高效异养硝化好氧反硝化菌株的分离鉴定与脱氮性能刘健楠副本

28220103()JournalofBeijingTechnologyandBusinessUniversity(NaturalScienceEdition)Vol.28No.2Mar.2010　　:1671-1513(2010)02-0018-06-刘健楠,　汪　苹,　尹明锐,　王　磊(北京工商大学化学与环境工程学院,北京　100048)　:利用BTB培养基从实验室驯化成熟的SBR反应器的活性污泥中筛选出一株高效异养硝化-好氧反硝化菌株WYLW-X06.通过对菌株WYLW-X06的形态观察、生理生化特征测定、Biolog鉴定,以及16SrDNA序列测定,认定菌株WYLW-X06是蜡状芽孢杆菌(Bacilluscereus).对菌株脱氮性能的研究结果表明:该菌株脱氮效果良好,氨氮去除率达到97.18%,总氮去除率96.63%,N2O气体总产量1.848987mg,N2O-N产量占总氮去除量的0.572%.:异养硝化-好氧反硝化;脱氮;N2O生物控逸:X172;TS264.2+3　　　　　:A:2009-10-20:“”(2007BAK36B07).:(1984—),,,,;　(1953—),,,,..　　,,[1-2].[3]:1),,.2),、,.OH-,pH.3),,COD.,,,,-[4-6].,-N2O.SBR-,、N2O,.1　1.1　SBR(TN80.0%～85.0%,CODCr85%～90%).1.2　(BTB)(g/L)[7]:20,KNO31,KH2PO41,FeC12·6H2O0.5,CaCl2·7H2O0.2,MgSO4·7H2O1,8.5,BTB(0.1BTB10mL)1mL,1mol/LNaOHpH7.0～7.3,121℃20min,.LB(g/L):KNO31,KH2PO41,FeCl2·6H2O0.05,CaCl2·7H2O0.02,MgSO4·7H2O1,8.5,121℃20min,.DM(g/L)[8]:KNO30.72,KH2PO41,MgSO4·7H2O1,2.8,18pH7.0,121℃20min,.(g/L):(NH4)2SO40.47,KH2PO41.0,FeCl2·6H2O0.5,CaCl2·7H2O0.2,Mg-SO4·7H2O1.0,4.08,121℃20min,.(g/L):(NH4)2SO40.47,5.62,50mL,121℃20min,.(g/L):K2HPO45.0,FeSO4·7H2O0.05,NaCl2.5,MgSO4·7H2O2.5,MnSO4·4H2O0.05,1L.1.3　1.3.1　菌株的筛选,0.1mLBTB,,30℃2～3d,,.,4℃.1.3.2　菌株的好氧反硝化性能测定(),100mL50mLLB,30℃、160r/min(DO5～7mg/L).10%()250mL200mLDM,30℃、160r/min,.(NO-3-N)(NO-2-N),TIN(TIN=NO-3-N+NO-2-N),.(、)(8000r/min),.1.3.3　菌株的异养硝化-好氧反硝化性能和脱氮产物N2O的测定[9-10]2.5L5LMinifors.16h5%.30℃、120r/min、pH9.0,pH1moL/LNaOH1moL/LHCl.,(NH+4-N)、(NO-3-N)(NO-2-N)OD600CODCr.TIN(TIN=NH+4-N+NO-3-N+NO-2-N),-.,N2O.(、、)CODCr(8000r/min),.1.4　1.4.1　形态观察,[11].1.4.2　生理生化鉴定《》[12].1.4.3　Biolog自动微生物鉴定Biolog[13]95,,.BUG+B,37℃16h,,(GN/GP-IF+T),,20%.8Biolog,150μL.37℃,24hBiolog.1.4.4　菌株的分子生物学鉴定[14]DNADNA().16SrDNA,27f(5′-AGAGTTTGATCATGGCTCAG-3′),1492r(5′-GGTTACCTTGTTACGACTT-3′),.PCR(50μL):10×PCRBuffer(15mmolMgCl)5μL,dNTPs4μL,27f1492r2.5μL,34μL,DNA2.5μL,Taq0.5μL.PCR:94℃4min;94℃,30s,52℃,80s,72℃,90s,30;72℃8min.PCR1.0%,.16SrDNAGen-Bank,CLUSTALX[15]、MEGA[16],Neighbor-Joining[17],19282　　　　　　:-Bootstrap.1.5　1.5.1　水体中各项指标的测定[18]GB7479—87,GB7480—87,GB7493—87N-(1-)-.:,721600nm.CODCrCODCr(5B-1,).1.5.2　气体N2O的测定GC-TCDN2O,:2h100mL,500mL1L.10mL(HAMILTON)GC-ECD.N2O:(TD)(350℃)、(TC)(50℃)、(fC)(20mL/min).Varian3800;10mci63Ni(ECD);3m×3mm,PorapakQ,80/100.N2ON2ON2O.N2O,:,,,N2O;x＊A=P·yA/kAN2O.2　2.1　WYLW-X0624h,3～5mm,、,.,,.,(10～13)μm×(3～4)μm,、.1.2.2　,1　WYLW-X06(20000×)Fig.1　ElectronmicroscopeofWYLW-X06strain(20000×),,.WYLW-X061.,WYLW-X06(Bacillus.sp).1　WYLW-X06Tab.1　PhysiologicalandbiochemicalcharacteristicsofstrainWYLW-X06WYLW-X06+-M-R+V-P-+WYLW-X06+++F2.3　