您好,欢迎访问三七文档
当前位置:首页 > 行业资料 > 其它行业文档 > 好氧反硝化细菌的筛选鉴定及其反硝化反应条件优化周莉
ActaAgriculturaeZhejiangensis,2011,23(5):942-947[J].,2011,23(5):942-947.周 莉1,2,汤江武2,王 新2,姚晓红2,吴逸飞2,葛向阳1,*(1,430070;2,310021):2010-12-27:(1983-),,,,,。E-mail:zhouli407@yahoo.com.cn*,,Tel:027-87281040;E-mail:gxy@mail.hzau.edu.cn :、,,、、pH、、。,35GC5,16SrDNA,(Pseudomonassp.)。、15∶1,1%,pH7.5,160rmin-130℃,,99.19%53.83%。:;;;:Q939.11+2 :A:1004-1524(2011)05-0942-06Screeningandidentificationofaerobicdenitrifiersandtheoptimizationofdenitrificationcon-ditionsZHOULi1,2,TANGJiang-wu2,WANGXin2,YAOXiao-hong2,WUYi-fei2,GEXiang-yang1,*(1CollegeofLifeScienceandTechnology,HuazhongAgriculturalUniversity,Wuhan430070,China;2InstituteofPlantProtectionandMicrobiology,ZhejiangAcademyofAgriculturalSciences,Hangzhou310021,China)Abstract:Aerobicdenitrifyingbacteriawereisolateddirectlyfromtheaquaculturewater,sludgeandruralrivers.Followingcharacterizingthephylogenyofthestrainswiththehighestdenitrifyingcapability,theeffectsofcarbonsource,C/Nratio,initialpH,inoculationquantity,rotationalspeedandtemperatureonthecharacteristicsofdeni-trificationwerestudied.Theresultsshowedthat35strainsofaerobicdenitrifierswereobtainedbyprimaryscreeningonplatesofBromothymolBluemedium.OnestrainnamedGC5,whichhadthehighestdenitrifyingcapabilitywasi-dentifiedaftersecondaryscreeningandwasdesignatedasPseudomonassp.basedontheanalysisof16SrDNAse-quenceandphylogeneticanalysis.Furthermore,theoptimumconditionsfordenitrificationwereasfollows:carbonsourcesethanol,C/Nratio15∶1,inoculationquantity1%,initialpH7.5,rotationalspeed160rmin-1,andtem-perature30℃.Undersuchcondition,theremovalrateofnitratenitrogenandtotalnitrogeninsimulationsewagewere99.19%and53.83%,respectively.Keywords:aerobicdenitrification;screening;denitrifyingcharacteristics;Pseudomonassp. ,、、、,。,,:,,,;,,,[1]。,Alcaligenesfaecalis,PseudomonasputidaP.stutzeri[2],,,。、,,。1 材料与方法1.1 、。100mL(9.63g,0.85g,1.36g,0.27g,1g,0.19g,1mL[3],1L,pH7.3,121℃,30min(300mL),30℃,160rmin-1。3d,5mL(3)。(BTB)(1g,1g,1g,0.05g,0.2g,1g,BTB1mL(1%,),20g,1L,pH7.3,121℃,30min),30℃2~3d,,,。LB(3),,,5000rmin-1,10min;,,(3)。1%(V/V)DM(KNO30.5gL-1、KH2PO41gL-1、MgSO47H2O1gL-1、2.8gL-1,121℃30min),30℃,160rmin-1,3d。1.2 1%(W/V),pH7.2~7.4,,,121℃30min。BTB,,37℃1~7d;,[4]。1.3 16S1.3.1 16SrDNA的PCR扩增和测序(BioerTechonl-ogyCo.Ltd)DNA,16SrDNA。27F(5′-AGAGTTGATCCTGGCTCAG-3′)1492R(5′-TACCTTGTTACGACTT-3′)。PCR25μL,:10×2.5μL(15mmolL-1Mg2+),dNTP(10mmolL-1)0.5μL,(10μmolL-1)0.4μL,DNA30ng,ExTaqDNA0.75U,ddH2O25μL。PCR:95℃4min,94℃1min,55℃1min,72℃1.5min,30,72℃8min。PCR1%,PCR1.5kb,PCR。1.3.2 系统发育树的构建1.5kbGenBankBLAST,Bioedit7.0MEGA4.0,Neighbor-Joining。