Enrichment and Isolation of a Mixed Bacterial ... - Semantic Scholar

4 downloads 0 Views 1007KB Size Report
stereoisomerism, where the enzymes released from bacterial system may be ac- ... dosulfan. These observations reflect that stereoisomerism and the release of.
Journal of Environmental Science and Health Part B, 41:81–96, 2006 C Taylor & Francis Inc. Copyright  ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601230500234935

Enrichment and Isolation of a Mixed Bacterial Culture for Complete Mineralization of Endosulfan Mathava Kumar and Ligy Philip Environmental and Water Resources Engineering Division, Department of Civil Engineering, Indian Institute of Technology Madras, Chennai, India In the present study, we isolated three novel bacterial species, namely, Staphylococcus sp., Bacillus circulans–I, and Bacillus circulans–II, from contaminated soil collected from the premises of a pesticide manufacturing industry. Batch experiments were conducted using both mixed and pure cultures to assess their potential for the degradation of aqueous endosulfan in aerobic and facultative anaerobic condition. The influence of supplementary carbon (dextrose) source on endosulfan degradation was also examined. After four weeks of incubation, mixed bacterial culture was able to degrade 71.82 ± 0.2% and 76.04 ± 0.2% of endosulfan in aerobic and facultative anaerobic conditions, respectively, with an initial endosulfan concentration of 50 mg l−1 . Addition of dextrose to the system amplified the endosulfan degradation efficiency by 13.36 ± 0.6% in aerobic system and 12.33 ± 0.6% in facultative anaerobic system. Pure culture studies were carried out to quantify the degradation potential of these individual species. Among the three species, Staphylococcus sp. utilized more beta endosulfan compared to alpha endosulfan in facultative anaerobic system, whereas Bacillus circulans–I and Bacillus circulans–II utilized more alpha endosulfan compared to beta endosulfan in aerobic system. In any of these degradation studies no known intermediate metabolites of endosulfan were observed. Key Words: Endosulfan; Mixed culture; Biodegradation; Enriched cultures; Staphylococcus sp.; Bacillus circulans; Mineralization.

INTRODUCTION Endosulfan (6,7,8,9,9a-hexahydro-6,9-methano,3,4-benzo (e. dioxathiepin-3oxide) is a chlorinated cyclodiene insecticide, acaricide widely used throughout

Received March 21, 2005. Address correspondence to Ligy Philip, Environmental and Water Resources Engineering Division, Department of Civil Engineering, Indian Institute of Technology Madras, Chennai, India; E-mail: [email protected]

81

82

Kumar and Philip

Figure 1: Molecular structure of endosulfan and its two isomers.

the world to control numerous insects and mites in many food and nonfood crops. Technical-grade endosulfan is a mixture of two stereo isomers, alpha and beta endosulfan (Fig. 1), in a ratio of 70:30. Both the isomers are extremely toxic to aqueous organisms. Because of its widespread usage and potential transport, endosulfan contamination is frequently found in the environment at considerable distances from the point of its original applications.[1−8] Detoxification of endosulfan through biological means is receiving serious attention as an alternative to existing methods, such as incineration and landfill.[8] Numerous studies have been reported regarding the isolation of pure and mixed cultures of bacteria and fungi [9–15] capable of degrading endosulfan. A bacterial coculture of two bacillus spp. was enriched from soil samples collected from a contaminated industrial area for the degradation of endosulfan.[12] The degradation of endosulfan and the formed metabolites during degradation by this bacterial coculture were evaluated by the reduction in toxicity against the test organism Tubifex tubifex Muller.[16] A bacterial strain, Klebsiella pneumoniae (KE-1), capable of degrading endosulfan without the formation of endosulfan sulfate was enriched from endosulfan polluted soil samples.[17] Even though mortality of the test organism and the reduction in toxicity (converting the parent compound into less toxic metabolites) is a sensitive end point for the assessment of ecotoxicological risk by the environmental pollutants, but none of these microbes were able to mineralize endosulfan to CO2 and other environmental friendly compounds.

