Rising levels of atmospheric carbon dioxide necessitate innovative biotechnological solutions to mitigate emissions while simultaneously producing sustainable energy carriers. Microbial CO₂ fixation offers a promising approach by utilizing acetogenic bacteria to convert CO₂ into high-value biofuels such as ethanol, butanol, and hexanol as renewable alternatives to fossil-derived chemicals. Clostridium muellerianum P21, a Gram-positive, rod-shaped acetogen, has demonstrated potential as a biocatalyst for CO₂ fixation into C₂-C₆ alcohols. However, its metabolic capabilities remain under-characterized, requiring an evaluation of key process parameters affecting its fermentative efficiency. This study investigates the influence of nutrient composition (Corn Steep Liquor [CSL] vs. Yeast Extract-based P11 medium), incubation temperature (27°C vs. 37°C), headspace pressure (10 vs. 20 psig), and carbon monoxide (CO) enrichment on the metabolic flux and product spectrum of strain P21. Batch fermentations were performed in 250 mL assay bottles under a H₂:CO₂ (3:1) gas phase at 125 rpm. Results demonstrated that increasing the cultivation temperature to 37°C led to a 2- to 4-fold enhancement in total alcohol production compared to 27°C. CSL-based medium supported enhanced fermentation performance, yielding a 2-fold increase in butanol and a 1.6-fold increase in hexanol compared to P11 medium, highlighting CSL's enriched nutrient profile for C₄-C₆ alcohol biosynthesis. While higher headspace pressure marginally improved gas-to-product conversion kinetics, its effect on final alcohol concentrations was minimal, particularly for butanol and hexanol. Under optimized conditions (CSL medium, 37°C, and CO-enriched gas phase), strain P21 achieved titer of ethanol, butanol, and hexanol of 9 g/L, 2 g/L, and 0.8 g/L, respectively. This is a 1.5- to 2-fold improvement over cultivation in the same medium at 27°C. Additionally, CO supplementation resulted in a 1.1-fold and 1.6-fold increase in butanol and hexanol yields, respectively. These findings underscore the metabolic versatility of C. muellerianum P21 and emphasize the critical role of process optimization in enhancing CO₂-to-alcohol conversion efficiency. Future research should focus on developing strategies to improve product selectivity, yield, and scalability, thereby advancing microbial CO₂ bio-utilization for sustainable biofuel production.
