一株路德维希肠杆菌的筛选及其对3-苯氧基苯甲酸的降解特性分析
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摘要
【目的】3-苯氧基苯甲酸(3-phenoxybenzoic acid,3-PBA)的消除是解决拟除虫菊酯类农药污染的关键,目的是从受拟除虫菊酯类农药污染植物根系土壤中分离出3-PBA高效降解菌株。【方法】采用富集驯化、筛选纯化方法,以3-PBA为唯一碳源、能源筛选3-PBA降解菌株;菌株鉴定采用形态、生理生化和16S r RNA序列分析法;并研究其生长降解动力学特性,最后采用Box-Behnken响应面分析确定最佳降解条件。【结果】从川北地区大豆根系土壤中筛选得到1株高效降解菌BPBA031,经鉴定为路德维希肠杆菌(Enterobacter ludwigii);该菌株耐3-PBA浓度达1600 mg/L,其生长降解过程分别符合Logistic生长动力学(μm=0.09149 h~(–1),X_m=1.1145)和一级降解动力学模型(k=0.02085,t_(1/2)=33.24 h);对3-PBA降解的最适条件为34–37°C、3-PBA浓度25–200 mg/L和p H 7.5–8.5;在35.19°C、30.0 mg/L 3-PBA和p H 7.58条件下,该菌株48 h对3-PBA的降解率达83.75%。【结论】路德维希肠杆菌BPBA031是1株高效3-PBA降解菌,可作为生物修复受3-PBA或拟除虫菊酯类农药污染环境的潜在菌株资源。
[Objective] Elimination of 3-phenoxybenzoic acid(3-PBA) is the key to solve the pyrethroid pesticides contamination environment. The aim of this study was to isolate efficient 3-PBA-degrading strains from the plant rhizosphere contaminated by pyrethroid pesticides. [Methods] 3-PBA-degrading strains was screened by using enrichment domestication, isolating and purification methods with 3-PBA used as the sole carbon source. Then the effcient 3-PBA-degrading strain was identified by morphological, physio-biochemical tests and 16 S rRNA sequence analysis, and its growth and degradation kinetics and degradation characteristics were studied. Moreover, the degradation conditions were optimized by the Box-Behnken response surface analysis. [Results] A novel 3-PBA-degrading strain BPBA031 isolated from the soybean rhizosphere in northern Sichuan Province was classified into Enterobacter ludwigii, which could be resistant to high concentration of 3-PBA(1600 mg/L). The growth of strain BPBA031 and 3-PBA degradation respectively followed the logistic growth kinetic model(μm=0.09149 h~(–1), X_m=1.1145) and first-order degradation kinetic model(k=0.02085, t_(1/2)=33.24 h). The optimum conditions for 3-PBA degradation were culture temprature of 34–37 °C, 3-PBA concentration of 25–200 mg/L and initial p H of 7.5–8.5. In addition, the degradation rate of 3-PBA was 83.75% at 35.19 °C, 3-PBA concentration of 30.0 mg/L and initial p H 7.58 after 48 h of incubation. [Conclusion] Enterobacter ludwigii BPBA031, a highly effcient 3-PBA-degrading strain, can be used as a potential strain resource for bioremediation of environment polluted by 3-PBA or pyrethroid pesticides.
[Objective] Elimination of 3-phenoxybenzoic acid(3-PBA) is the key to solve the pyrethroid pesticides contamination environment. The aim of this study was to isolate efficient 3-PBA-degrading strains from the plant rhizosphere contaminated by pyrethroid pesticides. [Methods] 3-PBA-degrading strains was screened by using enrichment domestication, isolating and purification methods with 3-PBA used as the sole carbon source. Then the effcient 3-PBA-degrading strain was identified by morphological, physio-biochemical tests and 16 S rRNA sequence analysis, and its growth and degradation kinetics and degradation characteristics were studied. Moreover, the degradation conditions were optimized by the Box-Behnken response surface analysis. [Results] A novel 3-PBA-degrading strain BPBA031 isolated from the soybean rhizosphere in northern Sichuan Province was classified into Enterobacter ludwigii, which could be resistant to high concentration of 3-PBA(1600 mg/L). The growth of strain BPBA031 and 3-PBA degradation respectively followed the logistic growth kinetic model(μm=0.09149 h~(–1), X_m=1.1145) and first-order degradation kinetic model(k=0.02085, t_(1/2)=33.24 h). The optimum conditions for 3-PBA degradation were culture temprature of 34–37 °C, 3-PBA concentration of 25–200 mg/L and initial p H of 7.5–8.5. In addition, the degradation rate of 3-PBA was 83.75% at 35.19 °C, 3-PBA concentration of 30.0 mg/L and initial p H 7.58 after 48 h of incubation. [Conclusion] Enterobacter ludwigii BPBA031, a highly effcient 3-PBA-degrading strain, can be used as a potential strain resource for bioremediation of environment polluted by 3-PBA or pyrethroid pesticides.
