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A review of bacterial degradation of pesticides

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DOI: 10.1071/SR9950925

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 Aust. J. Soil Res., 1995, 33, 925-42

A Review of Bacterial Degradation of Pesticides

J. Aislabie and G. Lloyd-Jones

Maanaki Whenua-Landcare Research, Private Bag 3127,
Hamilton, New Zealand.

Abstract
Pesticide fate in the environment is affected by microbial activity. Some pesticides are readily
degraded by microorganisms, others have proven to be recalcitrant. A diverse group of
bacteria, including members of the genera Alcaligenes, Flavobacterium, Pseudomonas and
Rhodococcus, metabolize pesticides. Microbial degradation depends not only on the presence of
microbes with the appropriate degradative enzymes, but also on a wide range of environmental
parameters. This review describes recent advances in biodegradation of pesticides by addressing
the biology and molecular characterization of some pesticide degrading bacteria.

Keywords: bacterial degradation, pesticides, molecular biology, biology.

Introduction
The fate of pesticides in the environment is determined by both biotic and
abiotic factors (Fig. 1). Pesticides are degraded in the environment principally
by the action of microorganisms, a process termed biodegradation, where
biodegradation is defined as the breakdown of a substance to smaller products

Pesticide

Fig. 1. Fate of pesticides in the environment.

0004-9573/95/060925$10.00
 Table 1. Pesticides, their common and chemical names, structures and uses
Common name Chemical name Chemical structure useA
Atrazine 2-chloro-4-ethylamino-6- Herbicide for broadleaf and grassy
iso-propylamine-s-triazine vegetation. Used on maize, sweet
H-~N~MOI(OIJI corn, forestry and established lucerne.

Carbofuran 2,3-dihydro-2,2-dimethyl-7- Systemic insecticide, acaricide and

benzofuranol methylcarbamate nematidcide.

Diazinon 0,O-diethyl 0-[&methyl-2-(1- Broadspectrum insecticide on grass

methylethyl)-4-pyrimidinyl] grub, indoor pests such as fleas and
phosphorothioate beetles, and fruit and vegetable pests.
Sheep dip.

(2,4-dichlorophenoxy)acetic Herbicide to control broadleaf weeds

acid *a in pasture and non-cropland areas.
cycocrc
a Controls most species of thistles and
buttercups.

DDT l,l,l-trichloro-2,2 bis- Insecticide applied to pasture to

(p-chlorophenyl) ethane control grassgrub. Use ceased in 1971.
Bacterial Degradation of Pesticides

caused by microorganisms or their enzymes (Atlas 1988). The rate at which

different pesticides are biodegraded varies widely. Some pesticides such as DDT
[l,l,l-trichloro-2,2-bis-(p-chlorophenyl)ethane] and dieldrin have proven to be
recalcitrant. Consequently they remain in the environment for a very long time
and are known to accumulate into food chains decades after their application
to soil (Aberdeen 1993; Kannan et al. 1994). Pesticides which are more readily
biodegradable, such as the organophosphates, are now used in preference to the
more persistent chlorinated pesticides. Others, such as atrazine and simazine, are
biodegradable at slow rates and may be leached from soil to ground water, where
they pose a threat to drinking water supplies (Kookana and Aylmore 1994). At
the other end of the scale, there are pesticides, such as carbofuran and diazinon,
that are readily biodegraded, and are broken down so rapidly under certain soil
conditions that they may not allow effective pest control (Felsot 1989).
To predict the fate of pesticides in soils it is important to have an understanding
of those microbes able to degrade pesticides, their activities and the factors
that limit their activity in situ. Microbial metabolism of pesticides has been
reviewed extensively (Hill and Wright 1978; McRae 1989; Somasundaram and
Coats 1990; Cork and Krueger 1991; Linn et al. 1993). In this article we
discuss both the factors influencing the biodegradation of pesticides, and review
recent reports of the bacterial metabolism of pesticides with an emphasis on
catabolic pathways involved, and the genetic basis for biodegradation. Some of
the pesticides discussed in this paper, their structure and uses, are described in
Table 1.
Factors that Effect Biodegradation of Pesticides
Presence of Appropriate Bacteria
I n situ degradation is dependent on the presence, numbers and activity level
of bacteria which possess the appropriate enzymatic capability. In addition,
environmental conditions must be suitable such that degradation can proceed.
Degradation rates are strongly influenced by a wide variety of environmental
factors (Scheme I ) , including soil type, moisture content, temperature, and pH,

Scheme 1. Factors that affect bacterial degradation of pesticides

The environment
Presence and number of appropriate microorganisms
Contact between the microbe and the substrate
pH, temperature and salinity
Available water
Oxygen tension and Redox potential
Nutrient availability
Presence of alternative carbon substrates
Light quality and intensity
Binding to surfaces
Alternative electron acceptors
The molecule
Chemical structure, molecular weight and
functional groups, e.g -CI, -CH3, -COOH, -OH
Concentration and toxicity
Solubility in water
 J. Aislabie and G. Lloyd-Jones

