Isolationg,Identification Of Chlorpyrifos Degrading Strains And Study On Its Metabolic Pathway

Chlorpyrifos is a mid-toxic organophosphorus pesticide.It is one of the most widely used pesticides in the world due to its broad insecticidal spectrum and high efficiency.However,its residue has caused the problems of environmental pollution and food safety.Therefore,isolating bacteria with high and stable chlorpyrifos-degrading ability and investigating its application in the bioremediation of chlorpyrifos contaminated environments have important theory and application value in solving the problem on the residue of chlorpyrifos.In the present study,two chlorpyrifos-degrading bacteria strains OP-1 and DT-1 were isolated from sludge of a pesticide factory which has produced organophosphorus pesticides for many years.According to their morphological,physiolohical and biochemical characters,and 16S rRNA gene phylogenetic analysis,strain OP-1 and strain DT-1 were identified as Burkholderia sp.and Cupriavidus sp.,respectively.The 16S rRNA gene of strain OP-1 had the highest similarities with B.glathei(=ATCC 29195T);B.sordidicola(=KCTC 12081T)and B.bryophila(=LMG 23644T)in genus Burkholderia,they were 97.4%?96.4%and 96.3%,respectively.Strain OP-1 contained C16:0(23.38%),C17:cyclo(21.61%),C18:1 ? 7c(16.59%)as its main cellular fatty acids.Its predominant ubiquinone was Q-8.Phosphatidylethanolamine(PE)was its predominant polar lipids.The DNA G+C content of its total DNA was 59.4 mol%,the DNA-DNA relatedness to B.glathei was 30%.On the base of the above characters,we propose that the strain OP-1T represents a novel species in the genus Burkholderia,and designated as Burkholderia zhejiangensis sp.nov.OP-1T(=CCTCC AB 2010354T=KCTC 23300T).Strain OP-1 could only hydrolyze chlorpyrifos to 3,5,6-trichloro-2-pyridinol(TCP);strain DT-1 could completely degrade chlorpyrifos.Therefore,strain DT-1 was used in the following study on its growth and degradation characteristics.The results showed that the optimal temperature and pH value for the growth of strain DT-1 were 30? and 7.0,respectively.The optimal carbon source and nitrogen source were fructose and peptone.The growth rate of strain DT-1 was positively correlated to the entilation quantity.The salt tolerance of DT-1 was 0-10 g·L-1.Strain DT-1 grew well in MSM medium with 100 mg·L-1 chlorpyrifos as the sole carbon source and completely degraded chlorpyrifos within 12 h.The optimal temperature and pH for degradation were 30 ? and 7.0,and the optimal concentration of NaCl was 5 g·L-1.The degradation efficiency increased positively with the increase of ventilation quantity and inoculation amount.The addition of metal ions could obviously inhibit the degradation of chlorpyrifos by strain DT-11 Co2+ and Ni2+ showed the strongest inhibitory effect.Strain DT-1 had a widely degradation spectrum,it could also degrade other organophosphorus pesticides such as parathion,methyl parathion and fenitrothion,but could not degrade methamidophos and dimethoate.By LC-MS,four metabolites(3,5,6-trichloro-2-pyridinol,5,6-dichloro-2-pyridinol,6-chloro-2-pyridinol and 2-pyridinol)were indentified during the degradation of chlorpyrifos.And the chlorpyrifos biochemical degradation pathway of strain DT-1 was proposed:Chloipyrifos was first hydrolyzed into TCP and diethylthiophospshate(DETP)by organophosphorus hydrolase,which was followed by three successive reductive dechlorination reaction,resulted into 5,6-dichloro-2-pyridinol,6-chloro-2-pyridinol and 2-pyridinol,respectively.Then pyridine ring was cleaved,till completely mineralized.Studied on the degradation characteristics of TCP by strain DT-1 was also carried out,the results showed that DT-1 could grow well in MSM medium with 50mg·L-1 TCP as the sole carbon source and completely degraded the TCP.The optimal degradation temperature and pH were 30? and 7.0.The degradation efficiency was high under the initial concentration lower than 50 mg·L-1.When the initial concentration was higher than 100 mg·L-1,DT-1 could not degrade TCP.The degradation efficiency increased positively with the increase of ventilation quantity and inoculation amount.Adding metal ions could inhibit the degradation of TCP by strain DT-1,among which,Co2+;Ni2+ and Mn2+ had the most serious inhibitory effect.Added different exogenous nutrient substances could promote the degradation to some degree,among which the addition of peptone showed the best promotion effect.The organophosphorus hydrolase gene mpd was cloned from strain DT-1,its length was 996 bp(No.JQ988072).It showed 99.7%similarity with the reported mpd gene(AF338729),at amino acids level the similarity was 99%.The mpd gene was cloned into pET29a,and expressed in the Escherichia coli BL21(DE3).The expressed protein was about 33 KD,which had the ability to hydrolyze of chlorpyrifos.The bioremediation of chlolrpyrifos contaminated soil by inoculating DT-1 was studied under laboratory condition;the results showed that strain DT-1 could promote the degradation of chlorpyrifos in soil.The optimal application condition was:inoculation cells of strain at 1 × 106 CFU g-1 dry soil;pH 7.0;30 ? and 35%of water content.Under this condition,strain DT-1 could degrade 100 mg-kg-1 chlorpyrifos in the soil within 30d.This result suggests that strain DT-1 has potential for use in bioremediation of chlorpyrifos-contaminated environments.