除草剂2,4-D降解菌株的分离、筛选与鉴定
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摘要
2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid,2,4-D)是一类广泛应用于单子叶作物田间杂草防除的除草剂,但其大量施用导致的环境残留已对生态环境造成严重威胁。通过富集培养的方法,从2,4-D污染土壤样品中筛选分离到85株耐受菌,16S rDNA分析显示其分属假单胞菌属(Pseudomonas)、芽孢杆菌属(Bacillus)、赖氨酸杆菌属(Lysinibacillus)、类芽孢杆菌属(Paenibacillus)、短杆菌属(Brevibacillus)、沙雷氏菌属(Serratia)、甲基杆菌属(Methylobacterium)、贪铜菌属(Cupriavidus)、红球菌属(Rhodococcus)、拉恩氏菌属(Rahnella)、葡萄球菌属(Staphylococcus)、阿氏肠杆菌属(Enterobacter)和分支杆菌属(Mycobacterium)。其中一株假单胞菌属细菌2,4-D最高耐受浓度达8 g/L。选取部分菌株以2,4-D为唯一碳源培养,通过检测2,4-D降解产物(4-aminoantipyrine显色测量和高效液相色谱)测定菌株的降解能力,并利用质谱(LC/MS、LC-QTOF)检测其代谢产物。共确定11株菌株具有较高的2,4-D降解能力,其中菌株L1、L2、L4和L6在培养72 h后2,4-D的利用率分别为17.3%、28.07%、38.97%和49.02%;LC/MS和LC-QTOF分析检测表明上述菌株将2,4-D脱去侧链基团转化为酚类物质,推测其降解主要为2,4-二氯苯酚(2,4-DCP)途径。
2,4-dichlorophenoxyacetic acid(2,4-D) is a systemic and broad-spectrum foliar herbicide which can control broadleaf weeds. But because of its frequent use, 2,4-D has resulted in considerable pollution to water and soil and thus threatened the ecological environment and human health. In this paper, 85 strains were isolated and screened from 2,4-D contaminated soil samples(inorganic salt medium with 2,4-D as the sole carbon source) by enrichment culture. 16 S rDNA analysis showed that it belongs to Pseudomonas, Bacillus, Lysinibacillus, Paenibacillus, Brevibacillus, Serratia, Methylobacterium, Cupriavidus, Rhodococcus, Larender(Rahnella), Staphylococcus, Enterobacter and Mycobacterium. The strain herbicide tolerance test showed that Pseudomonas can tolerate a higher concentration of 2,4-D with a maximum tolerated concentration of 8 g/L. Some strains were selected to be cultured with 2,4-D as the sole carbon source. The degradation ability of the strain was determined by detecting 2,4-D degradation products(4-aminoantipyrine colorimetric measurement and high performance liquid chromatography), the LC/MS and LC-QTOF were used to detected its metabolites. A total of 11 strains were identified with highly 2,4-D degradation ability. The utilization rate of L1, L2, L4 and L6 cultured for 72 h was 17.3%, 28.07%, 38.97% and 49.02% respectively. The preliminary analysis and structural prediction of the metabolites of the above selected strains were carried out by LC/MS and LC-QTOF. The results showed that a total of 4 strains could convert 2,4-D de-chained groups into phenolic substances, presumably the product was 2,4-dichlorophenol(2,4-DCP).