all of which affect microbial activity. For degradation to proceed, bacteria, or

their enzymes, must be in contact with the pesticide, and the pesticide or its
metabolites must be transported into the cell. Some bacteria display chemotactic
responses to substrates, which may play an important role in the environment;
others grow in a filamentous form towards potential substrates (Fewson 1988).
To date, most studies of microbial pesticide degradation have focused on
surface soils. Biodegradation of pesticides in the rhizosphere of plants and in
the soil subsurface has not been addressed in any detail, nor has the influence of
soil heterogeneity and spatial variability (Linn et al. 1993). In the rhizosphere,
surrounding plant roots, microbial activity is enhanced, for example p-nitrophenol,
the hydrolysis product of parathion (Fig. 6), was mineralized more rapidly in the
rhizosphere of rice than in unplanted soil under non-flooded and flooded conditions
(Reddy and Sethunathan 1994). Although it is becoming increasingly apparent
that substantial numbers of metabolically diverse bacteria exist in subsurface
environments, their ability to metabolize pesticides is virtually unknown (Dobbins
et al. 1992). Metabolism of atrazine has been detected in subsoils but the microbes
involved were not isolated (McMahon et al. 1992). Parkin and Shelton (1992)
investigated the degradation rates of carbofuran in corn fields. The degradation
rates were much higher in the planted furrow than between the rows of corn. It was
postulated that differences in degradation rates may be due to either the method of
pesticide application or alternatively to the increased available carbon in the plant
rhizosphere, which may enhance microbial activity under appropriate conditions.
The time lapse before the biodegradation of some pesticides proceeds, i.e. a lag
in which no significant decomposition is observed, is referred to as the acclimation
period. Several mechanisms have been proposed to explain acclimation (Chen
and Alexander 1989). The acclimation period may reflect the time taken for
the pesticide-degrading population to increase to such a level that enhanced
degradation rates occur. Following the application of 2,4-D to soils, mineralization
rates are initially slow, becoming more rapid over time, with concomitant increases
in the numbers of 2,4-D-degrading microorganisms (Robertson and Alexander
1994). Robertson and Alexander (1994) also reported that the same effect was
detected following the application of propham and glyphosate to soils. Acclimation
periods may also arise from adverse environmental conditions which may not
be conducive to microbial activity. Alternatively, not all microbial communities
possess the ability to degrade a pesticide before the first application of that
pesticide. In some instances biodegradation will require a new trait to develop
within the community, for example, the evolution of catabolic genes, by either a
relaxation of substrate specificity or inducer specificity of existing enzymes, or
by the acquisition of specific enzymes by genetic exchange (van der Meer 1994).
Eventually in such populations it is anticipated that there will be sufficient
advantageous changes to existing genes, and genetic exchange between strains,
either located on plasmids or transposable elements, to allow a cell to utilize the
novel compound/pesticide (Kellogg et al. 1981; Chen and Alexander 1989). Such
change will give the strain a selective advantage, and may allow this strain to
predominate within the community, hence magnifying the new trait such that it
becomes part of the community genome. This is not the birth of a new gene,
but the adaptation of pre-existing genes to transform a compound novel to that
community.
Bacterial Degradation of Pesticides

Chemical Structure
Virtually all molecules of biogenic origin are readily degraded by microorganisms,
given favourable growth conditions. Synthetic molecules such as pesticides often
contain functional groups such as chlorine substituents and novel arrangements
rarely found in nature which increase recalcitrance, thus leading to greater
persistence. DDT, for example, proved to be recalcitrant yet its non-chlorinated
analogue diphenylmethane was readily degraded (Alexander 1977). Other
molecules that have proven to be recalcitrant due to the presence of chlorine
substitutions include dieldrin and aldrin. The location and number of chlorine
substituents on a pesticide molecule will affect recalcitrance; for example,
2,4,5-T [2,4,5-trich1orophenoxy)aceticacid] is more recalcitrant than 2,4-D [(2,4-
dichlorophenoxy)acetic acid] (Atlas 1988). With the realization that chlorinated
pesticides are recalcitrant, moves were made in the 1970s to use more readily
degraded organophosphate pesticides.

Biological Availability
In soils, biodegradation of pesticides can be limited by the poor availability
of the chemical. A number of factors influence availability, though effects are
primarily due to either low solubility and/or strong adsorption to the soil matrix
(Linn et al. 1993). The chemical structure of a pesticide, its size and functional
groups all determine the water solubility of the compound. Some pesticides are
highly soluble, and others are applied as their water-soluble salts or amides to
enhance dispersal. Less soluble pesticides are applied as an emulsion or suspension
of fine solids in an aqueous carrier (07Conner1994). Methods of application aim to
provide an even dispersal of the pesticide; this can aid biodegradation. For growth
at the expense of pesticides that have low solubilities in water, microorganisms
may require some physiological adaptations such as surfactant production. Two
Bacillus spp. have been reported to produce emulsifying agents when grown in
the presence of the organophosphate fenthion (Pate1 and Gopinathan 1986).
In soils, pesticide molecules may bind tightly to soil organic matter, therefore
reducing biological availability. Pesticide sorption can either enhance or decrease
microbial degradation rates in soil. The concentration of pesticide applied to the
soil may be toxic, but once bound to soil an apparent reduction in toxicity is
seen allowing biodegradation to proceed. The degradation of carbofuran sorbed
to three different soils was studied by Singh and Sethunathan (1992); degradation
proceeded much more quickly in alluvial soils than in the soils rich in organic
matter. As carbofuran was desorbed much more quickly from the alluvial soil,
it appears that degradation rates of soil-sorbed carbofuran were related to its
desorption. It is possible that pretreating contaminated soils with surfactants
may be a mechanism for enhancing the solubility of the pesticide and hence its
ease of biodegradation. This approach has proved effective for enhancing the
degradation of the hydrocarbon phenanthrene in soil (Aronstein et al. 1991).

Nutrient Availability
Availability of essential nutrients such as carbon, nitrogen, phosphorus and
oxygen may also limit the rate at which bacteria degrade pesticides. The
 J. Aislabie and G. Lloyd-Jones

concentration of the pesticide must not be so high as to be toxic, nor so low such
that biodegradation does not proceed due to a lack of induction of appropriate
degradative enzymes and uptake mechanisms, failure to induce sufficient enzyme
activities, or problems in providing sufficient energy even for cell maintenance
(Fewson 1988; Pahm and Alexander 1993). Some pesticides, such as 2,PD
and carbofuran, serve as a carbon source for bacterial growth, however, in the
presence of alternative readily degradable carbon sources, bacteria are less likely
to degrade the pesticide. Other pesticides, for example DDT, are not utilized
as a carbon source and are biodegraded co-metabolically, hence an alternative
carbon source is required as the growth substrate.
When pesticides are applied in a mixture, varying effects have been observed.
Addition of one compound may shorten or lengthen the acclimation period needed
before another compound is degraded. Parathion amendment to soils treated with
EPTC and butylate inhibited the degradation of the two herbicides (Behki and
Khan 1991), and the degradation of EPTC by Rhodococcus TE1 was inhibited by
parathion (Behki and Khan 1991). Yet the simultaneous degradation of 2,4-D
and mecoprop by mixed bacterial cultures has been observed (Hallberg et al.
1991).
Until recently, degradation of many pesticides has only been studied under
aerobic conditions. The significance of anaerobic degradation, and of chlorinated
compounds in particular, has become more apparent. Degradation of 2,4,5-T under
anaerobic conditions was described by Gibson and Sulflita (1990), proceeding
readily in the presence of alternative electron acceptors. An anaerobic isolate,
Desulfomonile tiedjei, derives energy from anaerobic dechlorination reactions (Mohn
and Tiedje 1991). It appears that dechlorination of more highly chlorinated
compounds is favoured under anaerobic conditions. Degradation of some pesticides
may proceed under alternating aerobic-anaerobic conditions, should alternative
electron acceptors be available. Other pesticides may require aerobic conditions for
aromatic ring cleavage reactions to proceed. Hydrolysis of diazinon, for example, can
occur under both aerobic and anaerobic conditions but subsequent ring cleavage of
the hydrolysis product IHMP (2-isopropyl-6-methyl-4-hydroxy-pyrimidine) requires
aerobic conditions (Reddy and Sethunathan 1994).

