含油废水处理工艺中细菌多样性李凤梅

＊李凤梅1,2　黄翠霞3　郭书海1＊＊　张玲妍1(1中国科学院沈阳应用生态研究所,沈阳110016;2中国科学院研究生院,北京100039;3铁岭师范高等专科学院,辽宁铁岭112000)　　从辽河油田曙光污水处理厂含油废水样品中直接抽提细菌总DNA,并对总DNA中16SrRNAV3可变区序列作PCR扩增、变性梯度凝胶电泳(DGGE),与常规水质分析相结合对系统中不同处理阶段细菌种群多样性和水质之间的关系进行分析。结果表明,处理系统的不同处理阶段细菌的多样性有明显差异,既存在共同的细菌种属,也存在着各自独特的细菌种属,且细菌种群的多样性与水质CODcr和总石油烃(TPH)的浓度呈负相关,细菌种群的多样性越高,CODcr和TPH的浓度越低,反之则越高。在处理过程中,随着样品中细菌多样性的增加,种群结构之间的相似性指数(Cs)逐渐升高,最后形成了稳定的种群结构。　含油废水;DGGE;水质;相似性指数　X172　　A　　1000-4890(2009)12-2574-05Bacterialdiversityinoilwastewaterduringitstreatmentprocess.LIFeng-mei1,2,HUANGCui-xia3,GUOShu-hai1,ZHANGLing-yan1(1InstituteofAppliedEcology,ChineseAcademyofSciences,Shenyang110016,China;2GraduateUniversityofChineseAcademyofSciences,Beijing100039,China;3TielingTeachersCollege,Tieling112000,Liaoning,China).ChineseJournalofEcology,2009,28(12):2574-2578.Abstract:ThetotalDNAwasdirectlyextractedfromtheoilwastewaterofShuguangWastewaterTreatmentPlantinLiaoheOilfield,andbythemethodsofPCR-DGGEandconventionalwaterqualityanalysis,therelationshipsbetweenthebacterialdiversityinthewastewaterduringitstreatmentprocessandthewaterqualitywereanalyzed.Thereweresignificantdifferencesinthebacterialcommunitiesatdifferenttreatmentstages,notonlysomecommonbacterialspecies,butalsotheuniquebacteriapopulation.ThebacterialdiversitywasnegativelycorrelatedwiththeCODcrandTPHconcentrationsinwastewater.ThehighertheCODcrandTPHconcentrations,thelowerthebacterialdiversitywas.Duringtreatmentprocess,thesimilaritycoefficient(Cs)ofbacterialcommunitiesinthewastewaterincreasedwithenhancingbacterialdiversity,andfinallyformedastablepopulationstructure.Keywords:oilwastewater;DGGE;waterquality;similaritycoefficient.＊(2004CB418501)(2009ZX07208-008)。＊＊E-mail:[e-mail]:2009-06-18　　:2009-08-24　　,,。、30～40,,,85%。,、,。,,(,2002)。,,,、,、(Brit-schgiGiovannoni,1991;Delongetal.,1993;Fuhr-manetal.,1993;MacNaughtonetal.,2001;AbedChineseJournalofEcology　2009,28(12):2574-2578　　　　　　　　　　　　　DOI:10.13292/j.1000-4890.2009.0395etal.,2002;,2005)。,。,DNA,PCR-DGGE16SrRNA,,,、,。1　1.1　,1。20068,,、3,1#()、2#(1)、3#(2)、4#(3)、5#()。,,-20℃。1.2　(,2002),COD,(TPH)。1.3　1.3.1　DNA　10000r·min-1,;DNA(1998)DNA;DNADNA,DNA;DNA1%,UVP-GDS8000,DNA,DNA。1.3.2　DNAPCR　DNA,16SrRNAV3(Gililianetal.,1998)。50μlGM5F-GC518R(10pmol·μl-1)2μl,dNTP(25mmol·L-1)4μl,10×buffer5μl,2μl,TaqDNA2.5U,。PCR94℃5min,2094℃1min,65℃～55℃1min72℃3min(0.5℃),1094℃1min,55℃1min72℃3min,72℃7min。PCRPTC-200PCR(Bio-Rad),1%。1.3.3　(DGGE)　Muyzer(1993),Bio-RadDcodeSystem16SrRNAV3DGGE。8%,30%～70%,50μl,60℃、200V、1×TAEBuffer4h。(Bassametal.,1991),。