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 [
]. 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 [
]. 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 [
]. 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 [
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 [
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 [
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 [
20,
61]. The combination of distinct enzymes, which is a strategy utilized to increase extraction yields [
20], must be carefully evaluated, since their interaction may have an antagonistic effect, opposite to the desired synergistic one [
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 [
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 [
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 [
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 [
67,
68]. Nevertheless, they have been studied with regard to lipid extraction from microalgae, as in Shankar et al. (2019) [
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 [
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) [
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) [
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) [
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 [
49].
An altogether distinct approach was that of Halim et al. (2019) [
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).