ANNOUNCEMENTS
Endosulfan, a broad-spectrum insecticide consists of the a-and b-isomers. Widespread use of the insecticide endosulfan has led to the environmental accumulation of its residues (typical value ranging from 1-6 ppm) including those of its metabolite endosulfan sulfate, which are persistent and toxic to several environmental species. Thus there is an urgent need to find ways to degrade and detoxify this compound. Also as the understanding of the ways in which this compound might be degrade is limited, the present study was to understand the different routes through which endosulfan might be degraded and isolate a suitable strain with a high ability to degrade endosulfan.
Initially agriculture soils (A1, A2 and A3) were collected and used for enrichment and isolate of endosulfan degrading strains using 3 different media Rich Medium (RM, endosulfan as a cosubstrate), Sulfur Free Medium (SFM, endosulfan as a sulfar source) and Minimal Salt Medium (MSM, endosulfan as a carbon source). However, out of the 18 enrichment cultures initiated only 2 initiated with A1 and A2 soils in RM (designated as Consortium 1 and Consortium 2) displayed endosulfan degradation with the formation of the intermediate endosulfan diol. The eight strains (RM1-RM8) isolated from these consortia displayed 11.5% - 46.7% degradation of endosulfan. However abiotic losses and formation of endosulfan diol was also observed control flasks that consisted of sterile medium. Another experiment was undertaken to observed the role of pH on the transformation of endosulfan. The results revealed that while abiotic losses occurred due to volatilization etc the specific chemical hydrolysis of endosulfan to endosulfan diol occurred at pH 7 and above. In order to minimize the chemical hydrolysis to endosulfan diol, the present RM was modified (M-RM) to include buffering capacity and reducing initial pH. In this case it was observed that while the chemical transformation of endosulfan to endosulfan diol was eliminated in sterile controls and the pH of the cultures were lower than in RM, the actual degradation capacities of these strains had further reduced in M-RM (4.25% to 32.5%) in comparison to those in RM. Moreover the pH of strain RM2 still was in the alkaline value of 7.3 and displayed the presence of endosulfan diol the indicating that degradation observed could still be attributed to abiological losses. Since it was unclear if strains RM2 would be able to remediate endosulfan in acidic soils due to the high buffering capacity of soils, at this point in the study another soil sample P1 was procured that had longer exposure to endosulfan contamination and higher residue content in comparison to the agriculture soils.
When the P1 soil sample was used to enrich for endosulfan degrading cultures, degrading consortia were developed in M-RM (Consortium 3), SFM (Consortium 4) and MSM (Consortium 5) only when 4 mg/1 endosulfan concentration was used indicating that the 10 mg/1 concentration was toxic to indigenous strains. Consortium 5 displayed highest endosulfan degradation followed by Consortium 4 and Consortium 3 along with the development of various intermediates in each case. Overall 10, 13 and 4 strains were isolated from Consortium 3, 4 and 5 respectively, including a single fungal strain from Consortium 3. Individual strains isolated from the various consortia were tested for their degradation capacities. It emerged that from Consortium 3 and Consortium 4 strain Pseudomonas aeruginosa M-RM6 and Pseudomonas aeruginosa SFM4 were the highest degraders from their respective consortia and degraded endosulgan by 42.9% and 43.7% respectively. Intermediates observed in the case of both these strains included endosulfan diol, endosulfan ether and endosulfan lactone. From Consortium 5 strain Achromobacter xylosoxidans ES9 displayed highest degradation efficiency of 54.5% amongst all isolated strains with the formation of endosulfan ether and endosulfan lactone.
Some isolates, including the fungal strain did produce endosulfan sulfate as an intermediate metabolite and so they were not taken forward as they also displayed lower degradation capacities.
Growth and degradation rate studied were undertaken for selected strains P.aeruginosa M-RM6 and P.aeruginosa SFM4 and A. xylosoxidans ES9 revealed that the latter exhibited highest degradation rates for both the a-and b-isomer, with the a-isomer being degraded to a greater extent. Strains P.aeruginosa M-RM6 and P.aeruginosa SFM4 degraded endosulfan by nearly comparable levels although strain P.aeruginosa SFM4 displayed preferential utilization for the b-isomer. As the best degrader of endosulfan strain A. xylosoxidans ES9 was chosen for optimization studies. Optimization of growth parameters revealed that increase in inoculum size (to 500 µl), addition of 0.05g/l yeast extract and a pre-existing concentration of tween 80 enhanced the degradation capacity of strain A. xylosoxidans ES9 to 85.8%. A time course study of the intermediates produced by A. xylosoxidans ES9 in MSM revealed the production of endosulfan lactone on day 4, the concentrations of which increased until day 8after which its levels declined until day 2.0 Endosulfan ether appeared on day 6 and its concentrations increased until day 10 after which they declined until the end of the incubation period. GCMS analysis confirmed the identity of the intermediates.
An attempt was also made to enrich and isolate for endosulfan sulfate degrading strains from PI soil using SFM and MSM. It was observed that although individual consortia degraded endosulfan sulfate none of the stains isolated from the consortia exhibited degradation capacity for endosulfan sulfate individually, indicating that key degrading strains were lost during isolation on plates.
Screening of the selected strains for the presence of the esd monooxygenase gene isolated previously in and endosulfan degrading Mycobacterium strain was examined by developing degenerate primers for the amplification of a 900 bp fragment of the original gene. PCR amplification in strains A. xylosoxidans ES9 and O. anthropi ES3 revealed single bands at 900 bp however multiple bands were observed in strains P.aeruginosa M-RM6 and P.aeruginosa SFM4 despite standardization of various parameters. Deduced amino acid sequence of the sequenced gene fragment from A. xylosoxidans ES9 revealed 33% homology to a monooxygenase of the dszC family while that of strain O. anthropi ES3 displayed homology of 29% to the monooxygenase of the esd gene.
In order to study the diversity of microbial flora in the P1 soil sample and well as in Consortium 3, Consortium 4 and Consortium 5 DGGE technique was used. It was observed that the PI soil sample had considerably more diversity that the individual consortia indicating enrichment bias during cultivable techniques. Amongst the 3 consortia result indicated that Consortium 5 enriched using MSM and endosulfan as the carbon source had a higher bacterial diversity than Consortium 3 and Consortium 4 developed using M-RM and SFM respectively. Similar results were also seen in culture dependent studies where the 4 strains isolated from MSM were of different genera.
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