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MICROBIAL TECHNOLOGY FOR BIOREMEDIATION OF ORGANOPHOSPHORUS PESTICIDES
ByKAROLIN K.P2012-11-165
SEMINAR REPORTSubmitted in partial fulfillment of the requirement of the
course MICRO 591
1
Department of Agricultural MicrobiologyCollege of Agriculture
VellayaniThiruvanathapuram
2014
DECLARATION
I hereby declare that this seminar report entitled
“Microbial Technology for Bioremediation of
Organophosphorus Pesticides” is a bonafide record of work
done by me during the course of MSc Ag. Programme.
Place: Date:
Karolin K.P
2012-11-165
2
CERTIFICATE
I hereby certify that this seminar report entitled
“Microbial Technology for Bioremediation of
Organophosphorus Pesticides” has been prepared by Ms.
KAROLIN K.P. (2012-11-165) for the partial fulfillment of
the M.Sc Ag. Programme
Place: Dr. K.S.MeenakumariDate: (Chairman, Advisory committee) Professor and HOD Department of Agricultural Microbiology. College of Agriculture, vellayani
3
4
SI NO. CONTENTS PAGE NUMBER
1 INTRODUCTION 5
2 ORGNOPHOSHORUS PESTICIDES 5
3 BIOREMEDIATION 10
4 CHLORPYRIFOS 17
5 DEGRADATION OF OP’s BY
MICROBIAL CONSORTIA
25
6 ENZYMATIC DEGRADATION OF
ORGANOPHOSPHORUS PESTICIDES
27
7 GENETIC BASIS OF OP DEGRADATION 29
8 SIGNIFICANCE OF MICROBIAL
TECHNOLOGY FOR BIOREMEDIATION
OF OP PESTICIDES
31
9 DISCUSSION 33
10 REFERENCES 34
11 ABSTRACT 46
5
INTRODUCTION
Pesticides constitute the key control strategy for crop
pest and disease management. Continuous application of
pesticides to the soil and aquatic system resulted in
health hazards and environmental pollution which has
triggered much public concern. The wide spread use of these
pesticides over the years has resulted in problems caused
by their interaction with the biological systems in the
environment. Pesticides will continue to be an
indispensable tool for the management of pests in the years
to come, as there is no suitable alternative to replace
them totally.
1. Organophosphorus pesticides
Organophoshorus compounds are the most widely used
insecticides, accounting for 34% of world-wide insecticide
sales. These compounds possess high mammalian toxicity and
it is therefore essential to remove them from the
environment. Considering the toxic effect of these
6
pesticides it is essential to remove them from the
environment employing suitable remedial measures. The first
commercialized OP insecticide Bladan, containing TEPP
(tetraethyl pyrophosphate) was synthesized by German
chemist Gerhard Schrader, who later synthesized insecticide
parathion in 1944 (Gallo and Lawryk 1991). Thus, their
development as insecticides took place in the late 1930s
and early1940s. Bioremediation exploiting microbial
technology is one of the recent techniques for
environmental clean-up. In this process heterotrophic
microorganisms breakdown hazardous compounds to obtain
carbon and energy. The first micro-organism that could
degrade organophoshorus compounds was isolated in 1973
(Dragun et al., 1984) and identified as Flavobacterium sp.
Since then several bacterial and a few fungal species have
been isolated which can degrade a wide range of
organophoshorus compounds in liquid cultures and soil
systems. The wide varieties of microorganisms have been
found to possess the enzyme catalyzing hydrolysis of7
organophoshates. This enhances the feasibility of using
bioremediation to treat organophosphate compounds. Moreover
application of molecular biology to genetically engineer
microorganism contain appropriate genes can potentially
contribute to making bioremediation more efficient.
1.1 Structure of organophosphorus pesticides
The general structure of OP pesticide is represented as:
R1 and R2 are the aryl or alkyl groups which either
directly bonded to phosphorous (P) as in case of
phosphinates or through oxygen (phosphates) or sulphur
(phosphothioates) atoms.
In phosphonates R1 is directly bonded to P but R2 is
bonded to oxygen. In phosphonothioates R1 is directly
bonded to P but R2 is bonded to sulphur atom.
In phosphoramidates both or atleast one R group is
attached to –NH2.
X is the ‘‘leaving group’’ (as it gets displaced upon
hydrolysis of OP).
8
Oxygen and sulphur (O/S) are directly attached to P by
a double bond
1.2 Use of Organophosphorus pesticides
Organophosphate pesticides are widely used in variety
of cereal crops, oil seed crops, vegetable crops and fruit
crops for effective control of insect such as termites,
jassids, aphids, white fly, leaf hoppers etc. They are also
used in some veterinary and human medicines to remove
parasitic insects, in various public hygiene products
generally for the control of cockroaches and termites
(Racke et al. 1994). Among them chlorpyrifos (O, O-diethyl-
O-3,5,6 trichloro-2-pyridylphosphorothioate) is being
extensively used in developing countries like India where
it was the fourth highest consumed pesticide after
monocrotophos, acephate and endosulfan, in the year 2000
(Ansaruddin, 2003).
1.3 Bioaccumulation of organophosphorus pesticides
The bioaccumulation is the primary cause of toxicity
of pesticides as their higher concentrations in biological9
systems leads health problems. The persistence of OP in
soil has also been related to the organic matter, clay
content and iron and/or aluminiumoxy(hydro)oxide content of
the soil (Weber, 1972). These have higher affinity to
absorb/adsorb the pesticides and act as a sink for such
hydrophobic compounds and discharge into other phases
included living organisms and plants (Boonsaner et al.
2002). Parathion, an organophosphate pesticide, has been
found to persist in soil for more than 16 year (Stewart et
al. 1971). The bioaccumulation of OPs is primarily related
to their molecular weight and aqueous phase solubility.
There are reports regarding their bioaccumulation in
living systems ranging from blue green algae to higher
systems. Chlorpyrifos due to their very low aqueous phase
solubility (2ppm) had the highest bioaccumulation factor in
the blue-green algae, aquatic plants, gold fish, and
mosquito fish and Mytilus galloprovincialis, a
Mediterranean mussel (Spacie and Hamelink, 1985; Lal et al.
1987). Thus, being at the top of the food chain humans10
indirectly gets much adversely affected by bioaccumulation
of chlorpyrifos and other pollutants in aquatic fauna
(Serrano et al.1997).
Chlorpyrifos has least water solubility and presence
of three chlorine atoms attached to its ring makes it more
resistant to microbial degradation. Due to these structural
features the limiting rate of degradation results in its
accumulation in environment (Volkering et al. 1998;
Angelova and Schamander 1999)
1.4 Fate of Organophosphate pesticides
Pesticides undergo various changes in environment
including their adsorption transmission and degradation
depending on the physicochemical nature of the pesticide
and the soil (Redondo et al. 1997). The predominant
processes involved in transformation of such molecules is
mediated by microbes (Vink and Van der Zee, 1997) followed
by photolysis or photodegradation and chemical
transformations (Roberts, 1998; Stangroom et al. 2000).