Enrichment and Isolation of Bacterial Culture

Endosulfan can be degraded by attacking the sulfide group by oxidation and/or hydrolysis to form the toxic metabolite endosulfan sulfate and less toxic endosulfan diol, respectively.[15] But the formation of intermediate compounds (metabolites) is mainly based on the metabolic activity of the specific culture and the environmental conditions. Many species were isolated and checked for the endosulfan degradation potential, but in no case endosulfan was completely mineralized. Also, not many studies were carried out on the behavior of individual pure cultures of a mixed consortium on endosulfan and its isomers degradation. This paper describes the enrichment and isolation of endosulfan mineralizing microbial cultures. The endosulfan degradation capacities of these microbes individually and as a group were evaluated under various conditions.

MATERIALS AND METHODS Chemicals High purity (99.4%) endosulfan, endosulfan sulfate, endosulfan ether, and endosulfan lactone were purchased from Sigma Aldrich Ltd., USA, and technical-grade endosulfan of 96% purity was purchased from EID Parry India Ltd., Chennai, India. Other chemical reagents and solvents used were of HPLC grade purchased from Ranbaxy Ltd., Chennai, India. The stock endosulfan solution of 1% was prepared in methanol and used for all the experiments. All the glassware used was supplied by Borosil, India, and before every experiment, all glassware was cleaned with distilled water and dried at 110◦ C for 5 h.

Sample Collection for Enrichment Studies Soil samples used in this study were collected from the premises of an endosulfan processing industry (EID Parry India Ltd., Chennai, India). Topsoil was collected from the first 15 cm and preserved at 4◦ C (at the site).The preserved soil samples were brought to the laboratory on the same day, and the microbial enrichment was started immediately.

Enrichment of Mixed Bacterial Culture A soil suspension (10 g /50 ml) was made in a nutrient broth (NB). The composition of NB is as follows: KH2 PO4 1.0 g, K2 HPO4 ; 1.0 g, NH4 NO3 ; 1.0 g, NaCl; 1.0 g, MgSO4 ·7H2 O; 0.2 g, CaCl2 ; 0.02 g, Fe (SO4 )3 ; 0.02 g, trace metal solution[12] 1 ml dissolved in 1 liter of distilled water; final pH of the minimal medium was adjusted to 7 using HCl and/or NaOH. The soil suspension was kept in an orbital shaker (Remi Instruments Ltd., India) set for 28 ± 2◦ C for 24 h at 150 rpm. The solid particles were allowed to settle for 1 h, and 1 ml of

83

84

Kumar and Philip

the supernatant was inoculated into a fresh 100 ml Erlenmeyer flask containing 10 ml of fresh NB spiked with 50 mg l−1 of endosulfan. The contents were incubated at 28 ± 2◦ C and 150 rpm for one week. Thereafter, 1 ml of the suspension was transferred into a fresh Erlenmeyer flask and cultured as above (at each culturing step, the concentration of endosulfan was increased, and at the end, the concentration of endosulfan was around 500 mg l−1 in the NB). After six transfers, a loopful of this inoculum was streaked onto agar plates and incubated for 24 h at 28 ± 2◦ C. Dilution and streaking of colonies with same morphology was carried out three times. The colonies with distinct morphology were streaked on agar slants for 24 h at 28 ± 2◦ C and refridgerated 4◦ C for further use.

Endosulfan Degradation Studies with Mixed Bacterial Culture To study the degradation of endosulfan, the mixed bacterial culture was grown in NB at 28 ± 2◦ C and 150 rpm for two to three days. After this, the cells were centrifuged at 5000 × g for 10 min and suspended in physiological saline water. The bacterial concentration in the system was estimated in terms of optical density using a UV spectrophotometer at a wavelength of 550 nm. About 75 mg l−1 (0.15 OD) of bacterial concentration was added to seven identical conical flasks (triplicate) containing 100 ml of NB that was amended with a technical grade endosulfan concentration of 50 ppm. The studies were carried out in aerobic and facultative anaerobic conditions for 28 days at 28 ± 2◦ C with occasional shaking. To maintain the facultative anaerobic condition, nitrogen gas was flushed to the conical flask and sealed with an air-tight septum. At the end of 2, 5, 7, 10, 14, 21, and 28 days, 5 ml of sample was collected using a syringe from each conical flask and analyzed for endosulfan concentration using gas chromatography (PerkinElmer Clarus 500) with ECD (electron capture detector).

