Effect of benzoic acid on metabolic fluxes in yeasts: A continuous‐culture study on the regulation of respiration and alcoholic fermentation

Abstract

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 (q O2) from 2.5 to as high as 19.5 mmol g−1 h−1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in (q O2) to 13 mmol g−1 h−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 of S. cerevisiae in ethanol‐limited cultures in the presence of benzoate also led to very high q O2 levels of 19–21 mmol g−1 h−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 and Hansenula polymorpha, growth in the presence of benzoate resulted in an increase in q O2 and, at high concentrations of benzoate, in aerobic fermentation. In contrast to S.Cerevisiae, the highest q O2 of these yeasts when growing at D = 0.10 h−1 in the presence of benzoate was equal to, or lower than the q O2 attainable at μ max 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.