Physiological and Environmental Factors Influencing Hydrogen Production by Unicellular Green Alga Monoraphidium sp. KMITL-1

With the growing global energy demand and the urgent need to reduce carbon emissions, hydrogen (H2) has emerged as a promising clean energy carrier. Among various biological H₂ production, green algae present a sustainable and eco-friendly alternative due to their ability to produce H₂ via photobiological pathways. This study aimed to investigate H2 production by unicellular green alga Monoraphidium sp. KMITL-1, isolated from hydroponic water at the Plant Tissue Culture Laboratory, King Mongkut’s Institute of Technology Ladkrabang. The taxonomic identity of the strain, belonging to the genus Monoraphidium within the Selenastraceae family, was confirmed through morphological observation and molecular characterization using 23S plastid rRNA gene sequencing. Various physiological and environmental parameters influencing H₂ production were evaluated, including cell age, cell density, nutrient deprivation, carbon source, pH, temperature, and light intensity. A 24-hour-old culture with an OD₇₅₀ of 0.8 exhibited a significant increase in H₂ production. The optimal medium was potassium-deprived Tris-acetate-phosphate (TAP-K) supplemented with glucose at a concentration of 350 mmol C-atom L⁻¹. The ideal environmental conditions for H₂ production were pH 7.2, a temperature of 30 °C, and a light intensity of 60 µmol photons m⁻² s⁻¹. Under these optimized conditions, Monoraphidium sp. KMITL-1 achieved a maximum H₂ production rate of 67.976 ± 1.096 µmol H₂ mg Chl⁻¹ h⁻¹ and a cumulative H₂ yield of 3,190.436 ± 2.219 µmol H₂ mg Chl⁻¹ after 72 h of incubation. These results highlight the potential of Monoraphidium sp. KMITL-1 for large-scale biohydrogen production and its applicability in the development of sustainable energy technologies. HIGHLIGHTS Monoraphidium sp. KMITL-1 was isolated and identified as a promising green alga for biohydrogen production. Potassium (K) deprivation significantly enhanced H₂ production by reducing O₂ evolution and increasing hydrogenase activity under anoxic conditions. Among various carbon sources, glucose supplementation yielded the highest H₂ production, with 350 mmol C-atom L⁻¹ identified as the optimal concentration. Maximum H₂ production was achieved under pH 7.2, 30 °C and light intensity of 60 µmol photons m⁻² s⁻¹. GRAPHICAL ABSTRACT