## 4 Biodegradable Solvents for Protein Extraction
### Deep Eutectic Solvents (DESs)
Thus, using natural products to prepare DESs (natural deep eutectic solvents or NADESs) will ensure low or negligible environmental impact and toxicity, as these new solvents are biodegraded >70% in 7 days [110], along with ubiquity and low cost [62]. A rigorous comparison of the key properties of ILs, DESs, and NADESs, has been recently performed by [PERSON] et al., highlighting that NADESs are more eco-friendly and exhibit lower toxicity than DESs, which in turn are better in these and other aspects such as biodegradability, cost, and synthesis facility than ILs [111]. NADESs have proved to be an ideal extraction media since many macromolecules such as DNA, proteins, carbohydrates, and other small molecules are highly soluble in some of these solvents such as 1,2-propanediol-choline chloride-water, glucose-choline chloride-water, or lactic acid-glucose-water, as demonstrated by [PERSON] et al. [112]. Analogous to ILs, the complete understanding of the underlying extraction mechanism is yet to be elucidated. Even though NADESs show numerous advantages and are considered capable of dissolving all sorts of biomolecules in living things [109], they also present some limitations, such as their high viscosity, which may prevent further industrial application. Nonetheless, this issue can be solved by increasing the operation temperature, or by adding water, which is preferred to maintain the structural activity of the proteins extracted [111]. \begin{table}
\begin{tabular}{c c c} \hline
**Type** & **General Formula** & **Terms** \\ \hline I—HBA and metal chloride & Cat\({}^{+}\) X\({}^{-}\) z MCL\({}_{n}\) & M = Zn, Sn, Fe, Al, Ga, In \\ II—HBA and metal chloride hydrate & Cat\({}^{+}\) X\({}^{-}\) z MCL\({}_{n}\) y H\({}_{2}\)O & M = Cr, Co, Cu, Ni, Fe \\ III—HBA and HBD & Cat\({}^{+}\) X\({}^{-}\) z RZ & Z = OH, COOH, CONH\({}_{2}\) \\ IV—Metal chloride and HBD & MCL\({}_{n}\) + RZ = MCL\({}_{n-1}\)\({}^{+}\) RZ + MCL\({}_{n+1}\) & M = Al, Zn; Z = OH, CONH\({}_{2}\), \\ V—Molecular compounds & Non-ionic DESs & Molecular substances \\ \hline \end{tabular}  
Cat\({}^{+}\): ammonium, phosphonium, or sulfonium cation. X\({}^{-}\): Lewis base (generally, a halide). HBA: hydrogen bond acceptor. HBD: hydrogen bond donor. R: alkyl rest. z: stoichiometric coefficient.  
\end{table} 
Table 1: General classification of DESs [102; 103]. Many works have proved the good performance of DESs to selectively extract proteins for several matrices, such as animal tissues or plant-derived sources like oilseeds [24; 113; 114; 115; 116; 117]. For instance, [PERSON] et al. achieved >90% of extraction efficiency for collagen proteins from cod skin, with the corresponding purity of 93%, using the single-DES formed by choline chloride and oxalic acid in optimum conditions [115]. Turning the attention to the research focused on matrices similar to microalgae biomass, it is worth mentioning [PERSON] and co-workers research, in which protein-rich extracts from rapeseed cake and evening primrose cake, residues derived from the oil industry were obtained using a choline chloride:glycerol DES, in 1:2 molar ratio. Once the proteins are in the DES phase, they can be precipitated using water as an anti-solvent, increasing the percentage of protein in the final extract up to 20% compared to the starting biomass [114]. However, most of the research is focused on DES-based ATPS, as it is usually the most efficient technique to recover and purify proteins due to the low interfacial tension of these systems. For instance, [PERSON] et al. developed a biphasic system based on a NADES containing betaine as HBA, urea as HBD, and water in a 1:2:1 molar ratio, reaching an extraction efficiency of 99.82% for BSA, and therefore showing ATPS are valuable tools for protein obtaining [117]. Regarding protein recovery from microalgae using DESs, only a couple of studies were found [118; 119]. Firstly, [PERSON] et al.