O工艺好氧单元中微生物群落结构

66Vol．6No．620126ChineseJournalofEnvironmentalEngineeringJun．2012PCR-DGGEA2/O1231131．3001912．3001913．300171PCR-DGGE。5γ、δ、α、εProteobacterias、Bacilli。γPseudomonassp．、Rheinheimerasp．、Citrobactersp．、Klebsiellasp．、Enterbacte-riaceae、Stenotrophomonasmaltophilia、Acinetobacter。unculturedPseudomonassp．、Halobacillussp．、Pseudomonassp．、Pseudomonasstutzeri、Acinetobactersp．。Halobacillussp．、Pseudomonassp．、Pseudomonasstutzeri、Acinetobactersp．。PCR-DGGEX172A1673-9108201206-1907-08ApplicationofPCR-DGGEtoanalysisofmicrobialcommunitystructureinaerobicunitofA2/OprocessLüJinghua12MaTing3ZhengXianqiang1DuanYunxia1LiZhaoyu31．ResearchandDevelopmentDepartmentofTianjinAcademyofEnvironmentalSciencesTianjin300191China2．TianjinFederationEnvironmentEngineeringDesignCo．Ltd．Tianjin300191China3．StateKeyLaboratoryofPetroleumMicrobiologyCollegeofLifeSciencesNankaiUniversityTianjin300171ChinaAbstractThedynamicprocessandthemicrobialcommunitystructureintheaerobicactivatedsludgesys-temoftheindustrialwastewatertreatmentweretracedbythePCR-DGGEtechnology．Theresultsshowthatthemicrobialcommunitystructurechangeswiththewaterqualityinthesystemandbecomesstableastheincubationtimeincreasing．ThemicrobialcommunitystructureismainlycomposedoffivecategorieswhichrespectivelyhavecloserelationshipswiththeγδαεProteobacteriasBacilli．γProteobacteriumisthemainmicrobialcommu-nitywhichincludingPseudomonassp．Rheinheimerasp．Citrobactersp．Klebsiellasp．EnterbacteriaceaeStenotrophomonasmaltophiliaAcinetobacter．FivebacteriaincludingunculturedPseudomonassp．Halobacillussp．Pseudomonassp．PseudomonasstutzeriAcinetobactersp．canexiststablyinthesystemandbecomedomi-nantmicroorganisms．Sotheeffectofwastewatertreatmentcanbeimprovedbyimprovingquantityandqualityofthedominantmicroorganismsinthesystem．KeywordsPCR-DGGEactivatedsludgesystemmicrobialcommunitystructuredominantmicroorganisms2008ZX07314-001511783112011－02－162011－06－021983～。[e-mail]1979FisherLermanDNA1-3。1993Muyzer4。PCR-DGGE。5PCR-DGGE2。6PCR-DGGE6。7。PCR-DGGE8、PCR-DGGE9、UASBPCR-DGGEUASB10。A2/ODNAPCR-DGGE。11．1、、、1。、0.6m×0.3m×0.35m0.2m×0.35m×0.4m2L/h12h。。4L/h。BOD。1．2DNA1331、47、414、428、514、528、614、628714DNA12。DNA－20℃。11。1.3PCRDGGEPCR16SrRNAV6～V8968F/1401R435bp。1PCR50μL10×PCRbufferMg2+2.5mmol/L5μLdNTPs2.5mmol/L5μLV6-8F-GC1320mmol/LCGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCACGGGCGGTGTGTAC1μLV6～8R20mmol/LACGGGCGGTGTGTAC1μLDNA1～10ngTaq0.5μL50μL。PCRTouchdownPCR1494℃5min94℃1min65℃1min72℃1min21℃2094℃1min55℃1min72℃1min1572℃10min。DGGEV6～8FATGGCTGTCGTCAGCTV6～8R。2PCR。