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为获得菌株发酵菊芋生产燃料乙醇的最佳方案,首先选取实验室保存的重组菌株R32对其产酶条件进行优化,其最高产菊粉酶活性为298.8U·mL-1,此时的最佳培养基配方为:YPG培养基为酵母粉1%(w/v),蛋白胨2%(w/v),甘油0.5%(v/v);YPM培养基为酵母粉1%(w/v),蛋白胨2%(w/v),甲醇1%(v/v);培养基pH为自然初始pH。然后选取酿酒酵母S.c和克鲁维酵母Klu,比较是否在添加重组菌株R32粗酶液条件下,两株酵母菌分别进行单独发酵和混合发酵时的产乙醇能力,以获得最佳的发酵组合。结果表明,酿酒酵母S.c和克鲁维酵母Klu在未添加重组菌株R32粗酶液时,混合一步发酵获得的乙醇含量较高,发酵84h时乙醇含量为11.37%。添加重组菌株R32粗酶液进行两步发酵时,2株酵母菌混合发酵72h时,乙醇含量为11.43%。2种发酵组合的最高乙醇含量以及各个发酵参数基本相同,虽然一步法发酵时间延长,但节省成本,操作简单,更适宜工业生产应用。最后对其进行正交试验优化,培养条件为菊粉浓度225g·L-1,脲素浓度40g·L-1,接种量15%,pH为5时,酿酒酵母菌S.c和克鲁维酵母Klu混合一步发酵法的最高乙醇体积比达11.82%。
In order to obtain the best protocol for producing fuel ethanol from strain Jerusalem artichoke, firstly, the recombinant strain R32 preserved in laboratory was used to optimize its enzyme production conditions, and its highest activity of inulinase was 298.8U · mL-1, at this time the best The medium of YPG was 1% (w / v) yeast extract, 2% peptone and 0.5% glycerol. YPM medium was 1% (w / v) , Peptone 2% (w / v) and methanol 1% (v / v); the medium pH is the natural initial pH. Then S. cerevisiae S.c and Kluyveromyces Klu were selected to compare whether ethanol production ability of the two yeasts were separately fermented and mixed fermented under the condition of adding R32 crude enzyme solution to obtain the best fermentation combination. The results showed that when S. cerevisiae S.c and Kluyveromyces Klu were not added with the recombinant R32 crude enzyme solution, the ethanol content obtained by the mixed one-step fermentation was higher, and the ethanol content was 11.37% after 84h fermentation. When two strains of yeast were added into the fermentation, the ethanol content was 11.43%. The highest ethanol content and the same fermentation parameters of the two fermentation combinations were basically the same. Although the one-step fermentation time was prolonged, the cost savings and the simple operation were more suitable for industrial production and application. At last, orthogonal experiments were carried out to optimize the conditions of inoculation with 225g · L-1 inulin concentration, 40g · L-1 urea concentration, 15% inoculum size and 5 pH. Saccharomyces cerevisiae Sc and Kluyveromyces Klu The highest ethanol volume ratio of mixed one-step fermentation reached 11.82%.