11
Thus, generally fate of pesticide involves both biological
and non-biological agents.
1.5 Transformation of OP pesticides
1.5.1 Abiotic transformation of OP pesticides
The abiotic processes involved either their transport
where parent compound remains unchanged and simply
transferred from one matrix to another depends on the
physicochemical properties of pesticides itself (Stangroom
et al. 2000) e.g. volatilization, leaching (Laabs et al.
2000), runoff (Moore et al. 2002), absorption and
adsorption of pesticides (Yu et al. 2006) or by abiotic
transformations by photodegradation (Walia et al. 1988) and
chemical hydrolysis (Liu et al. 2001). Volatilization can
transform pesticides from liquid or solid into gaseous form
depending on temperature of the surface and air currents in
that area (Yates et al. 2002; Haith et al. 2002). The run
off/soil erosion causes movement of pesticides either on
their dissolution in the water or through attachment with
soil particles or sediments and add a fairly large part12
(e.g., 5–30% for chlorpyrifos) of the overall mass transfer
from ground soil to surface water aquifers (Bailey et al.
1974; Richards and Baker, 1993; Wood and Stark, 2002; Luo
and Zhang 2009). Leaching also plays role in transmission
of higher water soluble pesticides to ground water through
fractures, root holes and earthworm burrows in earth crust
(Stagnitti et al. 1994; Magri and Haith, 2009). Pesticides
can also be absorbed/adsorption by plants and soils depend
on their water solubility, soil characteristics and
properties of pesticide itself (Gan et al. 1996; Trapp,
2000; Davis et al. 2002; Paranychianakis et al. 2006;
Johnson et al. 2007).
1.6 Biotic transformation of OP pesticides
Xenobiotic compounds like organophosphate pesticides
are man made compounds and were not previously present in
nature. Consequently the natural microflora does not have
potential to metabolize these pesticides due to lack of
enzyme and proper transport processes. But over the year
due to excessive use of xenobiotic compounds microbes have13
evolved the new degradation pathways resulting in
accelerated degradation of such compounds (Seffernick and
Wackett, 2001; Johnson and Spain, 2003). For instance
Seffernick et al. in 2001 suggests that enzyme Atrazine
chlorohydrolase (AtzA) responsible for dechlorination of
herbicide atrazine under strong selective pressure was
evolved from enzyme melamine deaminase (TriA) and is 98%
similar to the TriA. The accelerated bioremediation under
natural conditions is also helped by transfer of genes
among different microbial cultures by transformation,
transduction and conjugation (Ghigo 2001 and Fux 2005).
2. Bioremediation
Bioremediation techniques for treatment of hazardous
waste have attracted attention over physical-chemical
processes because of their environmentally sound and cost
effective characteristics. Bioremediation is a clean-up of
pollution from soil, ground water, surface water and air,
using biological, usually microbiological processes (Philip
et al., 2001). Biological processes generally produce14
benign end-products, unlike conventional physical-chemical
processes which often create other potential pollution
hazards, such as emission of toxic byproducts from
incineration and leaching of toxic substances from land
disposal. By properly establishing and maintaining
microbial communities and environmental conditions
necessary for biodegradation, most hazardous organic
compounds can be biologically detoxified or mineralized.
A waste is considered as hazardous when it poses a
substantial present or potential danger to human health or
the environment regardless of the form of the waste. In the
United States, a waste is called hazardous when it has any
of the characteristics of corrosivity, ignitability,
reactivity, or toxicity. Nowadays, sources of hazardous
wastes are not limited to large chemical companies but
include a wide range of industrial, agricultural, and
household activities. Amounts produced by various sources
vary widely.
15
The publication of Silent Spring by Rachel Carson in
1962, which emphasized the ecological consequences of
pesticide contamination that were widely ignored, began a
great public debate on pesticide usage. She reported the
hazards of the pesticide DDT (dichloro-diphenyl-trichloro-
ethane), the most powerful pesticide in world history. DDT,
an organochlorine, was originally synthesized in 1874, but
it was in 1939 that DDT was discovered to have insecticide
properties by a Swiss scientist, Paul Müller, who was later
awarded the Nobel Prize. In 1945, DDT was released for
civilian use to become a miracle compound with its
capability of killing hundreds of different kinds of
insects at once. However, extensive usage of DDT created
problems in the environment and in humans because of its
toxicity and its bioaccumulation in fatty tissue. In 1970,
Norway and Sweden banned DDT, and in 1972 the US followed
suit. However, DDT is still used in some countries.
2.1 Factors affecting degradation of OPs
16
The most important parameters involved in pesticide
biodegradation are pesticide concentration, inoculum size,
pH, temperature and its bioavailability, (Karpouzas and
Walker, 2000; Singh et al. 2006; Getzin, 1981; Chapman and
Chapman, 1986; Singh et al., 2002).
2.2.1 Effect of substrate concentration
The chlorpyrifos concentration found in the upper 10
mm of the soil sediment after its application was observed
to be the highest (Brock et al. 1992). Cink and Coats
(1993) observed that after the use of agricultural
application rate of 10 mg kg-1 of chlorpyrifos 5% of
chlorpyrifos persisted in the soil. However, at termite
infestation sites where chlorpyrifos might be used at
higher level almost 57% of appended chlorpyrifos remained
in soil. The ability of microbes to degrade a pollutant
depends on the available concentration of polluting
chemicals, as high concentrations are usually toxic for
microbial degraders and low concentrations may not be able
to induce the enzymes involved in degradation (Block et al.17
1993; Morra, 1996). Menon et al. (2005) reported delayed
dehydrogenase activity in the soil after chlorpyrifos
application at 0.20 μg g-1 indicated inhibition of
microbial growth at the polluted site. A similar
observation was reported by Pandey and Singh (2004) where a
dose of 4 L/hm2 chlorpyrifos showed a short-term inhibitory
effect on the total microbial population. Shan et al.
(2006) also indicated that the application of chlorpyrifos
lead to decrease in bacterial, fungal and actinomycete
populations with increasing chlorpyrifos concentration (2,
4, and 10 mg kg-1) in the soil. Hua et al. (2009) reported
that soil ammended with chlorpyrifos at the initial level
of 4, 8, and 12 mg kg-1, the chlorpyrifos was degraded
after 35 days with average half-live of 14.3, 16.7, and
18.0 respectively. The initial inhibition of soil microbial
communities was followed by recolonization of soil
microbial communities after two weeks.
