好氧反硝化细菌N61亚硝酸盐还原活性的研究陈松

微生物学通报JUL20,2008,35(7):1007~1010Microbiology©2008byInstituteofMicrobiology,[e-mail]*通讯作者：Tel:[phone];Fax:[phone];:[e-mail]收稿日期：2007-11-12;接受日期：2008-01-11研究报告好氧反硝化细菌N6-1亚硝酸盐还原活性的研究陈松洪秀娟黄雷鸣窦洁周长林*(210009)摘要:针对本实验室筛选得到的1株好氧反硝化细菌(AerobicDenitrifyingBacteria)N6-1,研究了培养条件和培养基组分对菌株亚硝酸盐还原活性的影响。结果表明,其最适培养条件为30℃、摇床转速120r/min、初始pH8.5,分别以NaNO2和乙酸钠为唯一氮源和碳源时,最适碳氮比为12;初始NaNO2浓度为2g/L时,培养20h后NO2−−N全部被还原,平均还原速率可达20.3mg/L·h;1.5%NaCl和1%蛋白胨对其亚硝酸盐还原活性没有明显影响;10L发酵罐培养24h后菌体浓度高达1.2×1011CFU/mL。关键词:好氧反硝化细菌,亚硝酸盐还原,还原速率StudyontheNitrite-reducingActivityofAerobicDenitrifyingBacterialStrainN6-1CHENSongHONGXiu-JuanHUANGLei-MingDOUJieZHOUChang-Lin*(SchoolofLifeScience&Technology,ChinaPharmaceuticalUniversity,Nanjing210009)Abstract:Thenitrite-reducingactivityofaerobicdenitrifyingbacterialstrainN6-1wasstudied.Itshowedthatthenitrite-reducingactivityreachedthehighestat30℃,120r/min,pH8.5andC/Nratio12,usingCH3COONaandNaNO2asthesolecarbonsourceandnitrogensource,respectively.WhentheinitialNaNO2concentrationwas2g/L,NO2−−Nwasreducedcompletelyafter20hourscultivationwiththereducingrateof20.3mg/L·h.Therewouldbenoeffectonitsnitrite-reducingactivityinthepresentof1.5%NaClor1%peptone.Thecellconcentrationcouldreach1.2×1011CFU/mLafter24hourscultivationin10Lfermentor.Keywords:Aerobicdenitrifyingbacteria,Nitrite-reducing,Reducingrate(DenitrifyingBacteria),O2,,,RobertsonKuenen[1](AerobicDenitrifyingBacteria),[2−5],,,1,DOI:10.13344/j.microbiol.china.2008.07.0191008微生物学通报2008,Vol.35,No.7材料与方法1.1材料1.1.1菌种：N6-1,1.1.2仪器：BiostatC10-3(B.BraunBiotechInternational,Germany);C-25IncubactorShaker(NewBrunswickScientific,USA);752N()1.1.3：1.2方法1.2.1培养基：NaNO20.5g,CH3COONa5.0g,MgSO4·7H2O0.03g,MnSO4·4H2O0.01g,FeSO4·7H2O0.01g,K2HPO40.75g,NaH2PO40.25g,1L,pH8.5MgSO4·7H2O0.03g,MnSO4·4H2O0.01g,FeSO4·7H2O0.01g,K2HPO40.75g,NaH2PO40.25g,NaNO20.5g~5g,,NaCl0g~35g,0g~10g,1L,pH6.0~10.01.2.2NO2−−N测定方法：N-(1-)-[6]1.2.3菌体浓度测定方法：[7]2结果与讨论2.1初始pH对亚硝酸盐还原活性的影响pH30,,O2,;,,,250mL100mL,120r/min,pH,pH,pH7.3N2,pH7.3N2O[8]pH(1),pH7.0~10.0,pH8.5图1初始pH对菌株亚硝酸盐还原活性的影响Fig.1EffectofinitialpHonthenitrite-reducingactivityofstrainN6-12.2碳源对亚硝酸盐还原活性的影响,5.0g/L,0.5g/LNaNO2,(1),,12hNO2−−N,2.3碳氮比对亚硝酸盐还原活性的影响,NaNO20.5g/L,C/N(),C/N(2),C/N12,12hNO2−−N,表1不同碳源培养时的NO2−−N还原率Table1ThereducingratioofNO2−−NwithdifferentcarbonsourcesReducingratio(%)Time(h)SodiumacetateGlucoseMannitolMaltoseSucroseStarchsoluble1210072.830.41.63.283.2241001001006.821.61003610010010038.053.210048100100100100100100:N6-11009表2不同C/N培养时的NO2−−N还原率Table2ThereducingratioofNO2−−NwithdifferentC/NC/N148121620Reducingratio(%)4.628.464.21001001002.4初始NaNO2浓度对还原活性的影响C/N12,NaNO2,NaNO2(2),NaNO24g/L,,NaNO25g/L,NaNO22g/L,NO2−−N,20.3mg/L·h(3)图2初始NaNO2浓度对菌株亚硝酸盐还原活性的影响Fig.2EffectofinitialNaNO2concentrationonthenitrite-reducingactivityofstrainN6-1表3不同初始NaNO2浓度时的NO2−−N平均还原速率Table3TheaveragereducingrateofNO2--Nwithdif-ferentinitialNaNO2concentrationInitialNaNO2concentra-tion(g/L)InitialNO2−−Nconcentra-tion(mg/L)ResidualNO2−−Nconcentra-tion(mg/L)Time(h)Averagereducingrate(mg/L·h)0.510101010.1120301216.9240602020.3360903616.9481205415.051014828603.12.5NaCl对亚硝酸盐还原活性的影响,0.5g/L,35g/LNaCl(3),NaCl15g/L,;NaCl,,,,图3NaCl对菌株亚硝酸盐还原活性的影响Fig.3EffectofNaClonthenitrite-reducingactivityofstrainN6-12.6蛋白胨对亚硝酸盐还原活性的影响,(AssimilatoryNitrateReduction),,(4),0g/L~10g/L,图4蛋白胨对菌株亚硝酸盐还原活性的影响Fig.4Effectofpeptoneonthenitrite-reducingactivityofstrainN6-12.7发酵罐培养与亚硝酸盐还原活性的研究,30100r/min0.05vvm5,(g/L)NaNO20.5,1010微生物学通报2008,Vol.35,No.7·7H2O0.03,MnSO4·4H2O0.01,FeSO4·7H2O0.01,K2HPO40.75,NaH2PO40.25,pH8.5NO2−−N10h,pH0h~10h(5),NaNO2,10h5mg/L·hNaNO2NaNO2NO2−−N,NaNO224h,1.2×1011CFU/mL,pO224h(6),24h,图5发酵过程中NaNO2浓度和pH变化Fig.5FermentationtimecurvesofNaNO2concentrationandpH图6发酵过程中菌浓度和pO2变化Fig.6FermentationtimecurvesofbacteriaconcentrationandpO23结论3,,,,1(NitriteOxidizingBacteria)N6[9],,,2,,,N6-1,,,,参考文献[1]RobertsonLA,KuenenJG.Aerobicdenitrification:acon-troversyrevived.ArchMicrobiol,1984,139:351−354.[2]..,2004,44(6):837−839.[3]..,2005,11(2):222−225.[4].().,2005,11(5):600−603.[5]..,2007,28(7):1548−1552.[6]...:,2002,pp.271−274.[7]..:,2004,p.86.[8]..:,2006,p.27.[9],,..,2005,32(5):56−61.