采用FISHDGGE和Clon省略短程脱氮系统中硝化菌群的比较分析曾薇

26520065　　　　　　ActaScientiaeCircumstantiaeVo.l26,No.5May,2006:(No.2003A06);(No.50521140075);SupportedbytheNewStarPlanProjectofBeijingScienceandTechnologyCommittee(No.2003A06);theKeyInternationalCooperativeProjectofNSFC(No.50521140075);TheOpenProjectofKeyLaboratoryofBeijing:　(1974—),,,();*(),E-mail:[e-mail]:ZENGWei(1974—),female,Ph.D.,associateprofessor;*Correspondingauthor,E-mail:[e-mail]　,　,,.2006.FISH、DGGECloning[J].,26(5):734-739ZengW,YangQ,ZhangSJ,etal.2006.Analysisofnitrifyingbacteriainshort-cutnitrification-denitrificationprocessesbyusingFISH,PCR-DGGEandCloning[J].ActaScientiaeCircumstantiae,26(5):734-739[]:FISH、DGGECloning曾　薇1,杨　庆1,张树军1,马　勇1,刘秀红2,彭永臻1,*,李　军31.北京工业大学环境与能源工程学院,北京1000222.哈尔滨工业大学市政环境工程学院,哈尔滨1500903.浙江工业大学建筑工程学院,杭州310014:2006-03-23　　　:2005-03-29:4(SBR、UASB-A/O、A/OSBR),Fish、PCR-DGGEPCR-Cloning-SequencingAOBNOB.Fish,4,AOBNOB,3%～12%;SBRNOB;A/ONitrospira(0.2%),UASB-A/ONitrobacteria(0.2%).PCR-DGGESBR、A/OUASB-A/O3AOBNitrosomonas-like.SBRPCR-Cloning-Sequencing,Nitrosomonas,60%Nitrosomonaseuropaea.:;AOB;FISH;PCR-DGGE;PCR-Cloning-Sequencing:0253-2468(2006)05-0734-06　　　:X703.1　　　:AAnalysisofnitrifyingbacteriainshort-cutnitrification-denitrificationprocessesbyusingFISH,PCR-DGGEandCloningZENGWei1,YANGQing1,ZHANGShujun1,MAYong1,LIUXiuhong2,PENGYongzhen1,*,LIJun31.KeyLaboratoryofBeijingWaterQualityScienceandWaterEnvironmentRecoveryEngineering,BeijingUniversityofTechnology,Beijing1000222.CollegeofMunicipalandEnvironmentalEngineering,HarbinInstituteofTechnology,Harbin1500903.CollegeofCivilEngineering,ZhejiangUniversityofIndusty,Hangzhou310014Received23March2006;　　　accepted29March2006Abstract:MolecularbiologytechniquesofFish,PCR-DGGEandPCR-Cloning-Sequencingwereusedtoqualitativelyandquantitativelyanalyzeammonia-oxidizingbacteria(AOB)andnitrite-oxidizingbacteria(NOB)inSBRlargepilot-scaleplant,UASB-A/Olab-scalereactor,A/Opilot-scaleplantandSBRlab-scalereactorwiththeperformanceofshort-cutnitrification-denitrificationtreatingactualwastewater.Fishresultsshowedthatintheabove4processes,AOBbecamedominantwithcomparedtoNOBandaccountedfor3%～12%oftotalbiomass.NoNOBwasdetectedinbothSBRpilot-scaleandlab-scalereactors.AverysmallamountofNitrospiraandNitrobacteriabelow0.2%wasfoundinA/Opilot-scaleplantandUASB-A/Olab-scalereactor,respectively.PCR-DGGEresultsshowedthattheAOBinSBRpilot-scale,A/Opilot-scaleandUASB-A/Olab-scaleprocesseswerephylogeneticallyrelatedtoNitrosomonas-likespecieswithameltingpointintherange30%to50%.PCR-Cloning-SequencingresultsshowedthatallclonesfromthesludgesampleoftheSBRlargepilot-scaleplantwereaffiliatedwithNitrosomonasand60%ofthecloneswereaffiliatedwithNitrosomonaseuropaea.Keywords:short-cutnitrification-denitrification,AOB,FISH,PCR-DGGE,PCR-Cloning-Sequencing　　