Differential analysis of pesticides biodegradation in soil using conventional and high-throughput technology

A potential pesticide degrading bacterial isolate (2D), showing maximum tolerance (450 ppm) for cypermethrin, fipronil, imidacloprid and sulfosulfuron was recovered from a pesticide contaminated agricultural field. The isolate degraded cypermethrin, imidacloprid, fipronil and sulfosulfuron in minimal salt medium with 94, 91, 89 and 86 % respectively as revealed by HPLC and GC analysis after 15 days of incubation. Presence of cyclobutane, pyrrolidine, chloroacetic acid, formic acid and decyl ester as major intermediate metabolites of cypermethrin biodegradation was observed in GC-MS analysis. Results based on 16S rDNA sequencing, and phylogenetic analysis showed maximum similarity of 2D with Bacillus cereus (MH341691). Stress responsive and catabolic/ pesticide degrading proteins were over expressed in the presence of cypermethrin in bacteria. Enzyme kinetics of laccase was deduced in the test isolate under normal and pesticide stress conditions. Amplification of laccase gene showed a major band of 1200bp. Maximum copy number of 16S rDNA was seenin uncontaminated soil as compared to pesticide contaminated soil using qRT-PCR. The metagenome sequencing revealed reduction in the population of proteobacteria in contaminated soil as compared to uncontaminated soil but showed dominance of actinobacteria, firmicutes and bacteriodates in pesticide spiked soil. Presence of some new phyla like chloroflexi, planctomycetes, verrucomicrobia was observed followed by extinction of acidobacteria and crenarchaeota in spiked soil. The present study highlights on the potential of 2D bacterial strain i.e., high tolerance level of pesticide, effective biodegradation rate, and presence of laccase gene in bacterial strain 2D, could become a potential biological agent for large-scale treatment of mixture of pesticide (cypermethrin, fipronil, imidacloprid and sulfosulfuron) in natural environment (soil and water).