Bacterial Metabolism of Pesticides

The degradation of pesticides in situ is usually achieved by a consortium of
microbes rather than a single species. Pure culture studies do, however, allow
the mechanisms by which the pesticide is metabolized to be elucidated. The
mechanisms for transport of the pesticide into the cell, degradation pathways and
induction and regulation of degradative pathways can be studied. Degradation
products are isolated and identified. Pure culture studies also allow the location
of genes involved in degradation of the pesticide; this can lead to the location of
the genes involved in pesticide degradation and the development of specific gene
probes for detection of these organisms in situ, precluding the need for prior
cultivation (Pickup 1991).
Traditional isolation methods, involving enrichment culture and plating
techniques, have been used to isolate many of the microbes that are able
to degrade pesticides in pure culture. The methods used are reviewed by Bartha
Bacterial Degradation of Pesticides

(1990). Sources of inocula for the enrichment cultures are generally soils that
have a previous history of treatment with pesticides (Pate1 and Gopinathan 1986;
Chaudhry and Ali 1988); other sources include cattle dip (Shelton and Somich
1988) and the fleece-rot lesions of sheep (Merritt et al. 1981) which have also
experienced a pre-exposure to the pesticide. One of the major difficulties in
isolating bacteria that are able to degrade pesticides is the chemical structure of the
pesticide which can often limit biodegradability. Bacteria utilize available organic
compounds. Therefore, by increasing the solubility of hydrophobic substrates by
the use of surfactants, or solvents in biphasic cultures (Ascon-Cabrera et al.
1993), or by dissolving the pesticide as its salt (for carboxylic acids and phenols),
the isolation of bacteria able to degrade these hydrophobic compounds can be
enhanced. Once isolated in pure culture and presumptively identified, the ability
of the bacteria to either utilize the pesticide as sole carbon source or, in some
cases as sole nitrogen source, or alternatively to co-metabolize it, is confirmed.
To understand the fate of pesticides in the environment it is important to
know the biochemical pathways utilized during microbial attack on the pesticide
(Somasundaram and Coats 1990). This information allows prediction of the fate
of not only the pesticide but also the metabolites generated. This information
can be crucial as microbial attack on a pesticide may result in metabolites that
are more toxic and more mobile than the parent molecule. Some intermediates
may accumulate in the environment, for example DDE a residue of DDT (Boul
et al. 1994); others may effect the rate of degradation of the parent molecule
(Somasundaram and Coats 1990).
Genetic studies of pesticide-degrading bacteria have centred on cloning and
characterization of specific enzymes involved in pesticide degradation. The
involvement of plasmid-encoded catabolic sequences in pesticide degradation has
been widely documented (Sayler et al. 1990). The potential for the spread of
plasmid-encoded catabolic genes has long been recognized and this phenomenon
may be linked to the poor efficacies of some readily degraded pesticides in certain
soils (Pemberton and Fisher 1977).
Many bacteria able to degrade pesticides have been isolated; some recent
isolates and those discussed in this paper are summarized in Table 2. McRae
(1989) prepared an extensive list of pesticide-degrading bacteria prior to 1987.
The following discussion describes the catabolism of a number of pesticides by
heterotrophic bacteria, along with any detailed genetic studies. The pesticides
selected are described in Table 1.

Organochlorines
The organochlorine pesticide DDT was used extensively in New Zealand between
1949 and 1970 to control pasture damage by grass grub (Costelytra zealandzca
White) and in Australia to control the cotton bollworm (Heliothis armigera)
(Pratchett 1990). Unfortunately, DDT and its residues still remain in soils more
than 20 years after use ceased. Consequently DDT residues have been transported
to water and sediments by surface runoff (McKenzie-Smith et al. 1994), and have
been accumulated into food chains (Kannaan et al. 1994). Agricultural practises
can influence the fate of pesticides. In irrigated agricultural soils, significantly
reduced DDT levels have been detected; this may be due to the enhanced activity
 J. Aislabie and G. Lloyd-Jones

Table 2. Examples of bacteria able to degrade pesticides in pure culture

Pesticide group Bacteria Reference
Chlorinated hydrocarbons
DDT Alcaligenes eutrophus Nadeau et al. (1994)

Phenoxy compounds
2,4-D Alcaligenes eutrophus Pemberton & Fisher (1977)
Flavobacterium Chaudhry & Huang (1988)
Arthrobacter Sandman and Loos (1988)
Pseudomonas cepacia Bhat et al. (1994)

Triazine
atrazine Norcardia Cook (1987)
Pseudomonas Cook (1987)
Pseudomonas Mandelbaum et al. (1995)
Rhodococcus Behki et al. (1993)
Rhodococcus Behki & Khan (1994)

Organophosphates
parathion Flavobacterium A T C C 27551 Sethunathan & Yoshida (1973)
Pseudomonas diminuta Serder et al. (1982)
diazinon Flavobacterium ATCC 27551 Sethunathan & Yoshida (1973)
fenthion Bacillus Patel & Gopinathan (1986)

