Techniques for Studying the Mechanism of Higher Alcohol Formation by Yeasts

Abstract

Procedures developed for research investigations of the mechanisms of formation of higher aliphatic alcohols or fusel oil components during alcoholic fermentation are described. Fermentations of 20 per cent glucose solutions by resting cells of mutant strains of Saccharomyces cerevisiae, lacking the normal ability to synthesize one or more particular amino acids, were used. The higher alcohol fraction was quantitatively concentrated by one or two procedures, fractional distillation coupled with ether extraction or close fractional distillation alone. The fraction was analyzed by gas chromatography on a 6 ft x ¼ in. column containing Ucon LB 385 as the stationary active phase operated with helium carrier gas at 100°C. The separation of active and isoamyl alcohols required a second column with glycerol packing and used at 75°C. The individual alcohols produced in fermentations of radioactive carbon-14 intermediates were separated by gas chromatography. Sufficient amount (about 0.5 mg) were obtained by condensation of the effluents from a peak from a single injection onto the column to permit radioactive counting by liquid scintillation.

The procedures are illustrated by reporting the results of an experiment in which glucose was fermented by a valine-, isoleucine-dependent mutant in the presence of α-aminobutyric acids, 1-C-14 and 2-C-14, as a source of α-ketobutyric acid. Of the higher alcohols the mutant produces only n-propanol and n-butanol. Both were labeled by the 2-C-14 acid, but neither by the 1-C-14 acid, since the carbon in the number one position is lost by decarboxylation when the α-keto acid is utilized by yeast to form an alcohol according to the Ehrlich scheme.