ON α-KETOGLUTARIC ACID FERMENTATION

Abstract

That α-Ketoglutaric acid fermentation is widely distributed in microorganisms was confirmed by experiments with shaken cultures of Pseudomonas fluorescens, Serratia marcescens, Bacillus megatherium, Bacillus natto (a variant of Bacillus subtilis), Bacterium succinicum, Gluconoacetobacter cerinum and a new Bacterium strain No. 84C (temporary name), isolated from soil by the authors. The maximum yield of α-Ketoglutaric acid among the species examined was obtained with Bacterium strain No. 84C which produced up to 56% of the acid for total glucose supplied in a 10% glucose concentration after 72 hours' incubation. A description of the morphological and physiological properties of this bacterium is presented.
With regard to the constituents of the fermentation medium, various nitrogen and carbon sources and their optimum concentrations, as well as the additive influences of metals, arsenite and vitamins, were examined, especially in relation to α-Ketoglutaric acid fermentation in Serratia marcescens and Bacterium strain No. 84C, both of which are considered to have value for industrial application. α-Ketoglutaric acid was formed from D-glucose, D-mannose, D-galactose, D-fructose, D-xylose, L-arabinose, L-rhamnose, K-gluconate and K-2-ketogluconate in Serratia marcescens No. 18.
In the case of Bacterium strain No. 84C, α-Ketoglutaric acid formation was tested with limited carbon sources involving of D-glucose, D-galactose, D-fructose, sucrose, maltose, and Ca-gluconate, and the results indicate that all substrates were utilized for acid formation.
The optimum range of glucose concentration in the medium was found to lie at 3-4% for the former strain; in the latter it was somewhat at a higher, 5-7%, and in this strain complete fermentation was possible even glucose concentration of 10%, provided that the medium was balanced in other components and an excess of CaCO₃ was added.
Among inorganic nitrogen sources, ammonium salts such as (NH₄)₂SO₄, NH₄H₂PO₄, or a mixture of the two, were found to be suitable, and a rather sharp peak seemed to exist in the range of limited concentration, 0.15-0.17% in the case of (NH₄)₂SO₄ in medium containing 7% glucose. The highest yield was obtained by using Bacterium strain No. 84C and a medium containing 0.135g (NH₄)₂SO₄ and 0.135g NH₄H₂PO₄ with a 10% concentration of glucose.
The addition of iron to the medium increased the acid yield in Serratia marcescens; the optimum quantity was found to be 0.1-0.6ppm Fe.
Remarkably interesting were the effects of adding arsenite and thiamin; these gave in optimum concentrations a marked increase in acid production in Serratia, while in Bacterium strain No. 84C no stimulative effect was observed.
From the investigation of chemical changes during fermentation and from the isolation of intermediates of the initial stages of fermentation, it may be said that α-Ketoglutaric acid formation in Pseudononas fluorescens No. 33F, Serratia marcescens No. 18 and Gluconoacetobacter cerinum most probably proceeds via a direct oxidative pathway as has been proposed by Koepsell et al. In Bacterium strain No. 84C, however, the Emden-Meyerhof scheme, or some alternative pathway involving a direct oxidation not involving 2-ketogluconate as an intermediate, are considered possible.