing colonization of the matrix surface (Kilonzo & Bergougnou, 2012). Adsorption is based on
weak forces like van der Waals forces, ionic interactions and hydrogen bonds which govern the
cell-support adhesion (Górecka & Jastrzębska, 2011). This frequently reversible process is simple,
efficient and cost effective. It instigates microbial metabolism and protects cells by preserving
their physiological activities (Martins et al., 2013). As it facilitates a direct interaction between
nutrients and the immobilized cells, it lacks the disadvantages borne by the entrapment and en-
capsulation mechanisms.

Matrix Materials

One of the vital decisions which guide the process of immobilization is the selection of matrix/support
material. For treatment of dye-rich wastewater, support materials need to possess the following charac-
teristics (Martins et al., 2013; Górecka & Jastrzębska, 2011):

• Insolubility
• Non-biodegradability
• Non toxic nature
• Light weight and flexibility

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• High mechanical and chemical stability

• High diffusivity
• High biomass retention
• Cost effectiveness

The matrix materials used in previous studies were classified into mainly two types, i.e. organic and
inorganic. Thermostability and resistance to microbial degradation were the reasons behind selection
of inorganic carriers for immobilization of microorganisms. Inorganic supports include clay, zeolite,
poly-acrylamide (PAM), polyvinyl alcohol (PVA), polysulphone beads, etc. They also display much
better mechanical performance in comparison to natural carriers. The organic carriers may comprise of
biopolymers, alginates, agro-industrial wastes like rice husk, wheat husk, saw dust, etc. or other biological
components. Such substrates are convenient to prepare and handle, nontoxic, cost effective and easily
available in large quantities. However, it is difficult to store these substances over long periods in aque-
ous solutions as the encapsulation of the immobilized microorganism may be easily broken during the
operation (Martins et al., 2013). In order to render additional stability to such carriers, various synthetic
plastics such as polypropylene, polyethylene, polyvinylchloride, poly-urethane are being extensively
explored in present studies (Martins et al., 2013). Studies on dye removal using immobilized organisms
on various supports are given in table 1.
In case of entrapment, it was seen that mechanical strength of the immobilized beads was often af-
fected by its repeated use for dye removal (Khan & Banerjee, 2010). In this concern, a rotating biologi-
cal contactor with a disk on which the bacterial specie was immobilized proved to be an improvisation
of this technique as it stabilized the dye-degradation activity of the strains over a longer period of time
(Khan & Banerjee, 2010).

5. OPTIMIZATION PROCEDURES

Experimental factors influencing efficient dye degradation using ICC include pH, temperature, salinity,
dye concentration and availability of oxygen during the process. Optimization of the different parameters
influencing this process of dye removal can be done using two mathematical models namely, Response
Surface Methodology and Artificial Neural Network.

Response Surface Methodology

Response surface methodology (RSM) is a collection of mathematical and statistical techniques imple-
mented for to optimization of a response (in this case, dye removal) which is influenced by several inde-
pendent variables (experimental parameters) through a series of experiments called runs. During these
experiments, a range of variable parameters are considered as input variables in order to determine the
reasons for changes in the output response. The purpose of application of RSM is aimed at reducing the
cost of expensive and repetitive analysis methods and their associated numerical noise. The response
can be recorded either graphically in the 3D space or as contour plots. RSM can be performed for the
optimization of both experimental and numerical outputs.

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Table 1. Studies on azo dye removal using bacterial cells immobilized on inorganic and organic supports

Type of Form of Matrix Bacteria Specie Method of Treated Reference

Matrix Immobilized Immobilization Contaminant
Agro waste Bleached cotton fabric Shewanella sp. • physical Remazol Black B Li and Guthrie,
adsorption method 2010
• chemical coupling
method
Alginates Sodium alginate beads Bacillus sp Encapsulation • Crystal violet, Kulandaivel et al.,
• Congo red, 2014
• Methylene blue
• Safranin
Alginates Sodium alginate beads Pseudomonas aeruginosa Encapsulation Procion blue 2G Saravanan et al.,
2013
Alginates Sodium alginate–kaolin Burkholderia Encapsulation Crystal Violet Cheng et al., 2012
gel beads vietnamiensis
Alginates • Calcium Alginate Proteus vulgaris Entrapment Reactive Blue 172 Saratale et al.,
beads 2011
• k-carrageenan
Alginates Calcium alginate beads Stenotrophomonas Entrapment SITEX Black Galai et al., 2010
maltophilia
Alginates Calcium alginate beads Bacillus subtilis Entrapment Reactive Blue 4 Binupriya et al.,
(RB) 2010
Alginates Calcium Alginate beads • Shewanella sp. Entrapment Acid Red 3R Su et al., 2009
• Pseudomonas sp.
• Aeromonas sp.
• Enterobacteriaceae sp.
• Actinobacterium sp.
Alginates Alginate–silicate sol– Pseudomonas luteola Entrapment Reactive Red 22 Chen and Lin,
gel beads 2007
Alginates Alginate beads Agrobacterium sp. Entrapment 4-aminobenzene- Singh et al., 2006
sulfonate
Cellular Aspergillus oryzae Pseudomonas sp. Adsorption Malachite Green Yang et al., 2011
Mass fungi cellular mass
Composite • Zeolite, Paenabacillus sp. Physical adsorption Reactive Black 5 Lim et al., 2011
• Activated Carbon
• Cement
Composite • Kaolin, • Enterobacter, Physical adsorption Acid Orange 7 Barragan et al.,
• Bentonite, • Pseudomonas 2007
• Powdered activated • Morganella
carbon
Corals Porites corals Aeromonas hydrophila Physical attachment • Reactive red 141 Chen et al., 2009b
• Reactive black 5
• Reactive blue 171
• Reactive yellow 84
• Reactive red 198
• Reactive green 19
Polyvinyl Phosphorylated PVA Sludge Entrapment RED RBN Chen et al., 2003
Alcohol gel beads
(PVA) Gel
Synthetic Polyacrylamide beads Proteus vulgaris Entrapment Reactive Blue 172 Saratale et al.,
beads 2011
Synthetic Polysulphone matrix Corynebacterium Entrapment Methylene blue Vijayaraghavan et
beads glutamicum al., 2008

continued on following page

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Table 1. Continued
Type of Form of Matrix Bacteria Specie Method of Treated Reference
Matrix Immobilized Immobilization Contaminant
Synthetic Polysulphone matrix Corynebacterium Entrapment Reactive Black 5 Vijayaraghavan
beads glutamicum and Yun, 2007
Synthetic Polyvinyl alcohol Consortium Entrapment Direct Fast Scarlet Fang et al., 2004
beads (PVA) beads 4BS
Synthetic Agar cubes Bacillus firmus Entrapment Polar red B Ogugbue et al.,
media 2010
Synthetic Marble chips • Bacillus vallismortis, Biofilm Direct Red 28 Tony et al., 2009
media • B. pumilus,
• B. cereus,
• B. subtilis, and
• B. megaterium

Artificial Neural Networks

Artificial Neural Networks (ANN), also known as neurocomputing, connectionism, or parallel distributed
processing (PDP), is an alternative approach applied to problems where the algorithmic and symbolic
approaches are inappropriate. The design of Artificial Neural Networks is based upon our available
knowledge of biological nervous systems, although they differ in every detail. An Artificial Neural
Network program is a parallel, distributed information processing structure comprised of interconnected
processing units. It is used to determine whether a designed experimental procedure follows the theoreti-
cally determined predictions. These types of mathematical models guide any experimental procedure
to maximum efficiency. These models are presently playing an extremely vital role in modern research.

6. FUTURE SCOPE

Efficient biological treatment of dye-rich wastewater using immobilized bacterial strains necessitates
special attention. The primary reason of successful treatment of recalcitrant xenobiotics with immobilized
cells is the rapid degradation of these compounds to those of relatively much lower toxicity. Besides a high
concentration of biomass also facilitates the tolerance of the immobilized species to the toxic substances
they are exposed to. Further studies are required to apply this technique to other streams of wastewater
bearing different environmental toxins like heavy metals, polyaromatic hydrocarbons, pharmaceutical
compounds, etc. Designing and implementation of treatment units for a wide scale application of this
technology also require more attention from researchers.

7. CONCLUSION

The treatment of dye rich wastewater has been a sizable challenge since ages and till date, no single,
environmentally benign and economically feasible method has been identified that can effectively reclaim
water for reuse. The selection of the most appropriate treatment procedure is also difficult due to the

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complex composition of such effluents. However, this necessitates the synergistic effect of combined
technologies to be carefully studied and implemented. Through this review, it may be concluded that the
following concerns need to be addressed in order to secure environmental protection:

• Utilization of more waste materials for immobilization of different microorganisms;

• Designing of appropriate bioreactors to carry out treatment of dye bearing wastewaters with ICC;
and
• Proper optimization of the procedure using mathematical and statistical models like RSM and
ANN.

It is also important to analyze the applicability of this procedure on a wide scale industrial basis in
order to treat large volumes of dye rich wastewater.

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KEY TERMS AND DEFINITIONS

Adsorption: Physical or chemical adhesion of atoms, ions, or molecules of any state (gaseous, liquid
or a dissolved solid) to surface of a substrate.
Azo Dyes: Dyes possessing the functional group R-N=N-R’ in their structure whereby R and R’ can
be either aryl or alkyl moities.
Bacteria: Prokaryotic microorganisms extending a few micrometres with spherical, rod-like or spiral
shapes.
Biodegradation: Reduction of toxic compounds to non-toxic forms using bacteria, fungi, or other
biological means.
Effluent: A toxic liquid or gaseous discharge from domestic or industrial activities.
Immobilization: Entrapping, encapsulation, crosslinking or adsorption of cells or enzymes on an
inert matrix of natural or synthetic origin.
Matrix: The primary constituent of a composite substance.
Optimization: Selection of the best conditions of a procedure from available alternatives for maxi-
mizing the yield.
Toxins: A poisonous substance, biological or chemical in nature.

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Chapter 14
Decolorization of Direct Blue:
14 Dye by Thermoalkalophilic
Aerobic Bacillus sp.

Shankara S. Vijayakumar M. H.
Government College for Women, Chintamani, Gulbarga University, India
India
Gaddad S. M.
Kotresha Dupadahalli Gulbarga University, India
KSPL Degree College, India

ABSTRACT
A thermo-alkalophilic bacterium isolated from textile mill effluent samples and identified as a Bacillus
sp., on the basis of biochemical tests. The selected bacterium showed high decolorization activity in
static condition as compared to shaking condition and the maximum 1000 mg l-1 Direct Blue-14 dye
decolorization was takes place in 72 h. The optimum physical parameters such as temperature 40-50
°C, pH 8.0 with 2.5% (w/v) of nitrogen source and 4% (w/v) glucose were required for the decoloriza-
tion of Direct Blue-14 from this bacterium. UV–Visible analyses and colorless bacterial cells suggested
that Bacillus sp. exhibited decolorizing activity through biodegradation, rather than inactive surface
adsorption. The degraded dye metabolites are analyzed by TLC and diazotization, carbylamines, Ames
test for individual metabolite indicates biotransformation of Direct Blue-14 into aromatic amine and
non-toxic aromatic metabolites. These results suggest that the isolated organism Bacillus sp. as a useful
tool to treat waste water containing azo dyes at static condition.

INTRODUCTION

Synthetic dyes have a wide application in the food, pharmaceutical, textile, leather, cosmetics and pa-
per industries due to their ease of production, fastness, and variety in colour compared to natural dyes.
More than 100,000 commercially available dyes are known and close to one million tons of these dyes
are produced annually worldwide (Adedayo et al., 2004, Saratale et al., 2011). Azo dyes are the largest
group of dyes used in textile industry constituting 60-70% of all dyestuffs produced. They have one or

DOI: 10.4018/978-1-4666-9734-8.ch014

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.
 
Decolorization of Direct Blue

more azo groups having aromatic rings mostly substituted by sulfonate groups. These complex aromatic
substituted structures make conjugated system and are responsible for intense color, high water solubil-
ity and resistance to degradation of azo dyes under natural conditions. Disposal of these dyes into the
environment causes serious damage, since they may significantly affect the photosynthetic activity of
hydrophytes by reducing light penetration (Aksu et al., 2007) and also they may be toxic to some aquatic
organisms due to their breakdown products (Hao et al., 2000; Mate & Pathade, 2012; Sahasrabudhe &
Pathade, 2012; Madhuri et al., 2014).
Dyes can be removed from waste water by chemical and physical methods including adsorption, co-
agulation–flocculation, oxidation and electrochemical methods (Lin & Peng, 1994; Lin & Peng, 1996).
However, both the physical and chemical methods have many disadvantages in application, such as
high-energy costs, high-sludge production, formation of by-products (Sarioglu et al., 2007). Conversely,
bioprocessing can overcome these defects because it is cost saving and environmentally benign. Fungi
(Acuner & Dilek, 2004; Jadhav et al., 2007; Asgher et al., 2008) and algae (Mohan et al., 2002; Daneshvar
et al., 2007) have been used in dye decolorization. Adsorption rather than degradation plays a major role
during the decolorization process by fungi and algae, as a result, the dyes remain in the environment. It is
well known that bacteria can degrade and even completely mineralize many reactive dyes under certain
conditions (Chen et al., 2003; Moosvi et al., 2005; Asad et al., 2007; Kapdan & Erten, 2007; Praveen
and Bhat, 2012; Saratale etal., 2011). Even better, the products of intermediate metabolism during the
decolorization process, such as aromatic amines, can be degraded by the hydroxylase and oxygenase
produced by bacteria (Pandey et al., 2007; Saratale et al., 2011; Shah, 2014).
In this study, a thermo-alkalophilic bacterium Bacillus sp. capable of decolorizing Direct Blue-14 was
isolated and in addition, the effects of various physical parameters (such as initial glucose concentration,
dye concentration, pH and temperature etc) on dye decolorization by the bacterium and qualitative tests
of dye metabolites were investigated (see Figure 1).

Materials and Methods

The azo dye Direct Blue-14 (DB-14) was obtained from Shailaja textile Industry Sholapur, Maharastra,
India.

Isolation of Dye Decolorizing Microorganisms

Dye decolorizing bacteria were isolated from different samples such as dye amended soil, Textile mill
effluents and other soil samples. The 1g of soil dissolved in physiological saline and inoculated into
Bushanell-Hass (BH) containing 0.1 g of dye DB-14 (MgSO4.7H2O 0.2, CaCl2- 0.02, KH2PO4 1, K2HPO4

Figure 1. Direct Blue- 14 (DB-14)

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Decolorization of Direct Blue

1, FeCl3.3H2O 0.05, peptone 1.5, pH 7.0 – 7.4) and the flasks were incubated at 37 oC under static con-
dition for 48 h. The decolorized flask culture was transferred to fresh BH medium to screen the strain
having color removing ability and the screening process was repeated in 5 times. The isolated bacterial
strains were tentatively characterized by cultural, morphological and biochemical tests and maintained
on nutrient agar at 4 °C.

Spectrophotometric Analysis

Aliquots of cell-free decolorized supernatant collected at regular intervals of time were used for
Spectrophotometric analysis (ANTHELE-SECOMAM France) at wavelength of 300- 700 nm. The dye
DB-14 absorbance maximum was at 590 nm. The efficiency of decolorization was expressed as the
percentage ratio of the decolorized dye concentration to that of the initial one.

Standardization of Process Parameters

Various operational and environmental parameters were standardized by varying a particular param-
eter and keeping other parameters constant by using 0.3 g l-1 of DB-14 in BH medium. The process
parameters standardized Dye concentration, pH, Temperature, Inoculum size and Nitrogen source.

Identification of Metabolites

The complete decolorized medium of Direct Blue-14 was centrifuged at 10,000 rpm for 15 min. The
pH was adjusted to 7 and 200 ml of the supernatant was extracted twice with 500 ml diethylether.
The remaining aqueous layer was acidified to pH 2 by 1N HCl and extracted twice with Diethylether
(500 ml). The acidic and basic extracted fractions were pooled and evaporated over anhydrous Na2SO4
and under reduced pressure at 30 oC. The residue was dissolved in 500 µl of Methanol.

Thin Layer Chromatography (TLC)

Preliminary identification was made by TLC of extracted compounds. The glass plate of 200 × 100 × 2
mm (length × breadth × thickness) coated with 40% (w / v) aqueous slurry of Silica Gel G with binder
were used for carrying out TLC, 10 µl of extracted fractions were loaded on Silica gel, dried and the
solvent system used were propanol:water:acetic acid (90: 9: 1 v/ v/ v) for developing the TLC plate.
The dye and products chromatogram was observed by exposing to long wavelength UV-Light (365
nm). The metabolites were identified by Diazotization and Carbylamine test.

Curing

The Bacillus sp. was precultured in BH medium with shaking for 18 h. the preculture was inoculated
into 5 ml of fresh BH medium containing 0.5 ml curing agent (Acridine Orange 1%), and incubated
for 24 h at 37 oC. After 24 h the culture was transferred to fresh BH medium containing 0.3 g of dye,
Simultaneously transferred to fresh BH medium containing 0.5 ml of 1% Acridine orange, the process
was repeated every 24 h up to 5 days. The culture was properly diluted with saline and 0.1 ml of diluted
culture was placed on Nutrient agar. The inoculated plate was incubated at 37 oC for 24 h. Each colony

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appearing on the agar plate was tested for its ability to decolorize the DB-14. The colony which did
not decolorize the dye is considered as a cured colony. The Curing frequency was calculated by colony
counts of the initial one, ie, the difference between the initial colony counts, (i) and the Final colony
count (f) of the sample. The Curing frequency was calculated by following formula.

Curing frequency (%) = Colonies (i) – Final (f) × 100/ Colonies (i)

Ames Test

Three plates were used synchronously and this test was carried out on the basis of described Ames
test (Maron & Ames, 1983).

