Degradacion de Organofosforados

Microbial degradationof organophosphorus compounds
Brajesh K. Singh
1
& Allan Walker
2
1
Environmental Sciences, Macaulay Institute, Craigiebuckler, Aberdeen and
2
Horticulture Research International, Wellesbourne, Warwick, UK
Correspondence: Brajesh Singh,
Environmental Sciences, Macaulay Institute,
Craigiebuckler, Aberdeen, AB15 8QH, UK.
Tel.: 144 1224 498200; fax: 44 1224
498207; e-mail: b.singh@macaulay.ac.uk
Received 16 June 2005; revised 24 November
2005; accepted 6 January 2006.
First published online April 2006.
doi:10.1111/j.1574-6976.2006.00018.x
Editor: Alexander Boronin
Keywords
organophosphorus compounds; microbial
degradation; metabolic pathways; detoxifying
enzymes; genetic basis; biotechnological
aspects.
Abstract
Synthetic organophosphorus compounds are used as pesticides, plasticizers, air
fuel ingredients and chemical warfare agents. Organophosphorus compounds are
the most widely used insecticides, accounting for an estimated 34% of world-wide
insecticide sales. Contamination of soil from pesticides as a result of their bulk
handling at the farmyard or following application in the eld or accidental release
may lead occasionally to contamination of surface and ground water. Several
reports suggest that a wide range of water and terrestrial ecosystems may be
contaminated with organophosphorus compounds. These compounds possess
high mammalian toxicity and it is therefore essential to remove them from the
environments. In addition, about 200 000 metric tons of nerve (chemical warfare)
agents have to be destroyed world-wide under Chemical Weapons Convention
(1993). Bioremediation can offer an efcient and cheap option for decontamina-
tion of polluted ecosystems and destruction of nerve agents. The rst micro-
organism that could degrade organophosphorus compounds was isolated in 1973
and identied as Flavobacterium sp. Since then several bacterial and a few fungal
species have been isolated which can degrade a wide range of organophosphorus
compounds in liquid cultures and soil systems. The biochemistry of organopho-
sphorus compound degradation by most of the bacteria seems to be identical, in
which a structurally similar enzyme called organophosphate hydrolase or phos-
photriesterase catalyzes the rst step of the degradation. organophosphate hydro-
lase encoding gene opd (organophosphate degrading) gene has been isolated from
geographically different regions and taxonomically different species. This gene has
been sequenced, cloned in different organisms, and altered for better activity and
stability. Recently, genes with similar function but different sequences have also
been isolated and characterized. Engineered microorganisms have been tested for
their ability to degrade different organophosphorus pollutants, including nerve
agents. In this article, we review and propose pathways for degradation of some
organophosphorus compounds by microorganisms. Isolation, characterization,
utilization and manipulation of the major detoxifying enzymes and the molecular
basis of degradation are discussed. The major achievements and technological
advancements towards bioremediation of organophosphorus compounds, limita-
tions of available technologies and future challenge are also discussed.
Introduction
The excessive use of natural resources and large scale
synthesis of xenobiotic compounds have generated a num-
ber of environmental problems such as contamination of air,
water and terrestrial ecosystems, harmful effects on different
biota, and disruption of biogeochemical cycling. At the
present time, the most widely used pesticides belong to the
organophosphorus group. The rst organophosphorus in-
secticide, tetraethyl pyrophosphate, was developed and used
in 1937 (Dragun et al., 1984). At the same time, two
chemical warfare agents (also called nerve agents), Tabun
and Sarin, were developed and produced. Later, several
other organophosphorus pesticides were developed and
commercialized. These pesticides are widely used world-
wide to control agricultural and household pests. Overall,
organophosphorus compounds account for 38% of total
pesticides used globally (Post, 1998). In the USA alone over
40 million kilos of organophosphorus are applied annually
(Mulchandani et al., 1999a; EPA, 2004). Glyphosate and
FEMS Microbiol Rev 30 (2006) 428471 c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
chlorpyrifos are the most widely used in the US and account
for 20% and 11% of total pesticide use, respectively (EPA,
2004). Organophosphorus compound poisoning is a world-
wide health problem with around 3 million poisonings
and 200 000 deaths annually (Karalliedde & Senanayake,
1999; Sogorb et al., 2004). The compounds have been
implicated in several nerve and muscular diseases in human
beings. Their acute adverse effects have been discussed by
Colborn et al. (1996) and Ragnarsdottir (2000). Immuno-
toxicity of organophosphorus compounds towards human
beings and wild-life has been reviewed by Galloway &
Handy (2003).
Continuous and excessive use of organophosphorus
compounds has led to the contamination of several ecosys-
tems in different parts of the world (EPA, 1995; McConnell
et al., 1999; Cisar & Snyder, 2000; Tse et al., 2004). For an
example, surveys revealed that 100% of sampled catchments
in Scotland and 75% of sampled aquatic sites in Wales were
contaminated with organophosphorus compounds used in
sheep dips (Boucard et al., 2004). Several organophosphorus
compounds are used on animals for the control of body
pests as several of them are fat soluble and can thus enter the
body readily through the skin and potentially nd their way
into meat and milk (MAFF/HSE, 1995). Contamination of
grains, vegetables and fruits with organophosphorus com-
pounds is also well documented (Pesticide Trust 1996;
National Consumer Council 1998). Another potential and
more dangerous source of organophosphorus contamina-
tion comes from chemical warfare agents. About 200 000
tons of extremely toxic organophosphorus chemical warfare
agents such as Sarin, Soman, and VX were manufactured
and are stored. As required by the Chemical Weapon
Convention (CWC) 1993, these stocks must be destroyed
within 10 years of ratication by the member states. Use of
micro-organisms in detoxication decontamination of or-
ganophosphorus compounds is considered a viable and
environment friendly approach.
The available literature on the microbial degradation of
xenobiotics indicates that most studies have considered
three aspects:
(1) The fundamental basis of biodegradation.
(2) Evolution and transfer of such activities among micro-
organisms.
(3) Bioremediation techniques to detoxify contaminated
environments (Singh et al., 1999).
However, the use of micro-organisms for bioremediation
requires an understanding of all physiological, microbiolo-
gical, ecological, biochemical and molecular aspects in-
volved in pollutant transformation (Iranzo et al., 2001).
There are two types of xenobiotics that cause environ-
mental concerns: (1) compounds that are persistent and
therefore provide long exposure to non-target organisms
such as lindane and DDT, and (2) compounds that are
biodegradable but mobile in soil and are toxic and therefore
have the potential to pollute ground water, such as carbo-
furan. Extensive and repeated use of the same pesticide
without any crop or pesticide rotation for a number of years
has occasionally resulted in unexpected failures to control
the target organisms. It has been demonstrated that a
fraction of the soil biota can develop the ability rapidly to
degrade certain soil-applied pesticides. This phenomenon
has been described as enhanced or accelerated biodegrada-
tion (Walker & Suett, 1986). The rst evidence of biodegra-
dation of pesticide affecting its efcacy was reported in 1971
(Sethunathan, 1971). However, it was not until the early to
mid 1980s that the wider implication of enhanced bio-
degradation became observable in the eld (Walker & Suett,
1986) and since then this phenomenon has been reported
for several other pesticides such as isofenphos (Chapman
et al., 1986), fenamiphos (Stiriling et al., 1992) and etho-
prophos (Karpouzas et al., 1999).
The practical signicance of enhanced bio-degradation
depends on a number of interactive factors like the use of the
pesticides (soil or foliage applied), the frequency of use, the
interval between successive applications and the stability of
the active microora without the presence of pesticides
(Kaufman et al., 1985). Recently, soil pH has been impli-
cated as a factor in enhanced degradation of atrazine in
different soils (Houot et al., 2000). This hypothesis has been
supported by recent reports of high enzymatic activity
(Acosta-Martinez & Tabatabai, 2000) and higher bacterial
activity at higher soil pH (Vidali, 2001). Sims et al. (2002)
suggested that soil pH may inuence the rate of degradation
by affecting the uptake of the herbicide by soil micro-
organisms. The problem of enhanced bio-degradation be-
came more acute, following the observation that a pesticide
can be degraded rapidly in soil from a eld to which it had
never been applied before but which had been exposed to a
pesticide from the same chemical group (Prakash et al.,
1996). This phenomenon is known as cross-adaptation.
Cross-adaptation of enhanced biodegradation has been
reported within many groups of pesticide, such as the
carbamates (Morel-Chevillet et al., 1996), dicarboximides
(Mitchell & Cain, 1996) and isothiocyanates (Warton et al.,
2002). On the other hand, only limited cross-adaptation for
enhanced biodegradation within the organophosphorus
class has been reported (Racke & Coats, 1988; Singh et al.,
2005). Cross-adaptation within groups is unpredictable and
may occur only in one direction. The positive side of this
problem is that micro-organisms isolated for degradation of
one compound can be used for bioremediation of other
compounds for which no known degrading microbial
system is known. This aspect is well established for organo-
phosphorus compounds where a parathion-degrading bac-
terium was able to degrade a wide range of other structurally
similar compounds including chemical warfare agents.
429 Microbial degradation of organophosphorus compounds
Isolation of pesticide degrading microorganisms is impor-
tant for three main reasons:
(1) To determine the mechanism of the intrinsic process of
microbial metabolism.
(2) To understand the mechanisms of gene/enzyme evolu-
tion.
(3) To use these microbes for the detoxication and decon-
tamination of polluted aquatic and terrestrial environ-
ments (bioremediation).
Several microorganisms have been isolated which are able
to utilize pesticides as a source of energy. There are some
examples of fungi including Trametes hirsutus, Phanero-
chaete chrysosporium, Phanerochaete sordia and Cyathus
bulleri that are able to degrade lindane and other pesticides
(Singh & Kuhad, 1999, 2000; Singh et al., 1999). However,
most evidence suggests that soil bacteria are the principal
components responsible for enhanced bio-degradation
(Walker & Roberts, 1993). Several pure bacterial isolates
with the ability to use specic pesticides as a sole source of
carbon, nitrogen or phosphorus have been isolated (Singh
et al., 1999, 2000).
On numerous occasions, mixed bacterial cultures with
pesticide degradation ability are isolated but their individual
components are unable to utilize the chemical as an energy
source when puried (Shelton & Somich, 1988; Mandel-
baum et al., 1993; De Souza et al., 1993; Roberts et al., 1993);
an example is the organophosphorus nematicide fenami-
phos (Ou & Thomas, 1994; Singh et al., 2003b). Several
other studies failed to obtain micro-organisms capable of
growing on specic chemicals. However, this failure does
not exclude biological involvement in degradation and
could be attributed to the selection and composition of the
liquid media under articial environments, strains requiring
special growth factors, or a major role of non-culturable
microorganisms (Walker & Roberts, 1993). A recent report
of growing previously non-culturable bacteria in the labora-
tory with a simulated natural environment (Kaeberlein
et al., 2002) may lead to isolation and characterization of
several new chemical-degrading bacteria.
The main aim of this article is to review the metabolic
pathways involved in organophosphorus compound degrada-
tion. Our understanding of the molecular basis of organopho-
sphorus degradation has progressed dramatically in recent
years. Additional information has become available by gen-
ome sequencing of several microorganisms and advancement
in molecular techniques. There is growing interest in devel-
oping biotechnological methods for clean up of contaminated
water and soil with organophosphorus compounds and to aid
in the destruction of large amounts of nerve agents. In this
article we also critically review recent biotechnological ad-
vancements in the development of bio-catalysts and bio-
sensors for organophosphorus compounds and their possible
application in bioremediation of contaminated ecosystems.
Chemistry and toxicology of
organophosphorus compounds
Most organophosphorus compounds are ester or thiol
derivatives of phosphoric, phosphonic or phosphoramidic
acid. Their general formula is presented in Fig. 1. R
1
and R
2
are mainly the aryl or alkyl group, which can be directly
attached to a phosphorus atom (phosphinates) or via
oxygen (phosphates) or a sulphur atom (phosphothioates).
In some cases, R
1
is directly bonded with phosphorus and R
2
with an oxygen or sulfur atom (phosphonates or thion
phosphonates, respectively). At least one of these two groups
is attached with un-, mono- or di-substituted amino groups
in phosphoramidates. The X group can be diverse and may
belong to a wide range of aliphatic, aromatic or heterocyclic
groups. The X group is also known as a leaving group
because on hydrolysis of the ester bond it is released from
phosphorus (Fig. 1) (Sogorb & Vilanova, 2002).
The mode of action of organophosphorus compounds
includes inhibition of neurotransmitter acetylcholine break-
down. Acetylcholine is required for the transmission of
nerve impulses in the brain, skeletal muscles and other areas
(Toole & Toole, 1995). However, after the transmission of
the impulse, the acetylcholine must be hydrolyzed to avoid
overstimulating or overwhelming the nervous system. This
breakdown of the acetylcholine is catalyzed by an enzyme
called acetylcholine esterase. Acetylcholine esterase converts
acetylcholine into choline and acetyl CoA by binding the
substrate at its active site at serine 203 to form an enzyme
substrate complex. Further reactions involve release of cho-
line from the complex and then rapid reaction of acylated
enzymes with water to produce acetic acid and the
Fig. 1. General formula of organophosphorus compounds and major
pathway of degradation.
430 B.K. Singh & A. Walker
regenerated acetylcholine esterase. It has been estimated that
one enzyme can hydrolyze 300 000 molecules of acetylcho-
line every minute (Ragnarsdottir, 2000).
Organophosphorus compounds inhibit the normal activ-
ity of the acetylcholine esterase by covalent bonding to the
enzyme, thereby changing its structure and function. They
bind to the serine 203 amino acid active site of acetylcholine
esterase. The leaving group binds to the positive hydrogen of
His 447 and breaks off the phosphate, leaving the enzyme
phosphorylated. The regeneration of phosphorylated acet-
ylcholine esterase is very slow and may take hours or days,
resulting in accumulation of acetylcholine at the synapses.
Nerves are then overstimulated and jammed (Manahan,
1992). This inhibition causes convulsion, paralysis and
nally death for insects and mammals (Ragnarsdottir,
2000).
Microbial degradation of
organophosphorus compounds
Use of organochlorine pesticides such as dichloro-diphenyl-
trichloroethane (DDT), lindane, etc., has been reduced
drastically in developed countries due to their long persis-
tence, tendency towards bioaccumulation and potential
toxicity towards non-target organisms. This group of com-
pounds has been replaced by the less persistent and more
effective organophosphorus compounds. However, most of
the organophosphorus compounds possess high mamma-
lian toxicity. Among the organophosphorus compounds,
glyphosate, chlorpyrifos, parathion, methyl parathion, dia-
zinon, coumaphos, monocrotophos, fenamiphos and pho-
rate have been used extensively and their efcacy and
environmental fate have been studied in detail. The chemical
and physical properties of some of these compounds are
listed in Table 1. The phosphorus is usually present either as
a phosphate ester or as a phosphonate. Being esters they
have many sites which are vulnerable to hydrolysis. The
principal reactions involved are hydrolysis, oxidation, alky-
lation and dealkylation (Singh et al., 1999). Microbial
degradation through hydrolysis of P-O-alkyl and P-O-aryl
bonds is considered the most signicant step in detoxica-
tion (Fig. 1). Both co-metabolic and bio-mineralization of
organophosphorus compounds by isolated bacteria have
been reported. A list of micro-organisms capable of degrad-
ing these compounds is presented in Table 2.
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 detoxication, studies on the further metabolism of
the phosphorus containing products have not been exten-
sive. Hypothetical phospho-ester hydrolysis steps can be
postulated, yielding mono-ester and nally inorganic phos-
phate, but this pathway has not been specically studied.
Analogous phospho-monoesterase and diesterase, which
degrade methyl and dimethyl phosphate, respectively, have
been reported in Klebsiella aerogenes (Wolfenden & Spence,
1967) and are produced only in the absence of inorganic
phosphate from the growth medium. The nal enzyme in
the postulated degradative pathway is bacterial alkaline
phosphatase, which can hydrolyze simple monoalkyl phos-
phates 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 phospho-
nates (Kertesz et al., 1994a). The way in which metabolism is
regulated depends very strongly on what role the organo-
phosphorus 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, 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 specic regulation mechan-
isms for the degradation pathways.
