Abstract
The increasing reliance on fossil fuels for global energy production has intensified greenhouse gas emissions, highlighting the need for sustainable energy alternatives. Hydrogen is considered a promising green fuel due to its high energy density and conversion efficiency. Among various production pathways, microalgae-based biohydrogen generation via biophotolysis is particularly attractive owing to its high biomass productivity, adaptability to diverse water sources, flue gas mitigation potential, and low land requirements.This study investigates the effects of key microalgal growth parameters on biohydrogen production by Chlorella sp. through biophotolysis. The impacts of nitrogen purging during the transition from aerobic to anaerobic conditions, different photoperiod regimes (continuous illumination, continuous darkness, and a light-dark cycle), glucose supplementation (5, 10, and 15 g L⁻(1)), and temperature (25, 30, and 35 °C) were systematically evaluated. Microalgal cell density was monitored during hydrogen production to elucidate its relationship with hydrogen yield. Initial experiments were conducted in 10 mL test tubes to identify optimal conditions, which were subsequently applied to scale-up experiments in a 1000 mL jacketed reactor. Nitrogen purging significantly enhanced hydrogen production by removing oxygen and activating hydrogenase, resulting in a peak hydrogen concentration of 11 ppm. Continuous illumination yielded higher hydrogen levels than darkness and light-dark cycling. Glucose addition substantially increased hydrogen production, with the highest yield observed at 15 g L⁻(1) (30 ppm). An optimal temperature of 30 °C also maximized hydrogen production. Under these conditions, hydrogen production increased as cell density decreased due to metabolic shifts. Scale-up experiments achieved a 405-fold increase in hydrogen yield, demonstrating the scalability potential of the process. These findings emphasize the importance of optimizing algal growth conditions to balance microalgal growth and biohydrogen production for future industrial applications.