Yeast xylose metabolism and xylitol production: Dissertation

A screening method was used for testing yeast strains in shake flask cultivations for their ability to convert xylose to xylitol. Of the 37 different strains studied by far the best were Candida guilliermondii C-6, C. tropicalis C-86 and C. tropicalis C-87. Of these strains C-6 was superior in a technical sense, being able to convert xylose to xylitol with a yield of 0.5 g g-1 at xylose concentrations at least up to 300 g l-1, whereas the other two strains did not tolerate xylose concentrations more than 120 g l-1. Fermentation kinetics in xylose conversion were studied more closely with the strain C-6 both in shake flasks and in a fermenter. Oxygen availability was the key process variable. In order to quantify its effect on yeast metabolism, oxygen transfer characteristics for both shake flasks and a fermenter were determined. The rate of specific xylose uptake by the yeast was independent of the oxygen transfer rate above a certain threshold value. The growth of the yeast could be limited by oxygen limitation, under which conditions a typical overflow metabolism resulted in very efficient xylitol production. Under optimum conditions for oxygen transfer the yield of xylitol from xylose was 0.74 g g-1 and the rate of specific xylitol production was about 0.22 g g-1h-1. An initial xylose concentration of 200 g l-1 slowed down the xylose conversion, but this effect could be avoided by a fed-batch fermentation, in which the xylose concentration was controlled to 40 - 50 g l-1. By this method the process time was decreased by 40 % and the yield of xylitol was increased from 0.6 to 0.78 g g-1 compared with a batch fermentation. The metabolism of xylitol could also be limited by addition of the glycolytic and TCA-cycle inhibitor furfuraldehyde at a concentration of 0.6 ml l-1 under which conditions the limitation by oxygen was less critical for xylitol production. Xylose metabolism was studied both by cultivation experiments and by simulation of a structured mathematical model. The model was constructed on the basis of the assumption of pseudo-steady-state of intracellular NADH, NADPH and ATP concentrations. The basis for xylitol accumulation appeared to be the high efficiency of the oxidative pentose phosphate cycle. This was verified by fermentation results, according to which the value of the respiratory qoutient rose up to 10. The values of the activities or the affinities of the first two enzymes in xylose metabolism, xylose reductase and xylitol dehydrogenase, could not explain xylitol accumulation. The activity of xylitol dehydrogenase was four to sixfold compared with that of xylose reductase, and the Km-value of xylitol dehydrogenase for xylitol was not higher than 60 mM. Xylose reductase was strictly specific for NADPH and xylitol dehydrogenase for NAD, which both favour xylitol accumulation under oxygen limitation. The structured mathematical model of xylose metabolism in the strain C-6 was combined to a model describing the performance of the fermenter. On the basis of the simulation using this combined model the fermentation could be optimized in relation to e.g. oxygen transfer. Xylitol production was also studied with a genetically modified Saccharomyces cerevisiae strain carrying a gene coding for xylose reductase in a vector under the constitutive S. cerevisiae PGK-promoter. By feeding this strain with a cosubstrate and xylose under carefully controlled conditions of dissolved oxygen concentration, yields of xylitol from xylose of over 0.95 g g-1 were achieved. Ethanol was used as the cosubstrate to regenerate the cofactor and for cell maintainance. The molar yield of xylitol on ethanol at the optimum dissolved oxygen concentration was about 1 mol mol-1. Thus about half of the reducing power produced from ethanol was used for the reduction of xylose. Glucose inhibited xylose uptake very efficiently and was therefore not a suitable cosubstrate.