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.     

Endosulfan Mineralization by Bacterial Isolates and Possible Degradation Pathway Identification

A bacterial consortium consists of three bacterial isolates, which rapidly mineralizes endosulfan, was enriched from an endosulfan-processing industrial surface soil. Batch experiments were conducted using bacterial consortium and its pure isolates for their potential degradation of endosulfan and its metabolites, i.e., endosulfan sulfate, endosulfan ether, and endosulfan lactone, in anaerobic condition. Endosulfan degradation was promising with bacterial consortium and pure isolates. Staphylococcus sp. preferably utilized beta endosulfan whereas other two Bacillus strains utilized more alpha endosulfan. The addition of supplementary carbon, i.e., dextrose, stimulated the endosulfan degradation efficiency in both the cases. Degradation of endosulfan ether and endosulfan lactone was promising with Bacillus circulans I and II whereas no endosulfan sulfate was degraded by any of these strains. From the present investigation, it was postulated that endosulfan was mineralized via hydrolysis pathway with the formation of carbenium ions and/or ethylcarboxylates, which later converted into simple hydrocarbons.     

Biodegradation of α and β endosulfan in broth medium and soil microcosm by bacterial strain Bordetella sp. B9

Bacterial strains were isolated from endosulfan treated soil to study the microbial degradation of this pesticide in broth medium and soil microcosm. The isolates were grown in minimal medium and screened for endosulfan degradation. The strain, which utilized endosulfan and showed maximum growth, was selected for detail studies. Maximum degrading capability in shake flask culture was shown by Bordetella sp. B9 which degraded 80% of α endosulfan and 86% of β endosulfan in 18 days. Soil microcosm study was also carried out using this strain in six different treatments. Endosulfan ether and endosulfan lactone were the main metabolites in broth culture, while in soil microcosm endosulfan sulfate was also found along with endosulfan ether and endosulfan lactone. This bacterial strain has a potential to be used for bioremediation of the contaminated sites.     

Klebsiella pneumoniae KE-1 degrades endosulfan without formation of the toxic metabolite,endosulfan sulfate

For bioremediation of toxic endosulfan, endosulfan degradation bacteria, which do not form toxic endosulfan sulfate, were isolated from various soil samples using endosulfan as sole carbon and energy source. Among the 40 isolated bacteria, strain KE-1, which was identified as Klebsiella pneumoniae by physiological and 16S rDNA sequence analysis, showed superior endosulfan degradation activity. Analysis of culture pH, growth, free sulfate and endosulfan and its metabolites demonstrated that KE-1 biologically degrades 8.72 microg endosulfan ml(-1) day(-1) when incubated with 93.9 microg ml(-1) endosulfan for 10 days without formation of toxic endosulfan sulfate. Our results suggest that K. pneumoniae KE-1 degraded endosulfan by a non-oxidative pathway and that strain KE-1 has potential as a biocatalyst for endosulfan bioremediation.     

Mycoremediation of Endosulfan and Its Metabolites in Aqueous Medium and Soil by Botryosphaeria laricina JAS6 and Aspergillus tamarii JAS9

Microbial degradation offers an efficient and ecofriendly approach to remove toxicants from the contaminated environments. Botryosphaeria laricina JAS6 and Aspergillus tamarii JAS9 were capable of degrading endosulfan and their metabolites which were isolated through enrichment technique. Both the strains were able to withstand an exposure of 1300 mg/L and showed luxuriant growth at 1000 mg/L of endosulfan. The change in pH in the culture broth was from 6.8 to 3.4 and 3.8 during growth kinetic studies of JAS6 and JAS9 strains, respectively upon biological degradation of endosulfan. The degradation of endosulfan by JAS6 and JAS9 strains were examined by HPLC. The biodegradation rate constant (k) and the initial concentration were reduced by 50% (DT₅₀) which was determined by first and pseudo first order kinetic models. In the present investigation it has been revealed that Botryosphaeria laricina JAS6 and Aspergillus tamarii JAS9 possessing endosulfan degrading capability are being reported for the first time. These findings confirm the degradation of endosulfan by JAS6 and JAS9 strains which were accompanied by significant reduction in the toxicity and could be used as remedial measure in contaminated environments.     

Kinetics of endosulfan degradation by Phanerochaete chrysosporium

The chlorinated pesticide, endosulfan, could be degraded by Phanerochaete chrysosporium under non-ligninolytic conditions, and this did not require direct contact with mycelium. The major metabolites formed were endosulfan sulfate and endosulfan diol. The rate of degradation depended on the initial concentration. With 2.5 mg endosulfan l^–1, degradation was at 0.23 mg l^–1 day^–1. The degradation could be described using a nonlinear rate expression that was similar to the Michaelis–Menten equation.     

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.     

