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Bioremediation

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140
Bioremediation
A Promising Xenobiotics Cleanup Technique
Thandapani Gomathi, M. Saranya, E. Radha, K. Vijayalakshmi,
P. Supriya Prasad and P.N. Sudha
PG and Research of Department of Chemistry, D.K.M. College for Women, Vellore, Tamil Nadu, India
140.1 ­Introduction
Ecosystems are frequently confronted with
­natural environmental variations and distur-
bances over time and geographic space. The
enormous industrial expansion that occurred
during the 19th and 20th centuries accounted
for various economic and technological
advances, profoundly changing the course of
humanity. The technological innovations
across different processes and products were
made with no proper diligence to the environ-
ment and involved excessive extraction, dis-
semination and disposal of materials into
the environment without proper precautions.
Not only industrialization, but also wars, and
intensive use of large-scale heavy metals and
synthetic xenobiotics, also led to many envi-
ronmental problems (Bayat et  al., 2015;
Akhtar et  al., 2003). There is a belief that
bioremediation will be a successful and eco-
nomic method to mold the environment as
pollution free for future generations. Thus this
chapter deals with the extensive importance
of bioremediation, its types, advantages, limi-
tations, and its applications.
Encyclopedia of Marine Biotechnology: Five Volume Set, First Edition. Edited by Se-Kwon Kim.
© 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.
3139
140.1.1  The Current Environment
The quality of life on Earth is linked inextrica-
bly to the overall quality of the environment.
In early times, we believed that we had an
unlimited abundance of land and resources;
today, however, the resources of the world
show, to greater or lesser degree, our careless-
ness and negligence in using them. Rapid
industrialization and urbanization over the
past decades have resulted in contamination of
all the components of the environment, that is
the air, the water, the soils, and even our food.
Enormous quantities of organic and inorganic
compounds are released into the environment
each year as a result of human activities. In
some cases, these releases are deliberate and
well regulated (e.g. industrial emissions) while
in other cases they are accidental (e.g. chemi-
cal or oil spills). Many of these compounds are
both toxic and persistent in terrestrial and
aquatic environments.
Groundwater is one of the most vital sources
of drinking water on earth. This major water
resource has been threatened by many anthro-
pogenic organic compounds. Water and soil
3140 140  Bioremediation: A Promising Xenobiotics Cleanup Technique
contamination resulting from industrial and
agricultural activities has caused concern in
recent years (Ha et  al., 2014). Various pollut-
ants entering the soil or water pose a huge
threat to human health and natural ecosystem
(Gong et al., 2009; Hu et al., 2013; Kavamura
and Esposito, 2010; Tang et al., 2014; Udeigwe
et al., 2011; Xu et al., 2012; Zeng et al., 2013a;
Zeng et al., 2013b). Main soil pollutants include
polycyclic aromatic hydrocarbons (PAHs),
petroleum and related products (Belanger,
2010; Chatterjee and Lefcovitch, 2010), pesti-
cides (Moreno-Medina et  al., 2014; Mesnage
et  al., 2014; Roberts and Karr, 2012), chloro-
phenols, and heavy metals (Herrera et  al.,
2008; Wojcieszyńska et  al., 2008; Liu et  al.,
2007). Over the years the indiscriminate use of
synthetic chemicals has released several such
organic contaminants which are recalcitrant to
natural degradation and may also turn hazard-
ous or toxic. Even if released into the sewers,
the compounds come back to the soil system
and the food chain through contaminated
sludge. Nevertheless, the next question is why
we should be worried about such pollutants.
140.1.2  Contaminants and their
Hazards
Over 347 000 chemicals are registered and regu-
lated via national and international authorities
(CHEMLIST), new chemicals enter the market
continuously, and the global volume of produc-
tion of chemicals is growing (CEFIC, 2016).
Many of these chemicals and their transforma-
tion products enter the aqueous environment
during their life cycle (Schwarzenbach et  al.,
2006). The contamination of the environment
is from petroleum products, pharmaceutical
compounds, chloro- and nitrophenols and their
derivatives, PAHs, organic dyes, pesticides and
heavy metals is a serious problem (Mohamed
et  al., 2016; Rodgers-Vieira et  al., 2015;
Smułek  et  al., 2015; Wasilkowski et  al., 2014;
Wojcieszyńska et al., 2014; Greń et al., 2010).
In the last decades, efforts have been made to
raise knowledge about the adverse effects of
contaminants such as legacy pesticides, poly-
chlorinated biphenyls (PCBs), PAHs, mer-
cury (Hg), arsenic (As), or cadmium (Cd) on
humans and animals worldwide, and new
methods are developed to analyze these com-
pounds in very different matrices and various
environmental media (Luzardo et  al., 2013;
Sharma et  al., 2014). Thus, numerous studies
have revealed that these toxic compounds,
individually and in combination, may contrib-
ute to the development of severe health prob-
lems such as cancer, immune suppression, or
genotoxic effects in humans, even with long-
term low-dose exposure (Bergman et al., 2012;
Jarvis et  al., 2014; WHO, 2003), and many of
them have demonstrated endocrine-disrupting
effects in both animals and humans (Camacho
et al., 2014; Kortenkamp et al., 2011).
Farmers make indiscriminate use of pesti-
cides to control pests and diseases (Okoffo
et  al., 2016), resulting in many health issues
among farmers and farm workers. Nearly 0.3
million farmers die each year; of these, 99% are
from developing countries (WHO, 2009).
Different health problems are reported due to
pesticides which include headache, stomach
pain, vomiting, skin allergies, asthma, etc.
(Khan et al., 2015; Bakhsh et al., 2016). In fact,
the use of organochlorine pesticides (OCPs)
and PCBs are now banned in most developed
countries, but they are still widespread in the
environment (Almeida-González et  al., 2012;
Kakuschke et  al., 2010; Luzardo et  al., 2014).
Although there are different routes of exposure
for humans to pollutants, it has been estab-
lished that ingestion of food contributes more
than 90% of total human exposure, and that
the fatty fraction of food represents the main
entrance to the human body (Darnerud et al.,
2006; Vazquez et al., 2015).
Heavy metals containing industrial effluent
cause health hazards to plants, animals,
aquatic life, and humans, increasing pressures
on the flora and fauna (Robin et  al., 2012).
Tannery effluent is highly polluted because it
contains imbalance-suspended solids, nitrogen,
conductivity, sulfate, sulfide and chromium,

