CARBON AND ELECTRON FLOW IN CLOSTRIDIUM-BUTYRICUM GROWN IN CHEMOSTAT CULTURE ON GLYCEROL AND ON GLUCOSE

The metabolism of Clostridium butyricum DSM 5431 was studied in chemos tat culture under carbon limitation using either glucose or glycerol. On glycerol, the enzymes glycerol dehydrogenase, diol dehydratase and 1,3-propanediol (1,3-PD) dehydrogenase constitute the branch point tha t partitions the carbon flux between the competing pathways, i.e. form ation of either 1,3-PD or acetate and butyrate. The increasing levels of these enzyme activities with increasing dilution rates (D) explaine d the constant proportion of glycerol conversion into 1,3-PD. The prod uction of acetate or butyrate constitutes another important branch poi nt and when D increased (i) large amounts of intracellular acetyl-CoA accumulated, (ii) the carbon flux switched from butyric acid to acetic acid, (iii) the specific activity of thiolase was not affected, sugge sting this enzyme may be the bottleneck for carbon flux to butyrate bi osynthesis providing an explanation for the accumulation of large amou nts of intracellular acetyl-CoA, and (iv) high levels of NADH were fou nd in the cell. Oxidation of NADH by 1,3-PD dehydrogenase was linked t o the production of 3-hydroxypropionaldehyde (3-HPA) by glycerol dehyd ratase. The fact that high intracellular concentrations of NADH were f ound means that diol dehydratase activity is the rate-limiting step in 1,3-PD formation, avoiding the accumulation of 3-HPA which is a very toxic compound. The specific rate of glucose catabolism (q(glucose) = 11.1 mmol h(-1) g(-1)) was around four times lower than the specific r ate of glycerol catabolism (q(glucose) = 57.4 mmol h(-1) g(-1)). On gl ucose-grown cells, reducing equivalents which are released in the glyc olytic pathway were reoxidized by the butyric pathway and the low spec ific formation rate of butyric acid led to an increase in the intracel lular level of acetyl-CoA and NADH. Carbon flow was higher on glycerol due to the reoxidation of NADH by both butyric and PD pathways.