植物纤维的酒精发酵
摘要
由于原油和天然气资源逐渐减少以及人们对经济稳定、环保效果和全球变暖的关注，用燃料乙醇替代汽油已经成为目前的一大热点。廉价的原料和高效转化工艺是燃料乙醇商业化生产的必要条件。植物纤维原料的水解液中木糖占还原糖的比例将近1／3，选育高效的木糖发酵菌是植物纤维原料生产酒精的重要技术关键之一。为提高木糖发酵能力，本文对自筛菌株的发酵性能进行研究，并以管囊酵母作为出发菌株对其进行了紫外和化学诱变处理。
对从自然界中筛选出的一株能利用木糖产酒精的菌株1125-3进行发酵性能的研究，研究了不同条件下自筛菌生长和发酵的规律。自筛菌株定向驯化后对木糖的利用率可达92.92％，酒精得率达12.88％。菌株1125-3的最适生长温度为30℃、pH值范围为4.6～5.0；最适发酵温度为37℃、pH值为6.0左右；用尿素作为单一氮源自筛菌1125-3的生长和发酵情况良好。自筛菌的混合糖发酵效果优于单纯木糖发酵，在2.5％葡萄糖与2.5％木糖做混合碳源发酵时，其酒精得率达到了纯葡萄糖发酵水平。
通过对嗜单宁管囊酵母的紫外和化学诱变，以酒精产量为筛选因子，最终筛选获得一株酒精发酵高产株Z30-4。菌株Z30-4在30℃、100rpm、摇瓶发酵72h，酒精产量可达13.33g·L~(-1)，酒精得率达81.15％，与出发菌株相比酒精产量和酒精得率均提高了3倍多。对木糖的利用率为71.42％，和原始菌株相比变化不大。
管囊酵母在传代过程中出现退化现象，适当降低培养基氮源含量，结合纯种筛选可以快速地恢复菌种的性状。经过一代复壮培养就可以使发酵周期较退化株缩短2／3，48h酒精产量是退化株的1.5倍，效果明显。
Using fuel ethanol as a substitute for gasoline, is expected to be a hotspot at present because of the diminishing of crude oil and natural gas resource, and the concerns related to economic stability, environmental impact and global warming. Cheap raw material and high ethanol yield are needed for commercial fuel-ethanol production . Xylose accounts for up to 30% of lignocellulose sugars, so effective xylose fermentation is one of the most critical features in the industrial ethanol production from lignocellulose. In order to get some high - yield strains, the main research contents of our task are optimization of ferment condition, carring on UV and chemistry mutagen to Pachysolen tannophilus, different mutant modes and screening factors were used.
The fermentation property of the wild yeast 1125-3, which can quickly and effectively ferment xylose to ethanol, was studied. After domestication and cultivation, the utilization ratio of xylose is 92.92%, the ethanol yield rate is 12.88%. Effects of culture condition, including temperature, pH value and nitrogen source on fermentation were investigated. The optimum temperature and pH value of the growth were 30℃and 4.6～5.0, respectively. The optimum temperature and pH value of the xylose fermentation were 37℃and 4.6～5.0, respectively. The growth and fermentation were good with urea as the nitrogen source. When a mixed sugar with 2.5% glucose and 2.5% xylose was present in the fermentation culture medium, the ethanol yield rate of strain 1125-3 can attain the pure glucose fermentation level.
Pachysolen tannophilus was taken as the the starting strain and the ethanol yield rate as screening factor, after UV and chemistry mutagen, a stable strain Z30-4 was obtained. After 72h of fermentation at 30°C and an agitation rate of 100rmp on a rotary shaker, the alcohol concentration is 13.33g·L~(-1) and the ethanol yield rate is 81.15%, which was increased by 200% compared with original strain. The utilization ratio of xylose is 71.42 %, as same as the original strain.
The fermentation quality of Pachysolen tannophilus degenerated during breeding. The degenerated strain was purified and rejuvenated by adjusting the kind of nitrogen source and the c/n. The fermentation cycle was shorten after rejuvenation. After 48h of fermentation, the ethanol fermentation power of the rejuvenated strains were about 150% of the original retrogressive strains.
对从自然界中筛选出的一株能利用木糖产酒精的菌株1125-3进行发酵性能的研究，研究了不同条件下自筛菌生长和发酵的规律。自筛菌株定向驯化后对木糖的利用率可达92.92％，酒精得率达12.88％。菌株1125-3的最适生长温度为30℃、pH值范围为4.6～5.0；最适发酵温度为37℃、pH值为6.0左右；用尿素作为单一氮源自筛菌1125-3的生长和发酵情况良好。自筛菌的混合糖发酵效果优于单纯木糖发酵，在2.5％葡萄糖与2.5％木糖做混合碳源发酵时，其酒精得率达到了纯葡萄糖发酵水平。
通过对嗜单宁管囊酵母的紫外和化学诱变，以酒精产量为筛选因子，最终筛选获得一株酒精发酵高产株Z30-4。菌株Z30-4在30℃、100rpm、摇瓶发酵72h，酒精产量可达13.33g·L~(-1)，酒精得率达81.15％，与出发菌株相比酒精产量和酒精得率均提高了3倍多。对木糖的利用率为71.42％，和原始菌株相比变化不大。
管囊酵母在传代过程中出现退化现象，适当降低培养基氮源含量，结合纯种筛选可以快速地恢复菌种的性状。经过一代复壮培养就可以使发酵周期较退化株缩短2／3，48h酒精产量是退化株的1.5倍，效果明显。
Using fuel ethanol as a substitute for gasoline, is expected to be a hotspot at present because of the diminishing of crude oil and natural gas resource, and the concerns related to economic stability, environmental impact and global warming. Cheap raw material and high ethanol yield are needed for commercial fuel-ethanol production . Xylose accounts for up to 30% of lignocellulose sugars, so effective xylose fermentation is one of the most critical features in the industrial ethanol production from lignocellulose. In order to get some high - yield strains, the main research contents of our task are optimization of ferment condition, carring on UV and chemistry mutagen to Pachysolen tannophilus, different mutant modes and screening factors were used.
