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Article

Room-Temperature Cell Disruption and Astaxanthin Recovery from Haematococcus lacustris Cysts Using Ultrathin α-Quartz Nanoplates and Ionic Liquids

by
Nakyeong Lee
1,†,
Aditya Lakshmi Narasimhan
2,†,
Gyuseop Moon
2,
Young-Eun Kim
2,
Myeonghwa Park
1,
Bolam Kim
1,
Rendi Mahadi
2,
Sungwook Chung
1,2,* and
You-Kwan Oh
1,2,*
1
Institute for Environment & Energy, Pusan National University, Busan 46241, Korea
2
School of Chemical Engineering, Pusan National University, Busan 46241, Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Appl. Sci. 2022, 12(4), 2210; https://doi.org/10.3390/app12042210
Submission received: 29 January 2022 / Revised: 15 February 2022 / Accepted: 17 February 2022 / Published: 20 February 2022
(This article belongs to the Section Applied Biosciences and Bioengineering)
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Versions Notes

Abstract

:
Ionic liquids (ILs) are new green solvents, which are widely used in lignocellulosic and microalgal biorefineries. However, high-temperature operating conditions limit their application in the extraction of heat-labile algal products, such as bioactive astaxanthin. In this study, we report the technical feasibility of room-temperature astaxanthin extraction from Haematococcus lacustris cysts with a thick and complex cell wall structure, by combining ultrathin α-quartz nanoplates (NPLs) with ethyl-3-methylimidazolium ([Emim])-based ILs. When four different [Emim]-based ILs with thiocyanate (SCN), diethylphosphate (DEP), HSO4, and Cl anions were applied to 90-day-old H. lacustris cysts at room temperature (~28 °C), the astaxanthin extraction efficiency was as low as 9.6–14.2%. Under sonication, α-quartz NPLs disrupted the cyst cell wall for a short duration (5 min). The astaxanthin extraction efficacies of a subsequent IL treatment improved significantly to 49.8% for [Emim] SCN, 60.0% for [Emim] DEP, 80.7% for [Emim] HSO4, and 74.3% for [Emim] Cl ions, which were 4.4, 6.1, 8.4, and 5.2 times higher than the extraction efficacy of only ILs, respectively. This finding suggests that α-quartz NPLs can serve as powerful cell-wall-disrupting agents for the room-temperature IL-mediated extraction of astaxanthin from robust algal cyst cells.

