产甲烷菌的分离纯化培养及其培养基对于菌株的选择作用

,(,100193):,,10,:;;:Q93-3:A:1671-7236(2010)06-0032-04(euryarchaeota),,,,,:2009-12-01:(1989-),,,,::,,,E-mail:[e-mail]:(2006A84);11.1产甲烷菌的典型生境,3:¹;º;»(,1991)1701m(,1990)1.2产甲烷菌的生物学地位,(,2003),,,Abstract:Tostudytherelationshipbetweenfluorosisandhepatocyteapoptosisandmorphologicalchangesinmeatducks,1608-day-oldTian-fumeatduckswerechosenandrandomlydividedintofourgroups,and0,350,450and550mgofsodiumfluoridewereaddedtoconventionaldietaryperkgrespectively.AfterchallengefiveduckswerekilledineachgroupeveryeightdayandTdT-mediateddUTPnickend-labeling(TUNEL)methodwasusedforexperimentalfluorosislivertissuehandlingandlabeling.Percentageofhepatocyteapoptosisandmorphologicalchangesweredetectedonthe24th,40thand56thdaywiththelightmicroscope.TUNELresultsshowedthatDABstainingpositivecellsappearedtypicalmorphologicalcharacteristicsandthepercentageofapoptosiscellincreasedobviouslycomparedwiththecontrolgroup.Fluorosisinmeatducksmainlycausedearlyandmiddlehepatocyteapoptosis.Withtimeextensionanddoseincrease,sodiumfluorideacceleratedapoptosisofhepato-cyteanddevelopedintonecrosis,indicatingthattoxiceffectsofexcessivefluorideinvivoiscloselyrelatedtohepatocyteapopto-sis.Keywords:meatduck;fluorosis;hepatocytes;apoptosis#32#201037625(,2007),74%(Lowe,2006);,(Moss,1993),,,22.1产甲烷菌的分类界定Balch19793,4,713,,200951031200(,2009)5Metha-nobacteriales,Methanococcales,Methanomicrobiales,Metha-nosarcinales,Methanopyrales(Liu,2008)2.2产甲烷菌的生理特征16SrRNA,,,:,,(,);,16SrRNA,pH,,;NH3,Ni()FeCoMoSe(,2008),MHS-B(Gar-cia,2000)F420-NADPHNADPH(,2005),-F420420nm,33.1产甲烷菌分离纯化历程,1950,,/0,Balch(1979),,,,/0,,Wolfe(1979),(Balch,1979;Fox,1979)3.2亨盖特厌氧滚管技术,,:¹4~5mL,47e;º(),39e;»,,CO2;¼(Hungate,1969)3.3现有技术的局限性,,,,,,,,,;,,#33#201037644.1产甲烷菌的主要营养代谢类型2:,(Ferry,1997),3:¹(hydrogenotrophicmethanogenesis),H2/CO2,50%;º(acetoclasticmetha-nogenesis),,,98%~99%,;»(methylotrophicmethanogene-sis),()()(,2002)4.2底物对于产甲烷菌的选择作用,,,Jarvis(2000),3Balch,,3,BRM9BRM16H2/CO2,3CM1H2/CO2,316SrDNA,BRM9BRM16CM1(Methanobacteriumformicicum),(Methanomicrobiummobile)(Methanosarcinabarkeri)(Jarvis,2000),H2/CO2;H2/CO2;H2/CO2(Boone,1988),,5,,(:Cr6+Cd2+Cu2+Zn2+Ni2+)(,2004)2-(BES)2-BESM,:-S-M(Muller,1993),,(Methanomicrobiummobile)(Methanobrevibactersmithii)(Methanosarcina)(Tewart,1997)(Methanobre-vibacterruminantium)(Metha-nomicrobiummobile),(Ungerfeld,2003)2-,(Methanosarcina)(Meth-anomicrobiummobile)(Sprott,1993),BES2-,,(Ungerfeld,2004),6,,,,,,#34#2010376,1,,.[J].,1991,6(3):62~68.2,,.[J].,2003,23(6):42~46.3,,.[J].,1990,2(2):12~19.4,.[J].,2002,75(1):181~184.5,,,.[J].,2007,29(2):58~63.6,,,.[J].,2008,31(3):5~8.7,,.[J].,2007,35(1):111~113.8,,,.Bt/0[J].,2004,10(1):63~67.9,,.[J].,2005,45(1):145~148.10,.[J].,2009,15(4):574~578.11NeetaA,,.[J].,2009,36(10):24.12BalchWE,WolfeRS.SpecificityandbiologicaldistributionofcoenzymeM(2-mercaptoethanesulfonicacid)[J].JBacteriol,1979,137(1):256~263.13BooneDR,WhitmanWB.Proposalofminimalstandardsforde-scribingnewtaxaofmethanogenicbacteria[J].IntJSystBacte-riol,1988,38:212~219.14FerryJG.Methane:smallmolecule,bigimpact[J].Science,1997,278(5342):13~14.15FoxGE,BalchWE,MagrumLJ,etal.Reevaluationofauniquebiologicalgroup[J].MicrobiolMolBiolRev,1979,43(2):260~269.16GarciaJL,PatelKC,OllivierB.Taxonomic:phylogeneticandecologicaldiversityofmethanogenicArchaea[J].Anaerobe,2000,6:205~226.17HungateRE.Arol-ltubemethodforcultivationofstrictanaer-obes[J].MethodsMicrobiol,1969,36:117~132.18JarvisGN,StromplC,BurgessDM,etal.Isolationandidentif-icationofruminalmethanogensfromgrazingcattle[J].CurrentMicrobiology,2000,40:327~332.19LiuYC,WilliamBW.Metabolic,phylogenetic,andecologicald-iversityofthemethanogenicArchaea[J].AnnNYAcadSci,2008,1125:171~189.20LoweDC1Globalchange:agreensourceofsurprise[J].Na-ture,2006,439(7073):148~149.21MossAR,Methane.Globalwarmingandproductionbyanimals[M].1st1Kingston,Kent,UK:ChalcombePublications,1993122MullerV,BlautM,GottschalkG.Bioenergeticsofmethanogene-sis.InMethanogenesis.Ecology,Physiology,Biochemistry&Geneticsed[M].FerryJG,NewYork:Chapman&Hall,1993,360~406.23SprottGD,BeveridgeTJ.microscopy.InMethanogenesis.E-cology,Physiology,Biochemistry&Geneticsed[M].FerryJG,NewYork:Chapman&Hall,1993,81~127.24TewartCS,FlintHJ,BryantMP.Therumenbacteria.InTheRumenMicrobialEcosystem[M].HobsonPN,StewartCS,London:BlackieAcademic&Professional,1997,10~72.25UngerfeldEM,RustSR,BooneDR,etal.Effectsofseveralin-hibitorsonpureculturesofruminalmethanogens[J].JournalofAppliedMicrobiology,2004,97:520~526.26UngerfeldEM,RustSR,BurnettR.Useofnovelalternativee-lectronsinkstoruminalmethanogenesis[J].ReproductionNutr-itionDevelopment,2003,43:189~202.IsolationandPurificationofMethanogensandtheirSelectiveActionofMediumsPANGDe-gong,YANGHong-jian(CollegeofAnimalScienceandTechnologyofChinaAgriculturalUniversity,Beijing100193,China)Abstract:Methanogenisakindofstrictlyanaerobicarchaeathatcantransformorganicmatterandinorganiccompoundsin-tomethaneandcarbondioxideinananaerobicfermentation.Methanogenesisusuallyoccursinthelaststepofanaerobicfermen-tationprocess.Beingthekeypartofthenaturecarboncycle,end