Here, a comprehensive investigation was conducted under various operational strategies aimed at enhancing biohydrogen production via dark fermentation, with a specific focus on the lactate metabolic pathway, using powdered cheese whey as a substrate. Initially, a batch configuration was tested to determine both the maximum hydrogen yield (100.2 ± 4.2 NmL H₂/g COD_fed) and the substrate (total carbohydrates) consumption efficiency (94.4 ± 0.8%). Subsequently, a transition to continuous operation was made by testing five different operational phases: control (I), incorporation of an inert support medium for biomass fixation (II), addition of carbon-coated, zero-valent iron nanoparticles (CC-nZVI NPs) at 100 mg/L (III), and supplementation of Fe₂O₃ nanoparticles at concentrations of 100 mg/L (IV) and 300 mg/L (V). The results emphasized the critical role of the support medium in stabilizing the continuous system. On the other hand, a remarkable increase of 10% in hydrogen productivity was observed with the addition of Fe₂O₃ NPs (300 mg/L). The analysis of the organic acids’ composition unveiled a positive correlation between high butyrate concentrations and improved volumetric hydrogen production rates (25 L H₂/L-d). Moreover, the presence of iron-based NPs effectively regulated the lactate concentration, maintaining it at low levels. Further exploration of the bacterial community dynamics revealed a mutually beneficial interaction between lactic acid bacteria (LAB) and hydrogen-producing bacteria (HPB) throughout the experimental process, with Prevotella, Clostridium, and Lactobacillus emerging as the predominant genera. In conclusion, this study highlighted the promising potential of nanoparticle addition as a tool for boosting biohydrogen productivity via lactate-driven dark fermentation.

Dark fermentation (DF) has gained increasing interest over the past two decades as a sustainable route for biohydrogen production; however, understanding how reproducible the process can be, both from macro- and microbiological perspectives, remains limited. This study assessed the reproducibility of a parallel continuous DF system using fruit-vegetable waste as a substrate under strictly controlled operational conditions. Three stirred-tank reactors were operated in parallel for 90 days, monitoring key process performance indicators. In addition to baseline operation, different process enhancement strategies were tested, including bioaugmentation, supplementation with nutrients and/or additional fermentable carbohydrates, and modification of key operational parameters such as pH and hydraulic retention time, all widely used in the field to improve DF performance. Microbial community structure was also analyzed to evaluate its reproducibility and potential relationship with process performance and metabolic patterns. Under these conditions, key performance indicators and core microbial features were reproducible to a large extent, yet full consistency across reactors was not achieved. During operation, unforeseen operational issues such as feed line clogging, pH control failures, and mixing interruptions were encountered. Despite these disturbances, the system maintained an average hydrogen productivity of 3.2 NL H₂/L-d, with peak values exceeding 6 NL H₂/L-d under optimal conditions. The dominant microbial core included Bacteroides, Lactobacillus, Veillonella, Enterococcus, Eubacterium, and Clostridium, though their relative abundances varied notably over time and between reactors. An inverse correlation was observed between lactate concentration in the fermentation broth and the amount of hydrogen produced, suggesting it can serve as a precursor for hydrogen. Overall, the findings presented here demonstrate that DF processes can be resilient and broadly reproducible. However, they also emphasize the sensitivity of these processes to operational disturbances and microbial shifts. This underscores the necessity for refined control strategies and further systematic research to translate these insights into stable, high-performance real-world systems.