Biodegradation Of Chlorpyrifos By Microbial Consortium And Its Study On Degradation Of Chlorpyrifos

Chlorpyrifos is a broad spectrum moderately toxic organophosphorus insecticide.In recent years,with the restrictions on the use of some highly toxic organophosphorus compounds,chlorpyrifos is increasingly used in the contrrol of insect pests of economically important crops,However,the large amount of chlorpyrifos used causes excessive accumulation in the soil and water,Leading to serious pollution to the environment.Compared with the traditional physical and chemical methods(photolysis,fenton oxidation,chemical oxidation,and heterogeneous photocatalytic degradation,etc.)to eliminate organic phosphorus pollution in the environment,bioremediation has the characteristics of high efficiency,lbw cost,non-pollution,and non-secondary residues.Therefore,it has become a hot topic in the field of environmental remediatioaIn this study,the activated sludge samples collected from the pesticide production company in Hubei Province was used for domestication that tolerant chlorpyrifos.After six months of domestication,the characteristics of microbial communities in the process of domestication of activated sludge were analyzed,and three strains of degrading chlorpyrifos and 3,5,6-trichloro-2-pyridinol(TCP)were screened from activated sludge.The strains were compounded to obtain a microbial consortium(MLCF7)that efficiently degraded chlorpyrifos.Further,the characteristics of degradation of chlorpyrifos by the microbial consortium were studied.The conditions for degrading chlorpyrifos by microbial consortium were optimized,the effect of microbial consortium on the degradation of TCP was discussed,and the metabolic intermediates of degrading chlorpyrifos were analyzed by HPLC and also characterized by FTIR The surface morphology of microbial consortium in the process of degrading chlorpyrifos was observed.The bioassay of the metabolites of chlorpyrifos by microbial consortium was performed.Finally,the effect of degrading chlorpyrifos in soil by microbial consortium was studied.Through high-throughput sequencing methods,two environmental factors,chlorpyrifos at different concentrations and activated sludge from different sources,were studied to affect the microbial population and community structure in activated sludge.The experimental results showed that with the increase of chlorpyrifos concentration during the acclimation process,some strains that effectively degraded chlorpyrifos in activated sludge were enriched and the community structure of the bacterial community was changed as well as the community structure of activated sludge samples from different sources also had significant differences.When the concentration of chlorpyrifos was 400 mg/L,the main dominant bacteria in activated sludge MLCa were Paracoccus sp,Enterobacter sp,and Methylobacillus sp,etc.in activated sludge MLCp were Klebsiella sp,Pseudoxanthomonas sp,and Achromobacter sp,etc.in activated sludge MLCs were Serratia sp,Bacillus sp,and Pseudomonas sp,etc.The suspensions of activated sludge(MLCa,MLCp,MLCs)were streaked on MSM plates with chlorpyrifos and TCP as the sole carbon source,and obtain seven strains(MLCp-1,MLCp-2,MLCp-3,MLCa-1,MLCa-2,MLCs-1 and MLCs-2)that simultaneously degrade chlorpyrifos and TCP,After 7 d cultivated,the degradation efficiency of MLCp-2,MLCa-1 and MLCs-1 from three different activated sludge single strains for 100 mg/L chlorpyrifos reached 51.85%,53.71%and 65.37%,respectively.The strains was identified by16S rRNA gene sequence analysis,Sequence alignment showed that MLCp-2 is possible belongs to Klebsiella sp,MLCa-1 is possible belongs to Paracoccus sp,and MLCs-1 is possible belongs to Serratia sp.Further,the three strains were compounded in a ratio of 1:1:1 to obtain a microbial consortium(MLCF7),the degradation efficiency of 100 mg/L chlorpyrifos by microbial consortium was significantly improved,the degradation rate reached 80.96%after 7 d cultivated.The conditions for the degradation of chlorpyrifos by the microbial consortium(MLCF7)were optimized.The results showed that MLCF7 completely degraded 50 mg/L chlorpyrifos within 7 days,while the degradation rate of 250 mg/L chlbrpyrifos was only about 30.54%.The optimum pH and temperature of MLCF7 degraded chlorpyrifos were 8 and 31 ? and the degradation rates were 88.79%and 86.77%,respectively.In addition,the addition of additional substances(glucose,tryptone and yeast extract)can promote the degradation of chlorpyrifos by MLCF7.MLCF7 also could degraded the intermediate metabolite TCP of chlorpyrifos,and the degradation efficiency of 50 mg/L TCP reached about 62%after 7 d cultivated.The mechanism of degradation of chlorpyrifos by MLCF7 was studied.The results of HPLC showed that from the beginning of degradation to the 3th d,the liquid-phase peak of TCP was increased gradually.At 3th d,the TCP peak reached its maximum and then decreased,and on the 7th day,the liquid-phase peaks of chlorpyrifos and TCP disappeared;Fourier transform infrared spectroscopy was used to characterize the degradation of intermediate metabolites of chlorpyrifos by MLCF7.After 7th days,the main characteristic peak of chlorpyrifos(836.95,954.59,1018.23,1405.85,and 1536.98 cm-1)disappeared,indicating that it was transformed into other small molecules;By electron microscopy,the surface morphology of MLCF7 cells was observed under different concentrations of chlorpyrifos treatment.When the concentration of chlorpyrifos was 500 mg/L,the morphology of MLCF7 cells deformed from elliptical to rod-shaped.Using Drosophila melanogaster W118 as an indicator organism to evaluate the toxicity of chlbrpyrifos degraded products.The results showed that chlorpyrifos degraded products for 3 days had a greatly reduced toxicity.The survival rate of Drosophila melanogaster W118 was 66.66%.While the blank is 80%,it can be seen that MLCF7 can degrade chlorpyrifos and produce very little toxic substances.The experimental results of MLCF7 degrading chlorpyrifos in soil suggested that the microbial consortium could effectively degrade chlorpyrifos in soil.After 42 days cultured,the concentration of chlorpyrifos in soil was reduced from the initial 300 mg/L to 25.71 mg/L,and the degradation efficiency was 91.43%.In summary,the three isolated strains of paracoccus sp,Klebsiella sp,and Serratiaspwere compounded to obtain the microbial consortium(MLCF7)which had better effects on chlorpyrifos degradation It can effectively degrade chlorpyrifos,and also degrade the intermediate metabolite TCP of chlorpyrifos as well as degrade chlorpyrifos to other substances with low toxicity.In addition,the microbial consortium can also degrade the high concentrations of chlorpyrifos in soil.Thus,it will have a good application scenario in the restoration of the environment contaminated by chlorpyrifos.