Art:10.1007/s11157 010 9215 6

Rev Environ Sci Biotechnol (2010) 9:215288
DOI 10.1007/s11157-010-9215-6
REVIEWS
A comprehensive overview of elements in bioremediation

Asha A. Juwarkar Sanjeev K. Singh
Ackmez Mudhoo
Published online: 29 August 2010

Springer Science+Business Media B.V. 2010
Abstract Sustainable development requires the

development and promotion of environmental management and a constant search for green technologies
to treat a wide range of aquatic and terrestrial habitats
contaminated by increasing anthropogenic activities.
Bioremediation is an increasingly popular alternative
to conventional methods for treating waste compounds
and media with the possibility to degrade contaminants using natural microbial activity mediated by
different consortia of microbial strains. Many studies
about bioremediation have been reported and the
scientific literature has revealed the progressive
emergence of various bioremediation techniques. In
this review, we discuss the various in situ and ex situ
bioremediation techniques and elaborate on the anaerobic digestion technology, phytoremediation, hyperaccumulation, composting and biosorption for their
effectiveness in the biotreatment, stabilization and
eventually overall remediation of contaminated strata
and environments. The review ends with a note on the
A. A. Juwarkar (&) S. K. Singh
Eco-Restoration Division, National Environmental
Engineering Research Institute (NEERI), Council
of Scientific and Industrial Research (CSIR),
Govt. of India, Nehru Marg, Nagpur 440 020,
Maharashtra, India
e-mail: aa_juwarkar@neeri.res.in
A. Mudhoo
Department of Chemical and Environmental Engineering,
Faculty of Engineering, University of Mauritius, Reduit,
Mauritius
recent advances genetic engineering and nanotechnology have had in improving bioremediation. Case
studies have also been extensively revisited to support
the discussions on biosorption of heavy metals, gene
probes used in molecular diagnostics, bioremediation
studies of contaminants in vadose soils, bioremediation of oil contaminated soils, bioremediation of
contaminants from mining sites, air sparging, slurry
phase bioremediation, phytoremediation studies for
pollutants and heavy metal hyperaccumulators, and
vermicomposting.
Keywords Bioremediation Green technology
Environmental contaminants Anaerobic
biotechnology Composting Phytoremediation
Biosorption
1 Introduction
The global environment is under great stress due to
urbanization and industrialization as well as population pressure on the limited natural resources. The
problems are compounded by drastic changes that
have been taking place in the lifestyle and habits of
people. The environmental problems are diverse and
sometimes specific with reference to time and space.
The nature and the magnitude of the problems are ever
changing, bringing new challenges and creating a
constant need for developing newer and more appropriate technologies. In this context, biotechnology has
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tremendous potential to cater for the needs and

holds hope for environmental protection, sustainability
and management (Hatti-Kaul et al. 2007; Azadi and
Ho 2010). While some applications such as bioremediation are direct applications of biotechnology
(Koenigsberg et al. 2005; Dowling and Doty 2009;
Sen and Chakrabarti 2009), there are many which are
indirectly beneficial for environmental remediation,
pollution prevention and waste treatment.
1.1 Environmental pollution and biotreatment

options
The problems of environment can be classified into the
following subheads as most of the problems can be
traced to one or more of the following either directly or
indirectly: Waste generation (sewage, wastewater,
kitchen waste, industrial waste, effluents, agricultural
waste, food waste) and use of chemicals for various
purposes in the form of insecticides, pesticides,
chemical fertilizers, toxic products and by-products
from chemical industries). Waste generation is a side
effect of consumption and production activities and
tends to increase with economic advance. What is of
concern is the increased presence of toxic chemicals
such as halogen aliphatics, aromatics, polychlorinated
biphenyls and other organic and inorganic pollutants
which may reach air, water or soil and affect the
environment in several ways, ultimately threatening
the self-regulating capacity of the biosphere (Sen and
Chakrabarti 2009; Prasad et al. 2010; Beltrame et al.
2010). They may be present in high levels at the points
of discharge or may remain low but can be highly toxic
for the receiving bodies. The underground water
sources are increasingly becoming contaminated. For
example, the underground water sources have been
permanently abandoned in the valley of the River Po in
north Italy due to industrial pollution. Some substances may reach environment in small concentrations but may be subjected to biomagnification or
bioaccumulation up the food chain, wherein their
concentrations increase as they pass through the food
chain (Davies et al. 2006; Kelly et al. 2007; Fatemi
and Baher 2009; Sharma et al. 2009; Takeuchi et al.
2009). Zhang et al. (2010) have recently detected
legacy pollutants, polychlorinated biphenyls (PCBs),
dichlorodiphenyl trichloroethane and its metabolites
(DDTs), and some emerging organhalogen pollutants,
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such as polybrominated diphenyl ethers (PBDEs),

hexabromobenzene (HBB), pentabromotoluene (PBT),
2,3,4,5,6-pentabromoethyl benzene (PBEB), 1,2bis (2,4,6-tribromophenoxy) ethane (BTBPE), and
dechlorane plus (DP) in an aquatic food chain
(invertebrates and fish) from an E-waste recycling
region in South China. Polychlorinated biphenyls,
DDTs, PBDEs, and HBB were detected in more than
90% of the samples, with respective concentrations
ranging from not detected (ND)32,000 ng/g lipid
weight, ND850 ng/g lipid weight, 81,300 ng/g
lipid weight, and 0.28240 ng/g lipid weight. Pentabromotoluene, PBEB, BTBPE, and DP were also
quantifiable in collected samples with a concentration
range of ND40 ng/g lipid weight. Earlier, Ozkoc
et al. (2007) had detected considerable levels of aldrin,
dieldrin, endrin, heptachlor epoxide, lindane, endosulphan sulphate, and HCB in sediment, mussel, and
seawater samples collected three times during
20012003 at nine sampling stations along the midBlack Sea coast of Turkey. The highest concentrations
of DDT metabolites measured in the sediment and
mussel samples were 35.9 and 14.0 ng/g wet weight
respectively.
There are three main approaches in dealing with
contaminated sites: Identification of the problem,
assessment of the nature and degree of the hazard,
and the best choice of remedial action. The need to
remediate these sites has led to the development of
new technologies that emphasize the detoxification
and destruction of the contaminants (Wang and Chen
2007; Weber 2007; Kulkarni et al. 2008; Busca et al.
2008) rather than the conventional approach of
disposal. Wang and Chen (2007) recently developed
a novel system of phytoremediation ex planta based on
the overexpression of a secretory laccase (Kunamneni
et al. 2008) that catalyzes the oxidation of various
aromatic compounds, including 2,4,6-trichlorophenol.
All the more, rapid developments in understanding
activated sludge processes and wastewater remediation warrant exploitation of different strategies for
studying their degradation and some of the biological
remediation terminologies such as bioleaching,
biosorption, bioaugmentation, biostimulation, biopulping, biodeterioration, biobleaching, bioaccumulation, biotransformation and bioattenuation are being
actively researched on (Whiteley and Lee 2006).
Enzyme technology has equally been receiving
increased attention. Hussain et al. (2009) have
reviewed the biotechnological approaches for enhancing the capability of microorganisms and plants
through the characterization and transfer of pesticide-degrading genes, induction of catabolic pathways, and display of cell surface enzymes, while
Theron et al. (2008) have performed a thorough review
of nanotechnology, the engineering and art of manipulating matter at the nanoscale (1100 nm), and have
highlighted the potential of novel nanomaterials for
treatment of surface water, groundwater, and wastewater contaminated by toxic metal ions, organic and
inorganic solutes, and microorganisms. Husain et al.
(2009) have analyzed the role of peroxidases in
the remediation and treatment of a wide spectrum
of aromatic pollutants. Peroxidases can catalyze
degradation/transformation of polycyclic aromatic
hydrocarbons (PAHs), PCBs, organochlorines, 2,4,6trinitrotoluene, phenolic compounds and dyes. These
enzymes are also capable of treating various types of
recalcitrant aromatic compounds in the presence of
redox mediators.
1.2 Bioremediation: definitions

Bioremediation, which is the use of microorganisms
consortia or microbial processes to degrade and
detoxify environmental contaminants (Margesin et al.
2007; de Lorenzo 2008; Zhao and Poh 2008; Singh
et al. 2008a), is also amongst these new technologies
which derives its scientific justification from the
emerging concept of Green Chemistry and Green
Engineering, and is a fast growing promising remediation technique increasingly being studied and applied
in practical use for pollutant clean-up. Vidali (2001)
has proposed the following classification of microorganisms involved in bioremediation processes:
Aerobic microbes bring about biodegradation in
the presence of oxygen with Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium being the aerobic bacteria recognized for their
degradative abilities. These microbes have often been
reported to degrade pesticides and hydrocarbons,
both alkanes and polyaromatic compounds. Many of
these bacteria use the contaminant as the sole source
of carbon and energy.
Anaerobic bacteria cause degradation in the
absence of oxygen. There is an increasing interest
in anaerobic bacteria used for bioremediation of
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PCBs in river sediments, dechlorination of the

solvent trichloroethylene (TCE), and chloroform.
Ligninolytic fungs are fungi such as the white rot
fungus Phanaerochaete chrysosporium and have the
ability to degrade an extremely diverse range of
persistent or toxic environmental pollutants.
Methylotrophs are aerobic bacteria that grow 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 inorganic compounds.
Advances in bioremediation harness molecular,
genetic, microbiology, and protein engineering tools
and rely on identification of novel metal-sequestering
peptides, rational and irrational pathway engineering,
and enzyme design (Singh et al. 2008a). In this
review, the various in situ and ex situ bioremediation
techniques namely anaerobic digestion technology,
phytoremediation, composting, bioaugmentation,
biostimulation and biosorption have been described
and discussed for their effectiveness in the biotreatment, stabilization and eventually overall remediation
of contaminated strata and environments. The last
segment of the review briefly revisits the potential
genetic engineering and nanotechnology have in
enhancing bioremediation. Case studies have also
been extensively revisited to support the discussions
on biosorption of heavy metals, gene probes used in
molecular diagnostics, bioremediation studies of
contaminants in vadose soils, bioremediation of oil
contaminated soils, bioremediation of contaminants
from mining sites, air sparging, slurry phase bioremediation, phytoremediation studies for pollutants
and heavy metal hyperaccumulators, and vermicomposting. Figure 1 highlights the elements in bioremediation that have been addressed and discussed in
this review.
1.3 Characteristics of bioremediation

Bioremediation techniques have been used for decontamination of surface and subsurface soils, freshwater
and marine systems, soils, groundwater and contaminated land ecosystems. However, the majority of
bioremediation technologies initially developed were
to treat petroleum hydrocarbon contamination to
immobilize contaminants or to transform them to
chemical products no longer hazardous to human
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Fig. 1 Break down of the

elements in bioremediation
discussed in this review
health and the environment. Where contaminants

pose no significant risk to water supply or surface
water bodies, biodegradation products will include
carbon dioxide, water and other compounds with
little deleterious effects on the environment (Baker
and Herson 1994).
Bioremediation of soils or any site may be enhanced
by fertilizing (adding nutrients such as carbon, nitrogen and phosphorous) and/or seeding with suitable
microbial populations. This is enhanced or engineered
bioremediation. Intrinsic bioremediation, which utilizes existing microbial communities, is often the most
cost effective method available for land decontamination. Even in the most contaminated soils, indigenous microbial activity can be enough to clean the soil
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effectively. Microbial communities within contaminated ecosystems tend to be dominated by those

organisms capable of utilizing or surviving toxic
contamination. These communities are typically less
diverse than those in non-stressed systems (Baker and
Herson 1994). Once the soil has been fertilized and/or
seeded, control of temperature, water oxygen content
can be used to speed up the process or reduce the
negative effects of factors such as air pollution. Soil
remediation has suspended the established technologies of excavation followed by either incineration or
landfilling.
Bioremediation techniques are cost effective as
compared to other technologies as indicated in
Table 1. Biological treatments compare favourably

Table 1 A comparison of soil remediation treatment costs
Treatment
Biological
Approximate cost
(()/tonne soil)
5170
Chemical
12600
Physical
20170
Solidification/stabilization
17171
Thermal
30750
with alternative methods. Treatment periods generally last from 2 to 48 months, about the same for
chemical or thermal methods. Physical processes
(soil washing and soil vapour extraction) are faster,
rarely lasting more than 1 year. Solidification is
almost instantaneous.
Bioremediation (when used in solution) doses not
require environmentally damaging processes such as
chemicals or heat treatment. It has beneficial effects
upon soil structure and fertility, but with limitation on
its effectiveness. These limitations may be summarized as follows:
Susceptibility to inhibition by other toxic contaminants such as heavy metals

Low biodegradability of some contaminants such
as chlorinated solvents
Possible residual contamination after treatment,
such as using hydrogen peroxide as an oxygen
provider
The potential formation of intermediate compounds which are more toxic than the original
treatment, such as dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane
(DDD) from the breakdown of DDT (Failey and
Scrivens 1994).
2 Green technology principles

Green technology, emanating directly from Green
Chemistry (or, environmentally benign chemistry)
may be described as the utilization of a set of principles
that reduces or eliminates the use or generation of
hazardous substances in the design, manufacture and
application of chemical products (Kidwai and Mohan
2005). In practice, Green Chemistry is taken to cover
a much broader range of issues than the definition
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suggests. As well as using and producing better

chemicals with less waste, Green Chemistry also
involves reducing other associated environmental
impacts, including reduction in the amount of energy
used in chemical processes (Kidwai and Mohan 2005).
Anastas and Warner (1998) have developed The
Twelve Principles of Green Chemistry that serve as
valuable and benchmark guidelines for practicing
chemists, researchers and engineers in developing and
assessing how green a synthesis, compound, process or
technology is. These principles are related to the
concepts of prevention, atom economy, less hazardous
chemical syntheses, designing safer chemicals, safer
solvents and auxiliaries, design for energy efficiency,
use of renewable feedstocks, reduce derivatives,
catalysis, design for degradation, real-time analysis
for pollution prevention and inherently safer chemistry
for accident prevention.
Green chemistry is an essential part of green
engineering. The definitions of green chemistry and
green engineering share many commonalities, and the
application of both chemistry and engineering principles is needed to advance the goals of environmental
sustainability (Kirchhoff 2003). A working definition
of green engineering proposed in Kirchhoff (2003) is
the design, commercialization, and use of processes
and products that are feasible and economical while
minimizing pollution at the source and risk to human
health and the environment. The link between green
chemistry and green engineering is strong in ensuring
that inputs and outputs, both for materials and energy
flows and budgeting, are as inherently safe as possible.
Whilst Green Chemistry focuses on the design of
chemical products and processes that reduce or
eliminate the use and generation of hazardous substances, it also lays down the ground plan for the
design of the green engineering technologies needed
to implement sustainable products, processes, and
systems (Kirchhoff 2003). The reader is in point of
fact directed to the following excellent publications
which present and discuss the salient aspects of
Green Chemistry and Green Engineering: Anastas
and Kirchhoff (2002), Anastas and Zimmerman
(2003), Anastas and Lankey (2000), Clark (2006),
Hofer and Bigorra (2007), Kirchhoff (2003), Lankey
and Anastas (2002), Ran et al. (2008), Tang et al.
(2008) and Tundo et al. (2000). The subsequent
discussions on bioremediation are contextualized
under the Green Technology concept.
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3 Merits and demerits of bioremediation

Although bioremediation is being engineered into a
novel and green technology, microorganisms have
been used routinely for the treatment and transformation of waste products for at least 100 years so far.
The municipal wastewater treatment industry which is
based on the exploitation of microorganisms in
controlled and engineered systems depends on the
metabolic activities of microorganisms which degrade
the organic matter in wastewaters arriving to wastewater treatment plants containing selected and acclimatized populations of microorganisms (Eckenfelder
1989; Vargas et al. 2000; Chen et al. 2005).
3.1 Merits of bioremediation

Bioremediation offers several advantages over the
conventional remediation techniques such as landfilling. Table 2 summarizes the chemical class and their
susceptibility to biodegradation. Often, bioremediation can be done on site, thereby eliminating transportation costs and liabilities. In many instances,
manufacturing and industrial use of the site can
continue while the bioremediation process is being
implemented. Bioremediation results in the decomposition of the waste and the long-term liability
associated with non-destructive treatment methods.
Finally, bioremediation can be coupled (i.e., integrated) with other treatment technologies into a
treatment chain allowing for the treatment of mixed
Table 2 Chemical classes

and their susceptibility to
bioremediation
Chemical class
and complex wastes (Yergeau et al. 2009; Goel et al.

2010; McMahon et al. 2008).
The use of renewable (waste) materials has also
boosted the bioremediation of waste streams (Deleu
and Paquot 2004). Residues such us cereals straw,
corn cobs, cotton stalks, various grasses and reed
stems, maize and sorghum stover, vine prunings,
sugarcane and tequila bagasse, coconut and banana
residues, corn husks, coffee pulp and coffee husk,
cottonseed and sunflower seed hulls, peanut shells,
rice husks, sunflower seed hulls, waste paper, wood
sawdust and chips, are some examples of residues
and by-products that can be recovered and upgraded
to higher value and useful products by chemical or
biological processes (Wang 1999; Fan et al. 2000;
Pandey et al. 2000a; Webb et al. 2004). In fact, the
chemical properties of such lignocellulosic agricultural residues make them a substrate of enormous
biotechnological value. They can be converted by
solid state fermentation (SSF) into various different
value-added products including mushrooms, animal
feed enriched with microbial biomass, compost to be
used as biofertilizer or biopesticide, enzymes, organic
acids, ethanol, flavours, biologically active secondary
metabolites and also for bioremediation of hazardous compounds, biological detoxification of agroindustrial residues and biopulping (Pandey et al.
2000b; Bennet et al. 2002; Sanchez et al. 2002; Kim
and Dale 2004; Nigam et al. 2004; Zervakis et al.
2005; Krishna 2005). SSF has been suggested for
upgrading and valorizing lignocellulosic residues
using basidiomycetous cultures, either through
Examples
Aromatic hydrocarbons
Benzene, toluene
Aerobic and anaerobic
Ketones and esters

Petroleum hydrocarbons
Acetone, MEK
Fuel oil
Aerobic and anaerobic

Aerobic
Chlorinated solvents
TCE, PCE
Aerobic (methanotrophs), anaerobic

(reductive dechlorination)
Polyaromatic
hydrocarbons
Anthracene, benzo
(a)pyrene, creosote
Aerobic
PCBs
Arochlors
Some evidence; not readily degradable
Organic cyanides
Aerobic
Metals
Cadmium
Not degradable experimental biosorption
Radioactive materials
Corrosives
Uranium, plutonium
Inorganic acids, caustics
Not biodegradable
Not biodegradable
Asbestos
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Biodegradability
Not biodegradable
protein enhancement and transformation of residues

into animal feed (Zadrazil 2000), or for enzyme
production (Revankar et al. 2007, Elisashvili et al.
2008). With specific reference to lignocellulolytic,
mushroom fungi like Pleurotus ostreatus and Trametes versicolor have been investigated for bioremediation and biodegradation of toxic and hazardous
compounds like caffeinated residues (Fan et al. 2000)
as well as toxic chemicals such as pesticides, PAHs
and PCBs and chlorinated ethenes (CIUs) in polluted
soils or contaminated groundwater (Perez et al. 2008;
Rigas et al. 2007).
3.2 Demerits of bioremediation
Like most treatment technologies, bioremediation
also has its limitations and disadvantages. Some
chemicals, e.g., highly chlorinated compounds and
heavy metals, are not readily amenable to biological
degradation and stabilization. Table 2 also summarizes the general categories of contaminants and their
relative susceptibility to biodegradation. In addition,
for some chemicals, microbial degradation may lead
to the production of more toxic or mobile substances
than the parent compound(s). For example, under
anaerobic conditions, TCE undergoes a series of
microbiologically mediated reactions resulting in the
sequential removal of chlorine atoms from the
molecule. This process is called reductive dehalogenation. The end product of this series of reactions
is vinyl chloride (VC), a known carcinogen. Thus, if
bioremediation is applied without a through understanding of the microbial processes involved and the
metabolic and chemical pathways, it could actually
lead to a worse situation than already exists in some
cases. Hence, bioremediation is a scientifically
intensive procedure, which must be tailored to the
site-specific conditions to minimize the effects of
environmental and kinetic constraints (Price et al.
2004; Beck and Jones 1995). Therefore, initial costs
for site assessment, characterization and feasibility
evaluation for bioremediation may be higher than the
costs associated with more conventional technologies
such as air stripping. As with remediation technologies, there is also the need for extensive monitoring
of the site during implementation of the project
(Sabean et al. 2009) to assess the effectiveness of the
bioremediation technique in its clean-up performance. Monitoring requirements may include some
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form of microbiological monitoring in addition to the

chemical monitoring associated with physical/chemical remediation techniques. Finally, there are regulatory constraints that impact on the implementation
of bioremediation (Talley and Sleeper 2006).
4 Bioremediation technologies
Bioremediation technologies can be broadly classified
as ex situ or in situ (Hatzinger et al. 2002; Talley and
Sleeper 2006). Table 3 summarizes the most commonly used bioremediation technologies. Ex situ
technologies are those treatment modalities which
involve the physical removal of the contaminated to
another area (possibly within the site) for treatment.
Bioreactors, landfarming, anaerobic digestion, composting, biosorption and some forms of solid-phase
treatment are all examples of ex situ treatment techniques. In contrast, in situ techniques involve treatment of the contaminated material in place. Bioventing
for the treatment of the contaminated soil and biostimulation of indigenous aquifer microorganisms are
examples of these treatment techniques. Although
some sites may be more easily controlled and maintained with ex situ configurations (Talley and Sleeper
2006), others are more effective with in situ treatment.
Table 3 Bioremediation treatment technologies

Bioaugmentation Addition of bacterial cultures to a
contaminated medium; frequently used
in bioreactors and ex situ systems
Biofilters
Use of microbial stripping columns to treat

air emission
Biostimulation
Stimulation of indigenous microbial

populations in soils and/ or ground water;
may be done in situ or ex situ
Bioreactors
Biodegradation in a container or reactor;

may be used to treat liquids or slurries
Bioventing
Method of treating contaminated soils

by drawing oxygen through the soil to
stimulate microbial growth and activity
Composting
Aerobic, thermophilic treatment process in

which contaminated material is mixed
with a bulking agent; can be done using
static piles, aerated piles, or continuously
fed reactors
Landfarming
Solid-phase treatment system for

contaminated soils; may be done in situ
or in a constructed soil treatment cell
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For example, many sites are located in industrial/

commercial areas, and these sites normally consist of
numerous structures interconnected by concrete and
asphalt. These physical barriers would make excavation extremely difficult, and if the contamination is
deep in the subsurface, excavation becomes too
expensive. As a result of these physical barriers, the
required excavation efforts may make ex situ biotreatment impracticable. Other factors could also have an
impact on the type of treatment. At a typical site, the
contamination is basically trapped below the surface.
To expose the contamination to the open environment
through excavation can result in potential health and
safety risks (Talley and Sleeper 2006). In addition, the
publics perception of the excavation of contaminants
could be negative, depending on the situation. All of
these conditions clearly favor in situ biotreatment.
Nonetheless, the key is to carefully consider the
parameters involved with each site before evaluating
which technique to use (Talley and Sleeper 2006).
4.1 Microbial consortia for bioremediation

Regardless of the exact nature of the treatment
technology, all bioremediation techniques depend on
having the right microorganisms in the right place
with the right environmental conditions for degradation to occur (Iranzo et al. 2001; Baxter and
Cummings 2006). The right microorganisms are
those bacteria or fungi that have the physiological
and metabolic capabilities to degrade the contaminants. Although it is generally accepted that more
than 80% of the total microorganisms are unknown
(Iranzo et al. 2001), reactions mediated by both the
known and the unknown microorganisms are already
employed in biotreatment and in bioremediation
(Hamer 1993). This consideration, together with the
potential use of engineered microorganisms, offers an
expanded time scale technology (Pieper and Reineke
2000). In many instances, these organisms will
already be present at the (indigenous microorganisms). In other circumstances, such as bioreactors
treating wastes with high concentrations of toxic
material. In order for the microorganisms to degrade
the contaminants, they must be in close proximity to
the contaminants; they must be in the right place.
Thus, the presence of toluene-degrading microorganisms in the surface soils at a site will be of little use
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for the remediation of a contaminant which is

biodegradable. If such populations are not present,
then some mechanisms must be engineered to bring
the microorganisms into contact with the contaminants. This may involve such techniques as flushing
the system to transport the contaminants to aboveground bioreactors (Litchfiled 2005), the addition of
surfactants to the subsurface to release adsorbed
contaminants and render them available to the microorganisms (Singh et al. 2007), or the introduction
and transport of the microorganisms to the contaminated area. Once the right microorganisms are
present in the right place, the environmental conditions must favor the metabolic activities of the
microorganisms. Such environmental factors as temperature, inorganic nutrients (primarily nitrogen and
phosphorus), electron acceptors (oxygen, nitrate, and
sulphate), and pH can be modified to optimize the
environment for bioremediation (Singh et al. 2006a;
Ge et al. 2004).
The objectives of the bioengineered remediation
treatment processes are analogous to those conventional biological treatment operations. With conventional biological treatment systems, a treatment vessel
is engineered to provide optimal conditions for the
microorganisms to grow. As a result of their growth,
the microorganisms will metabolize the compound(s)
of interest, usually resulting in the production of
innocuous end products (Ahuja and Kumar 2003). An
example of this concept would be a wastewater
treatment facility. For this process, the conditions in a
treatment vessel (i.e., a large tank) are optimized (pH
is adjusted and provisions to control flow rates to
provide adequate contact time are optimized) to
promote biodegradation of the organic materials in
the wastewater. For a bioengineered treatment system,
instead of utilizing a manufactured container to
accommodate the treatment process, the soil environment could be bioengineered to create an in-place
treatment vessel and to provide optimal growth
conditions for the indigenous microorganisms present.
The effective application of this type of biological
treatment can result in the complete breakdown of the
contaminant(s) to innocuous end products in many
instances (Barnabe et al. 2009). Pyridine and pyridine
based products are of major concern as environmental
pollutants due to their recalcitrant, persistent, toxic
and teratogenic nature. Lodha et al. (2008) have
studied the biodegradation of pyridine by an isolated
consortium/strain under aerobic condition. Batch

experiment results revealed that at lower initial
pyridine concentrations (120 mg/l), almost complete
degradation was observed whereas at higher concentration (3050 mg/l), the degradation efficiency was
dropped significantly. Bioaugmentation of the isolated consortium/strain into the activated sludge
consortium in different quantity had been also done
and the effect of bioaugmentation on degradation has
been studied. This showed that as the quantity of
bioaugmentation increased, the degradation of pyridine also increased. Prasanna et al. (2008) have
studied the bioremediation of soil-bound anthracene
studied in a series of bio-slurry phase reactors
operated in periodic discontinuous/sequencing batch
mode under anoxicaerobicanoxic microenvironment using native soil microflora. Five reactors were
operated for a total cycle period of 144 h at soil
loading rate of 16.66 kg soil/m/day at 30 2C
temperature. The control reactor (without microflora)
showed negligible degradation of anthracene due to
the absence of biological activity, while the performance of the bio-slurry system with respect to
anthracene degradation was found to depend on both
substrate loading rate and bioaugmentation. Subsequent application of bioaugmentation however
showed positive influence on the rate of degradation
of anthracene. All the more, phytoremediation has
been used as an emerging technology for remediation
of soil contamination with PAHs, ubiquitous persistent environmental pollutants derived from natural
and anthropogenic processes, in the last decade. In
this respect, Xu et al. (2009) carried out a pot
experiment to investigate the potential of phytoremediation of pyrene from spiked soils planted with white
clover (Trifolium repens) in the greenhouse with a
series of pyrene concentrations ranging from 4.22 to
365.38 mg/kg. Their results showed that growth of
white clover on pyrene contaminated soils was not
affected. The removal of pyrene from the spiked soils
planted with white clover was obviously higher than
that from the unplanted soils. At the end of the
experiment (60 days), the average removal ratio of
pyrene in the spiked soils with white clover was 77%,
which was 31 and 57% higher than those of the
controls with or without microbes, respectively,
thereby supporting that the removal of pyrene in the
contaminated soils was feasible using T. repens.
Lately, Osman et al. (2009) investigated the
223
bioremediation of the nematicide, oxamyl, applied at

