采用FISHDGGE和Cloning对短程脱氮系统中硝化菌群的比较分析

26520065ActaScientiaeCircumstantiaeVol.26,No.5May,2006:(No.2003A06);(No.50521140075);SupportedbytheNewStarPlanProjectofBeijingScienceandTechnologyCommittee(No.2003A06);theKeyInternationalCooperativeProjectofNSFC(No.50521140075);TheOpenProjectofKeyLaboratoryofBeijing:(1974),,,();3(),E2mail:[e-mail]:ZENGWei(1974),female,Ph.D.,associateprofessor;3Correspondingauthor,E2mail:[e-mail],,,.2006.FISHDGGECloning[J].,26(5):734-739ZengW,YangQ,ZhangSJ,etal.2006.Analysisofnitrifyingbacteriainshort2cutnitrification2denitrificationprocessesbyusingFISH,PCR2DGGEandCloning[J].ActaScientiaeCircumstantiae,26(5):734-739[]:FISHDGGECloning1,1,1,1,2,1,3,31.,1000222.,1500903.,310014:2006203223:2005203229:4(SBRUASB2A/OA/OSBR),FishPCR2DGGEPCR2Cloning2SequencingAOBNOB.Fish,4,AOBNOB,3%12%;SBRNOB;A/ONitrospira(0.2%),UASB2A/ONitrobacteria(0.2%).PCR2DGGESBRA/OUASB2A/O3AOBNitrosomonas2like.SBRPCR2Cloning2Sequencing,Nitrosomonas,60%Nitrosomonaseuropaea.:;AOB;FISH;PCR2DGGE;PCR2Cloning2Sequencing:025322468(2006)0520734206:X70311:AAnalysisofnitrifyingbacteriainshort2cutnitrification2denitrificationprocessesbyusingFISH,PCR2DGGEandCloningZENGWei1,YANGQing1,ZHANGShujun1,MAYong1,LIUXiuhong2,PENGYongzhen1,3,LIJun31.KeyLaboratoryofBeijingWaterQualityScienceandWaterEnvironmentRecoveryEngineering,BeijingUniversityofTechnology,Beijing1000222.CollegeofMunicipalandEnvironmentalEngineering,HarbinInstituteofTechnology,Harbin1500903.CollegeofCivilEngineering,ZhejiangUniversityofIndusty,Hangzhou310014Received23March2006;accepted29March2006Abstract:MolecularbiologytechniquesofFish,PCR2DGGEandPCR2Cloning2Sequencingwereusedtoqualitativelyandquantitativelyanalyzeammonia2oxidizingbacteria(AOB)andnitrite2oxidizingbacteria(NOB)inSBRlargepilot2scaleplant,UASB2A/Olab2scalereactor,A/Opilot2scaleplantandSBRlab2scalereactorwiththeperformanceofshort2cutnitrification2denitrificationtreatingactualwastewater.Fishresultsshowedthatintheabove4processes,AOBbecamedominantwithcomparedtoNOBandaccountedfor3%12%oftotalbiomass.NoNOBwasdetectedinbothSBRpilot2scaleandlab2scalereactors.AverysmallamountofNitrospiraandNitrobacteriabelow0.2%wasfoundinA/Opilot2scaleplantandUASB2A/Olab2scalereactor,respectively.PCR2DGGEresultsshowedthattheAOBinSBRpilot2scale,A/Opilot2scaleandUASB2A/Olab2scaleprocesseswerephylogeneticallyrelatedtoNitrosomonas2likespecieswithameltingpointintherange30%to50%.PCR2Cloning2SequencingresultsshowedthatallclonesfromthesludgesampleoftheSBRlargepilot2scaleplantwereaffiliatedwithNitrosomonasand60%ofthecloneswereaffiliatedwithNitrosomonaseuropaea.Keywords:short2cutnitrification2denitrification,AOB,FISH,PCR2DGGE,PCR2Cloning2Sequencing,,5:FISH,DGGECloning.PCR(Fluorescencein-situhybridization,FISH),(Kaneetal.,1993)(Wagneretal.,1994)(Christenssonetal.,1998)(Damisetal.,2001).,.AOB(ammonia2oxidizingbacteria,)NOB(nitrite2oxidizingbacteria,).AOBNOB.,,(Zengetal.,2004;Maetal.,2004;,2004).,,,.1(Materialsandmetbods)1.14.(1)SBR:54m3,13,95%,HRT;(2)UASB2A/O:A/O15L,98%,(FA)HRT;(3)A/O:300L,1520d,2223,DO0.57mgL-1.80%,DOHRT;(4)SBR:12L,28,DO2mgL-1,95%,HRT.UASB2A/O,3.1.2FishAmannFish.4%PFA,423h.1min,,50%80%98%3min.0.9molL-1NaCl20mmolL-1Tris/HCl,0101%SDS(FA1),pH7.2.,462h.1.,4820min.,2025(OLYMPUSBX52)(LeicaQWINSoftware).1FishTable116SrRNA-targetedoligonucleotideprobesusedFAEUB3mixFITCEubacteriaNSO122535%Cy3Ammonia-oxidizing2ProteobacteriaNIT340%Cy3NitrobacteriaNtspa66235%Cy3Nitrospira3EUBmixEUB338EUB338EUB338=111,FA,FA.1.3PCR2DGGEFastDNAspinkitforsoil(Bio101,USA)DNA.PCRAmoA21F2Clamp(52Clamp2GGGGTTTCTACTGGTGGT23)AmoA22R2TC(52CCCCTCTGCAAAGCCTTCTTC23),AOB.PCR(50L):35.5LdH2O,5LPCR,1L(50pmolL),1L(50pmolL),0.5L5UTaqDNA,5LdNTP(1.25mmolL-1),2LDNA.PCR:9510min,35(9415s,5520s,722min),725min.DcodeSystem(BioRad)AmoAPCRDGGE.230%70%,100V601TAE15h.VistraGreen20min,FluorImage595.1.4PCR2Cloning2sequencingPCRAmoA21F(52GGGGTT53726TCTACTGGTGGT23)AmoA22R(52CCCCTCKGSAAAGCCTTCTTC23),AOB.PCR(50L):1PCR,0.25molL-1,0.25molL-1,1125UTaqDNA,0.2mmolL-1dNTPs.PCR1.3.PCR(QIAquickPCRpurificationkit,QIAGEN),pDriveCloning(QIAGENPCRCloningkitligationProtocol).QIAGENEZ(QIAGENPCRCloningkitTransformationProtocol).,3718h..M13AmoA,M13PCR,8(E8E9F8E12F11F12C9D8)M13PCR(ABI3100).AutoAssembler(AppliedBiosystems,maconly)DDBJ(),BLASTsearch.BLAST,AmoA,FASTA,DDBJ,ClustalW.2(Resnlts)2.1FishAOBNOB4Fish:SBR(95%)AOB3%,NOB;UASB2A/O(98%)AOB4%,NOB012%;A/O(80%)AOB5%,NOB0.2%;SBR(95%)AOB12%,NOB.Fish,80%,AOB,AOBNOB,NOB.Fish12.1AOBFISH(FITCEUBmix,Eubacteria;Cy3NSO1225,2AOB.A:SBR;B:SBR;C:A/O;D:UASB2A/O)Fig.1FISHresultsforAOB(FITClabeledEUBmixtargetforEubacteria,Cy3labeledNSO1225targetfor2AOB.A:SBRpilot2scale;B:SBRlab2scale;C:A/Opilot2scale;D:UASB2A/Olab2scale)6375:FISH,DGGECloningNOB,A/OUASB2A/ONOB,NOB.A/ONOBNitrospira,UASB2A/ONOBNitrobacteria.2NOBFISH(FITCEUBmix,Eubacteria.A:UASB2A/O,Cy3NIT3,Nitrobacteria;B:A/O,Cy3Ntspa662,Nitrospira)Fig.2FISHresultsforNOB(FITClabeledEUBmixtargetforEubacteria.A:UASB2A/Olab-scale,Cy3labeledNIT3targetforNitrobacteria;B:A/Opilot2scale,Cy3labeledNtspa662targetforNitrospira)3DGGEFi