Gene cloning and molecular characterization of a two-enzyme system catalyzing the oxidative detoxification of beta-endosulfan

The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent beta-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of beta-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of beta-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either alpha-endosulfan or the metabolite of endosulfan, endosulfan sulfate.     

Enrichment of an Endosulfan-Degrading Mixed Bacterial Culture

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.     

Kinetics of Endosulfan Biodegradation by Stenotrophomonas maltophilia EN-1 Isolated From Pesticide-Contaminated Soil

Endosulfan is one of the persistent organochlorinated pesticides used extensively throughout the world, particularly in the developing countries. Its microbial metabolic transformation product endosulfan sulphate is more toxic and persistent than the parent compound itself. In order to completely mineralize endosulfan, augmentation of soil microbial community with efficient endosulfan degradation properties could a potentially viable option. In the present study, endosulfan degrading bacterium was isolated from the agriculture-contaminated soil of Shujaabad, Multan, Pakistan by using enrichment technique. The isolated bacterial strain EN-1 (Endosulfan-1) was identified as S. maltophilia by 16S rRNA sequencing and biochemical analysis. EN-1 has demonstrated the ability to utilize endosulfan as sole sulfur source. Kinetics of endosulfan degradation was studied at various initial concentrations ranges from 5 mg/L to 100 mg/L by growth dependent and growth independent kinetic models. Biodegradation kinetics revealed that the bacterium was highly efficient in endosulfan degradation. The average values of kinetic constants i.e. K_s, and µ_max were 13.73 mg/L and 0.210 h^?1 respectively, while µ_max/K_s ratio was 0.015. Addition of sulfur decreased the rate of degradation as the µ_max/K_s was observed to reduce. GC-MS analysis revealed that the bacterium metabolised the endosulfan into non-toxic metabolite i.e. endosulfan diol. The study instigates a complete elucidation of degradation process for commercial applications.     

Enrichment of an endosulfan-degrading mixed bacterial culture

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.     

Biodegradation of endosulfan and endosulfan sulfate by <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> strain C8B in broth medium

Endosulfan is one of the most widely used wide spectrum cyclodiene organochlorine insecticide. In environment, endosulfan can undergo either oxidation or hydrolysis reaction to form endosulfan sulfate and endosulfan diol respectively. Endosulfan sulfate is as toxic and as persistent as its parent isomers. In the present study, endosulfan degrading bacteria were isolated from soil through selective enrichment technique using sulfur free medium with endosulfan as sole sulfur source. Out of the 8 isolated bacterial strains, strain C8B was found to be the most efficient endosulfan degrader, degrading 94.12% α-endosulfan and 84.52% β-endosulfan. The bacterial strain was identified as Achromobacter xylosoxidans strain C8B on the basis of 16S rDNA sequence similarity. Achromobacter xylosoxidans strain C8B was also found to degrade 80.10% endosulfan sulfate using it as sulfur source. No known metabolites were found to be formed in the culture media during the entire course of degradation. Besides, the bacterial strain was found to degrade all the known endosulfan metabolites. There was marked increase in the quantity of released CO₂ from the culture media with endosulfan as sulfur source as compared to MgSO₄ suggesting that the bacterial strain, Achromobacter xylosoxidans strain C8B probably degraded endosulfan completely through the formation of endosulfan ether.     

First isolation and characterization of a bacterial strain that biotransforms the herbicide mesotrione

AIM: The aim of this study was to find and characterize a fungal or bacterial strain capable of metabolizing mesotrione, a new selective herbicide for control of broad-leaved weeds in maize. METHODS AND RESULTS: This strain was isolated from cloud water and showed close phylogenetic relationship with strains belonging to the Bacillus genus, based on 16S rRNA gene alignment. Kinetics of mesotrione degradation were monitored by high-performance liquid chromatography and in situ(1)H nuclear magnetic resonance spectroscopy at different concentrations. Mesotrione was completely biotransformed even at 5 mmol l(-1) concentration. 2-Amino-4-methylsulfonyl benzoic acid (AMBA) was identified as one of the metabolites, but was not the major one. CONCLUSIONS: This study reports the first rapid mesotrione biotransformation by a pure bacterial strain and the formation of several metabolites including AMBA. SIGNIFICANCE AND IMPACT OF THE STUDY: This bacterium isolated from cloud water is the first pure strain capable of rapidly degrading mesotrione.     

