Regulation of carbon and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in ph osphate-limited chemostat culture by changing the overall degree of reducti on of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-pr opanediol became the major product, the specific rate of acid formation dec reased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydro xyacetone kinase feeding glycerol into the central metabolism and (ii) an o xygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol d ehydrogenase involved in propanediol formation. The redirection of the elec tron flow from hydrogen to NADH formation was associated with a sharp decre ase in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/fr ee CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional e nzyme to operate so as to reduce NAD in this culture. The decrease in aceta te and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in th e acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increas e in the ATP/ADP ratio in the culture grown with glucose and glycerol compa red with that in the culture grown with glucose alone. As previously report ed for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucai lle, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyric am is inverted (more acidic inside) when the in vivo activity of hydrogenas e is decreased (cultures grown on glucose-glycerol mixture). For both cultu res, the stoichiometry of the H+ ATPase was shown to remain constant and eq ual to 3 protons exported per molecule of ATP consumed.