Extraction of Astaxanthin and Lutein from Microalga Haematococcus pluvialis in the Red Phase Using CO2 Supercritical Fluid Extraction Technology with Ethanol as Co-Solvent

Astaxanthin and lutein, antioxidants used in nutraceutics and cosmetics, can be extracted from several microalgal species. In this work, investigations on astaxanthin and lutein extraction from Haematococcus pluvialis (H. pluvialis) in the red phase were carried out by means of the supercritical fluid extraction (SFE) technique, in which CO2 supercritical fluid was used as the extracting solvent with ethanol as the co-solvent. The experimental activity was performed using a bench-scale reactor in semi-batch configuration with varying extraction times (20, 40, 60, and 80 min), temperatures (50, 65, and 80 °C) and pressures (100, 400, and 550 bar). Moreover, the performance of CO2 SFE with ethanol was compared to that without ethanol. The results show that the highest astaxanthin and lutein recoveries were found at 65 °C and 550 bar, with ~18.5 mg/g dry weight (~92%) astaxanthin and ~7.15 mg/g dry weight (~93%) lutein. The highest astaxanthin purity and the highest lutein purity were found at 80 °C and 400 bar, and at 65 °C and 550 bar, respectively.


Introduction
Currently, the interest in microalgae is on the rise, as the knowledge of their benefits in several aspects of human life increases. Astaxanthin and lutein, high-value molecules for many industrial sectors, e.g., pharmaceuticals, nutraceuticals, additive/functional foods, natural medicine, and cosmetics, can be extracted from microalgal species [1,2]. Microalgae can live both in freshwater and seawater environments, using their photosynthesis activity to convert atmospheric carbon dioxide into oxygen and sugars in the presence of sunlight [3]. During their life cycle, they can be used for carbon dioxide sequestration; in fact, for each kilogram of dry biomass, about 1.8-2 kg of CO 2 can be captured. olive oil), observing the increase in astaxanthin recovery and the decrease in extraction time in the presence of a co-solvent (astaxanthin recovery = 92%, at 55 • C, 8 MPa, and 15 min). Lutein extraction via CO 2 -SFE in the presence of co-solvents from other microalgal species, such as Chlorella [54,55] and Scenedesmus [56], was also investigated. CO 2 -SFE extraction from microalgae is the starting point for the production of natural compounds with a sustainable approach and a low environmental impact.
In this paper, astaxanthin and lutein extraction from H. pluvialis in the red phase (HPR) was investigated using CO 2 -SFE technology with ethanol as the co-solvent. The effects of temperature (50-80 • C) and pressure (100-550 bar) on recovery and purity over extraction time (20-80 min), i.e., four extraction cycles of 20 min each, were tested by keeping flow rates of carbon dioxide and ethanol constant at 3.62 g/min and 1 mL/min, respectively, using a bench-scale experimental apparatus. Moreover, at the same operative conditions, a comparison between the experimental findings from CO 2 -SFE with ethanol and the experimental findings from CO 2 -SFE without ethanol was carried out. Experimental findings from CO 2 -SFE without ethanol were published in a previous study by Di Sanzo et al [57].

Extraction Yield
Total extraction yields for each operative condition are summarized in Table 1, in which the total yields extracted without co-solvent are also included. The results are expressed as mg of extract per gram of dry weight of HPR, and values were obtained at the end of the extraction procedure. In the presence of a co-solvent, the total extraction yield ranged from 207.67 mg/g (T = 80 • C, P = 100 bar) to 292.70 mg/g (T = 65 • C, P = 400 bar); without co-solvent, the total extraction yield varied in the range of 0.1 mg/g (T = 50 • C, P = 100 bar) to 277.1 mg/g (T = 65 • C, P = 400 bar). As shown, with and without co-solvent, the highest total extraction yield was found at the same operative conditions (T = 65 • C, P = 400 bar); however, CO 2 -SFE extraction with a co-solvent increased the highest total extraction yield from 277.1 mg/g to 292.7 mg/g. Data in Table 1 show that the total extraction yield increases as temperature and pressure increase, until a maximum is reached, before a subsequent decrease. This result is due to counteracting phenomena: increasing the pressure increases the CO 2 density (the solvation power of the fluids [56]), but a pressure too high may obstruct the diffusion of supercritical fluid into the matrix; increasing the temperature decreases the viscosity of the solvent improving the mass transfer, but a temperature too high may degrade the extracted compounds [48,58,59].

