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.

The use of metal nanoparticles (NPs) to enhance hydrogen production in dark fermentation (DF) has become a pioneering field of interest. In particular, iron-based nanoparticles (FeNPs) play a pivotal role in enhancing the activity of metalloenzymes and optimizing feedstock utilization, resulting in improved hydrogen production. This study investigated the effect of FeNPs (magnetite) supplementation at three different concentrations of 50, 100, and 200 ppm in a continuous dark fermenter for the production of hydrogen from rice straw acid hydrolysate. The highest hydrogen production rate of 2.6 ± 0.3 NL H₂/L-d was achieved with the addition of 100 ppm of nanoparticles, representing a 53% increase compared to the condition without FeNPs addition. This improvement was driven by a microbial community in which Clostridium was the major dominant genus. In addition, increasing the nanoparticle concentration to 100 ppm resulted in an increase in butyrate concentration to 2.0 ± 0.1 g/L, which is 43% higher than the butyrate concentration without FeNPs. However, when the NP concentration was increased to 200 ppm, the hydrogen production rate decreased to 1.6 ± 0.2 NL H₂/L-d. This study can serve as a guideline for future research aimed at evaluating the effects of FeNPs in continuous dark fermentation systems. This work highlights the potential benefits and challenges associated with the use of FeNPs, paving the way for future studies to optimize their application and improve the efficiency of dark fermentation processes.