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 step-
wise 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
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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 feasi-
bility studies and process-oriented, multi-cycle operation on industrial-grade feedstocks,
confirming that feeding-driven inhibition control can ensure robust performance without
substrate purification.