Bacterial Cell Density Measurement The mixed bacterial culture was grown to mid log phase in NB, and the cells were centrifuged at 5000 × g for 10 min. The bacterial pellets were washed twice and suspended in 100 ml of saline water (0.8% NaCl). About 0.1, 0.2, 0.3, 0.4, and 0.5 ml of above set solution was diluted and made up to 10 ml using fresh saline water. From each dilution, 3 ml of sample was filtered through Millipore filter paper (0.45 µm) in triplicate, and the filter paper was kept in the oven at 104◦ C for 3 h. The difference in weight (average weight) of the filter paper was recorded as bacterial cell density. The optical density of the corresponding dilutions was found by UV digital spectrometer in fixed-point measurement at a wavelength of 550 nm. Using bacterial cell density and the optical density, a standard graph was prepared, and thereafter the bacterial cell density in the samples was calculated using the standard graph.

Enrichment and Isolation of Bacterial Culture

Figure 2: Chromatograph of endosulfan and its metabolites at standard operating condition: 1. n-hexane, 2. endosulfan ether, 3. endosulfan lactone, 4. alpha endosulfan, 5. beta endosulfan, 6. endosulfan sulfate.

Endosulfan Degradation Studies with Pure Cultures The pure cultures were grown separately in nutrient medium at 28 ± 2◦ C and 150 rpm for two to three days. The grown cells were centrifuged, quantified as above, and the same bacterial concentration (75 mg l−1 , OD 0.15 at 550 nm) was added to nine identical conical flasks (triplicate) containing 100 ml of NB amended with 5 ppm of technical-grade endosulfan. The study was conducted in aerobic condition at 28 ± 2◦ C with occasional shaking, and the samples were collected at 0, 2, 5, 7, 10, and 14 days. The collected samples were analyzed for endosulfan and bacterial cell concentrations. Occasionally, the endosulfan concentration in bacterial cells was also measured by digesting and extracting the endosulfan using n-hexane.

Analysis of Endosulfan Endosulfan containing sample (5 ml) was extracted with 10 ml of n-hexane and shaken vigorously for 15 min in a standard separating funnel with Teflon stopper. The water layer was decanted carefully, and the supernatant was extracted with 5 ml of n-hexane two more times. Finally, the extracted sample was dehydrated by passing through anhydrous sodium sulfate and analyzed in PerkinElmer Clarus 500 gas chromatograph with electron capture detection (GC/ECD) equipped with autosampler, an on-column, split/splitless capillary injection system, and with PerkinElmer (PE)-35 capillary column (30 m × 0.53 mm × 0.5 µm film thickness). The operating conditions were as follows. The column was held initially at a temperature of 120◦ C for 1 min, then at 30◦ C min−1 to 180◦ C, then at 20◦ C min−1 to 240◦ C, and finally held at that temperature for 3 min. The temperature of injector and detector were maintained at 260◦ C and 300◦ C, respectively. Nitrogen was used as a carrier gas at a flow rate of 30 ml min−1 , and the injections were made in the split mode with a split ratio of 1:10. Under these conditions the retention time for alpha endosulfan, beta endosulfan, endosulfan sulfate, endosulfan ether, and endosulfan lactone was 6.31 min, 9.36 min, 11.95 min, 3.72 min, and 6.42 min, respectively (Fig. 2).

85

86

Kumar and Philip

Identification of Bacterial Cultures The bacterial strains were isolated from the mixed culture by repeated streaking on agar plates. The isolates were separated based on their morphology. Each colony was carefully separated and streaked on fresh agar plates, and this was repeated three times. Thereafter, the cultures were streaked on agar slants and preserved at 4◦ C for further experiments. The agar slants in triplicate were sent to Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India, for identification.

