Studies on the energy metabolism during anaerobic fermentation of glucose by baker's yeast

As a result of the intimate association of ADP phosphorylation with alcoholic fermentation, resulting in the synthesis of 2 mole ATP per mole glucose fermented, it may be calculated that a minimum of 672 mucal heat development may be expected for every mm-3 CO2 developed during alcoholic fermentation. When all ATP produced would be fully de-phosphorylated to ADP + Pi (e.g. by ATP-ase activity) a maximum heat development of 1200 mucal per mm-3 CO2 could be expected. Using the LKB-Flow-Microcalorimeter for measurement of heat development and at the same time the Warburg technique for measuring CO2 development during anaerobic glucose fermentation of a baker's yeast suspension, the heat development per mm-3 CO2 produced was calculated over a fermentation period of 90 min. Maintenance of strict anaerobic conditions in the Flow-Microcalorimeter vessel was complicated by diffusion of traces of oxygen via the Teflon transport lines, resulting in excessive heat development values, not representative for the alcoholic fermentation. This problem could be circumvented by removal of traces of oxygen by means of addition of the enzyme glucose-oxidase. Poisoning the respiratory enzyme system of the yeast by addition of KCN or azide, or using respiratory-deficient mutants of the yeast also resulted in heat development values, inherent with alcoholic fermentation. The values obtained were very close to the minimum of 672 mucal per mm-3 CO2, at least during the initial phases of fermentation, indicating that ADP regeneration from ATP, essential for maintaining the high fermentation rate, is not primarily the result of ATP-ase activity, but must be due to participation of ATP in energy-requiring synthetic reactions.