Anammox反应器的启动及其菌群演变的研究胡宝兰

21720057TransactionsoftheCSAEVol.21　No.7July　2005Anammox胡宝兰1,郑　平1,胡安辉1,周尚兴2,丁革胜2(1.,310029;　2.,322002)　:,,,(Anammox),。,,;,。,,、,。PCR-DGGE,,。:硝化;厌氧氨氧化;细菌多样性;DGGE:X703.1　　　　:A　　　　:1002-6819(2005)07-0107-04,　,,.Anammox[J].,2005,21(7):107-110.HuBaolan,ZhengPing,HuAnhui,etal.Start-upofanammoxreactorandevolvementofbacterialcolonies[J].TransactionsoftheCSAE,2005,21(7):107-110.(inChinesewithEnglishabstract):2004-07-04　:2005-05-16:(30070017);(2003C13005):(1972-),,,,,。　,310029。Email:[e-mail]:　(1962-),,,,,。　,310029。Email:[e-mail]0　　,“”,、,。,“”“”,。、、,[1]。,。(Anaerobicammoniaoxidation,Anammox)(NH+4),(NO-2),N2。,,40%,,[1-3]。,,11d[4],。(Anammox),。,,,。Anammox,,。1　1.1　,:TSS()39.87g/L,VSS()15.6g/L,VSS/TSS39.12%,pH6.94。,2.0L,1.7L。,1000mL。,。,。30℃。Anammox1。图1　厌氧氨氧化反应器系统Fig.1　Anammoxbiofilmreactorsystem1.2　,,NH+4-N。,,,(DO),。,107NH+4-NNO-2-N(1∶1～1.3),Anammox。1.3　MPN()Anammox[5]:VandeGraaf(g/L),KHCO31.25;NaH2PO40.05;MgSO4·7H2O0.2;CaCl2·2H2O0.3;2mL,10mmol/LNH+4-N10mmol/LNO-2-N。10mL/。。[6]:,10mL/。Griess。[4]:VandeGraaf(g/L),30mmol/LNH+4-N。5mL/。GriessH2SO4-。[7]:,10mL/。。1.4　1)DNA,Hepes(N-2--N-2-)/KHCO3(75mmol·L-1/5mmol·L-1)3,,,Hepes/KHCO3。K717,。2)PCRPCR()DNA,50μL(5μL;Mg2+3μL;dNTP4μL;3μL;3μL;3μL;Taq0.6μL;28.4μL。PCR:338F(5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCAG-3′)518R(5′-ATTACCGCGGCTGCTGG-3′)[8];PCR:94℃5min→94℃1min;53℃1min;72℃2min;30→72℃5min。3),,。1):,0.75mm,16cm×10cm。10%,10%APS()80μL,TEMED(N,N,N′,N′-)15μL。33.8%～50.0%。(7mol/L40%(v/v))。2):15～20μL30μLPCR,。3):60℃155V,1×TAE330min(“Running”“Max”)。4):①10%20min;②1%HNO310min(),DNA;③,5min/;④500μL/1000mL0.2%AgNO3,20min;⑤10s;⑥250μL/1000mL2.5%Na2CO3,;⑦。5):BIO-RAD。1.5　1)NH+4-N:-[9]。2)NO-2-N:N-(1-)-[9]。3)TSSVSS:[9]。4)pH:pHS-9v。2　2.1　,NH++-N,30～50mg/L。,。,,。,(NH+4-N50～140mg/L),。(HRT)1d,,;9d,95%(2)。10d,130.96mg/L·d,130.19mg/L·d(3),99%,0.77mg/L(2)。20,,。,。图2　富集培育硝化污泥阶段反应器运行性能Fig.2　Reactorperformanceduringcultivationofnitrifyingsludge2.2　Anammox,,(),,,()[10]。,,,1082005　,,。,。17d～22d,,。,88.02%90.53%(45)。图3　富集培育硝化污泥阶段反应器容积效率Fig.3　Volumetricefficiencyofreactorduringcultivationofnitrifyingsludge图4　启动厌氧氨氧化后反应器的氨去除性能Fig.4　Ammoniaremovalperformanceofreactorduringanaerobicammoniaoxidation图5　启动厌氧氨氧化后反应器的亚硝氮去除性能Fig.5　Nitriteremovalperformanceofreactorduringanaerobicammoniaoxidation,[1]。Anammox,1∶0.02,,。30d,1∶(0.9～1.4),1∶(1.2±0.2)[4],。,,,,c[11]。2.3　,,。1,,,,,1.1×109/mL9.5×107/mL,,9.5×107/mL4.5×106/mL。,,。,,1.5×105/mL3.0×105/mL,,,3.0×105/mL2.5×106/mL。,,,00.3×103/mL,,0.4×103/mL。4,,3。,3。