Statistical Analysis

All analysis was conducted in triplicate and results presented here are the mean of triplicate ± stan-
dard deviations (SD).

Results

Isolation and Identification

We have isolated 20 bacterial strains as a dye decolorizing micro-organisms (Table 1). Out of 20, one strain
DBS-3 was found to be most efficient in that it decolorized 0.1 g of DB-14 within 16 h of incubation.

Table 1. Isolation of Direct Blue-14 decolorizing bacteria from different sources

Isolates Source Time for Complete Decolorization (h)

DBS-1 Dye amended soil 72
DBS-2 Dye amended soil 44
DBS-3 Dye amended soil 16***
DBS-4 Textile mill effluents 52
DBS-5 Textile mill effluents 48
DBS-6 Saw waste 56
DBS-7 Red soil 52
DBS-8 Black soil 64
DBS-9 Textile mill effluents 120
DBS-10 Textile mill effluents 30**
DBS-11 Field soil 72
DBS-12 Textile mill effluents 32*
DBS-13 Sewage waste water 48
DBS-14 Dye amended soil 44
DBS-15 Pond water 52
DBS-16 Red soil 32*
DBS-17 Black soil 68
DBS-18 Textile mill effluents 56
DBS-19 Dye amended soil 40*
DBS-20 Dye waste water 68

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This isolate has been tentatively identified as Bacillus sp. on the basis of morphology and biochemical
tests such as irregular, white, opaque, waxy growth on nutrient agar, gram positive, rod with central
endospore, motile, catalase, starch hydrolysis, casein hydrolysis, NO3 reduction and oxidase positive.

Decolorization of Dye DB-14 by Bacillus sp.

The complete dye DB-14 decolorized medium was centrifuged, to separate the spent medium and bacte-
rial cell biomass. The spent medium and bacterial cell biomass were independently inoculated into the
fresh medium containing dye DB-14 and incubated at 37 oC under static condition. The decolorization
of DB-14 was observed in inoculated bacterial cell biomass not in inoculated spent medium indicates
that takes place by bacterium Bacillus sp.

Decolorization Assays

A spectrophotometric scanning (300–700 nm) of the DB-14 dye showed a single peak at 590 nm. The
dye decolorization was represented by a decrease in absorbance and the absorbance spectral pattern
of each recovered dye solution was not similar to that of the initial one (Figure 2). Furthermore, after
decolorization, at 16-28 h the bacterial cell mass was slightly colored and the same bacterial cell mass
was clear at 32 h. It clearly indicated that the decolorization was initiated by adsorption then degrada-
tion takes place and this indicates that the observed dye decolorization is due to the biological activity
of the bacterium Bacillus sp.

Figure 2. Spectral scans (300–700 nm) of the culture supernatant containing dye Direct Blue-14 (100
mg l-1) at different time intervals. Samples were centrifuged at 8000 rev/min for 10 min (ay 0; 6h; 12h;
18h; 24h).

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Decolorization of Direct Blue

Effect of Dye Concentration, Temperature, pH and

Inoculum Size on Dye Decolorization

Effect of dye concentration (Figure 3) on the decolorization ability of the bacterium was studied by
inoculation of the bacterium to BH medium supplemented with different concentrations of DB-14 (50
- 1000 mg l-1). The dye concentration at 50 mg l-1 complete decolorization was observed at 12 h. Even
with the dye concentration as high as 1000 mg l-1, nearly 87% of dye was reduced after 68 h and nearly
complete decolorization was observed at 72 h and the 0.3 g l-1 of dye DB-14 were used for further stan-
dardization of various operational and physical parameters.
One of the most important parameters for dye decolorization is temperature. In order to determine
the optimum temperature for dye decolorization using DB-14 a temperature range of 25-60 °C was ex-
amined. Figure 4 shows that the percentage decolorization increased with increase in temperature from
25 to 45 °C and 100% dye decolorization was observed at 45 °C and temperature between 40–50 °C was
optimum for removal of DB-14. Complete decolorization of DB-14 was observed even at 60 °C, though
taking a bit longer time (72 h). The results showed that Bacillus sp. is thermophilic in nature, exhibiting
color removal activity even above 50 °C.
The optimum pH for maximum rate of dye decolorization was performed using DB-14 (300 mg l-1)
over a pH range of 5.5–10 (Figure 5) and optimum pH required for the dye decolorization is pH 8.0.
Further decrease or increase in pH gradually decreased the dye decolorization efficiency. The complete
color removal was observed between pH 5.5 to 10.0 and at pH 10,0 it takes 96 h for complete decolor-
ization. This shows that the isolated Bacillus sp. was alkalophilic in nature.
The color removal was observed by increasing the Inoculum size 1 to 10 ml (Figure 6) by Bacillus
sp. and 1 ml of inoculum takes 28 h for complete dye decolorization. Further increase in the inoculum
size above 6 ml, rate of dye decolorization is constant and it takes 12 h for complete decolorization.

Figure 3. Effect of dye concentration on Decolorization of Direct Blue-14 by Bacillus sp.

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Decolorization of Direct Blue

Figure 4. Effect of temperature on Decolorization of Direct Blue-14 by Bacillus sp.

Figure 5. Effect of pH on Decolorization of Direct Blue-14 by Bacillus sp.

Nitrogen and Carbon Source

The effect of nitrogen and carbon source was studied (0.25-2.5 g l-1) using peptone as a nitrogen source
and glucose, fructose, sucrose as a carbon source (Figure 7). The nitrogen source concentration at 0.25-
1.25 g l-1 gradually increases in the dye decolorization. At 0.25 g l-1 it takes 72 h for complete removal
of color, while a concentration of 1.25 - 1.5 g l-1 takes 28 h. At higher nitrogen concentration of 1.75
to 2.0 g l-1 color removal was at 20 -24 h. Further increase in the concentration 2.0-2.5 g l-1 of nitrogen
source did not increase in reduction of color by Bacillus sp. Nitrogen source was found to be essential
for decolorization (Figure 7).

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Decolorization of Direct Blue

Figure 6. Effect of inoculum size on Decolorization of Direct Blue-14 by Bacillus sp.

Figure 7. Effect of nitrogen and monosaccharide source on Decolorization of Direct Blue-14 by Bacillus sp.

An additional carbon source was investigated. The Figure 7 showed color reduction did not improve
with the addition of Glucose, Sucrose and Fructose in absence of nitrogen source. At lower 0.25 g l-1 and
higher concentrations 2.5 g l-1 of glucose, fructose & sucrose did not influence the decolorization activity.
These results indicated the nitrogen source were essential for decolorization activity.

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Isolation and Identification of Metabolites

The metabolites of DB-14 by Bacillus sp. were extracted by solvent extraction. The extracted was loaded
to on a TLC (silica gel plate) and developed by Propanol: Water: Acetic acid (90: 9: 1 v/v) solvent system.
The developed plate was dried and observed under UV-light. The 4 bluish spots were detected on a TLC
plate the (Figure 8) and Table 2 show Rf values of different spots. The results of UV-Visible scanning,
diazotization, carbylamine and Ames tests are show in Table 2.

Curing

The Table 3 and Figure 9 show frequency of curing and the time taken for complete decolorization by
Bacillus sp. The frequency of curing observed was only 0.9% on 1st d and 4.87% on 5th d of curing. The
complete color removal was observed form 1st d to 5th d at 28 h in cured Bacillus sp. The Bacillus sp

Figure 8. A). Direct Blue-14 Dye, B). Direct Blue-14 Dye degraded products

Table 2. Characteristics of Direct Blue-14 dye degradation products

S. No Dye Rf values λmax Diazotization test Carbylamine Test Ames Test

1. Direct Blue-14 0.14 590 --- ----- -
2. Dye metabolites
a 0.80 393 +ve +ve +ve
b 0.60 305 +ve +ve +ve
c 0.45 240 -ve -ve -ve
d 0.21 285 -ve -ve -ve

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Decolorization of Direct Blue

Table 3. Curing of Bacillus sp. with Acridine orange

Curing Days No of Colonies Time Taken for Decolorization % of Curing

Control 410 28 0.0
I 406 28 0.9
II 402 28 1.91
III 398 28 2.98
IV 395 28 3.65
V 390 28 4.87

Figure 9. Curing of Bacillus by an Acidine orange

retains the decolorization activity even on the 5th day and these results indicate that the decolorizing
genes were present on bacterial genome and not on a plasmid. This investigation showed the genomic
genes were responsible for decolorization by Bacillus sp.

Discussion

Biological methods are simple to use and low cost involved in operation and only the microorganisms are
the promising agents in remediation of the environment. In the present investigation we are reporting the
decolorization of Direct Blue-14 (DB-14) by Bacillus sp. under aerobic condition. A spectrophotomet-
ric scanning (300–700 nm) of the DB-14 dye showed a single peak at 590 nm. The dye decolorization
was represented by a decrease in absorbance and the absorbance spectral pattern of each recovered dye
solution was not similar to that of the initial one. A general decrease of this kind is usually attributed to
dye degradation rather than adsorption (Wang et al., 2009; Tripathi & Srivastava 2011). Furthermore,
after decolorization, there was no visible dye adsorbed to the biomass and less than 0.5% of the initial
colour was recovered by methanol extraction, suggesting that the observed dye decolorization is due to
the biological activity of the bacterium Bacillus sp. (Chen et al., 2003; Vijayakumar et al., 2006).

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Decolorization of Direct Blue

When the effect of different initial dye concentrations of DB-14 on decolorization was observed
using 50, 100, 200 300, 400, 500, 600, 700, 800, 900 and 1000 mg l-1 and the required times to reach a
maximum decolorization extent were 12, 16, 20, 24, 36, 40, 52, 56, 60, 68, and 72 h, respectively (Fig.
2). It indicates that with increase of the initial dye concentration, the decolorization extent over the
same time interval decreased. Chang & Kuo, (2000) reported that E. coli could remove Reactive Red
22 up to 0.2 g l-1 and Psuedomonas sp. was capable of decolorizing 30-80% of 0.1-0.5 g l-1of solution of
5 Azo dyes (Mali et al., 2000). Saratale et al. (2009) observed that 200 mg l-1 optimum dye concentra-
tions during their study on biodegradation of textile dyes and Bhatt et al. (2012) reported DN1 culture
decolorizes Reactive Red HE8b dye 200 mg l-1 concentration. Micrococcus glutamicus NCIM 2168 was
able to decolourize the dye up to 300 mg l-1 in proportion to the time and takes 70 hours to decolourize
66.08% of 500 mg l-1 of the dye above this concentration the isolate was unable to grow (Madhuri et
al., 2014). It was reported that dye decolorization can be strongly inhibited when a high concentration
dyestuff was used to examine the poisonous effect of the dye on the degrading microorganisms (Kalme
et al., 2007; Khehra et al., 2005).
One of the most important parameters for dye decolorization is temperature. The mesophilic range is
traditionally used, since it is generally thought that maintaining high temperature would be uneconomical,
whilst degradation within the psychrophilic range is too slow (Keharia et al., 2004). In order to determine
the optimum temperature for dye decolorization using DB-14 at temperature range of 25–60 °C was
examined. The dye decolourization was increases with increase in temperature 25–45 °C and above the
45 °C dye decolorization decreases, Which might be due to the loss of cell viability or deactivation of the
enzymes responsible for decolorization (Cetin & Donmez, 2006; Panswad & Luangdilok, 2000). Bhatta
et al., 2012 reported that 40 ºC (91.5%) optimum temperature for the decolorization of Reactive Red
HE8b by DN1 bacterium and Wang and Yuen (2011) suggested optimum temperature at 40 ˚C during
their study on decolorization of the azo dye by bacterial isolate and at higher temperature percentage of
decolorization was rapidly decreased (Sahasrabudhe & Pathade, 2011). The maximum decolorization
was takes place at optimum temperature 37 °C (Mathur & Kumar, 2013, Shah et al., 2013). The 100%
dye decolorization was takes place at 45 °C in 108 h (300 mg l-1) and these results indicates that the
Bacillus sp. was thermophilic in nature.
The best decolorization was achieved at pH 8.0 with 100% decolorization in 120 h. This could be due
to the fact that the optimum pH for the growth of Bacillus sp. The decolorization extent was observed at
lower and higher pH and the rate of color removal was much lower at acidic and alkaline pH conditions.
pH has a major effect on the efficiency of dye decolorization and the optimal pH for color removal is
often between 6.0 and 10.0 (Chen et al., 2003; Guo et al., 2007; Kilic et al., 2007). The pH tolerance
of decolorizing bacteria is quite important because reactive azo dyes bind to cotton fibers by addition
or substitution mechanisms under alkaline conditions and at high temperatures (Aksu, 2003). Bacte-
rial culture generally exhibit maximum decolorization at pH near 7.0 (Shah, 2014) and potent bacterial
culture DN1 gave maximum decolorization at pH 6.5. Tripathi and Srivastava (2012) showed pH 6.9 for
biodegradation of orange G by a novel bacterial isolated bacterial strain Bacillus megaterium ITBHU01
and M. glutamicus requires optimum pH 5 for the decolorization of Reactive Red 195 (Madhuri et al.,
2014). P.putida decolourized methyl red in the range of 6.5-7.5 maximum being 7 (Mathur & Kumar,
2013) and these results indicate that the our bacterium Bacillus sp. was alkalophilic in nature.
The aerobic decolorization DB-14 by Bacillus sp. was much faster at static conditions compared
to shaking conditions. The several studies reported at static conditions for decolorization by Proteus

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Decolorization of Direct Blue

mirabilis and Enterbacter agglomerans (Chen et al., 1999; Moutaouakkil et al., 2004; Shah, 2014) and
Nermeen et al., (2011) reported decolorization of red dye by Streptomyces globosus under static and
shaking condition.
The decolorization of DB-14 along with organic nitrogen and carbon though Bacillus sp. was observed.
The presence of organic nitrogen source like peptone the rate of color reduction was increased with the
concentration of organic nitrogen 0.25 g l-1 to 1.25 g l-1 and above 2.0 g l-1 it was constant. Praveenkumar
& Bhat, (2012) have reported similar reports of increase in the efficiency of decolorization with increase
in the organic nitrogen concentration and many report shows the maximum decolorization of dyes takes
place in presence of nitrogen source (Bhatt et al., 2012; Ponraj et al., 2011) and Dong & Zhou, (2003);
Vijaya et al., (2003) have reported the decolorization of reactive dyes in presence of peptone. These
investigations pointed nitrogen source was essential for decolorization of azo dyes. The decolorization of
DB-14 was investigated in absence of Nitrogen by using different carbon source (Glucose, Fructose and
Sucrose) there is on color reduction was observed. The addition of Glucose or Sucrose was completely
inhibited in color reduction reported by (Chen et al., 1999; Shah, 2014).
The biodegradation products of DB-14 were loaded on TLC plate and developed by Propanol: Water:
Acetic acid (90: 9: 1 v/v) solvent system. The developed TLC plate have shown 4 spots under UV-light
observation with Rf values of 0.21, 0.45, 0.60, and 0.80 because of biodegradation dye DB-14 by Bacil-
lus sp. Further metabolites were characterised by chemical tests such as Diazotization, Carbylamines
and Ames test. The spots with Rf values 0.80 and 0.60 are positive to diazotization, carbylamine and
Ames tests. Whereas Rf values 0.45 and 0.21 showed negative result to the diazotization, carbylamine
and Ames tests. These results indicate that our isolated bacterium is potential for the degradation azo
dyes and converted into aromatic compounds without amine group. Form the curing study indicates that
the decolorization gene was present on chromosomal genome and still decolorization occurred even at
5th of curing but the plasmid was last at 3rd day.

CONCLUSION

Isolated bacterium Bacillus sp was thermo alkalophilic in nature and Direct Blue-14 degraded into four
metabolites out of which two are aromatic amines and two are non toxic aromatic compounds without
amine group. These results suggest that the isolated organism Bacillus sp. as a useful tool to treat waste
water containing azo dyes at static condition.

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Chapter 15
Fighting Ecomafias:
The Role of Biotech Networks
in Achieving Sustainability

Nadia Di Paola Adele Caldarelli

University of Naples Federico II, Italy University of Naples Federico II, Italy

Rosanna Spanò Roberto Vona

University of Naples Federico II, Italy University of Naples Federico II, Italy

ABSTRACT
Innovation processes are becoming increasingly central, and newer industries have become already less
resource-intensive in comparison with the traditional ones. Nevertheless, this alone does not assure sus-
tainability, which requires a step further towards economically viable, environmentally compatible, and
socially responsible behaviours. This chapter addresses the issues relating to sustainable development
to provide a critical discussion on the potential role played by networking relationships in the biotech
field. For the purposes of the study, we employ the co-management and multi-stakeholder perspectives.
We demonstrate that the biotechnology research results may be enhanced thanks to cooperation dynam-
ics and interactions among heterogeneous actors, with undeniable cultural and social positive impacts.
Also, we discuss social implications and open concerns, both with regard to the relationships within
innovative networks and between institutional professional actors, allowing the identification of any grey
areas and limitations, especially relevant to policy makers.

1. INTRODUCTION

Over the recent years, institutions, policy makers, and scientists have devoted huge attention to the prob-
lems relating to environmental degradation, pollution, and ecological variations, as the consequences
of uncontrolled progress and prosperity, putting effort in the realization of plans and activities towards
sustainable development. Clearly, sustainable development is a complex and difficult challenge for hu-
manity, as attaining sustainability involves the consideration of many, heterogeneous, multifaceted, and
sometimes contrasting fundamental issues, as well as a broad range of stakeholders at local, regional

DOI: 10.4018/978-1-4666-9734-8.ch015

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.
 