RESULTS AND DISCUSSION Degradation Studies in Aerobic and Facultative Anaerobic Conditions with Mixed Culture Endosulfan degradation studies were conducted at an initial endosulfan concentration of 50 mg l−1 amended in 200 ml of NB with 75 mg l−1 (OD 0.15 at 550 nm) of mixed bacterial culture in a batch system for a period of 28 days. At regular intervals, samples were collected from the aerobic and facultative anaerobic system and analyzed for endosulfan concentration in GC/ECD. The decrease in concentration of endosulfan in the system was considered as microbial degradation. The GC analysis and optical density measurements confirmed substantial removal of endosulfan with simultaneous increase in bacterial mass. Previous researchers reported that the removal of endosulfan took place simultaneously with the formation of intermediate metabolites like endosulfan sulfate, endosulfan lactone, endosulfan ether, endosulfan hydroxy ether, and endosulfan monoaldehyde. In the present study, none of the reported endosulfan metabolites were accumulated in the system. The metabolites might have formed and immediately degraded by the microbial consortium, or the pathway of endosulfan degradation might be entirely different. In the initial stages of the study, degradation of endosulfan in facultative anaerobic and aerobic systems were almost the same, and around 50% of the initial concentration was degraded within the first 7 to 10 days. The enriched mixed bacterial culture was able to degrade/mineralize alpha and beta isomers of endosulfan. At the end of 28 days, 10.01 mg l−1 of alpha endosulfan and 4.08 mg l−1 of beta endosulfan were remaining in the aerobic system, which corresponds to a total endosulfan degradation efficiency of 71.82 ± 0.2% with a maximum bacterial cell density of 355 mg l−1 at OD550 . The concentrations of alpha and beta endosulfan remaining and their percentage degradation in both aerobic and facultative anaerobic systems are shown in Figures 3a and 3b. The endosulfan degradation efficiency of the mixed culture was 71.4 ± 0.15% and 72.8 ± 0.05% for alpha and beta isomers, respectively, at the end of four weeks of incubation. The rate of alpha and beta endosulfan degradation was more in the first week,

Enrichment and Isolation of Bacterial Culture

Figure 3: Kinetics of alpha and beta endosulfan degradation in aerobic system (a) and facultative anaerobic system (b).

87

88

Kumar and Philip

and it reduced considerably in the second and third week of incubation. After that, the rate of degradation was insignificant, and the residual alpha and beta endosulfan concentration remained almost the same (Figs. 3a and 3b). However, the total endosulfan degradation at the end of 28 days was more in facultative anaerobic system compared to aerobic system, even though the bacterial cell density in the facultative anaerobic system was less compared to aerobic system. It is reported that the occurrence of dehalogenation is faster in anaerobic process. Almost 76.04 ± 0.2% of the initial endosulfan concentration disappeared from facultative anaerobic system (Fig. 4), which is 5.9 ± 0.6% more than that of aerobic system. Also in facultative anaerobic system, half of the initial endosulfan concentration was degraded within the first 7 to 10 days. Degradation of alpha and beta endosulfan was almost the same in aerobic system, but in the case of facultative anaerobic system the percentage of alpha endosulfan degradation (76.51 ± 0.15%) was slightly more compared to degradation of beta endosulfan (74.91 ± 0.05%). This may be due to stereoisomerism, where the enzymes released from bacterial system may be active toward one of the stereo isomers. While studying endosulfan degradation by various microbes, it is reported that fungal cultures removed more alpha endosulfan compared to beta endosulfan, while bacteria degraded more beta endosulfan compared to alpha endosulfan.[8] But Awasthi, Manickam, and Kumar[12] reported that degradation of beta endosulfan was less compared to alpha endosulfan. These observations reflect that stereoisomerism and the release of enzymes from the bacterial/fungal systems play a major role in degrading endosulfan. Sutherland et al.[15] reported that degradation of endosulfan can be achieved via oxidation and hydrolysis pathways, but it leads to the formation of toxic endosulfan sulfate and less toxic endosulfan diol, respectively. But throughout the present study, the intermediate metabolites (endosulfan sulfate, endosulfan lactone, endosulfan diol, endosulfan ether, endosulfan hydroxy ether, and endosulfan monoaldehyde) reported by previous researchers [11,15,16,18,19] were not observed. In this study, the loss due to dissipation was checked by the help of control reactors (without bacterial culture and with endosulfan). It was observed that the abiotic loss in the system was negligible (Fig. 5). During the study, samples were collected at regular intervals from aerobic and facultative anaerobic systems, and the bacterial culture was separated by centrifugation. The bacterial pellets were suspended in distilled water and mixed thoroughly in a vortex mixer, sonicated, extracted with n-hexane, and analyzed for endosulfan concentration. The endosulfan concentration in bacterial cell extracts was negligible (0.01 mg l−1 ). This result shows that endosulfan was used completely by the bacterial culture for metabolic activity, and the chance of bioaccumulation of endosulfan as the reason for disappearance of endosulfan from the NB can be ruled out.

