Microbial oils are lipids produced by oleaginous microorganisms, which can be used as a potential feedstock for oleochemical production. The oleaginous yeast Rhodotorula toruloides can co-produce microbial oils and high-value compounds from low-cost substrates, such as xylose and acetic acid (from hemicellulosic hydrolysates) and raw glycerol (a byproduct of biodiesel production). One step towards economic viability is identifying the best conditions for lipid production, primarily the most suitable carbon-to-nitrogen ratio (C/N). Here, we aimed to identify the best conditions and cultivation mode for lipid production by R. toruloides using various low-cost substrates and a range of C/N ratios (60, 80, 100, and 120). Turbidostat mode was used to achieve a steady state at the maximal specific growth rate and to avoid continuously changing environmental conditions (i.e., C/N ratio) that inherently occur in batch mode. Regardless of the carbon source, higher C/N ratios increased lipid yields (up to 60% on xylose at a C/N of 120) but decreased the specific growth rate. Growth on glycerol resulted in the highest specific growth and lipid production (0.085 g lipids/gDW*h) rates at C/Ns between 60 and 100. We went on to study lipid production using glycerol in both batch and fed-batch modes, which resulted in lower specific lipid production rates compared with turbisdostat, however, fed batch is superior in terms of biomass production and lipid titers. By combining the data we obtained in these experiments with a genome-scale metabolic model of R. toruloides, we identified targets for improvements in lipid production that could be carried out either by metabolic engineering or process optimization.

Microbial oil is a potential substitute for vegetable oils in the biodiesel industry. Efforts to obtain cheap carbon sources for the cultivation of lipid-producing microorganisms comprise an active research area. This work aimed to extract the hemicellulose fraction from Eucalyptus uograndis and to use its hydrolysate as a carbon source for Rhodotorula toruloides (an oleaginous yeast) cultivation for microbial oil production. Hemicellulose hydrothermal extractions were performed at different temperatures, times, and ratios of solid to liquid (S/L). Temperature and time showed a stronger effect on the solubilization of hemicellulose. Hemicellulose extraction at 155 °C, 195 min, and an S/L ratio of 1/2 resulted in a hydrolysate with a xylose content of 37.0 g/l. R. toruloides cultivation in this hydrolysate showed that initial pH had a strong influence on cell growth. At an initial pH of 6.2, cells grew to 6.0 g/l of biomass with a lipid content of 50%. Therefore, we believe that E. urograndis hemicellulose hydrolysate could be a potential substrate for R. toruloides for lipid production based on the biorefinery concept.