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OLAND16SrDNADGGE张丹,刘耀平,徐慧,张颖,陈冠雄＊(,110016):(),,DGGE()16SrDNAPCR,:OLAND,,,。FISH(),OLAND,(Nitrosomonas),OLAND72.5%。:;;;;;中图分类号:X172 文献标识码:A 文章编号:1004-311X(2003)05-0001-03DGGEAnalysisof16SrDNAofNitrifyingBacteriainOLANDBiologicalNRemovalSystemZHANGDan,LIUYao-ping,XUHui,ZHANGYing,CHENGuan-xiong(InsitituteofAppliedEcology,AcademicSinica,Shenyang110016,PRC)Abstract:Inordertoinvestigatethemicrobialcommunitydiversityofnitrifyingbacteria(ammoniumoxidizingbacteriaandnitriteoxidizingbacte-ria)andtheirdynamicruleschangedwiththedecreaseofDOinOLANDsystem,andthentoestablishapracticalmolecularmonitoringtechnologysuitableforidentifyingthefunctionalmicrobialcommunityofautotrophicNremovalsystem,thespecificproductsof16SrDNAPCRamplificationofnitrifyingbacteriawereanalyzedwithDGGEtechnique.Itwasfoundthattherewasagreatdealofdifferencebetweenammoniumoxidizingbacte-riaandnitriteoxidizingbacteriaonthedynamicrulesofmicrobialcommunitychangedwiththedropofDO.TheDOhadagreatereffectonammo-niumoxidizingbacteriathannitriteoxidizingbacteria.Simultaneously,attheoxygenlimitednitritationstageofOLANDsystem,nitrosomonaswasprovedtobethedominantammoniumoxidizingbacteriabyFISHtechnique,whichaccountedfor72.5%oftotalbacteriainthereactorofthisstage.Keywords:biologicalNremoval;DGGE;nitrifyingbacteria;molecularmonitoring;dissolvedoxygen;Nitrosomonas:2003-04-21;:2003-05-30:(BIL0003);;(20022015):(1975-),,,,,4;＊(1939-),,,、、,,3,,。 OLAND(Oxygen-LimitedAutotrophicNitrificationandDenitri-fication,-),(0.1～0.3mgL),50%NH+4NO-2,(NH+4NO-2=1∶1.2±0.2),,[1]。,OLAND。,NH+4NO-2,NitrosomonasNitrosospira;NO-2NO-3,NitrobacterNitrospira[2]。,NH+4,[1]。,,、MPN(MostProbableNumber),。,DGGE(DenaturingGradientGelElectrophoresis)、FISH(FluorescenceInSituHybridization),[3,4],。OLAND,0.1～0.3mgL,,DGGE(),,OLANDOLAND。1 1.1 实验材料及仪器DNA(WinzardDNAclean-upsystem,Promega,USA);PCR(PCRcoresystem,Promega,USA);40%,50×TAE,2%BisAA,10%APS,TEMED,()(Bio-rad,USA);,3×PBS,4%(AppliedChemCo.,Germany)。PCR(GeneAmpPCRsystem9600,AppliedBiosystem,USA);Bio-radDGeneSystem(Bio-rad,USA);(ZeissAxioskopII,Germany)。1.2 实验方法1.2.1 ,6,,。,,0.1mgL,(NH+4NO-2=1:1.2±0.2)。:35℃、pH7.8～8.2、1d、∞,100r/min。1.2.2 ,,0d()、15d、21d、37d、49d、57d、67d72d4ml(1),2mlDNA,2mlrRNA。1 (d)(mgL)(mgNL)NH+4-NNO-2-NNO-3-N0NDNDNDND15400.91094521280.249203728180068490.52984606.5570.33883805.8670.14324234.7720.14324453.4 ND:1.2.3 DNADNA[5]。DNA-20℃,。DNAPromegaDNA(WinzardDNAclean-upsystem,Promega,USA)。