Isolation And Identification Of A Fluoroglycofen Ethyl-Degrading Strain And Its Degradation Characteristics And Application Research

In this study, a highly efficient fluoroglycofen ethyl degrading strain was isolated from the pesticide wastewater treatment pool by enrichment and separation technology, which could utilize fluoroglycofen ethyl as the sole carbon source for growth and the strain was named as MBWY-1. It was finally identified as Mycobacterium phocaicum according to its physiological and biochemical properties and combined with16S rRNA gene sequence analysis of the strain and its homology comparison.The stain MBWY-1growed fastest after17h when cultured in LB liquid medium, and gradually into stable growth period after19h. The optimum temperature and pH for the growth of strain MBWY-1were30℃and6.5～7.0, respectively. More liquid volume was better for the growth of the strain MBWY-1. Ventilation had effect on the growth of the strain; larger dissolved oxygen in the water was more benefit for the growth. The concentration of NaCl had great impact on the growth of stain MBWY-1, it was gradually inhibited with the concentration of NaCl increased, and the strain was hardly grow when the concentration of NaCl attended4.0%as a result of the high-salt stress. The strain could grow in most of the organic carbon source in the test, and it utilize best in LB medium with xylose, fructose and glucose,while use little of the inorganic carbon source such as potassium carbonate and sodium bicarbonate. The strain could highly utilize all forms of nitrogen source in the test, while had a highest utilization of peptone. The strain were resistant to chloramphenicol, carbenicillin, amoxicillin, and was sensitive to ampicillin, streptomycin, gentamicin while has no resistance to kanamycin.The performance of fluoroglycofen ethyl degradation by MBWY-1showed that when the inoculation rate was5%, the concentration of fluoroglycofen ethyl was100mg/L, the degradation rate of fluoroglycofen ethyl by MBWY-1reached51.9%and91.6%after32h and96h respectively when cultured in MSM with the growth of the strain indicated that the strain could use fluoroglycofen ethyl as the sole carbon source; The optimum temperature and pH for fluoroglycofen ethyl degradation by strain MBWY-1were30℃and6.0～7.0, respectively. The larger amount of dissolved oxygen, the better degradation rate of fluoroglycofen ethyl. Improving inoculation rate speeded up the degradation of fluoroglycofen ethyl. With the increasing initial concentration of fluoroglycofen ethyl, the degradation rate of fluoroglycofen ethyl increased; the lower initial concentration, the easier utilization of fluoroglycofen ethyl by the strain. All of the addition heavy metals (expect for Ba2+, Cu2+and Ag+) reduced the degradation efficiency of fluoroglycofen ethyl in a certain extent when the concentration was0.1mM/L. The inhibition became significant when the concentration attened1OmM/L in which Co2+、Cu2+、Ag+had the greatest effect on the degradation.1,5,20mg/Kg of fluoroglycofen ethyl were added into the blank soil respectively, the average recoveries of fluoroglycofen ethyl was between87.8%and92.3%, relative standard deviation of soil recoveries of fluoroglycofen ethyl was between1.76%to4.41%. The inoculation of strain MBWY-1into soil that treated with fluoroglycofen ethyl showed a higher degradation rate than in uninoculated soil regardless of the soil sterilized or non-sterilized. The indigenous microorganisms play a role in fluoroglycofen ethyl degradation as synergistic role with additional MBWY-1.The metabolites were detected by MS/MS,{5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrophenylacyl} hydroxyacetic acid, acifluorfen,5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrobenzoate,5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-hydroxyl,3-chloro-4-hydroxyl benzotrifluoride were detected with the degradation of fluoroglycofen ethyl, thus indicating the metabolic pathway. The first and second steps involved in the degradation of fluoroglycofen ethyl are the scission of the ester bond to form acifluorfen. Acifluorfen was subsequently decarboxylated to5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrobenzoate. And further reduction of nitryl to hydroxyl. And the last step by strain was to form3-chloro-4-hydroxyl benzotrifluoride and p-dihydroxybenzene following the cleavage of ether-bond. The substrate spectrum degradation test results showed that the strain could only degrade fluoroglycofen ethyl while the other two diphenyl ether herbicides (acifluorfen and fomesafen) could not be degraded.The conditions of embedding in sodium alginate immobilized strainswere studied, the results showed that when the concentration of sodium alginate was4.0%, the concentration of CaCl2was3.0%, immobilized time was16h,the mechanical strength of obtained immobilized beads were best. When the packet bacteria were5.0%, it had the best degradation of fluoroglycofen ether by strain MBWY-1.Even the immobilized bacteria had a wider tolerance range of pH and temperature.