2,4-dichlorophenoxyacetic acid(2,4-D) is a systemic and broad-spectrum foliar herbicide which can control broadleaf weeds. But because of its frequent use, 2,4-D has resulted in considerable pollution to water and soil and thus threatened the ecological environment and human health. In this paper, 85 strains were isolated and screened from 2,4-D contaminated soil samples(inorganic salt medium with 2,4-D as the sole carbon source) by enrichment culture. 16 S rDNA analysis showed that it belongs to Pseudomonas, Bacillus, Lysinibacillus, Paenibacillus, Brevibacillus, Serratia, Methylobacterium, Cupriavidus, Rhodococcus, Larender(Rahnella), Staphylococcus, Enterobacter and Mycobacterium. The strain herbicide tolerance test showed that Pseudomonas can tolerate a higher concentration of 2,4-D with a maximum tolerated concentration of 8 g/L. Some strains were selected to be cultured with 2,4-D as the sole carbon source. The degradation ability of the strain was determined by detecting 2,4-D degradation products(4-aminoantipyrine colorimetric measurement and high performance liquid chromatography), the LC/MS and LC-QTOF were used to detected its metabolites. A total of 11 strains were identified with highly 2,4-D degradation ability. The utilization rate of L1, L2, L4 and L6 cultured for 72 h was 17.3%, 28.07%, 38.97% and 49.02% respectively. The preliminary analysis and structural prediction of the metabolites of the above selected strains were carried out by LC/MS and LC-QTOF. The results showed that a total of 4 strains could convert 2,4-D de-chained groups into phenolic substances, presumably the product was 2,4-dichlorophenol(2,4-DCP).
引文
[1] Islam F,Wang J,Farooq M A,et al..Potential impact of the herbicide 2,4-dichlorophenoxyacetic acid on human and ecosystems[J].Environ.Intern.,2018,111:332-351.
[2] Walters J.Environmental fate of 2,4-Dichlorophenoxyacetic acid environmental monitoring and pest management,department of pesticide regulation[R/OL].https://mafiadoc.com/environmental-fate-of-24-dichlorophenoxyacetic-acid-california-_597735321723dde18bca2bed.html.
[3] Ordaz-Guillén Y,Galíndez-Mayer C J,Ruiz-Ordaz N,et al..Evaluating the degradation of the herbicides picloram and 2,4-D in a compartmentalized reactive biobarrier with internal liquid recirculation[J].Environ.Sci.Poll.Res.,2014,21(14):8765-8773.
[4] Chinalia F A,Killham K S.2,4-Dichlorophenoxyacetic acid (2,4-D) biodegradation in river sediments of Northeast-Scotland and its effect on the microbial communities (PLFA and DGGE)[J].Chemosphere,2006,64(10):1675-1683.
[5] Gaultier J,Farenhorst A,Cathcart J,et al..Degradation of [carboxyl-14C] 2,4-D and [ring-U-14C] 2,4-D in 114 agricultural soils as affected by soil organic carbon content[J].Soil Biol.Biochem.,2008,40(1):217-227.
[6] Kearns J P,Wellborn L S,Summers R S,et al..2,4-D adsorption to biochars:Effect of preparation conditions on equilibrium adsorption capacity and comparison with commercial activated carbon literature data[J].Water Res.,2014,62:20-28.
[7] Shareef K,Shaw G.Sorption kinetics of 2,4-D and carbaryl in selected agricultural soils of northern Iraq:Application of a dual-rate model[J].Chemosphere,2008,72(1):8-15.
[8] Mountassif D,Kabine M,Mounchid K,et al..Biochemical and histological alterations of cellular metabolism from jerboa (Jaculus orientalis) by 2,4-Dichlorophenoxyacetic acid:Effects on D-3-hydroxybutyrate dehydrogenase[J].Pest.Biochem.Physiol.,2008,90(2):87-96.
[9] Boivin A,Amellal S,Schiavon M,et al..2,4-Dichlorophenoxyacetic acid (2,4-D) sorption and degradation dynamics in three agricultural soils[J].Environ.Poll.,2005,138(1):92-99.
[10] Lappin H M,Greaves M P,Slater J H.Degradation of the herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] by a synergistic microbial community[J].Appl.Environ.Microbiol.,1985,49(2):429-433.
[11] Itoh K,Kanda R,Sumita Y,et al..tfdA-like genes in 2,4-dichlorophenoxyacetic acid-degrading bacteria belonging to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in α-Proteobacteria[J].Appl.Environ.Microbiol.,2002,68(7):3449-3454.
[12] Liu Y J,Liu S J,Drake H L,et al..Consumers of 4-chloro-2-methylphenoxyacetic acid from agricultural soil and drilosphere harbor cadA,r/sdpA,and tfdA-like gene encoding oxygenases[J].FEMS Microbiol.Ecol.,2013,86(1):114-129.
