Candida utilis CBS 621 exhibits the Kluyver effect for maltose, i.e. this yeast can respire maltose and is able to ferment glucose, but is unable to ferment maltose. When glucose was pulsed to a maltose-grown, oxygen-limited chemostat culture of C. utilis, ethanol formation from glucose started almost instantaneously, indicating that the enzymes needed for alcoholic fermentation are expressed in maltose-grown cells. However, the addition of glucose inhibited maltose metabolism. To eliminate a possible catabolite inhibition and/or repression of enzyme activities involved in maltose metabolism, the effect of simultaneously feeding glucose and maltose to an oxygen-limited, maltose-grown chemostat culture was studied. In this case, the glucose concentration in the culture remained below 0.1 mM, which makes glucose catabolite repression unlikely. Nevertheless, maltose metabolism appeared to cease when the culture was switched to the mixed feed. Based on the outcome of the mixed-substrate studies, it was postulated that the Kluyver effect may be caused by feedback inhibition of maltose utilization by ethanol, the product of fermentative maltose metabolism. If ethanol suppresses the utilization of non-fermentable disaccharides, this would provide a phenomenological explanation for the occurrence of the Kluyver effect: accumulation would then not occur and the rate of maltose metabolism would be tuned to the culture's respiratory capacity. This hypothesis was tested by studying growth of C. utilis CBS 621 and Debaryomyces castellii CBS 2923 in aerobic batch cultures on mixtures of sugars and ethanol. With both yeasts diauxic growth was indeed observed on mixtures of ethanol and a disaccharide that gives rise to the Kluyver effect, with ethanol being the preferred substrate. In contrast, sugars which could be fermented were either utilized simultaneously with ethanol or preferred over this substrate.

Growth and metabolite formation were studied in oxygen-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 growing on glucose or maltose at a dilution rate of 0.1 h-1. With either glucose or maltose S. cerevisiae could be grown under dual limitation of oxygen and sugar. Respiration and alcoholic fermentation occurred simultaneously and the catabolite fluxes through these processes were dependent on the magnitude of the oxygen feed. C. utilis could also be grown under dual limitation of glucose and oxygen. However, at very low oxygen feed rates (i.e. below 4 mmol 1-1 h-1) growth was limited by oxygen only, as indicated by the high residual glucose concentration in the culture. In contrast to S. cerevisiae, C. utilis could not be grown anaerobically at a dilution rate of 0.1 h-1. With C. utilis absence of oxygen resulted in wash-out, despite the presence of ergosterol and Tween-80 in the growth medium. The behaviour of C. utilis with respect to maltose utilization in oxygen-limited cultures was remarkable alcoholic fermentation did not occur and the amount of maltose metabolized was dependent on the oxygen supply. Oxygen-limited cultures of C. utilis growing on maltose always contained high residual sugar concentrations. These observations throw new light on the so-called Kluyver effect. Apparently, maltose is a non-fermentable sugar for C. utilis CBS 621, despite the fact that it can serve as a substrate for growth of this facultatively fermentative yeast. This is not due to the absence of key enzymes of alcoholic fermentation. Pyruvate decarboxylase and alcohol dehydrogenase were present at high levels in maltose-utilizing cells of C. utilis grown under oxygen limitation. It is concluded that the Kluyver effect, in C. utilis growing on maltose, results from a regulatory mechanism that prevents the sugar from being fermented. Oxygen is not a key factor in this phenomenon since under oxygen limitation alcoholic fermentation of maltose was not triggered.

Saccharomyces cerevisiae T23C (pda1::Tn5ble) is an isogenic gene replacement mutant of the wild-type strain S. cerevisiae T23D. The mutation causes a complete loss of pyruvate dehydrogenase activity. Pyruvate metabolism in this pyruvate-dehydrogenase-negative (Pdh-) strain was investigated in aerobic glucose-limited chemostat cultures, grown at a dilution rate of 0.10 h-1, and compared with the metabolism in the isogenic wild-type strain. Under these conditions, growth of the Pdh- strain was fully respiratory. Enzyme activities in cell-free extracts indicated that the enzymes pyruvate decarboxylase. acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase could provide a functional bypass of the pyruvate dehydrogenase complex. Since this metabolic sequence involves ATP hydrolysis in the acetyl-CoA synthetase reaction, a negative effect of the pda1::Tn5ble mutation on the growth efficiency was anticipated. Indeed, the biomass yield of the Pdh- strain [0.44 g biomass (g glucose)-1] was significantly lower than that of wild-type S. cerevisiae [0.52 g biomass (g glucose)-1]. The effect of the mutation on biomass yield could be quantitatively explained in terms of a lower ATP yield from glucose catabolism and an increased ATP requirement for the synthesis of acetyl-CoA used in anabolism. Control experiments showed that the pda1::Tn5ble mutation did not affect biomass yield in ethanol-limited chemostat cultures. The results support the view that, during aerobic glucose-limited growth of S. cerevisiae at low growth rates, the pyruvate dehydrogenase complex accounts for the major part of the pyruvate flux. Moreover, it is concluded that hydrolysis of pyrophosphate formed in the acetyl-CoA synthetase reaction does not contribute significantly to energy transduction in this yeast. Respiratory-deficient cells did not contribute to glucose metabolism in the chemostat cultures and were probably formed upon plating.

A simple method is described for the preparation of d-xylulose. It consists of the isomerization of d-xylose with xylose isomerase (EC 5.3.1.5), yielding an equilibrium mixture of d-xylulose and d-xylose. This is followed by the quantitative oxidation of residual d-xylose to d-xylonic acid with immobilized A. calcoaceticus cells. A combination of methanol precipitation and ion exchange is used for the removal of xylonic acid. This procedure offers many advantages over existing methods for the preparation of d-xylulose. The purity of the final product compares favorably to that of a commercial d-xylulose preparation.