1. Introduction

Astaxanthin (3,3′-dihydroxy-β,β′-carotene-4,4′-dione), a secondary ketocarotenoid phytopigment, is widely used in the cosmetic, aquaculture, food, and pharmaceutical industries [1,2]. The potent antioxidant activity of astaxanthin has preventive effects against cancer, diabetes, cardiovascular disease, ulcers, and inflammation through the enhancement of the immune response [3,4]. Astaxanthin has been proposed as a potential geroprotector that may affect the rate of human aging and extend cell longevity [5]. This biomolecule can induce the synthesis of hyaluronan (hyaluronic acid), which is related to skin hydration [6]. Haematococcus lacustris (formerly, H. pluvialis; [7]), a unicellular freshwater green microalga, is considered an important source of astaxanthin because of its high astaxanthin content (~4% of dry weight) and potential for mass cultivation in open-pond and photobioreactor systems [8,9].
Astaxanthin accumulates in H. lacustris as neutral lipid bodies, primarily in mono-and di-ester forms with fatty acids, during the physiological development of dormant aplanospores (cysts) from vegetative palmella cells under conditions of stress, such as nutrient deprivation, pH shock, high salinity, strong light irradiance, and nanoparticle exposure [4,10,11]. In particular, cell encystment is accompanied by the formation of a rigid multilayered cell wall structure. The cell wall thickness (~4 μm) of H. lacustris cysts is significantly greater than that of Chlamydomonas (~351 nm), which is extensively used in various algal biorefineries [12,13,14].
To destroy the cell wall of mature H. lacustris cysts, harsh chemical treatments, such as hydrochloric acid administered at high temperatures [15], and highly energy-intensive mechanical methods, such as high-pressure homogenization [16], are applied extensively. However, excessive physicochemical stress can degrade the molecular structure of astaxanthin, significantly reducing its antioxidative activity [3,17]. Therefore, efficient cell disruption and astaxanthin extraction are considered important technical issues affecting the overall economics of H. lacustris biorefineries [9,18].
Ionic liquids (ILs) are new “green” solvents with several unique properties, such as good thermal and chemical stability, low volatility, and high hydrolytic activity [19,20]. In particular, ILs can be custom-designed and synthesized as asymmetric organic cations and inorganic or organic anions, and they have been widely applied in lignocellulosic and algal bioenergy biorefineries. However, ILs generally require high-temperature operating conditions (~140 °C) to decompose the cell walls of various algal species, including Chlamydomonas, Chlorella, Nannochloropsis, and Haematococcus [17,19,20,21,22,23]. This implies that despite the strong cell-wall-disrupting effect of ILs, their use is not always feasible in the extraction of heat-sensitive bioactive algal products, including astaxanthin. For example, Choi et al. [17] found that the extraction yields of lipids and astaxanthin were significantly different, at 60–82% and 11–17%, respectively, when 1-ethyl-3-methylimidazolium [Emim]-based ILs with bis(trifluoromethylsulfonyl)imide and acetate anions were applied to H. lacustris cyst biomass at 80 °C.
Recently, we reported the synthesis of ultrathin α-quartz nanoplates (NPLs) using a novel template-free hydrothermal method; we demonstrated their disruptive effect on algal cell walls for subsequent dichloromethane/methanol (DCM/MeOH) solvent extraction [13]. Here, we aimed to investigate the technical feasibility of room-temperature IL-based astaxanthin extraction from rigid H. lacustris cysts using α-quartz NPLs as cell-wall disrupters. This approach may help minimize the use of harsh inorganic acids and/or organic solvents and reduce the need for energy-intensive mechanical processing through the development of environment-friendly H. lacustris biorefining processes.

2. Materials and Methods

2.1. Materials

Ultrathin α-quartz NPLs (lateral size, 1.14 ± 0.32 μm; thickness, 7.7 ± 0.6 nm) were synthesized from monodisperse amorphous silica nanoparticles (average diameter: 60 nm) according to a previously reported template-free hydrothermal protocol [13]. The synthesized white α-quartz NPL powder was stored at room temperature (~28 °C) until the algae experiments.
Four types of [Emim]-based ILs with HSO4, diethylphosphate (DEP), thiocyanate (SCN), and Cl anions (purity ≥ 95%) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Astaxanthin was used as obtained from the supplier (Sigma-Aldrich). All other solvents and chemicals used were of analytical grade, purchased from Junsei (Tokyo, Japan), Daejung (Siheung-si, Korea), Samchun (Seoul, Korea), or Sigma Aldrich, and used as received. Purified and deionized water was obtained using a Millipore Milli-Q system (Tokyo, Japan).

2.2. Algal Cultivation

The H. lacustris NIES-144 strain, obtained from the National Institute for Environmental Studies (NIES), University of Tokyo, Japan, was used in this study. The alga was photoautotrophically cultivated in a photosynthetic NIES-C medium [11]. The medium was sterilized using a 0.2 μm mixed cellulose ester membrane filter after adjusting the pH to 7.5. One colony of H. lacustris from the agar plate culture was transferred aseptically to a 250 mL Erlenmeyer flask with a porous silicon stopper (working volume: 150 mL). The flask culture was performed in a shaking incubator under continuous illumination (ca. 90 µmol/m2/s; 25 °C, 150 rpm; ISF-7100RF, Jeio Tech, Daejeon, Korea). Sequentially, during the photosynthetic flask cultivations, vegetative biflagellate motile cells lost their flagella, expanded in volume, and changed into non-motile palmella cells. Under nitrogen-nutrient deprivation conditions, astaxanthin biosynthesis was induced in the palmella cells, giving them a brownish-green color. Eventually, these cells were transformed into bright red, astaxanthin-rich cyst cells. In general, the intensity of red color in the H. lacustris cells and the amount of astaxanthin are proportional [24] After 90 days of culture, mature red H. lacustris cyst cells were harvested by centrifugation (3000 rpm for 10 min; Combi-514R, Hanil Science Inc., Daejeon, Korea) and lyophilized for 4 days (FD5512, IlShin BioBase Co., Yangju-si, Korea). The freeze-dried algal cells were stored in a vacuum bag at −20 °C before the astaxanthin extraction experiments. Detailed media composition and culture conditions have been reported earlier [11,13].