2.2.2 Effect of inoculum size
18
Ramadan et al. 1990 observed that at low inoculum
levels, <104 cells g-1 of soil, significantly lower number
of inoculated cells could survive the initial exposure to
effectively carry out pesticide degradation. Comeau et al.
(1993) suggested that for the bioremediation of pesticide
contaminated sites the inoculum level in the range of 106–
108 Cells g-1 of soil was adequate. A higher initial
inoculum level can counterbalance the initial decline in
viable cell number due to various environmental factors and
toxic substrate concentrations, so that the survivors can
multiply and degrade the pesticides (Comeau et al. 1993;
Duquenne et al. 1996). Rousseaux et al. (2003) also
reported that inoculum size of Chelatobacter heintzii Cit 1
is one of the important factors for efficient
biodegradation of applied atrazine. Singh et al. 2006
observed that inoculum size has a noticible effect on
biodegradation of fenamiphos and chlorpyrifos. Fenamiphos
was not degraded when soils was inoculated with less than
105 cells g-1. Similarly, degradation of chlorpyrifos by19
Enterobacter sp. below an inoculum density 103 cells g-1
was not observed
2.2.3 Effect of pH
Many organophosphorus insecticides are succeptable to
enhanced basecatalyzed hydrolysis at pH values above 7.5
(Greenhalgh et al. 1980). The change of rate of hydrolysis
is about a factor of 10 for each pH unit (Schwarzen bach et
al. 1993). Singh et al. 2003 reported that chlorpyrifos
degradation in the acidic soils with pH 4.7 was slow where
the half-life of chlorpyrifos was 256 days. The
chlorpyrifos degradation was rapid in alkaline soils (pH
7.7 and 8.4) with a half-life of 16 days and leads to
formation of TCP up to 10 mg kg-1. Initially, it was
assumed that the high rate of chlorpyrifos degradation in
alkaline soils was due to chemical hydrolysis. In general,
higher the pH higher is the rate of hydrolysis of OP
pesticides) which may be due to higher copy numbers of opd
(organophosphate degrading) gene (Sparks, 1989; Singh et
al. 2003; Singh et al. 2003). Wang et al. (2006) had also20
reported that chlorpyrifos degradation rate by B.
laterosporus DSP was increased with increase in pH from 7.0
to 9.0. Al-Qurainy and Abdel-Megeed 2009 observed the
effect of pH on two OP pesticides malathion and dimethoate.
They reported complete degradation of malathion and
dimethoate by Pseudomonas frederiksbergensis at pH 7.0
after 6 days of incubation with half lives accounted by 3
and 2.3 days respectively. However, when the medium pH was
set at 8.0, biodegradation began on the first day and
complete degradation was observed after 3 days. Wang et al.
(2005) reported that the biodegradation rates of
chlorpyrifos in the pH range 6.5–9.0 by Fusarium LK. ex Fx.
WZ-I were higher.
2.2.4 Effect of solubility/bioavailability
Many researchers have reported that high organic
matter content of the soil leads to an absorption of
pesticide to soil particles resulting in lower
bioavailability of organophosphorus pesticides and hence
decreases their degradation rate (Barriuso et al. 1992;21
Weber and Huang, 1996; Karpouzas and Walker, 2000; Nelson
et al. 2000; Ben-Hur et al. 2003). Knuth and Heinis (1992)
and Brock et al. (1992) also reported high absorption of
chlorpyrifos to sediments in static aquatic systems
sediments due to the hydrophobic nature of aquatic
sediments and lower solubility of chlorpyrifos in aqueous
phase. Similarly, Civilini, 1994 reported that it is easy
to remove more water soluble lighter hydrophilic compounds
than heavier hydro-phobic PAHs. In general pesticides
having low water solubility are less susceptible to
accelerated degradation due to their limited dissolution
rates (Alexander, 1999). The fate and behavior of pesticide
in the environment is determined by its solubility, half-
life and partitioning coefficients (Neitsch et al. 2005).
The hydrophobic compounds become non-available to microbial
degraders as these compounds get entrapped in nanopores of
the solid phase of organic matter (Arbeli and Fuentes
2007). However, many researchers used chemical surfactant
or biosurfactants produced by microorganisms to desorb22
chemical compounds from soil organic matter so as to make
them bioavailable for their consumption (Aronstein et al.
1991; Brown and Jaffe, 2006; Zhou and Zhu, 2008).
2.2 Microbial degradation/transformations of
Organophosphorus pesticides
Use of organochlorine pesticides such as dichloro-
diphenyl-trichloroethane (DDT), lindane, etc., has been
reduced drastically in developed countries due to their
long persistence, tendency towards bioaccumulation and
potential toxicity towards non-target organisms. This group
of compounds has been replaced by the less persistent and
more effective organophosphorus compounds. However, most of
the organophosphorus compounds possess high mammalian
toxicity. Among the organophosphorus compounds, glyphosate,
chlorpyrifos, parathion, methyl parathion, diazinon,
coumaphos, monocrotophos, fenamiphos and phorate have been
used extensively and their efficacy and environmental fate
have been studied in detail. The phosphorus is usually
present either as a phosphate ester or as a phosphonate.23
Being esters they have many sites which are vulnerable to
hydrolysis. The principal reactions involved are
hydrolysis, oxidation, alkylation and dealkylation (Singh et
al., 1999). Microbial degradation through hydrolysis of P-O-
alkyl and P-O-aryl bonds is considered the most significant
step in detoxification. Both co-metabolic and bio-
mineralization of organophosphorus compounds by isolated
bacteria have been reported.
Hydrolysis of organophosphorus compounds leads to a
reduction in their mammalian toxicity by several orders of
magnitude. Since most of the research has been directed
towards detoxification, studies on the further metabolism
of the phosphorus containing products have not been
extensive. Hypothetical phospho-ester hydrolysis steps can
be postulated, yielding mono-ester and finally inorganic
phosphate, but this pathway has not been specifically
studied. Analogous phospho-monoesterase and diesterase,
which degrade methyl and dimethyl phosphate, respectively,
have been reported in Klebsiella aerogenes (Wolfenden & Spence,24
1967) and are produced only in the absence of inorganic
phosphate from the growth medium. The final enzyme in the
postulated degradative pathway is bacterial alkaline
phosphatase, which can hydrolyze simple monoalkyl
phosphates and is also regulated by the level of phosphate
available to the cell (Wolfenden & Spence, 1967). A similar
mechanism of metabolism has been reported for phosphonates
(Kertesz et al., 1994a). The way in which metabolism is
regulated depends very strongly on what role the
organophosphorus compound plays for the particular
organisms studied. Most often these compounds are used to
supply only a single element (carbon, phosphorus or sulfur)
and the relevant gene cannot be expressed as a response to
starvation for another of these elements (Kertesz et al.,
1994a). For example, a strain of Pseudomonas stutzeri isolated
to utilize parathion as a carbon source released the
diethylphosphorothioanate products quantitatively and could
not metabolize them further, even when alternative source
of phosphorus or sulfur were removed (Daughton & Hsieh,25
1977). Similarly, a variety of isolates that could use
phosphorothionate and phosphorodithionate pesticides as a
sole source of phosphorus were unable to utilize these
compounds as a source of carbon (Rosenberg & Alexander,
1979). Shelton (1988) isolated a consortium that could use
diethylthiophosphoric acid as a carbon source but was
unable to utilize it as a source of phosphorus or
sulfur. Kertesz et al. (1994a) explained possible underlying
reasons for this phenomenon. They suggested that the
conditions under which environmental isolates enriched were
crucial in selecting for strains not only with the desired
degradative enzyme systems but also with specific
regulation mechanisms for the degradation pathways.
