Isolation,identification,Degradation Characteristics And Degradation Gene Of Bifenthrin-degrading Bacteria

Pyrethroid pesticides are a kind of broad-spectrum high-efficiency bionic pesticides,which are a new class of pesticides following organophosphorus,organochlorine and carbamate.They account for about 20% of the world market,and are widely used in agricultural production and controlling household pests.The widespread use of pyrethroid pesticides has resulted in a large amount of residues in the environment.The residues of pyrethroids in the environment pollute the aquatic system along with surface runoff,resulting in the decline of aquatic product quality and ultimately harm human health through the amplification of food chain and food web.Microbial degradation is the main way to solve the problem of pesticide residues.In this study,bifenthrin with high insecticidal activity and long residual period was selected as the target pesticide to screen and identify microbial strains that can degrade bifenthrin from the environment,and its degradation characteristics and degradation genes were studied.The main findings are as follows:1.Using bifenthrin as the only carbon source,through enrichment and domestication,three strains of bacteria with good degradation effect on bifenthrin were screened from the sludge collected from the outlet of pesticide plant and sewage treatment plant,and numbered as E1,F1 and F2 respectively.Through second-generation sequencing,E1 was Acinetobacter schindleri and named as Acinetobacter schindleri-E1,F1 was Serratia marcescens which named as Serratia marcescens-F1,F2 was identified as Acinetobacter johnsonii and named as Acinetobacter johnsonii-F2.2.The assembly and functional annotation of the second generation sequencing genome data showed that the metabolic genes of the three strains were similar in distribution.Through KEGG database annotation,it was found that proteins related to signal and cell transport accounted for the highest proportion.The COG annotation gene families of the three strains were similar in distribution,and the maximum number of functional genes was above 600.The percentage distribution of GO database annotation was also similar,with metabolic genes,catalytic genes and binding genes accounting for the highest proportion.The number of proteins annotated by the F1 strain was 177% that of the other two strains.The abundant functional annotation of the genome provides strong support for the subsequent search for degrading genes.3.The effects of temperature,p H and inoculation amount on the degradation of bifenthrin by E1,F1 and F2 were studied.The results showed that in the range of 20-40℃,the degradation capacity of the three strains to bifenthrin the strongest when incubation temperature at 30℃.The three strains showed the highest degradation efficiency when the p H value of the culture medium was 7.The results were consistent with the external conditions suitable for the growth of the strain.The degradation capacity of the three strains of bifenthrin increased first and then decreased with the increase of the inoculation amount.Finally,the optimal inoculation ranges of E1,F1 and F2 were determined to be 0.5-0.9 g/L,0.1-0.5 g/L and 0.3-0.7 g/L,respectively.4.Through response surface method,the effects of culture temperature,p H of culture medium and inoculation amount on the degradation of bifenthrin by three strains were studied and predicted the optimal degradation conditions of the three strains.The results showed that three factors had significant interaction effects on the degradation of bifenthrin by the strains.The effects of temperature,p H and inoculum on the ability of three strains to degrade bifenthrin were: p H> temperature> inoculum.The predicted degradation conditions of strains E1 and F2 were very similar: the optimal culture temperature was 30℃,and the p H of the culture medium was 7.3 and 7.1,respectively.The inoculation amounts were 0.67g/L and 0.53 g/L,respectively.Under the optimum degradation conditions,the degradation rates of E1 and F2 were 65.26% and 85.84%.When the p H of strain F1 was 7.6,the temperature was 29℃ and the inoculation amount was 0.34 g/L,the highest degradation efficiency of p-bifenthrin was 90.30%.5.By comparing the genomic data of strain F2 with the pyrethroid degrading genes in the database,the 10 genes with the highest similarity were obtained.F2＿MGA-2674 was the most up-regulated gene when bifenthrin was the only carbon source.After heterologous expression of F2＿MGA-2674 gene,the degradation rate of bifenthrin(1 mg/L)in the culture medium was 34% after 7 days,which was significantly different from that of the control without access to the gene fragment.It was preliminarily determined that this gene was related to degradation.