B-vitamin supplementation unlocks DHA production of Aurantiochytrium limacinum on molasses-based fermentation residue.

Various marine protists inhabiting high-salinity environments can convert organic nutrients into high-value biomolecules or biomass, making them suitable agents for valorizing saline organic residue from fermentation waste streams, such as condensed molasses fermentation solubles (CMS). This study established a screening method to identify mixotrophic or heterotrophic marine protists suitable for CMS valorization and to assess their bioconversion potential. Growth-performance and nutrient-replacement assays were evaluated based on relative colony coverage on agar plates and final cell density in liquid culture. When applied to the selected strain, Aurantiochytrium limacinum, CMS supplementation was shown to enhance glucose consumption and biomass production; however, it did not increase the production of fatty acids such as docosahexaenoic acid (DHA). Reducing medium salinity did not rescue the impaired lipid biosynthesis, whereas supplementation with vitamins B1, B7, and B12 restored fatty acid production in a dose-dependent manner. These results indicate that vitamin-associated cofactors are major determinants of fatty acid biosynthesis under CMS-based cultivation. Our findings demonstrate that molasses-derived residues can serve as substrates for the production of highly unsaturated fatty acids. They also provide evidence that B-vitamin cofactors play an important role in the fatty acid biosynthetic pathway of thraustochytrids. KEY POINTS: • CMS supported robust growth but did not promote proportional lipid synthesis in Aurantiochytrium limacinum. • B-vitamin insufficiency (B₁, B₇, and B₁₂) was identified as the primary factor limiting the biosynthesis of fatty acids. • Supplementation with B-vitamin cofactors restored fatty-acid and DHA biosynthesis in a dose-dependent manner.