Expression and inducibility of endosulfan metabolizing gene in Rhodococcus strain isolated from earthworm gut microflora for its application in bioremediation

The metabolizing potential of a bacterial strain Rhodococcus MTCC 6716, isolated from the gut of an Indian earthworm (Metaphire posthuma) was studied for endosulfan bioremediation. In the present work, the optimum conditions for the maximum growth, kinetic of endosulfan degradation, regression equation, half life and correlation coefficient were studied. Endosulfan induced alterations in the expression of mRNA and protein of specific endosulfan metabolizing marker gene (Esd) was studied. Maximum growth of bacteria was observed at pH 7.0, 30°C and 0.085 M sodium chloride concentration in a liquid culture medium. Endosulfan was degraded by Rhodococcus strain up to 97.23% within 15 days without producing toxic metabolite and with strong correlation coefficient (-0.728) and half life 5.99 days. Endosulfan degradation was mediated through gene(s) present in genomic DNA. Expression of marker gene was found endosulfan concentration dependent. The results suggest that this novel strain (Rhodococcus) may be utilized for bioremediation of endosulfan.     

Enrichment of a microbial culture capable of degrading endosulphate, the toxic metabolite of endosulfan

AIMS: The aim of this study was to isolate a source of enzymes capable of degrading endosulphate (endosulfan sulphate), the toxic metabolite of the pesticide endosulfan. METHODS AND RESULTS: A microbial broth culture capable of degrading endosulphate was enriched from endosulfan-contaminated soil by providing the metabolite as the sole source of sulphur in broth culture. No microbial growth was observed in the absence of endosulphate. In the presence of endosulphate, growth of the culture occurred with the concomitant formation of three chlorine-containing compounds. Thin layer chromatography and gas chromatography--mass spectral analysis identified these metabolites as endosulfan monoaldehyde, 1,2,3,4,7,7-hexachloro-5,6-bis(methylene)bicyclo[2.2.1]-2-heptene and 1,2,3,4,7,7-hexachloro-5-hydroxymethylene-6-methylenebicyclo[2.2.1]-2-heptene. The second and third compounds have not been reported in previous metabolic studies. The enriched culture was also able to utilize alpha- and beta-endosulfan as sulphur sources, each producing the hydrolysis product endosulfan monoaldehyde as the sole chlorine-containing metabolite. Alpha-endosulfan was more readily hydrolysed than the beta-isomer. CONCLUSIONS: This study isolated a mixed microbial culture capable of degrading endosulphate. The products of degradation were characterized as novel endosulfan metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation of a mixed microbial culture that is potentially a valuable source of hydrolysing enzymes for use in enzymatic bioremediation, particularly of endosulphate and alpha-endosulfan residues.     

Biodegradation of the organochlorine insecticide, endosulfan, and the toxic metabolite, endosulfan sulfate, by Klebsiella oxytoca KE-8

Biodegradation of endosulfan, a chlorinated cyclodiene insecticide, is generally accompanied by production of the more toxic and more persistent metabolite, endosulfan sulfate. Since our reported endosulfan degrader, Klebsiella pneumoniae KE-1, failed to degrade endosulfan sulfate, we tried to isolate an endosulfan sulfate degrader from endosulfan-polluted soils. Through repetitive enrichment and successive subculture using mineral salt medium containing endosulfan or endosulfan sulfate as the sole source of carbon and energy, we isolated a bacterium capable of degrading endosulfan sulfate as well as endosulfan. The bacterium KE-8 was identified as Klebsiella oxytoca from the results of 16S rDNA sequence analysis. In biodegradation assays with KE-8 using mineral salt medium containing endosulfan (150 mg l^–1) or endosulfan sulfate (173 mg l^–1), the biomass was rapidly increased to an optical density at 550 nm of 1.9 in 4 days and the degradation constants for - and -endosulfan, and endosulfan sulfate were 0.3084, 0.2983 and 0.2465 day^–1, respectively. Analysis of the metabolites further suggested that K. oxytoca KE-8 has high potential as a biocatalyst for bioremediation of endosulfan and/or endosulfan sulfate.     

