Effects of iron nanoparticles and electro-stimulation on biohydrogen production by Clostridium pasteurianum

Biological hydrogen (H₂) production is a promising alternative to fossil-based energy processes, and strategies capable of improving H₂ yields are required to enhance its industrial feasibility. In this study, iron nanoparticles supported on carbon black (Fe/CB) were evaluated, for the first time, alone and in combination with electro-stimulated fermentation (EF), to enhance H₂ production by Clostridium pasteurianum DSMZ 525. Suspended Fe/CB and Fe/CB immobilized on the anode were employed and compared with traditional fermentation (TF) and EF. Under TF conditions, suspended and immobilized Fe/CB increased H₂ production by 16.7% and 62.5%, respectively, while increasing organic acid concentrations by 3.6- and 4.7-fold. The highest H₂ concentration (129.82 mmol L⁻¹) and H₂ yield were achieved under EF (0.4 V) without Fe/CB. Under this condition, organic acid and cell biomass concentrations decreased by 83% and twofold, respectively. In contrast, the addition of suspended or immobilized Fe/CB during EF decreased H₂ production by 29% and 24%, respectively, while substantially increasing organic acid concentrations. Overall, Fe/CB enhanced H₂ production during TF, particularly when immobilized on the anode, whereas EF alone proved to be the most effective strategy for maximizing H₂ production. These findings demonstrate that electro-stimulation can redirect microbial metabolism toward H₂ evolution, while Fe/CB supplementation may be more suitable for promoting the formation of value-added fermentation products. The results provide new insights into the combined use of conductive materials and electro-fermentation for controlling product selectivity in Clostridium-based bioprocesses. KEY POINTS: Suspended iron nanoparticles increase hydrogen production by 16.7% in fermentation.While immobilized iron nanoparticles increase hydrogen production by 62.5%.Electro-stimulation yields more hydrogen than iron-assisted electro-stimulation.