海洋石油降解微生物的分离鉴定

274Vol.27,No.420057ACTAOCEANOLOGICASINICAJuly20051,2,23(11,361005;21,361005):2004207201;:2004210221.:(02302);(202CCA05000);(40376041).:(1980),,,.E2mail:[e-mail]3:E2mail:[e-mail]:,,9,1,.16SrDNA93111.111(M25),B25,,9512%,.M23,.B25M22,B25.,.:;;;:Q948:A:0253241932(2005)04201142071,.8,100t.,,.[1],,[2,3]..,,.,..,(Pseudomonas)(Enterobact2er)(Acinetobacter)(Corynebac2terium)[4,5];,[6].[710],.,[5,11,12].,,.2211.120018;220024.212;(PAHs):MERCK2Schuchardt,96%;Sigma,98%;Fluka,97%.(MMC):NaCl=24g/dm3;MgSO47H2O=710g/dm3;NH4NO3=1g/dm3;KCl=017g/dm3;KH2PO4=210g/dm3;Na2HPO4=310g/dm3,pH714,,PAHs.0122m.LB:NaCl=30g/dm3;10g/dm3;5g/dm3,pH710.2131cm31%(v/v)100cm3MMC.28,200r/min45d1cm3100cm3.8,,2848h.214,(40%).:LB28,260r/min,1cm3MMC.50cm3MMC1g,LB,200r/min,287d.2%(w/v)MMC.[13,14],.1cm3.3.21550cm350mg/dm3PAHsMMC,214.200r/min,2810d.,(HPLC)[15].21616SrDNADNAPCR[16].16SrDNAPCR16SF(AGAGTTTGATCCTGGCTCAG)16SR(ACG2GCTACCTTGTTACGACT),1500bp.TaqDNAMBI.PCRU210;pUCm2T,.BLAST(),DNAMAN(Version511)Genbank,DNAMAN[17]PHYLIP(316211a)[18](maximum2parsimony)[19][20].1000(boot2strap).217Genbank,MonF(TCAAY2ACMGSNCAYGARCT)MonR(CCGTART2GYTCNAYRTARTT),420bp.50dm35dm310PCRbuffer4dm32mmol/dm3dNTPs,2dm310mol/dm350ng2UTaq.216.3311,10(1),1B21,B22,B23,B25,B26;2M21,M22,M23,M24,M25.,M23,9.,245114:1G+,1B25,4G+(1).1M21,+M22,-M23,/M24,-M25,-B21,+B22,+B23,+B25,-B26,+2M23.:.M23,(1)..1M23M23,312PAHs2%(w/v)MMC.200r/min,287d,1B21(3417%)B22(2719%)B23(3319%)B25(4618%)B26(3310%).5,3513%,B25,153418%(2a).2M25(2a),41,35%42%,4314%,(2b).,M21,M23,B21,B25,B26,.,,(2%),(7d).22%MMC7d.2aB21,B22,B23,B25,B265;2bM21M255B25M25PAHs,B25PAHs61127,M25,PAHs,,M2510d1515%.,M25.,,.M25.313,1525.4d3d200mm3,,,,1,(4d)B23,B25,B26,B25,B23,B26.2,M21,M22,M23,M24M25,M23,M21M22.314916SrDNA,1B21,B22,B26,B23,B25;2M21,M22,M24,M25,.39.3B21BacillusthuringiensisATCC10972TBacilluscereusATCC14579T,9916%9918%.B21,(Bacilluscereus).B22,B26M21Bacillussubtilissubsp.spizizeniiNR2RLB223049TBacillusmojavensisJF22,99%,JF22McInerney[21],B22,B26M21,JF22(Bacillusmojavens2is).B23StaphylococcusarlettaeATCC43957T,9913%,B23(Staphylococcusarlettae).B25,AlcanivoraxvenustensisISO4T,9512%.G+CITSDNA2DNA(),B25(Alcanivorax).M22PseudomonasstutzeriATCC17682Pseudo2monasbalearicaTG23,9913%9812%,M22(Pseudomonasstutzeri).M24Alcaligenesxylosoxi2danssubsp.denitrificansDSM30026Alcali2genesxylosoxidanssubsp.xylosoxidansATCC27061,9817%9715%.