Immunodetection of Dichlorodiphenyltrichloroethane and its biodegradation by microbial consortium

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.