2.3. Total Astaxanthin Content Estimation

The total astaxanthin content in the lyophilized H. lacustris biomass was measured by bead-beater-assisted ethyl acetate extraction followed by high-performance liquid chromatography (HPLC), with a slight modification to a previously reported protocol [13]. Briefly, 5 mg of algal cells were mixed with 1 mL of ethyl acetate and 1.0 g of glass beads (1.5 mm diameter; Daihan Scientific, Wonju-si, Korea) in a 2.0 bead-beating tube (Biofact, Daejeon, Korea). The algal solution was vigorously homogenized using a Fastprep-24 5GTM bead beater for three cycles (6 m/s, each 30 s; MP Biomedicals, Santa Ana, CA, USA) and centrifuged at 10,000 rpm for 5 min (Sorvall Legend Micro 17R, Thermo Scientific, Waltham, MA, USA). Bead-beater-assisted solvent extraction was repeated for three additional rounds until all the red algal cells turned colorless. The astaxanthin-containing ethyl acetate layer was separated by centrifugation and filtered using a 0.2 μm membrane. Next, 0.1 mL of the ethyl acetate solution was mixed with 0.9 mL of DCM/MeOH (1:1, v/v) (containing 0.025 N NaOH) in a fresh tube and analyzed using an HPLC instrument equipped with a diode-array detector (Agilent 1260 infinity, Hewlett-Packard, Santa Clara, CA, USA) and a YMC Carotenoid column (250 mm × 4.6 mm, 5 μm; YMC Inc., Wilmington, NC, USA). Detailed HPLC conditions have been reported previously [11,13]. The total astaxanthin content of the 90-day-old H. lacustris cyst cells was estimated to be 26.2 ± 1.9 mg/g cell.

2.4. Cell Wall Disruption and Astaxanthin Extraction Using α-Quartz NPLs and ILs

Cell wall disruption and astaxanthin extraction from H. lacustris cyst cells were performed using only IL or α-quartz NPL-assisted IL treatment. The IL types and concentrations were selected based on a previous study on H. lacustris biomass [17]. In the absence of α-quartz NPLs, approximately 10 mg of lyophilized H. lacustris cysts were treated with 1.5 mL of 33.3% (v/v) IL/water solution in a 12 mL Pyrex-glass tube with a Teflon-sealed screw stopper. The algal solutions were incubated for 2 h at five different temperatures (room temperature [ca. 28 °C], 40, 60, 80, and 100 °C) using a heating block (HS R200, Humas, Daejeon, Korea). For astaxanthin recovery, the reaction mixture was mixed with 0.75 mL of ethyl acetate and incubated for 30 min in a tube rotator at 50 rpm (Fine PCR, Gunpo-si, Korea) at room temperature. The astaxanthin-containing ethyl acetate layer was separated by centrifugation (3000 rpm for 5 min). The astaxanthin concentration of the ethyl acetate solution was estimated based on the correlation between the optical density at 474 nm, as measured using UV/Vis spectrophotometry (Optizen 3220UV, Mecasys Co., Daejeon, Korea), and data from HPLC-based astaxanthin quantification (refer to Section 2.3. Total Astaxanthin Content Estimation for the detailed method). For the control experiment, astaxanthin was extracted from the lyophilized H. lacustris cyst biomass using only ethyl acetate without IL treatment.
For NPL-assisted IL-mediated extraction at the ambient temperature (~28 °C), 1 mL of the α-quartz NPL solution (1000 mg/L; [13]) was added to approximately 10 mg of H. lacustris cysts under the conditions. To facilitate sufficient physical contact between H. lacustris cysts and α-quartz NPLs, the algal solution was incubated in an ultrasonic bath (40 kHz, 100 W; 2510-DTH, Branson, Danbury, CT, USA) for 5 min, as described previously [13]. To extract astaxanthin, 33.3% (v/v) IL in an aqueous solution was supplied. As described above, the algal solution was incubated at room temperature for 2 h, mixed with ethyl acetate for 30 min, separated by centrifugation, and analyzed using a UV/Vis spectrophotometer. For the control experiment, astaxanthin was extracted from H. lacustris cysts using only NPL or IL, with sonication for 5 min. Astaxanthin recovery efficiency was expressed as a percentage of the total astaxanthin content obtained, as described above.