Enrichment and Isolation of Bacterial Culture

Figure 4: Degradation of endosulfan in aerobic and facultative anaerobic systems without supplementary carbon.

Endosulfan is highly immiscible in water. In order to get higher concentration in water, methanol was used as a solvent. The addition of methanol to the system created more chemical oxygen demand and also acted as a carbon source for the microbes. But methanol is a less preferred substrate for the microbial consortia compared to dextrose, which is evident from the growth kinetics (Fig. 5).

Influence of Supplementary Carbon on Endosulfan Biodegradation The degradation studies were repeated with the addition of dextrose (supplementary carbon) to check the influence of auxiliary carbon on endosulfan degradation. Awasthi, Manickam, and Kumar[12] reported that the addition of glucose did not increase the degradation efficiency of endosulfan. Hence, dextrose was used as an auxiliary carbon source in the present study. The addition of dextrose (1 g l−1 ) increased the concentration of bacterial cells and the degradation efficiency of endosulfan from 71.82 ± 0.2% to 81.42 ± 0.2% in aerobic system and from 76.04 ± 0.2% to 85.42 ± 0.2% in facultative anaerobic system (Fig. 5). Significant increase in bacterial cell density was observed in the first

89

90

Kumar and Philip

Figure 5: Degradation of endosulfan in aerobic and facultative anaerobic systems with supplementary carbon source.

two days due to the addition of dextrose (Fig. 5), which increased the endosulfan degradation efficiency from the initial stages itself. The enhancement in the degradation efficiency due to supplementary carbon was 13.36 ± 0.6% in aerobic system and 12.33 ± 0.6% in facultative anaerobic system, respectively. In aerobic system, without dextrose, alpha and beta endosulfan degradation efficiency was 71.4 ± 0.15% and 72.8 ± 0.05%, respectively. The addition of dextrose (aerobic co-metabolic system) to the aerobic system amplified the alpha endosulfan degradation by 15.56 ± 0.5% (71.4 ± 0.15% to 82.51 ± 0.15%) and beta endosulfan degradation by 8.3 ± 0.15% (72.8 ± 0.05% to 78.86 ± 0.05%). On the other hand, in facultative anaerobic system the degradation of both the isomers was reached around 85.5%, and the amplification in alpha and beta endosulfan degradation efficiency was 11.58 ± 0.5% (76.51 ± 0.15% to 85.37 ± 0.15%) and 14.15 ± 0.15% (74.93 ± 0.05% to 85.53 ± 0.05%), respectively (Fig. 6). From the results it is clear that the addition of supplementary carbon increased the degradation rate of both alpha and beta endosulfan in aerobic and facultative anaerobic systems. Several researchers have observed that the addition of auxiliary carbon to the system having xenobiotic compounds increased the biodegradation

Enrichment and Isolation of Bacterial Culture

Figure 6: Degradation of alpha and beta endosulfan in aerobic and facultative anaerobic systems with supplementary carbon.

potential of bacterial and fungal cultures. But some of the researchers observed no such increase in degradation efficiency due to the addition of secondary carbon.[12,20] The addition of methanol to the system (as a solvent) would create more chemical oxygen demand and also act as a carbon source for the microbes. In such cases, the addition of supplementary carbon source to the system may not be useful to the microbes if the microbes are able to use methanol effectively, and obviously in such cases, no increase in degradation efficiency can be observed. The mixed bacterial consortium used in the present study was preferring dextrose over methanol as the substrate, which was evident from the growth kinetics (results not shown). The maximum specific growth rate of the mixed culture was 0.0395 h−1 while using dextrose, whereas the specific growth rate was only 0.0137 h−1 when methanol was the auxiliary carbon source. The use of multiple substrates did not change the metabolism of the cultures, and during degradation of endosulfan no intermediate metabolites were observed. Isolation of relevant enzymes and genes from the mixed bacterial culture and comparison with those of other endosulfan degrading bacteria may give insight into the pathway/mechanism of endosulfan degradation. The investigations are in progress for determining the pathway of endosulfan degradation by the isolated pure strains as well as the mixed bacterial consortium.

