Biohydrogen production via dark fermentation of brewery wastewater and sewage sludge: Influence of substrate mixture on process kinetics and microbial community

Industrial wastewaters with high organic loads are increasingly recognized as underexploited resource streams, and dark fermentation offers a promising route for simultaneous hydrogen and volatile fatty acid production. However, process stability remains limited by substrate imbalance and microbial competition. This study examined the co-fermentation of brewery wastewater (BWW) and sewage sludge (SS) at different mixing ratios to assess substrate complementarity, biohydrogen yield, and biodegradability under batch biochemical hydrogen potential assays via dark fermentation. Partial degradation and solubilization of organic matter were observed, with SS-rich mixtures promoting hydrolysis of particulate fractions, whereas BWW-rich substrates showed greater net soluble consumption. The highest volatile fatty acid concentration (over 8200 mg/L) was obtained with a 50:50 BWW-SS mixture, highlighting the role of substrate complementarity in intermediate metabolite accumulation . Increasing BWW content enhanced hydrogen production and yield, reaching 90.8 NmL H2 /g VS . A kinetic model based on a diauxic growth pattern successfully described the two-stages biohydrogen production profiles, reflecting the contribution of distinct metabolic pathways to hydrogen generation. The kinetic analysis revealed that the highest hydrogen production rate was obtained with the 50:50 (v/v) BWW–SS mixture. Microbial analysis showed a stable Eubacteria:Archaea ratio of approximately 70:30 across all conditions, indicating that variations in soluble load did not significantly alter domain-level distribution. Increased BWW content reduced hydrogen-consuming methanogens and promoted butyrate-associated Eubacteria.