好氧反硝化菌株的鉴定及其反硝化特性研究王弘宇

1,2,1＊,1,1,1,1(1.,　150090;2.,　430072):1C3,.,C3,90%.,16SrDNA,C3(Pseudomonassp.),.C3,pH30℃7.0.,C3.CNC3,5.5～6.0,.:;;16SrDNA;:X172;X703　:A　:0250-3301(2007)07-1548-05:2006-07-27;:2006-09-06:(50521140075);(GB05C202):(1976～),,,,E-mail:[e-mail]＊,E-mail:[e-mail]cterizationofaBacterialStrainC3CapableofAerobicDenitrificationWANGHong-yu1,2,MAFang1,SUJun-feng1,ZUOWei1,ZHANGXian-xu1,ZHANGJia1(1.SchoolofMunicipalandEnvironmentalEngineering,HarbinInstituteofTechnology,Harbin150090,China;2.DepartmentofMunicipalEngineering,WuhanUniversity,Wuhan430072,China)Abstract:AbacterialstrainC3screenedfromtheactivatedsludgewasfoundcapableofaerobicdenitrificationandthedenitrifyingcapabilityofthestrainwasstudiedinbatchcultureunderaerobiccondition.TheresultsshowedthatthenitrateintheculturecouldbeefficientlyremovedbystrainC3andthenitrogenremovalratewasuptoabove90%.Accordingtothemorphologicalobservation,physiologicalbiochemicaltestandsequenceanalysisofthe16SrDNA,strainC3wasidentifiedasPseudomonassp..Andthephylogenticpositionofthestrainwasperformedbasedonthephylogenetictree.ThefactorsaffectingaerobicdenitrificatrionbystrainC3werealsodiscussed.TheresultsindicatedthatthemostsuitabletemperatureandpHvalueforaerobicdenitrificationwere30℃and7.0,respectively.ThedenitrificationperformanceofstrainC3wasalmostnotaffectedbythepresenceofoxygenandthestrainC3hadahighertoleranceofdissolvedoxygenconcentrationthanotheraerobicdenitrifiersreportedpreviously.TheoptimalCNratiowas5.5～6.0andnearlycompletedenitrificationcanbeobtainedattherangeofCNratio.Keywords:aerobicdenitrifier;biologicalnitrogenremoval;16SrDNAsequencing;phylogeneticanalysis　　,.2.[1],,、NO、N2O.[2].,,,[3].,,,Thiosphaerapantotropha,Alcaligenesfaecalis,Pseudomonasnautical,Thaureamechernichensis,PseudomonasstutzeriMicrovirgulaaerodenitrificans[4～7]..,.C3,、,.1　1.1　,,C3.1.2　DM[5](gL):Na2HPO4·7H2O7.9;28720077　　　　　　ENVIRONMENTALSCIENCEVol.28,No.7Jul.,2007DOI:10.13227/j.hjkx.2007.07.025KH2PO41.5;NH4Cl0.3;MgSO4·7H2O0.1;4.7;2mL;pH7.0～7.5;KNO3.(gL):EDTA50.0;ZnSO42.2;CaCl25.5;MnCl2·4H2O5.06;FeSO4·7H2O5.0;(NH4)6Mo7O2·4H2O1.1;CuSO4·5H2O1.57;CoCl2·6H2O1.61;pH=7.0.1.3　[8,9].1.4　16SrDNAPCR、DNA16SrDNA,[10].BSF820:5′-AGAGTTTGATCCTGGCTCAG-3′(Escherichiacoli8～27);BSR154120:5′-AAGGAGGTGATCCAGCCGCA-3′(Escherichiacoli1541～1522)..PCR[11](50μL):10×PCR5μL,MgCl2(25mmolL)4μL,dNTP2μL,BSF820BSR1541201μL,DNA1μL,Taq(10000UmL)0.5μL,35.5μL.PCR:①94℃2min;②94℃1min,56℃1min,72℃2min;③229;④72℃10min;⑤60℃10min.PCR.C316SrDNAGenBank,CLUSTALX、BIOEDITMEGA,Neighbor-Joining[12],Bootstrap.1.5　