高效厌氧产甲烷颗粒污泥微生物多样性及定量化研究孙寓姣

,＊,,,(,　100084):,(DGGE)、(FISH)(RTQ-PCR)、,:,;,;,;.:;;PCR-DGGE;FISH;RTQ-PCR:X176;X172　:A　:0250-3301(2006)11-2354-04:2005-12-30;:2006-03-07:(863)(2002AA601190):(1975～),,,,E-mail:[e-mail]＊,E-mail:[e-mail]DifferentMicroorganismsinMethanogenicGranularSludgeSUNYu-jiao,ZUOJian-e,XINGWei,LIJian-ping,LUYi-qiong(DepartmentofEnvironmentalScienceandEngineering,TsinghuaUniversity,Beijing100084,China)Abstract:PCR-DGGE,FISHandRTQ-PCRtechniqueswereusedtostudythemicrobialcommunitystructureandquantityofdifferentmicroorganismsinthemethanogenicgranuleindifferentphasesfromalab-scaleanaerobicreactor.Theresultsindicatedthatastheorganicloadingrateofthereactorincreasing,thearchaeacommunitychangedmoresignificantlythanbacterialcommunity.Mostbacteriawerelocatedintheoutlayerofgranule,whilemostarchaeawerelocatedintheinnerlayer.Thequantityofarchaeawasalittlelessthanbacteria,andthequantityofmethanosaetaincreasedsignificantly.Keywords:methanogenicgranulesludge;microbialcommunitystructure;PCR-DGGE;FISH;RTQ-PCR　　,,(),(),..、.,[1]、[2～4]、[5][6],[7,8].,PCR-DGGE,FISH,RTQ-PCR[9]、、.1　1.1　,15L,10L,5L;2.04m.,EGSB.,、、,COD∶N∶P400∶2.5～5∶1.pH6.8～7.2,35℃.,3,A:1d,;B:25d,(OLR)20kg/(m3·d),COD96%;C:75d,OLR35kg/(m3·d),COD90%.1.2　1.2.1　PCR-DGGEDNADP301.DNA,GCP338f-p518rGC[11]2711200611　　　　　　ENVIRONMENTALSCIENCEVol.27,No.11Nov.,2006DOI:10.13227/j.hjkx.2006.11.040Arc109f-Arc344rGC[12]PCR.Bio-radDGGEPCR,8%,45%～60%,60℃,60V,1×TAE16h,TE20min,.1.2.2　FISH,4%,PBS,PBS100%-20℃(6).,60℃,,8μm,,.CY3()EUB338(5′ACTCCTACGGGAGGCAG3′)FITC()ARC915(5′GTGCTCCCCCGCCAATTCCT3′),,46℃,25%35%[10].1.2.3　RTQ-PCR、、3P338f-p518r[11]、Arc109f-Arc344r[12]、518f-MX825r[13],SYBRGreen(FP201)PCR(BioerFQD-33A)(:94℃3min,94℃10s,57℃20s,72℃30s,32).16SrDNAT;16SrDNA46-825T.TA,,0.5μL,PCR,,:1×106copies/mL,1×107copies/mL,1×108copies/mL,1×109copies/mL.2　2.1　3DNA,PCR,DGGE,3DGGE,1.1(a),(A),,,;BC,,,、,.,,B、C,,25d,;50d,,.1　3DGGEFig.1　DGGEprofileofarchaeaandbacteriainthreesiuagesamples1(b),,();,,.,12,B,2;C1.1,,,,;,,(2.2),,,,235511　　　　　　,,.,,,,,.1,,,,.,,,.[14].2.2　EUB338-cy3ARC915-fitc3,2.,A;B、C,,.,3,,,,.,,,,7.0kg/(m3·d),5d,20.0kg/(m3·d),75d,35.0kg/(m3·d).,、.、;,,,,(pH、ORP、),,.EUB338(),ARC915()2　(×200)Fig.2　Spacialdistributionofbacteriaandarchaeainthreemethanogenicgranulesamples　　,,、,,.2.3　,,.,2,.,PCR3、.,A、B、C、16SrRNA,16SrRNA[15],、16SrRNA(4.1、1.5、2.0),3,,A、B、C,1.00×1010、1.61×1010、0.48×1010/mL;0.77×1010、1.09×1010、4.99×109/mL;0.99×109、1.35×109、1.25×109/mL.A、B、C43.72%∶56.28%、40.45%∶59.55%、51.23%∶48.76%;12.6%、12.35%、25.05%(2356　　　　　　273).,,,();,,12.6%,35.0kg/(m3·d)25.05%,100%.,,,;,,,,.3　、Fig.3　Relativeabundanceofbacteria,archaeaandmethanosaetainthreegranulesamples3　(1),,、,.