C/N ratio and carbon source-dependent lipid production profiling in Rhodotorula toruloides

Abstract

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.

Keywords: Alternative substrates; Biorefineries; Carbon to nitrogen ratio; Flux balance analysis; Genome-scale metabolic model; Microbial oil; Rhodotorula toruloides.

Fig. 1

R. toruloides turbidostat cultivations under different C/N ratios and carbon sources (blue circles, acetic acid; black squares, glycerol; gray diamonds, xylose). a Specific growth rate (1/h); b lipid yield (g-lipid/gDW). Numeric values are highlighted for the highest lipid yields for every carbon source studied

Fig. 2

Effect of C/N ratio on yields and specific biomass and lipid production rates on the turbidostat cultivation of R. toruloides using different carbon sources. Biomass yield (Y_sx), lipid yield on substrate (Y_ls), and volumetric biomass (q_BM) and specific lipid (r_LIP) production rates are presented

Fig. 3

Specific CO₂ (circles) and O₂ (squares) production rates for acetic acid (blue), glycerol (black), and xylose (gray) growing R. toruloides strain under various C/N ratios (a). Note that negative production indicates consumption. RQ values for the same conditions (data points) and FBA predicted values (dotted lines; b)

Fig. 4

Condition-dependent flux predictions in R. toruloides turbidostat cultivations on different carbon sources: a glycerol, b acetic acid, and c xylose. Fluxes are calculated using flux balance analysis on R. toruloides genome-scale model. Arrow width represents proportional flux values relative to carbon uptake flux. Precise values and reaction names can be found from Supplementary Table S6. Gly, glycerol; Xyl, xylose; Ace, acetic acid; PPP, pentose phosphate pathway; DHAP, dihydroxyacetone phosphate; Pyr, pyruvate; Cit, citrate; TCA, tricarboxylic acid cycle; Ace-CoA, acetil-CoA; Ace-P, acetyl phosphate; G3P, glycerol 3-phosphate; OAA, oxaloacetate

Fig. 5

R. toruloides fed-batch fermentation on glycerol, where CO₂ (black line with filled circles), OD (red line), and glycerol concentration in the reactor (open blue circles) are pictured. Red rectangle indicates the period when glycerol feeding with a constant specific growth rate of 0.05 1/h was applied (experimental set-up no. 3, “Materials and methods”)