VOL. 12 (1953) CO2 TURNOVER IN FERMENTATION 217 



using cultures 19, 34W, and 49W but low fixation values were obtained. Among other 

 factors tested was supplementation of the growth medium with B vitamins, and with 

 the bacteria from such media somewhat better fixation was obtained; representative 

 results are shown in Table VIII. A complete analysis of the products was not attempted 

 in these fermentations. The non-volatile and votatile acids were titrated and the pro- 

 pionate and succinate were isolated by chromatography. It is seen in Fermentation No. 

 21 and No. 23 that the results are very similar to those previously obtained with P. 

 pentosaceum^. For example in No. 21 the specific activity of the propionate (9.85 cpm//iM) 

 was half that of the final COg (19.7 cpm/jitAf) and there was considerable dilution of the 

 added ^^CO^- In this case of the calculated COgused was 52.5 and the ^^COg produced 

 was 30.3 as compared to approximately 34.1 required by the scheme of Fig. i. It is 

 noted in Fermentation 20 that a really substantial amount of COg was fixed, 31.4 vaM, 

 and the carboxyls of succinate and propionate have a high specific acticity. Nevertheless 

 the dilution of the^^COg was not large and the calculated CO 2 produced was only ii.o milf 

 per 100 milf of fermented glycerol. This shows that there can be formation of propionate 

 and also substantial fixation of CO 2 into propionate without a large turnover of CO 2. 



At present it is not possible to give an adequate explanation of the variation in 

 capacity of different cell preparations of fix CO 2. It is usually has been assumed that the 

 primary fixation reaction in the propionic acid bacteria is in oxalacetate. Actually no 

 studies have been made with these bacteria on CO 2 fixation in oxalacetate and sub- 

 sequent to the original proposal a number of other mechanisms for fixation have been 

 found which may play a part. Interest in the possibility that fixation of COg may occur 

 by combination with propionates^ has been stimulated by the recent studies of Barban 

 AND Ajl22, Delwiche et aiy^, Larsen*", Lardy^^, and our investigation on randomiza- 

 tion of the isotope of propionate-3-^^C and incorporation of the latter into succinate. An 

 important question is whether or not the CO 2 is fixed directly in propionate to yield 

 succinate. Lardy^^ has proposed a very intriguing scheme for CO 2 fixation in which 

 the propionate is activated by formation of an acyl linkage with the sulfhydryl group 

 on the enzyme. The methyl group is pictured as reacting with a second sulfhydryl thus 

 forming a ring structure. This is followed by a cleavage of the acyl linkage. Carbonic 

 acid is activated by reacting with a sulfhydryl and combination with the propionate 

 then occurs to yield succinate. It is apparent that this scheme offers possibilities of 

 explaining the formation of a "C^" from succinate that is not in equilibrium with CO2 

 and also a mechanism for randomization of the a and ^ carbons propionate. It is of 

 interest that fixation of CO 2 in oxalacetate, malate and succinate could in each case 

 occur by an identical C3 -(- CO 2 reaction in the sulfhydryl complex, the major difference 

 in the reaction brought about by the enzymes being the stage at which the C4-sulfhydryl 

 complex is cleaved. Thus oxidation of propionate to the pyruvate level might occur as 

 the sulfhydryl complex and fixation take place at the oxalacetate level. The cleavage 

 of the C^ acid might occur only after succinate is formed by reduction of the formed 

 oxalacetate. The non-equilibration of succinate with fumarate and malate as found by 

 Barban and Ajl^^ might thus be explained. 



The variation in the capacity of different cell preparations to fix or exchange CO 2 

 may lie in their ability to catalyze the reaction "C^" ^f^ COg or to form the "Cj" sulfhy- 

 dryl complex. 



It is of interest that CO 2 is fixed only in the carboxyl group whereas formaldehyde 

 is fixed in every position of propionate, succinate, and acetate^^. Thus the "C^" formed 



References p. 221J222. 