copper, cadmium and manganese, biological
oxygen demand (BOD) and chemical oxy-
gen demand (COD) (Mondal et  al., 2005;
Zahid et  al., 2006). Chrome powder and
chrome liquor are applied in the tanning
industry, and are highly toxic heavy metals
(Cr6+) which cause water pollution (Sing,
1994), where a lot of (> 170 000 tons) chro-
mium wastes are released to the surroundings
(Kamaludeen et  al., 2003). They cause health
hazards since they can easily enter biological
cell membranes (Chaudhary et  al., 2003).
Tanned skin-cut wastes (SCW) which are
used to produce feeds and fertilizers, are the
direct phenomenon of chromium toxicity
(Rafiqullah et  al., 2008). Lead and cadmium
which are major contaminants found in the
environment, are extremely poisonous to
human(s), animals, plants, and microbes and
can damage cell membranes, alter particular-
ity of enzymes, and destroy the structure of
DNA. This toxicity is created by the displace-
ment of essential metals from their native
binding sites or ligand interactions (Olaniran
et al., 2013).
140.1.3  Need for Bioremediation
As far as the solution to this problem is con-
cerned, there can be two options: prevention or
cure. As the common saying insists, “preven-
tion is always better than cure,” and the former
should always be practiced well. Even if the
switching to “cleaner processes” (i.e. preven-
tion) is by and large accepted, we still face the
toxicity and environmental persistence of
xenobiotic compounds in the transitional
phase. Therefore, there is a need to develop a
wide variety of physicochemical and biological
techniques that can remediate the hazardous
contaminants from the environment without
causing further damage. The conventional
techniques of incineration/landfills, etc., basi-
cally transfer pollutants from one medium to
another, are expensive, and energy demand-
ing. Thus the challenge today is to develop
technologies that consume fewer resources,
140.2 ­Bioremediatio 3141
incorporate recycling and reuse of components
thereby reducing the production of wastes
while maintaining/improving the efficiency
(Fulekar, 2009).
The world requires a higher production of
food and an increase of industrial activity in
order to satisfy basic needs of the population.
A consequence of these anthropogenic activi-
ties is that areas contaminated with toxic
organic and inorganic compounds have been
detected worldwide. This simultaneous con-
tamination, known as co-contamination, rep-
resents the real current challenge of grey
biotechnology. Bioremediation which is an
attractive and potential alternative for treat-
ment of contaminated sites, paved a way for all
the needs. Bioremediation of organic com-
pounds and heavy metals has been shown to be
successful, although at the moment each pro-
cess has generally been performed singly.
Certainly, a multifunctional biological process
is needed for bioremediation of co-contami-
nated sites.
Hence bioremediation requires the develop-
ment of our knowledge of microbial popula-
tions, their interactions with the natural
environment and contaminants, the increase
of their genetic capabilities to degrade contam-
inants, and long-term field studies of new eco-
nomical bioremediation techniques that can
increase the potential for significant advances.
There is no doubt that bioremediation is
needed and can lead to the protection and
preservation of natural resources we have used
for the next generation. Let us now examine in
detail what exactly the term bioremediation
implies and the host of technologies that this
term encompasses.
140.2 ­Bioremediation
Bioremediation comes from two words, bios,
meaning life, and remediate, meaning to deci-
pher an issue. The scientific world can boast
that science was behind many of the most
important events in the history of humanity.
3142 140  Bioremediation: A Promising Xenobiotics Cleanup Technique
The application of scientific principles to the
processing of materials by biological agents to
generate a better quality of life is generally
known as biotechnology. Environmental bio-
technology, usually bioremediation, may be
defined as the “use of ­living organisms to clean
up pollutants from soil, water, or wastewater”
(EPA, 2016). Overall, bioremediation may be
considered as a “treatment that uses naturally
occurring organisms to transform hazard-
ous  substances into less toxic substances.”
Bioremediation depends on the presence of
specific microorganisms, i.e. yeast, fungi, or
bacteria in the correct amounts and combina-
tion and in the appropriate environmental
conditions (Strong and Burgess, 2008).
140.2.1  Principles of Bioremediation
The most important principle of bioremedia-
tion is that microorganisms (mainly bacteria)
can be used to destroy hazardous contami-
nants or transform them into less harmful
forms. Although existing microorganisms can
detoxify a vast array of contaminants, some
compounds are more easily degraded than oth-
ers. The microorganisms act against the con-
taminants only when they have access to a
variety of materials – compounds to help them
generate energy and nutrients to build more
cells. More often, bioremediation requires the
construction of engineered systems to supply
microbe-stimulating materials  –  a process
called engineered bioremediation. Engineered
bioremediation relies on accelerating the
desired biodegradation reactions by encourag-
ing the growth of more organisms, as well as
by optimizing the environment in which the
organisms must carry out the detoxification
reactions.
The suitability of a site for bioremediation
depends not only on the contaminant’s biodeg-
radability, but also on the site’s geological and
chemical characteristics. When deciding
whether a site is suitable for bioremediation, it
is important to realize that no single set of site
characteristics will favor bioremediation of
all contaminants. For example, certain com-
pounds can only be degraded when oxygen
is  absent, but destruction of others requires
that oxygen be present. The keys for site-
characteristic bioremediation and engineered
bioremediations are consistent ground water
flow throughout the seasons; the presence of
minerals that can prevent pH changes; high
concentrations of either oxygen, nitrate, sul-
fate, or ferric iron; permeability of the subsur-
face to fluids; uniformity of the subsurface;
and relatively low (less than 10 000 mg kg–1
solids) residual concentrations of nonaqueous-
phase contaminants. In addition, one must
consider how the bioremediation system may
perform under variable and not perfectly
known conditions. A scheme that works opti-
mally under specific conditions but poorly
­otherwise may be inappropriate for in situ
bioremediation.
140.2.2  Factors of Bioremediation
The control for bioremediation processes is a
complex system of many factors. These fac-
tors include the existence of a microbial pop-
ulation capable of degrading the pollutants,
the availability of contaminants to the micro-
bial population, chemical composition, water
activity, ratio of carbon to nitrogen, minerals,
surfactant, pH, moisture, sources of nitrogen,
particle size, and amount of inoculum, and
antimicrobial agents; the presence of interac-
tions between microorganisms are consid-
ered as chemical, physical, and biological
factors (Eira, 2003). The main environmental
factors encompass temperature, humidity,
luminosity, and air composition of the sur-
rounding substrate, such as concentration of
oxygen and carbon dioxide (AMGA, 2004).
For the treatment of polluted sites, support
materials need to present several properties,
such as being non-biodegradable, non-toxic,
and non-polluting, as well as presenting
high mechanical and chemical stability, high

Table 140.1  Factors affecting bioremediation.
Intrinsic factors Extrinsic factors
●● Composition of substrates ○○ Temperature
●● Sources of nitrogen ○○ Humidity
●● Ratio of carbon to ○○ Luminosity
nitrogen (C : N)
○○ Air composition
pH
○○ Envase
●●
●● Moisture
●● Minerals
●● Particle size
●● Levels of spawning
●● Surfactant
­ iffusivity, minimum attachment with other
d of mechanical treatments;
organisms, and preferably low cost (Martins
et al., 2013).
Intrinsic and extrinsic factors which affect
the bioremediation process are given in
Table 140.1.
Nitrogen source is a major factor (Singh
et  al., 2008) that affects bioremediation.
Nitrogen is important in protein, nucleic acid,
purine, pyrimidine, and polysaccharide syn-
thesis (Drozdowski et al., 2010; Abdullah et al.,
2015), constituents of the cell wall of  many
fungi, which are composed of b (1–4)-linked
unit of N-acetylglucosamine (Miles and
Chang, 1997) and may be added in the form of
ammonium nitrate or organic nitrogen (Chang
and Miles, 2004; Gil-Ramírez et al., 2013). It is
generally accepted that pH is a predominant
variable in bioremediation efficiency of PAHs
and heavy metals. Microorganisms will be
affected by pH since the optimal pH for differ-
ent species is varying. Moreover, pH has
impacts on the redox and solubility of heavy
metals (Brito et  al., 2015; Bamforth and
Singleton, 2005). For most fungi, the wide
humidity range is 20–70% (Pandey et al., 2001).
According to Chang and Miles (2004) and Li
et al. (2015), the appropriate humidity during
the darkened spawn-running and mycelia
stimulation should encompass a range between
60–75% and 85–97%, respectively, in the
140.2 ­Bioremediatio 3143
e­ nvironment, enabling a satisfactory growth of
Pleurotus spp. High humidity is favorable for
pining and fruiting (Pandey et al., 2008).
140.2.3  Advantages of Bioremediation
Bioremediation is eco-friendly, non-invasive,
cheaper than conventional methods, and is a
permanent solution that can end with degra-
dation or transformation of environmental
contaminants into harmless or less-toxic forms
(Garbisu and Alkorta, 2003; Perelo, 2010, Kulik
et al., 2006; Xu and Lu, 2010):
●● In situ, passive, solar-driven costs 10 to 20%
●● Transfer is faster than natural attenuation;
●● High public acceptance;
●● Fewer air and water emissions;
●● Generate less secondary wastes;
●● Soils remain in place and are usable follow-
ing treatment;
●● Phytovolatilized contaminants could be
transformed to less-toxic forms (e.g. elemen-
tal mercury and dimethyl selenites);
●● Phytovolatilization accelerates degradation
processes;
●● Phytostabilization circumvents the removal
of soil with low cost and is less disruptive
than other remedial technologies;
●● Revegetation enhances ecosystem restoration.
140.2.4  Limitations of Bioremediation
Bioremediation efficiency depends on environ-
mental conditions which permit optimal
microbial growth and activity; it involves the
manipulation of environmental conditions to
enhance microbial growth and faster degrada-
tion. There are several afactors that can lower
the effectiveness of traditional bioremediation
approaches. The bioremediation process is
highly dependent on specific environmental
factors; temperature, pH, moisture content,
presence of electron acceptors, local microbial
flora, contaminant type, and other such param-
3144 140  Bioremediation: A Promising Xenobiotics Cleanup Technique

eters require specific biodegradation tech- amount of mineral nutrients and also requires
niques to be developed for specific treatment temperature on which maximum microbes can
sites. survive i.e. 20°C to 30°C. Besides these, some
Some common limitations to biodegradation other factors affect the bioremediation such as
are: solubility of waste, nature and chemical com-
position of waste, oxidation–reduction poten-
●● Shallow soils, streams, and groundwater;
tial of waste, and microbial interaction with
●● High concentrations of hazardous materials
this. Hence the researchers should research
can be toxic to plants;
genetically different type of microbes which
●● Mass transfer limitations associated with
can also work on slightly adverse condition.
other biotreatments;
Therefore, bioremediation is still considered as
●● Slower than mechanical treatments;
a developing technology to regulate the day-to-
●● Only effective for moderately hydrophobic
day environmental problems faced by humans
contaminants;
residing in an area.
●● Toxicity and bioavailability of degradation
products is not known;
Contaminants may be mobilized into the
●●
140.3 ­Environmental Factors
groundwater;
Potential for contaminants to enter food
A disturbance is any process that removes bio-
●●

chain through animal consumption;

mass from a community, such as fire, flood,
Unfamiliar to many regulators;
drought, or predation. Disturbances occur over
●●

