Combination of Two-Stage Continuous Feeding and Optimized Synthetic Medium Increases Lipid Production in Lipomyces starkeyi

The oleaginous yeast Lipomyces starkeyi is recognized for its remarkable lipid accumulation under nitrogen-limited conditions. However, precise control of microbial lipid production in L. starkeyi remains challenging due to the complexity of nutrient media. We developed a two-stage fed-batch fermentation process using a well-defined synthetic medium in a 5-L bioreactor. In the first stage, the specific growth rate was maintained at a designated level by maximizing the cell density through optimizing the feeding rate, molar carbon-to-nitrogen (C/N) ratio, and phosphate concentration in feeding media, achieving a high cell density of 213 ± 10 × 10⁷ cells mL⁻¹. In the second stage, we optimized the molar C/N ratio in the feeding medium for lipid production and achieved high biomass (130 ± 5 g L⁻¹), lipid titer (88 ± 6 g L⁻¹), and lipid content (67% ± 2% of dry cellular weight). Our approach yielded a high lipid titer, comparable to the highest reported value of 68 g L⁻¹ achieved in a nutrient medium, by optimizing cultivation conditions with a synthetic medium in L. starkeyi. This highlights the importance of well-established yet powerful bioprocess approaches for the precise control of microbial cultivation.