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16SrDNA李建婷1,纪树兰1*,刘志培2,秦振平1,刘缨2,杨媛媛11.,1001242.,100101:16SrDNA.82(700bp),Blast,,7,,B(B-Proteobacteria)(Sphingobacteria),34116%30150%;CandidatedivisionTM7(Flavobacteria)C(C-Proteobacteria),9176%,7132%7132%;(Actinobacteria)A(A-Proteobacteria),4188%1122%.,(Acidovorax)(Pseudomonas),CODCrMicropruinaglycogenica,(Comamonadaceae).:;16SrDNA;:X172:A:1001-6929(2009)10-1218-06AnalysisofBacterialCompositionofAerobicGranularSludgewith16SrDNACloneLibraryLIJian-ting1,JIShu-lan1,LIUZhi-pei2,QINZhen-ping1,LIUYing2,YANGYuan-yuan11.CollegeofEnvironmentandEnergyEngineering,BeijingUniversityofTechnology,Beijing100124,China2.InstituteofMicrobiology,ChineseAcademyofSciences,Beijing100101,ChinaAbstract:Thebacterialdiversityofanaerobicgranularsludgewasstudiedbytheconstructionofa16SrDNAclonelibrary.82cloneswererandomlyselectedfromthelibrary.Thecorrespondingpartial16SrDNAgene(ca.700bp)wassequencedandblasted.Theresultsindicatedthatthebacterialcommunityintheaerobicgranularsludgewashighlydiverse,andcouldbedividedintosevenmajorgroups.ThelargestfractionswereB-ProteobacteriaandSphingobacteria,whichaccountedfor34116%and30150%ofthetotalclones,respectively.TheportionsofCandidatedivisionTM7,FlavobacteriaandC-Proteobacteriawere9176%,7132%,and7132%,respectively.Therelativelylowerportionswere4188%Actinobacteriaand1122%A-Proteobacteria.Theresultofsequenceanalysisalsoshowsthattherearenotonlybacteriaimportantfortheformingandstableoperationofaerobicgranularsludge(forexample,Acidovorax,Pseudomonas),butalsobacteriabeneficialtotheremovalofCODCrandammonia-nitrogen(forexample,Micropruinaglycogenica,Comamonadaceae)intheaerobicgranularsludge.Keywords:aerobicgranularsludge;16SrDNAclonelibrary;bacteriacomposition:2009-01-17:2009-04-09:(20876003);(8042007):(1983-),,,tingting-510@163.com.*,(1953-),,,,,jshl@bjut.edu.cn.[1],,,.,.(DGGE)[2-4],,,[5],.[6].16SrDNA16SrDNA2210200910ResearchofEnvironmentalSciencesVol.22,No.10Oct.,2009DOI10.13198/j.res.2009.10.108.lijt.018,[7-10],.,.16SrDNA,.11.1SBR,:Q(CODCr)1000mgPL,Q(C)BQ(N)BQ(P)100B5B1,pH7101:360min(10min,1min,10min,339min),70%.5g,11-20e.1Table1OperationconditionandsludgepropertyCODCrNH4+-NQP(mgPL)P%QP(mgPL)P%PmmSVIP(mLPg)16~229717~98.316~199113~92162~318~211.2DNA();(Omega);pGM-T();TaqDNA(Japan,Takala);dNTP(Japan,Takala);T4DNA(Germany,Promega).TGL-20M();PCR(Germany,Biometra);BINTA2020D().1.31.3.1DNA5g,5mLA(100mmolPLTris,25mmolPLEDTA,pH810),(4e,10000@g)2min,;5mLA,5min;100LL(100mgPmL),,37e2~3h;015molPLEDTA750LL,2molPLNaCl250LL,20%SDS500LL,015g,5min,(4e,10000@g)8min,;5mL,5mLV()BV()1B15mL;015mL(5molPL),10mLDNA1h;70%DNA,3min,(4e,10000@g)5min[11];100LLTEDNA.1.3.2DNADNADNA,DNA.1.3.3PCRDNA,16SrDNA27F1492RPCR.PCR:10@buffer10LL,dNTP(10mmolPL)2LL,27F(30LmolPL)2LL,1492R(30LmolPL)2LL,TaqDNA(5UPLL)1LL,DNA2LL,ddH2O100LL,.PCR:95e5min;94e1min,55e1min,72e115min,3072e10min.1.3.416SrDNAPCR,pGM18-T,DH5a,Amp-XgalPIPTG16SrDNA.