燃料酒精高产酵母菌株选育及应用研究
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摘要
从来源不同的基质中分离得到115株酵母菌，经初筛、复筛和酒精发酵试验，最终获得一株适宜以玉米为原料，可进行浓醪酒精发酵的高产酵母菌株，编号为SP-48。进行了以下生理生化实验：发酵糖类试验，可发酵葡萄糖、蔗糖、麦芽糖、半乳糖，不发酵乳糖；同化碳源试验，可同化棉子糖、半乳糖、麦芽糖、海藻糖、蔗糖，不同化纤维二糖、肌醇、核糖、赤藓糖醇、鼠李糖、乳糖、木糖、核糖醇、柠檬酸、琥珀酸、甘露醇、可溶性淀粉、阿拉伯糖；同化氮源试验，不能同化硝酸盐、盐酸乙胺、盐酸尸胺：可在无维生素培养基上生长；可在50％(w/w)葡萄糖酵母膏培养基中生长；37℃下可生长；不能在100ppm放线菌酮中生长。并对照模式菌株的生理特性，鉴定SP-48菌株属于酵母属(Saccharomyces)的酿酒酵母(Saccharomyces cerevisiae)。
本研究确定了SP-48生长发酵的最佳生长温度、pH；研究了温度、pH和接种量对生长和发酵曲线的影响：研究了SP-48菌株的耐酸碱；耐酒精；耐盐；耐糖和耐发酵副产物特性。结果表明，其最适生长温度为30℃，最适生长pH为4.5；最适发酵温度为30℃，最适发酵pH为4.5；温度和接种量对生长和发酵曲线的影响显著，pH对生长和发酵曲线的影响不显著；耐酒精能力为20％(v/v)，可在含18％(v/v)酒精的培养基中发酵；耐盐能力为7％；可在50％(w/w)的葡萄糖溶液中发酵；并对乙酸、乳酸和甘油有一定的耐受能力。
采用四因素三水平正交试验确定了SP-48菌株的浓醪酒精发酵工艺。浓醪发酵的最佳工艺条件为：料水比为1：2，糖化酶用量为300IU/g原料，接种量为6％(v/v)，初始pH值为4.5。按照不同的料水比例，应用10L发酵罐进行小试的实验数据表明，料水比为1：2时，72h的酒精产量为16.2％(v/v)，淀粉利用率为90.8％，比ADY在同样条件下产量高出15.7％；料水比为1：3时，72h的酒精产量为12.0％(v/v)，淀粉利用率为89.4％，比ADY在同样条件下产量高出6.2％；料水比为1：4时，72h的酒精产量为9.3％(v/v)，淀粉利用率为86.2％，比ADY在同样条件下产量高出9.4％。
115 isolates out of different environments were screened for fuel ethanol-producing yeast with high yield. Among them, the strain SP-48, which was able to use maize as fermentation substate to produce maximally alcohol, was identified as Saccharomyces cerevisiae based on its biophysiological and biochemical characteristics, and employed for further studies.
S. cerevisiae strain SP-48 grew the best in 36℃ and a pH of 4.5, whereas its optimum fermentation temperture and pH was 30℃ and 4.5, respectively. It was seen that temperature and initial inoculum's concentration were 2 important factors in affecting the growth and fermentation of strain SP-48. S. cerevisiae SP-48 showed good tolerances to various stresses including acids, alkalis, and osmotic pressure. Also, strain SP-48 showed better resistances to high concentration alcohol and byproducts than the produced-commercially ADY (active dried yeast).
If 33.3%(w/v) concentration of crude corn in fermenting material, 300 units alucoamylse per gram of raw corn, 6%(v/v) inoculum size of the yeast, and initial pH of 4.5 were fixed, orthogonal test indicated that strain SP-48 produced the highest amount of alcohol after incubated at 30℃ for 72 hours. Moreover, if strain SP-48 was inoculated to 10 L fermentter and controlled under the optimal conditions metioned above for alcohol production, the yield of alcohol at the end of fermentation was 16.2%(v/v). In the case of 25%(v/v) of the raw ground corn concentration, this strain produced only an alcohol level of 12.0%(v/v). In the case of 20%(v/v) of the raw ground corn concentration, the alcohol amount produced by strain SP-48 was only 9.3%(v/v).
本研究确定了SP-48生长发酵的最佳生长温度、pH；研究了温度、pH和接种量对生长和发酵曲线的影响：研究了SP-48菌株的耐酸碱；耐酒精；耐盐；耐糖和耐发酵副产物特性。结果表明，其最适生长温度为30℃，最适生长pH为4.5；最适发酵温度为30℃，最适发酵pH为4.5；温度和接种量对生长和发酵曲线的影响显著，pH对生长和发酵曲线的影响不显著；耐酒精能力为20％(v/v)，可在含18％(v/v)酒精的培养基中发酵；耐盐能力为7％；可在50％(w/w)的葡萄糖溶液中发酵；并对乙酸、乳酸和甘油有一定的耐受能力。
采用四因素三水平正交试验确定了SP-48菌株的浓醪酒精发酵工艺。浓醪发酵的最佳工艺条件为：料水比为1：2，糖化酶用量为300IU/g原料，接种量为6％(v/v)，初始pH值为4.5。按照不同的料水比例，应用10L发酵罐进行小试的实验数据表明，料水比为1：2时，72h的酒精产量为16.2％(v/v)，淀粉利用率为90.8％，比ADY在同样条件下产量高出15.7％；料水比为1：3时，72h的酒精产量为12.0％(v/v)，淀粉利用率为89.4％，比ADY在同样条件下产量高出6.2％；料水比为1：4时，72h的酒精产量为9.3％(v/v)，淀粉利用率为86.2％，比ADY在同样条件下产量高出9.4％。
115 isolates out of different environments were screened for fuel ethanol-producing yeast with high yield. Among them, the strain SP-48, which was able to use maize as fermentation substate to produce maximally alcohol, was identified as Saccharomyces cerevisiae based on its biophysiological and biochemical characteristics, and employed for further studies.
S. cerevisiae strain SP-48 grew the best in 36℃ and a pH of 4.5, whereas its optimum fermentation temperture and pH was 30℃ and 4.5, respectively. It was seen that temperature and initial inoculum's concentration were 2 important factors in affecting the growth and fermentation of strain SP-48. S. cerevisiae SP-48 showed good tolerances to various stresses including acids, alkalis, and osmotic pressure. Also, strain SP-48 showed better resistances to high concentration alcohol and byproducts than the produced-commercially ADY (active dried yeast).
If 33.3%(w/v) concentration of crude corn in fermenting material, 300 units alucoamylse per gram of raw corn, 6%(v/v) inoculum size of the yeast, and initial pH of 4.5 were fixed, orthogonal test indicated that strain SP-48 produced the highest amount of alcohol after incubated at 30℃ for 72 hours. Moreover, if strain SP-48 was inoculated to 10 L fermentter and controlled under the optimal conditions metioned above for alcohol production, the yield of alcohol at the end of fermentation was 16.2%(v/v). In the case of 25%(v/v) of the raw ground corn concentration, this strain produced only an alcohol level of 12.0%(v/v). In the case of 20%(v/v) of the raw ground corn concentration, the alcohol amount produced by strain SP-48 was only 9.3%(v/v).
引文
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