## 4 Biodegradable Solvents for Protein Extraction
### Deep Eutectic Solvents (DESs)  
To avoid the problems associated with ILs, scientists turned their interest into a newly emerging type of solvents, DESs, which was first described in 2003 by [PERSON] et al. [95]. DESs are liquid mixtures at room temperature, formed by the combination of two solids (or a solid and a liquid) in a specific molar ratio, for which the eutectic point temperature is below that of an ideal mixture, much lower than the compounds separately [96]. The liquid obtained shows exceptional physicochemical properties similar to ILs [97]. Generally, the eutectic point is found in a significant depression, around 150 \({}^{\circ}\)C lower than if the mixture had an ideal behavior [98], and it is thought to be caused by the charge delocalization produced between the molecules mixed [99]. Some authors consider DESs as a subclass of ILs. However, most agree that DESs are a different kind of solvent because they are not formed exclusively by ionic species (existing DESs with non-ionic compounds), and no chemical reaction is involved in their preparation [17; 62; 98; 100]. DESs have two components: a hydrogen bond donor (HBD) or a metal halide and a hydrogen bond acceptor (HBA). The hydrogen bond interaction between those components forms the eutectic mixture [98]. The variety of those two groups is enormous, so it is estimated there could be around 10\({}^{6}\)-10\({}^{8}\) possible combinations to form DESs [101]. Depending on the chemical nature of the components, DESs are classified into five distinct types (Table 1) [102; 103]. However, most of the research has focused on Type III: DESs consisting of a quaternary ammonium salt as HBA (usually choline chloride)and a suitable hydrogen bond donor such as alcohols, carboxylic acids, or amides [104]. This is because type III shows a stronger hydrogen bond interaction, which directly affects the physicochemical properties of DESs [105]. As mentioned, DESs have similar physicochemical properties compared to ILs, since they present low volatility and vapor pressure (slightly larger than ILs), thermal stability, and tunable polarity, which allow solubilization of various molecules, including proteins [104]. The separation mechanism is meant to imply several forces such as hydrophilic-hydrophobic or electrostatic interactions, although it is thought that their excellent protein extraction capabilities are driven by the formation of protein-DES aggregates [24; 106]. Contrary to ILs, DESs starting materials are cheap, easy to produce, and do not entail associated pollution (most are allowed for human consumption). DESs are also more biodegradable than ILs (>69.3% in 28 days, according to [PERSON] et al. [107]), but the nature of the ions highly determines their biodegradability [72]. Moreover, DESs are considered mixtures rather than pure compounds [96], so there is no need for by-product removal as no chemical reaction has occurred (process with 100% of the atom economy, as opposed to ILs). Due to their outstanding properties, such as great biodegradability, comparable physicochemical properties of ILs, and small or no toxicity, DESs are applied in many fields such as electrochemistry, organic and inorganic chemistry. In addition, DESs are being used in separation strategies replacing the role of ILs, as they are considered a greener alternative [108]. However, as reported by [PERSON] et al. [61], not only the greenness of ILs must be questioned. Some DESs should also be critically reevaluated, as they encompass some downsides such as hygroscopicity, toxicity, regenerability and renewability, and decomposition at high temperatures. To circumvent some of these problems, [PERSON] et al. found that a DES-like liquid could be formed when mixing natural primary cellular metabolites such as choline, sugars, betaine, alcohols, organic acids, and amino acids [109].