Addition of benzoate to the medium reservoir of glucose‐limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 growing at a dilution rate (D) of 0.10 h−1 resulted in a decrease in the biomass yield, and an increase in the specific oxygen uptake rate (qO2) from 2.5 to as high as 19.5 mmol g−1h−1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in (qO2) to 13 mmol g−1h−1. The stimulatory effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration was not enhanced by benzoate. Cells could only gradually adapt to growth in the presence of benzoate: a pulse of benzoate given directly to the culture resulted in wash‐out. As the presence of benzoate in cultures growing at low dilution rates resulted in large changes in the catabolic glucose flux, it was of interest of study the effect of benzoate on the residual glucose concentration in the fermenter as well as on the level of some selected enzymes. At D=0.10 h−1, the residual glucose concentration increased proportionally with increasing benzoate concentration. This suggests that modulation of the glucose flux mainly occurs via a change in the entracellular glucose concentration rather than by synthesis of an additional amount of carriers. Also various intracellular enzyme levels were not positively correlated with the rate of respiration. A notable exception was citrate synthase: its level increased with increasing respiration rate. Growth ofS. cerevisiae in ethanol‐limited cultures in the presence of benzoate also led to very high qO2 levels of 19–21 mmol g−1h−1. During growth on glucose as well as on ethanol, the presence of benzoate coincided with an increase in the mitochondrial volume up to one quarter of the total cellular volume. Also with the Crabtree‐negative yeasts Candida utilis, Kluyveromyces marxianus andHansenula polymorpha, growth in the presence of benzoate resulted in an increase in qO2 and, at high concentrations of benzoate, in aerobic fermentation. In contrast to S.Cerevisiae, the highest qO2 of these yeasts when growing at D = 0.10 h−1 in the presence of benzoate was equal to, or lower than the qO2 attainable at μmax without benzoate. Enzyme activities that were repressed by glucose in S. cerevisiae also declined in K.Marxianus when the glucose flux was increased by the presence of benzoate. The maximal aerobic fermentation rate at D = 0.10 h−1 of the Crabtree‐negative yeasts at high benzoate concentrations was considerably lower than for S. cerevisiae. This is probably due to the fact that under aerobic conditions these yeasts are unable to raise the low basal pyruvate decarboxylase level: cultivation without benzoate under oxygen‐limited conditions resulted in rates of alcoholic fermentation and levels of pyruvate decarboxylase comparable to those of S. cerevisiae.

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 pmax of 0.31 h-l and a K, for glucose of 0.55 mM. At a dilution rate of 0.10 h-l, a maximal yield of 0.10 g biomass (g glucose)-' 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-l, the yield of the S . cereuisiae 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)-' for optimal growth. Lower or higher concentrations of this fatty acid, supplied as Tween 80, resulted in uncoupling of dissimilatory and assimilatory processes.

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-' glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-'. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of 02 g (dry weight)-' h-. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-'. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to jLmax. we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-'. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases. The observed pattern of metabolite production in chemostat cultures is in agreement with the conditions under which these insufficiencies can be calculated to occur. The uncoupling effect of weak acids on respiration and the associated triggering of alcoholic fermentation were confirmed in chemostat experiments in which the yeast was grown in the presence of propionate. In this case, the maximal respiratory activity was attained at a dilution rate of 0.30 h-1, above which alc...

Growth yields of Saccharomyces cerevisiae and Candida utilis in carbon-limited chemostat cultures were evaluated. The yields on ethanol and acetate were much lower in S. cerevisiae, in line with earlier reports that site I phosphorylation is absent in this yeast. However, during aerobic growth on glucose both organisms had the same cell yield. This can be attributed to two factors: -S. cerevisiae had a lower protein content than C. utilis; -uptake of glucose by C. utilis requires energy whereas in S. cerevisiae it occurs via facilitated diffusion. Theoretical calculations showed that, as a result of these two factors, the ATP requirement for biomass formation in C. utilis is 35% higher than in S. cerevisiae (theoretical YATP values of 20.8 and 28.1, respectively). The experimental YATP for anaerobic growth of S. cerevisiae on glucose was 16 g biomass-mol ATP -1.In vivo P/O-ratios can be calculated for aerobic growth on ethanol and acetate, provided that the gap between the theoretical and experimental ATP requirements as observed for growth on glucose is taken into account. This was done in two ways: -via the assumption that the gap is independent of the growth substrate (i.e. a fixed amount of ATP bridges the difference between the theoretical and experimental values). -alternatively, on the assumption that the difference is a fraction of the total ATP expenditure, that is dependent on the substrate. Calculations of P/O-ratios for growth of both yeasts on glucose, ethanol, and acetate made clear that only by assuming a fixed difference between theoretical and experimental ATP requirements, the P/O-ratios are more or less independent of the growth substrate. These P/O-ratios are approximately 30% lower than the calculated mechanistic values.

The energetics of Saccharomyces cerenisiae 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)-'. 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.

Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography. The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose. Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21). Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme. The activity with NADH was approx. 70% of that with NADPH for the various aldose substrates. NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction. These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol.

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 relevant 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.

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