豆腐废水UASB反应器中的原核生物多样性及主要功能菌群张春杨

44　220044ActaMicrobiologicaSinicaVol.44April　No.22004:(30025001);“863”(2001AA227131)＊。TelFax:86-10-62558320;E-mail:[e-mail]:(1975-),,,,。E-mail:[e-mail]:2003-06-17,:2003-09-29UASB张春杨　刘晓黎　东秀珠＊(　　100080)　:16SrRNA,UASB,MPN。,33%16SrRNA,,1.1×109mL4.5×108mL。GCδ-,16SrRNA22%9%,4.5×107ml。,16SrRNA12%。。,。:UASB,,,:Q939　　:A　　:0001-6209(2004)02-0141-07　　、、SO42-、NO3-、Fe3+,(Hydrolyzingandfermentingbacteria),(Syntrophicacetogens),(Methanogens)[1]。76%(、),,()()。,、。,、、,,UASB(Up-flowAnaerobicSludgeBlanket),UASB(Granularsludge),,[2]。COD、[3]。“”UASB,COD11.5kgm30.3kgm3,97.4%,。UASB16SrRNA,,,。1　材料和方法1.1　“”UASB,DNA。1.2　1.2.1　:TaqDNA、PCR;T4DNA、pUCm-TMBI-Fermentas。1.2.2　:[4]20mmolL,20mmolL10mmolL、;20mmolLH2CO2(80∶20)(1.3×105Pa)DOI:10.13343/j.cnki.wsxb.2004.02.003。25mL(5mL),。,1.01×105PaN2CO2(80∶20)。1.3　16SrRNA1.3.1　DNA:DNA[5],DNA,,(10mmolL,pH7.2)。30500.3mm～0.5mm,(10mmolLTris-HCl,pH7.5,50mmolLEDTA,0.5molLNaCl),(Cycle0.5,Amplitude90,220V,2A)1.5min。1.3.2　16SrRNAPCR:DNA,530F1490R16SrRNA。530F(5′-GTGCCAGCAGGCCGCGG-3′)1490R(5′-GGT-TACCTTGTTACGACTT-3′)(Es-cherichiacoli)16SrDNA514～5291491～1509[5]。PCR:95℃5min;94℃1min,50℃1min,72℃1.5min,30;72℃10min。PCR6(25μL),1%(wv)PCR,EB。1.0kbPCR。T4DNApUCm-T,E.coliDH5α。(100μgmL)[6],,16SrDNA。1.3.3　:16SrRNA,。CHECK-CHEMERA。16SrDNA(Operationaltaxonomicunits,OTU)。OTUBLASTGenBank[7]。DNA-MAN(version4.0),,Bootstrap。1.4　MPN[8]。,N2,10,。37℃3。;。MPN[9]。1.5　GC[10],N230cm3min。50℃220℃。HPLC[11],60%40%,0.5mLmin。2　结果2.1　16SrRNA1.0kb16SrRNA。25OTUs58(1)。OTUs16SrRNA(1～4),5819(33%),。39(67%),GC(22%)、δ-(9%)(12%)。。1　16SrRNATable1　Distributionof16SrRNAgeneclonesobtainedinthegranularsludgeGroupNo.ofOTUsNo.ofclonesPercentageoftotalclonesArchaea　EuryarchaeotaBacteria　Gram-positivebacteria　HighG+Csubdivision　LowG+CsubdivisionBacteroidesCytophagaSpirochaetalesProteobacteria　DeltasubdivisionGreennon-sulfurbacteriaCandidatedivisionOP8CandidatedivisionOP1151622431119113335752332225591293　　2.1.1　(Archaea):142　　　　　　　　44　1916SrRNA,5OTUs(Euryarchaeota)(1)。G4-13(1358)(Methanobacteriumbry-antii)OTU,95%～97%。4OTUs(Methanobacte-riumformicicum),(Methanosaetacon-cilii),(Methanosarcinamazei)(Methanocorpusculumparvum),16SrRNA97%。1　OTUsFig.1　PhylogenetictreeoftheOTUsandtheirrelativesinEuryarchaeotaofthedomainArchaea“G”referstothegranularclonesrepresentingdifferentOTUs.Numbersinparenthesesrepresentthesequences'accessionnumberinGenBank.Numbersinsquarebracketsindicatetheclonenumberoutofthetotalclones.Thenumberateachbranchpointsisthepercentagesupportedbybootstrap.Bar,5%sequencedivergence.2.1.2　(Gram-positivebacteria):1416SrRNA,7OTUs。