Abstract

BackgroundBio-based succinic acid holds promise as a sustainable platform chemical. Its production through microbial fermentation concurs with the fixation of CO2, through the carboxylation of phosphoenolpyruvate. Here, we studied the effect of the available CO2 on the metabolism of Pseudoclostridium thermosuccinogenes, the only known succinate producing thermophile. Batch cultivations in bioreactors sparged with 1 and 20% CO2 were conducted that allowed us to carefully study the effect of CO2 limitation.ResultsFormate yield was greatly reduced at low CO2 concentrations, signifying a switch from pyruvate formate lyase (PFL) to pyruvate:ferredoxin oxidoreductase (PFOR) for acetyl-CoA formation. The corresponding increase in endogenous CO2 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. CO2 limitation also prompted the formation of significant amounts of ethanol, which is only marginally produced during CO2 excess. Altogether, the changes in fermentation product yields result in increased ferredoxin and NAD+ reduction, and increased NADPH oxidation during CO2 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 CO2 limitation, yielded only ambiguous results regarding the known (trans) hydrogenation mechanisms.ConclusionsThe results hinted at a decreased NAD+/NADH ratio, which could ultimately be responsible for the stress observed during CO2 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.

Highlights

  • Bio-based succinic acid holds promise as a sustainable platform chemical

  • In order to investigate the effect of the available CO2 on the formation of succinic acid and other fermentation products by P. thermosuccinogenes, a series of bottle cultivations was conducted with medium containing different NaHCO3 concentrations

  • Through bioreactor cultivations of Pseudoclostridium thermosuccinogenes sparged with 1 and 20% CO2 we studied the effect of CO2 limitation on its metabolism

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Summary

Introduction

Bio-based succinic acid holds promise as a sustainable platform chemical. Its production through microbial fermentation concurs with the fixation of CO2, through the carboxylation of phosphoenolpyruvate. The strictly anaerobic Pseudoclostridium thermosuccinogenes is the only known thermophile to produce succinic acid as one of its major fermentation products (along with acetic acid, lactic acid, formic acid, ethanol, and hydrogen gas) [3, 4]. It is a close relative of the much better studied Hungateiclostridium thermocellum and Hungateiclostridium cellulolyticum, both efficient cellulose degraders that produce ethanol; Pseudoclostridium thermosuccinogenes is incapable of cellulose degradation, instead it is able to grow rapidly on inulin, a fructose polymer (as well as a range of C5 or C6 monosaccharides). Other reactions in the central metabolism that could be affected by different CO2 concentrations include those catalyzed by malic enzyme (ME) and pyruvate ferredoxin oxidoreductase (PFOR), facilitating the oxidative decarboxylation of malate to pyruvate, and that of pyruvate to acetyl-CoA, respectively

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