不同碳源强化生物除磷系统的PCRDGGE分析

39420074JOURNALOFHARBININSTITUTEOFTECHNOLOGYVol39No4Apr.2007PCR-DGGE刘亚男1,2,夏圣骥3,于水利1(1.,150090,Emai:[e-mail];2.,201620;3,200092):实验考察了不同碳源强化生物聚磷系统(EBPR)中的聚磷菌群,对3个不同碳源(1号实际生活污水2号葡萄糖和3号乙酸钠)的序批式反应器(SBR)强化生物聚磷系统聚磷菌的16SrDNA特异性聚合酶链式反应(PCR)扩增产物进行了变形梯度凝胶电泳(DGGE)分析.结果表明,以生活污水为碳源的1号反应器具有数量最多的优势种群,而以葡萄糖和乙酸钠为碳源的2号3号反应器的生物多样性较少.3个反应系统的微生物种群各有异同.具有除磷作用的未被培养细菌(条带34,AF527584AF502204)为3个反应器所共有的生物量较多的优势种群,且其在以生活污水为碳源的1号反应系统中相对生物量最多.:强化生物除磷;碳源;PCR-DGGE;聚磷菌;16SrDNA:X703:A:0367-6234(2007)04-0589-04Applicationofpolymerasechainreaction-denaturinggradientgelelectrophoresistoanalyzebiologicalphosphorusremovalsystemsfedwithdifferentcarbonsourcesLIUYanan1,2,XIAShengji3,YUShuili1(1.SchoolofMunicipalandEnvironmentalEngineering,HarbinInstituteofTechnology,Harbin150090,China,Emai:[e-mail];2.SchoolofEnvironmentalScience&Engineering,DonghuaUniversity,Shanghai200051.China;3.StateKeylaboratoryofPollutionControlandResourcesReuse,TongjiUniversity,Shanghai200092,China)Abstract:Themicrobialcommunitiesofphosphate-accumulatingorganismscultivatedwithdifferentcarbonsourceswereinvestigated.Threesequencingbatchreactorswereoperatedsuppliedwithsewage,glucoseandsodiumacetateascarbonsourcesrespectively.Basedontheresultsofdenaturinggradientgelelectrophoresis(DGGE)ofpolymerasechainreaction-amplified16SrDNAfragments,itwasdemonstratedthatthesystemfedwithrealsewagehadthemostbiodiversity,whiletheothertwofedwithpurecarbonsourcehadless.Threereactorshadbothcommonanddifferentbacteria.Bacteriacorrespondingtobands3and4(AF527584AF502204)whichwerereportedtohavephosphorusremovalabilityweredominantinallthreesystems,andtheNo.1systemsuppliedwithrealsewagehadthelargestamountofthem.Keywords:enhancedbiologicalphosphorusremova;lcarbonsource;PCR-DGGE;phosphate-accumulatingbacteria;16SrDNA:2005-05-31.:(113-10-0044065).:(1978),,,;(1962),,,.(DGGE).1993Muyzer[1].[2~8].,.,DGGE.PCR-DGGE,3/SBR.1材料与方法3SBR,18L.11SBR1.,24h.1,2,3.8h:150min,15min;180min;.1CODcrP(KH2PO4)NH3-N(NH4Cl)TOCCODMg(MgCl2)Cu(CuSO4)Ca(CaCl2)Mn(MnSO4)Zn(ZnCl2)1326.87.535.1104.7326.82589.51540202.4600100.150.10.1103741.41540347.4600100.150.10.110SBR,.MLSS3000mg/L,,(SRT)7d.222,pH7002(05MHCl05MNaOH).12MLSSCODPO43--PNH4+-N[9],TOCTOC-VCPN(SHIMADZU),NiandongWang[10](GC-MS,6890NAgilen;t5973NAgilen,t,HP-5,30mlength,025mmID,FID).13PCR-DGGE1).,Ahn[11]().10000g,45min(AnkeGL-20G-II,),3TE(10mmol/LTris-HC,l1mol/LEDTA,pH80)5min.3,-20.2)DNA.DNATsai[12].DNA,PCR,.3)PCR.PCR341907(Escherichiacoli)16SrDNA[11],EUB341f(5-CCTACGGGAGGCAGCAG-3),5(5-CGCCCGCCGCGCGGCGGCGGGCGGGGCGGCACGGGGGG-3)[11],UNIV907r(5-CCCCGTCAATTCCTTTGAGTTT-3).PCRPCR(BIO-RAD),PCR[13]:945min;801min,1LTaq;651min;721min,319:941min,641min(21),723min;9:941min,551min,723min;1:941min,551min,7210min,4.PCR50L,105L,1L(10mmolL-1),dNTP4L(25mmolL-1),50ng1U.3LPCR,1%100V30min,05TAE,(EB).4)DGGE.