91

92

Kumar and Philip

Bacterial Identification The bacterial cultures isolated from the mixed microbial consortium were identified as Staphylococcus sp., Bacillus circulans–I, and Bacillus circulans– II through Microbial Type Culture Collection (MTCC), Chandigarh, India, as MTCC 6801, MTCC 6802, and MTCC 6803. Also, these cultures are preserved in the gene bank of MTCC. All cultures were Gram-positive endosporeforming rods (except Staphylococcus sp. (non-endospore-forming cocci) and round shaped. All the cultures can hydrolyze starch and grow in the presence of 2.5% NaCl. Negative tests include indole formation, citrate utilization, and growth in 10% NaCl. The favorable growth temperature lies between 25◦ C and 42◦ C and can grow favorably in a range of pH between 5 and 11. Some of the other characteristics of the cultures are given in Table 1.

Degradation Studies with Pure Cultures Under Aerobic Condition From the mixed culture studies it was observed that the utilization of alpha endosulfan was more in aerobic condition, and utilization of beta endosulfan was more in facultative anaerobic condition. The main objective of pure culture study was to investigate the endosulfan degradation ability of individual cultures and their rate of alpha and beta endosulfan degradation.

Table 1: Characteristics of bacterial culture. Character Parameter Configuration Gram’s reaction Shape Size Arrangements Endospore Shape Motility Growth under anaerobic condition Gas production from glucose Casein hydrolysis Starch hydrolysis Urea hydrolysis Nitrate reduction Nitrite reduction H2 S production Cytochrome oxidase Catalase test Oxidation/fermentation (O/F) Gelatin hydrolysis Arginine dihydrolase

Staphylococcus sp.

Bacillus circulans–I

Bacillus circulans–II

Round + ve Cocci

Round + ve Rods Long Single + Oval + − − ± + − + + − − + − + +

Round + ve Rods Long Single + Oval + − − ± + − + + − + + − + +

Groups − ± + − ± + − − − − − + F − +

Enrichment and Isolation of Bacterial Culture

Figure 7: Degradation of total endosulfan by pure cultures.

The degradation of endosulfan was almost the same with all three pure cultures studied. At the end of 14 days of incubation, total endosulfan degradation efficiency of 87.8 ± 0.2%, 88.1 ± 0.2%, and 88.4 ± 0.2%, was achieved by Staphylococcus sp., Bacillus circulans I and II with corresponding final bacterial cell density of 285 mg l−1 , 275 mg l−1 , and 280 mg l−1 , respectively (Fig. 7). Alpha endosulfan was utilized up to 93.3 ± 0.15%, and 93.4 ± 0.15% by Bacillus circulans I and II , respectively, at the end of 14 days (Fig. 8a), which was 3.74 ± 0.5% and 3.84 ± 0.5% more compared to the degradation efficiency achieved by Staphylococcus sp. (89.95%). On the other hand, beta endosulfan was utilized more by Staphylococcus sp. that was 9.14 ± 0.15% and 8.04 ± 0.15% more compared to the degradation efficiency achieved by Bacillus circulans I and II. At the end of 14 days of incubation 82.9 ± 0.05%, 75.96 ± 0.05% and 76.73 ± 0.05% of beta endosulfan degradation efficiency was achieved by Staphylococcus sp., Bacillus circulans I and II, respectively (Fig. 8b). These results were in good agreement with our earlier observations. It is evident from the bacterial identification tests that the growth of Staphylococcus sp. is more favorable in facultative anaerobic condition compared to other Bacillus cultures. Staphylococcus sp. might be mainly responsible for the degradation of endosulfan in

93

94

Kumar and Philip

Figure 8: Degradation of alpha endosulfan (a) and beta endosulfan (b) by pure cultures.

Enrichment and Isolation of Bacterial Culture

facultative anaerobic system whereas Bacillus circulans I and II degraded majority of endosulfan in aerobic system.

CONCLUSION The isolated mixed bacterial culture was able to mineralize endosulfan without the formation of any intermediate metabolites reported by previous researchers. Also, the culture was able to work in both aerobic and facultative anaerobic environments. Staphylococcus sp. utilized more beta endosulfan in facultative anaerobic system, whereas Bacillus circulans I and II preferred more alpha endosulfan in aerobic system. The addition of dextrose increased the degradation efficiency of endosulfan in both aerobic and facultative anaerobic systems. These microbial consortiums can effectively be used to degrade endosulfan from contaminated soils, sediments, and wastewaters.