,,DOI:牨牥牨牫牰牱牨牤jhjkxxb牪牥牥牰牥牭牥牥牰5　:FISH,DGGECloning.PCR(Fluorescencein-situhybridization,FISH),(Kaneetal.,1993)、(Wagneretal.,1994)、(Christenssonetal.,1998)、(Damisetal.,2001).,.AOB(ammonia-oxidizingbacteria,)NOB(nitrite-oxidizingbacteria,).AOBNOB.,,“”、(Zengetal.,2004;Maetal.,2004;,2004).,,,.1　(Materialsandmetbods)1.1　活性污泥样品4.(1)SBR:54m3,13℃,95%,HRT;(2)UASB-A/O:A/O15L,98%,(FA)HRT;(3)A/O:300L,15～20d,22～23℃,DO0.57mgL-1.80%,DOHRT;(4)SBR:12L,28℃,DO2mgL-1,95%,HRT.UASB-A/O,3.1.2　Fish及寡核苷酸探针AmannFish.4%PFA,4℃2～3h.1min,,50%、80%98%3min.0.9molL-1NaCl、20mmolL-1Tris/HCl,0.01%SDS(FA1),pH7.2.,46℃2h.1.,48℃20min.,20～25(OLYMPUSBX52)(LeicaQWINSoftware).1　FishTable1　16SrRNA-targetedoligonucleotideprobesusedFA　　EUB*mix—FITCEubacteriaNSO122535%Cy3Ammonia-oxidizingβ-ProteobacteriaNIT340%Cy3NitrobacteriaNtspa66235%Cy3Nitrospira　　*EUBmixEUB338∶EUB338Ⅱ∶EUB338Ⅲ=1∶1∶1,FA,FA.1.3　PCR-DGGEFastDNAspinkitforsoil(Bio101,USA)DNA.PCRAmoA-1F-Clamp(5’-Clamp-GGGGTTTCTACTGGTGGT-3’)AmoA-2R-TC(5’-CCCCTCTGCAAAGCCTTCTTC-3’),AOB.PCR(50μL):35.5μLdH2O,5μLPCR,1μL(50pmolμL),1μL(50pmolμL),0.5μL5UTaqDNA,5μLdNTP(1.25mmolL-1),2μLDNA.PCR:95℃10min,35(94℃15s,55℃20s,72℃2min),72℃5min.DcodeSystem(BioRad)AmoAPCRDGGE.230%70%,100V、60℃、1×TAE15h.VistraGreen20min,FluorImage595.1.4　PCR-Cloning-sequencingPCRAmoA-1F(5’-GGGGTT735　　　　　　　　　　26TCTACTGGTGGT-3’)AmoA-2R(5’-CCCCTCKGSAAAGCCTTCTTC-3’),AOB.PCR(50μL):1×PCR,0.25μmolL-1,0.25μmolL-1,1.25UTaqDNA,0.2mmolL-1dNTPs.PCR1.3.PCR(QIAquickPCRpurificationkit,QIAGEN),pDriveCloning(QIAGENPCRCloningkitligationProtocol).QIAGENEZ(QIAGENPCRCloningkitTransformationProtocol).,37℃18h..M13AmoA,M13PCR,8(E8、E9、F8、E12、F11、F12、C9、D8)M13PCR(ABI3100).AutoAssembler(AppliedBiosystems,maconly)DDBJ(),BLASTsearch.BLAST,AmoA,FASTA,DDBJ,ClustaWl.2　(Resnlts)2.1　Fish方法对AOB与NOB的初步定量分析4Fish:SBR(95%)AOB3%,NOB;UASB-A/O(98%)AOB4%,NOB0.2%;A/O(80%)AOB5%,NOB0.2%;SBR(95%)AOB12%,NOB.Fish,80%,AOB,AOBNOB,NOB.Fish1、2.1　AOBFISH(FITCEUBmix,Eubacteria;Cy3NSO1225,β-AOB.A:SBR;B:SBR;C:A/O;D:UASB-A/O)Fig.1　FISHresultsforAOB(FITClabeledEUBmixtargetforEubacteria,Cy3labeledNSO1225targetforβ-AOB.A:SBRpilot-scale;B:SBRlab-scale;C:A/Opilot-scale;D:UASB-A/Olab-scale)7365　:FISH,DGGECloning　　NOB,A/OUASB-A/ONOB,NOB.A/ONOBNitrospira,UASB-A/ONOBNitrobacteria.2　NOBFISH(FITCEUBmix