Table 1. History, toxicity and half-life of some organophosphorus
pesticides
Name Type
Year of
introduction
Mammalian
LD50
(mg kg
1
)
Half-life
soil
(days)
Chlorpyrifos Insecticide 1965 135163 10120
Parathion Insecticide 1947 210 30180
Methyl parathion Insecticide 1949 330 25130
Glyphosate Herbicide 1971 35305600 30174
Coumaphos Acaricide 1952 1641 241400
Fenamiphos Nematicide 1967 610 2890
Monocrotophos Insecticide 1965 1820 4060
Dicrotophos Insecticide 1965 1522 4560
Diazinon Insecticide 1953 80300 1121
Dimethoate Insecticide 1955 160387 241
Fenitrothion Insecticide 1959 1700 1228
Ethoprophos Nematicide 1966 146170 330
Table 2. Microorganisms isolated for the degradation of organophosphorus compounds
Compound Microorganisms Mode of degradation Reference
Chlorpyrifos Bacteria
Enterobacter sp. Catabolic (C, P) Singh et al. (2003c)
Flavobacterium sp. ATCC27551 Co-metabolic Mallick et al. (1999)
Pseudomonas diminuta Co-metabolic Serdar et al. (1982)
Micrococcus sp. Co-metabolic Guha et al. (1997)
Fungi
Phanerochaete chrysosporium Catabolic (C) Bumpus et al. (1993)
Hypholama fascicularae ND Bending et al. (2002)
Coriolus versicolor ND Bending et al. (2002)
Aspergillus sp. Catabolic (P) Obojska et al. (2002)
Trichoderma harzianum Catabolic (P) Omar (1998)
Pencillium brevicompactum Catabolic (P) Omar (1998)
Parathion Bacteria
Flavobacterium sp. ATCC27551 Co-metabolic Sethunathan & Yoshida (1973)
Pseudomonas stutzeri Co-metabolic Daughton & Hsieh (1977)
Arthrobacter spp. Co-metabolic Nelson et al. (1982)
Agrobacterium radiobacter Co-metabolic Horne et al. (2002b)
Bacillus spp. Co-metabolic Nelson et al. (1982)
Pseudomonas sp. Catabolic (C, N) Siddaramappa et al. (1973)
Pseudomonas spp. Catabolic (P) Rosenberg & Alexander (1979)
Arthrobacter sp. Catabolic (C) Nelson et al. (1982)
Xanthomonas sp. Catabolic (C) Rosenberg & Alexander (1979)
Methyl parathion
Pseudomonas sp. Co-metabolic Chaudry et al. (1988)
Bacillus sp. Co-metabolic Sharmila et al. (1989)
Plesimonas spM6 Co-metabolic Zhongli et al. (2001)
Pseudomonas putida Catabolic (C) Rani & Lalitha-kumari (1994)
Pseudomonas sp. A3 Catabolic (C, N) Zhongli et al. (2002)
Pseudomonas sp. WBC Catabolic (C, N) Yali et al. (2002)
Flavobacterium balustinum Catabolic (C) Somara & Siddavattam (1995)
Glyphosate Bacteria
Pseudomonas ssp. Catabolic (P) Kertesz et al. (1994a)
Alcaligene sp. Catabolic (P) Tolbot et al. (1984)
Bacillus megaterium 2BLW Catabolic (P) Quinn et al. (1989)
Rhizobium sp. Catabolic (P) Liu et al. (1991)
Agrobacterium sp. Catabolic (P) Wacket et al. (1987)
Arthrobacter sp. GLP Catabolic (P) Pipke et al. (1987)
Arthrobacter atrocyaneus Catabolic (P) Pike & Amrhein (1988)
Geobacillus caldoxylosilyticus T20 Catabolic (P) Obojska et al. (2002)
Flavobacterium sp. Catabolic (P) Balthazor & Hallas (1986)
Fungi
Penicillium citrium Co-metabolic Pothuluri et al. (1998)
Pencillium natatum catabolic (P) Pothuluri et al. (1992)
Penicillium chrysogenum Catabolic (N) Klimek et al. (2001)
Trichoderma viridae Catabolic (P) Zboinska et al. (1992b)
Scopulariopsis spand Catabolic (P) Zboinska et al. (1992b)
Aspergillus niger Catabolic (P) Zboinska et al. (1992b)
Alternaria alternata Catabolic (N) Lipok et al. (2003)
Coumaphos
Nocardiodes simplex NRRL B24074 Co-metabolic Mulbry (2000)
Agrobacterium radiobacter P230 Co-metabolic Horne et al. (2002b)
Pseudomonas monteilli Co-metabolic Horne et al. (2002c)
Flavobacterium sp. Co-metabolic Adhya et al. (1981)
Nocardia strain B-1 Catabolic (C) Mulbry (1992)
Chlorpyrifos
Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl)
phosphorothioate) is one of the most widely used insecti-
cides effective against a broad spectrum of insect pests of
economically important crops. It is effective by contact,
ingestion and vapour action but is not systemically active. It
is used for the control of mosquitoes (larvae and adults),
ies, various soil and many foliar crop pests and household
pests. It is also used for ectoparasite control on cattle and
sheep. It has low solubility in water (2 mg L
1
) but is readily
soluble in most organic solvents. It has a high soil sorption
co-efcient (Racke, 1993) and is stable under normal storage
conditions. Chlorpyrifos is dened as a moderately toxic
compound having acute oral LD
50
; 135163 mg kg
1
for rat
and 500 mg kg
1
for guinea pig.
The environmental fate of chlorpyrifos has been studied
extensively. Degradation in soil involves both chemical
hydrolysis and microbial activity. The half-life of chlorpyr-
ifos in soil varies from 10 to 120 days (Getzin, 1981; Racke
et al., 1988) with 3,5,6-trichloro-2-pyridinol (TCP) as the
major degradation product. This large variation in half-life
has been attributed to different environmental factors, the
most important of which are soil pH, temperature, moisture
content, organic carbon content and pesticide formulation
(Getzin, 1981a, b; Chapman & Chapman, 1986). Initially,
the high rate of chlorpyrifos degradation in soils with
alkaline pH was attributed to chemical hydrolysis. Later,
Racke et al. (1996) concluded that the relationship between
high soil pH and chemical hydrolysis was weak and that
other factors like soil silt content might be important in
determining environmental fate.
Unlike other organophosphorus compounds, chlorpyri-
fos has been reported to be resistant to the phenomenon of
enhanced degradation (Racke et al., 1990). There have been
no reports of enhanced degradation of chlorpyrifos since its
rst use in 1965 until recently. It was suggested that the
accumulation of TCP, which has anti-microbial properties,
acts as a buffer in the soil and prevents the proliferation of
chlorpyrifos degrading microorganisms (Racke et al., 1990).
However, Robertson et al. (1998) suggested that chemical
hydrolysis of chlorpyrifos and enhanced degradation of TCP
can result in loss of efcacy of the insecticide against
termites in sugar cane elds in Australia. Attempts to
introduce enhanced degradation in the laboratory or in the
eld by repeated application have failed (Racke et al., 1990;
Mallick et al., 1999).
In recent experiments, we found that the degradation of
chlorpyrifos was very slow in acidic soils but that the rate of
degradation increased considerably with an increase in soil
pH. However, in 90 days of incubation, there was no
difference between soils in release of
14
CO
2
from the
pyridine ring despite the large differences in degradation
rate. Repeated applications of chlorpyrifos did not affect
Monocrotophos
Pseudomonas spp. Catabolic (C) Bhadbhade et al. (2002b)
Bacillus spp. Catabolic (C) Rangaswamy & Venkateswaralu (1992)
Arthrobacter spp. Catabolic (C) Bhadbhade et al. (2002b)
Pseudomonas mendocina Catabolic (C) Bhadbhade et al. (2002a)
Bacillus megaterium Catabolic (C) Bhadbhade et al. (2002b)
Arthrobacter atrocyaneus Catabolic (C) Bhadbhade et al. (2002b)
Pseudomonas aeruginosa F10B Catabolic (P) Singh & Singh (2003)
Clavibacter michiganense SBL11 Catabolic (P) Singh & Singh (2003)
Fenitrothion
Flavobacterium sp. Co-metabolic Adhya et al. (1981)
Arthrobacter aurescenes TW17 Catabolic (C) Ohshiro et al. (1996)
Burkholderia sp. NF100 Catabolic (C) Hayatsu et al. (2000)
Diazinon
Flavobacterium sp. Catabolic (P) Sethunathan & Yoshida (1973)
Pseudomonas spp. Co-metabolic Rosenberg & Alexander (1979)
Arthrobacter spp. Co-metabolic Barik et al. (1979)
Chemical warfare agents
G Agent Pseudomonas diminuta Co-metabolic Mulbry & Rainina (1998)
Altermonas spp. Co-metabolic DeFrank et al. (1993)
V Agent Pseudomonas diminuta Co-metabolic Mulbry & Rainina (1998)
Pleurotus ostreatus (fungus) Co-metabolic Yang et al. (1990)
Symbol in brackets after mode of degradation represents the type of nutrient that the pesticide provides to degrading microorganisms. C, carbon; N,
nitrogen; P, phosphorus.
either the degradation rate or degradation kinetics, suggest-
ing that repeated treatment did not result in enhanced
degradation. Fumigation of soil samples completely inhib-
ited hydrolysis of chlorpyrifos, suggesting an involvement of
soil micro-organisms (Singh et al., 2003c). Chlorpyrifos has
been reported previously to be resistant to enhanced degra-
dation. Given the tremendous adaptability of the soil
microbial community for degradation of a wide variety of
synthetic compounds, Racke et al. (1990) cited three possi-
ble reasons why a specic pesticide might not be susceptible
to enhanced degradation. One possibility is an inability of
the microora to initiate degradation of the parent pesticide
easily. This may be due to factors such as steric hindrance of
enzymes by functional groups, electronic stability against
hydrolysis or lack of weak links in the molecule (Alexander,
1965; Niemi et al., 1987). The pesticide may also be unavail-
able for uptake and degradation by soil microorganisms due
to strong sorption to organic surfaces in the soil (Orgam
et al., 1985). However, these reasons cannot explain the
present results because chlorpyrifos is rapidly hydrolyzed by
the soil bacterial community in alkaline soils. The second
possibility is that the soil environmental conditions may in
some way inhibit the development or expression of en-
hanced degradation. This also cannot explain the present
results because repeated treatment of the same soil samples
resulted in enhanced degradation of fenamiphos (Singh
et al., 2003b). A third possibility is that the soil micro-
organisms cannot benecially catabolize pesticide metabo-
lites. In these circumstances co-metabolism may occur (e.g.
hydrolysis of parent pesticides), but the microbial metabo-
lism of the degradation products is not possible. This is the
case with such relatively recalcitrant pesticides as DDT and
alachlor, which are converted to products that are them-
selves quite resistant to further metabolism (Tiedje &
Hagedorn, 1975). From our experiments we concluded that
in high pH soils, the microbial community transforms
chlorpyrifos co-metabolically into TCP. However, TCP con-
tains three chlorine atoms on the pyridinol ring. To break
this ring, chlorine atoms have to be removed (Feng et al.,
1997), and free chlorine has toxic effects on the micro-
organisms. Thus TCP metabolism may be toxic to micro-
organisms. Similar results were obtained by Price et al.
(2001) in a eld where degradation of chlorpyrifos was
strongly related with soil pH but degradation was mediated
by soil micro-organisms. Later, Singh et al. (2003c) sug-
gested that chlorpyrifos is degraded by non-specic and
non-inducible enzyme systems produced in high pH soils.
This suggests that chlorpyrifos is co-metabolically hydro-
lyzed to TCP and that because the TCP has toxic effects,
normally enhanced degradation does not occur. Although
Shelton & Doherty (1997) in their model proposed a
signicant role of bioavailability in degradation of xenobio-
tics, the toxic effect of TCP seems to be a realistic explana-
tion of its resistance to enhanced degradation because TCP
has high water solubility and therefore is bioavailable for the
degradation. However, repeated treatment with chlorpyrifos
over many years in an Australian soil resulted in develop-
ment of some opportunist microorganisms with the cap-
ability to use the toxic compound as has been reported with
organochlorine compounds (Robertson et al., 1998; Singh
et al., 2000). This adaptation can provide them with a
competitive advantage over other microbes in terms of
sources of energy. Further studies found higher copy num-
bers of opd (organophosphate degrading) gene in higher pH
soils (Singh et al., 2003a, c).
In most cases described to date, the aerobic bacteria tend
to transform chlorpyrifos by hydrolysis to produce
diethylthiophosphoric acid (DETP) and TCP, which in turn
accumulate in the culture medium without further metabo-
lism. This transformation reaction removes chlorpyrifos and
its mammalian toxicity but yields compounds that are not
metabolized by the microorganisms that produce them
(Richins et al., 1997; Mallick et al., 1999; Horne et al.,
2002b; Wang et al., 2002b).
Chlorpyrifos has been reported to be degraded co-meta-
bolically in liquid media by Flavobacterium sp. and Pseudo-
monas diminuta, which were initially isolated from a
diazinon treated eld and by parathion enrichment, respec-
tively (Sethunathan & Yoshida, 1973; Serdar et al., 1982).
However, these microbes do not utilize chlorpyrifos as a
source of carbon. A Micrococcus sp. was isolated from a
malathion enriched soil which was later reported to degrade
chlorpyrifos in liquid media (Guha et al., 1997). We have
isolated an Enterobacter sp. from a soil from Australia
showing enhanced degradation of chlorpyrifos. This bacter-
ium degrades chlorpyrifos to DETP and TCP and utilizes
DETP as a source of carbon and phosphorus (Singh et al.,
2003c, 2004). Cook et al. (1978a) isolated several bacteria
from sewage sludge that were able to use dialkylthiopho-
sphonic acid as a sole source of phosphorus. One of these
organisms, Pseudomonas acidovorans, was able to use DETP
as a sole source of sulfur (Cook et al., 1980). Another
signicant observation was the utilization of organopho-
sphorus insecticides as a source of phosphorus by Entero-
bacter sp. (Singh et al., 2003c, 2004). Sethunathan & Yoshida
(1973) isolated a Flavobacterium sp. that could use diazinon
as a source of carbon. However, Flavobacterium was not able
to use other organophosphorus pesticides as a source of
either phosphorus or carbon. Similarly, a variety of isolates
that could use phosphorothionate or phosphorodithionate
compounds as a sole source of phosphorus were unable to
degrade these compounds as a source of carbon (Rosenberg
& Alexander, 1979). Shelton (1988) isolated a consortium
that could use DETP as a carbon source but was unable to
degrade it when presented as source of phosphorus or sulfur.
It is believed that the conditions under which environmental
isolates are enriched are crucial in selecting for strains not
only with the desired degradative enzymes systems, but also
with the specic regulation mechanisms for the degradation
pathways (Kertesz et al., 1994a).
Studies on further metabolism and identication of
intermediate products of the phosphorus containing pro-
ducts have not been extensive. The postulated pathway steps
include hydrolysis, yielding monoester and nally inorganic
phosphate (Fig. 2). Bacterial phosphodiesterase has been
puried from a wide range of organisms including Escher-
ichia coli (Imamura et al., 1996), Haemophilus inuenzae
(Macfadyen et al., 1988), and Burkholderia caryophylli
PG2982 (Dotson et al., 1996). The phosphodiesterase from
the rst two bacteria are similar in sequence and both
moderate intracellular cyclic AMP levels. However, the
phosphodiesterase from B. caryophilli has a different se-
quence from that in the rst two bacteria (Dotson et al.,
1996). This enzyme could not be assigned a clear function
but was thought to play a role in xenobiotic degradation
pathways because it degraded glycerol glyphosate. However,
until recently no phosphodiesterase had been isolated or
characterized which could utilize xenobiotic degradation
products such as diethyl phosphate and diethyl phospho-
nate. A novel phosphodiesterase was isolated and cloned
from Delftia acidovorans which has both mono- and di-
esterase activity (Tehara & Keasling, 2003). This enzyme
allows D. acidovorans to use diethyl phosphonate as a sole
source of phosphorus under phosphorus limiting condi-
tions. The nal enzyme in the postulated degradative path-
way is alkaline phosphatase, which can hydrolyze simple
monoalkyl phosphates (Neidhardt et al., 1996).
Since only one bacterium has been isolated so far which
can degrade TCP in liquid medium, little literature is
available on microbial metabolism of TCP. Feng et al.
(1997) isolated a Pseudomonas sp. which can mineralize
TCP in liquid medium. Later the same group, on the basis of
combined experiments with photolysis and microbial de-
gradation, suggested that TCP was metabolized by a Pseu-
domonas sp. by a reductive dechlorination pathway (Feng
et al., 1998). In this pathway, TCP is rst reductively
dechlorinated into chlorodihydro-2-pyridone, which is
further dechlorinated to tetra-hydro-2-pyridone. Ring clea-
vage of this compound resulted in formation of maleamide
semialdehyde, which is metabolized to water, carbon diox-
ide, and ammonium ions. Microbial degradation of analo-
gous compounds such as pyridine and hydroxypyridine has
been researched and reviewed extensively (Shukla, 1984;
Sims & OLoughlin, 1989; Kaiser et al., 1996). Several micro-
organisms were reported to degrade hydroxypyridine (Kai-
ser et al., 1996). Cain et al. (1974) reported that 2- or 3-
hydroxypyridine was oxidized to 2,5-dihydroxypyridine and
production of maleamic acid occurred later through ring
cleavage. Oxygen atoms used to transform 4-hydroxypyr-
idine via 3,4-dihydroxypyridine were derived from water
molecules by hydroxypyridine hydrolase (Watson et al.,
1974). It is likely that TCP is metabolized in a similar
manner as one of the metabolites of TCP was identied to
have similar structure to 2-hydroxypyridine.
Fungal mineralization of chlorpyrifos by Phanerochaete
chrysosporium was reported by Bumpus et al. (1993).