引文
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[4]Watkins DJ,Fortenberry GZ,Sánchez BN,Barr DB,Panuwet P,Schnaas L,Osorio-Valencia E,Solano-González M,Ettinger AS,Hernández-ávila M,Hu H,Téllez-Rojo MM,Meeker JD.Urinary 3-phenoxybenzoic acid(3-PBA)levels among pregnant women in Mexico City:distribution and relationships with child neurodevelopment.Environmental Research,2016,147:307–313.
[5]Sun H,Chen W,Xu XL,Ding Z,Chen XD,Wang XR.Pyrethroid and their metabolite,3-phenoxybenzoic acid showed similar(anti)estrogenic activity in human and rat estrogen receptorα-mediated reporter gene assays.Environmental Toxicology and Pharmacology,2014,37(1):371–377.
[6]Mc Coy MR,Yang Z,Fu X,Ahn KC,Gee SJ,Bom DC,Zhong P,Chang D,Hammock BD.Monitoring of total type II pyrethroid pesticides in citrus oils and water by converting to a common product 3-phenoxybenzoic acid.Journal of Agricultural and Food Chemistry,2012,60(20):5065–5070.
[7]Liu Y,Wu AH,Hu J,Lin MM,Wen MT,Zhang X,Xu CX,Hu XD,Zhong JF,Jiao LX,Xie YJ,Zhang CZ,Yu XY,Liang Y,Liu XJ.Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.Analytical Biochemistry,2015,483(1):7–11.
[8]Yuan C,Wang C,Gao SQ,Kong TT,Chen L,Li XF,Song L,Wang YB.Effects of permethrin,cypermethrin and3-phenoxybenzoic acid on rat sperm motility in vitro evaluated with computer-assisted sperm analysis.Toxicology in vitro,2010,24(2):382–386.
[9]Sun H,Xu XL,Xu LC,Song L,Hong X,Chen JF,Cui LB,Wang XR.Antiandrogenic activity of pyrethroid pesticides and their metabolite in reporter gene assay.Chemosphere,2007,66(3):474–479.
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[12]Tang J,Yao K,Liu SL,Jia DY,Chi YL,Zeng CY,Wu S.Biodegradation of 3-phenoxybenzoic acid by a novel Sphingomonas SP.SC-1.Fresenius Environmental Bulletin,2013,22(5):1564–1572.
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[14]Chen SH,Hu MY,Liu JJ,Zhong GH,Yang L,Rizwan-ul-Haq M,Han HT.Biodegradation of beta-cypermethrin and3-phenoxybenzoic acid by a novel Ochrobactrum lupini DG-S-01.Journal of Hazardous Materials,2011,187(1/3):433–440.
[15]Zhang J,Lang ZF,Zheng JW,Hang BJ,Duan XQ,He J,Li SP.Sphingobium jiangsuense sp.nov.,a 3-phenoxybenzoic acid-degrading bacterium isolated from a wastewater treatment system.International Journal of Systematic and Evolutionary Microbiology,2012,62(Pt 4):800–805.
[16]Chen SH,Yang L,Hu MY,Liu JJ.Biodegradation of fenvalerate and 3-phenoxybenzoic acid by a novel Stenotrophomonas sp.strain ZS-S-01 and its use in bioremediation of contaminated soils.Applied Microbiology and Biotechnology,2011,90(2):755–767.
