The rising demand for polyunsaturated fatty acids, coupled with the decline of traditional fish-based sources, highlights Ulkenia visurgensis Lng2, a newly isolated thraustochytrid strain, as a promising alternative for sustainable polyunsaturated fatty acids production. Using Response Surface Methodology to optimize medium composition and culture conditions, biomass production was improved to 11.64 g/L-120% higher than previous studies-with 35% of the biomass consisting of total lipids. Of the total fatty acids, 40% corresponded to polyunsaturated fatty acids, including 27% of docosahexaenoic acid, 4% of eicosapentaenoic acid, and 7% of arachidonic acid. Alternative media, such as wastewaters from corn and fish meal processing, were also evaluated. Biomass yields in wastewaters-based media (30% v/v) were lower due to limited nutrient availability, achieving between 2.96 g/L and 2.28 g/L, with the strain showing mostly around 8% carbon and nitrogen assimilation. Morphological changes, including increased vegetative and multinucleated cells, were also observed. Lipid content decreased in wastewater-based cultures, yet PUFAs constituted 47% of total fatty acids, with EPA nearly doubling, DHA remaining stable and decreased ARA content. These results shed light on how U. visurgensis Lng2 adapts to nutrient-limited environments and offer valuable insights for developing sustainable PUFA production using industrial by-products as alternative media.