Maximizing Omega-3 production in Chlorella sp. KLSc59 through central composite design-driven cultivation and...

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.