Bioprospecting of chlorpyrifos degrading bacteria for bioremediation of apple orchard soils of Himachal Pradesh
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Date
2015
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YSPU
Abstract
Organophosphorus compounds are the most widely used insecticides, accounting for an estimated 34% of worldwide insecticide sales. Chlorpyrifos is a non-systemic organophosphorus insecticide, with broad spectrum insecticidal activity. Contamination of soil from pesticides as a result of their bulk handling at the farmyard or following application in the field or accidental release may lead occasionally to contamination of surface and ground water. The present study was carried out to explore the status of indigenous chlorpyrifos degrading bacteria in the apple orchard soils in the state of Himachal Pradesh, which harbours plenty of diverse forms of microbial life, which has remained largely unexplored till date. The pH and electrical conductivity (EC) of the thirty soil samples from apple orchards were recorded and ranged from 6.55-
7.58 and 0.24-0.39 dSm-1 respectively. The percent organic carbon (% OC), nitrogen (N), phosphorus (P) and potassium (K) content ranged from 0.66-0.116%, 302-486 Kg/ha, 12.8-23.6 Kg/ha and 255-371 Kg/ha respectively. A total of 72 bacterial isolates were isolated from 30 soil samples using enrichment technique and mineral salt medium. These 72 isolates were rescreened on selective medium i.e. eosin-methylene blue agar (EMBA) containing 50 mg/l chlorpyrifos and only 15 isolates were able to change the colour of colonies from creamish/yellowish/whitish/transparent to red, which was observed as an indication of chlorpyrifos degradative ability of these bacterial isolates. These 15 bacterial isolates were characterized
biochemically alongwith organophosphorus hydrolase activity and 6 bacterial isolates exhibiting highest extracellular OPH activity of 0.012-0.165 U/ml were selected. Molecular characterization of selected isolates was carried out using RAPD-PCR and 16S rrna gene technology. Only 5 bacterial isolates were amplified by universal primers and in silico analysis of 16S rrna gene sequence led to identification of these bacterial isolates as Pseudomonas indoloxydans ASK3.2, Pseudomonas aeruginosa ASR1.1, Pseudomonas resinovorans AST2.2, Pseudomonas otitidis AST3.2 and Pseudomonas stutzeri ASM1.1.
Two bacterial strains Pseudomonas indoloxydans strain ASK3.2 and Pseudomonas resinovorans strain AST2.2 exhibited mottled growth in form of colonies on agar plates fortified with 1600 mg/l concentration of chlorpyrifos and these two bacterial strains were selected for optimization and biodegradation experiments. In both strains highest OPH activity was observed at pH 8.0, temperature 37°C, after 48hrs in presence of glucose as carbon source. Activity of extracellular OPH enzyme in both the strains was found to be enhanced in presence of Co+2 metal ions, while Cu+2, Mg+2 and Fe+2 ions had showed lesser effect on OPH activity. GC-FID results confirmed that Pseudomonas indoloxydans strain ASK3.2 is a more efficient degrader as 82.72% chlorpyrifos was degraded successfully as compared to 43.90% by Pseudomonas resinovorans strain AST2.2. GC-MS analysis results confirm that peak area/height in both test samples was found to be lower than in control sample, indicating decreasing amount of chlorpyrifos due to its degradation into smaller intermediate compounds. Only one metabolite i.e. phosphoric acid, diethyl 3,5,6-trichloro-2-pyridyl ester (C9H11Cl3NO4P) with CAS no. 5598-15-2 was identified by NIST library search in both test samples at RT 11.10 and RT 11.11 respectively.
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Keywords
bacteria, sampling, enzymes, selection, pesticides, genes, poultry equipment, fungi, soil sampling, dna