Biological processes are often highly spe-

vastly different ranges in terms of magnitudes
●●

cific. For instance, anaerobic bacteria used

as well as distances and time periods (Levin,
for bioremediation of polychlorinated biphe-
1992) and are both the cause and product of
nyls in river sediments, dechlorination of
natural fluctuations in death rates, species
the solvent trichloroethylene. The white rot
assemblages, and biomass densities within an
fungus Phanaerochaete chrysosporium have
ecological community.
the ability to degrade an extremely diverse
range of persistent or toxic environmental
pollutants; 140.3.1 Nutrients
Research is needed to develop and engineer
Although the microorganisms are present in
●●

bioremediation technologies that are appro-

contaminated soil, they cannot necessarily be
priate for sites with complex mixtures of
there in the numbers required for bioremedia-
contaminants that are not evenly dispersed
tion of the site. The proper and adequate sup-
in the environment;
ply of nutrients is essential for any organism’s
Bioremediation often takes longer than
growth and survival. Microalgae require three
●●

other treatment options, such as excavation

major constituents, carbon, nitrogen, and
and removal of soil or incineration;
phosphorus, for their growth. Their growth
Regulatory uncertainty remains regarding
and activity must be stimulated. Biostimulation
●●

acceptable performance criteria for bioreme-

usually involves the addition of nutrients and
diation and there are no acceptable end-
oxygen to help indigenous microorganisms.
points for bioremediation treatments
These nutrients are the basic building blocks
There are some concerns that the products
of life and allow microbes to create the neces-
●●

of biodegradation may be more persistent or

toxic than the parent compound.
For proper growth of microorganism it
requires favorable pH condition and sufficient
sary enzymes to break down the contaminants.
All of them will need nitrogen, phosphorus,
and carbon. Carbon is the most basic element of
living forms and is needed in greater quantities

than other elements. Carbon is supplemented
as bicarbonates. In addition to hydrogen, oxy-
gen, and nitrogen it constitutes about 95% of
the weight of cells. The nitrogen is provided in
the form of nitrates and also in soluble form
like ammonia and urea. Some microalgal spe-
cies also have the ability to assimilate atmos-
pheric nitrogen in the surroundings (Hsieh
and Wu, 2009; Zhu et  al., 2014). Similarly,
phosphorus is another major nutrient in the
growth of microalgae since it forms the con-
stituents of energy transduction and also
nucleic acids.
Phosphorus and sulfur contribute with 70%
of the remainders. The nutritional require-
ment of carbon to nitrogen ratio is 10 : 1 and
carbon to phosphorus is 30 : 1. A lower amount
of phosphorus was found to cause lipid accu-
mulation in microalgae (Liang et  al., 2013;
Rodolfi et al., 2009). In addition to these major
constituents, algae also require some minor
constituents such as iron, magnesium, cal-
cium, potassium, chlorine, and sodium and
trace elements such as boron, copper, zinc,
molybdenum, cobalt, and selenium (Martin-
Jezequel et al., 2000).
140.3.2  Environmental Requirements
Environmental factors play a crucial role in
the  growth of microorganisms. They highly
depend on the light and nutrients which
enhance the process of photosynthesis, thereby
producing food and synthesizing essential
compounds for their survival. Microbial
growth and activity are readily affected by pH,
temperature, and moisture (Walker, 1954).
Temperature is an important environmental
condition that affects the growth rate of micro-
organisms, cell size, biochemical composition,
and nutrient requirements. The temperature
should be neither lower nor higher than the
optimal level (Renaud et al., 2002). Temperature
affects biochemical reaction rates, and the rates
of many of them double for each 10°C rise in
temperature. Above a certain temperature,
however, the cells die. Plastic covering can be
140.4 ­Types of Bioremediatio 3145
used to enhance solar warming in late spring,
summer, and autumn.
Although microorganisms have been also
isolated in extreme conditions, most of them
grow optimally over a narrow range, so it is
important to achieve optimal conditions. pH is
one of the important parameters that deter-
mines the solubility of carbon dioxide and
essential nutrients which determine the
growth factor of microorganisms (Hansen,
2002; Gensemer et al., 1993). At higher pH the
availability of carbon will be reduced since
there will be no free carbons but the carbon-
ates (Rotatore and Colman, 1991). At alkaline
pH, the flexibility of mother cells increases,
causing a delayed rupture and liberation of
daughter cells, thereby increasing the time
required for the cell cycle (Guckert et al., 1992).
If the soil has too much acid it is possible to
rinse the pH by adding lime.
Available water is essential for all living
organisms and irrigation is needed to achieve
the optimal moisture level. Soil structure con-
trols the effective delivery of air, water, and
nutrients. To improve soil structure, materials
such as gypsum or organic matter can be
applied. Low soil permeability can impede
movement of water, nutrients, and oxygen;
hence, soils with low permeability may not be
appropriate for in situ cleanup techniques.
140.4 ­Types of Bioremediation
Biomremediation can be undertaken via two
different modes: in situ and ex situ.
140.4.1  In Situ Bioremediation/
Passive Bioremediation
In situ bioremediation can be described as the
process whereby organic pollutants are biologi-
cally degraded under natural conditions to
either carbon dioxide and water or an attenu-
ated transformation product. It is a low-cost,
low-maintenance, environmentally friendly,
and sustainable approach for the cleanup of
3146 140  Bioremediation: A Promising Xenobiotics Cleanup Technique
polluted sites. When remediation is performed
in situ soil is not excavated, leading to impor-
tant cost savings due to the elimination of
costs of excavation and transportation of the
contaminated soil to the treatment facilities
(Saadoun, 2013). In situ bioremediation also
presents some limitations: it is not suitable for
all soils, complete degradation is difficult to
achieve, and natural conditions (temperature)
are difficult to control (Silion and Cordoneanu,
2003). In situ bioremediation under anaerobic
conditions may also be enhanced by the provi-
sion of electron acceptors, such as nitrate or
sulfate (Sherwood et  al., 1995; Freedman
et al., 1995).
140.4.2  Ex Situ Bioremediation/
Off-site Bioremediation
Ex situ bioremediation techniques are usually
aerobic and involve the excavation or removal
of contaminated soil from ground or sediments
using solid or slurry-phase systems. During the
ex situ processes the contaminated medium is
excavated or extracted and moved to the pro-
cess location. This process requires excavation
of contaminated soil or pumping of groundwa-
ter to facilitate microbial degradation. This
technique has more disadvantages than advan-
tages. Liquids can be cleaned in constructed
wetlands while semi-solid or solid wastes can
be cleaned in slurry bioreactors. Solid contami-
nations are biodegraded through land farming,
composting, and biopiles (Xu and Lu, 2010;
Tomei and Daugulis, 2013).
140.5 ­Bioremediation
Techniques
140.5.1 Bioaugmentation
Bioaugmentation is the injection of native or
non-native microbes with specific catabolic
abilities into the contaminated environment in
order to supplement the indigenous population
and to speed up or enable the degradation of
pollutants. In instances in which a competent
degradative population either does not exist or
in which the time needed for adaptation/accli-
mation may be too long, microbial inoculants
may be added to promote and/or enhance
bioremediation rates (Singleton, 1994; Bouwer
and Zehnder, 1993; Liu and Suflita, 1993). This
technique is referred to as bioaugmentation
and may involve the addition of natural isolates
or genetically engineered microorganisms
(GEMS). Concern about the potential negative
impacts of release of GEMS to the environment
has resulted in strict international control, with
GEM augmentation studies consequently being
confined to contained laboratory-based biore-
actor applications.
The efficacy of bioaugmentation is a subject
of discussion where both positive and negative
results have been reported. Bioaugmentation
has proven successful for remediation of PAHs
in sediments with poor or lacking intrinsic deg-
radation potential (Major et  al., 2002; Juhasz
and Naidu, 2000), while other studies demon-
strated that bioaugmentation did not enhance
biodegradation significantly compared to natu-
ral attenuation (Tam and Wong, 2008).
One of the main problems in applying bio-
augmentation is to ensure the survival and
activity of the introduced organisms in the
environment. Bioaugmentation can be inhib-
ited by a variety of factors including pH and
redox, the presence of toxic contaminants,
concentration and bioavailability of contami-
nants, or the absence of key co-substrates. But
the key factor to successful bioaugmentation
is the selection of the appropriate bacterial
strain for this purpose. When selecting the
strain for augmentation purposes, the kind of
microbial communities present in the source
habitat should be considered (Thompson
et al., 2005).
140.5.2 Bioreactors
The term bioreactor refers to a vessel where
the  biological degradation of contaminants is
performed in fully controlled conditions, i.e.

