Construction Of Chlorthalonil Pesticide Degrading Flora And Optimization Of Its Degradation Condition

The organochlorine pesticide chlorothalonil is one of the most widely used high-efficiency fungicides in the last century,but it can not only pollute the soil ecosystem,but also enter the food chain and reach the human body,endangering human health.Using a more environmentally friendly,safe and efficient microbial degradation method to remove the residual chlorothalonil pesticide in the soil environment has important practical significance for alleviating soil pollution and protecting human health.In this study,the gradient domestication method was used to isolate and screen the strains that could degrade the pesticide chlorothalonil from the farmland soil polluted by the pesticide chlorothalonil for a long time,and the combination of strains with stronger degradation ability was selected to obtain the superior degradation ability of efficiently degrading chlorothalonil.Bacteria to study the degradation effect of the dominant degrading bacteria under different influencing factors,and then optimize the degradation conditions through response surface design,and finally prepare a chlorothalonil degrading agent,and evaluate the effect of the degrading agent on the actual pollution by pesticide chlorothalonil.The soil remediation effect,by analyzing the changes in the soil flora structure,explored the feasibility of the flora restoration of chlorothalonil-contaminated soil.The main research results are as follows:(1)A total of 6 strains with strong degrading effect on the pesticide chlorothalonil were screened after separation,and they were numbered as B-1,B-2,B-3,B-4,B-5,B-6,among which,the strains were The degradation rates of chlorothalonil in 7 days for B-2,B-3 and B-4 were 74.83%,70.41%and 85.01%,respectively.The three strains were identified as belonging to Bacillus sp.,Klebsiella aerogenes and Stenotrophomonas maltophilia strain.,its Gen Bank accession numbers are ON548919,ON556406 and ON556407,respectively.(2)The optimal growth environment conditions of the dominant strains were determined respectively.The strain B-2 was in the adaptation period within the first 4 hours,the logarithmic growth period within 4-24 hours,and reached the stable period after 23 hours.The growth environment condition is 35℃,pH=7;strain B-3 is in the logarithmic growth phase within 0-12h,and reaches the maximum growth amount at the 13th hour,the growth amount of strain B-3 at20℃,pH=5 Maximum;strain B-4 is in the logarithmic growth phase from 0 to13h,and reaches stability in the 24th hour.The optimum growth environment of strain B-4 is 25℃,pH=7.(3)A bacterial colony composed of B-2,B-3,and B-4 was constructed,and the bacterial colony composed of three strains mixed in equal volumes through different combinations had the best degradation effect on chlorothalonil;The degradation effect of the bacteria group under different influencing factors was studied.The study found that under the conditions of pH 7,temperature 30℃,pesticide concentration 10mg/L,and dosage ratio of 1:2:1,chlorothalonil had a good degradation effect.(4)On the basis of the single factor experiment,the response surface conditions were optimized for pH,temperature,pesticide concentration,and dosage ratio,and the experimental results were regressed to the kernel by the Design Expert software,and the quadratic equation fitting of the influence of the independent variables on the response value was obtained.Kernel model:Y=65.82+2.32A-0.84B+2.17C+6.28D-2.89AB-3.10AC+2.82AD+4.03BC-2.70BD+2.85CD-3.77A~2-4.23B~2-15.32C~2-4.80D~2.Through the analysis,the optimal degradation conditions are:when the pesticide concentration is17.95mg/L,the temperature is 25.73°C,the pH is 7.12,and the dosage ratio is1:2:1,the bacteria will degrade chlorothalonil The rate reached 70.44%.(5)The constructed dominant flora has a good degradation and restoration effect on the farmland soil polluted by spraying chlorothalonil.After adding bacterial agent for 10 days,the degradation rate of 100mL/kg of chlorothalonil can reach 53.28%.Through metagenomic classification and sequencing of soil microbial communities before and after restoration,it was found that the strains in the dominant flora could grow and reproduce,and the structure of the bacterial community in the soil changed after restoration.Among them,the bacteria with strong organochlorine pesticide degradation ability The abundance of strains Proteobacteria,Actinobacteria and Firmicutes showed an obvious increasing trend.