Steering the product spectrum in high-pressure anaerobic processes: CO₂ partial pressure as a novel tool in biorefinery concepts

Background: Elevated CO₂ partial pressure (pCO₂) has been proposed as a potential steering parameter for selective carboxylate production in mixed culture fermentation. It is anticipated that intermediate product spectrum and production rates, as well as changes in the microbial community, are (in)directly influenced by elevated pCO₂. However, it remains unclear how pCO₂ interacts with other operational conditions, namely substrate specificity, substrate-to-biomass (S/X) ratio and the presence of an additional electron donor, and what effect pCO₂ has on the exact composition of fermentation products. Here, we investigated possible steering effects of elevated pCO₂ combined with (1) mixed substrate (glycerol/glucose) provision; (2) subsequent increments in substrate concentration to increase the S/X ratio; and (3) formate as an additional electron donor. Results: Metabolite predominance, e.g., propionate vs. butyrate/acetate, and cell density, depended on interaction effects between pCO₂–S/X ratio and pCO₂–formate. Individual substrate consumption rates were negatively impacted by the interaction effect between pCO₂–S/X ratio and were not re-established after lowering the S/X ratio and adding formate. The product spectrum was influenced by the microbial community composition, which in turn, was modified by substrate type and the interaction effect between pCO₂–formate. High propionate and butyrate levels strongly correlated with Negativicutes and Clostridia predominance, respectively. After subsequent pressurized fermentation phases, the interaction effect between pCO₂–formate enabled a shift from propionate towards succinate production when mixed substrate was provided. Conclusions: Overall, interaction effects between elevated pCO₂, substrate specificity, high S/X ratio and availability of reducing equivalents from formate, rather than an isolated pCO₂ effect, modified the proportionality of propionate, butyrate and acetate in pressurized mixed substrate fermentations at the expense of reduced consumption rates and increased lag-phases. The interaction effect between elevated pCO₂ and formate was beneficial for succinate production and biomass growth with a glycerol/glucose mixture as the substrate. The positive effect may be attributed to the availability of extra reducing equivalents, likely enhanced carbon fixating activity and hindered propionate conversion due to increased concentration of undissociated carboxylic acids.