处理垃圾渗滤液复合式膜生物反应器中微生物群落结构的演变和分析李军

36820108JOURNALOFBEIJINGUNIVERSITYOFTECHNOLOGYVol．36No．8Aug．20101001243DNAV6－V8GC968F/1492RPCRPCRDGGE．DGGE．2．PCR－DGGEX703.1A0254－0037201008－1111－072009-06-29．2008ZX07314－0088092007．1964—．．．hybridmembranebioreactorHMBR、、、、1-2．PCR－DGGE．DGGE．1993Muyzer3DGGE．4DGGE．Rowan52PCR．MBR．MBR．36DNA16SrDNADGGE．MBR．201011.1、1．2．3．4．5．6．pH7．8．9．10．11．12．13．14．15．16．17．U1Fig．1Thedevicesketchofreactor1．A－O．A/O24L6L18L．125mm．5cm．．3．．2200mg/LpH7.8ρCOD3500mg/L．30℃ρDO1.0mg/LHRT4d．0.1～0.25kg/m3·d2．31∶97d5∶536d59d61～6－20℃．NO－2-N/NO－x-N95%90%COD50%．1.2DNADNAZhou6SDS．650μL120mmol/LpH=8.0100μLpH=8.0TE230μLTNSρNaCl=100mmol/L10%SDSρTris－HCl=500mmol/LpH=8.050W30s60℃10min15000r/min15℃3min．1200μL2mL．480μL7.5mol/L5min15000r/min15℃10min．2700μL21.5mLeppendorf490mL100%－20℃．4℃DNA2d25℃15000r/min30min．－20℃70%500μLDNA．15000r/min4℃5min．DNA50μLElutionBufferDNA4℃4h．21.5mLDNA11.5mL5μL1%．1.3PCRDNAElutionBuffer10PCR．．16SrDNA968f5'-AACGCGAAGAACCTTAC-3'1401r5'-CGGTGTGTACAAGACCC-211183'DNAPCR968f5'40bpGC5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCCCGGGGGG-3'PCRr-TaqDNATaKaRa12.5μL1μL50μmol/LDNA1μL9.5μL25μL．PCRPCRMJPTC－200Bio－RadUSAPCR795℃3min2095℃1min60～50℃45s10.5℃72℃1min1095℃1min50℃1min72℃1min72℃7min4℃．PCR1%．1.4DGGEPCRDGGE35%～55%8%20mL．100V60℃12h．15mLSYBR－Gold1∶10000．40minUPV．PCR－DGGE．1.5DGGEDGGE．DGGE8Ri=Li/Lt1RiiLiiLtDGGE1．2C9-10Cij=2×100Lij/Li+Lj2CijijLijijLiiLjj．22.12．10dρNH+4-NNH+4-N80%．ρNH+4-N200mg/L．、DO、pHNO－2-NNO－3-NNO－2-N90%ρNO－3-N50mg/L．3COD55%．2500～4500mg/L．2.2DNAPCRDNA1%EBDNA23kb4．PCR1%PCR433bp5.311120102、Fig．2Waterqualityoftreatedwastewatertheremovalrateofammonianitrogenandthenitriteaccumulationrateinthebioreactor3CODFig．3ThechangeofCODinthebioreactor4DNAFig．4MapofDNAafteragarosegelelectrophoresis5PCRFig．5MapofPCRproductsafteragarosegelelectrophoresis2.36DGGEFig．6DGGEprofileofthemicrobialcommunitiesinthereactorfPCRDGGEDecodeBio－Rad15mLSYBR－Gold1∶10000UPV．PCR－DGGEGenBank6．12．1、264、612、13．．1、210d．1、3、52、4、6．411181RTable1RichnessvalueRofmicrobialcommunityindifferentsamples123456/67712813R0.350.410.410.700.470.762CTable2ComparableindexCofmicrobialcommunityindifferentsamples1234561100627722714226210071535350377711004280504225342100508057153805010067642505080671006bc2．a．gi2．f．k．2.417GenBankBlast3．316SrDNATable3Resultsof16SrDNAsequencesusingBLASTinGenBank/%aAM292279Nitrobactersp．Natopartial16SrRNAgenestrainNato96bEF076127UnculturedChloroflexibacteriumcloneAD00716SribosomalRNAgenepartialsequence97cAY856379Nitrosomonassp．