Screening of soil fungi for in vitro degradation of endosulfan

Intensive use of endosulfan has resulted in contamination of soil and water environments at various sites in Pakistan. This study was conducted to isolate efficient endosulfan-degrading fungal strains from contaminated soils. Sixteen fungal strains were isolated from fifteen specific sites by employing enrichment techniques while using endosulfan as a sole sulfur source, and tested for their potential to degrade endosulfan. Among these fungal strains, Chaetosartorya stromatoides, Aspergillus terricola, and Aspergillus terreus degraded both α- and β-endosulfan upto 75% in addition to ∼20% abiotic degradation of the spiked amount (100 mg l⁻¹) in the broth within 12 days of incubation. Biodegradation of endosulfan by soil fungi was accompanied by a substantial decrease in pH of the broth from 7.0 to 3.2. The major metabolic product was endosulfan diol along with very low concentrations of endosulfan ether. Maximum biodegradation of endosulfan by these selected fungal strains was found at an initial broth pH of 6, incubation temperature of 30°C and under agitation conditions. This study indicates that the isolated strains carried efficient enzyme systems required for bioremediation of endosulfan-contaminated soil and water environments.     

Degradation of pyrene, benz[a]anthracene, and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site.

The degradation of three polycyclic aromatic hydrocarbons (PAH), pyrene (PYR), benz[a]anthracene (BAA), and benzo[a]pyrene (BaP), by Mycobacterium sp. strain RJGII-135 was studied. The bacterium was isolated from an abandoned coal gasification site soil by analog enrichment techniques and found to mineralize [14C]PYR. Further degradation studies with PYR showed three metabolites formed by Mycobacterium sp. strain RJGII-135, including 4,5-phenanthrene-dicarboxylic acid not previously isolated, 4-phenanthrene-carboxylic acid, and 4,5-pyrene-dihydrodiol. At least two dihydrodiols, 5,6-BAA-dihydrodiol and 10,11-BAA-dihydrodiol, were confirmed by high-resolution mass spectral and fluorescence analyses as products of the biodegradation of BAA by Mycobacterium sp. strain RJGII-135. Additionally, a cleavage product of BAA was also isolated. Mass spectra and fluorescence data support two different routes for the degradation of BaP by Mycobacterium sp. strain RJGII-135. The 7,8-BaP-dihydrodiol and three cleavage products of BaP, including 4,5-chrysene-dicarboxylic acid and a dihydro-pyrene-carboxylic acid metabolite, have been isolated and identified as degradation products formed by Mycobacterium sp. strain RJGII-135. These latter results represent the first example of the isolation of BaP ring fission products formed by a bacterial isolate. We propose that while this bacterium appears to attack only one site of the PYR molecule, it is capable of degrading different sites of the BAA and BaP molecules, and although the sites of attack may be different, the ability of this bacterium to degrade these PAH is well supported. The proposed pathways for biodegradation of these compounds by this Mycobacterium sp. strain RJGII-135 support the dioxygenase enzymatic processes reported previously for other bacteria. Microorganisms like Mycobacterium sp. strain RJGII-135 will be invaluable in attaining the goal of remediation of sites containing mixtures of these PAH.     

Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons

AIM: The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism. METHODS AND RESULTS: The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture. CONCLUSIONS: This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.     

Biodegradation of organochlorine pesticide, endosulfan, by a fungal soil isolate, Aspergillus niger

