Isolation, Identification Of Herbicides Intermediate (4-Hydroxyphenoxy)Propanoate -Degrading Bacteria And Its Degradation Rathway

The modernization of agricultural has led to abusing herbicide. Environment pollution problems caused by abusing herbicide get more and more attention by people. Low efficiency absorption of herbicides result in a large amount of residual soil after rain eluviation. As widespread disintegrators in nature, variety of adaptability microbes play important roles of repair tools of soil ecological. Therefore herbicides-degradating microbe has become a study hotspot in recent years. Aryloxyphenoxypropionate is one of the most applicate herbicides in the world in recent years. The sale of the herbicide in 2011 has accounted for 2.1% in global pesticide market, accounting for 4.8% in the total herbicide market. Herbicide application reduces farmland weeds harm as well as causes the environmental pollution by residual. In our work, the fenoxaprop-ethyl degradation intermediates (R)-(+)-2-(4-HydroxyPhenoxy)Propanoate (HPP) was used as substrates to isolate and screen of degradation microorganisms from sludge of long-term contaminated chemical plant. Based on the research in metabolism and the genetic background of the microorganism, we intended to provide metabolic research and microbial resources for the ecological restoration of herbicide pollution soil and herbicide resistance study of genetically modified (gm) crops.Three high efficiency HPP-degradating strains were isolated from long-term contaminated chemical plant sludge and named MEPE0129, MEPE0902 and MEPE0903, respectively. Based on phenotypic, physiological and biochemical characteristics and 16S rRNA gene phylogenetic analysis, MEPE0129, MEPE0902 and MEPE0903 were preliminary classed as Rhodospirillaceae gen. sp., Delftia sp. and Pigmentiphaga sp.. MEPE0129 MEPE0902 and MEPEO903 are all aerobic strains. The optimum growth temperature of three strains are 37℃, the optimum growth pH are 6.0-7.0,7.0 and 7.0 respectively. When concentration of NaCl is less than 3%, MEPE0129 MEPE0902 and MEPE0903 grew well.Result of 16S rRNA gene homology indicates 91.17% of highest homology between MEPE0129 and reported strain. Phylogenetic analyses show that MEPE0129 form a new branch in Rhodospirillaceae. It’s different from model strains of other genera in the family. The Biolog, API, fatty acid content of G+C mol%, polar ester, and respiratory quinone of MEPE0129 were step analyzed. The DNA G+C content of strain is 65.1 mol%. The main respiratory quinone is Q10 (75%). The main fatty acid group of MEPE0129 is consist of C18:1 ω7c, C16:0 and C19:0 cyclo ω8c. Result of compared with other type strains suggest that MEPE0129 own unique physiological and biochemical characteristics. Thus a new genera with type strain MEPE0129 is proposed and named Taonella mepensis (=CICC 10529T= CCTCC AB 2012861T=KACC 16940T).Sole carbon source, growth, optimum temperature, optimum pH and experiment of degradation conditions in a gradient of inoculation, initial concentration, substrates and metal ions was carried on MEPE0129, MEPE0902 and MEPE0903. Experiment results indicate that MEPE0129 can grow when make use of 50 mg-L"1 in 13 d, with the highest growth OD600nm of 0.1. MEPE0902 have the ability to utilize as high as 450 mg·L-1 HPP·grow within 14 h, with corresponding OD600nm of 0.53 rising from 0.027. The MEPE0903 can degrade 96% of 50 mg·L-1 HPP within 8 days. Optimum degradation temperature of MEPE0129, MEPE0902 and MEPE0903 are respective 28℃,37℃and 30℃. MEPE0129 prefer low range temperature below 28℃. Optimal degradation pH of three bacterias was 8.0,7.0 and 8.0. Three strains have great degradation effect in slightly alkaline environment. HPP gradient initial concentration experiments show that MEPE0129 can degrade 125 mg·L-1 HPP within 6 d. MEPE0902 would degrade as high as 500 mg·L-1 HPP within 15 h. Different from MEPE0129 and MEPE0902, MEPE0903 can merely degrade 50 mg·L-1 HPP. When HPP is equal to or greater than 100 mg·L-1, MEPE0903 lose degradation ability completely. Experimental results of gradient initial inoculation of three strains show that HPP degradation efficiency within the scope of selected inoculation increase with inoculation. The metal ions Cu2+and Ni+ inhibit the degradation of three degradation strains, and Co2+have remarkable inhibitory effect for MEPE0902.Degradation of single hydroquinone (HQ), the mixture of HPP and HQ and degradative intermediate of HPP in MEPE0902 were studied.50 mg·L-1 HQ was converted into the unknown intermediate which couldn’t be metabolize thoroughly. Meanwhile, in the metabolism experiments of the mixture of HQ and HPP, we observed none impact between HQ metabolism and HPP metabolism. Finally, the concentration process of intermediate experiment indicates that a novel metabolic pathway of HPP in MEPE0902 which is mediated by microscale HQ.For research multiple possible of HPP degradation pathways of MEPE0129 and MEPE0902, hydroxyquinol ring-cleavage activity were analyzed in degradation strains. Crude extract experiment indicated that MEPE0129 and MEPE0902 own hydroxyquinol ring-cleavage activity. By PCR with conservative primer in MEPE0129 genome, we got fragment of hydroxyquinol 1,2-dioxygenase gene. Then by using Sefa-PCR and Tail-PCR, 6.8 k gene cluster of aromatic degradation was acquired. Complete MEPE0129 hydroxyquinol 1,2-dioxygenase gene hppC was cloned and constructed to pET-29a vector. Express HppC activity is lower than hydroxyquinol 1,2-dioxygenase in crude extract, suggesting that other hydroxyquinol 1,2-dioxygenase may exist in MEPE0129.