Chlorpyrifos was hydrolyzed and then the pyridinyl ring
underwent cleavage before being converted to carbon diox-
ide and water. Degradation of chlorpyrifos in biobed
composting substrate by two other white-rot fungi, Hypho-
loma fascicularae and Coriolus versicolor, was observed
(Bending et al., 2002). Degradation of a wide range of
xenobiotic compounds by white-rot fungi is well documen-
ted (Kuhad et al., 1997; Singh & Kuhad, 1999, 2000; Singh
et al., 1999). These organisms have been reported to degrade
several persistent aromatic compounds by ring cleavage
(Armenante et al., 1994; Reddy & Gold, 2000). The multi-
step pathway of pentachlorphenol degradation by the white-
rot fungus Phanerochaete chrysosporium is initiated by lignin
peroxidase and manganese peroxidase, producing tetra-
chloro-1-4-benzoquinone, which is further metabolized by
two parallel but cross-linked pathways. The tetrachloroben-
zoquinone is reduced to tetrachlorodihydroxybenzene,
which can undergo four successive dechlorinations to pro-
duce 1,4-hydroquinone. This is then hydroxylated to pro-
duce the nal aromatic metabolite, 1,2,4-trihydroxybenzene.
Alternatively the tetrachlorobenzoquinone converts to
2,3,5-trichlorotrihydroxybenzene, which undergoes succes-
sive reductive dechlorination to produce 1,2,4-trihydroxy-
benzene. At several points, hydroxylation reaction converts
chlorinated dihydroxybenzene to chlorinated trihydroxy-
benzene, linking two pathways. The 1,2,4-trihydroxyben-
zene is ring cleaved to produce CO
2
and water (Reddy &
Gold, 2000). Mineralization of TCP by white-rot fungi is
possible via reductive de-chlorination. White-rot fungi have
been reported previously to use this transformation step to
degrade other chlorinated compounds such as pentachlor-
ophenol (Aiken & Logan, 1996) and hexachlorocyclohexane
(Mougin et al., 1996; Singh & Kuhad, 1999, 2000). Degrada-
tion of several polychlorinated compounds by white-rot
fungi suggests that they produce a range of isoenzymes with
a wide range of substrate specicity. Several species of
Aspergillus, Trichoderma harzianum and Penicillium brevi-
compactum were reported to utilize chlorpyrifos as sources
of phosphorus and sulfur (Omar, 1998) (Table 1). On the
basis of the above discussion, the authors propose possible
pathways for microbial degradation of chlorpyrifos (Fig. 2).
Parathion
Parathion (O,O-diethyl-O-p-nitrophenyl phosphorothio-
ate) is one of the most toxic insecticides registered with the
US Environmental Protection Agency (EPA). Extreme toxi-
city with ease of exposure has resulted in numerous human
and non-target species deaths in several developing coun-
tries (McConnell et al., 1999). The microbial degradation of
parathion has received extensive attention among the orga-
nophosphorus compounds because of its widespread use
and the ready detection of its hydrolytic product (p-nitro-
phenol). Parathion is rapidly degraded in biologically active
Fig. 2. Proposed pathways for chlorpyrifos
degradation by microorganisms. The scheme is
based on articles cited in the text. When the
conversion of one compound to another is
believed to occur through a series of inter
mediates, the steps are indicated by dotted
arrows. DETP, diethylthiophosphate; TCP,
trichloropyridinol.
soil. A proportional increase in the bacterial population in
soils was observed with an increase in the concentration of
parathion added (Nelson, 1982). Flooded soil conditions
favoured hydrolysis of parathion and release of
14
CO
2
from
ring labelled parathion in the rhizosphere of rice seedlings
(Reddy & Sethunathan, 1983).
Several species of bacteria have been isolated either from
parathion enrichment or other organophosphate enriched
environments, which can hydrolyze parathion (Table 2)
(Munnecke et al., 1982; Kertesz et al., 1994a; Racke et al.,
1996). Both mineralization, where parathion was used as a
source of carbon (Munnecke & Hsieh, 1976; Rani & Lalitha-
kumari, 1994) or phosphorus (Rosenberg & Alexander,
1979), and co-metabolic hydrolysis (Serdar et al., 1982;
Horne et al., 2002b) have been reported. Sethunathan &
Yoshida (1973) isolated the rst organophosphorus degrad-
ing bacterium, Flavobacterium sp., that could degrade para-
thion and diazinon. Siddaramappa et al. (1973) isolated a
Pseudomonas sp. that was able to hydrolyze parathion and
utilize the hydrolysis product p-nitrophenol as a carbon or
nitrogen source. Later, P. stutzeri was isolated, which can
hydrolyze parathion although p-nitrophenol was metabo-
lized by a separate bacterium (Daughton & Hsieh, 1977).
Rosenberg & Alexander (1979) isolated two Pseudomonas
ssp. that were able to hydrolyze a number of organopho-
sphorus compounds including parathion, and to use the
ionic cleavage products as a sole source of phosphorus.
Several species of Bacillus and Arthrobacter have been
isolated that were capable of hydrolyzing parathion; one of
the Arthrobacter strains was also able to utilize p-nitrophenol
as a sole source of carbon (Nelson, 1982). A Pseudomonas sp.
and a Xanthomonas sp. were isolated which can hydrolyze
parathion and can further metabolize p-nitrophenol (Tche-
let et al., 1993). A Moraxella sp. can use p-nitrophenol as the
sole source of carbon and nitrogen (Spain & Gibson, 1991).
This bacterium degrades p-nitrophenol to p-benzoquinone
using the enzyme p-nitrophenol monooxygenase. p-Benzo-
quinone is transformed to hydroquinone by a reductase
(Spain & Gibson, 1991). Candida parapsilosis has been
reported to produce hydroquinine 1,2-dioxygenase, which
converts hydroquinone to cis,trans-4-hydroxymuconic
semialdehyde. This is then metabolized to maleylacetate by
semialdehyde dehydrogenase. Maleylacetate is converted to
3-oxoadipate by a reductase, which is nally metabolized to
intermediary metabolites of the tricarboxylic acid (TCA)
cycle (Carnett, 2002). A Pseudomonas putida strain was
found to metabolize p-nitrophenol to hydroquinone and
1,2,4-benzenetriol, which was further cleaved by benzene-
triol oxygenase to maleylacetate (Rani & Lalitha-kumari,
1994). A similar pathway of p-nitrophenol degradation was
reported in Pseudomonas cepacia that can utilize p-nitro-
phenol as a source of carbon and nitrogen (Prakash et al.,
1996).
A different pathway of degradation was reported in
Arthrobacter sp. strain JS443 and Arthrobacter protophormiae
RHJ100 where p-nitrophenol was mineralized via p-nitroca-
techol. Nitrocatechol is converted to 1,2,4-benzenetriol by
benzotriol dehydrogenase, which in turn is directly con-
verted to maleylacetate by benzotriol dioxygenase (Jain et al.,
1994; Bhushan et al., 2000a; Chauhan et al., 2000). Recently,
a consortium of two Pseudomonas ssp. (strains S1 and S2)
was isolated which can also metabolize p-nitrophenol via
p-nitrocatechol (Qureshi & Purohit, 2002). The analogous
compound 3-methyl-4-nitrophenol has also been reported
to be metabolized by Ralstonia sp. via catechol formation
(Bhushan et al., 2000b). A Nocardia sp. was reported to
produce p-nitrophenol-2-hydroxylase, which catalyzes trans-
formation of p-nitrophenol to p-nitrocatechol (Mitra &
Vaidyanathan, 1984). A mono-oxygenase from a Moraxella
sp. that releases nitrite from p-nitrophenol has been partially
puried (Spain & Gibson, 1991). A soluble nitrophenol
oxygenase was puried from P. putida B2 that converts
ortho-nitrophenol to catechol and nitrite (Zeyer & Kocher,
1988). A novel monooxygenase was characterized from
Bacillus sphaericus that catalyzes the rst two steps of the
degradation of p-nitrophenol via p-nitrocatechol and benzo-
triol. This enzyme consists of two components, a reductase
and oxygenase, and catalyzes two sequential mono-oxygena-
tion reactions that convert p-nitrophenol to benzotriol. The
rst reaction converts p-nitrophenol to p-nitrocatechol and
the second removes the nitro group (Kadiyala & Spain,
1998). A pentachlorophenol degrading Sphingomonas
sp. UG30 was found to degrade p-nitrophenol. Apentachloro-
phenol-monooxygenase was puried from this bacterium
that can catalyze the hydroxylation of p-nitrocatechol to
benzotriol (Leung et al., 1999). A hydroxyquinol (benzo-
triol) ring cleavage dioxygenase was isolated and character-
ized from p-nitrophenol degrading Arthrobacter sp. strain
JS443. The gene encoding this dioxygenase (npdB) was
found to be in the same gene cluster as reductase (npdA1)
and oxygenase (npdA2) components of the p-nitrophenol
mono-oxygenase, maleylacetate reductase (npdC), and a
regulatory protein (npdR) (Zylstra et al., 2000; Parales
et al., 2002). Rhodococcus strain PN1 and Rhodococcus
erythropolis HL PM-1, which degrade 2,4-dinitrophenol
and p-nitrophenol, were reported to contain an npd gene
cluster including npdC (encoding hydride transferase I),
npdG (encoding the NADPH-dependent F420 reductase)
and npdI (encoding hydride transferase II). It was observed
that npdG and npdI genes have the same function as the
homologous genes (Heiss et al., 2003). Recently, a novel gene
called orf243 was reported from Flavobacterium sp. orf243
which is transposon based and is linked with the opd gene
(Siddavattam et al., 2003). This gene encodes a protein with
homology to a family of aromatic compound hydrolases and
is able to degrade p-nitrophenol.
Although in most of the studies on microbial degradation
of parathion, the rst reaction was hydrolysis of the phos-
photriester bond, there have been reports of different
degradation pathways. In one study, degradation of para-
thion by a mixed culture and a Bacillus sp. (Sharmila et al.,
1989) was shown to occur by reduction of the nitrogroup
that was later hydrolyzed to p-aminophenol. Another report
of conversion of parathion to paraoxon before hydrolysis of
phosphotriester bond was reported in a mixed bacterial
culture (Tomlin, 2000).
Studies on the degradation of methyl parathion (O,O-
dimethyl-O-p-nitrophenyl phosphorothioate) have also
been reported. Methyl and ethyl parathion have identical
chemical structures except for the ethyl groups of the P
chain of parathion, which are replaced by methyl groups as
evident by the name of the compound. A Pseudomonas sp.
was isolated that can co-metabolically degrade methyl para-
thion (Chaudry et al., 1988). Rani & Lalitha-kumari (1994)
isolated P. putida that could hydrolyze methyl parathion and
utilize p-nitrophenol as a source of energy. A Bacillus sp. was
reported to degrade methyl parathion by both hydrolysis
and nitro group reduction (Sharmila et al., 1989). Utiliza-
tion of methyl parathion by Flavobacterium balustinum as
the sole source of carbon was observed earlier (Somara &
Siddavattam, 1995). In this bacterium the opd gene was
found to be linked with a novel gene involved in degradation
of p-nitrophenol (Siddavattam et al., 2003). Degradation of
methyl parathion by a Pseudomonas sp. in soil and on
sodium alginate beads was reported (Ramanathan & La-
lithakumari, 1996). Co-metabolic degradation of methyl
parathion by Plesimonas sp. strain M6 was observed (Zhon-
gli et al., 2001) which was mediated by a novel degrading
gene. They also isolated Pseudomonas sp. A3 which can
utilize p-nitrophenol as sole source of carbon and nitrogen.
This isolate can also utilize a series of aromatic compounds
as a sole source of carbon (Zhongli et al., 2002). Another
strain of Pseudomonas sp. WBC was isolated from polluted
soils around a Chinese pesticide factory. The isolate was
capable of complete degradation of methyl parathion and
could utilize it as sole source of carbon and nitrogen (Yali
et al., 2002). The hydrolysis product of methyl parathion is
also p-nitrophenol, for which the degradation pathways
have already been described. The different proposed path-
ways of parathion and methyl degradation are presented
in Fig. 3.
Glyphosate
Glyphosate (N-(phosphonomethyl) glycine) is a globally
used broad-spectrum herbicide. It is a representative of the
phosphonic acid group of compounds, which is character-
ized by a direct carbon to phosphorus (CP) bond. The CP
linkage is chemically and thermally very stable and renders
the molecule much more resistant to non-biological degra-
dation in the environment than its analogues with O-P
linkage (Hayes et al., 2000). Mode of action of glyphosate
includes inhibition of the plant enzyme 5-enol-pyruvyl-
shikimate-3-phosphate synthase, which catalyzes synthesis
of aromatic amino acids (Fisher et al., 1984; Cole, 1985).
Glyphosate is moderately persistent with a half-life of
30170 days (Tomlin, 2000). Microbial degradation is
considered to be the most important of the transformation
processes controlling its persistence in soil (Araujo et al.,
2003). It was observed that mineralization of glyphosate is
related to both the activity and biomass of soil micro-
organisms (Wiren-Lehr et al., 1997). Microbial degradation
of glyphosate produces the major metabolite aminomethyl
phosphonic acid and ultimately leads to the production of
CO
2
, phosphate and water (Forlani et al., 1999; Araujo
et al., 2003). Several species of bacteria have been isolated
from previously treated and untreated environments, which
can degrade glyphosate either co-metabolically or as a
source of phosphorus. There has been no report of the
utilization of glyphosate as a source of carbon or nitrogen
(Dick & Quinn, 1995). Several species of Pseudomonas have
been isolated which can degrade glyphosate (Moore et al.,
1983; Tolbot et al., 1984; Jacob et al., 1988; Quinn et al.,
1989). Similarly, a Flavobacterium sp. (Balthazor & Hallas,
1986), an Alcaligenes sp. (Tolbot et al., 1984), Bacillus
megaterium strain 2BLW (Quinn et al., 1989), several species
of Rhizobium (Liu et al., 1991), three species of Agrobacter-
ium (Wacket et al., 1987; Liu et al., 1991) and an Arthro-
bacter sp. (Pipke et al., 1987) have also been reported to
degrade this herbicide (Table 2).
Three different pathways for CP bond cleavage have
been reported for the use of phosphonate as a source of
phosphorus for growth.
The phosphonatase pathway is involved in degra-
dation of alpha carbon substituted phosphonates, which are
primarily naturally occurring phosphonates such as
2-aminoethylphosphonates that have been reported in
Bacillus cereus (Lee et al., 1992b), and Pseudomonas aeru-
ginosa (Lacoste et al., 1993), Salmonella typhimurium and
several other organisms (Jiang et al., 1995). In a two-step
process, this pathway leads to the cleavage of the CP bond
by a hydrolysis reaction requiring an adjacent carbonyl
group. 2-Aminoethylphosphonate is converted to phosp-
honoacetaldehyde by a specic transaminase, which is
further degraded to acetaldehyde by phosphonatase.
The CP lyase pathway is involved in the cleavage of
both substituted and unsubstituted phosphonates such as
methylphosphonates (Lee et al., 1992b).The phospho-
noacetate hydrolase pathway specically degrades phospho-
noacetate and appears to have evolved for phosphonate use
as a carbon source. This enzyme catalyzes the hydrolysis of
phosphonoacetate ;to acetate and inorganic phosphonates
via metal cation-assisted PC bond cleavage (McMullan &
Quinn, 1994; McGrath et al., 1995). Glyphosate has been
found to be degraded by the second of these pathways.
Two different pathways of glyphosate degradation are
presented in Fig. 4. Arthrobacter sp. GLP-1 and Pseudomonas
sp. PG2982 degrade glyphosate by initial cleavage of the CP
bond, resulting in the production of sarcosine (N-methyl-
glycine) by CP lyase activity (Moore et al., 1983; Shinabar-
ger & Braymer, 1984; Pipke et al., 1987; Liu et al., 1991; Dick
& Quinn, 1995). Rhizobium meliloti has also been reported
to degrade glyphosate by this pathway but, unlike other
bacteria, it has only one CP lyase, which is able to degrade a
wide range of phosphonates (Park & Hausinger, 1995). The
sarcosine formed is further degraded to the amino acid
glycine and a C
1
-unit, which is incorporated into purines,
and the amino acids serine, cysteine, methionine and
histidine (Pipke et al., 1987). The second pathway involves
the conversion of glyphosate to aminomethylphosphonic
acid (AMPA) by the loss of a C
2
unit. This compound is then
dephosphorylated by CP lyase and further broken down by
subsequent steps to methylamine and formaldehyde (Pike &
Amrhein, 1988; Lerbs et al., 1990). An identical pathway has
Fig. 3. Different pathways of parathion and
methyl parathion degradation by microorgan-
isms. When the conversion of one compound to
another is believed to occur through a series of
intermediates, the steps are indicated by dotted
arrows. DATP, dialkylthiophosphate.
been observed in Arthrobacter atrocyaneus (Pike & Amrhein,
1988) and Flavobacterium sp. (Balthazor & Hallas, 1986;
Pipke et al., 1987). Recently, a thermophile, Geobacillus
caldoxylosilyticus T20 was isolated from a central heating
system which also degrades glyphosate by this pathway,
utilizing the compound as a sole source of phosphorus
(Obojska et al., 2002). A halophilic bacterium, Chromohalo-
bacter marismortui, isolated from soil beneath a road
gritting salt pile was capable of utilizing several organopho-
sphonates including aminomethyl phosphonic acid as a
source of phosphorus (Hayes et al., 2000). Utilization of
aminoalkylphosphonates as a source of nitrogen by different
bacterial isolates has been reported (McMullan & Quinn,
1994; Ternana & McMullan, 2000). Pseudomonas uorescens
was reported to utilize a diverse range of organophospho-
nates as sources of carbon, nitrogen and phosphorus
(Zboinska et al., 1992a). A strain of Kluyveromyces fragilis
has been shown to utilize AMPA as a source of nitrogen
(Ternana & McMullan, 2000). Strains of Streptomyces were
also reported to degrade and utilize several organopho-
sphonate compounds as sources of carbon and nitrogen.
