Oleaginous fungi are promising platforms for sustainable production of lipids and very-long-chain fatty acids, including nervonic acid (NA; 24:1 n-9), whose natural availability is limited despite its importance. Following the identification of a promising strain in a screening study, Mortierella capitata CBS 110633 was selected for stepwise evaluation of cultivation conditions in shake-flask cultures. The effects of glucose concentration and nitrogen source composition on biomass formation, lipid accumulation, and NA enrichment were evaluated. Increasing glucose concentration primarily enhanced lipid accumulation, from approximately 4.8 to 8.7 g L⁻¹, whereas nitrogen source composition was associated with differences in fatty acid distribution and NA enrichment. Under conditions favoring NA enrichment, with 60 g L⁻¹ glucose, 4 g L⁻¹ yeast extract, urea supplementation, and 14 days of cultivation, NA enrichment reached 9.5% of total fatty acids. In contrast, the highest lipid production (8.7 g L⁻¹) and estimated NA yield (504.7 mg L⁻¹) were obtained using 60 g L⁻¹ glucose supplemented with yeast extract and peptone (4 g L⁻¹ each) after 14 days. These findings indicate that NA enrichment and total lipid accumulation respond differently to cultivation conditions and highlight M. capitata CBS 110633 as a promising microbial platform for further investigation and development of NA production.

Arachidonic acid (AA) is a high-value long-chain polyunsaturated fatty acid commonly produced by submerged fermentation of oleaginous fungi. Solid-state fermentation (SSF) using low-cost agro-industrial by-products represents a promising alternative approach for sustainable microbial lipid production. In this study, spent malt, a by-product of the brewing industry, was evaluated as a solid substrate for AA production by Mortierella alpina under SSF conditions. Four M. alpina strains were screened for growth, lipid accumulation, and AA production, and M. alpina 959 was selected as the most promising strain. The effects of nitrogen supplementation and oil incorporation on biomass formation, lipid accumulation, and AA productivity were subsequently investigated. Sunflower oil incorporation into spent malt-based substrates improved AA productivity under the most favorable conditions, resulting in an AA proportion of 38.6% of total fatty acids and 102.5 mg AA g⁻¹ of final dry mass of the SSF system (FDW-SC). Comparison of selected vegetable oils further demonstrated that the lipid source affected AA production, while having only a limited effect on fungal growth and total lipid accumulation. Overall, this study demonstrates the feasibility of spent malt-based SSF for AA production by M. alpina and highlights the valorization of brewery spent malt as a low-cost agro-industrial by-product for the production of value-added microbial lipids.