parameters such as temperature, pH, aeration,
and stirring rates are known and controlled.
Bioreactor is the treatment of contaminated
soil or groundwater in a controlled environ-
ment to optimize degradation, such as an in
situ bioreactor landfill or biological permeable
reactive barrier (biobarrier) or an ex situ batch-
or continuous-feed reactor. Organisms grow-
ing in bioreactors may be submerged in a liquid
medium or may be attached to the surface of a
solid medium and suspended or immobilized.
Immobilization is a general term describing a
wide variety of cell or particle attachments
or  entrapments (IUPAC, Compendum of
Chemical Terminology, 2006) It can be applied
to basically all types of biocatalysis including
enzymes, cellular organelles, animal, and plant
cells (Lopez et  al., 1997). Immobilization is
useful for continuously operated processes,
since the organisms will not be removed with
the reactor effluent, but is limited in scale
because the microbes are only present on the
surfaces of the vessel. Bioreactors have also
been widely applied for treatment of VOC-
contaminated gases (such as monoaromatic
hydrocarbons) (Pedersen and Arvin, 1995;
Bielefeldt and Stensel, 1999; Lu et al., 2002).
140.5.3 Bioventing
Bioventing is a promising technology that
encourages the natural in situ biodegradation
of any aerobically degradable compounds in
soil by providing oxygen to indigenous soil
microorganisms. Bioventing combines soil
venting, which removes volatile components
from the soil, with bioremediation, which uses
the oxygen from the vented air for biodegrada-
tion of the remaining organic contaminants.
The volatilization of low molecular weight,
high vapour pressure compounds can be maxi-
mized using a greater volume of air or short
travel distances between injection and extrac-
tion wells (Dupont et al., 1991). The bioventing
process can be maximized by using lower air
flow rates, longer flow paths, and optimizing
conditions for microbial growth through the
140.5 ­Bioremediation Technique 3147
addition of moisture and inorganic nutrients.
It works for simple hydrocarbons and can be
used where the contamination is deep under
the surface (Tao and Mancl, 2013).
140.5.4 Composting
Composting is a process of piling contami-
nated soil organic substances such as manure
or agricultural wastes. The added organic
material supports the development of a rich
microbial population and elevates the temper-
ature of the pile. Stimulation of microbial
growth with added nutrients results in effec-
tive biodegradation in a relatively short period
of time. Composting involves mixing the con-
taminated soil with a bulking agent such as
straw, hay, or corncobs to make it easier to
deliver the optimum levels of air and water to
the microorganisms. Mechanically agitated
composting involves the placement of the con-
taminated soil in treatment vessels where it is
mixed to achieve aeration. In window com-
posting, the soil is placed in long piles knows
as windows and periodically mixed by tractors
(Cunningham and Philip, 2000). Compost
remediation is known to have faster cleanup
results since cleanup can be estimated in terms
of weeks instead of months.
140.5.5  Land Farming
Land farming involves the spreading of con-
taminated soil over an area and either applying
specialized bacteria or allowing indigenous
bacteria to metabolize the contaminants
(Glenn, 1998). Land farming is a method in
which contaminated soil is spread over a pre-
pared bed along with some fertilizers and occa-
sionally rotated. It stimulates the activity of
bacteria and enhances the degradation of oil.
The criteria for determining proper site loca-
tion includes a minimum separation distance
of three feet between the ground surface and
the seasonable high groundwater table, and
the slope of the land does not exceed 8% (Tao
and Mancl, 2013).
3148 140  Bioremediation: A Promising Xenobiotics Cleanup Technique
140.5.6 Biopiling
Biopiling is a hybrid of land farming and com-
posting. Essentially, engineered cells are con-
structed as aerated, composted piles. Typically
used for treatment of surface contamination
with petroleum hydrocarbons, biopiles 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 aero-
bic and anaerobic microorganisms (U.S. EPA
handbook).
140.5.7 Biostimulation
Biostimulation attempts to increase the
indigenous microorganisms and accelerate the
natural biodegradation rates by improving
nutrients and the bioavailability of contami-
nants in cold environments. It involves the
management of a naturally occurring micro-
bial population to monitor or provide an envi-
ronment that optimizes the growth and activity
of microbes. Methods of biostmulation include
biorenting, air sparging, nutrient addition, and
oxygenation (Riggle, 1997). Additives are usu-
ally added to the subsurface through injection
wells. Subsurface characteristics such as
groundwater velocity, hydraulic conductivity
of the subsurface, and lithology of the subsur-
face are important in developing a biostimula-
tion system (Vidali, 2001).
If the correct, naturally occurring microbes
are present in sufficient numbers and types,
microorganisms can break down the waste
effectively. The most widely used technique is
to add inorganic nutrients or oleophilic fertiliz-
ers, which reportedly has a positive effect for oil
decontamination in cold ecosystems (Allard
and Neilson, 1997). Biostimulation is popularly
believed to stimulate hydrocarbon-degrading
biomass and further influence the relative com-
position of microbial communities. Studies
have been conducted on the biodegradation of
oil in the cold Arctic (Pritchard and Costa,
1991) and Antarctic (Kerry, 1993), all reporting
a favorable effect of nutrient addition when
aeration was provided.
140.6 ­Organisms Involved
in Bioremediation
Bioremediation technology mainly involves
the stimulation of microorganisms, i.e. yeast,
fungi, or bacteria to rapidly degrade hazardous
organic pollutants to environmentally safe lev-
els in soils, sediments, substances, materials,
and groundwater (Strong and Burgess, 2008).
The effectiveness of bioremediation was sig-
nificantly determined from the interactions of
microbes with the environment and pollutants
(Tiedje, 1993) and those microbes can be either
naturally present in the site of bioremediation
or isolated from other sites and inoculated arti-
ficially (Vidali, 2001). In recent years, the
enhancement of microbial degradation as a
means of bringing about the in situ cleanup of
contaminated soils has spurred much research
(Jean-Marc Bollag et al., 1994). In the bioreme-
diation process, the enhancement of natural
detoxification of contaminated environments
has been achieved by the stimulation of the
activity of indigenous microorganisms by the
addition of nutrients, regulation of redox con-
ditions, optimization of pH conditions, etc.
Some other approaches that are still in early
stages of testing include (a) inoculation of the
sites with microorganisms of specific biotrans-
forming abilities; (b) application of immobi-
lized enzymes; and (c) use of plants to contain
or transform pollutants (phytoremediation).
Microbes are nature’s ultimate garbage dis-
posal, devouring the dead, decomposing, and
inert material that litters the Earth’s surface. In
recent years, various scientists have been
designing or deploying microbes to purge sites
of contaminants such as PCBs, oil, radioac-
tive  waste, gasoline, and mercury, and new
bioremediation research appears regularly.
A  microorganism or microbe is a micro-
scopic  organism, which may be single-celled
(Madigan and Martinko, 2006) or multicellular,

