Real-Time Respirometric Monitoring of Hydrogen Utilization in Iron-Assisted Hydrogenotrophic Denitrification Systems: Performance Evaluation and Mechanistic Insights

Abstract This study evaluated real-time respirometric monitoring of hydrogen utilization in iron-assisted hydrogenotrophic denitrification systems. A biological respirometer system (BRS) was used to measure real-time hydrogen consumption and its relationship to nitrate reduction. Batch reactors were operated under hydrogen-only (H-2) and iron-assisted (I-2) conditions, with abiotic controls (H-1 and I-1). Complete nitrate removal (50 mg NO3–-N L–1) was achieved in both biological reactors within 60–72 h, with removal rates of 0.70–0.75 mg L–1 h–1. The Fe-assisted reactor required significantly less external hydrogen input (28.13–29.58 mL per cycle) than the hydrogen-only system (32.97–36.98 mL), corresponding to an approximately 15–20% reduction in external hydrogen demand; nitrate removal rates were statistically similar between the two systems, indicating that this benefit reflects a lower external electron-donor requirement rather than enhanced denitrification kinetics. Kinetic analysis showed zero-order behavior (R2 = 0.97–0.99), indicating that nitrate reduction was primarily controlled by biofilm activity and hydrogen availability rather than substrate concentration. Microbial community analysis revealed enrichment of hydrogenotrophic and redox-adaptive taxa, with greater abundance of iron-responsive populations in the Fe-assisted system. Iron wool corrosion is proposed to have supplied additional hydrogen and Fe2+ as supplementary electron sources; enhanced electron transport within the biofilm is offered as a possible, but not directly demonstrated, contributing mechanism. The BRS enabled continuous, dynamic monitoring of hydrogen consumption, providing new insights for process optimization and improved nitrate removal efficiency in autotrophic denitrification systems.