Isolation And Identification Of Clothianidin Degrading Strain And Its Application Basis In Soil Pollution Remediation

Recent years,greenhouses are widely used around the world.Greenhouse has economic benefits and also has environmental pollution problem.The abuse of pesticides not only has damaged the quality of the soil,but also polluted the air,water bodies,and even entered the human body through the food chain,endangering human health.Clothianidin is a relatively small molecule with a high water solubility and low volatility,and has a long half-life in soil,which was used in greenhouses frequently.In terms of chemical properties and environmental persistence,the residue of clothianidin could leach and move into surface water and groundwater.In addition,the residue of clothianidin also can be toxic to nervous system of non-target species such as honeybee,birds,and aquatic invertebrates.Therefore,the research on bioremediation technology of clothianidin pesticide contaminated soil is of great significance.This study took 10 years of vegetable greenhouse soil as the experiment object and determinated clothianidin as a target pesticide by sampling survey.The effects of clothianidin residues on soil pH,soil enzyme activity and soil microbial quantity were studied by soil ecological simulation test in the laboratory.The research constructed and screened bacterial consortia SCAH by enrichment culture and directed domestication.The degradation characteristics and degradation mechanism of the bacterial consortia SCAH were studied.The bioremediation effect of the bacterial consortia SCAH on the clothianidin contaminated soil was studied by comprehensive analysis of the residual changes of clothianidin in different treated soils.The main test results are as follows:Different concentrations of clothianidin showed inhibition effect on soil pH.5 mg kg^–1and 20 mg kg^–1 clothianidin showed a promoting effect on invertase activity,but 50 mg kg^–1clothianidin showed inhibition effect.Different concentrations of clothianidin showed a trend of"promoting–inhibition–recovery"on urease activity.Low concentration of clothianidin showed a trend of"inhibition–promotion–inhibition"on catalase activity,but high concentration clothianidin showed inhibition on catalase activity.For alkaline phosphatase,different concentrations of clothianidin showed inhibition.Different concentrations of clothianidin showed the effect of"inhibition–promoting"on soil bacteria and fungi,but showed strong inhibition on actinomycetes,and the greater the concentration of clothianidin,the stronger the inhibition.Five high-efficiency degrading bacteria were isolated and screened from the soil of Tianshui vegetable greenhouse in Gansu Province and identified as Ochrobactrum anthropi,Enterobacter sp,Acinetobacter johnsonii,Pseudomonas putida and Stenotrophomonas maltophilia,respectively.In order to improve the degradation efficiency of clothianidin,the bacterial consortia SCAH was constructed and screened.In the mineral salt medium,79.3%of clothianidin(500 mg L^–1)was degraded by bacterial consortia SCAH after 15 days.The characteristics of the bacterial consortia SCAH degradation of clothianidin were investigated.The results showed that the degradation rate of clothianidin was the highest at pH 6.0,temperature of 35°C and inoculum of 3%.After 10 days of culture,the degradation rate reached 69.8%.Based on the results of LC-MS,the proposed metabolic pathway of clothianidin by the consortia SCAH was speculated.The major metabolic reactions of clothianidin were concluded to be as follows:（1）transformation of the nitroimino moiety to the urea compound N-（2-chloro-1,3-thiazole-5-ylmethyl）-N￠-methylurea（TZMU）.（2）The cleavage of the N–N bond in a part of the nitroguanidine and the cleavage of the C–Cl bond in the thiazole formed the N-（1,3-thiazole-5-ylmethyl）-N′-methylguanidine（TZMG）,and was further metabolized to 5-amino-methlthiazol（MTZ）via cleavage of the C–N bond between the thiazolylmethyl moiety and the nitroguanidine moiety.In the soil simulation study,it was found that higher degradation rates were shown in the combination of bioaugmentation and biostimulation compared with other treatment groups.After 45 days,the degradation rate of clothianidin achieved 95.7%.The residue of clothianidin in soil was determined by high performance liquid chromatography.The minimum detection concentration was 0.01 mg kg^–1,the recovery was84.2%⁸6.4%,and the relative standard deviation was 5.5%⁷.4%.The results were reliable and met the requirements of pesticide residue analysis.The residual amount of clothianidin in four different treatment soils gradually decreased with time,which accorded with the first-order kinetic equation.The degradation rate constant（k）observed in soil were0.008 mg kg^–1 d^–10.066 mg kg^–1 d^–1,and the half-life is 10.6⁹2.4 days.