REFERENCES 1. Kannan, S.T.; Sengupta, R. Organochlorine residues in zooplankton of Saurashtra coast, India. Mar. Pollut. Bull. 1987, 18, 92–104. 2. Rajendran, R.B.; Karunagaran, V.M.; Babu, S.; Subramanian, A.N.; Mohan, D. Levels of chlorinated insecticide in fishes from the Bay of Bengal. Mar. Pollut. Bull. 1992, 24, 567–570. 3. Mansingh, A.; Wilson, A. Insecticide contamination of Jamaican environment. Baseline studies on the status of Kingston Harbour. Mar. Pollut. Bull. 1995, 30, 640–645. 4. Miles, C.J.; Pfeuffer, R.J. Pesticides in canals of south Florida. Arch. Environ. Contam. Toxicol. 1997, 32, 337–345. 5. Shailaja, M.S.; Nair, M. Seasonal differences in organochlorine pesticide concentrations of zooplankton and fish in the Arabian Sea. Mar. Pollut. Bull. 1997, 44, 263–274. 6. Sujatha, C.H.; Nair, S.M.; Chacko, J. Determination and distribution of endosulfan and malathion in an Indian estuary. Water Res. 1999, 33(1), 109–114. 7. Bhattacharya, B.; Sarkar, S.; Mukherjee, N. Organochlorine pesticide residues in sediments of a tropical mangrove estuary, India: Implications for monitoring Environ. Int. 2003, 29, 587–592. 8. Siddique, T.; Okeke, B.C.; Arshad, A.; Frankenberger, W.T., Jr. Enrichment and isolation of endosulfan-degrading microorganisms. J. Environ. Qual. 2003, 32, 47–54. 9. Miles, J.R.W.; Moy, P. Degradation of endosulfan and its metabolites by a mixed culture of soil microorganisms. Bull. Environ. Contam. Toxicol. 1979, 23, 13–19. 10. Mukherjee, I.; Gopal, M. Degradation of beta-endosulfan by Aspergillus niger. Toxicol. Environ. Chem. 1994, 46, 217–221. 11. Kullman, S.W.; Matsumura, F. Metabolic pathway utilized by Phenerochete chrysosporium for degradation of the cyclodine pesticide endosulfan. Appl. Environ. Microbiol. 1996, 62, 593–600. 12. Awasthi, N.; Manickam, N.; Kumar, A. Biodegradation of endosulfan by a bacterial co-culture. Bull. Environ. Contam. Toxicol. 1997, 59, 928–934.

95

96

Kumar and Philip 13. Guerin, T.F. The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments. Environ. Pollut. 1999, 106, 13–21. 14. Shetty, P.K.; Mitra, J.; Murthy, N.B.K.; Namitha, K.K.; Savitha, K.N.; Raghu, K. Biodegradation of cyclodiene insecticide endosulfan by mucor-thermo-hyalospora MTCC 1384. Current Sci. 2000, 79(9), 1381–1383. 15. Sutherland, T.; Horne, I.; Lacey, M.; Harcourt, R.; Russell, R.; Oakeshott, J. Enrichment of an endosulfan-degrading mixed bacterial culture. Appl. Env. Microbiol. 2000, 66(7), 2822–2828. 16. Awasthi, N.; Singh, A.K.; Jain, R.K.; Khangarot, B.S.; Kumar, A. Degradation and detoxification of endosulfan isomers by a defined co-culture of two Bacillus strains. Appl. Microbiol. Biotechnol. 2003, 62(2–3):279–283. 17. Kwon, G.; Kim, J.; Kim, T.; Sohn, H.; Koh, S.; Shin, K.; Kim, D. Klebsiella pneumoniae KE-1 degrades endosulfan without formation of the toxic metabolite, endosulfan sulfate. FEMS Microbiol. Lett. 2002, 215, 255–259. 18. Martens, R. Degradation of (8–9 Environ. Microbiol. 1976, 31, 853–858.

14

C) endosulfan by soil microorganisms. Appl.

19. Katayama, A.; Matsumura, F. Degradation of organochlorine pesticides, particularly endosulfan by Trichoderma harzianum. Environ. Toxicol. Chem. 1993, 12, 1059– 1065. 20. Awasthi, N.; Ahuja, R.; Kumar, A. Factors influencing the degradation of soil applied endosulfan isomers. Soil. Biol. Biochem. 2000, 32 (11–12), 1697–1705.