Nannochloropsis is a genus of microalgae comprising seven described species, wherein individuals are characterized by being non-motile, presenting a spherical morphology and diameters ranging from 2 to 8 µm ^[1][2][3][2,5,6]. Furthermore, these microalgae are also widely recognized for presenting high polyunsaturated fatty acids (PUFA) contents, particularly that of the omega-3 fatty acid eicosapentaenoic acid (EPA, C20:5n3) ^{[4][5][6][7][8][9]}[7,8,9,10,11,12]. As all microalgae, Nannochloropsis have different composition lipids, which may be divided into polar (phospholipids, glycolipids, and betaine lipids) and non-polar (acylglycerols, sterols and free fatty acids) ^[10][11][13,14]. The PUFA present in these microalgae are mainly located in polar lipids, namely phospholipids, while their content in neutral triacylglycerols (TAG) is lower ^[5][12][8,15]. This is an advantage, since omega-3 PUFA are more stable and possess higher bioavailability when in the form of polar lipids (particularly phospholipids) ^[9][13][12,16].

In order to obtain the compounds of interest from microalgae cells, present in the cell wall itself or intracellularly, extraction must be performed ^[14][15][3,17]. The main objective of extraction techniques is to obtain a high yield of the desired compounds without jeopardizing quality and activity, as well as to preserve co-products, minimize the amount of energy spent and waste generation, optimize the process (operational temperature, pressure, carrying capacity, side reactions and separations) and be scalable ^[16][18].

Traditionally, fatty acids were obtained from microalgae via the conventional solvent extraction techniques. These techniques include solid–liquid and liquid–liquid extractions, in which organic solvents, such as hexane, toluene, dichloromethane, acetone and others are utilized ^[16][17][18,19]. However, nowadays, there is a generalized opinion that the solvents used in the extraction processes should be safe, inexpensive and nontoxic ^[16][18][19][18,20,21].

The extraction of biologically active compounds (namely fatty acids) from microalgae, such as Nannochloropsis, has been performed utilizing several different technologies which include ultrasounds ^[5][20][8,27], microwaves ^[21][22][28,29], super- and subcritical fluids ^[23][24][30,31], and high pressure ^[25][32], among others. Extraction processes may entail a very important step, that is, the pretreatment of the biomass, to increase/improve the extraction yield, obtained by disrupting the cell wall ^[16][18]. Cell wall rigidity can inhibit conventional organic solvents, such as hexane, to enter the cell, preventing or hindering the contact between the solvent and the intracellular compounds ^[26][24]. As such, pretreatment makes the bioactive compounds present in the cells more bioavailable ^[27][26]. Cell wall disruption techniques can be of mechanical, chemical, physical or enzymatic nature, such as high pressure homogenization, alkaline lysis, ultrasonication, and enzymatic hydrolysis ^{[28][29][30][31]}[33,34,35,36]. Nannochloropsis microalgae have been described as possessing a rigid, robust, cell wall ^[4][25][7,32], resulting from its composition, consisting of an outer hydrophobic trilaminar sheat algaenan-based layer and an inner cellulose-based layer (linked by struts to the plasma membrane), which hinder the extraction of intracellular compounds ^{[1][4][18][27][30]}[2,7,20,26,35]. Thus, pretreatments to disrupt cell wall are desired/required to increase extraction yields.

2. Extraction Technologies

2.1. Microwave-Assisted Extraction (MAE)

Microwave technology can, as previously mentioned, be used to enhance yields of bioactive compounds by disrupting cell walls and is used as a complement in the extraction methodology. Microwaves are the alternating current signals with frequencies varying from 0.3 to 300 GHz, which transform electromagnetic energy into heat with the polarity of compounds. Polar compounds will realign in the direction of the electric field and, when the microwave field alters, they will rotate in high speed. If ions are present, ions will migrate as the electric field alters. Electromagnetic energy is then transformed into heat by the friction between the compounds or the ions ^[32][37]. Microwave heating is a non-contact heat source, heating the overall target reactants simultaneously as compared to conductive heating ^[33][38]. The heat source can penetrate into the biomaterial, interacting with polar molecules, such as water, and heat the entire sample uniformly ^[34][39]. The increase in temperature will cause the evaporation of water molecules, which will apply pressure on the cell walls. This will rupture the cell walls, which will release the intracellular components into the medium. The utilization of microwaves also facilitates extraction as hydrogen bounds are disrupted and dissolved ions, by migration, increase the penetration of solvent into the matrix ^[16][18]. Microwave-assisted extraction is considered to be a rapid and cost-effective method of obtaining bioactive compounds ^[16][32][34][18,37,39], and has been frequently used to extract different compounds from microalgae.