2.5. Other Analytical Methods

Algal cell growth was monitored by evaluating the cell number density using an improved Neubauer hemocytometer (Marienfeld, Germany) [11]. Light intensity and pH were measured using a quantum photometer (LI-250A; Li-Cor Inc., Lincoln, NE, USA) and a pH meter (HM-30R; TOADKK, Tokyo, Japan), respectively. Changes in the morphological appearance of H. lacustris cysts during cell disruption and astaxanthin extraction treatments were observed using bright-field and fluorescence microscopy (Axio Imager A2; Carl Zeiss, Jena, Germany). To analyze the penetration of IL into H. lacustris cysts, blue fluorescence signals were detected using a DAPI filter set (excitation: 335–389 nm; emission: 420–470 nm), and the intensity was measured using the AxioVision software (Carl Zeiss, Jena, Germany) after washing the algal samples with purified water [14].

3. Results and Discussion

3.1. 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 °C [17].
Figure 1 shows the effect of temperature (28–100 °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 ± 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 °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, Desai 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 °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 °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 °C) was significantly different from the performance expected based on the yield from 80 °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] Cl 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 °C. However, when the temperature was further increased to 100 °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 Choi 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]. High temperatures not only induce a build-up of internal pressure via the heating effect but also promote the hydrolytic activity of thermally stable ILs on the algal cell wall. Collectively, these effects can lead to the effective disruption of the algal cell wall structure through physical and chemical means [18,29]. However, as shown in Figure 1, a careful balance should be maintained between the astaxanthin-extracting capability of the IL solvent and the thermal sensitivity of astaxanthin. In addition, given the energy required for the maintenance of a high temperature and the scale-up of the algal biorefinery process, mild temperatures are preferred if high-efficiency astaxanthin extraction can be achieved [9].

3.2. Room-Temperature IL-Mediated Astaxanthin Extraction Using α-Quartz NPLs

To improve room-temperature astaxanthin extraction using [Emim]-based ILs, ultrathin α-quartz NPLs were introduced to lacerate the rigid cell walls of 90-day-old H. lacustris cysts, based on findings from a previous report [13]. Brief incubation of the H. lacustris solution for 5 min in an ultrasonic water bath facilitated sufficient contact between the algal cells and the α-quartz NPLs. As a control, when the algal cysts were treated with α-quartz NPLs followed by extraction with only with ethyl acetate for 2 h, the astaxanthin recovery efficiency was very low (4.7 ± 0.1%), indicating that the cell-wall-disrupting effect of NPLs was not conducive to ethyl acetate extraction (Figure 2). Five-minute sonication of the algal solution without α-quartz NPLs helped increase the astaxanthin recovery efficiency of ILs to 24.0–38.6%, irrespective of the IL type, compared to the recovery efficiency achieved with IL treatment alone (9.6–14.2%, Figure 1). This improvement is attributed to partial cell wall disruption by the high-pressure bubbles and acoustic cavitation effects and, consequently, the enhancement of IL permeability [9]. However, it should be noted that excessive sonication can lead to severe biodegradation and oxidation of bioactive astaxanthin by localized heating and free radical formation [3,18]. In particular, free radicals readily break the hydroxyl and C=C bonds of the astaxanthin structure.
Notably, using α-quartz NPLs in room-temperature IL-mediated extraction, the astaxanthin recovery from H. lacustris cysts improved significantly (49.8% for [Emim] SCN, 60.0% for [Emim] DEP, 80.7% for [Emim] HSO4, and 74.3% for [Emim] Cl) (Figure 2). These values were 4.4, 6.1, 8.4, and 5.2 times higher, respectively than obtained with the IL treatment alone (Figure 1). This finding clearly demonstrated that the cell-wall-lacerating effect of α-quartz NPLs increased the cell wall permeability and astaxanthin extraction potential of the four [Emim]-based ILs from rigid H. lacustris cysts. It also suggests that α-quartz NPLs can be considered as effective cell-wall-disrupting tools for the application of custom-designed ILs in the extraction of various products, including edible oils, fragrances, biopolymers, biodiesel, bio-jet fuel, bioethanol, and biogas, using algal biorefineries [2,20,30,31].

