Applying metabolic flux analysis to hydrogen fermentation using a metabolic network constructed for anaerobic mixed cultures

In this study, a mixed-cultural metabolic network for anaerobic digestion that included the concept of a “universal bacterium” was constructed, and metabolic flux analysis (MFA) applying this network was conducted to evaluate the flow of electrons and materials during H₂ fermentation under various conditions. The MFA results from two H₂ fermenters feeding glucose with (GP) or without (GA) the addition of peptone suggest that hydraulic retention time (HRT) presents a significant impact on hydrogen production, and the reversed trends could be observed at HRTs below and above 4 h. From the MFA results of lactate/acetate-fed H₂ fermenter, the highest flux of H₂ production is associated with more significant acetate consumption and the following pathways toward the anaplerotic reactions cycle that produces NADH. The occurrence of acetogenesis in the H₂ fermenters using various types of bioethanol-fermented residues (BEFRs) was also identified according to the MFA results. By analyzing the MFA results of all 49 sets of data from H₂ fermenters via Pearson's correlation, it was revealed that the flux of H₂ production positively correlates to the reduction of ferredoxin with pyruvate oxidation, acetate formation, and acetate emission when lactate was produced in the system. On the contrary, negative relationships were found between the flux of H₂ production and these three fluxes. The extended application of MFA provides additional information, including the fluxes between intracellular metabolites, and the information has the potential to be used in decision-making systems during the future operation of anaerobic processes by connecting operational parameters.