对降解喹啉的厌氧生物反应器中重要功能菌群的鉴定刘彬彬

26520065　　　　　　ACTAECOLOGICASINICAVol.26,No.5May,2006刘彬彬1,张　峰2,冯晓西2,刘勇弟2,张晓君1,赵立平1,＊(1.,　200240;2.,　200037):863(2001AA214131);(30470061):2005-12-15;:2006-03-15:(1978～),,,,.E-mail:[e-mail]＊Correspondingauthor.E-mail:[e-mail]:JamesBorneman(DepartmentofPlantPathology,UniversityofCalifornia,Riverside),Foundationitem:TheprojectwassupprotedbyHighTech.DevelopmentProgramofChina(863)(No.2001AA214131);NationalNaturalScienceFoundationofChina(No.30470061)Receiveddate:2005-12-15;Accepteddate:2006-03-15Biography:LIUBin-Bin,Ph.D.candidate,mainlyengagedinmicrobialecology.E-mail:[e-mail]:,。,,。DGGE,。16SrDNA,,。DGGE,,GammaProteobacteriaDesulfobacterpostgatei,。:;;DGGE;16SrDNA:1000-0933(2006)05-1390-06　:Q143,Q938　:AIdentificationoffunctionallyimportantmicroorganismsinalab-scaleanaerobicbiofilmreactorforquinoline-degradationLIUBin-Bin1,ZHANGFeng2,FENGXiao-Xi2,LIUYong-Di2,ZHANGXiao-Jun1,ZHAOLi-Ping1,＊(1.LaboratoryofMolecularMicrobialEcologyandEcogenomics,CollegeofLifeScienceandBiotechnology,ShanghaiJiaotongUniversity,Shanghai200240,China;2.CollegeofResourceScienceandEnvironmentalEngineering,EasternChinaUniversityofScienceandTechnology,Shanghai200237,China).ActaEcologicaSinica,2006,26(5):1390～1395.Abstract:Microbialcommunitystructureinalab-scalequinoline-degradinganaerobicbiofilmreactorwasdissectedincomparisonwiththeseedingsludgetoidentifythefunctionallyimportantmembers.SampleswerecollectedfromthebiofilmwhenthereactorreachedthestablequinolineandCODremovalefficiency.TotalDNAwasextractedfromthesamples.16SrDNAv3regionwasamplifiedandDGGEanalysiswasperformed.ThedominantbandswereexcisedandthenucleotidesequencesoftherRNAgenesweredetermined.Atthesametime,anear-fulllength16SrDNAlibrarywasconstructed.Nucleotidesequencesof100randomlyselectedclonesweredeterminedandidentifiedbynearestneighboranalysis.ComparisonanalysiswiththeseedingsludgeindicatedasignificantincreaseoftheGammaProteobacteriaandDesulfobacterpostgateiduringtheacclimationperiodinthereactor,thissuggestedthatthesemicroorganismsmaybefunctionallyimportantforquinoline-degradationunderanaerobicconditions.Keywords:biofilm;anaerobicreactor;DGGE;16SrDNAclonelibrary　　,,。,。,[1]。,,,。,[2]。,,。,Chouari[3]。.,,。[4,5]。,,,,,,。。,DNA、,rRNA[6]。,。,[7],。,,[8,9]。,。,[10,11]。,,DGGE,,,,。1　1.1　18L,。。,、、(NH4)2SO4K2HPO4。40mgL,、、150∶5∶1。(ESseries,IWAKI,Japan)24h,(YH,China)30℃。6,,5dCOD。CODGreenberg[12]。HPLC(highperformanceliquidchromatography,GC7890,Techcomp,ShanghaiChina)。pH(DO)pH(pHS-3C,Leici,China)(Oxi330i,WTW,German)。