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Previous Article | Next Article 
Applied and Environmental Microbiology, July 2000, p. 2822-2828, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Enrichment of an Endosulfan-Degrading Mixed Bacterial
Culture
Tara D.
Sutherland,*
Irene
Horne,
Michael J.
Lacey,
Rebecca L.
Harcourt,
Robyn J.
Russell, and
John G.
Oakeshott
CSIRO Entomology, Canberra ACT 2601, Australia
Received 3 February 2000/Accepted 2 May 2000
 |
ABSTRACT |
An endosulfan-degrading mixed bacterial culture was enriched from
soil with a history of endosulfan exposure. Enrichment was obtained by
using the insecticide as the sole source of sulfur. Chemical hydrolysis
was minimized by using strongly buffered culture medium (pH 6.6), and
the detergent Tween 80 was included to emulsify the insecticide,
thereby increasing the amount of endosulfan in contact with the
bacteria. No growth occurred in control cultures in the absence of
endosulfan. Degradation of the insecticide occurred concomitant with
bacterial growth. The compound was both oxidized and hydrolyzed. The
oxidation reaction favored the alpha isomer and produced
endosulfate, a terminal pathway product. Hydrolysis involved a novel
intermediate, tentatively identified as endosulfan monoaldehyde on the
basis of gas chromatography-mass spectrometry and chemical
derivatization results. The accumulation and decline of metabolites
suggest that the parent compound was hydrolyzed to the putative
monoaldehyde, thereby releasing the sulfite moiety required for growth.
The monoaldehyde was then oxidized to endosulfan hydroxyether and
further metabolized to (a) polar product(s). The cytochrome P450
inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or
the formation of other metabolites. These results suggest that this
mixed culture is worth investigating as a source of
endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of
endosulfan residues.
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INTRODUCTION |
The chlorinated cyclic sulfite
diester endosulfan
(Thiodan, bicyclo-[2.2.1]-2-heptene-5,6-bisoxymethylene sulfite) is a broad-spectrum insecticide that has been used extensively for over 30 years on a
variety of crops. Endosulfan is often classified as a cyclodiene and
has the same primary action and target site as other cyclodienes (3). However, it has chemical and physical properties
significantly different from other cyclodiene insecticides that affect
both its environmental and biological fates. In particular, endosulfan has a relatively reactive cyclic sulfite diester group (32) and, as a consequence, its environmental persistence is lower than that of other cyclodienes, albeit still higher than that of
many other insecticides. Since the deregistration in many countries of
most cyclodiene insecticides, the ongoing availability of endosulfan has become important as an alternative option in resistance
management strategies of pest species. Additionally, compared to
many other available insecticides, it has low toxicity to many
species of beneficial insects, mites, and spiders (10).
However, endosulfan is extremely toxic to fish and aquatic
invertebrates and it has been implicated increasingly in mammalian
gonadal toxicity (28-31), genotoxicity (4), and
neurotoxicity (24). These environmental and health concerns
have led to an interest in postapplication detoxification of the insecticide.
The aim of this research is the investigation of an enzymatic method
for endosulfan detoxification. Enzymatic detoxification of pesticides
is receiving serious attention as an alternative to existing methods,
such as incineration and landfill. In particular, enzymatic
insecticide bioremediation is the focus of extensive study after
the isolation of a phosphotriesterase capable of detoxifying a range of
organophosphate compounds from several bacterial species (for review,
see reference 7 and references within). An essential initial step in the investigation of an enzymatic method for endosulfan detoxification is the definitive identification of a biological source
of endosulfan-degrading activity. Numerous studies have described the
degradation of endosulfan in soils (14, 26), soil inocula
(11, 21, 27), mixed microbial cultures (1), and isolated microorganisms (9, 13, 17, 20, 22). The compound is degraded by attack at the sulfite group via both
oxidation and hydrolysis to form the toxic endosulfate (endosulfan
sulfate) and the nontoxic endodiol (endosulfan diol),
respectively. The formation of endosulfate is thought to occur
only through biological transformation, whereas hydrolysis to the diol
occurs readily at alkaline pH (20). Many studies describing
degradation of endosulfan in microbial cultures do not
differentiate between chemical and biological hydrolysis, as culturing
often leads to an increase in pH of the growth medium (20,
21). In addition to stringent pH controls, the detection of
metabolites is important for the confirmation of degradation, as losses
of endosulfan from culture media or soils occur readily by
volatilization and adsorption to surfaces (12).
