Influence of H2:CO2 ratio and low CO supplementation on product distribution and carboxylate reduction in Clostridium autoethanogenum

Gas fermentation using H 2 :CO 2 and CO as feedstocks offers a sustainable alternative to fossil fuel-derived production of chemicals. Clostridium autoethanogenum is an industrially relevant acetogen for this process, but the effects of gas compositions – particularly H 2 :CO 2 ratios – on product distribution are still unclear, hindering optimisation. Previous studies have not provided comprehensive analysis of metabolic responses during the coutilisation of multiple gaseous substrates in batch cultures. This study investigates how H 2 :CO 2 ratios and low-level CO supplementation steer C. autoethanogenum metabolism, specifically influencing acetogenic and solventogenic pathways (acetate and ethanol production) and exogenous carboxylate reduction (conversion of butyrate to butanol). Batch cultures were conducted with H 2 :CO 2 ratios of 1:1, 2.5:1, and 4:1, CO supplementation of 0%, 2%, and 5%, and butyrate addition of 0, 10, and 20 mM. Substrate consumption, product generation, and biomass were monitored over time. Flux balance analysis (FBA) was employed to estimate the changes in internal fluxes during different stages of the culture. Ethanol production positively correlated with initial H 2 :CO 2 ratios, increasing from 0.7 mM ± 0.6 mM at 1:1 to 6.0 mM ±1.8 mM at 4:1. Acetate concentrations remained stable at 24.7 mM ± 4.7 mM. Higher initial CO pressures boosted biomass and CO uptake rates. Butyrate addition accelerated butanol but delayed ethanol production, indicating competition for reducing equivalents. Overall, this study demonstrates how H 2 :CO 2 :CO ratios can be strategically adjusted to steer C. autoethanogenum metabolism. The combination of factorial and Box-Behnken experimental design provided a flexible approach to generate data that can guide gas fermentation strategies toward targeted chemical production.