Carbamates
carbofuran Achromobacter Karns et al. (1986)
Pseudomonas Chaudhry & Ali (1988)
Flavo bacterium Chaudhry & Ali (1988)
Flavobacterium Head et al. (1992)
EPTC Arthrobacter Tam et al. (1987)
Rhodococcus Behki et al. (1993)
Rhodococcus Behki & Khan (1994)

of anaerobic bacteria in wet soils (Boul et al. 1994). Bacterial transformation

of DDT has been known to occur under anaerobic conditions, and numerous
bacteria able to co-metabolically metabolize DDT by dechlorination to DDD
under anaerobic conditions have been reported (Fig. 2) (McRae 1989). Recently,
an aerobic bacterium, Alcaligenes eutrophus A5, isolated for its ability to grow
on 4-chlorobiphenyl as sole carbon source, was shown to partially metabolize
low levels (1 ppm) of both the o,pt- and p,pt-DDT isomers when incubated at
high cell-density in resting cell cultures (Fig. 2) (Nadeau et al. 1994). The
mechanism for attack on DDT by this bacterium appears to be analogous to
the 4-chlorobiphenyl degradation pathway, and probably results from the actions
of the enzymes specific for 4-chlorobiphenyl degradation. DDT appears to
be oxidized by a dioxygenase to yield a dihydroxy derivative that undergoes
meta cleavage, ultimately yielding 4-chlorobenzoic acid. The dihydroxy-DDT
intermediate and 4-chlorobenzoic acid were isolated as intermediates from resting
cell incubations. This is the first report of aerobic metabolism of DDT by a
bacterium, although the significance of resting, high cell-density incubations to
Bacterial Degradation of Pesticides

Anaerobic
/
DDD

DDA

- c o o - i c
acid
Q
COOH

Fig. 2. Proposed bacterialdegradation pathways for DDT.Key: DDT;l,l,l-trichloro-

2,2-bis(p-chloropheny1)ethane. DDD; 1,l-dichloro-2,2-bis(p-chloropheny1)ethane.
DDE; l,l-bis(p-chlorophenyl)-2-dichloroethylene. DDA; bis(p-chloropheny1)acetic
acid. DBP;p,p-dichlorobenzophenone.

environmental conditions is unclear, as is the significance, and prevalence, of

strains such as this in DDT-contaminated soils.

Atrazine is an important member of the s-triazine herbicide family which is

used in Australia and New Zealand to control some annual grasses and most
broadleaf weeds in crops such as maize, sorghum, sweetcorn and linseed. Intensive
use of atrazine and its relative persistence indicate its potential as a pollutant of
both ground waters and surface waters. This persistance could have implications
for the reuse of water, as highlighted in an Australian study where residual
atrazine in irrigation water on sensitive crops has been investigated (Bowmer
1991). Relatively few bacterial cultures able to metabolize atrazine in pure culture
have been reported. Nocardia and Pseudomonas species enriched from atrazine-
contaminated soil utilized one or more of the side chains of atrazine aerobically
 J. Aislabie and G. Lloyd-Jones

as the sole source of carbon and energy. The metabolites deethylatrazine and
deisopropylatrazine (Fig. 3) were shown to accumulate. These metabolites have
also been detected in soil. A proposed pathway for their subsequent degradation
was described by Cook (1987). Recently a Pseudomonas able to mineralize
atrazine has been enriched from soil; this organism used atrazine as the sole
source of nitrogen with sodium citrate as the carbon source (Mandelbaum et al.
1995).

/ \
Atrazine

(H3C)2HCHN H2N

Deethylatrazine Deisopropylatrazine

Fig. 3. Initial reactions in the metabolism of atrazine by bacteria.

Rhodococcus spp. that degrade the herbicide EPTC have been demonstrated
to metabolize atrazine (Behki et al. 1993; Behki and Khan 1994). One of the
strains, Rhodococcus TE1 degrades other triazine herbicides including simazine,
propazine and cyanazine. Plasmid-borne genes were implicated in the ability of
Rhodococcus TE1 to dealkylate atrazine (Behki et al. 1993).
Carbamates
Carbofuran is a carbamate pesticide used in Australia to control pests of
cotton. Microbial degradation of this compound has been studied in some
detail. The efficacy of carbofuran in the field is decreased on reapplication due
to the phenomenon of enhanced degradation. It was proposed that bacteria
present in the field are able t o utilize the pesticide as a substrate for growth
and that their numbers are enhanced on subsequent applications. A single
hydrolytic step is sufficient to inactivate carbamate insecticides (Cain and Head
1991). Bacteria, belonging to the genera Achromobacter, Pseudomonas and
Flavobacterium (Table 2), are able to utilize carbofuran as a growth substrate,
and have been isolated from soils with a previous history of treatment with
carbofuran. Many of the isolates described utilize methylamine, which is released
by the hydrolyses of the N-methylcarbamate ester linkage of carbofuran by
carbofuran hydrolase, as either sole carbon or sole nitrogen source (Fig. 4). In
some instances, the product, carbofuran phenol, was seen to accumulate (Karns
et al. 1986; Chaudhry and Ali 1988); in others, mineralization of carbofuran
Bacterial Degradation of Pesticides

\ O CH3 Carbofuran
@CH3

Fig. 4. Initial reaction in the metabolism of

carbofuran by bacteria.

\ O CH3 Carbofuran phenol

&CH3

occurs (Chaudhry and Ali 1988; Head et al. 1992). No intermediates downstream
of carbofuran phenol have been reported. Chaudhry and Ali (1988) have isolated
some strains that mineralize carbofuran but were unable to utilize carbofuran
phenol and the authors have postulated that an alternative pathway for degradation
of carbofuran is employed by these strains. Some of the isolates are also able to
metabolize other carbamate pesticides such as carbaryl and aldicarb (Chaudhry
and Ali 1988).
Genes involved in the degradation of carbamates have been isolated and cloned
from Achromobacter sp. W M l l by Tomasek and Karns (1989) and Rhodococcus
sp. strain NI86/21 by Nagy et al. (1995). The gene encoding for carbofuran
hydrolase in Achromobacter sp. W M l l was cloned and designated the mcd gene
(methyl carbamate degradation); the enzyme has a broad spectrum of activity
towards carbamates. The distribution of this gene in the environment has not
been addressed, though homologous hydrolases are probably widespread, and
result in the low persistence and efficacy of carbamate pesticides. Plasmid-borne
genes have been implicated in the complete mineralization of carbofuran by an
unidentified soil bacterium by Head et al. (1992). Similarly, plasmid involvement
has also been implicated in the degradation of the carbamate herbicide EPTC
by an Arthrobacter strain (Tam et al. 1987). Recently a Rhodococcus sp. Strain
NI86/21, which degrades EPTC, has been developed as a biosafening agent (Nagy
et al. 1995). Molecular characterization of this isolate demonstrated that both
its ability to degrade EPTC and its application as a biosafener were associated
with the presence of a gene cluster that codes for an aldehyde dehydrogenase and
cytochrome P-450 specific for thiocarbamate degradation. They are the major
thiocarbamate-induced enzymes in Rhodococcus sp. Strain NI86/21.
Phenoxyacetates
The phenoxyacetic acid herbicides 2,4,-D and 2,4,5-T have been used in New
Zealand to control nuisance vegetation such as gorse, thistles and buttercups.
Unlike the more recalcitrant 2,4,5-T, microbial metabolism of 2,4-D has been
studied for many years. A diverse group of aerobic bacteria are reported to
utilize it as the sole carbon source. These include species belonging to the
 J. Aislabie and G. Lloyd-Jones