Fighting Ecomafias

and global levels (Lostarnau et al., 2011; Lafreniere et al., 2013). What should be noted is that, at any
level, science and technology play a crucial role to achieve sustainability.Moreover, political decisions
assisted by societal support and coordinated policies are essential. Hence, it is clear that to ensure suc-
cessful initiatives for industrial sustainability, there is the need for a global and holistic view encompass-
ing economic, social and environmental aspects. In this regard, it is important to acknowledge that the
industrial production globally is progressively attempting to reduce the adverse impact of its activities
on the environment. However, the use of processes and technologies devoted to prevent pollution, rather
than barely intervening with a restorative approach, have become a priority only very recently (Council
Directive, 1996; Allen & Sinclair Rosselot, 1997; World Bank, 1999; EPA, 2003). In such a complex
context, innovation processes are becoming increasingly central, and newer industries such as micro-
electronics, telecommunications and biotechnology are already less resource intensive in comparison
with the traditional ones (Kristensen, 1986; OECD, 1989; Rigaux, 1997). Nevertheless, this alone does
not assure sustainability, which requires a step further towards economically viable, environmentally
compatible, and socially responsible behaviours (OECD, 1998; UNEP, 1999; Wong, 2001). In particular,
biotechnologies have gained plenty of faith and credit by public opinion and governments worldwide. The
extraordinary development of biotechnology creates expectations and hopes for a tangible and continuous
improvement of the quality of life. Also, it brings intimate and profound reflections that should balance
the tension towards innovation, with the power of direct intervention owned by the protagonists of the
scientific research. In addition, the improvements may allow the strengthening of the protection of the
critical variables for the competitive advantage, more efficient operational processes, less polluting air
emissions and waste products, and product innovations likely to achieve effective differentiation strategies.
These can be considered concrete objectives to pursue but, despite the extraordinary and unquestion-
able ability of biotechnology to producing radical innovations and improving life conditions, it is quite
clear that there precious elements to support a full expression of their potential are still hardly lacking.
This chapter addresses the issues relating to sustainable development to provide a critical discussion on
the potential role played by biotechnologies in practically pursuing the expectations for technological,
socio-economic, political and cultural changes. The aim of the paper is to deepen the issues relating
to the possible contribution of the networks operating in the biotech field to social and environmental
sustainability, and to better understand the role of the diverse research actors and the variety of issues
that characterize the structural and relational complexity of biotechnology.
The idea that we put forward is that effective networking relationships in the biotech field can contrib-
ute to sustainability, in its broadest sense, not only by reducing the negative impacts of human/industrial
activities and the restoration of contaminated territories, but also by creating a territorial and corporate
competitive advantage to prevent and reduce the danger of criminal infiltrations in economic and produc-
tive activities. More specifically, building on the above mentioned themes, the chapter assumes a new
and challenging point of view concerning the positive potential of biotechnology and biotech companies
to settle the threatens deriving to human health and environmental safety from the increasing infiltration
of organized crime in the current economy. Bearing in mind the rising importance of biotechnology, our
contribution deepens the debate on the issues relating to the new discoveries, pointing out their central
role to ensure environmental sustainability, also in the light of the threats deriving from organized
crime activities and infiltration in the economy, that over the years have damaged a number of areas
and territories threatening their environmental and ecological safety. In fact, despite the development of
a complex set of norms, standards, and operational suggestions, the debate on the issues relating to the
crucial need for purifying companies by criminal infiltration, preserving the corporate going concern,

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and introducing social and environmental values in the management of the entities, is still rather than
being concluded (Donato et al., 2013). Also, the real awareness of the solutions available to restore/pre-
serve the destroyed eco-systems and the compromised health of certain areas, appears still limited and
jeopardized. For the purposes of the study, we employa multi-stakeholder perspective. Indeed, the natural
resources management involves multiple and complex systems, characterized by horizontal connections
between organizations operating at the same level, and vertical links between organizations, stakehold-
ers, and the government (Berkes, 2009). In such a complex environment, it is unlikely that only one of
the parties involved possess all the knowledge needed for the management of natural resources. On the
contrary, knowledge and capabilities owned at the different levels are complementary to achieve shared
goals (Reid et al. 2006). Thus, the actors involved should “co-produce” knowledge (Davidson-Hunt &
O’Flaherty, 2007), and need systems and mechanisms to ensure participation and dialogue.
Hence, by adopting a critical approach, we will examine extant literature to depict the characteristics
of the various actors operating in the field, to provide a framework useful for the achievement ofthe
above-mentioned conditions, and to find out what are the factors that influence effective systems rela-
tionships and an enhancement of knowledge production and knowledge transfer, towards an improved
sustainability. In this regard, it is worth specifying that the systemic view adopted in the study allows us
to provide evidences that not only look at the issues relating to the “corporate social/environmental sus-
tainability”, but also point out the crucial questions of the “systems social/environmental sustainability”.

2. CRIMINAL ORGANIZATIONS INFILTRATIONS

IN THE ECONOMIC ENVIRONMENT

The Case of Toxic Wastes Disposal

It is well known that organized crime infiltrations in the economy represent a growing phenomenon
that poses tremendous threats not only to legitimate firms and competitiveness, but also, from a broader
perspective, to health and environmental safety. As a consequence, these issues have raised increasing
attention over the last years, especially with reference to any actions suitable to face such a societal prob-
lem. Indeed, what should be noted is that globalization and market development have certainly favoured
the effort by criminal organizations to invest in legal business activities, determining an ever-increasing
expansion of entrepreneurial crime rooted in the domestic markets, but also spread at the international
level (Giordano, 2014). Entrepreneurial crime is mainly based on the recognized major benefits that
criminal groups can derive from the business activity (Vona, 2014; Donato et al., 2013).
It is worth mentioning that the reliance upon the entity as an instrument of wealth for the mafia groups
has been the subject of a progressive evolution over times. In fact, while at the beginning the relationship
between the criminal organizations and the economic context was mainly limited to activities of extortion
–where the entrepreneur running a legal business was either a victim or a colluded actor –the last twenty
years have been characterized by the appearance of a newer and complex figure. The criminal entrepreneur
manages a company which capital is conferred by the criminal organization, and derived from its core
business based on a broad range of illegal activities, such as drug, waste and human traffics, prostitution,
corruption, army, money laundering, and so on. Criminal entrepreneurship represents the deepest level of
penetration of mafia groups in the business context, and relies upon highly qualified actors specifically
skilled to run a business activity that does not differ from that of other companies, except in the origin

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of capital. Clearly, a first element of advantage comes from the fact that the never-ending cash-flows
coming from the illegal businesses of the criminal organizations ensures that the entity can avoid the
recourse to debt, and can also sell products at a lower price in comparison to competitors. Furthermore,
when the illegal capitals are incorporated in the circuit of production, criminal organizations are able
to distance such capitals from the illicit origin, and they can also realize further profits to re-invest both
in the illegal and the legal businesses (Di Paola & Spanò, 2014). Bearing in mind this framework that
efficiently summarizes how criminal entrepreneurship generally operates, it is important to focus on
the crucial question relating to the criminal business activities concerned with the toxic waste disposal,
given the immense impact that these determine at thesocietal level. In fact, the toxic waste disposal is an
area where the above-cited organizations, especially in the Southern Italy, have largely operated over the
last 20 years. In particular, at the beginning the illegal waste disposal was prominently focused on urban
garbage, but with the rise in the production of hazardous wastes criminal organizations recognized an
even more profitable and challenging opportunity, that now represents an ever-expanding industry. The
illegal disposal facilities run by mafia-type organizations easily spread across the country for two main
reasons, i.e. the cost savings for the entities and the absence of any regional limitations that facilitated
the development of trafficking routes. For a purpose of clarity, it is important to highlight that the illegal
disposal of waste can follow different routes (see Costa, 2014):

1. Toxic wastes can be hidden by falsifying the documents so that they result as commercial products.
2. Toxic wastes can be hidden by falsifying the documents so that they are indicated as non-toxic
wastes.
3. Toxic wastes can be melt with non-toxic waste to hide the first ones, thus disposing these at a lower
cost.
4. The documents relating to toxic wastes can be transferred for a number of times between different
places, until these are s fully declassified and hidden and, in the end, illegally disposed at reduced
costs.
5. Toxic wastes can be melt with inert materials from constructions and/or excavation rocks.
6. Toxic wastes can be disposed through the burial in caves realized specifically to this aim (this hap-
pened especially in Campania, given the wide control of the territories by criminal organizations
that facilitated this praxis).
7. Toxic wastes can be illegally used for agricultural purposes by falsifying the needed certifications
8. Toxic wastes can be disposed through the above-cited techniques, but exploiting international roots,
and directing these actions towards underdeveloped economic areas or territories with a high rate
of corruption.

It is crucial to highlight that a common feature that characterizes this broad range of methodologies
is the harmful effect on agriculture, environment and health. Indeed, directly or indirectly each of these
solutions produces undeniable negative consequences that are not only related to the above-mentioned
areas, but can also exert a negative impact on the reputation, the economy and the competitiveness of
certain territories. In addition, another distinctive aspect of these various methods is that to ensure the
success of the illicit mechanisms activated there is the need of a well-articulated criminal chain, in some
cases even characterized by a huge administrative and managerial complexity. Moreover, special features
characterize the “Eco-mafias” in comparison to the traditional ones. Environmental crime is peculiar

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in comparison to traditionally organized crime, as it is really uncommon that violence is perpetrated,

people involved do not usually have previous criminal records and are not part of huge organizational
structures, despite having strong relationships with them. Nevertheless, the intimidating behaviours,
typical of criminal organizations, are usually adopted. That said, it is fundamental to underline that a
vast amount of special and toxic wastes ends its path in an unsafe way for the environment, strongly
contributing to the pollution and the deterioration in the health of the population. In this regard, the
Italian law 136/2010, that has to be interpreted in the light of a broader intervention against organized
crime, states that for the organized activities for the illicit traffic of waste the jurisdiction is assigned to
the District Antimafia Directorate (DirezioneDistrettualeAntimafia, DDA henceforth). Such relatively
recent innovation highlights Regulator’s worries in relation to this particular crime, and the awareness
of the need for the specific investigative methodologies and techniques that distinguish DDA. On this
general basis, it is therefore clear why in the wake of this progressively dangerous situation, a number of
legislative interventions have been realized to identify the criminal entrepreneurs, and to deprive them
of the capital accumulated through the company. A primary question to take into account in relation
to the norms available for the environmental crime is the usefulness of the investigation in this sphere.
Over the years, the regulator has recognized the inadequacy of the command and control approach
introduced in 1982, and progressively switched towards a preventive perspective. The main idea is to
develop an effective contrast against environmental crime by limiting at the origin any damages, rather
than intervening only in a subsequent moment. Hence the punitive ex post logic is now regarded as a
residual solution limited to a “last resort” role applicable only when the preventive protection tools are
not able to avoid the above-cited phenomena of environmental crime and pollution.
In particular, the pre-existing approach – the command and control one – based on authoritative instru-
ments, was undermined by a practical incapability to rigorously apply the legal requirements. Nevertheless,
the sanctioning logic cannot be abandoned because it is not always possible to prevent environmental
crime, due to the increasing complexity that characterizes the context in which such criminal actions are
played. Thus, since 2006 EU regulators, and especially the Italian one, rendered available both admin-
istrative and penal sanctions leading to the “administrative-depenalized environmental offense” that, in
any case, is not free from critique. In fact, in countries characterized by weak administrative apparatus
and high corruption, the use of administrative sanctions is likely to lead to a restriction of the controls,
and to situations in which the controller and controlled actors may coincide. Also, the deterrent effect
of an administrative penalty is usually lower than the immoderate profits deriving fromthese illegal
activities. This creates the premises to further reflect upon the opportunity to give penal relevance to
environmental crimes that put in serious danger, if not damaging, environmental resources, given the lack
of an organic corpus to regulate the whole of the phenomenon. Indeed, there is the need to find a balance
between the demand for ex ante protection for the environmental good, and the protection ex post, which
is now neglected. In addition to the regulatory issues – that despite being important to comprehend the
whole of the phenomenon are out of the scope of this study – a further and extremely important point
to consider is related to the non-normative tools employable to prevent environmental crime and/or to
face its consequences. In this regard the idea that the research puts forward is that effective networking
relationships in the biotech field can contribute to sustainability, in its broadest sense, not only by reduc-
ing the negative impacts of human/industrial activities and the restoration of contaminated territories,
but also by creating a territorial and corporate competitive advantage to prevent and reduce the danger
of criminal infiltrations in economic and productive activities. More specifically, as the following sec-

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tion will better clarify, the paper assumes a new and challenging point of view concerning the positive
potential of biotechnology and biotech companies to settle the threatens deriving to human health and
environmental safety from the increasing infiltration of organized crime in the economy.
In fact, over the last years there has been an increasing attempt by regulators and policy makers to
enhance the legal provisions for the prevention of environmental crimes and the management of the enti-
ties seized from Camorra and Mafia (Di Paola & Spanò, 2014). However, the knowledge of the solutions
available to restore/preserve the eco-systems and the health of certain areas appears still limited and
jeopardized. In this context is crucial to highlight that the function of biotechnology to solve this kind
of problems is absolutely fundamental, and is not restricted to an ex post intervention, as the available
options could be helpful also ex ante to stimulate competitive and sustainable production, thus removing
any factors likely to facilitate criminal infiltrations in the companies.

3. ENVIRONMENTAL SUSTAINABILITY AND THE

POTENTIAL ROLE OF BIOTECHNOLOGY

An unavoidable starting point to highlight the crucial importance of biotechnology to prevent environ-
mental crime and/or to face its consequences is to clarify what industrial environmental sustainability
practically entails. For the purposes of this chapter, sustainable development is understood as a “pro-
cess of change in which the exploitation of resources, the direction of investments, the orientation of
technological development, and institutional change are all in harmony and enhance both current and
future potential to meet human needs and aspirations... (It is) meeting the needs of the present without
compromising the ability of future generations to meet their own needs”, as defined by World Commis-
sion on Environment and Development (Brundtland, 1987; Gavrilescu & Chisti, 2005).
Sustainable development is a complex and difficult challenge for humanity and has raised the attention
of institutions, policy makers, and scientists on heterogeneous, multifaceted, and sometimes contrast-
ing questions relating to environmental degradation, pollution, and ecological variations. All of these,
in the awareness that to ensure successful initiatives, there is the need for a global and holistic view
encompassing economic, social and environmental aspects. In this respect, it is significant to recognize
that industrial production globally is trying to reduce the adverse impact of its activities on the environ-
ment, but the use of processes and technologies to prevent pollution, rather than intervening barely in
a restorative mode, have become a priority only very recently (Allen & Sinclair Rosselot, 1997; World
Bank, 1999; EPA, 2003; Vona, 2014). However, especially focusing on biotechnology, it provides wholly
novel opportunities for sustainable production, not only to eliminate pollutants from the environment,
but also to inhibit pollution since the beginning. The potential of biotechnology as a tool to effectively
manage the processes to restore any contaminated territories, as the consequence of activities such as
the illegal disposal of toxic wastes, and as a tool to prevent such events, represents an interesting issue
which to date has been only marginally addressed. The role of biotechnology is crucial from the waste
management perspective, as it would not only reduce the fraction to be allocated to the landfill, but would
also allow to replenish the organic products within the chain after their use, to rebalance the system, and
to ensure environmental sustainability (Hamer, 2003). Green biotechnologies represent cost-effective
and environmentally sustainable technologies and, despite having already proven their valuability in the
treatment of wastes in the textile and mining industries, their diffusion in treating wastes is not really
spread (Van Wyk, 2001). Specially, the ways through which biotechnologies could positively contribute

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to waste management are the aerobic (e.g., composting) and anaerobic (e.g., digestion) bio-treatments.
However, there are no procedures that ensure the complete removal of organisms and pathogens from
the treated waste materials, thus stressing the need for the development of technologies that can con-
tribute to the sanitization effectiveness of the process as a whole (Hamer, 2003). In addition, it is worth
noting that biotechnologies spread could also lead to “improving the quality of wastes”, increasing
their biodegradable part thanks to the development of new materials. A crucial aspect to consider is the
potential and importance of biotechnologies for environmental restoration, since the development of
remedies for contaminated sites is a matter of absolute relevance, and biotechnologies provide in-situ
treatments (microremediation, phytoremediation, vermiremediation, and so on) that do not require the
use of chemical reagents (Burlakovs, Vircavs, 2011; Sinha, Valani, Sinha, Singh, & Herat 2009), and
appear less expensive and environmental friendly.
The recovery processes start from the identification of contaminated sites, and then develop through
a range of technologies for the control of pollutants and possibly their elimination. The choice of the
most appropriate procedures is very complex: it involves considerations relating to technology tools and
knowledge available, as well as a huge amount of financial resources. The potential of various biotech-
nological techniques to carry out these complex processes is really valuable due to their effectiveness
and the cost savings that they allow in the long-term. Also, it is interesting to highlight that the employ-
ment of biotech innovations within companies is known as a tool that favours competitive advantage and
financial sustainability. Consequently, it is possible to argue that small and medium enterprises that rely
upon such innovations are more likely to reach a competitive advantage, thus escaping criminal infiltra-
tions, and qualifying the cited solutions as preventive mechanisms in the fight against environmental
crime and criminal organizations. In this regard, the idea that we put forward is that effective networking
relationships in the biotech field can contribute to sustainability, in its broadest sense, not only by reduc-
ing the negative impacts of human/industrial activities and the restoration of contaminated territories,
but also by creating a territorial and corporate competitive advantage to prevent and reduce the danger
of criminal infiltrations in economic and productive activities. Environmental and health safety, from
the sustainability perspective and thanks to biotech innovation, strongly require the positive interaction
of the following different factors: the creation and spread diffusion of new knowledge and its practical
application in the biotechnologies, thanks to the technology transfer activities (among them, the creation
of biotech start-ups will have particular relevance), right actions undertaken by Institutions, regulators
and policy makers, and the effective contribution, above all in critical areas and territories of Public
Prosecutors, Police Forces, and Professionals such as accountants.
In other words, this chapter assumes that in order to achieve environmental health and safety, the
production of new knowledge and technology transfer mechanisms are crucial. In order to achieve this
goal, they require the interaction between a wider range of actors and institutions that are engaged in
the process from the outset (Perkmann, Tartari, McKelvey, Autio, Broström, D’Este, & Sobrero, 2013).
Nevertheless, literature shows that the virtuous processes of knowledge creation and transfer in the field
of biotechnology are not activated whenever different subjects interact with each other (D’Este & Patel,
2007). On the contrary, special conditions are necessary both in relation to the type of relationship es-
tablished, and to the type of knowledge transferred, so that collaborative processes can bring benefits to
the parties involved (Ponomariov & Boardman, 2008). The enlargement of the network to further actors,
that have different features and goals, makes the issue even more complex. In addition, in relation to
the role of Institutions, regulators and policy makers, as well as Public Prosecutors, Police Forces, and
Professionals such as accountants, it is crucial to highlight that there the need for a coordinated effort

