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1,1,CriddleCraig2,1,1,3(1.,100084;2.DepartmentofCivilandEnvironmentalEngineering,StanfordUniversity,Stanford,CA9430524020,USA;3.,100044):(AOB),AOB,AOB(amoA)(T2RFLP),69AOB.T2RFLPAOB,(T2RF)354491291bp.T2RFLP,AOB,AOB.amoA,AOBNitrosomonasspp.,Nitrosospiraspp..Nitrosomonasspp.(max),AOB.:(AOB);(T2RFLP);(amoA);:X172:A:025023301(2009)1023002205:2008211211;:2009203204:(2006BAC19B01202):(1980),,,,E2mail:wangxiaohui05@mails.tsinghua.edu.cn3,E2mail:xhwen@tsinghua.edu.cnCommunityStructuresofAmmonia2oxidizingBacteriainActivatedSludgeofWastewaterTreatmentPlantsWANGXiao2hui1,WENXiang2hua1,CriddleCraig2,ZHANGJie1,YANGNing2ning1,ZHAOYin3(1.DepartmentofEnvironmentalScienceandEngineering,TsinghuaUniversity,Beijing100084,China;2.DepartmentofCivilandEnvironmentalEngineering,StanfordUniversity,StanfordCA9430524020,USA;3.BeijingDrainageGroupCo.Ltd.,Beijing100044,China)Abstract:Inordertoinvestigatethecommunitystructureofammoniaoxidizingbacteria(AOB)inwastewatertreatmentplantsandtheinfluenceoftreatmentprocessandsystemscaleonAOBcommunity.WeinvestigatedAOBcommunitiesinactivatedsludgescollectedfrom9wastewatertreatmentsystems,usingspecificPCRfollowedbyterminalrestrictionfragmentlengthpolymorphism(T2RFLP),cloning,andsequencingofamoAgenes.TheT2RFLPfingerprintsanalysisshowsthattherearesomedifferencesamongtheAOBcommunitystructuresfromdifferentwastewatertreatmentsystems,andthedominantterminalrestrictionfragments(T2RFs)are354,491and291bp.TheT2RFLPprofilesandclusteranalysismayalsoindicatetheAOBstructuresareslightlyaffectedbythetreatmentprocess,whiletheyareinfluencedbythesystemscaletosomeextent.PhylogeneticanalysisofclonedamoAgeneshowsclearlythatallthedominantAOBinthesystemswereNitrosomonasspp.,notNitrosospiraspp..ThereasonmaybeexplainedasthatNitrosomonasspp.havehighermaxthanNitrosomonasspp..ThisgrowthadvantagemayfavortheNitrosomonasspp.ratherthanNitrosomonasspp.beingprevailinactivatedsludge.Keywords:ammonia2oxidizingbacteria(AOB);terminalrestrictionfragmentlengthpolymorphism(T2RFLP);amoA;wastewatertreatmentplant.2.,.(AOB),NH+4NO-2,(NOB),NO-2NO-3.,AOB,pH(DO)(SRT)(HRT),,[1].AOB,[2].,,011%10%[3];,.,[4].,16SrRNA,,(FISH)(DGGE)T2RFLP[5].T2RFLP[6],[712].3010200910ENVIRONMENTALSCIENCEVol.30,No.10Oct.,2009,AOB,AOBNitrosomonas[1,2,1317],AOBNitrosospira[18].,.[2,15]DO[9]SRT[1]HRT.T2RFLP,AOB,,AOB,.11.169,A1A2D1D2D3AD.9D2,.1,9,A2BD1D2A2O,A1A2O,D3AO,CE(CAS),F.A1A2CE,.9,1.20073,,115mL,,.(14000rPmin),-20.1.2DNAFastDNARSpinKitforSoil(Qbiogene,)DNA.