2,4-dichloromuconic acid Fig. 5. Proposed pathway for

bacterial degradation of 2,4-D.

trans-2-chlorodiene lactone

cis-2-chlorodiene lactone

2-chloromaleylacetic acid

genera Alcalzgenes, Arthrobacter, Flavobacterium and Pseudomonas. The pathway

for bacterial catabolism of 2,4-D (Fig. 5) was first elucidated over 20 years
ago. Evans et al. (1971) demonstrated that 2,4-dichlorophenoxyacetic acid is
first converted to 2,4-dichlorophenol, then hydroxylated to 3,5-dichlorocatechol
which is subsequently metabolized by a modifiedortho-cleavage pathway to
chloromaleylacetic acid (Fig. 5). The bacterial degradation of 2,4,5-T via 2,4-D
has been detected under anaerobic conditions in methanogenic aquifer slurries
(Gibson and Sulflita 1990).
Bacterial Degradation of Pesticides

The involvement of plasmids in the degradation of 2,4-D was first demonstrated by

Pemberton and Fisher (1977). A particularly detailed understanding of the genetics
of 2,4-D degradation has resulted from the study of the 2,4-dichlorophenoxyacetic
acid (2,4-D) degrading strain Alcaligenes eutrophus JMP134(pJP4) (Don and
Pemberton 1985). Subsequent studies on the genetics of the 2,4-D degradative
pathway, encoded on the catabolic plasmid pJP4, have allowed the genetic details
to be elucidated. These studies have demonstrated that the genes involved in 2,4-D
degradation, designated tfd 2,4-dichlorophenoxyacetic acid), are arranged in three
differentially regulated operons (Fig. 5 ) : tfdA-encoding 2,4-dichlorophenoxyacetate
monooxygenase (Streber et al. 1987); tfdB-encoding 2,4-dichlorophenol hydroxylase
(Kaphammer and Olsen 1990); and tfdCDEF-encoding chlorocatechol-1,2-
dioxygenase, chloromuconate cycloisomerase, chlorodiene lactone isomerase and
chlorodienelactone hydrolase, respectively (Kaphammer et al. 1990).
The information gleaned from such detailed genetic studies of a particular
catabolic pathway has allowed the molecular ecology of the tfd genes in soils
to be studied, making use of molecular biological techniques (Neilson et al.
1992; Holben et al. 1992). More recently these approaches have been used to
reveal the diversity of these genes, their persistence, and competitiveness amongst
2,4-D-degrading bacteria in 2,4-D-treated soils (Ka et al. 1994a-c).

0CH2CH3
Diazinon DETP

Parathion 4-Nitrophenol DETP

H
H3CH2C0CP
/ II 0

' C I
0 -'
How
' Cl
+
PCHpCH3
HO-P=S
\0CH2CH3
CH3 CH3
Coumaphos Chlorferon DETP

Fig. 6. Initial reaction in the metabolism of the organophosphate pesticides ( a )

diazinon (b) parathion and (c) coumaphos. Key: IMPH; 2-isopropyl-4-methyl-6-
hydroxypyrimidine. DETP; diethyl thiophosphoric acid.
 .I. Aislabie and G. Lloyd-Jones

Organophosphates
Organophosphates such as diazinon are used in Australia and New Zealand
for the control of flystrike (ovine cutaneous myiasis), an invasion of the skin
of sheep by blowfly larvae (Rammell and Bentley 1988). They are also used
in cotton to control pests.Flavobacterium sp. (ATCC 27551) able to utilize the
organophosphate compounds parathion and diazinon as the sole carbon source
was isolated from paddy water (Sethunathan and Yoshida 1973). Parathion
is inactivated by a single enzymic reaction catalysed by parathion hydrolase
(also called phosphotriesterase); this enzyme has a broad substrate range, being
able to hydrolyse a number of related compounds including diazinon (Brown
1980). Parathion hydrolase cleaves the phosphodiester linkage of parathion to
form DETP (0,O-diethylthiophosphoric acid) and p-nitrophenol (Fig. 6). The
gene encoding for this enzyme, designated opd (organophosphate degradation),
has been isolated from strains derived from diverse geographic areas (Mulbry
et al. 1986; Chaudhry et al. 1988). The opd gene was first isolated from a
parathion-degrading strain Pseudomonas diminuta MG by Serdar and Gibson
(1985); the gene was homologous to that found in a Flavobacterium sp. (ATCC
27551) (Mulbry e t al. 1986). Homology toopd genes was also detected by Chaudhry
et al. (1988) in a methyl parathion-degrading Pseudomonas sp. isolated in the
U.S.A. Subsequent sequencing of the opd genes has confirmed the high degree
of homology between the opd genes derived from Pseudomonas diminuta MG
(McDaniel et al. 1988; Serdar et al. 1989) and Flavobacterium sp. (ATCC 27551)
(Harper et al. 1988; Mulbry and Karns 1989), as originally inferred by DNA
hybridization.