1　Table1　MeasuredresultsofthenumberoffourbacterialcoloniesindifferentoperationalphasesAnammox1.1×1093.0×1061.5×1050Anammox9.5×1074.5×1063.0×1050.3×103Anammox4.5×1067.5×1062.5×1060.4×1032.4　PCR-DGGE(-),,Anammox。(61)、3.5(62)7(63)Anammox,,,AD,B、C、EF,。　　　　　　1:　　　　　　2:3　　　　　　3:7　　　　　　:图6　Anammox反应器内DNA的PCR-DGGE分析Fig.6　PCR-DGGE(polymerasechainreaction-denaturinggradientgelelectrophoresis)analysisoftheDNAfromanammoxreactor109　7:Anammox3　,;,。,,,、。,。PCR-DGGE,。,,。[　　　][1]　　,,.[M].:,2004:61-71.[2]　MulderA,VandeGraafAA,RobertsonA,etal.Anaerobicammoniumoxidationdiscoveredinadenitrifyingfluidizedbedreactor[J].FEMSMicrobiolEcol,1995,16:177-183.[3]　ChristianF,MarcB,PhilippH.Biologicaltreatmentofammonium-richwastewaterbypartialnitritationandsubsequentanaerobicammoniumoxidation(anammox)inapilotplant[J].JournalofBiotechnology,2002,99:295-306.[4]　StrousM,HeijnenJJ,KuenenJG,etal.Thesequencingbatchreactorasapowerfultooltostudyveryslowlygrowingmicroorganisms[J].AppliedandEnvironmentalMicrobiology,1998,50(5):589-596.[5]　VandeGraafAA,MulderA,BruijnPD,etal.Anaerobicoxidationofammoniumisabiologicallymediatedprocess[J].ApplEnvionMicrobiol,1995,61(4):1246-1251.[5]　NicolaisenMH,RamsingNB.Denaturinggradientgelelectrophoresis(DGGE)approachestostudythediversityofammonia-oxidizingbacteria[J].JournalofMicrobiologicalMethods,2002,50:189-203.[6]　,.[M].:,1985:227-238.[7]　,.[M].:,1999:251-267.[8]　ZhangD,LiuYP,XuH,etal.DGGEAnalysisof16SrDNAofnitrifyingbacteriainOLANDbiologicalNremovalsystem[J].Biotechnology,2003,13(5):1-5.[9]　.()[M].:,1989.[10]BockE,SchmidtI,StuvenR.NitrogenlosscausedbydenitrifyingNitrosomonascellsusingammoniumorhydrogenaselectrondonorsandnitriteaselectronacceptor[J].ArchMicrobiol,1995,163:16-20.[11]JettenMSM,StrousM,vandePas-SchoonenKT,etal.Theanaerobicoxidationofammonium[J].FEMSMicrobiologyReviews,1999,22:421-437.Start-upofanammoxreactorandevolvementofbacterialcoloniesHuBaolan1,ZhengPing1,HuAnhui1,ZhouShangxing2,DingGesheng2(1.DepartmentofEnvironmentalEngineering,ZhejiangUniversity,Hangzhou310029,China;2.ZhejiangMifengGroupCo.Ltd,Yiwu322002,China)Abstract:Thenitrificationbiofilmwascultivatedwiththeactivatedsludgefrommunicipalwastewatertreatmentplant.Undercontrolledconditionsthenitrificationbiofilmwassuccessfullytransittedfromnitrificationtoanaerobicammoniaoxidation(Anammox).Duringthewholeprocessthenumberofheterotrophicbacteriadecreased,butthenumberofnitrifyingbacteria,denitrifyingbacteriaandanammoxbacteriaincreased.Theincreaseofthreebacterialcolonieswassupposedtorelatetoanammox.TheresultfromPCR-DGGE(polymerasechainreaction-denatu