| High density cultivation is one of the important researches in fermentation industry recently.The progress in a series of R&D programs about breeding of ethanol yeast strain,developing of high density yeast cells cultivation and ethanol fermentation technology,enhancing of alcohol tolerance ability for ethanol yeast, eliminating of poison effect and inhibition of high osmotic pressure substrates on yeast cells,is satisfied to improve the efficiency of ethanol production and to speed up fuel ethanol industrialization.The breakthrough of alcohol fermentation technology is of great significance for reducing environmental pollution, promoting green energy popularization,and guaranteeing national energy sources security.In this thesis,writer employed excellent yeast strains,has achieved succeed in ethanol yeast cells density cultivation and alcohol over-production in per unit fermentation liquid volume by the optimized process of fed-batch culture and constant feeding culture.Meanwhile,the performances of ethanol yeast employed in alcohol tolerance,ethanol fermentation productivity and reusable fermentation ability were investigated experimentally.Main work contents and research results are as follows:(1) The cell increase batch cultivation of Saccharomyces cerevisiae As2.458 is investigated.Under the optimum conditions of cell growth:rotation speed 150rpm, 28℃,initial sugar concentrations 40 g·L-1,GLy concentrations 3 g·L-1,the yeast cell concentration reached 9.01 g·L-1,the alcohol concentration of broth up to 11.7%fermented in glucose concentrations 250 g·L-1,the alcohol concentrations produced is increased 24.5%than that of common density cells batch fermentation. These results showed that increasing cell concentrations of fermentation broth can significantly increase the concentration of alcohol and the alcohol productivity of per fermentation unit volume.But the ability of alcohol tolerance and the lethal concentration of S.cerevisiae As2.458 were lower comparatively.Moreover,the yeast cells can not grow normally and metabolize when exposed to surrounding with 10%alcohol concentration.The fermented performance of Saccharomyces cerevisiae As2.458 needs to be improved ulteriorly.(2) Affection of batch culture,fed-batch culture and constant feeding culture on S. cerevisiae 4-S cell growth were investigated.The results showed that:the fed-batch culture or constant feeding culture(feeding rate 0.133 g·L-1·min-1) was beneficial to S.cerevisia 4-S cells grow fleetly and accumulate high cells concentration,and the cells concentration of broth reached 71.82 g·L-1 and 82.01 g·L-1 respectively after 44hrs fermentation run.Compared to batch culture,the cells density is improved by 155.68%and 191.95%separately in above culture modes.(3) The experimental investigation has been performed in the alcohol tolerance performance of high density yeast.The research results showed that the high density yeast can respiratory and metabolism normally under 12%(v/v) alcohol concentrations,and the viability of high density yeast cells can remain 42%after impacted 2 hrs by 18%(v/v) alcohol liquid.The supplement with 3 g·L-1GLy,0.5 g·L-1(NH4)2SO4 and 2.6 g·L-1 KH2PO4 can enhance the alcohol tolerance performance of high density yeast,and the viability of yeast cells can reach to 57%after impacted 2 hrs by 18%(v/v) alcohol liquid.(4) With the Plackett-Burman designing experiment,the two key factors affected ferment performance of high density yeast cells were validated,they are glucose concentrations and alcohol concentrations outside cells.The extremum points of key factors,substrate concentration 350 g·L-1 and alcohol concentration 11.6%(v/v),were optimized by response surface method and experiment data analysis.Research the fermentation performance of high density yeast cells.The experimental results showed that alcohol concentrations of broth can reach 16.23%with high cell concentrations 82.01 g·L-1 fermented in glucose concentrations 350 g·L-1.Compared to batch fermentation with common cells density 28.01g·L-1,the fermentation run hours employed high density yeast cells was shorted 12hrs,and the alcohol concentrations produced was improved by 24.4%.(5) The experimentation and investigation on the repeatable fermentation performance of alcohol yeast have been accomplished.The research results showed that the alcohol concentrations produced by alcohol yeast in the start four batches were keeping more than 13%,and it showed a good repeatable fermentation capacity.But the fermentation capacity enters a downward trend following the fifth batch running.Activation culture of yeast cells for alcohol repeatable fermentation showed that the alcohol concentrations produced in the start seven batches broth can kept at 13%or more,and the alcohol concentrations produced in following batches decreased dramatically.Obviously,the activation of yeast cell in the intermission of per fermentation batch can improve the yeast capacity of repeatable fermentation.But the activation process for yeast cells would increase the cost of alcohol fermentation.Consequently,in the fermentation process of alcohol production,the ideal for alcohol yeast cells recycle used directly four batches was a reasonable choose and economy method.
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