Concluding Statements
Problems are encountered with the use of pesticides, with the pendulum
swinging from persistence of organochlorines to the low efficacy and rapid
biodegradation of the non-chlorinated replacements. This raises two different
problems which need to be addressed: removal of persistent contaminants on
the one hand, whilst predicting and controlling enhanced biodegradation on the
other.
Firstly, how can we decontaminate soils containing high levels of persistent
pesticides? The slow rates of degradation of the organochlorine pesticide DDT,
and its residue DDE, resulted in their accumulation into food chains. This
is due to the lack of degradation or alternatively the extremely slow rate
at which this compound is metabolized. To overcome this persistence, novel
solutions have to be sought. As DDT is insoluble in water and bound to
organic matter, pretreatment with a surfactant may enhance degradation rates.
Pesticide leaching into ground water also requires addressing. Atrazine and its
metabolites can leach into ground water; it is therefore important to determine
where degradation occurs i n situ. As results to date indicate that biodegradation
rates for atrazine are minimal in subsurface environments (McMahon et al.
1992), it is essential that the pesticide remains in the topsoil where there
is a considerably larger microbial population and the potential for pesticide
degradation is enhanced.
Bacterial Degradation of Pesticides

Farm management practices also affect the rate at which pesticides are degraded.
Irrigation of soiIs has been shown to enhance degradation of DDT to DDD,
thought to be due to the creation of anaerobic microsites, though in the case of
atrazine this may also facilitate the transport of the pesticide and its metabolites
to ground water.
The problem of low efficacy of pesticides can in reality be controlled by
good management practices, although for some problem soils a microbiological
evaluation of the capabilities of the indigenous microorganisms may be required.
Classical microbiological methodologies, or even DNA probing studies directed at
the genes for the key enzymatic steps in the inactivation of the pesticide, may
give a better understanding of when poor efficacy may be problematical, and
therefore allow for a more informed management of pesticide application leading
to decreased usage.

Acknowledgments
The authors wish to acknowledge the assistance of David Hunter who prepared
some of the figures for this article. This work was supported by funding from
the Foundation for Research Science and Technology, New Zealand.

References
Aberdeen, I. (1993). Litigation over dieldrin in Victoria. Agricultural Science May, 42-5.
Alexander, M. (1977). 'Introduction to Soil Microbiology.' 2nd Edn. (John Wiley: New York.)
Aronstein, B. N., Calvillo, Y. M., and Alexander, M. (1991). Effect of surfactants at low
concentrations on the desorption and biodegradation of sorbed aromatic compounds in
soil. Environmental Science and Technology 25, 1728-31.
Ascon-Cabrera, M., and Lebeault, J-M. (1993). Selection of xenobiotic-degrading microor-
ganisms in a biphasic aqueous-organic system. Applied and Enviromental Microbiology 59,
1717-24.
Atlas, R.M. (1988). 'Microbiology. Fundamentals and Applications.' 2nd Edn. (MacMillan:
New York.)
Bartha, R. (1990). Isolation of microbes that metabolize xenobiotic compounds. In 'Isolation
of Biotechnological Organisms from Nature'. ( Ed. D. P. Labeda.) pp. 283-305. (McGraw
Hill: New York.)
Behki, R. M., and Khan, S. U. (1991). Inhibitory effect of parathion on the bacterial degradation
of EPTC. Journal of Agriculture and Food Chemistry 39, 805-8.
Behki, R. M., and Khan, S. U. (1994). Degradation of atrazine, propazine, and simazine by
Rhodococcus strain B-30. Journal of Agn'culture and Food Chemistry 42, 1237-41.
Behki, R. M., Topp, E., Dick, W., and Germon, P. (1993). Metabolism of the herbicide
atrazine by Rhodococcus strains. Applied and Environmental Microbiology 59, 1955-59.
Bhat, M. A., Tsuda, M., Horiike, K., Nozaki, M., Vaidyanathan, C. S., and Nakazawa, T.
(1994). Identification and characterisation of a new plasmid carrying genes for degradation of
2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV9O. Applied and Environmental
Microbiology 60, 307-12.
Boul, H. L., Garnham, M. L., Hucker, D., Baird, D., and Aislabie, J. (1994). The influences of
agricultural practices on the levels of DDT and its residues in soil. Environmental Science
and Technology 28, 1397-1402.
Bowmer, K. H. (1991). Atrazine persistence and toxicity in two irrigated soils in Australia.
Australian Journal of Soil Research 29, 339-50.
Brown, K. A. (1980). Phosphotriesterases of Flavobacterium sp. Soil Biology and Biochemistry
12, 105-12.
Cain, R. B., and Head, I. M. (1991). Enhanced degradation of pesticides: its biochemical
and molecular biological basis. In 'Pesticides in Soils and Water: Current Perspectives'. A
BCPC Vol. 47. (Ed. A.Walker.) pp. 23-40.
 J . Aislabie and G. Lloyd-Jones