6 l/ha in sandy soil cultivated with tomato and
amended with different animal manures at the recommended dose of 2.5 tons/ha. By the end of the experiment (28 days), the dissipation percentage of oxamyl
reached about 99% in the case of bovine manureamended soil, and this rate of disappearance was 1.76
times higher than in unamended-soil, while poultry
and sheep manures enhanced the dissipation rate by
1.52 and 1.44 times, respectively. The results of
Osman et al. (2009) demonstrated that animal manures
may offer an efficient, cheap, safe, and friendly
bioremediator for pesticide-polluted soil.
4.2 Approach to bioremediation techniques
The successful implementation of bioengineered
remediation techniques will involve a multidisciplinary approach requiring input from individuals with
expertise in microbiology, chemistry, geology, soil
science, environmental engineering and chemical
engineering. In order to use bioengineering successfully for the remediation of environmental contamination problems, the first step is to obtain a through
understanding of the matrix characteristics of the
media to be treated and the properties (physical,
chemical and microbiological) of the contaminant(s).
Lai et al. (2007) stress that the performance monitoring of applied remediation technologies is an
important part of site remediation. It involves periodic measurement of site parameters to evaluate
whether the remediation technologies perform as
expected or to determine the termination date of
remediation projects. According to Lai et al. (2007),
performance monitoring can be a difficult undertaking if there are no well-defined and measurable
remediation objectives, such as a reduction in mass
discharge rate from a contaminant source. The
monitoring requirements for a bioremediation treatment system shall most reasonably comprise the
following analyses and inspections.
Daily: Inspection of the system components, i.e.,
piping, pumps and valves; monitoring of pH, dissolved oxygen, temperature, and mineral nutrient
levels within the treatment system, and monitoring
flow rates and pumping rates.
Monthly: Monitoring the following parameters
within the treatment system and in the off-site
monitoring wells: contaminant concentration, aerobic
123
224
heterotrophic bacterial population density, pH, dissolved oxygen, temperature and available mineral
nutrient concentrations.
Quarterly: perform a series of soil boring and
analysis for the following parameters: contaminant
concentration, aerobic heterophic bacterial population density, pH, soil moisture and available mineral
nutrient concentrations. Subsequently, any adjustment in the bioremediation technique will be made
accordingly to the treatment system based on the
results from these analyses so as to enhance the
bioremediation performance.
All the more, through the advances in gene technology, bioremediation is now in a position to take
advantage of genomic-driven strategies to analyze,
monitor and assess its course by considering multiple
microorganisms with various genomes, expressed transcripts and proteins (Stenuit et al. 2009). High-throughput methodologies, including microarrays, fingerprinting
(Karpouzas and Singh 2010), real-time polymerase chain
reaction (PCR) (Baek et al. 2009), genotypic profiling,
ultrafast genome pyrosequencing, metagenomics, metatranscriptomics, metaproteomics and metabolomics
(Desai et al. 2010; Jerez 2009), show great promise in
environmental interventions against recalcitrant contaminants such as 2,4,6-trinitrotoluene (TNT) that have been
studying for many years. The emerging genomic and
metagenomic methodologies now allow environmental
researchers and engineers to promote and restore environmental health in impacted sites, monitor remediation
activities, identify key microbial players and processes,
and finally compile an intelligent, site-specific and
pollutant(s)-specific database of genes for targeted use
in bioremediation (Stenuit et al. 2009).
5 Bioremediation techniques for contaminated

sites
Soil is one of the key resources for sustainability and
survival, and its degradation caused by willful or
accidental contamination from industrial sources or
degradation caused by salination and waterlogging is
a great matter of concern. Land degradation is
recognized as the loss of the fertility or potential
utility by changes in irreplaceable features or communities of organisms. Bacteria and fungi are natural
recyclers capable of transforming natural and
123
synthetic chemicals into sources of energy and raw

materials for their own growth. This implies that
biological processes supplement chemical or physical
remediation processes and that is why bioremediation
is becoming important for the clean-up of contaminated soils all around the world.
Contamination of soils can occur through the
accidental release of materials on the surface or
through the direct introduction of contaminants into
the subsurface, as in the case of leaking underground
storage tanks. From the perspective of remediation, the
soil environment can be divided into two zones:
shallow surface soils and subsurface (vadose) soils.
Shallow surface soils usually include the upper 13 ft
of the environment. These soils represent the region of
the environment typically included in the agronomic
definition of soils. They are easily modified and are
generally more amenable to remediation than deeper
vadose soils. Operationally, surface soils can be
defined as those soils which can be excavated or
treated by surface amendments not requiring the
installation of wells. Vadose soils are those soils
which lie between the surface soils and the water table
or aquifer. Vadose soils are generally unsaturated,
although there may be pockets of water saturated soil
within the vadose zone, particularly in the area of the
root zone and in the capillary fringe at the surface of
the water table. In addition, there may be inclusions of
low permeability materials such as clay lenses within
the vadose zone, which can become saturated with
water. Unlike surface soils, vadose soils are often not
amenable to excavation or surface treatment; rather,
modification of such soils usually involves the use of
infiltration galleries, injection wells, or other engineered means for introducing materials.
5.1 Characterization of contaminated sites by

molecular diagnostic
A prerequisite for any remediation strategy is the
characterization of the sites with regards to various
factors that may affect bioremediation, particularly
characterization of indigenous bacterial communities
(Hjeitzer and Sayler 1993). Knowledge of the types,
concentrations and activities of biodegradative bacteria and of the processes that control their activities
is important in at least two regards:
225
as part of site characterization for determining

appropriate remediation strategies; and
as part of monitoring the progress and effectiveness of bioremediation.
Although rarely used, concentrations of specific

bacteria may also serve as indicators of remediation
endpoints i.e., as indicators of remediation sufficient to declare a site clean. Identification and
enumeration of individual bacterial species are usually deemed too time consuming and laborious for
routine analysis, and is usually dispensed with in
favor of evaluation of biodegradative potential of
samples in laboratory microcosms.
Among the limitations of site characterization by
bench top microcosms are the lack of sensitivity;
inaccuracy due to an inability to cultivate most soil
bacteria in the laboratory; deviation from what is
actually occurring in the field; and cumbersome
experimental setups. In addition, many sites are
contaminated with mixtures of compounds, thereby

greatly increasing the numbers of analyses required for
sufficient site characterization. A relatively new
approach, molecular diagnostic (Sayler and Layton
1990; Ritalahti et al. 2005; Katsoyiannis et al. 2007),
provides an alternative to traditional laboratory microcosms and has great potential to provide important
information on resident microbial communities that
would be unavailable from traditional laboratory
microcosm studies. The most commonly applied
molecular genetic approach to site characterization is
so-called molecular diagnostic, referring to the use
of cloned genes of interest (gene probes) to measure
concentrations of similar genes in microbial DNA or
RNA directly extracted from the soil sample (Yagi and
Madsen 2009; NGuessan et al. 2010) (Fig. 2).
Molecular diagnostics utilizes information regarding the structure and activities of resident microbial
communities contained in the communitys nucleic
Soil sample
Addition of
bioluminescent
reporter strains
Cell lysis and

extraction of
nucleic acids
Measurement of
light production
BIOAVAILABILITY
OF SUBSTRATE
Purification
of DNA
Hybridization with
gene probes
BIODEGRADATIVE
POTENTIAL
Purification
of RNA
Hybridization with
gene probes
IN SITU
ACTIVITY
Fig. 2 Flow chart of sample characterization by molecular diagnostics
123
226
acid to characterize the biodegradative potential of a

site. Much of the information required for accurate
site characterization is encoded in the microbial
communitys DNA, and analyzing DNA directly
isolated from samples may access much of this
information. Utilizing DNA as an indicator molecule
in this way bypasses the need for laboratory cultivation, and therefore bypasses much of the bias and
uncertainty associated with laboratory incubations. In
addition, molecular diagnostics can be a very rapid
and accurate means of simultaneously screening
numerous samples, thereby increasing the efficiency
with which samples may be processed.
Gene probes cloned from bacterial pathways for
metabolisms of the pollutant of interest may be used
against a large number of samples in a relatively short
period of time, simultaneously providing information
on the specific pathways present and the concentrations of bacteria possessing the genes. Selected
examples of gene probes are listed in Table 4.
Information regarding specific pathway(s) involved
in the bioremediation process will increase understanding of specific environmental factors that may be
manipulated for optimization of then bioremediation
process. The approach may be quantitative, and the
relative concentration of specific genes in a given
sample is related to the potential for that sample to
biodegrade the compound of interest. This information
will aid scientists and engineers in developing and
monitoring remediation strategies appropriate for the
site and the types of bacteria available for remediation.
The potential for biodegradation to occur is
indicated by the concentration of the gene of interest
within the community DNA, and this information
may be accessed by using DNA as the target for gene
probes. This approach dose not yield information
regarding the activity of these genes, or serve as an

indication of in situ biodegradation rates. The activity
of the genes is expressed as RNA, and using RNA as
a target for gene probes may, in some cases, correlate
with biodegradation rates. Virtually all studies
attempting to correlate biodegradation rates with
mRNA concentrations have involved correlating
nahA (naphthalene biodegradation rates (Fleming
et al. 1993; Sanseverino et al. 1993). Using RNA as
a target in such studies may be limited for some genes
due to the very high turnover rate of some mRNAs,
but nahA transcripts have correlated well with
biodegradation rates in a number of soils.
Some advantages of molecular genetic applications
to site characterization are a rapid characterization of
numerous samples; the simultaneous characterization
of multiple biodegradative pathways from mixedwaste sites; acquisition of information regarding the
specific biochemical pathway most likely to dominate
remediation process; possibility for a rapid assessment
of the progress of biostimulation strategies; and a rapid
quantitative assessment of indicators of remediation
endpoints.
As with any microbial method, molecular diagnostic has limitations, although most of these are
being overcome with time. The greatest limitation to
date is that the gene of interest must be known. Soils
harbor a great diversity of bacteria (perhaps as many
as 10,000 distinct species), most of which have never
been cultivated in the laboratory. Laboratory cultivation is an obvious prerequisite for studying the
genetics of biodegradation, and it is likely that we
currently know very little of the diversity of genes
involved in biodegradation of many organic pollutants. This lack of information limits the number of
gene probes available for use, and in many case
Table 4 Example of gene probes used in molecular diagnostics

Target compound
Target enzyme
Target gene
Host strain
Reference
Toluene, TCE
Toluene-4-monoxygenase
Tom A
Burkholderia cepacia G4
Shield et al. (1989)
Toluene, TCE
Toluene, TCE
Toluene dioxygenase
Toluene-2-monoxygenase
TodC1C2BA
tmoABCDE
Pseudomonas putida F1
Pseudomonas mendocina KR1
Zylstra and Gibson (1989)

Yen et al. (1991)
BTEX compounds
Catechol 2,3 dioxygenase
xylE
Pseudomonas putida mt-2
Assinder and Williams (1990)
TCE
Soluble methane
monooxygenase
mmoB
Methylosinus trichsporium
0B3b
Tsien and Hanson (1992)
Naphthalene
Naphthalene dioxygenase
nahAcd
Pseudomonas putida G7
Simon et al. (1993)
123
molecular diagnostic may underestimate both the

numbers of bacteria involved in bioremediation and
types of biodegradative pathways involved.
It is not reasonable to over-emphasize the great
diversity of soil bacteria and our relative ignorance of
the genetic and biochemical diversity present in the
soil. A recent example of the failure of molecular
diagnostics to accurately describe the biodegradative
potential of a site involved a dominant and undescribed toluene pathway. In this case, DNA was
isolated from soil that rapidly mineralized toluene, and
analyzed with gene probes specific to all five toluene
pathways known at that time. No positive signals were
observed with any of the gene probes against the soil
DNA, indicating that these genes were not likely to
represent the dominant toluene degrading bacteria if
they were present at all in the samples. Toluene
metabolizing bacteria were isolated from the soil, and
none of these harbored genes similar to the five known
pathways. The dominant toluene degrading bacterial
strain in this soil was later identified as a Rhodococcus
sp., a gram positive species that shares relatively low
similarity at the DNA level to all cloned genes
involved in toluene metabolism. Virtually all that is
known of the genetics and biochemistry of biodegradation was learned from gram negative species such as
Pseudomonas. These species grow rapidly in standard
enrichment cultures and frequently out grow other
species that may be equally or more important in the
soil. Until more is known of the diversity of biodegradative bacteria in soil, the general applicability of
molecular diagnostic will be limited.
5.2 Treatment of contaminated vadose soils

Treatment of contaminated vadose soils several feet
below the surface is a challenging task which usually
involves some types of in situ treatment system(s).
Electronic acceptors, inorganic nutrients, and other
supplements (i.e., bacterial cultures) are introduced,
if necessary, into the subsurface environment to
stimulate microbial degradation of the contaminants.
Proper controls must be included in the system to
ensure that the contaminants do not migrate.
Water was the first medium used to transport
materials throughout the vadose zone. In this approach
to bioremediation, hydraulic control of the site is first
established. This typically involves installation of a
227
series of injection wells or trenches and recovery

wells. Alteration of the water table level also may be
undertaken. Nutrients and oxygen dissolved in water
are injected into the subsurface environment. As the
water percolates through the subsurface, nutrients and
oxygen are delivered to the microorganisms, stimulating biodegradation of the contaminant. The material
which leaches through the vadose zone into the
saturated zone is captured and pumped to the surface,
where it is treated, if necessary, and recirculated into
the system. This type of treatment system is fundamentally the same as treatment systems designed for in
situ bioremediation of contaminated aquifers. Often,
contaminants in the vadose zone and concomitant
contamination of groundwater are treated as a single
unit. Several research works have reported the success
of contaminated vadose soils clean-up by bioremediation. Table 5 summarizes some of these most recent
examples.
5.3 Bioremediation of oil contaminated media

Large areas of earth surface including land and water
are contaminated with oil-derived compounds and
toxic chemicals. More than 2 million tonnes of oil are
estimated to enter the sea each year. 50% from
industrial effluents, sewage and river overflows and
the rest from non-tanker shipping and natural seepage
from below sea floor. Only about 18% comes from
refineries, off-shore operations and tanker activities.
Oil contamination is easy to detect unlike other
pollutants. Most oils are relatively less toxic to
environment but can affect bird and aquatic animal
life very seriously. Three main types of bioremediation
technologies that are currently being developed for
treatment of oil spills are: (a) addition of nutrients to
open water oil slicks, (b) addition of microbes to oiled
shorelines, and (c) addition of nutrients and/or
microbes to open water slicks. Just adding oil to an
environment will stimulate growth of indigenous
microbes as oil acts as a source of carbon. But there
is a lag period, which can be several days to several
weeks, before they can effectively degrade oil. During
this period, certain fractions initially toxic to microbes
undergo weathering. On March 23, 1989 about 11
million gallons of Prudhoe Bay crude oil were spilled
into Prince William Sound, Alaska from the tanker
Exxon Valdez. The spilled oil spread over 350 miles of
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228
Table 5 Example of bioremediation studies of contaminants in vadose soils

Contaminant(s)
Technology/technique employed
Bioremediation performance
Reference
Perchlorate
Experiments performed in soil slurries With no external carbon source added to Gal et al. (2008)
with sediments taken from the
the slurry of soil from land surface, all
contaminated site with native
perchlorate was removed after 134 days
microbial communities along
of incubation
the contaminated vadose zone
Average perchlorate-reduction rate using
natural organic matter as a carbon
source was 0.45 mg/day, while the
average rate using acetate as an external
carbon source was 7.2 mg/day
Perchlorate and nitrate
Gaseous electron donor injection

technology
Laboratory microcosm studies

Evans and Trute
demonstrated that hydrogen and ethanol
(2006)
promoted nitrate and perchlorate
reduction in vadose zone soil
Nitrate removal in the column studies, up
to 100%, was best promoted by ethyl
acetate
Up to 39% perchlorate removal was
achieved with ethanol and was limited
by insufficient incubation time
Chromate
Test involved injecting hydrogen

sulphide, diluted in air, into
contaminated vadose zone sediment
to reduce Cr(VI) to Cr(III)
All Cr(VI) concentrations measured in the Thornton et al.

posttest samples were well below the
(2007)
remedial goal and regulatory limit of
30 mg/kg
In addition, the field test demonstrated
that vadose zone treatment of
contamination could be safely
conducted using diluted hydrogen
sulphide gas mixtures
Naphthalene at a creosotecontaminated
Combined remediation mechanisms

of volatilization and biodegradation
Soil gas sampling showed that more than Andersen et al.

90% of the naphthalene vapors were
(2008)
biodegraded aerobically within
510 cm above the water table
Toluene
Radiation-resistant bacterium
Deinococcus radiodurans was
engineered for toluene degradation
by cloned expression of tod and xyl
genes of Pseudomonas putida
Complete oxidation of the toluene by the Brim et al.

engineered bacteria under both minimal
(2006)
and complex nutrient conditions
Atrazine [6-chloro-N-ethylN0 -isopropyl-1,3,5triazine-2,4-diamine] and

cyanazine {2-[[4-chloro6-(ethylamino)-1,3,
5-triazin-2-yl] amino]-2methylpropanenitrile}
Overall temporal sampling (0342 days)

Combined chemicalbiological
revealed atrazine and cyanazine
approach comprising
concentrations decreased by 7991%
Fe0 ? FeSO4 ? emulsified soybean
[Glycine max (L.) Merr.] oil (EOS),
EOS remediation scenarios
shoreline. About 1520% was lost by initial weathering due to volatilization. Mostly aliphatic hydrocarbons of less than 12 carbon atoms, aromatic
hydrocarbons such as benzene, toluene, xylene and
methyl-substituted naphthalenes were lost this way.
123
Waria et al.
(2009)
The weathered oil was black in colour. Initial tests

were conducted following which bioremediation was
successfully undertaken to overcome this problem.
Table 6 presents a digest of recent studies conducted
to bioremediate oil contaminated soils.
Tsai et al. (2009)

Three-stage treatment train system to remediate fuel Results from the column study indicate that approximately
oil-contaminated soils: first stage of biodegradable
80% of TPH in soil (with initial concentration of 50,000 mg/
surfactant and groundwater flushing followed by
kg) could be removed after the Simple GreenTM (SG)
[50 pore volumes (PVs)] followed by groundwater (30PVs)
the Fenton-like oxidation, ending with application
flushing. The Fenton-like oxidation (with 6% of H2O2
of enhanced bioremediation for the removal of
addition) was able to remove another 15% of TPH. Observed
residual total petroleum
first-order reaction rate constant of TPH oxidation was
2.74 9 10-2/min, and the half-life was 25 min during the
first 40 min of reaction
Bacterial consortium tested for biodegradation

potential
Efficacy of Candida catenulata CM1, was evaluated After 13 days of composting, 84% of the initial petroleum
Joo et al. (2008)
during composting of a mixture containing food
hydrocarbon was degraded in composting mixes containing
waste and diesel-contaminated soil
a powdered form of CM1 (CM1-solid), compared with 48%
of removal ratio in control reactor without inoculum
PAH degradation efficacy in presence of Salix

viminalis was investigated in a greenhouse
experiment
Fuel oil-contaminated soils
Oil contaminated and

pristine soils from Signy
Island (South Orkney
Islands, Antarctica)
Diesel-contaminated soil
PAH in an aged creosotecontaminated soil
Presence of S. viminalis or the surfactant enhanced PAH

degradation, primarily by a rhizosphere effect on the
microbial activity in the former case and by increased
bioavailability in the latter case
Phenanthrene and pyrene were degraded 100 and 80%,

respectively, in the presence of plants but only 68 and 63%
without plants
Pseudomonas species was dominant as soil bacteria in both

bioaugmented and biostimulated microcosms
Addition of water to oil contaminated soil (hydration) also

enhanced oil degradation, although less than the other
treatments
Hultgren et al. (2010)
Of the 300 isolates cultured, Pseudomonas strain ST41 grew on Stallwood et al. (2005)
the widest range of hydrocarbons at 4C. Microcosm
experiments showed that at 4C the levels of oil degradation
increased, relative to the controls, with (i) the addition
of ST41 to the existing soil microbial population, (ii) the
addition of nutrients and to the greatest extent with (iii)
a combination of both treatments
P. aeruginosa strains were more efficient than B. subtilis strain

in reducing the TPH content from the medium
Bioaugmentation of TPH contaminated microcosm with

Das and Mukherjee (2007)
P. aeruginosa M and NM consortia and B. subtilis strain
showed a significant reduction of TPH levels in treated soil as
compared to control soil at the end of experiment (120 days)
Bacterial degradation in shake flask tests using

Bacillus subtilis DM-04 and Pseudomonas
aeruginosa M and NM strains
Reference
Crude petroleum-oil
hydrocarbons
Technique employed
Oil/Oil fraction
Table 6 Resume of recent studies on bioremediation of oil contaminated soils

229
123
230
5.4 Bioremediation of mine spoil dumps

The rapid increase in industrialization in all sectors
has led to degradation of natural resources i.e., air,
water and soil. Mineral exploitation (mining) is
second only to agriculture as the worlds oldest and
most important industry and its operation leads to a
number of environmental problems namely deforestation, removal of fertile top soil, unstable slopes
prone to sliding and erosion, siltation of water bodies
due to wash off of mineral overburden dumps, air
pollution due to discharge of dust, ground vibration
and finally the socio-economic status of local people.
As a result of mining activities, significant areas of
land are degraded and undesirable materials in the
form of overburden dumps, tailings and ash dams
replace the existing ecosystems. The overburden
materials (solid wastes) thus produced are physically
and structurally unstable, prone to subsidence and
chemically as well as hydrogically unsuitable for
plant growth. The degraded lands are devoid of
nutritive and supportive capacity for biomass development. Gradual increase in such landscapes due to
intensive mining activities adversely impacts aquatic,
land and atmospheric ecosystems. Ecological amelioration of these ecovulnerable systems is a challenging task as the top soil suitable for plant growth,
which takes a number of decades to be produced have
been disturbed due to mining and buried deep down
the biologically unproductive surface.
Thus, realizing the major physical, chemical and
biological constraints in biorestoration, an Integrated
Biotechnological Approach (IBA) was developed
(Juwarkar et al. 2000) to restore the natures pattern
of air, water and land blending stable and diverse
ecosystem comprising of different components of
flora and fauna. An IBA is a biocompatible technology which comprised of isolation and inoculation of
site specific specialized nitrogen fixing strains of
Bradyrhizobium and Azotobacter species and nutrient
mobilizing vesicular arbuscular mycorrhizal spores of
Glomus and Gigaspora species in combination with
industrial waste material available near the vicinity of
mine site used as organic amendments to ameliorate
the mine spoil and encourage revegetation. Different
plant species of ecological and economical importance can be planted on mine spoil dumps using
appropriate blends of organic waste along with site
specific nitrogen fixing bacteria and endomycorrhizal
123
fungi. Besides this the plants having medicinal value

can be planted on the sites. Different types of grasses
for slope stabilization can be used on large scale to
prevent runoff and erosion of dumps. Thus, IBA is an
ecofriendly technology for holistic restoration of lost
biological diversity of the mined out areas and
commercial utilization of such degraded landscapes
through plantation of ecologically and economically
important plant species. The technology has been
successfully demonstrated and commercialized to
solve problems of different mining sectors and thermal
power (fly ash dump reclamation). Table 7 presents a
summary of some selected studies conducted on the
bioremediation of mine related contaminants.
6 In situ bioremediation
6.1 Bioventing
In bioventing, the aerobic biodegradation of soil
contamination is stimulated by delivery of oxygen to
the subsurface. This is accomplished by injecting or
extracting air through unsaturated soil in a passive
system. This technology is designed primarily to treat
soil contamination by fuels, non-halogenated volatile
organic compounds (VOCs) and semi-volatile organic
compounds (SVOCs), pesticides and herbicides. The
process may be applied to halogeneted organics, but is
less effective. Bioventing typically costs around $15
per cubic yard of soil and uses simple, inexpensive,
low-maintenance equipment that can be left unattended for long periods of time. Also, the technology
tends to enjoy good public acceptance.
The technology requires the presence of indigenous organisms capable of degrading the contaminants of interest, as well as nutrients necessary for
growth. Also, it is necessary that the contaminants be
available to the organisms, and not tightly sorbed to
soil particles. Bioventing is not as effective in treating
areas where the water table is high, and soils with
very low moisture content. Lastly, the technology is
not applicable in sites where high concentrations of
inorganic salts, heavy metals, or organic compounds
are present, as these hinder microbial growth. However, some studies have demonstrated the merits of
bioventing as a bioremediation technique. Mller
et al. (1996) have investigated the effects of
231
Table 7 Studies on remediation of contaminants from mining sites