These strains were capable of degrading glyphosate in
phosphate-free media via CP bond cleavage accompanied
by sarcosine formation (Obojska et al., 1999). Streptomyces
morookaensis DSM 40565 could degrade aminoalkylpho-
sphonate as a sole source of nitrogen and phosphorus
(Obojska & Lejczak, 2003). Alkyl amines are intermediate
degradation products for several xenobiotics such as carbo-
furan, atrazine, and monocrotophos and have been reported
to serve as a source of energy for different micro-organisms
(Strong et al., 2002). Use of methylamine as a source of
carbon is widespread in nature (Hanson & Hanson, 1996;
Trabue et al., 2001).
Fungi play an important role in degradation of xenobio-
tics and biospheres (Pothuluri et al., 1998, 1992) including
glyphosate. Probably the rst fungal degradation of glypho-
sate by Penicillium citrinum was reported by Zboinska et al.
(1992b). Penicillium notatum can utilize the herbicide as a
source of phosphorus and can degrade it by the amino-
methyl phosphonic acid pathway (Bujacz et al., 1995).
Strains of Trichoderma harzianum, Scopulariopsis spand and
Aspergillus niger were able to degrade glyphosate and
aminomethyl phosphonic acid in the laboratory (Krzysko-
Lupicka et al., 1997). The rst report of utilization of
glyphosate as a source of nitrogen by a microorganism was
reported for Penicillium chrysogenum (Klimek et al., 2001).
The fungal cells were found to lack detectable nitrogen
reductase activity and therefore this isolate seemed to lack
Fig. 4. Pathways of microbial degradation for
glyphosate. AMPA, aminomethyl phosphonic
acid.
the ability to convert nitrate to ammonium. Recently,
Alternaria alternata, a plant pathogen, was found to utilize
glyphosate as a source of nitrogen (Lipok et al., 2003).
The above observations suggest that glyphosate is de-
graded by several soil microorganisms, and different steps of
the degradation involve different microorganisms which
utilize different degradation products as different sources of
energy. The possible pathways of glyphosate degradation are
presented in Fig. 4.
Coumaphos
Coumaphos (O,O-diethyl-O-(3-chloro-4-methyl-2-oxo-
2H-1-benzo-pyran-7-yl) phosphorothioate) is used as an
acaricide for the control of cattle ticks. It is widely used by
different government agencies for tick eradication and
quarantine purposes. The primary tool used in the eradica-
tion programme is a series of dipping vats placed at border
crossing points. The cattle are induced to jump into the deep
end of the vat, resulting in their complete immersion in
coumaphos. They then swim the length of the vat and climb
out to other end. There are around 42 vats in the USA alone
and each vat contains about 15 000 L of coumaphos suspen-
sion at the rate of 1600 mg L
1
(42% active ingredient, a.i.)
(Shelton & Somich, 1988; Mulbry et al., 1998). The vats are
cleaned and recharged every 2 years to keep the concentra-
tion of acaricide at a desirable level. These operations
generate approximately 460 000 L of concentrated insecti-
cide waste yearly in USA alone (Mulbry et al., 1996). A
similar programme within Mexico is thought to produce a
much larger volume. Coumaphos is comparatively persis-
tent in soil, with a half-life of about 300 days (Kearney et al.,
1986) and it possesses a very high mammalian toxicity.
Because of these characteristics, it requires a safe and
effective method for disposal. Rapid degradation of couma-
phos was observed in several cattle-dipping vats, resulting in
loss of efcacy against cattle ticks (Shelton & Karns, 1988).
Under aerobic conditions, experiments with radiolabelled
coumaphos demonstrated that the aromatic portion of the
molecule is susceptible to mineralization by bacteria in
problematic vat dips (loss of efcacy). Three morphologi-
cally distinct bacteria (designated B-1, B-2 and B-3) that
could metabolize coumaphos were isolated from a problem
vat dip (Shelton & Somich, 1988). All these bacteria hydro-
lyzed coumaphos to DETP and chlorferon. Chlorferon was
further metabolized by B-1 and B-2 to a-chloro-b-methyl-
2,3,4-trihydroxy-trans-cinnamic acid (CMTC). Further ex-
periments demonstrated that B-1 was capable of mineraliz-
ing and incorporating the aromatic portion of the
coumaphos molecule into biomass, but this was inhibited
by the accumulation of metabolites that was due apparently
to the inefcient metabolism of a chlorinated intermediate.
Combination of B-1 with another organism from the vat,
designated strain B-4, which metabolized these inhibitory
products, yielded a stable two-member consortium able to
grow at the expense of coumaphos (Shelton & Haperman-
Somich, 1991). No further study on the degradation path-
way or metabolite identication has been carried out.
Ralstonia sp. LD35 has been reported to degrade an
analogous compound, 3,4-dihydroxycinnamic acid via ben-
zoic acid (Gioia et al., 2001). A similar breakdown pathway
for the propenoic side chain of substituted cinnamic acid
molecule, p-coumaric acid, has been observed in Pseudomo-
nas sp. (Tse et al., 2004) and Acinetobacter strains (Delneri
et al., 1995). These bacteria use p-coumaric acid as the
source of carbon. In the rst step, they convert p-coumaric
acid into p-hydroxybenzoic acid which is then transformed
to protocatechuic acid and integrated to the TCA cycle via
the b-ketodipate pathway. Many bacteria degrade substi-
tuted cinnamic acid by decarboxylation of side chains.
Enzymes and genes responsible for such degradation have
been puried and characterized (Degrassi et al., 1995;
Barthelmebs et al., 2000). Streptomyces setonii (Sutherland
et al., 1983) and Rhodopseudomonas palustris (Harwood &
Gibson, 1988) have been shown to degrade cinnamic and 4-
coumaric acids to their corresponding benzoic acid deriva-
tives. Several other bacteria follow the same pathway for
degradation of substituted cinnamic acids. Monooxygenase
and dioxygenase catalyze the formation of the 2-, 3-, and 4-
hydroxy derivatives as substituted acid and/or substituted
catechol (Peng et al., 2003).
The b-oxidation pathway has been proposed for the
degradation of substituted cinnamic acids by Pseudomonas
putida (Zenk et al., 1980). This pathway, which is analogous
to the b-oxidation of fatty acids, is thought to include
thiolytic cleavage of 4-hydroxy-3-methoxy-b-ketopropinyl-
CoA to yield acetyl CoA and vanillyl CoA, which is catalyzed
by b-ketoacyl CoA thiolase. The pathway subsequently leads
to ring ssion and requires several co-factors including ATP,
CoA and NAD
1
(Zenk et al., 1980). Under anaerobic
conditions, coumaphos undergoes reductive dechlorination
to form potasan (Mulbry et al., 1998).
Nocardia sp. strain B-1 was reported to degrade couma-
phos by a different gene enzyme system to the known opd
gene (Mulbry, 1992). Another microorganism, Nocardiodes
simplex NRRL B-24074, was found to have a distinct
enzymes system for coumaphos degradation (Mulbry,
2000). Horne et al. (2002b) isolated an Agrobacterium
radiobacter P230 capable of hydrolyzing coumaphos from
an enrichment culture containing organophosphorus as the
sole source of phosphorus. This bacterium degrades couma-
phos by hydrolysis of the phosphotriester bond. Pseudomo-
nas monteilli was isolated which can hydrolyze coumaphos
as well as its oxo analogue coroxon but it can utilize only
coroxon as a sole source of phosphorus, not coumaphos or
its hydrolysis product DETP. This bacterium degrades
coumaphos and diazinon but not parathion (Horne et al.,
2002a). Coumaphos is degraded by the other microorgan-
isms like Flavobacterium sp. (Sethunathan & Yoshida, 1973),
P. diminuta (Serdar et al., 1982), and Enterobacter sp. B-14
(Singh et al., 2004), which were isolated for their ability to
degrade other organophosphorus compounds. This obser-
vation suggests that these microorganisms produce several
isoenzymes or broad-specicity enzymes that can degrade a
range of organophosphorus compounds. The proposed
pathway of microbial degradation of coumaphos is shown
in Fig. 5.
Fenamiphos
Fenamiphos (ethyl 4-methylthio-m-tolyl isopropylpho-
sphoramidate) is an organophosphorate used extensively
for the control of soil nematodes. It is systemic, active
against ecto- and endo-parasitic, cyst forming and root-
knot nematodes, and is recommended for application at
520 kg a.i.ha
-1
. Its solubility at room temperature is
700 mg L
1
water. The acute oral LD
50
is 15.319.4
mg kg
1
for rats, 10 mg kg
1
for dogs and 75100 mg kg
1
for guinea pigs (Tomlin, 2000).
Fig. 5. Proposed pathways for microbial
degradation of coumaphos. The scheme is based
on articles cited in the text. When the conver
sion of one compound to another is believed
to occur through a series of intermediates, the
steps are indicated by dotted arrows. DETP,
diethylthiophosphate; CMTC, chloromethyl
trihydroxy cinnamic acid.
Although, there have been reports of enhanced degrada-
tion of fenamiphos, the mechanism of degradation has
received little attention. Fenamiphos is oxidized rapidly to
fenamiphos sulfoxide (FSO) which in turn is oxidized to
fenamiphos sulfone (FSO
2
). As FSO and FSO
2
, have nema-
ticidal activity and toxicity similar to fenamiphos (Waggoner
& Khasawinah, 1974), degradation and persistence studies
usually include estimation of total toxic residue, which is the
combination of the two oxidation products along with
parent compound. The half-life in soil for fenamiphos and
its metabolites (total toxic residues) varies from 30 days to 90
days (Johnson, 1998). More rapid rates of degradation in soil
repeatedly treated with the fenamiphos in the laboratory
have been reported (Chung & Ou, 1996) and enhanced
degradation of fenamiphos in the eld has been observed in
many countries (Stiriling et al., 1992; Smelt et al., 1996;
Meghraj et al., 1999). It was suggested that 34 years were
necessary before the accelerated degradation of fenamiphos
declined in a sandy soil in a temperate region (Ou, 1991).
Fenamiphos rapidly disappears from both enhanced and
non-enhanced soils but FSO
2
is rarely formed in enhanced
soils (Ou, 1991). This suggests that enhanced bio-degrada-
tion of fenamiphos total toxic residue was due to an increase
in the disappearance rate of FSO in soil samples collected
from eld sites treated one or two consecutive times with
fenamiphos (Davis et al., 1993). In a recent study of soil
samples from a eld in the UK, which had similar physical
characteristics except for soil pH, the degradation rate of
fenamiphos increased with the increase in pH. Repeated
application of fenamiphos slowed down the rate of degrada-
tion in acidic soils, and in the neutral pH soil, three
consecutive treatments did not result in the development of
enhanced degradation of fenamiphos. However, in the two
alkaline soils, a second treatment with fenamiphos led to
enhanced degradation (Singh et al., 2003b). Chung & Ou
(1996) have tried to shed light on the mechanism of
fenamiphos degradation in soils that showed enhanced
degradation. They reported that fenamiphos is degraded
into FSO which in turn is rapidly degraded into FSO-
phenol, which is subsequently mineralized into CO
2
. There-
fore in enhanced soil, degradation of fenamiphos (total toxic
residue) is rapid because it misses one step, FSO to FSO
2
. In
enhanced UK soils, fenamiphos was rapidly oxidized to FSO,
which in turn, was quickly degraded. The major fenamiphos
metabolites identied were FSO and FSO-phenol. No FSO
2
was detected in the enhanced soil samples (Singh et al.,
2003b). However, in two Australian soils, a different me-
chanism of fenamiphos degradation was observed where the
nematicide was directly converted to fenamiphos phenol,
suggesting that the rst oxidation step was replaced by
hydrolysis (Singh et al., 2003b).
Ou & Thomas (1994) isolated the rst microbial con-
sortium with six different bacterial species that degraded
fenamiphos in liquid culture. A pure culture of Brevibacter-
ium sp. MM1 was isolated which hydrolyzed fenamiphos
and its hydrolysis products but did not utilize these chemi-
cals as energy sources (Megharaj et al., 2003). Two different
consortia from Australian soils, made up of ve and four
different bacterial strains, were isolated [B. K. Singh, un-
published]. Both consortia could utilize fenamiphos as sole
sources of carbon and nitrogen. In contrast to the con-
sortium isolated by Ou & Thomas (1994), the two Austra-
lian consortia (CRF and BEP) did not require any
supplementary nutrient source for fenamiphos degradation
and were active in liquid media in the absence of mineral
surfaces (Singh et al., 2003b). These microbial systems were
found to mineralize fenamiphos or its oxidative metabolites
by hydrolysis as a rst step. The hydrolytic product fenami-
phos phenol, FSO-phenol or fenamiphos sulfone phenol
(FSO
2
-OH) can be further degraded by desulfonation. Three
modes of desulfonation are reported for aromatic sulfo-
nates: desulfonation (a) before, (b) during or (c) after ring
cleavage (Kertesz et al., 1994a). Mode (a) is considered to be
most common pathway of desulfonation in the environ-
ment. In this pathway, the target compound is oxygenated
by a multi-component oxygenase, yielding an unstable
sulfono cis-diol, which then spontaneously re-aromatizes
to the corresponding catechol with the loss of sulte. An
enzyme which catalyzes this reaction in toluene sulfonate
and benzene sulfonate has been isolated from an Alcaligenes
sp. (Thurnheer et al., 1986, 1990). In Pseudomonas putida
S-313, a broad-spectrum monooxygenolytic sulfonatase
catalyzes the conversion of sulfonate to a phenol with
incorporation of one oxygen atom from molecular oxygen
(Kertesz et al., 1994b). Alcaligenes sp. strain O-1 is reported
to contain two different desulfonative pathways where the
initial desulfonation is catalyzed by different dioxygenase
enzyme systems. One enzyme system can degrade 2-amino-
benzenesulfonate, benzene sulfonate and 4-toluene sulfo-
nate but the other one can degrade only the last two
compounds (Junker et al., 1994). Hydrogenophaga palleronii
S1 has been reported to degrade 4-carbo-4-sulfoazobenzene
by the 4-sulfocatechol pathway via the formation of 4-
aminobenzenesulfate (Vickers, 2002). Another proposed
pathway is transformation of toluene sulfonate to hydroxy
toluene by toluenesulfonate monooxygenase. Pseudomonas
putida strain S-313 catalyzes toluene sulfonate desulfona-
tion, which can serve as its sole source of sulfur and leaves 4-
hydroxytoluene unmetabolized. However 4-hydroxy toluene
is a metabolite that is readily catabolized by other bacteria
via the toluene pathway (Eisenmaan & McLeish, 2002).
Another toluene sulfonate degrading bacterium, Coma-
monas testosteroni T-2, was found to contain a degrading
gene on a plasmid (Hooper et al., 1990). Simple alkane
sulfonates are utilized by Pseudomonas sp. as a carbon source
where crude cell extract catalyzes the oxidation of the
a-carbon atom of alkanesulfonate to an aldehyde bisulte
adduct. This adduct then degrades to produce the corre-
sponding aldehyde and sulte. The substrate range for this
reaction has been reported to be relatively broad where
hydroxy-, methyl-, and alkenyl-substituted compounds are
all transformed (Thysse & Wanders, 1974). Degradation of
alkylsulfate proceeds via initial hydrolysis of the sulfate ester
linkage and subsequent oxidation of the released alkanol
(Kertesz et al., 1994a). Pseudomonas sp. C
12
B and a strain of
Comamonas terrigena were reported to utilize a range of
alkylsulfates as a source of carbon (Payne & Faisal, 1963;
Fitzgerald et al., 1977). Five different alkylsulfatases were
characterized from Pseudomonas sp. C
12
B and two from C.
terrigena (Dodgson et al., 1982). On the basis of the above
studies, we propose the microbial degradation pathways for
fenamiphos as presented in Fig. 6.
Other organophosphorus pesticides
Several other organophosphorus compounds have been
used extensively for pest control. Diazinon, monocrotophos,
malathion, dimethoate, etc., are being used world-wide.
Several species of bacteria have been isolated and character-
ized that can degrade these compounds in liquid medium
and soils (Table 2).
Fig. 6. Proposed pathways for fenamiphos
degradation by microorganisms. The scheme
is based on articles cited in the text. FSO,
fenamiphos sulfoxide; FSO
2
, fenamiphos
sulfone; FSO-phenol, fenamiphos sulfoxide
phenol; FSO
2
-OH, fenamiphos sulfone phenol;
F phenol, fenamiphos phenol.
Monocrotophos ((3-hydroxy-N-methyl-cis-crotonamide)
dimethyl phosphate) is widely used to control aphids, leaf
hoppers, mites and other foliage pests. It has been classied
as extremely hazardous, with an LD
50
value of 20 mg kg
1
for mammals. The half-life of monocrotophos in soil was
reported to be 4060 days (Tomlin, 2000). Monocrotophos
is easily soluble in water and therefore has potential to
contaminate ground water. Together with its high mamma-
lian toxicity, these characteristics make monocrotophos an
ideal compound for decontamination and detoxication.
