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34420134ENVIRONMENTALSCIENCEVol．34No．4Apr．2013123341*41121.1000852.1000493.1000224.050018．16SrDNA、PLFAPCR、．ProteobacteriaBacteroidete．．PCRAOBNOBAOBNOB3AOBNOB2．16SrDNAPLFAPCRX172A0250-3301201304-1448-092012-06-192012-10-12KZZD-EW-09-3KZCX2-YW-JC407-3KSCX2-YW-G-054-21975～E-mailzhiruizh@163．com*E-mailzhbai@rcees．ac．cnComparingMicrobialCommunityofHighAmmoniaWastewaterandMunicipalSewageinaPartialNitrificationSystemZHAOZhi-rui12MABin3ZHANGShu-jun3LIBin4BAIZhi-hui1WANGXiao-hui4ZHUANGGuo-qiang1ZHANGHong-xun121.ResearchCenterforEco-EnvironmentalSciencesChineseAcademyofSciencesBeijing100085China2.UniversityofChineseAcademyofSciencesBeijing100049China3.BeijingDrainageGroupCo．Ltd．Beijing100022China4.CollegeofEnvironmentalScienceandEngineeringHebeiUniversityofScienceandTechnologyShijiazhuang050018ChinaAbstractNitritationisanimportantpartofthebiologicalnitrogenremovalprocessandtheperformanceoftheprocesswasdeterminedbythemicrobialcommunitystructure．Toexplorethemicrobialadaptabilitytodifferentsewagethemicrobialdiversityandtheamountofbacteriawereinvestigatedinahighammoniawastewatertreatmentprocessandasewagetreatmentprocessusingtheclonelibraryofbacterial16SrDNAthephospholipidfattyacidmethodPLFAandthequantitativePCR．TheclonelibraryresultsshowedthattherewasasignificantlydifferenceinbacterialcommunitystructureofthesetwoprocessesalthoughthedominantbacteriabelongtotheProteobacteriaandBacteroidetethereweremoreclustersinthesewagetreatmentprocess．ThePLFAsresultsshowedthatthemicrobialdiversityindexandtheevennessindexofthehighammoniumwastewatertreatmentprocessweresignificantlylow．ThequantitativePCRresultsshowedthatamountsofammoniaoxidizingbacteriaAOBandnitrite-oxidizingbacteriaNOBinthehighammoniumwastewatertreatmentprocesswerehigherthantheseinsewagetreatmentprocess．ThecopynumberofAOBwashigherthanthecopynumberofNOBinthehighammoniawastewatertreatmentprocessbythreeordersmagnitude．ThecopynumberofAOBwashigherthanthecopynumberofNOBinsewagetreatmentprocessbytwoordersofmagnitude．Keywordspartialnitritationbiologicalnitrogenremoval16SrDNAclonelibraryphospholipidfattyacidPLFAquantitativePCR1234．56．78．AOBNOB9．410h．10．AOBNOB．AOBNOB11．．FAFNA1213．．．AOBDO14．16SrDNA．PCRAOBNOB．11.1/A/O．A/O24L615．A/O28L7161．．．．A/O26±1℃．、12．1/A/OFig．1Schematicdiagramofplug-flowanoxic/oxicA/Oprocess1A/O1Table1RunningconditionsofA/OreactorQ/L·d－1HRT/hSRT/dDO828.2100.411843.134.50.961QHRTSRTDO2A/O1Table2InfluentqualityandrunningeffectofA/Oreactor/mg·L－1COD/mg·L－1MLSS/mg·L－1SVI/mL·g－1NAR/%42.08～53.1647.70121.0～165.7142.63500～5000430074～777584.59～93.9990.60291～387337100～1711282500～3500306452～957795.0～98.396.61MLSSSVINAR1.21.2.1DNAPCRDNA0.5gDNAOmegaUSADNA40ng·μL－1．