Microorganisms have increasingly been investigated as a source of
xenobiotic-degrading enzymes (5). We are interested in the
isolation of an endosulfan-degrading bacterium for further investigation into enzymatic endosulfan bioremediation. Using endosulfan as the only available sulfur source, we enriched soil inocula for microorganisms capable of releasing the sulfur from endosulfan, thereby providing a source of sulfur for growth. Since removal of the sulfur moiety dramatically decreases vertebrate toxicity
of endosulfan (8, 10), this results in concurrent detoxification of the insecticide. We report here on the resultant mixed bacterial culture that, to our knowledge, is the first reported enrichment of an endosulfan-degrading microbial culture. The culture degrades endosulfan to produce a novel metabolite not reported to occur
as a result of chemical hydrolysis. These results suggest this mixed
culture is a potential source of an enzymatic bioremediating agent.
| |
MATERIALS AND METHODS |
Soil.
The soil used in this study was collected from a
cotton field near Narrabri, New South Wales, Australia, at the end of
the growing season. The field had generally received several
applications of endosulfan in the summer months for at least the
previous 5 years. The soil was fertile grey clay at pH 7.5. Topsoil was
collected from the first 15 cm, air dried, and stored at 4°C for up
to 1 month prior to enrichment.
Isolation of soil bacteria.
Soil (approximately 15 g)
was first enriched for endosulfan-degrading organisms by the addition
of 2 mg of technical-grade endosulfan in 100 µl of acetone to
remoistened soil, followed by incubation in the dark at room
temperature for 1 month. Further enrichment was then achieved by
initiating shake flask enrichment cultures from these samples by using
endosulfan as the only added source of sulfur. The enrichment medium
(pH 6.6 to 6.8) consisted of 20 mg of technical-grade endosulfan (99%
pure), 0.05% Tween 80, 2.0 g of KH2PO4,
7.5 g of K2HPO4, 1.0 g of
NH4Cl, 0.5 g of NaCl, 1.0 g of glucose, 0.1 g of MgCl2, 0.86 mg of p-amino benzoic acid,
0.86 mg of nicotinic acid, and 10 ml of a trace element solution per
liter. The stock trace element solution contained 20 mg of
(NH4)6Mo7O24 · 4H2O, 50 mg of H3BO3, 30 mg of
ZnCl2, 3 mg of CoCl2 · 6H2O,
10 mg of (CH3COO)2Cu · H2O,
and 20 mg of FeCl2 · 6H2O per liter.
According to impurity data provided by Sigma Chemical Co. Castle Hill,
New South Wales, Australia) the maximum limit of sulfite/sulfate
contamination in the enrichment medium was less than 0.4 × 10
3 g liter1. Approximately 1 g of
endosulfan-enriched soil was inoculated into 50 ml of enrichment media
and cultured in a 400-ml Erlenmeyer flask on a rotary shaker (200 rpm)
at 28°C for up to 14 days. Substrate levels were measured using
thin-layer chromatography (TLC), and when approximately 50% of the
endosulfan had degraded relative to sterile controls, 5 ml of the
culture was transferred into 50 ml of fresh enrichment medium. Ten
different soil samples were enriched for endosulfan-degrading activity.
An endosulfan-degrading culture was obtained from only one of these
samples. After approximately six transfers into enrichment media,
cultures were transferred into sulfur-free media (see below) for
further enrichment.
A sulfur-free medium was also designed because contaminating sulfur in
the enrichment medium could promote culture growth, resulting in
increases in optical density at 595 nm of the culture from 0.05 to 0.3. A second soil culture was initiated for the sole purpose of preparing
medium free of contaminating sulfur. Sulfur-free medium was prepared by
growing the second soil culture overnight in enrichment medium without
endosulfan and then removing cells by centrifugation and filtering the
supernatant through a 0.22-µm-pore-size filter. After inoculation of
this medium with either the endosulfan-degrading culture or
Escherichia coli TG1, no growth was observed until the
addition of a source of sulfur. After the addition of either 50 µM
sodium sulfite or magnesium sulfate, both the endosulfan-degrading
culture and the E. coli TG1 culture were able to grow to an
optical density at 595 nm of at least 0.8. The sterility of the
sulfur-free medium was confirmed by the absence of growth when aliquots
were incubated on rich medium agar plates. After initial enrichment,
the endosulfan-degrading culture was maintained in sulfur-free medium
with endosulfan as the only sulfur source. Rich medium agar used in
this study included low-salt Luria broth (LB; 10 g of tryptone
liter1, 5 g of yeast extract liter1,
0.5 g of NaCl liter1, 15 g of Noble agar
liter1) and BUGM (Oxoid, Melbourne, Victoria, Australia).
