Supercritical carbon dioxide extraction of lipids and carotenoids from Rhodotorula toruloides CBS 14 in comparison...

Oil from oleaginous yeasts has the potential to replace non-sustainable vegetable oil as raw material to produce food, feed, biofuels, or biochemicals. Co-produced compounds like carotenoids may be helpful to obtain economically viable bioprocesses. Identifying appropriate extraction methods is a bottleneck both for establishing oleaginous yeasts as cell factories for both oil and carotenoids production and for analysis of intracellular compounds like lipids and carotenoids. We conducted extractions using supercritical carbon dioxide (SC-CO₂) and conventional solvent methods to extract and analyze lipids and carotenoids from R. toruloides CBS 14 cells grown on wheat straw hydrolysate. The lipid extracts were analyzed using gas chromatography (GC), and the carotenoids were identified and quantified using ultra-high-performance liquid chromatography (UHPLC).

Four main carotenoids in the extracts from both extraction methods were identified including β-carotene, γ-carotene, torularhodin, and torulene. Interestingly, torularhodin was the major carotenoid extracted using SC-CO₂ extraction, followed by torulene. This was different from the conventional acetone extraction method, where β-carotene was the main carotenoid. After the conventional extraction, torularhodin and torulene underwent degradation due to the saponification step, which was necessary to remove lipids before UHPLC analysis. The total carotenoid concentration obtained from SC-CO₂ extraction was 332.09 ± 27.32 μg/g dry weight compared to 19.9 ± 2.74 μg/g dry weight in acetone extraction. A small amount of carotenoids was observed to be lost into the lipid extract, but this loss was not as substantial as that seen with acetone extraction. Additionally, the total lipid content in samples extracted using SC-CO₂ was significantly lower than that obtained using the conventional Folch method. GC analysis revealed that oleic acid was the major fatty acid in both lipid extracts, followed by palmitic acid and linoleic acid. Notably, the proportion of unsaturated fatty acids was higher in the extracts from the SC-CO₂ method compared to the conventional method.

These findings indicate that the SC-CO₂ extraction method outperformed conventional methods by preserving the integrity of unsaturated lipids and retaining an abundance of carotenoids, resulting in high-quality extracts.