Mass analysis was used to determine the amount of acetate which is totally synthesized from 13CO2 during fermentations by Clostridium formicoaceticum, C. acidiurici, C. cylindrosporum, Butyribacterium rettgeri, and Diplococcus glycinophilus. In the fermentation of fructose by C. formicoaceticum, 27% of the acetate was found to be totally synthesized from CO2, and the remaining acetate was unlabeled, having been formed from fructose. Evidence is presented that the purine-fermenting organisms, C. acidiurici and C. cylindrosporum, totally synthesized about 9% of the acetate from CO2, and that the methyl group of an additional 9% was formed from CO2. The remaining acetate was formed from the carbons of the purine and not via CO2. It has been postulated that the fermentation of the purines and synthesis of acetate from CO2 both occur via derivatives of tetrahydrofolate. Evidence is presented that a compartmentalization of these folate intermediates is required if both the purine degradation and the CO2 utilization involve identical intermediates. Neither B. rettgeri nor D. glycinophilus incorporated sufficient 13CO2 into acetate to allow determination of the types of acetate by mass analysis, although they did incorporate labeled 14C02 in both positions of acetate. I Present address:

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Whole-cell preparations of Clostridium thermoaceticum were exposed to a short pulse of '4CO2 under conditions in which double-labeled acetate was synthesized. Radioactive methyltetrahydrofolate monoglutamate, diglutamate, and triglutamates were isolated from extracts of the cells. The radioactivity was found to be exclusively in the five methyl position. The specific activities of the methyltetrahydrofolate derivatives were very high and were in accord with the proposal that methyltetrahydrofolates are the precursors of the methyl of acetate. A new method of separation of folates employing QAE-Sephadex chromatography and a linear gradient with triethylammonium bicarbonate is presented which completely resolves the common folate monoglutamates and, upon freeze-drying, yields saltfree preparations.

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