## 2 Microalgae Biorefinery: Biomass Treatment  
According to [PERSON] et al., the biorefinery is a \"process to obtain biofuels, energy and high-value products through biomass transformation and process equipment\" [18]. In the case of microalgal biorefinery, microalgae biomass is used as feedstock. Different steps are involved in the process, including cultivation, harvesting, cell disruption, component recovery, and final product purification [19]. The development of these new industries is faced with critical challenges; as an example, industrial photobioreactors for cultivation in synthetic media have high costs and reach low biomass concentrations. At the same time, current downstream processes are designed for a single main product, and the rest of the microalgae biomass is frequently \"waste\" that is not useful [11]. Because of all this, it is necessary to develop more efficient technologies to achieve a sustainable process for microalgal cultivation, but also to profit and valorize all the components of this microalgal biomass, establishing a fractional biorefinery process. This sequential valorization main concern is the release of different fractions without damaging the other fractions to attain an integral recovery of the biomass components [18]. The first step for the microalgae valorization is the cell disruption (Figure 1), which is a critical process for obtaining the desired biocomponents from intracellular contents [20]. Microalgae cells present a high resistance to intracellular extraction, usually attributed to their rigid cell walls [21]. This cell wall consists of a microfibrillar network composed of diverse polysaccharides, proteins (mainly glycoproteins), and a biopolymer called algaenan, which is very difficult to degrade [14]. This cell disruption can be achieved by several processes, which can be classified into physical (e.g., bead milling, high-pressure homogenization, microwave irradiation, ultrasonication, pulse electric field), chemical (e.g., acid, alkali, and thermal hydrolysis) and biological methods (e.g., enzymatic hydrolysis) [16]. However, most of these techniques require harsh conditions (such as chemical methods), which allow obtaining high release efficiencies but at the expense of degrading the released bioproducts, resulting in low recovery yields. Thus, the use of mild cell disruption will maintain the biomolecule's activity and functionality to provide high-quality products [20]. Moreover, in order to take advantage of all the algal biomass and achieve a sustainable process, cell wall components (including some types of proteins) may themselves also be target molecules within the biorefinery process [15]. Once the cellular disruption occurs (Figure 1), the biomolecules contained inside the cell are released (proteins, carbohydrates, or lipids), usually employing an extraction solvent. Those soluble biomolecules will be in a liquid phase, whose composition will depend on the cell disruption method and extraction conditions. Therefore, a recovery step is needed to isolate the required product from the other released compounds [16]. In the case of protein recovery, there are many possible techniques, including chemical precipitation, electrophoresis, membrane filtration, chromatography, or solvent extraction [23]. Although some of these methods provide good separation results, they have essential disadvantages such as low resolution, use of toxic solvents, presence of plenty of impurities, time-consuming process, and the use of harsh conditions such as high temperatures or energy consumption, which may lead to protein denaturation [23; 24]. Furthermore, a single methodology usually cannot achieve the desired purification effect. Among all these alternatives, solvent-extraction stands as an attractive technique because it is well known, efficient, and easily scalable. Nonetheless, the use of traditional toxic organic solvents can lead to changes in the stereochemistry and degradation of the released proteins [12].