3. Chlorpyrifos
Currently among the various groups of pesticides,
organophosphates form the major, accounting for more than
36 percent of the total world market (Kanekar et al.,
2004).Among the insecticides, monocrotophos, quinalphos and
chlorpyrifos top the list of organophosphorus insecticides26
in Indian market. In India the estimated consumption of
technical grade chlorpyrifos during 2002-03 was 5000 MT
(Singhal, 2003). In the light of restricted or banned use
of organochlorine compounds, chlorpyrifos is gaining
importance in Agriculture. It is a broad spectrum pesticide
displaying insecticidal activity against a wide range of
insects and pests. The most commonly available formulations
include emulsifiable concentrates (EC), granules (GR) and
wettable powders (WP). Aerial application of chlorpyrifos
is a common method followed against surface feeding insects
of cotton, rice, mustard, bengal gram etc. (Dhawan and
Simwat, 1996; Gupta et al., 2001). Soil applications are used
for control of root damaging insect larvae attacking crops
such as vegetables, cardamom, tobacco, cole crops,
groundnut and onion (Rouchaud et al., 1991; Bhatnagar and
Gupta, 1992).The practice of application of 1-2%
concentration of chlorpyrifos to soil surrounding building
structures against termite invasion has aggravated the
residue problems (Sundararaj et al., 2003).27
In soil, chlorpyrifos may remain biologically active
for periods ranging from days to months. It is moderately
persistent in nature as its residues were detected in soil
even after 3 months and hence causes potential
environmental hazards (Chapman et al., 1984). Extensive use
of chlorpyrifos contaminates air, groundwater, rivers,
lakes, rainwater and fog water. The contamination has been
found up to about 24 kilometers from the site of
application. Considerable residues of chlorpyrifos were
found in tomatoes (Aysal et al., 1999), cotton seed (Blossom
et al., 2004) and oil of oil seed crop like groundnut,
safflower and mustard (Bhatnagar and Gupta, 1998; Gupta et
al., 2001). It is speculated that the bioaccumulation ability
of chlorpyrifos and other organophosphorus pesticides in
living tissues may spell a potential environmental risk to
marine organisms and humans as well (Serrano et al., 1997;
Tilak et al., 2004).
When organophoshates are released in to the
environment, their fate is decided by various environmental28
conditions and microbial degradation is the key factor for
the disappearance of these pesticides, since they possess
the unique ability to completely mineralize many aliphatic,
aromatic and heterocyclic compounds.
3.1 Microbiological transformation of chlorpyrifos and its
metabolites
In general, microorganisms demonstrate considerable
capacity for the metabolism of many pesticides. Although
they are capable of catalyzing similar metabolic reactions
as mammals and plants, they possess the unique ability to
completely mineralize many aliphatic, aromatic, and
heterocyclic compounds. There are two major types of
microbial degradation of organic chemicals. The first,
termed catabolism is a type of degradation in which the
organic chemical or a portion thereof is completely
degraded (e.g. mineralized) and the energy or nutrient
gained contributes to cell growth. The second, incidental
metabolism or cometabolism, involves the partial
degradation of an organic chemical with no net benefit to29
the organism, the compound being merely caught up in some
metabolic pathway during the normal metabolic activities of
the microorganisms (Racke 1993).Studies conducted in soil
have generally reported significantly longer dissipation
half–lives under sterilized versus natural conditions and
led to the conclusion that microbial activities are
important in the degradation of chlorpyrifos in soil (Miles
et al. 1984). Evidence from soil degradation studies
indicates that cleavage and mineralization of the
heterocyclic ring occurs in soil due to the activities of
microorganisms (Racke &Coats 1990). However, the singularly
most important microbial role in the chlorpyrifos
degradation pathway may be the further metabolism and
mineralization of 3, 5, 6- trichloro-2-pyrinidinol (TCP)
and 3, 5, 6-trichloro-2-methoxypyridine (TMP) metabolites
(Racke 1993).
Microbial enzymes have been shown to hydrolyze
chlorpyrifos under controlled conditions. Munnecke and his
co-worker in 1975 first reported the ability of parathion30
hydrolase, an organophosphorus ester-hydrolyzing enzyme
isolated from a mixed microbial culture, to hydrolyze
chlorpyrifos.
Jones and Hastings (1981) reported the metabolism of
50-ppm chlorpyrifos in cultures of several forest fungi
(Trichoderma harzianum, Penicillium vermiculatum, and Mucor sp.).
After 28 days, chlorpyrifos and its metabolite TCP were
present in all cultures at levels of 2-5 % and 1-14% of
applied, respectively. Ivashina (1986) studied chlorpyrifos
degradation by several microbial cultures maintained in
liquid media containing 10 ppm chlorpyrifos. Dissipation
was more rapid in a sucrose-supplemented media containing
Trichoderma sp. and glucose supplemented media containing
Bacillus sp. than in control media containing no
microorganisms. Chlorpyrifos disappeared from the microbial
cultures in a linear fashion over a 2-week period. Lal and
Lal (1987) observed some degree of degradation by the yeast
Saccharomyces cervisiae. Only half the initial chlorpyrifos was
recovered 12 h after the cultures were inoculated with 1-1031
ppm. The possible metabolism by two lactic acid bacteria
(Lactobacillus bulgaricus and Streptococcus thermophilus) was reported
by Shaker et al. (1988). The synthetic culture medium, in
which the bacteria were grown initially contained 7.4 ppm,
but displayed a 72-83% loss in chlorpyrifos after 96 h.
Havens and Rase (1991) circulated a 0.25 % aqueous (EC)
solution of chlorpyrifos through a packed column containing
immobilized parathion hydrolase enzyme obtained from
Pseudomonas diminuta. Approximately 25 % of the initial dose
was degraded after 3 h of constant recirculation through
the column. Strains of Aspergillus flavus and Aspergillus niger
isolated from agricultural soil with previous history of
chlorpyrifos use were, also reported to biomineralise
chlorpyrifos in liquid culture medium (Swati & Singh 2002).
