Isolation And Identification Of Chlorpyrifos Degrading Strains And Optimization Of Degradation Conditions

As an insecticide,chlorpyrifos belongs to the organophosphorus group according to the classification of pesticides,and has a broad spectrum.Chlorpyrifos has the characteristics of high efficiency and low toxicity,but its half-life is longer^[1].Chlorpyrifos residues can still be detected in soils where chlorpyrifos has not been used for a long time,and chlorpyrifos residues can also be found in crops.The environmental pollution caused by chlorpyrifos has attracted people's attention.The degradation of chlorpyrifos in the environment is mainly divided into three types,which are the use of physical degradation,chemical degradation and biodegradation to degrade chlorpyrifos^[2].Among them,biodegradation is the degradation of chlorpyrifos through the secretion of relevant degrading enzymes by microorganisms.In this study,a 16S rDNA high-throughput sequencing technology was used to establish a soil microbial library of phosphorus-contaminated farmland in Changbai Mountain,Jilin Province and analyze its microbial diversity;select chlorpyrifos according to the library design medium formula Efficient degradation of strains;strains Pseudomonas aeruginosa AY-1 and Pantoea PB-1were obtained,and their properties were characterized to determine the optimal growth conditions and optimal degradation conditions;and explored the broad spectrum of pesticide substrates of the strain Pseudomonas aeruginosa AY-1 feature.The results of the study are as follows:(1)Using 16S rDNA high-throughput sequencing to analyze the microbial diversity in farmland soilAfter 16S rDNA high-throughput sequencing,it was found that there is a diversity of species among different farmland soil samples.The dominant bacterial genera were Burkholderia,Arthrobacter,Bradyrhizobium,Gemmatimonas,Rhizomicrobium,Pseudomonas,etc.,but in different soil samples,the composition of the microbial community is different,so in the subsequent isolation and screening of chlorpyrifos efficient degrading bacteria,different genus,Design different media to obtain different groups of strains.(2)Chlorpyrifos degrading bacteria were isolated and screened from farmland soilTwo strains of chlorpyrifos-degrading bacteria AY-1 and PB-1 were isolated from farmland soil by the method of combining enrichment culture and laboratory adaptive evolution.Through morphological observation of the two strains,identification of related physiological and biochemical aspects as well as The 16S rDNA molecules were sequenced,and preliminary conclusions were reached after preliminary identification.The two strains were members of Pseudomonas aeruginosa and Pantoea,named Pseudomonas aeruginosa AY-1 and Pantoea PB-1.(3)Optimize the growth conditions of Pseudomonas aeruginosa AY-1 and the conditions for degradation of chlorpyrifosThe conditions for degradation of chlorpyrifos by the strain were optimized through single factor experiment and orthogonal experiment.The optimum degradation conditions were 35?,p H=8,substrate concentration 100 mg/L,and degradation efficiency 75.09%;using different carbon and nitrogen sources As a substrate to study its effect on the growth of Pseudomonas aeruginosa AY-1,it was found that the most suitable carbon and nitrogen sources of strain Pseudomonas aeruginosa AY-1 were glucose and ammonium sulfate.(4)Preliminary study on the degradation mechanism of chlorpyrifosThe intracellular and extracellular extracts of Pseudomonas aeruginosa AY-1 were extracted,and the degradation efficiency of chlorpyrifos was tested respectively.The results showed that the degradation efficiency of intracellular products was higher.It was preliminarily determined that the degrading enzymes of AY-1 chlorpyrifos were mainly intracellular enzymes;Analysis of the degradation products of chlorpyrifos by strain AY-1 revealed three main degradation products,namely DETP(diethyl phosphorothoate),TCP(3,5,6-trichloro-2-pyridinol)and DEP(diethyl phosphate),based on the results of this experiment,it is inferred that the pathway of the strain Pseudomonas aeruginosa AY-1 to degrade chlorpyrifos is:chlorpyrifos is hydrolyzed by hydrolase to obtain TCP and DETP,and then DETP is desulfurized to produce DEP.