DNA16SrDNA27f/1492rPCR15．PCR50μL10×buffer5μLdNTPs10mmol·L－11μL27f、1492r10μmol·L－11μLTaq2.5U0.5μLDNA0.5μL20ng94413450μL．95℃3min95℃1min56℃1min72℃2min3072℃10min4℃．1.2.23SDNAPCRPCRpGEM-TeasyvectorPromegaUSA．2×RapidLigationBuffer5μLpGEM-Teasyvector50ng·μL－11μLPCR75ng3∶1T4DNAligase1μL4℃．DH5αTiangenAMPIPTGX-GalLB37℃12hAMPLBPCR1500bp．230T7/SP6PCRRsalPromegaUSAHhalPromegaUSA1BioeditGenBankclustalx1.83DoCompleteAlignmentBootstrapN-Jtree1000MEGA4.1．1.2.3SYBRGreenAOBEub338/Nso122516NOBNSR1113/FNSR1264R1716SrDNAPCRAOB59×1010NOB3.61×1010．DNARealMasterMixSYBRGreenTIANGENNPCRMx3000PPCRGenetimes．25μL0.5μL10μmol·L－12μLDNA9μL2.5×MasterMix3．Ct．AOBNOBCt．1.2.4PLFAsPLFAsPLFAsPLFAs．Bligh-Dyer185g0.01gPLFA．ctaianteiso-CCiso-CCbr10Me10cyαβ—OH2、3Ci130a130i140i150a150i160i170a170141ω5c151ω6c161ω7c161ω7t161ω5c181ω9c181ω7c181ω7t182ω6c183ω6c183ω3c1920．PLFAs．SPSS13.0．Shannon-WienerHH'．PielouE．H=－Σpilnpipi=ni/NE=H/HmaxHHmaxHmax=lnSSniiN21．22.10541436OTUs23OTUs5．Bacteroidetes26.9%Chloroflexi13.7%Spirochaetes12.3%Firmicutes4.7%．Proteobacteria42.4%Snaidr22Proteobacteria．Proteobacteriaβ-β-Proteobacteria22.2%γ-γ-Proteobacteria1.9%δ-δ-Proteobacteria4.7%α-α-Proteobacteria9.9%ε-ε-Proteobacteria3.7%．2Fig．2Phylogenetictreeofthebacteriaofactivesludgeinthehighammonianitrogenprocess2.2221555OTU．45OTU723.26%1.4%4.68%．Actinobacteria1.86%．Planctomycetes3.72%．Denitrifyingbacterium5.56%．52.1%．Proteobacteriaβ-、δ-γ-、α-23.72%、2.8%、14.88%、10.7%．Unculturedbacterium7.46%．、45.26%．1541343Fig．3Phylogenetictreeoftheproteobacteriaofactivesludgeinthehighammonianitrogenprocess、．①β-23AOBNitrosomonasNitrosospira24②γ-NOBNitrococcus③δ-Desulfobacterales、Desulfoarculaceae、NOBNitrospina25．α、β、γ、δ．Nitrobacter．．．254144Fig．4Phylogenetictreeofthebacteriaofactivesludgeinthemunicipalwastewaterprocess．NO－2NH+4anaerobicammoniumoxidationANAMMOX．26DO．2.3PCRrRNA27PCR．16SrDNA3541345Fig．5PhylogenetictreeoftheproteobacteriaofactivesludgeinthemunicipalwastewaterprocessAOB6.69×108NOB3.58×10516SrDNAAOB3.17×106NOB4.15×104．AOBNOB．AOB2NOB1AOBNOB3NOB．AOBNOB．AOB．AOB．454142.4PLFAsPLFAs．9.43nmol·g－112.0nmol·g－1．6．HE3．126Fig．6Comparisonofsamplebiomass3Table3ComparisonofthediversityindexandEvennessindexofsamplesH1.241.41E0.6370.7253．．31、、、、α-Proteobacteria、β-Proteobacteria、γ-Proteobacteriaδ-Proteobacteria．、．2AOBAOB/NOB．．1RabalaisNNTurnerREScaviaD．BeyondscienceintopolicyGulfofMexicohypoxiaandtheMississippiRiverJ．