Microbial oils also called single cell oils are lipids synthesized by microorganisms exceeding 20% of the dry weight of the cell. The aim of this work was to investigate the possibility to apply a rapeseed post-frying oil from fish fillets as a carbon source in growth medium for Yarrowia lipolytica oleaginous yeast species in order to synthesize a microbial oil. The key contribution of this work was the solution that provides a sustainable method for valorization of post-frying waste oil. A shaken batch cultures were provided and the influence of triacylglycerides hydrolysis on yeast growth was evaluated. In conclusion, the post-frying rapeseed oil seemed to be an easily utilizing carbon source by the yeast. Regardless of the method of lipid substrate pre-treatment, the yeast strain preferentially accumulated oleic acid (C18: 1) from 52.07 to 66.62% and linoleic acid (C18: 2) from 12.98 to 24.10%. To our knowledge, this is the first report of using the oxygen nanobubbles as an unconventional method of aerating the culture medium containing lipid carbon sources. The use of water oxygenated with nano-sized bubbles to prepare culture media resulted in obtaining a higher yield of biomass compared to the biomass yield in distilled water based medium.