| The survival and development of people facing the most serious challenges is Energy, resources and environmental issues in the21st century. It is also the major factors restricting China’s social and economic development. It is imminent that we should develop and utilize of a green renewable new energy. Bio-ethanol as the renewable green energy is becoming the most promising liquid fuel. Biomass is transformated to fuel ethanol by microbial fermentation. Yeast is the earliest and most important industrial ethanol production microorganisms, but it also has many problems in fermentation process. Such as low fermentation temperature simultaneous saccharification and fermentation, cannot taking advantage of the pentose in the hemicellulose hydrolyzate, and poor tolerance to ethanol. These problems is the key for biomass conversion processes.In this study, we selected28yeast strains from the soil samples of Yuanjiang, Kunming, Yunnan Province, it is separated by sample enrichment and tablet separation. There are9yeast strains growing at45℃well. L27strain can torerant to48℃and capable of high-yield ethanol. Their colony morphology, physiological, biochemical and, molecular biological identification to determine its position on the taxonomy by JA Barnett’s yeast characteristics and identification manual, we identified L27yeast strain as Kluyveromyces marxianus of genus Kluyveromyces through JA Barnett’s yeast characteristics and identification manual. L27yeast strain was subjected to molecular identification in D1/D2+ITS region. Thus, we confirmed that L27yeast strain was Kluyveromyces marxianus of genus Kluyveromyces on molecular level.We(studied glucose, xylose fermentation characteristics of strain L27. Ethanol productions of strain L27are higher than other temperatures at45℃, ethanol production is42.32g/1at10%glucose concentration. Xylose as the substrate for fermentation, the ethanol productions of strain L27is4.98g/1at2%xylose concentration. In the fermentation of different concentrations of xylose, the highest ethanol yield is0.28g/g, the conversion rate is27.71%at6%xylose concentration.When mixed sugar fermentation strains L27priority to use of glucose, glucose will inhibition xylose utilization. The study of corncob hemicellulose hydrolyzate fermentation ethanol. The corncob dilute sulfuric acid pretreatment conditions is optimized by CCD central composite design. The final xylose yield is response value and determine the best pretreatment conditions. The optimal hydrolysis condition is treated by0.66%dilute sulfuric acid at120℃and1.5h, the xylose concentration is29.74g/1. The ethanol production is9.73g/1, reducing sugar utilization rate is84.29%,when L27strain ferment in detoxificated hemicellulose hydrolyzate. When the corncob is is treated by1.0%dilute sulfuric acid at125℃and2.0h, the cellulose content is72.68%at this time, the hemicellulose content is0.29%, the lignin content is11.37%, and ash is0.54g.We overexpression6-trehalose phosphate synthase gene to improve the tolerance of the yeast alcohol. We constructed the yease expression vector pYES3-tpsl and transformed into Saccharomyces cererisiae INVSc cells. The trehalose content, ethanol tolerance and fermentation ethanol production is researched after6-trehalose phosphate synthase gene overexpression. The results show that the trehalose content of recombinant strain has been higher than the wild-type strain with rising ethanol concentration. The recombinant strain can grow at12%ethanol concentration, but the wild-type strain cannot. The ethanol yield of the wild-type strain is35.97g/1, the ethanol yield of the recombinant strains is37.98g/1, the recombinant strain is5.59%higher than the wild-type strain when L27strain ferment by10%glucose as a substrate. Therefore we believe that the the tpsl gene overexpression can increase the ethanol tolerance of the yeast. |
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