Rangaswamy & Venkateswaralu (1992) isolated a monocro-
tophos degrading Bacillus sp. from previously treated soil.
Megharaj et al. (1987) isolated monocrotophos degrading
algae from soil. Two different algae, Aulosira fertilissima
ARM 68 and Nostoc muscorum ARM 221, were found to
utilize monocrotophos as a sole source of phosphorus
(Subramanian et al., 1994). Pseudomonas aeruginosa F10B
and Clavibacter michiganense ssp. insidiosum SBL 11 were
isolated from soil. These bacteria can utilize monocrotophos
as a phosphorus source but not as a carbon source (Singh &
Singh, 2003). Two species of Pseudomonas, three species of
Bacillus and three species of Arthrobacter were isolated from
soils, which can utilize monocrotophos as a sole source of
carbon (Table 2). Further studies demonstrated that Pseu-
domonas mendocina is the most efcient monocrotophos
degrader among the isolated bacteria and its degrading
capability is plasmid based (Bhadbhade et al., 2002a). The
same group isolated another 17 bacterial isolates from
previously exposed soils which can mineralize monocroto-
phos in liquid culture (Bhadbhade et al., 2002b). The two
most versatile degraders, Bacillus megaterium and A. atro-
cyaneus, were chosen for further studies on the biochemical
mechanisms and pathways of monocrotophos degrada-
tion. Phosphatase activities were observed in both cul-
tures, and it was suggested that the phosphates identied
may be mono- and dimethyl phosphates (Bhadbhade et al.,
2002b). Dimethyl- and monomethyl phosphates were
involved as intermediates in monocrotophos degradation
in plants and animals (Menzer & Cassida, 1965; Muck,
1994). Another intermediate identied during monocroto-
phos degradation was methylamine, produced by an esterase
enzyme. This esterase could be an amidase capable of
selecting amides as substrates since esterases sometimes
attack the amide bond (Hassal, 1990). Similar pathways of
degradation were reported for dicrotophos, which is rst
demethylated to monocrotophos and then further degraded
to methyl amine (Eto, 1974). As with most of the other
organophosphorus compounds, the rst degradation step
of monocrotophos should involve hydrolysis, which
could produce N-methyl acetoacetamide and dimethyl
phosphate (Beynon et al., 1973). Further degradation
of N-methyl acetoacetamide produced valeric acid in A.
atrocyaneus and acetic acid in B. megaterium (Bhadbhade
et al., 2002b). Acetic acid is the key intermediate of the
glycolytic pathway in microorganisms. The pathway of
dicrotophos- and monocrotophos degradation is shown in
Fig. 7.
Degradation of fenitrothion (O,O-dimethyl O-4-nitro-m-
tolyl phosphorothioate), a widely used insecticide, by Bur-
kholderia sp. strain NF100 was reported (Hayatsu et al.,
2000). This strain utilized fenitrothion as a source of carbon
with the help of two plasmids. The rst plasmid (pNF2) was
found to catalyze the hydrolysis of fenitrothion to 3-methyl-
4-nitrophenol. The nitro group from this compound
was oxidatively removed to form methylhydroquinone,
which was further metabolized by the second plasmid
(pNF2) (Hayatsu et al., 2000). This bacterium was also
found to degrade p-nitrophenol as a source of energy.
Methylhydroquinone may be degraded by ring ssion as
one of the two methods described for p-nitrophenol
degradation in the section dealings with parathion. p-
Nitrophenol degrading Ralstonia sp. SJ98 was reported to
have chemotaxis towards 3-methyl-4-nitrophenol and to
utilize it as a source of carbon. This strain degrades 3-
methyl-4-nitrophenol by the formation of catechol (Bhush-
an et al., 2000b).
Microbial degradation of various other organopho-
sphorus compounds has been documented. Diazinon de-
gradation by a Flavobacterium sp. was reported in 1973
(Sethunathan & Yoshida, 1973). Two Pseudomonas spp.
isolated from sewage sludge were found to degrade diazinon
in a culture medium (Rosenberg & Alexander, 1979). Two
strains of Arthrobacter sp. were reported to hydrolyze
diazinon (Barik et al., 1979). Dimethoate degradation was
reported to be carried out by a plasmid based gene of
P. aeruginosa MCMB-427 (Deshpande et al., 2001). A novel
dimethoate degrading enzyme was puried and character-
ized from a strain of the fungus Aspergillus niger. This
enzyme was found to degrade all compounds containing
PS linkage like malathion and fermothion but not com-
pounds with the PO linkage (Liu et al., 2001).
Utilization of ethoprophos as a sole source of carbon by P.
putida has been observed (Karpouzas et al., 2000). Isolation
and metabolism of cadusafos by Sphingomonas paucimobilis
and Flavobacterium sp. have been reported recently (Kar-
pouzas et al., 2005). Similarly, several species of bacteria
were isolated from different environments which degrade
organophosphorus compounds in laboratory cultures and
in soils (Singh et al., 1999). Microorganisms isolated from
enrichment of one organophosphorus compound can de-
grade other structurally similar compounds. For example,
Flavobacterium sp. and P. diminuta were isolated by diazinon
and parathion enrichment but they can degrade a wide
range of other organophosphorus compounds such couma-
phos, methyl parathion, chlorpyrifos and nerve agents
(Adhya et al., 1981; Singh et al., 1999).
Chemical warfare agents
Among lethal chemical warfare agents, the nerve agents have
played a dominant role since the Second World War. Nerve
agents acquired their name because they affect the function-
ing of nerve impulses like other organophosphorus com-
pounds. The nerve agents are a group of particularly toxic
warfare agents. There are ve major substances that are
classied as nerve agents and they can be divided into two
main groups:
(1) G agents, including Tabun (GA), Sarin (GB), Soman
(GD) and cyclohexyl methylphosphonouoridate
(commonly referred to as cyclosarin or GF).
(2) Vagent, represented by VX.
G agents are usually non-persistent volatile liquids
whereas VX is highly persistent, non-volatile and much
more active than any of the G agents. Physical and chemical
properties of these nerve agents are listed in Table 3. Munro
et al. (1994) described the acute and chronic toxicity of
nerve agents. In another review by this group, they listed the
different sources, fate and toxicity of degradation products
of chemical warfare agents (Munro et al., 1999).
It is estimated that about 30 000 tons in USA and about
200 000 tons nerve agents globally have to be destroyed
under the Chemical Weapons Convention (CWC), 1993. As
of 30 January 2002, 175 states have made CWC commit-
ments. CWC bans the use of chemical weapons but more
signicantly also bans their development, production,
stockpiling and transfer and requires that all existing stocks
be destroyed by the member states within 10 years of
ratication. Other known chemical warfare agents concen-
trations include Japanese chemical weapons munitions
abandoned in China in 1945, and an estimated 100 000 tons
of German chemical weapons munitions that were dumped
O
O
O
C
H
3
C
CH
3
CH
3
C
Dicrotophos
H
C N
P
O
O
O
C
H
3
C
CH
3
CH
3
CH
3
C
MCP
H H
C N
P
H
3
CO
H
3
CO
O
C C C N
H H
O
OH
HO
P
Dimethyl phosphate N-Methyl acetoacetamide
Methylamine
H
3
CO
H
3
CO
O
OH
Phosphoric acid
Phosphate HCHO
Valeric acid Acetic acid
CH
3
(Ch
2
)
3
COOH CH
3
COOH
P
HO
HO
CO
3
O
O C C
H
COOH
NH
4
+
P
H
3
CO
CH
3
NH
2
H
3
CO
H
3
C
H
3
CO
H
3
CO
Formaldehyde
C
1
metabolic cycle
Fig. 7. Pathways for microbial degradation
of dicrotophos and monocrotophos (MCP) .
into the Baltic Sea at the end of World War II. Prior to 1969,
the US army disposed of chemical weapons by open pit
burning, evaporative atmospheric dilution, burial and pla-
cement of munitions in concrete cofns for ocean dumping.
In the 1970s, alkaline hydrolysis replaced the above methods
for destroying nerve agents. Later, due to the resistance of
GB to alkaline hydrolysis, the incineration method was
adopted for destroying all groups of chemical weapons.
However, due to strong opposition to incineration by
environmentalist and local populations, this method of
chemical warfare agents destruction was stalled in the USA
and republics of the former Soviet Union. Consequently,
there is a need to nd alternative remediation methods that
can provide an environmentally safe and economically
viable solution. In this section, we review the environmental
fate of important nerve agents and the pathways of micro-
bial degradation.
Tabun (GA)
It is believed that Tabun (name given by its inventor), or GA
(ethyl N,N-dimethylphosphoroamidocyanidate), was the
rst nerve agent ever discovered. It was manufactured in
1937 in Germany, although large scale production and
stockpiling started in 1942 (Robinson, 1967). GA is the
American denomination of Tabun. It enters the body mainly
through the respiratory tract and the primary action is on
the respiratory system. It can also cause vision impairment
through its anti-acetylcholine esterase activity. GA has a
high water solubility but is also readily soluble in organic
solvents and can therefore easily penetrate skin (Munro
et al., 1999).
GA contains a cyanide group and is subject to hydrolysis.
Under neutral and acidic conditions, the rst step, which is
very rapid, includes formation of O-ethyl N,N-dimethyl
amidophosphoric acid and hydrogen cyanide. The subse-
quent hydrolytic step, which is comparatively slow, is
hydrolysis of O-ethyl N,N-dimethyl amidophosphoric acid
to dimethylphosphoramidate and then nally to phosphoric
acid. Under acidic conditions, hydrolysis to ethylphosphor-
ylcyanide and dimethylamine occurs. The nal product of
all pathways is phosphoric acid. Several different metabolites
were identied in soil exposed to GA. DAgostino & Provost
(1992) identied 16 different compounds from a soil con-
taminated with GA. However, several of them were impu-
rities and some were degradation products.
The chemical structure of GA suggests that it contains
several possible microbial degradation sites. The initial steps
are potentially O-dealkylation and C-dealkylation, nitrile
hydrolysis and N-dealkylation (Morrill et al., 1985). No
specic microorganism has been isolated for exclusive GA
degradation from natural environments but P. diminuta
isolated for degradation of other organophosphorus com-
pounds can degrade several chemical warfare agents includ-
ing GA (Mulbry & Rainina, 1998). DeFrank et al. (1993)
isolated several strains of Alteromonas that can effectively
degrade all G nerve agents. As with other organophosphorus
compounds, the complete degradation of GA is likely to
produce phosphoric acid. Several bacterial species have been
reported to cleave CP bonds. The mechanism and asso-
ciated micro-organisms have been described in detail under
the section dealing with glyphosate.
Several intermediate metabolites of GA have been identi-
ed from soils that include dimethylamine and triethyl
phosphate (Sanches et al., 1993; Verschueren, 1996), and
diethyl dimethylphosphoramidate (Munro et al., 1999).
These compounds are readily biodegradable (Verschueren,
1996). Degradation and utilization of alkylamine as a source
of energy is widespread in natural environments as dis-
cussed for glyphosate degradation. On the basis of the above
details, a proposed pathway for GA degradation is presented
in Fig. 8.
GB
GB (isopropyl methylphosphonouoridate) is a highly toxic
nerve agent rst produced in Germany in 1937 (Bakshi et al.,
2000). The term Sarin is an acronym of its discoverers
(Gerhard Schrader, Ambros Rudriger and Van der Linde).
Immediate death from exposure occurs because of respira-
tory tract failure (Rickett et al., 1986). Other routes of
exposure include the gastro-intestinal tract and skin absorp-
tion (Spruit et al., 2000). GB was implicated in terrorist
attacks in 1994 and 1995 in Japan, which caused death and
Table 3. Chemical, physical and biological properties of some organophosphorus chemical warfare agents
Name
First made
(Year)
Vapour pressure
(mmHg)
Volatility
(mg m
3
)
Solubility in
water (g L
1
)
Lethal dose
Breathing
(mgmin
1
m
3
)
Skin
(mg individual
-1
)
Tabun (GA) 1936 0.07 600 98 150400 10001700
Sarin (GB) 1938 2.9 17 000 00 75100 10001700
Soman (GD) 1944 0.3 3900 21 3550 50100
VX 1952 0.0007 10 30 10 610
injured many people (Abu-Qare & Abou-donia, 2002).
People exposed to GB during the incident in Japan reported
symptoms such as darkness of vision, ocular pain, dyspnoea
and headache. A review on GB effects on health is available
elsewhere (Abu-Qare & Abou-donia, 2002). GB is non-
persistent, volatile and completely soluble in water and
subject to acid and alkaline hydrolysis (Munro et al., 1999).
Like other xenobiotics, the fate of GB in soil includes
biotic and abiotic degradation, evaporation and leaching.
More than 90% of GB added to soil was reported to be
degraded within 5 days (Small, 1984); however, degradation
is comparatively slow at low temperature (Morrill et al.,
1985; Sanches et al., 1993). As discussed for GA, several
bacteria have been reported to degrade G agents including
GB. The major metabolites identied for GB degradation
are isopropylmethylphosphonic acid (IMPA) and methyl
phosphonic acid (MPA) (Mulbry & Rainina, 1998; Munro
et al., 1999). Chemically, IMPA is extremely stable and is
predicted to have a half-life of over 1900 years (Rosenblatt
et al., 1975). IMPA is relatively resistant to bacterial degra-
dation. However, two bacterial species, P. testosteroni and
Pseudomonas melophthora have been reported to degrade
Fig. 8. Possible pathways for microbial degrada-
tion of GA. The scheme is based on articles cited
in the text.
IMPA to MPA (Daughton et al., 1979). These bacteria
metabolize IMPA via cleavage of the CP bonds to methane
and inorganic phosphorus compounds. Zhang et al. (1999)
reported biotransformation of IMPA by a microbial con-
sortium. Four mixed cultures were acclimated to IMPA. Two
of these cultures, namely APG and SX microorganisms, used
IMPA as the sole source of phosphorus. The intermediate
metabolites were identied as MPA and inorganic phos-
phates. Although attempts to use IMPA as a source of
carbon to support microbial growth were not successful, in
a bioreactor 85 mg L
1
IMPA was decreased to non-detect-
able level within 6075 h. MPA is also susceptible to CP
lyase producing bacteria (Zhang et al., 1999). Use of MPA as
a source of phosphorus by a P. putida has been observed
(Cook et al., 1978b). Several other bacteria were reported to
possess CP lyases and they have been described for
glyphosate degradation. The microbial degradation pathway
for GB is presented in Fig. 9.
GD
Soman, or GD (pinacolyl methylphosphonouoridate), is
structurally similar to GB. GD was the given identier of
Soman post war (American denomination, GC was already
in medical use) when the information relating to Soman was
recovered by old Soviet Union in 1949. Its volatility is
intermediate between GA and GB. It is less water soluble
and more lipid soluble than the other two G agents, which
results in more rapid skin penetration and greater toxicity
(Munro et al., 1999). Like other G agents, GD is subject to
hydrolysis but the rate of hydrolysis is ve times slower than
GA (Hambrook et al., 1971). The rst step in hydrolysis is
uoride removal to form pinacolyl methylphosphonic acid
(PMPA), which is then slowly degraded to MPA and
pinacolyl alcohol (Kingery & Allen, 1995). No data were
found on the fate of PMPA or pincolyl alcohol in the
environment. It is assumed that, like alkyl methylphospho-
nic acid, PMPA is probably resistant to degradation (Munro
et al., 1999). However, like other G agents, GD is hydrolyzed
by P. diminuta and several strains of Alteromonas (DeFrank
et al., 1993). Similarly, IMPA degrading consortia were able
to degrade PMPA as a sole source of phosphorus. In
successive batch experiments using immobilized cells,
PMPA level decreased from 164 mg L
1
to below the detec-
tion limit within 60 h (Zhang et al., 1999). The proposed
microbial degradation pathway is presented in Fig. 9.
VX
O-ethyl-S[2-(di-isopropylamino) ethyl] methylphospho-
nothioate was rst discovered by British scientists. Later,
the US produced it in large quantities under code name VX.
It is a moderately persistent nerve agent characterized by a
PS bond and, therefore, it belongs to the phosphorothio-
lates group. It is less volatile than G agents and does not
evaporate easily (Munro et al., 1999). VX is soluble in water
(30 g L
1
at 25 1C) and is relatively resistant to hydrolysis.
However, at acidic and extreme alkaline pH, cleavage of the
PS bond predominates, resulting in formation of ethyl
methylphosphonic acid (EMPA) and diisopropylethyl mer-
captoamine (DIEM). The latter can be oxidized to bis (2-
diisopropylaminoethyl) disulde (BIAEDS) (Yang et al.,
1993). At neutral and alkaline pH, the common pathway of
hydrolysis includes cleavage of CO bonds to ethanol and S-
(2-diisopropyl aminoethyl) methyl phosphonothioate
(DIAEMP). The half-life of VX in water at pH 7 and 25 1C
is 1742 days (Clark, 1989). Laboratory and eld studies on
the fate of nerve agents demonstrated that loss is due to a
Fig. 9. Microbial degradation pathway for GB and GD. IMPA, isopropyl-
methyl phosphonic acid; PMPA, pinacolylmethyl phosphonic acid; MPA,
methyl phosphonic acid.
combination of abiotic and biotic processes such as evapora-
tion, hydrolysis and microbial degradation. According to one
study, 90% of added VX was lost from soil in 15 days (Small,
1984). Diethyl methyl phosphonate and BIAEDS were ex-
tracted when VX was added to soil (Sanches et al., 1993). In
other studies, EMPA and DIEM were found to be major
metabolites in soils (Kaaijk & Frijlink, 1977; Omar, 1998).
