Isolation,identification And Degrading Characteristics Of A Clofentezine-degrading Bacterium S-8

Clofentezine,the chemical structure is 3,6-double（2-chlorophenyl）^-1,2,4,5-four triazine,is an organic nitrogen heterocyclic acaricide.It appears red in high concentration of clofentezine solution,and it is a kind of acaricide with the advantages of highly effective,safe,broad-spectrum and long residual activity.Clofentezine is mainly used to prevent apple spider mite,citrus rust mite,hawthorn spider mite,citrus four spotted yellow spider,tetranychus mite,eriophyes oleivorus and other plant pest mites.Clofentezine is moderately toxic,and it shows high initial activity and residual activity in the environment.The residual clofentezine in environment can transfer in food chain through bioaccumulation,which finally causes huge risk with food safety and human health.In recent years,great concern and interest have been raised about the residue and repair of clofentezine.Microbial degradation has become the main method during the remediation of pesticide residue pollution because of its advantages of environmental friendliness,high efficiency and economic feasibility.However,few studies on the microbial degradation of clofentezine have been reported.This study aimed to isolating the efficient clofentezine-degrading bacteria and researching the degradation characteristics of clofentezine,which can provide the strain resource and theoretical basis for the bioremediation of the clofentezine.This study used the enrichment method to isolate a clofentezine-degrading strain from a long-term pesticide manufacturer of clofentezine,and this strain was named as S-8,this clofentezine-degrading strain was first discovered.Based on physiological&biochemical characteristics and 16S rRNA gene sequence analysis,strain S-8 was preliminarily identified as Mycobacteriu sp.It was found that it could grow well in the condition of pH 6.0～8.0,the optimum temperature and pH for its growth was 30℃ and 7.0,respectively.strain S-8 grew well in NaCl concentration of 5～20 g/L;Cd²⁺,Cu²⁺or Ni²⁺（2mM）had a strong inhibitory effect on the growth of strain S-8.The growth of strain S-8 was affected when the liquid volume was larger than 150 mL in the 250 mL Erlenmeyer flasks,which showed that the decline of aeration could affect the growth of strain S-8.In the enrichment medium,strain S-8 could degrade 30 mg/L clofentezine in 60 h,the optimum temperature and pH for the degradation of clofentezine was 30℃ and 7.0,respectively.Without the addition of metal ions,the degradation rate of degrading clofentezine by strain S-8 was 92.38%,after adding Cu²⁺at 2 mM,the degradation rate was 18.6%,which had a strong inhibitory effect on the degradation of clofentezine;Ni²⁺and Cd²⁺（2 mM）also had a certain inhibitory effect,the degradation rate was 42.39%and 41.76%,respectively.Without adding carbon source,the degradation rate was 3.08%,after adding 1 g/L starch or glucose,the degradation effect would be improved obviously,the the degradation rate was 94.05%and 93.07%,respectively..By HPLC and MS/MS,the metabolites were detected during the degradation of clofentezine by strain S-8,The metabolite was found as 3,6-bis（2-chlorophenyl）^-1,2-two hydrogen^-1,2,4,5-four triazine,the-N=N-double bond of clofentezine was reducted by hydrogenation.It was done for the toxicity tests of the metabolite.When clofentezine and metabolite at the same concentration level were added in the medium,the growth of Saccharomyces cerevisiae was significantly different at P<0.05.And it was found that the metabolite biological toxicity was significantly lower than that of clofentezine.Strain S-8 had the detoxification during the degradation of clofentezine.The degradation characteristics of clofentezine by the crude enzyme of strain S-8 were studied.It was concluded that the enzyme system is 2 mL crude enzyme solution with 30 mg/L clofentezine reacting about 100 min.NADH was a more suitable electron donor.The crude enzyme of strain S-8 was stable when the temperature was lower than 37℃.Otherwise,it was gradually decreased.In the further study,the optimal reaction temperature range was 30～42℃ and the optimum pH was 7.0.Degradation effect of clofentezine by the crude enzyme of strain S-8 could be promoted by Fe²⁺ or Ca²⁺ at 1.0 mM concentration,and the relative enzyme activity was increased 13.56%and 15.62%,respectively.It could be strongly inhibited by Cu²⁺,Co²⁺ or Mn²⁺at 1.0 mM concentration,the relative enzyme activity decreased 78.79%,89.7%and 94.55%,respectively.EDTA at 100 mM could almost completely inhibit the activity of the crude enzyme of strain S-8,and the relative enzyme activity was 4.47%.Under anaerobic conditions,the crude enzyme activity（92.93%）was higher than that in the aerobic environment（85.97%）for 30 mg/L clofentezine in 30 h.The Vmax/Km about the crude enzyme of strain S-8 towards clofentezine was 10.79 min^-1·mg^-1.