1.2.4 16SrDNAPCRDNA,PCR[6]CTO189f(5'GGAGRAAAGCAGGGGATCG3')CTO654r(5'CTAGCYTTGTAGTTTCAAACGC3'),[7]NIT3(5'CCTGTGCTCCATGCTCCG3',)P338f(5'ACTCCTACGGGAGGCAG3'),16SrDNAPCR。PCR:94℃1min,57℃1min,72℃2min,35;PCR:94℃1.5min,65℃0.5min,72℃1min,30。PCR,40bpGCP338fP518r(5'ATTACCGCGGCTGCTGG3')PCR[8]。PCR:95℃1min,53℃1min,72℃2min,30。1%PCR。1.2.5 DGGEBio-radDGGEGCPCR。8%,45%60%,60°C,38V,1×TAE16h,SYBRGreenI(1:10000,135:1200310 BIOTECHNOLOGY Vol.13,No.5:1Oct.2003DOI:10.16519/j.cnki.1004-311x.2003.05.001Rockland,USA)10min,(VilbertLourmat,France)。1.2.6 FISH,4%,2,100%,-20℃。1.2.7 :NSO-190[9]、(Nitrosomonas)NSM156[9](Nitrosospira)NSV443[9],(-Cy3,-Cy5,-FLUOS)。1 DNA1.2.8 2～10μL,45℃,50%、80%100%3min,。,1.5～3h(46℃)。,,,48℃10min,,。1.2.9 AxioskopII(CarlZeiss,Germany)、,Microimage4.0。2 2.1 16SrDNA巢式PCR扩增DNA,DNA(1)。DNA,PCR[10]16SrDNAPCR。8PCR,。DGGE,,PCR,PCR,,220bp(2)。2 16SrDNAPCRMarker-100bp;Blank:DNA。2.2 16SrDNAPCR扩增产物的DGGE分析DGGEDNA(≤500bp)。5'30～50bpGC,PCR,GCPCR,()。DNADNA,DNA。PCR,PCR,DGGE,3、4。3 DGGE(A,B,C)4 DGGEDGGE(3、4),,。,,A,,,。,,,,1C,COLAND。。,,。5 OLANDFISH630×,ANSO190-Fluos;BNSM156-Cy3;C。2.3 氨氧化菌的FISH分析OLAND,NSO190-(Nitrosomonas)(Nitrosospira)NSM156、NSV443,FISH。Microimage4.0FISH(5),OLAND(72d),72.5%,。2 135OLAND,DGGEC。3 DGGE、FISH,、,、、。,OLAND,。,OLAND,,,。FISH,OLAND,(Nitrosomonasgenus),OLAND72.5%。“”,DNA,。:[1]KuaiL,VerstraeteW.Ammoniumremovalbytheoxygenlimitedautotrophicnitrificationdenitrification(OLAND)system[J].ApplEnvironMicrobiol,1998,64(11):4500-4506.[2]FochtDD,VerstraeteW.Biochemicalecologyofnitrificationanddenitrifica-tion[J].AdvMicrobEcol,1977,1(1):135-142.[3]JettenMSM,WagnerM.Microbiologyandapplicationoftheanaerobicam-moniumoxidation(anammox)process[J].CurrentopinionBiotechnol,2001,12:283-288.[4]LogemannS,SchantlJandBijvankS,etal.Molecularmicrobialdiversityinnitrifyingreactorsystemwithoutsludgeretention[J].FEMSMicrobilEcol,1998,27:239-249.[5]BoonN,GorisJandDeVosP,etal.Bioaugmentationofactivatedsludgebyanindigenous3-chloroanilinedegradingComamonastestosteronistrain,I2gfp[J].ApplEnvironMicrobiol,2000,66(7):2906-2913.[6]KowalchukGA,BodelierPLEandHeiligGHJ,etal.Communityanalysisofammonium-oxidizingbacterium,inrelationtooxygenavailabilityinsoilsandroot-oxygenatedsediments,usingPCR,DGGEandoligonucleotideprobehy-bridisation[J].FEMSMicrobiolEcol,1998,24:339-350.[7]ReganJM,HarringtonGWandNogueraDR.Ammonia-andnitrite-oxi-dizingbacterialcommunitiesinapilot-scalechloraminateddrinkingwaterdis-tributionsystem[J].ApplEnvrionMicrobiol,2002,68(1):73-81.[8]vreasL,ForneyLandDaaeFL,etal.Distributionofbacterioplanktoninmeromicticlakesaelevannet,a