94℃5min94℃30s55℃30s72℃30s30。72℃10min。PCR2%1mg/L10min。1Fig.1Modeloftreatmentplant80916PCR-DGGEA2/O1.4DGGE1PCRDGGE。6%∶=37．5∶140%～60%100%7mol/L40%。1×TAE20mmol/LTris10mmol/L0．5mmol/LEDTApH7．460℃160V4h。1mg/LEB15min3min。BIO-RADQUANTITYONE。CSDicecoeffi-cientDGGE。1．51．5mLEP50μLDNA。DNAPCR1．32PCRPCR。22．1DNAPCR2．1．12。2Fig．2Effectofwastewatertreatmentduringsystemoperation2COD。CODCOD6COD。2．1．2DNAPCRDNA3。310DNA3DNAFig．3ElectrophoreticimageoftotalDNAextractedfromdifferentactivatedsludgesamples23kbDNADNA。PCR4450bpPCR。4PCRFig．4PCRproductsofeachsample2．22．2．1DGGE10DGGE5。5DGGEFig.5DGGEelectrophoreticimageofdifferentsamples510。5abcde5105。5Pseudomonassp．、Halobacillussp．、Pseudomonassp．、Pseudomonasstutzeri、Acinetobactersp．。90916106。6111。1。DGGE。。56。6Fig.6Comparisonpictureoflanes2．2．2Thecompletelinkage7。1039%。59%63%。7ThecompletelinkageFig.7Dendrogramcomputedbycompletelinkagearithmetic2．2．3DGGEPCR。NCBIHQ833591～HQ833641NCBI。105127。2．2．4GC-MS4。1021、213、4239%59%63%。1331COD1310mg/L、、PAHs、ε、δ、γ、、α。247—428COD983mg/L1γPseudomonassp．、、Stenotrophomonasmaltophilia。。3514—614COD893mg/L、、PAHs、Haloba-cillussp．γPseudomonassp．、Pseud-omonasstutzeri。ε、δγ。4628—728COD465mg/L3839、108γProteobacterium、Citrobactersp．Rheinheimerasp．Rheinheimerasp．。2．2．55189。515γ、δ、α、εProteobacterias、Ba-cilli。γ、Rhein-heimerasp．、Citrobactersp．、Klebsiellasp．、Enterbacte-riacea、Stenotrophomonasmaltophilia、Acinetobacter。01916PCR-DGGEA2/O8、α、δ、ε、Fig．8AnalysisofphylogenetictreeofbacteriuminactivatedsludgesamplesBacilli、AlphaProteobacteriumunt、Dettaproteobacterium、EpsilonProteobacterium、Unculturebacterium3515γ、δ、α、εProteobacterias、BacilliγProteobacteriasPseudomonassp．、Halobacillussp．、Pseudomonassp．、Pseudomonasstutzeri、Acinetobactersp．。。Pseudomonas、、、15-20。Halobacillus21。Acinetobacter、、、22-25。Citrobactersp．26。Klebsiellasp．、27-30。Stenotrophomonasmaltophilia、3132。。GC-MS、、PAHs、。434。119169γFig．9AnalysisofphylogenetictreeofbacteriuminactivatedsludgesamplesGammaProteobacterium1、213、4239%59%63%。γProteobacterias。4PCR-DGGE15γ、δ、α、εProteobacterias、Bacilliγ。Pseudo-monassp．、Halobacillussp．、Pseudomonassp．、Pseud-omonasstutzeri、Acinetobactersp．。221916PCR-DGGEA2/O。1FischerS．G．LermanL．S．DNAfragmentsdifferingbysinglebase-pairsubstitutionsareseparatedindenaturinggradientgelsCorrespondencewithmeltingtheory．Proc．Natl．Acad．Sci．U．S．A．19838061579-15832LermanL．S．FischerS．G．HurleyI．etal．Se-quence-determinedDNAseparations．Annu．Rev．Bio-phys．Bioeng．198413399-4233MyersR．M．FischerS．G．LermanL．S．etal．Near-lyallsinglebasesubstitutionsinDNAfragmentsjoinedtoaGC-clampcanbedetect