Abstract

Degenerated strains of Clostridium acetobutylicum, lacking solventogenic pathways, enable continuous biohydrogen and organic acid production. This study investigated the influence of medium pH (5.5, 6.0, and 6.5) on metabolic distribution and biohydrogen production in continuous cultures of a degenerated strain. Experimental data on substrate consumption and product formation were integrated into a genome-scale metabolic model using the Pheflux algorithm to infer intracellular flux distributions and cofactor dynamics. Steady-state conditions were achieved at all pH values. Acetic and butyric acids were the predominant metabolites at pH 5.5 and 6.0, while at pH 6.5, lactic acid production became dominant. Biohydrogen production peaked at pH 6.0 with a volumetric rate of 12.78 LH2 center dot L-1 reactor center dot d-1 and a yield of 2.41 mol H2/mol glucose. Lower yields were observed at pH 5.5 and 6.5, with thelatter condition showing significant carbon redirection toward lactic acid synthesis, consuming NADH and reducing biohydrogen generation. In silico analyses revealed pH-dependent shifts in ATP generation, NADH utilization, and proton flux. At low pH, increased ATPase activity compensated for intracellular acid stress, while at higher pH, lactate production enhanced glycolytic flux, resulting in higher ATP production rates but lower efficiency. These findings demonstrate that external pH critically modulates metabolic pathways, cofactor balance, and energy efficiency, highlighting pH control's importance in optimizing biohydrogen yield and productivity in continuous fermentations using degenerated strains of C. acetobutylicum.