Isolation, Identification And Degradative Properties Of Bacterial Strains For Chlorpyrifos

Organophosphorus is one of the most widely used insecticides in the world. It could contaminate the environment and harm the health of human while improve the crop yield. There were many studies conducted on the microbial remediation of chlorpyrifos, and most of them focused on the isolation, characterization, degrading abilities of pesticide-degrading strains. But the degradative efficiation of some of microorganisms is lower, so it is necessary to expand the resource of biodegradative bacterium.In this study, we isolated three chlorpyrifos-degrading bacterial strains by enrichment culturing and screening and found that all of them could use chlorpyrifos as the carbon source. Then we studied the chlorpyrifos-degrading strain CP1, including its characterization, growth curve, optimized the main factors which influence the chlorpyrifos-degrading efficiency, metabolites analysis and the bidegradation gene clone. The main results are as follows:1 From the activated sludge samples obtained from pesticide manufacturing plant, three chlorpyrifos -degrading bacterial bacterial strains were isolated and named in CP1, CP6 and CP7, respectively. Then based on the results of morphology, and 16S rDNA analysis, the CP1 was identified as Ochrobactrum anthropi, the CP6 was identified as Pseudomonas sp., the CP7 was identified as Burkholderia sp. Their GenBank accession numbers are HQ589348, JF280144 and JF280145, respectively.2 Both the orthogonal experimental design and Box?Behnken design were employed to optimized the main factors which influences the chlorpyrifos -degrading efficiency by strain CP1. The optimum conditions for chlorpyrifos biodegradation were as follows: the initial concentration of chlorpyrifos was 100 mg/L, the pH was 7.0, and the culturing temperature was 28.5℃. Under the optimum condition, the biodegradation efficiency of chlorpyrifos increased from 70.26% to 75.18%. Therefore, the optimization of chlorpyrifos-degrading condition could improve the biodegradation efficiency of chlorpyrifos by Ochrobactrum anthropi strain CP1.3 The main metabolite of chlorpyrifos-degrading strain CP1 was analysed by chromatography and mass spectrometry. There was a new metabolite instead of TCP, which as the metabolite of chlorpyrifos- degrading strains in past reports. It could be induced that the new metabolite was produced because of the break of P-S bond of chlorpyrifos. The result showed that there maybe a new gene in the strain CP1, which could code a new degradation enzyme for chlorpyrifos-degrading.4 The plasmid amplification of strain CP1 was employed to determine the location of gene. The result indicated that the degrading gene in strain CP1 might not locate in the plasmid. SDS-PAGE analysis of the total proteins of bacterial strain under induced and noninduced cultures indicated that the degrading enzymes in strain CP1 might constitutively expressed.