Medium composition, temperature, and CO supplementation control CO2 conversion to C2-C6 alcohols by Clostridium muellerianum P21.

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.