Contaminants/
contaminated media
Technique employed
Reference
Fuel oil
contaminated
mixtures of soil
and sawdust
Feasibility of aerated in-vessel

composting at a laboratory scale as
a bioremediation technology to
clean-up contaminated desert
mining soils (fuel
concentration [ 50,000 mg kg-1)
and sawdust (fuel
concentration [ 225,000 mg/kg) in
the Atacama Region
The highest (59%) and the lowest (35%)

contaminant removals were observed in
the contaminated sawdust and
contaminated soil reactors after 56 days
of treatment, respectively
Godoy-Faundez
et al. (2008)
Column experiments were carried out

to evaluate the feasibility of using
humic acid (HA) to mobilize arsenic
and heavy metals
It was found that the HA could significantly

enhance the mobilization of arsenic and
heavy metals simultaneously from the
mine tailings. After a 70-pore-volumeflushing, the mobilization of arsenic,
copper, lead and zinc reached 97, 35, 838
and 224 mg/kg, respectively
Arsenic and heavy

metals (i.e., Cu, Pb
and Zn) from
oxidized PbZn
mine tailings
samples
Results of this research indicate that

bioremediation of an aged contaminated
mixture of desert mining soil and sawdust
with fuel oil is feasible
Wang and
Mulligan
(2009)
Use of HA in arsenic and heavy metal

remediation was deduced to show
promise as an environmentally benign
and possible effective remedial option to
reduce and avoid further contamination
bioventing, nutrient addition and inoculation with an

oil-degrading bacterium on biodegradation of diesel
oil in unsaturated soil in a mesocosm system consisting
of 6 soil compartments each containing 6 m3 of
naturally contaminated soil mixed 1:1 with silica sand,
resulting in a diesel oil content of approximately
2,000 mg/kg. Biodegradation was monitored over
112 days by determining the actual diesel oil content
of the soil and by respirometric tests, and it was
observed that the oil composition changed following
degradation resulting in the unresolved complex
mixture constituting up to 96% of the total oil content
at the end of the experimental period. Lately, Shewfelt
et al. (2005) have conducted experiemnets using
small-scale respirometers containing gasoline-contaminated soil from an active remediation site to
determine the effects of soil water content, nitrogen
content, nitrogen form, and the composition of the
microbial population on the gasoline biodegradation
rate. Results indicated that optimum bioventing conditions were 18 wt.% soil water content, C:N = 10:1,
using NH4?Sui et al. (2006) have studied the
cometabolic bioventing for removal of TCE in the
unsaturated zone in a soil column study using methane
as growth substrate, and the experimental data showed
that a total TCE remediation efficiency of over 95%

was obtained with a volatilization -to- biodegradation
ratio of TCE being about 7:1.
6.2 Biostimulation
Biodegradation in the subsurface may be stimulated
by addition of water-based solutions carrying nutrients, electron acceptor or other amendments. These
technologies are designed primarily to treat soil and
groundwater contamination by fuels, non-halogeneted VOCs, SVOCs, pesticides, and herbicides. These
processes may be applied to halogeneted organics,
but are sometimes less effective. Although the costs
of biostimulation technologies vary tremendously
from site to site, these technologies tend to be
amongst the cheapest alternatives when applicable.
The technology requires the presence of indigenous
organisms capable of degrading the contaminants of
interest. Also, it is necessary that the contaminants be
available to the organisms, and not tightly sorbed to
soil particles. With specific reference to chlorine
containing contaminants, the successful application
of bioaugmentation requires consideration of a number of additional factors including:
123
232
1.
2.
3.
the availability of a sufficient amount of culture

to facilitate complete dechlorination of the target
contaminant;
the presence of co-contaminants that may affect
biodegradation; and,
the added cost and benefit of adding bacterial
cultures.
Biostimulation is not applicable in sites where high

concentrations of inorganic salts, heavy metals, or
organic compounds are present, as these hinder microbial growth. Lastly, the calculation of water-based
solutions through the soil may increase contaminant
mobility and necessitate treatment of underlying groundwater. Preferential colonization by microbes may occur
causing clogging of nutrient and water injection wells.
Recent studies on the application of biostimulation
for degrading a variety of contaminants unanimously
advocate the merit of this technique. Krishnani et al.
(2009) have used molecular methods based on
sequencing of clone libraries to provide sequence and
the phylogenetic information of ammonia oxidizing
bacteria (AOB). Ammonia monooxygenase (amoA)
gene, which catalyzed the oxidation of ammonia to
hydroxyl amine in the initial rate-determining step of
nitrification was targeted for detection and characterization of AOB using gene-specific primers. The use of
a matrix prepared from abundantly available lignocellulosic agrowaste-bagasse has successfully been demonstrated for biostimulation of AOB in aquaculture
environment by supplementing nutritional requirement facilitating the biofilm mode of growth of the
autotrophic consortia, the applicatiom of the results of
this study could be useful in enhanced predictability
and reliability of the treatment of ammonia in brackishwater aquaculture. Dafale et al. (2008) have identified the strains viz. Pseudomonas aeruginosa and
Bacillus circulans and other unidentified laboratory
isolates (NAD1 and NAD6) to be predominantly
present in a microbial consortium acclimatized from
activated sludge from a textile effluent treatment plant
to effectively decolorize RB5 dye solutions. The
optimum inoculums concentration for maximum
decolorization were found to be 15 ml of 1,800 mg/
l MLSS and 37C, respectively. Overall, the effectiveness of the acclimatized biomass under optimized
conditions towards decolorization of two types of
synthetic dye bath wastewaters that were prepared
using chosen azo dyes has been demonstrated.
123
Hirschorn et al. (2007) have reported based on stable

carbon isotope analysis that the dechlorination of TCE
was occurring in in situ biostimulation pilot test areas
during biostimulation. Garcia-Blanco et al. (2007)
have assessed the effectiveness of biostimulation in
restoring an oil-contaminated coastal marsh dominated
by Spartina alterniflora under north-temperate conditions through nitrogen and phosphorus addition for
accelerating oil disappearance, and then have equally
determined the role of nutrients in enhancing restoration in the absence of wetland plants, and the rate at
which the stressed salt marsh recovered. It was
reported that GCMS resolved alkanes and aromatics
degraded substantially ([90 and [80%, respectively)
after 20 weeks with no loss of TPH. Earlier, Hamdi
et al. (2007) had studied the degradation of spiked
anthracene (ANT), pyrene (PYR) and benzo[a]pyrene
P
(B[a]P) in soil (3,000 mg
3 PAHs/kg dry soil) in
aerobically incubated microcosms for 120 days. The
applied treatments aimed at enhancing PAH removal
from the heavily contaminated soils were: (i) bioaugmentation by adding aged PAH-contaminated soil
(ACS) containing activated indigenous degraders; and
(ii) combined bioaugmentation/biostimulation by
incorporating sewage sludge compost (SSC) and
decaying rice straw (DRS). Hamdi et al. (2007)
reported that the adopted treatments produced higher
PAH dissipation rates than those observed in unamended PAH-spiked soils, especially for ANT and
PYR in the presence of DRS or ACS ([96%). Later,
Salinas-Martnez et al. (2008) have studied the biostimulation of the native microbial consortium as a novel
application of the heap leaching technique to bioremediate mining soils contaminated with hydrocarbons.
Two genera, Flavobacterium and Aspergillus, were
identified as the primary microorganisms that degraded
hydrocarbons in the polluted soil. The main outcomes
showed that of the rates tested, biodegradation was
most efficient at a flow rate of 200 ml/h, and the heap
leaching technique demonstrated good efficiency in
the column and pile arrangement, with a 2% soil-sand
mixture lowering the TPH concentration from 61,000
to 1,800 mg/kg (98.5%) in 15 days.
6.3 Air-sparging
Air-sparging stimulates aerobic biodegradation of
contaminated groundwater by delivery of oxygen to
233
the subsurface (Johnson et al. 2007; Tsai 2008). This

is accomplished by injecting air below the water
table. This technology is designed primarily to treat
groundwater contamination by fuels, non-halogenated VOCs, SVOCs, pesticides, organics, and herbicides. Air sparging has also been demonstrated to
be an innovative groundwater remediation technology capable of restoring aquifers that have been
polluted by volatile and (or) biodegradable contaminants, such as petroleum hydrocarbons (Heron et al.
2002; Gidarakos and Aivalioti 2008). The process
may be applied to halogenated organics, but is less
effective. Air-sparging can cost less than $1 per
1,000 l in favorable situations and tends to be among
the cheapest remedial alternatives when applicable.
The technology uses simple, inexpensive, low-maintenance equipment that can be left unattended for
long periods of time. Also, the technology tends to
enjoy good public acceptance. The technology
requires the presence of indigenous organisms capable of degrading the contaminants of interest, as well
as nutrients necessary for growth. Also, it is necessary that the contaminants be available to the
organisms, and not tightly sorbed to soil particles.
Air sparing is not applicable in sites where high
concentrations of inorganic salts, heavy metals, or
organic compounds are present, as hinder microbial
growth. Table 8 reports some studies on the application of air sparging to bioremediate contaminated
media.
while non-destructive processes cause a reduction in

contaminant concentrations (Khan et al. 2004).
Target contaminants for natural attenuation include
fuels, non-halogenated VOCs, SVOCs, pesticides and
herbicides. The process may be applied to halogenated
organics, but it requires longer treatment times. Also,
the technology is applicable to especially hydrophobic
contaminants such as high molecular weight PAHs that
tend to sorb very tightly to soil particles and have very
low rates of migration. Often, communities of adopted
degraders will mineralize such contaminants quickly
after they desorb from soil particles. The costs of
natural attenuation are associated with modeling
contaminant migration, degradation rates to determine
its feasibility, and evaluation and monitoring costs to
confirm adequate performance (). Although these costs
tend to be low compared with other remedial alternatives, the public is often suspicious of natural attenuation due to the impression that nothing is being done.
Some very important observations related to the
performance of natural attenuation technology are
(Khan et al. 2004): it is a relatively simple technology
compared to other remediation technologies; it can be
carried out with little or no site disruption; it often
requires more time to achieve clean-up goals than other
conventional remediation methods; it requires a longterm monitoring program and the program duration
affects the cost; if natural attenuation rates are too
slow, the pollution/contaminant plume could migrate;
and it is difficult to predict with high reliability the
performance of natural attenuation.
6.4 Natural attenuation
6.5 Landfarming
Natural attenuation is a proactive approach that

focuses on the verification and monitoring of natural
remediation processes (Khan et al. 2004). Also known
as passive remediation, in situ bioremediation, intrinsic remediation, bioattenuation, and intrinsic bioremediation, natural attenuation is an in situ treatment
method that uses natural processes to contain the
spread of contamination from chemical spills and to
reduce that concentration and amount of pollutants at
contaminated sites (Boparai et al. 2008; Khan et al.
2004). This means the environmental contaminants are
undisturbed while natural attenuation works on them.
Natural attenuation processes are often categorized as
destructive or non-destructive (Gelman and Binstock
2008). Destructive processes destroy the contaminant,
This technology involves the application of contaminated material that has been excavated onto the soil
surface and periodically tilled to mix and aerate the
material (Maciel et al. 2009; Harmsen et al. 2007).
The contaminants are degraded, transformed and
immobilized by means of biotic and abiotic reactions
(Rubinos et al. 2007). Sometimes, in cases of very
shallow contamination, the top layer of the site may
simply be tilled without requiring any excavation.
Liners or other methods may be used to control
leachate. This technology is designed primarily to
treat soil contamination by fuels, PAHs, non-halogenated VOCs, SVOCs, pesticides, and herbicides. The
process may be applied to halogenated organics, but
is less effective. Although the technology is very
123
123
Laboratory and numerical investigations
using a pulsed air sparging system
Groundwater contaminated with

trichloroethylene TCE in a sandy
aquifer
Reference
Sparging air at ambient temperature through the contaminated Mohamed et al. (2007)
soil could remove NAPLs, but employing hot air sparging
could provide higher contaminant removal efficiency, by
about 9%
Aqueous concentrations for TCE were still much higher than Kim et al. (2007)
the maximum contaminant level in spite of successful
removal of 95% of residual TCE, implying that it would be
more appropriate to apply air sparging combined with other
remediation technologies such as bioremediation for
remediation of TCE-contaminated groundwater
In the air sparged control zone, TCE was removed at only two
monitoring locations nearest the sparge-well, to
concentrations of 15 and 60 lg/l
In the propane-stimulated zone, c-DCE concentrations

decreased below the detection limit (1 lg/l and TCE
concentrations ranged from less than 530 lg/l, representing
removals of 9097%
TCE, cis-dichloroethene (c-DCE); and dissolved oxygen

concentration levels decreased in proportion with propane
usage, with c-DCE decreasing more rapidly than TCE
Chlorinated aliphatic hydrocarbons

(CAHs) in groundwater
Co-metabolic air sparging (CAS)

demonstration using propane as the
cometabolic substrate.
Pulsed operation of the installed system lasted about

5 months and the results of frequent groundwater sampling
and analysis indicated an important decline in total TPH
and BTEX concentrations of up to 99%
Tovanabootr et al. (2001)
Gidarakos and Aivalioti

(2008)
Air sparging assisted stirred tank reactors. SDS removed more than 80% crude oil from non-weathered Urum et al. (2005)
Two surfactants (rhamnolipid and
soil samples, whilst rhamnolipid showed similar oil removal
sodium dodecyl sulfate, SDS) were
at the third and fourth levels of the parameters tested
tested and the effects of different
The approach of soil washing was noted to be effective
parameters (temperature, surfactant
in reducing the amount of oil in soil
concentrations, washing time, volume/
mass ratio) were investigated under
varying washing modes namely, stirring
only, air sparging only and the
combination of stirring and air sparging
Petroleum hydrocarbon contaminated Pulsed operation of an in-well air

aquifers
sparging
Removal of crude oil from soil
Removal of volatile contaminant from Laboratory-scale surfactant-enhanced air SEAS was significantly more efficient than conventional air Kim et al. (2009)
an aquifer model and mass removal sparging (SEAS) was evaluated versus
sparging for removing perchloroethene. For SEAS, about 78
of dense non-aqueous phase liquid
conventional air sparging. Surfactant
and 75% of total perchloroethene mass was depleted from the
(DNAPL); Perchloroethene was the
used was sodium dodecylbenzene
flow chamber at 350 and 150 mg/l SDBS
test contaminant
sulfonate (SDBS)
Water saturated contaminated soil

Air sparging conditions
under air sparging conditions for
different NAPLs and soil properties
Description of method(s) employed
Contaminated media
Table 8 Studies on the application of air sparging for bioremediation
234
235
simple and inexpensive, it does require large space,

and reduction in contaminant concentrations may
sometimes be due to volatilization rather than biodegradation (Sanscartier et al. 2010; Souza et al. 2009).
Souza et al. (2009) have used Allium cepa bioassays
to assess landfarming and landfarming with rice hulls
amendment before and after hydrocarbons biodegradation assay in the laboratory. It has been reported that
after 108 days of biodegradation, the landfarming
reached the rate of 26.30 mmol of CO2 released and
the concentration of TPHs decreased by 27%. Landfarming treated with rice hulls had the highest release
of CO2, 110.9 mmol, associated with a remarkable
reduction in TPHs concentration, 59%, thereby showing that the use of rice hulls accelerated the biodegradation efficacy of landfarming to improve the
efficiency of bioremediation processes. In their study,
Marn et al. (2005) assessed the ability landfarming to
reduce the total hydrocarbon content added to soil with
refinery sludge in low rain and high temperature
conditions. It was seen that 80% of the hydrocarbons
were eliminated in 11 months, half of this reduction
taking place during the first 3 months. Rubinos et al.
(2007) treated a soil heavily contaminated ([5,000 mg
kg-1) with hexachlorocyclohexane (HCH) isomers
derived from lindane production using the landfarming
technique and observed significant decreases of the
a- and c-HCH isomers woth up to 89 and 82% of
the initial concentration, respectively, at the end of the
11 months. In this respect, the aerobic landfarming
appeared to be a viable and cost effective bioremediation treatment technology for soils contaminated with
a- and c-HCH isomers on large scales. Clark and
Boopathy (2007) equally concluded from their study
that landfarming from bench-scale studies could be
promisingly transferred to full-scale application.
Lately, Sanscartier et al. (2009) have investigated the
bioremediation of weathered medium- to high-molecular weight petroleum hydrocarbons (HCs) in Polar
regions. Their findings suggested that temperature and
low moisture content had affected the biodegradation
of HCs but with volatilization possibly predominating
in the field.
and the technology has yet to be extensively proven

in the marketplace. Because of this, most information
about phytoremediation comes mainly from field and
laboratory research (Table 9). However, the potential
of phytoremediation for cheap, simple and effective
soil and groundwater remediation is generating
considerable interest.
Phytoremediation may be used for remediation of
soil and groundwater contaminated with toxic heavy
metals, radionuclides, organic contaminants such as
chlorinated solvents, BTEX compounds, non-aromatic
petroleum hydrocarbons, nitrotoluene ammunition
wastes, and excess nutrients (Schnoor et al. 1995).
Table 10 summarizes some studies which have been
conducted to remove heavy metals from contaminated
media by phtyoremediation and Table 11 presents the
findings of research on the application of phytoremediation for organic pollutants.
Other applications of phytoremediation include
landfill caps, buffer zones for agricultural runoff and
even drinking water and industrial wastewater treatment. Phytoremediation may also be used as a final
polishing step, in conjunction with other treatment
technologies. While indeed promising, the applicability of phytoremediation is limited by several factors.
First, it is essential that the contaminated site of
interest is able to support plant growth. This requires
suitable climate, soil characteristics such as pH and
texture, and adequate water and nutrients. Second,
because plant roots only go so deep, phytoremediation
is practical only in situations where contamination is
shallow (less than 5 m), although in some situations
with deeper contamination it may be used in conjunction with other technologies. Third, since the time
requirements for phytoremediation are sometimes
long relative to some conventional technologies such
as landfilling and incineration, it is not suitable for
situations requiring rapid treatment. Plants facilitate
remediation via several mechanisms:
6.6 Phytoremediation
3.
Using plants in soil and groundwater remediation

(i.e., phytoremediation) is a relatively new concept
4.
1.
2.
Direct uptake, and incorporation of contaminants

into plant biomass
Immobilization, or phytostabilization of contaminants in the subsurface
Release plant enzymes into the rhizosphere that
act directly on the contaminants
Stimulation of microbially mediated degradation
in the rhizosphere
123
236
Table 9 Plants used in phytoremediation studies for pollutants

Pollutant(s)
Plant species used
Reference
Nitrogen, phosphorus
Thalia geniculata f. rheumoides Shuey, Oenenathe

javanica (Blume) DC. Flamingo, Phyla lanceolata
(Michx.) Greene
Polomski et al. (2008)
Cadmium, copper, arsenic
Lolium perenne cv Elka
Sidoli OConnor et al.

(2003)
Cadmium, copper, lead, zinc
Paulownia tomentosa
Doumett et al. (2008)
2,4,6-trinitrotoluene
Vetiver grass (Vetiveria zizanioides)
Makris et al. (2007)
Anthracene in mycorrhizospheric soil
Ryegrass (Lolium multiflorum)
Korade and Fulekar (2008)
Phenol
2,6-dinitrotoluene
Vetiver (Vetiveria zizanoides L. Nash)

Arabidopsis thaliana
Singh et al. (2008b)

Yoon et al. (2007)
Arsenic species such as arsenate
Spider brake (Pteris cretica L.) plants
Ebbs et al. (2010)
Arsenic
Nugget marigold, a triploid hybrid between American

(Tagetes erecta L.) and French (Tagetes patula)
marigolds
Chintakovid et al. (2008)
Cadmium, chromium, nickel, iron,

arsenic
Helianthus annuus (sunflower)
January et al. (2008)
Recalcitrant PAHs
Fescue (Festuca arundinacea), switchgrass (Panicum

virgatum), zucchini (Curcubita pepo Raven)
Cofield et al. (2007)
Dibenzofuran-contaminated soil
Bermuda grass (Cynodon dactylon), bent grass (Agrostis

palustris Huds.), lawn grass (Zoysia japonica), white
clover (Trifolium repens L.)
Canola (Brassica napus var. Hyola 420), tall fescue
(Festuca arundinacea var. Au Triumph), salado grass
(Sporobulus airoides), cordgrass (Spartina patens var.
Flageo).
Wang and Oyaizu (2009)
Selenium-laden drainage sediments
Soil contaminated with diesel fuel
Lead, copper, zinc, cadmium
Scots Pine (Pinus sylvestris), Poplar (Populus

Palmroth et al. (2002)
deltoides 9 Wettsteinii), Red fescue, Festuca rubra;
Smooth meadowgrass, Poa pratensis, Perennial ryegrass,
Lolium perenne), White clover, Trifolium repens and Pea,
Pisum sativum
Vetiver grass Vetiveria zizanioides
Chen et al. (2004)
6.6.1 Phytoremediation of inorganic contaminants

Remediation of metal-contaminated soils and groundwater is another potentially promising application of
phytoremediation. Given that metals cannot be chemically transformed, and they can be toxic to microorganisms, metal contamination is not readily amenable
to in situ treatment strategies such as microbially
mediated remediation. Treatment of metal contamination therefore calls for either extraction or immobilization, and conventional treatment strategies, such as
incineration, landfilling, leaching, and chemical fixation are often prohibitively expensive. Additionally,
landfilling and incineration are often hampered by
public hostility. Understandably, cost-effective treatments for metal contamination are desperately needed.
123
Banuelos and Lin (2005)
This has stimulated considerable interest in using the

natural ability of some plants to accumulate (and
hyperaccumulate) metals in their tissues. Phytoremediation technologies that exploit this trait include
phytoextraction, rhizofiltration, phytostabilization,
phytodegradation and phytovolatilization (Salt et al.
1995).
6.6.2 Phytoextraction
Phytoextraction makes use of metal accumulating
plants to transport and concentrate metals in harvestable roots and shoots in order to remove metals from
soil. Typically, multiple crops of metal-accumulators
could be grown, followed by harvest and processing of
the plant material, which could involve reclamation of
Cd-hyperaccumulator Rorippa
globosa (Turcz.)
Nickel phytoextraction from four

types of Ni-contaminated soils
by Ni-hyperaccumulators
Alyssum corsicum, Alyssum
murale, and nonhyperaccumulators radish,
mustard
Maize (Gold Dent), soybean (Enrei After 2 months cultivation, the Gold Dent maize and Milyang 23 rice shoots
and Suzuyutaka), and rice
took up 20.229.5 and 18.520.2% of the 0.1 mol/l HCl-extractable Cu,
(Nipponbare and Milyang 23)
10.037.3 and 8.534.3% of the DTPA-extractable Cu, and 2.46.5 and
were pot-grown under aerobic
2.15.9% of the total Cu, respectively, in the two soils analyzed
soil with low to moderate Cu
contamination
Cadmium-contaminated soils
Nickel-contaminated soils
Copper
Arsenic
T. diversifolia mopped up substantial concentrations of Pb in the above-ground Adesodun et al.

biomass compared to concentrations in the roots. The concentrations in the leaf (2010)
compartment were 87.3, 71.3, and 71.5 mg/kg at 4, 6, and 8 weeks after
planting (AP), respectively. In roots, it was 99.4, 97.4, and 77.7 mg/kg.
Observations with H. annuus followed the pattern found with T. diversifolia,
showing significant accumulation of Pb in the above-ground biomass
Murakami and Ae
(2009)
Ni concentration in shoot of A. corsicum and A. murale was significantly higher Qiu et al. (2008)
than radish or mustard in all tested soils. A. corsicum and A. murale removed
much more Ni from Mojiang soil (total Ni 1,062 mg/kg) than common
vegetables, but mustard extracted most Ni from Xinyi soil (total Ni 107 mg/kg)
107.0 and 150.1 mg/kg of the Cd accumulated in stems and leaves, respectively, Wei and Zhou (2006)
when soil Cd added was concentrated to 25.0 mg/kg. The Cd-removing ratio
by shoots of R. globosa harvested at the flowering phase was up to 71.4% of
that at the mature phase
Arundo donax for phytoextraction Increasing As concentration in nutrient solution caused an increase in shoot and Mirza et al. (2010)
of arsenic from synthetic
root biomass without toxicity symptoms in A. donax growing under a range of
wastewater
As concentration from 50 to 600 lg/l. The As doses up to 600 lg/l did not
affect the growth of A. donax. It was suggested that A. donax plants may be
employed to treat contaminated waters containing arsenic concentrations up to
600 lg/l
Contaminants were added as lead Two species of sunflower

Tithonia diversifolia and
nitrate (Pb (NO3)2) and zinc
nitrate (Zn (NO3)2) at 400 mg/kg Helianthus annuus
which represents upper critical
soil concentration for both Pb
and Zn
Thlaspi caerulescens as a
phytoextraction plant
Cadmium and zinc
The periodic use of phytoextraction with T. caerulescens to maintain soils

Maxted et al. (2007)
below statutory metal concentration limits, when modern sewage sludges are
repeatedly applied, seems very attractive given the non-intrusive and
cost-effective nature of the process
Fenugreek (Trigonella
The Cr concentration in fenugreek, spinach, and raya increased with increasing Dheri et al. (2007)
foenumgraecum L.), spinach
level of added Cr in both soils. Cr in both shoot and root was highest in raya,
(Spinacia oleracea L.), and raya followed by spinach and fenugreek. The overall mean uptake of Cr in shoot
(Brassica campestris L.)
was almost four times and in root was about two times higher in raya compared
to fenugreek. The findings indicated that family Cruciferae (raya) was most
tolerant to Cr toxicity, followed by chenopodiacea (spinach) and Leguminosae
(fenugreek)
Reference
Chromium-contaminated soils
Removal performance
Plant(s) used
Heavy metal(s)
Table 10 Heavy metal removal by phtyoremediation: removal conditions and performance

237
123
123
Phytolacca americana (pokeweed) P. americana not showed remarkable tolerance to Mn. Maximum Mn
Min et al. (2007)
concentration in the leaf dry matter was 8,000 mg/g on Xiangtan Mn tailings
wastelands. P. americana was characterized by a high translocation factor of
more than 10.76. Under nutrient solution culture conditions, manganese
concentration in the shoots increased with increasing external Mn levels, and
reached a maximum concentration of Mn in leaves at 47.06 g/kg. Pokeweed
was thus classified as a new manganese hyperaccumulator plant
Cobalt given to soybean (Glycine Results showed higher concentration (Co level (100200 mg/kg) in the soil)
Jayakumar and Jaleel
max) plants in pot culture by soil resulted in maximum accumulation in all parts of soybean plants, while the low (2009)
drenching method
concentrations of cobalt (50 mg/kg Co level) in the soil didnt show any
significant effect
Manganese
Cobalt
Water hyacinth (Eichhornia

crassipes (Mart.) Solms)
Manganese (1 mg/l Mn from

synthetic wastewaters in
constructed wetlands)
Phytoremediation mainly due to phytoextraction substantially contributed to

Kularatne et al.
manganese removal. However, chemical precipitation was absent, suggesting
(2009)
that manganese has a higher solubility in the given average pH (6.27.1)
conditions in constructed wetlands
Cold vapor Atomic Absorption Spectroscopy confirmed an increase of mercury Skinner et al.
within the plant root tissue and a corresponding decrease of mercury in the
(2007)
water. All species of plants appeared to reduce mercury concentrations
in the water via root uptake and accumulation. Water lettuce and water
hyacinth appeared to be the most effective, followed by taro and zebra rush,
respectively
Water hyacinth (Eichornia

crassipes), water lettuce (Pistia
stratiotes), zebra rush (Scirpus
tabernaemontani) and taro
(Colocasia esculenta)
Reference
Mercury
Removal performance
Plant(s) used
Heavy metal(s)
Table 10 continued
238
Plant(s) used
The Kazakhstan maximum acceptable concentration
for DDT and HCH metabolites in plant tissue is
20 lg/kg. Species in this category included:
Artemisia annua L., Kochia sieversiana (Pall.) C.A.
Mey. Kochia scoparia (L.) Schrad., and Xanthium
strumarium L
P450 genes CYP1A1, CYP2B6, and CYP2C19 in rice

plants (Oryza sativa cv. Nipponbare) introduced
using the plasmid pIKBACH
Lemna minor (L. minor), Elodea canadensis
(E. canadensis) and Cabomba aquatica
(C. aquatica)
Pea (Pisum sativum), with a genetically tagged

bacterial endophyte
Three cultivars of zucchini (Cucurbita pepo spp.

pepo cv Costata Romanesco, Goldrush, Raven)
Water hyacinth (Eichhornia crassipes)
Organic pollutant(s)
Obsolete pesticidesPOPs pesticides

including metabolites of DDT
(dichlorodiphenyltrichloroethane) and
isomers of HCH (hexachlorocyclohexane)
Herbicides atrazine and metolachlor
Copper sulphate (fungicide), flazasulfuron

(herbicide) and dimethomorph (fungicide)
Selective systemic herbicide 2,4dichlorophenoxyacetic
Weathered p,p0 -DDE in soil
Dicofol (an organochlorine pesticide)
Table 11 Application of phytoremediation for removal of organic pollutants

Reference
After 10 days of incubation in nutrient solution at

Xia (2008)
251C, the remaining dicofol which was spiked
initially at 1 mg/l was 0.05 and 0.26 mg/l in the nonsterile planted and non-sterile unplanted, 0.07 and
0.31 mg/l in the sterile planted and sterile unplanted
treatments, respectively
Total amount of contaminant phytoextracted during the White et al.

