Cornelis Verduyn

Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures.

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in anaerobic glucose-limited chemostat cultures in a mineral medium supplemented with ergosterol and Tween 80. The organism had a mu max of 0.31 h-1 and a Ks for glucose of 0.55 mM. At a dilution rate of 0.10 h-1, a maximal yield of 0.10 g biomass (g glucose)-1 was observed. The yield steadily declined with increasing dilution rates, so a maintenance coefficient for anaerobic growth could not be estimated At a dilution rate of 0.10 h-1, the yield of the S. cerevisiae strain H1022 was considerably higher than for CBS 8066, despite a similar cell composition. The major difference between the two yeast strains was that S. cerevisiae H1022 did not produce acetate, suggesting that the observed difference in cell yield may be ascribed to an uncoupling effect of acetic acid. The absence of acetate formation in H1022 correlated with a relatively high level of acetyl-CoA synthetase. The uncoupling effect of weak acids on anaerobic growth was confirmed in experiments in which a weak acid (acetate or propionate) was added to the medium feed. This resulted in a reduction in yield and an increase in specific ethanol production. Both yeasts required approximately 35 mg oleic acid (g biomass)-1 for optimal growth. Lower or higher concentrations of this fatty acid, supplied as Tween 80, resulted in uncoupling of dissimilatory and assimilatory processes.

Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures.

The energetics of Saccharomyces cerevisiae were studied in anaerobic glucose-limited chemostat cultures via an analysis of biomass and metabolite production. The observed YATP was dependent on the composition of the biomass, the production of acetate, the extracellular pH, and the provision of an adequate amount of fatty acid in the medium. Under optimal growth conditions, the YATP was approximately 16 g biomass (mol ATP formed)-1. This is much higher than previously reported for batch cultures. Addition of acetic acid or propionic acid lowered the YATP. A linear correlation was found between the energy required to compensate for import of protons and the amount of acid added. This energy requirement may be regarded as a maintenance energy, since it was independent of the dilution rate at a given acid concentration.

Physiology of yeasts in relation to biomass yields

The stoichiometric limit to the biomass yield (maximal assimilation of the carbon source) is determined by the amount of CO2 lost in anabolism and the amount of carbon source required for generation of NADPH. This stoichiometric limit may be reached when yeasts utilize formate as an additional energy source. Factors affecting the biomass yield on single substrates are discussed under the following headings:- Energy requirement for biomass formation (YATP). YATP depends strongly on the nature of the carbon source.- Cell composition. The macroscopic composition of the biomass, and in particular the protein content, has a considerable effect on the ATP requirement for biomass formation. Hence, determination of for instance the protein content of biomass is rolevant in studies on bioenergetics.- Transport of the carbon source. Active (i.e. energy-requiring) transport, which occurs for a number of sugars and polyols, may contribute significantly to the calculated theoretical ATP requirement for biomass formation.- P/O-ratio. The efficiency of mitochondrial energy generation has a strong effect on the cell yield. The P/O-ratio is determined to a major extent by the number of proton-translocating sites in the mitochondrial respiratory chain.- Maintenance and environmental factors. Factors such as osmotic stress, heavy metals, oxygen and carbon dioxide pressures, temperature and pH affect the yield of yeasts. Various mechanisms may be involved, often affecting the maintenance energy requirement.- Metabolites such as ethanol and weak acids. Ethanol increases the permeability of the plasma membrane, whereas weak acids can act as proton conductors.- Energy content of the growth substrate. It has often been attempted in the literature to predict the biomass yield by correlating the energy content of the carbon source (represented by the degree of reduction) to the biomass yield or the percentage assimilation of the carbon source. An analysis of biomass yields of Candida utilis on a large number of carbon sources indicates that the biomass yield is mainly determined by the biochemical pathways leading to biomass formation, rather than by the energy content of the substrate.

Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode

SummaryAn alcohol electrode was constructed which consisted of an oxygen probe onto which alcohol oxidase was immobilized. This enzyme electrode was used, in combination with a reference oxygen electrode, to study the short-term kinetics of alcoholic fermentation by aerobic yeast suspensions after pulsing with glucose. The results demonstrate that this device is an excellent tool in obtaining quantitative data on the short-term expression of the Crabtree effect in yeasts.Samples from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae not producing ethanol, immediately (within 2 min) exhibited aerobic alcoholic fermentation after being pulsed with excess glucose. With chemostat-grown Candida utilis, however, ethanol production was not detectable even at high sugar concentrations. The Crabtree effect in S. cerevisiae was studied in more detail with commercial bakers yeast. Ethanol formation occurred only at initial glucose concentrations exceeding 150 mg·l-1, and the rate of alcoholic fermentation increased with increasing glucose concentrations up to 1,000 mg·l-1 glucose.Similar experiments with batch cultures of certain ‘non-fermentative’ yeasts revealed that these organisms are capable of alcoholic fermentation. Thus, even under fully aerobic conditions, Hansenula nonfermentans and Candida buffonii produced ethanol after being pulsed with glucose. In C. buffonii ethanol formation was already apparent at very low glucose concentrations (10 mg·l-1) and alcoholic fermentation even proceeded at a higher rate than in S. cerevisiae. With Rhodotorula rubra, however, the rate of ethanol formation was below the detection limit, i.e., less than 0.1 mmol·g cells-1·h-1.