(14000rPmin)2,DNA.1.3AOBPCRAOB,aomAamoA21F(52GGGGTTTCTACTGGTGGT23)amoA22R(52CCCCTCKGSAAAGCCTTCTTC23;KGT;SCG).PCR50L,1LDNA,5L10,4LdNTP(215mmolPL),(10pmolPL)1L0125LTaqDNA(5UPL,TaKaRa,),35175L.PCR:945min;9460s,5190s,7290s,40;7210min.T2RFLP,AOBamoA21F5FAM,PCRDNA.QIAquickPCRPurificationKit(Qiagen,).1.3T2RFLP1Table1PerformanceconditionofwastewatertreatmentsystemsA1A2BCD1D2D3EFA2POA2POA2POCASA2POA2POAPOCAS+++++BODPmgL-12542542251524117518118175265186139NH+42NPmgL-152185218561445194415361666TNPmgL-17014521552195215791352107BODPmgL-110181019819917111791981278182NH+42NPmgL-17183196515510111984100NO-22NPmgL-15131211901185145515216501930128NO-32NPmgL-1817281210184121431801116124138TNPmgL-121162015161521145151117122115PmgL-1282133305630SRTPd131316126DOPmgL-131612156Taq(TaKaRa,)AOBPCR,655h.20L,PCR10L,1L,102L,10BSA(200gPmL)2L,5L.[19],ABI3130(AppliedBiosysterms,),500LIZ.50RFU,T2RFLP300310:GeneMapversion317.T2RFs50bp,.1.4AOBPCR,pGEM2TEasy(Promega,),EscherichiacoliDH5.,T7SP6PCR.,AOBPCR,PCRT2RFLP,AOB,().1.5aomAGenBank(),.Mega410(Neibor2joining).aomAGenBank:FJ4230022FJ423027.1.6SPSS1310(classify2hierarchicalclusteranalysis)T2RFLP.T2RF,,T2RF().22.1T2RFLPT2RFLP1.AOBT2RF.354bp491bp,354bp,T2RF.219bpCD1D3EF.CET2RF,T2RF,334bp483bp.[9,20],AOBT2RF219354491bp.T2RF,bp1AOBT2RFLPFig.1AOBT2RFLPprofilesofdifferentwastewatertreatmentsystems2.2AOBA1A2,,A2PO,A2PO,T2RFLP,T2RF219354bp.400330D1D3,,A2PO,APO,T2RFLP,T2RF219354491bp,2A1A2,D1D3.,AOB.Rowan[15](BAF)AOB.Dytczak[21],PSBRSBRAOB,AOBNitrosomonas,AOBNitrosospira.,AOB,A2POA2PO,A2POAPO,,AOB.2AOBT2RFLPFig.2ClusteranalysisofAOBfromdifferentwastewatertreatmentsystems2.3AOBD1D2,A2PO,,.1T2RFLP,D1T2RF219354491bp,D2T2RF354491bp,2,.,AOB,AOB,AOB.Rowan[22],AOB,,AOB.Curtis[23],,.,,().3AOBamoAFig.3PhylogenetictreebasedoncomparisonofamoAgeneofAOB2.4AOBT2RFLP(T2RF),A1BCD1EFAOB,T2RFLP.AOB,T2RFLP.,GenBankBLAST,.,AOBNitrosomonas,[1,9,13,15,2425].3.T2RFLP,219bpNitrosomonaseuropaeacluster,354bpNitrosomonasoligotrophaculster,491bpNitrosomonascommunis500310:culster.,Nitrosomonas,N.europaea,max01088h-1,Nitrosospiramax0103301035h-1.NitrosomonasAOB[20].,,Nitrosospira[2].3(1)T2RFLP,AOB,T2RF354491219bp.(2)A2POA2POAOB,A2POAOAOB,,,AOB.(3)AOB,AOB,AOB.(4),,AOBNitrosomonas.:[1]HallinS,LydmarkP,KokaljS,etal.Communitysurveyofammonia2oxidizingbacteriainfull2scaleactivatedsludgeprocesseswithdifferentsolidsretentiontime[J].JournalofAppliedMicrobiology,2005,99(3):6292640.[2]LimpiyakornT,ShinoharaY,KurisuF,etal.Communitiesofammonia2oxidizingbacteriainactivatedsludgeofvarioussewagetreatmentplantsinTokyo[J].FemsMicrobiolog