Characterization Of Chlorpyrifos-Degrading Bacterium H27 And Its Application In Soil Remediation

As a highly effective and moderately toxic organophosphate pesticide,chlorpyrifos is widely used in agricultural production.With the large use of chlorpyrifos,its excessive accumulation in the water and soil environment will cause the problem of pesticide residue pollution,which will adversely affect the ecological environment and threaten human health.Among the methods for eliminating pesticide pollution,compared with traditional physical and chemical methods,bioremediation has gradually become a research hotspot for environmental pollution control due to its advantages such as high efficiency,low cost,and no secondary residue.So far,there have been some reports on microbial degradation of chlorpyrifos,but there are few studies on its degradation mechanism.Therefore,it is of great significance to isolate and obtain chlorpyrifos-degrading microorganisms and study their degradation characteristics and related mechanisms,which will enrich the resources of chlorpyrifos-degrading bacteria and accumulate theoretical data for pesticide pollution repair.In this study,a strain of chlorpyrifos degrading bacteria H27 was isolated and identified from farmland soil and identified.The characteristics of chlorpyrifos degradation were further studied.In order to improve the degradation rate,the degradation conditions of chlorpyrifos were optimized by response surface analysis.The mechanism of degradation of chlorpyrifos by degrading bacteria was discussed through degradation pathways,degrading enzymes and degrading genes,and then the degrading bacteria were applied to chlorpyrifoscontaminated soil restoration.The Biolog method was used to analyze the effects of degrading bacteria on soil microorganisms.The main contents are as follows:(1)Isolation and identification of degrading bacteria.In this study,a strain H27 capable of growing with chlorpyrifos as carbon source was isolated and screened.It was found that the colonies of the plate were round,milky white,translucent,with neat edges,bulging in the middle,and thicker.The strain was rod-shaped through scanning electron microscopy,which produced spores,no flagella,and could not move.The 16 S r DNA gene sequence alignment was performed on the strain.The strain was identified as a Bacillus bacterium.(2)Study on optimization of degradation conditions and degradation characteristics of degrading bacteria.Studies on the degradation characteristics of chlorpyrifos-degrading bacteria have shown that culture time,temperature,and p H all have an effect on chlorpyrifos degradation.The effects of different incubation time,temperature and p H on the degradation effect of degrading bacteria H27 were determined by single factor degradation conditions experiments.The optimal time to obtain chlorpyrifos degradation was 48 h,the optimal temperature was 25 ?,and the optimal p H was 7.0.Using Box-Behnken experimental design and response surface method analysis,the degradation conditions of degrading bacteria H27 were optimized.The results showed that when the initial concentration of chlorpyrifos was 25 mg·L-1,the optimal degradation conditions were 54 h degradation time,p H 7.2 and temperature 24 ?,and the degradation rate of chlorpyrifos could reach 88.96%.(3)Preliminary exploration of degradation mechanism.Studies on degradation mechanisms include analysis of degradation pathways,mapping of degrading enzymes,and research of degrading genes.The liquid chromatographic analysis of chlorpyrifos microbial degradation products has shown that TCP is the main degradation product during chlorpyrifos degradation,and no other degradation products were detected by liquid quality analysis.The crude enzymes of intracellular,extracellular and periplasmic enzymes of H27 were extracted,and the degradation characteristics of three types of degrading crude enzymes on chlorpyrifos were studied.The results showed that the degradation rate of the crude enzyme was the highest,which could reach 49.6%.It can be inferred that the key enzyme in the degradation of chlorpyrifos by the degrading bacteria H27 was the intracellular enzyme.The optimum p H for the enzymatic degradation of chlorpyrifos is 7.5,the optimum temperature is 25 ?,and the degradation enzyme has a good degradation activity in the test range of 25 to 40 ? and p H 6.0 to 9.5.p H is well tolerated.Primers were designed for amplification based on the existing degradation genes mpd and opd.The results showed that the strain does not contain mpd and opd-related gene sequences,and may be a new degradation enzyme gene.(4)Remediation application of degrading bacteria in soil and its effect on soil microorganisms.The laboratory simulated chlorpyrifos-contaminated soil and used bacteria H27 to repair chlorpyrifos-contaminated soil.The results showed that the degradation rate of chlorpyrifos increased rapidly during the first 5 days of treatment,and the maximum degradation rate was 56.4%,after which the degradation rate tended to increase.The Biolog method was used to evaluate the changes of soil microorganisms during the process of degrading bacteria restoration,and the Shannon index,Mc Intosh index,and Simpson index were used to compare the functional diversity of soil microbial communities under different treatments.The results show that the application of alien degrading bacteria can increase the richness of soil microbial species to a certain extent during the process of repairing contaminated soil,but due to its prominent species status,the number structure and diversity of species in the soil have changed.