Chaudhry, G. R., and Ali A. N. (1988). Bacterial metabolism of carbofuran. Applied and
Environmental Microbiology 54, 1414-19.
Chaudhry, G. R., and Huang G. H. (1988). Isolation and characterization of a new plasmid from
a Flavobacterium sp. which carries the genes for degradation of 2,4-dichlorophenoxyacetate.
Journal of Bacteriology 170, 3897-902.
Chaudhry, G. R., Ali, A. N., and Wheeler, W. B. (1988). Isolation of a methyl parathion-
degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a
Flavobacterium sp. Applied and Environmental Microbiology 54, 288-93.
Chen, S., and Alexander, M. (1989). Reasons for the acclimation of 2,4-D biodegradation in
lake water. Journal of Environmental Quality 18, 153-6.
Cook, A. M. (1987). Biodegradation of s-triazine xenobiotics. FEMS Microbiology Reviews 46,
93-116.
Cork, D. J., and Krueger, J. P. (1991). Microbial transformations of herbicides and pesticides.
Advances i n Applied Microbiology 36, 1-66.
Dobbins, D. C., Aelion, C. M., and Pfaender, F. (1992). Subsurface, terrestrial microbial
ecology and biodegradation of organic chemicals: a review. Critical Reviews i n Environmental
Control 22, 67-136.
Don, R. H., and Pemberton, J. M. (1985). Genetical and physical map of the 2,4-
dichlorophenoxyacetic acid degradative plasmid PJP4. Journal of Bacteriology 161, 466-8.
Evans, W. C., Smith, B. S. W., Fernley, H. N., and Davies, J. I. (1971). Bacterial metabolism
of 2,4-dichlorophenoxyacetic acid. Biochemistry Journal 122, 543-51.
Felsot, A. S. (1989). Enhanced biodegradation of insecticides in soil: implications for
agroecosystems. Annual Review of Entomology 34, 453-76.
Fewson, C. A. (1988). Biodegradation of xenobiotic and other persistent compounds: the
causes of recalcitrance. Trends i n Biotechnology 6, 148-53.
Gibson, S. A., and Sulflita J . M. (1990). Anaerobic biodegradation of 2,4,5-trichlorophenoxy-
oacetic acid in samples from a methanogenic aquifer: stimulation by short-chain organic
acids and alcohols. Applied and Environmental Microbiology 56, 1825-32.
Hallberg, K. B., Kelly, M. P., and Tuovinen, 0 . H. (1991). Simultaneous degradation of the
hebicides 2,4-dichlorophenoxyacetic acid and 2-(2-methyl-4-ch1orophenoxy)propionic acid
by mixed bacterial cultures. Current Microbiology 23, 65-9.
Harper, L. L., McDaniel, C. S., Miller, C. E., and Wild J . R. (1988). Dissimilar plasmids
isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain
identical opd genes. Applied and Environmental Microbiology 54, 2586-9.
Head, I. M., Cain, R. B., and Suett, D. L. (1992). Characterization of a carbofuran-degrading
bacterium and investigation of the role of plasmids in catabolism of the insecticide
carbofuran. Archives of Microbiology 158, 302-8.
Hill, I. R., and Wright, S. J. L. (1978). 'Pesticide Microbiology.' (Academic Press: London.)
Holben, W. E., Schroeter, B. M., Calabrece, V. G. M., Olsen, R. H., Kukor, J. J., Biederbeck, V.
O., Smith, A. E., and Teidje, J. M. (1992). Gene probe analysis of soil microbial populations
selected by amendment with 2,4-dichlorophenoxyacetic acid. Applied and Environmental
Microbiology 58, 3941-8.
Ka, J. O., Holben, W. E., and Teidje, J . M. (1994a). Genetic and phenotypic diversity of
2,4-dichlorophenoxyaceticacid (2,4-D)-degrading bacteria isolated from 2,4-D-treated field
soils. Applied and Environmental Microbiology 60, 1106-15.
Ka, J. O., Holben, W. E., and Teidje, J. M. (1994b). Use of gene probes to aid in recovery and
identification of functionally dominant 2,4-dichlorophenoxyaceticacid-degrading populations
in soil. Applied and Environmental Microbiology 60, 1116-20.
Ka, J . O., Holben, W. E., and Teidje, J. M. (1994~).Analysis of competition in soil among
2,4-dichlorophenoxyaceticacid-degrading bacteria. Applied and Environmental Microbiology
60, 1121-8.
Kannan, K., Tanabe, S., Williams, R. J., and Tatsukawa, R. (1994). Persistant organochlorine
residues in foodstuffs from Australia, Papua New Guinea and the Solomon Islands:
contamination levels and dietary exposure. The Science of the Total Environment 153,
29-49.
Kaphammer, B., Kukor, J. J., and Olsen, R. H. (1990). Regulation of tfdCDEF by tfdR of
the 2,4-dichlorophenoxyaceticacid degradative plasmid PJP4. Journal of Bacteriology 172,
2280-6.
Bacterial Degradation of Pesticides

Kaphammer, B., and Olsen, R. H. (1990). Cloning and characterisation of tfdS, the repressor-
activator gene of tfdB, from the 2,4-dichlorophenoxyacetic acid catabolic plasmid PJP4.
Journal of Bacteriology 172, 5856-62.
Karns, J. S., Mulbry, W. W., Nelson, J. O., and Kearney P. C. (1986). Metabolism of
carbofuran by a pure bacterial culture. Pesticide Biochemistry and Physiology 25, 211-17.
Kellogg, S. T., Chatterjee, D. K., and Chakrabarty, A. M. (1981) Plasmid-assisted molecular
breeding: New technique for enhanced biodegradation of persistent toxic chemicals. Science
214, 1133-5.
Kilbane, J. J., Charterjee, D. K., Karns, J. S., Kellogg, S. T., and Chakrabarty, A. M. (1982).
Biodegradation of 2,4,5-trichlorophenoxyaceticacid by a pure culture of Pseudomonas
cepacia. Applied and Environmental Microbiology 44, 72-8.
Kookana, R. S., and Aylmore, L. A. G. (1994). Estimating the pollution potential of pesticides
to ground water. Australian Journal of Soil Research 32, 1141-55.
Linn, D. M., Carski, T . H., Brusseau, M. L., and Chang, F-H. (1993). Sorption and degradation
of pesticides and organic chemicals in soils. SSSA Special Publication No. 32. Soil Science
Society of America.
McDaniel, C. S., Harper, L. L., and Wild, J. R. (1988). Cloning and sequencing of a
plasmid-bourne gene (opd ) encoding a phosphotriesterase. Journal of Bacteriology 170,
2306-1 1.
McKenzie-Smith, F., Tiller, D., and Allen, D. (1994). Organochlorine pesticide residues in
water and sediments from the Ovens and King Rivers, north-east Victoria, Australia.
Archives of Environmental Contamination and Toxicology 26, 483-90.
McMahon, P. B., Chapelle, F. H., and Jagucki, M. L. (1992). Atrazine mineralization potential of
alluvial-aquifer sediments under aerobic conditions. Environmental Science and Technology
26, 1556-9.
McRae, I. C. (1989). Microbial metabolism of pesticides and structurally related compounds.
Reviews of Environmental Contamination and Toxicology 109, 1-87.
Mandelbaum, R. T., Allan, D. L., and Wackett, L. P. (1995). Isolation and characterisation
of a Pseudomonas sp. that mineralizes the s-triazine herbicide atrazine. Applied and
Environmental Microbiology 61, 1451-7.
van der Meer, J. R. (1994). Genetic adaptation of bacteria to chlorinated aromatic compounds.
FEMS Microbiology Reviews 15, 239-49.
Merritt, G. C., Watts J . E., and McDougall, K. (1981). In vitro degradation of organophosphorus
insecticides byPseudomonas aerzlginosa isolated from fleece-rot lesions of sheep. Australian
Veterinary Journal 57, 531.
Mohn, W. W., and Tiedje, J. M. (1991). Evidence for chemiosmotic coupling of reductive
dechlorination and ATP synthesis in Desulphomonile tiedjei. Archives of Microbiology 157,
1-6.
Mulbry, W. W., and Karns, J . S. (1989). Parathion hydrolase specified by the Flavobacterium
opd gene: relationship between gene and protein. Journal of Bacteriology 171, 6740-6.
Mulbry, W. W., Karns, J. S., Kearney, P. C., Nelson, J. O., McDaniel, C. S., and Wild, J. R.
(1986). Identification of a plasmid-bourne parathion hydrolase gene from Flavobacterium sp.
by southern hybridization with opd from Pseudomonas diminuta. Applied and Environmental
Microbiology 51, 926-30.
Nadeau, L. J., Menn, F-M., Breen, A., and Sayler, G. S. (1994). Aerobic degradation of
l,l,l-trichloro-2,2-bis(4-chloropheny1)ethane (DDT) by Alcaligenes eutrophus A5. Applied
and Environmental Microbiology 60, 51-5.
Nagy, I., Schoofs, G., Compernolle, F., Proost, P., Vanderleyden, J., and de Mot, R. (1995).
Degradation of the thiocarbamate herbicide EPTC (8-ethyl dipropylcarbamothioate) and
biosafening by Rhodococcus sp. Strain NI86/21 involve an inducible cytochrome P-450
system and aldehyde dehydrogenase. Journal of Bacteriology 177, 676-87.
Neilson, J. W., Josephson, K. L., Pillai, S. D., and Pepper, I. L. (1992). Polymerase chain
reaction and gene probe detection of the 2,4-dichlorophenoxyaceticacid degradation plasmid
PJP4. Applied and Environmental Microbiology 58, 1271-5.
OIConner, B. (1994). 'Novachem Manual: A Guide to Plant Protection in New Zealand.'
(Swiftprint: Palmerston North.)
Pahm, M. A., and Alexander, M. (1993). Selecting inocula for the biodegradation of organic
compounds at low concentrations. Microbial Ecology 25, 275-86.
 J. Aislabie and G. Lloyd-Jones