,。1.2　DNA[13],[13]。1.2.1　　5TENP(50mmolLTris,20mmolLEDTA,100mmolLNaCl0.01gmlPVP,pH10)(5min)。11821rmin(10000g),4℃5min,。5PBS(8gLNaCl,0.2gLKCl,1.44gLNa2HPO4,0.24gLKH2PO4,pH7.4),,(213915　:　)。PBS,10ml,4ml1ml,DNA,-70℃。1.2.2　DNA　1.5ml,11821rmin(10000g),4℃5min,50～100mg,200μl(100mmolLTris,100mmolLEDTA,200mmolLNaCl,1%PVP,2%CTAB,pH8.0),2,5min,。200μlSDS(2%SDS),5min,。13479rmin(13000g)10min,。400μl。13479rmin(13000g)15min,。400μl(1∶1)400μl1,0.6,-20℃,13987rmin(14000g)20min,。70%,30min,100μl。1.5μlRNaseA(20mgml)37℃20min。DNA-20℃。1.3　16SrRNAV3PCR(DGGE)16SrRNAV3P2,P3[14]。PCRMuyzer[14]。PCR(DGGE)(Dcodesystem,Bio-Rad)。35%～60%(100%7molL40%)。8%,1×Tris-Acetate-EDTA(TAE)(pH8.4)。200V、60℃200min,SYBRgreenⅠ(Amresco)。DNAUVI(UVItec,Cambridge,UnitedKingdom)。,、、。1.4　16SrRNA,16SrRNAP0,P6[15]。PCRDicello[15]。PCRpGEMT-easy(Promega),(BIO-RAD)DH5α。100。1.5　100P0,100,,500bp,99%,100(OTU,provisionaloperationaltaxonomicunits)。OTUT7SP6。RDP(chimera)[16]。,Kemp[17]。,(pseudo-librariy)。,Kemp[18]estimators,Schao1[19,20]SACE[21]。,Kemp[18]。,GenBank(http:),。(Phylogenetictree)。1.6　(Accessionnumber)DGGE16SrRNAGenBankDQ296464-DQ296465DQ296466-DQ296474。2　2.1　6,,pH7.0。(DO)0.1mgL。5dCOD,53.6%,COD60.4%。2.2　DGGEDNA,16SrDNAV3,DGGE1。(a～e),,。GenBank1392　　　　26(http:),aPseudomonassp.PCP2(AF326380),bDesulfobacterpostgatei(AF418180)。(c～e)3[13],ab,,,ab。,。＊BandsGenBankRelatedbacterialsequencesSimilarity(%)AccessionNo.aPseudomonassp.PCR2(AF326380)99DQ296464bDesulfobacterpostgatei(AF418180)99DQ296465cUnculturedbetaProteobacterium(AY133064)95AY945926dUnculturedBacteroidetesbacterium(AJ575722)97AY945927eUnculturedbacteriumPHOS_HE36(AF314435)97AY945928　　＊BandswereexcisedfromDGGEgelshownleft1　DGGEFig.1　DGGEanalysisof16SrDNAv3regionamplifiedfromtheseedingsludgeandthebiofilmintheanaerobicreactor.Thedenaturinggradientwasfrom35%to60%.Table1showstheresultofthesequenceanalysisofbandsobtainedfromDGGEgel1:Lane1:Seedingsludge;2:Lane2:AnaerobicReactor2.3　16SrRNA[13],97(3),(phylotypesrichness)。Kemp[17](progressivesampling)。2,,estimator,。2　KempSACESchaolFig.2　PredictednumbersofphylotypesbasedontheSACEandSChaolversusthesubsamplesinthelibrary.Thelibraryreachedanasymptoticmaximum,indicatingthatthislibrarywaslargeenoughtoyieldstableestimatesofphylotyperichness2.4　99%,9OTU,OTU,GenBank,。(Phylogenetictree)3。OTU(AR2AR1,54.6%26.8%)GenBankGammaProteobacteria(AY972868)Desulfobacterpostgatei(AF418180)。3　,、,,16SrDNA13935　:　GammaProteobacteriaDesulfobacterpostgatei。DGGE,aPseudomonassp.PCP2,GammaProteobacteria,GammaProteob