Continuous Fermentation Of Pentose By Pichia Stipitis With Low-pH Treatments

Nowadays the society of human being are faced many crises, such as surplus population, environmental pollution, deficient resources, irremediable diseases, and so on. People want to establish a continuously developable system in order to release from these crises. The development and appliance of high-technology including biotechnology plays an important role during those processes. Lignocellulosic materials containing cellulose, hemicellulose, and lignin as their main constituents are the most abundant renewable organic resource present on earth. The production of fuel alcohol using the inexpensive and renewable sugar of biomass is one of the most effective methods to resolve energy crisis in the world.This dissertation studied effects of culture conditions on growth of Pichia stipitis and pentose fermentation by it, substance balance and metabolic analysis of carbon during glucose/xylose fermentation by establishing steady and continuous cultures of P. stipitis, technique of restraining contaminative germ in the two-stage continuous fermentation with methods of immobilized cells and low-pH treatment, which based on continuous cultures of free yeasts using glucose/xylose mixture to simulate lignocellulosic hydrolyzates. Major experimental results are as follows:Effect of nitrogen source on growth of P. stipitis and xylose fermentation by the same yeast has been discussed. The growth and xylose fermentation were as good with urea as a sole nitrogen source as with peptone as a sole nitrogen source. With peptone, urea, ammonium sulphate and potassium nitrate as a sole nitrogen source and using 40.0 g/L xylose as substrate, after 36 h culture the ethanol concentration was 17.29, 15.07, 13.76 and 13.46 g/L, while the ratio of ethanol yield to theoretical yield was 97.30, 94.79, 78.03 and 84.84 %, respectively.Effect of pH value and temperature for xylose fermentation by P. stipitis has also been discussed. The optimum pH value and temperature range of the growth of P. stipitis were 25-30℃ : and 4.6-5.8. When the culture temperature was 25℃ and 35℃ :, the optimum pH value range of the xylose fermentation by P. stipitis was 3.4-4.2 and 3.8-5.0, respectively.Two continuous cultures with P. stipitis at different dilution rates have been established. One was under anaerobic condition with 30.0g/L glucose as substrate; the other was underoxygen-limited condition with 45.0 g/L glucose/xylose mixture as the basic carbon source. The same rule of the two cultures was that the concentration of yeasts, ethanol and the ratios of sugar consumed were reducing with the enhancing of dilution rates. In the anaerobic culture the ethanol productivity reached a maximum of 1.110g/Lh when the dilution rate was equal to 0.14 h-1, while the optimism ethanol productivity was 0.790 g/L-h when the dilution rate was at 0.06 h-1 in the oxygen-limited culture.A 400 h chemostat continuous culture was divided into 4 phases by adding different sugar concentration during each phase. Four steady states were formed. The balance computation and metabolism analysis of carbon were studied. The carbon recovery ratios of the four steady states were 118.03, 105.59, 113.51 and 94.72 %, respectively. Roughly 41.19 % of carbon in the mixture substrate of 15.0 g/L xylose and 30.0 g/L glucose was turned into ethanol, while only 34.46 % of carbon in the mixture substrate of 30.0 g/L xylose and 30.0 g/L glucose converted into ethanol.At a dilution rate of 0.08 h"\ free cells of P. stipitis were cultured in a single-stage continuous reactor with different sugar concentration of glucose/xylose mixture with a sugar ratio of 2:1. When substrate concentration was increased, the residual sugar concentration of the effluent went up, and the ethanol concentration was slightly increased. When the substrate concentration was more than 60.0 g/L, the ethanol concentration of the effluent was between 14.5-15.5 g/L and the ratio of ethanol yield to theoretical yield was 79.8-85.4 %.Free cells of P. stipitis were cultured in a two-stage continuous reactor with glucose/xylose mixtu...