ERICPCR指纹图谱技术对低温下SUFR反应器中菌群结构分析

28120081ActaScientiaeCircumstantiaeVo.l28,No.1Jan.,2008:(No.90510020);(No.50378095);;(No.CSTC2007BB6160)SupportedbytheStateKeyProgramofNationalNaturalScienceofChina(No.90510020),theNationalNaturalScienceFoundationofChina(No.50378095),theOpenProgramofChongqingKeyLaboratoryofWaterEnvironmentalSafetyandEcoenvironmentsinThreeGorgesReservoirRegionandtheNaturalScienceFoundationofChongqing(No.CSTC2007BB6160):(1963),,(),Emai:[e-mail];*()Biography:YEJiangyu(1963),male,associateprofessor(Ph.D.),Emai:[e-mail];*Correspondingauthor,,,.2008.ERICPCRSUFR[J].,28(1):101-107YeJY,WangTJ,LuoGY,etal.2008.AnalysisofthemicrobialcommunitystructureofaSpiralupflowreactor(SUFR)atlowtemperaturebyERICPCRfingerprinting[J].ActaScientiaeCircumstantiae,28(1):101-107ERICPCRSUFR叶姜瑜1,2,*,王图锦2,罗固源1,2,季铁军21.三峡库区生态环境教育部重点实验室,重庆4000452.重庆大学城市建设与环境工程学院,重庆400045:20061121:20070820:20071129:ERICPCR(SpiralUp-FlowReactor,SUFR).,,;5,,TNTP72.35%93.28%,.,,.,SUFR,.:SUFR;ERICPCR;;;:02532468(2008)0110107:X172:AAnalysisofthemicrobialcommunitystructureofaSpiralupflowreactor(SUFR)atlowtemperaturebyERICPCRfingerprintingYEJiangyu1,2,*,WANGTujin2,LUOGuyuan1,2,JITiejun21.KeyLaboratoryofEcoenvironmentsofThreeGorgesReservoirRegionundertheStateMinistryofEducation,Chongqing4000452.CollegeofCityConstructionandEnvironmentalEngineering,ChongqingUniversity,Chongqing400045Received21November2006;receivedinrevisedform20August2007;accepted29November2007Abstract:ThemicrobialcommunitystructureofactivatedsludgeinaSpiralupflowreactor(SUFR)operatedunderlowtemperatureconditionswasinvestigatedbyERICPCRfingerprinting.TheresultsindicatedahighdiversityofthebacterialcommunityintheSUFRreactoratlowtemperatureandsomeapparentlydominantbacteria.At5,thebacterialdiversityandthenumberofpredominantbacteriadecreased,buttheremovalefficiencyofTNandTPstillreached72.35%and93.28%respectively,sotheSUFRreactormaintaineditsnormalfunction.ComparativeanalysisofthebacterialcommunityatlowtemperatureandnormaltemperatureshowedthattheSUFRreactorproduceddifferentkindsofpredominantbacteriaatdifferenttemperatures,andthediversityofthepredominantbacteriaatnormaltemperaturewasmuchgreaterthanthatatlowtemperature.Therefore,theSUFRreactor,withitsspiralupdesign,facilitatesgrowsofdifferentkindsoffunctionalbacteriaatdifferenttemperatures,whichallowsittomaintaingoodnitrogenandphosphorusremovalatlowtemperatures.Keywords:SpiralUpFlowReactor;ERICPCRfingerprinting;activatedsludge;microbialcommunitystructure;nitrogenandphosphorusremoval281(Introduction)3(),,.,20~30,15,5.20~40,15,,.,.,,,(,2000).SUFR,,,(,2004;2005);,,.,ERICPCR.