[13] 韩丽珍,赵德刚,罗信旭.除草剂 2,4-D 对土壤微生物类群的影响[J].贵州农业科学,2014,42(2):85-88.
[14] Kumar A,Trefault N,Olaniran A O.Microbial degradation of 2,4-dichlorophenoxyacetic acid:Insight into the enzymes and catabolic genes involved,their regulation and biotechnological implications[J].Crit.Rev.Microbiol.,2016,42(2):194-208.
[15] 卫亚红,张晓燕,曲东.2,4-D 除草剂降解菌的分离及其生物学特性的研究[J].农业环境科学学报,2007,26(6):2183-2188.
[16] Zaprasis A,Liu Y J,Liu S J,et al..Abundance of novel and diverse tfdA-like genes,encoding putative phenoxyalkanoic acid herbicide-degrading dioxygenases,in soil [J].Appl.Environ.Microbiol.,2010,76(1):119-128.
[17] González A J,Gallego A,Gemini V L,et al..Degradation and detoxification of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) by an indigenous Delftia sp.strain in batch and continuous systems[J].Intern.Biodet.Biodeg.,2012,66(1):8-13.
[18] Zabaloy M C,Gómez M A.Isolation and characterization of indigenous 2,4-D herbicide degrading bacteria from an agricultural soil in proximity of Sauce Grande River,Argentina[J].Ann.Microbiol.,2014,64(3):969-974.
[19] Han L,Zhao D,Li C.Isolation and 2,4-D-degrading characteristics of Cupriavidus campinensis BJ71 [J].Brazil.J.Microbiol.,2015,46(2):433-441.
[20] Chang Y C,Reddy M V,Umemoto H,et al..Bio-augmentation of Cupriavidus sp.CY-1 into 2,4-D contaminated soil:microbial community analysis by culture dependent and independent techniques[J].PLoS ONE,2015,10(12):e0145057.
[21] Dai Y,Li N,Zhao Q,et al..Bioremediation using Novosphingobium strain DY4 for 2,4-dichlorophenoxyacetic acid-contaminated soil and impact on microbial community structure[J].Biodegradation,2015,26(2):161-170.
[22] González-Cuna S,Galíndez-Mayer J,Ruiz-Ordaz N,et al..Aerobic biofilm reactor for treating a commercial formulation of the herbicides 2,4-D and dicamba:Biodegradation kinetics and biofilm bacterial diversity[J].Intern.Biodet.Biodeg.,2016,107:123-131.
[23] Quan X,Ma J,Xiong W,et al..Bioaugmentation of half-matured granular sludge with special microbial culture promoted establishment of 2,4-dichlorophenoxyacetic acid degrading aerobic granules[J].Bioproc.Biosyst.Engin.,2015,38(6):1081-1090.
[24] Xia Z Y,Zhang L,Zhao Y,et al..Biodegradation of the herbicide 2,4-dichlorophenoxyacetic acid by a new isolated strain of Achromobacter sp.LZ35 [J].Curr.Microbiol.,2017,74(2):193-202.
[25] Loos M,Roberts R N,Alexander M.Formation of 2,4-dichlorophenol and 2,4-dichloroanisole from 2,4-dichlorophenoxyacetate by Arthrobacter sp.[J].Canad.J.Microbiol.,1967,13(6):691-699.
[26] Rodríguez-Cruz M S,B?lum J,Shaw L J,et al..Biodegradation of the herbicide mecoprop-p with soil depth and its relationship with class III tfdA genes[J].Soil Biol.Biochem.,2010,42(1):32-39.
[27] Balajee S,Mahadevan A.Dissimilation of 2,4-dichlorophenoxyacetic acid by Azotobacter chroococcum[J].Xenobiotica,1990,20(6):607-617.
[28] Dorn E,Knackmuss H J.Chemical structure and biodegradability of halogenated aromatic compounds.Substituent effects on 1,2-dioxygenation of catechol[J].Biochem.J.,1978,174(1):85-94.
[29] Newby D T,Gentry T J,Pepper I L.Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors[J].Appl.Environ.Microbiol.,2000,66(8):3399-3407.
