Lipids and carotenoids production by oleaginous yeasts from lignocellulose biomass

Lipids and carotenoids produced by oleaginous yeasts have potential to replace fossil resources for the manufacture of biodiesel, oleochemicals, colorants and pharmaceuticals. Using lignocellulosic biomass as carbon source for yeast cultivation can potentially lead to a sustainable pathway to produce these valuable compounds with reduced costs. To support the development of this process, a technical route was proposed and carried out on a laboratory scale with the aim of identifying critical obstacles and finding possible solutions to them.
The experiments started with the selection of the best yeast strain for use in this process. Rhodosporidium toruloides was selected as the best producer of lipids and carotenoids among a total of 6 oleaginous yeasts that were cultivated in wheat straw hydrolysates. However, the growth of this yeast was affected by the presence of inhibitors in the hydrolysate, which are generated during the pretreatment of wheat straw to obtain fermentable sugars. Detoxification of the hydrolysate with activated charcoal can improve the performance of the yeast; however, it increases the production costs.
To avoid the detoxification step, the strategy of adaptive laboratory evolution was adopted to increase the tolerance of the yeast to the inhibitors derived from lignocellulosic biomass. Ten evolved strains were obtained after the evolution. When compared to the wild-type starting strain, the evolved strains presented a significant reduction in the lag phase (up to 72 h) when cultivated in hydrolysate-based medium. Moreover, the ability of the evolved strains to accumulate lipids and carotenoids was also improved.
Further research was conducted to explain the genetic variations behind the evolution. Whole genome sequencing analysis indicated that the wild-type strain contained abundant tolerance-related genes, which provided a background that allowed the strain to evolve in biomass-derived inhibitors, resulting in distinct resistance phenotypes. Several important mutations occurred on genes encoding for major facilitator superfamily transporters, stress response proteins, ATP-binding cassette transporters and oxidoreductases, indicating that the improved tolerance in these mutants may be related to efficiency of transporters, stress response and anti-oxidation capacity.
To better understand the effect of inhibitors on the growth of the wild-type and evolved strains, cultivations were performed in media containing different concentrations of inhibitor compounds and the specific growth rate and lag phase of the strains were compared. Benzoic acid in concentrations higher than 3 mM completely inhibited the growth of the strains, while acetic acid and levulinic acid were less toxic. However, when used in combination, a Plackett-Burman experimental design revealed that furfural was the most potent inhibitor affecting the growth of R. toruloides, followed by vanillin and 5-hydroxymethylfurfural.
The lack of an effective and feasible downstream processing to recover lipids and carotenoids from oleaginous yeast is another barrier to overcome for the implementation of this process on a large scale. In this thesis, a saponification-based downstream processing was developed, which allowed achieving 85.9±3.9% of lipid recovery yield with 99.9±0.0% purity, and 78.7±2.4% of carotenoid recovery yield with 81.5±0.5% purity. At the end, efforts were also done to select appropriate fermentation conditions able to maximize the production of lipids and carotenoids by the evolved yeast cultivated in wheat straw hydrolysate. Performing the fermentation at 17 °C and using 3.5 g/L of inoculum resulted in an improved simultaneous production of lipids and carotenoids.
In conclusion, the main achievements obtained in this thesis include: (1) The possibility of producing microbial lipids and carotenoids using lignocellulosic biomass as carbon source for fermentation was demonstrated in small scale, and a yeast strain with great potential for utilization in this process was identified. (2) Robust strains of the oleaginous yeast R. toruloides with improved ability to tolerate biomass-derived inhibitors were developed. (3) The fully annotated genome reference of the wild-type strain of R. toruloides was provided, and the whole genome sequence of the evolved strains, as well as the probable mutations responsible for the improved tolerance were revealed. (4) The main biomass-derived inhibitory compounds affecting the growth performance of R. toruloides were identified. (5) A simple and effective downstream processing method for lipid and carotenoid recovery from oleaginous yeast was developed. These findings contribute with new knowledge and information to support the development of a sustainable technology for the production of lipids and carotenoids by oleaginous yeasts from lignocellulosic biomass able to be implemented on a large scale.