16SrDNA克隆文库法分析B省略滤池处理城市污水的细菌多样性研究窦娜莎

3110201110ActaScientiaeCircumstantiaeVol．31No．10Oct．2011No．2008ZX07010-008-04No．2009GGB01012No．JK09-17SupportedbytheMajorProjectsonControlandRectificationofWaterBodyPollutionNo．2008ZX07010-008-04theScientificandTechnologicalProjectofShandongProvinceNo．2009GGB01012andtheScienceandTechnologyProjectsonConstructioninQingdaoNo．JK09-171983—[e-mail]*[e-mail]1983—[e-mail]*[e-mail]tyrbiologicalaeratedfilterofmunicipalwastewaterby16SrDNAJ．ActaScientiaeCircumstantiae31102117-212416SrDNABiostyr*2661002010-12-102011-01-192011-01-2116SrDNABiostyrBAF．16SrDNA501．5kbBLAST．BiostyrBAF4173β-proteobacteriumγ-proteobacteriumBacteroidetes．26OTUMEGABiostyr．Biostyr16SrDNA0253-2468201110-2117-08X703．1ABacteriadiversityinabiostyrbiologicalaeratedfilterofmunicipalwastewaterby16SrDNADOUNashaWANGLin*CollegeofEnvironmentalScienceandEngineeringOceanUniversityofChinaQingdao266100Received10December2010receivedinrevisedform19January2011accepted21January2011AbstractThebacterialdiversityinaBiostyrbiologicalaeratedfilterBAFwasstudiedby16SrDNAcloningandsequencingmethod．Fiftycloneswererandomlyselectedandtheir16SrDNAgenesca．1500bpweresequencedandblasted．TheresultsindicatedthatthebacterialdiversityintheBiostyrBAFwasveryhigh41clonesbelongto7differentpublishedphylaand3clonesbelongtounknownphyla．ThedominantbacterialcommunityintheBiostyrBAFwereβ-proteobacteriaγ-proteobacteriaandBacteroidetes．UsingMEGAsoftware26OTUphylotypeswereselectedfromthe44sequencedclonesandthephylogeneticanalysisalsodemonstratedhighbacterialdiversityintheBiostyrBAF．KeywordsBiostyrbiologicalaeratedfiltermunicipalwastewater16SrDNAlibrarybacterialdiversity1IntroductionbiologicalaeratedfilterBAF207080．8090．、BAF、．90BAFBAFBiostyrOTV—biostyreneDOI:10.13671/j.hjkxxb.2011.10.017311g·cm－3．BAFXieetal．2005、Zubairetal．2008、20082006．、、．1985PacePaceetal．1986．BiostyrBAF16SrDNABiostyr．2Materialsandmethods2．1Biostyr14t813．81m×16．70m×8．12m231m27．7m·h－1biostyrene6mm13．5m7200m3．BiostyrBAF1、、、、．．．1BiostyrBAF2008Fig．1SchematicoftheBiostyrBAF2．2BiostyrBAF3h100mm300mm．BiostyrBAF1、2．1BiostyrTable1TheoperationparametersoftheBiostyrprocess/m3·m－2·h－1/kg·m－3·d－1/kg·m－3·d－1/℃1．131．240．451∶6．0814．581121016SrDNABiostyr2BiostyrTtable2TheinfluentandeffluentofthebiostyrprocessCOD/mg·L－1COD/mg·L－1CODNH+4-N/mg·L－1NH+4-N/mg·L－1NH+4-NSS/mg·L－1SS/mg·L－1SS137．456．658．8%38．817．056．2%44．414．467．6%2．3DNADNA“--SDS”Heetal．