Endosulfan is a chlorinated pesticide widely used in India for the protection of cotton, tea, sugarcane and vegetables. The persistence of endosulfan in environment and toxic effects on biota necessitate its removal. The role of soil fungi in recycling organic matter prompted us to attempt biodegradation of endosulfan using fungi. This study aims at enrichment, isolation and screening of fungi capable of metabolizing endosulfan. In all, 16 fungal isolates were obtained by enrichment of soil samples that had seems exposed to endosulfan before. Isolates were screened by a gradient plate assay, and results were confirmed by broth assay. On the basis of tolerance to endosulfan, an isolate, identified as Aspergillus niger was selected for further studies. The culture could tolerate 400 mg ml⁻¹ of technical grade endosulfan. Complete disappearance of endosulfan was seen on 12 days of incubation. Evolution of carbon dioxide during endosulfan metabolism has indicated the complete mineralization of endosulfan. Change in pH of culture broth to acidic range supported the biological transformation. Thin layer chromography (TLC) analyses revealed the formation of various intermediates of endosulfan metabolism including endosulfan diol, endosulfan sulfate, and an unidentified metabolite. The toxic intermediate, endosulfan sulfate, was also metabolized, further resulting in complete mineralization of endosulfan. Direct desulfurization of endosulfan sulfate or a novel pathway could be the mechanism of endosulfan and endosulfan sulfate degradation in Aspergillus niger. The fungal strain isolated by us could prove valuable for bioremediation of endosulfan contaminated soils and waters.     

Evidence that RDX biodegradation by Rhodococcus strain DN22 is plasmid-borne and involves a cytochrome p-450

AIMS: To investigate the biodegradation of the explosive compound RDX in Rhodococcus strain DN22, a bacterium previously isolated for its ability to grow on RDX as sole nitrogen source. METHODS AND RESULTS: Analysis of the rates of RDX degradation and nitrite production indicated that 2 mol nitrite were produced per mole RDX degraded. Cells of strain DN22 had the highest activity against RDX during the exponential phase and low activity in the stationary phase. Nitrite production from RDX was inhibited by metyrapone, menadione, piperonyl butoxide, n-octylamine and carbon monoxide and inducible by pyrrolidine, pyridine and atrazine. Acridine orange treatment yielded RDX-minus derivatives of strain DN22 at a curing rate of 1.5% and all of the cured derivatives had lost a large plasmid. CONCLUSIONS: RDX biodegradation in strain DN22 appears to involve a plasmid-encoded cytochrome p-450 enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: Plasmid-borne RDX degradation genes could potentially be transferred between bacteria. Our research into RDX metabolism in strain DN22 will facilitate future applications of this bacterium for bioremediation.     

Microcosmic study of endosulfan degradation by Paenibacillus sp. ISTP10 and its toxicological evaluation using mammalian cell line

In the present study, an endosulfan degrading strain Paenibacillus sp. ISTP10 was isolated from activated sludge. Soil microcosms were set up with endosulfan (60 mg kg⁻¹ of dry soil) to evaluate the degradation potency of the strain. Soil samples from the microcosms were collected at regular intervals and the organic compounds were extracted with hexane. GC–MS analysis of the soil extract showed the formation of metabolites of endosulfan such as endosulfan diol and endosulfan ether confirming that the strain degrades endosulfan via a hydrolytic pathway. Methyl tetrazolium (MTT) assay for cytotoxicity and alkaline comet assay for genotoxicity were carried out in human hepato-carcinoma cell line HepG2 to evaluate the toxic potential of endosulfan and its degraded metabolites. The bacterium reduced toxicity as determined by an increase in LC₅₀ value by 75.86 fold and a reduction in Olive Tail Moment by 21 fold after 30 days of treatment. The by-products of degradation were found to be less toxic than the parent compound showing the biodegradation and detoxification potential of endosulfan by Paenibacillus sp. ISTP10.     

Optimization of environmental parameters for biodegradation of alpha and beta endosulfan in soil slurry by Pseudomonas aeruginosa