Further degradation of EMPA resulted in formation of MPA
(Omar, 1998). EMPA can be used as a phosphorus source for
natural microbial systems (Cook et al., 1978b; Mulbry &
Rainina, 1998). Diethyl dimethylpyrophosphonate, diisopro-
pylaminoethanol (DIPAE), diisopropylamine (DIPA) ethyl-
methylphosphonothioic acid (EMPTA) were reported as
other possible intermediate metabolites (Munro et al., 1999).
VX is resistant to microbial hydrolysis. It cannot be
hydrolyzed by any of the strains of Alteromonas isolated for
the degradation of G agents. OPH of P. diminuta is found to
be active against VX and Russian VX but its activity is less
than 0.1% toward VXas compared with parathion. However,
site-specic mutagenesis in OPH resulted to a 33% increased
activity against VX (Gopal et al., 2000). It was noted that VX
could be rapidly degraded by chemical oxidation of the PS
bond using various peroxides (Yang et al., 1993) and mono-
magnesium perphthalate (Amitai et al., 1998). Oxidative
hydrolysis of VX produces EMPA and dialkylaminoethane-
sulfonate as compared with the corresponding alkylthion
hydrolytic product formed via the hydrolysis pathway. EMPA
has been reported to be degraded as a source of phosphorus
by two glyphosate-degrading bacteria, Burkholderia caryo-
philli and Pseudomonas testosteronis (Elashvili & DeFrank,
2001). A partially puried enzyme from B. caryophilli
bacterium has shown a broad specicity towards neutralized
nerve agents, including GF, GB, GD, VX and Russian VX
(Elashvili & DeFrank, 2001).
Oxidative hydrolysis of VX by the enzyme laccase from a
white-rot fungus, Pleurotus ostreatus was observed. The
mechanism of such degradation is not fully understood. It
was suggested that the sulfur atom is oxidized followed by
cleavage of the PS bond (Yang et al., 1990). The nitrogen
atom at the b position to the carbon bound to the sulfur
atom was assumed to play an important role in the
enzymatic reaction. One suggested pathway is the formation
of an N-oxide intermediate in the N,N dialkyl aminoethyl
moiety at alkaline pH that may affect the cleavage of the PS
bond. Cleavage of the SC bond may also occur forming O-
ethyl methyl phosphorothoic acid and 2-diisopropylami-
noethanol (Yang et al., 1990). The proposed pathways of VX
degradation are shown in Fig. 10.
Detoxifying enzymes
Microbial enzymes that can hydrolyze organophosphorus
compounds have been identied and characterized from
different microbial species. The hydrolysis of organopho-
sphorus compounds leads to a decrease in mammalian
toxicity by several order of magnitudes and therefore this
step is also called detoxication. An excellent review on the
role of bacterial enzymes in detoxication of organopho-
sphorus nerve agents has been published recently (Raushel,
2002). Consequently, in the present article, we only review
the characteristics, improvement and utility of a few of the
most extensively studied organophosphorus hydrolyzing
enzymes. Several bacterial and fungal isolates with novel
enzyme/gene systems are reported (Table 4). However,
despite the apparent diversity of the enzyme systems, most
studies of organophosphorus degrading enzymes have fo-
cused on organophosphorus hydrolase (OPH) and organo-
phosphorus acid anhydrolase (OPAA), which are among the
most extensively studied enzymes in the biological sciences.
Organophosphorus hydrolase has been isolated from
several bacteria (Serdar et al., 1982; Mulbry & Karns, 1989a;
Singh et al., 1999). Among bacterial enzymes, OPH from P.
diminuta has the widest range of substrate specicity and,
therefore, has received most attention. OPH is a dimer of
two identical subunits containing 336 amino acid residues
(Dumas et al., 1989) that folds into a (ab)
8
-barrel motif
(Gerlt & Raushel, 2003). Each subunit contains a binuclear
zinc situated at the C-terminal portion. The two zinc atoms
are separated by about 3.4 A
and linked to the protein

through the side chain of His 55, His 57, His 201, His 230,
Asp 301 and a carboxylated Lys 169. Both the Lys 169 and
the water molecule (or hydroxide ion) act to bridge the two
zinc ions together (Benning et al., 2001). A schematic
diagram of the structure of the binuclear metal centre within
the active site of OPH is presented in Fig. 11. It has a
molecular weight of 72 kDa.
Organophosphorus compounds bind to the binuclear
metal centre within the active site via co-ordination of the
phosphoryl oxygen to the b-metal ion. This interaction
weakens the binding of the linking hydroxide to the b-metal.
The metal-oxygen interaction polarizes the phosphoryl
oxygen bond and creates a more electrophilic phosphorus
centre. Subsequent nucleophilic attack by the bound hydro-
xide is assisted by proton abstraction from Asp 301. The
hydroxide attacks the phosphorus centre, resulting in weak-
ening of the bond to the leaving group (Raushel, 2002). A
working model for the OPH reaction mechanism is shown
in Fig. 12. In summary, the role of one metal ion in the
active site of OPH is to increase the electrophilicity of the
phosphorus centre through co-ordination with the non-
ester oxygen atom of the substrate, whereas the second metal
ion acts as a promoter of the attacking nucleophile (Efr-
menko & Sergeeva, 2001). However, questions regarding the
mechanisms of catalytic activity remained unanswered. The
pKa value of the bridging solvent is not known; it is believed
to be determined by variation of the kinetic parameters with
pH. In addition, it has still to be resolved whether the two
Zn ions within the active site have distinct functions or
whether they act in tandem (Raushel, 2002).
Organophosphorus hydrolase has a wide range of sub-
strate specicity. It hydrolyzes PO, PF, and PS bonds to
different extents (Table 4). The lowest specicity is for the
PS bond. However, the enzyme does not catalyze the
cleavage of carbonyl groups such as those found in p-
nitrophenyl acetate. Similarly, organophosphate diesters are
very poor substrates. The K
cat
and VK
m
values for the
H
3
C
H
3
C
O
P
S
O
P
S N H
5
C
2
O
HO
O
P
O
O
P
S OH
HO
HO
O
P
OH HO
HO
O
P
OH
H
3
C
H
5
C
2
O
O
O
S HO
CH
2
VX
HS
CH
2
DIEM
EMPA
BIAEDS
DIPAE
DIPA
HOH
2
C
H
5
C
2
O
CO
2
+ H
2
O NH
4
+
+ CO
2
+ H
2
O
H
3
C
EMPTA
HOC
2
H
4
sulfurous acid
phosphoric acid
DIAEMP
CH (CH
3
)
2
CH (CH
3
)
2
N CH
2
CH
2
CH (CH
3
)
2
CH (CH
3
)
2
CH (CH
3
)
2
CH (CH
3
)
2
CH
3
CH
2
OH
Ethanol
N CH
2
CH
2
CH (CH
3
)
2
CH (CH
3
)
2
N
N
N H
CH
2
CH (CH
3
)
2
CH (CH
3
)
2
CH (CH
3
)
2
CH (CH
3
)
2
N CH
2
CH
2
CH (CH
3
)
2
CH (CH
3
)
2
C
2
H
5
OH
Fig. 10. Proposed pathways for VX degradation by microorganisms. The scheme is based on articles cited in the text. When the conversion of one
compound to another is believed to occur through a series of intermediates, the steps are indicated by dotted arrows. DIAEMP, diisopropyl aminoethyl
methyl phosphonothioate; DIEM, diisopropylethyl mercaptoamine; EMPA, ethylmethyl phosphonic acid; BIAEDS, bis-diisopropyl aminomethyl disulde;
DIAPAE, diisopropyl amino ethanol; DIPA, diisopropyl amine; EMPTA, ethylmethyl phosphonothioic acid.
hydrolysis of paraxon by the enzyme have been determined
to be nearly 10
4
s
1
and 10
8
M
1
s
1
, respectively (Omburo
et al., 1992).
The effects of metal substitution on the catalytic activity
of OPH were studied by removing the native metal (Zn)
from puried OPH and reconstitution with a series of
divalent cations which include Co, Cd, Cu, Fe, Mn and Ni
(Omburo et al., 1992; Di Sioudi et al., 1999; Benning et al.,
2001). Further enzymatic assays showed that Co
21
had the
greatest activity against paraoxon (Omburo et al., 1992). It
was suggested that divalent cations increased the activity of
enzyme by assisting folding of expressed enzyme in the
medium (Manavathi et al., 2005). Site specic mutagenesis
was used to substitute the original histidinyl residues at
positions 254 and 257. Of these mutant enzymes, H254R
(histidine at 254 was replaced with arginine) and H257L
(histidine at 257 was substituted with leucine) demonstrated
a four- to ve-fold higher catalytic activity against the PS
bond (VX and demeton-S). Other mutants also showed
higher activity against Soman and other nerve agents (Di
Sioudi et al., 1999).
The hydrolysis of asymmetric organophosphorus com-
pounds catalyzed by OPH is stereoselective (Lewis et al.,
1988; Chae et al., 1994). For example, OPH degrades the Sp
isomer of EPN 21 times faster than the Rp isomer. Similarly
Sp isomers of acephate and methamidophos are catalyzed
preferentially by OPH (Hong & Raushel, 1999). To achieve a
practical solution to nerve agent contamination, the enzyme
should be able to degrade the racemic mixture, as both
isomers are usually present in compounds such as Soman,
GB and VX. This aim was achieved by rational modication
to the substrate binding activities. The size and shape of
these binding subsites were remolded through a rational
restructuring via site-directed modication (Wu et al., 2001;
Raushel, 2002). Preferential degradation of the Sp isomer of
the EPN by OPH presumably arises because the bulkier
phenyl substituent is better accommodated in the large
subsite and the ethyl group within the small subsite of OPH
for the Spenantiomer. To increase the degradation of the
Rp-enantiomer, the small subsite was expanded. This was
achieved by the substitution of Phe132, Ser308 and Ile106
to glycine and/or alanine. With these mutants, the
Table 4. Organophosphorus degrading microbial enzymes
Enzyme Origin MW Structure
Bond cleavage
PO PF PS PC
Bacterial
OPH Pseudomonas diminuta 72 Dimer 1 1 1 1
OPAA Alteromonas spp. 5060 Monomer 1 1 1
OPDA A. radiobacter 70 Dimer 1 1 1 1
ADPase Nocardia sp. 43 Monomer 1 ND ND
AMPP Escherichia coli 52 Tetramer 1 ND ND ND
HOCA Pseudomonas monteilli 19 Monomer 1 ND ND
SC-OPH SC strain 67 Tetramer 1 ND ND
NS-OPH Nocardiodes simplex 45 Monomer 1 ND ND ND
PEH Burkholderia caryophilli 58 Tetramer 1
CP lyase Pseudomonas spp. 200 ND 1
Phosphonatase Bacillus cereus 37 Dimer 1
Fungal
A-OPH Aspergillus niger 67 Monomer ND 1 ND
P-OPH Penicillium lilacinum 60 Monomer 1 ND 1 ND
Laccase Pleurotus ostreatus ND ND 1 ND
1, positive activity; , no activity; ND, not determined; MW, molecular weight.
Fig. 11. Representation of the structure of the binuclear centre within
the active site of the bacterial organophosphorus hydrolase (reproduced
with permission from Benning et al., 2001).
stereoselectivity for the Sp and Rp enantiomers for EPN
decreased from 21 : 1 to 1 : 1.3 (Chen-Goodspeed et al.,
2001; Wu et al., 2001).
The course of evolution of OPH in bacteria remains an
unresolved question. Organophosphorus compounds were
released as pesticides after World War II and it is difcult to
understand how these OPH catalytic activities appeared so
quickly. One suggestion is that this enzyme was already
present in the environment before the application of orga-
nophosphorus pesticides. This hypothesis has gained im-
portance following isolation of similar OPH from
phylogenetically and geographically different micro-organ-
isms (Mulbry et al., 1986; Somara et al., 2002). The recent
nding of OPH encoding genes in a eld soil which has
never been exposed to this group of pesticides supports this
hypothesis (Singh et al., 2003c). Another possibility is that
this enzyme has evolved new substrate specicity from pre-
existing enzymes as it has been shown that OPH (phospho-
triesterase) could acquire phosphodiesterase activity by
alteration of only one amino acid (Shim et al., 1998). Urease
has been found to have carbamylated lysine as a bridging
ligand with binuclear Ni at the active site (Park & Hausinger,
1995). The binuclear centre of urease and OPH was found to
be remarkably similar. However, the chemical nature of the
active sites of these enzymes is quite different (Raushel,
2002). A larger group of enzymes with similar active site
architecture has been identied (Holm & Sander, 1997).
Interestingly this superfamily also includes atrazine chlor-
ohydrolase.
A similar enzyme, OPDA, has been isolated from A.
radiobacter and was found to have 90% homology to OPH
at the amino acid level and a very similar overall secondary
structure (Horne et al., 2002b; Yang et al., 2003). Despite
these similarities, the two enzymes have different substrate
specicities. There is about a 30-sequence difference be-
tween OPH and OPDA. The largest group consists of 19
residues at the C-terminus. In addition, two regions with
signicant difference in OPDA from OPH are a large pocket
at the active site and another in the region of the protein that
is responsible for binding phenyl ethanol (an inhibitor) in
OPH. Apart from the sequence difference, the water struc-
ture in this region differs in the two enzymes. There are two
water molecules that form a network of hydrogen bonds in
OPDA. The equivalent residues in OPH could not form the
same hydrogen bonds but were found to be stabilized by the
presence of phenyl ethanol. These differences at the active
site of OPDA likely to give it a preference for substrates that
have shorter alkyl substituents. Further studies using site-
specic mutagenesis in OPH gave a series of mutants that
had activities similar to those of OPDA. Yang et al. (2003)
argued that this alteration in the active site gave substrate
specicity and represented the progressive natural evolution
of the enzyme from OPH to OPDA. However, the observa-
tion that alterations of amino acids in the active site
increases the activity of enzymes emphasizes that enzyme
efciency depends on several factors and that evolution can
take place in many ways.
Another enzyme that has received considerable attention
recently, for detoxication of organophosphorus nerve
agents is OPAA. A highly active OPAA from Alteromonas
undina was isolated and puried and is composed of a single
polypeptide with molecular weight 53 kDa (Cheng et al.,
1993). However, another OPAA isolated from Alteromonas
sp. JD6.5 is composed of 517 amino acids with molecular
weight of 60 kDa. However, one from Alteromonas halo-
planktis contains 440 amino acids (Cheng et al., 1996, 1997)
with molecular weight 50 kDa. The 10 kDa difference be-
tween OPAAs of these two Alteromonas spp was found to be
Fig. 12. Working model for the catalytic me-
chanism for hydrolysis of organophosphorus
nerve agents by organophosphorus hydrolase
(reproduced with permission from Raushel,
2002).
due to the presence of an extended C-terminal region in the
JD 6.5 enzyme (DeFrank & White, 2002). The three-dimen-
sional structure of this enzyme is not yet known. It possesses
low catalytic activity against PO but high activity against
PF bonds. OPAA also displays stereoselectivity towards the
chiral phosphorus centre by displaying preference for the
Rp-enantiomers. It also exhibits an additional preference for
the stereochemical conguration at the chiral carbon centre
of the Soman analogue (Hill et al., 2001). This observation
was conrmed by a recent study, which found that OPAAs
along with wild-type phosphotriesterase catalyze preferen-
tially the hydrolysis of (1) GF isomer in a racemic mixture
(Harvey et al., 2005). However, OPAAs from different
species of Alteromonas have demonstrated wide variation in
catalytic activity, with the highest activity observed with the
enzyme obtained from Alteromonas sp. J.D.6.5 (DeFrank &
White, 2002). OPAA from Alteromonas sp. JD6.5 has high
degree of homology at amino acid level with E. coli X-Pro
dipeptidase (48%) and E. coli aminopeptidase P (31%).
Further molecular and biochemical analyses of OPAA have
established that this enzyme is a prolidase; a type of
dipeptidase cleaving dipeptide bond with a prolyl residue at
the carboxyl terminus (Cheng et al., 1999). Although the
native function of OPAA is not yet known, it has been
suggested to play an important role in cellular dipeptide
metabolism because all OPAAs were found to have activity
against several dipeptides (DeFrank & White, 2002). Mole-
cular modelling studies with Soman and Leu-Pro revealed
that the three-dimensional structure and electrostatic den-
sity maps of the two are nearly identical (DeFrank & White,
2002). This explains why several dipeptidase enzymes have
catalytic activity against organophosphorus compounds.
Determination of the crystal structure of OPAA may con-
rm this hypothesis. Both prokaryotes and several eukar-
yotes have been found to possess this enzyme (Mazur, 1946;
Hoskin et al., 1999), which suggests that OPAA is not a
newly evolved enzyme (Cheng et al., 1999). In a recent study,
aminopeptidase P (AMPP) was found to catalyze the
hydrolysis of a wide range of organophosphate triesters.
