Effect of nitrogen limitation and carbon sources on hydrogen and lipid production by Chlorella pyrenoidosa

Biofuel (including H₂ and lipids) from green algae is an attractive way to produce renewable energy. Under nitrogen-limitation, Chlorella pyrenoidosa produced both H₂ and lipids. In this study, the effects of nitrogen and carbon sources on the growth and the co-production of H₂ and lipids in C. pyrenoidosa were examined, and the regulatory mechanisms including photosynthetic electron transport, NADPH production and acetyl-CoA metabolism were investigated. The peak H₂ yield (241.42 mL L⁻¹) was found in 5 % nitrogen concentration (0.35 mM NH₄Cl) treatment, with the total lipid content being 45.0 % of dry weight. Nitrogen-limitation decreased PSII activity, inhibiting photosynthetic O₂ evolution, and thereby creating favorable anaerobic conditions for enhanced H₂ production. Excess energy in the photosynthetic electron transport chain prompted hydrogenase to consume surplus electrons as the terminal electron acceptor and produce H₂. Furthermore, nitrogen-limitation induced overreduction on the PSII acceptor side, resulting in the accumulation of NADPH, which provided reducing power for fatty acids synthesis. The addition of acetic acid enhanced the growth, H₂ production, and lipid accumulation in C. pyrenoidosa, with a recommended concentration of 2 mL L⁻¹. It was proposed that acetic acid inhibited photosynthesis, subsequently decreasing photosynthetic O₂ evolution. Additionally, acetic acid was converted into acetyl-CoA to provide the precursor for fatty acid synthesis, and it also promoted the generation of NADH, which provided the reducing power required for both fatty acid synthesis and H₂ production. These results highlight the positive roles of nitrogen limitation and acetic acid in enhancing H₂ and lipid production in C. pyrenoidosa and provide a basis for improving Chlorella's energy production efficiency through artificial regulation.