## 4 Biodegradable Solvents for Protein Extraction
### Solvent Recovery Options
Nonetheless, little research has been reported about the recovery problem of these new biodegradable solvents. For example, [PERSON] et al. used a sodium dodecyl sulfate solution as a stripping reagent to recover the DESs employed in a K\({}_{2}\)HPO\({}_{4}\)-ATPS system (choline chloride-urea, tetramethylammonium chloride-urea, tetrapyammonium bromide-urea, and choline chloride-methylurea). However, they did not succeed, due to their hydrophilic character [24]. [PERSON] et al. managed to back-extract the proteins from a DES-ATPS (K\({}_{3}\)PO\({}_{4}\) and betaine-urea) by forming a new ATPS with fresh K\({}_{3}\)PO\({}_{4}\) aqueous phase mixed with ethanol (to improve the phase-forming ability of the DES). The ATPS created was shaken mechanically for 12 min, and then the DES phase was collected. Unfortunately, the highest back-extraction efficiency obtained was relatively low (32.66%) [117]. In turn, [PERSON] et al. studied the extraction efficiency for BSA and trypsin with a DES-ATPS formed by K\({}_{2}\)HPO\({}_{4}\) and choline chloride-glycerol, obtaining extraction yields of 98.71 and 94.36%, respectively. Then, to obtain the BSA from the DES-rich phase, 1.8 g of K\({}_{2}\)HPO\({}_{4}\) were added to 0.27 mL to the DES-rich top phase, forming a new DES-ATPS, fixing the ion strength by adding 3% solution of NaCl, they obtain a highest back-extraction yield of 32.96% [116]. Similarly, [PERSON] et al. study BSA's extraction and back extraction efficiencies for two DES: tetramethylammonium chloride-urea (TMAC-U) and tetramethylammonium chloride-glycerol-urea ternary (TMAC-G-U). Once the extraction was performed and the DES phase-separated, they carried out the back extraction by adding a solution of K\({}_{2}\)HPO\({}_{4}\) (0.70 g mL\({}^{-1}\)) to create a new ATPS system. For TMAC-G-U, the back extraction efficiency achieved was 71.88%, while for the TMAC-U, only a modest 21.02% was reached. Thus, [PERSON] et al. confirmed that the main handicap for back-extraction was the high interfacial mass transfer resistance between the two phases, as the addition of glycerol reduced the mass transfer resistance. Furthermore, they also checked the extraction capability of the DES used, showing that it still can be used for further experiments (97.99% of extraction efficiency for new BSA added) [113]. Regarding ILs, there is even less investigation. The research discussed in Section 4.1 from [PERSON] et al. studied the recovery of the IL by using a precipitating agent (ammonium sulfate), concluding that it could be reused up to three times, although the extraction capabilities were reduced [81]. Alternatively, [PERSON] et al. studied the recycling of the IL employed for selective lipid extraction through anti-solvent precipitation, adding methanol to the biomass-IL mixture. Then, the precipitated IL was separated from the biomass by centrifugation, and finally, the methanol was eliminated via evaporation. Although chlorophyll was also recovered along with the IL, they obtained an average recovery of 98.0%, showing a similar performance in further extraction cycles. Thus, more studies focusing on the recovery of other impurities should be performed [123]. Furthermore, in the context of a biorefinery, these solvent recovery processes have not been put into effect at an industrial level, although given the low toxicity of NADESs, they might not need to be recovered, which would diminish the overall cost of the process [124]. Therefore, despite the bright present and future of biodegradable solvents, there is an urgent need to develop green, reliable, and straightforward methods that will enable us to recover and purify the proteins and that allows correct recycling and regeneration of the solvent used so that the environmental impact is minimized or negligible. The current state of the art is in its infancy, and there are very few studies dealing with protein purification or solvent recovery issues.