62 day growing season ranged from 0.722.9%
(2006)
Toxicity of the contaminants was the same for all the Olette et al.
aquatic plants studied and occurred in this descending (2008)
order of toxicity:
flazasulfuron [ copper [ dimethomorph. L. minor
had the most efficient uptake capacity, followed by
E. canadensis and then C. aquatica. The maximum
removal rate of copper, flazasulfuron and
dimethomorph was 30, 27 and 11 lg/g fresh weight/
day, respectively
Results showed that the strain tested had actively
Germaine
et al. (2006)
colonized inoculated plants internally (and in the
rhizosphere). Inoculated plants showed a higher
capacity for 2,4-dichlorophenoxyacetic acid removal
from soil and showed no 2,4-dichlorophenoxyacetic
acid accumulation in their aerial tissues
The transgenic rice plants (pIKBACH rice plants)

Kawahigashi
became more tolerant toward various herbicides than et al. (2006)
nontransgenic Nipponbare rice plants
Three species exceeded the MAC by up to 90 times

Nurzhanova
including A. annua, Ambrosia artemisiifolia L., and
et al. (2010)
Erigeron canadensis L. Most pesticides accumulated
in the root systems; however, among the species
investigated, K. scoparia, A. annua, Barbarea
vulgaris W. T. Aiton, and A. artemisiifolia
demonstrated capabilities to translocate pesticides
from roots to aboveground tissues
Removal performance

239
123
The removal yields during the 4-day test periods varied Dosnonfrom 10 to 18% and 712% for dimethomorph and
Olette et al.
pyrimethanil, respectively. The maximum removal
(2009)
rate during the 4-day test period was 48 lg/g fresh
weight (FW) for dimethomorph and 33 lg/g FW for
pyrimethanil. L. minor and S. polyrhiza showed the
highest removal efficiency for the two fungicides
Five macrophyte speciesL. minor, S. polyrhiza,
C. aquatica, C. palustris and E. canadensis
Two fungicidesdimethomorph
and pyrimethanil
Dordio et al.
(2009)
At a concentration of 20 lg/l, Typha had removed
[50% of CA within the first 48 h, reaching a
maximum of 80% by the end of the assay.
Experimental conditions assured that
photodegradation, adsorption to vessel walls and
microbial degradation did not contribute to the
removal
Typha spp.
Clofibric acid (CA which is a metabolite
of blood lipid regulator drugs)
Reference
Plant(s) used
Removal performance
Organic pollutant(s)
Table 11 continued
240
123
metals or simply incineration and disposal. The

mechanisms of root uptake and transport within the
plants are poorly understood. Solubilized metal ions
may enter the root by two pathways: apoplastically
(extracellularly), or symplastically (intracellularly)
(Salt et al. 1995). Metal ions tend to enter plant cells
via metal ion carriers or channels in energy dependent,
saturable process (Clarkson and Luttge 1989). Nonessential heavy metals may compete with essential
metals for these transmembrane carriers, which may
explain the ability of some non-essential metals to
enter the cell against the concentration gradient (Salt
et al. 1995). Upon entering the roots, metals can either
be stored or transported to the shoot. Xylem transport
is thought to be responsible for transport to the shoot,
although metals may disperse throughout the shoot via
the phloem (Salt et al. 1995). Several wild plants with
the ability to accumulate very large concentrations of
metals in their roots and shoots have been identified
and are termed as hyperaccumulators (Table 12).
Approximately 400 hyperaccumulator species have
been identified, according to the analysis of fieldcollected specimens. Metal hyperaccumulators are
interesting model organisms to study for the development of a phytoremediation technology, the use of
plants to remove pollutant metals from soils (Kramer
et al. 1997).
Botanists first recognized the metal-accumulating
ability of the genus Thlaspi over a century ago. Some
have suggested this ability may have evolved as a
defense against herbivores (Baker et al. 1994). Baker
and co-workers were able to grow some individual
Thlaspi caerulscens plants that accumulated more
than 30,000 mg of Zn and over 1,000 mg Pb per
gram dried biomass (Baker et al. 1994). They also
showed that the five isolated populations of the plant
were adapted in tolerating and accumulating metals
not present in the parent soil, which suggested the
mechanisms of tolerance and accumulation may be
similar for different metals.
While wild metal-accumulators such as T. caerulescens have shown an impressive ability to
accumulate metals, they tend to be slow growing
and small in size. The use of such plants for
phytoextraction may therefore require an unreasonable number of harvests to decontaminate a given
site. Ideally, a plant well suited for phytoextraction
should tolerate and accumulate metals, grow rapidly,
and have the potential to produce a high biomass.
241
Table 12 Heavy metal hyperaccumulators

Metal
Hyperaccumulator plant species
References
Arsenic
Pteris vittata, Pityrogramma calomelanos
Wang et al. (2002), Lombi et al. (2002), Tongbin et al. (2002),

Francesconi et al. (2002), Tu et al. (2003)
Cadmium
Thlaspi caerulescens, tumbleweed (Salsola kali),

Solanum nigrum L
Pence et al. (2000), de la Rosa et al. (2004), Wei et al. (2006)
Zinc
Thlaspi caerulescens, Arabidopsis halleri,

Thlaspi praecox Wulfen
Nickel
Alyssum lesbiacum, Alyssum bertolonii, Thlaspi

goesingense, Berkheya coddii, Sebertia acuminate,
Alyssum murale
Shen et al. (1997), Pence et al. (2000), Zhao et al. (2000),

Sarret et al. (2002), Vogel-Mikus et al. (2006)
Kramer et al. (1997), Kupper et al. (2001), Robinson et al.
(1997), Sagner et al. (1998), Bernal et al. (1994)
Lead
Thlaspi praecox Wulfen
Vogel-Mikus et al. (2006)
Copper
Aeolanthus biformifolius (Labiatae); Commelina

communis
Malaisse et al. (1997), Wang et al. (2004)
In the attempt to find such a plant, Dushenkov and

co-workers have evaluated the heavy-metal accumulating abilities of several high biomass crop species
such as Brassica juncea (Indian mustard) (Dushenkov et al. 1995). Although Thlaspi caerulescens has a
higher tolerance for the heavy metals tested and
demonstrated a higher ratio of metal accumulation to
plant mass. Brassica juncea produces 20 times more
biomass. They found Brassica juncea to be especially
adapted in accumulating lead. One strain was able to
accumulate Pb at up to 3.5% dry weight in the shoots
suggesting that a crop of such plants could extract
630 kg ha-1 of Pb in above ground biomass with a
single harvest, more if some root material was
harvested as well (Dushenkov et al. 1995). It is
important to note however, that these experiments
were done hydroponically. In soil, the property of Pb
to bind to clay soil particles and organic matter, and
its inclusion in insoluble precipitates significantly
reduces the bioavailability of Pb to the plant
(Dushenkov et al. 1995). This is true of other metals
as well. For this reason, biological mechanisms that
enhance metal bioavailability are being investigated.
For instance, in response to nutrient deficiencies,
plants can secrete metal-chelating molecules (phytosiderophores) that chelate and solubilize soil-bound
metals such as Fe, Mn, Cu, and Zn (Salt et al. 1995).
Also, plants can reduce soil bound metal ions by
specific plasma membrane-bind metal reductases; and
they can adjust soil pH, which decreases metal
adsorption, by exuding protons (Salt et al. 1995).
Lastly, the presence of some microorganisms in the
rhizosphere has been shown to enhance metal

availability.
Arsenic hyperaccumulators: Arsenic hyperaccumulators bioconcentrate arsenic over 2,000 mg/kg in
plant tissues (Bondada and Ma 2003). In addition, a
good arsenic phytoextraction species should accumulate more arsenic in shoots than in roots because for
an easy harvest or removal of arsenicladen
aboveground biomass. The concentration of the
contaminant is generally very high in these plants
when grown in contaminated media. To compare the
levels of bioconcentation and distribution of arsenic
in plants a bioconcentration factor (BF) and transfer
factor (TF) can be used. The BF of arsenic is the ratio
of the arsenic concentration in plant to the concentration in soil, while the TF is the ratio of the arsenic
concentration in roots to the concentration in shoots.
Ma et al. (2001) discovered an arsenic-hyperaccumulator species-Chinese brake fern (Pteris vittata)
that accumulated arsenic in the shoots to a concentration as high as 22,000 mg/kg (Huang et al. 2004).
Research has demonstrated that other species in the
Pteris genus also hyperaccumulate arsenic in their
shoots. Greenhouse studies (Salido et al. 2003)
indicated that P. vittata accumulated an arsenic
concentration in the above ground plant tissue more
than 200-fold higher than most other plant species
tested using arsenic-contaminated soil. In addition,
this species grows rapidly and generates substantial
amounts of biomass, thus making P. vittata an
excellent candidate to rapidly remove arsenic from
arsenic-contaminated environments. The Chinese
123
242
brake fern is a primitive plant which thrives on

arsenic, doubling its biomass in 1 week when
subjected to 100 mg/l arsenic. The striking difference
between P. vittata and arsenic non-accumulators is
the remarkable transport of arsenic from roots to
shoots in P. vittata, accumulating up to 95% of the
arsenic in the above-ground tissue (Doucleff and
Terry 2002). Many other ferns in the Pteris genus like
P. longifolia, P. cretica and P. umbrosa (Zhao et al.
2002), as well as a non-Pteris fern, Pityrogramma
calomelanos (Visoottiviseth et al. 2002) have also
been found to hyperaccumulate arsenic. However, not
all members of the Pteris genus are able to hyperaccumulate arsenic. Meharg (2003) found that Pteris
tremula and Pteris stramina do not hyperaccumulate
arsenic. To date the only non-Pteris fern to exhibit
this ability is Pityrogramma calomelanos (Francesconi et al. 2002). Srivastava et al. (2006) showed
that Pteris biaurita L., P. quadriaurita Retz and
P. ryukyuensis Tagawa could be used as hyperaccumulators of arsenic with the average arsenic concentration ranging from 1,770 to 3,650 mg/kg dry
weight (DW) in the fronds and 182507 mg/kg DW
in the roots of P. cretica, P. biaurita, P. quadriaurita
and P. ryukyuensis after having been grown in
100 mg As/kg soil.
Soil amendments that can increase metal availability are also being studied. Chelating agents have
been investigated by Blaylock, Raskin and co-workers for their ability to prevent precipitation and
sorption of metals (Dushenkov et al. 1995). B. juncea
seedling grown for 4 weeks in soil treated with a
chelating agent accumulated 875 mg/kg DW Cd in
the shoot, compared to 164 mg/kg DW Cd by
seedlings grown in untreated soils. Additionally,
numerous studies have demonstrated that lowering
soil pH increases metal availability.
6.6.3 Rhizofiltration
Rhizofiltration is defined as the use of plant roots to
absorb, precipitate, and concentrate toxic metals from
water (Salt et al. 1995). In engineered systems, metalcontaminated water may be passed through a network
of roots, which can then be harvested, dried and either
combusted and discarded or subjected to process to
reclaim metals from plant biomass. Given its potential
for treating high volumes of water contaminated with
low concentration of metals, rhizofiltration enthusiasts
123
assert that it will be a cost-effective treatment

for everything from industrial wastewater, to agricultural runoff, to contaminated surface water and
groundwater.
The mechanisms for root accumulation include: (1)
surface sorption, in which physical/chemical processes
such as chelation and ion exchange lead to sorption by
the root; (2) biological processes, including intracellular uptake, vacuolar deposition, and translocation to
the shoots (Chaney 1983); and (3) root remediated
precipitation, which probably involves the release of
root exudates (Dushenkov et al. 1995). Surface sorption tends to be the fastest of these, especially in the
case of Pb, and rootremediated precipitation the
slowest, although the relative importance of these
different mechanisms is dependent on concentration.
At low concentration surface sorption dominates, at
higher concentrations, however, when sites for sorption are saturated, biological processes and precipitation assume more importance. These mechanisms also
differ between metals and plant species.
Dushenkov and co-workers have tested the ability
of hydroponically grown plants to remove toxic
metals from aqueous waste streams (Dushenkov et al.
1995). The ability of the high biomass crop plant
Brassica juncea to accumulate Pb in the roots was
compared to 24 other plant species such as Helanthus
annus (sunflower), and various grasses such as
colonial bentgrass and Poapratensis (Kentucky bluegrass). While all the species tested demonstrated
significant root accumulation of Pb, Brassica juncea
(14% dry weight Pb in the roots) possessed the most
favorable combination of metal accumulating ability
and high-biomass production. Additionally, Brassica
juncea was shown to accumulate significant amounts
of Cu2? Cd2?, Cr 2?, Ni 2? and Zn2? as well. Given
that the magnitude of metals accumulated by way of
surface sorption is proportional to root mass, the
ability of Brassica juncea to generate a large mass of
roots quickly and economically makes it a promising
candidate for rhizofiltration.
6.6.4 Phytostabilization
Phytostabilization of inorganics involve the use
of metal tolerant plants to reduce the mobility of
metals in the subsurface. Soils contaminated with
toxic metals lack vegetation due to either physical
disturbance or toxic effects of the contamination.
Metal contamination of exposed soils is often more

mobile due to leaching and transport by wind and
water. Metal tolerant plants may be useful in reducing
metal mobility that results from these mechanisms.
This strategy has been used successfully to stabilize
metalliferous mine-wastes by a group in Liverpool
(Cunningham and Berti 1993). Also, Salt and
co-workers were able to demonstrate that seedlings
of B. juncea were able to reduce the level of Pb
leached from contaminated soils into the groundwater
(Salt et al. 1995).
At the NEERI, Nagpur, extensive work on phytostabilization of coal mine dumps, manganese mine
dumps, fly ash dumps and metalliferous mine wastes
has been carried out using IBA (Juwarkar et al. 2000).
Legumes are especially well suited for reestablishing
vegetation and stabilizing degraded and metal contaminated soils. This is due to several factors: first,
they accumulate nitrogen in a mineralizable form in
symbiosis with rhizobia, which then becomes available to non-leguminous plants; second, they are able to
grow in low nutrient conditions; and third, they are
able to colonize barren habitats which are subject to
strong winds and flooding (Jha et al. 1995). Jha and
co-workers studied how this rhizobial symbiosis
influences the ability of several wild legumes to
revegetate an unreclaimed limestone quarry. They
found that seeds encapsulated with polyacrylamideentrapped rhizobia showed higher establishment, survival, and subsequent growth than uninnoculated
seedlings. Mycorrhizae inoculation to leguminous
and nonleguminous tree species of resulted in rapid
reclamation of mine spoil dumps (Juwarkar et al. 1992,
1997, 2000).
6.6.5 Phytodegradation
Phytodegradation is the breakdown of organics, taken
up by the plant to simpler molecules that are incorporated into the plant tissues (Chaudhry et al. 1998).
Plants contain enzymes that can breakdown and
convert ammunition wastes, chlorinated solvents
(such as trichloroethylene) and other herbicides. The
enzymes include usually dehalogenases, oxygenases
and reductases (Black 1995). Rhizodegradation is the
breakdown of organics in the soil through microbial
activity of the root zone (rhizosphere). Soil microorganisms can utilize organic pollutants as their carbon
and energy sources. Indeed, all phytoremediation
243
processes or technologies are not exclusive and may

be used simultaneously. For example, a constructed
treatment wetland may involve all the phytoremediation processes for the cleanup of wastewaters contaminated with both metals and organic compounds.
6.6.6 Phytovolatilization
Phytovolatilization involves the use of plants and
plant-associated soil microbes to take up contaminants
from the soil, transform them into volatile forms and
release them into the atmosphere (Lin 2008). Phytovolatilization occurs as growing trees and other plants
take up water and the organic and inorganic contaminants. Metalloids, such as selenium, arsenic, and tin,
can be methylated to volatile compounds or mercury
that can be biologically transformed to elemental Hg.
Phytovolatilization has been primarily used for the
removal of mercury and selenium.
7 Ex situ bioremediation
7.1 Composting
Composting is the biochemical degradation of organic
materials to a sanitary, nuisance-free, humus-like
material (Kulcu and Yildiz 2004). Composting has
been defined as a controlled microbial aerobic
decomposition process with the formation of stabilized organic materials that may be used as soil
conditioner (Negro et al. 1999). The main factors in
the control of a composting process include environmental parameters (temperature, moisture content, pH
and aeration) and substrate nature parameters (C/N
ratio, particle size, and nutrient content) (Diaz et al.
2002; Artola et al. 2009). Aerobic composting is the
decomposition of organic substrates in the presence of
sufficient oxygen (Agnew and Leonard 2003). The
main products of the biological metabolism are
carbon dioxide, water and considerable amounts of
heat (Ghaly et al. 2006). Various factors correlate with
each other physically, chemically and biologically in
complicated composting processes (Agnew and Leonard 2003). A slight change in a single factor may cause
a drastic avalanche of metabolic and physical changes
in the overall process. In other words, there may be
extremely strong non-linearities involved in these
processes (Seki 2000). These processes occur in
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244
matrix of organic particles and interconnected pores,

and the pores are partially filled with air, aqueous
solution, or a combination of the two (Richard et al.
2006). A multitude of microorganisms and their
enzymes is responsible for the biodegradation process
(Fogarty and Tuovinen 1991), resulting in a complex
biochemicalmicrobial system.
Because of its complicated and dynamic nature, the
composting process is one of the most intractable
processes from an engineering point of view although
the macroscopic process kinetics have been well
engineered to date to remediate a wide variety of
organic wastes namely municipal solids wastes, poultry litter, wastes vegetables, food processing residuals,
and sludge from wastewater treatment plants and other
sludge generating processes. Under optimal conditions, composting proceeds from the psychrophilic
state through three phases: (a) the mesophilic or
moderate-temperature phase, (b) the thermophilic or
high temperature phase, and (c) the cooling and
maturation phase which lasts for several months
(Mohee et al. 2008). The first, second and third phases
are referred to as the active stage in which heat is
produced (Ghaly et al. 2006). This active stage is
governed by the basic principles of heat and mass
transfer (Keener et al. 1993; Mudhoo and Mohee
2008) and by the biological constraints of living
microorganisms.
Many anthropogenic organic contaminants entering
the environment are not fully degraded during treatment and eventually accumulate in biosolids (Ang
et al. 2005; Bhandari and Xia 2005). Due their
relatively low water solubility and high lipophilicity
(Bhandari and Xia 2005), organic contaminants easily
partition into biosolids resulting in their accumulation
in biosolids at concentrations several orders of magnitude greater than influent concentrations (Govind
et al. 1991). The following sections now present and
discuss selected research findings for the application of
composting in bioremediating such organic contaminants, namely, PAHS, petroleum-based hydrocarbons,
phenol derivatives, polychlorinated biphenyls, phthalic acid esters (PAEs) and pesticides.
2005). In the process, a major fraction of the nutrients

contained in the organic matter is converted to more
bioavailable forms. The first step in vermicomposting
occurs when earthworms break the substrate down to
small fragments prior to ingesting the substrate
(Gajalakshmi and Abbasi 2008). This increases the
surface area of the substrate, facilitating microbial
and enzymatic actions. The substrate is then ingested
and goes through a process of enzymatic digestion
brought about by numerous species of bacteria and
enzymes present in the worms gut (Gajalakshmi and
Abbasi 2008).
Due to their biological, chemical and physical
actions, earthworms can be directly employed to
promote biodegradation of organic contaminants in
bioremediation processes (Hickman and Reid 2008).
Earthworms have been shown to aerate and bioturbate soils and thence improve their nutritional status
and fertility, which are normally variables known to
limit bioremediation rates (Hickman and Reid 2008).
Earthworms also hinder processes during which
organic contaminants bind to soils, and thus promote
the dispersion and bioavailablity of organic contaminants to the degrading microorganisms (Hickman
and Reid 2008). Earthworms in general are tolerant to
many chemical contaminants including heavy metals
and organic pollutants in soil and can bio-accumulate
them in their tissues (Sinha et al. 2008). Earthworms
species like Eisenia fetida, Eisenia tetraedra, Lumbricus terrestris, Lumbricus rubellus and Allobophora chlorotica have been found to remove heavy
metals (Cd, Pb, Cu and Hg) pesticides and lipophilic
organic micropollutants like the PAH from the soil
(Sinha et al. 2008). Therefore, by using these
excellent properties of earthworms, the vermicomposting process has been employed to degrade organic
pollutants like PAHs, PCBs (Contreras-Ramos et al.
2009), atrazine and metamitron (Forouzangohar et al.
2005). Table 13 summarizes a few studies where
vermicomposting has been employed to bioremediate
such contaminants.
7.1.1 Vermistabilisation
Polycyclic aromatic hydrocarbons are a class of

organic compounds that have accumulated in the
natural environment mainly as a result of anthropogenic activities such as the combustion of fossil fuels
(Bamforth and Singleton 2005; Johnsen et al. 2005).
Vermicomposting is the term given to the process of

conversion of biodegradable matter by earthworms
into vermicompost (Garg et al. 2006; Tognetti et al.
123
7.1.2 PAHs remediation by composting
245
Table 13 Degradation of chemical contaminants through vermicomposting

Contaminant(s)/contaminate
media
Earthworm
species
Vermistabilizaion performnace
References
Polychlorinated biphenyls
Eisenia
fetida
Results demonstrated that earthworms survived and reproduced

in the presence of contaminated media
Tharakan et al.
(2004)
Biomass increase decreased rapidly with increasing mass

fraction of sludge, and biomass increased ranged from 103%
in the negative control to biomass reduction of 54 with 75%
sludge
Gas chromatography results demonstrated an 80% reduction
in PCB level in all vermicomposting bioreactors
Beverage industry bio sludge
Eisenia
fetida
Degradation of 50:50 mixture of bio sludge and cattle dung

could be achieved in 75 days when worms were inoculated
at 25 g kg-1 feed mixture
Distillery industry sludge mixed

with a bulking agent (cow
dung)
Perionyx
Inoculated earthworms could maximize the decomposition and
excavatus
mineralization rate when sludge was used with appropriate
bulking material for earthworm feed
Singh et al.
(2010)
Suthar and
Singh (2008)
Vermicomposting also caused significant reduction in total

concentration of metals: Zn (15.139.6%), Fe (5.229.8%),
Mn (2.636.5%) and Cu (8.639.6%) in sludge
Phenanthrene, anthracene and
benzo(a)pyrene
Eisenia
fetida
Average anthracene removal by the autochthonous

microorganisms was 23, 77% for phenanthrene and 13%
for benzo(a)pyrene, while it was 51% for anthracene, 47%
for benzo(a)pyrene and 100% for phenanthrene in soil with
earthworms. At 50 and 100 mg phenanthrene/kg E. fetida
survival was 91 and 83%, but at 150 mg/kg all died within
15 days. Survival of E. fetida in soil amended with
anthracene B 1,000 mg/kg and benzo(a)pyrene B 150 mg/kg
was higher than 80% and without weight loss compared to the
untreated soil
The increasing use of fossil fuels and their combustion products by human beings during the two past
centuries raises several questions about PAHs hazards for living organisms. First, apart from accidental
oil spills leading to massive pollutions, the precise
origin of trace PAHs, e.g., natural versus anthropogenic, has rarely been clear traced. Second, the
toxicity of PAHs, like other hazardous chemicals,
requires their bioavailability. And since most PAHs
are highly hydrophobic (Wild and Jones 1992), their
pathways of transfer through geological and biological media are far from being comprehensively
understood. Third, explicit correlations between
PAH sources and carcinogenic effects have been
reported only for intense exposure to PAHs such as
for coalmine workers. PAHs structure and stability
stand in the way of their biodegradation by microorganisms (fungi and bacteria). Biodegradation is
slow and is a function of environmental parameters
such as oxygen, water and nutriment contents.
Interest has continuously surrounded the occurrence
ContrerasRamos et al.
(2006)
and distribution of PAHs for many decades due to

their potentially harmful effects to human health
(Juhasz and Naidu 2000). Although various physicochemical methods have been used to remove these
compounds from our environment, they have many
limitations (Samanta et al. 2002). This concern has
prompted researchers to address ways to detoxify and
remove these organic compounds from the natural
environment (Bamforth and Singleton 2005). Bioremediation is one approach that has been used to
remediate contaminated land and waters, and that has
promoted the natural attenuation of the contaminants
using the in situ microbial community of the site.
PAHs are recalcitrant and can persist in the
environment for long periods, but are conducive to
biodegradation by certain enzymes found in bacteria
and fungi (Juhasz and Naidu 2000; Ang et al. 2005).
In the past several years, several oxidoreductases
such as laccases and cytochrome P450 monooxygenases have been exploited for the enzymatic degradation of PAHs. Composting has been applied as a
123
246
bioremediation technique for degrading toxic organic

compounds and perhaps lowering their persistence
and toxicity in organic residues and wastes (Barker
and Bryson 2002). The biochemical and physicochemical processes of remediation in composts are
similar to those that usually occur biologically in soil.
However, composting may accelerate the destruction
of organic contaminants since metabolic temperatures
developed are generally higher in composts than in
soils. This remediation characteristic of composts and
composting matrices has been successfully explored
and exploited for the degradation of PAHs.
Al-Daher et al. (2001) selected the bioremediation
technique involving the use of composting soil piles
from among the most appropriate methods and
evaluated its performance to remediate PAHs on a
pilot scale. Soil piles were constructed from the
contaminated soil after amendment with necessary
soil additives and the piles were subjected to regular
irrigation and turning, and a monitoring program was
carried out, including monthly soil sample collection
from each pile for the measurement of petroleum
hydrocarbon PAHs, soil microbial counts, mineral
and metal concentrations. Al-Daher et al. (2001)
found that the composting soil pile treatment resulted
in the reduction of up to 59% total extractable matter
of oil contamination within 8 months of the composting process. More interestingly, Reid et al. (2002)
studied the catabolism of phenanthrene within mushroom compost resulting from its incubation with (1)
phenanthrene, and (2) PAH-contaminated soil. Respirometers measuring mineralization of freshly added
14
C-9-phenanthere were used to evaluate the induction of phenanthrene-catabolism. Where pure phenanthrene spiked at a concentration of 400 mg/kg wet
weight was used to induce phenanthrene-catabolism
in compost, induction was measurable, with maximal
mineralization observed after 7 weeks phenanthrenecompost contact time. Where PAH-contaminated soil
was used to induce phenanthrene-catabolism in uninduced compost, induction was observed after
5 weeks soil-compost contact time. Microcosm-scale
amelioration of soil contaminated with 14C-phenanthrene (aged in soil for 516 days prior to incubation
with compost) indicated that both induced (using pure
phenanthrene) and uninduced mushroom composts
were equally able to promote degradation of this soilassociated contaminant. After 111 days incubation
time, 42.7% loss of soil-associated phenanthrene was
123
observed in the induced-compost soil mixture, while

