分子生物学在废水系统硝化细菌群落特征分析中的应用进展黄燕

246200811WATERRESOURCESPROTECTIONVol.24No.6Nov.2008　　:“”(2006):(1982—),,,,。E-mail:[e-mail]:,,E-mail:[e-mail]　,,,　,　,　(,　200062):较系统地介绍了与环境微生物相关的分子生物学研究方法,如克隆文库分析法、分子杂交技术、遗传指纹技术等,及其这些相关技术在硝化细菌群落结构与分布、多样性、动态性分析中的应用进展。结果表明,分子生物学技术在研究硝化细菌群落特征中发挥了重要的作用。:硝化细菌;群落特征;分子生物学;综述:Q71　　　:A　　　:1004-6933(2008)06-0012-05ProgressintheapplicationofmolecularbiologytoanalysisofcommunitycharacteristicsofnitrifyingbacteriainwastewaterHUANGYan,HUANGMin-sheng,DAIXing-chun,ZHUYong,GAOShang,GAOYan(SchoolofResourcesandEnvironmentSciences,EastChinaNormalUniversity,Shanghai200062,China)Abstract:Asystematicreviewofmolecularbiologyresearchmethodsappliedinenvironmentalmicrobiologyisgiven,includingclonelibraryprofiling,molecularhybridizationandgeneticfingerprinting.Progressintheapplicationofthesemethodstotheanalysisofcommunitycompositionanddistribution,diversityanddynamicanalysisofnitrifyingbacteriaisalsopresented.Theresultsshowthatmolecularbiologytechniquesplayanimportantroleinthestudyofthecommunitycharacteristicsofnitrifyingbacteria.Keywords:nitrifyingbacteria;communitycharacteristics;molecularbiology;applicationprogress　　,,,,。,。、,、,。,。,,[1]。:(AmmoniaOxidizingBacteria,AOB),(NitriteOxidizingBacteria,NOB)。,,,。,。,、、,、,。,,、、·12·,、,。,,,。1　　　,,、、,。,。,,,,,DNA/RNA、。,,。1.1　(clonelibraryprofiling),,,PCR。,,,,。,16SrRNA/DNA。16S。,,16SrRNA,16SrRNA,,,[2]。1.2　(molecularhybridization),。,(、DIG),。,。(FISH)、PCR(IS-PCR)、(Microarrays)。,(FISH)DNARNA、[3-4]。FISH、、、。1.3　PCR(geneticfingerprinting)　　,。。。DNADNAPCR。DNA[5],PCR,、GC、Taq,。DNA,。,16S/18SrRNADGGE/TGGE,。,(RAPD)、rDNA(ARDRA)、(RFLP/T-RFLP)。1.4　PCR(real-timePCR)real-timePCR,PCR,PCR,。,real-timePCR3:SYBRGreenTaqmanMolecularBeacon。PCR,PCR,,,,,·13·。PCR,、、、、。2　　　、、,;,;。,,,。2.1　DGGEPCR,,,。[6]PCR-DGGE,。β,Nitrosomonassp.。,AOB,,,。Pynaert[7](RBC),,(AOB),Nitrosomonassp.。Tsuneda[8]DGGE,Nitrosococcusmobilis,0.125kg·m-3·d-10.82kg·m-3·d-1,3,Nitrosomonasmarina,Nitrosospira,Nitrosomonas。PCR(NestedPCR)DGGE,,。,Philips[9]PCR0.01%。Boon[10]PCRDGGE15。Bacteria、α-Proteobacteria,Actinomycetes,(AOB),CMethanotrophsAcidobacteriumPCR-DGGE,。,16S/18SrRNA(restrictionfragmentlengthpolymorphism,RFLP),。[11]PCRDNA,16SrDNA,HhaⅠRsaⅠRFLP,353RFLP,94%,3%。RFLP,GenBank,(Nitrosomonas),,Nitrosomonas。[12]DNA,16SrDNA,16SrDNA,。,,NitrosamonasNitrospira。,。2.2　,FISH、PCR;,。,。Jang[13]SBR(:1±0.35mm～1.3±0.45mm)(AOB),FISH:AOB,300pm。Tsuneda[8]FISHPCR·14·(amoA),。,FISH,rRNA,PCR。,PCR。[14]16SrRNANSO190,proteobacteriaβ。NIT,proteobacteriaα。NSO190NITFAM,HEX。,,。,,。[15]FISH、PCR,AUSB。:,(AOB),(NOB),;,AOB。