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.     

Comparison of colorimetric and chemiluminescent enzyme‐linked immunosorbent assay for the detection of endosulfan in food samples

Pesticides have become part of food protection since their inception. Endosulfan, an organochlorine insecticide, has been used against insect pests such as whiteflies, aphids, red spiders and mites. Methods of immunochemical assays have been devised for the determination and analysis of pesticides and commonly used for the analysis of contaminants in food, water, soil and body fluids. Chicken IgY antibodies raised against endosulfan haptens were used for the detection of endosulfan. We have compared colorimetric (CO) and chemiluminescence (CL) enzyme‐linked immunosorbent assay (ELISA) techniques for the detection of endosulfan isomers in a food matrix. CL ELISA assay was found to be more sensitive than CO assay. The mean recovery was 81.2–95.6% for α‐ and β‐endosulfan‐spiked food samples with 2.8–4.6% relative standard deviation. The detection of the endosulfan isomers was linear in the range 100 µg/mL–5 fg/mL, with a limit of detection at 100 µg/mL and 5 fg/mL for the CL ELISA method and 100 µg/mL and 1 ng/mL for the CO ELISA method respectively. These methods can be used for the rapid and reliable detection of organochlorine pesticide endosulfan. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Degradation of lindane and endosulfan by fungi, fungal and bacterial laccases

The ability of two white-rot fungi (Trametes versicolor and Pleurotus ostreatus) and one brown-rot fungus (Gloeophyllum trabeum) to degrade two organochlorine insecticides, lindane and endosulfan, in liquid cultures was studied and dead fungal biomass was examined for adsorption of both insecticides from liquid medium. Lindane and endosulfan were also treated with fungal laccase and bacterial protein CotA, which has laccase activities. The amount of degraded lindane and endosulfan increased with their exposure period in the liquid cultures of both examined white-rot fungi. Endosulfan was transformed to endosulfan sulphate by T. versicolor and P. ostreatus. A small amount of endosulfan ether was also detected and its origin was examined. Degradation of lindane and endosulfan by a brown rot G. trabeum did not occur. Mycelial biomasses of all examined fungi have been found to adsorb lindane and endosulfan and adsorption onto fungal biomass should therefore be considered as a possible mechanism of pollutant removal when fungal degradation potentials are studied. Bacterial protein CotA performed more efficient degradation of lindane and endosulfan than fungal laccase and has shown potential for bioremediation of organic pollutants.     

Isolation and characterization of a Mycobacterium strain that metabolizes the insecticide endosulfan

AIM: The aim of this study was to isolate and characterize a bacterium capable of metabolizing endosulfan. METHODS AND RESULTS: A endosulfan-degrading bacterium (strain ESD) was isolated from soil inoculum after repeated culture with the insecticide as the sole source of sulfur. Analysis of its 16S rRNA gene sequence, and morphological and physiological characteristics revealed it to be a new fast-growing Mycobacterium, closely related to other Mycobacterium species with xenobiotic-degrading capabilities. Degradation of endosulfan by strain ESD involved both oxidative and sulfur-separation reactions. Strain ESD did not degrade endosulfan when sulfite, sulphate or methionine were present in the medium along with the insecticide. Partial degradation occurred when the culture was grown, with endosulfan, in the presence of MOPS (3-(N-morpholino)propane sulphonic acid), DMSO (dimethyl sulfoxide), cysteine or sulphonane and complete degradation occurred in the presence of gutathione. When both beta-endosulfan and low levels of sulphate were provided as the only sources of sulfur, biphasic exponential growth was observed with endosulfan metabolism being restricted to the latter phase of exponential growth. CONCLUSIONS: This study isolated a Mycobacterium strain (strain ESD) capable of metabolizing endosulfan by both oxidative and sulfur-separation reactions. The endosulfan-degrading reactions are a result of the sulfur-starvation response of this bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: This describes the isolation of a Mycobacterium strain capable of degrading the insecticide endosulfan. This bacterium is a valuable source of enzymes for use in enzymatic bioremediation of endosulfan residues.     

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.     

