高效硝化细菌的分离与鉴定

2008,Vol.25No.4Chemistry&Bioengineering60:2006年湖北省科技厅重大项目(S135):2007-11-26:余敦耀(1979-),男,湖北大冶人,助理工程师,硕士研究生,研究方向为污水处理;通讯联系人:邱雁临,女,教授,博士生导师E-mail:[e-mail],[e-mail]1,1,2(11湖北工业大学生物工程学院,湖北武汉430068;21华中农业大学食品科学技术学院,湖北武汉430070):采用连续进水方式进行硝化细菌富集培养,筛选得到了一株硝化强度达到1517mg#L-1#d-1的硝化细菌N-5通过形态学特征亚硝酸氧化酶(Nor)电泳分析一般生理生化特征的初步鉴定等方法对筛选菌株进行鉴定,依据5伯杰氏细菌鉴定手册6进行分类,确定筛选的N-5为硝化球菌属(Nitrococcus)中的活动硝化球菌(Nitrococcusmobils):硝化细菌;分离;鉴定;亚硝酸氧化酶:Q9391114104:A:1672-5425(2008)04-0060-04,,[1],,,,564,NitrobacterNitrococcusNitrospinaNitrospira[2][3],,,,1111::24cm@5cm,;,;A-,AR:015g,150mL10%,Ñ,;A-011g,50mL,,150mL10%,Ò,112(g):K2HPO4#3H2O1,MgSO4#7H2O0103,CaCl20175,FeSO4#7H2O0103,(NH4)2SO4015,1000mL,pH(g):NaNO21,MgSO4#7H2O0103,MnSO4#4H2O0101,K2HPO4#3H2O0175,NaH2PO4#2H2O0125,1,1000mL,pH(g):NaNO21,MgSO4#7H2O0103,MnSO4#4H2O0101,K2HPO4#3H2O0175,NaH2PO4#2H2O0125,1,1000mL,pH71011310cm10g,2000mL,10cm@10cm@20cm,0165m3#h-1,1(28e)pH(710),,[4],2L,NO-2,NO-3,:/20084611Fig.1Testequipmentfornitrobacteriaenrichmentculture,NO-2,,,,11430mL/300mL20,28e12NO-2(g#L-1)123456,140r#min-1,28e30mL/300mL,630mL/300mL430mL/300mL230mL/300mLN-5115[5]01003mg#L-1,012mg#L-1,(mg#L-1#d-1)=c1-c0t,c1,mg#L-1;c0,mg#L-1;t,min11611611N-511612(Nor)SDS(Nor),NorNO-2NO-3,NO-3NO-2Nor,Nor,,NorcNorb,Norb,C,C,cNorb,cNor[6],,cNor,,C,qNor,qNor[7]2001,Gesche[8]Nor,,SDS-1161356[9]2211NO-2540nm(OD),22NO-2Fig.2StandardcurveofNO-22123,N-3N-9N-10S-1S-4;N-7S-3S-9;,12213NO-2121g#L-12g#L-13g#L-14g#L-15g#L-16g#L-111,N-1N-2N-4N-5S-2S-5NO-24g#L-1NO-25g:/2008462#L-1NO-26g#L-1N-1N-2N-4N-5S-2S-561NO-212Tab11Growthsituationsof12strainsunderdifferentNO-2concentration/g#L-1123456N-1+++++++++-N-2+++++++++-N-4+++++++++-N-5+++++++++-N-6+++++---N-8++++++---S-2+++++++++-S-5+++++++++-S-6++++++---S-7+++++---S-8+++----S-10+++++++--:/++0,/+0,/-0214630mL/300mL5d,540nmNO-2,2265d/mg#L-1#d-1Tab.2Nitrationintensitiesof6strainsculturedfor5days/mg#L-1#d-1N-1N-2N-4N-5S-2S-52914627153181332614330148201162,65d,N-1N-2N-5S-229146mg#L-1#d-127153mg#L-1#d-126143mg#L-1#d-130148mg#L-1#d-1,N-4S-5,N-1N-2N-5S-24N-1N-2N-5S-2430mL/300mL5d(2),540nmNO-2,3345d/mg#L-1#d-1Tab13Nitrationintensitiesof4strainsculturedfor5days/mg#L-1#d-1N-1N-2N-5S-2311122610930116201323,N-1N-5,,N-1N-5N-1N-530mL/300mL5d(3),540nmNO-2,4425d/mg#L-1#d-1Tab14Nitrationintensitiesof2strainsculturedfor5days/mg#L-1#d-1N-1N-521142301424,N