(BIO-RAD),PCR25L25LLoadingdye05TAE5903930%~70%,100V,1440min,60.,EB10min,(BIO-RADGelDoc1000).Gel-ProAnalyzer,.5).DGGE13.2结果与讨论213,3.3,94d904%,2,294d969%.,,40d,.Liu[14],[15].1,,5mg/L,70d,.223DGGE221DNA3DNA(1).DNA13DNA23kb,DNA.3DNA,PCR[13]16SrDNA(2),DNA2316SrDNAPCR.,500bp.DGGE.22.216SrDNAPCRDGGEPCRDGGE,3.DGGE16SrDNAPCR,DNA,.DGGE,DGGE.DGGE,[1].1,DGGE11,23,7.,GenBank,MolecularEvolutionaryGeneticsAnalysis(version30),4.,1234Flavobacterium,345914,:PCR-DGGE,McMahon(AF527584,AF502204).5891013.56Bacillales,7101112Actinobacteridae,8-protebacteria,913-protebacteria.FlavobacteriumBacillales(1~6)3,-protebacteria-protebacteria(8913)1.223Gel-ProAnalyzer,(5).3,(34,42%~46%),3.13316SrDNADGGE;2Bacillales;3Flavobacterium.4DGGE16SrDNA59239533结论1)1,23.3.2)(34,AF527584AF502204)3,1.3)2Bacillales,3Flavobacterium.-protebacteria-protebacteria(8913)1.参考文献:[1]MUYZERG,DeWAALEC,UITTERLINDENAG.Profilingofcomplexmicrobialpopulationsbydenaturinggradientgelelectrophoresisanalysisofpolymerasechainreaction-amplifiedgenescodingfor16SrRNA[J].ApplEnvironMicrobio,l1993,59:695-700.[2]FERRISMJ,MUYZERG,WARDDM.Denaturinggradientgelelectrophoresisprofilesof16SrRNA-definedpopulationsinhabitingahotspringmicrobialmatcommunity[J].ApplEnvironMicrobio,l1996,62(2):340-346.[3]ARAYAR,TANIK,TAKAGIT,etal.BacterialactivityandcommunitycompositioninstreamwaterandbiofilmfromanurbanriverdeterminedbyfluorescentinsituhybridizationandDGGEanalysis[J].FEMSMicrobiolEco,l2003,43:111-119.[4]SCHFERH,SERVAISP,MUYZERG.Successionalchangesinthegeneticdiversityofamarinebacterialassemblageduringconfinement[J].ArchMicrobio,l2000,173:138-145.[5]MLLERAK,WESTERGAARDK,CHRISTENSENS,etal.Thediversityandfunctionofsoilmicrobialcommunitiesexposedtodifferentdisturbances[J].MicrobEco,l2002,44:49-58.[6]KOWALCHUKGA,GERARDSS,WOLDENDROPJW.DetectionandcharacterizationfungalinfectionsofAmmophilaarenaria(Marramgrass)rootsbydenaturinggradientgelelectrophoresisofspecificallyamplified18SrDNA[J].ApplEnvironMicrobio,l1997,63(10):3858-3865.[7]JOHWANA,TOMAOTAKAD,SATOSHIT,etal.Characterizationofdenitrifyingphosphate-accumulatingorganismscultivatedunderdifferentelectronacceptorconditionsusingpolymerasechainreaction-denaturinggradientgelelectrophoresisassay[J].WatRes,2002,36:403-412.[8]WENTL