Abstract

This study evaluates the effects of seven biochemical stimulants, ferulic acid, malic acid, L-carnitine, ergothioneine, magnesium sulfate, trehalose, and betaine, on biomass accumulation, total lipid content, and docosahexaenoic acid (DHA) production in Aurantiochytrium sp. ATCC PRA-276 under controlled fermentation conditions. The compounds were selected based on their reported roles in antioxidant defense, carbon flux regulation, osmoprotection, or methyl group donation, with the aim of assessing whether targeted, non-GMO supplementation could modestly enhance microbial productivity. Compared with the control, supplementation with L-carnitine and ferulic acid resulted in the greatest enhancement of DHA production, increasing DHA concentration by 31.7% and 29.2%, respectively. These treatments were also associated with statistically significant, albeit moderate, increases in total lipid accumulation and biomass production. These responses indicate correlated improvements in overall cellular productivity rather than isolated effects on lipid biosynthesis. DHA concentrations were quantified following lipid extraction and transesterification using GC-based analysis; however, comprehensive fatty acid compositional profiling (e.g., saturated, monounsaturated, and polyunsaturated fatty acid distributions or DHA-to-total lipid ratios) was not performed. Although direct mechanistic assays were not performed, the observed trends are consistent with known biochemical functions related to redox balance, cofactor availability, and stress adaptation. A preliminary cost-efficiency analysis identified malic acid as the most economical stimulant for DHA enhancement, whereas ergothioneine was the least cost-effective despite measurable biological effects. Collectively, these findings demonstrate that biochemical stimulation can provide incremental yet reproducible gains in DHA production and lipid accumulation. This work supports the use of targeted biochemical supplementation as a scalable, non-GMO strategy for microbial omega-3 production and establishes a foundation for future optimization through combinatorial supplementation, multi-omics validation, and process engineering.

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