【作者】 韩阳；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 遗传学， 2007， 硕士

【摘要】 随着石油资源趋于匮乏和全球环境污染问题的日益加重,各国纷纷开始开发新型能源。燃料乙醇是目前为止最理想的石油替代能源,它的生产方法以发酵为主。菌种的优劣对发酵效果的影响非常大,能够筛选出具有优良性状的菌株及对菌株进行改良,对于降低生产成本,乃至实现酒精的大规模工业化生产,解决能源危机都有着重大意义。本文从自然界多种基质中采样,分离得到69株酵母菌,经TTC法一级筛、耐酒精能力二级筛和产酒精能力三级筛,最终获得4株产酒精能力强且能耐受一定酒精浓度的酵母菌,分别编号为Y-1、Y-2、Y-3和Y-4。根据《The Yeast:a Taxonomic Study》(第三版)提供的鉴定方法,进行了发酵糖试验、同化碳源试验、同化氮源试验、无维生素生长试验、50%葡萄糖-酵母膏生长试验、37℃生长试验、放线酮抗性试验等生理生化试验,并对照模式菌株的生理生化特性,鉴定4株酵母菌分别为:Y-1属于酵母属(Saccharomyces);Y-2为清酒假丝酵母(Candida sake);Y-3、Y-4均为酿酒酵母(Saccharomyces cerevisiae)。本研究分别确定了Y-1、Y-2、Y-3、Y-4的最适生长温度、最适生长pH值;研究了温度、pH值、接种量对Y-1、Y-2、Y-3、Y-4生长的影响。结果表明,Y-1、Y-2、Y-3、Y-4的最适生长温度分别为30℃、32℃、30℃、30℃;最适生长pH值分别为5.5、5.0、4.5、4.5;接种量对Y-1、Y-2、Y-3、Y-4的生长影响显著;温度对Y-1的生长影响不显著,对Y-2、Y-3、Y-4的生长影响显著;pH值对Y-1、Y-3、Y-4的生长影响不显著,对Y-2的生长影响显著。用重铬酸钾比色法分别确定了Y-1、Y-2、Y-3、Y-4的最适发酵温度、最适发酵pH值;研究了温度、pH值、接种量对Y-1、Y-2、Y-3、Y-4的发酵曲线的影响。结果表明,Y-1、Y-2、Y-3、Y-4的最适发酵温度分别为32℃、32℃、30℃、30℃;最适发酵pH值分别为5.5、5.0、4.5、4.0;温度对Y-1、Y-2、Y-4的发酵曲线影响显著,对Y-3的发酵曲线影响不显著;pH值对Y-1、Y-2、Y-3的发酵曲线影响显著,对Y-4的发酵曲线影响不显著;接种量对Y-1、Y-2、Y-3、Y-4的发酵曲线影响不显著。针对所获得的高产酒精酵母野生菌株Y-1、Y-2、Y-3、Y-4,研究它们的耐酒精特性。在生长培养基中添加不等量的酒精,测定Y-1、Y-2、Y-3、Y-4在培养10 h后的细胞死亡率,以此方法得出Y-1、Y-2、Y-3、Y-4的酒精耐受度均在20%左右。Y-1、Y-2、Y-3、Y-4在最适发酵温度、最适发酵pH值和2×107个/mL接种量的条件下摇瓶发酵72 h后,最终发酵液中的酒精含量(v/v)分别为10.44%、11.46%、10.92%和10.84%。更多还原

【Abstract】 Many countries started to develop the new energy for the reason that the oil resource became deficient day by day and the environmental pollution became serious. The fuel ethyl alcohol is at present up to the most ideal petroleum substitution energy, and it is mainly produced by fermentation. The strain’s quality affects a lot on the fermentation. It is so important for reducing the production cost and developing large-scale industrialization production of alcohol that the strains with fine character can be screened and improved.69 isolates out of different environments were screened for alcohol-producing yeast with high yield. After the stair-screening by TTC means、the second-screening for high alcohol tolerance and the third-screening for high yield of alcohol, 4 strains of yeast with high yield of alcohol and tolerance were obtained which were named Y-1, Y-2, Y-3 and Y-4. With the methods that were mentioned in The Yeast: a Taxonomic Study(3rdedition), such as saccharide-fermentation experiment, carbon source assimilation experiment, nitrogen source assimilation, non-vitamin growth experiment, 50% glucose-yeast extract growth experiment, 37℃growth experiment and cycloheximide-resistance experiment, Y-1 was classified into Saccharomyces; Y-2 was identified as Candida sake and Y-3、Y-4 as Saccharomyces cerevisiae.And then, the study focused on the growth characteristic of the 4 stains. The optimum growth temperature of Y-1, Y-2, Y-3 and Y-4 separately was 30℃、32℃、30℃、30℃; the optimum growth pH value of the 4 stains separately was 5.5、5.0、4.5、4.5; the initial inoculum was an important factor in affecting the growth of Y-1, Y-2, Y-3 and Y-4; temperature affected a little on the growth of Y-1 but a lot on the growth of Y-2, Y-3 and Y-4; pH value affected a little on the growth of Y-1, Y-3 and Y-4 but a lot on Y-2’s.With the potassium dichromate colorimetry, the optimum fermentation temperature of Y-1, Y-2, Y-3 and Y-4 separately was 32℃、32℃、30℃、30℃; the optimum fermentation pH value of Y-1, Y-2, Y-3 and Y-4 separately was 5.5、5.0、4.5、4.0; the temperature affected a lot on the fermentation curve of Y-1, Y-2 and Y-4, but a little on the Y-3’s; pH value affected a lot on the fermentation curve of Y-1, Y-2 and Y-3, but a little on the Y-4’s; the initial inoculum was not the important factor for the fermentation of the 4 strains.The 4 strains were inoculated into the medium with different content of alcohol in it, then the alcohol tolerance came out with the death rate of the 4 strains. The alcohol tolerance of the 4 strains was about 20% (v/v).On the optimum condition for fermentation, Y-1, Y-2, Y-3 and Y-4 were fermented for 72 h in the bottle, after then, the alcohol content (v/v) in the liquid of Y-1, Y-2, Y-3 and Y-4 reached 10.44%, 11.46%, 10.92% and 10.84%.更多还原