Astaxanthin Recovery
The effects of pressure (100-550 bar), temperature (50-80 • C), and extraction time (extraction cycle number; 20-80 min) on the cumulative recovery of astaxanthin are reported in Figures 1 and 2.   decreasing, probably due to thermal degradation [60][61][62]. Results show that the maximum astaxanthin recovery was obtained at the highest pressure (550 bar) and at an intermediate temperature (65 °C). It is worth pointing out that, under the best extraction conditions, a negligible influence of extraction time was basically observed, as, at 20 min, an astaxanthin extraction yield of about 18.4 mg/gd was achieved, with respect to an astaxanthin extraction yield of about 18.5 mg/gd reached at 80 min. Pan et al. [63] investigated the effects of H. pluvialis loading, CO2 flow rate, time, pressure, and temperature of extraction, as well as ethanol loading, on astaxanthin extraction, observing a yield of about 71% at the following operating conditions: temperature = 50 °C, pressure = 310 bar, CO2 flow rate = 6 L/min, time = 160 min, and H. pluvialis loading = 6.5 g. Machmudah et al. [53] reported an extraction yield of about 80% at a pressure of 400 bar and a temperature of 343 K, with a CO2 flow rate of 3 mL/min and an ethanol concentration of 5%; a comparable finding was found by the same research group at a pressure of 550 bar and a temperature of 343 K, with CO2 flow rate of 3 mL/min without ethanol. It is worth highlighting that, in this study, an astaxanthin yield of about 93% at 550 bar and 65 °C, with a CO2 flow rate of 3.62 g/min and an ethanol flow rate of 1 mL/min was observed.
The comparison between cumulative recovery of astaxanthin with and without co-solvent [57] is reported in Table 2. As shown, the maximum recovery of astaxanthin (~94%) was found without At 50 • C (Figure 1a), with an extraction time of 20 min (first extraction cycle), increasing pressure from 100 to 550 bar resulted in astaxanthin recovery growing from~3.4 mg/g d (~17%) to~5.4 mg/g d (~27%). With an extraction time of 80 min (four extraction cycles), astaxanthin extraction yield increased from~4.9 mg/g d (~25%) to~5.6 mg/g d (~28%). Increasing extraction time from 20 min to 80 min, at 100 bar, astaxanthin recovery increased from~3.4 mg/g d to~4.9 mg/g d ; at 400 bar, astaxanthin recovery slightly increased from~4.8 mg/g d to~5.3 mg/g d ; at 550 bar, astaxanthin recovery slightly increased from~5.4 mg/g d to~5.6 mg/g d . At 65 • C ( Figure 1b) and at 80 • C (Figure 1c), for the first extraction cycle, astaxanthin recovery reached a maximum at~18.4 mg/g d (~92%) at 65 • C and 550 bar, while this value was only~0.2 mg/g d (~1%) at 80 • C and 100 bar. However, it is worth highlighting that, at 65 • C and 550 bar, astaxanthin recovery was relatively unaffected by the extraction time.
For each operative condition, the astaxanthin yield at the end of a cycle of extraction is shown in Figure 2. This figure clearly shows that, whatever the pressure, astaxanthin recovery increased with extraction temperature, reaching a maximum at 65 • C, corresponding to~18.5 mg/g d (~93%), before decreasing, probably due to thermal degradation [60][61][62]. Results show that the maximum astaxanthin recovery was obtained at the highest pressure (550 bar) and at an intermediate temperature (65 • C). It is worth pointing out that, under the best extraction conditions, a negligible influence of extraction time was basically observed, as, at 20 min, an astaxanthin extraction yield of about 18.4 mg/g d was achieved, with respect to an astaxanthin extraction yield of about 18.5 mg/g d reached at 80 min.
Pan et al. [63] investigated the effects of H. pluvialis loading, CO 2 flow rate, time, pressure, and temperature of extraction, as well as ethanol loading, on astaxanthin extraction, observing a yield of about 71% at the following operating conditions: temperature = 50 • C, pressure = 310 bar, CO 2 flow rate = 6 L/min, time = 160 min, and H. pluvialis loading = 6.5 g. Machmudah et al. [53] reported an extraction yield of about 80% at a pressure of 400 bar and a temperature of 343 K, with a CO 2 flow rate of 3 mL/min and an ethanol concentration of 5%; a comparable finding was found by the same research group at a pressure of 550 bar and a temperature of 343 K, with CO 2 flow rate of 3 mL/min without ethanol. It is worth highlighting that, in this study, an astaxanthin yield of about 93% at 550 bar and 65 • C, with a CO 2 flow rate of 3.62 g/min and an ethanol flow rate of 1 mL/min was observed.
The comparison between cumulative recovery of astaxanthin with and without co-solvent [57] is reported in Table 2. As shown, the maximum recovery of astaxanthin (~94%) was found without co-solvent at 550 bar and 50 • C. However, a comparable result was achieved with co-solvent (~92%) at 550 bar and 65 • C. The purity of astaxanthin for each stage of extraction, under different operative conditions, with and without co-solvent is reported in Table 3. With co-solvent, the highest purity (~18%) was achieved with an extraction time of 40 min at 80 • C and 400 bar. Comparable findings were observed by Reyes et al. [64], who investigated the effect of ethanol content in the CO 2 flow on astaxanthin extraction from H. pluvialis by CO 2 -SFE, observing a maximum purity of about 23% at 70 • C and 275 bar with an ethanol content in the CO 2 flow of 13% (w/w). Without co-solvent, the highest purity (~34%) was found with an extraction time of 80 min at the same pressure and temperature. As shown in Table 3, an effective improvement of the extraction of astaxanthin in the presence of co-solvent was not observed, despite CO 2 -SFE with ethanol showing a valuable level of astaxanthin recovery. Moreover, a reduction in purity was found; this result may be explained by considering that, in the presence of ethanol, the total extraction yield increased, reducing the purity of astaxanthin.

