## 4 Biodegradable Solvents for Protein Extraction
### Ionic Liquids (ILs)  
ILs are liquid salt mixtures at room temperature [73]. This low melt temperature is a result of their low-charge density and low symmetry ions of the organic salts, along with ionic interactions, hydrogen bonds, and dipole-dipole intermolecular forces [74]. Generally, to obtain salts in the liquid state, very high temperatures are required to disrupt the strong ionic interactions. However, the strong electrostatic interactions of ILs lead to a lowering of the melting point below 100 \({}^{\circ}\)C [75]. Although there are many options for organic salts, cations are usually exclusively organic derivatives, while anions might have either organic or inorganic nature [76]. Because of their molecular structure, ILs possess outstanding physicochemical properties compared to organic solvents: low vapor pressure (<1 Pa), high conductivity, moderate thermal stability, and great extraction capacities for many organic and inorganic compounds [77]. They also have a wide electrochemical window, and valuable polarity properties, depending on the ions used [78]. Moreover, thanks to their physicochemical characteristics, some ILs (such as 1-tetradyl-3-methylimidazolium bromide, or 1-ethoxyethanol-3-methylimidazolium chloride, etc.) have demonstrated promising results in guaranteeing the stability and activity of proteins, as shown in the review published by [PERSON] et al. [79]. Nevertheless, details of the mechanism of action of how the IL interacts with proteins to extract them still need to be elucidated [72]. The first IL was synthesized in 1914 by [PERSON], yet until 2007, they were not explicitly applied to extract biocompounds [76]. To synthesize an IL, diluted solutions of each ion must be prepared and mixed correctly. When the reaction ends (which usually takes up to 48 h and needs 25-100 \({}^{\circ}\)C), the solid residue is removed and washed with an organic solvent. As stated by [PERSON] et al., ILs have been used extensively to extract natural products [80]. However, attention must be paid to the ion components chosen to maximize the analyte-solvent interactions, leading to a more efficient process [72]. Extraction of proteins from microalgae biomass can be carried out by single IL solvent extraction or through an ATPS system. Single IL extraction is usually aided with a complementary technique such as ultrasonication or microwave. [PERSON] et al. evaluated different ILs and their mixture for obtaining phycobiliproteins from _Spirulina platensis_[81]. The authors extracted the highest protein concentration using an equimolar mixture of 2-hydroxyethylammonium acetate and 2-hydroxyethylammonium formate using an ultrasonication bath (25 \({}^{\circ}\)C and 25 kHz) and a solvent:biomass ratio of 7.93 mL g\({}^{-1}\). The pH was fixed at 6.50, and the extraction time was set to 30 min. The protein recovered in a more significant amount was allophyocyanin, attaining values of 6.34 mg g\({}^{-1}\), but also 5.95 mg g\({}^{-1}\) of phycocyanin and 2.62 mg g\({}^{-1}\) of phycoerythrin were recovered. As another example, [PERSON] et al. also extracted proteins and astaxanthin by treating the liquid microalgae extract from _Haematococcus pluvialis_ with the commercial trihexyl(tetradylphosphonium bistrifamide ionic liquid and a microgel-stabilized emulsion. They reached an average extraction efficiency greater than 90% using a solvent biomass ratio of 3:7, a microgel particle concentration of 1 g L\({}^{-1}\), and mixing time of 120 min; the proteins recovered preserved their native state after separation [82]. Similarly, [PERSON] et al. employed an IL-buffer (cholinium 2-hydroxy-3-morpholinopropanesulfonate) at pH of 7.00 combined with ultrasonication (400 W; 24 kHz; 5 s on/5 s off pulse mode) to obtain proteins from _Chorella vulgaris_. Proteins were recovered in a single step operation (extraction from the cell and purification occurred simultaneously) with an almost complete recovery yield of 95.0 \(\pm\) 1.9% [83]. Instead of ultrasonication, [PERSON] et al.