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[18]Zhu YT,Li JL,Yao K,Zhao N,Zhou K,Hu XJ,Zou LK,Han XF,Liu AP,Liu SL.Degradation of 3-phenoxybenzoic acid by a filamentous fungus Aspergillus oryzae M-4 strain with self-protection transformation.Applied Microbiology and Biotechnology,2016,100(22):9773–9786.
[19]Wang Y,Chen H,Liu YX,Ren RP,Lv YK.An adsorption-release-biodegradation system for simultaneous biodegradation of phenol and ammonium in phenol-rich wastewater.Bioresource Technology,2016,211:711–719.
[20]Pinyakong O,Habe H,Supaka N,Pinpanichkarn P,Juntongjin K,Yoshida T,Furihata K,Nojiri H,Yamane H,Omori T.Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp.strain P2.FEMS Microbiology Letters,2000,191(1):115–121.
[21]Liu B,Tang J,Chen TT,Shi Y,Zeng L,Zeng CY.Isolation and identification of two pyrethroid pesticides degradation strains and research of degradative capabilities.Science and Technology of Food Industry,2017,38(4):214–219,224.(in Chinese)刘波,唐洁,陈廷廷,史颖,曾林,曾朝懿.两株拟除虫菊酯类农药降解菌的分离鉴定及其降解能力的研究.食品工业科技,2017,38(4):214–219,224.
[22]Zhang YX,Meng XJ,Chai TY.Characterization of phenol-degrading Rhodococcus sp.strain P1 from coking wastewater.Acta Microbiologica Sinica,2013,53(10):1117–1124.(in Chinese)张玉秀,蒙小俊,柴团耀.苯酚降解菌红球菌(Rhodococcus sp.)P1的鉴定及其在焦化废水中的应用.微生物学报,2013,53(10):1117–1124.
[23]Ren L,Shi YH,Jia Y,Yao XS,Nahurira R,Mi CX,Yan YC.Biodegradation characteristics and kinetics of p-nitrophenol by strain Arthrobacter sp.CN2.Environmental Science,2015,36(5):1757–1762.(in Chinese)任磊,史延华,贾阳,姚雪松,Nahurira R,弥春霞,闫艳春.菌株Arthrobacter sp.CN2降解对硝基苯酚的特性与动力学.环境科学,2015,36(5):1757–1762.
[24]Hoffmann H,Stindl S,Ludwig W,Stumpf A,Mehlen A,Heesemann J,Monget D,Schleifer KH,Roggenkamp A.Reassignment of Enterobacter dissolvens to Enterobacter cloacae as E.cloacae subspecies dissolvens comb.nov.and emended description of Enterobacter asburiae and Enterobacter kobei.Systematic and Applied Microbiology,2005,28(3):196–205.
[25]Lu LL,Xiao M,Xu XD.Enterobacter agglomerans B1producingβ-galactosidase with transglycosylation activity:screening,identification,fermentation conditions,and galacto-oligosaccharides synthesis.Acta Microbiologica Sinica,2008,48(1):38–44.(in Chinese)卢丽丽,肖敏,徐晓东.转糖基β-半乳糖苷酶产生菌Enterobacter agglomerans B1:筛选鉴定、发酵产酶及其合成低聚半乳糖的研究.微生物学报,2008,48(1):38–44.
[26]Shoebitz M,Ribaudo CM,Pardo MA,Cantore ML,Ciampi L,CuráJA.Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere.Soil Biology and Biochemistry,2009,41(9):1768–1774.
[27]Haritash AK,Kaushik CP.Biodegradation aspects of polycyclic aromatic hydrocarbons(PAHs):a review.Journal of Hazardous Materials,2009,169(1/3):1–15.
[28]Hoffmann H,Stindl S,Stumpf A,Mehlen A,Monget D,Heesemann J,Schleifer KH,Roggenkamp A.Description of Enterobacter ludwigii sp.nov.,a novel Enterobacter species of clinical relevance.Systematic and Applied Microbiology,2005,28(3):206–212.