36.7% loss of soil-associated phenanthrene was
observed in the uninduced-compost soil mixture.
Antizar-Ladislao et al. (2005) investigated the biodegradation of 16 United States Environmental Protection Agency (USEPA)listed PAHs (Fu et al.
2003) present in contaminated soil from a manufactured gas plant site using laboratory-scale invessel
compostingbioremediation reactors over 8 weeks.
Antizar-Ladislao et al. (2005) found that temperature
and amendment ratio were important operating parameters for PAH removal for invessel composting
bioremediation of aged coal tar-contaminated soil and
thereafter recommended that when conventional composting processes using temperature profiles to meet
regulatory requirements for pathogen control need to
be used, these should be preferably started with a
prolonged mesophilic stage followed by thermophilic,
cooling, and maturation stages. More recent studies on
the application of composting to degrade PAHs have
been conclusive and in concert with the findings of
earlier studies. Plaza et al. (2009) have investigated the
binding of phenanthrene and pyrene, by humic acids
(HAs) isolated from an organic substrate at different
stages of composting and a soil using a batch
fluorescence quenching method and the modified
Freundlich model. With respect to soil HA, the organic
substrate HA fractions were characterized by larger
binding affinities for both phenanthrene and pyrene.
Further, Plaza et al. (2009) found that the isotherm
deviation from linearity was larger for soil HA than for
organic substrate HAs, indicating a larger heterogeneity of binding sites in the former. The composting
process decreased the binding affinity and increased
the heterogeneity of binding sites of HAs and hence
Plaza et al. (2009) inferred that the changes undergone
by the HA fraction during composting may be
expected to contribute to facilitate microbial accessibility to PAHs. The results obtained also suggested
that bioremediation of PAH-contaminated soils with
matured compost, rather than with fresh organic
amendments, may result in faster and more effective
clean-up. The beneficial use of compost to bioremediate PAHs was further evidenced from the findings of
Yuan et al. (2009). Yuan et al. (2009) have studied the
biodegradation of phenanthrene and pyrene in compost and compost-amended soil. The degradation rate
of phenanthrene was found to be more than that of
pyrene. The degradation of the PAHs was enhanced
when the two species were present simultaneously in

the soil, thereby suggesting some kind of mutually
supported synergistic effect which favored their individual degradation rate since the addition of either of
the two types of compost (straw and animal manure)
individually enhanced PAH degradation. Further to
analyze the effect of compost size, compost samples
were separated into fractions with various particle size
ranges, which spanned 250, 50105, 105500 and
5002,000 lm. Yuan et al. (2009) observed that the
compost fractions with smaller particle sizes demonstrated higher PAH degradation rates but the when the
different compost fractions were added to soil, compost particle size had no significant effect on the rate of
PAH degradation. Of the microorganisms isolated
from the soil-compost mixtures, Arthrobacter nicotianae, Pseudomonas fluorescens, and Bordetella Petrii,
respectively, demonstrated the best degradation ability
for the PAHs studied.
7.1.3 Petroleum-based hydrocarbons remediation

by composting
Total Petroleum Hydrocarbons is a term used to
describe a broad family of several hundred chemical
compounds that originally come from crude oil. Crude
oils can vary in how much of each chemical they
contain, and so can the petroleum products that are
made from crude oils. Some are clear or light-colored
liquids that evaporate easily, and others are thick, dark
liquids or semi-solids that do not evaporate. Many of
these products have characteristic gasoline, kerosene,
or oily odors. Because modern society uses so many
petroleum-based products (gasoline, kerosene, fuel
oil, mineral oil, and asphalt), contamination of the
environment by them is potentially widespread. Contamination caused by petroleum products contains a
variety of these hydrocarbons. Because they are found
in a complex mixture, it is not usually practical to
measure each one individually and treat them separately with complete remediation. The amount of TPH
found in a sample is useful as a general indicator of
petroleum contamination at that site.
Composting of contaminated soil in biopiles is an
ex situ technology, where OM such as bark chips are
added to contaminated soil as a bulking agent.
Composting of lubricating oil-contaminated soil was
performed in field scale (5 9 40 m3) using bark chips
247
as the bulking agent, and two commercially available

mixed microbial inocula as well as the effect of the
level of added nutrients (nitrogen, potassium and
phosphorus) were tested by (Jrgensen et al. 2000).
Jrgensen et al. (2000) also performed the composting
of diesel oil-contaminated soil at one level of nutrient
addition and with no inoculum. Jrgensen et al. (2000)
noted that the mineral oil degradation rate was most
rapid during the first months of the composting
process, and it followed a typical first order degradation curve. During these 5 months, composting of the
mineral oil had decreased in all piles with lubrication
oil from approximately 2,400700 mg/kg dry weight,
which was about 70% of the mineral oil content.
Correspondingly, the mineral oil content in the pile
with diesel oil-contaminated soil decreased with 71%
from 700 to 200 mg/kg dry weight. In this type of
treatment with addition of a large amount of OM, the
general microbial activity as measured by soil respiration was enhanced and no particular effect of added
inocula was observed, thereby advocating the suitability of composting to bioremediate diesel oilcontaminated soil. Namkoong et al. (2002) conducted
research to find the appropriate mix ratio of organic
amendments for enhancing diesel oil degradation
during contaminated soil composting by adding sewage sludge or compost as an amendment for supplementing OM for composting of the contaminated soil.
Namkoong et al. (2002) thereafter found that the
degradation of diesel oil was significantly enhanced by
the addition of these organic amendments relative to
straight soil. The degradation rates of TPH and nalkanes were found to be greatest at the ratio of 2:1 of
contaminated soil to organic amendments on wet
weight basis. The work of Marn et al. (2006)
ascertains the efficacy of composting as a low cost
technology bioremediation technique for reducing the
hydrocarbon content of oil refinery sludge with a large
total hydrocarbon content of 250300 g/kg, in semiarid conditions. The composting system designed by
Marn et al. (2006), which involved open air piles
turned periodically over a period of 3 months, proved
to be inexpensive and reliable. The influence on
hydrocarbon biodegradation of adding wood shavings
as bulking agent and inoculation of the composting
piles with pig slurry was also studied. Marn et al.
(2006) determined that the most effective composting
treatment was the one in which the bulking agent was
added, where the initial hydrocarbon content was
123
248
reduced by 60% in 3 months as compared with the

32% reduction achieved without bulking agent.
Although, spiking the piles with an organic fertilizer
did not significantly improve the degree of hydrocarbon degradation, Marn et al. (2006) concluded that the
composting process without doubt led to the biodegradation of toxic compounds.
Mihial et al. (2006) determined that bioremediation
by composting was a suitable alternative for the
remediation of soil in and around a pit contaminated
with petroleum waste comprising used oil, gasoline,
diesel fuel and paint thinners. Mihial et al. (2006)
conducted a bench scale treatability study to assess the
potential for successful bioremediation of the site
using composting. They set up two reactors each with
ammonium phosphate fertilizer as the nutrient amendment using a mixture of grass clippings and sheep
manure in one reactor to determine if the composting
process could be accelerated by the addition of these
abundantly available waste materials. Based on the
results of the treatability study, the half-life of the
petroleum hydrocarbons at the subject site was estimated to be 36.3 and 121.6 days with and without the
addition of grass clippings and sheep manure, respectively. It was estimated that it would take approximately 192 and 643 days to remediate the soil and
lower reduce the TPH to 1,000 mg/l using the
amendments of the reactors, respectively. Atagana
(2008) inoculated the contaminated soil with sewage
sludge and incubated for the mix for 19 months.
Compost heaps were set up in triplicates on wood
pallets covered with double layers of nylon straw
sheets and control experiments which contained
contaminated soil and wood chips but without sewage
sludge were set up in triplicate, and the concentrations
of selected hydrocarbons in the contaminated soil were
measured monthly during the incubation period.
Atagana (2008) noted a typical composting performance through the temperature rise up to about 58C
in the sewage sludge compost within 60 days of
incubation, while temperature in the control fluctuated
between 15 and 35C throughout the incubation
period. All the more, TPH was reduced by 17% in
the control experiments and up to 99% in the sewage
sludge compost at the end of the incubation period.
Much promisingly as being a reliable bioremediation
technique, the composting process reduced the concentrations of the TPH by up to 100% within the same
period.
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7.1.4 Phenol derivatives

Phenol is both a synthetically and naturally produced
aromatic compound. Microorganisms capable of
degrading phenol are common and include both
aerobes and anaerobes (van Schie and Young 2000).
Many aerobic phenol-degrading microorganisms have
been isolated and the pathways for the aerobic degradation of phenol are now established (van Schie and
Young 2000). The first steps include oxygenation of
phenol by phenol hydroxylase enzymes to form
catechol, followed by ring cleavage adjacent to or in
between the two hydroxyl groups of catechol. Phenol
can also be degraded under anaerobic conditions, but
this process is less well understood and documented,
and only a few anaerobic phenol-degrading bacteria
have been isolated to date (van Schie and Young 2000).
A number of practical applications exist for microbial
phenol degradation and these comprise the exploitation
of anaerobic phenol-degrading bacteria in the in situ
bioremediation of creosote-contaminated subsurface
environments, and the use of phenol as a co-substrate
for aerobic phenol-degrading bacteria to enhance in situ
biodegradation of chlorinated solvents. Chlorophenols
have been introduced into the environment through
their use as biocides and as by-products of chlorine
bleaching in the pulp and paper industry (Field and
Sierra-Alvarez 2008). Chlorophenols are subject to
both anaerobic and aerobic metabolism (AntizarLadislao and Galil 2003). Under anaerobic conditions,
chlorinated phenols can undergo reductive dechlorination when suitable electron-donating substrates are
available (Field and Sierra-Alvarez 2008). Under
aerobic conditions, both lower and higher chlorinated
phenols can serve as sole electron and carbon sources
supporting growth. Two main strategies are used by
aerobic bacteria for the degradation of chlorophenols.
Lower chlorinated phenols are initially attacked by
monooxygenases yielding chlorocatechols as the first
intermediates whilst polychlorinated phenols are
converted to chlorohydroquinones as the initial intermediates. Fungi and some bacteria are additionally
known that co-metabolize chlorinated phenols (Field
and Sierra-Alvarez 2008). These microbial degradation mechanisms have gradually been put to use for
the remediation of phenol- and phenol derivativescontaminated soil and other strata through composting.
Some of the most conclusive studies where
composting has been applied to bioremediate phenol
contaminated strata are now discussed. Laine et al.

(1997) have studied the fate of chlorophenols during
the composting of sawmill soil and impregnated wood
to see whether chlorophenols, in addition to mineralization, would form any harmful metabolites. The
toxicity assessed by luminescent bacteria tests
decreased during the composting, and it followed the
chlorophenol concentrations in the compost piles. The
threshold value for chlorophenol toxicity appeared to
be 200 mg of total chlorophenols/kg dry weight.
Based on the results obtained, Laine et al. (1997)
deduced that the toxicity tests were a quick and
promising tool for assessing the toxicity changes in
chlorophenol-contaminated soil but were not sensitive
enough to detect the concentrations that would meet
the remediation criteria for clean-up of chlorophenolcontaminated soil, which in this case was 10 mg/
kg dry weight total chlorophenols. In conclusion,
Laine et al. (1997) found that no harmful metabolites
were formed during composting of chlorophenolcontaminated soil, but the existing ones such as
polychlorinated dibenzo-p-dioxins and dibenzofurans
(PCDD/Fs) compounds were not removed during the
biological treatment. The results of biotransformation
studies suggested that the 3040% of the carbon in
chlorophenols that disappeared but did not mineralize
during the composting process most likely was built
into the bacterial biomass. Das and Xia (2008)
characterized the transformation kinetics of 4-NP
and its isomers during biosolids composting. Five
distinctive 4-NP isomer groups with structures relative
to a- and b-carbons of the alkyl chain were identified in
biosolids. Composting biosolids mixed with wood
shaving at a dry weight percentage ratio of 43:57 (C:N
ratio of 65:1) removed 80% of the total 4-NP within
2 weeks of the composting experiments. Das and Xia
(2008) have also found that isomers with a-methyla-propyl structure transformed significantly slower
than those with less branched tertiary a-carbon and
those with secondary a-carbon, suggesting isomerspecific degradation of 4-NP during biosolids
composting.
7.1.5 PCBs
Polychlorinated biphenyls (PCBs), that can be mixtures of up to 209 congeners, were first manufactured
in 1929 (Bhandari and Xia 2005) and these are
249
among the most widely detected chemicals in wastewater residual biosolids. Although PCBs are no
longer produced in the United States because they
build up in the environment and can cause harmful
health effects, they are still in use in many other
countries. Polychlorinated dibenzodioxins and polychlorinated dibenzofurans (dioxins) (Fu et al. 2003)
consist of 210 different compounds which have
similar chemical properties (Bhandari and Xia
2005). This class of compounds is persistent, toxic,
and bioaccumulative. They are generated as byproducts during incomplete combustion of chlorine containing wastes like municipal solid waste, sewage
sludge, and hospital and hazardous wastes (Bhandari
and Xia 2005). PCBs were widely used in the past
and now contaminate many industrial and natural
areas.
PCBs can be degraded by microorganisms via a
metacleavage pathway to yield tricarboxylic acid
cycle intermediate and (chloro)benzoate (CBA). The
initial step in the aerobic biodegradation of PCBs is
the dioxygenation of PCB congeners by the biphenyl
dioxygenase enzyme (Ang et al. 2005). In this step,
the enzyme catalyzes the incorporation of two
hydroxyl groups into the aromatic ring of a PCB
congener, which increases the reactivity of the PCBs,
rendering them more susceptible to enzymatic ring
fission reactions (Bruhlmann and Chen 1999).
Only one research has been reported in the literature
where composting has been applied for bioremediating
PCBs. Michel et al. (2001) determined the effects of
soil to amendment ratio on PCB degradation when a
PCB-contaminated soil from a former paper mill was
mixed with a yard trimmings amendment and composted in field scale piles. Temperature, oxygen
concentrations, and a number of other environmental
parameters that usually influence microbial activity
during composting were monitored. The PCBs in the
contaminated soil had a concentration of 16 mg/kg dry
weight and an average of 4 chlorines per biphenyl. The
soil was composted with five levels of yard trimmings
amendment (1482% by weight) in pilot scale compost piles of volume 25 m3 and turned once every
month. Michel et al. (2001) observed that up to a 40%
loss of PCBs with amendment levels of 60 and 82%.
Also, congener specific PCB analysis indicated that
less chlorinated PCB congeners (13 chlorines per
biphenyl) were preferentially degraded during the
composting process. On the other hand, bench-scale
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250
studies indicated that less than 1% of the PCBs in the

contaminated soil were volatilized from composts
during incubation with forced aeration at 55C. In
conclusion, Michel et al. (2001) observed PCB loss
during the composting of the PCB-contaminated soil
and this appeared to be for the most part due to
biodegradation, rather than volatilization.
7.1.6 Phthalic acid esters
Phthalic acid esters (PAEs) or phthalate esters are
manufactured in large quantities and have been used in
the production of plastics. Di-(2-ethylhexyl) phthalate
(DEHP), the most widely used phthalate ester, is
persistent during sewage treatment and readily accumulates in sediments and lipid tissues in aquatic
organisms (Bhandari and Xia 2005). DEHP, a suspected endocrine disruptor (Hoyer 2001) has been
reported in a variety of media including water,
atmospheric deposition, sediments, soil, biosolids,
biota, and food products (Fu et al. 2003; Bluthgen
2000). Among the PAEs targeted by the USEPA as
priority pollutants, DEHP is the major pollutant
identified at high concentrations level in lagooning
sludge at about 28.67 mg/kg and in activated sludge at
about 6.26 mg/kg. Other PAEs, such as di-butyl
phthalate (DBP) and di-methyl phthalate (DMP) show
very low concentrations (Amir et al. 2005).
Several studies have been carried out to assess the
biodegradability and bioremediation of phthalate
esters by composting and results have so far been
promising. Marttinen et al. (2004) studied the potential
of composting and aeration to remove DEHP from
municipal sewage sludge with raw sludge and anaerobically digested sludge. They found that composting
removed 58% of the DEHP of the raw sludge and 34%
of that of the anaerobically digested sludge during
85 days stabilization in compost bins, while a comparable removal for the anaerobically digested sludge
was achieved in a rotary drum composter in 28 days.
Although DEHP removal was greater from raw sludge
compost than anaerobically digested sludge compost,
the total and volatile solids removals were similar in
the two composts. Moreover, Marttinen et al. (2004)
determined that in the aeration process mode of raw
sludge at 20C, the DEHP removals were 3341 and
5062% in 7 and 28 days, respectively. The pool of
results hence collected by Marttinen et al. (2004)
indicated that both composting and aeration have the
123
potential to reduce the DEHP contents typically found

in sewage sludges to levels acceptable for agricultural
use. On a similar note, in assessing sludge composting
as a bioremediation approach for DEHP, Gibson et al.
(2007) investigated the impact of pilot-scale composting and drying of sludge on the physicochemical
characteristics and on the concentrations of some
organic contaminants. During the 143-day composting
experiments, OM content fell by 22% and moisture by
50%. Concentrations of 4-nonylphenols fell by 88%
and DEHP by 60%, and these losses continued
throughout the procedure. The drying process was
much shorter and lasted only 40 days, yet OM content
decreased by 27% and moisture by 85%. Losses of
4-NPs (39%) and DEHP (22%) were less than in
composting and stopped when the moisture content
quasi stabilized. Gibson et al. (2007) concluded that
composting would be the method of choice for
reducing organic contaminants but this bioremediation
technique requires much longer times than drying.
Cheng et al. (2008) also came to similar inferences as
Amir et al. (2005) when investigating the potential
degradation of DEHP and OM of sewage sludge by
composting using laboratory reactors at different
operating conditions. At the end of composting, Cheng
et al. (2008) observed that the total DEHP degradation
was more than 85% in all conditions and the total
carbon reduction varied from 7.6 to 11.8%. Cheng et al.
(2008) deduced that the degradation kinetics of DEHP
in thermophilic phase and the phase thereafter were
modeled by first order and fractional power kinetics,
respectively.
7.1.7 Bioremediation of pesticides
Chemical pesticides1 have consistently demonstrated
their merit by increasing the global agricultural
productivity (Ecobichon 2001), reducing insectborne, endemic diseases and protecting plantations,
forests and harvested wood (Ecobichon 2000). As of
date, pesticides are more valued in developing
According to the United States Environmental Protection

Agency (US EPA) (1999), the term pesticide is a broad
nonspecific term covering a large number of substances
including, insecticides, herbicides and fungicides, though
often misunderstood to refer only to insecticides.
countries, particularly those in tropical regions seeking to enter the global economy by providing offseason fresh fruits and vegetables to countries in
more temperate climates (Ecobichon 2001). However, the continuous use of pesticides has caused
severe irreversible damage to the environment,
caused human ill-health, negatively impacted on
agricultural production and reduced agricultural sustainability (Wilson and Tisdell 2001).
Traditional methods of pesticide remediation
which are however relatively costly include excavation and/or chemical oxidation processes (for example, photocatalysis, ozonation and iron-catalyzed
Fentons reaction) or thermal processes (for example
low temperature themal desorption, incineration). On
the other hand, bioremediation and phytoremediation
are the biotic processes that are sometimes employed
for the remediation of pesticides contaminated sites
(Lynch and Moffat 2005). The use of phytotechnologies to remediate these more persistent pesticides is
only emerging (Chaudhry et al. 2002; Zhuang et al.
2007). Still, difficulties persist, including the potential phytotoxicity of some herbicides (Eullaffroy and
Vernet 2003; Van Eerd et al. 2003) that were
originally developed but destroyed plant material.
Typically the mechanisms involved in pesticide
phytoremediation are phytodegradation, rhizodegradation, and phytovolatilization. As a form of low cost
clean-up bioremediation option, composting and
biobeds2 are increasingly being assessed as an
approach to remediate pesticides. Some studies have
been carried out to this end and they unanimously are
in favor of composting. The fate of the widely used
lawn care herbicide 2,4-dichlorophenoxyacetic acid
(2, 4-D) during the composting of yard trimmings
consisting of primarily leaves and grass is an
important unexplored question. In their study, Michel
et al. (1995) determined the extent of 2, 4-D
mineralization, incorporation into humic matter,
volatilization, and sorption during the composting
of yard trimmings. Yard trimmings (2:1 [wt/wt]
leavesgrass) were amended with 14C-ring-labeled 2,
A biobed in its simplest form is a rectangular lined pit,

11.3 m deep, filled with a mixture of topsoil, peat-free
compost and straw in a ratio of 1:1:2, respectively and turfed
over. Biobeds filter out pesticides and use enhanced microbial
activity to break them down.
251
4-D (17 mg/kg dry weight) and composted in a

temperature-controlled laboratory scale compost system. During composting, thermophilic microbes were
numerically dominant, reaching a maximum of
2 9 1011/g. At the end of composting, 46% of the
OM present in the yard trimmings was lost and the
compost was stable, with an oxygen uptake rate of
0.09 mg O2/g OM/h, and was well humified. Michel
et al. (1995) also observed that the mineralization of
the OM temporally paralleled the mineralization of
2,4-D. In the final compost, 47% of the added 2,4-D
carbon was mineralized, about 23% was complexed
with high-molecular-weight humic acids while about
20% remained bound. With very little volatilization of
2,4-D occurred during the composting process, Michel
et al. (1995) noted with interest that their results
indicated an active mineralization of 2,4-D at composting temperatures of 60C. To elucidate the hazard
potential of compost application, Hartlieb et al. (2003)
amended municipal biowaste with 14C labelled pyrene
and simazine, which they incubated in a pilot-scale
composting simulation system. A mass balance incorporating the mineralization, metabolism and sorption
of the two model substances was then established over
a period of 370 days. Hartlieb et al. (2003) found that
the results wee quite different for the two chemicals
thereby reflecting their intrinsic properties during their
degradation in the composting environment. Ghaly
et al. (2007) have evaluated the effectiveness of invessel thermophilic composting on the destruction of
pirimiphos-methyl (O-(2-diethylamine-6-methylpyrimidin-4-yl) O,O-dimethyl phosphorothioate). Pirimiphos-methyl is an insecticide with both contact and
fumigant action and shows activity against a wide
variety of insects including ants, beetles, caterpillars,
cockroaches, fleas, flies, mites, mosquitoes and moths.
With a half-life of 117 days in water, 180270 days on
greens and seeds, pirimiphos-methyl has been reported
to cause cholinesterase inhibition in humans which at
high dose rates results in nausea, dizziness, and
confusion and at high exposure due to accidents and
major spills results in respiratory paralysis and death.
The bioreactor for the composting process studied by
Ghaly et al. (2007) was operated on a mixture of
tomato plant residues, wood shavings and municipal
solid compost. Ghaly et al. (2007) found that the
composting process successfully destroyed 8189% of
pirimiphos-methyl within the first 54 h of the composting process, while the complete destruction of the
123
252
pesticide required approximately 440 h. Ghaly et al.

(2007) also inferred that a number of physical,
chemical and biological mechanisms contribute to
the degradation of pirimiphos-methyl in the environment and these consist of mineralization, abiotic
transformations, adsorption, leaching, humification,
and volatization. During composting of greenhouse
wastes, in particular, the degradation of pirimiphosmethyl is accelerated by high temperatures developed
during the thermophilic stage of the process, OM
content, moisture of the compost matrix and level of
biological activity. Delgado-Moreno and Pena (2009)
amended a typical calcareous agricultural soil of the
Mediterranean area contaminated with four triazine
herbicides with olive cake, compost and vermicompost of olive cake at rates four times higher than the
agronomic dose in order to stimulate the biodegradation of simazine, terbuthylazine, cyanazine and prometryn, and thereafter observed that the residual
herbicide concentrations at the end of the degradation
assay showed no significant differences between non
amended and amended soil. However, interestingly,
Delgado-Moreno and Pena (2009) found that the
addition of compost and vermicompost had enhanced
the biological degradation rate of triazines during the
first week of incubation, with half-lives ranging from 5
to 18 days for the amended soils.
7.2 Controlled solid phase biotreatment
These processes include prepared treatment beds,
biotreatment cells, and soil piles, biopiles or composting matrices. Moisture, heat, nutrients, oxygen,
and pH can be controlled to enhance biodegradation.
These technologies differ from landfarming in that
treatment processes are often enclosed to control offgases. Typically, excavated material is mixed with
soil amendments and placed on a treatment area that
includes leachate collection systems and some of
aeration. The costs of these techniques vary widely,
but are among the expensive ones when applicable.
Some prepared bed bioremediation techniques
involved the continuous spray application of a
nutrient solution into the soil and collection and
recycle of the drainage from the soil pile. The
drainage itself may be treated in a slurry-phase
bioreactor before recycling. Vendors have developed
proprietary nutrient and additive formulations and
methods for incorporating the formulation into the
123
soil to stimulate biodegradation. Target contaminants

include non-halogenated VOCs and SVOCs. Pesticides also can be treated, but the process may be less
effective and may be applicable only to some
compounds within these contaminant groups. Like
landfarming, these technologies require a lot of space,
and excavation of contaminated material is required.
One advantage, however, of contained ex situ methods
is that toxic byproducts or metabolites formed during
the biodegradation process (e.g., vinyl chloride from
TCE) are contained.
7.3 Slurry phase bioremediation
These technologies involve the treatment of excavated
contaminated soils in the controlled environment of a
bioreactor. Excavated soil is processed to separate
stones and rubble, then mixed with water to a
predetermined concentration dependent upon the
concentration of the contaminants, the rate of biodegradation, and the physical nature of the soils. Usually
slurries contain from 10 to 40% solids. Electron
acceptors and nutrients are added to the reactor, and
parameters such as pH and temperature are controlled
to optimize biological processes. Also, the reactor may
be inoculated with specialized organisms if a suitable
population is not present. Both aerobic and anaerobic
reaction environments may be used. Target contaminants include petrochemicals, solvents, pesticides,
wood preservatives, explosives, petroleum hydrocarbons and other organic chemicals. Bioreactors are
favored over in situ biological techniques for heterogeneous soils, low permeability soils, areas where
underlying groundwater would be difficult to capture,
or when faster treatment times are required. Like solid
phase ex situ treatments, they have the advantage of
containing toxic metabolites such as vinyl chloride.
Slurry phase treatment tends to be faster, but more
expensive, than controlled solid phase treatment.
Table 14 highlights some findings of recent studies
which have demonstrated the promise of slurry phase
bioremediation of organic contaminants and soils.
8 Anaerobic digestion biotechnology

Anaerobic processes are defined as biological processes in which organic matter is metabolized in an
environment free of dissolved oxygen or its precursors
SETTLE, and 3 h of DECANT with chlorpyrifos

concentrations of 3,000, 6,000 and 12,000 lg/g
Venkata
Mohan et al.
(2004)
TNT (2,4,6-trinitrotoluene)- Molasses and starch used to estimate the effect of cocontaminated soil
substrate on anaerobic slurry phase bioremediation
Carbofuran degrader isolated from carbofuran

phytoremediated soil, Burkholderia cepacia PCL3
(PCL3) immobilized on corncob used as the inoculum
Degradation efficiency of TNT in molasses and starch addition was
approximately 97 and 87%, which is 5060% higher than 38%
without co-substrate addition. Molasses and starch addition
enhanced TNT degradation
In et al.
(2008)
Carbofuran (20 mg/kg soil) Bioslurry phase sequencing batch reactor. A 2-L laboratory Bioaugmentation treatment (addition of PCL3) gave the highest
Plangklang
percentage of carbofuran removal (96.97%), followed by
contaminated soil
glass bottle was used as a bioreactor with a working
and
bioaugmentation together with biostimulation (addition of molasses) Reungsang
volume of 1.5 l at room temperature (27 2C). One
total cycle period of the SBR was comprised of 1 h of fill treatment (88.23%), suggesting that bioremediation was an effective (2010)
technology for removing carbofuran in contaminated soil
phase, 82 h of react phase, and 1 h of decant phase
Depletion of around 47 and 57% of NAP and CBZ within 24 h,

