## 3 Results and Discussion  
### High-Temperature Astaxanthin Extraction Using ILs  
In _H. lacustris_ cysts, astaxanthin is present primarily in mono- and di-ester forms with long-chain fatty acids in intracellular neutral lipid droplets [4; 11]. This implies that lipid-extracting solvents can be effective for the co-extraction of lipophilic astaxanthin biomolecules. To investigate the room-temperature extractability of astaxanthin with IL, we selected four [Emim]-based ILs with HSO4, DEP, SCN, and Cl anions, which showed various lipid extraction performances (efficiency: 95.1%, 64.3%, 34.8%, and 34.8%, respectively) from 18-day-old _H. lacustris_ cysts at 80 \({}^{\circ}\)C [17]. Figure 1 shows the effect of temperature (28-100 \({}^{\circ}\)C) on the efficiency of astaxanthin recovery from mature 90-day-old _H. lacustris_ cysts using [Emim]-based ILs. Astaxanthin recovery was expressed in terms of a percentage of the total astaxanthin content (26.2 \(\pm\) 1.9 mg/g cell) measured using bead-beater-assisted solvent extraction [11]. Owing to its low toxicity and high biodegradability, ethyl acetate has been recommended for the extraction of lipophilic bioactive compounds in the food industry [25; 26]. However, as a control, when _H. lacustris_ cysts were treated using only ethyl acetate at 28 \({}^{\circ}\)C for 2 h, almost no astaxanthin was recovered (efficiency, 1.4%). This implies that simple ethyl acetate extraction is not effective in disrupting the rigid three-layer cell wall structure of _H. lacustris_ cysts. Similarly, [PERSON] et al. [21] reported an astaxanthin extraction yield of less than 5% from spray-dried _H. lacustris_ biomass using ethyl acetate at a high temperature (65 \({}^{\circ}\)C). Overall, regardless of the IL type, a two-step treatment involving IL-mediated extraction and ethyl acetate recovery from 90-day-old _H. lacustris_ cysts improved the astaxanthin yield (9.6-14.2%) at 28 \({}^{\circ}\)C compared to that in the ethyl acetate-treated control (1.4%). However, the astaxanthin extraction performance of selected ILs at a high temperature (40 to 100 \({}^{\circ}\)C) was significantly different from the performance expected based on the yield from 80 \({}^{\circ}\)C-lipid-extraction from 18-day-old _H. lacustris_ cysts (efficiency, 34.8-95.1%; [17]). The astaxanthin recovery efficiencies of the [Emim] DEP, [Emim] SCN, and [Emim] CI ILs were less than 10%, regardless of the temperature and IL type. Only the yield obtained with [Emim] HSO4 (astaxanthin recovery, 97.7%) was comparable to previous lipid extraction yields (astaxanthin recovery, 95.1%; [17]) at 80 \({}^{\circ}\)C. However, when the temperature was further increased to 100 \({}^{\circ}\)C, the astaxanthin recovery efficiency of [Emim] HSO4 decreased by 8%. Hydrophilic imidazolium-based ILs have been proposed to destroy the cellulose network of algal cell walls based on glycosidic bond hydrolysis event through IL and hydroxide ion formation from water dissociation [9,17]. However, the detailed chemical mechanism according to the type of anion in [Emim]-based ILs has not been elucidated yet. This could be an important topic of further investigation for the extension of IL technology in algal biorefinery. The extractability of ILs may depend on the developmental state and robustness of the cell wall during cyst morphogenesis in _H. lacustris_ (i.e., 90-day-old cysts in this study and 18-day-old cysts in the study by [PERSON] et al. [17]). Nevertheless, Figure 1 results suggest that the optimal conditions for lipid and astaxanthin extraction by ILs may be different and that higher temperatures do not necessarily promote the recovery of heat-sensitive astaxanthin from _H. lacustris_ cysts. At elevated temperatures, different ILs have been used to permeabilize the cell walls of various algae, including _Haematococcus_, _Chlamydomonas_, _Chlorella_, _Dunaliella_, and _Scenedesmus_[17,21,23,27,28].