Yu et al. (2006) isolated and characterized a fungal strain
capable of degrading chlorpyrifos.18S rDNA gene analysis
revealed that they showed that the fungal strain had a high
level of homology (99%) to those from other Verticillium
species. They found that the degradation of chlorpyrifos in32
by the fungal strain in mineral salt medium increased
almost linearly with increasing concentrations of
chlorpyrifos (r 2 =0.9999), suggesting that the degradation
is subjected to pseudo-first order kinetics. With the first
order kinetic function, the DT50 of chlorpyrifos at
concentrations of 1, 10, and 100 mg/l, were calculated to
be 2.03, 2.93, and 3.49 days, respectively. In the controls
the hydrolysis percentages of chlorpyrifos were found to be
less than 5%. They further used the cell free extracts of
the strain to detoxify chlorpyrifos in vegetables and
reported that the cell free extracts of the fungus can used
for enhanced degradation in vegetables.
Some evidence also indicates that, the metabolites of
chlorpyrifos are also degraded and mineralized by soil
microorganisms. Several researchers have noted the
extensive mineralization of TCP and TMP to carbon dioxide
in soil. Racke et al. (1988) reported that approximately
65-85 % of TCP applied (5ppm) to several soils was
mineralized within 14 days. The initially accelerating rate33
of mineralization observed in these soils was indicative of
microbial enzyme induction or adaptation. Racke and Robbins
(1991) probed a suite of soils for evidence of the presence
of TCP-catabolizing microorganisms. Of the 25 soils
investigated, only two displayed significant degradation of
TCP within 21 days of inoculation into mineral salts medium
containing 5-ppm TCP as the sole carbon source. Isolation a
pure culture of bacteria capable of using 3, 5, 6-
trichloro-2-pyridinol (TCP) as the sole source of carbon
and energy under aerobic conditions was reported for the
first time by Feng and his co-workers in 1998. The
bacterium was identified as a Pseudomonas sp. and designated
as ATCC 700113. The TCP degradation yielded CO2, chloride
and some unidentified polar metabolites. They further
reported that the degradation of the parent compound, TCP,
by the Pseudomonas sp. appeared to involve a reductive de-
chlorination mechanism.
3.2 Microbial degradation of chlorpyrifos
34
When organophosphates are released in to the
environment, their fate is decided by various environmental
conditions and microbial degradation. Microbial degradation
is the key factor for the disappearance of these
pesticides. Micro organisms possess the unique ability to
completely mineralize many aliphatic, aromatic and
heterocyclic compounds. Several studies conducted in soil
indicated significantly longer dissipation half-lives under
sterilized versus natural conditions, and led to the
conclusion that microbial activities are important in
degradation of chlorpyriphos (Getzin, 1981a; Miles et al.,
1983). Schmimmel et al. (1983), based on laboratory
degradation studies with aqueous solution and sediments,
concluded that microorganisms play an important role.
Cleavage and mineralization of heterocyclic ring occur in
soil due to activities of microorganisms (Somasundaram et
al., 1987; Racke et al., 1988).
In literature there are many reports regarding
screening and isolation of microbes capable of degrading35
pollutants under laboratory conditions. However, their use
at the contaminated sites under field scale application has
not been successful (Pilon-Smits 2005; Dua et al. 2002;
Kuiper et al. 2004). The reasons behind this include the
competition faced from the natural microflora and
microfauna of the soil, suboptimal nutrition or nutritional
deficiency leading to low microbial growth, non
availability or less bioavailability of the pollutant
desired to be degraded and the growth inhibitory
concentration of pollutant itself (Kuiper et al. 2004;
Dillewijn et al.2007). Shan et al. (2006) reported the
suppressed growth of bacterial, fungal, and actinomycete
populations in the presence of chlorpyrifos (10 mg kg-1).
Vischetti et al. (2007) reported reduction in soil
microbial biomass in an Italian soil field by 25% and 50%
after chlorpyrifos treatment at 10 mg kg-1and 50 mg kg-1,
respectively. Inspite of these limitations, microbial
degradation of organophosphate pesticides is an important
process responsible for their biotic degradation in36
environment (Felsot, 1989). There are reports regarding
ability of microbes to degrade pesticides co-metabolically
or as source of carbon, nitrogen and phosphorous.
3.2.1 Bacterial degradation of chlorpyrifos
Bacteria are dominantly involved in accelerated
biodegradation of pesticides (Racke and Coats, 1990). The
bacterial strains from different taxonomic groups with
potential to degrade the organophosphorus insecticides have
been reported (Yasouri, 2006; Li et al., 2008).
Sethunathan and Yoshida (1973) reported Flavobacterium
sp. having the ability to degrade chlorpyrifos in liquid
medium by cometabolism. Similarly, Serdar et al. in 1982
isolated Pseudomonas diminuta degrading chlorpyrifos co-
metabolically rather than as a source of carbon On the
other hand, Ohshiro et al. (1996) reported that Arthrobacter
sp. strain B- 5 can use chlorpyrifos as a substrate rather
than a co-metabolite. Singh et al. (2003) isolated six
chlorpyrifos degrading bacteria capable of degrading
chlorpyrifos in both liquid medium and soil. Dutta et al.37
(2004) observed increase in net microbial biomass carbon
(MBC) in chlorpyrifos treated soils as compared to the
control containing no chlorpyrifos. Singh et al. (2004)
also reported degradation of chlorpyrifos by pure culture
Enterobacter sp. B-14 in liquid as well as in soils. Wang et
al. (2006) reported degradation of chlorpyrifos by pure
culture of Bacillus laterosporus DSP. Li et al. (2008) reported
isolation of chlorpyrifos degrading bacterial strains Dsp-
2, Dsp-4, Dsp-6 and Dsp-7 identified as Sphingomonas sp.,
Stenotrophomonas sp., Bacillus sp. and Brevundimonas sp.
respectively and few other strains distinguished as members
of Pseudomonas sp. From chlorpyrifos-contaminated samples.
There are many chlorpyrifos degraders reported but
very few degraders are known to degrade the compound at
higher rates. Mallick et al. (1999) reported complete
degradation of 10 mg l-1 of chlorpyrifos in the mineral
salts medium by Flavobacterium sp. ATCC 27551 and Arthrobacter
sp. within 24 h and 48 h respectively. As described
earlier, the major limitation in the process of38
chlorpyrifos degradation is the formation an anti-microbial
compound 3,5,6-trichloro-2-pyridinol (TCP) which may also
affect the growth of chlorpyrifos-transforming
microorganisms (Racke et al. 1990). A report by Racke and
Coats (1990) indicate that transformation of 30 mg kg-1 of
chlorpyrifos in the soil resulted in production of TCP
which repress the proliferation of microbes introduced into
the soil. The accelerated degradation of chlorpyrifos was
observed either due to the ability of degraders to tolerate
TCP or their potential to mineralize TCP efficiently at a
rate higher rapidly than the rate of its formation in the
medium.