Parkin, T . B., and Shelton, D. R. (1992). Spatial and temporal variability of carbofuran
degradation in soil. Journal of Environmental Quality 21, 672-8.
Patel, M. N., and Gopinathan, K. P. (1986). Lysozymesensitive bioemulsifier for immiscible
organophosphorus pesticides. Applied and Environmental Microbiology 52, 1224-6.
Pemberton, J. M., and Fisher, P. R. (1977). 2,4-D plasmids and persistence. Nature 268,
732-3.
Pickup, R. W. (1991). Development of molecular methods for the detection of specific bacteria
in the environment. Journal of General Microbiology 137, 1009-19.
Pratchett, D. (1990). DDT concentrations in cattle grazing irrigated pastures in the Ord River
irrigation area in Western Australia. Australian Veterinary Journal 67, 423.
Rammell, C. R., and Bentley, G. R. (1988). Organophosphate residues in the wool of sheep
dipped for flystrike control. New Zealand Journal of Agriculture 31, 151-4.
Reddy, R., and Sethunathan, N. (1994). Mineralization of p-nitrophenol in the rhizosphere of
rice. Agriculture, Ecosystems and Environment 47, 313-17.
Robertson, B. K., and Alexander, M. (1994). Growth-linked and cometabolic biodegradation:
possible reason for occurrence or absence of accelerated pesticide biodegradation. Pesticide
Science 41, 311-18.
Sandmann, E. R. I. C., and Loos, M. A. (1988). Aromatic metabolism by a 2,4-D degrading
Arthrobacter sp. Canadian Journal of Microbiology 34, 125-30.
Sayler, G. S., Hooper, S. W., Layton, A. C., and King, J. M. H. (1990). Catabolic plasmids
of environmental and ecological significance. Microbial Ecology 19, 1-20.
Serdar, C. M., and Gibson, D. T . (1985). Enzymic hydrolysis of organophosphates: cloning
and expression of a parathion hydrolase gene from Pseudomonas diminuta. Bio/Technology
3, 567-71.
Serdar, C. M., Gibson, D. T., Munnecke, and D. M., Lancaster, J. H. (1982). Plasmid
involvement in parathion hydrolysis by Pseudomonas diminuta. Applied and Environmental
Microbiology 44, 246-9.
Serdar, C. M., Murdock, D. C., and Rohde, M. F. (1989). Parathion hydrolase gene from
Pseudomonas diminuta MG: subcloning, complete nucleotide sequence and expression of
the mature portion of the enzyme in Escherichia coli. Bio/Technology 7, 1151-5.
Sethunathan, N., and Yoshida, Y. (1973). A Flavobacterium that degrades diazinon and
parathion. Canadian Journal of Microbiology 19, 873-5.
Shelton, D. R., and Somich, C. J. (1988). Isolation and characterisation of coumaphos-
metabolizing bacteria from cattle dip. Applied and Environmental Microbiology 54, 2566-71.
Singh, N., and Sethunathan, N. (1992). Degradation of soil-sorbed carbofuran by an enrichment
culture from carbofuran-retreated Azolla plot. Journal of Agriculture and Food Chemistry
40, 1062-5.
Somasundaram, L., and Coats, J. R. (1990). Influence of pesticide metabolites on the
development of enhanced biodegradation. In 'Enhanced Biodegradation of Pesticides in
the Environment'. (Eds. K. D. Racke and J. R. Coats.) (American Chemical Society,
Washington, D.C.)
Streber, W. R., Timmis, K. N., and Zonk, M. H. (1987). Analysis cloning and high level
expression of 2,4-dichlorophenoxyacetic acid monooxygenase gene t f d A of Alcaligenes
eutrophus JMP134. Journal of Bacteriology 169, 2950-5
Tam, A. C., Behki, R. M., and Khan, S. U. (1987). Isolation and characterisation of an S-
ethyl-N,N-dipropylthiocarbamatedegradingArthrobacter strain and evidence for plasmid-
associated S-ethyl-N,N-dipropylthiocarbamate degradation. Applied and Environmental
Microbiology 53, 1088-93.
Tomasek, P. H., and Karns, J. S. (1989). Cloning of a carbofuran hydrolase gene from
Achromobacter sp. W M l l l and its expression in Gram-negative bacteria. Jowrnal of
Bacteriology 171, 4038-44.

Manuscript received 1 February 1995, accepted 6 June 1995

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