1株具硫酸盐还原功能的柠檬酸杆菌的分离及其生理特性研究杨丽平

31320103　　　　　　ENVIRONMENTALSCIENCEVol.31,No.3Mar.,201011,2,3,4,1,2,3,1,2,3,5,1,2,3,1,2,3＊(1.,　510070;2.,　510070;3.,　510070;4.,　330045;5.,　510275):(UASB)、1SR3.、16SrDNA,SR3(Citrobactersp.).:,(dsr);,37℃,pH8.0,Cr(Ⅵ)0.4～0.8mmolSO2-4Cr(Ⅵ).Cr(Ⅵ)1mmol..:;;;:X172　:A　:0250-3301(2010)03-0815-06:2009-04-28;:2009-07-10:(863)(2006AA06Z322);(2005A30401002);(2008C13G041);(9351007002000001):(1985～),,,,E-mail:[e-mail]＊,E-mail:[e-mail]3YANGLi-ping1,2,3,4,ZHENGXiao-hong1,2,3,ZENGGuo-qu1,2,3,5,XUMei-ying1,2,3,SUNGuo-ping1,2,3(1.GuangdongInstituteofMicrobiology,Guangzhou510070,China;2.GuangdongProvincialKeyLaboratoryofMicrobialCultureCollectionandApplication,Guangzhou510070,China;3.GuangdongOpenLaboratoryofAppliedMicrobiology,Guangzhou510070,China;4.CollegeofBioscienceBioengineering,JiangxiAgriculturalUniversity,Nanchang330045,China;5.SchoolofEnvironmentalScienceandEngineering,SunYat-senUniversity,Guangzhou510275,China)Abstract:Asulfatereducingbacterium,designatedstrain`SR3',wasisolatedfromsludgeofasulfate-reducingup-flowanaerobicsludgebed(UASB)reactorfortreatinghighconcentrationsulfatewastewater.ItwasidentifiedasCitrobactersp.baseduponthephenotypic-characteristicsandphysiologicalpropertiesaswellastheanalysisofthesequenceof16SrDNA.Thestraincouldreducesulfateunderanaerobicandmicro-aerobicconditions.Thedissimilatorysulphitereductasegene(dsr)wasalsoamplifiedfromthisstrainsgenomicDNAusingspecificdsrgeneprimers.Inaerobicconditions,thestraincouldntreducesulfate,butexhibitedahighestgrowthrate.Inanaerobicconditions,theoptimalgrowthconditionsforthisstainweretemperature37℃andinitialpH8.0.Underthisconditions,thestraincouldreducedbothSO2-4andCr(Ⅵ)atinitialCr(Ⅵ)concentrationsof0.4-0.8mmolsynchronously.ItstoleranceabilitytoCr(Ⅵ)concentrationsreaches1.0mmol.Thisisthefirstreportaboutanfacultativeanaerobewithsulfatereducingfunctionanddissimilatorysulphitereductasegene(dsr).Keywords:sulfate-reducing;Citrobactersp.;characteristics;dissimilatorysulphitereductasegene(dsr)　　、、、、SO2-4.,(SRB)(H2S),,H2S,,[1],.SO2-4,.,.,.,、、,.(sulfatereducingbacteria,SRB)SO2-4H2S,SRB[2].,SRB(Desulfovibrio)、(Desulfotomaculum)(Desulfomonas)1240[3].DOI:10.13227/j.hjkx.2010.03.008　　　　　　31,SRB、、、[4～8]..1932,Werkman[9],,(Citrobacterfreundii).,,13,[10～12].2007,Barton[13]“Sulphate-ReducingBacteria”.1SR3,.,.1　1.1　(UASB).1.2　:K2HPO40.5g;NH4Cl1.0g;Na2SO40.5g;CaCl20.1g;MgSO42.0g;3.5g;1.0g.1000mL,121℃20min.0.03g;C0.01g.1.3　1mL250mL,100mL,3min.