Sustained iron-nanoparticle exposure reveals concentration-dependent enhancement and inhibition during continuous hydrogen production from brewer's spent grain hydrolysate

Iron-based nanoparticles can enhance dark fermentation, but sustained-exposure effects remain unclear. This study aimed to investigate continuous supplementation with iron (III) oxide nanoparticles (FeNPs) during dark-fermentative hydrogen production from brewer's spent grain (BSG) hydrolysate. A continuous stirred-tank reactor was operated sequentially without FeNPs and at 50, 100, and 200 mg FeNPs/L. Hydrogen productivity was highest at 100 mg FeNPs/L, reaching 8.4 L H 2 /L-d (0 °C, 1 atm), 15% above nanoparticle-free operation. Substrate utilization and acetate concentration were highest, while Clostridium remained dominant. At 200 mg FeNPs/L, sustained exposure coincided with persistent productivity decline and a community shift from Clostridium toward Lentilactobacillus . Preliminary estimates showed greater hydrogen energy recovery at 100 mg FeNPs/L, but the gain did not offset single-use laboratory-grade FeNP costs. These findings identify sustained FeNP exposure as a key variable in continuous operation and provide a process-level basis for jointly evaluating hydrogen productivity, reactor stability, and material cost.