Chemicals.
Technical-grade endosulfan (99% pure) for
bacterial growth was a gift from Hoechst Schering AgrEvo Pty Ltd.
Technical-grade endosulfan (used commercially) is a mixture of two
diastereoisomers: alpha-endosulfan and
beta-endosulfan in a ratio of 7:3. With the exception of
endosulfan diacetate, insecticide and metabolite standards (at least
99% pure) were purchased from Chem. Services Inc. (West Chester, Pa.).
Endosulfan diacetate was synthesized by peracetylation of endosulfan
diol with acetic anhydride in dry pyridine at 80°C for 1 h and
purified by silica chromatography. The O-benzyl oxime of
endosulfan monoaldehyde was prepared by the reaction of the putative
aldehyde (recovered by thin-layer chromatography [TLC] on alumina)
with a fivefold excess of benzylhydroxylamine hydrochloride (Alltech,
Baulkham Hills, New South Wales, Australia) in dry pyridine at room
temperature for 8 h. All other chemicals used were of at least
reagent grade.
TLC.
Cultures were extracted with equal volumes of ethyl
acetate. The organic phase was passed through a 6-cm MgSO4
column in a Pasteur pipette stoppered with glass wool to remove any
residual water, gently evaporated under a dry nitrogen stream,
dissolved in acetone, and then applied to neutral aluminium oxide
F254 TLC plates (Alltech). The plates were developed in
either petroleum ether-acetone (4:1) or chloroform-ethyl acetate (3:1).
Rf values for endosulfan and its metabolites in
these solvent systems are given in Table
1. The aqueous phase was reduced to
dryness by rotary evaporation, and the resultant residue was extracted
with dichloromethane (DCM) to recover any hydrophilic metabolites. The
DCM-soluble products were spotted onto TLC plates as described above
and developed in methanol. Chlorine-containing constituents were
visualized by spraying plates with silver nitrate-saturated methanol
and then exposing them to UV light. The lower limit of detection of
this method for endosulfan and metabolites containing the
hexachlorinated ring structure was 0.1 µg (data not shown). As
detection is based on formation of silver chloride, dechlorinated metabolites will have a detection limit relative to the level of
dechlorination.
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TABLE 1.
Rf values for TLC with different
solvent systems and retention times of FID GC for endosulfan isomers
and metabolites
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GC and GC-mass spectrometry (GC-MS) analysis.
As endosulfan
and its chlorine-containing metabolites are strongly electronegative,
previous studies have employed electron-capture GC for detection of
these compounds. As we demonstrate in the present study, flame
ionization detection (FID) can replace this if preliminary steps are
used to recover the metabolites selectively. In addition, FID enables
the use of DCM for clean and efficient solvent extraction.
Cultures (15 ml) were extracted with DCM (10 ml), and the organic phase
was dried with MgSO4, as described above. The solution of
endosulfan and its lipophilic metabolites was diluted with hexane to
yield a 20% hexane-DCM solution, which was applied to a 5-cm silica
column (DCC silica gel, 63-200; Aldrich) within a Pasteur pipette. The
column was flushed with a further 3 ml of 20% hexane-DCM. Control
experiments demonstrated that endosulfan hydroxyether and endodiol were
the only metabolites retained by the silica under these conditions.
Endosulfan diacetate (50 µg) was added as an internal standard to the
combined eluate and washings, which were then concentrated to 25 µl
under a gentle stream of nitrogen before storage at 
20°C and
subsequent GC analysis using FID.
The more polar metabolites, endosulfan hydroxyether and endodiol, were
subsequently eluted from the silica column with 10% methanol-DCM (8 ml). Endosulfate (40 µg) was added as the internal standard, and the
recovered solution was evaporated to near dryness under a gentle stream
of nitrogen. The residue was taken up in DCM (10 µl), and
bis(trimethylsilyl)trifluoroacetamide (BSTFA; 25 µl) was added with
initial vortex mixing to silylate the metabolites (6 h, room
temperature) before storage at 20°C and GC analysis.