[29]Paauw A,Caspers MPM,Schuren FHJ,Hall MALV,Delétoile A,Montijn RC,Verhoef J,Fluit AC.Genomic diversity within the Enterobacter cloacae complex.PLo S One,2008,3(8):e3018.
[30]Kwon TY,Shim SM,Heo MY,An DH,Shin KS,Lee JH.Isolation and characterization of exopolysaccharide-producing bacteria from Korean fermented vegetables.Korean Journal of Microbiology and Biotechnology,2007,35(3):191–195.
[31]Pau-Roblot C,Lequart-Pillon M,Apanga L,Pilard S,Courtois J,Pawlicki-Jullian N.Structural features and bioremediation activity of an exopolysaccharide produced by a strain of Enterobacter ludwigii isolated in the Chernobyl exclusion zone.Carbohydrate Polymers,2013,93(1):154–162.
[32]Yousaf S,Afzal M,Reichenauer TG,Brady CL,Sessitsch A.Hydrocarbon degradation,plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains.Environmental Pollution,2011,159(10):2675–2683.
[33]Neria-González I,Wang ET,Ramírez F,Romero JM,Hernández-Rodríguez C.Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico.Anaerobe,2006,12(3):122–133.
[34]Singh B,Bhat TK,Sharma OP,Kanwar SS,Rahi P,Gulati A.Isolation of tannase-producing Enterobacter ludwigii GRT-1from the rumen of migratory goats.Small Ruminant Research,2012,102(2/3):172–176.
[35]Yang M,Cai J,Wang CG,Du X,Lin JG.Characterization of endo-β-mannanase from Enterobacter ludwigii MY271 and application in pulp industry.Bioprocess and Biosystems Engineering,2017,40(1):35–43.
[36]Li HX,Lin H,You SH,Xu XY,Xia SQ.Effect of p H on reductive degradation of para-chloronitrobenzene by autohydrogenotrophic microorganisms.Acta Scientiae Circumstantiae,2015,35(7):2083–2089.(in Chinese)李海翔,林华,游少鸿,徐晓茵,夏四清.p H对氢基质自养微生物还原降解对氯硝基苯的影响.环境科学学报,2015,35(7):2083–2089.
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[2]Zhang C,Jia L,Wang SH,Qu J,Li K,Xu LL,Shi YH,Yan YC.Biodegradation of beta-cypermethrin by two Serratia spp.with different cell surface hydrophobicity.Bioresource Technology,2010,101(10):3423–3429.
[3]Chuang JC,Van Emon JM,Trejo RM,Durnford J.Biological monitoring of 3-phenoxybenzoic acid in urine by an enzyme-linked immunosorbent assay.Talanta,2011,83(5):1317–1323.
[4]Watkins DJ,Fortenberry GZ,Sánchez BN,Barr DB,Panuwet P,Schnaas L,Osorio-Valencia E,Solano-González M,Ettinger AS,Hernández-ávila M,Hu H,Téllez-Rojo MM,Meeker JD.Urinary 3-phenoxybenzoic acid(3-PBA)levels among pregnant women in Mexico City:distribution and relationships with child neurodevelopment.Environmental Research,2016,147:307–313.
[5]Sun H,Chen W,Xu XL,Ding Z,Chen XD,Wang XR.Pyrethroid and their metabolite,3-phenoxybenzoic acid showed similar(anti)estrogenic activity in human and rat estrogen receptorα-mediated reporter gene assays.Environmental Toxicology and Pharmacology,2014,37(1):371–377.
[6]Mc Coy MR,Yang Z,Fu X,Ahn KC,Gee SJ,Bom DC,Zhong P,Chang D,Hammock BD.Monitoring of total type II pyrethroid pesticides in citrus oils and water by converting to a common product 3-phenoxybenzoic acid.Journal of Agricultural and Food Chemistry,2012,60(20):5065–5070.
[7]Liu Y,Wu AH,Hu J,Lin MM,Wen MT,Zhang X,Xu CX,Hu XD,Zhong JF,Jiao LX,Xie YJ,Zhang CZ,Yu XY,Liang Y,Liu XJ.Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.Analytical Biochemistry,2015,483(1):7–11.