3.3. Microscopic Observation and NPL-Assisted IL-Mediated Extraction Process

Figure 3 illustrates the changes observed in the cysts microscopically and provides the schematic representation of astaxanthin extraction from H. lacustris cysts using α-quartz NPLs and [Emim] HSO4 at room temperature. When mature 90-day-old H. lacustris cysts (Figure 3a) were treated with α-quartz NPLs with sonication for 5 min, the intracellular circular domain-containing red astaxanthin-rich lipids appeared slightly smaller under a light microscope (Figure 3b). Furthermore, the region between the outer perimeter of the red domain and the cell wall exhibited differential interference contrast imaging, primarily because of the light scattering effects of α-quartz NPLs passing through the cell wall [13]. After the subsequent introduction of [Emim] HSO4, the astaxanthin-containing lipid droplets began to emerge from the cyst cell, owing to the lacerating effect of α-quartz NPLs (Figure 3c). Astaxanthin could be recovered from the upper ethyl acetate phase after the addition of ethyl acetate to the IL-treated algal solution, followed by centrifugation (Figure 3d). Astaxanthin-extracted and/or broken cells were observed at the interface of the two-phase solvent system (Figure 3e).
IL uptake by H. lacustris cyst cells was further analyzed by fluorescence microscopy based on the fluorescence of [Emim] HSO4 at approximately 450 nm. Several areas of weak blue fluorescence were observed inside the IL-treated cysts not treated with α-quartz NPLs. Instead, the outer layer of the cyst cell exhibited a strong blue fluorescence owing to the binding of [Emim] HSO4, which indicated a significant barrier effect of [Emim] HSO4 on the algal cell wall (Figure 4a). In contrast, H. lacustris cyst cells subjected to NPL-assisted IL treatment were able to sufficiently absorb [Emim] HSO4 and emitted stronger cytoplasmic blue fluorescence than cells treated with IL alone. The average blue fluorescence intensity of the former was approximately 4.3 times higher than that of the latter (Figure 4b). Along with the astaxanthin recovery results (Figure 2), these results demonstrate the synergistic effect of α-quartz NPLs and [Emim]-based ILs in astaxanthin extraction from H. lacustris cysts with a rigid cell wall structure.
Various IL solvents were used to extract astaxanthin from H. lacustris cyst biomass at different temperatures. Of note, the astaxanthin extractability of IL is highly dependent on the state of the algal biomass, such as cultivation conditions, aging, and the drying methods used, as well as the extraction parameters, such as IL concentration, temperature, and processing time. Desai et al. [21] reported 77% astaxanthin recovery from spray-dried H. lacustris cysts using 1-butyl-3-methylimidazolium [Bmim] dibutylphosphate at 55 °C. Liu et al. [30] reported 91% astaxanthin extraction from commercial H. lacustris powder by applying [Bmim] Cl at 70 °C. Notably, Choi et al. [17] reported almost complete astaxanthin recovery from lyophilized 18-day-old H. lacustris cysts using [Emim] CH3SO3 and [Emim] (CF3SO2)2N under optimized conditions (6.7% [v/v] IL/water mixture and treatment for 1 h at 30 °C).
In this study, relatively moderate recovery efficiency of 49.8–80.7% was obtained from 90-day-old H. lacustris cysts by room-temperature treatment with [Emim]-based ILs with SCN, DEP, HSO4, and Cl anions (Figure 2). Multiple solvent extractions and/or NPL reuse strategies can be considered to maximize astaxanthin recovery, as demonstrated previously for H. lacustris cyst biomass [13,32]. Detailed instrumental analysis such as HPLC-mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy on the cis and trans isomer composition of the extracted astaxanthin is required to accurately evaluate the effect of room-temperature extraction of bioactive astaxanthin [33]. Furthermore, the reduction of nanomaterial synthesis cost, minimization of chemical contamination in the final product, process optimization, and scale-up can be important research topics for the practical application of NPL-assisted IL-mediated extraction in H. lacustris biorefineries.

