Background Nannochloropsis is a typical oil-producing microalga characterized by rapid growth and high eicosapentaenoic acid (EPA) content. However, its large-scale application is currently limited by low biomass yield and insufficient EPA accumulation, highlighting the need to optimize cultivation conditions to improve productivity.

Objective This study aims to optimize the cultivation conditions of N. gaditana FACHB-2541 to enhance its eicosapentaenoic acid (EPA) production, providing theoretical support and technical guidance for its large-scale, high-value applications in aquaculture, food, and pharmaceutical industries.

Methods Using f/2 medium as the initial medium, the effects of different nitrogen sources sodium nitrate (the control group), urea, ammonium chloride, and ammonium acetate, nitrogen concentrations (50, 100, 150 and 200 mg/L), and light intensities 75, 150 and 200 µmol/(m²·s) on the growth, lipid accumulation, and EPA biosynthesis of N. gaditana FACHB-2541 were investigated. A single-factor experimental design was employed, and multiple parameters including biomass, lipid content, and EPA yield were evaluated to determine the optimal cultivation conditions.

Results The results showed that the set levels of cultivation parameters (nitrogen sources, nitrogen concentration and light intensity) had significant effects on the growth performance and EPA production of N. gaditana FACHB-2541. Among the tested nitrogen sources, ammonium acetate was more efficiently utilized by N. gaditana FACHB-2541, resulting in faster growth and higher EPA accumulation. At a concentration of 150 mg/L nitrogen (ammonium acetate), N. gaditana FACHB-2541 achieved the highest biomass and EPA yield. Moreover, a light intensity of 75 µmol/(m²·s) was more favorable for growth and EPA production compared to higher intensities 150 and 200 µmol/(m²·s). Under the optimized conditions (ammonium acetate as the nitrogen source at 150 mg/L; light intensity 75 µmol/(m²·s), the microalga exhibited the maximum biomass of 2.10 g/L and EPA yield of 156.22 mg/L; which were 0.45 and 0.31 times more than those in the pre-optimization conditions.

Conclusion N. gaditana FACHB-2541 under the optimized cultivation conditions was the potential candidate to exploit high-valued EPA production. This studys provide a solid theoretical basis and practical parameters for enhancing the production efficiency of N. gaditana, especially for applications requiring high-value EPA output. The optimized cultivation strategy proposed here offers operational simplicity and scalability, making it promising for industrial-scale cultivation in sectors such as aquaculture, nutraceuticals, and functional food development.