(EnterobacterialRepetitiveIntergenicConsensus,ERIC)126bp.ERICPCR(44bp),,DNA.,(,2003).ERICPCRSUFR,,.2(Materialsandmethods)2.1SUFR系统装置与运行SUFR(,2004):,,,,.,,.:HRT9h,100%~150%,200%~250%,SRT20d.2.2样品采集2005111420051212,SUFR,1,5,5~17.200643,20~26.2hDNA.2.3水质分析1.1Table1WatercompositionandanalyticalmethodsCODCr(mg!L-1)HACHCODPO3-4P(mg!L-1)TP(mg!L-1),NH+4N(mg!L-1)TN(mg!L-1),2.4活性污泥总DNA的提取10mL8mL,8000r!min-1410min,;8mL,,8000r!min-1410min,,3.,2mLDNA(50mmol!L-1trisbase,20mmol!L-1EDTA,100mmol!L-1NaC,l0.01g!mL-1,pH=10),40LK(10mg!mL-1),,37225r!min-130min.,2mL5%SDS,652h,20min1.8000r!min-110min,1021:ERICPCRSUFR.DNA(1∀1),.8000r!min-15min,,3.,,8000r!min-15min,,3.40L3mol!L-14mLDNA,-2030min10000r!min-120min.2mL70%,10000r!min-110min,,.DNA100L,RnaseA3L(10mg!mL-1),3720minRNA.DNADNA(),DNA-20.2.5ERICPCR扩增ERIC(Versalovicetal.,1991),.ERICP1:5#ATGTAAGCTCCTGGGGATTCAC3#,ERICP2:5#AAGTAAGTGACTGGGGTGAGCG3#.ERICPCR:25L,DNA60ng,12.5pmo,ldNTP200pmol!L-1,PCR(10mmol!L-1TrisHC1,50mmol!L-1KC1,2.5mmol!L-1MgC12,pH=8.3)2UDNA().TEDNA.:955min;941min;501min30s;721min30s,35,728min.ERICPCR:ERICPCR(10L)1.5%,TBE(0.1mol!L-1TrisHC1,0.1mol!L-1,0.002mol!L-1EDTA,pH=8.0),4V!cm-1,1h,1KbDNA,.2.6ERICPCRERICPCRQuantityOne4.5.0.Shannon∃s(H#).:H#=-%(ni/N)ln(ni/N)(1),ni,N.completemethod.Sorenson(pairwisesimilaritycoefficien,tCs)ERICPCR.Cs=2j/(a+b)&100%(2),aERICPCR,bERICPCR,j2.2Sorenson0,2DNASorenson100%.3(Results)3.1低温期水质分析结果1,SUFR.3.45,,,TNTP7235%93.28%.5,.TNTP92.17%9837%,.1CODFig.1TNTPandCODremovaleffectatlowtemperature3.2低温期时SUFR反应器菌群结构特征ERICPCR,,,.2,5ERICPCR,,,.2,,,1032854,3.2ERIC-PCR(M:1kbladderDNAMarker;Nc:;An:;Ax:;Ox:)Fig.2TheERICPCRfingerprintingofactivatedsludgeatlowtemperature3.3不同温度条件下菌群指纹图谱比较34ERICPCR,,;,,.34ERICPCR(M:1kbladderDNAMarker;Nc:;An:;Ax:;Ox:)Fig.3ERICPCRfingerprintingofactivatedsludgecollectedatfourthweekatlowtemperatureandatnormaltemperature3.4常温下不同反应池不同采样点的菌群结构特征4,SUFR,.,.,3,.4ERICPCR(M:1kbladderDNAMarker;Nc:;An:;Ax:;Ox:)Fig.4ERICPCRfingerprintingofactivatedsludgeatnormaltemperature4(Discussion)4.1低温期SUFR反应器脱氮除磷效果分析1,SUFR.45,,,.1,,,,.(,1994;HelmerCetal.,1998;ChoiEetal.,1998;BactensDetal,1999),,.SUFR,SUFR,1041:ERICPCRSUFR.,,,(,2005).4.2低温期SUFR反应器菌群多样性分析2,SUFR.∋(,(,2004),,SUFR.,(Minoetal.,2000;Jeonetal.,2003;Seviouretal.,2003).ERICPCR,FISHDGGETRFLP,,,,!,,G+C.55,3,4,,.,,,5,.5Fig.5ShannonindexoftheactivatedsludgemicrobialcommunityatlowtemperatureSUFR,,,.,SUFR,;,(,2004).4.3低温期时SUFR反应器的优势菌群分析2,3,2.25,45;,,,,,1,2,2,,.,2.5,,1,3,.(Crocettieta