Fermentation of Pyruvic Acid by Clostridium botulinum. ∗

Abstract

It has been reported 1 that the fermentation of glucose by Types A and B Clostridium botulinum differs from the majority of bacterial fermentations in that ethyl alcohol and carbon dioxide are the main products of the fermentation, only small amounts of acetic and lactic acids together with traces of hydrogen being formed. This study has been extended to include the fermentation of pyruvic acid, which may be an intermediate compound in the fermentation of glucose and also in the degradation of amino acids such as alanine, by Cl. botulinum. 2

The experiments reported below were carried out with washed suspensions of Type A (E-43) Cl. botulinum although studies with Type B gave essentially the same results. Glucose-broth (800 ml) containing 0.1% Difco yeast extract was inoculated with 1.0 ml of a beef-brain culture of the organism and incubated for 20 hours at 37°C in a McIntosh and Fildes anaerobic jar. The culture was then centrifuged and the cells suspended in distilled water. The suspension was diluted with an equal volume of M/7.5 phosphate buffer and placed in the central chamber of Warburg vessels. A rapid stream of O₂-free H₂ was passed through the vessels for 10 minutes and they were then equilibrated 10 minutes before tipping in the sodium pyruvate from the side-arm. CO₂ production was determined at 37°C by the Warburg technic, the initial and final bound CO₂ being determined following the addition of 10% sulfuric acid to the contents of separate Warburg vessels. In the semi-macro experiments, Warburg vessels of 40 ml capacity were employed with Clerici fluid in the manometers.

It was observed that pyruvic acid is rapidly decarboxylated by washed suspensions of Cl. botulinum, Qco₂ values of 25-30 being observed under optimal conditions.