The addition of the internal standards to the fractions enabled both
qualitative assessment of the metabolites from their relative retention
times by GC and quantitative evaluation of the metabolic pathways.
Losses from volatilization and extraction efficiencies ranged from 15 to 40% (depending on length of time incubated, media composition, and
compound) and were calculated by comparison with stocks of known
concentration. GC was performed using a Varian model 3300 with a cool
on-column injector, an FID, and a computer with data acquisition and
processing software. The capillary column was 5% phenyl methylsilicone
(SE54, Alltech Econocap, 30 m by 0.32 mm [inside diameter],
0.25-µm film thickness) with a helium flow rate of 2 ml
min1. The column was preceded by a retention gap of
deactivated silica (2 m) to preserve the integrity of the column. A
typical temperature program for analysis of the endosulfan metabolites
comprised an initial period after injection of 2 min at 40°C and a
temperature gradient of 20°C min1 to 200°C for 10 min, followed by a temperature gradient of 10°C min1 to
300°C.
The identities of the known metabolites in the fractions were confirmed
by GC-MS using a VG Trio 2000 mass spectrometer interfaced to a
Hewlett-Packard 5890 gas chromatograph (cool on-column injector), with
VG MassLynx software for control and data acquisition. The GC column
was 5% phenyl methylsilicone (SE54, Alltech Econocap, 30 m by
0.32 mm [inside diameter], 0.5-µm film thickness), with a helium
flow rate of 1 ml min1. Ionization modes used for MS of
the metabolites were either electron ionization (EI; 70 eV) or
positive-ion chemical ionization (PCI; ammonia reagent gas; source
pressure, 60 Pa). The molecular and fragment ions were generally
represented by peak distributions over several masses because their
respective chlorine compositions included the additional natural
isotope 37Cl.
| |
RESULTS |
Enrichment of microorganisms.
After approximately six rounds
of successive subculturing in enrichment media followed by four rounds
in sulfur-free media, TLC analysis and optical density measurements of
the enrichment culture confirmed substantial disappearance of
endosulfan with a simultaneous increase in bacterial mass. The culture
was incapable of growth in sulfur-free medium without the addition of a
sulfur source (Fig. 1). Addition of
either alpha-, beta-, or technical-grade endosulfan promoted growth to various degrees. No significant growth
was seen in the absence of endosulfan, and endosulfate could not
substitute for endosulfan as a utilizable source of sulfur (Fig. 1).
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FIG. 1.
Growth of a bacterial culture after enrichment for
endosulfan degradative ability in sulfur-free medium containing
endosulfan isomers (50 µM). OD595, optical density at 595 nm.
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When dilutions of the enrichment culture were incubated on agar plates
for 48 h, two distinct colony types became apparent on enrichment
medium agar and five were revealed on rich medium agar (LB and BUGM).
Inoculation of sulfur-free medium broth containing endosulfan with
isolates or combinations of isolates, after purification on agar
plates, failed to produce a culture capable of growth. However, growth
was observed when "scrapes" from the initial dilution plating on
either medium type were used to inoculate sulfur-free medium broth
containing endosulfan. Many separate components of the culture may be
required for metabolism of endosulfan, including enzymes involved in
hydrolysis, sulfite oxidation, and the provision of nutrients not
supplied in the minimal medium. The inability of combinations of
isolates to utilize endosulfan as a sulfur source suggested that a key
species in the degradative process either did not form obvious colonies
on agar plates or lost its ability to contribute to endosulfan
metabolism after passage on rich media.
Continuous subculturing in sulfur-free medium led to an increase in
rates of endosulfan disappearance, with no detectable levels of
endosulfan remaining after 4 days by the twentieth subculture as
compared to 8 days after the tenth subculturing. Degradative ability
was retained in cultures initiated from frozen 20% glycerol stocks after several months at 80°C.
Characterization of endosulfan metabolites.
TLC and GC
analysis indicated the disappearance of both diastereomers of
endosulfan and the concomitant formation of endosulfan metabolites. The
known metabolites of endosulfan are not diastereomeric. Three
metabolites were identified as endosulfan hydroxyether, endosulfate,
and endodiol on the basis of comigration with authentic standards on
TLC plates developed in different solvent systems, coincident retention
times on GC (Table 1), and structural confirmation by GC-MS (data not
shown). A single additional metabolite, with mobility on TLC plates
similar to that of endosulfate, was also detected. MS analysis (70-eV
EI) of the compound after purification by TLC indicated a molecular ion
with an m/z of 342 (35C6), isomeric
with that of endosulfan ether (Fig. 2).