There are reports regarding degradation of both
chlorpyrifos and TCP in aqueous phase (Feng et al. 1998;
Mallick et al. 1999; Horne et al. 2002; Bondarenko et al.
2004). A Stenotrophomonas sp. isolated by Yang et al. (2006)
was found to be a degrader of both chlorpyrifos and TCP. On
the other hand, Singh et al. (2004) isolated Enterobacter sp.
capable of degrading chlorpyrifos was not able to degrade39
TCP but utilize diethylthiophosphate as carbon and
phosphorus source. The Enterobacter species in this case
showed the tolerance against TCP even at higher
concentrations (150 mg l-1), which might be the reason of
effective chlorpyrifos degradation.
3.2.2 Fungal degradation of chlorpyrifos
There are not many reports of OP degradation by fungal
species as compared to those by their bacterial
counterparts. Furthermore the organophosphates degradation
rate by fungal isolates was found to be slower. Jones and
Hastings (1981) reported 95% to 98% degradation of 50 ppm
chlorpyrifos by a group of forest fungi namely Trichoderma
harzianum, Penicillium vermiculatum, and Mucor sp. after 28 days
of incubation along with accumulation of its metabolite
TCP. Bumpus et al. (1993) reported a fungal strain
Phanerochaete chrysosporium able to mineralize only 26.6% of
added chlorpyrifos after 18 days of incubation. Bending et
al. (2002) reported Hypholoma fasciculare and Coriolus versicolor
degraded chlorpyrifos in soil bio-bed after 42 days.40
Studies had also reported the chlorpyrifos degradation in
soil by Aspergillus sp., Trichoderma sp. (Liu et al. 2003) and
Fusarium sp. (Wang et al. 2005).
4. Degradation of OPs by microbial consortia
Most of the lab scale microcosm based in-situ
bioremediation studies involving addition of pure cultures
to polluted soil are prone to problem because of poor
survival or low activity of these cultures in the natural
environmental conditions. Munnecke and Hsieh (1974)
reported a mixed bacterial consortium consisting of
Pseudomonas sp., Xanthomonas sp., Azotomonas sp. and a
Brevibacterium sp. capable of hydrolysing 50 mg l-1 of
parathion. The biodegradation of pesticides, such as 4-
nitrophenol (Laha & Petrova 1998), endosulfan (Awasthi et
al. 2000), 1,3- dichloropropene (Ou et al. 2001) and
diazonin degradation (Cycon et al. 2009) by a microbial
consortium has been reported.
The entire pesticide degradation pathways generally may
not be present in individual species. However, different41
components of microbial consortia can work in concerted
manner to acheive effeciant degradation of these compounds
(Macek et al. 2000; Kuiper et al. 2004; Chaudhry et al.
2005). The rhizosphere soil contains 10–100 times more
microbes than un-vegetated soil due to presence of plant
exudates such as sugars, organic acids, and larger organic
compounds in the soil (Lynch 1990; Kumar et al. 2006).
However, there are certain factors those can interfere with
the enrichment process for development of degradative
consortium including (a) the complex molecular and
structural features of the degrading compound that may
limit its degradability e.g. polyhalogenated compounds
(Wackett et al. 1994), (b) natural dominance of a non-
productive metabolic pathway (Oh and Bartha 1997), (c) low
frequency of an essential degradative gene (Shapir et al.
1998), (d) poor bioavailability e.g. polycyclic aromatic
hydrocarbons (Bastiaens et al. 2000) and (c) production of
recalcitrant intermediates (Van Hylckama Vlieg and Janssen
2001). The problem may be overcome by developing42
genetically engineered microbial strains or by developing
an efficient consortium from natural degraders. The
metabolic synergism between different microbial species
encourages the biodegradation of recalcitrant molecules via
aerobic and anaerobic reactions. Gilbert et al. 2003
developed a consortium comprised of two engineered strains,
Escherichia coli SD2 with plasmids encoding a gene for parathion
hydrolase and Pseudomonas putida KT2440 having pSB337 plasmid
contained a p-nitrophenol-inducible operon encoding the
genes for p-nitrophenol mineralization, to hydrolyze 500 μM
of organophosphate insecticide parathion without the
accumulation of p-nitrophenol in suspended culture.
Vidya Lakshmi et al. 2008 developed a microbial
consortium consisting of Pseudomonas fluorescence, Brucella
melitensis, Bacillus subtilis, Bacillus cereus, Klebsiella sp., Serratia marcescens
and Pseudomonas aeruginosa supported 75–87% degradation of
chlorpyrifos after 20 days of incubation.
Immobilization of the pure cultures and consortium may
help to improve bioremediation potential as immobilized43
cells have prolonged microbial cell viability ranging from
weeks to months and improved capacity to tolerate higher
concentrations of pollutants (Richins et al. 2000; Chen and
Georgiou 2002). Karamanev et al. 1998 immobilized bacterial
consortium in alginate beads and on tezontle (a porous
igneous rock) by biofilm for the removal of a pesticide
mixture form liquid medium composed of methyl-parathion and
tetrachlorvinphos.
5. Enzymatic degradation of organophosphorus pesticides
Microorganisms degrading xenobiotic chemicals are
equipped with elaborate enzyme systems. Biodegradation of
organophosphates involves activities of phosphatase,
esterase, hydrolase and oxygenase enzymes. Munnecke (1976)
observed that crude cell free extract from a mixed
bacterial culture growing on parathion, hydrolysed the same
at a rate of 416 n mol/min/mg of protein. The enzyme
phosphotriesterase (parathion hydrolase) was also reported
from Streptomyces lividans (Rowland et al., 1991) partially
purified enzyme can use as a field inoculum to44
decontamination of soil. Parathion hydrolase is also
isolated from Pseudomonas diminuta, Monomeric, spherical
protein having molecular weight of 39,000 Hydrolyze
paraxon, cyanophos, fensulfothion and coumaphos. (Dumas et
al., 1989).The enzyme cloned from a Flavobacterium sp. Into
Streptomyces lividans was secreted at high levels and consisted
of a single polypeptide with an apparent molecular weight
of 35,000. Substrate specificity showed kms of 68 micro M
for parathion and 599 micro M for methyl parathion.
Biodegradation of organophosphorus pesticides by surface
expressed organophosphorus hydrolase was studied by Richins
et al. (1997). Organophosphorus hydrolase (OPH) was displayed
and anchored on to the surface of Escherichia coli using an Lpp-
OmpA fusion system. OPHs are members of the amidohydrolase
superfamily and share the same (α–β) 8 barrel structural
folds and an active site with two transition metal ions,
such as zinc, iron, manganese or cobalt. The OPHs purified
from B. diminuta and Flavobacterium sp. have identical, or very
45
similar, amino-acid sequences (Serdar et al., 1982; Mulbry,
1989; Siddavattam et al., 2003).