Lutein Recovery
The effects of pressure, temperature, and extraction time (extraction cycle number) on total lutein recovery are reported in Figure 3.    At 50 • C (Figure 3a) with an extraction time of 20 min (first extraction cycle), increasing pressure from 100 to 550 bar resulted in lutein recovery growing from~3.3 mg/g d (~43%) to~3.7 mg/g d (~49%). With an extraction time of 80 min (four extraction cycles), lutein extraction yield increased from~3.9 mg/g d (~50%) to~5.0 mg/g d (~65%). Increasing extraction time from 20 min to 80 min, at 100 bar, lutein recovery slightly increased from~3.3 mg/g d (~43%) to~3.9 mg/g d (~50%); at 400 bar, lutein recovery slightly increased from~3.7 mg/g d (~48%) to~4.1 mg/g d (~54%); at 550 bar, lutein recovery increased from~3.7 mg/g d (~48%) to~5.0 mg/g d (~65%). At 65 • C (Figure 3b) and at 80 • C (Figure 3c), the influence of extraction time on lutein recovery was quite similar to that observed at 50 • C ( Figure 3a); however, the maximum lutein recovery, close to 7.15 mg/g d (~93%) was reached in the second extraction cycle at 65 • C and 550 bar, before decreasing to~0.4 mg/g d (~6%) at 80 • C and 100 bar. Moreover, it is worth highlighting that, at the best extraction conditions (65 • C and 550 bar), starting from 40 min, the effect of extraction time was negligible ( Figure 3b); therefore, a 40 min extraction was sufficient to achieve the maximum lutein extraction yield.
For each operative condition, the lutein yield at the end of a complete cycle of extraction is shown in Figure 4. This figure clearly shows that, at the pressure of 100 bar, almost the same recovery value was obtained at 50 • C and 65 • C, while, at higher extraction pressures, temperature played a crucial role, as lutein recovery increased with temperature, reaching a maximum at 65 • C before decreasing, probably due to the thermal instability of carotenoids, as reported by several authors [63,65]. At the best extraction conditions, it is possible to assume that about 93% of lutein was extracted. At 50 °C (Figure 3a) with an extraction time of 20 min (first extraction cycle), increasing pressure from 100 to 550 bar resulted in lutein recovery growing from ~3.3 mg/gd (~43%) to ~3.7 mg/gd (~49%). With an extraction time of 80 min (four extraction cycles), lutein extraction yield increased from ~3.9 mg/gd (~50%) to ~5.0 mg/gd (~65%). Increasing extraction time from 20 min to 80 min, at 100 bar, lutein recovery slightly increased from ~3.3 mg/gd (~43%) to ~3.9 mg/gd (~50%); at 400 bar, lutein recovery slightly increased from ~3.7 mg/gd (~48%) to ~4.1 mg/gd (~54%); at 550 bar, lutein recovery increased from ~3.7 mg/gd (~48%) to ~5.0 mg/gd (~65%). At 65 °C (Figure 3b) and at 80 °C (Figure 3c), the influence of extraction time on lutein recovery was quite similar to that observed at 50 °C ( Figure 3a); however, the maximum lutein recovery, close to 7.15 mg/gd (~93%) was reached in the second extraction cycle at 65 °C and 550 bar, before decreasing to ~0.4 mg/gd (~6%) at 80 °C and 100 bar. Moreover, it is worth highlighting that, at the best extraction conditions (65 °C and 550 bar), starting from 40 min, the effect of extraction time was negligible ( Figure 3b); therefore, a 40 min extraction was sufficient to achieve the maximum lutein extraction yield.