The fragmentation pattern was also similar to that obtained with
endosulfan ether, except for the absence of a prominent fragment ion
with an m/z of 69 derived from the pentacyclic ether moiety.
An analogous ion with an m/z of 85 was observed in the 70-eV
EI mass spectrum of endosulfan hydroxyether (data not shown). Thus, the
molecular structure of the novel isomer does not include a pentacyclic
ether ring.
The PCI mass spectrum [PCI(NH3)] of the novel metabolite
displayed the molecular parent ions [M+H]+ and
[M+NH4]+ with m/z of 341 and 358, respectively (data not shown), confirming the molecular mass
(35Cl6) indicated previously in the EI mass
spectrum (Fig. 2). The preliminary evidence indicated that the
molecular structure of the novel isomer was that of endosulfan
monoaldehyde (Fig. 3). The PCI mass
spectrum of the metabolite also displayed fragment ions indicating
consecutive losses of two molecules of HCl from [M+H]+
ions. Since the most probable site for gas phase proton attachment in
the putative structure would be the carbonyl oxygen atom, the initial
HCl loss may be rationalized as elimination of the reagent proton
together with the vicinal bridgehead chlorine atom via a favored
six-centered transition structure.
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FIG. 3.
Proposed pathway for metabolism of endosulfan by the
microbial culture described in this paper.
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Support for the structure of the novel metabolite is provided by the
observation that it forms an O-benzyl oxime derivative. Although the expected molecular ion is absent in its 70-eV EI mass
spectrum (Fig. 2), an [MCH3]+ ion with an
m/z of 430 is present (data not shown), indicating a
relative molecular mass of 445 (35Cl6) for the
derivative and substantiating a monoaldehyde structure for the metabolite.
Formation of endosulfan metabolites by the culture.
TLC
analysis of the culture allowed qualitative comparison of the amounts
of each product during the growth cycle after the tenth subculture in
sulfur-free medium (Table 2). Endosulfate appeared rapidly in the growing culture, followed by the putative monoaldehyde, hydroxyether, and then small amounts of the diol. The
endosulfate continued to accumulate until both isomers of the parent
compound had disappeared and then remained at constant levels in the
medium. Of the other metabolites, levels of the putative monoaldehyde
decreased first, followed by the hydroxyether and then the diol.
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TABLE 2.
Qualitative TLC analysis of endosulfan isomers and
metabolites present in organic extracts from different stages of the
culture growth cyclea
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Table 3 shows quantitation by GC analysis
of various chlorinated products in DCM extracts from cultures at one
time point in log phase growth during the twelfth subculture in
sulfur-free medium as a percentage of the added sulfur-containing
substrate (corrected for extraction efficiency and volatilization from
the medium). Four sulfur-containing substrates were tested separately, each added to an initial concentration of 50 µM: technical-grade endosulfan, alpha-endosulfan, beta-endosulfan,
and endosulfate. The final column of Table 3 shows the amount of
substrate that is not accounted for by products in the organic extract.
Significant amounts of chlorinated product(s) were detected by TLC in
the DCM extract of the dried residue from the aqueous fraction. While we were unable to analyze this by GC or GC-MS, it corresponded semi-quantitatively to the amount of product unaccounted for in the
organic fraction.
The predominant metabolite detected in cultures grown with
alpha-endosulfan as the only sulfur source was endosulfate
(16.2%) (Table 3). The putative endosulfan monoaldehyde, endosulfan
hydroxyether, and endodiol were detected at much lower levels
(<0.5%). Conversely, less endosulfate was detected in cultures grown
with the beta isomer (4.2%) and the other metabolites were
all detected at comparatively higher levels (1.4 to 3.5%). In cultures
grown in the presence of beta-endosulfan, the majority of
metabolites was found in the DCM extract of residue from the aqueous
fraction after organic extraction. This was attributed to further
metabolism of the described products. No growth was observed in
cultures grown with endosulfate (Fig. 1), and the added endosulfate was
recovered from the media after 7 days (data not shown).
Despite their initial accumulation and then disappearance in cultures
grown in the presence of endosulfan, endodiol and endosulfan hydroxyether were not degraded when added to cultures grown in sulfur-free media with 50 µM added sulfite (data not shown). This may
be due to an inability of these more polar compounds to traverse the
cell membrane and therefore an inability to interact with their
respective degradative enzymes. However, endodiol and endosulfan hydroxyether have been degraded by inocula from soil (27).
