处理不同废水MBR系统中微生物群落结构的比较张斌

MBR1,2,1＊,1,1,1(1.,　300072;2.,　300050):,,4MBRDNA,PCR-DGGE,.,MBR,,,,Shannon0.770.78.,Proteobacteria(8OUTs)Bacillus(2OUTs).,;MBR,,2UnculturedAchromobactersp.Uncultureddenitrifyingbacterium,.:;16SrDNA;PCR-DGGE;;:X172　:A　:0250-3301(2008)10-2944-06:2007-10-31;:2008-01-08:(07JCZDJC02100):(1980～),,,,,E-mail:[e-mail]＊,E-mail:[e-mail]CommunityStructureinMBRsTreatingDifferentWastewaterZHANGBin1,2,SUNBao-sheng1,LIUHui-na1,LIUXian-hua1,JIMin1(1.SchoolofEnvironmentalScienceandTechnology,TianjinUniversity,Tianjin300072,China;2.InstituteofHygieneandEnvironmentalMedicine,AcademyofMilitaryMedicalSciences,Tianjin300050,China)Abstract:Inordertoinvestigatethemicrobialcommunitystructuresindifferentmembranebioreactors,totalbacterialgenomicDNAwasextractedfrombiomassinfourMBRstreatingdifferentwastewater.Themicrobialcommunitystructureswerestudiedbypolymerasechainreaction-denaturinggradientgelelectrophoresis(PCR-DGGE)andcloning-sequencing.Furthermore,andthesequenceswereusedforhomologyanalysisandthentwophylogenetictreeswereconstructed.ResultsindicatethateachMBRownsitsspecificecologicalcommunityafteralong-termofoperation.Influentwastewatercompositionhasasignificantimpactontotalbacterialcommunitystructures.Populationdiversity,whichisinthebioreactorfedwithmorecomplexcomponentswastewater,ishigherthanthoseofotherMBRs.TheShannonIndexare0.77and0.78.ProteobacteriaandBacillus,whichcontain8OUTsand2OUTs,aretheclimaxcommunitiesintheinvestigableMBRs.Theprofilesofammonia-oxidizingbacterialcommunityinthefourreactorsareverysimilar.TheremayexistseveralmodesofnitrificationanddenitrificationinMBRbecausesequencesrevealedthatthereexistmultitudenitrifyingorganisms,inwhichNitrosomonasarethemostpredominant,andtwodenitrifyingbacterium(unculturedAchromobactersp.anduncultureddenitrifyingbacterium)inreactors.Keywords:MBR;16SrDNA;PCR-DGGE;microbialcommunitystructure;ammonia-oxidizingbacteria　　(MBR),,,、[1,2].MBR90%[3],[4,5],.-(PCR-DGGE)[6]DNARNA,,.、、.,,,.,MBR,.MBR,[7～9].,PCR-DGGEMBR2910200810　　　　　　ENVIRONMENTALSCIENCEVol.29,No.10Oct.,2008DOI:10.13227/j.hjkx.2008.10.039,GenBank,,,、.1　1.1　1.、SS、、[10].TOCTOC-VCPH.,　　　　　　1　Table1　OperationalconditionsandStatusoftheMBRsmg·L-1TOCBOD5A、50～10035～6515～25—、SS100%、TOC80%～95%、70%～80%DO4.0～7.0mgL,HRT=6hB150～250180～27535～504.5～5.5SS95%～100%、TOC90%～95%、60%～70%DO6.5～7.5mgL,HRT=5hC100～130100～2507～15—、SS100%、TOC80%～92%、60%～70%DO5.5～7.5mgL,HRT=5hD100～25050～7530～401～3SS90%～100%、TOC85%～90%、80%～95%DO4.5～6.5mgL,HRT=5hDNA.1.2　DNA、、DNA,23kb,,PCR,[11].1.3　