Optimizing Cyanobacterial Hydrogen Production:Metabolic and Genetic Strategies with Glycerol Supplementation

Ayshat M Bozieva ¹ Mahipal S. Rao ^2,3 Makhmadyusuf K. Khasimov ⁴ Maria A. Sinetova ¹ Roman A. Voloshin ¹ Dmitry O. Dunikov ⁵ Anatoly A. Tsygankov ⁴ Yoong Kit Leong ⁶ Jo-Shu Chang ⁷ Suleyman I Allakhverdiev ^1* Barry D Bruce ^2,3,8*

• ¹ Institute of Plant Physiology, Russian Academy of Sciences (RAS), Moscow, Moscow Oblast, Russia
• ² The University of Tennessee, Knoxville, Knoxville, United States
• ³ Department of Biochemistry and Cellular and Molecular Biology, College of Arts and Sciences, The University of Tennessee, Knoxville, Knoxville, Tennessee, United States
• ⁴ Institute of Basic Biological Problems (RAS), Pushchino, Russia
• ⁵ Joint Institute for High Temperatures (RAS), Moscow, Moscow Oblast, Russia
• ⁶ Department of Chemical Engineering, School of Materials Science and Engineering, Tsinghua University, Beijing, China
• ⁷ Center for Smart Sustainable Circular Economy, Tunghai University, Taichung, Taiwan
• ⁸ Department of Chemical and Biomolecular Engineering, Tickle College of Engineering, University of Tennessee, Knoxville, Knoxville, Tennessee, United States

Developing sustainable hydrogen production is essential for advancing renewable energy and re-ducing fossil fuel dependence. Cyanobacteria, which harness solar energy through photosynthesis, offer a promising biological platform for hydrogen generation. However, maximizing yields re-quires targeted metabolic and genetic interventions. This study investigates four cyanobacterial species, assessing the effects of photosynthesis inhibitors and glycerol as an exogenous carbon source. Our analysis reveals that nitrogen-fixing Dolichospermum sp. achieves significantly higher and sustained hydrogen production when supplemented with glycerol, far surpassing previous benchmarks. Notably, Dolichospermum reached a maximum hydrogen production rate of 132.3 µmol H₂/mg Chl a/h—a 30-fold increase over rates achieved with DCMU, a common photosynthe-sis inhibitor. Genomic analysis identified key transporter proteins that may further optimize hydro-gen output. These findings demonstrate how metabolic and genetic modifications can enhance biohydrogen efficiency, positioning cyanobacteria as a viable, renewable hydrogen source with significant potential for global energy sustainability.