| During the use of fossil fuels, it would be inevitably that the pollution to the natural environment. Since the lack of fossil energy and the pollution brought by application of fossil energy was exhausted, a new energy which is renewable and clean is related to the development of global economy, and to the development of human society as well. As an ideal biofuel with high renewability and little pollution, bioethanol has become a hot spot among both research scholars and governments concern. Currently, the main method for fuel ethanol production is fermentation. However, the intractable problems such as ethanol stress, contamination and so on, arise during the fermentation process, which blocked further realize the mass production of fuel ethanol. The accumulation of ethanol during the fermentation can inhibit the growth and metabolism of Saccharomyces cerevisiae and eventually lead to the reduce of ethanol yield and production efficiency. Although S. cerevisiae can tolerate a certain degree of ethanol, with the fermentation time going on, The accumulation of ethanol, the lack of nutrients and the deterioration fermentation environment would lead to the serious degree of inhibition. Throughout the fermentation process, the growth of Saccharomyces cerevisiae and the production of ethanol is mutually independent and integral, which means that the growth of Saccharomyces cerevisiae must adapt to the ethanol stress of environment and that to realize high production of ethanol is inseparable from Saccharomyces cerevisiae.This paper aims to study the response of yeast cells in different phases to the ethanol stress to further obtain high ethanol tolerance ability of Saccharomyces cerevisiae. Growth curve, cell diameter and broth pH in the fermentation process were measured and analyzed. The cells harvested from 2 h,8 h, and 16 h of the batch fermentation process were chosen to represent the cells at lag phase, exponential phase and stationary phase. The contents of trehalose, ergosterol and fatty acids of cells at different phases were also determined and compared. The results showed that the contents of trehalose and ergosterol increases with ethanol concentration. The content trehalose and the contents and composition of fatty acids obviously changed with ethanol concentration increased. In addition, the expression of genes related to metabolisms of trehalose and fatty acids of cells at different phases were determined. The results showed that expression of trehalose decomposition gene (ATH1, NTHl and NTH2) was restrained at exponential phase. The expression of trehalose synthase gene (TPS1 and TPS2) increased at stationary phase, But the expression of trehalose synthase gene (TPS3 and TSL1) decreased at stationary phase. On the other hand, the expression of saturated fatty acids (SFAs) synthase gene was higher at exponential phase and stationary phase than that at lag phase. While the expression of fatty acid dehydrogenase (OLE1) increased at exponential phase and decreased at stationary phase than that at lag phase. Thus the determination of yeast cell membrane structure shown that with the increase of the ethanol stress, yeast cell membrane structure became loose, and the liquidity of cell membrane enhanced.On this basis, metabonomics analysis was taken to cells at different phases to get a further understanding to yeast metabolism in fermentation process. The results showed that different the metabolism of Saccharomyces cerevisiae cells at different phases changed obviously. The amino acid metabolism of cells at exponential phase was strong, while of those at stationary phase, metabolism of sugar and organic acid was strong.All those experimental results showed that the cell membrane of Saccharomyces cerevisiae yeast played an important role in ethanol stress resistance. With the ethanol stress enhanced, cell membrane structure became loose and the liquidity of it increased. At the same time, the metabolism of cells at different phases showed its own characteristics and different from each other. |
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