Gas uptake stoichiometry governs carbon partitioning in syngas-fermenting Clostridium autoethanogenum

Given the current global environmental challenges, waste biomass is an attractive renewable resource for circular economies. Gasification of biomass yields syngas (CO, CO ₂ , and H ₂ ) that is a suitable feedstock for gas fermentation in biomanufacturing of fuels and chemicals using acetogen microbes. While it is generally known that syngas composition influences both acetogen growth and process performance, we are lacking a consistent dataset quantifying these effects under controlled fermentation conditions. Here, we mapped the metabolic response of the model-acetogen Clostridium autoethanogenum to seven synthetic syngas mixtures during exponential batch growth in bioreactor fermentations. Notably, distinct gas compositions resulted in different fermentation profiles, affecting both growth and metabolite production. Maximum specific growth rates ranged within 0.05 0.13 h ^-1 , with slower growth for low-CO mixtures. While acetate and ethanol production yields varied between 20–133 and 76–353 mmol per gram dry cell weight, respectively, minor production of 2,3-butanediol was detected. All syngas mixtures supported co-utilization of CO and H ₂ , though gas uptake stoichiometry only moderately correlated with syngas content. Importantly, gas uptake stoichiometry strongly influenced carbon partitioning, with higher relative H ₂ uptake reducing CO ₂ loss or even realizing CO ₂ fixation together with increasing carbon flow towards metabolites. Interestingly, higher syngas H ₂ content favored ethanol and 2,3-butanediol production, while higher H ₂ :CO uptake ratios increased total flux through the Wood–Ljungdahl pathway rather than selectively favoring reduced by-products. Our results are valuable for a better understanding of syngas composition effects on the acetogen biocatalyst and for process engineering towards optimizing gas fermentation performance.