FISH法对垃圾渗滤液硝化生物膜发育的观察与分析张丽娟

　200734(2)FISH＊张丽娟1,2,3　许玫英1　孙国萍1＊＊(　　510070)1(　　510301)2　(　　100049)3　　＊(No.015017)(No.2004A30404002;2002C31605)(No.2006Z3-E0461)＊＊　Tel:[phone],E-mail:[e-mail]:2006-05-12,:2006-08-02:。(FISH),,(SEM)。,,,,。7d,60%,20d,40%;50d,,,35%,106d155d。,,。30mg/L70mg/L,,30%。:,,FISH,:Q938.1;X703.1　　:A　　:0253-2654(2007)02-0241-05FluorescenceinSituHybridizationAnalysisofNitrifyingBiofilmsinLandfillLeachateAeratedTreatingSystem＊ZHANGLi-Juan1,2,3　XUMei-Ying1　SUNGuo-Ping1＊＊(GuangdongInstituteofMicrobiology,GuangdongProvincialKeyLaboratoryofMicrobialCultureCollectionandApplication,Guangzhou510070)1(SouthChinaSeaInstituteofOceanology,ChineseAcademyofSciences,Guangzhou510301)2(GraduateSchooloftheChineseAcademyofSciences,Beijing100049)3Abstract:Thecultivationandrapiddetectionofammonia-oxidizershavebeennodusindebuggingandmonitoringofhighammoniaconcen-trationwastewatertreatmentengineering.Inthisresearch,landfillleachatebiofilmsandsuspendedactivesludgeindifferentoperationperi-odsweresampledinGuangzhouDatianshanlandfillleachatetreatingsystem.Theinsituspacialorganizationofammonia-oxidizingβ-pro-teobacteriaofsampleswasinvestigatedbyusingfluorescenceinsituhybridization(FISH)andwithscanelectronmicroscope(SEM).FISHandSEMobservationrevealedthatinthestartupperiod,micro-organismsinitiallyattachedonthelacunosepartofcarriermatrix.Thebacte-riatotalquantitykeptgrowinginthebiofilmsof7,20,50,106,155day.Theproportionofammonia-oxidizingβ-proteobacteriaisapprox-imately60%amongallbiomassintheearly7thdayand40%inthe20thdaysamples,whichmeansthatammonia-oxidizersfirstlyinhabitonthesurfaceofthecarriermatrixasthedominantcomponentofthecommunityintheprocessofbiofilmforming.Thebiofilmwasmatureinthe50daysamplesdetectedbyFISHandSEM.Theproportionofammonia-oxidizerattachedonthesurfaceofcarrierkeptarelativelysteadyvalueofapproximately35%amongallbiomassinthe50thday,106thdayand155thdaybiofilmssamples.Theproportionofammonia-oxi-dizersinthesuspendedactivesludgesamplingatthesametimewithbiofilmsamplesisunstableandfarbelowtheproportioninbiofilmgrownonthecarriersurface.Theproportionofammonia-oxidizersinsuspendedactivesludgedeclinedobviouslywhentheconcentrationofNH4-Nintheeffluentsincreasedto70mg/L,buttheproportionofammonia-oxidizersinbiofilmswasnotaffectedandkeptarelativelysteadyvalueofapproximately30%,meanwhile,theammonia-oxidizersstillworkwellinthetreatingsystem.TheseresultsindicatethattheapplicationofFISHprovidearapidmethodfordetectionofnitrifyingbacteriaandanacademicreferencefordebuggingofenvironmentalengineering.Keywords:Landfillleachate,Biofilm,FISH,Ammonia-oxidizingbacteria·241·DOI:10.13344/j.microbiol.china.2007.02.011200734(2)　　、、、[1,2]。。,。,,,。,,,,,[3]。MPN,,。(Fluorescenceinsituhybridiza-tionFISH),,,,。[4～8]。FISH,[4]、[5～8],,FISH[6,8];FISH[5]。,。,,。,。FISH,,。1　1.1　。,、、(ABR)、、。500m3/d,3,:COD,3800mg/L;NH4-N,1300mg/L;:COD,900mg/L;NH4-N,13mg/L。99%。,。7d,20d,50d,106d155d。24h,。1.2　FISH[9]1.2.1　DNA:DNA5′FITCEUB338,EUB338-Ⅱ,EUB338-Ⅲ[10](),,5′ROXNSO190[11](),β-proteobacteria16srRNA。DAPI()。1.2.2　:(1):,,,50mL,1×PBS,,3～4;4%,,4℃5h。,1×PBS,,3～4;98%∶1×PBS(1∶1),-20℃。(2):[9]。1.2.3　:[9]。1.2.4　:LEICADMRA2,LEICACW4000FISH,LEICAQ500IW。·242·　200734(2)1.3　,30%,50%,70%,90%,100%3,,3。,,,。1　FISH(100×)2　,-,。2.1　FISHDAPI,,。,EUB338,EUB338Ⅱ,EUB338Ⅲ,,,1。FISH,7d·243·200734(2),。20d。,,,。,,。3　(100×),。7d20d,EUB33890%;NSO1907d60%,20d40%(2)。,7d～20d,,。50d,90%,35%(2)。,50d,,。,2　◆,□。(3),7d;20d,;50d,,,,,FISH·244·　200734(2);106d155d,。,50d,。2.2　,(2),。50d106d(4),2%,50d106d,35%,。,,,,[3],FISH,,。4　106dFISH(100×),2,,,,。,,。2.3　,,50d,30～40mg/L,,,50d70mg/L,,6d10mg/L,155d。,FISH,,(2),50d155d,。,,。,,FISH,,。FISH,,、。[1]LiXZ,ZhaoQL,HaoXD.WasteManage,1999,19:409～15.[2]BaunA,LedinA,ReitzelLA,etal.WaterRes,2004,38(18):3845～3858.[3]HoilijokiTH,KettunenRH,RintalaJA.WaterRes,2000,34:1435～1446.[4],,.,2003,23(2):192～195.[5]OkabeS,SatohH,WatanabeY.ApplEnivonMicrobiol,1999,65(7):3182～3191.[6]YouaSJ,HsubCL,ChuangcSH,etal.WaterRes,2003,37:2281～2290.[7]TsunedaS,NaganoT,HoshinoT,etal.WaterRes,2003,37:4965～4973.[8]PerssonF,WikT,SorenssonF,etal.WaterRes,2002,36:1439～1448.[9]AmannRI.Insituidentificationofmicroorganismsbywholecellhy-brizationwithrRNAtargetednucleicacidprobes.MolecularMicrobi-alEcologyManual,part3.3.6KluwerAcademic,Dordrecht,1995.1～15.[10]DaimsH,BruhlA,AmannR,etal.SystApplMicrobiol,1999,22:434～444.[11]MobarryBK,WagnerM,UrbainV,etal.ApplEnvironMicro-biol,1996,62(6):2156～2162.·245·