Biodegradation of α- and β-endosulfan by soil bacteria

Extensive applications of persistent organochlorine pesticides like endosulfan on cotton have led to the contamination of soil and water environments at several sites in Pakistan. Microbial degradation offers an effective approach to remove such toxicants from the environment. This study reports the isolation of highly efficient endosulfan degrading bacterial strains from soil. A total of 29 bacterial strains were isolated through enrichment technique from 15 specific sites using endosulfan as sole sulfur source. The strains differed substantially in their potential to degrade endosulfan in vitro ranging from 40 to 93% of the spiked amount (100 mg l⁻¹). During the initial 3 days of incubation, there was very little degradation but it got accelerated as the incubation period proceeded. Biodegradation of endosulfan by these bacteria also resulted in substantial decrease in pH of the broth from 8.2 to 3.7 within 14 days of incubation. The utilization of endosulfan was accompanied by increased optical densities (OD₅₉₅) of the broth ranging from 0.511 to 0.890. High performance liquid chromatography analyses revealed that endosulfan diol and endosulfan ether were among the products of endosulfan metabolism by these bacterial strains while endosulfan sulfate, a persistent and toxic metabolite of endosulfan, was not detected in any case. The presence of endosulfan diol and endosulfan ether in the bacterial metabolites was further confirmed by GC-MS. Abiotic degradation contributed up to 21% of the spiked amount. The three bacterial strains, Pseudomonas spinosa, P. aeruginosa, and Burkholderia cepacia, were the most efficient degraders of both α- and β-endosulfan as they consumed more than 90% of the spiked amount (100 mg l⁻¹) in the broth within 14 days of incubation. Maximum biodegradation by these three selected efficient bacterial strains was observed at an initial pH of 8.0 and at an incubation temperature of 30°C. The results of this study may imply that these bacterial strains could be employed for bioremediation of endosulfan polluted soil and water environments.     

Biodegradability and biodegradation pathways of endosulfan and endosulfan sulfate

Endosulfan and endosulfan sulfate are persistent organic pollutants that cause serious environmental problems. Although these compounds are already prohibited in many countries, residues can be detected in soils with a history of endosulfan application. Endosulfan is transformed in the environment into endosulfan sulfate, which is a toxic and persistent metabolite. However, some microorganisms can degrade endosulfan without producing endosulfan sulfate, and some can degrade endosulfan sulfate. Therefore, biodegradation has the potential to clean up soil contaminated with endosulfan. In this review, we provide an overview of aerobic endosulfan degradation by bacteria and fungi, and a summary of recent advances and prospects in this research field.     

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.     

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 endosulfan during substrate preparation and cultivation of Pleurotus pulmonarius

Summary Endosulfan is an insecticide used on many vegetable crops. In mushroom cultivation, vegetable materials used as a growth substrate may contain residues of endosulfan that may accumulate in the final mushroom biomass. After preparing the substrate, it is subjected to pasteurization and/or composting and then inoculated with the desired fungus. The purpose of this research was to determine the rate and extent of endosulfan reduction from a grass substrate that was either composted or sterilized by autoclaving. In addition, the rate and extent of removal of endosulfan from substrate colonized with Pleurotus pulmonarius was determined. The degradation of 65 mg/kg endosulfan was analyzed on both, the substrate preparation and the culture of P. pulmonarius on the grass Digitaria decumbens. During composting in presence of Ca(OH)₂ for 120 h, the concentrations of α and β endosulfan were reduced by 61.4 and 49.5% respectively, significantly higher compared with the control (without Ca(OH)_2,) in which the reduction was 38.5%. After sterilization the concentration of α and β endosulfan was reduced by 84.8 and 87.5% respectively. After the colonization of substrate by P. pulmonarius (15 days after spawning) α and β endosulfan were reduced by 96% and at the end of cultivation (35 days after spawning) were reduced by 99%. When carpophores were analyzed, residues of α and β endosulfan were observed between 0.019–0.084 mg/kg. The results showed that α and β endosulfan were partially removed during the preparation of substrate and entirely eliminated during fungal colonization on the substrate.     

Biodegradation and bioremediation of endosulfan contaminated soil

Among the three mixed bacterial culture AE, BE, and CE, developed by enrichment technique with endosulfan as sole carbon source, consortium CE was found to be the most efficient with 72% and 87% degradation of alpha-endosulfan and beta-endosulfan, respectively, in 20 days. In soil microcosm, consortium AE, BE and CE degraded alpha-endosulfan by 57%, 88% and 91%, respectively, whereas beta-endosulfan was degraded by 4%, 60% and 67% after 30 days. Ochrobacterum sp., Arthrobacter sp., and Burkholderia sp., isolated and identified on the basis of 16s rDNA gene sequence, individually showed in situ biodegradation of alpha-endosulfan in contaminated soil microcosm by 61, 73, and 74, respectively, whereas degradation of beta-endosulfan was 63, 75, and 62, respectively, after 6 weeks of incubation over the control which showed 26% and 23 % degradation of alpha-endosulfan and beta-endosulfan, respectively. Population survival of Ochrobacterum sp., Arthrobacter sp., and Burkholderia sp., by plate count on Luria Broth with carbenicillin showed 75-88% survival of these isolates as compared to 36-48% of survival obtained from PCR fingerprinting. Arthrobacter sp. oxidized endosulfan to endosulfan sulfate which was further metabolized but no known metabolite of endosulfan sulfate was detected.