Study On Breeding Of Xylose Fermentation Yeast To Produce Ethanol

As nonrenewable fossil fuel reserves dwindle and its price is climbing up continuously, the idea of production of ethanol from renewable plant biomass is increasingly more attractive. D-xylose comprise nearly one-third of the reducing sugars of lignocellulosics hydrolysate, the economic exploitation of lignocellulosics for ethanol production will thus significantly be enhanced by the efficient utilization of D-xylose. Some yeasts are able to ferment xylose, but ethanol concentration and yield are correspondingly low. In an attempt to extend and improve the possibilities for fermenting xylose with yeasts, Pichia stipitis was taken as starting strain, a better strain M-1 was screened by UV and the optimal fermentation conditions were studied.Effect of the culture age, the enzyme concentration, temperature, enzymatic hydrolysis time and osmotants on the formation and regeneration of protoplast from Pichia stipitis were studied, three-factor and three-level orthogonal experiments were designed to study the single factor including enzymes concentration, temperature and enzymatic hydrolysis time, the optimum conditions for Pichia stipitis protoplast generation and regeneration were determined: hypha age was 5h, enzyme concentration was 1.5%, enzymatic hydrolysis time was 1.5h, enzymatic temperature was 28 ℃ and sorbitol was selected as osmotants, under the optimum condition, the generation quotiety and regeneration quotiety were 84% and 4.5% respectively.The relation of lethality of Pichia stipitis protoplast and UV light irradiated dose was also examined in this study, the results suggested that when UV light irradiated dose was 20 watt, distance was 30cm and irradiated time was 120 seconds, the lethality of protoplast was 72%, The regenerated protoplast was primarily screened under the condition above.A strain M-1 was achieved after two turns of screened, the alcohol concentration of this strain was 6.67g/L after 48 hours fermentation, which was increased by 49% compared with original strain 4.47g/L, the residue of sugar of M-1 and parent strain were 0.45g/L, 0.61g/L respectively. The effect of serial passage on the genetic stability was studied and the results demonstrated that compared with original strain, the fermentation ability of M-l from 1^st to 20^th passage did not vary significantly.In addition, the growth and ferment characteristics of M-1 were examined in this paper, including growth temperature, pH, fermentation temperature, pH, and rotating rate for M-1 and studied their affection on growth and fermentation curve. The optimal conditions for M-1 growth was 28℃, 5.0(pH), The optimal conditions for M-1 fermentation was 28℃, 5.0(pH), and rotating rate 90r/min.A four-factor and three-level orthogonal experiment was designed to study the...