(2)、.(3);,(),.:[1]FangHP,ChuiHK,LiYY.Effectofdegradationkineticsonthemicrostructureofanaerobicbiogranules[J].WaterSci.Technol.,1995,32(8):165～172.[2]BatstoneDJ,KellerL.Variationofbulkpropertiesofanaerobicgranuleswithwastewatertype[J].Wat.Res.,2001,35(7):1723～1729.[3]FangHP,ChuiHK,LiYY,etal.PerformanceandgranulecharacteristicsofUASBprocesstreatingwastewaterwithhydrolyzedproteins[J].WaterSci.Technol.,1994,30(1):55～63.[4],,,.[M].:,2002.19～65.[5]MacLeodFA,GuiotSR,CostertonJW.Layeredstructureofbacterialaggregatesproducedinanupflowanaerobicsludgebedandfilterreactor[J].Appl.Environ.Microbiol.,1990,56(6):1598～607.[6],,.[M].:,1993.243～275.[7]WuJerhorng,LiuWentao,TsengIcheng,etal.Characterizationofmicrobialconsortiainaterephthalate-degradinganaerobicgranularsludgesystem[J].Microbiology,2001,147:373～382.[8]Wen-TsoLiu,On-ChimChanc,HerbertHP.Characterizationofmicrobialcommunityingranularsludgetreatingbrewerywastewater[J].WaterRes.,2002,36(7):1767～1775.[9]BustinSA.AbsolutequantificationofmRNAusingreal-timereversetranscriptionpolymerasechainreactionassays[J].JournalofMolecularEndocrinology,2000,25(1):169～193.[10]BercovierHO,Kafri,SelaS.MycobacteriapossessasurprisinglysmallnumberofribosomalRNAgenesinrelationtothesizeoftheirgenome[J].Biochem.Biophys.Res.Commun.,1986,136(5):1136～1141.[11]OverasL,ForneyL,DaeFL.Distributionofbacterioplanktoninmeromicticlakesaelevanner,asdeterminedbydenatureinggradientgelelectrophoresisofPCR-amplifyiedgenefragmentscodingfor16SrRNA[J].Appl.Envrion.Microbeol.,1997,63(8):3367～3373.[12]RegingG,PeterHJ,WernerL.DiversityandStructureoftheMethanogenicCommunityinAnoxicRicePaddySoilMicrocosmsasExaminedbyCultivationandDirect16SrRNAGeneSequenceRetrieval[J].AmericanSocietyforMicrobiology,1998,64(3):960～969.[13]StahlDA,AmannR.Developmentandapplicationofnucleicacidprobes[A].In:NucleicAcidTechniquesinBacterialSystematics[C].WileyChichester,1991.205～248.[14]RoestK,HeiligHG,SmidtH.Communityanalysisofafull-scaleanaerobicbioreactortreatingpapermillwastewater[J].Syst.Appl.Microbiol.,2005,28:175～185.[15]SchmidtTM,DeLongEF,PaceNR.Analysisofamarinepicoplanktoncommunityby16SrRNAgenecloningandsequencing[J].Bacteriol.,1991,173:4371～4378.235711