Rodrguezrespectively. Complete depletion of NAP and around 48% for CBZ Rodrguez
were achieved within 72 h in sludge solid cultures with 38% bulking et al. (2010)
material
The degradation rate was rapid at lower substrate concentration and

12,000 lg/g of substrate concentration was found to be inhibitory
Bioslurry reactor adopting sequencing batch mode (anoxic At 3,000 lg/g of chlorpyrifos concentration, 91% was degraded
aerobicanoxic) operation
after 72 h of the cycle period, whereas in the case of 6,000 lg/g
Reactor operation was monitored for a total cycle period of of chlorpyrifos, 82.5% was degraded. However, for 12,000 lg/g
of chlorpyrifos, only 14.5% degradation was observed
72 h consisting of 3 h of FILL, 64 h REACT, 2 h of
The reactor was operated with a total cycle period of 120 h

comprising 3 h of fill, 114 h of react and 3 h of decant
phases
Bioslurry cultures spiked

Growth and activity of the white-rot fungus Trametes
with naproxene (NAP)
versicolor on sewage sludge were assessed in bioslurry
and carbamazepine (CBZ) and solid-phase systems
Chlorpyrifos contaminated
soil using native mixed
microflora
Bio-slurry phase reactor operated in the sequencing batch Control reactor (without ETP micro-flora) showed 23% of substrate Rama Krishna
mode under anoxicaerobicanoxic micro-environment
removal efficiency. The reactor augmented with mixed ETP micro- et al. (2006)
Effect of augmentation with effluent treatment plant (ETP) flora showed enhanced performance with more than 90% of
micro-flora on the process performance of the reactor was substrate removal efficiency within 72 h of the cycle period
Pendimethalin
contaminated soil
also evaluated
Bio-slurry phase reactors operated in periodic

Control reactor (killed control) showed only 6% of pyrene degradation Venkata
discontinuous batch mode under anoxicaerobicanoxic while the non-augmented reactor showed an efficiency of 34%
Mohan et al.
anoxic microenvironment
(substrate degradation rate (SDR)0.0165 g pyrene/kg soil/day).
(2008)
In the case of augmented reactors, the system operated with low
substrate loading rate (SLR) showed a pyrene degradation efficiency
of almost 90% (SDR0.04 g pyrene /kg soil/day) and the reactor
with high SLR showed 50% (SDR0.025 g pyrene /kg soil/day)
of pyrene degradation
Reference
Simulated pyrenecontaminated soil
Bioprocess feature(s)
Contaminated media/
contaminants
Table 14 Example of slurry phase bioremediation: Process features and remediation potential

253
123
The performance of the bio-slurry system with respect to anthracene

degradation was found to depend on both substrate loading rate and
Five reactors were operated for a total cycle period of 144 h bioaugmentation
(6 days) at soil loading rate of 16.66 kg soil/m/day at
Application of bioaugmentation showed positive influence on the rate
30 2C temperature
of degradation of anthracene
Series of bio-slurry phase reactors operated in periodic

discontinuous/sequencing batch mode under anoxic
aerobicanoxic microenvironment using native soil
microflora
Soil-bound anthracene
The control reactor (without microflora) showed negligible

Prasanna et al.
degradation of anthracene due to the absence of biological activity (2008)
Slurry phase bioreactor operated in sequencing batch

mode (anoxicaerobicanoxic microenvironments)
Soil contaminated with
pendimethalin
Bioprocess feature(s)
Contaminated media/
contaminants
Table 14 continued
At 1:20 soilwater ratio, the slurry phase system showed enhanced Venkata
degradation of substrate (629 lg pendimethalin/g soil). The removal Mohan et al.
efficiency of pendimethalin in the reactors was dependent on the
(2007)
mass-transfer rates of the substrate from the soil to the aqueous
phase
Reference
254
123
(Khanal 2008). The anaerobic process is classified as

either anaerobic fermentation (Valdez-Vazquez et al.
2005; Ren et al. 2006) or anaerobic respiration
(Rhoads et al. 2005) depending on the type of electron
acceptors (Khanal 2008).
In an anaerobic fermentation, organic matter is
catabolized in the absence of an external electron
acceptor by facultative anaerobes through internally
balanced oxidationreduction reactions under dark
conditions (Khanal 2008; Vatsala et al. 2008). The
product generated during the process accepts the
electrons released during the breakdown of organic
matter. Thus, organic matter acts as both electron
donor and acceptor. During the fermentation reactions,
the substrate is only partially oxidized, and therefore,
only a small amount of the energy stored in the
substrate is conserved (Khanal 2008). The major
portion of the adenosine triphosphate (ATP) or energy
is generated by substrate-level phosphorylation (Sgarbi et al. 2009; Lemire et al. 2009; Atlante et al. 2005).
Anaerobic respiration on the other hand requires
external electron acceptors for the disposal of electrons released during the degradation of organic
matter. The electron acceptors in this case could be
CO2, SO42- or NO3-. Both substrate-level phosphorylation and oxidative phosphorylation generate energy
(or ATP) (Khanal 2008). The energy released under
such a condition is much greater than anaerobic
fermentation (Skoog et al. 2007). Skoog et al. (2007)
have reported that at in situ geochemical conditions
where large numbers of heterotrophic microorganisms
inhabit hydrothermal systems, for aldose being reacted
upon by these microbial populations, fermentation
yields 220420 kJ/mol of energy while anaerobic
respiration releases 5002,400 kJ/mol.
Anaerobic biotechnology is becoming widely
popular due to its potential to produce renewable
biofuels and value-added products from low-value
feedstock such as waste streams (Khanal 2008). In
addition, it provides an opportunity for the removal of
pollutants from liquid and solid wastes more economically than the aerobic processes (Marttinen et al.
2003; Khanal 2008). The merits of anaerobic digestion technology are a recovery of bioenergy and
biofuels, recovery of value-added products and waste
treatment. Although the anaerobic process has many
inherent benefits, it is not a panacea for the treatment
of all types of wastewaters and sludges (Khanal
2008). Some of the limitations of anaerobic treatment
system are: long start-up time, long recovery time,

specific nutrients and trace metal requirements, more
susceptible to changes in environmental conditions,
treatment of high-sulphate wastewater and constant
meticulous operational attention.
8.1 Anaerobic digestion chemistry
The anaerobic digestion process is characterized by
a series of biochemical transformations brought on
by different consortia of bacteria (Fantozzi and
Buratti 2009). The anaerobic digestion of organic
matter basically follows the following stages: hydrolysis, acidogenesis, acetogenesis and methanogenesis
(Appels et al. 2008; Vavilin et al. 2008; Fountoulakis
et al. 2008; Fantozzi and Buratti 2009). Despite the
successive steps, hydrolysis is generally considered as
rate limiting (Appels et al. 2008) and the rate of
hydrolysis depends on the pH, temperature, composition and concentration of intermediate compounds
(Fantozzi and Buratti 2009). The hydrolysis step
degrades both insoluble organic material and high
molecular weight compounds such as lipids, polysaccharides, proteins and nucleic acids, into soluble
organic substances (e.g., amino acids and fatty acids)
(Appels et al. 2008) by extracellular hydrolytic
enzymes produced by hydrolytic bacteria and then
dissolved into solution. The components formed
during hydrolysis are further split during acidogenesis,
the second step. Volatile fatty acids, alcohols (Fantozzi
and Buratti 2009) are produced by acidogenic bacteria
(Bengtsson et al. 2008) along with ammonia, carbon
dioxide, hydrogen sulpide and other by-products
(Goblos et al. 2008). This phase is accompanied by
decrease of pH due to production of acids and protonic
acidification. If the reactor is overloaded, low pH value
may inhibit the process (Chen et al. 2008b). The main
species identified as responsible for the biological
hydrogen production during the acidogenesis of the
carbohydrates are Enterobacter, Bacillus and Clostridium (Davila-Vazquez et al. 2008; Cai et al. 2009).
The third stage in anaerobic digestion is acetogenesis,
where the higher organic acids and alcohols produced
by acidogenesis (Shida et al. 2009) are further digested
by acetogens to produce mainly acetic acid as well as
CO2 and H2. This conversion is controlled to a large
extent by the partial pressure of H2 in the mixture
(Appels et al. 2008). The final stage of methanogenesis
produces methane (Tatsuzawa et al. 2006) by two
255
groups of methanogenic bacteria (Narihiro and Sekiguchi 2007): the first group splits acetate into methane
and carbon dioxide and the second group uses hydrogen as electron donor and carbon dioxide as acceptor to
produce methane. The bacteria involved in the methanogenesis stage are sensitive to low as well as to high
pH, which must be kept within a range of 6.58.
8.2 Sludge digestion
Sludge treatment has long become the one of the most
challenging problems in wastewater treatment plants
(Zhang et al. 2007; Yu et al. 2008). As a result of the
wide application and utilization of the waste activated
sludge process, excess sludge presents a serious
disposal problem (Neyens and Baeyens 2003; Hao
et al. 2007). The management of excess activated
sludge also imposes great economic costs on the
operation and maintenance of wastewater treatment
plants and hence represents in itself significant
technical challenges (Li et al. 2008) as a results of
environmental, economic, social and legal factors
(Chu et al. 2009). Many efforts have been devoted to
reduce the excess sludge burden (Naddeo et al. 2009)
by treatments such as digestion and dewatering. Some
sludge treatment technologies include pre-treatment
and sludge minimization, anaerobic digestion, aerobic
digestion, alkaline stabilization, composting, dewatering, drying and innovative technologies (Fitzmorris
et al. 2009). Anaerobic digestion has now become a
commonly applied biological process for stabilization
of sewage sludges (Arnaiz et al. 2006; Aitken et al.
2005). The process is more beneficial among several
sludge stabilization methods by reason of it having be
able to produce a net energy gain (Mao et al. 2004; Lu
et al. 2008; Bohn et al. 2007) in the form of methane
gas leading to cost-effectiveness (Mao et al. 2004).
The biodegradability of waste sludge can be
improved by using thermal energy (Bougrier et al.
2008), enzymes and bacteria (Li et al. 2009), ozonation
(Zhang et al. 2009; Dytczak et al. 2007), acidification,
alkaline addition (Lopez Torres and Espinosa Llorens
2008), high pressure homogenization (Kidak et al.
2009), mechanical disintegration and ultrasound (Chu
et al. 2001) pre-treatments. Some investigations have
discussed the combined treatment of alkaline addition
and ultrasound. Among these processes of physical
pre-treatments, ultrasonication is viewed as an environmentally and economically sound pretreatment
123
256
(Show et al. 2007; Mao and Show 2007) which exhibits

the benefit of not being hazardous to the environment,
and hence being green (Chu et al. 2001; Cintas and
Luche 1999; Nikolopoulos et al. 2006).
8.3 Anaerobic biotechnology and pollutant

remediation
During the last recent years, research interest has also
been growing in the study and application of anaerobic digestion for the degradation and elimination of
pollutants such as dyes (Senthilkumar et al. 2009),
PAHs (Bernal-Martinez et al. 2007), highly chlorinated hydrocarbons and xenobiotics (Zhang and
Bennett 2005), adsorbable organic halides (Savant
et al. 2006) and pesticides (dos Santos et al. 2007).
Very recently, Baczynski and Pleissner (2010) have
used methanogenic granular sludge and wastewater
fermented sludge as inocula for batch tests of anaerobic bioremediation of chlorinated pesticide contaminated soil, and their results obtained for both types
of biomass were similar wit 80 to over 90% of chexachlorocyclohexane (c-HCH), 1,1,1-trichloro-2,2bis-(4-methoxyphenyl)ethane (methoxychlor) and 1,1,
1-trichloro-2,2-bis-(4-chlorophenyl)ethane (DDT)
removed in 4-6 weeks. Bernal-Martinez et al. (2009)
have assessed the removal of PAH naturally present in
sludge by continuous anaerobic digestion with recirculation of ozonated sludge. Recirculation of ozonated digested sludge allowed enhancing PAH removals
with the highest efficiency obtained with the highest
ozone dose (0.11 g O3/g TS). In another study,
Bernal-Martnez et al. (2005) investigated the combined effects of anaerobic digestion and ozonation in
reducing sewage sludge production, and it was
ozonation of anaerobically digested sludge improved
the PAH removal rate up to 61%. In their study,
Fuchedzhieva et al. (2008) have tested Bacillus cereus
isolated from municipal wastewater treatment plant to
assess the efficiency of two anionic surfactants, a
chemical surfactant and a biosurfactant during fluoranthene biodegradation under anaerobic methanogenic conditions (linear alkyl benzene sulphonates
(LAS) and rhamnolipid-biosurfactant complex from
Pseudomonas sp. PS-17, respectively). Biodegradation of fluoranthene was monitored by GC/MS for a
period up to 12th day. No change in the fluoranthene
concentration was registered after 7th day. While it
123
was reported that the rhamnolipid-biosurfactant had

inhibited the cell growth and had no effect on the
biodegradation rate, LAS enhanced the cell growth as
well as the fluoranthene biodegradation, thereby
demonstrating the latter surfactants promise as an
agent for facilitating the process of anaerobic PAH
biodegradation under methanogenic conditions.
9 Biosorption of heavy metals

Wastewaters from various industries, such as metal
finishing, electroplating, plastics, pigments and mining, contain several heavy metals of health and
environmental concern, such as cadmium, copper,
chromium, zinc and nickel (Dang et al. 2009).
Industrial wastewater containing heavy metals is a
threat to the public health because of the accumulation
of the heavy metals in the aquatic life which is
transferred to human bodies through the food chain.
All the more, nowadays, an increasing number of
hazardous organic compounds together with variable
levels of heavy metals ions are also being discharged
into the environment (Aksu 2005). Most of the organic
pollutants are degraded or detoxificated by physical,
chemical and biological treatments before released
into the environment. Although the biological treatments are a removal process for some organic compounds, their products of biodegradation may also be
hazardous. Moreover, some non-degradable compounds like the heavy metals ions discharged into the
environment along with the treated compounds can
cause problems due to non-degradability, bioaccumulation, biomagnification and transport to long distances. As a result, some organic molecules and the
heavy metals ions are not biodegradable and persist in
the environment.
9.1 Heavy metals removal

Conventional methods for the removal of the heavy
metals ions from wastewaters include chemical
precipitation, electroflotation, ion exchange, reverse
osmosis and adsorption onto activated carbon
(Cimino et al. 2005). But due to operational demerits,
high cost of the treatment and the generation of
toxic chemical sludges, some new technologies have
been tried for a long time (Elouear et al. 2008).
Among them less expensive non-conventional adsorbents like apple waste (Maranon and Sastre 1991),
peanut hull carbon (Periasamy and Namasivayam
1995), agricultural wastes (Azab and Peterson 1989)
and red mud (Apak et al. 1998) are being investigated
for the removal of ions like the Cd and Ni ions. Sud
et al. (2008) propose the use of agricultural waste
materials as bioadsorbents of heavy metals as a low
cost and highly efficient technology, because the
functional groups present in agricultural waste biomass (acetamido, alcoholic, carbonyl, phenolic,
amido, amino and sulphydryl groups) have affinity
for heavy metals ions to form metal complexes or
chelates that immobilize the contaminants through
reactions of chemisorption, complexation, adsorption
on surface, diffusion through pores and ion exchange.
As a result, researchers and engineers, all alike, have
been oriented toward the practical use of adsorbents
for the treatment of wastewater polluted by heavy
metals (Kocasoy and Guvener 2009).
Many agricultural wastes, including barks,
manures, and composts, contain high levels of lignocellulosic materials. Harman et al. (2007) have
hypothesized that the lignin fraction, which contains
numerous reactive groups, would be highly effective
in binding and removing heavy metals ions from
contaminated water, and, further, that the absorptive
capabilities of the materials would be strongly affected
by the pH of the solution. A series of materials have
been tested by Harman et al. (2007), and, at pH levels
above about 5.5, these materials were highly effective
in removing heavy metals ions, generally as large or
larger than nickel, but ineffective in removal of lighter
ions such as sodium or magnesium. Various barks
were generally observed to be the most effective and
were capable of removing more than 90% of iron,
copper, or lead from solutions in simple shake flask
experiments. Harman et al. (2007) also highlighted
that materials that retain cellular structures and that
have high lignin contents were highly effective with
barks possessing these properties. At alkaline pH
levels, many heavy metals ions precipitate, but three
separate lines of evidence from the extensive study of
Harman et al. (2007) indicate that ions were removed
from aqueous solutions by absorption to barks rather
than by precipitation. At acidic pH levels, they also
were partially effective in removal of the oxyanion
chromate. The study of Harman et al. (2007) hence
underpinned that biosorption is becoming a promising
257
alternative to replace or supplement the present

removal processes of pollutants from wastewaters
and other contaminated aqueous media.
9.2 Removal of heavy metals by biosorption

Among these pollutants consisting of dyes, phenolics,
herbicides, hormones and pesticides, heavy metals
ions have recently been of great and renewed concern
because of the extreme toxicity and/or persistency in
the environment. Biosorption is the binding and
concentration of adsorbate(s) from aqueous solutions
(even very dilute ones) by certain types of inactive,
dead, microbial biomass. The major advantages of
biosorption over conventional treatment methods
include: low cost, high efficiency, minimization of
chemical or biological sludge, regeneration of biosorbents and possibility of metal recovery (Sud et al.
2008). Another powerful technology is adsorption of
heavy metals by activated carbon for treating domestic
and industrial wastewater. However the high cost of
activated carbon and its loss during the regeneration
restricts its application. Since the 1990s the adsorption
of heavy metals ions by low cost renewable organic
materials has gained momentum. Recently attention
has been diverted towards the biomaterials which are
byproducts or the wastes from large scale industrial
operations and agricultural waste materials.
Hence, research on biosorption of heavy metals,
intrinsically guided by Green Chemistry, has led to the
identification of a number of microbial biomass types
that are extremely effective in concentrating metals.
Some types of biomass are waste byproducts of largescale industrial fermentations while other metal-binding biomass types can be readily harvested from the
oceans. These biomass types can accumulate in excess
of 25% of their dry weight in deposited heavy metals:
Pb, Cd, U, Cu, Zn, Cr and others. Some biosorbents
can bind and collect a wide range of heavy metals with
no specific priority, whereas others are specific for
certain types of metals. When choosing the biomass
for metal biosorption experiments, its origin is a major
factor to be considered. In general terms, biomass can
come from industrial wastes which should be obtained
free of charge, organisms that can be obtained easily in
large amounts in nature (e.g., bacteria, yeast, algae) or
fast-growing organisms that are specifically cultivated
or propagated for biosorption purposes (crab shells,
123
258
Table 15 Biosorption studies for heavy metals

Heavy metal
Biosorbents
References
Cadmium
Black gram husk (Cicer arientinum), Rice polish

agricultural waste, Orange wastes from orange juice
production processes, Wheat bran, Pretreated
rice husk (RRH), Red algae (Ceramium virgatum)
Saeed and Iqbal (2003), Singh et al. (2006b),

Perez-Marn et al. (2007), Kumar and Bandyopadhyay
(2006), Sar and Tuzen (2008)
Chromium
Neurospora crassa fungal biomass, Mucilaginous

seeds of Ocimum basilicum, Sargassum sp. algae,
Turbinaria ornata seaweed, Helianthus annuus
(sunflower) stem waste
Tunali et al. (2005), Melo and DSouza (2004),

Vieira et al. (2008), Aravindhan et al. (2004),
Jain et al. (2009)
Copper
Lichen biomass of Cladonia rangiformis hoffm.,

Sphaerotilus natans immobilised in polysulfone
matrices, Marne alga Sargassum sp., Spent-grain,
Grape stalks
Marne alga Gracilaria Corticata
Ekmekyapar et al. (2006), Beolchini et al. (2003),

Da Silva et al. (2002), Lu and Gibb (2008),
Machado et al. (2003), Esmaeili et al. (2008)
Nickel
Loofa sponge-immobilized biomass of Chlorella

sorokiniana, Sargassum wightii seaweed, Cone
biomass of Thuja orientalis, Marne green alga
Ulva reticulata, Biomass and silica-immobilized
biomass of Medicago sativa (alfalfa)
Akhtar et al. (2004), Vijayaraghavan et al. (2005a, b),

Malkoc (2006), Gardea-Torresdey et al. (1996)
Lead
Candida albicans, Rhodotorula glutinis yeast,

Powder of mature leaves of the Neem
(Azadirachta indica) tree, Green algae
Cladophora fascicularis, Formaldehyde
polymerized banana stem
Baysal et al. (2009), Cho and Kim (2003),

Bhattacharyya and Sharma (2004),
Deng et al. (2007), Noeline et al. (2005)
Zinc
Azadirachta indica bark, Orange peel cellulose with

Phanerochaete chrysosporium immobilized
Ca-alginate beads, Gossypium hirsutum (Cotton)
waste biomass, Mature leaves and stem bark
of the Neem (Azadirachta indica) tree
King et al. (2008), Lai et al. (2008),

Riaz et al. (2009), Arshad et al. (2008)
seaweeds). Research on biosorption (examples of

which are given in Table 15) is revealing that it is
sometimes a complex phenomenon where the metallic
species could be deposited in the solid biosorbent
through various sorption processes, such as ion
exchange, complexation, chelation, microprecipitation and oxidation/reduction.
9.3 Scientific basis of biosorption

Several important aspects of biosorption for heavy
metal removal need to be considered when exploring
this emerging bioremediation technique for optimization purposes. The underlying principles of biosorption for removal of metal ions, the kinetics of
mass transfer during the process of biosorption
of metal ions, the theory and models that can be
used to describe the mass transfer process and the
123
thermodynamics of biosorption of heavy metals onto

biomass and the models which can be used to
quantify metal-biomass interactions at equilibrium,
all are key knowledge areas in biosorption science
which have been hence so far relatively well
presented, dicussed and reviewed in the literature.
The reader is earnestly directed to more comprehensive and extensive reviews by Davis et al. (2003),
Figueira et al. (2000), Loukidou et al. (2004), Naja
and Volesky (2006), Vijayaraghavan and Yun (2008)
and Volesky (2001) where these scientific aspects of
biosorption have excellently and extensively been
reported.
A biosorption process can be performed via
several modes (Vijayaraghavan and Yun 2008); of
which, batch and continuous modes of operation are
frequently employed to conduct laboratory scale
biosorption processes. Although most industrial
applications prefer a continuous mode of operation,
batch experiments have to be used to evaluate the

required fundamental information, such as biosorbent
efficiency, optimum experimental conditions, biosorption rate and possibility of biomass regeneration.
The factors influencing bacterial batch biosorption
are solution pH, temperature, ionic strength, biosorbent dosage, biosorbent size, initial solute concentration and agitation rate (Vijayaraghavan and Yun
2008).
9.4 Models for biosorption
Within the literature, the Langmuir and Freundlich
models (two-parameter models) have been used to
describe biosorption isotherm. The models are simple, well-established and have physical meaning and
are easily interpretable, which are some of the
important reasons for their frequent and extensive
use (Vijayaraghavan and Yun 2008). Some other twoparameter models widely used for describing biosorption isotherms include the Temkin isotherm, the
DubininRadushkevich model, the RedlichPeterson
model, the Sips model, the Khan model, the Radke
Prausnitz model and the Toth model. Of these threeparameter models, the RedlichPeterson and Sips
models have been used with most success. Preetha
and Viruthagiri (2007) have repoted the biosorption
of chromium using suspended and immobilized cells
of Rhizopus arrhizus by evaluating the physicochemical parameters of the solution such as initial
chromium ion concentration in both batch and packed
bed reactor. Besides the Langmuir, Freundlich and
RedlichPeterson adsorption isotherm models which
fitted accurately with the experimental data, the
Thomas model, AdamsBohart and Wolborska models were also used to represent the dynamic sorption
of chromium using immobilized beads. Preetha and
Viruthagiri (2007) deduced that the Thomas model
represented well the sorption of chromium at different residence times whilst the AdamsBohart model
was fitted better at the initial part of the breakthrough,
with the Wolborska model also representing the
sorption of chromium accurately.
Mechanistic models have been proposed to describe
solute adsorption onto the surfaces of biomass. The
development of a mechanistic model is usually based
on preliminary biomass characterization, with the
formulation of a set of hypothesized reactions between
the sorbent sites and solutes, which also considers the
259
particular solution chemistry of the solutes. Mechanistic models can often be characterized by the
different degrees of complexity or accuracy in a
system description to account for the surface heterogeneity and other factors that contribute to non-ideal
adsorption phenomena. Mechanistic modeling of biosorption has been attempted in several investigations,
with significant success.
Mathematical models that can describe the behaviour of a batch biosorption process operated under
different experimental conditions are very useful for
scale up studies or process optimization (Loukidou
et al. 2004). Over 20 models have been reported in
the literature, all of which have attempted to quantitatively describe the kinetic behavior during the
adsorption process. Each adsorption kinetic model
has its own limitations, which are derived according
to specific experimental and theoretical assumptions.
Even though they violate the fundamental assumptions, many adsorption models have been used to
successfully test experimental biosorption data. Of
these, pseudo-first and pseudo-second order models
(Eqs. 1, 2, respectively) have often been used to
describe biosorption kinetic data.

