Alginate-bentonite immobilization with zero-valent iron nanoparticles enhances dark fermentative hydrogen production

Dark fermentation is a light-independent route for biohydrogen production, but conventional alginate immobilization may constrain mass transfer. Bentonite is an abundant, low-cost porous clay with potential to improve fermentation performance. However, its application in immobilized dark fermentation remains insufficiently investigated. This study systematically evaluated alginate-bentonite composition for immobilizing Clostridium butyricum CGS5, examined the further incorporation of zero-valent iron nanoparticles (Fe⁰NPs), and assessed the selected formulation under semi-continuous operation. Beads containing 1.125 % (w/v) sodium alginate and 1.05 % (w/v) bentonite achieved hydrogen accumulation of 409.27 ± 5.61 mL and a hydrogen yield of 1.79 ± 0.01 mol H₂/mol glucose, representing increases of approximately 70 % and 44 %, respectively, relative to suspended cells. Most bentonite-containing formulations exhibited greater resistance to compressive deformation than pure alginate beads, alongside changes in morphology and swelling behavior. Incorporating 50 mg/L Fe⁰NPs into the selected formulation further increased hydrogen accumulation to 459.17 ± 2.40 mL, an 8.1 % improvement over the Fe⁰NP-free immobilized control, without significant improvements in hydrogen production rate or yield. Following an initial 24-h batch phase, semi-continuous operation with 50 % medium replacement every 16 h prolonged hydrogen production across four feeding cycles, yielding 667.91 mL over 88 h. However, cycle-specific output varied and remained below the initial batch total. These findings support alginate-bentonite immobilization for enhanced batch biohydrogen production and identify an additional benefit of Fe⁰NP incorporation for hydrogen accumulation, while longer-term operational performance remains to be established.