The fermentation property of the wild yeast 1125-3, which can quickly and effectively ferment xylose to ethanol, was studied. After domestication and cultivation, the utilization ratio of xylose is 92.92%, the ethanol yield rate is 12.88%. Effects of culture condition, including temperature, pH value and nitrogen source on fermentation were investigated. The optimum temperature and pH value of the growth were 30℃and 4.6～5.0, respectively. The optimum temperature and pH value of the xylose fermentation were 37℃and 4.6～5.0, respectively. The growth and fermentation were good with urea as the nitrogen source. When a mixed sugar with 2.5% glucose and 2.5% xylose was present in the fermentation culture medium, the ethanol yield rate of strain 1125-3 can attain the pure glucose fermentation level.
Pachysolen tannophilus was taken as the the starting strain and the ethanol yield rate as screening factor, after UV and chemistry mutagen, a stable strain Z30-4 was obtained. After 72h of fermentation at 30°C and an agitation rate of 100rmp on a rotary shaker, the alcohol concentration is 13.33g·L~(-1) and the ethanol yield rate is 81.15%, which was increased by 200% compared with original strain. The utilization ratio of xylose is 71.42 %, as same as the original strain.
The fermentation quality of Pachysolen tannophilus degenerated during breeding. The degenerated strain was purified and rejuvenated by adjusting the kind of nitrogen source and the c/n. The fermentation cycle was shorten after rejuvenation. After 48h of fermentation, the ethanol fermentation power of the rejuvenated strains were about 150% of the original retrogressive strains.
引文
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[3] 张远欣．燃料乙醇的发展状况[J]．甘肃科技，2005，21(4)：117～119
[4] George. P P, Tammy K S. Limiting factors in the simultaneous saccharification and fermentation process for conversion of cellulosic biomass to fuel ethanol [J]. Appl biochem biotechnol, 1995, 51-52:117-124
[5] Arnaldo V. The Brazilian ethanol experiences fuel as fuel for transportation[R].The Word bank. Biomass Energy Workshop and Exhibition, 2003
[6] Goldenberg J, Coelho S T, Nastari P M, et al. Ethanol leering curve-the Brazilian experience [J]. Biomass and bioenergy, 2004, 26:301-304
[7] Foley. Renewable energy in third world energy assistance [M].Energy Policy, 1992
[8] Goldenberg J, Monaco L, Marcedo Ⅰ. The Brazilian fuel-alcohol program [J]. Renewable Energy Sources for Fuels and Electricity, 1991
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[10] 魏忠贺．探索中国燃料乙醇发展之路[J]．中国石油和化工经济分析，2007，03：46-49
[11] 姚向君，田宜水．生物质能资源洁净转化利用技术[M]．北京：化学工业出版社，2005．162
[12] 黄忠水，纪威．我国开发燃料酒精的综合效益分析[J]．节能，2001，(12)：3-7
[13] Reddy V, Yang Y. Biofibers from agricultural by products for applications [J]. Trends in Biotechnology, 2005, 23(1): 22-27
[14] 魏述众．生物化学[M]．北京：中国轻工业出版社，2002：26-27
[15] 王超，章超桦．酶解纤维素类物质生产燃料酒精的研究进展[J]．节能，2003，12：6-9
[16] 高洁，汤烈贵．纤维素科学[M]．北京：科学出版社，1999
[17] 夏延斌．食品化学[M]．北京中：国农业出版社，2004：49-50
[18] 吴坤，张世敏，朱显峰．木质素生物降解研究进展[J]．河南农业大学报，2000，34(4)：349-354
[19] 蒋挺大，木质素．资源化技术丛书[M]．北京：化学工业出版社，2001．2
[20] Anil lachke. Biofule from D-xylose-the second most abundant sugar[J]. Resonance, 2002, 5:50-58
[21] 张继泉，王瑞明，孙玉英．利用木质纤维素生产燃料酒精的研究进展[J]．酿酒科技，2003，(1)：39-42
[22] 杨雪霞，陈洪章，李佐虎．玉米秸秆氨化汽爆处理及其固体发酵[J]．过程工程学报，2001，1(1)：86-89
[23] 陈洪章，李佐虎．秸秆蒸汽爆破的研究[J]．纤维素科学与技术，1998，7(2)：60-67
[24] 李步海，孙小梅，郝文颖．酶解蔗渣蒸汽预处理研究[J]．中南民族学院学报(自然科学版)，1995，14(1)：37-39
[25] 陈洪章，李佐虎．生化工程新理念及其技术范例[J]．中国生物工程杂志，2002，22(3)：74-77
[26] 詹怀宇．纤维化学与物理[M]．北京：科学出版社，2005．132
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