## 3 Protein Recovery
### Solvent Extraction  
As mentioned above, solvents can be used to release the proteins from inside the biomass cell, but they can also be employed to selectively obtain proteins from the released compounds through conventional liquid-liquid extraction (LLE). This separation technique consists of the analyte's migration to the extracting phase, relying on the different affinity for the medium (measured by the distribution constant, \(K_{D}\)) and the concentration gradient [54]. This section will discuss classical and new emerging solvents for protein LLE, along with innovative solution combination strategies. Commonly employed solvents for protein recovery are hydrocarbons, alkalis, acetone, alcohols, and chloroalkanes [55]. Even though these solvents are low-cost and have good solubility capacities [56], they also present important drawbacks such as long operational times, high solvents and energy consumption, and the high odds of separated proteins damage [57]. Furthermore, most of them are volatile organic compounds (VOCs) derived from non-renewable sources such as petroleum, being both detrimental to human health (as the product obtained might introduce pollutants) and the earth's well-being [56, 58]. The use exclusively of classical solvents to obtain proteins from microalgae is not common. However, several studies reported their use in combination with greener options such as aqueous two-phase systems, in which the organic solvent volume is significantly reduced [59]. Due to the drawbacks of classical solvents, alternative strategies are needed to achieve more sustainable processes, with reduced energy consumption and lower presence of hazardous substances [60]. One approach to accomplish these goals consists of replacing classical for green solvents, which must be cheap, non-toxic, safe, stable, biodegradable, renewable, and atom economic in their synthesis; however, telling which solvents are green is still a controversial issue [61]. Moreover, most typical green solvents are not preferred for protein recovery because proteins are not very soluble in water [56] nor supercritical fluids such as CO\({}_{2}\)[62]. Thus, the aim is to find greener solvent options for LLE, and one class of solvents that have shown promising results in the recovery of bioactive compounds are the so-called biodegradable solvents, which are not necessarily fully green (although every green solvent must be biodegradable). The advantage is that defining what a biodegradable solvent is can be quantified in a rigorous manner [63, 64]. These solvents can be combined with innovative assisted technologies such as microwave, or ultrasound, to achieve an optimum purification process [55; 62]. This is the case for ionic liquids (ILs) and deep eutectic solvents (DESs), whose advantages and applications will be examined in Section 4. Another possibility is to combine two different solutions to carry an LLE process, as applied in an aqueous two-phase system (ATPS), which can also be found in literature as aqueous biphasic system (ABS) [65]. As its name indicates, ATPS consists of two aqueous phases that are immiscible due to the repulsive behavior of their components [66; 67]. Compared to classical single solvent extraction, ATPSs allow obtaining in one step proteins in high concentration, saving time and costs, as the equilibrium is rapidly reached [68]. One phase is usually formed by a water-soluble polymer solution or a hydrophilic organic solvent (such as alcohols or organic acids), while the other is generally a salt solution [69]. ATPSs allow mild operation conditions, reducing the chance of denaturation or loss of structural activity of the proteins [69]. Moreover, ATPSs enable continuous mode operation conditions, so they are easy to scale up, unlike other techniques, restricted to batch mode [65]. ATPS containing conventional organic solvents has proved potential for microalgae protein purification; for example, [PERSON] et al. [59] prepared an ATPS formed by aqueous solutions of ammonium sulfate and 2-propanol; or [PERSON] et al.