Effects of CO(2) limitation on the metabolism of Pseudoclostridium thermosuccinogenes

BACKGROUND: Bio-based succinic acid holds promise as a sustainable platform chemical. Its production through microbial fermentation concurs with the fixation of CO(2), through the carboxylation of phosphoenolpyruvate. Here, we studied the effect of the available CO(2) on the metabolism of Pseudoclostridium thermosuccinogenes, the only known succinate producing thermophile. Batch cultivations in bioreactors sparged with 1 and 20% CO(2) were conducted that allowed us to carefully study the effect of CO(2) limitation. RESULTS: Formate yield was greatly reduced at low CO(2) concentrations, signifying a switch from pyruvate formate lyase (PFL) to pyruvate:ferredoxin oxidoreductase (PFOR) for acetyl-CoA formation. The corresponding increase in endogenous CO(2) production (by PFOR) enabled succinic acid production to be largely maintained as its yield was reduced by only 26%, thus also maintaining the concomitant NADH re-oxidation, essential for regenerating NAD(+) for glycolysis. Acetate yield was slightly reduced as well, while that of lactate was slightly increased. CO(2) limitation also prompted the formation of significant amounts of ethanol, which is only marginally produced during CO(2) excess. Altogether, the changes in fermentation product yields result in increased ferredoxin and NAD(+) reduction, and increased NADPH oxidation during CO(2) limitation, which must be linked to reshuffled (trans) hydrogenation mechanisms of those cofactors, in order to keep them balanced. RNA sequencing, to investigate transcriptional effects of CO(2) limitation, yielded only ambiguous results regarding the known (trans) hydrogenation mechanisms. CONCLUSIONS: The results hinted at a decreased NAD(+)/NADH ratio, which could ultimately be responsible for the stress observed during CO(2) limitation. Clear overexpression of an alcohol dehydrogenase (adhE) was observed, which may explain the increased ethanol production, while no changes were seen for PFL and PFOR expression that could explain the anticipated switch based on the fermentation results.