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Immunodetection of Dichlorodiphenyltrichloroethane and its biodegradation by microbial consortium
Deepthi, N. (2010) Immunodetection of Dichlorodiphenyltrichloroethane and its biodegradation by microbial consortium. PhD thesis, University of Mysore.
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Abstract
1, 1, 1- trichloro- 2, 2- bis- (4- chlorophenyl) ethane (DDT) is one of the highly persistent and recalcitrant organochlorine pesticides synthesized and used by man. This pesticide has been listed as one of the highly toxic chemical by EPA. It is shown to have mutagenic, carcinogenic and teratogenic effects on man. With continuous usage, the pesticide enters the human body via food chain. Residues of DDT have been detected in soil, water and air. Residues of DDT are generally analyzed by thin layer chromatography, gas chromatography which are expensive, labour intensive, require extensive clean up and are nor suited for on field assay. Immunoassay is highly sensitive, specific, on- field method, does not need sample clean up and has high throughput of samples. In our lab we attempted to develop an immunoassay for the detection of DDT by using both rabbit and avian antibodies. DDT levels up to 1 ng could be detected by Dot-ELISA. By signal amplification technique the detection limit could by improved to 1 pg level. A microbial consortium capable of degrading DDT was isolated in the laboratory by long term enrichment. The consortium was made of 10 bacterial isolates of which 7 belonged to Pseudomonas sp. and one each of Flavobacterium , Vibrio and Burkholderia sp. Conditions were optimized by response surface methodology. The maximum predicted percentage degradation of 96.69, 96.37, 97.97, 92.44 and 98.19 was obtained respectively for 5, 10, 20, 30 and 35 ppm initial DDT concentrations at different pH levels 7.82, 6.59, 6.92, 7.06 and 8.00 with an incubation temperature of 25 0C and inoculum concentration of 1500 mg protein/ mL. The degradation of DDT by the microbial consortium increased with time. The degradation reached 95% by 72 h of incubation. Primers designed for identifying DDT-dehydrohalogenase genes gave positive amplification with genomic DNA and plasmid DNA of Flavobacterium sp. The PCR product of genomic DNA of Flavobacterium sp. was ligated to pTZ57R/T vector and cloned in to E.coli DH5a. The sequence analysis of Flavobacterium species T6 DDT-dehydrohalogenase gene PCR amplified insert indicated that the protein had 272 residues. DDT- dhl 1 gene had 24- 26% identity with Ps. putida genome. The DDT-dhl 1 gene showed homology with acetolactate v synthase gene sequence. The southern blot analysis of genomic DNA from Flavobacterium species for the DDT- dhl 1 probe was done. The genomic DNA of Flavobacterium species yielded positive hybridisation. The cloned PCR product was expressed in E. coli BL21 using pET28a vector. The expressed protein showed positive amplification in western blot analysis.
| Item Type: | Thesis (PhD) |
|---|---|
| Uncontrolled Keywords: | DDT, organochlorine pesticides, immunodetection,DDT, Degradation |
| Subjects: | 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 26 Pesticide Chemistry 500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 04 Biosynthesis |
| Divisions: | Fermentation Technology and Bioengineering |
| Depositing User: | Food Sci. & Technol. Information Services |
| Date Deposited: | 01 Mar 2011 04:52 |
| Last Modified: | 28 Dec 2011 10:21 |
| URI: | http://ir.cftri.res.in/id/eprint/9941 |
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