[8]Yuan C,Wang C,Gao SQ,Kong TT,Chen L,Li XF,Song L,Wang YB.Effects of permethrin,cypermethrin and3-phenoxybenzoic acid on rat sperm motility in vitro evaluated with computer-assisted sperm analysis.Toxicology in vitro,2010,24(2):382–386.
[9]Sun H,Xu XL,Xu LC,Song L,Hong X,Chen JF,Cui LB,Wang XR.Antiandrogenic activity of pyrethroid pesticides and their metabolite in reporter gene assay.Chemosphere,2007,66(3):474–479.
[10]Topp E,Akhtar MH.Mineralization of 3-phenoxybenzoate by a two-membered bacterial co-culture.Canadian Journal of Microbiology,1990,36(7):495–499.
[11]Vidal JLM,Plaza-Bola?os P,Romero-González R,Frenich AG.Determination of pesticide transformation products:a review of extraction and detection methods.Journal of Chromatography A,2009,1216(40):6767–6788.
[12]Tang J,Yao K,Liu SL,Jia DY,Chi YL,Zeng CY,Wu S.Biodegradation of 3-phenoxybenzoic acid by a novel Sphingomonas SP.SC-1.Fresenius Environmental Bulletin,2013,22(5):1564–1572.
[13]Halden RU,Tepp SM,Halden BG,Dwyer DF.Degradation of3-phenoxybenzoic acid in soil by Pseudomonas pseudoalcaligenes POB310(p POB)and two modified Pseudomonas strains.Applied and Environmental Microbiology,1999,65(8):3354–3359.
[14]Chen SH,Hu MY,Liu JJ,Zhong GH,Yang L,Rizwan-ul-Haq M,Han HT.Biodegradation of beta-cypermethrin and3-phenoxybenzoic acid by a novel Ochrobactrum lupini DG-S-01.Journal of Hazardous Materials,2011,187(1/3):433–440.
[15]Zhang J,Lang ZF,Zheng JW,Hang BJ,Duan XQ,He J,Li SP.Sphingobium jiangsuense sp.nov.,a 3-phenoxybenzoic acid-degrading bacterium isolated from a wastewater treatment system.International Journal of Systematic and Evolutionary Microbiology,2012,62(Pt 4):800–805.
[16]Chen SH,Yang L,Hu MY,Liu JJ.Biodegradation of fenvalerate and 3-phenoxybenzoic acid by a novel Stenotrophomonas sp.strain ZS-S-01 and its use in bioremediation of contaminated soils.Applied Microbiology and Biotechnology,2011,90(2):755–767.
[17]Chen SH,Hu W,Xiao Y,Deng YY,Jia JW,Hu MY.Degradation of 3-phenoxybenzoic acid by a Bacillus sp.PLo S One,2012,7(11):e50456.
[18]Zhu YT,Li JL,Yao K,Zhao N,Zhou K,Hu XJ,Zou LK,Han XF,Liu AP,Liu SL.Degradation of 3-phenoxybenzoic acid by a filamentous fungus Aspergillus oryzae M-4 strain with self-protection transformation.Applied Microbiology and Biotechnology,2016,100(22):9773–9786.
[19]Wang Y,Chen H,Liu YX,Ren RP,Lv YK.An adsorption-release-biodegradation system for simultaneous biodegradation of phenol and ammonium in phenol-rich wastewater.Bioresource Technology,2016,211:711–719.
[20]Pinyakong O,Habe H,Supaka N,Pinpanichkarn P,Juntongjin K,Yoshida T,Furihata K,Nojiri H,Yamane H,Omori T.Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp.strain P2.FEMS Microbiology Letters,2000,191(1):115–121.
[21]Liu B,Tang J,Chen TT,Shi Y,Zeng L,Zeng CY.Isolation and identification of two pyrethroid pesticides degradation strains and research of degradative capabilities.Science and Technology of Food Industry,2017,38(4):214–219,224.(in Chinese)刘波,唐洁,陈廷廷,史颖,曾林,曾朝懿.两株拟除虫菊酯类农药降解菌的分离鉴定及其降解能力的研究.食品工业科技,2017,38(4):214–219,224.