For each operative condition, the lutein yield at the end of a complete cycle of extraction is shown in Figure 4. This figure clearly shows that, at the pressure of 100 bar, almost the same recovery value was obtained at 50 °C and 65 °C, while, at higher extraction pressures, temperature played a crucial role, as lutein recovery increased with temperature, reaching a maximum at 65 °C before decreasing, probably due to the thermal instability of carotenoids, as reported by several authors [63,65]. At the best extraction conditions, it is possible to assume that about 93% of lutein was extracted. To the best of the authors' knowledge, few papers focusing on lutein extraction from H. pluvialis via supercritical fluid extraction were published. Nobre et al. [52] reported lutein recovery from H. pluvialis close to 92% at a pressure of 300 bar and a temperature of 60 °C, using CO2-SFE with ethanol as a co-solvent (ethanol 10%/CO2 90%, (v/v)). As shown, our experimental findings are relatively in line with literature data.
The comparison between the cumulative recovery of lutein with and without co-solvent [57] is reported in Table 4. As shown, the presence of ethanol strongly improved the extraction of lutein, as the highest recovery increased from about 51% without co-solvent (P = 550 bar, T = 50 °C) to about 93% with co-solvent (P = 550 bar, T = 65 °C).  To the best of the authors' knowledge, few papers focusing on lutein extraction from H. pluvialis via supercritical fluid extraction were published. Nobre et al. [52] reported lutein recovery from H. pluvialis close to 92% at a pressure of 300 bar and a temperature of 60 • C, using CO 2 -SFE with ethanol as a co-solvent (ethanol 10%/CO 2 90%, (v/v)). As shown, our experimental findings are relatively in line with literature data.
The comparison between the cumulative recovery of lutein with and without co-solvent [57] is reported in Table 4. As shown, the presence of ethanol strongly improved the extraction of lutein, as the highest recovery increased from about 51% without co-solvent (P = 550 bar, T = 50 • C) to about 93% with co-solvent (P = 550 bar, T = 65 • C). The purity of lutein for each stage of extraction, under different operative conditions, with and without co-solvent is reported in Table 5. With co-solvent, the highest purity (~16%) was achieved with an extraction time of 40 min at 65 • C and 550 bar, which are the same conditions that resulted in the highest lutein recovery, as shown in Figure 3. Without co-solvent, the highest purity (~7.5%) was found with an extraction time of 80 min at 400 bar and 50 • C. As shown, ethanol was effective for the enhancement of lutein purity, as the maximum purity increased from about 7.5% to about 16%.