OZK1116SribosomalRNAgenepartialsequence97dFJ529954UnculturedbacteriumcloneMABRDTU116SribosomalRNAgenepartialsequence98eAJ551095Hyphomicrobiumsp．wp14partial16SrRNAgeneisolatewp1499fEU312975ProteobacteriumBF-416SribosomalRNAgenepartialsequence99gAF169545Bacillussp．NRS-81016SribosomalRNAgenepartialsequence96hEU179737PseudomonasputidastrainMG-Y216SribosomalRNAgenepartialsequence95iDQ414434UnculturedNitrospirasp．clone1fromactivatedsludge16SribosomalRNAgenepartialsequence98jAF338213NitrosococcusoceanistrainC-2716SribosomalRNAgenepartialsequence99kFJ393072UnculturedAeromonassp．cloneMFC-B162-B0216SribosomalRNAgenegenepartialsequence98lEU652562UnculturedbacteriumcloneC8S-13716SribosomalRNAgenepartialsequence100mDQ640676UnculturedBacteroidetesbacteriumcloneSkagenf9316SribosomalRNAgenepartialsequence98nU82826CastellanielladenitrificansstrainNKNTAU16SribosomalRNAgenecompletesequence97oAJ005450Alcaligenesdefragrans16SrRNAgenestrain65Phen98pDQ822531BacteriumQM4616SribosomalRNAgenepartialsequence96qEU107758Comamonassp．P3-316SribosomalRNAgenepartialsequence9751112010395%．aicj．eHyphomicrobiumsp．．nCastellanielladenitrificanssp．oAlcaligenessp．2．．qComamonassp．11．hPseudomonassp．kUnculturedAeromonassp．12．．31．．2．3PCR－DGGEMBR．MBR、．1THOMASHMURRERJ．FoulingcharacteristicsofmembranefiltrationinmembranebioreactorsJ．MembraneTechnology2000122610-13．2ROSENBERGERSKRIIGERUWITZIGRetal．PerformanceofabioreactorwithsubmergedmembranesforaerobictreatmentofmunicipalwastewaterJ．WaterResearch2002362413-420．3MUYZERGUITTERLINDENAG．Profilingofcomplexmicrobialpopulationsbydenaturinggradientgelelectrophoresisanalysisofpolymerasechainreactionamplifiedgenescodingfor16SrRNAJ．App1iedandEnvironmentalMicrobio1ogy199359695-700．4．PCR－DGGEJ．200425674-79．WANGFengFUYi-gangXIASi-qingetal．Characteristicsofmunicipalsewagechem-bioflocculationtreatmentprocessbyusingPCR-DGGEtechnologyJ．ChineseJournalofEnvironmentalScience200425674-79．inChinese5ROWANAKSNAPEJRFEARNSIDEDetal．Compositionanddiversityofammonia-oxidisingbacterialcommunitiesinwastewatertreatmentreactorsofdifferentdesigntreatingidenticalwastewaterJ．FEMSMicrobiologyEcology200343195-206.6ZHOUJBRUNSMATIEDJEJM．DNArecoveryfromsoilsofdiversecompositionJ．AppliedMicrobiologyandBiotechnology1996622316-322．7BAEWBAEKSCCHUNGJetal．OptimaloperationfactorsfornitriteaccumulationinbatchreactorsJ．Biodegradation200212359-366.8．PCR-DGGESBBRJ．20072851095-1101．61118XIAOYongYANGZhao-huiZENGGuang-mingetal．BacterialdiversityinsequencingbatchbiofilmreactorSBBRforlandfillleachatetreatmentusingPCR-DGGEJ．EnvironmentalScience20072851095-1101．inChinese9GILLIANDCSPEKSNIJDERAGCLZWAR