[30] Streber W R,Timmis K N,Zenk M H.Analysis,cloning,and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134[J].J.Bacteriol.,1987,169(7):2950-2955.
[31] Aspray T J,Hansen S K,Burns R G.A soil-based microbial biofilm exposed to 2,4-D:bacterial community development and establishment of conjugative plasmid pJP4[J].FEMS Microbiol.Ecol.,2005,54(2):317-327.
[32] Annadurai G,Juang R S,Lee D J.Microbiological degradation of phenol using mixed liquors of Pseudomonas putida and activated sludge [J].Waste Manag.,2002,22(7):703-710.
[33] Olaniran A O,Singh L,Kumar A,et al..Aerobic degradation of 2,4-dichlorophenoxyacetic acid and other chlorophenols by Pseudomonas strains indigenous to contaminated soil in South Africa:Growth kinetics and degradation pathway[J].Appl.Biochem.Microbiol.,2017,53(2):209-216.
[34] Chetverikov S P,Sharipov D A,Korshunova T Y,et al..Degradation of perfluorooctanyl sulfonate by strain Pseudomonas plecoglossicida 2,4-D [J].Appl.Biochem.Microbiol.,2017,53(5):533-538.
[35] Nwinyi O C,Ajayi O O,Amund O O.Degradation of polynuclear aromatic hydrocarbons by two strains of Pseudomonas[J].Brazil.J.Microbiol.,2016,47(3):551-562.
[36] Zylstra G J,McCombie W R,Gibson D T,et al..Toluene degradation by Pseudomonas putida F1:genetic organization of the tod operon[J].Appl.Environ.Microbiol.,1988,54(6):1498-1503.
[37] Short K A,King R J,Seidler R J,et al..Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301 (pR0103),a constitutive degrader of 2,4-dichlorophenoxyacetate[J].Mol.Ecol.,1992,1(2):89-94.
[38] Park H S,Lim S J,Chang Y K,et al..Degradation of chloronitrobenzenes by a coculture of Pseudomonas putida and Rhodococcus sp.[J].Appl.Environ.Microbiol.,1999,65(3):1083-1091.
[39] Peng P,Yang H,Jia R,et al..Biodegradation of dioxin by a newly isolated Rhodococcus sp.with the involvement of self-transmissible plasmids[J].Appl.Microbiol.Biotechnol.,2013,97(12):5585-5595.
[40] Cuadrado V,Gomila M,Merini L,et al..Cupriavidus pampae sp.nov.,a novel herbicide-degrading bacterium isolated from agricultural soil [J].Int.J.Syst.Evol.Microbiol.,2010,60(11):2606-2612.
[2] Walters J.Environmental fate of 2,4-Dichlorophenoxyacetic acid environmental monitoring and pest management,department of pesticide regulation[R/OL].https://mafiadoc.com/environmental-fate-of-24-dichlorophenoxyacetic-acid-california-_597735321723dde18bca2bed.html.
[3] Ordaz-Guillén Y,Galíndez-Mayer C J,Ruiz-Ordaz N,et al..Evaluating the degradation of the herbicides picloram and 2,4-D in a compartmentalized reactive biobarrier with internal liquid recirculation[J].Environ.Sci.Poll.Res.,2014,21(14):8765-8773.
[4] Chinalia F A,Killham K S.2,4-Dichlorophenoxyacetic acid (2,4-D) biodegradation in river sediments of Northeast-Scotland and its effect on the microbial communities (PLFA and DGGE)[J].Chemosphere,2006,64(10):1675-1683.
[5] Gaultier J,Farenhorst A,Cathcart J,et al..Degradation of [carboxyl-14C] 2,4-D and [ring-U-14C] 2,4-D in 114 agricultural soils as affected by soil organic carbon content[J].Soil Biol.Biochem.,2008,40(1):217-227.
[6] Kearns J P,Wellborn L S,Summers R S,et al..2,4-D adsorption to biochars:Effect of preparation conditions on equilibrium adsorption capacity and comparison with commercial activated carbon literature data[J].Water Res.,2014,62:20-28.
[7] Shareef K,Shaw G.Sorption kinetics of 2,4-D and carbaryl in selected agricultural soils of northern Iraq:Application of a dual-rate model[J].Chemosphere,2008,72(1):8-15.
