Isolation,Degrading Characteristics Of Chlorothalonil And Its Isomer-degrading Bacteria,and Identification Of The Dehalogenases

Chlorothalonil(2,4,5,6-tetrachloroisophthalonitrile),a kind of broad-spectrum,non-absorptive,protective fungicide,has two isomers(o-tetrachlorophthalonitrile and p-tetrachlorophthalonitrile).Compared with o-tetrachlorophthalonitrile and p-tetrachlorophthalonitrile,chlorothalonil has a better solubility in water.O-tetrachlorophthalonitrile,an important intermediate in organic synthesis,is widely used in the production of pesticides,pharmaceuticals and fuels.Chlorothalonil has been frequently detected in ecosystems and is highly toxic to fish,birds and aquatic invertebrates.To date,contamination caused by the wide use of chlorothalonil and its isomers has aroused the concern of the scientific community However,the reductive dehalogenation of chlorothalonil by aerobic microorganisms has not been extensively studied and no strains capable of degrading the isomers of chlorothalonil have been reported.Biodegradation of chlorothalonil and its isomers by microorganisms is a simple,reliable,effective and environmental friendly means for the clean up the sites contaminated by chlorothalonil and its isomers.Therefore,isolation of microbial strains efficient in the degradation of chlorothalonil and its isomers,study of the degrading characteristics and its dehalogenation mechanism,and elucidation of ecological effect during the bioremediation process have important theoretical significance and application potential.In this study,through long-term selective culture medium supplemented with chlorothalonil or o-tetrachlorophthalonitrile,two degrading bacterial strains named as BLM18 and BSQ-1,respectively,were isolated from the soil samples inside and nearby the factory manufacting chlorothalonil.These isolated strains were identified based on morphological,physiological and biochemical tests with referencing to the Bergey's Manual of Determinative Bacteriology combined with 16S rRNA gene sequence analysis.Based on these analyses,the two strains were identified as Massilia sp.and Pseudochrobactrum sp.,respectively.Massilia sp.BLM18 was isolated from the soil of a chlorothaloni-producing factory in Xinyi in Jiangsu province.Strain BLM18 was capable of degrading chlorothalonil.Physiological and biochemical experimeidal results showed that its optimum growth temperature was 30? and the optimum initial pH was 7.0.The isolate grew best when the NaCl concentration was 1%.Strain BLM18 could make full use of lactose,but could not utilize magnesium acetate,xylose,dextrin or sucrose as the sole carbon source.Strain BLM18 was not resistant to common antibiotics.Moreover,strain BLM18 could degrade 50 mg/L chlorothalonil completely within 64 hours,while could not degrade the isomers of chlorothalonic,o-tetrachlorophthalonitrile and p-tetrachlorophthalonitrile.The optimum degradation temperature of strain BLM18 was 30?,but the degradation also occurred at 20?40?.Besides,0.2 mmol/L Fe3+,Mg2+ or Ca2+ could enhance the degradation rate of chlorothalonil,while,Ni2+,Co2+ and Zn2+(0.2 mmol/L)had no significant effects on the degradation rate.In strain BLM18,the initial metabolic pathway of chlorothalonil was identified as the reductive dehalogenation pathway by GC/MS analysis of the metabolite metylthiotrichloroisophtalonitrile.Pseudochrobactrum sp.BSQ-1 was isolated from soil outside of a chlorothalonil-producing factory in Liyang of Jiangsu province.Strain BSQ-1 was capable of simultaneously degrading chlorothalonil and its isomers.Physiological and biochemical results showed that,the optimum growth temperature of strain BSQ-1 was 30?,and the optimum initial pH was 7.0.When the concentration of NaCl was 1%,the isolate grew best.However,strain BSQ-1 could not utilize o-tetrachlorophthalonitrile as the sole carbon source for growth.Strain BSQ-1 could completely degrade 30 mg/L o-tetrachlorophthalonitrile within 72 hours.The optimum degradation temperature was 30?.Besides,the addition of 0.2 mmol/L Co2+;Zn2+ or Mg2+ could increase the degradation rate by 30%,20%and 20%,respectively,while,the addition of Cu2+,Ca2+,Pb2+,Ni2+ or Fe3+(0.2 mmol/L)did not inhibit the degradation of o-tetrachlorophthalonitrile.The metabolic pathway of o-tetrachlorophthalonitrile in strain BSQ-1 was identified as the hydrolytic dehalogenation by the analysis of MS/MS and degradation experiments under anaerobic and aerobic conditions.The substituted hydroxyl group was determined to be at the para-position by NMR measurement and confirmed by the crystallography.The degradation rates of strain BSQ-1 towards chlorothalonil was faster than the other two isomers,and the degradation rate of p-tetrachlorophthalonitrile was the slowest.In strain BSQ-1,the primers according to the chd gene was designed to amplify the gene involved in the degradation of o-tetrachlorophthalonitrile.The amplified fragment from strain BSQ-1 showd 100%similarity to chd gene and was confirmed to have hydrolytic dehalogenation activities to o-tetrachlorophthalonitrile as well as chlorothalonil.Therefore,the chd gene was the specific hydrolytic dehalogenase gene for the dehalogenation of o-tetrachlorophthalonitrile.The key gene in strain BLM18 that was involved in the reductive dehalogenation of chlorothalonil was also investigated through a gel chromatography method.Through analysis and comparison,a chlorothalonil reductive dechlorinase gene,cpoA,was iedntified.The activities of CpoA against chlorothalonil were different when different kinds of cofactors were present.CpoA could not dehalogenate the other two isomers of chlorothalonil.The temperature and pH value had significant influence on the enzymatic characteristics of CpoA.The activity of CpoA was the best at 35?.When the temperature was at 30?37?,the activity of CpoA could be maintained above 80%;but when the temperature was below 20? the activity of CpoA was less than 50%and when the temperature was higher than 45?,the activity of CpoA was less than 20%.In the ecological study during the bioremediation of chlorothalonil contaminated soils,the strains BSQ-1 and BLM18 could degrade 50 mg/kg chlorothalonil to an undetectable level and could degrade 60%of 100 mg/kg chlorothalonil within 35 days.In comparison,the chlorothalonil degradation rate by strain BSQ-1 was quicker than that of strain BLM18.Meanwhile we analyzed the microbial community change of proportion in the level of phylum and genus,PCA diagram and UPGMA clustering tree of the soil samples.These circumstances indicated the feasibility of bioremediation by strains BLM18 and BSQ-1 towards soils contaminated with chlorothalonil.