Endosulfan strongly adsorbs to microorganisms, with the majority of the
insecticide being associated with the cell membrane rather than
the growth medium (22, 25). Hence, degradation of endosulfan
presumably leads to an accumulation of products within the cell,
facilitating their further degradation.
Effect of piperonyl butoxide.
Inclusion of 1 and 10 µM
piperonyl butoxide (PBO, a known cytochrome P450 inhibitor) in
sulfur-free medium with endosulfan did not prevent the formation of any
metabolites, including endosulfate, nor did it significantly inhibit
growth (data not shown). While culture growth was approximately halved
by the addition of 100 µM PBO, the formation of metabolites was not
qualitatively affected (data not shown).
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DISCUSSION |
Enzymatic bioremediation of insecticides is receiving considerable
attention, particularly since the extensive characterization of a
phosphotriesterase enzyme capable of detoxifying a range of
organophosphate compounds (7). The basis of similar
investigations for enzymes capable of detoxifying other classes of
insecticides requires a source of enzymes for catalytic detoxification.
This study describes the enrichment of a culture of soil bacteria
capable of degrading endosulfan. Enrichment was achieved and maintained by providing endosulfan as the only sulfur source. Endosulfan is a poor
biological energy source, as it contains only six potential reducing
electrons and previous attempts to enrich for endosulfan-degrading microorganisms using the insecticide as a carbon source have been unsuccessful (11). However, endosulfan has a relatively
reactive cyclic sulfite diester group (32). In this study,
microorganisms were selected for their ability to release the sulfite
group from endosulfan and to use this as a source of sulfur for growth.
This selection procedure enriches for a culture capable of either the direct hydrolysis of endosulfan or the oxidation of the insecticide followed by its hydrolysis. The degradation products in our culture indicate that both hydrolysis and oxidation reactions are occurring. However, the accumulation of endosulfate and the inability of the
culture to grow when this is provided as the sole sulfur source indicate that we have selected for the direct hydrolysis of endosulfan.
The strategy for enrichment also addressed the issues that endosulfan
is virtually insoluble in water and spontaneously hydrolyzes at
alkaline pH. A study into the distribution of the compound in sterile
microbial broth showed that it concentrated at the glass-medium
interface and that the inclusion of Tween 80 (a mixture of oleic,
linoleic, palmitic, and stearic acids) resulted in dispersion of the
pesticide (12). Therefore, we included Tween 80 in the enrichment broth to emulsify endosulfan, thereby increasing the amount
of insecticide in contact with the soil bacteria. This detergent has
previously been used to solubilize pyrethroids during the isolation of
microorganisms capable of metabolizing permethrin (19).
Endosulfan is susceptible to alkaline hydrolysis (20), with
approximately 10-fold increases in hydrolysis occurring with each
increase in pH unit. Many previous studies have been unable to
differentiate between chemical and biological hydrolysis of endosulfan
because microbial growth has led to increases in the alkalinity of the
culture medium (20, 21). To minimize nonbiological hydrolysis, the enrichment medium was buffered to pH 6.6 and cultures were monitored constantly to ensure that growth did not decrease hydrogen ion concentrations. We observed detectable levels of endodiol
(>0.1 ppm) in sterile media inoculated with 50 µM endosulfan at pH
7.2 after 4 days (data not shown) and recommend that the pH of the
medium for biodegradation studies of endosulfan be maintained below pH
7.0.
The formation and decay of metabolites led us to propose a pathway of
endosulfan metabolism by the culture (Fig. 3). According to this
pathway, the parent compound is either oxidized or hydrolyzed. The
oxidation reaction is favored for the alpha isomer and
produces endosulfate. Preferential oxidation of this isomer has been
previously reported, and it is thought that it contributes a
disproportionate amount of endosulfate found in the environment
(2, 6, 21). Cytochrome P450 monooxygenases are known to
contribute to the oxidation of many sulfur-containing pesticides. In
contrast to other studies investigating endosulfan oxidation (13,
16, 17), the cytochrome P450 inhibitor PBO does not prevent the formation of endosulfate by this culture. While this does not exclude
the possibility that oxidation is catalyzed by a cytochrome P450
monooxygenase, as PBO is not a universal inhibitor of these enzymes, it
suggests that the reaction is being catalyzed by a type of oxidase that
is different to that in the previous studies.
