Limonene-enabled green extraction of omega-3 rich lipids from wet Schizochytrium sp. with potential for sustainable food ingredient applications

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Sustainable extraction of omega-3 lipids from wet microbial biomass remains a significant challenge, and the potential of limonene as a green solvent for this purpose has not yet been systematically investigated. This study addresses this gap by evaluating limonene-based solvent systems for extracting docosahexaenoic acid (DHA) from wet Schizochytrium sp. biomass, eliminating the need for energy intensive drying. To the best of our knowledge, this is the first study to optimize a limonene-based extraction system for DHA recovery from wet Schizochytrium sp. biomass. Limonene alone recovered only ∼50% of DHA from wet biomass compared to the conventional chloroform:methanol (C:M 2:1 v/v) system. However, a binary solvent system of limonene and methanol, optimized using Response Surface Methodology (Box–Behnken design), produced a highly significant model ( p = 0.001; R² = 92%; adjusted R² = 87.8%; predicted R² = 75.6%). Under optimized conditions (limonene:methanol 1:1.1 v/v, 80°C, 1.5 h), experimental evaluation achieved 112 ± 0.6% DHA recovery, closely matching the predicted 118%. Ethanol was also tested as a food-grade alternative, achieving 99% DHA recovery, statistically comparable to the conventional system. These findings demonstrate that limonene-based solvent systems enable environmentally responsible lipid recovery from wet biomass, providing a pathway toward more sustainable, scalable, and bio-based processing strategies for omega-3 food ingredients. Future work should address solvent recycling and co-extraction of high-value compounds such as astaxanthin to improve process economics.