4. Conclusions

A low astaxanthin extraction efficiency of 9.6–14.2% was achieved from 90-day-old H. lacustris cysts (with a rigid and complex cell wall structure), when four different [Emim]-based ILs with SCN, DEP, HSO4, and Cl anions were applied at room temperature (~28 °C). Under 5-min sonication, α-Quartz NPLs disrupted the cyst cell wall, significantly improving the astaxanthin extraction efficiency of the room-temperature [Emim]-based ILs, regardless of the IL type (a 4.4–8.4-fold improvement compared to the yield in the control group). Astaxanthin-containing lipids were recovered by simple phase separation after subsequent ethyl acetate addition and centrifugation. The findings of our study suggest that α-quartz NPLs can be used as effective cell-wall-disrupting agents for the room-temperature IL-mediated extraction of bioactive compounds from various robust microalgae without significant thermal and chemical degradation of the target product.

Author Contributions

Conceptualization, Y.-K.O. and S.C.; Methodology, N.L., A.L.N., G.M., Y.-E.K. and M.P.; Investigation, N.L. and A.L.N.; Resources, G.M. and Y.-E.K.; Data Curation, B.K. and R.M.; Writing—Original Draft Preparation, N.L., A.L.N. and Y.-K.O.; Writing—Review & Editing, Y.-K.O. and S.C.; Visualization, B.K. and R.M.; Supervision, Y.-K.O. and S.C.; Project Administration, Y.-K.O.; Funding Acquisition, Y.-K.O. and S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the National Research Foundation of Korea [grant number NRF-2019R1A2C100346313] funded by the Ministry of Science and ICT, the Basic Science Research Program through the National Research Foundation of Korea [NRF-2019R1F1A1060060] funded by the Ministry of Education of Korea, the Research/Development Program of the Korea Institute of Energy Research [KIERC0-2424], and the 2020 BK21 FOUR Program of Pusan National University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Effect of temperature on astaxanthin recovery efficiency from 90-day-old Haematococcus lacustris cysts using four different ethyl-3-methylimidazolium ([Emim])-based ionic liquids (ILs) with thiocyanate (SCN), HSO4, diethylphosphate (DEP), and Cl anions for 2 h. Astaxanthin was recovered by subsequent ethyl acetate (EA) extraction at room temperature (ca. 28 °C) for 30 min. As a control experiment, H. lacustris cyst cells were treated only with EA for 2.5 h. The asterisk (*) represents room temperature.
Figure 1. Effect of temperature on astaxanthin recovery efficiency from 90-day-old Haematococcus lacustris cysts using four different ethyl-3-methylimidazolium ([Emim])-based ionic liquids (ILs) with thiocyanate (SCN), HSO4, diethylphosphate (DEP), and Cl anions for 2 h. Astaxanthin was recovered by subsequent ethyl acetate (EA) extraction at room temperature (ca. 28 °C) for 30 min. As a control experiment, H. lacustris cyst cells were treated only with EA for 2.5 h. The asterisk (*) represents room temperature.
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Figure 2. Astaxanthin recovery efficiency from 90-day-old H. lacustris cysts using 5-min-sonication-assisted α-quartz nanoplate (NPL) pretreatment followed by treatment with four different [Emim]-based ILs with SCN, HSO4, DEP, and Cl anions for 2 h at room temperature (ca. 28 °C). As controls, the cyst cells were treated using only NPL or IL in a sonication bath for 5 min. Astaxanthin was recovered by subsequent ethyl acetate extraction at room temperature for 30 min.
Figure 2. Astaxanthin recovery efficiency from 90-day-old H. lacustris cysts using 5-min-sonication-assisted α-quartz nanoplate (NPL) pretreatment followed by treatment with four different [Emim]-based ILs with SCN, HSO4, DEP, and Cl anions for 2 h at room temperature (ca. 28 °C). As controls, the cyst cells were treated using only NPL or IL in a sonication bath for 5 min. Astaxanthin was recovered by subsequent ethyl acetate extraction at room temperature for 30 min.