Aim: To determine optimal environmental conditions for achieving biodegradation of α‐ and β‐endosulfan in soil slurries following inoculation with an endosulfan degrading strain of Pseudomonas aeruginosa. Methods and Results: Parameters that were investigated included soil texture, soil slurry: water ratios, initial inoculum size, pH, incubation temperature, aeration, and the use of exogenous sources of organic and amino acids. The results showed that endosulfan degradation was most effectively achieved at an initial inoculum size of 600 μl (OD = 0·86), incubation temperature of 30°C, in aerated slurries at pH 8, in loam soil. Under these conditions, the bacterium removed more than 85% of spiked α‐ and β‐endosulfan (100 mg l^?1) after 16 days. Abiotic degradation in noninoculated control medium within same incubation period was about 16%. Biodegradation of endosulfan varied in different textured soils, being more rapid in course textured soil than in fine textured soil. Increasing the soil contents in the slurry above 15% resulted in less biodegradation of endosulfan. Exogenous application of organic acids (citric acid and acetic acid) and amino acids (l ‐methionine and l ‐cystein) had stimulatory and inhibitory effects, respectively, on biodegradation of endosulfan. Conclusion: The results of this study demonstrated that biodegradation of endosulfan by Ps. aeruginosa in soil sediments enhanced significantly under optimized environmental conditions. Significance and Impact of the Study: Endosulfan is a commonly used pesticide that can contaminate soil, wetlands and groundwater. Our study demonstrates that bioaugmentation of contaminated soils with an endosulfan degrading bacterium under optimized conditions provides an effective bioremediation strategy.     

Degradation of [8,9,-14C]endosulfan by soil microorganisms.

Twenty-eight soil fungi, 49 soil bacteria, and 10 actinomycetes were tested as to their ability to degrade the insecticide endosulfan. Using 14C-labeled material, the qualitative as well as the quantitative formation of metabolities, as well as of 14CO2, could be followed. Sixteen fungi, 15 bacteria, and 3 actinomycetes were found capable of metabolizing more than 30% of the applied endosulfan. The major metabolities detected were endosulfate, formed by oxidation of the sulfite group, and endodiol, formed by hydrolysis of the ester bond. The majority of highly active fungi formed endosulfate as the major metabolite, whereas the majority of active bacteria formed endodiol. In addition to endosulfate and endodiol, individual cultures contained small quantities of endohydroxyether and two unidentified products. The very small quantities of 14CO2 evolved from cultures indicated that an extensive mineralization of the carbon skeleton of endosulfan did not occur.     

Degradation of [8,9,-14C]endosulfan by soil microorganisms.

Twenty-eight soil fungi, 49 soil bacteria, and 10 actinomycetes were tested as to their ability to degrade the insecticide endosulfan. Using 14C-labeled material, the qualitative as well as the quantitative formation of metabolities, as well as of 14CO2, could be followed. Sixteen fungi, 15 bacteria, and 3 actinomycetes were found capable of metabolizing more than 30% of the applied endosulfan. The major metabolities detected were endosulfate, formed by oxidation of the sulfite group, and endodiol, formed by hydrolysis of the ester bond. The majority of highly active fungi formed endosulfate as the major metabolite, whereas the majority of active bacteria formed endodiol. In addition to endosulfate and endodiol, individual cultures contained small quantities of endohydroxyether and two unidentified products. The very small quantities of 14CO2 evolved from cultures indicated that an extensive mineralization of the carbon skeleton of endosulfan did not occur.     

Isolation and characterization of a Pseudomonas sp. strain IITR01 capable of degrading α‐endosulfan and endosulfan sulfate

Aim: To isolate bacteria capable of degrading endosulfan (ES) and the more toxic ES sulfate and to characterize their metabolites. Methods and Results: A Pseudomonas sp. strain IITR01 capable of degrading α‐ES and toxic ES sulfate was isolated using technical‐ES through enrichment culture techniques. No growth and no degradation were observed using β‐ES. Thin‐layer chromatography and gas chromatography‐mass spectrum analysis revealed the disappearance of both α‐ES and ES sulfate and the formation of hydroxylated products ES diol, ether and lactone. We show here for the first time the formation of aforementioned metabolites in contrast to ES hemisulfate yielded by an Arthrobacter sp. Metabolism of α‐ES and endosulfate was also observed using the crude cell extract of IITR01. The molecular mass of protein induced during the degradation of α‐ES and sulfate as substrate was found to be approximately 150 kDa as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Conclusion: We describe characterization of bacterium capable of degrading α‐ES and ES sulfate but not β‐ES. Genetic investigation suggests that a gene nonhomologous to the reported esd may be present in the strain IITR01. Significance and Impact of the Study: This study describes toxic ES degradation by a Pseudomonas species that may be utilized for the bioremediation of the industrial soils contaminated with ES residues.