Comparison with Literature for Recovery of Astaxanthin and Lutein Using CO 2 -SFE with Co-Solvent
The comparison between the operative conditions allowing the maximum recovery of astaxanthin and lutein defined in the present work and data available in literature is reported in Table 6. As shown, the supercritical extraction process is an effective technology for the extraction of astaxanthin and lutein, with recoveries higher than 90% for both compounds. In particular, the operative conditions identified in our study, including the type of pre-treatment, produced the highest recovery of astaxanthin and lutein. However, as shown in Figures 1 and 3, in terms of the effect of extraction time on recovery, it is worth highlighting that, at the operative conditions with which the maximum recoveries were achieved (65 • C; 550 bar), the recoveries were relatively unaffected by extraction time. Therefore, the maximum recoveries of astaxanthin and lutein were found during the first extraction cycle (20 min) and the second extraction cycle (40 min), respectively, and they did not vary along with the increase in extraction time (Figures 1 and 3). This study a Ethanol/vegetable oils mentioned in the column served as a co-solvent in the extraction; b operating pressure; c operating temperature; d total extraction time; e recovery at optimum conditions; § n.a. = not available; # at the operative conditions with which the maximum recoveries were achieved (65 • C; 550 bar), the recoveries were not affected by extraction time.

Samples and Chemicals
Experimental activity was carried out using lyophilized H. pluvialis microalgae in the red phase (HPR) provided by Micoperi Blue Growth (Ravenna, Italy). HPR has a mesh particle sieve size lower than 50 µm, with a total content of astaxanthin of 20.0 mg/g dry weight, corresponding to 2% (w/w) dry weight, which was measured using the method proposed by Li et al. [67]. Since no standard method to measure lutein content in HPR is reported in the literature, lutein content was measured using the same method proposed by Li et al. [67], except that lutein measurement was carried out using ultra (u)HPLC analysis. A value of about 7.7 mg/g dry weight, equal to 38.5% (w/w) astaxanthin content was found, in agreement with Vidhyavathi et al. [68], who found that, for the HPR cystic phase, lutein was about 30-40% (w/w) astaxanthin content. HPR was stored at −20 • C in a vacuum-sealed plastic bag to avoid degradation, and was brought to atmospheric conditions before extraction. All chemicals and standards (astaxanthin and lutein) were of analytical grade and purchased from Sigma Aldrich (St. Louis, MO, USA). All other reagents were of uHPLC grade unless otherwise stated. Carbon dioxide (industrial grade) with a purity of 99.999% was purchased from Rivoira (Bari, Italy).

Experimental Set-Up
The experimental set-up, shown in Figure 5, was a laboratory-scale equipment with a reactor volume of 30 mL [64]. The bench-scale experimental apparatus for CO 2 -SFE was equipped with a heater in order to achieve temperatures up to 250 • C, and a pumping system for the compression of CO 2 up to 680 bar. Two vessels were located inside the module: the first was used as a CO 2 pre-heater and the second was used for extraction. In the extraction vessel, two pressure control systems-inlet and outlet valves (Wika Transmitter, Klingenberg, Germany), with precision of 0.6 mbar-were installed, whereas the CO 2 flow rate was controlled using a flow meter LPN/S80 AL G 2.5 (SACOFGAS, Milano, Italy). The inlet flow rate was adjustable up to 25 mL/min and the flow control was carried out on the expanded gas. Temperature was monitored using thermocouples, while the inlet and outlet flow streams were controlled using micrometric valves. The experimental apparatus was also equipped with a specific line for supplying co-solvent, using a syringe pump (Speed SFE Modifier Pump Module-PN 7170, Allentown, PA, USA) to compress the co-solvent up to 680 bar and to regulate the flow rate up to 10 mL/min. The experimental apparatus was also equipped with acoustic and visual high-pressure alerts and, as the primary security system, a rupture disc was installed. All parameters of the process were controlled using a distributed control system (DCS). Before each extraction stage, HPR was mechanically pre-treated using the Mixer Mill (Retsch MM400, Haan, Germany) with three steel spheres of 1 cm for 5 min at 400 rpm, in which 1.4 g of HPR was mixed with 0.8 g of diatom land [69].
For each experimental test, four extraction cycles of 20 min each (extraction time = 20-80 min) The experimental apparatus was also equipped with acoustic and visual high-pressure alerts and, as the primary security system, a rupture disc was installed. All parameters of the process were controlled using a distributed control system (DCS). Before each extraction stage, HPR was mechanically pre-treated using the Mixer Mill (Retsch MM400, Haan, Germany) with three steel spheres of 1 cm for 5 min at 400 rpm, in which 1.4 g of HPR was mixed with 0.8 g of diatom land [69].
For each experimental test, four extraction cycles of 20 min each (extraction time = 20-80 min) were carried out. Carbon dioxide and ethanol flow rates were kept constant at 3.62 g/min and 1 mL/min, respectively. Temperature (T) and pressure (P) were varied in the ranges of 50-80 • C and 100-550 bar, respectively. The experimental plan, in which HPR biomass loading was included, is reported in the Table 7. The effects of pressure and temperature on astaxanthin and lutein extraction were reported in terms of cumulative recovery and purity [57]. Cumulative recovery was expressed both as mg of the compound extracted for g of dry HRP biomass loading (Equation (1)) and as percentage of the total content (Equation (2)). Purity was calculated as the ratio between the recovery (mg/g) and the total extraction yield (Equation (3)).

