Isolation And Identification & Degrading Mechanism Of Diphenyl Ethers Herbicide-Degrading Bacteria

Diphenylether herbicide is one of the widely used herbicides in our country, including fomesafen, lactofen, fluoroglycofen-ethyl, acifluorfen and so on. Just as other pesticides, diphenylether herbicides will pose influence on organisms and ecosystem when they are put into the entironment.A strain of bacterium designated as Strain ZB-1, capable of degrading fomesafen, was isolated from contaminated soils by fomesafen. Strain ZB-1 was identified preliminarily as Lysinibacillus sp. based on its physiological and biochemical characters and the result of the 16S rRNA homologue sequenceBiological properties were analyzed. The optimal pH value for the growth of strain ZB-1 was 6.0-9.0, and the optimal pH was 6.0. The optimal temperature was 30℃. The optimal osmotic pressure for strain ZB-1 was 1-3%. The optimal medium for the growth of strain ZB-1 was glucose as carbon source and organic nitrogen as nitrogen source.The strain ZB-1 could utilize fomesafen as the sole carbon source for growth, and the total degradation rate was 81.32% after 7 d of inoculation in mineral salts medium. The optimum temperature for fomesafen degradation by strain ZB-1 was 30℃. The effect of fomesafen concentration on degradation was also examined. During the degradation of fomesafen, some major metabolites were analyzed by LC-MS and GC. The proposal metabolic pathways had been deduced as follows:reduction, acetylation of nitro groups, dechlorination and cleavage of the S-N bond when fomesafen is degrade by strain ZB-1.A strain of bacterium designated as Strain LY-2, capable of degrading lactofen, was isolated from contaminated soils by lactofen. Based on its physiological and biochemical characters and the result of the 16S rRNA homologue sequence, Strain LY-2 was identified preliminarily as Brevundimonas sp.Strain LY-2 could degrade 81.8% of 50 mg L-1 lactofen after 5 days. During the degradation of lactofen, the hydrolysis of the ester bond of the alkanoic side chain of lactofen, followed by the reduction of nitro group and acetylation of amino derivative, dechlorination and the reduction of acetyl and carboxyl when lactofen is degrade by strain LY-2.Strain ZB-1 was applied to treat fomesafen contaminated soil and the results showed that strain ZB-1 could effectively degrade fomesafen residues, which could provide theoretic evidences for the in situ bioremediation.A novel qualitative analysis method of lactofen hydrolase was established. A lactofen hydrolase from cell-free extract of the strain LY-2 was purified 113.3-fold to homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and PAGE-electrophoretic elution with 6.83% recovery. SDS-PAGE gels produced a single band for esterase with a molecular mass of 25±1 kDa indicating that it was a monomer. The purified enzyme was optimally active at pH 7 and 20℃, and the Km and Vmax for lactofen hydrolysis were 0.81μM and 1.26 nmol min-1mg-1, respectively.