(Bugbox,UK)30℃,.5,1%.1.4　10-2～10-8,(Bugbox,UK)30℃.5～6d、,.1.5　,[14]16SrDNA.1.5.1　:(),(Bugbox,UK)30℃72h.:,(Bugbox,UK)30℃,.1.5.2　、[14],BIOLOG.1.5.3　16SrDNAdsrAB16SrDNAPCR:,,[15]DNA.16SrDNA27F1492RPCR16SrDNA[16],(dissimilatorysulphitereductasegene,dsr)DSR1F(5′-ACSCACTGGAAGCACG-3′)DSR4R(5′-GTGTAGCAGTTACCGCA-3′)dsrABPCR[17].PCR.:BLASTGenBank/EMBL/DDBJ,ClustalX,(NeighbourJoining)(PHYLIP,Version315)[18].1.6　1.6.1　,、150r·min-1、、pH,D600.D600,.1.6.2　[Cr(Ⅵ)]0.4、0.8、1.0、1.21.6mmol[Cr(Ⅵ)]200mL,1%7d,Cr(Ⅵ).1.7　、Cr(Ⅵ)、pH[19].8163:12　2.1　、.1,SR3..2.2　SR32.2.1　SR330℃3d,,,,1～2mm.,,(0.5～0.6)μm×(2.0～2.2)μm,(1).,PHB.2.2.2　1　SR3Fig.1　ElectronmicrographofSR3　　SR3:,、、、、.、、.4℃,45℃.Biolog,SR3,、D-、D,L-、α-D-、、D,L-、L-、、、.SR3BIOLOG,(Citrobacteramalonaticus),0.56,.2.2.3　16SrRNABLASTSR316SrDNAGenBank,2(a),SR3Citrobacteramalonaticus95%.BLASTSR3dsrGenBank,2(b),SR3dsrDesulfovibriodesulfuricans99%.16SrDNA,SR3.Desulfovibriodesulfuricans99%,,.2　16SrDNAdsrSR3Fig.2　PhylogeneticanalysisofstrainsofSR3basedon16SrDNAanddsrgene2.3　SR3,SR3.2.3.1　SR3、SR3,20mmol7d,3～5.817　　　　　　313　、pHFig.3　GrowthofSR3andsulfateremovalrate,pHasafunctionoftimeinanaerobiccondition4　、pHFig.4　GrowthofSR3andsulfateremovalrate,pHasafunctionoftimeinmicro-aerobiccondition5　Fig.5　GrowthofSR3inaerobiccondition,.92%,2d54%,20%.,,..,3d,D6000.36,3dD6001.3,42hD6002.1..pH.7d,pH6.67.8;7d,pH6.68.8.2,pH.,Na+、SO2-4HS-、S2-H2S,Na+.Na+.2.3.2　SR3(20、30、37、4045℃),72h,D600.637℃.2.3.3　pHSR3pH(4、5、6、7、8、9)72h.pH8183:16　Fig.6　EffectsoftemperatureongrowthofSR37.,pH6～9,pH4,,2.3.4　[Cr(Ⅵ)]Cr(Ⅵ),7d,8.Cr(Ⅵ)≤0.8mmol,7d,Cr(Ⅵ),Cr(Ⅵ),87.7%47.5%.,SO2-4※S2-8e,SO2-487.7%　　　7　pHFig.7　EffectsofpHvalueongrowthofSR347.5%,SO2-464.16mmol.Cr(Ⅵ)※Cr(Ⅲ)3e,Cr(Ⅵ)0.4mmol0.8mmol1.2mmol.Cr(Ⅵ).Cr(Ⅵ)=1.0mmol,7d,Cr(Ⅵ),11%16%,Cr(Ⅵ).Cr(Ⅵ)1.2mmol,7dCr(Ⅵ).8　Cr(Ⅵ)Cr(Ⅵ)Fig.8　VariationofsulfateandCr(Ⅵ)underdifferentconcentrationofCr(Ⅵ)3　,,40[13]..Angeles-Cha[20]Citrobactersp.H2CO2,(SRB).Neria-Gonzalez[21],H2S.Citrobactersp..Qiu[22]1Citrobactersp.,,Cu..819　　　　　　31,SR3,.SR3.Desulfovibriodesulfuricans99%,,.16SrDNA,SR3Citrobacteramalonaticus95%,,1mmolCr(Ⅵ).SR3、pH,,,.4　(1)UASB1SR3.、16SrDNA,(Citrobactersp.);SR3.(2),,.37℃,pH6～9,1.0mmolCr(Ⅵ),Cr(Ⅵ)0.4～0.8mmolSO2-4Cr(Ⅵ).Cr(Ⅵ).:[1]　SipmaJ,JanssenAJH,Hulshoff-PolLW,etal.DevelopmentofanovelprocessforthebiologicalconversionofH2Sandmethanethioltoelementalsulfur[J].BiotechnolBioeng,2003,82(1):1-11.[2]　.[M].:,2004.[3]　,.[M].:,2004.300-301.[4]　KaksonenAH,Riekkola-VanhanenML,PuhakkaJA.Optimizationofmetalsulphideprecipitationinfluidized-bedtreatmentofacidi