Cumulative recovery
where W C,i (mg) is the weight of the compound extracted for each extraction cycle, W B (g) is the weight of HPR biomass loading (Table 7), W T (mg/g) is the total content of the compound (Section 3.1; 20.0 mg/g dry weight for astaxanthin; 7.7 mg/g dry weight for lutein), and W E (mg/g) is the total extraction yield (Table 1). Each experimental condition was investigated in triplicate.

Analytical Methods
After each extraction cycle, the sample was collected in an amber vial and subjected to basic hydrolysis in the presence of NaOH (saponification), in order to remove lipids and chlorophylls from the sample, avoiding the overlap of the spectra with the species present in the carotenoid family [56]. Specifically, saponification was carried out by adding 1 mL of NaOH solution in methanol (0.05 M) to 5 mL of extract. This solution was left in the dark in an inert atmosphere for 7 h. Once this step was completed, the sample was neutralized with 3 mL of an NH 4 Cl solution in methanol (0.05 M). After saponification, astaxanthin and lutein were measured using a uHPLC Agilent 1290 Infinity II with a Zorbax reverse-phase C18 column with methanol/water (95:5, v/v) as a mobile phase solvent, while the sample was dissolved in a mixture of methanol/chloroform (90:10 containing 0.1% butylated hydroxytoluene (BHT) as an antioxidant). The flow rate and column temperature were kept constant at 0.4 mL/min and 28 • C, respectively [69]. The extraction procedure was detailed previously in Di Sanzo et al. [57].

Conclusions
In the present work, the effects of time (20-80 min), temperature (50-80 • C), and pressure (100-550 bar) on astaxanthin and lutein extraction from H. pluvialis in the red phase via CO 2 -SFE with ethanol (GRAS solvent) as a co-solvent were investigated. Moreover, a comparison of CO 2 -SFE with without ethanol was also carried out.
The results highlight that, for both astaxanthin and lutein, temperature played a crucial role in both extraction yield and carotenoid degradation. At 65 • C and 550 bar, astaxanthin and lutein extraction yields were maximized, avoiding carotenoid thermal degradation. Moreover, at the best extraction conditions (65 • C and 550 bar), a negligible effect of extraction time was observed; therefore, to basically achieve the maximum recovery, an extraction time of 20 min was sufficient for astaxanthin, while an extraction time of 40 min was required for lutein. These conditions led to an astaxanthin extraction yield of about 92%, and a lutein extraction yield of about 93%. From the comparison between experimental findings and literature data, it is possible to see that the operative conditions identified in our study, including the type of pre-treatment, produced the highest recovery of astaxanthin and lutein. In terms of purity of astaxanthin, similar results were achieved.
By comparing the performance of CO 2 -SFE with and without ethanol, it is worth highlighting that a real effectiveness of ethanol as a co-solvent for astaxanthin extraction was not observed, with the drawback of a reduction in astaxanthin purity. On the other hand, the presence of ethanol was found to be effective for the extraction of lutein, as an enhancement was observed both in terms of recovery and purity.