## 4 Biodegradable Solvents for Protein Extraction
### Ionic Liquids (ILs)
applied bead milling as a cell disruption technique and managed to separate lipids of carbohydrates and proteins from _Neochloris oleabundans_. They used a 40% solution of tributylmethylphosphonium methyl sulfate, a biomass concentration of 6.67 g L\({}^{-1}\) at 45 \({}^{\circ}\)C after 30 min. After centrifugation, the lipids were obtained in the solvent phase (68% recovery yield of total lipids). The proteins and carbohydrates remained in the microalgae biomass, which was subsequently mechanically disrupted, leading to an aqueous phase rich in those components, achieving 80% recovery yield for proteins and 77% for carbohydrates [84]. Concerning IL-based ATPS, there is more research when compared to single ILs extraction, as it combines the advantages of using ionic liquids with the benefits of following an ATPS approach: short extraction time, low viscosity, and options of recyclability [85]. The most common IL-based ATPS is composed of one ionic liquid and an aqueous polymer or salt solution [86; 87; 88]. For instance, [PERSON] et al. studied the fractionation of several microalgal biomolecules: proteins, carbohydrates, lipids, and pigments, from _Neochlorisoleabundans_, grown in saline freshwater conditions [86]. After cell disruption, it was treated with three different ILs-ATPSs with a polymer-rich phase. A multistep fractionation process was implemented, and the system formed by PPG400-Ch DHp provided the best results, attaining an 82% of protein recovery in the IL phase, along with pigments (98%) and carbohydrates (93%), while lipids and starch were isolated from the interface, with recovery yields of 79 and 71%, respectively. Then, they proposed a further purification step applying ultrafiltration, selectively obtaining 80% of proteins from sugars and IL, allowing the recycling of the solvent. [PERSON] et al. also concluded that the electrostatic interaction between proteins and the IL might be the driving force of the separation process. Using salts instead of polymers ([C8\({}_{\rm{mim}}\)]Cl--K\({}_{3}\)PO\({}_{4}\)) to _lochrysis sablana_, [PERSON] et al. achieved a selective and efficient carbohydrate-protein separation, with 100% of proteins in the top phase, and 71% of carbohydrates in the bottom phase [87]. [PERSON] et al. also employed an IL-salt AIPS, consisted of Iolilyte 221 PG as IL, and potassium citrate tribasic monohydrate as salt, obtaining protein extracts with industrial relevance from crude microalgae _Neochloris oleabundans_ and _Tetraselmis suecia_ applying mild conditions (pH = 7.0 and room temperature) [88]. The number of published articles concerning ILs grew until 2016, when concerns about their toxicity surpassed their assumed benefits. Even though ILs do not pollute the air due to their low vapor pressure at room temperature, some of the ions used have displayed ecotoxicological effects, flammability, and have proved to be poorly biodegradable [89]. However, not all ILs are indeed equally harmful. Some choline-based ILs derived from biomaterials (Bio-ILs) have shown low toxicity and high stability [90], substituting the cation (typically pyridinium or imidazolium) by choline. Nonetheless, the appearance of Bio-ILs has not prevailed among scientist investigations [91; 92]. Furthermore, synthesizing ILs is expensive compared to classical solvents [93], mainly due to the starting products, which are obtained from fossil fuels sources, making the process less green, as waste is created indirectly [94]. Other important aspects of any green extraction are the recovery of the value-added compounds (hindered by their low vapor pressures) and the capacity of the solvent to be recovered and reused. These issues remain a challenge, and there is a need for further research, as generally, studies focused only on measuring protein content on the IL and are not concerned about these aspects (more information in Section 4.3) [76]. These drawbacks prevent the widespread ILs in the industry and engender the need for alternative green options.