[8] Mountassif D,Kabine M,Mounchid K,et al..Biochemical and histological alterations of cellular metabolism from jerboa (Jaculus orientalis) by 2,4-Dichlorophenoxyacetic acid:Effects on D-3-hydroxybutyrate dehydrogenase[J].Pest.Biochem.Physiol.,2008,90(2):87-96.
[9] Boivin A,Amellal S,Schiavon M,et al..2,4-Dichlorophenoxyacetic acid (2,4-D) sorption and degradation dynamics in three agricultural soils[J].Environ.Poll.,2005,138(1):92-99.
[10] Lappin H M,Greaves M P,Slater J H.Degradation of the herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] by a synergistic microbial community[J].Appl.Environ.Microbiol.,1985,49(2):429-433.
[11] Itoh K,Kanda R,Sumita Y,et al..tfdA-like genes in 2,4-dichlorophenoxyacetic acid-degrading bacteria belonging to the Bradyrhizobium-Agromonas-Nitrobacter-Afipia cluster in α-Proteobacteria[J].Appl.Environ.Microbiol.,2002,68(7):3449-3454.
[12] Liu Y J,Liu S J,Drake H L,et al..Consumers of 4-chloro-2-methylphenoxyacetic acid from agricultural soil and drilosphere harbor cadA,r/sdpA,and tfdA-like gene encoding oxygenases[J].FEMS Microbiol.Ecol.,2013,86(1):114-129.
[13] 韩丽珍,赵德刚,罗信旭.除草剂 2,4-D 对土壤微生物类群的影响[J].贵州农业科学,2014,42(2):85-88.
[14] Kumar A,Trefault N,Olaniran A O.Microbial degradation of 2,4-dichlorophenoxyacetic acid:Insight into the enzymes and catabolic genes involved,their regulation and biotechnological implications[J].Crit.Rev.Microbiol.,2016,42(2):194-208.
[15] 卫亚红,张晓燕,曲东.2,4-D 除草剂降解菌的分离及其生物学特性的研究[J].农业环境科学学报,2007,26(6):2183-2188.
[16] Zaprasis A,Liu Y J,Liu S J,et al..Abundance of novel and diverse tfdA-like genes,encoding putative phenoxyalkanoic acid herbicide-degrading dioxygenases,in soil [J].Appl.Environ.Microbiol.,2010,76(1):119-128.
[17] González A J,Gallego A,Gemini V L,et al..Degradation and detoxification of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) by an indigenous Delftia sp.strain in batch and continuous systems[J].Intern.Biodet.Biodeg.,2012,66(1):8-13.
[18] Zabaloy M C,Gómez M A.Isolation and characterization of indigenous 2,4-D herbicide degrading bacteria from an agricultural soil in proximity of Sauce Grande River,Argentina[J].Ann.Microbiol.,2014,64(3):969-974.
[19] Han L,Zhao D,Li C.Isolation and 2,4-D-degrading characteristics of Cupriavidus campinensis BJ71 [J].Brazil.J.Microbiol.,2015,46(2):433-441.
[20] Chang Y C,Reddy M V,Umemoto H,et al..Bio-augmentation of Cupriavidus sp.CY-1 into 2,4-D contaminated soil:microbial community analysis by culture dependent and independent techniques[J].PLoS ONE,2015,10(12):e0145057.
[21] Dai Y,Li N,Zhao Q,et al..Bioremediation using Novosphingobium strain DY4 for 2,4-dichlorophenoxyacetic acid-contaminated soil and impact on microbial community structure[J].Biodegradation,2015,26(2):161-170.
[22] González-Cuna S,Galíndez-Mayer J,Ruiz-Ordaz N,et al..Aerobic biofilm reactor for treating a commercial formulation of the herbicides 2,4-D and dicamba:Biodegradation kinetics and biofilm bacterial diversity[J].Intern.Biodet.Biodeg.,2016,107:123-131.
