Optimizing specific dark fermentation strategies for enhanced bio-hydrogen production from different agricultural wastes

The integration of the dark fermentation process into existing anaerobic digestion plants, leading to the implementation of two stage processes, may enable the sequential recovery of biohydrogen and biomethane from agricultural residues. This study evaluated semi-continuous dark fermentation of cattle manure, corn residue, and soybean residue at hydraulic retention times of 3–10 days, including co-fermentation and digestate recirculation strategies. Cattle manure did not support stable biohydrogen production under the tested conditions, producing no detectable hydrogen and rapidly shifting towards methanogenesis. CR showed only transient biohydrogen peaks, followed by process instability associated with pH decrease, nutrient limitation, and methanogenic competition. Soybean residue supported stable hydrogen production at HRT 3 d, reaching 4.7 NmLH2/gVS. The best performance was obtained by soybean/CR co-fermentation, which achieved 12.4 NmLH2/gVS, 67.8 NmLgas/gVS, and 0.32 gCODVFA/gVS. VFA production was strongly substrate-dependent, with CR at HRT 7 d reaching the highest VFA yield among single-substrate trials. Biomethane potential tests showed that fermented effluents retained relevant residual energy potential, with fermented CR increasing biomethane recovery by up to 34% compared with the untreated substrate. Overall, the results demonstrate that substrate selection and nutrients balance are key factors for implementing two-stage anaerobic digestion of agricultural residues.