[22]Zhang YX,Meng XJ,Chai TY.Characterization of phenol-degrading Rhodococcus sp.strain P1 from coking wastewater.Acta Microbiologica Sinica,2013,53(10):1117–1124.(in Chinese)张玉秀,蒙小俊,柴团耀.苯酚降解菌红球菌(Rhodococcus sp.)P1的鉴定及其在焦化废水中的应用.微生物学报,2013,53(10):1117–1124.
[23]Ren L,Shi YH,Jia Y,Yao XS,Nahurira R,Mi CX,Yan YC.Biodegradation characteristics and kinetics of p-nitrophenol by strain Arthrobacter sp.CN2.Environmental Science,2015,36(5):1757–1762.(in Chinese)任磊,史延华,贾阳,姚雪松,Nahurira R,弥春霞,闫艳春.菌株Arthrobacter sp.CN2降解对硝基苯酚的特性与动力学.环境科学,2015,36(5):1757–1762.
[24]Hoffmann H,Stindl S,Ludwig W,Stumpf A,Mehlen A,Heesemann J,Monget D,Schleifer KH,Roggenkamp A.Reassignment of Enterobacter dissolvens to Enterobacter cloacae as E.cloacae subspecies dissolvens comb.nov.and emended description of Enterobacter asburiae and Enterobacter kobei.Systematic and Applied Microbiology,2005,28(3):196–205.
[25]Lu LL,Xiao M,Xu XD.Enterobacter agglomerans B1producingβ-galactosidase with transglycosylation activity:screening,identification,fermentation conditions,and galacto-oligosaccharides synthesis.Acta Microbiologica Sinica,2008,48(1):38–44.(in Chinese)卢丽丽,肖敏,徐晓东.转糖基β-半乳糖苷酶产生菌Enterobacter agglomerans B1:筛选鉴定、发酵产酶及其合成低聚半乳糖的研究.微生物学报,2008,48(1):38–44.
[26]Shoebitz M,Ribaudo CM,Pardo MA,Cantore ML,Ciampi L,CuráJA.Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere.Soil Biology and Biochemistry,2009,41(9):1768–1774.
[27]Haritash AK,Kaushik CP.Biodegradation aspects of polycyclic aromatic hydrocarbons(PAHs):a review.Journal of Hazardous Materials,2009,169(1/3):1–15.
[28]Hoffmann H,Stindl S,Stumpf A,Mehlen A,Monget D,Heesemann J,Schleifer KH,Roggenkamp A.Description of Enterobacter ludwigii sp.nov.,a novel Enterobacter species of clinical relevance.Systematic and Applied Microbiology,2005,28(3):206–212.
[29]Paauw A,Caspers MPM,Schuren FHJ,Hall MALV,Delétoile A,Montijn RC,Verhoef J,Fluit AC.Genomic diversity within the Enterobacter cloacae complex.PLo S One,2008,3(8):e3018.
[30]Kwon TY,Shim SM,Heo MY,An DH,Shin KS,Lee JH.Isolation and characterization of exopolysaccharide-producing bacteria from Korean fermented vegetables.Korean Journal of Microbiology and Biotechnology,2007,35(3):191–195.
[31]Pau-Roblot C,Lequart-Pillon M,Apanga L,Pilard S,Courtois J,Pawlicki-Jullian N.Structural features and bioremediation activity of an exopolysaccharide produced by a strain of Enterobacter ludwigii isolated in the Chernobyl exclusion zone.Carbohydrate Polymers,2013,93(1):154–162.
[32]Yousaf S,Afzal M,Reichenauer TG,Brady CL,Sessitsch A.Hydrocarbon degradation,plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains.Environmental Pollution,2011,159(10):2675–2683.
[33]Neria-González I,Wang ET,Ramírez F,Romero JM,Hernández-Rodríguez C.Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico.Anaerobe,2006,12(3):122–133.
[34]Singh B,Bhat TK,Sharma OP,Kanwar SS,Rahi P,Gulati A.Isolation of tannase-producing Enterobacter ludwigii GRT-1from the rumen of migratory goats.Small Ruminant Research,2012,102(2/3):172–176.
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