发酵产氢细菌分离培养的厌氧实验操作技术

©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(,150090):,.,,,Hungate..,550.:;;;;:X703:A:0367-6234(2004)12-1589-04Theexperimentalanaerobicoperationofisolationandcultureonhydrogen2producingandfermentativebacteriaLIYong2feng,RENNan2qi,CHENYing,LIJian2zheng,HULi2jie,ZHENGGuo2xiang(SchoolofMunicipalandEnvironmentalEngineering,HarbinInstituteofTechnology,Harbin150090,China)Abstract:Isolationandcultureofhydrogen2producingandfermentativebacteriaisanimportantfoundationonbiohydrogenproductionprocess.Therearecomplicatedoperationandcompositioninpresentanaerobictechniquesandculturemedia.Hungatetechniquewasimprovedandplateofculturebottlewasestablishedbycomparinganaerobicmethodsandbacteriumgrowth.Isolationandenrichmentculturemediawerecon2firmedbythetestofdifferentcompositionandthespeciesandamountofhydrogen2producingandfermenta2tivebacterium.550bacteriumstrainswereisolatedbytheanaerobicoperation.Keywords:biohydrogenproduction;hydrogen2producingandfermentativebacteria;isolationandculture;anaerobicoperation;culturemedia:2003-12-10.:(50125823);(G2000026402);(HIT.MD2002125).:(1961-),,;(1959-),,,,.,,.Hungate[1],[2-4].,;,,.,[5-14],,,23[611].,,,Kumar[11][5],.,.©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[5],.11210ml,,10,1h30min,,10,.20d,MPN[15].1.31ml()9ml,10-110-25,,10-1310-18.,.,(Hungate).-25.1.4[5].1.411Hungate[1]Hungate.(),,35,.11412[1618]11412113ml,,.1141212,,().5,,(),,..11413,,().,,.012015%,.,,..11511511[9]LM-1.11512HPB-1(g/l):,20;+(110+310);,210;,110;NaCl,310;K2HPO4,110;MgCl2,0105;FeS2O417H2O,0105;L-,015;LM-1[9];(012%);110210ml;,2010g.HPB-2(g/l):LM-1[9];01025g/l0101g/l,10ml.LM-1[9].2010,HPB-1.HPB-3(g/l):HPB-1[9],HPB-1[9],:(NiCL216H2O,01001;CaCl2.H2O,0101,ZnCl2,0102),HPB-1[9].HPB-LR:HPB-1HPB-2HPB-3LM-1,HPB-LR,(g/l):,2010;,110;,310;,210;,110;NaCl2,310;K2HPO4,110;L-,015;(,01025;0101,NICl216H2O,01001;Ca2Cl212H2O,0101,ZnCl2,0102)10ml;(012%)1ml;202510g;pH410415.11611530min,,120r/min.095136©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(99,99%).,.(,),.10ml,(1015cm),.21112.,,.45,10-1010-15,.,,.21113(,),,,..,,25..SIGMA,.400.21221211[20]5g/l,25g/l,;3ml,2cm;,013015ml,;35,,.,Hungatl.20.212.2[7]11512,(120),,,,23,,.10.,,.,,..213120.1.113cm..013015ml,,.(115),120.12.1,.214LM-1[9].,,CN..:,[21].:[21],,1g/l3g/l195112,:©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[9],PH(PH410415)[21].Mg2+,(ATP+NADP).,,,MgCl2MgSO47H2O,SO42-.Fe2+,.L-,,,[5].2152.,,.HPB-LR.(),,LM-1,4g/l1g/l3g/l,.PHPH415.2(ml-1)(A600nm)LM-111510122180HPB-111110113145HPB-211010132110HPB-321010114120HPB-LR1161011412031),Hungate.Hungate,,.2)--,25,.3)LM-1,(),,HPB-LR.4)HPB-LR.,.,.:[1]HUNGATERE.Methodsinmicrobiology[M].NewYork:Academicpress,1969.117-132.[2],.[M].:,2001.[3],.[J].,1994,21(2):119-120.[4],.-[J].(),1998,21(4):363-367.[5],,,.[J].,2003,24(1):80-84.[6],,.[J].,2003,35(4):398-402.[7]TANISHOS,SHIATALY.Continuoushydrogenpro2ductionfrommolassesbythebacteriumEnterobacteraerogenes[J].IntJHydrogenEnergy,1994,19(10):807-812.[8]TAGUCHIF,MIZUKAMIN,HASEGAWAK.Micro2bialconversionofarabinosandxylosetohydrogenbyanewlyisolatedClostridiumsp[J].CanJMicrobiol,1994,40:228-233.[9]TAGUCHIF,CHANGJD,MIZUKAMIN,etal.Iso2lationofahydrogen-producingbacterium,ClostridiumbeijerinckiistrainAM21B,fromtermites[J].CanJMi2crobiol,1993,39:726-730.[10]KUMARN,DASD.Enhancementofhydrogenpro2ductionbyEnterobactercloacaeIIT-BTO8[J].ProcBiochem,2000,35:589-593.[11]OOSHIMAH,TAKAKUWAS,KATSUDAT,etal.Productionofhydrogenbyahydrogenase2deficientmu2tantofRhodobactercapsulatus[J].JFermerntBioeng,1998,85(5):470-475.(1694)295136©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(q12)=1+k2(q12Ru),().(3):k(x)=3j1(x)/x,j1(x),Rg,RsRu,.22,,.,(2).(1)(2),(1),(2).,(2)q12Rs=mp(m=1,2,,)1,,qs=/Rs.(3),(2):1,,q12,q12qu=/Ru,[12],Rs=3/5Ru,qu=0177/Rs,q12,quqs,qus=qu-qs=0172/Rs.(2),qu1=/Rs,(3)qs1=4149/Rs=3145/Ru,qu1qs1.,(2)(3):1)q12;2).,.2.:[1]SATZH.CriticalbehaviorinfinitetemperatureQCD[J].PhysRep,1982,88(5):349-364.[2]BJORKENJD.Highlyrelativisticnucleus2nucleuscolli2sions:thecentralrapidityregion[J].PhysRevD,1983,27(1):140-151.[3]WEINERRM.Bosoninterferometryinhighenergyphysics[J].PhysRep,2000,327(5):249-346.[4]WIEDEMANNUA,HEINZU.Particleinterferometryforrelativisticheavyioncollisions[J].PhysRep,1999,319(4):145-230.[5]BOALDH,GELBKECK,JENNINGSBK.Intensityinterferometryinsubatomicphysics[J].RevModPhys,1990,62(3):553-303.[6]GYULASSYM,KAUFFMANNSK,WILSONLW.Pioninterferometryofnuclearcollisions.I.theory[J].PhysRevC,1979,20(6):2267-2292.[7]ZAJCWA,BISTIRLICHJA,BOSSINGHAMRR,etal.Two-pioncorrelationsinheavyioncollisions[J].PhysRevC,1984,29(6):2173-2187.[8],,,.2[J].,2001,25(2):149-153.[9],,,.2[J].,2000,24(6):524-527.[10],.2[J].,1999,23(11):1097-1101.[11].3[J].,1998,22(10):903-909.[12].2[J