AMPP belongs to the family of protein-specic peptidases
and catalyzes the cleavage of amino-terminal X-Pro peptide
bonds (Jao et al., 2004). This enzyme possesses many
similarities with OPAA, such as two divalent (Mn
21
) ions,
which are critical for maximal activity of AMPP. Replace-
ment of the Mn
21
ion with other divalent ions except Co
21
resulted in the loss of catalytic activity (Jao et al., 2004).
AMPP is a tetramer with each sub-unit composed of a pita
bread fold of the C-terminus domain (Wilce et al., 1998).
The active site of AMPP is located at the C-terminal portion
of the b-sheet with the two manganese ions separated by
33 A
. The two ions are co-ordinated by Asp 260, Asp 271,

His 354, Glu 383, Glu 406 and two water molecules. Awater
molecule or hydroxide ion bridging between the two metal
ions is believed to be strongly activated, and acts as the
nucleophile in the attack on the scissile peptide bond
XaaPro. A single amino acid mutation with hydrophobic
side chains such as R153W, R153L, R370L increased the
hydrolysis rates towards most of the organophosphorus
substrates compared to the wild-type enzyme (Jao et al.,
2004). This result suggests that the further protein engineer-
ing of AMPP may signicantly enhance the cleavage of PO
bond in a variety of organophosphorus compounds.
Other structurally and functionally different organopho-
sphorus degrading enzymes have been reported. Three
unique parathion hydrolases were isolated, puried and
characterized from gram-negative bacterial isolates. One
cytosolic hydrolase described as an ADPase (aryldialkylpho-
sphatase) from Nocardia sp. strain B-1 was composed of a
single sub-unit of approximately 43 kDa (Mulbry, 1992).
Another hydrolase from strain SC was membrane bound
and is composed of four identical sub-units of 67 kDa.
While having some common features such as constitutive
production and similar temperature optima around 40 1C,
the substrate specicity and structure of these enzymes
differ one from another, and also from the other known
OPHs (Mulbry & Karns, 1989a). A unique phosphotriester-
ase has been characterized from Nocardioides simplex NRRL
B-24074. The puried enzyme is monomeric, has a native
molecular weight of 45 kDa, is constitutively expressed and
located in the cytoplasm. This enzyme is quite distinct with
respect to its activity towards different substrates and also in
its stimulation or inhibition by divalent cations and dithio-
threitol (Mulbry, 2000). Another novel phosphotriesterase
HocA (hydrolysis of caroxon) was isolated from P. monteilli
(Horne et al., 2002c). This enzyme was required by the host
for phosphate metabolism and was suggested to be evolved
from phosphodi- or mono-esterase. HocA (19 kDa) does
not require a metal ion for its catalytic activity but was
reported to be less efcient at hydrolyzing organopho-
sphorus compounds than other reported microbial phos-
photriesterases (Horne et al., 2002c). HocA is not a
metalloenzyme and its activity is controlled by the presence
of phosphate in the medium.
There have been a number of reports on isolation and
purication of organophosphorus hydrolyzing enzymes
from pure isolates or from mixed cultures of bacteria. Only
a few organophosphorus hydrolyzing enzymes have been
reported from fungi. Degradation of phosphorothiolates by
a broad-spectrum fungal enzyme, laccase (phenol oxidase)
from a white-rot fungus P. ostreatus was reported (Amitai
et al., 1998). This is a signicant observation as this enzyme
attacks PS bond, which is comparatively resistant to OPH
and OPAA cleavage. Laccase was observed to be capable of
complete and rapid degradation of VX and Russian VX
(Amitai et al., 1998). Several white-rot fungi are capable of
organophosphorus degradation (Table 2), and it will be
interesting to know if the degradation capability of all white-
rot fungi towards organophosphorus compounds is
mediated by the presence of laccase, or whether different
fungi possess different enzyme systems. A novel organopho-
sphorus degrading fungal enzyme (A-OPH) was isolated
from A. niger ZHY256 that could hydrolyze a range of PS
bonds containing organophosphorus compounds (Liu et al.,
2001). This 67-kDa enzyme has found to have optimal pH at
7 with thiol and sulfhydryl groups in the active catalytic site.
A-OPH does not require divalent cations for activation;
however, Cu
21
was found to activate its activity. Another
novel organophosphorus-hydrolyzing enzyme was puried
from Penicillium lilacinum BP303. Interestingly, this peni-
cillium OPH (P-OPH) was found to degrade various
organophosphorus compounds by cleaving both PO and
PS linkages (Liu et al., 2004). The molecular mass of P-
OPH is 60 kDa with optimal activity at pH 7.5. The puried
enzyme was reported to be a member of a cysteine hydrolase
group and similar to A-OPH and OPAA. Despite having
several similar structural components, P-OPH is different
from OPH, OPAA and A-OPH in its catalytic activity. P-
OPH degraded all organophosphorus compounds with PO
and PS linkage, whereas OPH in Flavobacterium sp. only
attacks PO bond and A-OPH splits only PS linkage.
The rst reported enzyme able to degrade the phospho-
nates, 2-phosphonoacetaldehyde hydrolase (phosphona-
tase), was isolated from B. cereus (La Nauze et al., 1970).
The isolated and puried phosphonatase showed optimal
activity at pH 8, required Mg
21
for its activity, and was
inhibited by sulfhydryl reagents (La Nauze et al., 1970).
Phosphonatase resembles alkaline phosphatase in many
properties but has narrow substrate specicity. Phosphona-
tase does not degrade phosphomonoesters and is not a
metalloenzyme (Kononova & Nesmeyanova, 2002). This
enzyme has been reported from several bacterial species
and can degrade a range of phosphonates including glypho-
sate (Baker et al., 1998). Further analysis suggests that
phosphonatases belong to a new family of hydrolase having
a high conservative aspartate residue in their active site to
which the phosphoryl group from a lysine residue of the
enzyme is transferred (Baker et al., 1998). Phosphonatase is
a homodimer of 3337 kDa subunits, and its active site is
mainly comprised of polar amino acid residues, which
suggests that phosphonatase may have a common origin to
the NAD dependent superfamily of dehalogenase, phospho-
tase and phosphomutase (Kononova & Nesmeyanova,
2002). Another interesting enzyme that can degrade phos-
phonates is CP lyase. There is one report suggesting partial
purication of this enzyme from Pseudomonas sp. GLC11
(Selvapandiyan & Bhatnagar, 1994). The molecular mass of
this enzyme was reported to be approximately 200 kDa and
it was found to be localized in the periplasmic space of
bacteria. However, subsequent reports suggest that CP
lyase manifests its activity only in cells and has never been
reliably found in cell-free extracts (Kononova & Nesmeya-
nova, 2002). This obstacle considerably limits the possibility
of understanding the mode of catalytic action of CP lyase.
A good review on the proposed mechanism of phosphonates
degradation by CP lyase on the basis of computer model-
ling and gene structures is available (Kononova & Nesmeya-
nova, 2002). In brief, the action is initiated by the generation
of a phosphonyl radical. Subsequent cleavage of this reactive
intermediate would lead to metaphosphate and alkyl moi-
eties as the corresponding alkenes. Abstraction of hydrogen
by an alkyl radical would yield the corresponding alkanes as
products, which is a specic feature of phosphonate degra-
dation.
Several microbial isolates have been reported to have
further novel enzyme/gene systems but most of these were
not isolated or puried such as CP lyase (Kertesz et al.,
1994a), methyl parathion hydrolase (Zhongli et al., 2001)
and chlorpyrifos degrading enzyme (Singh et al., 2004).
Most of the enzymatic studies were carried out to improve
the catalytic activity of OPH and OPAA by protein or
genetic engineering. Nonetheless, a few novel enzymes have
been puried recently and these enzymes differed in mole-
cular mass, substrate specicity and, sensitivity to chemicals
(Table 4). Improvements of known enzymes should con-
tinue but this trend of characterizing new and diverse
enzymes from prokaryotes and eukaryotes needs to be
sustained to nd the best bioremedial enzymes with optimal
activity in detergents and in the presence of metal ions, and
which have a broad pH and temperature optima. Discovery
of diverse microbial enzymes will also facilitate understand-
ing of the evolutionary structurefunction relationship of
organophosphorus-degrading enzymes.
Genetic basis of organophosphorus
degradation
The rst described organophosphorus degrading (opd) gene
was found in P. diminuta, and was shown to be present on a
plasmid (Serdar et al., 1982). The plasmid size was 66 kb and
was termed pCMS1. By cloning into different plasmids and
into the broad range cloning vector, it was shown that a
1.5 kb BamHI fragment with single restriction sites for SalI,
PstI and XhoI encoded this enzyme (Serder & Gibson, 1985).
Mulbry et al. (1987) found that the opd gene from Flavo-
bacterium sp. strain ATCC 27551 was encoded on a 43-kb
plasmid (pPDL2) and had a similar restriction map to the
opd gene from P. diminuta. Southern hybridization experi-
ments demonstrated that the opd gene from the two bacteria
possessed signicant homology. This nding of homologous
genes on two non-homologous plasmids from two phylo-
genetically and temporally different bacteria isolated from
different geographical regions suggests that the gene may be
a mobile genetic element or transposon (Mulbry et al.,
1987). Sequencing of the opd gene proved that the gene
from both bacteria had identical sequences (Harper et al.,
1988). Later, the nucleotide sequence of the opd gene from P.
diminuta was determined and a single open read frame
located (Serder et al., 1989). The opd gene from Flavobacter-
ium sp. consists of 1693 base pairs with one open reading
frame (Mulbry & Karns, 1989b). The opd gene has been
cloned into various bacterial strains (Serder & Gibson,
1985), actinomycetes (Steiert et al., 1989), fungi (Xu et al.,
1996) and insect cells (Dumas et al., 1989). Several other
bacteria have opd genes with almost identical nucleotide
sequences (Chaudry et al., 1988; Somara et al., 2002). Horne
et al. (2002b) reported a similar opd gene from A. radio-
bacter P230 isolated in Australia for coumaphos degrada-
tion, which was chromosome based. This gene, called opdA,
was approximately 88% identical at the nucleotide level to
opd (Horne et al., 2002b). Sequencing of the whole genome
revealed the presence of opd-like genes in Mycobacterium
tuberculosis (Philipp et al., 1996) and E. coli (Blattner et al.,
1997), which supports the hypothesis that the opd gene may
be transposon based. However, this evidence has been
provided only recently (Siddavattam et al., 2003) where a
complete sequence of a region of plasmid pPDL2 from
Flavobacterium sp. is reported, which has identical restric-
tion patterns to opd containing plasmid pCMS1 of P.
diminuta (Fig. 13). The opd gene was found to be anked
by an insertion sequence, ISF1sp1 (encoding a complete
istAB operon), which is a member of the IS21 family, and
downstream by a Tn3-like element (tnpA and tnpR) encod-
ing a transposase and a resolvase. It was also observed that
adjacent to opd, but transcribed in the opposite direction, is
an open reading frame (orf243) which encodes a polypep-
tide of 27 kDa that plays a role in the degradation of p-
nitrophenol (the major degradation product of parathion
and methylparathion). A 2.5-kb region upstream of the opd
gene contains two ORFs transcribed in the same direction as
opd, which have signicant homology to the IstA and IstB
genes. Similarly, Horne et al. (2003) reported that opdA in A.
radiobacter P230 is transposable. A tnpA gene was found
upstream of the opdA. The two genes are anked by
insertion sequences which resembles Tn 610 transposon
from Mycobacterium fortuitum. Two additional putative
ORFs separate opdA and tnpA, and the deduced translation
products show similarity to two proteins encoded on the
Geobacillus stearothermophilus IS5376 (Horne et al., 2003).
These observations of linkage of the opd/opdA genes to IS
elements and transposase genes supports the idea that the
widespread distribution of the opd gene could be due to its
lateral transfer by a combination of transposition and
plasmid transfer (Siddavattam et al., 2003).
The evolutionary origin of the opd gene is presently not
known. However, it has been argued that the closest homo-
logues of the IstA, IstB, and tnpR genes are all found in
strains of Agrobacterium tumefaciens and an opd gene has
recently been reported from a strain of A. radiobacter
(Horne et al., 2002b). These similarities could indicate an
evolutionary origin for these genes in Agrobacterium (Sid-
davattam et al., 2003). Another hypothesis is that the gene
was present in the environment long before organopho-
sphorus compounds were commercialized. The presence of
genes similar to opd in several bacteria that have never been
exposed to this group of compounds also supports this
argument (Philipp et al., 1996; Blattner et al., 1997; Richins
et al., 1997). Recently, higher copy numbers of the opd gene
were observed in higher pH soils (Singh et al., 2003a, c).
Fig. 13. Map of the sequence region from pPDL2 showing eight ORFs identied together with the location of the IR sequences that ank ISF1sp1 (&).
The extent of the plasmid subclones (pWWM1079, pWWM44 and pSM1) used as a starting material for sequencing is indicated and the region
identied previously as homologous to plasmid pCMS1 of Pseudomonas diminuta is indicated by a dashed arrow. The complete opd gene cluster (on
pSM2) is included. BamH1, EcoRI, HindIII, and PstI sites in the sequence are indicated by B, E, H and P, respectively. pWWM1079 is a recombinant
plasmid that contains an entire conserved region of opd gene cluster located downstream of the opd gene, pWWM44 is a recombinant plasmid that
contains an entire conserved region of opd gene cluster located upstream of the opd gene. Isolation and religation of the larger HindIII fragment from
pWWM1079 gave a plasmid (pSM1) carrying part of orf243 with tnpA and tnpR. The conserved region upstreamof opd was isolated on a 5.2-kb HindIII
fragment from plasmid pWWM44 and cloned into pSM1 in the correct orientation to reconstitute the entire region, thereby giving pSM2 (reproduced
and adapted with permission from Siddavattam et al. (2003)).
This observation has considerable signicance because this
eld had not been exposed to organophosphorus com-
pounds. It is worth mentioning that OPH has optimal
activity at higher pH. It is possible that opd or its ancestor
has some important function to play at high pH. It is
unlikely that such gene/enzyme systems evolved to protect
against anticholinesterase compounds since bacteria do not
contain acetylcholinesterases but their widespread distribu-
tion suggests that the gene/enzyme system does serve an
important function. One suggested function is the role of an
opd-like gene in phosphate metabolism (Horne et al.,
2002b). These genes may have evolved from pre-existing
mono-phosphatase or phosphodiesterase as it has been
shown that OPH (phosphotriesterase) could acquire phos-
phodiesterase activity by the change of only one amino acid
(Shim et al., 1998). Isolation, characterization and cloning
of a novel phosphodiesterase (which degrades organopho-
sphate xenobiotics) gene from Delftia acidovorans is strong
evidence to support this hypothesis (Tehara & Keasling,
2003). This gene shows sequence similarity to cyclic AMP
(cAMP) phosphodiesterase and cyclic nucleotide phospho-
diesterases and exhibits activity on cAMP in vivo when the
gene is expressed in E. coli, suggesting that it may have
evolved from a common ancestor of the cAMP gene and
may regulate cAMP levels in bacterial cells (Tehara &
Keasling, 2003). Further evidence in the form of the gene
structure and function relationships is required to reach to a
denitive conclusion.
Another organophosphorus degrading gene which has
received considerable attention is opaA, rst isolated and
cloned from Alteromonas sp. JD6.5 (Cheng et al., 1996,
1997). In spite of functional similarity with the opd gene,
no sequence homology was found between them. One ORF
of 1552 nucleotides was identied that codes for OPAA. The
OPAA enzyme has amino acid sequence similarity with that
of E. coli AMPP and human prolidase. It is believed that
opaA and the prolidase gene may have evolved from the
same ancestral gene and may play a role in bacterial peptide
metabolism (Cheng et al., 1996). However, the role of pepP
gene (encodes for AMPP) in organophosphorus degrada-
tion has also been reported (Jao et al., 2004).
Genes for phosphonate degradation have received con-
siderable attention due to their commercial exploitation in
genetically modied crops. Seventeen open reading frames
(phnA to phnQ) were reported to be involved in phospho-
nate uptake and degradation by E. coli (Chen et al., 1990).
Further study suggested that the genes phn ABQ were not
involved in degradation and that the phn operon therefore
consisted of 14 genes which are transcribed from a single
promoter preceding the phnC gene. On the basis of sequence
analysis and comparison with known motifs, reading frames
phnCDE have been proposed to form a phosphonate trans-
port complex, whereas phnF and phnO may be involved in
regulation. Two genes (phnNP) are not required for phos-
phonate use and may encode accessory proteins for the CP
lyase. The remaining seven genes (phnG-M) were suggested
to be involved in the CP lyase complex itself (Metcalf &
Wanner, 1991). Parker et al. (1999) reported the presence of
a homologous part of the E. coli phn gene cluster in
Sinorhizobium meliloti. By cloning, phnGHIJK genes were
identied in S. meliloti. However, several genes from phn
cluster of E. coli were not detected in S. meliloti, despite the
fact that S. meliloti appeared to have a broader substrate
specicity. This observation suggests that not all genes in
phn cluster may be required for phosphonate metabolism or
that these genes are functionally redundant in S. meliloti.
