The rapid expansion of biodiesel production has resulted in the accumulation of substantial quantities of crude glycerol, posing a significant challenge for biofuel industry, due to its low viscosity and associated environmental concerns. Oleaginous microorganisms have gained attention in recent years, owing to their ability to utilize a wide spectrum of industrial by-products, including crude glycerol, for the synthesis of intracellular lipids and other high-value secondary metabolites. In this study, the potential of four novel Rhodotorula strains to exploit crude glycerol for the concurrent production of intracellular lipids and carotenoids was examined. Key parameters affecting microbial growth were assessed, including waste purity, tolerance to low viscosity, varying carbon-to-nitrogen (C/N) ratios (90:1 and 120:1) and high glycerol concentration (140g/L).
All strains were able to grow on unrefined crude glycerol, without pH adjustment, under both elevated glycerol levels and low C/N conditions. Biomass accumulation, intracellular lipid content and total carotenoid production were quantified across all conditions. Under pure glycerol conditions, maximum biomass, lipid and carotenoid yields reached 29.74 g/L, 13.97g/L and 931.98 μg/g respectively. In contrast, crude glycerol supported enhanced carotenoid biosynthesis under both C/N ratios, with peak values reaching 1214.81 μg/g, while the 90:1 C/N ratio favored higher biomass and lipid production. These findings highlight the potential of Rhodotorula spp. in valorizing crude glycerol through a low-input, multi-product
bioprocess. Optimization of cultivation parameters could further improve yields, contributing to both the economic feasibility and environmental sustainability of biodiesel industry by-product management.

Maria Karpeli, Eleni Naziri, Dimitris Sarris; Laboratory of Physico-Chemical and Biotechnological Valorization of Food By-Products Department of Food Science and Nutrition, School of the Environment, University of the Aegean