-5N-19mg#L-1#d-1,,N-552155N-55,5,,N-5517134mg#L-1#d-115178mg#L-1#d-115101mg#L-1#d-114173mg#L-1#d-115164mg#L-1#d-1,1517mg#L-1#d-1N-5,,5,,21621611N-5,,,21612(5)5Tab15PhysiologicalbiochemistrypreliminaryappraisalresultsH2SMR-VP+++-+-+--++-------------N-5+--+++++-++-:/200846356,N-5(Nitrococcus)(Ni-trococcusmobils)21613(Nor)SDSSDS-,33(Nor)SDSFig.3TheSDSelectrophoretogramsofNor3,1#2#3#35kDa65kDa110kDa(Nor)116~130kDa65kDa32kDa,SDS-(Nor)3(1)3NO-2,N-5,30142mg#L-1#d-1(2)N-5517134mg#L-1#d-115178mg#L-1#d-115101mg#L-1#d-114173mg#L-1#d-115164mg#L-1#d-1,1517mg#L-1#d-1N-5,5,[10]10(1014~1510mg#L-1#d-1)Sauthier(4411g#m-3#d-1)N-5,(3)SDS-(Nor)(4)N-5(Nor),56,(Nitro-coccus)(Nitrococcusmobils):[1],,.[J].,2006,18(1):75-77.[2]StaleyJT,BryantMP,PfennigN,etal.BergeypsManualofSystemat-icBacteriology[M].Williams&Wilkins,2001:1807-1835.[3].[J].,2002,14(6):348-350.[4],,,.VE[J].,2004,22(4):95-99.[5].[J].,2004,31(3):130-134.[6]JettenMSM,LogemannS,MuyzerG,etal.Novelprinciplesinthemicrobialconversionofnitrogencompounds[J].AntonievanLeeuwenhoek,1997,71(1-2):75-93.[7]JettenMSM,PeterDeBruijn,KuenenJG.Hydroxylamineme-tabolisminPseudomonasPB16:Involvementofanovelhydroxyla-mineoxidoreductase[J].AntonievanLeeuwenhoek,1997,71(1-2):69-74.[8]GescheBraker,HectorL,Ayala-De-lRioAllanHD,etal.Com-munitystructureofdenitrifiersbacteria,andArchaeaalongredoxgradientsinpacificnorthwestmarinesedimentsbyterminalre-strictionfragmentlengthpolymorphismanalysisofamplifiedn-itritereductase(nirS)and16SrRNAgenes[J].ApplEnvironM-icrobiol,2001,67(4):1893-1901.[9]RE,NE,,56,.()[M].:,1984:622-651.[10],,,.[J].,2004,10(6):786-789.IsolationandIdentificationofHigh-efficientNitrobacteriaYUDun-yao1,QIUYan-lin1,ZHUYing2(11SchoolofBioengineering,HubeiUniversityofTechnology,Wuhan430068,China;21CollegeofFoodScienceandTechnology,HuazhongAgriculturalUniversity,Wuhan430070,China)Abstract:Withcontinuouswaterinletmethod,anitrobacteriumstrainN-5,whosenitrationintensityreached1517mg#L-1#d-1,wasscreenedbyenrichmentculture.Throughthemorphologicalcharacterization,nitrousacidoxydase(Nor)analysisandgeneralphysiologicalbiochemistrypreliminaryappraisal,thestrainN-5wasidentified.ItwasdeterminedasNitrococcusmobilsofNitrococcusbyclassificationonthebasisof3BergypsManualofSystematicBacteriology4.Keywords:nitrobacterium;isolation;identification;nitrousacidoxydase(Nor)