BiologBiolog,WYLW-X069544,51.,:(Bacilluscereus)0.644.Biolog2.2.4　WYLW-X062.4.1　菌株WYLW-X06的16SrDNA的PCR扩增与测序WYLW-X06DNA,16SrDNA(27f1492r)PCR,1.5kb,2.16SrDNA,WYLW-X0616SrDNA1440,GenBank(NCBI):GU116563.20()　　　　　　　　　　　　　201032　WYLW-X06BiologTab.2　BiologcharacteristicsofstrainWYLW-X06-α-+β-++--+40+80+N--D-+N--β-D-+-L-+D---D-+D-+L--D--D---+α-D-+m--α-D---++D--D--D-+D--α--D--β--D--3--D-+α--D--β--D--α--D--+D-+D-/-L--D-+--D--()-+D--D-++-D---α--β-+γ-+p--α--α-+-D--L-+D-+L---+-+-/-N--L+L--D--L-+L--+L--L---L-+L--L-+-2,3--/+-2′-++++5′-+5′-+5′-+6--D--1--α-D--6--D--D-L-α-+2.4.2　菌株WYLW-X06的系统进化分析GenBank8WYLW-X06,3.3,WYLW-X06,99%,WYLW-X06.16SrDNABiolog,WYLW-X06.2.5　WYLW-X062.5.1　菌株WYLW-X06的好氧反硝化性能研究KNO3,,78mg/L,C/N15,DO120r/min,pH7.0,30℃,4d.WYLW-X064.21282　　　　　　:-2　WYLW-X0616SrDNAPCRFig.2　Electrophoresismapofcloningprocedureof16SrDNAfromWYLW-X063　WYLW-X06Fig.3　ConsensusphylogenetictreeforstrainWYLW-X064　WYLW-X06Fig.4　AerobicdenitrificationofstrainWYLW-X064,78mg/L2.09mg/L,97.32%,96.35%..WYLW-X06,.2.5.2　菌株WYLW-X06的异养硝化好氧反硝化性能和脱氮产物N2O的研究(NH4)2SO4,,85mg/L,COD/N20,DO120r/min,pH7.0,30℃,58h,2h.WYLW-X065.5　WYLW-X06Fig.5　EffectofstrainWYLW-X06onnitrogenandcarbonsourceconcentrationinthedenitrificationculturemediumunderaerobicincubation5,,97.19%,96.63%,.,4h,OD600,56h0.838.,,.,,.CODCr1565.20mg/L165.6mg/L,1399.6mg/L,COD89.4%.CODCr,,CODCr.1.5.2WYLW-X06N2O,,N2O1.848790mg.N2O,N2O,.30℃,N2O2.62×105Pa,101325Pa,x＊A=P·yA/kAN2O0.000197mg.,WYLW-X0622()　　　　　　　　　　　　　20103N2O1.848987mg.N2O-NTIN0.572%.(TIN=NH+4-N+NO-2-N+NO-3-N)WYLW-X06N2O,:WYLW-X06N2O,N2O.3　　1)BTB,--WYLW-X06.、、Biolog,16SrDNA,WYLW-X06(Bacilluscereus).2),-WYLW-X06,.WYLW-X06,97.19%,.,,,※※※-,N2O,N2O..3),Biolog16SrDNA.,,,.:[1]　,.[J].,2008,31(2):25-27.[2]　,,.、[J].,2008,20(3):334-338.[3]　,,,.[J].,2006(7):153-156.[4]　PatureauD,ZumsteinE,DelgenesJP,etal.Aerobicdenitrificationisolationfromdiversenaturalandmanagedecosystems[J].MicrobEcol,2000,39:145-152.[5]　,,,.[J].,2009,29(1):47-52.[6]　,,.N2O[J].,2005,6(12):42-47.[7]　TakayaN,MariaAntoninaBCS,YasushiS,etal.Aer-obiedenitrifieationbacteriathatproducelowlevelsofni-trousoxide[J].AppandEnvirMicrobiol,2003,69(6):3152-3157.[8]　,,,.[J].,2007(1):11-13.[9]　,,.-[J].,2008,3(21):121125.[10]　AndersonIC,PothM,HomsteadJ,etal.Acompari-sonofNOandN2Oproductionbytheautotrophicnitrifi-ernitorsomonaseu-ropaeaandtheheterotrophicnitrifieralcaligenesfaecalis[J].AppandEnvirMicrobiol,1993,59(11):35253533.[11]　,,.[J].,2005,24(4):440-440.[12]　,.[M].:,2006.[13]　,,.Biolog-[J].,2006,32(5):50-54.[14]　CW,GS.PCR[M].:,2006.[15]　JeannmouginF,ThompsonJD,GouyM