,、.2L(1),,20.25μm.1LDM,10%,,,3Lmin3min,,.,,,,.,,(DO8～10mgL).:,,;,.,,.1,,2,pHDO,NH+4-N、NO-3-NNO-2-N.1　Fig.1　Experimentalsetupforaerobicdenitrification1.6　NO-2-N:N-(1-)-;NO-3-N:;(N2):AgilentHP4890D,GS-03-106,:He20mLmin,0.08MPa,60℃,120℃,120℃;:,721480nm;(DO):YSI5000DO;pHORP:320pH.2　2.1　C3,,0.5μm×2.0μm,.,,,,.C32.2.2　C3:,,,、15497:2　C3Fig.2　ElectronandmicroscopyphotographofstrainC3、、、、、,-(V.P.)、(M.P.)、、,.2.3　16SrDNAGENEBANKC3,BIOEDITPHYLIP,Neighbor-Joining16SrDNA,3.3.C3Pseudomonassp.SN1,Pseudomonasstutzeri,Pseudomonasputida,PseudomonaschloritidismutansPseudomonascf..C3Pseudomonas,C3Pseudomonassp.SN1,16SrDNA99%.3　16SrDNAC3Fig.3　Unrootedphylogenetictreesbasedonthepartial16SrDNAsequencesofstrainC3andrelatedbacteria2.4　(1)C3,30℃.NO-3-N、NO-2-N,C3(4).4,C39h,NO-3-N110.98mgL2.31mgL.,,9h28.40mgL,NO-2-N.,.12h,0.4mgL..4,,,..4　C3Fig.4　AerobicdenitrificationperformanceofstrainC32.5　pHpH,,30℃.5,pHC3.pH,.pH7,92.71%.pH869.04%;pH959.58%.(pH7～9)C3.(pH≤6)(pH≥10),,.pH,,.,C3pH7.0.1550　　　　　　285　pHC3Fig.5　InfluenceofpHonaerobicdenitrificationbystrainC32.6　,,.20、25、30、35、40℃5,C3,24h,,6.6,C3.NO-3-N120mgL,20℃,40%.25℃,78%.30～35℃,90%,30℃93.39%.40℃,75.26%.,25～35℃,C3.,,.6　C3Fig.6　InfluenceoftemperatureonaerobicdenitrificationbystrainC32.7　DO,DO.,DO,DO24hDO.30℃.1C3.1,,120mgL,30℃24h,C390%.,DOC3.,,NO-3NO-2,.,DOC3,DO2.3～11.3mgL,,.,C3,,Wilson[13].[14～16],C3.1　DOC3Table1　DenitrificationeffectsofstrainC3atvariousDOconcentrationsDOmg·L-124hNO-3-Nmg·L-1NO-2-Nmg·L-1NO-3-Nmg·L-1NO-2-Nmg·L-1%11.3117.391.3410.200.6190.898.5118.521.3210.660.2990.865.6116.830.988.670.9891.813.2117.911.158.120.5992.682.3118.391.417.250.2693.732.8　CNCN.CNC3,,(TOC)720mgL,(TOCN),CN1、2、3、4、5、5.5、6、915.7C3.7,CN1,20.48%;CN5.5,90.4%.CN5.5,CN.,,.CN6,94.3%,15517:,.CN5.5,.,.,,.,,,.,5.5～6.0.7　C3Fig.7　EffectoftheCNratioondenitrificationperformancebystrainC33　(1)1,、16SrDNA(Pseudomonassp.).(2)C3,.,,.,90%.(3)C3pH7.0,30℃.DOC3,DO2.3～11.3mgL,.CN,5.5～6.0.:[1]ZumftWG,ViebrocA,KornerH.Biochemicalandphysiologicalaspectsofdenitrfication[A].In:ColeJA,FergusonSJ.(eds).TheNitrogenandSulphurCycles[M].Cambridge:PressSyndicate　　　　oftheUniversityofCambridge,1988.245～279.[2]BoninP,GilewiczM,BertrandJC.EffectsofoxygenoneachstepofdenitrificationonPseudomonasnautica[J].CanJMicrobiol,1989,35:1061～1064.[3]RobertsonLA,KuenenJG.CombinedheterotrophicnitrificationandaerobicdenitrficationinThiosphaerapantotrophaandotherbacteria[J].AntonieVanLeeuwenhoek,1990,57:139～152.[4]PatureauD,GodonJJ,DabertP,etal.Microvirgulaaerodenit