Biodiesel derived from lipids has proven to be a promising alternative when it comes to transportation fuels. However, its development and use have been hindered by low lipid yields and high production costs when compared to e.g., fossil fuels. Therefore, an increasing interest has emerged in investigating new and economically favorable methods for producing lipids that can be used for biodiesel production.
In this thesis, two time-studies were conducted to enlighten the optimal fermentation setup (Separate Hydrolysis and Fermentation (SHF) vs. Simultaneous Saccharification and Fermentation (SSF)) when using the organic fraction of municipal solid waste (OFMSW) as the feedstock while having an economic and sustainable point of view. The biomass hydrolysates were produced by enzymatic hydrolysis for 24 hours of the untreated waste to obtain the optimal sugar accessibility after which the non-detoxified hydrolysate was adopted as media for shake-flask fermentations. The maximum total lipid production that was extracted pr liter waste media after fermentation with Cutaneotrichosporon oleaginosus was 34.53 g/L for SHF after 4 days and 35.30 g/L for SSF after 7 days. This is an increase in the lipid content of 10.20 g/L and 9.67 g/L using SHF and SSF, respectively, compared to the OFMSW media (50 % w/v), which corresponds to 61.41 g of lipids being produced from each kg of dry OFMSW for SHF and 57.80 g of lipids produced pr kg dry OFMSW for SSF. Furthermore, a new vector system, pFLEXI-hyg-yfp, was developed with the aim of enhancing the future possibilities of genetic engineering of C. oleaginosus. The plasmid was successfully used for transformation of C. oleaginosus with Agrobacterium tumefaciens mediated transformation (ATMT), the obtained mutants were validated for successful integration by colony PCR and an optimized protocol for the ATMT procedure of C. oleaginosus was established. Lastly, the effect of random integration on the level of expression was investigated by measuring the yfp fluorescence over time of several different C. oleaginosus mutants.