Qt Qe 1 eKt
1

1
Qt Qe 1
2
1 Qe Pt
where Qe is the amount of solute sorbed at equilibrium (mg/g); Qt the amount of solute sorbed at time
t (mg/g); K the first order equilibrium rate constant
(min-1) and P the second order equilibrium rate
constant (g/mg/min). In most published cases involving biosorption, the pseudo-first order equation was
found to not fit well over the entire contact time
range, but was generally applicable over the initial
periods of the sorption process.
9.5 Mechanisms of biosorption
Different metal-binding mechanisms have been postulated to be active in biosorption metal uptake such
as chemisorption by ion-exchange, complexation,
coordination, chelation; physical adsorption and
microprecipitation (Volesky 2001). There are also
possible oxidationreduction reactions taking place
in the biosorbent. Due to the complexity of biomaterials and biosorbents, it is also plausible that at
123
260
least some of these mechanisms are acting simultaneously to varying extents depending on the biosorbent
composition, surface properties and functional chemical groups, and the solution environment (Volesky
2001). Biomass materials offer several molecular
groups that are known to offer ion exchange sites,
carboxyl, sulphate, phosphate, and amine, could be the
main ones (Volesky 2001). Ion-exchange is an important concept in biosorption, because it explains many
of the observations made during heavy metal uptake
experiments (Davis et al. 2003). It should be pointed
out that the term ion-exchange does not explicitly
identify the binding mechanism, rather it is used here
as an umbrella term to describe the experimental
observations (Davis et al. 2003). The precise binding
mechanism(s) may range from physical (i.e., electrostatic or Londonvan der Waals forces) to chemical
binding (i.e., ionic and covalent).
10 Factors influencing bioremediation

The microbial population follows a growth cycle
comprising the three distinct phases namely the lag
phase, exponential phase, stationary phase, and death
phase (Brul et al. 2008; Chong et al. 2008; Akerlund
et al. 1995). In the lag phase there is a delay in the
microbial population growth until the microbes have
become acclimatized to the substrate(s) (Bai et al.
2009a, b; Saravanan et al. 2008), which in many
instances are the contaminants/pollutants under remediation, and surrounding conditions. The microbes
cannot consume the food source until they have
developed the required enzymes and metabolites
necessary to break down the contaminant (Talley and
Sleeper 2006). After the necessary enzymes and
metabolites have been produced, the microbes enter
the exponential phase of growth (Rahman et al. 2006).
The rate of exponential growth is influenced by
environmental conditions as well as by characteristics
of the organism itself. However, exponential growth
cannot occur indefinitely. Generally, either an essential nutrient for growth is used up or some waste
product of the organism builds up to such a level that
the exponential growth is inhibited and ultimately
ceases (Mulchandani et al. 1989; Okpokwasili and
Nweke 2006). At this point the microbial population
reached the stationary phase, where there is no net
increase or decrease in microbial cell populations.
123
Dependent on the possible build up of environmental

toxins or depletion of bioavailable substrates (Yates
and Smotzer 2007), the microbial population may
enter the death phase and the viable number of
microbes will decrease. Based on this growth cycle,
almost all organic compounds are degradable given
the proper environmental, physicochemical and time
conditions (Talley and Sleeper 2006). However, a
range of physical, chemical and biochemical conditions or materials can interfere with bioremediation
rates. Some of them can be controlled or modified
while some are difficult to control. The most salient
factors are discussed below.
10.1 pH
pH values\3 and[9 or 10 as well as sudden changes
in the pH of the waste/treatment system matrix can
significantly inhibit microbial growth by interfering
with the microbial metabolism, gas solubility in soil
water, nutrients availability and bioavailability in soil
water, and heavy metal solubilities (Agarry et al.
2008). Most natural environments have values of pH
between 5.0 and 9.0, and as a result this range is
optimal for microbial enhanced biodegradation of
waste contamination. This pH range is maintained by a
natural buffering capacity that exists in most fertile
native soils due to the presence of carbonates and other
minerals (Robinson et al. 2009). However, this
buffering capacity can be depleted over time as a
result of acidic byproducts of degradation (Komnitsas
et al. 2004). The majority of bacteria exhibit growth
optima at or near neutral pH (Andreas and Ekelund
2005) whilst most soils are acidic throughout the
world. Treatment commonly known as liming involves
the addition of finely ground agricultural limestone,
calcium hydroxide, calcium carbonate or magnesium
carbonate during tilling and mixing of the upper layers
to keep the pH in a favorable range for optimal
microbial metabolism. This treatment may affect the
solubility, bioavailability and the chemical form of the
organic pollutants and of soil macro- and micronutrients. Fungi are generally more resistant to acidic
soils than soil and aquifer bacteria.
10.2 Temperature
Temperature affects (a) the bacterial metabolism (b)
microbial growth rates (c) the soil matrix and (d)
physic-chemical state of the contaminants. Generally

in situ bioremediation is carried under mesophilic
condition (2040C). Even for laboratory studies,
bacteria with potential remediation value have
focused on mesophilic species because of the species,
of cultivation and relatively short doubling times
(Kuntz et al. 2008; Abid et al. 2007). The rate of
biochemical reactions in cells increases with temperature up to a maximum, above which the rate of
activity declines as enzyme denaturation occurs and
organisms either die or become less active (TrasarCepeda et al. 2007). Low temperatures seldom kill the
microbes and with warming the microbes typically
recover. Temperature also affects gas solubilities and
must be taken into account when designing a remediation system. In compost heaps or biopiles, the
temperature in the center of the soil/sediment may
reach 70C or higher during the initial active phase
and thermophilic bacteria can be seen performing
under such conditions (Bongochgetsakul and Ishida
2007; Eklind et al. 2007). In situ technology for soils
in tropical countries where the soil temperature may
exceed 50C merits further investigation. Even at
0.28.3C, purging of contaminated groundwater,
enrichment with nutrients and hydrogen peroxide
have been carried successfully. Even modest increases
in temperature can significantly increase bioremediation rates. Melin et al. (1998) have mineralized
groundwater contaminants including 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP), and
pentachlorophenol (PCP) in three aerobic fluidizedbed reactors (FBRs) employing sand, volcanite, and
diatomaceous earth as biomass carriers. The effect of
temperature on the chlorophenol degradation kinetics
was studied in FBR batch tests at temperatures
ranging from 4 to 16.5C. Melin et al. (1998) reported
that the specific maximum degradation rates for TCP
and TeCP varied with temperature from 0.46 9 10-3
to 31 9 10-3 mg/mgVS/h and Ks varied from zero to
7.1 mg/l, while the specific degradation rates for PCP
degradation varied with temperature from 0.24 9
10-3 to 1.7 9 10-3 mg/mgVS/h and were always
lower than for other chlorophenols. Use of the
Arrhenius equation described the temperature effects
on biodegradation of chlorophenols, and in the studied
temperature range, Melin et al. (1998) deduced that a
10C increase in temperature generally resulted in
over seven times higher degradation rates. Ferguson
et al. (2003) studied the effects of temperature on the
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hydrocarbon mineralisation rate in Antarctic terrestrial

sediments. 14C-labelled octadecane was added to
nutrient amended microcosms that were incubated
over a range of temperatures between -2 and 42C.
Ferguson et al. (2003) found a positive correlation
between temperature and mineralisation rate, with the
fastest rates occurring in samples incubated at the
highest temperatures. The main implications for
bioremediation in Antarctica from the study of Ferguson et al. (2003) have been that a high-temperature
treatment would yield the most rapid biodegradation
of the contaminant. Still, Coulon et al. (2005) have
conducted mesocosm studies using sub-Antarctic soil
artificially contaminated with diesel or crude oil in
Kerguelen Archipelago in an attempt to evaluate the
potential of a bioremediation approach in high latitude
environments. All mesocosms were sampled on a
regular basis over 6 months period, and it was found
that soils responded positively to temperature increase
from 4 to 20C, and to the addition of a commercial
oleophilic fertilizer containing N and P. Both factors
were seen to have increased the hydrocarbon-degrading microbial abundance and total petroleum hydrocarbons (TPH) degradation. The major inferences
from the study of Coulon et al. (2005) were that the
bioremediation of hydrocarbon-contaminated subAntarctic soil appeared to be feasible, and various
engineering strategies, such as heating or amending
the soil could accelerate hydrocarbon degradation.
Still, a number of techniques are used to increase in
situ soil remediation applications. These include use of
mulches, plastic covers, vegetation cover to moderate
fluctuations in soil temperature. In cold climates,
steam may be injected to raise the soil temperature or
heating return water. Temperatures of compost heaps
can be increased by irrigation with heated waters.
For very cold environments, above ground liquid
and slurry bioreactors, where the temperature can be
optimized, are the only choices.
10.3 Metals
Metals can inhibit various cellular processes and their
effects are often concentration-dependent (Salanitro
et al. 1997; Sani et al. 2001; Alisi et al. 2009). Metal
toxicity for microbes will usually involve specific
chemical reactivity. Metals such as copper, silver,
and mercury are typically very toxic particularly as
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262
ions, while metals such as lead, barium and iron are

usually benign to the microbes at levels typically
encountered. The nutrient metals are usually found
naturally in the necessary amounts for plants and
microbes in fertile soils (Khan 2005). The principal
inorganic nutrients are nitrogen and phosphorus;
however, trace amounts of potassium, calcium, sulphur, magnesium, iron, and manganese are also
required for optimum biological growth (Rajeshwari
et al. 2000). The availability and/or toxicity of these
metals to the microbes is usually dependent on pH,
with the metals becoming more mobile/available at
higher values of pH.
Metals can be actively accumulated by certain
microorganisms and plant species (Kamal et al. 2004).
Living cells can adsorb metals and concentrate inorganics within the cell and although heavy metals may
not be metabolically essential, they are taken up by the
biomass as a side effect of the normal metabolic
activity of the cell (Teng et al. 2008; Azcon et al.
2009). Activated biomass removes metals from solution by a variety of mechanisms which include ion
exchange at the cell walls, complexation reactions at
the cell walls, and intra- and extra-cellular complexation reactions. Inactivated biomass removes metals
primarily by adsorbing metals to the ionic groups
either on the cell surface (Powell et al. 1999;
Ahluwalia and Goyal 2007) or in the polysaccharide
coating found on most forms of bacteria (Das et al.
2007). The metal ions are bound by exchange of
functional groups or by sorption on polymers. Adsorption is therefore a procedure of choice for treating
industrial effluents, and a useful tool for protecting the
environment when used to bioremediate contaminated
aqueous media (Crini 2005). Adsorption on natural
polymers and their derivatives are known to remove
pollutants from water. The increasing number of
publications on adsorption of toxic compounds by
modified polysaccharides (over 3,500 since 2000) has
shown the recent increasing interest in the synthesis of
new low-cost adsorbents used in wastewater treatment. In this context, Crini (2005) has performed an
excellent review of the latest developments in the
synthesis of adsorbents containing polysaccharides, in
particular modified biopolymers derived from chitin,
chitosan, starch and cyclodextrin. New polysaccharide
based-materials have described and their advantages
for the removal of pollutants from the wastewater
thoroughly discussed.
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10.4 Toxic compounds

Just as contaminant concentrations that are too low can
complicate bioremediation (Sikdar et al. 1998), high
aqueous-phase concentrations of some contaminants
can create problems (Volkering et al. 1997). At high
concentrations, some chemicals are toxic to microbes,
even if the same chemical is readily degraded at lower
concentrations (Ramos et al. 2009). Toxicity prevents
or slows metabolic reactions and often prevents the
growth of new biomass needed to stimulate rapid
contaminant removal (Agarry et al. 2008). The degree
and mechanisms of toxicity vary with specific toxicants, their concentration, and the exposed microorganisms. Microbial cells cease to function when at
least one of the essential steps in their numerous
physiological processes is blocked. The blockage may
result from gross physical disruption of the cell
structure or competitive binding of a single enzyme
essential for metabolizing the toxicant (Agarry et al.
2008; Talley and Sleeper 2006). By design, some
organic compounds are toxic to targeted life forms
such as insects and plants, and may also be toxic to
microbes. These compounds include herbicides, pesticides, rodenticides, fungicides and insecticides.
In addition, some classes of inorganic compounds
such as cyanides and azides are toxic to many
microbes (Talley and Sleeper 2006; Gijzen et al.
2000); however, these compounds may be degraded
following a period of microbial adaption (Marsolek
et al. 2007; Kwon and Yeom 2009). In this respect,
certain studies have indeed shown the fungal biodegradation of cyanide and microbial adaption to such
toxic compounds. Dumestre et al. (1997) identified a
fungus identified as Fusarium solani IHEM 8026 as a
good potential for cyanide biodegradation under
alkaline conditions (pH 9.210.7). Results of K14 CN
biodegradation studies had showed that the fungal
metabolism seemed to proceed by a two-step hydrolytic mechanism with the first reaction involving the
conversion of cyanide to formamide by a cyanidehydrolyzing enzyme, cyanide hydratase (EC 4.2.1.66),
and thereafter a second reaction consisting of the
conversion of formamide to formate, which was
ociated with fungal growth. Earlier, Shah and Aust
(1993) had demonstrated the mineralization of potassium cyanide and various other cyanide salts (Fe, Cu,
Zn, Cd and Cr) by the white rot fungus Phanerochaete
chrysosporium with a 1.5 mmol/L potassium cyanide
solution having a rate of mineralization was about

0.17 mmol/L/day. P. chrysosporium also mineralized
[14C]cyanide contaminated soil (3,000 mmol/L/day)
using ground corn cobs as nutrient (10 mg/l/day).
Cyanide was oxidized to the cyanyl radical by a lignin
peroxidase from P. chrysosporium. Lately, Gurbuz
et al. (2009) have examined cyanide effluent degradation by Scenedesmus obliquus. Gold mill effluents
containing cyanide concentration of 77.9 mg/l was fed
to batch unit to examine the ability of S. obliquus for
degrading cyanide. Cyanide was reduced down to
6 mg/l in 77 h. Gurbuz et al. (2009) reported that the
cells had well adapted to high pH and the effluent
contained cyanide and the metals. All the more,
chlorinated aromatic compounds are biorecalcitrant,
and in particular, 2,4,5-trichlorophenol demonstrates
greater resistance to biodegradation than other trichlorophenols and is also a known uncoupler of the
electron transport chain (Marsolek et al. 2007). In this
respect, Marsolek et al. (2007) have investigated the
biorecalcitrance, inhibition, and more specifically the
adaptation to 2,4,5-trichlorophenol by aerobic mixed
microbial communities. Although it was initially
observed that 2,4,5-trichlorophenol was strongly
resistant to biodegradation at concentrations greater
than 40 lmol/L, and inhibited to respiration in direct
proportion to 2,4,5-trichlorophenol concentration, the
microbial communities later showed consistent adaptation patterns to 2,4,5-trichlorophenol at concentrations of 10 and 20 lmol/L.
263
mineralogy. Rocky conditions, low permeability,

complex mineralogy and water logged or arid conditions are not favourable to bioremediation (Bali
et al. 2002). Vinas et al. (2005) have examined the
bacterial community dynamics and biodegradation
processes in a highly creosote-contaminated soil
undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial
community, the number of heterotrophs and PAH
degraders, and the TPH and PAH concentrations were
monitored during the bioremediation process. While
TPH and PAHs were significantly degraded in all
treatments, Vinas et al. (2005) maintained the moisture content and aeration were the key factors
associated with the PAH bioremediation. Holden
et al. (1997) had earlier quantified the effects of
matric and solute waterpotential on toluene biodegradation by Pseudomonasputida mt-2, a bacterial
strain originally isolated from soil. Across the matric
potential range of 01.5 MPa, growth rates were
maximal for P. putida at -0.25 MPa and further
reductions in the matric potential resulted in concomitant reductions in growth rates. Growth rates
were constant over the solute potential range
01.0 MPa and lower at -1.5 MPa. This specific
study revealed that for P. putida, slightly negative
matric potentials facilitate faster growth rates on
toluene but more negative water potentials result in
slower growth. Also, the toluene utilization rate per
cell mass was observed to be highest without matric
water stress and was unaffected by solute potential.
10.5 Water content and geological characters

10.6 Nutrient availability
Water contents, and especially water availability,
influences bioremediation rates (Boopathy 2000). Li
et al. (1997) have reported that a lack in the effect
from bioremediation could be attributed to poor soil
water sorption, which was negatively influenced by
hydrocarbon residuals. This study hence supported
that the soil-water relation is one of the most
important factors in assessing endpoint of bioremediated soils for plant growth. Water in soils or
sediments may not be available to microorganisms
because it is absorbed by solid substances or tied up
as water of hydration to dissolved solutes. This can be
solved by irrigating the contaminated soils, compost
heaps and/or biopiles. In general, in situ degradation
rates are enhanced when the soil is granular or porous
with a relatively high permeability and uniform
Nutrients are generally supplemented in both in situ

and ex situ bioremediation of soils, sediments, ground
and surface waters for the promoting the bioremediation rates (Aspray et al. 2007; Liu et al. 2009).
Nutrient requirement depends on the nature of
contaminants and the extent to which the polluted
site has been subjected to agricultural use. Remediation of petroleum hydrocarbon contaminated sites
typically requires nitrogen, phosphorus. Chen et al.
(2008a) have reported that addition of excess ferric
iron combined with limited nitrate could promote the
in situ bioremediation of benzene, toluene, ethylbenzene and xylene isomers and trimethylbenzene isomers in the Borden aquifer and possibly for other sites
contaminated by hydrocarbons. Zhou et al. (2009)
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264
have investigated the effect of phosphorus concentration on PAH dissipation in the rhizosphere of
mycorrhizal plants in a pot experiment using two
plant species, alfalfa (Medicago sativa) and tall fescue
(Festuca arundinacea), The major finding was the
significant positive impact of mycorrhizal plants on
the dissipation of high molecular weight PAH in highwater low-phosphorus treatment. Earlier, El-Bestawy
and Albrechtsen (2007) investigated the mineralization and/or degradation of the phenoxy herbicide
mecoprop (MCPP) by a group of soil bacteria under
the effects of nutrient amendments. Five different
species of Pseudomonas (P. paucimobilis, P. aeruginosa, P. mallei, P. pseudomallei, and P. pickettii)
were isolated for the MCPP mineralization and/or
removal. Significant variations in the removal percentages of MCPP by either mineralization or biodegradation were observed. Also, the highest MCPP
mineralization and degradation by the selected Pseudomonas spp. were achieved by their inactive (dead)
followed by active-rich cultures with both inoculated
in nutrient-rich medium, confirming the positive
effects of nutrient amendments and sterilization on
MCPP decontamination. Brresen and Rike (2007)
have studied the effects of increased salinity (ionic
strength) and varying concentrations of nutrient and
soil moisture on hexadecane mineralization in a
hydrocarbon contaminated and nutrient deficient high
Arctic soil were assessed. Ammonium chloride
(NH4Cl) was added to give nitrogen concentrations
ranging from 0 to 1,000 mg NH4-N/kg soil, corresponding to molar cation concentrations of NH4?
from 0 to 71 mmol/kg soil. Soil samples with
combinations of NH4? and Na? were also included,
and the soil moisture content varied from 10 to 20%. It
was found that the fertilizing with NH4-N had
increased the total hexadecane mineralization compared to unfertilized soil at all concentrations investigated, and that the highest mineralization rates were
found in soil samples added 50200 mg NH4-N/kg at
10% moisture, where 5058 mg hexadecane/kg/day
had been mineralized.
10.7 External electron availability

Biostimulation through substrate addition is commonly practiced to support co-metabolic biodegradation processes. Addition of stimulatory substrates to
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enhance bacterial growth and metabolic activity

through enhanced electron transfer processes of
between electron donor and electron acceptors has
also been used in bio-augmentation experiments
involving both environmental clean-up and agricultural applications (Aboul-Kassim and Simoneit
2001).
Oxygen is used as an electron acceptor to increase
bioremediation activity (Boopathy 2000). A number
of anaerobic bacteria can break down a variety of
aliphatic and aromatic organic compounds both of
natural and anthropogenic origin wholly or partially by
denitrifying bacteria by sulphate, iron, and molybdenum reducers and by methanogenic consortia. Efforts
are being made to use anaerobic bacteria for breaking
down petroleum contaminated groundwater in oil
refinery sites in the presence of nitrates. Benzene,
toluene, ethyl benzene, xylene (BTEX) and chlorinated aliphatic and aromatic compounds have successfully removed. Methanogenic bacteria can degrade
chlorinated ethanes such as tetrachloroethane, trichloroethane, dichloroethane, perchloroethylene, carbon
tetrachloride, chloroform, tetrachloromethane, alkylbenzenes and a number of chlorinated aromatic
compounds. BTEX bioremediation projects often
focus on overcoming limitations to natural degradative
processes associated with the insufficient supply of
inorganic nutrients and electron acceptors. However,
other limitations associated with the presence and
expression of appropriate microbial catabolic capacities may also hinder the effectiveness of bioremediation. Thus, while subsurface addition of oxygen or
nitrate has proven sufficient to remove BTEX below
detection levels it has been only marginally effective at
some sites (Aboul-Kassim and Simoneit 2001). Dou
et al. (2008) have reported an effective anaerobic
BTEX biodegradation under nitrate and sulphate
reducing conditions by the mixed bacterial consortium
that were enriched from gasoline contaminated soil.
Under the conditions of using nitrate or sulphate as
reducing acceptor, the degradation rates of the six
tested substrates decreased with toluene [ ethylbenzene [ m-xylene [ o-xylene [ benzene [ p-xylene.
Drzyzga et al. (2002) carried out a sediment column
study to demonstrate the bioremediation of chloroethene- and nickel-contaminated sediment in a single
anaerobic step under sulfate-reducing conditions. By
stimulating the activity of sulphate-reducing bacteria
by the addition of sulphate as supplementary electron
acceptor, complex anaerobic communities were maintained with lactate as electron donor (with or without
methanol), which achieved complete dehalogenation
of tetra- and tri-chloroethenes (PCE and TCE) to
ethene and ethane. A few weeks after sulphate
addition, production of sulphide had increased, indicating an increasing activity of sulphate-reducing
bacteria. Hence, it may be deduced that microbial
activity stimulated under sulphate-reducing conditions
can have a beneficial effect on both the precipitation of
heavy metals and the complete dechlorination of
organochlorines as a result of the strongly negative
redox potential created by the activity of sulphatereducing bacteria. Regarding nitrate as a stimulant in
bioremediation, Lee et al. (2007) reported that triethyl
phosphate (TEP) treated along with NO3-, was most
effective for the biodegradation of diesel, this being
possible since TEP could be delivered more efficiently
to the target zones and with less phosphorus loss than
KH2PO4.
10.8 Bioavailability of pollutants

Biology in regards to bioremediation refers to the
intrinsic ability of the biota to assimilate and metabolize the contaminant (Pignatello 2009), and matrix
effects include the ways in which the biodegradation
is influenced by the interactions of the soil with the
biota and the contaminants. Bacteria in soils are
predominantly attached to soil particles, and so will
be constrained by this attachment and by the physicochemical properties of the surface (Pignatello 2009).
Contaminants interact with soils in complex ways
through sorption and mass transfer resistance that
generally impede their availability to organisms. For
example, anthropogenic organic polymers such as
polystyrene and polyvinyl chloride are highly
recalcitrant because of their insolubility and the lack
of extracellular microbial enzymes capable of catalyzing depolymerization. However, non-polymer
degrading bacteria and actinomycetes are able to
degrade oligomeric polystyrene fragments and low
molecular weight fragments of lignin resulting from
fungal attacks on the lignin polymer.
Raj et al. (2007) have isolated eight bacterial
strains on kraft lignin (KL) containing mineral salt
medium (L-MSM) agar with glucose and peptone
from the sludge of pulp and paper mill. Out of these,
265
ITRC-S8 was selected for KL degradation, because

of its fast growth at the highest tested KL concentration and use of various lignin-related low molecular weight aromatic compounds (LMWACs) as sole
source of carbon and energy. Significant reduction in
colour and KL content in subsequent incubations
have been reported and the degradation of KL by
bacterium was confirmed by gas chromatography
mass spectrometry (GCMS) analysis indicating
formation of several LMWACs such as t-cinnamic
acid, 3,4,5-trimethoxy benzaldehyde and ferulic acid
as degradation products, which were not present in
the uninoculated sample, which led to think of the
biochemical modification of the KL polymer to a
single monomer unit. Recently, Trinh Tan et al.
(2008) have investigated the aerobic biological
degradation of the synthetic aliphaticaromatic
co-polyester EcoflexTM (BASF) by 29 strains of
enzyme-producing soil bacteria, fungi and yeasts at
moderate environmental conditions. It was found
that the aliphaticaromatic co-polyester could be
degraded by a number of different microorganisms
and the bacteria studied preferentially degraded the
bonds between aliphatic components of the copolymer and the rate of biodegradation of oligomers was
appreciably faster than that for the polymer chains.
Using GCMS techniques, Trinh Tan et al. (2008)
identified the degradation intermediates as the
monomers of the co-polyester, and gel permeation
chromatography was able to suggest exo-enzyme
type degradation, whereby the microbes had hydrolysed the ester bonds at the termini of the polymeric
chains preferentially.
All the more, low water solubility and a tendency
to adsorb to particulate matter in soils and sediments
are factors that can severely limit in situ biodegradation of many sediments contaminated with organic
contaminants, polychlorinated and polycyclic aromatic hydrocarbons. Consequently, the rates of
desorption and dissolution of contaminants in the
water phase can be improved by adding surfactants
(either biosurfactants or synthetic detergents) to the
contaminated zone (Mulligan 2009). Biosurfactants
are surface active compounds having a wide range of
industrial applications such as enhanced oil recovery,
lubricants, bioremediation of pollutants and food
processing. The structures of these complex molecules include lipopeptides, glycolipids, polysaccharide protein complexes, fatty acids and phospholipids.
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266
Optimal production of biosurfactant (glycolipid) by