G4-17GC(Thermoleiphilumalbum)(Thermoleiphilumminutum),6OTUs(13)GC。,OTU(G2-14),2OTUs(7),(Syntrophothermus)、(Syntroph-omonas)、(Syntrophospora)(Pelospora)16SrRNA93%～95%。3OTUs(Aminomonas)、(Desulfotomac-ulum)(Bacillus),88%～93%(2)。2.1.3　δ-(δ-Proteobacteria):5δ-,4OTUs,。OTU(G4-8)(Syntrophobacter)96%～99%。OTU(Smithella)(Syntrophus),(S.propionica),99%。2OTUs(G3-7G3-19),(88%～90%),,SJA-162(3)。2.1.4　(Greennon-sulfurbacteria):7,3OTUs。OTU(Dehalococ-cidessp.FL2)90%。OTUEub4,95%。OTUSHA-3197%,1,2-(4)。2.1.5　:2OTUs93%95%143　2:UASB2　OTUsFig.2　PhylogenetictreeoftheOTUsandtheirrelativesamongGram-positivebacteriaTheannotationwasthesameasinFig.1.-(BacteroidesCyto-phaga)。2OTUs(Spiro-chaetales)88%,KB43。2OTUs(G2-7G4-2)90%95%OP8OP11。2.2　MPN,(2),UASB,,;,。2　Table2　NumerationofdifferenttrophicgroupsinthegranulesusingMPNmethodTrophicgroupsCounts(CellsmL)ⅠSyntrophicacetogensdegradingorganicacids　　Syntrophicpropionate-degradingbacteria　　Syntrophicbenzoate-degradingbacteria　　Syntrophicbutyrate-degradingbacteriaⅡMethanogens　　Acetoclasticmethanogens　　Hydrogenotrophicmethanogens7.0×1069.0×1064.5×1074.5×1081.1×109　　3　讨论144　　　　　　　　44　3　δ-OTUsFig.3　PhylogenetictreeoftheOTUsandtheirrelativesamongδ-ProteobacteriaTheannotationwasthesameasinFig.1.16SrRNA,()UASB,。,。[12]、[13][14]、,。,16SrRNA(1358),95%～97%。。,GCδ---[13,15,16]。2OTUs(758),16SrRNA95%,。4OTUs--。,()。16SrRNA,。[14,17],,[18]。16SrRNA--,。OP8OP11,95%。,:DNA;16SrRNADNAPCR[19,20]。145　2:UASB4　OTUsFig.4　PhylogenetictreeoftheOTUsandtheirrelativesamongotherbacteriabranchesTheannotationwasthesameasinFig.1.。,,PCR。16SrRNA。[1]　ThieleJH,ZeikusJG.Interactionsbetweenhydrogen-andformate-producingbacteriaandmethanogensduringanaerobicdigestion.In:EricksonLE,etal,ed.HandbookonAnaerobicFermentations.NewYork:MarcelDekker,1988,537-595.[2]　HulshoffPLW,ZeeuwWJ,VelzeboerCTM,etal.GranulationinUASBreactor.WaterSciTechnol,1983,15:291-304.[3]　LiuS,HuJ,GuX.AnaerobicsludgegranulationinaUASBreactortreatingprotein-containingwastewater.WaterTreatment,1992,7:297-306.[4]　ZehnderAJB,WuhrmannK.PhysiologyofaMethanobacteriumstrainAZ.ArchMicrobiol,1977,111:199-205.146　　　　　　　　44　[5]　WeisburgWG,BarnsSM,PelletierDA,etal.16SribosomalDNAamplificationforphylogeneticstudy.JBacteriol,1991,173:697-703.[6]　SambrookJ,FritschEF,ManiatisT.MolecularCloning:aLabora-toryManual,2nded.NewYork:ColdSpringHarborLaboratoryPress,1989,1.85-1.86.[7]　AltschulSF,GishW,MillerW,etal.Basiclocalalignmentsearchtool.JMolBiol,1990,215:403-410.[8]　WuW,JainMK,deMacarioEC,etal.Microbialcompos