反硝化细菌的分离筛选及其反硝化特性的初步研究曾庆武

2752008　10　　　　　　　JournalofHuazhongAgriculturalUniversityVol.27　No.5Oct.2008,616～620:2007-10-12＊“863”(2003AA601060)＊＊.E-mail:[e-mail],,1980,,430070曾庆武　梁运祥　葛向阳＊＊(华中农业大学农业微生物学国家重点实验室,武汉430070)　、202。、,1A13,16SrDNA,(Bacilluslicheniformis)。DNF409,30%。(,SAS9.1.3),25mg/L,CODMn35.1mg/L,32.5℃,6.2×106cfu/mL,114.2h,99%。　;;;;　Q939.9　　　A　　　　1000-2421(2008)05-0616-05　　,,、,、,,,,,。[1],。,,,。,。,:,,。。,1A13,,。　　[2]():2g、0.2g、0.5g、20g、1000mL;pH7.2。:1.8%。:LB。　DNF4091。　、、、、。[3-4],10mL1g100mL,30℃2d,10,,LB,。　1)。,,。,25mg/L,300mL,。2)。LB,28℃,　5:　200r/min20h,。3)。1%(1,),3,。3d、,。、,。。=-(+)×100%　[5]。[6]DNA,P0:5′-AGAGTTTGATCCTGGCTCAG-3′,Pc3:5′-GGTTACCTTGTTACGACTT-3′,16SrDNA,,GenBankBlast。　1)CODMn。[7],LBCODMn10.5mg/L,18.0mg/L,33.0mg/L,6000r/min6min,,,1%(106cfu/mL)1.41),28℃,3d、,。2)、pH、。[7],CODMn20℃、28℃、37℃,;1.61)pH6.0、7.0、8.0,28℃,pH;1.61)105、106、107cfu/mL,28℃,。　1.41)LBCODMn18mg/L,1%,12h、24h1%,3d,、,[8]。　2,(,SAS9.1.3),CODMn、、、,,45。,CODMnLB;,300mL,2.1%,6.3mL(2.6×108cfu/mL),,CODMn,106cfu/mL,,24h2,106～107cfu/mL。1。1　Table1　ListoffactorsandlevelsFactorsNameLevels-2-1012X1CODMn/(mg·L-1)15.022.530.037.545.0X2Temperature/℃2026323844X3Time/h487296120144X4Bacterialconcentration/%0.11.12.13.14.1　;N-(1-)-;CODMn[9]。　　,1)。,,202。,2A13。DNF409CODMn25mg/L,,,。。,,1、、A13,。2。617　　27　2　Table2　ResultsofdenitrificationabilityTreatmentNitrate/(mg·L-1)Nitrite/(mg·L-1)Percentageofdenitrification/%Control28.560.02A131.257.3070.1DNF4093.189.8054.6　　2)A13。A13LB,LB,,3。700bp16SrDNA,GenBankBlast,(Bacilluslicheniformis)。3　DNF409A131)Table3　ExperimentresultsofphysiologicalandbiochemicalpropertiesaboutDNF409andA13DNF409A132)++3)++4)++5)++6)-+7)15)V.P8)-+9)++10)--11)16)17)12)++13)++14)++　1)“+”,“-”“+”showspositive,“-”negative;2)Gram'sstain;3)Gelatin-liquefaction;4)Nitratedisoxidation;5)Nitritedisoxidation;6)Methylred;7)Oxidationandfermentationforglucose;8)Vogesproskauer;9)Indoleproducing;10)Lipase;11)Litmusmilk;12)H2Sproducing;13)H2O2;14)Amylohydrolys-is;15)Fermentationtype;16)Peptonization;17)Acidproducingandconcreting　,,,　　1)CODMnA13。CODMn,,CODMn(4),,CODMn10.5mg/L,2(Bacilluslicheniformis),CODMn18.0mg/L,2,,CODMn33.0mg/L,。4　CODMnA13Table4　EffectofCODMnonpercentageofdenitrificationaboutA13CODMn/(mg·L-1)Percentageofdenitrification/%A13DNF40910.51.20.918.018.223.733.098.792.6　　2)A13。5。5,CODMn33.0mg/L,20℃,,,28℃37℃,。:CODMn,20℃、28℃、37℃。CODMn,28℃37℃,20℃。5　A13Table5　EffectoftemperatureonpercentageofdenitrificationaboutA13Temperature/℃Percentageofdenitrification/%A13DNF4092077.170.62899.993.13798.692.6　　3)pHA13。,6.08.0pH,,,pH。