2005①5mLTENP3min2min3②．②SDS15mL37℃1h15mL10%SDS10min．③DNA15mLTris-2、+1、1200μL3mol·L－115mLDNA－20℃30min20min5mL70%10min．DNA150μL4．5μLRnasea10mg·mL－137℃15～20minRNA－20℃．PCR27F5'-AGAGTTTGATTCCTGGCTCAG-3'1472R5'-ACGGCTACCTTGTTACGACT-3'．PCR．PCR50μL10×5μL25mmol·L－1Mg2+3μL10mmol·L－1dNTP1μL5μL+5μL25ng·μL－1DNA2μL5U·μL－1TaqDNA0．5μL28．5μL．PCR95℃5min94℃1min50℃1min72℃1．5min3072℃10min．16SrDNA、PCRDNATAKARAJapan．pMD19-TTAKARAJapanDH5ααX－gal．．16SrDNA．DNA16SrDNA50LB/AmpLA37℃37℃．http//．ncbi．nlm．nih．gov/BLAST/．MEGA．．3Results3．1DNAPCR2DNA1、2DNAPCR．3PCRPCRDNA1．5kb16SrDNAPCR16SrDNA．2DNAFig．2ElectrophoresisoftheextractedDNA9112313PCRFig．3ElectrophoresisofthePCRresults3．216SrDNABiostyr16SrDNA5064416SrDNABLAST4426OTUBLAST3．3rRNA55α、β、γ、δε．3β-proteobacterium1710OTUsγ-proteobacterium85OTUsα-proteobacterium43OTUsδ-proteobacterium121OTU．Bacteroidetes63OTUs．3．3MEGAOTU26bootstrap＞50%bootstrap100444proteobacterium、Firmicutes、BacteroidetesNitrospirae．1～19Nitrospirasp．16SrRNAgeneGammaproteobacteriumGammaproteobacterium2～8Comamonassp．Sphaerotilussp．Comamonassp2～10Sphaerotilussp．Ideonellasp．Ideonellasp．．3．4、．．5a5b．．．4Discussion4．1Biostyrproteobacterium70%．．rRNA55α、β、γ、δε．β-γ-/COD．Biostyrβ-38.6%γ-18．2%COD．δ-CODBiostyrδ-4．5%DO＞7．BiostyrBacteroidetessp．0212314Fig．4Thephylogeneticanalysisofbacteria13．6%．、、、Hilletal．2007．Bacteroidetes200616SrDNA97%Bacteroidetes67%GenBank16SrDNA97%Biostyr．22121016SrDNABiostyr5BAFFig．5SEMphotosofthebiofilmonthemediasurfaceoftheBAF4．2BiostyrCODNH+4-N137.4mg·L－138．8mg·L－1CODNH+4-N58．8%56．2%1BiostyrCODDNA2、4．1～17Acidovoraxsp．96%Acidovoraxsp．68Heetal．20052～4Sphaerotilussp．99%Sphaerotilussp．、Fe2+Fe3+20022～14Xanthomonassp．97%Xanthomonassp．2～16Flavobacteriumsp．99%Flavobacteriumsp．Koizumietal．2003．Biostyr．．．2～17Nitrosomonassp．97%2～14Nitrospirasp．97%．BiostyrTNBiostyrTN14．8%．1～5Hyphomicrobiumdenitrificans99%2～12Comamonassp．93%．7mg·L－124h、．．5Conclusions1BiostyrBiostyr/β-、γ-．2Biostyrβ-proteobacterium38．6%γ-proteobacterium18．2%Bacteroidetes13．6%α-proteobacterium9．1%δ-proteobacteriumNitrospirae4．5%Firmicutes3212312．3%9．1%．3Biostyr、、．1966—．5、15．、、、[e-mail]sphorusremovalandchangesinmicrobialcommunitystructureinamembranebioreactorEffectofdifferentcarbonsourcesJ．WaterResearch42198-210．2002．J．25643-46ChenJFXuXPLiHZ．2002．GeneralsituationinsphaerotilusstudyJ．EnvironmentalScienceandTechnology25643-46inChineseHeJZXuZHJaneH．2005．PrelysiswashingimprovesDNAextractionfromaforestsoilJ．SoliBiologyBioc