discovered by Antonie van Leeuwenhoek in
the 1670s (Lane Nick, 2015). The commonly
used organisms in bioremediation are bacteria
and fungi, and the more recently discovered
group of organisms, the archaea, which pos-
sess unique potential in the bioremediation
process. Numerous microorganisms such as
Mycrobacterium, Nocardia, Bacillins, Berijer­
inckia, Acinethobacter, Penicillium, Pseu­
domonas, Serratio, Methylosinus, Acaligenes,
Actinobacter, Flavobacterium, Nitrosomonas,
Rhizoctomia, Phanerochaete, Mycococcus,
Arthrobacter, Trametes, Xanthofacter, etc., carry
out biodegradation in many different environ-
ments are they were identified as active mem-
bers of microbial consortiums (Ravindra Singh
et al., 2014). In order to get effective bioremedi-
ation, the microorganisms must enzymatically
attack the pollutants and convert them into the
harmless products (Sharma, 2012).
140.6.1 Bacteria
Bacteria are widely diverse organisms which
make excellent players in biodegradation and
bioremediation. Pseudomonas putida is a
Gram-negative soil bacterium which is
involved in the bioremediation of toluene and
is also capable of degrading naphthalene, a
product of petroleum refining, in contami-
nated soils. Dechloromonas aromatica is a rod-
shaped bacterium which can oxidize aromatics
including benzoate, chlorobenzoate, toluene,
chlorate, or nitrate and also has the ability to
oxidize benzene anaerobically. D. aromatic is
especially useful for in situ bioremediation
of  this substance and this was mainly due
to the high propensity of benzene contamina-
tion, especially in ground and surface water
(Dechloromonas Aromatica RCB, 2016).
Methylibium petroleiphilum (formally known
as PM1 strain) bacterium is capable of biore-
mediating the methyl tert-butyl ether (MTBE)
using the contaminant as the sole carbon and
energy source (Jessica, 1999). Alcanivorax bor­
kumensis is a marine rod-shaped bacterium
which grows rapidly in environments damaged
140.6  ­Organisms Involved in Bioremediatio 3149
by oil and also has been used to aid in cleaning
the more than 830 000 gallons of oil from the
Deepwater Horizon oil spill in the Gulf of
Mexico (Biello, 2010).
Reported results have shown that many
organisms (prokaryotes and eukaryotes) also
have a natural capacity to biosorb toxic heavy
metal ions (Singh et  al., 2014). Depending
upon whether they are eukaryotes or prokary-
otes, microorganisms interact with heavy
metal ions and among them the eukaryotes are
more sensitive to metal toxicity than prokary-
otes (Perpetuo et  al., 2011). The active extru-
sion of metal, transformation into other
chemical species with reduced toxicity, and the
intracellular chelation (in eukaryotes) by vari-
ous metal-binding peptides were found to be
the possible modes of interaction of microor-
ganisms with toxic heavy metals. Certain
examples of the bacteria which play an impor-
tant strategic role in bioremediation treat-
ments for heavy metals were the Kocuria flava
(Achal et  al., 2011), Arthrobacter spp.(Roane
et  al., 2001), Burkholderia spp.(Jiang et  al.,
2008), Bacillus cereus (Kanmani et  al., 2012),
Pseudomonas veronii (Vullo et  al., 2008), and
Sporosarcina ginsengisoli (Achal et al., 2012).
140.6.2 Fungi
Because of their participation in the cycling of
elements through decomposition and transfor-
mation of organic and inorganic materials,
fungi have fundamentally played some impor-
tant roles in the bioremediation process. Fungi
have an advantage over bacteria in some cases,
not just in metabolic versatility but also in
their environmental resilience. A fungus
named Phanerochaete chrysosporium was dis-
covered to metabolize multiple key environ-
mental pollutants such as pesticides, PAHs,
dioxins, carbon tetrachloride, and many other
pollutants. The most important feature of
these fungi is their enzymatic functional abil-
ity to metabolize complex chemicals such as
lignin. Certain versatile species of fungi uti-
lized in the bioremediation process were the
3150 140  Bioremediation: A Promising Xenobiotics Cleanup Technique
white rot fungi (Gadd, 2001; Harms, 2011),
Aspergillus fumigatus (Ramasamy et al., 2011),
Penicillium canescens (Say et  al., 2003) and
Aspergillus versicolor (Tastan et al., 2010).
140.6.3 Archaea
Archaea constitute a domain and kingdom of
single-celled microorganisms and these
microbes (archaea; singular archaeon) are
prokaryotes having no cell nucleus or any
other membrane-bound organelles in their
cells. Research demonstrated the important
fact that the four extreme halophilic strains of
archaea (belonging to genus Halobacterium,
Haloferax, and Halococcus) have the potential
to carry out biodegradation at high tempera-
tures, in the range of 40–45°C (Al-Mailem
et  al., 2010), which is advantageous because
hydrocarbons have higher solubility and
­bioavailability at these higher temperatures
(Margesin and Schinne, 2001). The four strains
studied were resistant to six different antibiot-
ics, including penicillin, streptomycin, and
cycloheximide, and this gave them the poten-
tial to carry out biodegradation in conditions
unfavorable for bacteria. Research suggests
other genera of archaea are also capable of bio-
degrading in hypersaline environments. Some
of the algaes used in bioremediation for treat-
ment purpose were Spirogyra spp., Cladophora
fascicularis (Deng et al., 2007), Spirullina spp.
(Mane and Bhosle, 2012), and Cladophora spp.
(Lee and Chang, 2011), and certain examples
of yeast used in bioremediation treatment
were Saccharomyces cerevisiae (Machado et al.,
2010) and Candida utilis (Kujan et al., 2006).
The mineralization of most hazardous com-
pounds cannot be carried out by microorgan-
isms individually. The complete mineralization
process involves synergism and co-metabolism
actions and it also results in a sequential degra-
dation by a consortium of microorganisms. An
amazing physiological versatility was shown
by natural communities of microorganisms in
various habitats and hence they are able to
metabolize and often mineralize an enormous
number of organic molecules. Certain commu-
nities of bacteria and fungi metabolize a multi-
tude of molecules that can be degraded are not
known, but as a result of microbial activity in
one environment or other, thousands are
known to be destroyed. Running a system
under anaerobic conditions (Colberg and
Young, 1995) may permit microbial organisms
to degrade otherwise recalcitrant molecules
but most bioremediation systems are run
under aerobic conditions.
The microorganisms have distinct and
unique roles in the detoxification of polluted
soil environments and these microorganisms
utilize the contaminants as nutrient or energy
sources in the bioremediation process (Hess
et  al., 1997; Agarwal, 1998; Tang et  al., 2007).
Many factors like the genetic potential and cer-
tain environmental factors such as temperture,
pH, and available nitrogen and phosphorus
sources influence microorganisms to use pol-
lutants as substrates or cometabolize, which
seems to determine the rate and the extent of
degradation (Neza Touhrey Joutley et al., 2013).
Since the microorganisms dwell in a variety of
environments they can be used for a variety of
purposes related to industrial microbiology but
are often difficult to isolate, screen, put through
strain development, etc. This bioremediation
technology relies mainly on promoting the
growth of specific microflora or microbial con-
sortia that are indigenous to the contaminated
sites that are able to perform desired activities.
The recent advances in bioremediation tech-
nology using microbial consortia has also been
found to be effective for treatment of pesticides
in soil. Geetha and Fulekar designed a surface
soil treatment unit wherein the bioremedia-
tion of commonly used pesticides, namely
chlorpyrifos, cypermethrin, fenvalerate, and
trichlopyr butoxyethyl ester at varying concen-
tration viz. 25, 50, and 100 mg kg–1, have been
carried out using cow-dung microbial consor-
tia under simulated environmental conditions.
The obtained results highlight the potential of
cow-dung slurry consortia for bioremediation
of soil contaminated with pesticides in surface

soil treatment unit (Geetha and Fulekar, 2008).
Singh and Fulekar (2009) isolated, cultured,
and identified Pseudomonas putida IFO 14671
from the cow-dung microbial consortia as a
high-potential phenol degrader and in addi-
tion they also proved the potential of cow-dung
microflora as a source of biomass for benzene
biodegradation in TPPB (Singh and Fulekar,
2009). Pseudomonas putida MHF, isolated and
identified from cow-dung microbial consor-
tium has also been utilized for biodegrada-
tion  of selected petroleum hydrocarbon
compounds  –  benzene, toluene, and o-xylene
(BTX)  –  and this was reported by Singh and
Fulekar (2010).
140.7 ­Applications of
Bioremediation
140.7.1 Bioremediation
of Heavy Metals
Heavy metal ions have lethal effects on all
forms of life and these enter the food chain
through the disposal of wastes in water chan-
nels. From among various metal ions, lead,
mercury, cadmium, and chromium(VI) are at
the top on the toxicity list (Volesky, 1994). Due
to non-biodegradability, metal ions accumu-
late and their amounts are increased along the
food chain. Heavy metals are known to induce
oxidative stress by generating high concentra-
tions of reactive oxygen species (ROS) such
as superoxide radical (O2•−), singlet oxygen
(1O2), and hydrogen peroxide (H2O2). These
species react very rapidly with lipids, nucleic
acids, pigments, and proteins and cause lipid
peroxidation, membrane damage, and inacti-
vation of enzymes, thus affecting cell viability
(Foyer, 1997).
Hence, their toxic effects are more pro-
nounced in animals at higher trophic levels.
Sources and toxicity of certain metal ions listed
in Table 140.2.
Heavy metal pollution represents an
­important problem due to its toxic effect and
140.7 ­Applications of Bioremediatio 3151
accumulation throughout the food chain which
leads to serious ecological and health hazards
as a result of their solubility and mobility. Metal
remediation through common physicochemi-
cal techniques is expensive and not eco-friendly.
Hence, biotechnological approaches have
received a great deal of attention as an alterna-
tive tool in recent years. The microorganisms
have been utilized for environmental remedia-
tion and recovery purposes and it is believed
that the most fitting microorganisms for
removal treatments are the ones isolated from
the same environment where they were natu-
rally selected. Genetic manipulation techniques
can be used to enhance the capacity of different
microorganisms (Steciow, 2005).
Applicability of growing bacterial/fungal/
algal cells for metal removal and the efforts
directed towards cell/process development to
make this option technically and economically
viable for the treatment of metal-rich effluents
have been reviewed by Malik (2004). Due to
their high sorption capacity and ready availa-
bility in practically unlimited quantity, the
algae were now developed as new biosorbent
material. Many scientists have turned their
research work to treat various industrial and
municipal wastewater using different types
of  microorganisms (Karin Larsdotter, 2006;
Buchanan et al., 2013). Many algae and bacte-
ria produce secretions that attract metals that
are toxic in high levels.
The moss C. Purpureus has been found to
accumulate metals, however, including cop-
per, lead, and zinc from contaminated soil
(Gibson, 2000). The metals are in effect
removed from the food chain by being bound
to the secretions. Bang et  al. (2000) reported
that expression of the thiosulfate reductase
gene from Salmonella typhimurium in
Escherichia coli led to increase of the efficiency
of the removal of heavy metals from solution
and accumulation of cadmium up to 150 mM
in 98% cells. Exploitation of genetic and bio-
chemical capacity of bacteria for remediation
of heavy metal pollution has been studied by
Valls and Lorenzo (2002), who explained the
Table 140.2  Sources and toxicity of heavy metals.
Metal Source
Arsenic Smelting, mining, energy production from fossil fuels,
rock sediments
Cadmium Laundrettes, Electroplating, smelting, alloy
manufacturing, plastic manufacturing, pigments, plastic,
mining, refining.
Chromium Electroplating, leather tanning, textile, dyeing,
(VI) electroplating, metal processing, wood preservatives,
paints and pigments, steel fabrication and canning
industry
Copper Printed circuit board manufacturing, electronics plating,
plating, wire drawing, copper polishing, paint
manufacturing, wood preservatives and printing
operations
Lead Electroplating, manufacturing of batteries, pigments,
ammunition, Fuel additive, roofing, fishing weights.
Mercury Weathering of mercuriferous areas, volcanic eruptions,
naturally caused forest fires, biogenic emissions, battery
production, fossil fuel burning, mining and metallurgical
processes, paint and chloralkali industries
Nickel Non-ferrous metal, mineral processing, paint
formulation, electroplating, porcelain enameling, copper
sulphate manufacture and steam-electric power plants.
Zinc Domestic wastes, galvanizing, batteries, paints,
fungicides, textiles, cosmetics, pulp, papermills and
pharmaceutics
Toxic Effect
Gastrointestinal symptoms, disturbances of
cardiovascular and nervous system functions,
bone marrow depression, haemolysis,
hepatomegaly, melanosis, polyneuropathy and
encephalopathy, liver tumor
Carcinogenic, renal disturbances, lung
insufficiency, bone lesions, cancer, hypertension,
Itai–Itai disease, weight loss.
Carcinogenic, mutagenic, teratogenic, epigastric
pain, nausea, vomiting, severe diarrhea,
producing lung tumors
Reproductive and developmental toxicity,
neurotoxicity, acute toxicity, dizziness, diarrhea.
Anaemia, brain damage, anorexia, malaise, loss
of appetite, diminishing IQ
Neurological and renal disturbances, impairment
of pulmonary function, corrosive to skin, eyes,
muscles, dermatitis, kidney damage
Chronic bronchitis, reduced lung function, lung
cancer
Short term “metal-fume fever,” gastrointestinal
distress, nausea and diarrhea
Reference
Chilvers and Peterson (1987),
Dudka and Markert (1992),
Robertson (1989)
Chen and Hao (1998), Godt et al.
(2006), Low et al. (2000), Sharma
(1995), Singh et al. (2006)
Dupont and Guillon (2003),
Granados-Correa and Serrano-
Gómez (2009), Kobya (2004),
Singh et al. (2009)
Chuah et al. (2005), Papandreou
et al. (2007), Yu et al. (2000)
Gaballah and Kilbertus (1998),
Low et al. (2000), Volesky (1993)
Boening (2000), Manohar et al.
(2002), Morel et al. (1998)
Akhtar et al. (2004), Ozturk
(2007)
WHO (2001)

