Combined Enhancement of Biohydrogen Production in the Halotolerant Cyanobacterium Aphanothece halophytica by Reducing Agents and Glucose Under Nitrogen-Deprived Dark Anaerobic Conditions

Hydrogen (H2) is a promising renewable energy carrier because of its high energy density and environmentally friendly characteristics. The halotolerant cyanobacterium Aphanothece halophytica is a potential biological H2 producer because of its ability to generate H2 via oxygen-sensitive hydrogenase activity under nitrogen-deprived dark anaerobic conditions. This study investigated the effects of various chemical supplements on H2 production by A. halophytica. Initial screening identified glucose, sodium dithionite, and dithiothreitol (DTT) as effective stimulants for enhancing H2 production. Optimization experiments established 10 mM glucose, 5 mM DTT, and 1 mM sodium dithionite as the optimal concentrations for H2 production. Combined supplementation under these optimized conditions resulted in the highest H2 production rate of 0.588 ± 0.028 mL·L−1·h−1 and maximum cumulative H2 yield of 8.445 ± 0.750 mL·L−1 after incubation for 72 h, representing increases of approximately 170–180%, compared with the untreated control. Simultaneously, maximum cumulative O2 production decreased from 20.176 ± 0.850 to 10.405 ± 0.480 mL·L−1, corresponding to approximately a 50% reduction. These findings demonstrate that simultaneous optimization of electron supply, oxygen minimization, and intracellular redox balance using selected stimulants effectively enhances H2 production by A. halophytica, providing a simple and practical strategy for improving biological H2 production.