Bacillus megaterium was obtained in 3L laboratory
scale fermenter when peanut oil cake (2%) was used
as carbon source (Thavasi et al. 2008). Carotenoids
are important natural pigments with a range of
applications as colorants, feed supplements and
neutraceuticals. Lycopene is a red coloured intermediate of the b-carotene biosynthetic pathway and is an
important dietary carotenoid. It is reported to inhibit
the harmful effect of ferric nitrilotriacetate on DNA
in rats and prevents liver necrosis. Lopez-Nieto et al.
(2004) reported the development of a semi-industrial
process (800 l fermentor) for lycopene production by
mated fermentation of Blakeslea trispora plus (?)
and minus (-) strains. This process describes the
critical requirement of soybean cake (44 g/l) as
nitrogen source for optimal lycopene production.
Mustard oil cake (6%) in the presence of Mg2? ions
is reported to improve lactic acid production ability
of agar-gel immobilized Lactobacillus casei after
48 h, when further addition of the substrate (whey
lactose) failed to maintain the process efficiency (Tuli
et al. 1985). Biosurfactants produced by microorganisms within soils and sediments have been shown to
enhance biodegradation rates. In a laboratory study to
assess the effect of adding either Pseudomonas
aeruginosa UG2 cells or the biosurfactants produced
by this microorganism on the biodegradation of a
hydrocarbon mixture in soil at 20C over a 2-month
incubation period, Jain et al. (1992) had observed that
the addition of 100 lg of UG2 biosurfactants g-1 soil
significantly enhanced the degradation of tetradecane,
hexadecene and pristane but not 2-methylnaphthalene, the most water-soluble of the hydrocarbons. The
effect of two nonionic surfactants of the alkylphenolethoxylate type, Arkopal N-300 and Sapogenat
T-300, on the bioavailability of PAH in manufactured
gas plant soil was evaluated in soil columns percolated by recirculating flushing water by Tiehm et al.
(1997). It was observed that both surfactants had
enhanced the mass transfer rate of sorbed PAH into
the aqueous phase due to solubilization and made it
more bioavailable for biodegradation. Bardi et al.
(2000) have analyzed the in vitro effect of cyclodextrins on the biodegradative activity of a microbial
population isolated from a petroleum-polluted soil, as
shown by the decrease of dodecane (C12), tetracosane (C24) anthracene and naphthalene added individually as the sole carbon source to mineral medium
123
liquid cultures. B-cyclodextrin was seen to have

accelerated the degradation of all four hydrocarbons,
particularly naphthalene, and influenced the growth
kinetics as shown by a higher biomass yield and
better utilization of hydrocarbon as a carbon and
energy source.
Lately, Whang et al. (2008) have investigated the
potential application of two biosurfactants, surfactin
(SF) and rhamnolipid (RL), for the biodegradation of
diesel-contaminated water and soil with a series of
bench-scale experiments. The rhamnolipid used in this
study was a commonly isolated glycolipid biosurfactant produced by Pseudomonas aeruginosa J4, while
the surfactin, was a lipoprotein type biosurfactant
produced by Bacillus subtilis ATCC 21332. It was
deduced that both biosurfactants had been able in
increasing the diesel solubility with increased biosurfactant addition. In the diesel/water batch experiments,
an addition of 40 mg l-1 of surfactin significantly
enhanced biomass growth (2,500 mg VSS l-1) as well
as increased the diesel biodegradation percentage to
94%, compared to batch experiments with no surfactin
addition (1,000 mg VSS/l and 40% biodegradation).
The addition of rhamnolipid to diesel/water systems
from 0 to 80 mg/l substantially increased biomass
growth and diesel biodegradation percentage from
1,000 to 2,500 mg VSS/l and 40100%, respectively.
Hence, the enhancing capability on both efficiency and
rate of diesel biodegradation in diesel/soil systems of
surfactin and rhamnolipid was clearly demonstrated by
Whang et al. (2008). Last but not least, Lai et al. (2009)
have recently developed a screening method to evaluate the oil removal capability of biosurfactants for oilcontaminated soils collected from a heavy oil-polluted
site using two biosurfactants (rhamnolipids and surfactin) and synthetic surfactants (Tween 80 and Triton
X-100). Their results have convincingly shown that
biosurfactants exhibited much higher TPH removal
efficiency than the synthetic ones examined. Lai et al.
(2009) also reported that the TPH removal efficiency
had increased with an increase in biosurfactant
concentration (from 0 to 0.2 mass %). Yet another
promising approach to improve bioremediation rates is
to add biodegradable solvents to assist desorption and
dissolution rates with consequent increase in the
biodegradation of the adsorbed pollutants (Ludmer
et al. 2009).
Zoller and Reznik (2006) have developed a
surfactant/surfactant-nutrient mix (SSNM) for
267
enhanced bioremediation methodologies for sustainable, in situ bioremediation of fuel-contaminated

aquifers. The major findings of this study were the
kerosenes maximum enhanced mobilization compared with that of deionized water when using SSNM
having composition of linear alkylbenzene sulphonate
(LABS): coco-amphodiacetate (containing N): surfactant-nutrient X (containing both N and P) at 0.15: 0.15:
0.05 g/l, respectively. The major effects of the SSNM
addition were reported to be the enhanced mobilization of the bulky nonaqueous phase liquid (NAPL) and
the enhanced desorbtion/ solubilization/dispersion of
the entrapped NAPL which, in turn, facilitated their
enhanced biodegradation. Sun et al. (2009) have
examined the laboratory use of aqueous ethyl lactateodified [S,S]-ethylenediaminedisuccinic acid (EDDS)
washing solutions for the simultaneous removal of
phenanthrene, pyrene, and Cu from contaminated soils.
Ethyl lactate demonstrated greater solubilization efficiency for phenanthrene and pyrene than ethanol. Thus,
ethyl lactate is believed to have a greater potential for
extracting PAHs from contaminated soils. Sun et al.
(2009) highlight that the addition of ethyl lactate in
EDDS solution (EDDS/Cu molar ratio = 2) efficiently
enhanced the extraction of the PAHs and also significantly increased the Cu removal from 34.8 to 42.9%.
The latter was mainly attributed to the fact that ethyl
lactate increased the stability constant for Cu-EDDS
complexes, hence shifting the degree of desorption of
Cu from soil.
and microcosms to evaluate groundwater bioremediation potential of tetrachloroethene (PCE) in the

presence of additional pollutants present. Reductive
dechlorination of PCE was studied under anaerobic
and aerobic conditions. It was observed that the
consortia derived from anaerobic sludge and amended
with electron donors quantitatively and incompletely
degraded PCE to cis-dichloroethylene, whereas in
reactors augmented with a dehalogenating culture
complete dechlorination of PCE occurred even in the
presence of additional toxic contaminants. Adebusoye
et al. (2008) have observed substantial metabolism of
2,3,4,5-tetrachlorobiphenyl (2,3,4,5-tetraCB) and
2,30 ,40 ,5-tetraCB by axenic cultures of Ralstonia sp.
SA-5 and Pseudomonas sp. SA-6 in the presence of
biphenyl supplementation, although, the strains were
unable to utilize tetrachlorobiphenyls as growth substrate. Ziagova et al. (2009) have reported a comparison of the ability of Staphylococcus xylosus to
degrade 2,4-dichlorophenol and 4-Cl-m-cresol in separate cultures. In this study, bacterial adaptation and
the continuous presence of glucose, as a conventional
carbon source, were found to stimulate the degrading
efficiency of S. xylosus. All the more, microbes can
sequentially remove halogen atoms from polluting
halogenated compounds wherein halogen atoms are
replaced by hydrogen under anaerobic conditions.
Here, halogen atoms serve as hydrogen acceptors and
hence dehalogenation involves co-metabolism and
provision of a growth promoting substance.
10.9 Co-metabolism
10.10 Gene expression
Co-metabolism is a process whereby microorganisms

involved in the metabolism of a growth promoting
substrate also transform other organic contaminants
which can be called as co-substrates (Prince 2010).
The latter are however not growth supporting if they
are provided as the only sources of carbon and
energy. This is one of the most important mechanisms involved in the transformation of chlorinated
organic aliphatic and aromatic contaminants by
microbes. Such co-metabolic transformation of
organic pollutants is an important process in both
aerobic and anaerobic environments namely bacterial
transformation of dichlorodiphenyl trichloroethane
DDT, PCBs (Abraham et al. 2002) and trichloroethylene. Volpe et al. (2007) have used batch reactors
The ability of indigenous microorganisms to degrade

organic pollutants is dependent on the expression of
the genes encoding the required enzymes. These genes
may not express if these substances are available in
very low concentrations. This can be overcome by
adding substances that are structurally related to the
organic pollutants which will act as inducers. Similarly, the presence of alternate carbon or energy source
may repress the expression of the degradative enzyme
needed to transform the target pollutant. For example,
addition of glucose or amino acids to aquifer samples
contaminated with toluene, ethylenedibromide, phenol and p-nitrophenol inhibits the degradation of these
contaminants because the microbes will prefer the
more easily degradable substrate.
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268
10.11 Bioaugmentation
Where degradative microbes do not exit or where the
process is too slow, microbial inoculates may be added
to enhance bioremediation rates. This technique is
known as bioaugmentation (Lima et al. 2009) and may
involve (a) an addition of natural isolates of bacteria or
(b) genetically engineered organisms (GEMs). There
are rigid rules governing the release of GEMs as there
is concern about their potential negative impacts on the
environment. The genetic patterns have evolved over
several decades and they are relatively stable. It is
believed that altered genomes have greater instability
and increase the chances of mutations, some of which
may not be safe. Bioaugmentation has met with
varying degrees of success. Gertler et al. (2009) have
applied an experimental prototype oil boom including
oil sorbents, slow-release fertilizers and biomass of the
Marne oil-degrading bacterium, Alcanivorax borkumensis, for sorption and degradation of heavy fuel
oil in a 500-L mesocosm experiment, and it was found
that growth of this obligate oil-degrading bacterium on
immobilized oil coincided with a 30-fold increase in
total respiration. Earlier, Bento et al. (2005) evaluated
the effect of bioaugmentation on the degradation of
TPH in soil. It was reported that bioaugmentation of
the contaminated soil showed the greatest degradation
in the light (72.7%) and heavy (75.2%) fractions of
TPH since the greatest microbial activity (dehydrogenase activity) had occurred with bioaugmentation up
to 3.3-fold. Jacques et al. (2008) have evaluated the
capacity of a defined microbial consortium (five
bacteria: Mycobacterium fortuitum, Bacillus cereus,
Microbacterium sp., Gordonia polyisoprenivorans,
Microbacteriaceae bacterium, Naphthalene-utilizing
bacterium; and a fungus identified as Fusarium
oxysporum) isolated from a PAHs contaminated
landfarm site to degrade and mineralize different
concentrations (0, 250, 500 and 1,000 mg/kg) of
anthracene, phenanthrene and pyrene in soil, and it
was found that the microbial consortium had degraded
on average, 99, 99 and 96% of the different concentrations of anthracene, phenanthrene and pyrene in the
soil, in 70 days, respectively. Domde et al. (2007)
equally reported a 52.2% removal of chemical oxygen
demand (COD) in a bioaugmented reactor while only
15.1% reduction of COD was observed in the reactor
without bioaugmentation. Domde et al. (2007) have
suggested that the gene pool of the bioaugmented
123
reactor had catabolic loci that could degrade accumulated intermediates, thereby improving the efficiency
of the oevrall system. Much recently, Teng et al.
(2010) have conducted a microcosm study to test the
bioremediation potential of Paracoccus sp. strain
HPD-2 on an aged PAH-contaminated soil. The
bioaugmented microcosms showed (a) a 23.2%
decrease in soil total PAH concentrations after
28 days, with a decline in average concentration from
9,942 to 7,638 lg/kg dry soil, and (b) higher counts of
culturable PAH-degrading bacteria, microbial biomass and enzyme activities were observed in bioaugmented soil.
11 Novel research trends in bioremediation

Bioremediation, an intimate branch of biotechnology,
in principle includes the use of microorganisms in
improving the condition of a contaminated site, with
most commonly bacteria being the degraders and other
organisms, such as soil animals or plant roots, playing
a role in disseminating the bacteria and, in providing
nutrients and co-substrates for the bacteria active in the
degradation processes (Romantschuk et al. 2000).
Bioremediation has, in principle, considerable public
support because it aims to enhance natural processes
and it is generally seen as environmentally appropriate. However, bioremediation rates are often considerably slower than physical methods such as removing
the contaminated material to a secure landfill (Prince
2010). In this respect, and in pursuit to improve the
performance of bioremediation processes, there have
been a number of different procedures that have been
tested more-or-less successfully with a view to
improve reliability, cost efficiency and bioremediation
rates. These methods range from minimal intervention, such as mere monitoring of intrinsic bioremediation, through in situ introduction of nutrients and/or
bacterial inocula or improvement of physico-chemical
conditions, or still excavation followed by on site or ex
situ composting in its different varieties.
However, modern biotechnology including genetic
engineering; culture of recombinant microorganisms,
cells of animals and plants; metabolic engineering;
hybridoma technology; bioelectronics; nanobiotechnology; protein engineering; transgenic animals and
plants; tissue and organ engineering; immunological
assays; genomics and proteomics; bioseparations and
bioreactor technologies (Gavrilescu and Chisti 2005)

have been gaining momentum in research and showing
much promise to improve bioremediation rates. Strategies for improving bioremediation efficiency using
genetic engineering consist in improving strains and
chemotactic ability, the use of mixed population
biofilms and optimization of physico-chemical conditions. For example, biofilms are assemblages of single
or multiple populations that are attached to abiotic or
biotic surfaces through extracellular polymeric substances (Singh et al. 2006a). Gene expression in
biofilm cells differs from planktonic stage expression
and these differentially expressed genes regulate
biofilm formation and development. Biofilm systems
have been shown to be especially suitable for the
treatment of recalcitrant compounds because of their
high microbial biomass and ability to immobilize
compounds (Singh et al. 2006a). All the more,
bioremediation is also facilitated and bioremediation
rates enhanced by gene transfer among biofilm organisms and by the increased bioavailability of pollutants
for degradation as a result of bacterial chemotaxis
(Singh et al. 2006a). Table 16 presents some of the
novel research trends and/or advances depicted in
bioremediation.
11.1 Genetically engineered microorganisms
(GEMs) and microbial systems
As in practically all microbial applications, the use of
genetic engineering to improve microbial capacities
opens many interesting possibilities to obtain new
species that are able to use or to degrade different
contaminants with high efficiency (Iranzo et al.
2001). In the case of bioremediation there is much
scientific work suggesting that engineered microorganisms have greater potential for environmental
clean-up than natural ones (Raskin 1996; Pieper and
Reineke 2000; Iranzo et al. 2001). Particular attention
is also being given to the genetic engineering of
bacteria using bacterial hemoglobin (VHb) for the
treatment of aromatic organic compounds under
hypoxic conditions (Urgun-Demirtas et al. 2006).
The application of VHb technology may advance
treatment of contaminated sites, where oxygen
availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic
pollutants (Urgun-Demirtas et al. 2006). However,
269
despite the many advantages of GEMs, there are still

concerns that their introduction into polluted sites to
enhance bioremediation may have adverse environmental effects, such as gene transfer.
A number of new recombinant DNA techniques
have been developed for genetically engineered
microorganisms for the biodegradation of environmental contaminants or for the synthesis of small
molecules (Keasling and Bang 1998). These techniques include new expression vectors to carry the
heterologous genes into the host organism, new
mechanisms to control gene expression, containment
mechanisms to control persistence of geneticallyengineered 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 (Keasling and Bang 1998). The application of
culture-independent molecular biological techniques
also offers new opportunities to better understand the
dynamics of microbial communities (Iwamoto and
Nasu 2001). Fluorescence in situ hybridization (FISH),
in situ PCR, and quantitative PCR are expected to be
powerful tools for bioremediation to detect and
enumerate the target bacteria that are directly related
to the degradation of contaminants, and thence better
engineer these for enhanced metabolisms related to
pollutants degradation (Iwamoto and Nasu 2001).
Nucleic acid based molecular techniques for fingerprinting the 16S ribosomal DNA (rDNA) of bacterial
cells, i.e., denaturing gradient gel electrophoresis
(DGGE) and terminal restriction fragment length
polymorphism (T-RFLP), have enabled the monitoring
of the changes in bacterial community in detail, and
such advanced molecular microbiological techniques
will definitely provide new insights into bioremediation in terms of process optimization, validation, and
the impact on the ecosystem, which are indispensable
data to make the technology reliable and safe (Iwamoto
and Nasu 2001).
Although plants have the inherent ability to detoxify
some xenobiotic pollutants, they generally lack the
catabolic pathway for complete degradation/mineralization of these compounds compared to microorganisms. Hence, transfer of genes involved in
xenobiotic degradation from microbes/other eukaryotes to plants may further enhance their potential
for remediation of such dangerous groups of compounds. Transgenic plants with enhanced potential for
123
270
Table 16 Recent research trends and advances reported in bioremediation

Bioremediation method
Outline of novel finding(s)
Reference
Treatment of sites contaminated

with chlorinated solvents
Results suggest that the reductive treatment of chlorinated

solvent sites with nano-scale zero-valent iron particles
might be enhanced by the concurrent or subsequent
participation of bacteria that exploit cathodic
depolarization and reductive dechlorination as metabolic
niches
Xiu et al. (2010)
Bioremediation process by biosorption

of effluents of wash process of the cotton
fabric by silver nanoparticles with the
bacterium Chromobacterium violaceum
The bacteria after biosorption were morphologically

transformed, but the normal morphology after a new
culture was completely restored. The process also allowed
the recovery of silver material that was leached into the
effluent for a reutilization avoiding any effect to the ecoenvironment
Duran et al. (2010)
Reduction and adsorption of Pb2? in aqueous

solutions
Nano-zero-valent iron was produced by a reduction method

and compared with commercial available zero-valent iron
powder for Pb2? removal from aqueous phase. In
comparison with Fluka zero-valent iron, nano-zero-valent
iron has much higher reactivity towards Pb2? and within
just 15 min 99.9% removal can be reached. Nano-zerovalent iron material has thus been demonstrated to have
great potential for heavy metal immobilization from
wastewater
Xi et al. (in press)
Bacterial degradation of organophosphates

(OPs)
Stenotrophomonas sp. strain YC-1, a native soil bacterium

that produces methyl parathion hydrolase (MPH), was
genetically engineered to possess a broader substrate
range (OPs). Results indicate that the broader substrate
specificity in combination with the rapid degradation rate
makes this engineered strain a promising candidate for in
situ remediation of OP-contaminated sites
Yang et al. (2010)
Fungal degradation of oily

sludge-contaminated soil
A novel yeast strain Candida digboiensis TERI ASN6 was

developed and could degrade 40 mg of eicosane in 50 ml
of minimal salts medium in 10 days and 72% of
heneicosane in 192 h at pH 3. The degradation of alkanes
yielded monocarboxylic acid intermediates while the
polycyclic aromatic hydrocarbon pyrene found in the
acidic oily sludge yielded the oxygenated intermediate
pyrenol
Sood et al. (2010)
The strain C. digboiensis could efficiently degrade the

acidic oily sludge on site because of its robust nature,
probably acquired by prolonged exposure to the
contaminants. Hence, the potential of Candida
digboiensis TERI ASN6 to bioremediate hydrocarbons
at low pH under field conditions has been demonstrated
detoxification of xenobiotics such as trichloro ethylene, pentachlorophenol, trinitro toluene, glycerol trinitrate, atrazine, ethylene dibromide, metolachlor and
hexahydro-1,3,5-trinitro-1,3,5-triazine are a few successful examples of utilization of transgenic technology (Eapen et al. 2007). Trees are already being used
for wastewater clean-up, for site stabilization, and as
barriers to subsurface flow of contaminated groundwater. Clonal propagation and the genetic tools of both
123
classical breeding and genetic engineering exist for a

number of both angiosperm and gymnosperm species,
opening the door to creation of tree remediation
cultivars (Stomp et al. 1993). Active research is also
underway to screen tree and plant species for their
enhanced ability to tolerate, take up, translocate,
sequester, and degrade organic compounds and heavy
metal ions. Chen and Wilson (1997) have evaluated
cells of a genetically engineered Escherichia coli
strain, JM109, which expresses metallothionein and a

Hg2? transport system after induction for their selectivity for Hg2? accumulation in the presence of
sodium, magnesium, or cadmium ions and their
sensitivity to pH or the presence of metal chelators
during Hg2? bioaccumulation. The genetically engineered E.coli cells in suspension were observed to have
accumulated Hg2? effectively at low concentrations
(020 lmol/l) over a broad range of pH (311). These
results suggested that the E. coli strain JM109 could be
used for selective removal of Hg2? from wastewater or
from contaminated solutions which are normally
resistant to common treatments. In a attempt to further
enhance the efficiency and potential of plants for
phytoremediation of mercury pollution, Nagata et al.
(2009) constructed a genetically engineered tobacco to
simultaneously express mercury transporter, mercury
transporter (MerT) and mercury chelator (Kiyono and
Pan-Hou 2006), polyphosphate (polyP) by integrating
bacterial merT gene in polyphosphate kinase gene
(ppk)-transgenic tobacco to evaluate its ability to
phytoremediate mercury. It was observed that the
integration of the merT gene into the ppk-transgenic
tobacco did not significantly affect the mercury
resistant phenotypes and polyp production but the
transgenic expression of MerT in ppk-transgenic
tobacco had resulted in an accelerated and enhanced
mercury uptake into tobacco. In addition, tobacco
expressing MerT and polyP accumulated significantly
more mercury than the ppk-transgenic tobacco from
medium containing a wide range of low concentrations
of Hg2?. Later, Deng et al. (2005) constructed a
genetically engineered E. coli SE5000 strain simultaneously expressing nickel transport system and metallothionein to accumulate Ni2? from aqueous solution.
Compared with 1.62 mg/g of Ni2? uptake capacity by
original host E. coli cells, the genetically engineered E.
coli could remarkably bind 7.14 mg/g Ni2?, and it
accumulated Ni2? effectively over a broad range of pH
(410), with an optimal pH at 8.6.
However, the vast majority of studies pertaining to
genetically engineered microbial bioremediation are
mostly supported by laboratory-based experimental
data (Sayler and Ripp 2000). In general, relatively few
examples of GEM applications in environmental
ecosystems exist, and unfortunately, the only manner
in which to fully address the competence of GEMs in
bioremediation efforts is through long-term field scale
studies whereby a reasonable pool of requisite
271
information for determining the overall effectiveness

and risks associated with GEM introduction into
natural ecosystems is acquired (Sayler and Ripp
2000).
11.2 Nanotechnology and bioremediation
Nanotechnology has contributed to the development
of a great diversity of materials as those used in
electronic, optoelectronic, biomedical, pharmaceutical, cosmetic, energy, catalytic, and materials applications. As a general definition, nanotechnology is
involved with objects on the nano scale, or materials
measuring between 1 and 100 nm (Duran 2008). In
future, modification and adaptation of nanotechnology will extend the quality and length of life
(Rajendran and Gunasekaran 2007). The social
benefits are significant from nanomaterials and the
new products are applicable to information technology, medicine, energy, and environment.
The emergence of nanotechnology presents a
number of potential environmental benefits. Most
environmental applications of nanotechnology fall
into three categories: (i) environmentally-benign and/
or sustainable products (e.g., green chemistry or
pollution prevention), (ii) remediation of materials
contaminated with hazardous substances, and (iii)
sensors for environmental agents (Tratnyek and
Johnson 2006). Some nanoparticles destroy contaminants, for instance, while others sequester them (Rao
and Murthy 2007, Telling et al. 2009). Carbon
nanotubes, for example, have been recognized for
their ability to adsorb dioxin much more strongly
than traditional activated carbon (Duran 2008). All
the more, the utilization of microbes for intracellular/
extracellular synthesis of nanoparticles with different
chemical composition, size/shapes and controlled
monodispersity can be a novel, economically viable
and eco-friendly strategy that can reduce toxic
chemicals in the conventional protocol.
Mace et al. (2006) have studied the assessment of
remediation of soil heavy metals with nano-particle
hydroxyapatite by the Toxicity Characteristic Leaching
Procedure by cultivation experiment. Ther results
indicated that nano-particle hydroxyapatite significantly
reduced the bioavailability of soil Cu and Zn when
compared with the control. The more nano-particle
hydroxyapatite were added, the more was the increasing
equilibrium time, and the more was the decreased
123
272
bioavailability of soil Cu and Zn, since soil pH was

significantly increased after the addition of nanoparticle hydroxyapatite, and heavy metals could adsorb
on nano-particle hydroxyapatite. In their study, Varanasi et al. (2007) have used nano-particles to remediate
PCB contaminated soil and an attempt was made to
maximize PCB destruction in each treatment step. Their
results showed that nano-particles did aid in the
dechlorination process and high PCB destruction
efficiencies could be achieved, with a minimum total
PCB destruction efficiency reported at 95%. Kanel et al.
(2007) have synthesized, characterized and tested
surface-modified iron nanoparticles (S-INP) for the
remediation of arsenite (As(III)), a well known toxic
groundwater contaminant of concern. The results using
S-INP pretreated 10 cm sand-packed columns containing *2 g of S-INP showed that 100% of As(III) was
removed from influent solutions at a flow rate 1.8 ml/
min containing 0.2, 0.5 and 1.0 mg/l As(III) for 9, 7 and
4 days providing 23.3, 20.7 and 10.4 l of arsenic free
water, respectively. In addition, it was found that 100%
of As(III) in 0.5 mg/l solution for the same flow rate was
removed by S-INP pretreated 50 cm sand packed
column containing 12 g of S-INP for more than
2.5 months providing 194.4 l of arsenic free water.
These results hence suggested that S-INP have great
potential to be used as a mobile, injectable reactive
material for in situ sandy groundwater aquifer treatment
of As(III). In their recent study, Elliott et al. (2008)
exposed groundwater and aquifer samples from a site
contaminated by hexachlorocyclohexanes (totaling
1,500 lg l-1)) to nanoscale iron particles to evaluate
the technology as a potential remediation method. Batch
experiments with 2.227.0 g/l iron nanoparticles
showed that more than 95% of the HCHs were removed
from solution within 48 h. Based on a survey of
literature of previously published work on a wide
variety of chlorinated organic solvents, the work of
Elliott et al. (2008) additionally demonstrated the
potential of zerovalent iron nanoparticles for treatment
and remediation of persistent organic pollutants (POPs).
cadmium, chromium, lead and zinc) are resistant to

degradation and represent an ongoing toxicological
threat to both wildlife and human beings. Bioremediation has grown into a green, attractive and promising
alternative to traditional physico-chemical techniques
for the remediation of these POPs at a contaminated
site, as it can be more cost-effective and it can
selectively degrade the pollutants without damaging
the site or its indigenous flora and fauna. However,
bioremediation technologies have had limited applications due to the constraints imposed by substrate and
environmental variability, and the limited biodegradative potential and viability of naturally occurring
microorganisms.
This review was not intended to address the much
voluminous literature on bioremediation, but rather to
revisit the basic of bioremediation and demonstrate
that the application of biotreatment is growing
rapidly due to its merits which outweigh the demerits.
The application of diverse bioremediation technologies must be based on sound and relilable scientific
data obtained in both fundamental as well as research
environmental laboratories. For the development of
bioremedial processes to succeed commercially, it is
essential to link different disciplines such as microbial ecology, biochemistry and microbial physiology,
together with biochemical and bioprocess engineering. In short, the key to successful bioremediation
resides in continuing to develop the scientific and
engineering work that provides the real bases for both
the technology and its evaluation; and simultaneously
in explaining and justifying the valid reasons which
allow scientists and engineeres to actually use these
technologies for the welfare and safety of a public
which is more and more concerned about the
environment and its protection.
Acknowledgments We wish to express our deepest gratitude
to all the researchers whose valuable data as reported in their
respective publications and cited in this review have been of
considerable significance in adding substance to this review.
We are also grateful to our other colleagues and the anonymous
reviewers whose constructive criticisms have benefited the
manuscript, and brought it to its present form.
12 Concluding remarks
Anthropogenic activities have caused widespread
pollution of the natural environment. A number of
organic pollutants, such as PAHs, PCBs and pesticides,
and inorganic pollutants (heavy metals like arsenic,
123
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Rev Environ Sci Biotechnol (2010) 9:215288

A comprehensive overview of elements in bioremediation

Published online: 29 August 2010

Abstract Sustainable development requires the

tremendous potential to cater for the needs and

1.1 Environmental pollution and biotreatment

Rev Environ Sci Biotechnol (2010) 9:215288

such as polybrominated diphenyl ethers (PBDEs),

Rev Environ Sci Biotechnol (2010) 9:215288

1.2 Bioremediation: definitions

PCBs in river sediments, dechlorination of the

1.3 Characteristics of bioremediation

Rev Environ Sci Biotechnol (2010) 9:215288

Fig. 1 Break down of the

health and the environment. Where contaminants

effectively. Microbial communities within contaminated ecosystems tend to be dominated by those

Rev Environ Sci Biotechnol (2010) 9:215288

Susceptibility to inhibition by other toxic contaminants such as heavy metals

2 Green technology principles

suggests. As well as using and producing better

Rev Environ Sci Biotechnol (2010) 9:215288

3 Merits and demerits of bioremediation

3.1 Merits of bioremediation

Table 2 Chemical classes

and complex wastes (Yergeau et al. 2009; Goel et al.

Aerobic and anaerobic

Ketones and esters

Aerobic and anaerobic

Aerobic (methanotrophs), anaerobic

Some evidence; not readily degradable

Not degradable experimental biosorption

Rev Environ Sci Biotechnol (2010) 9:215288

protein enhancement and transformation of residues

form of microbiological monitoring in addition to the

Table 3 Bioremediation treatment technologies

Use of microbial stripping columns to treat

Stimulation of indigenous microbial

Biodegradation in a container or reactor;

Method of treating contaminated soils

Aerobic, thermophilic treatment process in

Solid-phase treatment system for

For example, many sites are located in industrial/

4.1 Microbial consortia for bioremediation

Rev Environ Sci Biotechnol (2010) 9:215288

for the remediation of a contaminant which is

Rev Environ Sci Biotechnol (2010) 9:215288

consortium/strain under aerobic condition. Batch

bioremediation of the nematicide, oxamyl, applied at

5 Bioremediation techniques for contaminated

Rev Environ Sci Biotechnol (2010) 9:215288

synthetic chemicals into sources of energy and raw

5.1 Characterization of contaminated sites by

Rev Environ Sci Biotechnol (2010) 9:215288

as part of site characterization for determining

Although rarely used, concentrations of specific

contaminated with mixtures of compounds, thereby

Cell lysis and

Fig. 2 Flow chart of sample characterization by molecular diagnostics

Rev Environ Sci Biotechnol (2010) 9:215288

acid to characterize the biodegradative potential of a

regarding the activity of these genes, or serve as an

Table 4 Example of gene probes used in molecular diagnostics

Shield et al. (1989)