A variant of OPH called OPDA (OP-degrading enzyme) has
been purified from A. radiobacter21. Production of OPH on cell
surface is host specific it is highly dependent on growth
conditions of microorganisms.OPH activity in Nocardia simplex
NRRL B-24074 located in the cytoplasm ( Mulbry ,2000). More
than 80 per cent of the activity was found to be located on
the cell surface. The precise conditions for surface
targeting or pesticide degradation were further studied by
Kaneva et al. (1998). Optimum OPH activity was observed when
cells were grown in Luria-Bertani buffered medium at 37°C.
The resulting culture grown under optimized conditions had
an eight fold increase in parathion degradation.
Sureshkumar et al. (1998) reported that the cell free extract
of Flavobacterium balustinum hydrolysed a variety of
organophosphorus pesticides like fenitrothion, quinalphos
and monocrotophos. The enzyme responsible for hydrolysis
was identified as a phosphotriesterase. Cho et al. (2002)46
observed that the effectiveness of degradation by OPH
varied dramatically ranging from highly efficient with
paraoxon to relatively slow with methyl parathion. A solid
phase top agar method based on detection of the yellow
product P-nitrophenol was developed for the rapid pre-
screening of potential variants with improved hydrolysis of
methyl parathion. One variant 22AII hydrolysed methyl
parathion 25 folds faster than did the wild strain.
Organophoshorus acid anhydrolase (OPAA) was isolated
from Alteromonas undina and Alteromonas haloplankti.(Chen et al.,
1993; Chen et al., 1998). Enzyme OPAA belongs to the
dipeptidase family and does not share enzyme or gene-
sequence similarity with OPH.
6. Genetic basis of OP degradation
Molecular biological studies have thrown light on
organophosphate degrading (opd) genes and involvement of
plasmid in degradation of organophosphates. Involvement of
plasmid in parathion degradation was reported by Serdar et
al. (1982). Pseudomonas diminuta cells grown for 48 hours47
contained 3400 U of parathion hydrolase activity per litre
of broth and the activity was found to be associated with
the plasmid pSC1 of molecular mass 44 x 106 daltons. In
Flavobacterium sp. strain ATCC 27551, a 43 kb plasmid
possessed significant homology to the opd sequence (Mulbry
et al., 1986). Chaudhary et al. (1988) described two mixed
bacterial cultures utilizing methyl parathion and
parathion. The hybridisation data showed that the DNA from
Pseudomonas sp. and from mixed culture had homology with opd
gene from a previously reported parathion hydrolysing
bacteria Flavobacterium sp.
Kumar et al. (1996) have reviewed microbial degradation
of pesticides with special emphasis on the role of
catabolic genes and the application of recombinant DNA
technology in the development of an organism which could
simultaneously degrade several xenobiotics. Possible
involvement of plasmids in degradation of malathion and
chlorpyriphos was reported by Guha et al. (1997). Two
plasmids harbouring strains of Micrococcus sp. (M-36 and AG-48
43) degraded malathion and parathion. Chen and Mulchandani
(1998) studied use of live biocatalysts for pesticides
detoxification. They discussed the use of a genetically
engineered E. coli with surface expressed organophosphorus
hydrolase and suggested the ultimate creation of super
biocatalyst capable of degrading several pesticides rapidly
and cost effectively. Moraxella sp. growing on P-nitrophenol
was genetically engineered for the simultaneous degradation
of OP pesticides and P-nitrophenol. The truncated ice
nucleation protein anchor was used to target the
organophosphorus hydrolase on to the surface and the
resulting Moraxella spp. degraded organophosphates rapidly,
all within an hour (Shimazu et al., 2001). Walker and
Keasling (2002) reported that transformation of Pseudomonas
putida with the plasmids harbouring opd and the p-
nitrophenol operons allowed the organism to utilize 0.8 mM
parathion as a source of carbon and energy.
Somara et al. (2002) amplified the opd gene of
Flavobacterium balustinum using PCR and the resulting product49
was cloned in to PUC 18. The protein sequence predicted
from the nucleotide sequence was almost identical to
parathion hydrolase. A transposon like organization of the
plasmid borne opd gene cluster in Flavobacterium sp. was
reported by Siddavattam et al. (2003).
Cho et al. (2004) selected five improved variants based on
the formation of clear halos on Luria-Bertani plates
overlaid with chlorpyriphos. Of these, B3561 exhibited a
725 fold increase in the K (cat)/K (m) value for
chlorpyriphos hydrolysis as well as enhanced hydrolysis
rates for several other OP compounds. Lei et al. (2005)
reported improved degradation of OP nerve agents by
Pseudomonas putida JS444 with surface expressed
organophosphorus hydrolase.
7. Significance of microbial technology for bioremediation
of OP pesticides
Among various pesticide used by farmers,
organophoshorus pesticides (OP) form the major group.
Although these are thought to be easily biodegradable,50
literature survey indicates that complete degradation or
mineralization of the pesticides is rarely achieved. Many
researchers have studied biodegradation of OP particularly
parathion, malathion in laboratory nutrient media wherein
it is easy to detect metabolites of degradation. However it
is difficult to detect metabolites in soils contaminated
with the pesticides. There is a mixed population in soil
and the pesticides are attacked by a number and variety of
microorganisms. The metabolites formed by one type of
microorganisms may be utilized by other group of organisms.
Some of the metabolites like acetic acid are assimilated by
the microbial cells. Such transient metabolites may not be
easily detected in soil. Depending upon solubility and
mobility, the pesticides and their metabolites may reach
deeper layers of soil. There is always uneven distribution
of the pesticides in soil. Many bind to soil particles.
Hence soil sampling for estimation of biodegradation of
pesticides becomes critical.
51
Since organophosphorus pesticides and some of their
metabolites are toxic, their degradation in contaminated
soil is necessary. Although indigenous soil microflora do
attack on the pesticide residue in soil, the process in
slow. To enhance the process in soil, it is desirable to
inoculate soils with cultures efficient in degradation of
such pesticides. This bio- augmentation of soil with
selected cultures will enhance bioremediation of
contaminated soil. Since industrial waste waters are one of
the source of pollution of water bodies. The studies so far
carried out suggest that micro-organisms endowed with this
property of degradation of toxic pollutants are a boon to
mankind.
