# Unconventional Extraction Methods of Oleaginous Yeast Cell Pretreatment and Disruption
## 1 Introduction  
Microphial lipids (SCO: single-cell oil) are produced by oleaginous bacteria, yeast, molds, and algae, so named because of their ability to accumulate lipids above 20% of cell dry weight. The type of lipids extracted from microorganisms depends mainly on the culture conditions and the microorganism species [1]. Plant sources can provide lipids of up to 50% of dry weight, while the bacterial species _Rhodococcus apacus_ can provide up to 87% of cellular dry weight, and the wild yeast strain _Yarrowia lipolytica_ up to 48% [2, 3]. Studies on the use of SCOs for biodiesel production are in progress [4, 5, 6]. The addition of SCOs has the potential to enrich and increase the nutritional value of food products as well as replace vegetable lipids (e.g., cocoa butter or palm oil) and certain free fatty acids (FFAs). Microbial lipids have been used as stabilizers, thickers, water binders, and emulsifiers [7]. SCOs are a rich source of essential FFAs, e.g., docosahexaneonic acid andarachidonic acid in infant formulas [8]. Microbial lipids are considered to play the role of soothing agents, emoliments, and laxatives. SCOs are also ingredients in products used in enteral and parenteral nutrition therapy [9]. _Y. lipolytica_ is a dimorphic oleogenic species, growing as mycelium and yeast-like cells. _Y. lipolytica_ has a high ability to utilize hydrophobic substrates, which makes it an interesting species for industrial applications. Relatively little is known about the structure of its cell wall, especially the cell wall proteins. The structure and composition of the yeast cell wall have been studied mainly in _Saccharomyces cerevisiae_. The cell wall is composed mainly of glucans, chitin, and glycoproteins. This structure shows high resistance to shear forces, indicating that its components are tightly linked [10; 11]. Extraction of lipids from plant and animal matrixes as well as microbial biomass can be carried out using a number of methods (Figure 1). Soxhlet developed a hexane-based extraction method that is widely used despite the major drawback of using organic solvents or the potential emission of toxic compounds during the procedure [12; 13]. The Bligh and Dyer method and Folch method are two of the best-known lipid extraction methods involving the use of a chloroform and methanol mixture [14; 15]. These methods are used to extract lipids from many food products such as avocados, eggs, mayonnaise, and fish [14; 16]. Still, a growing interest in unconventional extraction methods can be observed. Pulsed Electric Field (PEF) is a technology where the delivery of a specific portion of energy creates pores in the cell membrane, which contributes to its permeability and increases the extraction efficiency of valuable components such as dyes, flavors, proteins, lipids, and antioxidants present in the organelles of living cells [17; 18]. Microwave-assisted extraction is used to heat solvents in contact with the sample, with better simultaneous recovery of analytes compared to conventional extraction techniques [19; 20]. The use of ultrasound (US) also contributes to accelerating the extraction process. The increase in efficiency is mainly due to the mechanical effects of acoustic cavitation, which increases both solvent penetration into and release of intracellular products by disrupting cell walls [21; 22]. A method that is highly energy efficient and also results in minimal product degradation is HPP (high-pressure processing). The mechanisms of cell destruction are variously  
Figure 1: Methods for disintegrating cell walls to extract lipid compounds. attributed to fluid shear stress [23]. This method may be practically applied to large-scale processing of microbial biomass, only if the energy requirement is low enough [24]. The aim of the present study was to evaluate the effectiveness of using unconventional methods to extract microbial oil from _Y. lipolytica_ yeast cells cultured in media with waste carbon sources (namely post-frying rapeseed oil or molasses).