Maximizing omega-3 production in Chlorella sp. KLSc59 through central composite design-driven cultivation and cellulase-assisted extraction

Chlorella sp. KLSc59 is a promising freshwater microalga for omega-3 oil production, but its application is limited by low biomass yield and inefficient lipid recovery. This study developed an integrated strategy to improve biomass production and omega-3 recovery. Carbon and nitrogen source screening in modified Tris-Acetate-Phosphate (TAP) medium identified acetic acid (CH3COOH) and ammonium nitrate (NH4NO3) as the best-performing carbon and nitrogen sources. Central Composite Design (CCD) was then applied to optimize CH3COOH and NH4NO3 concentrations and pH. The resulting M2 formulation (33.9 mM CH3COOH, 16.1 mM NH4NO3, pH 6.6) gave the highest specific growth rate (0.20 h−1) and biomass concentration (1.16 g L−1) among the tested cultivation conditions. A second CCD was applied to optimize cell disruption by varying cellulase loading and sonication duration, identifying D6 (1,000 International Units (IU) g−1 DCW cellulase; 150 s sonication) as the best-performing extraction condition within the tested range for total and omega-3 fatty acid recovery. Among the tested comparative conditions, M2CaD6 yielded the highest omega-3 content and yield, increasing from 0.37 to 6.22 µg mg−1 DCW and from 0.74 to 14.90 mg L−1, respectively, corresponding to 17- and 20-fold increases over the previous TAP-CaCl2 cultivation and extraction method (PD0). Reducing NH4NO3 in M2 by half increased linoleic acid content, whereas higher light intensity (50 µmol m−2 s−1) reduced cellular total fatty acid accumulation. Overall, coordinated optimization of cultivation, CaCl2 induction, and enzymatic–mechanical disruption improved omega-3 production from Chlorella sp. KLSc59 and provides a basis for further process development.