4)A13。:,(6)CODMn18mg/L,107cfu/mL,。:CODMn10mg/L,107cfu/mL,;CODMn30mg/L,105cfu/mL,。6　A13Table6　EffectofbacterialconcentrationonpercentageofdenitrificationaboutA13/(cfu·mL-1)BacterialconcentrationPercentageofdenitrification/%A13DNF40910524.330.510634.029.910799.897.2　2DNF409A13618　5:　,DNF409,,A13,DNF409。DNF409,A13。,,1。1　A13DNF409Fig.1　ExperimentresultsofcombinationapplicationofA13andDNF409　1.A13　A13applieddirectly;2.DNF409DNF409applieddirectly;3.A13DNF409A13andDNF409appliedtogether;4.A13A13applieddou-bly;5.DNF409DNF409applieddoubly;6.A13,12hDNF409A13appliedfirst,thenDNF409applied12hlater;7.DNF409,12hA13DNF409appliedfirst,thenA13applied12hlater;8.A13,24hDNF409A13appliedfirst,thenDNF409applied24hlater;9.DNF409,24hA13DNF409appliedfirst,thenA13ap-plied24hlater,,,。3,2,2(4,5),。DNF409,24hA13(9),,,30%。,。　CODMn、、、,,7。SAS9.1.3,CODMn、、,2～6d,。Y1=9841.218+1232.407X1+394.595X2+783.350X4-638.550X12-967.129X1X4-512.220X22,CODMn35.1mg/L,32.5℃,114.2h,2.4%(6.24×106cfu/mL),0.0001,,。,,3,,。7　Table7　ExperimentdesignandresultsofresponsesurfaceanalysisRunX1X2X3X4/%Percentageofdenitrification1-1-1-1-163.62-1-1-1195.03-1-11-178.54-1-11199.05-11-1-178.86-11-1199.87-111-176.28-111197.491-1-1-198.3101-1-1199.9111-11-199.2121-11199.61311-1-199.81411-1199.915111-199.016111199.817-200066.918200099.8190-20077.720020097.02100-2099.822002099.623000-294.324000299.925000099.826000099.327000099.528000099.729000098.330000099.631000099.9　　[12],,,;,CODMn100mg/L;,CODMn20mg/L;,,CODMn。619　　27　,,CODMn20mg/L,[10-12]。,,,DNF409,1DNF409A13,。,,DNF409,24hA13,。,:CODMn[13]。CODMn10mg/L,2;CODMn18.0mg/L,,,;CODMn30.0mg/L,2。[1]　,,.[J].,2003,22(3):247-250.[2]　,.[M].:,2002:69-77;251-266.[3]　,.1[J].,2006,25(5):530-534.[4]　,,.[J].:,2005,28(10):119-123.[5]　,.[M].:,2001:62-65.[6]　SAMBROOKJ,RUSSELLDW.Molecularcloning:alaborato-rymanual[M].3rded.NewYork:ColdSpringHarborLabora-toryPress,2001:27-30.[7]　,,.[M].:,2004:55-74.[8]　,,.-[J].,2006,29(3):9-10.[9]　《》.[M].3.:,1989.[10]DE-KLEINCAM,VAN-LOGTESTIJNRSP.DenitrificationinthetopsoilofmanagedgrasslandsinTheNetherlandsinre-lationtosoiltypeandfertilizerlevel[J].PlantandSoil,1994,163:33-44.[11]MAHOODT,ALIR,MALIKKA,etal.Seasonalpatternofdenitrificationunderanirrigatedwheat-maizecroppingsystemfertilizedwithureaandfarmyardmanureindifferentcombina-tions[J].BiolFertilSoils,2005,42:1-9.[12]KENNETHJ,FORSHAY,EMILYH,etal.Rapidnitratelossanddenitrificationinatemperateriverfloodplain[J].Biogco-chemistry,2005,75:43-64.[13],,.FY99-01[J].,2006,25(4):404-407.ScreeningofDenitrobacteriaandPreliminarySt