(Alcaligenesxylosoxidanssubsp.denitrificans)2003Coenye(Alcaligenesdenitrificans)[22],M24.Gen2bankM25UnculturedalphaproteobacteriumcloneP_wp0211,9815%,PAHs,M25.Genbank,M25ThalassospiralucentensisDSM14000T,9413%,M25P_wp0211(Thalassospiralucentensis).916SrDNAGenbank.3159,B25M22425424bp.BLAST,(NYXEHYG[L/M]).M22PseudomonasfluorescensDSM50106(AlkB)9312%,B25GenbankBurkholde2riacepaciaRR10AlkB,6415%,.B25M22GenbankAY683534AY780807.4,.10,1(M23)3(B22,B26,M21)1(M22)17114:3B21B26M21M2516SrDNAGenbank,Genbank(B21)1(M24)1(B23)1(B25)1(M25),B25M25..,[23],,B25,4,.,.,.,C8C16.,,1B23()2M21(),;,,1B25()2M23().Hara[6],,B2536,,6000m,B25,..:[1]DALINGPS,BRANDVIKPJ.Characterizationofcrudeoilforenvironmentalpurposes[J].OilandChemicalPollution,1990,7(3):199224.[2]ATLASRM,ATLASMC.Biodegradationofoilandbioremediationofoilspills[J].CurrOpinBiotechnol,1991,2(3):440443.[3]BALBAMT,AWADHIAN,DAHERAR.Bioremediationofoilcontaminatedsoil:microbiologicalmethodsforfeasibilityassessmentandeldevaluation[J].JMicrobiolMethods,1998,32(2):155164.81127[4]SAADOUNI.Isolationandcharacterizationofbacteriafromcrudepetroleumoilcontaminatedsoilandtheirpotentialtodegradedieselfu2el[J].JBasicMicrobiol,2002,42(6):420428.[5]RAHMANKS,RAHMANT,LAKSHMANAPERUMALSAMYP,etal.Occurrenceofcrudeoildegradingbacteriaingasolineanddieselstationsoils[J].JBasicMicrobiol,2002,42(4):284291.[6]HARAA,SYUTSUBOK,HARAYAMAS.Alcanivoraxwhichprevailsinoil2contaminatedseawaterexhibitsbroadsubstratespecifici2tyforalkanedegradation[J].EnvironMicrobiol,2003,5(9):746753.[7],,.[J].,2001,21(1):8488.[8],,,.Phenotypiccharacteristicsandphylogeneticanalysisofcrudeoildegradingbacteria[J].BiodiversityScience,2002,10(2):202207.[9],,,.:.[J].,1999,21(6):116123.[10],,,.:.[J].,1997,19(6):6876.[11]RICHARDJY,VOGELTM.Characterizationofasoilbacterialconsortiumcapableofdegradingdieselfuel[J].InternationalBiodeteri2oration&Biodegradation,1999,44(23):93100.[12]BUNDYJG,PATONGI,CAMPBELLCD.Microbialcommunitiesindifferentsoiltypesdonotconvergeafterdieselcontamination[J].ApplMicrobiol,2002,92(2):276288.[13],.[A].[M].:,1992.5558.[14]IJAHUJJ,UKPELI.BiodegradationofcrudeoilbyBacillusstrains28Aand61Bisolatedfromoilspilledsoil[J].WasteManage2ment,1992,12(1):5560.[15],,,.PAHs[J].,2004,23(3):6164.[16]AUSUBELFM,BRENTR,KINGSTON,RE.ShortProtocolsinMolecularBiology[M],3rded.NewYork:JohnWiley&SonsInc,1995.39.[17]SAITOUN,NEIM.Theneighbor2joiningmethod:anewmethodforreconstructingphylogenetictrees[J].MolBiolEvol,1987,4(4):406425.[18]FELSENSTEINJ.PHYLIP.PhylogenyInferencePackage[M],version3.6.Seattle:DepartmentofGenetics,UniversityofWashing2ton,2004.[19]FITCHWM.Towarddefiningthecourseofevolution:minimum