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by these actors. In particular, an interesting question pertains the role of the above-cited subjects in sup-
porting the juridical action and in practically handling the tools that the norms have rendered available
for contrasting the criminal organizations’ infiltrations within the national economy. In this regard, it is
worth noting that the joint action of at least three categories of professionals, such as judges, accountants
as judicial administrators, and operational forces is crucial to ensure the success of any of these proce-
dures. Hence, a thought-provoking question to deepen is related to the ways through which the above
cited categories of Institutional actors and professionals, characterized by very different backgrounds,
interact and if such joint tasks find effective support in extant legislation of the phenomena investigated.
Moreover, with special reference to the issues relating to environmental crime and eco-mafias, the ef-
fectiveness of the regulatory frameworks available, of the institutional actions, and of the collaboration
between different subjects, assumes even more relevance. Indeed, when biotech innovation is regarded
as a fundamental tool to contrast the organized crime at multiple levels, it is important to consider that
successful initiatives in this sense can strongly benefit not only from a well managed and a well-organized
network but also, according to a broadest conception, from an effective system that encompasses all the
subjects recalled above.
From this perspective the issues relating to co-management assume particular relevance. The co-
management, which is the public-private joint management of common resources, may be defined as
the response to a number of top-down management policies of public resources (Driessen et al., 2012).
In an eco-systemic perspective, the active involvement of the stakeholders is considered as a useful and
practical solution to manage public resources, so as to reduce conflicts among the actors and opportun-
ism (Berkes, 2009). The recent scientific literature defines legitimacy as one of the primary outputs of
collaborations. In other words, the interactions among stakeholders seem to promote mutual understand-
ing and ultimately lead to their mutual agreement. This is especially true when they belong to different
worlds (such as individuals and professionals, government agencies, non-governmental organizations,
businesses, universities and research institutions, foundations). The following section will deepen the
issues raised about the importance of the formation of a hyper-expanded network by specifically focus-
ing on the current concerns and the possible routes to follow. Indeed, from a stricter technological point
of view, we already had the opportunity to see how biotechnologies may contribute to the treatment of
waste. In the following section, we will focus on co-management and governance aspects, which affect
the direct involvement ofbiotechnologies in the context of the waste management and its “supply chain”.
More specifically, we propose a cooperative model for the waste treatment, and discuss two main im-
plications: the enhanced effectiveness of the biotechnology research results, thanks to the cooperation
dynamics activated; the cultural and social positive impacts, arising from the stakeholder interactions,
especially between the industry and the research side, also highlighting the main concerns that it is worth
considering in relation to innovative and professional networks.

4. TOWARDS A HYPER-EXPANDED NETWORK: ISSUES AND CONCERNS

Innovative Networks and Biotechnologies

In order to address the issues relating to co-management it is useful to start by referring to the distinction
between open and closed science proposed by Merton (1973). Open science is typically exercised by
scientists in the academia, whichare interested in developing public knowledge, and possibly publish-

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ing it. On the other hand, closed scienceis frequently exerted by research-based firms, seeking for the
protection of scientific results to reach the commercial exploitation of the discoveries. Both open and
closed sciences are called to benefit largely from the creation of networking relationships and consortia.
That said, it is important to stress that in research-intensive sectorsconsortia are not very common. A
consortium involves a number of actors with different characteristics, which interact with each other
within a single entity with some defined objectives, and maintain their independence and individuality.
In a research consortium, the actors involved in open science and closed science co-exist, share resources
and their goals converge. The consortium, therefore, may be the vehicle through which cooperation
between biotechnology firms and other private and public players in the waste management chain is
guaranteed and institutionalized. In this sense, the opportunistic behaviours may be discouraged, and
the creation of shared values and social norms encouraged (Nooteboom 2002; Gilsing & Nooteboom,
2005). The formation of a consortium for the waste management, involving biotech companies, is likely
to enablemutual learning mechanisms that encourage the exploration and the exploitation (Oliver, 2009).
Moreover, it is important to notice that according to Sandstrom et al. (2014), each supply chain has its
crucial actors, and therefore the selection of those participating in the consortium is extremely relevant.
Hence, the degree of heterogeneity of the actors and the involvement of the leading institutional repre-
sentatives are essential, and the presence of at least one person in charge to coordinate the interactions
within the network is highly recommended.
What should be noted is that the business of biotechnology is characterized by a high scientific voca-
tion: biotechnology firms are usually created to commercialize research products, above all in the areas
of molecular biology and protein chemistry. This peculiarity makes them unique from different points of
view. Oliver (2009), for example, describes them as small-size firms, which often arise from the research
of famous scientists and the foresight of venture capitalists. Such firms have some peculiar features, in
terms of knowledge generation and exchange mechanisms because knowledge, in this particular business
context, is fuelled by direct interactions and can hardly be acquired on the market (Grant, 1996). In the
biotechnology field, collaborative relationships are essential to innovate, and take place both within the
individual firm and among the firms. In the seminal work by Oliver and Liebeskind (1997), the authors
focuson the newly established biotechnology companies. They mainly recognize three network schemes:
the intra-organizational one, between individuals, which involves scientists and technology transfer offices
within the University; the inter-organizational one, between individuals, involving scientists in univer-
sity and in biotechnology firms; the inter-organizational level, between companies, connecting the new
biotechnology companies with the university and the other companies already operating in the market.
Among the just mentioned subjects, there are crossed, and repeated relationships that make their networks
of relationships mutually interconnected. The authors, therefore, argue that the rules and the policies that
guide their organizationscondition the nature of the relationships, between both individuals and organi-
zations, also highlighting that the former are often guided by social or professional ethosand values. In
this regard, Granovetter (1985) defines embeddedness as the link between the economic exchanges that
takes place between some actors and their network of social relations. The context in which companies
operate sets the rules and the threats they are facing, as well as the opportunities to exploit. Therefore,
it is deeply connected to the strategic and operational decisions of the companies and the way through
which they interact.Indeed, previous studies show that the original institutional environment influences
how every experience of co-management starts and develops (Ansell and Gash, 2008). However, once
that the collaboration processes start, then they reform the rules of the game, trough an iterative mecha-
nism that helps to create dialogue and trust between the public and private actors involved (Sandstrom

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et al., 2014). In a research that relates to the stakeholder acceptance in coastal areas co-management
processes, the authors show that it is easier to start virtuous processes of collaboration when the actors
involved have some previous positive experiences of co-management. This suggests the potentially
strong social value of biotechnology within the economic systems. The biotechnology industry, in fact,
is traditionally linked to successful cooperative experiences, and involves by itself public and private
actors together, much more than any other industry (Peters, Groenewegen, & Fiebelkorn, 1998).Thus,
the exchange of knowledge and the interaction between biotechnology firms and the other players in
the economic system can help to harmonize the behaviours, balancing or softening the differences, in
a process of continuous improvement. All this appears to be highly desirable in contexts permeated by
some fairly widespread conditions of lawlessness, such as waste management.

Professional Networks

As already highlighted, over the last years there has been an increasing attempt by regulators and policy
makers to enhance the legal provisions for the management of the entities seized from the Camorra and
the Mafia. However, despite the development of a complex set of norms, standards, and operational
suggestions, the debate on the issues relating to the crucial need for purifying companies by criminal
infiltration, introducing social and environmental values in the management is still rather than being
concluded (Donato et al., 2013). In this regard, the joint action of at least three categories of profession-
als, such as judges, accountants as judicial administrators, and operational forces is crucial to ensure the
success of any procedures. Hence, the interactions between these professionals, characterized by very
different backgrounds, are crucial and need to find effective support in extant legislation. To ensure
effective collaboration between different professional groups it is important to supersede their typical
closure (Abbott, 1988) by fostering hybridization processes that allow one of the groups (or every group)
to acquiring new skills and admitting different points of view. It is interesting to highlight that despite
the importance of these issues and the huge amount of research that has investigated the role and nature
of professions in general (e.g., Carr Saunders & Wilson, 1934; Parsons, 1939; Parsons, 1951; Barber,
1963; Hickson & Thomas, 1969; Freidson, 1994; Hargreaves & Goodson; 1996), and of the accounting
profession in particular (e.g., Abbott, 1988; Sikka & Willmott, 1995; Martini & Zan, 2001; West, 2003;
Amaduzzi, 2004; Edwards, 2009), research on this topic is underdeveloped and still at the embryonic
stage. With reference to the formation of professional groups Abbot (1988) claims that sociological
research on professions has been dominated by a concern with their organizational structure, and with
processes of professionalization that are studied as a medium and outcome of this structure. In contrast,
he highlights that the professional development should be considered by looking at the link between a
profession and its work, and on the ways in which the boundaries of such jurisdiction are negotiated,
attacked and defended (Sikka & Willmott, 1995). In this regard, what should be noted is that Abbott in
his work raises an interesting issue in relation to the importance of interprofessional competition in the
process of professional formation and development. This view, despite the limits highlighted by Sikka
and Willmott (1995) discussing Abbot’s model, was and is still embraced by several scholars (e.g.,
Dezalay, 1989, 1991; Neu, 1991, Barone et al., 2013) focusing either on the nature of the accounting
profession or on the processes of professionalization over times and within different countries, from
both the historical and the regulative perspectives.
However, the idea that we put forward is rather different. In fact, for the purposes of this research,
the focus will not merely be on the formation of the accounting profession or on the characteristics of

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accountants and their role in society, largely discussed to date by many scholars (e.g., Carr Saunders &
Wilson, 1934; Parsons, 1939; Parsons, 1951; Barber, 1963; Hickson & Thomas, 1969; Freidson, 1994;
Hargreaves & Goodson; 1996; Abbott, 1988; Sikka & Willmott, 1995; Martini & Zan, 2001; West, 2003;
Amaduzzi, 2004; Edwards, 2009; Dezalay, 1989, 1991; Neu, 1991, Barone et al., 2013). On the contrary,
we claim here that an interesting and quite neglected theme in this field is related to the evolving nature
of the accounting profession in view of the fast changing social and economic landscapes, and the im-
portance of a kind of hybridisation of their role and tasks. Indeed, the upcoming roles of accountants in
the current economy, such as in the case of the management of the entities seized from Camorra, poses
the question as to whether this groups of professionals can be able to supersede their typical closure
(Abbott, 1988; Sikka & Willmott, 1995), and to actively cooperate with other professional groups, with
different jurisdictions and from diverse backgrounds, towards the achievement of common aims.
On these grounds, and broadening the previous reported view to the whole of the accounting field,
two fundamental concerns need to be highlighted. First, the success of hybridisation processes is strongly
reliant upon a participative approach and the creation of a common language between the different groups
Thus, the provision of formal and informal tools and practices to foster this process deserves more at-
tention. Second, given the fast evolving role and tasks of the accountants that has characterized the last
years, pushing them to operate at the border line of their typical jurisdictions (when for example they are
employed as judicial administrators), the issues relating to their ability to embrace different views and to
interact with other groups of professionals, need to be seriously taken into consideration. To better depict
the main implications of such a complexity, it is useful to refer to the issues relating to the management
of the entities seized form the Camorra by analysing the Italian situation. Without specifically entering
the content of the norms available, it is interesting to note that the Italian regulation for the management
of the sized assets (and of course entities), has increased over the years by progressively enriching the
number of measures available and their applicability (in relation to both subjects to be persecuted and
geographical areas of reference, also including cross-boarder criminal actions), and by highlighting
the importance of the use of confiscated assets for social purposes. Such a process culminated with the
D.lgs. 159/2011 (CodiceAntimafia - Antimafia Code) that in the initial intentions was aimed at provid-
ing a systematic and organic revision of the existing norms towards more effective actions to contrast
the organized crime. However, it is possible to argue that the Antimafia Code (henceforth Code) is still
affected by several limitations that comprise the effectiveness of the actions to contrast criminal infiltra-
tions in the national economy.
The first, and probably most evident concern is related to the fact that the Code allows a dual binary.
Indeed, a first set o measures is mainly based on the provisions of the art. 12sexies of the law 365/1992,
applied along the process to ascertain the criminal responsibility, within which the adoption of preventive
measures for the assets and their administration is assigned to the judges that proceed (GIP - judge for
the preliminary investigations, GUP - judge for the preliminary hearings, Court, and Court of Appeal).
On the other hand, a second set of measures, the assets preventive measures applied as a consequence
of a procedure undertaken by a specialized Court which, receiving a proposal by the Public Prosecutor,
the Police Commissioner, the Head of the Antimafia Investigative Agency (DIA), undertakes further
investigation through the judiciary police, can dispose the seizure of the assets, deliberates about the
requests to revoke the seizure, and through a delegate judge administers the sized assets until the final
confiscation. The greater effectiveness of the prevention measures is widely accepted, not only for the
presence of a specialized Court that administers the assets with continuity, but also for their more rapid
and broad applicability: the process is simplified, seizure and confiscation can also be adopted against

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persons suspected of membership in a Mafia-type association or of having committed serious offenses

(with no need to prove the conviction beyond any reasonable doubt), and in the event of death of the
subject proposed (in a criminal trial where the offense is extinguished and the seizure shall be revoked).
However, a first lack in the current regulation, despite the attempt to use the two options in a coordi-
nated manner, is that there are still overlaps when the second procedure starts and replaces the first one,
both in terms of subjects responsible for the procedure and sharing of the information, that although
practically agreed, need to be further and clearly regulated. Moreover, in case that the preventive measures
are disposed when the criminal procedures are on-going, it is likely that for various reasons (as someone
argued among other also political reasons can exert a certain influence) the judicial administrator chosen
at the beginning of the process can be replaced with a different subject. Such a circumstance creates a
problem in that it is not sure that the new judicial administrator will follow the pathway traced by the first
one. This generates a strategic discontinuity in the seized entity that seems even more problematic in the
light of the economic-financial imbalances that characterize the entity over the first phase of the judicial
administration, related to the process of emergence of legality. Also, the judicial administrator has a huge
amount of communication and information to manage to fulfil the accountability requirements of the two
different legal organs entitled to the two parallel procedures. This led the majority of the accountants to
consider the Antimafia Code as a “work in progress” that needs additional fittings and implementation
rather than a definitive answer to the call for an organic antimafia legislation. Broadly speaking, another
kind of limitation can be ascribed to the need for greater connection between the Code and the Civil law,
with especial reference to the corpus of norms that refers to insolvency and bankruptcy, and the fiscal
regulation, as well as to the legal provisions relating to the governance model of the different types of
companies. Indeed, accountants enrolled as judicial administrators for the seized entities often have to
face a series of challenges relating to the above cited issues, that enlighten the need to put more effort
in clarifying a comprehensive and less subjective way to handle any problems upcoming when criminal
law and civil law are characterized by different perspectives on certain questions. In fact, the main issue
raised by these people during the dialogues was that sic stantibus rebus they find it very difficult to carry
out their task towards an effective management of the seized entities, since too many aspects are left to
subjective and emotional decision-making, and in a constant atmosphere of uncertainty.
Another element of debate is associated to the nature and the correct interpretation of the role and
the tasks that the judicial administrator has to carry out, given the contrasting views around the legal and
practical implications of this figure. In this regard, it is important to highlight that the need to preserve
the going concern implies a complex management approach to avoid the deterioration of the entity and to
not incur in responsibilities of any type. Moreover, the progressive evolution of the judicial administra-
tor from a role of “guardian” to a role of “manager/entrepreneur”, poses the premises for an additional
difficulty that cannot be underestimated and can be identified with the business risk that in the first case
should be avoided, but in the second represents an intrinsic and unavoidable element of the activity. It
is worth remarking that such a dichotomy has not been settled within the Antimafia Code, that despite
enlarging the judicial administrator’s functions, do not clarify the essence of his role according to the
above-cited issues. As a consequence, the Code is formally more articulated but, in practical terms, does
not define a coherent and clear profile of the judicial administrator who incorporates plenty of different
figures in just one. Arguably, the practice will define this better, despite, or probably because of, the
new regulation, but still leaving unresolved the problems relating to subjectivity and relativity that do
not grant a homogeneous approach to the problem. Finally, it is important to enlighten that the majority
of the judicial administrators see themselves as professionals that are quickly becoming entrepreneurs,

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suffering a major change of their personal and work life, and struggling with a multidisciplinary and
multifaceted knowledge that they are required to acquire in short times. From this perspective, and in
the light of the above-cited difficulties, their call for more systematic regulative action seems even
more acceptable and reasonable. On this basis, it is possible to assert that the Antimafia Code, despite
representing aa good starting point needs toincorporate newer aspects that are still neglected and that,
consequently threaten the effectiveness of the regulation in supporting accountant’s hybridization towards
an effective management of the seized entities. Clearly, these kinds of improvements will be possible
only encompassing a broader view and adopting a more cooperative approach by regulators.
Only if the problems relating to the interaction between the professional groups re-called in this sub-
section will be effectively managed it will be possible to realize a fully successful interaction between
them and the innovative biotech networks, in the fight against organized crime towards sustainability.

