Design of experiments based optimization of pH and carbon dioxide to hydrogen ratio for enhanced acetate and biomass formation by Clostridium ljungdahlii

In gas fermentation processes, pH value, and gas composition represent key parameters determining efficiency and product formation. This study focuses on optimizing these cultivation parameters using a multivariate framework to increase biomass and acetate formation of fermentations using the model acetogen Clostridium ljungdahlii and carbon dioxide (CO 2 ) and hydrogen (H 2 ) as sole gaseous substrates. A design of experiments (DoE) approach with the two main influencing factors spanning a design space of pH 5.0–6.5 and CO 2 :H 2 of 1:1–1:3 was applied to build a predictive regression model. This model predicted an ideal CO 2 :H 2 ratio of 1:3 and optimal pH of 5.27 for biomass and 5.4 for acetate formation, respectively. This aligns well with experimental observations, where a maximal acetate titer of 27.8 g/L and an optical density (OD 600 ) of 1.1 was achieved. Combined with off-gas analysis, carbon and hydrogen balances showed over 94% utilization of fixed gases for acetate production. Furthermore, by transferring the process to a continuous operation mode, the acetate production rate could be increased up to 2 g/L/h. It could be shown that the pH value has the strongest influence on process performance and that consistent high production rates could be achieved by optimizing the process parameters and process mode.