The molecular-genetic analyses suggest that the process of
phosphonate degradation involves a multi-component sys-
tem with constituents localized in the membrane and
periplasm. This may explain the failure by several research
groups to isolate and purify cell free CP lyase despite
considerable effort. Expression of the genes for phosphonate
degradation is controlled by phosphorus supply to the cell
and they have been suggested to be the part of the pho
regulon on the basis of their similarity to promoter se-
quences (Mulbry & Karns, 1989b) and the requirement of
regulatory genes (Wacket et al., 1987). Two other novel
genes involved in the degradation and utilization of glypho-
sate, glpA and glpB, were isolated and sequenced from
Pseudomonas pseudomallei. The gene glpA (1260 bp long)
encodes an enzyme (phosphotransferase) of 420 amino
acids, which confers increased tolerance to glyphosate. The
gene glpB encodes a protein (309 amino acids long) with the
ability to break the NC bond of glyphosate to yield
aminomethyl phosphonic acid (Penaloza-Vazquez et al.,
1995). Another gene involved in glyphosate metabolism,
pehA (encodes PEH), was cloned and sequenced from B.
caryophilli PG2982 (Dotson et al., 1996).
Several other genes with similar or identical function but
totally different nucleotide sequences have been reported
(Table 5). A methyl parathion degrading (mpd) gene was
isolated from Plesiomonas sp. strain M6 (Zhongli et al.,
2001). Sequencing and cloning of mpd revealed that this
gene is 1061 bp long and encodes a 35-kDa product. When
the mpd nucleotide sequence and predicted protein se-
quence were compared with those in the Genbank database,
no region of extensive DNA homology was observed. The
highest similarity with predicted protein sequence was
found to be 31% with beta-lactamase, suggesting signicant
novelty of the gene-enzyme system. Another novel gene,
adpB (which encodes ADPase), for organophosphorus de-
gradation was obtained from Nocardia strain (Mulbry,
1992). This 1600 bp long gene does not share homology
with any of the other known genes involved in organopho-
sphorus degradation. Horne et al. (2002c) isolated and
cloned a gene called hocA (hydrolysis of caroxon) gene from
P. monteilli, consisting of 501 bp; it has a different sequence
from all other known organophosphate degrading genes.
The hocA gene encodes a 19-kDa protein that can degrade a
range of oxon and thion organophosphorus compounds.
Increased expression of hocA was observed from an integra-
tive hocAlacZ fusion when the culture was grown in the
absence of phosphate, suggesting that it might be part of the
pho regulon. This structural diversity and functional simi-
larity suggests that these genes have evolved from different
ancestors, but that the structural similarity of xenobiotics to
natural compounds, and the constant mutation in the
bacterial genome and rapid doubling time play an impor-
tant role in the evolution of the gene/enzyme systems for
xenobiotic degradation.
Biotechnological advancements
Biotechnological aspects of the degradation of organopho-
sphorus compounds have received considerable attention
recently due partly to their high mammalian toxicity
and partly to the requirements of the CWC. Several com-
pounds need sensible detoxication and disposal techniques
because of their bulk usage, storage and widespread use.
Current methods for detoxifying these compounds mainly
rely on incineration and landlls. Incineration of chemical
warfare agents has received strong and sustained opposition
from the public and environmental groups because of
potentially toxic emissions. This process is also very costly,
as it requires considerable amounts of energy to reach the
high temperatures needed to destroy the pollutants. Land-
lls provide an adequate short term solution but leaching of
pollutants to ground water is a major source of concern.
Bioremediation with micro-organisms is therefore an at-
tractive alternative to these conventional techniques for
pollutant disposal.
Munneck (1976) rst reported the potential use of para-
thion hydrolase producing bacteria for the detoxi-
cation and disposal of organophosphorus compounds.
Later, successful use of OPH producing bacteria for com-
plete destruction of coumaphos in cattle-dip waste was
reported (Kearney et al., 1986; Karns et al., 1987). The use
of a consortium of microbes in a lter bioreactor for
destruction of coumaphos has been very successful. Two
units, each capable of treating 15 000 litres of waste
cattle-dip at a time, have been operational since 1996.
The US Department of Agriculture has been using these
units for treatment of coumaphos waste generated under its
cattle fever tick eradication programme (Mulbry et al.,
1998). The use of whole living cells for bioremediation
presents some difculties such as delivery of fresh
inocula and nutrient composition. To avoid these difcul-
ties, the use of cell free OPH was carried out successfully
(Karns et al., 1998). It was observed that addition of non-
ionic detergents and cobalt salts increased the efciency of
OPH in waste cattle-dips. Both native and recombinant
OPHs, immobilized on a nylon membrane, powder and
tubing (Caldwell & Raushel, 1991a), silica beads and glass
(Caldwell & Raushel, 1991b) have been used for the detox-
ication of organophosphorus compounds. OPH from P.
diminuta was immobilized based on the formation of non-
composite protein-silicone polymers and was a highly
active, stable and versatile biocatalyst for the liquid and gas
phase detoxication of organophosphorus compounds. It
was fabricated as monoliths, sheets, thick lms, granulates
or monoporous foams (Gill & Ballesteros, 2000). OPH and
OPAA were also incorporated into an aqueous re-ghting
Table 5. List of genes, their origin, vector, and gene products involved in degradation of organophosphorus compounds
Gene Organism(s) Location Encoded enzyme Reference
opd Pseudomonas diminuta Plasmid OPH Serder et al. (1989)
Flavobacterium sp. Plasmid OPH Mulbry et al. (1986)
Flavobacterium balustinum Plasmid OPH Somara & Siddavattam (1995)
Pseudomonas sp. Plasmid OPH Chaudry et al. (1988)
opaA Alteromonas sp. JD6.5 Chromosome OPAA Cheng et al. (1996)
Alteromonas haloplanktis Chromosome OPAA Cheng et al. (1997)
Alteromonas undina Chromosome OPAA Cheng et al. (1996)
opdA Agrobacterium radiobacter Chromosome OPDA Horne et al. (2002b)
hocA Pseudomonas monteilli Chromosome ND Horne et al. (2002c)
mpd Plesiomonas sp. Chromosome ND Zhongli et al. (2001)
adpB Nocardia sp. B-1 Chromosome ADPase Mulbry (1992)
PdeA Delftia acidovorans Chromosome Phospho diesterase Tehara & Keasling (2003)
PepA Escherichia coli Chromosome AMPP Jao et al. (2004)
Phn Escherichia coli Chromosome Phosphonatase Chen et al. (1990)
Sinorhizobium meliloti Chromosome Parker et al. (1999)
glp A&B Pseudomonas pseuodomallei Chromosome CP lyase Penaloza-Vazquez et al. (1995)
pehA Burkholderia caryophilli Chromosome PEH Dotson et al. (1996)
foam which was active and stable (Cheng et al., 1999;
Raushel, 2002).
Recently, considerable efforts have gone into the develop-
ment of OPH biosensors to detect contamination of orga-
nophosphorus compounds. This bioanalytical technique
provides rapid, cost effective and in-eld monitoring of
contaminants. The use of OPH in this technique has
advantages over acetylcholine esterase and enzyme-linked
immunoassays (ELISA). ELISA can be sensitive but like
most immunoassays, targets a single compound and re-
quires multisteps. Acetylcholine esterase based enzyme
inhibitions are well suited for screening applications; how-
ever, due to their irreversible inhibition by organopho-
sphorus compounds, they are not particularly well suited
for process control monitoring applications that require
rapid and repeated measurements (Chough et al., 2002).
Because OPH based biosensors respond to organopho-
sphorus compounds as substrates rather than as inhibitors,
they show considerable potential for applications that
require repetitive analysis (White & Harmon, 2005). Two
assay formats were used with OPH for biosensors: potentio-
metric measurement of local pH change (Mulchandani
et al., 1998, 1999b) and amperometric measurement of
electroactive enzyme products (Wang et al., 1999; Chough
et al., 2002). A dual amperometric and potentiometric ow-
injection biosensor detection system was developed recently
which used different physical transducers simultaneously in
connection with OPH. This enhanced the output and
allowed discrimination between various organophosphorus
compounds (Wang et al., 2002a). This biosensor, which
combines the advantages of both the amperometric device
and potentiometric detection, displays well-dened signals
from the oxidized leaving group and has been accomplished
with silicon-based pH sensitive electrolyte-insulation-semi-
conductor transducers (Wang et al., 2002a, 2003). While
offering a fast response, such enzyme biosensors have
limitations in terms of the number of samples that can be
handled and discriminated among organophosphorus com-
pounds. To overcome this problem, an on-chip enzymatic
assay for screening organophosphorus nerve agents, based
on pre-column reaction of OPH, electrophoretic separation
of the phosphonic acid products, and their contactless
conductivity detection has been developed (Wang et al.,
2004). On-chip enzymatic assays combine the selectivity and
amplication features of biocatalytic reactions with the
analytic features and versatility of microchip devices. This
new microsystem holds promise for eld screening of
organophosphorus compounds with the advantages of
speed/warning, efciency, portability, sample size and cost.
Information regarding the structure and function of
enzymes and pathways involved in biodegradation will
provide opportunities for improving enzyme activities.
Catalytic mechanism and enzyme properties can be ma-
nipulated by site-directed mutagenesis guided by computer-
modelled three-dimensional structure of enzymes (Cheng
et al., 1999). Site-directed mutagenesis was successfully used
to enhance the activity of OPH against racemic mixtures of
organophosphorus enantiomers. The size and shape of the
substrate binding subsites were remoulded through rational
restructuring via site-directed mutagenesis (Wu et al., 2001;
Raushel, 2002). However, rational design can fail sometimes
due to unexpected inuences exerted by substituted amino
acids. Another limitation imposed by this rational approach
is that only a limited sequence space can be explored at one
time. Irrational approaches such as DNA shufing, random
priming and staggered extension processes have been sug-
gested as preferable alternatives to direct the evolution of
enzymes (Cheng et al., 1999). DNA shufing was success-
fully used to isolate an improved variant of opd cloned E.
coli, which can degrade methyl parathion 25 times faster
than the wild type (Cho et al., 2002). However use of
enzymes for detoxication of pesticides is not a cost-
effective process. It was expected that genetic engineering
would provide a means for cheaper production of microbial
enzymes. The OPH encoding gene opd has been cloned
under different promoters to increase the amount of OPH
produced by cloned bacteria (Cheng et al., 1999).
It was suggested that the use of growing or non-growing
whole cells immobilized onto supports could offer cheaper
and more effective options. The major problem associated
with whole cell bioreactors is mass transport limitation of
substrate across the cell membrane where OPH resides
(Mulchandani et al., 1999a). Uptake of organophosphorus
compounds as a rate limiting factor has been reported by
several groups (Hung & Liao, 1996; Elashvili et al., 1998).
This barrier of substrate transport can be overcome by
treating cells with permeabilizing agents such as EDTA and
DMSO. However, several enzymes are sensitive to such
treatment and immobilized viable cells cannot be subjected
to permeabilization (Mulchandani et al., 1999a). To over-
come this difculty, OPH was successfully anchored and
displayed onto the surface of E. coli using the same
LppOmpA fusion system used for beta-lactamase (Richins
et al., 1997). Whole cells with surface expressed OPH had
seven times higher activity than whole cells expressing
similar amounts of OPH intracellularly. A genetically en-
gineered E. coli expressing both OPH and cellulose-binding
domain on the cell surface was constructed, enabling the
simultaneous hydrolysis of organophosphorus nerve agents
and immobilization via specic adsorption to cellulose
(Wang et al., 2002b). OPH was expressed on the surface by
the use of a truncated ice-nucleation protein-fusion system
while the cellulose-binding domain was surface anchored by
the LppOmpA fusion system.
Microorganisms that can completely degrade and miner-
alize whole molecules of organophosphorus compounds
have not yet been reported, but a diverse set of organisms
has been isolated that are capable of collectively mineralizing
these compounds. For example, some microbes can rapidly
hydrolyze parathion and methyl parathion and utilize DETP
as a source of carbon or phosphorus but quantitatively
produce p-nitrophenol as a by-product. However, other
bacteria can utilize p-nitrophenol as a source of energy. A
micro-organism engineered to complete mineralization of
organophosphorus compounds would avoid the generation
of toxic hydrolytic products. This goal was achieved by the
genetic engineering of p-nitrophenol mineralizing Moraxella
sp., which was transformed with the opd gene. The truncated
ice nucleation protein anchor was used to express the OPH
onto the surface of the bacterium, overcoming the potential
substrate uptake limitation (Shimazu et al., 2001). In
another study, Walker & Keasling (2002) engineered P.
putida KT 2442 to use parathion as a source of carbon and
energy. Two separate plasmids, one harbouring a native opd
gene (pAWW04) and another harbouring an operon encod-
ing enzymes for p-nitrophenol transformation to b-ketoa-
dipate (pSB337), were introduced into P. putida; the
plasmids enabled the bacterium to utilize 0.8 mM parathion
as a source of carbon. Recently, an E. coli expressing
phosphotriesterase from A. radiobacter (opdA) and glycer-
olphosphodiesterase from Enterobacter aerogenes (GpdQ),
which can use methyl parathion as a source of phosphorus,
was used to screen for mutants with enhanced activity. This
process of directed evolution produced a variant with
increased protein expression and increased activity against
organophosphorus (McLoughlin et al., 2005). The intro-
duction of all degradative genes into a single organism
allows for future optimization of gene expression and the
potentials to utilize further directed evolution to optimize
degradation rates and minimize the metabolic burden
placed on the cell.
Perspectives
Degradation of organophosphorus compounds has at-
tracted considerable attention because of their widespread
use as pesticides, their high mammalian toxicity, and the
CWC (1993). A large number of microorganisms have been
isolated and characterized that can degrade organopho-
sphorus compounds by mineralization or co-metabolism.
Some microorganisms can degrade several compounds and
some can degrade only one or few structurally similar
organophosphorus compounds. Because hydrolysis of orga-
nophosphorus compounds reduces mammalian toxicity by
several orders of magnitude, the environmental fate of
degradation products has not received much attention from
the scientic community. Complete pathways of parathion
and glyphosate degradation are known but the pathways for
several other organophosphorus compounds are not yet
fully understood. This area of research needs concerted
efforts as degradation products of several compounds are
pollutants and may have deleterious effects on the environ-
ment and non-target organisms.
Organophosphorus degrading enzyme OPH has been
characterized, its three-dimensional structure determined
and its catalytic activity elucidated. Site-specic mutagenesis
has been carried out successfully to increase the catalytic
activity against poor substrates, and to decrease the stereo-
selectivity of the enzyme. In addition to the potential
bioremedial use of microbes and enzymes for dealing with
organophosphorus contamination in the environment,
there has been considerable interest in the use of organo-
phosphorus degrading enzymes prophylactically and ther-
apeutically for organophosphorus poisonings (Sogorb et al.,
2004; Petrikovics et al., 1999, 2000a, b). Future areas of
research include increasing enzyme activity against poor
substrates and improving enzyme catalytic activities in
mixtures of chemicals.
Sequencing and structure determination of new proteins
will provide missing links to relate and elucidate evolution
mechanisms. Determining the three-dimensional structure
of OPAA would be a major boost for furthering studies on
the manipulation of enzymatic activity, which in turn may
help in developing efcient enzymatic destruction methods
for chemical warfare agents, as this enzyme has higher
catalytic activity towards G-agents.
The biodegradation of chemical warfare agents has re-
cently been a major area of research because of the urgency
to destroy all stocks by 2007. Recent efforts have provided
successful laboratory results. However, only a few isolated
microorganisms have the capacity to degrade chemical
warfare agents. A comprehensive screening of microbial
dipeptidase activity from different sources may provide
new gene/enzyme systems with higher activities against G-
agents or V-agents. For example, screening of anaerobic
microorganisms and extremophiles may be useful but
this so far has received little attention for organophos-
phorus compound degradation. Another challenge for
the scientic community is scaling-up of laboratory success
to the eld because of differential behaviour of iso-
lated micro-organisms in the environment and also because
stockpiles of chemical warfare agents contain a mixture
of different chemical contaminants and degradation pro-
ducts. The application of genetic engineering and biochem-
ical techniques to improve and evolve natural
biodegradative capabilities will ultimately create strains
capable of degrading complex mixtures of compounds. For
example, micro-organisms were isolated which can utilise
several neutralized chemical warfare agents as a phosphorus
source but require addition of excess nitrogen and carbon
which are rate limiting. Introducing cells containing CP
lyase activity in consortia or CP lyase gene in degrading
microorganisms might accelerate the overall degradation
process.
Acknowledgements
Work in BKS laboratory is supported by a grant from the
Scottish Executive Environment and Rural Affairs Depart-
ment (SEERAD). We are grateful to Ms Pat Carnegie for
drawing chemical structures and pathways. We also thank
Drs Pete Millard, Charlie Shand, Colin Campbell (MI), Alun
Morgan, Gary Bending (HRI), and Michael Kertesz (Uni-
versity of Manchester) for helpful discussions.
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Degradacion de Organofosforados

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Degradacion de Organofosforados

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Microbial degradationof organophosphorus compounds

and linked to the protein

. The two ions are co-ordinated by Asp 260, Asp 271,

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