CO2 availability shapes acetate and ethanol production in Clostridium aceticum: a transcriptomics-guided study

Clostridium aceticum , a model acetogen utilizing the Wood–Ljungdahl pathway (WLP), offers a promising microbial platform for converting CO 2 -rich syngas into value-added biofuels. In this study, we systematically investigated the effects of varying CO 2 concentrations on fermentation kinetics and global transcriptional responses of C. aceticum . Acetate production increased steadily with higher CO 2 levels, reaching 12.3 g/L at 45% CO 2 , whereas ethanol accumulation peaked at 25% CO 2 , suggesting a metabolic trade-off between acid and alcohol formation. Elevated CO 2 enhanced biomass formation and product titer, but accelerated acidification, thereby constraining cell growth at very high concentrations. Comparative transcriptomics revealed 246 differentially expressed genes, with key CO 2 fixation genes ( acsA1 and fdhF ) and ethanol biosynthesis genes ( ald and adh ) upregulated, while ATP-dependent steps of the WLP, such as fhs , were repressed, reflecting intrinsic energy limitations. Notably, the upregulation of L-serine dehydratase and formate-acetyltransferase implicated serine metabolism as an alternative route to acetyl-CoA, providing formate and CO 2 to support the WLP. Guided by these insights, exogenous L-serine supplementation increased intracellular ATP by 1.95-fold, and enhanced acetate and ethanol titer by 23.3% and 12.5%, respectively. These findings highlight CO 2 availability as a central regulator of carbon flux and energy metabolism in C. aceticum and propose amino acid supplementation as a practical strategy to improve syngas fermentation efficiency.