Adapted Feeding Strategies Enable Efficient Growth and Lipid Accumulation Using Untreated Crude Glycerol in Transition Scale with Cutaneotrichosporon oleaginosum ATCC 20509

Yeasts such as Cutaneotrichosporon oleaginosum can convert low-value side streams into single-cell oils with fatty acid profiles comparable to vegetable oils. Crude glycerol (CG), a byproduct of biodiesel production, offers a cost-effective substrate, but its variable impurity load often causes strong growth inhibition. In this study, two untreated industrial CG batches were characterized and evaluated in 2.5 L and 19 L stirred-tank fermentations. Direct batch cultivation on CG resulted in no measurable growth, whereas an adapted stepwise feeding strategy effectively mitigated early inhibition and restored biomass formation, metabolic activity, and lipid accumulation. In 2.5 L cultivations, apparent growth rates up to 0.51 h−1 and volumetric productivities up to 0.22 g L−1 h−1 were achieved, with lipid contents of ~30% and oleate-dominated fatty acid profiles. Fatty acid profiles remained oleate-dominated (~53–55% C18:1). Transition-scale (19 L) repeated-batch fermentations confirmed process robustness across > 640 h of operation, during which lipid content (~30–36%) and fatty acid composition (oleate ~51–53%) remained stable despite pronounced substrate-batch variability and increasing nitrogen limitation. These results demonstrate that untreated CG can be reliably valorized for lipid production using scalable feeding strategies without prior detoxification. This closes a gap between laboratory-scale feasibility studies and process-oriented, multi-cycle operation on industrial-grade feedstocks, confirming that feeding-driven inhibition control can ensure robust performance without substrate purification.