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Figure 3. Microscopic images and schematic illustrations of cell wall disruption and room-temperature astaxanthin (AXT) extraction from 90-day-old Haematococcus lacustris cysts using α-quartz NPLs and [Emim] HSO4: (a) Initial mature cyst cell. (b) Intact cyst cell after 5-min NPL treatment in a sonication bath. The intracellular contrast difference indicates the presence of α-quartz NPLs inside the cyst. (c) Cyst cells after subsequent IL treatment for 2 h. The black arrow indicates that red-AXT-containing lipid droplets were extracted from lacerations caused by α-quartz NPLs in the cell wall. (d) AXT recovery with EA. After centrifugation, the reaction tube shows AXT in the EA phase and [Emim] HSO4 and α-quartz NPLs in the distilled water (DW) phase. (e) Broken and empty cells were collected at the interface between the EA and aqueous phases. Scale bars represent 20 µm.
Figure 3. Microscopic images and schematic illustrations of cell wall disruption and room-temperature astaxanthin (AXT) extraction from 90-day-old Haematococcus lacustris cysts using α-quartz NPLs and [Emim] HSO4: (a) Initial mature cyst cell. (b) Intact cyst cell after 5-min NPL treatment in a sonication bath. The intracellular contrast difference indicates the presence of α-quartz NPLs inside the cyst. (c) Cyst cells after subsequent IL treatment for 2 h. The black arrow indicates that red-AXT-containing lipid droplets were extracted from lacerations caused by α-quartz NPLs in the cell wall. (d) AXT recovery with EA. After centrifugation, the reaction tube shows AXT in the EA phase and [Emim] HSO4 and α-quartz NPLs in the distilled water (DW) phase. (e) Broken and empty cells were collected at the interface between the EA and aqueous phases. Scale bars represent 20 µm.
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Figure 4. (a) Microscopic images of Haematococcus lacustris cysts treated with [Emim] HSO4 and with or without α-quartz NPLs obtained using optical and fluorescence microscopy. Intracellular blue fluorescence under the DAPI filter represents [Emim] HSO4 uptake by the algal cells. Scale bars represent 20 µm. (b) Average blue fluorescence intensities were obtained as a function of cell length from several fluorescence images (a).
Figure 4. (a) Microscopic images of Haematococcus lacustris cysts treated with [Emim] HSO4 and with or without α-quartz NPLs obtained using optical and fluorescence microscopy. Intracellular blue fluorescence under the DAPI filter represents [Emim] HSO4 uptake by the algal cells. Scale bars represent 20 µm. (b) Average blue fluorescence intensities were obtained as a function of cell length from several fluorescence images (a).
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Lee, N.; Narasimhan, A.L.; Moon, G.; Kim, Y.-E.; Park, M.; Kim, B.; Mahadi, R.; Chung, S.; Oh, Y.-K. Room-Temperature Cell Disruption and Astaxanthin Recovery from Haematococcus lacustris Cysts Using Ultrathin α-Quartz Nanoplates and Ionic Liquids. Appl. Sci. 2022, 12, 2210. https://doi.org/10.3390/app12042210

AMA Style

Lee N, Narasimhan AL, Moon G, Kim Y-E, Park M, Kim B, Mahadi R, Chung S, Oh Y-K. Room-Temperature Cell Disruption and Astaxanthin Recovery from Haematococcus lacustris Cysts Using Ultrathin α-Quartz Nanoplates and Ionic Liquids. Applied Sciences. 2022; 12(4):2210. https://doi.org/10.3390/app12042210

Chicago/Turabian Style

Lee, Nakyeong, Aditya Lakshmi Narasimhan, Gyuseop Moon, Young-Eun Kim, Myeonghwa Park, Bolam Kim, Rendi Mahadi, Sungwook Chung, and You-Kwan Oh. 2022. "Room-Temperature Cell Disruption and Astaxanthin Recovery from Haematococcus lacustris Cysts Using Ultrathin α-Quartz Nanoplates and Ionic Liquids" Applied Sciences 12, no. 4: 2210. https://doi.org/10.3390/app12042210

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