[23] Quan X,Ma J,Xiong W,et al..Bioaugmentation of half-matured granular sludge with special microbial culture promoted establishment of 2,4-dichlorophenoxyacetic acid degrading aerobic granules[J].Bioproc.Biosyst.Engin.,2015,38(6):1081-1090.
[24] Xia Z Y,Zhang L,Zhao Y,et al..Biodegradation of the herbicide 2,4-dichlorophenoxyacetic acid by a new isolated strain of Achromobacter sp.LZ35 [J].Curr.Microbiol.,2017,74(2):193-202.
[25] Loos M,Roberts R N,Alexander M.Formation of 2,4-dichlorophenol and 2,4-dichloroanisole from 2,4-dichlorophenoxyacetate by Arthrobacter sp.[J].Canad.J.Microbiol.,1967,13(6):691-699.
[26] Rodríguez-Cruz M S,B?lum J,Shaw L J,et al..Biodegradation of the herbicide mecoprop-p with soil depth and its relationship with class III tfdA genes[J].Soil Biol.Biochem.,2010,42(1):32-39.
[27] Balajee S,Mahadevan A.Dissimilation of 2,4-dichlorophenoxyacetic acid by Azotobacter chroococcum[J].Xenobiotica,1990,20(6):607-617.
[28] Dorn E,Knackmuss H J.Chemical structure and biodegradability of halogenated aromatic compounds.Substituent effects on 1,2-dioxygenation of catechol[J].Biochem.J.,1978,174(1):85-94.
[29] Newby D T,Gentry T J,Pepper I L.Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors[J].Appl.Environ.Microbiol.,2000,66(8):3399-3407.
[30] Streber W R,Timmis K N,Zenk M H.Analysis,cloning,and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134[J].J.Bacteriol.,1987,169(7):2950-2955.
[31] Aspray T J,Hansen S K,Burns R G.A soil-based microbial biofilm exposed to 2,4-D:bacterial community development and establishment of conjugative plasmid pJP4[J].FEMS Microbiol.Ecol.,2005,54(2):317-327.
[32] Annadurai G,Juang R S,Lee D J.Microbiological degradation of phenol using mixed liquors of Pseudomonas putida and activated sludge [J].Waste Manag.,2002,22(7):703-710.
[33] Olaniran A O,Singh L,Kumar A,et al..Aerobic degradation of 2,4-dichlorophenoxyacetic acid and other chlorophenols by Pseudomonas strains indigenous to contaminated soil in South Africa:Growth kinetics and degradation pathway[J].Appl.Biochem.Microbiol.,2017,53(2):209-216.
[34] Chetverikov S P,Sharipov D A,Korshunova T Y,et al..Degradation of perfluorooctanyl sulfonate by strain Pseudomonas plecoglossicida 2,4-D [J].Appl.Biochem.Microbiol.,2017,53(5):533-538.
[35] Nwinyi O C,Ajayi O O,Amund O O.Degradation of polynuclear aromatic hydrocarbons by two strains of Pseudomonas[J].Brazil.J.Microbiol.,2016,47(3):551-562.
[36] Zylstra G J,McCombie W R,Gibson D T,et al..Toluene degradation by Pseudomonas putida F1:genetic organization of the tod operon[J].Appl.Environ.Microbiol.,1988,54(6):1498-1503.
[37] Short K A,King R J,Seidler R J,et al..Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301 (pR0103),a constitutive degrader of 2,4-dichlorophenoxyacetate[J].Mol.Ecol.,1992,1(2):89-94.
[38] Park H S,Lim S J,Chang Y K,et al..Degradation of chloronitrobenzenes by a coculture of Pseudomonas putida and Rhodococcus sp.[J].Appl.Environ.Microbiol.,1999,65(3):1083-1091.
[39] Peng P,Yang H,Jia R,et al..Biodegradation of dioxin by a newly isolated Rhodococcus sp.with the involvement of self-transmissible plasmids[J].Appl.Microbiol.Biotechnol.,2013,97(12):5585-5595.
[40] Cuadrado V,Gomila M,Merini L,et al..Cupriavidus pampae sp.nov.,a novel herbicide-degrading bacterium isolated from agricultural soil [J].Int.J.Syst.Evol.Microbiol.,2010,60(11):2606-2612.