Microbial lipid production using renewable feedstock shows great promise for the biodiesel industry. In this study, the ability of a lipid-engineered Yarrowia lipolytica strain JMY4086 to produce lipids using molasses and crude glycerol under different oxygenation conditions and at different inoculum densities was evaluated in fed-batch cultures. The greatest lipid content, 31% of CDW, was obtained using a low-density inoculum, a constant agitation rate of 800 rpm, and an oxygenation rate of 1.5 L/min. When the strain was cultured for 450 h in a chemostat containing a nitrogen-limited medium (dilution rate of 0.01 h−1; 250 g/L crude glycerol), volumetric lipid productivity was 0.43 g/L/h and biomass yield was 60 g CDW/L. The coefficient of lipid yield to glycerol consumption (Y L/gly) and the coefficient of lipid yield to biomass yield (Y L/X ) were equal to 0.1 and 0.4, respectively. These results indicate that lipids may be produced using renewable feedstock, thus providing a means of decreasing the cost of biodiesel production. Furthermore, using molasses for biomass production and recycling glycerol from the biodiesel industry should allow biolipids to be sustainably produced.

Production of valuable metabolites by Yarrowia lipolytica using renewable raw materials is of major interest for sustainable food and energy. Galactose is a monosaccharide found in galactomannans, hemicelluloses, gums, and pectins. Yarrowia lipolytica was found to express all the Leloir pathway genes for galactose utilization, which encode fully functional proteins. Gene organization and regulation in Y. lipolytica resembles filamentous fungi rather than Saccharomyces cerevisiae. After Y. lipolytica was grown on mixture of glucose and galactose, it was then able to metabolize galactose, including when glucose concentrations were higher than 4 g/L. However, glucose was still the preferred carbon source. Nonetheless, a strain overexpressing the four ylGAL genes of the Leloir pathway was able to efficiently use galactose as its sole carbon source. This mutant was used to produce citric acid and lipids from galactose; the yields were comparable to or greater than that obtained for the parental strain (W29) on glucose. The construction of a Y. lipolytica strain able to produce citric acid and lipids from galactose is a very important step in bypassing issues related to the use of food-based substrates in industrial applications.

Invertases are important enzymes used in the food industry. Despite many studies on the invertase-encoding SUC2 gene expression in the industrial yeast Yarrowia lipolytica, no biochemical characteristics of this enzyme expressed as heterologous protein have been provided. Here, two isoforms of extracellular invertase produced by Y. lipolytica were detected using ion-exchange chromatography. Specific activities of 226.45 and 432.66 U/mg for the first and second isoform, respectively, were determined. Basic characteristics of this enzyme were similar to the one isolated from Saccharomyces cerevisiae (optimum pH and temperature, metal ions inhibition, substrate specificity and fructooligosaccharides (FOS) biosynthesis). The apparent differences were higher K_M for sucrose (67 mM) and lower molecular mass (66 kDa) resulting from lower N-glycosylation level (9.1% of mass). The N-glycan structures determined by MALDI-TOF and HPLC represented high mannose structures, though with much shorter chains than hypermannosylated glycans from S. cerevisiae. Furthermore, galactose was detected as the modifying sugar in the glycan structures of invertase expressed in Y. lipolytica. N-glycans did not affect invertase activity but were important for its oligomerization. The expressed enzyme aggregated into dimers, tetramers, hexamers, and octamers, as well as structures of higher molecular mass, which might be decamers, which have not been described so far in the literature.