Effect of Operational Parameters on Dark Fermentative Hydrogen Production and Volatile Fatty Acids from Agro-Industrial By-Products

The purpose of this study was to examine how hydraulic retention time (HRT) influences biohydrogen generation and the formation of end-products during the co-digestion of olive mill wastewater (OMW), cheese whey (CW), and sewage sludge (SS) mixed in a 40:40:20 (v/v/v) ratio. The relationship between the substrates, resulting metabolites, and microbial communities was also explored. Continuous fermentation trials were carried out under both mesophilic (37 °C) and thermophilic conditions using HRTs of 12, 24 and 48 h. Acetic, propionic, and butyric acids were identified as the main end-products. The highest hydrogen production rate (4.4 ± 0.5 L H2/Lreactor/day) occurred under thermophilic conditions at an HRT of 24 h, whereas under mesophilic operation at the same HRT the hydrogen production reached 3.0 ± 0.3 L H2/Lreactor/day. In contrast, the greatest accumulation of volatile fatty acids (VFAs) was observed under mesophilic conditions (10.02 g/L), while thermophilic operation at 24 h HRT resulted in 5.54 g/L of total VFAs. The improved performance under thermophilic fermentation is likely linked to the suppression of hydrogen-consuming bacteria at elevated temperatures, which favors rapid hydrogen producers. Microbial community analysis indicated dominance of Firmicutes and persistent Lactobacillus prevalence across conditions. Shorter HRT at 37 °C promoted community diversification with genera such as Olsenella, Dialister, and Prevotella increasing in relative contribution. Under thermophilic operation, consortia remained Lactobacillus-dominant but showed significant temporal restructuring. The predominance of acetic acid (~2.80 g/L) and butyric acid (~2.60 g/L) indicates that hydrogen generation mainly followed the acetic and butyric pathways. This study reveals how targeted control of HRT and temperature can steer microbial communities toward highly hydrogen-productive consortia in the continuous dark fermentation of mixed agro-industrial wastes.