VOL. 12 (1953) CO2 TURNOVER IN FERMENTATION 2ig 



CO 2 in oxalacetate by chicken liver enzymes involves phosphopyruvate and the reaction 

 occurs as follows : 



Phosphopyruvate + COg + ADP ?^ Oxalacetate + ATP 



The fluoride intensitive mechanism might be responsible for the randomization of 

 propionate-3-^^C and the reaction through which propionate is formed or equilibrated 

 with C*-dicarboxylic acids. As noted previously, this reaction may be more complex 

 than a direct reversible decarboxylation of succinate. 



The other explanation is that only one mechanism of fixation is involved and that 

 the inhibition is never complete but is sufficient to reduce the fixation to a point where 

 there no longer is a net uptake of COg. 



At the present time there is insufficient data to decide between these two alternatives 

 or other possibilities which have not been discussed. 



There is one other point of interest in the fluoride experiments. This is the fact that 

 that the formation of the non-reducing carbohydrate from glucose*^ is inhibited by 

 fluoride^®. At present nothing is known about this compound. 



METHODS 



The following designations have been assigned to the various strains of propionic acid bacteria 

 used in these fermentations: 19 P. shermanii, 34W P. arabinosum, 49W P. pentosaceum. 



The procedures and methods employed in the fermentations reported in Tables I and II are 

 the same as those used by Wood and Werkman^'*. 



The analytical procedures employed for the experiments reported in Tables III, IV, VI, and VII 

 were as follows: 



At the conclusion of the incubation period the medium was made acid with 5 N HgSO^ and the 

 COg was swept out with Ng. The COgwas trapped in 3 N sodium hydroxide in a weighed COj collector. 

 After the COg was removed, the bacteria were centrifuged in a Sorval centrifuge, and were washed 

 by suspending in 20 ml of distilled water and recentrifuged. This washing was repeated twice. The 

 solutions were neutralized, combined, and diluted to volume. 



The unfermented polyhydric alcohols were determined on an aliquot by the periodate method**, 

 the pyruvic acid by eerie sulfate oxidation*^ and the residual glucose by the Somogyi method*^. The 

 remainder of the solution was distilled to one-half its volume and the neutral volatile alcohols were 

 determined in the distillate*^. 



The residue of this distillation was acidified to congo red and extracted continuously with ether 

 32-48 hrs. The volatile acids were removed from the extract by twelve volume distillation and were 

 titrated. The respective acids were separated on a silica gel column according to the method described 

 by Elsden*^, with some modification. The dried sodium salts were converted to the acid with 200 per 

 cent, excess oi t, N sulfuric acid, and the excess water removed by mixing with silica (Merck's CP 

 powdered). The dry powder was put on the column and the propionic and acetic acids removed with 

 2.5 per cent butanol in chloroform. Thirty per cent, butanol in chloroform was used to remove the 

 formic acid. The eluates from the column was collected in a dilute sodium hydroxide solution and 

 separated from the solvent by shaking in a separatory funnel. The volatile acids were regenerated by 

 acid steam distillation and titrated. 



The formic acid fraction from such a column was not pure, being contaminated with acetic acid 

 and pyruvic acid (the latter in the fermentation of pyruvate). It was rechromatogrammed on a silica 

 column, using an external indicator (phenolphthalein) according to the method given by Isherwood*^. 

 That the acid titrated was formic acid was demonstrated by the specific analytical method for formic 

 acid^". It was further identified by oxidation to COg with mercuric oxide. 



The acetic acid obtained from the column was rechromatogrammed to remove all traces of 

 contaminating propionic acid. 



The non-volatile acids were separated on a silica column using the external indicator method**. 

 The solvents were removed from the solution by the procedure described for volatile acids and the 

 acid was recovered by ether extraction, and titrated. It was acidified to congo red and oxidized at 

 100° C with potassium permanganate and the acid was again obtained by ether extraction. It was 

 then subhmed under high vacuum at 1 15 to 125° C. This procedure gave pure succinate as judged by 



References p. 22i\222. 

 14 