Conclusion
The pollution of the environment by pesticides is a
consequence of the continuous agricultural expansion,
combined with the population increase. Pesticides are used
in sizeable areas and applied to soil surfaces and
accumulate beneath the ground surface, reaching rivers and52
seas. The natural microbiota is continuously exposed to
pesticides therefore, it is no surprise that these
microorganisms, that inhabit in polluted environments, are
armed with resistance by catabolic processes to remove the
toxic compounds. Biological degradation by organisms
(fungi, bacteria, viruses, protozoa) can efficiently remove
pesticides from the environment, especially
organochlorines, organophosphates and carbamates used in
agriculture. The enzymatic degradation of synthetic
pesticides with microorganisms represents the most
important strategy for the pollutant removal, in comparison
with non-enzymatic processes. The degradation of persistent
chemical substances by microorganisms in the natural
environment has revealed a larger number of enzymatic
reactions with high bioremediation potential. These
biocatalysts can be obtained in quantities by recombinant
DNA technology, expression of enzymes, or indigenous
organisms, which are employed in the field for removing
pesticides from polluted areas. The microorganisms53
contribute significantly for the removal of toxic
pesticides used in agriculture and in the absence of
enzymatic reactions many cultivable areas would be
impracticable for agriculture.
Isolation and characterization of pesticide degrading
microorganisms is crucial for enhancing our understanding
of the array of mechanisms and biodegradative pathways
relating to their enhanced degradation in the environment.
Bioremediation technologies are aimed in the process of
degrading toxic compounds and related nerve agents using
organophosphorus hydrolase enzymes. Future studies on the
genes responsible for enhanced biodegradation will enable
us to elucidate the exact degradative pathway involved in
its microbial biodegradation.
DISCUSSION
Q) Is any microorganisms produce toxic products in to soil?
54
Ans) No. microorganisms use the soil as its food source
they use the enzymes and other soil related materials
for its growth and metabolism.
Q) What is the relevance of bioremediation in kerala?
Ans) we can use this technology for the waste water
treatment.
Q) Can we use this technology for purify ganga river?
Ans) of course we can use this technology for purify ganga
also.
Q) Is bioremediation is mainly for pesticide degradation?
Ans) No. bioremediation is the technology mainly used for
all type of xenobiotic compounds; it’s not only for
pesticides.
Q) What is meant by abiotic factors? How they involving in
bioremediation?
Ans) abiotic factors mainly include the photodegradation
and chemical hydrolysis of xenobiotics. They degrade
the xenobiotics and produce them to simpler molecular
55
forms that can easily degraded by microorganisms as
carbondioxide and hydrogen, methane etc
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KERALA AGRICULTURAL UNIVERSITYCOLLEGE OF AGRICULTURE, VELLAYANI
Agricultural MicrobiologyMicro 591
ABSTRACT
KAROLIN K.P.16 -11- 13
2012 – 11-16510 – 11 AM
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MICROBIAL TECHNOLOGY FOR BIOREMEDIATION OF ORGANOPHOSPHORUSPESTICIDES
Pesticides constitute the key control strategy forcrop pest and disease management. Continuous application ofpesticides to the soil and aquatic system resulted inhealth hazards and environmental pollution which hastriggered much public concern. The wide spread use of thesepesticides over the years has resulted in problems causedby their interaction with the biological systems in theenvironment. Pesticides will continue to be anindispensable tool for the management of pests in the yearsto come, as there is no suitable alternative to replacethem totally.Organophosphorus pesticides
Organophosphorus compounds are the most widely usedinsecticides, accounting for 34% of world-wide insecticidesales. These compounds possess high mammalian toxicity andit is therefore essential to remove them from theenvironment. Considering the toxic effect of thesepesticides it is essential to remove them from theenvironment employing suitable remedial measures.Bioremediation exploiting microbial technology is one ofthe recent techniques for environmental clean-up. In thisprocess heterotrophic microorganisms breakdown hazardouscompounds to obtain carbon and energy. The first micro-organism that could degrade organophosphorus compounds wasisolated in 1973 (Dragun et al., 1984) and identifiedas Flavobacterium sp. Since then several bacterial and a fewfungal species have been isolated which can degrade a widerange of organophosphorus compounds in liquid cultures andsoil systems.
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Pesticide transformationsAbiotic
The abiotic processes involved either their transportwhere parent compound remains unchanged and simplytransferred from one matrix to another depending on thephysicochemical properties of pesticides itself. Apart fromvolatilization, leaching, runoff, absorption and adsorptionof pesticides photodegradation, chemical hydrolysis etc.help in transformations.
Biotic Xenobiotic compounds like organophosphate pesticides
are man made compounds and were not previously present innature. Consequently the natural microflora does not havepotential to metabolize these pesticides due to lack ofenzyme and proper transport processes. Over the years, dueto excessive use of xenobiotic compounds microbes haveevolved new degradation pathways resulting in accelerateddegradation of such compounds.Bioremediation
Technology used for clean-up of pollutants from soil,groundwater, surface water and air, using biological,usually microbiological process (Philp et al., 2000).Microbiological transformation of chlorpyrifos and itsmetabolites
Chlorpyrifos is one of the world's most widely usedorganophosphorus pesticides in agriculture. Exposure tochlorpyrifos and its metabolites have been related to avariety of nerve disorders in humans. Microbial degradationis considered to be an efficient and cost effective methodfor decontamination of toxic organophosphorus pesticidesfrom the environment. Chlorpyrifos previously shown to be
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immune to enhanced biodegradation has now been proved toundergo enhanced microbe mediated decay into less harmfuland non-toxic metabolites, under a set of favourableabiotic conditions. Recently, research activities in thisarea has shown that a diverse range of microorganisms areresponsible for chlorpyrifos degradation.
Bacteria are dominantly involved in acceleratedbiodegradation of pesticides(Racke and Coats, 1990).Thebacterial strains from different taxonomic groups withpotential to degrade the organophosphorus insecticides havebeen reported (Yasouri,2006; Li et al., 2008).
Co-metabolic process of OP degradation by lignolyticor cytochrome 450 associated enzymes of fungi is alsoprevalent (Fernado and Aust,1994;Yadav et.al.,2003)
Degradation of OPs by microbial consortiaThe entire pesticide degradation pathways generally
may not be present in individual species. However,different components of microbial consortia can work inconcerted manner to achieve efficient degradation ofdifferent metabolites.Genetic basis of OP degradation
Molecular biological studies have thrown light onorganophosphate degrading (opd) genes and involvement ofplasmid in degradation of organophosphates.Conclusion
Isolation and characterization of pesticide degradingmicroorganisms is crucial for enhancing our understandingof the array of mechanisms and biodegradative pathwaysrelating to their enhanced degradation in the environment.Bioremediation technologies are aimed in the process ofdegrading toxic compounds and related nerve agents using
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organophosphorus hydrolase enzymes. Future studies on thegenes responsible for enhanced biodegradation will enableus to elucidate the exact degradative pathway involved inits microbial biodegradation. References
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80
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