DNA16SrDNAV3PCR1.3.1　PCR16SrDNAV3F357-GCR518,.PCR[6].100μL:10～100ng,10μL10×PCRbuffer(with20mmolLMgCl2),200μmolLdNTPs,0.5μmolL,2.5UExTaq,.PCR(BIOBASICINC.Cat.No.BS363),40μLBuffer.1.3.2　PCRCTO189fCTO654r,[12].PCRPCR,F357-GCR518,.,,.PCR1.5%.1.4　PCRDGGEC.B.S.SCIENTIFICDGGE-2001,8%,35%～55%,60℃,150V,1×TAE6.5h,,.1.5　Shannon-WeaverShannon(H),:H=-∑PilgPi,HDGGE,QuantityOne,Pi=niN,ni,N.1.6　DGGE20μLddH2O,4℃.,F357R518PCR,55℃,1.3.1.PCR1.5%.PCRpGEM-T(PromegapGEM®-TVectorSystemI),E.coliTOP10,X-gal、IPTGAmpLB16h(37℃),LB,37℃,,PCR(Invitrogen).1.7　BioEdit(V7.0.5)294510:MBRGenBank(http:),BLAST,.DNAMAN(V5.2.2).2　2.1　DGGE1(a),.Shannon2.,(,、),,、.,,.1(a),MBR,.1(a)Band1、2、3、8、12;,ABand4,BBand5,CBand6,DBand7、10.23,Band9、11.,,Band3、8AB,CD;Band1、12AD,BC.4Band2MBR,MBR..2,ABShannon,C,[8],,,Shannon[13].C(SV30≥80),.D[7],,(a)DGGE;(b)DGGE;M:MarkerDL20001　MBRDGGEFig.1　DGGEprofileofbacteriaandammoniaoxidizingbacteriainthedifferentMBRs2　MBRShannonFig.2　ShannonindexofmicrobialpopulationinthedifferentMBRs.,.1(a),GenBank,,2,3.DGGE(OTU),2,GenBank,Proteobacteria(8OUTs)Bacillus(2OUTs),Wagner[14].2946　　　　　　29　　　2　16SrDNATable2　Resultsofsomepartial16SrDNAsequencesusingBLASTinGenBankNCBIGenBank%1DEZDG1X8015AJ853605Unculturedbacteriumpartial16SrRNAgene1002DF0U3A7V01RDQ805501Unculturedbacteriumpartial16SrRNAgene953DDJ075E0015EF681114AeromonashydrophilaisolateAHPN3-516SribosomalRNAgene,partialsequence994DRRM385A014DQ886173UnculturedBacteroidetesbacteriumcloneedNE916SrRNAgene,partialsequence995DF3NEPVU015EF645247Pseudomonasplecoglossicida1007DF5NND3801RAM697386Unculturedbacteriumpartial16SrRNAgene1009DF4UVH61014EU031774Comamonassp.BS2716SribosomalRNAgene,partialsequence9910DS015KJ9012AY960268UnculturedEnterobacteriaceaebacteriumclone90316SrRNAgene,partialsequence9911EFR978X1012AY803983Bacillussp.HPC4016SribosomalRNAgene,partialsequence10012EFTKWUH801REF584540Bacillussp.JDM-2-216SribosomalRNAgene,partialsequence100aEFEBYEG1012AY543664UnculturedNitrosomonassp.clone20SB216SribosomalRNAgene,partialsequence94bDFSRZF75012EF042985UnculturedNitrosomonassp.cloneH316SribosomalRNAgene,partialsequence99cDFRYFY8N015AY543074UnculturedNitrosomonassp.clone15BAFln116SribosomalRNAgene,partialsequence98dEETRUD3Y01REF042993UnculturedNitrosospirasp.clone47-216SribosomalRNAgene,partialsequence94eEF5GPMVJ016AF351233UnculturedbetaProteobacteriumclone4-7016SribosomalRNAgene,partialsequence100fDFTPASSN015CP000127NitrosococcusoceaniATCC1970794gEFC8REFY012EF033497UnculturedAchromobactersp.cloneDGR1-p1c