5. CONCLUSION

This study is grounded on the idea that the problems relating to environmental degradation, pollution, and
ecological variations, as the consequences of uncontrolled progress and prosperity, represent issues that
still deserve further investigation, above all in relation to sustainable development. In fact, sustainable
development involves the consideration of many, heterogeneous, multifaceted, and sometimes contrast-
ing fundamental problems, as well as a broad range of stakeholders at local, regional and global levels
(Lostarnau et al., 2011; Lafreniere et al., 2013).
What should be noted is that at any level, science and technology play a crucial role to achieve sus-
tainability, and also political decisions assisted by societal support and coordinated policies are essential.
Hence, a global and holistic view encompassing economic, social and environmental aspects becomes
crucial. Also, innovation processes are increasingly central, and biotechnologies have gained in particular
plenty of faith and credit by public opinion and governments worldwide, over the last years.
However, despite the extraordinary and unquestionable ability of biotechnology to producing radical
innovations and improving life conditions, it is quite clear that there is still the lack of precious elements
to support a full expression of their potential. This chapter addressed the issues relating to sustainable
development to provide a critical discussion on the potential role played by biotechnologies in practi-
cally pursuing the expectations for technological, socio-economic, political and cultural changes. The
aim of the paper was to deepen the issues relating to the possible contribution of the networks operating
in the biotech field to social and environmental sustainability, and to contribute to better understand the
role of the diverse research actors and the variety of issues that characterize the structural and relational
complexity of biotechnology. The idea that we put forward is that effective networking relationships
in the biotech field can contribute to sustainability, in its broadest sense. More specifically, the chapter
assumed a new and challenging point of view concerning the positive potential of biotechnology and
biotech companies to settle the threatens deriving to human health and environmental safety from the
increasing infiltration of organized crime in the current economy. In particular, the chapter has described
how biotechnologies may contribute to the treatment of waste. Also it has discussed the issues relating
to co-management and governance aspects in relation to the direct involvement of biotechnologies in
the context of the waste management and its “supply chain”. In these regard, we demonstrated that the
biotechnology research results may be enhanced and reinforced thanks to the activation of cooperation
dynamics and that the stakeholder interactions among heterogeneous actors, especially taking into con-

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sideration the industry and the research side, have cultural and social positive impacts. Also, we discussed
social implications and open concerns, both in relation to the relationships within innovative networks
and between institutional professional actors, allowing the identification of any grey areas and limitations.
On this basis, the chapter highlights the importance of a cooperative model for the waste treatment,
centred on theidea of a kind ofhyper-network.Indeed, thanks to the multi-stakeholder perspective and
the examination of the extant literature, this work provides a framework useful to find out what are the
factors that influence effective systems relationships and an enhancement of knowledge production and
knowledge transfer, towards an improved sustainability. In this regard, it is worth specifying that the
above-cited systemic view adopted in the study allowed us to provide evidences that look at the issues
relating to the “corporate social/environmental sustainability”, and also to the crucial questions of the
“systems social/environmental sustainability”.

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Fighting Ecomafias

KEY TERMS AND DEFINITIONS

Biotechnology: Use of biological materials to make useful processes or products.

Co-Management: Public-private joint management of common resources.
Ecomafias: Illegal activities carried out by criminal organizations, which are causing damage to the
environment.
Environment: The surroundings of a dynamic system.
Network: Set of related and interacting actors.
Sustainable Development: process of change aimed at meeting human needs.
Toxic Waste: A waste material harmful to living things and the environment.

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Chapter 16
Effective Waste Water
Treatment and its Management
Sakthivel Lakshmana Prabu Ruckmani Kandasamy
Anna University, BIT Campus, Tiruchirappalli, Anna University, BIT Campus, Tiruchirappalli,
India India

TNK Suriyaprakash Thirumurugan Rathinasabapathy

Al Shifa College of Pharmacy, India International Medical University (IMU),
Malaysia

ABSTRACT
Worldwide there is an increasing industrialization leads to increased disposal of uncontrolled waste
products into the environment which made the environment more pollute and creates hazards. Industrial
wastewater is having a major role in the environmental pollution. The major physical, chemical and
biological products of the wastewater are solid content, organic matter, in-organic compounds, deter-
gents, soap, cleaning products, metals, gases, volatile compounds, numerous pathogenic microorgan-
isms, nutrients and toxic compounds. Untreated wastewater can cause various environment pollutions
problems such as eutrophication or oxygen depletion in the environment. Hence a effective wastewater
treatment process and its management is necessary to reduce the contaminants in the permissible levels
in the treated waste streams. The final outcome of an effective wastewater treatment and its management
is to ensure and provide an appropriate environment protection to the living things and public human
beings in the world.

INTRODUCTION

Wastewater is a combination of water and water-carried wastes originating from homes, commercial,
industrial facilities and from various institutions. Untreated wastewater generally contains more than
99% of water, but the remainder contains high levels of organic material, small amounts of inorganic
ions, numerous pathogenic microorganisms, nutrients and toxic compounds leads to environmental pol-
lution and health hazards.

DOI: 10.4018/978-1-4666-9734-8.ch016

Copyright © 2016, IGI Global. Copying or distributing in print or electronic forms without written permission of IGI Global is prohibited.

Effective Waste Water Treatment and its Management

Wastewater treatment is becomes a critical one due to its water resources, disposal costs, regulations
which reduces the wastewater contaminant levels in the disposal waste water streams. The critical goal
of waste water treatment and its management is to protect the environment, public health and at last the
socio-economic concerns (Tchobanoglous & Burton 1991).
Hence, waste water must be treated suitably to meet the required standard before it is getting disposed
to protect the environment from the pollution and prevent the health hazards of the human beings.
Choosing an appropriate waste water treatment technology is depends upon the nature of the contents
in the waste water.
Selecting a suitable waste water treatment process is depends upon:

• How clean the final water effluent from our plant must be.
• The quantities and nature of the influent water we need to treat.
• The physical and chemical properties of the pollutants we need to remove or render to neutral in
the effluent water.
• The physical, chemical and thermodynamic properties of the solid wastes generated from treating
water. (Cheremisinff, 2002)

Treatment of Wastewater

Generally wastewater treatment is carried out in four stages. They are:

1. Preliminary treatment
2. Primary treatment
3. Secondary treatment
4. Tertiary treatment (Bozkurt et al., 2015; Kalbar et al., 2012; Garrido et al., 2011; Al-Rekabi et al.,
2007)

VARIOUS TREATMENT PROCEDURES

Preliminary Treatment

Solids and oil with grease are combined to form sludge. Preliminary treatment is the first process which
makes the effluent suitable for further process by reducing the non-favourable wastewater characteristics
like large solids and rags, abrasive grit, odours and in certain cases, unacceptably high peak hydraulic
or organic loadings.
Preliminary treatment processes consist of screening/filtration as physical unit operation which re-
moves the coarse suspended matter and flotation for the removal of oil and grease. Other preliminary
treatment operations are comminution, removal of greases and oils, flow equalization, equalization and
neutralization of acid wastes and alkaline wastes.

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1. Screening

The screening of is one of the old technique used to remove the solids, gross pollutants from the waste
water through the use of a porous medium by downstream equipment process. The porous shape may
be of any shape, but in general circular or rectangular shape is used. In screening process various larger
suspended and floating materials such as fibres, paper, rags, string substances and other materials are
removed, before entering into the primary settling tanks.
Conventional screening process is carried in two stages. In first stage coarse screens such as metal
bars or heavy wires spaced 25 to 50 mm apart is used in the waste water flow channel to remove the
coarse/denser materials. Bar screens consist of vertical or inclined steel bars distributed equally across
a channel through which wastewater flows.
In the second stage 0.8 to 6.0 mm fine screen size opening is used to remove the finer particles. Fine
screens consist of various types of screen media, including slotted perforated plates, wire mesh, woven
wire cloth and wedge-shaped wire.
The accumulated solid waste materials are frequently removed by screening process to avoid the
clogging/choking of the screen filters. Various screen categories with its size of openings and its ap-
plications in preliminary treatment process are given in Table 1 (Davis, 2013; Waste-water treatment
technologies: A general review. United Nation, NY, 2003).

2. Comminution

Comminutors are used to pulverize large floating material into small one in the waste flow, where the
screening technique is impractical. Generally comminutors are installed between the grit chamber and the
primary settling tanks to remove the pulverized materials, which reduces odours, flies and unsightliness.

Table 1. Different screening technique in wastewater treatment

Screen Category Size of Openings Application Types of Screens

(mm)
Coarse Screen ≥6 Remove large solids, Manually cleaned bar screens/trash racks
rags, and debris. Mechanically cleaned bar screens/trash racks
Chain or cable driven with front or back cleaning
Reciprocating rake screens
Catenary screens
Continuous self-cleaning screens
Fine Screen 1.5-6 Reduce suspended Rotary-drum screens
solids to primary Rotary-drum screens with outward or inward flow
treatment levels Rotary-vertical-disk screens
Inclined revolving disc screens
Travelling water screens
Endless band screen
Vibrating screens
Very fine screen 0.2-5 Reduce suspended ---
solids to primary
treatment levels
Micro fine screen 0.001-0.3 Upgrade secondary ---
effluent to tertiary
standards

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Two types of comminutors are used in the comminution process, such as Rotating cutter comminutor
and Oscillating cutter comminutor (Waste-water treatment technologies: A general review. United Na-
tion, NY, 2003).

3. Removal of Greases and Oils

In primary treatment the water is left to stand so that oil and grease can form an insoluble layer on the
surface of the water. Grease traps can be removed from the surface of the water, whereas gravity or simple
skimming methods can also be used to remove the grease and oil (Abd El-Gawad, 2014).

4. Flow Equalization

Flow equalization is a technique used to improve the effectiveness of secondary and advanced wastewater
treatment processes by leveling out operation parameters such as flow, pollutant levels and temperature
to achieve constant flow to minimizing the downstream effects during the treatment process.
There are four type of flow equalization processes are available.

1. Alternating flow diversion

2. Intermittent flow diversion
3. Completely mixed, combined flow
4. Completely mixed, mixed flow

In waste water treatment process flow equalization can be applied in various locations in the treat-
ment plants. The locations are

• Near the head end of the treatment works

• Prior to discharge into a water body
• Prior to advanced waste treatment operations

5. Equalisation

Different properties of waste materials may be produced and discharged into the waste water stream at
different intervals in an industry, hence waste water might having different characteristic properties from
time to time. So, uniform treatment can’t be possible to treat the waste water, unless the waste water
has uniform properties. The receiving or initial treatment tanks of a facility often serve as equalization
tanks. To attain the uniform properties wastes need to equalize in waste treatment facilities by holding
waste streams in a tank for a certain time period to attain uniform properties which provides easier to the
treatment. Introducing uniform waste stream to the treatment system controls the treatment operations
resulting to produce more predictable and uniform treatment results. Operation parameters of waste
water treatment processes are to improve its effectiveness which is based on the equalization technique,
a leveling out operation by checking the flow, pH, BOD and pollutant levels. During equalisation pro-
cess solids present in the waste will settle along with heavy metals. The size, shape and its dimension of
equalisation tanks vary depends upon the quantity of waste and mode of discharge of the waste materials.

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The equalisation tank should have a capacity to hold the volume of waste produced up to the completion
of a cycle of operations. Usually equalization tanks are equipped with agitators or aerators for mixing
are preferred to prevent the settling of suspended solids to the bottom of the tank and to provide uniform
effluent for the effective treatment. The agitators are normally placed at the centre line of the length
of the tank spaced equidistantly, whereas aeration prevents settling of solids at the bottom of the tank.
Systematic diagram of equalization tank is shown in Figure 1.
Uniform effluent can be obtained by proper mixing in the equalization tank by:

1. Appropriate distribution and baffling

2. Mechanical agitation
3. Aeration

6. Neutralisation of Acid Wastes and Alkaline Wastes

Wastewater might have a wide range of pH depends upon the type of waste added into the waste water
stream. Neutralization is required in the untreated waste waters to eliminate either high or low pH values
prior to such as biological treatment. Generally chemical treatment process such as chemical precipi-
tation is utilized along with the neutralization to adjust the pH of the waste water. Various chemical
precipitating agents includes lime, sodium hydroxide (caustic), soda ash, sodium sulfide, and ferrous
sulfate. Neutralization is performed by adding acids such as sulfuric acid or hydrochloric acid, to reduce
pH, and alkalies, such as sodium hydroxides, to raise pH values. Neutralization may be performed in a
holding tank, rapid mix tank, or an equalization tank to control the pH of the discharge between 6 and 9
(Waste-water treatment technologies: A general review. United Nation, NY, 2003; Cheremisinff, 1995;
Spellman, 2004; Wastewater treatment technologies; Sincero, & Sincero, 2003; Drinan, & Whiting,
2001; Subramanyam, 2005). Systematic neutralization tank is shown in Figure 2.

Figure 2. Systematic diagram of Neutralization

Figure 1. Systematic diagram of equalization tank
tank

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PRIMARY TREATMENT

Primary treatment acts as a precursor for secondary treatment which makes the effluent suitable for fur-
ther downstream biological treatment by separating the solids as sludge. In primary treatment solids are
removed as sludge by the physical operations such as screening and sedimentation, but doesn’t remove
or degrade the dissolved organic matter. Some circumstance depends upon the nature of the effluent pre-
aeration or mechanical flocculation with chemical additions can be used to enhance primary treatment.

1. Sedimentation

Sedimentation is one of the unit operation in wastewater treatment involves settling of solid particles
based on gravitational force in a suspended mixture. In this way the gritty particles, particulate and
biological substances in the sludge, and chemical precipitates are removed. Sedimentation takes place
in a settling tank, also referred to as a clarifier. There are three main designs, namely, horizontal flow,
solids contact and inclined surface (Sperling, 2007).

2. Flocculation/Coagulation

Flocculation is a process of stirring or agitation of chemically-treated water to induce coagulation. Both

flocculation and coagulation process are interchangeable one. In general “coagulation” is the reduction
of the net electrical repulsive forces at particle surfaces by addition of coagulating chemicals, whereas
“flocculation” is the agglomeration of the destabilized particles by chemical joining and bridging.
Flocculation usually consists of a rapid mix tank which enhances the sedimentation or filtration treat-
ment processes results increased settling rates. Coagulating agents are added in the waste water to initiate
the coagulation. Once coagulation over, the coagulated wastewater flows into a flocculation basin where
slow mixing of the waste occurs, which allows the flocs to absorb and entrap the particles and other
suspended materials to agglomerate into heavier, more settleable/filterable solids by bring them down.
Generally three types of chemicals are used in the coagulation/flocculation treatment processes. They
are inorganic electrolytes, natural organic polymers and synthetic polyelectrolytes.
Commonly employed coagulating agents in the waste water treatment process are:

1. Alum (Aluminium sulphate)

2. Ferric sulphate
3. Chlorinated copper
4. Ferric chloride

Among these, alum is the most popular coagulant used in the wastewater treatment (Waste-water
treatment technologies: A general review. United Nation, NY, 2003; Wastewater treatment technologies;
O’Melia, 1972; Amirthanrajah, & Mills, 1982; Davis, 2013).

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3. Flotation

Flotation is a unit operation used to remove solid or liquid particles from a liquid phase by introducing
a fine gas, usually air bubbles. The gas bubbles either adhere to the liquid or are trapped in the particle
structure of the suspended solids, raising the buoyant force of the combined particle and gas bubbles.
Flotation is the process of introducing fine bubbles into the tank to encourage suspended particles to
rise to the surface of the tank. When gas bubbles are introduced into the wastewater, gas bubbles either
adhere to the liquid or are trapped in the particle structure of the suspended solids, thereby reducing their
specific gravity and causing them to float. Flotation process is used to remove the suspended solids and
oil and grease from oily wastewater by reducing the sedimentation times of suspended particles; which
have a specific gravity closer to that of water (solids particles which has the specific gravity slightly
greater than water whereas the oil/grease particles have specific gravity slightly less than water. Various
components of a flotation process include a centrifugal pump, a retention tank, an air compressor, and
a flotation tank. When the air introduced into fine bubbles, the bubbles are trapped inside the liquids
or solids to form sludge flocs, which raises the buoyant force of the combined particle and gas bubbles
and then floating them to the surface. The float is continuously swept from the tank surface. Flotation is
mainly used to remove suspended matter and to concentrate biological sludge. Various flotation methods
utilized in the waste water treatment process are dissolved air-flotation, air-flotation, vacuum flotation
and chemical additives.
The mechanism involved in the formation of sludge flocs includes:

1. Attachment of the bubbles on the particle surface

2. Collision between a bubble and a particle
3. Agglomeration of individual particles or a floc structure as the bubbles rise
4. Absorption of the bubbles into a floc structure as it forms (Waste-water treatment technologies: A
general review. United Nation, NY, 2003; Wastewater treatment technologies; Spellman, 2010 &
2001; Qasim, 1999)

SECONDARY TREATMENT

Secondary treatment acts as a precursor for tertiary treatment which makes the processes are used to
convert the finely divided and dissolved organic matter in wastewater into flocculent settle able organic
and inorganic solids by chemical decomposition catalyzed by microorganisms. The untreated and es-
caped soluble, colloidal organics and suspended solids from the primary treatment are further treated
in secondary treatment to reduce the Biological oxygen demand (BOD) and chemical oxygen demand
(COD) through biological process.
In the secondary treatment process, micro-organisms, particularly bacteria, convert the colloidal
and dissolved carbonaceous organic matter largely converted to ‘biogas’, a mixture of CH4 and CO2.
Secondary treatment process operation involves trickling filtration, activated sludge process, oxidation
ditch and oxidation ponds.

