Bed-to-feeding chamber height ratio: A promising design criterion for fixed-film dark fermentative biohydrogen reactors

The role of the fixed-bed in anaerobic structured-bed reactors for biohydrogen production remains paradoxical, as high substrate conversion and bioH 2 evolution are typically confined to the initial compartments, yet the bed's complete removal impairs performance. This study investigated the impact of the bed-to-feeding chamber height ratio, proposed as a promising design criterion for adjusting bed height relative to the feeding chamber, on biohydrogen evolution. Four reactors were operated with progressively reduced bed heights while maintaining equivalent operating conditions and geometrical features in the feeding chamber. Results demonstrated that the bed-to-feeding chamber height ratio critically governed fermentation performance. Best-performing condition was achieved by removing one-quarter of the bed zone, which successfully increased the hydrogen flow rate by 91% compared to the conventional anaerobic structured-bed reactor, as well as yielding 30 to 61% higher hydrogen than the other conditions investigated, regardless of the levels of overall organic loading rate and hydraulic retention time applied. The bed-to-feeding chamber height ratios significantly shaped the microbial community structure and metabolic pathways. Higher bed-to-feeding chamber height ratios favored the selection of Enterobacter , driving acetate and biohydrogen production, while lower ratios promoted a predominance of Thermoanaerobacterium and Bacillus , shifting metabolism towards lactic acid production. Spatial and temporal metabolite analyses revealed that the best-performing bed height enabled metabolic versatility, including lactate cross-feeding, which supported high bioH 2 yields even under propionic acid accumulation. Finally, this study pioneered the use of the bed-to-feeding chamber height ratio as a potential key design parameter for maximizing bioH 2 production in dark fermentative fixed-film reactors focused on energy recovery from carbohydrate-rich wastewater.