PCRDGGE法分析温度对A2O系统硝化菌群结构的影响陶芳

　520099()JournalofEastChinaNormalUniversity(NaturalScience)No.5　Sept.2009:1000-5641(2009)05-0053-10PCR-DGGEA2/O陶　芳,　黄　燕,　高　尚,　黄民生,　陈　诚(,　200062):A2/O,.A2/ODNA,DNADNA,DNADGGE,,.,AOB25℃,,NH+4-N95%;Nitrobacter20℃,,NH+4-N95%;Nitrospira,,15～20℃,NH+4-N75%～95%.:A2/O;　PCR-DGGE;　;　;　:X172　　:A　:2008-09　:(2007-007)　:,,,.E-mail:[e-mail].　:,,,,.E-mail:[e-mail]-DGGEtoanalyzetheeffectoftemperatureonstructureofnitrifyingbacteriainA2/OsystemTAOFang,　HUANGYan,　GAOShang,　HUANGMin-sheng,　CHENCheng(DepartmentofEnvironmentScience,EastChinaNormalUniversity,Shanghai　200062,China)Abstract:　TheeffectsoftemperatureonthestructureofnitrifyingbacteriainA2/Osystemwerestud-iedforthepurposeoftechniqueimprovementsandsystemregulations.TotalmicrobialDNAwasextrac-tedfromsampleswhichwerecollectedfromreactor,thentargetDNAfragmentswereamplifiedfromthetotalDNAusingspecificbacterialprimer.ThetargetDNAwereusedfordenaturinggradientgelelectro-phoresis(DGGE)analysis.ResultsindicatedthatthechangesoftemperaturecouldaffectthecommunitystructureandtheShannondiversityindexofnitrifyingbacteria.ThestructureofAOBcommunitywasstablewhenthetemperaturewashigherthan25℃,theremovalrateofNH+4-Nwasover95%inthiscondition;ThestructureofNitrobactercommunitywasstablewhenthetemperaturewashigherthan20℃,theremovalrateofNH+4-Ncanreach95%inthiscondition;ThestructureofNitrospiracommu-nitychangedgreatlywhenthetemperaturechanged,comparativelyspeaking,thecommunitystructurewasmoststablewhenthetemperaturewasbetween15℃to20℃,theremovalrateofNH+4-Nwasbe-tween75%to95%inthiscondition.()2009Keywords:　A2/O;　PCR-DGGE;　temperatureeffect;　nitrifyingbacteria;　communitystruc-ture0　引　　言A2/O,,,[1,2].(DenaturingGradientGelElectrophoresis,DGGE)PCR,,,[3,4],,.PCR-DGGEA2/O,,--,,[5].,PCR-DGGEA2/O、.1　材料与方法1.1　A2/OA2/O(1).(EH-B20VC-20EY1),2.4L/h,:6h,2h,2h;F/M()0.12gBOD5/(gMLSS·d),(BT100-1J),(R)2,(r)1/2;2～4mg/L,15d,2.5h.1　Fig.1　SchematicdiagramofA2/Oprocess545,:PCR-DGGEA2/OA2/O,,12～34℃,10d,DNA,(2007310-2007710)1,2.1　Tab.1　Samplingdateandtemperature123456789101112(/)3/103/203/304/104/204/305/105/205/306/106/307/10/℃13.313.620.118.717.118.121.924.525.224.928.633.22　Tab.2　InfluentcharacteristicspHCOD/(mg·L-1)NH+4-N/(mg·L-1)TN/(mg·L-1)NO-3-N/(mg·L-1)TP/(mg·L-1)7.8～8.4220～18018～2438～420.2～0.61～4820021400.421.2　DNA(1)　10000r/min5min,1.5mL,200μLSolutionSUS;400μLSolutionLYS0.3g,,30min,12000r/min5min,1.5mL.