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分类号密级UDC编号中国科学院研究生院硕士学位论文酿酒酵母和运动发酵单胞菌木薯乙醇发酵条件研究周玲玲指导教师赵海研究员中国科学院成都生物研究所申请学位级别理学硕士学科专业名称环境科学论文提交日期2008年5月论文答辩日期2008年5月培养单位中国科学院成都生物研究所学位授予单位中国科学院研究生院答辩委员会主席ETHANOLFERMENTATIONFROMCASSAVABYSaccharomycescerevisiaeandZymomonasmobilisADISSERTATIONSUBMITTEDTOGRADUATEUNIVERSITYOFTHECHINESEACADEMYOFSCIENCESINCANDIDATEFORMASTERDEGREEOFSCIENCECANDIDATE:ZHOULINGLINGADVISOR:Prof.ZHAOHAICENTERFORAPPLIEDANDENVIRONMENTALMICROBIOLOGYCHENGDUINSTITUTEOFBIOLOGYTHECHINESEACADEMYOFSCIENCESMay，2008II中文摘要酿酒酵母和运动发酵单胞菌木薯乙醇发酵条件研究周玲玲(环境科学)指导老师：赵海研究员(中国科学院成都生物研究所，成都，610041)摘要：本文结合我国燃料乙醇发展的方针政策，以酿酒酵母和运动发酵单胞菌为菌种研究其在非粮能源作物木薯中乙醇发酵的情况，为木薯原料更好地应用于生产中提供了理论依据。酿酒酵母木薯高浓度乙醇发酵的研究。实验采用的木薯干淀粉含量约70-75%。以酿酒酵母为菌种进行高浓度乙醇发酵的工艺条件研究，昀佳条件为：木薯干粉碎细度为35目，料水比1：2，α-淀粉酶用量0.09KNU/g淀粉，蒸煮温度85℃，蒸煮时间15min。采用30℃同步糖化发酵工艺，糖化酶用量为3.4AGU/g淀粉，发酵时间30h。在10L发酵罐中，乙醇质量比达127.88g/kg，发酵效率为88.28%，发酵强度4.263g/kg/h，100L中试研究中乙醇浓度为127.75g/kg，发酵强度4.258g/kg/h。利用高效液相色谱对发酵液中残糖进行了分析，证明葡萄糖、果糖等单糖已完全被菌体利用，剩余糖为二糖，三糖等不可发酵的低聚糖。运动发酵单胞菌快速乙醇发酵的研究。对实验室保藏的8株运动发酵单胞菌进行比较，选择发酵速度昀快的Zymomonasmobilis232B进行研究。该菌在纯葡萄糖中的昀佳发酵条件为：葡萄糖浓度18%，起始pH6-7，发酵温度30℃，发酵时间18h，乙醇浓度88g/kg。在以木薯为底物同步糖化快速乙醇发酵中，采用FullFactorial设计和昀速上升实验确定了培养基成分中的2个显著性因子及其昀适浓度：酵母粉4g/kg，硫酸铵0.8g/kg。在昀适培养基条件下，对木薯料水比和糖化酶用量进行了优化，得到Z.mobilis232B木薯乙醇发酵昀佳料水比1：3，糖化酶浓度4AGU/g淀粉，乙醇发酵4.915g/kg/h。利用高效液相色谱对发酵液中残糖进行了分析，剩余糖为二糖，三糖等，但成分较酵母发酵后复杂。关键词：乙醇发酵；木薯；酿酒酵母；运动发酵单胞I硕士学位论文酿酒酵母和运动发酵单胞菌木薯乙醇发酵条件研究II英文摘要EthanolFermentationfromCassavabySaccharomycescerevisiaeandZymomonasmobilisZhouLingling(EnvironmentalScience)DirectedbyProf.ZhaoHai(ChengduInstituteofBiology,CAS,Chengdu,610041)Abstract:Accordingtothefuelethanoldevelopmentplansandpoliciesinourcountry,theethanolproductionfromcassavabySaccharomycescerevisiaeandZymomonasmobiliswasstudied.Itprovidedtheoreticalbasisforethanolfermentationbycassavainindustry.Part1isthestudyofVHG(veryhighgravity)ethanolfermentationbySaccharomycescerevisiae.Thecontentofstarchincassavawas70-75%.Comparedwiththeperformancesunderdifferentexperimentalconditions,thefollowingoptimalconditionsforVHGfermentationwereobtained:Granulesizeofdrycassava35mashes,hydromodulusofcassavatowaterat1:2,α-amylaseenzymedosage0.09KNU/gstarch,cookingtemperature85℃for15min,usingtheSSFprocess(simultaneoussaccharificationandfermentation)andtheamountofglucoamylase3.4AGU/gstarch.Accordingly,thefinalethanolconcentrationwasupto127.88g/kg;theethanolyieldreached88.28%,andethanolproductivitywas4.263g/kg/hafter30h.Whenthefermentationscaleexpandedto100L,thefinalethanolconcentrationwas127.75g/kg,andtheethanolproductivitywas4.258g/kg/hin30h.Theresidualsugarwasanalyzedbyhighperformanceliquidchromatography,andprovedthattherewasnoglucoseandfructose.TheresidualreducingsugarwassomeunfermentableoligosaccharidePart2isthestudyoftherapidethanolproductionbyZymomonasmobilis.Comparewithothersevenstains,Zymomonasmobilis232Bwasselectedforresearch.Theoptimumconditioninglucosemediumwasasfollow:glucoseconcentration18%,initialpH6-7,andfermentationtemperature30℃.Theethanolconcentrationwas88g/kgin18h.Afterthat,rapidethanolproductionfromcassavainSSFbyZymomonasmobilis232Bwasstudied.ThroughaseriesofexperimentsaidedbyFullFactorialDesignandsteepestascentsearch,theoptimalconcentrationyeastextractandammoniumsulfateweredetermined:4g/kgand0.8g/kg,each.Underoptimummediumconditions,theoptimalhydromodulusofcassavatowaterandglucoamylasedosageswereobtained:hydromodulusofcassavatowaterat1:3andglucoamylasedosages4AGU/gstarch.Theethanolproductionreached4.915g/kg/h.TheresidualsugarwasanalyzedbyHPLC,andprovedthattheresidualreducingIII硕士学位论文酿酒酵母和运动发酵单胞菌木薯乙醇发酵条件研究sugarwassomeunfermentableoligosaccharide，butthecomponentsweremorecomplexthanthatfermentationbySaccharomycescerevisiae.Keywords:ethanolfermentation;cassava;Saccharomycescerevisiae;ZymomonasmobilisIV目录目录第一章研究背景与研究内容.........................................11.1木薯的基本特性.................................................11.2木薯乙醇发酵的意义.............................................21.3研究内容.......................................................21.4实验设计流程图.................................................3参考文献...........................................................4第二章酿酒酵母木薯高浓度乙醇发酵研究..............................52.1引言...........................................................52.2材料与方法.....................................................52.2.1菌种.......................................................52.2.2培养基成分.................................................52.2.3主要试剂与仪器.............................................62.2.4培养方法...................................................62.2.5分析方法...................................................72.2.5.1菌体OD值的测定.........................................72.2.5.2还原糖浓度的测定.......................................72.2.5.3总糖浓度的测定.........................................72.2.5.4残糖成分分析方法.......................................72.2.5.5乙醇浓度的测定.........................................72.2.5.6pH值的测定.............................................82.2.5.7粘度的测定.............................................82.2.6计算公式...................................................8