Continuous ethanol production in a two-stage, immobilized and suspended cell bioreactor

High levels of ethanol production and high sugar conversion to ethanol were accomplished with a two-stage, continuous-flow bioreactor, containing immobilized and suspended cells of Saccharomyces cerevisiae. Sufficient aeration to maintain respiratory-efficient cells under fermentation conditions was the critical factor in maintaining an active population of ethanol producing yeast cells. Thus, the combination of the immobilized cells and the high density of the suspended cells stabilized the continuous fermentation for long-term performance. The lipid composition of the suspended cells from the reactor was affected by the cultivation conditions of aeration or non-aeration. Sterols and unsaturated fatty acid syntheses of the suspended cells were increased significantly under aerated conditions as compared to non-aerated condition. However, the immobilized cells grown under aerated and non-aerated conditions showed the respiratory-deficient characteristics and similar patterns in the lipid compositions, but the immobilized cells had an adaptive ability to a high ethanol concentration environment through the synthesis of a broader range of sterol components and increasing levels of unsaturated fatty acids. It was confirmed that sterols and unsaturated fatty acids were essential components for both high levels of ethanol production and the high ethanol tolerance of yeast cells. The relationship of ergosterol to alcohol dehydrogenase activity during ethanol biosynthesis in S. cerevisiae was not established. Ergosterol and oleic acid stimulated cell growth and ethanol production, but specific alcohol dehydrogenase activity was not affected by supplementation with these lipids. There was no difference in the alcohol dehydrogenase activity between the cells grown under aerated and non-aerated conditions. However, at a higher ethanol concentration under non-aerated conditions, ergosterol and oleic acid supplements stimulated cell growth and ethanol production as well as the specific alcohol dehydrogenase activity. Only one alcohol dehydrogenase isozyme band was detected on starch gel electrophoresis regardless of the cultivated conditions under aeration or non-aeration, indicating that a fermentative state exists in both systems. Alcohol dehydrogenase activity was not correlated to ethanol production, but high cell density and the appropriate physiological state of the cells are more important for ethanol production than the level of alcohol dehydrogenase activity of the cells.