您好,欢迎访问三七文档
当前位置:首页 > 商业/管理/HR > 质量控制/管理 > 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:xixieugene@163.com. :,,,,.E-mail:mshuang@des.ecnu.edu.cn.ApplicationofPCR-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%~
本文标题:PCRDGGE法分析温度对A2O系统硝化菌群结构的影响陶芳
链接地址:https://www.777doc.com/doc-6539784 .html