1.4 1.4.1 不同碳源对GC5反硝化活性的影响DM,、、、,0.585gL-1。:1%、30℃,160rmin-1、100mL(500mL)。3。。3d,。1.4.2 碳氮比、初始pH值和接种量对GC5反硝化活性的影响 DM,(3∶1,6∶1,8∶1,10∶1,15∶1,30∶150∶1),pH(5.0,943 ,.5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,10.0)(0.1%,0.2%,0.5%,1.0%2.0%)3。、pHGC5,1.4.1。1.4.3 转数和温度对GC5反硝化活性的影响DM。(0,80,120,160,220rmin-1)(25℃,30℃,35℃,40℃)2,GC5。1.4.1。1.4.4 反硝化条件的正交优化,GC5、pH,L9(34),43,、pH。1.5 GB7479—87,,GB7493—87N-(1-)-,GB11894—89。2 结果与分析2.1 、35。14(1)。1,NB13,NQ2,NQY3,NQY4,NQY6GC5688%,20%~50%。,GC5,48h95.9%,45.3%,。2.2 GC5。3d,;1,,。,1 Table1 Theresultsofquantitativetestforaerobicdenitrifi-cationstrains NO-2-N NH+4-N /%/%CK00.70——NB37.4122.9853.710NB41.697.8556.8943.34NB1314.2524.5188.0422.40NBY83.2722.3975.9527.92NBY92.4310.4470.7029.58NBY1129.6439.7079.810NQ233.4445.3588.180NQ32.919.7351.3727.66NQ615.6421.5053.440NQY322.9017.6589.0122.80NQY413.7317.2990.9139.16NQY627.3014.5089.759.04NQY1116.6615.6180.6416.76GC57.0916.9995.9045.30,。GC5。2.3 GC5GC516SrDNAGen-BankBlast,599%,(1)。1,GC5、,99%,Pseudamonassp.。,[5-8]Pseudamonassp.。2.4 2.4.1 碳源对GC5反硝化活性的影响GC5(2-a),,,90.99%,GC5。2.4.2 碳氮比对GC5反硝化活性的影响,[9]。944 23 5(20119)1 菌株GC5的16SrDNA系统发育树Fig.1Phylogenetictreebasedon16SrDNAsequenceofstrainGC5GC5(2-b),,。10∶1,97%。。2.4.3 初始pH值对GC5反硝化活性的影响pH,(3-a)。pH5.0,GC5,pH7.5~10.0,92%,GC5pH,。pH7.5,93.41%,GC5。2.4.4 接种量对GC5反硝化活性的影响 ,,GC5。3-b,0.5%,98.53%。0.1%,51.34%。。2.4.5 转数对GC5反硝化活性的影响,[10]。4-a,0~160rmin-1,96%。220rmin-1,74.74%,,GC5。945 ,.2.4.6 温度对GC5反硝化活性的影响4-b,GC5。30~40℃,90%。,30℃,96.27%。2.5 GC54 不同转数和温度对菌株GC5反硝化活性的影响Fig.4DenitrificationactivityofstrainGC5underdifferentrotationratesandtemperature ,GC5、pH。(2),GC5C,,A3B1C3,1.0%、pH7.5、15∶1,98.20%。3(5),GC5A3B3C3,1.0%,pH8.5、15∶1。,pH(32),2,3,3。(3),A3B1C3A3B3C3,99.19%53.83%,,GC5:、15∶1、pH7.5,1%、160rmin-1、30℃。2 GC5Table2 Theorthogonalexperimentdesignofdenitrifyinga-bilitydeterminationforthestrainGC5A(%)B(pH)C()/%10.17.58∶173.7020.18.010∶176.5030.18.515∶189.3340.57.510∶178.4050.58.015∶195.1060.58.58∶173.8371.07.515∶198.2081.08.08∶169.0591.08.510∶181.93T1239.53250.30216.58T2247.33240.65236.83T3249.18245.09282.63R9.659.6566.05t179.8483.4372.19t282.4480.2278.94t383.0681.7094.21r3.223.2222.025 因素水平趋势图Fig.5Thetrendchartoffactorlevels946 23 5(20119)3 Table3 Theresultsofverifyingtest/%/%A3B1C398.7151.86A3B1C399.3555.74A3B1C399.5153.8999.1953.83A3B3C393.1250.50A3B3C392.5748.53A3B3C391.4549.0892.3849.373 讨论、,BTB35。,14,14,GC5。16SrDNA,GC5Pseudamonassp.,99%,Pseudamonassp.。,GC5,10∶1,97%;0.5%,98%。,,15∶1、pH7.5,1%、160rmin-130℃,99.19%,53.83%,。。Kim[2]Pseudomonasp
本文标题:好氧反硝化细菌的筛选鉴定及其反硝化反应条件优化周莉
链接地址:https://www.777doc.com/doc-6569095 .html