(2)DNA　120μLSolutionBID,1mLTIP“3SDNA”(),5min,3S2mL,12000r/min1min,,600μLWashSolution,10000r/min1min,10000r/min2min,;30μLTE(pH8,37℃),2min,12000r/min1min;20μLTE,DNA.5μL1.0%.1.3　PCR(1)3,[6-8]DNAPCR,DGGE.3　DGGETab.3　AmplificationprimersofDGGEAOBNitrospiraNitrobacterV3RegionCTO189fAB/CTO189fCP338fP338fP338f-GCCTO653rNtspa0685Nb1000rP518r(2)PCR,50μLPCR:TaqDNA0.5μL,105μL,Mg2+15mmol/L,dNTP(2.5mmol/Leach)1μL,0.8μL(25μmol/L),DNA15ng,50μL.(3)PCR　95℃10min,20(94℃1min,65～55℃1min,72℃2min),10(94℃30s,55℃30s,72℃60s),55()200972℃10min.PCR1.0%.1.4　(DGGE)(1)　,8%,10%APS80μL,TEMEN18μL.30%～60%(100%7mol/L40%),[9].(2)PCR　,,PCR30μL15μL.(3)　20～40V30min,150～160V,60℃5～6h.,.(4)　DGGEGelDoc2000(Bio-rad),SmartView.2　结果与分析2.1　DNADNADNA1.0%,DNA(2(a)),.260nm280nmOD260/OD2801.7～1.9,DNA,RNA,.2　DNA(a),AOB(b),Nitrospira(c)Nitrobacter(d)Fig.2　TotalDNA(a),AOBPCRproducts(b),NitrospiraPCRproducts(c)andNitrobacterPCRproducts(d)afteragarosegeleletrophoresis565,:PCR-DGGEA2/O2.2　DNAPCRPCR1%EB,,2(b)-(d),PCRPCR.AOB450bp,NOB(Nitrite-OxidizingBacteria)NitrospiraNi-trobacter[10],Nitrospira300bp,Nitrobacter450bp,.2.3　NH+4-NA2/O,NH+4-NA2/O,A2/ONH+4-N.3,20℃,A2/ONH+4-N70%～83%,75.8%,;20～25℃,NH+4-N,95%;25℃,NH+4-N,95%,25～30℃[11].5,(CODBOD5),,.4NO-3-NNO-3-N,A2/O,NO-3-N,,.2.4　DGGE(1)AOB(AOB)16SrDNAPCRDGGE5,1-12(1),,AOB1-AOB8.5,AOB2,AOB4AOB6,3A2/O.AOB3AOB5,,22℃,;,AOB7AOB857()2009,.,AOB,.5　AOBFig.5　EffectoftemperatureoncommunitystructureofAOB6,1-5,0.92～1.00,20℃AOB,;6-120.909～1.000,.20℃,AOB,,NH+4-N,95%(3).6　AOBDNAFig.6　DNAUPGMAdendrogramofAOBunderdifferenttemperature7,,AOB,AOB,.,AOB,,,AOB,,[12].(2)NitrobacterNitrobacter16SrDNAPCRDGGE8,Nb1-Nb9.9.585,:PCR-DGGEA2/O7　AOBFig.7　ShannonindexofAOBflora8,Nitrobacter,,Nb7,Nb8Nb9,.,,Nb5314℃,Nb1Nb4420℃,Nb2,Nb3Nb63420℃.8　NitrobacterFig.8　EffectoftemperatureoncommunitystructureofNitrobacter9,1-6NitrobacterDNA,12～20℃,Nitrobacter;20℃,NitrobacterDNA100%,,Nb7,Nb8Nb9,,NH+4-N95%(3).NitrobacterDNA10,3,,Nitrobacter.,NitrobacterNb7,Nb8Nb9,,Nitrobacter.(3)Nitrospira59()2009Nitrospira16SrDNAPCRDGGE11,Np1-Np11,12,13.　　11,Nitrospira,,,Nitrobacter.Np2,,25℃.Np5,Np6Np113,15℃.Np1,Np8Np94518～35℃,.12,1-3、4-78-12,3,Nitrospira,15℃,15～20℃23℃,.4-7,15～20℃Nitrospira,NH4+-N75%～95%(3).　　DNA,1,DNA,(13).,,1DNA30%,,Nitrospira,.605,:PCR-DGGEA2/O13NitrospiraFig.13　ShannonindexofNitrospiraflora13Nitrospira,3,450.6～0.8,,6,70.3.25℃Nitrospira,.3　结　　论,A2/O(AOBNOB),13.3～33.2℃,120d,.(1)PCR-DGGEA2/OAOBNOB.,A2/O.(2).AOB25℃,,25℃.,AOB,,,NH+4-N,95%.Nitrobacter10～20℃,,20℃,,,NH+4-N95%.Nitrospira,,,,15～20℃,NH+4-N75%～95%.18℃,NitrobacterNitrospira,NOBNitrobacter,18℃,,.,AOB25℃,NH+4-N,95%.Nitrobacter20℃,NH+4-N,95%.Nitrospira15～20℃,NH+4-N75%～