Isolation, Identification Of Aryloxyphenoxypropionate And Chloroacetamide Herbicides-Degrading Bacteria, Cloning And Expression Of Degradation Gene And Research Of Metabolic Pathway

The environmental pollution of extensive use of herbicides caused the people’s growing concern. The means and methods to solve pollution continue to be made and updated, and microbial degradation of herbicides has become a research hotspot in recent years. Aryloxyphenoxypropionate herbicides and chloroacetanilide herbicides are two of the most used herbicides except glyphosate in China. The variety and extensive use of these two herbicides caused the serve environmental pollution as well as processed the weeds in the farmland. In this study, the fenoxaprop-ethyl and acetochlor were used as substrates and the aryloxyphenoxypropionate herbicides and chloroacetanilide herbicides degrading-microorganisms were isolated and identified in order to provide the theoretical basis and microbial resources for bioremendiation and research of herbicide resistant transgenic crops of these two type herbicides.1. Isolation, identification and degradation characteristics of fenoxaprop-ethyl (FE)-degrading bacteriaAn efficient FE-degrading strain T1was isolated from the long-term pesticide-contaminated soil and identified as Rhodococcus sp. based on morphology, physiological and biochemical characteristics as well as16S rDNA sequence analysis. The optimum growth temperature and pH of strain T1were25℃and7.0, aerobic growth and the cell growth is good when the NaCl concentration is less than3%.The metabolite fenoxaprop acid (FA) was identified by HPLC/MS analysis and this strain converted FE by cleavage of the ester bond, but it could not further degrade FA. Strain T1could utilize ethanol for growth, which produced during the degradation of FE and the optimum temperature and pH were30℃and8.0for FE degradation, respectively. Under these conditions, strain T1could degrade94%of100mg-L"1FE within24h at5% inoculation and it could also efficiently degrade a variety of aryloxyphenoxy propionate herbicides including quizalofop-p-ethyl, cyhalofop-butyl, clodinafop-propargyl and haloxyfop-R-methyl, the degradation spectrum is very wide.2. Cloning, expression and enzymatic properties of fenoxaprop-ethyl hydrolase gene fehTwo positive clones containing the esterase gene were screened from the genomic library of Rhobococcus sp. T1, which constructed by shotgun and used trbutyrin as the substrate, and clones pTT2with fenoxaprop-ethyl hydrolysis activity. FE hydrolase gene feh was successfully amplified from the positive clone pTT2by using overlap extension PCR, and the gene expression vector pET29a-feh was successfully constructed. The feh could express in E.coli BL21(DE3) largely and the pure recombinant enzyme Feh was obtained after purifying by Ni-NTA.The optimum temperature and pH value of Feh is50℃and9.5, which with a good pH stability and thermal stability. Feh could also hydrolyze the p-nitrophenyl esters and the triglyceride substances, but the hydrolysis was affected by carbon chain length. Km and Vmax of Feh for fenoxaprop were0.37mmol·L-1and1.08mmol·min-1·mg-1, respectively.3. Isolation and identification of acetochlor-degrading bacteriaThe acetochlor-degrading enrichment culture T3and T4were obtained from the activated sludge of acetochlor plant by using acetochlor as sole carbon source in the mineral medium, which could completely degrade100mg-L"1acetochlor in six days. T3and T4could also degrade butachlor and transform propanil to new product, but they could not degrade metazachlor and pretilachlor. T3and T4were found with the rich diversity of bacteria by RFLP and plate culture medium.Acetochlor-degrading bacteria strain T3-1, T3-6, T4-7and MEA3-1were progressively isolated from the enrichment culture T3and T4, which could degrade acetochlor completely by the cooperation among them. The metabolic pathway of acetochlor by these bacteria is strain T3-1transforming acetochlor to2’-methyl-6’-ethyl-2-chloroacetanilide (CMEPA), and then CMEPA would be degrade to2-methyl-6-ethyl aniline (MEA) by strain T3-6and T4-7, and at last MEA could be completely degraded by strain MEA3-1. Strain T3-1, T3-6and MEA3-1were identified as Rhodococcus sp., Delftia sp. and Sphingobium sp. based on morphology, physiological and biochemical characteristics as well as16S rDNA sequence analysis, respectively.4. Degradation characteristics of acetochlor-degrading bacteria and the combined degradation of acetochlorStrain T3-1could use the metabolites of acetochlor as sole carbon source for growth. The optimum temperature and pH were37℃and7.0for acetochlor degradation, respectively. Under these conditions, strain T3-1could degrade95.5%of200mg-L-1acetochlor within14h at5%inoculation. Strain T3-1could also efficiently degrade butachlor, but could not degrade pretilachlor, propisochlor and metazachlor. Strain T3-6could not use MEA or chloroacetic acid as sole carbon source for growth, which generated from CMEPA degradation and the optimum temperature and pH were30℃and7.0for CMEPA degradation, respectively. Under these conditions, strain T3-6could completely transform500mg-L"1CMEPA to MEA and chloroacetic acid within10h at5%inoculation. Strain T3-6could also degrade aniline and catechol, and convert hydroquinone partially. Strain MEA3-1could use MEA as sole carbon source for growth and minerilize MEA completely. The optimum temperature and pH were also30℃and7.0for MEA degradation, respectively. Under these conditions, strain MEA3-1could completely degrade50mg·L-1MEA within10h at5%inoculation. Strain MEA3-1could not degrade aniline, and catechol and hydroquinone could only partially transformed by it.At temperature of30℃and pH7.0, more than80%of200mg-L-1acetochlor could be completely mineralize by the cooperation of strain T3-1, T3-6and MEA3-1within24h and the degradation rate is much higher than other reported strains. Therefore, the complex culture made by the three strains has great potential utility for the bioremediation of acetochlor and butachlor contaminated environment.