VOL. 12 (1953) CO 2 TURNOVER IN FERMENTATION 211 



fumarate or malate. In the presence of ^^C-propionate the succinate became labelled. 

 The rate of the reaction was not studied so the quantitative significance is difficult to 

 judge. 



It was decided to reinvestigate CO 2 turnover and fixation to determine if the 

 turnover was sufficient to account for the formation of all the propionate by decarboxy- 

 lation of succinate. For this purpose, the fermentations were conducted under partial 

 vacuum in flasks 5 to 10 times the volume of the reaction mixture. The flasks were 

 fitted with ground glass joints and were shaken continuously to ensure equilibration 

 between the liquid and gas phases. ^^COg-NaH^^COg of known specific activity and 

 amount was introduced into the closed system and from the dilution of the ^'^COg the 

 CO 2 turned over in the reaction was estimated. 



Calculations and assumptions 



Barker et al.^^ have pointed out that the total carbon dioxide production (CO 2 total) 

 must exceed the observed or net carbon dioxide production (CO 2 observed) t>y the amount 

 used for synthetic reactions (CO 2 used) ^•^• 



'-''-^2 total ^= '-'^2 observed i '-''-'2 used 



If the CO 2 observed is negative, the above equation still applies. 



The following equation has been derived by Barker et al^^ for the purpose of 

 calculation of the COg used and thus the COg total- 



Where: ^ is a proportionality factor [A x CO 2 observed = CO 2 used) 

 Xj= the total counts in the initial CO 2 

 Xf=^ the total counts in the final CO 2 

 Vi = the mil/ of initial CO 2 

 Vf= the mM of final CO 2 



The following assumptions are inherent in the derivation of the equation : 



1. That A as defined above applies, i.e. the fermentation is uniform (stoichiometric) 

 during the entire course of the reaction and that for every mole of CO 2 observed (^ef produc- 

 tion or net utilization) there is a proportional amount of CO 2 used- 



2. That there are no secondary conversions of the end products that cause a change 

 in the ^"^C concentration of the COg. 



3. That the CO2 inside and outside of the cell are in isotopic equilibrium at all times. 



4. That the fermenting system does not differentiate between compounds containing 

 ^-C and those containing^^C. 



The validity of each of these assumptions as applied to the propionic acid ferment- 

 ation will be considered briefly. 



The assumption that the fermentation is uniform may not be completely valid under 

 some conditions. This has been shown by Wood et al.^' ^^ in fermentations of glucose 

 and glycerol with proliferating cells grown over a long period of time (0 to 30 days) .The 

 present fermentations were conducted for a relatively short period of time and though 

 a thorough study was not made there was no indication of a gross variation of products 

 in fermentations of short duration with resting cells. 

 References p. 221I222. 