The culture is unable to utilize endosulfate as a sulfur
source, and it accumulates as a terminal pathway product as a result of
endosulfan oxidation. While an inability to transport the more polar
compound into the cell may contribute to this, the accumulation of
endosulfate in cultures provided with endosulfan suggests the absence
of an enzyme capable of hydrolyzing the oxidized compound. The
different oxidation states of the sulfur in endosulfan and endosulfate
make it unlikely that the same enzyme will be capable of releasing the
sulfur-containing moiety from both.
According to our proposed pathway, enzymatic hydrolysis of endosulfan
forms the monoaldehyde and releases sulfite. Both isomers are
substrates for this reaction, although the rapid oxidation of the
alpha-endosulfan makes it difficult to estimate the rate of
hydrolysis of this isomer. Because of the strong selection pressure
imposed on the culture, release of sulfur from endosulfan does not have
to be energetically favorable. The subsequent metabolism of
non-sulfur-containing metabolites is presumably driven by energy demands. We propose that oxidative cyclization of the monoaldehyde leads to endosulfan hydroxyether, which is further metabolized to polar
products. According to this pathway, the low levels of endodiol we
detected in the later stages of culture growth are a result of chemical
hydrolysis of the parent compound despite incubation in the slightly
acidic medium. The pathway of metabolism we propose for our culture is
substantially different from the degradation pathway in inocula from
sandy-loam soil (21), tobacco leaf (6), and
white-rot fungi (17), and the detoxification pathway of the
Indian honey bee (23). The pathway proposed in these other
studies involves a double hydration to produce endodiol and then a
dehydration to produce endosulfan ether. While insects are under
pressure to detoxify the insecticide, endosulfan is not toxic to plants
(18), bacteria, or soil fungi (20); hence, the
degradation observed in the other studies is most likely the result of cometabolism.
Although the monoaldehyde has not been reported previously, a putative
dialdehyde metabolite of endosulfan has been characterized in white-rot
fungi (17). In that case, however, it was inferred that the
dialdehyde was derived by oxidation of initially formed endosulfan
diol, with endosulfan hydroxyether as the intermediate.
From this study, we cannot predict the prevalence or relevance of this
pathway in the soil environment. We were successful in enriching
endosulfan-degrading organisms from only 1 out of 10 soil samples, but
our method relies on bacteria being able to grow in the minimal media;
hence, the number of culturable bacteria is severely restricted. We are
also unable to predict if the endosulfan-degrading organism would
utilize endosulfan as a sulfur source in the soil environment. The
majority of the sulfur content of soils is found in an organic form,
with over 95% present as sulfonates and sulfate esters. As a result,
it is expected that soil bacteria will have numerous enzymes capable of
releasing sulfur from organic compounds. Studies to date confirm this
(for review, see reference 15).
The formation of the novel monoaldehyde product further supports our
conclusion that the degradation we are observing is biological, as this
compound has not been described as a product of chemical degradation
(10). After approximately 25 rounds of subculturing, the
culture metabolizes 50 µM endosulfan to undetectable levels in less
than 4 days. This rate of degradation is significantly higher than
those previously measured for bacteria (1, 11, 20, 21, 27),
especially considering that chemical hydrolysis is not thought to be a
significant contributing factor. Other studies have provided sufficient
nutrient sources, and the biological transformation observed is
presumably cometabolism. Our study differs from previous studies by the
application of strong selection pressure on the culture to release the
sulfur moiety from the insecticide, allowing us to enrich for the
degradative activity. We are currently further characterizing the
hydrolytic ability of this culture as a potential enzymatic
bioremediating agent for endosulfan.
| |
ACKNOWLEDGMENTS |
We are grateful for the financial support of the Cotton Research
and Development Corporation (CSE 77C), the Horticultural Research and
Development Corporation (HG97340), and Orica Australia Pty Ltd. We
thank Hoechst Schering AgrEvo Pty Ltd for providing technical-grade endosulfan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CSIRO
Entomology, GPO Box 1700, Canberra ACT 2601, Australia. Phone: 61 2 6246 4157. Fax: 61 2 6246 4173. E-mail:
Tara.Sutherland{at}ento.csiro.au.
| |
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Applied and Environmental Microbiology, July 2000, p. 2822-2828, Vol. 66, No. 7
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Abstract
Introduction