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1. Activated Sludge Process

The activated sludge process is an aerobic biological treatment process employs suspended-growth
aerobic microorganisms to biodegrade organic contaminants. This activated sludge process is one of the
most valuable biological processes to treat the industrial wastes. A suspension of aerobic microorgan-
ism is utilized by mechanical mixing or turbulence induced in an aeration basin by diffused aerators in
the waste water to mix the contents. In this process, industrial waste or sewage is continuously exposed
and subjected to biological degradation. A series of biochemical reactions takes place in the aeration
basins, which degrades the organic matters and generate new biomass. Microbial mass of bacteria, fungi,
protozoa, rotifers and nematodes are used in the activated sludge process. They include carbon oxidizers,
nitrogen oxidisers, floc formers but primarily gram-negative in strain. The nature of organic matter in
the waste is the determining factor to identify exact type and species of bacteria or other microorgan-
isms in the slimes.
Microorganisms oxidize the soluble and suspended organic materials into carbon dioxide and water
using the available supplied oxygen. Oxygen is an important one to oxidize the organic materials. For
effective oxidation atleast a residual of 0.5mg/l of oxygen should be present at all times which can be
supplied either by mechanical or diffused aeration systems.
The amount of Oxygen requirements are based on the amount required for biodegradation of organic
matter and for endogenous respiration of the microorganisms. The turbid effluents due to the growth of
filamentious bacteria than the other required bacteria and protozoa when anaerobic conditions devel-
oped due the reduced supply of oxygen. The retarding of floc compaction and settling is observed in
filamentous bacterial growth.
After the specific time duration, the treated mixture is passed to a settling tank, where the microor-
ganisms are separated from the treated water. A portion of the settled sludge is recycled to the aeration
basin to maintain a required concentration of microorganisms in the process. The remaining settled
solids is collected as waste and discorded. For effective oxidation process the microorganisms should
supply with adequate nutrient (nitrogen and phosphors) levels must be available to the biomass. Lack of
these nutrients can impair biological activity and result in reduced removal efficiencies. Nitrogen and
phosphorus are supplied by the addition of either urea or mono ammonium or diammonium hydrogen
phosphate. Other than nitrogen and phosphorous various nutrient elements such as potassium, calcium,
magnesium are usually present in the waste. Inorganic substances such as iron, molybdenum and cobalt
are also required in trace quantities. Temperature, pH and oxidation-reduction potential are the other
factors having important role in the activated sludge process. The optimum pH range for the activated
sludge process is between 6.5 and 9.0; if the pH value is below 6.5 fungi will compete with the bacteria.
Hence, for effective oxidation of the organic matters by bacteria, pH measurement must be made at
regular intervals in the aeration tank.
Various operating variable have a role on the effectiveness of the activated sludge process, such as
organic loading, sludge retention time, hydraulic or aeration detention time, oxygen requirements levels
in the aeration tanks, Regulation of the amount of returning activated sludge and Control of the waste
activated sludge. Role of bacteria in the activated sludge process is shown in Figure 3.

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Figure 3. Role of bacteria in the Activated Sludge process

2. Aerated Lagoons

Aerated lagoons are large holding tanks used to oxidize the dissolved organics by mechanical aeration
by surface, turbine or diffused aeration technique. Generally floating aerator technique is used to supply
the required quantity of oxygen and mixing power. Aerated lagoons, the internal surface is lined with
cement or butyl rubber or polythene used. In this process the waste materials are treated for 2 to 6 days
depends upon the contaminant present in the effluent. During this time duration, around 90% of biological
oxygen demand is removed by oxidation of organic matter and to form a denser flocculent sludge. This
flocculent sludge is removed in a settling basin before final effluent discharge. After complete process,
bacteria might be present in the final effluent, hence further biological purification is required.

3. Trickling Filtration

Trickling filtration is an aerobic fixed-film biological treatment process for the removal of organic mat-
ters from the wastewater. In this process consists of a structure of highly packed with inert permeable
such as rock, wood, or plastic. The treated wastewater is spread/sprinkled over the upper surface of the
medium by moving sprinklers using either a fixed spray nozzle system or a rotating distribution system.
The inert medium develops a biological slime layer, that absorbs and biodegrades organic pollutants by
percolation. Air flows through the filter by convection, thereby providing the oxygen needed to maintain
aerobic conditions. The microbial slimes formed on the bottom of the bed oxidize the wastewater. The
thickness of the slime layer increases due to microbial growth. During this process, a gelatinous film of
the microbial growth is formed on the whole surface of the bed which absorbs the dissolved and colloidal
organic matter and degrades the organic matters. In trickling filtration process, organic concentration,
temperature of the waste, size of the bed stone, microbial surface area, depth of the filter, retention time
and oxygen transfer are having the important role in the removal of organic matters. The microbial layer
formed at the bottom of the bed is very sensitive to temperature, and the metabolic activities are directly
proportional to the temperature of the waste passing through the filter. A flow diagram of trickling filter
is shown in Figure 4.
Trickling filters are classified depending upon the organic loading. They are:

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Figure 4. Flow diagram of Trickling filter

Low-rate: In this the filters has a media bed depth of 1.5 to 3 meters
High-rate: In this the filters may have a bed depth from 1 to 9 meters.

4. Oxidation Ditch

Oxidation Ditch is a modified Activated Sludge process, which consists of a ring- or oval-shaped channel
and is equipped with mechanical aeration devices. In this aeration is done by using an aeration system
and mixing done by using brush rotors placed across a race-track-shaped basin. Filtered waste-water
enters the ditch at one end, is aerated by rotors and circulates. Oxidation ditches normally operate in
prolonged aeration mode with longer detention and solids retention time are provided.

5. Oxidation Ponds

The liquid is treated by bacteria which break down the organic matter remaining in solution and then it
is sent to oxidation ponds. Oxidation pond treatment is a very economical secondary treatment process.
The effluent entering the ponds is a mixture of primary and secondary treated effluent. Oxidation pond
is a large shallow pond in which wastes are added at one end where heterotrophic bacteria continue the
breakdown of the organics in which the required oxygen is supplied by the algae for its metabolism.
The composition of the effluent is varies during the year. In summer generally primary treated efflu-
ent, whereas in winter more secondary treated effluent is used. The carbon dioxide that is released by
bacteria will be utilized by algae for the photosynthesis reaction. High penetrative photosynthesis of
algae, powerful mixing and natural aeration are needed in effective treatment of wastes. These aerobic
and anaerobic processes enable the purification of waste water.
Solids present in the waste are getting settled at the bottom of the pond. In this oxygen bond solid
layer acts as anaerobic phase whereas the top liquid layer acts as aerobic phase. In the pond, algae use
solar energy to produce oxygen from carbon dioxide and water, and bacteria use oxygen to break down
the remaining organics to simple molecules such as carbon dioxide and ammonia.

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The rate of purification is depends upon the amount of the algae present in the pond due to the re-
lease of more amount of oxygen released by the algae. The growth of algae is enhanced by the nutrients
present in the waste. After the treatment the effluent is removed at another end. During this process
maximum amount of BOD is reduced and no longer is BOD treatment required for the effluent. If there
is any lacking of oxygen in the pond, the anaerobic conditions is created in the ponds leads to breakdown
of waste and produce foul odour in the pond (Technology Transfer Manual of Industrial Wastewater
Treatment, Overseas Environmental Cooperation Center, Japan, March 2003; Biological wastewater
treatment; Boari, et al., 1997; Wastewater treatment technology and application in Industrial facilities;
Hoffmann, 1998; Wang, et al., 2009; Melin, et al., 2006; Henze, et al., 2008; Van Haandel, & Van der
Lubbe, 2012; Kalbar, et al., 2012; Sewage treatment; Verbyla, & Mihelcic, 2015; Mara, & Johnson,
2007; Muga, & Mihelcic, 2008’; Mahajan, 2007). The chemical reaction taken place in oxidation ponds
is shown in Figure 5.

TERTIARY TREATMENT

Tertiary treatment is the final treatment process in the waste water treatment to remove significant
amounts of nitrogen, phosphorus, heavy metals, biodegradable organics, bacteria and viruses for further.
This treatment process is also to remove the dissolved inorganic solids, bacteria and traces of organics.
Removal of bacteria especially of faecal origin is achieved by keeping the effluents from secondary
biological treatment units in maturation ponds for specified periods. If the final effluent still contains
enough bacteria, then it is chlorinated. The tertiary treatment process operation involves chemical co-
agulation, flocculation and sedimentation, followed by filtration and activated carbon. Ion exchange
and reverse osmosis for specific ion removal or for dissolved solids reduction are less frequently used.
Inorganic dissolved solids are removed by the following methods.

1. Evaporation

It is a process utilized only when the recovered solids or concentrations solutions are reused. This process
is specifically useful for the concentration of radioactive waste and other wastes of smaller volume. In
this process waste effluent is evaporated by boiling and vaporising the water phase. The degree of evapo-

Figure 5. Chemical reaction of Oxidation Ponds

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ration is greatly dependent upon climatic conditions. This process is generally available only to those
facilities located in arid regions (Waste-water treatment technologies: A general review. United Nation,
NY, 2003; Wastewater treatment technologies; Evaporation - A Wastewater Treatment Alternative).

2. Reverse Osmosis

Reverse osmosis (RO) is a process for separating dissolved solids and metals from water, and it is useful
only when the solute molecules are same size as the solvent molecule. Osmosis is the process of diffu-
sion of a solvent (such as water) across a semi-permeable membrane from lower concentrated solution
into higher concentrated solution. Whereas in the reverse osmosis process, pressure is applied which
is greater than the normal osmotic pressure is applied in the higher concentration region to force the
purified water pass through the membrane and into the lesser concentrated solution through permeation.
The effluent liquid waste containing dissolved solids is passed over the surface of a semi permeable
membrane and at a pressure, which leaves the concentrated liquor on the surface of the semi permeable
members. In RO the low molecular-weight solutes such as salts, surfactants do not pass through the
membrane, which are called as concentrate. These concentrates are recirculate through the membrane
until the flow of permeate drops.
Various factors such as dissolved solids concentration and temperature of the feed stream, the applied
pressure, and the type of membrane selected having the important role in the separation of solids in RO.
Selectivity for water over ions, permeation rate and durability are the other factors need to be considered
while selecting the semi-permeable membrane in the RO. Generally Cellulose acetate and polyamide-
hydrazide membranes are used for the effective separation (Matsuura, 1993; Cruver, & Nusbaum, 1974;
Xiao, et al., 2014).

3. Membrane Separation

Various membrane separation processes such as reverse osmosis, ultrafiltration, dialysis, piezodialysis
and electrodialysis are used; whereas dialysis and piezodialysis techniques are not having any significant
role in the waste water treatment. In the dialysis technique separation of solutes from the solution is
based on the difference in the rates of diffusion. In electrodialysis gradient electrical potential is used
as driving force and an ion exchange membrane as discriminating barrier. Electrodialysis technique is
used to remove the ionic constituent in the wastewater treatment process (Davis, 2013; Water Environ-
ment Federation, 2005).

4. Chemical Precipitation

Chemical precipitation technique is used to remove metal compounds from wastewater by forming a
chemical precipitation of soluble metallic ions into insoluble form as precipitated product. Coagulation/
flocculation processes are used in conjunction with chemical precipitation to enhance the removal of
suspended and colloidal materials by forming an agglomeration and convent into more readily settle
able flocs and removed by liquid filtration or by clarification technique.
Commonly the metal ions are present as hydroxides, sulfides or carbonate forms. Commonly most
of the heavy metals are precipitated as hydroxide at elevated pH. Chemical precipitation process taken
place in two steps.

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1. Addition of chemical precipitating agents: Precipitating agents are added into the wastewater and
mixed by means of mechanical mixture to induce the formation of insoluble metal precipitants.
2. Filtration or clarification: The metal precipitates are removed from the wastewater through filtration
or clarification technique. If clarification technique is used, a flocculent is sometimes added to aid
the settling process. The degree of clarification in the separation of the precipitates is depends on
the quantity of chemicals used.

Various chemicals are used as precipitants, but selection of suitable precipitants is based on perfor-
mance, reliability and cost. Commonly used precipitants in the wastewater treatment are lime, sodium
hydroxide (caustic), soda ash, sodium sulfide, and ferrous sulfate. Lime is the cheapest and effective
precipitating agent. Chemicals such as sulfuric acid and phosphoric acid are used for the pH adjustment,
whereas polyelectrolytes are used as flocculation aids in the wastewater treatment. Various factors such
as chemical interactions, temperature, pH, solubility of waste contaminants, and mixing effects have an
important in the formation of chemical precipitation.
There are two type of precipitation technique such as Hydroxide precipitation and sulfide precipitation
are used in wastewater treatment process, often combination technique is used to remove the optimal metal.
Hydroxide precipitation: This technique is used to remove the metals like antimony, arsenic, chro-
mium, copper, lead, mercury, nickel and zinc.
Sulfide precipitation: This technique is used to remove the metals like lead, copper, silver, cadmium,
zinc, mercury, nickel, thallium, arsenic, antimony and vanadium (Waste-water treatment technologies:
A general review. United Nation, NY, 2003; Wastewater treatment technologies; Davis, 2013).

5. Ion Exchange

It is general technique used to remove the heavy metals from the low–concentration waste streams, such
as electroplating wastewater, is ion exchange. This process is used only when the metal contaminants
and exchanged salts are recovered and reused. In this technique, the waste water stream is passed through
a bed of resin (cationic or anionic or mixed) containing ionic charge on its surface; which are used for
exchange of same charge of ions in the wastewater. Selection of ion exchange technique in waste water
treatment is dependent upon the contaminant to be removed and for water softening. Often, individual
resin beds containing different resins are arranged in series, in specific treatment purpose mixed bed
system is used. Generally polystyrene copolymerized with divinylbenzene resin is used for the treatment.
Ion exchange process is taken place in four steps.

1. Treatment: Wastewater is passed through the resin bed and ions are exchanged until pollutant
breakthrough occurs.
2. Backwash: Backwash the bed to remove the suspended solids on the surface.
3. Regeneration: The resin bed is treated with higher concentrated acidic or alkaline solution to
charge the resin originally present in the resin bed.
4. Rinsing: The resin bed is rinsed to remove the residual regeneration solution present in the surface
of the resin (Gregory & Dhond, 1972).

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ANALYTICAL TECHNIQUES

After the tertiary treatment wastewater can have different elements. The amount of elements such as phos-
phorous, nitrogen, dissolved oxygen, nitrite, nitrate, sulphide, might be present in the treated wastewater,
and it’s needed to be quantified. Various analytical techniques are available to detect the residuals in the
treated waste water. The chosen analytical technique should be very specific to determine the residual
present in the treated waste water even in very low levels to ensure the effluent purity and provide the
assistance to the other stages for the treatment process.
Various instruments are used in the determination of residues in the treated waste water. They are:

1. Atomic absorption spectroscopy (AA)

2. Gas Chromatography (GC)
3. Inductively coupled plasma (ICP)

Atomic Absorption Spectroscopy (AAS)

AAS is used principally for the quantitative determination of metal elements in aqueous and solid samples.
AAS deals with the absorption of specific wavelength of radiation by neutral atoms in the ground state. In
the Atomic absorption spectroscopy, solution of metallic salt sample is atomising to form fine droplets.
Due to thermal energy the solvent in the droplet evaporates leaving fine residues, which are converted
into neutral atoms. These neutral atoms absorbs the light passing through the atoms present in the vapour
stage and measure the amount of light absorbed by the atoms. The amount of light absorbed depend-
ing on the amount of element present in the vapour. Various lamps are used which are depends upon
the element to be determined in the treated waste water. This technique is used for detecting cadmium,
chromium, copper, lead, silver and zinc (Willard, et al., 1989). Schematic diagram of Atomic absorption
spectroscopy is shown in Figure 6.

Gas Chromatography

Gas Chromatography consists of Gas solid chromatography (GSC) and Gas liquid chromatography
(GLC). In GSC the principle of separation is adsorption, whereas in GLC the principle of separation is

Figure 6. Schematic diagram of Atomic Absorption Spectroscopy

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partition. In GLC, gas is used as mobile phase and liquid which is coated onto a solid support is used as
stationary phase. In GLC, sample is converted into vapour and mixed with gaseous mobile phase. The
components are separated based on their partition coefficient. The component which is less soluble in the
stationary phase, travels faster and eluted out first and the components which is having more soluble will
be eluted later (Willard, et al., 1989). Schematic diagram of Gas chromatography is shown in Figure 7.

Inductively Coupled Plasma (ICP)

ICP is one of the commonly used instruments for the determination of various metal elements such as
arsenic, cadmium, chromium, copper, lead, silver and zinc. In this technique the sample is atomized
and injected in a chamber packed with argon plasma, to convert the sample solution into an aerosol
and pass into central channel to vaporize the sample. The vaporized sample liberates the atoms, due to
collision excitation, atoms are promoted to higher energy excited states. The excited atomic and ionic
excited species return to the ground state with the light emission of a photon, this photons have a char-
acteristic energies that can be measured. The total number of the proton is directed proportional to the
concentration of the element present in the sample. The intensity of the emission band is compared with
the previously measured intensities of the known concentration of the elements, by interpolation in the
calibration curve we can able to measure the concentration of the given sample. Thus the wavelength
of the photons from the element has a characteristic emission band which can be used to identify the
elements in the treated waste water (Hou, & Jones, 2000; Stefansson, et al., 2007). Schematic diagram
of inductively coupled plasma is shown in Figure 8.

Total Phosphorous

Normally phosphorous is occurs in the fifth oxidation level. In wastewater phosphorous is present as
phosphates, whereas the phosphorous atom combined with four oxygen atoms (PO4-3). Phosphorous is the
limiting nutrient for the plant growth. Generally phosphates are originate from various sources such as

Figure 7. Schematic diagram of Gas Chromatography

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Figure 8. Systematic diagram of inductively coupled plasma

cleaning solutions, fertilizers and biological wastes. If concentration of phosphate in high level persists,
algae and other aquatic life will grow by decreasing the amount of dissolved oxygen by increasing the
decay of organic matter.
The amount of phosphorous in the treated wastewater can be determined by colorimetric method.
The determination of phosphorous is done in three steps:

1. Oxidise the phosphate into orthophosphate using the mixture of sulphuric acid and nitric acid.
2. Formation of yellow complex by the reaction between the orthophosate with ammonium molybdate
and potassium antimonyl tartrate in presence of acid.
3. Reduction of yellow complex with ascorbic acid to molybedenum blue.