2.2. Ultrasound-Assisted Extraction (UAE)

Ultrasound-assisted extraction is another technology utilized to disrupt cell walls, and consequently increase extraction yields, which is sometimes used simultaneously with MAE. This technology is based on the cavitation phenomenon. When a liquid is submitted to ultrasounds, cavitation bubbles are generated, which can create implosive collapse ^[35][46]. The intense sonication of liquid generates soundwaves that propagate into the liquid, resulting in alternate high-pressure and low-pressure cycles. Cavitation is the phenomenon resulting from the violent collapse of small vacuum bubbles, generated in the low-pressure cycle, during the high-pressure cycle. During cavitation, shearing forces are formed around the cells by the high pressure and high speed liquid jets, resulting in the cell structure being “mechanically” broken, thereby improving material transfer ^[34][39]. This reduces particle size and increases the contact between the solvent and the compounds to be extracted ^[16][18]. The enhancement in extraction yields is attributed to the microstreaming and heightened mass transfer by cavitation and bubbles collapse, which result in the destruction of the cells ^[36][47]. This technology can, as previously mentioned, improve extraction yields and reduce the amount of solvent utilized, and the extraction time and costs, as there is a reduction in the temperature needed for the extraction process ^[36][37][47,48].

2.3. Supercritical Fluid Extraction (SFE)

Supercritical fluid extraction (SFE), together with pressurized liquid extraction (PLE), is probably the most widely employed extraction technique for obtaining bioactive components from natural sources ^[38][52]. Supercritical fluid extraction utilizes the solvents above their critical pressures and temperatures ^[16][38][18,52]. As the solvent power of a supercritical fluid is a function of density, it can be varied by changing the extraction pressure and temperature, enabling it to be suitable as an extraction solvent ^[34][39]. In the conditions utilized in SFE, supercritical fluids (SCFs) possess particular physicochemical properties between gases and liquids, generally acquiring higher density than a gas, yet maintaining similar viscosities and diffusivities ^[38][52]. The density of the SCF is like that of a liquid, and it can be altered by changing the temperature and pressure. The low viscosity and high diffusivity of SCFs generates better transport properties, when compared to liquids ^[16][18]. Although different solvents can be utilized, the most utilized solvent is carbon dioxide (CO₂), due to its moderate critical pressure (7.4 MPa) and low critical temperature (31.1 °C) ^[34][39]. Supercritical CO₂ (SC-CO₂) has several advantages, as it has mild critical conditions, and is nontoxic, nonflammable, nonexplosive and noncorrosive. Additionally, it is easily available and cheap, and is easily separated from the extract, inert to the product. Carbon dioxide, being a gas at room temperature, can be easily removed from the extract, when compared to other extraction techniques ^[16][38][18,52]. Another advantage is that the properties of SCFs can be adjusted with pressure and temperature changes, which directly influences density, making the technique very selective, which is a major advantage when the objective is the extraction of compounds from complex matrices. This technique has also the advantage of the possibility of, during decompression, performing fractioning just by utilizing two or more decompression steps, which is useful to separate components in the extract ^[38][52].

2.4. Pressurized Liquid Extraction (PLE)

Pressurized liquid extraction (PLE), also referred to as pressurized fluid extraction (PFE), pressurized hot-solvent extraction (PHSE) or accelerated solvent extraction (ASE), is an extraction technology based on the utilization of pressurized solvents at high temperatures, although always below their critical points, under conditions that maintain the solvents in the liquid state during the extraction process. When water is utilized as the extraction solvent, the general principles and instrumental requirements are the same, although other important parameters have significant influence, and the technology can be denominated as subcritical water extraction (SCWE), superheated water extraction (SHWE) or pressurized hot-water extraction (PWE) ^[38][52]. Pressurized liquid extraction conditions provide an enhanced mass-transfer rate, an increased solubility of the compounds to be extracted, and a decrease in solvent viscosity and surface tension ^[38][39][23,52]. The lower solvent viscosity and surface tension will allow the solvent to penetrate more easily into the matrix, reaching deeper areas and increasing the surface contact, which will improve the mass transfer to the solvent, resulting in an increased extraction rate ^[38][52]. As previously mentioned, when water is utilized as the solvent, the extraction is also affected by the dielectric constant (ε) of water. When water is heated at high temperatures while remaining in the liquid state, ε, which is a measure of the polarity of the solvent, is significantly reduced ^[24][38][31,52]. If this value is decreased to values close to the ones of organic solvents (when heated), water can be presented as a useful alternative. Even though this may not be possible for all applications, SWE can be seen as the “greenest” of the PLEs ^[38][52].

2.5. Enzyme-Assisted Extraction (EAE)

Enzyme-assisted extraction (EAE) is yet another technology/technique which has been utilized to obtain fatty acids from microalgae. As previously mentioned, microalgae possess cell walls which, dependent on their composition, may hinder the access of the extraction solvent to the intracellular compounds. In this sense, there is the need to rupture or, at least, disrupt the cell wall so that extraction of such compounds may be achieved. The algaenan/cellulose wall of Nannochloropsis is particularly resistant to chemical or mechanical treatments ^[40][60] and in many cases there is a need to apply combinations of these to increase the extraction potential as described in the previous sections. A rather promising alternative strategy to overcome this constraint is the use of enzymes which, according to their nature, may hydrolyze the cell wall structural components. This will damage the cell wall integrity, thereby providing easier access of the extraction solvent to the intracellular compounds, as well as promoting their leakage ^[41][42][61,62]. In this sense, for Nannochloropsis microalgae, a cellulase can be applied, envisioning the degradation of the inner cellulose-based layer. The main treatment parameters which impact the enzyme, and consequently extraction efficiency, are the enzyme dosage, pH, temperature, time, and the homogenization (agitation) speed ^[18][41][20,61]. The combination of distinct enzymes, which is a strategy utilized to increase extraction yields ^[18][20], must be carefully evaluated, since their interaction may have an antagonistic effect, opposite to the desired synergistic one ^[41][61].

