Pentose utilization in yeasts: Physiology and biochemistry

Abstract: The fermentative performance of bacteria, yeasts, and filamentous fungi was investigated in a pentose (xylose)-rich lignocellulosic hydrolyzate. The filamentous fungus Fusarium oxysporum and the xylose-fermenting yeast Pichia stipitis were found to be very sensitive to the inhibiting hydrolyzate. Recombinant xylose-utilizing Saccharomyces cerevisiae showed very poor ethanol formation from xylose; xylitol being the major product formed. The highest ethanol yields were obtained with recombinant Escherichia coli KO11, however, for maximal ethanol yield detoxification of the hydrolyzate was required. The influence of oxygen on the regulation of carbohydrate metabolism in the xylose-fermenting yeast P. stipitis CBS 6054 was investigated. A low and well-controlled level of oxygenation has been found to be required for efficient ethanol formation from xylose by the xylose-fermenting yeasts. The requirement of oxygen is frequently ascribed to the apparent redox imbalance which develops under anaerobic conditions due to the difference in co-factor utilization of the two first enzymes in the xylose metabolism, further reflected in xylitol excretion. However, a low and well controlled level of oxygenation for maximal ethanol production from glucose was also demonstrated, suggesting that the oxygen requirement is not only due to the dual co-factor utilization, but also serves other purposes. Cyanide-insensitive and salicyl hydroxamic acid-sensitive respiration (CIR) was found in P. stipitis. CIR is suggested to act as a redox sink preventing xylitol formation in P. stipitis under oxygen-limited xylose fermentations. Xylitol metabolism by P. stipitis CBS 6054 was strictly respiratory and ethanol was not formed under any conditions. The absence of ethanol formation was not due to a lack of fermentative enzymes, since the addition of glucose to xylitol-pregrown cells resulted in ethanol formation. Xylitol was not metabolized under anaerobic conditions, whereas ethanol was formed both from xylose and glucose. The in vitro pyruvate decarboxylase activity was shown to increase with decreasing oxygenation. Xylulose fermentation by four strains of S. cerevisiae, P. stipitis CBS 6054 and Candida shehatae NJ 23, was compared under anaerobic batch cultivations. In addition to ethanol, polyols such as xylitol and arabinitol were formed by all yeasts. The S. cerevisiae strains showed xylulose consumption rates 20- to 60-fold lower than the glucose consumption rates. Co-utilization of xylulose and glucose by S. cerevisiae ATCC 24860 in chemostat culture decreased the ethanol yield compared with utilization of glucose alone, due to increased acetate and arabinitol formation. The formation of acetate and arabinitol involves reduction and oxidation of co-factors and indicates an altered redox flux during xylulose fermentation.