(C):C=[1-(nlPN)]@100%,N;n1.1.3.5DNA16SrDNA82(),16SrDNA27F,GenBankBlast,121910:16SrDNAMEGA311,(Neighbor-JoiningAnalysis).22.1DNAPCRDNA,20kb,DNA,DNA16SrDNAPCR.DNA,16SrDNAPCR,1.1,2PCR,PCR,DNA10PCR,DNA1500bp./ReconditioningPCR0DNA,,16SrDNA.P:M);1)0;2)10;3)100;4)10001DNAPCRFig.1ThePCRresultselectrophoresisofthedifferentdilutionDNAtemplate2.216SrDNA82(700bp),83%,GenBankBlast,(OperationalTaxonomicUnit,OTU),30OTU,OTU212,GenBank16SrDNA100%,88%,66(80152%)95%.,,.B(B-Proteobacteria),28(9OTU);(Sphingobacteria),25(6OTU);CandidatedivisionTM7,8(2OTU);(Flavobacteria),6(4OTU);C(C-Proteobacteria)A(A-Proteobacteria),6(3OTU)1(1OTU);(Actinobacteria),4(2OTU).B-Proteobacteria,A-Proteobacteria,C-Proteobacteria,E-Proteobacteria,G+C,(Chlamydiae)(Planctomyces)(5212%,115%,115%,1119%,715%,115%115%)[12].B-Proteobacteria,A-Proteobacteria,C-Proteobacteria,Sphingobacteria,CandidatedivisionTM7,FlavobacteriaActinobacteria7(34116%,1122%,7132%,30150%,9176%,7132%4188%).B-Proteobacteria,B-Proteobacteria,C-Proteobacteria,Q(C)PQ(P).[13-14],SBR,Q(C)PQ(P)(10~50),C-Proteobacteria,B-Proteobacteria,C-ProteobacteriaB-Proteobacteria.Q(C)PQ(P)100,Q(CODCr)(1000?50)mgPL,Q(C)PQ(P).B-Proteobacteria,C-Proteobacteria.B-Proteobacteria16(19152%)Acidovorax,Acidovorax[15].C-Proteobacteria4(4188%)Pseudomonas100%,Pseudomonas(EPS)[2],.4OTU,6(7132%)(Flavobacteria),Flavobacteria[2],,.122022216SrDNATable2Bacterial16SrDNAcloningresultsofaerobicgranularsludgeOTUOTUPP%EMBLGenBankNCBIP%OTU11315186FJ645568Acidovoraxsp.AY56657999OTU456110FJ645565ComamonadaceaebacteriumSB1AJ60633398OTU522144FJ645564AquincolatertiaricarbonisDQ65648999OTU1122144FJ645558Acidovoraxsp.EU375646100OTU1622144FJ645553UnculturedsludgebacteriumAF23472699B-ProteobacteriaOTU1711122FJ645552UnculturedsludgebacteriumAY68909396OTU2011122FJ645549Thauerasp.EU037291100OTU2211122FJ645547PelomonassaccharophilaAM50143297OTU2911122FJ645540UnculturedAcidovoraxsp.DQ23416393OTU344188FJ645566Pseudomonassp.AY779523100OTU1811122FJ645551Pseudoxanthomonassp.DQ33759797C-ProteobacteriaOTU3011122FJ645539GammaproteobacteriumAM41193992OTU2311122FJ645546UnculturedalphaproteobacteriumFM25361399A-ProteobacteriaOTU71113142FJ645562Sphingobacteriaceae