Propionate metabolism in Saccharomyces cerevisiae : implications for the metabolon hypothesis

Aerobic, glucose-limited chemostat of Saccharomyces cerevisiae CBS 8066 co-metabolized propionate when this compound was added to the reservoir medium. Co-metabolism of propionate led to an increase of the biomass and protein yields. Attempts to grow S. cerevisiae on propionate as a sole source of carbon and energy were not successful. Activities of propionyl-CoA synthetase in cell-free extracts were sufficient to account for the rates of propionate consumption observed in the chemostat cultures. Activities of propionyl-CoA carboxylase, a key enzyme of the methylmalonyl-CoA pathway of propionate metabolism, were negligible. In contrast, activities of 2-methylcitrate synthase, a key enzyme activity of the 2-methylcitrate pathway of propionate metabolism, increased substantially with increasing propionate-to-glucose ratios in the reservoir media, and were sufficient to account for the propionate consumption rates observed in the chemostat cultures. This suggested that the 2-methylcitrate pathway is the major pathway of propionate metabolism in S. cerevisiae. In the literature, labelling patterns observed after incubation of this yeast with [3-13C]propionate have been interpreted as evidence for channelling of tricarboxylic acid (TCA) cycle intermediates, possibly as a consequence of the organization of TCA cycle enzymes in a metabolon. However, this interpretation of 13C-labelling patterns rested on the assumption that propionate metabolism in S. cerevisiae occurs via the methylmalonyl-CoA pathway. Since the distribution of 13C in alanine reported in the literature is fully compatible with a major role of the 2-methylcitrate pathway in propionate metabolism, it cannot be interpreted as evidence for the existence of a TCA cycle metabolon in S. cerevisiae.

Utilization of methanol by a catalase-negative mutant of Hansenula polymorpha

SummaryIn methanol-utilizing yeasts, catalase is an essential enzyme for the destruction of hydrogen peroxide generated by methanol oxidase (E.C. 1.1.3.13). It was found however that a catalase-negative mutant of Hansenula polymorpha is able to consume methanol in the presence of glucose in continuous cultures. At a dilution rate of 0.1 h-1, stable continuous cultures could be obtained during growth on methanol/glucose mixtures with a molar ratio of methanol/glucose between 0 to 2.4. In these cultures methanol oxidase was induced up to a level of 40% of that obtained in the wild-type strain. The hydrogen peroxide-decomposition activity of the mutant was studied in more detail by pulsing methanol to samples of steady-state cultures. Only after the addition of excess methanol the hydrogen peroxide-decomposing system became saturated, and the cells excreted hydrogen peroxide. This was accompanied by excretion of formaldehyde and a rapid loss of viability. The presence of extracellular catalase during a methanol pulse prevented the loss of viability. The nature of the alternative hydrogen peroxide-decomposing enzyme system remains to be elucidated. Its capacity strongly depended on the cultivation conditions and pretreatment of the cells. Cells grown on formaldehyde/glucose mixtures showed a lower methanol tolerance than those grown on the methanol/glucose mixtures. Freeze-drying of cells drastically enhanced the excretion of hydrogen peroxide, probably as a result of an inactivation of the decomposing system.

Production of catalase-free alcohol oxidase by Hansenula polymorpha

SummaryMany of the potential technical applications of alcohol oxidase (MOX; EC 1.1.3.13) are limited by the presence of high activities of catalase in the enzyme preparations. In order to circumvent laborious and costly purification or inactivation procedures, the induction of MOX in a catalase-negative mutant of Hansenula polymorpha has been studied. Emphasis was laid on the induction of activities of MOX and the dissimilatory enzymes in continuous cultures grown on various mixtures of formate/glucose and formaldehyde/glucose. In continuous cultures of the catalase-negative mutant grown on these mixtures, MOX can be induced efficiently. To obtain a stable and productive process, the ratio of the substrates is of critical importance. The optimal ratios of the mixtures for the catalase-negative strain for formate/glucose and formaldehyde/glucose were 3:1 and 1–2:1, respectively. Under identical cultivation conditions the wild-type strain showed similar induction patterns for MOX and the dissimilatory enzymes formaldehyde dehydrogenase (FaDH) and formate dehydrogenase (FoDH). The MOX levels in the catalase-negative strain were approx. 50% of those in the wild-type strain.

NAD(P)H-dependent aldose reductase from the xylose-fermenting yeast Pichia stipitis

BRUINENBERG, P. M,, VAN D1JKEN, J. P., KUENEN, J. G. and SCHEFFERS, W. A. 1985a. Critical parameters in the isolation of mitochondria from Candida utilis grown in continuous culture. J. Gen. Microbiol. 131: 1035-1042. BRUINENBERG, P. M., VAN D1JKEN, J. P., KUENEN, J. G. and SCHEFr~RS, W. A. 1985b. Oxidation of NADH and NADPH by mitochondria from the yeast Candida utilis. J. Gen. Microbiol. 131: 1043-1051.

The utility of alcohol oxidase for alcohol assays

Efficient anaerobic wastewater treatment in UASB-reactors (Lettinga et al., 1980) requires a sludge possessing good methanogenic activities and good settling characteristics. Under certain conditions a granular sludge was developed in this type of reactor, meeting both these requirements. Our research is focused on the microbiological aspects of this granulation. A method was developed for determination of the methane production rate by gas chromatography. Using the usual anaerobic techniques, small amounts of sludge were suspended into a phosphate-bicarbonate buffer (pH 7) in 130-ml serum-vials under a CO2-atmosphere. Gas analyses were performed using a pressure-lock syringe to prevent problems with pressure build-up. For the determination of maximal methanogenic activities on a variety of substrates this method provides a rapid and reliable tool. It may also be of use for the control of sludges after serious overloading, or after their exposure to toxic compounds. Granular and non-granular methanogenic sludge was grown on wastewater from a sugar factory. There were no significant differences between the methanogenic activity patterns of the two types of sludge as measured on formate, acetate, propionate and ethanol. On sucrose, however, though not a direct substrate for methanogenic bacteria, differences were found suggesting a possible role of carbohydrate-decomposing organisms in the formation of a granular methanogenic sludge.