advantages of biological approaches which
includes higher specificity, suitability, and
potential for genetic improvement. Degradation
of dyes is also brought about by some anaero-
bic bacteria and fungi (Colberg 1995).
Chlorella vulgaris beads were found to be
more efficient in heavy metal removal from
sewage than free cells (Abdel Hameed, 2002).
The observed results indicate that the effi-
ciency in iron, nickel, and zinc removal was
higher in the immobilized cells than the free
cells by 27, 23, and 25%, respectively. Abdel
Hameed examines the possibility of using
Chlorella vulgaris cells in repeated lead
adsorption/desorption cycles. Lead adsorp-
tion mainly occurs via the alginate matrix and
a minor contribution was made by algae.
Consistently high lead removal (> 90%) and
recovery (about 100%) were achieved. Two
marine algae, Thalassiosira weissflogii and
Thalassiosira pseudonana, produce phyto-
chelatins in great amounts due to the higher
activity of phytochelatin synthase, which has
greater affinity for the glutathione substrate or
metal ions (Semane et al., 2010).
The application of living bacteria (Gadd,
2000; Diels et  al., 1999) and biopolymers
(Gutnick and Bach, 2000) has been recently
incorporated into the concept of biosorption.
Biosorbents may be viewed as natural ionex-
change materials that primarily contain weakly
acidic and basic groups (Kratochvil and Volesky,
1998). Bacillus circulans strain EB1, a heavy-
metal-resistant bacterium, has been reported to
remove manganese, zinc, copper, nickel, and
cobalt during its active growth cycle (Yilmaz,
2003). A number of chromium-reducing bac-
teria, including Pseudomonas, Escherichia,
Bacillus, Desulfovibrio, Desulfomicrobium, and
Desulfotomaculum (Cheung and Gu, 2003;
Jong and Parry, 2003), Shewanella alga (Guha
et  al., 2001) and Pyrobaculum islandicum
(Kashefi and Lovley, 2000) have been studied.
The passive removal of toxic heavy metals by
brown marine algae via biosorption has been
reported by Davis et al. (2003), who attributed
this property to cell wall polysaccharides like
140.7 ­Applications of Bioremediatio 3153
alginate and fucoidan. The need for economi-
cal, effective, and safe methods for removal of
heavy metals from wastewater has directed
attention to extracellular polysaccharide or
exopolysaccharide (EPS) produced by algae,
bacteria, fungi, and yeast (Bender et al., 1994;
Salehizadeh and Shojaosadati, 2001; Volesky
and Holan, 1995). Few reports on metal chela-
tion by bacterial EPS are available today. The
adsorption of heavy metals by EPS is non-met-
abolic, energy independent, and can be caused
by interaction between metal cations and neg-
ative charge of acidic functional groups of EPS
(Kim et al., 1996). EPS are also recommended
as surface-active agents for heavy metal
removal (Kaplan et  al., 1987; Norberger and
Enfors, 1982).
A novel polysaccharide from Bacillus fir­
mus  is reported to remove metal ions like
lead,  copper, and zinc from aqueous solu-
tion  (Salehizadeh and Shojaosadati, 2003).
Enterobacter cloaceae, an EPS-producing
marine bacterium, was tested for its Cr(VI) tol-
erance and chelation. X-Ray Fluorescence
(XRF) spectroscopy analysis of both the bio-
mass and the EPS revealed that 60–70% chro-
mium was accumulated by this bacterium
(Iyer et  al., 2004). Based on the encouraging
results, studies on the biosorption capacity of
the same culture for different heavy metals
viz., cadmium (Cd), cobalt (Co), mercury (Hg),
and copper (Cu), were conducted.
The brown algae represent an especially effi-
cient and resilient class of biosorbents relative
to other biomass types. Fortunately, due to
their economic value in many industrial appli-
cations, there is much information about their
basic structure and biochemical constitution.
An investigation was done on the potential
use  of the immobilized Scenedesmus quadri­
cauda in Ca-alginate to remove Cu, Zn, and Ni
by Bayramoglu and Arýca. Reported results
showed that the qmax values for Cu, Zn, and Ni
were 75.6 mg g–1, 55.2 mg g–1, and 30.4 mg g–1.
The sorption process followed second-order
kinetics (Bayramoglu and Arýca 2009). Studies
evaluated the efficiency of the immobilized
3154 140  Bioremediation: A Promising Xenobiotics Cleanup Technique
brown alga Fucus vesiculosus in alginate xero-
gels for uptake of heavy metals. This in turn
makes the interpretation of the sorption behav-
iors and elucidation of metal-binding mecha-
nisms more rigorous. The order Laminariales
and Fucales of the brown algae (division
Phaeophyta) are the most important groups of
algae in the field of biosorption because of the
abundance of their cell wall matrix polysac-
charides and extracellular polymers.
Microcystis has proven efficient in metal
removal from single bimetallic as well as mul-
timetallic conditions. The blue-green alga
Microcystis, grown under continuous culture
in the presence of chromium (VI), cadmium
(II) and copper (II), as a single-metal species
and as mixtures of two or three metals in a
laboratory-scale system, indicate that the
Chromium (VI) and cadmium (II) had a posi-
tive interaction that increased the removal per-
centages of both these metals, whereas this
work could not detect any interaction with
copper (II). The investigation of heavy metal
removal using Synechocystis salina reveals that
almost 60% Cr, 66% Fe, 70% Ni, 77% Hg, 65%
Ca2+, 63% Mg2+, and 78% total hardness was
reduced in 15 days of treatment.
140.7.2 Bioremediation
of Degraded Soil
Bioremediation based on the presence of
microorganisms in the soil is an optimum
method in the remediation of soils contami-
nated with carbonate organic compounds,
being successfully used in remediation of soils
contaminated with crude oil and petroleum
products. Fauna and microorganisms present
in the soil, represented by protozoa, algae,
fungi, actinomycetes, and bacteria, play an
important role in the soil’s biological activity
(Donlon and Bauder, 2012; Gallizia et al., 2003).
Hydrocarbon-degrading bacteria and fungi
are widely distributed in marine, freshwa-
ter,  and soil habitats. Similarly, hydrocarbon-
degrading cyanobacteria have been reported
(Lliros et  al., 2003; Chaillan et  al., 2004),
although contrasting reports indicated that
growth of mats built by cyanobacteria in the
Saudi coast led to preservation of oil residues
(Barth, 2003). Typical bacterial groups already
known for their capacity to degrade hydrocar-
bons include Pseudomonas sp., Marinobacter
sp., Alcanivorax sp., Microbulbifer sp., Sphin­
gomonas sp., Micrococcus sp., Cellulomonas sp.,
Dietzia sp., and Gordonia sp. (Brito et al., 2006).
Molds belonging to the genera Aspergillus sp.,
Penicillium sp., Fusarium sp., Amorphoteca sp.,
Neosartorya sp., Paecilomyces sp., Talaromyces
sp., and Graphium sp., and the yeasts Candida
sp., Yarrowia sp., and Pichia sp. Have been
implicated in hydrocarbon degradation
(Chaillan et al., 2004).
The phylum includes phenotypically diverse
organisms which exhibit a wide variety of mor-
phologies that range from cocci to highly dif-
ferentiated mycelia and spore production
which could be advantageous for long-distance
dispersal. The taxon exhibits a cosmopolitan
distribution, with members of the group being
widely distributed in aquatic and terrestrial
ecosystems (Goodfellow et al., 2012).
Specially in soil, they are important organ-
isms mediating the formation of soil organic
matter. Different lifestyles are encountered
among actinobacteria, saprophytic free-living
aquatic and soil actinobacteria, as well as plant
commensals, nitrogen-fixing symbionts, gastro-
intestinal tract inhabitants, and animal and
plant pathogens (Fiedler et al., 2005; Goodfellow
et al., 2012; Hassanshahian et al., 2014).
140.7.3 Bioremediation
of Marine Oil Spills
Bioremediation is a potentially important option
for dealing with marine oil spills. Oil-degrading
microorganisms are indigenous to the world’s
oceans, but environmental constraints limit
their activity and so a bioremediation strategy
must be tailored to local conditions.
A study focused on the relationship between
indigenous and seeded microbial cultures.
The results showed that seeding with local or