2.6. Ionic Liquids (ILs) and Deep Eutectic Solvents (DES)

Recently, there has been a demand for solvents able to extract lipids, among other compounds, from distinct “matrices”, including microalgae, without having such a detrimental environmental impact as the conventionally utilized organic solvents. This has prompted researchers to explore other types of solvents, which include ionic liquids (ILs) and deep eutectic solvents (DES). Ionic liquids are a class of solvents which, as aforementioned, have been studied as alternatives to the conventionally used solvents to extract several compounds from microalgae. The ILs are solutions of salts that present melting temperatures below 100 °C, some of which may still even be liquid (molten) at room temperature, and their composition comprises both anions and cations, hence their designation ^{[4][43][44][45]}[7,65,66,67]. These solvents’ properties can be manipulated by combination and permutation of the anions and cations comprised therein, which endow solvents with distinct polarity, thermal stability, hydrophobicity and viscosity, that can be tailored according to the specific goal for which they are intended ^[14][43][45][3,65,67]. Furthermore, within ILs there is a subclass denominated switchable solvents, of which there are two types, namely switchable polarity solvents (SPS) and switchable hydrophilicity solvents (SHS), which can reversibly change the characteristics in response to a stimulus/trigger ^[44][45][46][66,67,68]. Although ILs have been considered in “green” extractions, there are significant environmental concerns regarding the utilization of these solvents due to the inefficient biodegradation and the potential use and production of toxic reagents in the synthesis of some ILs ^[45][46][67,68]. Nevertheless, they have been studied with regard to lipid extraction from microalgae, as in Shankar et al. (2019) ^[47][69], in which protic ILs (a subtype of ILs) have been utilized to extract lipids from N. oculata. The authors found that, in comparison with the conventional Bligh and Dyer (1959) method ^[48][49], extraction via ILs (in combination with a posterior microwaves treatment), in particular butyrolactam hexanoate, increased lipid yield by 34.9%, with a lower content of pigments, which is a positive trait when fatty acid extraction is concerned. Moreover, the study revealed that extraction was more efficient when biomass was hydrated, which is also favorable to the implementation of the technology, since a drying step is circumvented.

2.7. Others

In addition to the aforementioned technologies, a myriad of other solutions, some more innovative than others, have been studied to extract lipids, including fatty acids, from Nannochloropsis microalgae. Wang et al. (2018) ^[49][73] explored the effect of screw extrusion on N. oceanica cell integrity and lipid recovery, and found that the treatment increased the amount of fatty acids, including PUFA, subsequently extracted using hexane. Moreover, a balance between screw speed and feed moisture was shown to be critical to achieve the highest yields. Chemical methods have also been applied to enhance the extraction of fatty acids from Nannochloropsis. Potassium hydroxide (KOH) was utilized by Park et al. (2020) ^[50][74] to assist the solvent extraction of lipids from N. oceanica. The study showed that inclusion of KOH in the extraction process enabled the removal of chlorophyll from the extract, which in turn resulted in an increased amount of fatty acid methyl esters (FAME). This resulted in an extract more suited for biodiesel production, and which could further be separated from the EPA comprised therein, so that it could be utilized in other products. One other chemical approach is osmotic shock. Physical processes are likely the most studied regarding fatty acids extraction from Nannochloropsis. Lorente et al. (2018) ^[51][76] explored steam explosion as a pretreatment to diminish structural integrity of the cells, and consequently enhance lipid extraction from N. gaditana. The technology was able to disrupt the cell walls, and increase the amount of lipids extracted using hexane as extraction solvent by 8-fold, thereby resulting in a yield of ca. 80% as compared to the conventional Bligh and Dyer (1959) method ^[48][49].  An altogether distinct approach was that of Halim et al. (2019) ^[52][90], which explored a mechanism of autolytic self-ingestion to decrease the thickness of the cell walls of Nannochloropsis microalgae (Nannochloropsis sp. and N. gaditana). The treatment consisted of a thermally coupled dark-anoxia incubation, which lead to the anaerobic metabolism being activated and the consequent consumption of sugar reserves. This resulted in the reduction of the polysaccharides comprised in the cellulose-based layer of the cell wall, whose thickness was then decreased to half. The process weakened the cells, which were then easier to rupture in subsequent treatments, as those previously mentioned for such purpose (in that specific case, high pressure homogenization).