Rhodosporidium Toruloides as Robust Yeasts for Advanced Biofuel Production Using Wood Hydrolysate

In response to increasing global energy demand as well as greenhouse emissions from petroleum fuels, sustainable and renewable sources have been intensely researched to produce biofuel. For instance, microbial lipids have been recognized as a potential feedstock for biofuel production due to their similarities with vegetable oils in terms of fatty acids. Typically, microorganisms capable of accumulating more than 20% lipids are known as oleaginous microorganisms. These microorganisms can thrive on various renewable substrates and biochemically convert excess carbon into lipids. One such example of a renewable substrate is lignocellulosic biomass, which produces hydrolysate containing hexoses and pentoses sugars upon pre-treatment and saccharification, and thus could be employed as a potential substrate for microbial lipids. However, wood hydrolysate presents several challenges such as low consumption of pentose sugars, and the presence of microbial growth inhibitors such as furans, organic acids and phenols. In this sense, Rhodosporidium toruloides, an oleaginous yeast, could be employed to produce lipids due to its ability to accumulate 50-70% of lipids, consume C5 sugars, and tolerate inhibitors. Thus, the present thesis explores the ability of R. toruloides-1588 to thrive on undetoxified hydrolysate derived from forestry residues (hardwood and softwood sawdust) and accumulate lipids. Additionally, several strategies were employed to increase the lipid titer such as carbon and nitrogen ratios, fed-batch fermentation, and carbohydrate supplementation such as crude glycerol, which resulted in maximum lipid accumulation of 56.3% (w/w) along with more than 90% consumption of carbohydrates. A life cycle assessment has been also performed to identify the hotspots in terms of energy consumption, greenhouse gas emission, and waste produced during the lipid production process. Lastly, the strain was accessed for its ability to thrive on microbial growth inhibitor such as furfural and use it as an energy source. Based on the above findings, the current dissertation concludes that R. toruloides-1588 can thrive on undetoxified wood hydrolysate, accumulate lipids that can serve as a feedstock for biofuel production and provide aid in the further development of biorefinery industries.