foreign oil-degrading bacteria did not lead to
enhancement of hydrocarbon degradation and
resulted in dramatic decreases in the numbers
of the predominant, indigenous, oil-degrading
bacteria. Whereas local microorganisms were
able to establish themselves rather easily in
the  Gulf coast sand, the foreign bacteria (the
German Arthrobacter strains, KCCG 351-355)
either decreased or did not survive at all. Still,
they contributed to hydrocarbon degradation
(Radwan et al., 1997).
One experiment analyzed the effectiveness of
a certain bioremediation agent in degrading the
oil spilled in the Arabian Gulf. The commer-
cially available bacterial product consisted of a
mixture of naturally occuring microorganisms.
The degradation of the oil was observed under
different concentrations of oil, added nutrients,
and added bacteria (Fayad et al., 1992).
Al-Mailem et  al. (2010) studied crude oil-
degrading bacteria in symbiosis with filamen-
tous cyanobacteria and their results show
that  some bacteria, such as Halomonas aqua­
marina,  Marinobacter hydrocarbonoclasticus,
Marinobacter sp., Dietzia maris, and Alcanivorax
can degrade crude oil and present in cyanobac-
teria (Al-Mailem et al., 2010; Al-Awadhi et al.,
2003). Oil slicks caused by oil tankers and petrol
leakage into the marine environment are now a
constantly occurring phenomenon. Several
microorganisms can utilize oil as a source of
food, and many of them produce potent surface
active compounds that can emulsify oil in water
and facilitate its removal. Unlike chemical sur-
factants, the microbial emulsifier is nontoxic
and biodegradable. The microorganisms capa-
ble of degrading petroleum include pseudomon-
ads, various corynebacteria, mycobacteria, and
some yeast (Prince, 2002).
140.7.4 Bioremediation
of Rubber Waste
Currently only a few microbial species have
been found that can remove sulfur from rub-
ber, leaving many strains and species to inves-
tigate. Shruthi et  al. 2012 have worked on a
140.7 ­Applications of Bioremediatio 3155
bacterial strain which was isolated from the
rubber-processing industry effluent and was
identified as Pseudomonas sp. The efficiency of
this bacterium to carry out bioremediation of
rubber-processing industry effluent was stud-
ied. Bacterial treatment of the wastewater
yielded substantial reduction in total solids
(73%), suspended solids (75.7%), dissolved sol-
ids (68.8%), BOD (72.1%), COD (79.4%), ammo-
nia (71.3%), and phosphate (68.8%) after 15
days of incubation. The results of this study
revealed that the Pseudomonas sp. isolated
from the effluent is efficient enough to degrade
the toxic components in the rubber-processing
industry effluent and therefore could effec-
tively be used for the treatment of these
effluents.
An effective biodegradation method will
consist of a multistage process utilizing several
different microbes and biochemical pathways.
The first step may involve detoxification of
scrap tire rubber with the fungus Recinicium
bicolour to remove additives that inhibit micro-
bial growth. After detoxification, a sulfur-oxi-
dizing bacterial species such as Thiobacillus
ferroxidans, or the sulfur-reducing archaeon
Pyrococcus furiosus, could be used to devulcan-
ize the rubber. This devulcanized rubber might
then be recycled. Devulcanized rubber is more
susceptible to enzymatic degradation by rub-
ber-metabolizing microorganisms, such as the
adhesively growing Gordonia species and clear
zone–forming Streptomyces species (Holst
et al., 1998).
The tread compound found on truck tires
can be partially degraded by a mutant Nocardia
strain (835A). This strain can mineralize or dis-
integrate NR polymers in tread compounds to
leave small black particles of inorganic fillers
and residual rubber (Tsuchii and Tokiwa,
2006). One of the toxic additives in rubber is
2-mercaptobenzothiazole (MBT), a compound
that is used as a vulcanization accelerator.
Metabolites produced and identified during
MBT biodegradation include benzothiazolyl-
sulphonate and 2-methlthiobenzothiazole
(MTBT), which are much less toxic. Therefore
3156 140  Bioremediation: A Promising Xenobiotics Cleanup Technique
using Rhodococcus sp. to metabolize MBT
could greatly improve the safety of tire disposal
and make rubber easier to biodegrade
(Haroune et al., 2004).
Christiansson et al. (1998) and Romine and
Romine (1998) have studied the rubber-
degrading capacities of known sulfur-oxidiz-
ing bacteria. In these experiments, three main
species of Thiobacillus bacteria have been used
to degrade rubber: T. ferrooxidans,T. thioparus,
and T. thiooxidans. The most efficient sulfur-
oxidizing species in terms of released sulfate
was found to be T. ferrooxidans (Christiansson
et  al., 1998) (Romine and Romine 1998).
Manukulasooriya et al. (2010) has worked on
rubber latex in contaminated effluent. Bacteria
were isolated from the industrial wastewater in
nutrient agar medium and 10 bacterial colo-
nies were selected. The selected bacteria were
able to grow in 100% latex. Most of the bacteria
screened for this analysis was aerobic and
Gram-negative. Bacterial DNA isolated from
these bacteria by two different methods were
good in quality and were amplified by primer
specific for Pseudomonas aeruginosa, resulting
in expected 162 bp PCR product. Treatment
techniques used to optimize the growth of the
bacteria gave considerable change in pH, tur-
bidity, and COD of the industrial wastewater
sample.
Nowaczyk and Domka (1999) had a signifi-
cant interest in microbiological degradation of
rubber effluent, and the microorganisms
which can develop in them have been rela-
tively poorly recognized. It has been recog-
nized that only a few species of bacteria can
cause degradation of rubber, mainly the
Pseudomonas species and the Actinomycetes
species from the genus Streptomyces (Nowaczyk
and Domka, 1999).
It was found that new rubber was able to
revulcanize more effectively with P. furiosus-
treated rubber than with untreated rubber.
This suggests that P. furiosus was able to break
sulfide bridges between polyisoprene chains,
and free up reactive sites where new cross-
links could form upon revulcanization. In
addition, Pyrococcus furiosus grew successfully
in the presence of NR, latex, and ethanol-
leached CGTR. However, P. furiosus did not
grow on fresh tire rubber, untreated CGTR, or
NR-containing accelerators and fillers
(Bredberg et al., 2001).
140.7.5 Bioremediation
of Agricultural Waste
“Green waste” (food, agroindustrial, and forest
residues) is a renowned valuable resource of
polyphenols. Natural polyphenols are rela-
tively efficient in the cleanup of environmental
pollutants based on their unique traits of
chelation, adsorption, reduction, complexa-
tion, nutrient cycling, antibacterial effects, and
plant growth promotion (Saranya Kuppusamy
et  al., 2015). Agricultural waste materials,
being economic and ecofriendly due to their
unique chemical composition, available in
abundance, renewable, low in cost, and more
efficient, seem to be a viable option for heavy
metal remediation. Green waste comprises
food, forestry, garden, agricultural, and biolog-
ical industrial wastes. It is estimated that 140
billion metric tons of biomass are generated
globally every year from agriculture (Centore
et al., 2014). Almost 60% of the total (6273 mil-
lion tonnes per annum) food production is lost
or wasted (Gustavsson et  al., 2011). Fallen
branches, leaves and flowers of trees, grass
clippings, tree and shrub prunings, and weeds
from forest management and landscaping are
left as trash. Husks, pomace, nut or seed shells,
straw, and residual stalks are generated stead-
ily by agroindustrial activities. Agricultural
materials, particularly those containing cellu-
lose, show potential metal biosorption capac-
ity. The basic components of the agricultural
waste materials biomass include hemicellu-
lose, lignin, extractives, lipids, proteins, simple
sugars, water hydrocarbons, and starch con-
taining a variety of functional groups that facil-
itate metal complexation, which helps for the
sequestering of heavy metals (Bailey et  al.,
1999; Hashem et al., 2005a,b).

