Medium composition, temperature, and CO supplementation control CO₂ conversion to C2-C6 alcohols by Clostridium...

Bioconversion of CO₂ into liquid fuels represents a promising strategy for advancing carbon-neutral biomanufacturing. Clostridium muellerianum (P21), a recently identified acetogen, has demonstrated the capability to convert CO₂ into C2-C6 alcohols. However, the process parameters affecting product distribution and chain elongation remain uncharacterized. This study evaluated the effects of medium composition, temperature, headspace pressure, and CO supplementation on CO₂ fermentation by strain P21. Batch fermentation in assay bottles showed that corn steep liquor (CSL) medium and elevated temperature (37°C) significantly improved gas utilization efficiency and increased alcohol titers 2-4 folds compared to P11 medium or 27°C (p < 0.05). CSL medium promoted higher biomass formation and enabled carbon-chain elongation toward C4-C6 products, while CO supplementation further increased solventogenesis, increasing butanol and hexanol formation by up to 1.6-fold relative to CO₂-only conditions. Under studied conditions (CSL medium, 37°C, CO-enriched gas), strain P21 produced ethanol (9 g/L), butanol (2 g/L) and hexanol (1 g/L). Scale-up in a 3-L CSTR confirmed process robustness, achieving ∼12.5 g/L ethanol and ∼2.5 g/L butanol. These results demonstrate the metabolic versatility of strain P21 and highlight the importance of targeted tuning of operating parameters in enhancing scalable CO₂-to-alcohol bioconversion for sustainable fuel and chemical production.