Fermentation of Pyruvic Acid by Bacteria of the Colon-Aerogenes Group. ∗

Abstract

In view of the intermediary rôle assigned to pyruvic acid, an understanding of its behavior is of importance in formulating schemes of dissimilation for bacteria of the colon-aerogenes group. The anaerobic dissimilation of pyruvic acid by organisms of this group is discussed in the present work.

Aubel 1 and de Graff and Le Fevre 2 report the products of the fermentation of pyruvic acid by Escherichia coli to be acetic and formic acids, carbon dioxide and hydrogen. Acetaldehyde is reported to be an intermediate.

Reynolds, 3 fermenting glucose with Esch. coli under conditions of forced aeration, recovered much of the carbon as pyruvic acid with corresponding decreases in lactic acid. The rise and fall of pyruvic acid during the fermentation of glucose by Citrobacter freundii further substantiates its position as an intermediate.

Cells were prepared by centrifuging 24-hour cultures, washing once with physiological salt solution, and resuspending in enough solution to make a paste. A medium consisting of 10 gm. glucose, 8 gm. peptone, 5 gm. disodium phosphate per liter gave good growth. The medium was sterilized at 20 pounds for 30 minutes. Phosphate buffer was sterilized separately and added to the glucose and peptone after autoclaving. Incubation took place at 30°C. The cultures were identified strains used for several years in this laboratory: Aerobacter indologenes, strain 23B, isolated and described by Burkey 4 ; Esch. coli, American Type Culture Collection No. 26; and C. freundii, strain described by Werkman and Gillen. 5

The dissimilations were conducted anaerobically in 300 ml. of 0.1M phosphate buffer at pH 6.2. The flask and absorption-train were flushed with oxygen-free nitrogen before inoculation.

Carbon dioxide was absorbed in Bowen potash bulbs and weighed. An aliquot of the fermentation-liquor was acidified to congo red with 25% H₂SO₄, and residual carbon dioxide was determined by heating and aerating under a reflux condenser for an hour. Hydrogen was determined with the Hempel buret.

Before analysis of the liquor, the cells were removed by adding 1 ml. of phosphotungstic acid (20% in 5% H₂SO₄) per 20 ml. of medium and centrifuging. Acetic and formic acids were determined according to Osburn, Wood, and Werkman 6 on the steam distillate of an aliquot. Lactic acid was determined by the method of Friedemann and Kendall 7 and 2,3-butylene glycol according to Brockmann and Werkman. 8