1.3.4　　Cs(Gilianetal.,1998):Cs=2j/(a+b)×100%,a1,b2,j。2,100%;,0。1.3.5　　Bassam(1991),DNA,PCR,DGGE,。GenBank,Blast,16SrRNA。1　Fig.1　ProcessingprocessofShuguangWastewaterTreatmentPlant2575:2　CODcrTPHFig.2　CODcrandTPHcontentsinwastewaterinShu-guangWastewaterTreatmentPlant2　2.1　CODcr(TPH),2。2,,CODcr,,TPHCODcr,(2008)。2.2　DNADNA,DNA,,DNATE1%。3,520kb,3#、4#5#DNA1#2#,。3　DNAFig.3　AgarosegelelectrophoresisofgenomeDNAextrac-tedfromsamples4　PCRV3DNAFig.4　AgarosegelelectrophoresisofV3regionamplifiedbyPCR2.3　16SrRNAPCR,PCRDNA,。DNA,100,GM5F-GC518RPCR,233bp16SrRNAV3,DGGE(4)。2.4　DGGE16SrRNAV3PCRDGGE,2,,,DNA。DGGE,,。,DGGE,。　　5,516SrRNAV3DGGE,;1#,,;2#～4#,,;5#,35,。,N342576　　　　　　　　　　　　　　　　　　　　　　　　　　　28　12　5　16SrRNAV3DNADGGEFig.5　16SrRNAgeneV3regionseparatedbyDGGE1-2:1#;3-4:3#;5-6:2#;7-8:4#;9-10:5#,,。N34,4(N8、N31、N33、N38)2#、3#、4#、5#,,5。,,N332#、3#,4#、5#,1#,。,4#5#1#、2#3#,2#3#。,24,CODcrTPH。(CODcrTPH),;,,,CODcrTPH,1#5#。,,,,,。2.5　,,,。DGGE,,(1)。:,1#5#5.56%,;4#5#83.33%,2#3#87.5%,4#5#、2#3#。1#～5#,1#5#、2#5#、3#5#4#5#,(2),,。2.6　5,N8、N31、N33、N34N38,DGGE5,GenBank,2。,N82577:1　(%)Tab.1　Similaritycoefficientofbacterialcommunityindif-ferentwastewatersamples1#2#3#4#2#253#2087.54#7.6943.7552.945#5.5633.3340.9183.332　DGGEDNATab.2　SequenceanalysisofDGGEgelextractionDNAGenBank(GenBank)(bp)(%)N8BacilluscirculansstrainBB-2(FJ605391)236100N31CurtobacteriumflaccumfaciensstrainsSAFR-008(AY167854)24099N33Arthrobacterglobiformisstrain21015(GQ199717)247100N34Pseudomonassp.HB01(AB376103)23397N38BacillussubtilisstrainFQ06(GQ360038)228100BacilluscirculansstrainBB-2(100%),N31CurtobacteriumflaccumfaciensstrainsSA-FR-008(99%),N33Arthrobacterglobiformisstrain21015(100%),N34Pseudomonassp.HB01(97%),N38BacillussubtilisstrainFQ06(100%)。3　　,CODcrTPH,(CODcrTPH),。、。,,,,,;,,。,,,,。　,,　,.2002..,42(4):478-483..2002..:.,　,,.2005..,25(12):3314-3322.　,,,.2008..,24(19):62-66.,.1998..:.AbedRMM,SafiNMD,KösterJ,etal.2002.Microbialdiver-sityofaheavilypollutedmicrobialmatanditscommunitychangesfollowingdegradationofpetroleumcompounds.Ap-pliedandEnvironmentalMicrobiology,68:1674-1683.BassamBJ,Caetano-AnollesG,GresshoffPM.1991.FastandsensitivesilverstainingofDNAinpolyacrylamindegels.AnnalsofBiochemistry,196:80-83.BritschgiTB,GiovannoniSJ.1991.PhyloeneticanalysisofanaturalmarinebacterioplanktonpopulationbyrRNAgenecloningandsequencing.AppliedandEnvironmentalMicro-biology,57:1707-1713.DelongEF,FranksDG,AlldredgeAL.1993.Phylogeneticdi-versityofaggregate-attachedvs.free-livingmarinebacterialassemblages.LimnologyandOceanography,38:924-934.FuhrmanJA,McCallumK,DavisAA.19