# Unconventional Extraction Methods of Oleaginous Yeast Cell Pretreatment and Disruption
## 3 Results  
### Effects of Conventional and Unconventional Extracting Methods for Y. lipolytica Yeast Cells  
#### 3.1.1 Comparison of Lipid Extraction and Leaching Efficiencies for Yeast Storage Lipids  
The preliminary studies were performed and described in the paper of [PERSON] et al. [27] and allowed for choosing non-conventional methods for further investigations. The effectiveness of the PEF, US, and HPP methods for extraction of microbial oil from _Y. lipolytica_ yeast cells was studied, and it has been proven that the use of PEF with a field strength of 200 J/g or HPP (1100 bar) can be an effective pretreatment technique for _Y. lipolytica_ yeast cells for the high yield extraction of intracellular lipids, providing the extraction method with organic solvents. There were bioreactor batch cultures provided in which two different carbon sources were used. In post-frying rapeseed oil medium, waste oil medium (WOM) cells accumulated storage lipids via an ex novo pathway, and in cultures with molasses (MM), there was expected de novo lipid synthesis. Figure 3 presents a microscopic image of colored yeast cells from both cultures. In the case of the WOM culture, the content of cellular lipids after 40 h was 0.21 g/g d.m., while in the case of the culture of MM, the value reached a lower value of 0.13 g/g d.m. Analysis of the preparation from culture WOM shows a higher amount of lipid bodies, in which lipids are accumulated. The obtained image confirms the obtained trend of intracellular lipid content in both of these culture variants. The results shown in Figure 4 present the efficiencies of yeast lipid extraction obtained after using the mechanical and chemical methods PEF, US, and HPP. The first scientific hypothesis assumed that those methods can effectively disrupt yeast cells. The control variant was an untreated biomass. The highest efficiency of microbial oil extraction using the Folch method was obtained after the previous treatment of the biomass with a PEF (28%). The use of Triton solution (chemical method with the use of surfactant), Triton in combination with shaking with glass beads (combined chemical and mechanical techniques), as well as high-pressure processing made it possible to achieve an efficiency of 6-11%. The use of glass beads (8%) and US (9%) did not increase the amount of lipids extracted. None of the methods applied in the study allowed for the extraction of lipidsfrom the interior of the disrupted cells. It was concluded that these methods cannot be considered as extraction methods but they are worth investigation as permeabilization techniques in pretreatment steps. This statement was studied on the basis of comparing the leaching efficiency of microbial lipids measured for yeast cells treated with the same techniques (Figure 5). As in the case of Folch extraction, six experimental options and a control variant were performed. The highest efficiency of lipid leaching using hexane was obtained by application of PEF. The yield obtained was 28%. The use of the other treatments made it possible to amount  
Figure 4: Lipid extraction yield from yeast cells pretreated with different methods. Means with the same capital letter (a, b) did not differ significantly (\(\alpha\) = 0.05). Figure 3: Microscopic images of stained _Y. lipolytica_ yeast cells with visible red lipid bodies, magnification x = 160.8: (**a**) cultured in post-frying rapeseed oil medium (WOM—waste oil medium); (**b**) cultured in medium with molasses (MM—molasses medium). Images were taken with confocal laser microscope. leaching efficiencies for storage lipids at a higher or the same efficiency as the variant control, which was ca. 6%. Finally, the efficiency of lipid extraction and leaching yield using the Folch method were compared (Figure 6). The PEF method had the highest result (92.86%). Cells treated with US were also characterized by a high ratio value (94.63%). On the other hand, the methods using glass beads, as well as surfactants, had a slightly higher ratio value when entrained with the control variant (63.59%).