The intensity of the blue colour is directed proportional to the concentration of the element present
in the sample. The concentration of the phosphorous as phosphate present in the treated waste water can
be determined from the standard curve.

Total Organic Nitrogen

In surface and ground water various concentrations of Organic nitrogen is present from free amino acids
and ammonia. Presence of ammonia in the waste water is an indication of sanitary pollution. Ammo-
nia is readily oxidized by bacteria and converts into nitrite and nitrate. Nitrogen present in the treated
wastewater can be determined by heating the sample in acid using copper sulphate as catalyst. The re-
action is continued until the complete conversion of nitrogen into NH4 + ions and then converted into
ammonia by treating with a base. The amount of ammonia can be determined by using an autoanalyser
with colorimetric detection.

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Dissolved Oxygen

Oxygen enters into the water by diffusion from the atmosphere and dissolved oxygen is a vital one for
various aquatic living things. The solubility of the oxygen in the water is directly proportional to the
partial pressure in the gas phase, to salt concentration and temperature. The amount of dissolved oxy-
gen in the water is affected by various factors such as temperature, flow (velocity of water flowing in a
stream), presence of aquatic plants, altitude and dissolved or suspended solids. The amount of dissolved
oxygen in the treated waste water can be determined by:

1. Oxygen selective electrode method

2. Redox reaction

Nitrite (NO2)

Biological breakdown of organic nitrogen (oxidation of ammonia or reduction of nitrate) produces

nitrites as an intermediate product in the water. Amount of nitrite in the wastewater is an indicative of
the pollution. Amount of nitrite in the treated wastewater can be determined by reacting the nitrite with
sulphanilamide to form diazonium compound (Griess-Ilosvay’s method). This diazonium compound is
reacts with N-(1-napthyl)ethylenediamine to form a colored azo dye, which can be measured spectro-
photometrically.

Nitrates

Nitrates are produced as end product in the water when biological breakdown of organic nitrogen oc-
curs. When water shows presence of excess amount of nitrates, it indicates the poor quality of water. The
quantity of nitrite present in the sample illustrates the amount of nitrate present in the treated wastewater.
By measure the difference of nitrite present on successful treatment gives the quantity of nitrate present
in treated waste water.

Hydrogen Sulfide

Sulfide as hydrogen sulphide which inhibits the aerobic respiration, muscle contractions, including
breathing, and promotes excess breakdown of glucose. Hydrogen sulfide level in the treated wastewater
increases when sulfate-reducing bacteria grow up in the anaerobic sludge of a pond. The amount of
sulphide present in the treated wastewater can be determined by reacting the H2S and N,N-dimethyl-
p-phenylene diamine to form methylene blue (a strongly coloured complex) in the presence of FeCl3.

Total Solids

Total solids include both the suspended and dissolved solids in the wastewater. These solids have an
important role in the conductivity and turbidity of the sample. The amount of the total solid present in the
treated wastewater can be measured by place a specific quantity of the sample in a previously weighed

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dish in a drying oven at the temperature between 103°C and 105°C to evaporate the water. The dish is
weighed frequently until get the constant weight, the difference between the initial and final weight of
the dish gives the total weight of the solid present in the sample.

pH

pH is defined as the negative logarithm of the hydrogen ion concentration. The pH value indicates the
amount of hydrogen ion present in the sample and to categorise the sample into acidic, basic or neutral
of the treated wastewater. pH of the treated wastewater can be measured by using an ion-sensitive elec-
trode known as a glass electrode.

Alkalinity

The alkalinity of the sample is mainly due to bicarbonate, carbonate and hydroxide ions in the water,
which increases the pH of the water above 4.5. Alkalinity of the sample indicates the buffering capacity
of water and its ability to resist a change in pH. Alkalinity is differs from pH, and it can be measured
by simple acid-base titration (amount of acid required to neutralize the sample) using a suitable color
indicator.

Five Day Biological Oxygen Demand (BOD5)

Biological Oxygen Demand measures the amount of oxygen utilized by organisms in the biochemical
oxidation of organic matter in a wastewater sample in a specified time (usually 5 days), and at a specified
temperature. BOD measurements are used as a measure of the organic strength of the water.
BOD of the sample can be determined by diluting the sample to get concentration of 2 mgL-1 O2
(approximately) and incubating the sample in a sterile condition at the temperature of 20 ± 1°C for five
days (allyl thiourea is added to the incubating material to prevent the oxidation of these compounds);
measure the dissolved oxygen content which gives the BOD value.

Chemical Oxygen Demand (COD)

Chemical Oxygen Demand measures the total quantity of oxidisable materials (organic and inorganic
matters) present in the sample. COD of the sample can be determined by oxidation reaction (using strong
oxidant like potassium dichromate). The amount of oxidant utilized for the oxidation of the sample gives
the value of COD (Janben, https://www.ecn.nl/docs/society/horizontal/hor16_nutrient.pdf; Elser, et al.,
2007; Lapa, et al., 2000; Park, & Noguera, 2004; Posadas, et al., 2015; Barker, et al., 1999; Ma, et al.,
2001; Interpreting Results From Additional Water Tests; Water quality parameters).

STANDARDS AND GUIDELINES

Globally various standards for the treated wastewater has been developed to ensure it’s physical and
chemical quality. Each country they have a standards for the treated wastewater, whereas the limits of
the elements are reviewed and updated to meet the global needs for the protection of ambient waters.

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Table 2. NPDES and EC EDR for Discharges from Wastewater Treatment Plants

Parameter NPDES EC EDR WD

30-Day Average 7-Day Average Percentage of Concentration Percentage of
Concentration Concentration Removal (mg/L) Removal
BOD 30mg/L 45mg/L 85 25 70-90
TSS 30mg/L 45mg/L 85 35-60 70-90
pH 6-9 -- -- -- --
COD -- -- -- 125 75
Total Nitrogen -- -- -- 10-15 70-80
Total Phosphorous -- -- -- 12 80

Various regulatory agencies mandate the control of water pollution. They also establish secondary
treatment standards, the agency which controls are United States Environmental Protection Agency
(USEPA) and National Pollutant Discharge Elimination System (NPDES). Table 2 shows limitations in
secondary waste water treatment plants and European Community Environmental Directive Requirements
(EC EDR) for discharges from urban waste water treatment (United States Environmental Protection
Agency, 2007; Eaton, & Franson, 2005). Various organizations of wastewater treatment standards are
given in Table 2.

CONCLUSION

The effluent from the various industries contains various matters such as organic material, numerous
pathogenic microorganisms, nutrients and toxic compounds which leads to environmental pollution and
hazards of health community of various living things. An optimized wastewater treatment process and
plant design needs to be developed to support the effective treatment process. The critical role of the
wastewater treatment process is to ensure that the effluent leaves the treatment plant should meet the
limitation of the environmental standard.
Hence, the treated effluent must ensure the safety, efficacy, quality of the treated wastewater and
regulatory requirements to protect the public health from the various effluent environmental pollutions.

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About the Contributors

Ashok K. Rathoure shares his knowledge and experience in the field of Environment Impact As-
sessment with a doctoral degree in Bioremediation for M/s Vardan EnviroNet Gurgaon. Previously he
was associated with En-vision Group Surat (En-vision Environmental Services and En-vision Enviro
Engineers Pvt. Ltd.) for EIA studies; Himachal Institute of Life Sciences Paonta and Beehive College of
Ad. Studies Dehradun for teaching to Biotechnology, Microbiology, Biochemistry and other biosciences
subjects. He has more than 8 years of working experience in various domains. Other than double master
and doctorate degree, Dr. Rathoure has received PG Diploma in Human Resource Management (HRM)
from Algappa University Karaikudi Tamilnadu. His area of research is environmental biotechnology and
publication includes 62 full length research papers in international and national journals of repute, 12
Course books from reputed publishers in India, 4 Research books and 7 Book chapters in Springer verlag
USA, CRC press Tayer & Francis Florida, Wageningen Academic Publishers, Netherlands, I.K. Publishers
Mumbai and Daya Publishers New Delhi, respectively. He had reviewed more than 70 research manu-
script for many international journals. He is member of APCBEES (Hong Kong), IACSIT (Singapore),
EFB (Spain), Society for Conservation Biology (Washington) and founder member of Scientific Planet
Society (Dehradun). He has supervised 24 research scholars (UG, PG and Diploma). Dr. Rathoure is also
associated as Editor-in-Chief for Octa Journal of Environmental Research, Managing Editor for Octa
Journal of Biosciences and Executive Editor for Scientific India Magazine. Dr. Rathoure has travelled
extensively in the major cities of Northern and Western India for his education and as a teacher, trainer
and taught to widen the horizon of knowledge and to sharpen his intellect. He is working as expert for
Ecology & Biodiversity and Water pollution Monitoring, Prevention & Control for EIA studies.

Vinod K. Dhatwalia is presently working as an Assistant Professor in Dept. of Chemistry, Uttaranchal

University, Dehradun, UK, India. He has completed his PhD from HNB Gharwal University, Srinagar,
Uttarakhand. He has also completed his Master’s degree in Biochemistry form same university. His area
of specialization is Biochemistry.

***

Priya Banerjee PhD Fellow at Department of Environmental Science, University of Calcutta, Kol-
kata, India. Topic of research includes synthesis, characterisation and application nanocomposites for
pollution remediation.



About the Contributors

Bikram Basak has received his PhD degree in Biotechnology from National Institute of Technology,
Durgapur, India. He did his MS and BS from University of North Bengal, India. He is currently serving
as an Assistant Professor at Department of Microbiology, North Bengal St. Xavier’s College, India. His
Research included bioremediation of phenolic wastes, polyaromatic hydrocarbons, and azo dyes.

Stephen Rathinaraj Benjamin is a research scholar at University of Federal Goias, Goiânia, Brazil,
Department of Pharmacy. He received his B. Pharm and M.Pharm from Tamilnadu Dr. MGR Medical
university, Chennai, Tamilnadu. He has also done Erasmus Mundus Master in Quality in Analytical
Laboratories (EMQAL) from University of Algarve, Faro, Portugal. He has six years of intense experi-
ence in laboratory techniques and instruments of pharmaceutical analysis. He has published various
research papers in national and international journals in the field of pharmaceutical technology.

Jatindra Nath Bhakta served as an Associate Professor and is associated with ICEE, University of
Kalyani, India. He received Ph. D. from University of Kalyani, India and worked as Postdoctoral Re-
search Fellow of the JSPS, Govt. of Japan and as Researcher Faculty at the Department of Environmental
Engineering and Research Institute of Molecular Genetics, Kochi University, Japan. He has research and
teaching experience with microbiology & probiotic bacteria, carbon sequestration & biofuel production,
bioremediation, metagenomic, wastewater treatment & recycling, fisheries & aquaculture and environ-
mental pollution. Along with other collaborators (>25 researchers from 11 countries) he has published
51 journal articles, 9 abstracts and 5 popular articles. He has been awarded as a senior research fellow
(SRF) by ICAR, RA by DST and DBT, Govt. of India and JSPS postdoctoral fellow by Japan Society
for the Promotion of Science (JSPS), Govt. of Japan. He successfully handled 7 projects and served as
research mentor for Ph. D., masters and graduate students. He also serves as editorial board members
for 7 international journals and as reviewer for 12 international journals.

Adele Caldarelli Professor in Accounting. Her research interests cover the areas of accounting,
management accounting and corporate social responsibility.

Aniruddha Chabukswar, M.Pharm, Ph.D, is presently working as Professor in the Department of

Pharmaceutical Chemistry at MAEER’s Maharashtra Institute of Pharmacy, MIT Campus, Kothrud,
Pune, (MS), India. He has 14 years of academic experience. He has expertise in fields of synthesis and
docking study in pharmaceuticals.

Mamta Chauhan is working in Industrial biotechnology with special focus on extremophiles.

Papita Das is the Assistant Professor at Department of Chemical Engineering, Jadavpur University.
Topics of research include biotechnology and chemical engineering.

Fabio de Lima graduated in Chemistry from the Universidade Estadual de Mato Grosso do Sul
(2007) and master’s degree in Chemistry from the Universidade Federal de Mato Grosso do Sul (2010).
PhD in Chemistry from the Universidade Federal de Mato Grosso do Sul (2010). He has experience in
Chemistry, acting on the following topics: paste electrode, carbendazim, voltammetry, biosensors and

413
 About the Contributors

linuron. Has experience in Chemistry with emphasis on Physical Chemistry (Electrochemistry), acting
on the following themes: nanostructured metal electrocatalysts for the oxygen reduction reaction (ORR),
enzymatic bioelectrocatalysis (direct charge transfer in graphene, computational chemistry and density
functional theory (DFT).

Apurba Dey is a full time Professor at the Department of Biotechnology, National Institute of Tech-
nology, Durgapur, India. He received his PhD from the Indian Institute of Technology, Delhi. He did
his Mech and BTech studies at Jadavpur University, India.

Nadia Di Paola is a Postdoctoral Research Fellow in Innovation Management in Biotechnologies and

Health. Her research interests cover the areas of management, entrepreneurship, technology venturing,
operations and logistics management.

Kotresha Dupadahalli currently working as a Principal, KSPL Degree College, Hospet, India and
previously worked as a DBT-Post doctoral fellow in Department of Biochemistry, Indian Institute of
Science; as a Post doctoral Fellow at the Institute for Research in Molecular Medicine, Malaysia; and as
a Post doctoral Research Fellow in Dept of Parasitology, Faculty of Medicine, Malaysia.

Sanjay P. Govindwar is Professor and Head of Department of Biochemistry, Shivaji University,

Kolhapur. For more than 10 years, Prof. Jadhav and her group has worked in the area of bioremedia-
tion of textile dyes. Phytoremediation strategies for the textile industry effluent have been successfully
demonstrated at a pilot scale. More than 150 research papers in the area of bioremediation have been
published in various international journals. Currently, his group is attempting to develop a wetland system
for the large scale treatment of textile industry effluent using phytoremediation approach.

Charu Gupta (Gold Medal; ICAR-NET, GATE qualified), Assistant Professor, Amity Institute of
Herbal Research & Studies, Amity University Uttar Pradesh carries with over 8 years of experience in
teaching and research at University level. She has published around 30 research and review papers in
SCI journals, authored 4 books and 70 abstracts in both national and international conferences. During
her tenure at Amity, she has filed several patents and related to the medicinal plants and microbiology.
Her research interests are in probiotics & utilization of agro-industrial wastes for production of value
added products.

Amol Uttam Hivrale is an Assistant Professor in the Department of Biotechnology, Shivaji Univer-
sity, Kolhapur. His Ph.D. research includes the proteomic studies of plant lectins. He received UGC and
Swedish Institute Scholarship at Uppsala University in Sweden during his Ph. D and published seven
research paper during his Ph. D.

Swati Jagdale, M.Pharm, Ph.D, M.B.A studied at University of Pune. She is presently working as
Professor and Head, Department of Pharmaceutics at MAEER’s Maharashtra Institute of Pharmacy,
MIT Campus, Kothrud, Pune, (MS), India. She has 14 years of academic experience. She has expertise
in field of formulation development of pharmaceuticals.

414
About the Contributors

Ruckmani Kandasamy is the Director, Centre for Excellence in Nanobio Translational REsearch
(CENTRE), an autonomous centre under Anna University, Chennai; Professor and Head, Department of
Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli, Tamilnadu. Dr. Ruckmani
has got her Ph.D. & M.Pharm from Jadavpur University, Kolkatta. She is the First lady from Tamil
Nadu to be awarded a Doctorate in Pharmacy. She has been awarded BOYSCAST Fellowship by DST,
Government of India for one year at Airway Disease and Nanomedicine Research Center, College of
Medicine, University of South Florida, USA. Dr. Ruckmani has received a grant of around Rs. 800
lakhs including DST sponsored National Facility for Drug Development. Dr. Ruckmani has won several
awards and recognitions to mention a few – Best Innovation Award 2013 with a cash prize of Rs.50,000
from Anna University, Chennai, Women Achiever Award 2010-2011 by Bharathidasan University,
Tiruchirappalli, Tamilnadu Young Women Scientist Award -2006 by Government of Tamilnadu. Shri
P. K. Das Memorial Best Faculty Award 2010-2011. Served as Officiating Vice chancellor of Anna
University of Technology, Tiruchirappalli for six months, Invited as External examiner for PhD viva
voce at The University of Nottingham Malaysia Campus, Kuala Lumpur, Prof. S. P. Thiagarajan and
Prof. S. Rajarajan Endowment Award 2009 for the outstanding patent filed in the field of bio-medicine
under the prizes and awards of Indian Association of Biomedical Scientists (IABMS). Dr. Ruckmani
has 20 years of teaching & research experience. She has been awarded one US patent, filed three Indian
patents and one PCT application. She has about 95 peer-reviewed publications to her credit, produced 14
PhDs and 12 scholars are under her supervision. Dr. Ruckmani is the regular invited speaker at various
AICTE,UGC,DST,DBT sponsored programmes.

Jatinder Kaur Katnoria is an Assistant Professor, Department of Botanical and Environmental

Sciences, Guru Nanak Dev University, Amritsar Specialization: Envrionmental Genotoxicity, Heavy
Metal Pollution, Air Pollution Monitoring.

Mandeep Kaur is a research scholar doing research on Air pollution.

