MBR中微生物群落结构的演变与分析

2811200811ActaScientiaeCircumstantiaeVol.28,No.11Nov.,2008:(No.07JCZDJC02100)SupportedbytheNaturalScienceFoundationofTianjin(No.07JCZDJC02100):(1980),,,E2mail:[e-mail];3(),E2mail:[e-mail]:ZHANGBin(1980),male,Ph.D.candidate,E2mail:[e-mail];3Correspondingauthor,E2mail:[e-mail],,,.2008.MBR[J].,28(11):2192-2199ZhangB,SunBS,JiM,etal.2008.Analysisandsuccessionofmicrobialcommunitystructureinamembranebioreactor[J].ActaScientiaeCircumstantiae,28(11):2192-2199MBR1,2,1,3,1,21.,3000722.,300050:2007212204:2008204229:2008208204:2,DNA,16SrDNAPCR2DGGEDNA,,.DGGE,17d,29.2%,MBR.,PseudomonasAeromonashydrophila,Bacillussp.EnterococcusfaecalisComamonassp.Fusobacteriumsp..UPGMADGGE3.,MBR,ProteobacteriaBacillus.(Comamonassp.).:2;;PCR2DGGE;:025322468(2008)1122192208:X703:AAnalysisandsuccessionofmicrobialcommunitystructureinamembranebioreactorZHANGBin1,2,SUNBaosheng1,3,JIMin1,ZHAOZuguo21.SchoolofEnvironmentalScienceandTechnology,TianjinUniversity,Tianjin3000722.InstituteofHygieneandEnvironmentalMedicine,AcademyofMilitaryMedicalSciences,Tianjin300050Received4December2007;receivedinrevisedform29April2008;accepted4August2008Abstract:Inordertorevealthesuccessionofmicrobialcommunitydiversityinamembranebioreactor(MBR),thegenomeDNAofsludgeatdifferentperiodwasdirectlyextractedthroughchemicalsplitting.PCR2amplified16SrDNAanddenaturinggradientgelelectrophoresis(DGGE)wereappliedtoobtaintheDNAfingerprintprofileofthemicrobialcommunities.Thebandsinthegelwereanalyzedbystatisticalmethodsandexcisedfromthegelforsequencing.Thesequenceswerethenusedforhomologyanalysisandphylogenetictreeswereconstructed.DGGEanalysisindicatedthatanoticeablechangetookplaceinmicrobialstructureafter17daysofMBRoperation.Thissuggeststhatthetreatmentprocessandinfluentwastewatercompositionhasasignificantimpactonbacterialcommunitystructures.PopulationssuchasPseudomonasandAeromonashydrophilawerethepredominantspeciesthroughoutthestudy,whileprimarycommunitiessuchasBacillussp.diedout,andsecondarymicrobialcommunitiessuchasEnterococcusfaecalisComamonassp.andFusobacteriumsp.increased.ClusteranalysisofDGGEbyUPGMA(unweightedpairgroupmethod,arithmeticmean)dividedalllanesintothreeclusters,whichcorrespondedtothreemanipulationperiodsduringthewholeexperiment.ThesequencesindicatedthatthephylogenetictreepresentedthelongergeneticdistanceamongthebacteriaintheMBRbiomass,whereProteobacteriaandBacilluswerethedominantspecies.Thepreponderantbacteria(suchasComamonassp.)thatincreasedduringthelaterstageofoperationhadagreaterimpactontheproductionandaccumulationofmembranepollutants.Keywords:MBR;microbialdiversity;PCR2DGGE;cloningandsequencing11:MBR1(Introduction)2(MembraneBioreactor,MBR),(ThomasHetal.,2000;RosenbergerSetal.,2002),.MBR,,,.MBR,F/M,(autolysis)(EPSSMP),(MengFGetal.,2006;,2007).,,MBR.,,.Rowan(2003)DGGE;LaPara(2002),,;Calli(2006)DGGE;(2006a;2006b),.MBR,MBR,PCR2DGGE,,GenBank;,.2(Materialsandmethods)2.1MBR30L,1.5m.,,1.TOC160180mgL-1,NH+42N4045mgL-1,5.05.5mgL-1.(,2007)1FP2TPVDF,0.65mm1.0mm,0.22m,1m2,1518.,,0.100.20m3h-1.6h,,10min,2min..1Table1Componentsandconcentrationofartificialwastewater/(mgL-1)400(NH4)2SO4220NaHCO350MgSO47H2O50KH2PO423CaCl22H2O10FeCl36H2O485d.10,1,,5d1;,,10d,.2.3912282Table2CharacteristicsofMBRatthesamplingtimes/dDO/(mgL-1)pH/(mgL-1)TOCMLSS/(mgL-1)MLVSS/MLSS/MPa1G17.87.120175.138.559960.540.045G28.27.362.934.949500.720.0611G37.97.342.733.662200.780.0817G47.57.520130.118.773400.800.1228G57.86.925.515.778600.810.1437G67.57.215.313.693000.820.1550G77.37.211.512.591200.850.1962G87.37.520111.811.2114400.820.2270G97.07.914.012.7135000.840.2780G106.87.8501311.512.3148400.830.33:3,2.2DNA,(\\)2DNA(,2008).,DNA23kb;,A260/A2801.761.79,DNA,PCR.2.3PCR16SrDNAV3F3572GCR518,PCR(MuyzerGetal.,1993),.100L:10100ng,10L10PCRbuffer(with20mmolL-1MgCl2),200molL-1dNTPs,0.5molL-1,215UExTaq,.PCR(BIOBASICINC.Cat.No.BS363)40LBuffer.2.416SrDNADGGEC.B.S.SCIENTIFICDGGE22001,8%,35%55%,60,150V,1TAE6.5h,,.2.5DGGE2.5.1Shannon2WeaverShannon(H),:H=-PilogPi(1)HDGGE,QuantityOne,Pi=ni/N,ni,N.2.5.2UPGMA,MBR(),DGGEUPGMA(UnweightedPair2GroupMethodUsingArithmeticaverages),.2.6DGGE20LddH2O,4.,F357R518PCR,55,2.3.PCR1.5%.PCRpGEM2T(PromegapGEMÒ2TVectorSystemI),E.coliTOP10,X2galIPTGAmpLB16h(37);LB,37,,PCR(Invitrogen).2.7BioEdit(v7.0.5)GenBank(),BLAST491211:MBR.DNAMAN(v5.2.2).3(Experimentalresults)MBR1016SrDNAV3341534PCR,DGGE,1.Shannon2.UPGMA3.5912281A,GenBank,,3,4.316SrDNATable3Resultsofsomepartial16SrDNAsequencesusingBLASTinGenBankNCBIGenBank1DEZDG1X8015AJ853605Unculturedbacteriumpartial16SrRNAgene100%2FR91MT65014EU071522UnculturedAcinetobacterspcloneEHFS1_S14e16SribosomalRNAgene,partialsequence99%4DF0U3A7V01RDQ805501UnculturedbacteriumcloneRL203_aai63e0116SrRNAgenepartialse2quence95%5JGYW3GH4012DQ444188Unculturedbacteriumclonecs9016SribosomalRNAgene,partialse2quence99%6FRDGD4GU014AY803983Bacillussp.HPC4016SribosomalRNAgene,partialsequence100%7EFTKWUH801REF584540Bacillussp.JDM222216SribosomalRNAgene,partialsequence100%8DF3NEPVU015EF645247Pseudomo