MBR活性污泥培养驯化过程中生物多样性研究于凤庆

32920129ActaScientiaeCircumstantiaeVol．32No．9Sep．2012No．07JCZDJC02100SupportedbytheNaturalScienceFoundationofTianjinNo．07JCZDJC021001984—[e-mail]*[e-mail]1984—[e-mail]*[e-mail]tyoftheMBRactivesludgecultivationprocessJ．ActaScientiaeCircumstantiae3292084-2090MBR11*12111．3000722．3151032011-11-172011-12-142011-12-19MBRConventionalactivatedsludgeCASMBR．DNAPCR-DGGE．CASMBRMBR．MBRPCR-DGGE0253-2468201209-2084-07X703AStudyonbiologicaldiversityoftheMBRactivesludgecultivationprocessYUFengqing1SUNBaosheng1*CHENYi12DUJiang1ZHOUQiangjian11．SchoolofEnvironmentScienceandTechnologyTianjinUniversityTianjin3000722．SinopecNingboEngineeringCompanyLimitedNingbo315103Received17November2011receivedinrevisedform14December2011accepted19December2011AbstractToinvestigatethesuccessionofthestructureofmicrobialcommunityandthemicrobialdiversityofMBRintheentirecultivationprocessthesludgeinthetrainingdomesticatedstageofanMBRreactorwasstudiedbyusingtheconventionalactivatesludgemethod．BasedontheconventionalwastewatersludgeindexestotalbacterialgenomicDNAindifferentstageswasextractedfromtheMBRsludgeandthePCR-DGGEandsimilarityanalysiswereapplied．ItwasshownthatintheprocessofcultivationofCASintheMBRreactorthechangeofdiversityandsuccessionprocedureofbacteriawereobviousandthesimilarityindexofbacteriaineachstageshowedthesuccession．Thecultivationofsludgewasasequencingprocesswhichwasduetotheself-modulationofthemicroorgannismstotheenvironmentchangesindifferentperiodoftimeformingthecommunitystructurethatwasbestfortheMBRprocess．KeywordsMBRsludgedomesticatedPCR-DGGEbiodiversity1Introduction、．———2008．．PCR-DGGEWagneretal．2002．MBR、F/MCASDOI:10.13671/j.hjkxxb.2012.09.0299MBRYukietal．2007CASMBR．MBRWitzigetal．20022008a2008．MBR．CASMBRMBR．2Materialsandmethods2．1MBR1．5m3·d－1．MBR5h7h-．MBR360．2μm15L·m－2·h－120∶1．2．2MBRA2/O．MLSS5798mg·L－1SV23%．MBRMLSS650mg·L－1．、NH4Cl、KH2PO4MBRC∶N∶P=100∶5∶1250mg·L－1COD．20d400m3．40dMBR3000mg·L－1600m360d2000m3．MBR1．1MBRTable1ThefeedingwaterqualityoftheMBRofareclaimedwastewatertreatmentplantmg·L－1CODCrNH+4-NTNTPSS260～40020～3520～403～5160～2202．3MBR23d6d110．、、MBR2．CODCr、NH+4-N、SS、MLSSGB11914—89HJ535—2009GB11901—89．2MBRTable2OperationconditionsandtreatmenteffectforvariousstagesofthecultivationanddomesticationprocessesDO/mg·L－1CODCrSSC1C2C33d9d15d4．54．74．520∶1～22∶145%～70%－－C4C521d27d3．93．819∶1～21∶160%～80%－－C6C733d39d3．23．120∶1～21∶170%～85%－－C8C9C1051d57d63d3．02．92．820∶1～23∶189%93%95%90%92%91%93%94%93%5802322．4DNA500mL30min50mL50mL6～10℃、9165g10min30mL10min30mLTE10min15mL5mLDNA．-\\-DNA2008．2．5DNAPCRDNAPCR．16SrRNAV3F357-GCR518240bpMuyzeretal．1993．50μL10～100ng5μL10×PCRbuffer20mmolMgCl210mmol·L－1dNTPs1μL0．5μmol·L－10．5μL2．5U·L－1Taq1μL50μL．PCR94℃5min2094℃1min65～55℃1min72℃1min0．5℃1094℃1min55℃1min72℃1min72℃8min．1．5%．2．6PCRDGGE6%～12%35%55%100%7mol·L－140%30min．10%PCR20μL．1×TAE6．5h150V、60℃．2．7DGGEDGGE16SrDNA1．5mL50μLTE4℃DNA3μLPCR．PCR1．5%．PCRDGGE．2．82．8．1Shannon-WienerQuantityOneV4．25DGGEShannon-Wiener．ShannonHH=－∑Si=1PilogPi=－∑Si=1ni/Nlogni/NPi=ni/NniNS．2．8．2UPGMAMBR10QuantityOneDGGE．3Resultsanddiscussion3．1MLSS、SVIMBR390mg·L－1MLSS、SVI、SVShannon1．1aMLSS350mg·L－1SVI42．1mL·g－126.3mL·g－1．7dMLSS650mg·L－1MLSS24d455mg·L－1SVI．．20d24dMLSSSVI27d117.2mL·g－168029MBR．Shannon1bC421d0．91．2010MBR．40d3000mg·L－1．MBRShannon27dCASMBA．50d5000mg·L－1SVI120mL·g－1MBR．1MBRMLSS、SVI、SV、ShannonFig．1ThechangeofMLSSSVISVandShannondiversityindexesatdifferentstagesofthecultivationanddomesticationprocesses3．2253．Bandb、c、d、eBandb、cAeromonasBandd、ePseudomonas4．MBR．Bandm、n、o．BandnComamonasMBR7802322MBRDGGEFig．2DGGEpatternsofdominantbacteriainthecultivationanddomesticationprocesses．Comamonasslime-EPScapsular-EPSBalaetal．2006．ZetaMBR2008a．．C1～C42BandA、B、CDGGE．DGGEDNADNA1．5%Muyzeretal19932008a．BandD、F、G．MBRMBR．MBRMBRMBRMBR．MBR“”MBRStamperetal．2003．BandDCitrobacter、MBRMBR．BandFBacillus．、2008b．BandbEC6．MBRF/M．2005EPS．3DGGETable3TheclassificationofthebacteriathroughDGGEpatternsinthecultivationanddomesticationprocessesBandabcdefBandmnoBandABCBandDFGBandbE88029MBRUnculturedbacterium、UnculturedPseudomonas、UnculturedammoniaAmann1995．416SrDNATable4Sequencinganalysisofpartofthemicrobialcommunityofthedominantbacteria/bpNCBIGenbankBanda157FJ973478FJ372598UnculturedbacteriumisolateDGGEgelbandWA1797%Bandb152FJ973479DQ059496Aeromonassp．HPC1379100%Bandc172FJ973480FJ005059Aeromonassp．enrichmentculturecloneGuo599%Bandd158FJ973481FJ755909Pseudomonasputida98%Bande155FJ973482EU909141UnculturedPseudomonassp．Cloneccmr00299%Bandf150FJ973483AB239734Unculturedammonia-oxidizingbacterium97%Bandn150FJ973484FJ824117ComamonastestosteronistrainLMCB01498%Bando157FJ973485AM711886UnculturedPseudomonassp．90%BandD153FJ973486DQ279752Citrobactersp．AzoR-599%BandF146FJ973487FJ820330Bacillussp．VKMK-3698%3C1～C10Fig．3ThesimilarityindexofthedominantbacteriafromC1toC103．3DGGE3C10．3MBRC10C135.6%MBR．MBR．10C1010．MBR．4DGGEFig．4TheClusteranalysisofDGGEpatternsofdominantba