Juretschko[16]16SrRNAFISH,-,Proteobacteria,β。You[17]FISHPCR-DGGETNCU-I()A2O,。TNCU-IA2O。PCR-DGGE,NitrosospiraNitrosospira,NitrosomonasTNCU-IRBC。,FISH,TNCU-IRBC、TNCU-IRBC、A2O10.3%、13.7%5.2%,2.5%、3.6%2.3%。,TNCU-IA2O3.22.6;2.9。2.3　DGGE,。Boon[18]DGGE,3-CA,,、4,12。[19],,(Anammox),PCR-DGGE。,,。,,、,,。,。[20](AOB)。2,AOB。,(MDS)DGGE,,,AOB,AOB,。,,[21]PCR-DGGE。,COD,64.38%,,。,21,,Nitrosospira.spNitrobacter.sp,Bacillus.sp、Pseudomonas.spPseudochrobactrum.sp。PCR,,,。Aoi[22]real-timeamoAmRNA,,·15·,,mRNA,。Harms[23]real-timePCR,。1a,。。Park[24]FISH。Tanaka[25]FISH。3　　、,,。,,,,:①,;②;③,。。:[1]KOWALCHUKGA,STEPHENJR.Ammonia-oxidizingbacteria:amodelformolecularmicrobialecology[J].AnnuRevMicrobiol,2001,55:485-529.[2]BORNEMANJ,HARTINRJ.PCRprimersthatamplifyfungalrRNAgenesfromenvironmentalsamples[J].ApplEnvironMicrobiol,2000,66(10):4356-4360.[3],,.[J].,2004,5(11):14-20.[4].[M].:,2002.[5]BOURRAINM,ACHOUAKW,URBANV,eta1.DNAextractionfromactivatedsludge[J].CurrMicrobiol,1999,38(6):3l5-3l9.[6],,,.[J].,2003,43(3):371-378.[7]PYNAERTK,SMETSBF,BEHEYDTD,etal.Start-upautotrophicnitrogenremovalreactorviasequentialbiocatalystaddition[J].EnvironSciTechnol,2004,38:1228-1235.[8]TSUNEDAS,TATSUON,HOSHINOT,etal.Characterizationofnitrifyinggranulesproducedinanaerobicupflowfluidizedbedreactor[J].WatRes,2003,37:4965-4973.[9]PHILIPSCJ,HARRISD,DOLLHOPFSL,etal.Effectsofagronomictreatmentsonstructureandfunctionofammonia-oxidizingcommunities[J].ApplEnvironMicrobiol,2000,66:5410-5418.[10]BOONN,WINDTWD,VERSTRAETEW,etal.EvaluationofnestedPCR-DGGE(denaturinggradientgelelectrophoresis)withgroup-specific16SrRNAprimersfortheanalysisofbacterialcommunitiesfromdifferentwastewatertreatmentplants[J].FEMSMicrobiolEcol,2002,39:101-112.[11],,,.16SrDNARFLP[J].,2006,43(4):635-641.[12],,.[J].,2007,13(1):104-107.[13]JANGA,YOONYoung-Han.Characterizationandevaluationofaerobicgranulesinsequencingbatchreactor[J].JournalofBiotechnology,2003,105:71-82.[14],,.[J].,2003,22(5):363-365.[15],,,.[J].,2006,27(9):1858-1861.[16]JURETSCHKOS,LOYA,LEHNERA,etal.Themicrobialcommunitycompositionofanitrifying-denitrifyingactivatedsludgefromanindustrialsewagetreatmentplantanalyzedbythefull-cyclerRNAapproach[J].SystematicApplMicrobiol,2002,25:84-99.[17]YOUSJ,HSUCL,CHUANGSH,etal.NitrificationefficiencyandnitrifyingbacteriaabundanceincombinedAS-RBCandA2Osystem[J].WaterResearch,2003,37:2281-2290.[18]BOONN,TOPEM,VERSTRAETEW,etal.Bioaugmentationasatooltoprotectthestructureandfunctionofanactivated-sludgemicrobialcommunityagainsta3-chloroanilineshockload[J].ApplEnvironMicro