Gaddad S. M has presently working as a Professor in Dept. of Microbiology, Gulbarga University,

Gulbarga, Karnataka, India. He completed his Ph.D in Gulbarga University, Gulbarga. He guided more
than ten Ph D students and published several papers in national and International Journals.

Aniruddha Mukhopadhayay is the Head and Associate Professor at Department of Environmental

Science, University of Calcutta. Topic of research includes ecotoxicology.

Avinash Kaur Nagpal Specialization: Genotoxicity, Biomonitoring, Plant Tissue Culture, Bioin-
formatics.

Manisha Nanda is presently working as a lecturer in Dept. of Biotechnology, Dolphin (PG) Institute
of Biomedical and Natural Sciences, Dehradun, UK, India. She has completed her Master’s Degree in
Biotechnology from HNB Garhwal University, Srinagar, Uttarakhand. She has also completed her PhD
from same university. Her area of specialization is Bio-remediation and Environmental Biotechnology.

415
 About the Contributors

Pankaj K. Pawar is presently working as an Associate Professor in the Department of Biochemistry,

Shivaji University, Kolhapur. His research is focused on the studies on ameliorative effects of various
herbal formulations on toxicants / pollutants induced aging in Eukaryotic organisms.

Sakthivel Lakshmana Prabu, associated with Anna University since 2009. He has reputed Pharma-
ceutical Industrial experience in quality assurance, validation and formulation development. Currently he
is a teaching faculty member in the Department of Pharmaceutical Technology, Bharathidasan Institute
of Technology, Anna University, Tiruchirappalli. Dr. S. Lakshman Prabu, involved in developing new
analytical methods and new formulations for various pharmaceutical substances. Dr. S. Lakshman Prabu,
is an editorial board member for four international journals and reviewer for eight reputed journals. Dr. S.
Lakshman Prabu, has published 77 technical papers, articles, presented 76 papers in various international
and national conferences, authors 6 book chapters and having 3 patents for his credit.

Dhan Prakash, has 35 years of research experience in life sciences, worked as guest scientist at
GSF-Munich, (GERMANY), Freiburg University, (GERMANY) and Ecole Normale Superieur, Lyon
(FRANCE); guided more than 25 Ph. D. theses, published 145 research papers in SCI journals, 28 book
chapters, 173 abstracts in conferences, filed 35 patents and edited 5 books.

Thirumurugan RathinasabapathyI completed my Ph.D degree in the area of medicinal chemistry

at Birla Institute of Technology and Science (BITS-Pilani), Pilani and presently working as a Lecturer in
the department of Pharmaceutical Chemistry, International Medical University, Kuala Lumpur, Malaysia.
My research experience is gathered from my Post-Doctoral Associate position in the department of Plant
Biology and Pathology, at Rutgers University, NJ, USA under the supervision of Prof. Ilya Raskin. I was
working in the MMV (Medicines for Malaria Venture) project mainly to identify and develop the novel
lead molecules from the natural plant source for the treatment of chloroquine resistant malaria and also
development of novel molecules of Homobrassinolide, a powerful, plant-derived anabolic / ergogenic
agent, with no androgenic side effects; could be used in a range of human muscle wasting disorders,
including those associated with both cancer and AIDS, as well as general aging (Sarcopenia). Currently
our team is working on the discovery of novel molecule for the treatment of MetS.

Niraj R. Rane is working as a Ph. D. student in the Department of Biochemistry, Shivaji University,
Kolhapur. His topic of research covers phytoremediation approaches for the textile industry effluents
and synthetic dyes.

Shankara S. has presently working as a Assistant Professor in Department of Microbiology, Govern-

ment College for Women, Chintamani, Karnataka, India. He completed his Ph.D in Gulbarga University,
Gulbarga.

Ashita Sharma Research Scholar, Genotoxicity, Air Pollution Monitoring, Heavy metals

Parjanya Kumar Shukla is young, inspiring academician and researcher of pharmaceutical Sciences.
He received his M. Pharm in Pharmaceutical Chemistry from department of Pharmaceutical Sciences,
Faculty of Health Sciences, Sam Higginbottom Institute of Agriculture Technology and Sciences, Deemed
University, Allahabad, in 2012 & Pursuing PhD from the same university. He is presently working as

416
About the Contributors

Assistant Professor in Krishnarpit Institute of Pharmacy, Allahabad. He has written 8 articles in various
highly indexed and referred National and International journals, one book and one book chapter in an
edited book. He has presented many papers in several Seminars and conferences.

Rajender Singh is involved in mushroom research and effective management of spent mushroom
substrate to avoid the environmental problems.

Rosanna Spanò is a Postdoctoral Research Fellow in Management Accounting and Innovation. Her
research interests cover the areas of accounting, management accounting and corporate social responsibility.

TNK Suriyaprakash, a proud alumni of Madurai Medical College, Madurai, Tamilnadu and BITS,
Pilani, Rajasthan, was awarded with PhD in Pharmaceutical Technology by the Tamilnadu Dr. MGR
Medical University, Chennai. Published nearly 50 research and review papers in reputed journals, has to
his credit authored 8 book chapters published in Elsevier journals. One of the chapters in InTech, Croatia
has crossed more than 13,000 downloads worldwide, maximum being USA followed by India and Great
Britain. Received grants totalling 29 lakhs from AICTE, MHRD, ISTE and ICMR, carried out many
research projects, conducted many Quality Improvement Programs in Tamilnadu and Kerala. Presently
working as Principal, Al Shifa College of Pharmacy, Perinthalmanna, one of the premier pharmacy
institutions in Kerala, for the past 3 years, after gaining 18 years of experience elsewhere.

Vijayakumar M. H. is working presently in LGC Promochem India Pvt Ltd, Bangalore, Karnataka,
India. He completed his Ph.D in Gulbarga University, Gulbarga. He worked as a Postdoctoral Fellow in
Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore.

Amita Verma presently working as Professor and Head in Department of Pharmaceutical Sciences,
Faculty of Health Sciences, Sam Higginbottom Institute of Agriculture Technology and Sciences, Deemed
University, Allahabad, U.P. She has qualified GATE 2002 and completed B. Pharm. from HNB Garhwal
University, Uttaranchal and also completed M. Pharm. & Ph. D from Hamdard University, New Delhi.
She has received International travel grant by Department of Sciences and Technology, New Delhi,
2015, selected for International training programme for women scientists/technologists sponsored by
DST, New Delhi in partnership with the Indo-US Science and Technology Forum (IUSSTF) and CoACh
International, University of Oregon, Eugene, USA, Aug, 2015, awarded EACS (European AIDS Clinical
society, Belgium) scholarship, 2015, selected Marquis Who’s Who in the world, USA, 2015, received
Bharat Jyoti Award 2015 by IIFS, New Delhi and also awarded Two months Summer Research Fellow-
ship, 2014 by Indian academy of Sciences (IAS) - Indian National Science (INSA)-National Academy
of Science India (NASI). She has published more than 85 research articles in various reputed journals.
She is member of editorial board of six journals and as reviewer of many national/ international journals.
She has guided 5 Ph. D thesis and 9 M. Pharm. thesis.

Roberto Vona Professor in Management. His research interests cover the areas of management,
entrepreneurship, technology venturing, operations and logistics management.

Aisha Zaman is B,Sc in Botany and M.Sc in Environmental Science in Calcutta University. She is
now pursuing her PhD in Dept of Chemical Engineering, Jadavpur University.

417
418

Index

A biological treatment 29, 37, 51, 54-55, 168, 171,

181, 267, 269, 273, 316-317, 319-320, 322
abiotic environment 28, 42 biomagnification 8, 11, 26, 187
acceptable daily intake 26 biomagnification of metals 26
acclimatization 75, 81-82, 88 biomass 26, 35, 37-39, 54, 56-64, 69, 87, 98, 114,
aerobic conditions 37, 142, 144, 153, 320 119-121, 141, 145, 151-153, 159, 161, 163,
agaricus bisporus 162, 164-166, 169, 171 165, 172, 180-181, 184, 187, 191-193, 217-
agricultural resources 215 218, 220, 223-224, 237, 246, 252, 268, 273,
agro-industrial wastes 161, 171, 271 283, 288, 319
Ames Test 96, 279, 282, 290 bioremediation 27, 32-34, 36-38, 40-42, 54, 89, 97-
anaerobic conditions 30, 37-38, 101, 142-143, 145, 105, 113-114, 116-117, 119-126, 141, 143-145,
147, 153, 184, 193, 319, 322 149-154, 158-160, 162, 165-166, 168-172, 178-
anthropogenic 2-4, 6-7, 9, 27, 30, 35, 41-42, 75-76, 180, 182, 184-186, 188, 190-195, 208, 220,
153, 178, 180 226, 236-238, 244-253, 266-267, 269
arabidopsis thaliana 218, 225-226 biosorbents 57-58, 60-62, 64, 66-68, 119
aromatic compounds 120, 153, 181, 184, 198, 204, biosorption capacity 57-58, 63, 269
206, 243-244, 267, 290 biosorption isotherm 65-66
aromatic structures 52, 159, 170 biosorption kinetics 63-64
ATpases 85-86, 96, 104 biotech field 295-296, 299, 301, 307
ATpases metal-transporter 96 biotechnology 34, 36, 39, 101, 105, 124, 161, 178-
azo dyes 55, 169, 266-269, 278-280, 289-290 179, 195-196, 244, 250, 295-296, 300-304,
307, 311
B brassica juncea 217, 223
bacterium bacillus 280, 283, 288-290
bioaccumulation 36-37, 56, 75, 114, 121, 152, 180,
C
187, 246-247 Cation Diffusion Facilitator (CDF) 85-86, 96
bioaugmentation 32, 36, 114, 165-168, 179, 188 chemical compounds 96, 142, 159, 170, 179-180
bioavailability 7, 87, 121, 150, 180, 182, 185-187, chemical precipitation 30, 32, 36, 42, 316, 323-324
191, 193, 196, 224, 245-246 chemical structure 52, 55, 59, 192, 206, 208, 267,
biodegradation 32-34, 36, 38, 40, 42, 51, 54-55, 269
97, 99, 101-105, 113-115, 120-121, 124, 142, chicken manure 159, 163
144-145, 150, 152-154, 158-159, 168, 179-180, chlorinated solvents 181-182, 226-227, 237
184-185, 187-193, 198, 202, 204, 206-208, climatic conditions 250, 323
228, 236, 248-249, 252, 266-268, 278-279, colour photography 159, 170
289-290, 319 co-management 295, 302-304, 307, 311
biological concentration factor 7, 26 competitive advantage 87, 296, 299, 301
biological interventions 236 composting 143-145, 150, 158-159, 162, 194, 301
biological transformation 42, 180, 182, 195-196
Index

D H
decolorization 59, 268, 279-281, 283-290 hazardous pollutants 97, 198
Denaturing Gradient Gel Electrophoresis (DGGE) hazard quotient 14, 26
87, 96, 125 heavy metals 1-9, 11, 14, 16-17, 26-39, 41-42, 61,
detoxifying mechanisms 81, 88, 96 63, 86-87, 98, 103-104, 114, 117, 119-120,
direct blue-14 279-281, 283-288, 290 124, 141-143, 149-152, 154, 160, 170, 178-
179, 182, 184, 186, 191-192, 215-216, 218-
E 219, 223, 237, 243-247, 273, 315, 322-324
hydraulic control 224, 228
ecological pyramid 26 hydroxy benzene 198
ecological variations 295, 300, 307
ecomafias 295, 311 I
effluent 29, 55, 67, 98, 121, 148, 159, 165, 170, 194,
199, 208, 266-267, 278-279, 313, 316-317, immobilized bacteria 267
320-323, 325, 330 industrial effluents 27, 29, 38, 40, 42, 54, 68-69, 98,
efflux pumps 104 159, 195, 267-268
electron acceptors 38, 143, 153, 193 inorganic minerals 28, 42
electrostatic interactions 31, 59
endophytic actinomyces 238-239 L
endophytic bacteria 237-239, 244-245, 248, 251,
253 lentinula edodes 158, 162, 169, 171-172
endophytic fungi 152, 154, 238, 242, 246-247, 250 ligninocellulolytic 158
endophytic microorganisms 236, 243, 251 lignin peroxidase 158, 161, 166, 168-170
enrichment factor 6, 8 liquid chromatography 98, 103, 325
environmental awareness 36, 42 living organisms 1-3, 6, 17, 28, 31, 34, 36, 56, 117,
environmental degradation 27, 51, 295, 300, 307 142, 181, 266
environmental pollutants 99, 103, 113, 116-117,
143, 153, 159-160, 162, 171-172, 193, 236 M
environmental pollution 33, 113, 154, 179, 216, 312, manganese-dependent peroxidase 168, 170
330 metabolic activity 31, 39, 99
environmental protection 2, 9, 153, 180, 202, 274, metabolite 40, 100, 123, 228, 239, 279
330 metal accumulation 117, 121, 149, 152, 219, 221
Environmental Protection Agency (EPA) 2, 9, 153, metallothioneins 40, 86, 96, 117
180, 202, 330 Metallothioneins (MT) 40, 86, 96, 117, 124
metal pollution index 9
F metal resistance 31, 40, 83, 85-86, 88-89, 96, 114,
functional genomics 102, 188 120, 246-247
metal-sequestration 96
G metal toxicity 34, 40, 75-77, 79-82, 84-89, 96, 191,
246
gel electrophoresis 87, 96, 102-103, 125 metal-transporter 96
genetically engineered microorganisms 36-37, 61, microbial community 75-76, 88, 98-99, 102, 125,
103, 113-114, 122-124, 190, 193 187-188, 191, 243
genetically modified 61, 68, 100, 114, 117, 119-120, microbial fuel cells 193
126, 141, 149-150, 225, 253 microbial metal-sequestration 96
genetic engineering 39, 61, 99-100, 105, 114, 116, microbial organisms 37, 142, 153
120, 124-126, 141, 149-150, 190, 217-218, 250 microbial response 75, 81, 89
genetic modifications 61, 69, 100, 116

419
 Index

microorganisms 27, 31-33, 35-40, 42, 51, 54-55, pleurotus ostreatus 163, 168, 171
59, 61, 68, 75-77, 79, 81-83, 85-89, 96-104, polyaromatic hydrocarbons 100, 162, 165, 169, 273
113-114, 117, 119-120, 122-125, 142-147, 149, polychlorinated biphenyls 143, 179-180, 226-227
153-154, 160, 162, 165, 178-182, 185, 187, polycyclic aromatic hydrocarbons 168, 179, 216,
190-196, 198-199, 201-202, 207, 220, 226, 226-227
236-237, 242-243, 245, 247-249, 251, 253, polymer 61, 152, 181
266-269, 271, 278, 280, 288-289, 312, 318- proteomics 97, 99, 101-105, 125, 188
319, 330 pyrolysis 141, 145-146, 154
molecular biology 33, 37, 98-99, 105, 114, 116, 242,
303 R
molecular genetics 125, 186, 195
mushroom cultivation 158-159, 161-163, 171-172 redox transformation 82-83, 96
mushrooms 152, 154, 158-159, 161-165, 168-171 reverse osmosis 30, 32, 36, 42, 53, 322-323
mushroom substrate 152, 158-159, 161, 163-165, rhizopus arrhizus 58-59, 121
168, 170-172 rhizoremediation 180, 192, 237

N S
natural materials 113 secondary treatment 317-318, 321, 330
natural recycling 170 social/environmental sustainability 297, 308
next generation sequencing 99 socio-economic development 27, 51
soil pollution 29-31, 215
O solar energy 220, 321
Spent Mushroom Substrate (SMS) 152, 158-159,
organic compounds 29, 35, 37, 54-55, 168, 181, 187, 161, 163-165, 168, 170-172
193, 198, 221, 226, 243-244, 250, 253 supply chain 302-303, 307
organic material 144, 312, 330 surface methodology 267, 271
organic matter 97, 142, 144-145, 148, 151, 185, 217, sustainable development 51, 295-296, 300, 307, 311
224, 246, 312, 317-321, 327, 329 synthetic chemical compounds 159, 170
organic waste 141, 150, 167, 204, 250 synthetic compounds 113
organometals 76, 87 synthetic dyes 52, 68, 159, 169-172, 267, 279
osmotic pressure 31, 323
T
P
tertiary treatment 318, 322, 325
pathogenic microorganisms 312, 330 toxicants 41, 56, 119
phenol 40, 102, 150-151, 153, 169, 181, 184, 193, toxicity 2-3, 9, 17, 31, 34, 37-38, 40-41, 51, 53, 67,
198-202, 204-208 75-77, 79-89, 96, 117, 150, 154, 179, 185-187,
phyto 215-216 191, 199, 201, 216, 243-244, 246, 273
phytodegradation 215, 226, 228, 237, 250 toxic metals 2-4, 6, 9, 16-17, 31, 35, 40-41, 81, 83-
phytoextraction 192, 215, 218-221, 223, 226, 228, 85, 87, 120, 216, 218, 222-225
237 toxic waste 298, 311
phytofiltration 215, 222 toxins 61, 123-124, 142, 216, 219, 236, 273, 278
phyto-remediation 152 transfer factor 7, 26
Phyto-Remediation 215
phytostabilization 215, 224-225, 228, 237 U
Phytostabilization 223-224
phytovolatilization 215, 225-226, 228, 237 ubiquitous nature 181
plasma incineration 141, 145, 154

420
Index

V wastewater 8, 29-33, 35-40, 42, 51-53, 56, 58-59,

62-63, 65, 68, 120, 147, 151, 158-160, 162,
vehicle emission 30-31 170, 181, 184, 193, 202, 207, 216, 222, 266-
vermicomposting 143, 150 270, 273-274, 312-318, 320, 322-330
vermiculture 150, 154 water-carried wastes 312

W X
waste management 27, 29, 141, 164, 168, 236, 300- xenobiotics 97, 99, 120, 150, 178, 187, 189, 193,
304, 307 216, 243-244, 249, 268, 273

421