Studies reveal that various agricultural waste
materials such as rice bran, rice husk, wheat
bran, wheat husk, sawdust of various plants,
bark of the trees, groundnut shells, coconut
shells, black gram husk, hazelnut shells, wal-
nut shells, cotton seed hulls, waste tea leaves,
Cassia fistula leaves, maize corn cob, jatropa
deoiled cakes, sugarcane bagasse, apple,
banana, orange peels, soybean hulls, grape
stalks, water hyacinth, sugar beet pulp, sun-
flower stalks, coffee beans, arjun nuts, cotton
stalks, etc. have been tried (Annadurai et  al.,
2002; Cimino et  al., 2000; Hashem et  al.,
2006a,b; Macchi et  al., 1986; Maranon and
Sastre, 1991; Mohanty et al., 2005; Orhan and
Bujukgungor, 1993; Reddad et  al., 2002; Tee
and Khan, 1988). These promising agricultural
waste materials are used in the removal of
metal ions either in their natural form or after
some physical or chemical modification. The
use of these low-cost biosorbents is recom-
mended since they are relatively cheap or of no
cost, easily available, renewable, and show
high affinity for heavy metals. Literature also
reveals that in some cases the modification of
the adsorbent increased the removal efficiency.
Further research is to be carried out to make
the process economically viable at industrial
scale with focus on metal recovery and regen-
eration of agricultural waste.
140.8 ­Phytoremediation
Phytoremediation has emerged as a most desir-
able technology, which uses plants for removal
of environmental pollutants or detoxification
to make them harmless (Cunningham and
Berti, 1993). The word “phytoremediation”
derives from the Greek phyton, meaning
“plant,” and Latin remedium, which means “to
remedy” or “to correct. Phytoremediation can
be cost-effective (a) for large sites with shallow
residual levels of contamination by organic,
nutrient, or metal pollutants, where contami-
nation does not pose an imminent danger and
only “polishing treatment” is required; (b)
140.8 ­Phytoremediatio 3157
where vegetation is used as a final cap and clo-
sure of the site (Schnoor et al., 1995).
Many living organisms can accumulate cer-
tain toxicants to body concentrations much
higher than present in their environments
(Nyangababo et  al., 2005b; Igwe et  al., 2008;
Kord et al., 2010). Thus, the use of plants for the
decontamination of heavy metals has attracted
growing attention because of several problems
associated with pollutant removal using con-
ventional methods. Substances that may be
subjected to phytoremediation include metals
(Pb, Zn, Cd, Cu, Ni, Hg), metalloids (As, Sb),
inorganic compounds (NO3- NH4+, PO43-), radi-
oactive chemical elements (U, Cs, Sr), petro-
leum hydrocarbons (BTEX), pesticides and
herbicides (atrazine, bentazone, chlorinated
and nitroaromatic compounds), explosives
(TNT, DNT), chlorinated solvents (TCE, PCE)
and industrial organic wastes (PCPs, PAHs),
and others (Ensley, 2000). Bioremediation
strategies have been proposed as an attractive
alternative owing to their low cost and high
efficiency. Phytoremediation essentially com-
prise six different strategies, though more than
one may be used by the plant simultaneously:
phytoextraction, phytodegradation, phytostim-
ulation, phytostabilization, phytovolatilization,
phytofiltration and rhizodegradation (Oh et al.,
2013a; Wang et al., 2003).
Using green plants as weapons, phytoreme-
diation is one of most economical and environ-
mentally friendly techniques to target the
organic and inorganic pollutants in the water,
soil, and air simultaneously. Phytoremediation
presents a series of advantages, such as rela-
tively low cost, easy to implement and main-
tain, 10–20% costs of mechanical treatment,
and it’s faster compared to natural attenuation.
The method also has disadvantages, such as a
longer period of time is required, and it is influ-
enced by soil and climate conditions (Rusu
et  al., 2007). Phytoremediation processes rely
on the ability of plants to take up and/or metab-
olize pollutants to less toxic substances. The
uptake, accumulation, and degradation of con-
taminants vary from plant to plant. The plants
3158 140  Bioremediation: A Promising Xenobiotics Cleanup Technique
used in phytoremediation are generally selected
on the basis of their growth rate and biomass,
their ability to tolerate and accumulate contam-
inants, the depth of their root zone, and their
potential to transpire groundwater (Oh et  al.,
2013b). The plants used in phytoremediation
should not only accumulate, degrade, or volatil-
ize the contaminants, but should also grow
quickly in a wide range of different conditions.
Although phytoremediation is a promising
technique to remove pollutants, it is still an
immature and developing technology to deal
with pollution problems.
Modern biogenic hydrocarbons are also
important carbon sources in the sea; many are
produced by planktonic algae, while others
are  volatilized by terrestrial plants and are
washed into the sea (National Research
Council, (2002). It is thus no surprise that oil-
degrading microorganisms are ubiquitous in
the world’s oceans (Prince, 2002). Recently, the
use of aquatic plants, especially micro- and
macroalgae, has received much attention due
to their ability to absorb metals and take up
toxic elements from the environment or render
them less harmful (Mitra et al., 2012).
Wang et  al. (2004) suggested that the most
important anthropogenic sources of mercury
pollution in aquatic environment are atmos-
pheric deposition, urban discharges, agricul-
tural material runoff, mining, fossil fuel use
and industrial discharges, burning of coal, and
pharmaceutical production. The brown algae
Phaeophyta are particularly efficient accumu-
lators of metals due to high levels of sulfated
polysaccharides and alginates within their cell
walls, for which metals show a strong affinity
(Davis et al., 2003).
Dunaliella salina, a green microalgae, have
high tendency for zinc accumulation followed
by copper and cobalt, and the lowest tendency
was for cadmium; this may be due to the impor-
tance of zinc for transferring hydrogen in photo-
synthesis (Liu et  al., 2002). Nagaraju and
Karimulla (2002) 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.) (Vijayarengan, 2005). Such
plants can be used successfully to clean up heavy
metal-polluted soils if their biomass and metal
content are large enough to complete remedia-
tion within a reasonable period (Ebbs and
Kochian, 1998).
Approximately 400 plant species from at
least 45 plant families have so far been reported
to hyperaccumulate metals (Lasat, 2000;
Ghosh and Singh, 2005). Some of the families
are Brassicaceae, Fabaceae, Euphorbiaceae,
Asterraceae, Lamiaceae, and Scrophulariaceae
(Salt et  al, 1998; Dushekov, 2003). Crops like
alpine pennycress (Thlaspi caerulescens),
Ipomea alpine, Haumaniastrum robertii,
Astragalus racemosus, and Sebertia acuminate
have very high bioaccumulation potential for
Cd/Zn, Cu, Co, Se, and Ni, respectively (Lasat,
2002). Willow (Salix viminalis L.), maize (Zea
mays L.), Indian mustard (Brassica juncea L.),
and sunflower (Helianthus annuus L.) have
reportedly shown high uptake and tolerance to
heavy metals (Schmidt, 2003).
The root of Indian mustard is 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 (Lone et al.,
2008). 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. Tang et  al. (2003) reported the
increase in uptake of copper by Indian mustard
and sunflower plant. Nehnevajova et al. (2005)
investigated that the highest metal concentra-
tion was found in leaves (shoot) of commercial
cultivars of sunflower plants grown on metal-
contaminated soil. Among the cultivated crops,
rape and sunflower revealed higher cadmium
concentrations in their shoots than in the roots.
Nascimento and Xing (2006) expressed
that  phytoextraction may be considered as a

commercial technology in the future. Jiang
et  al. (2004) determined the growth perfor-
mance and ability for Cu phytoextraction of
Elsholtzia splendens. Zhang et  al. (2009)
expressed that as Cd phytoextraction is
observed by maize, the percentage of exchange-
able form of Cd decreased in the planted soil.
Similar findings of decrease in Cd level in soil
planted with maize have also been reported by
Mojiri (2011). Abhilash et  al. (2009) investi-
gated the potential of Limnocharis flava (L.)
Buchenau, grown for phytofiltration of Cd in
polluted water with low concentrations of Cd
in a hydroponic experiment. Banuelos (2000)
perceived that some plants were able to trans-
form Se in the form of dimethylselenide and
dimethyldiselenide in high-selenium media.
Unlike other remediation techniques, once the
contaminants have been removed via volatili-
zation, 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).
140.9 ­Conclusion
Considerable research on bioremediation now
provides the necessary body of knowledge to
understand their biotechnological potentials.
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