- Email: [email protected]
Supercritical fluid extraction of lutein from Scenedesmus cultured in an autotrophical photobioreactor
Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 53–57
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Supercritical fluid extraction of lutein from Scenedesmus cultured in an autotrophical photobioreactor Hong-Wei Yen *, Wei-Cheng Chiang, Cheng-Hsiung Sun Department of Chemical Engineering, Tunghai University, Taichung, Taiwan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 May 2011 Received in revised form 14 July 2011 Accepted 31 July 2011 Available online 19 October 2011
Lutein is a known health food supplement which is mainly extracted from marigold through the conventional solvent extraction route. The results of lutein recovery by the supercritical fluid extraction (SFE) of CO2 from Scenedesmus powder are reported here. The collected Scenedesmus biomass is cultured in an inner-illuminated photobioreactor at a light intensity of 350–400 mmol/m2 s. The increase of pressure and temperature in the SFE operation enhances the lutein recovery yield. However, the enhancement resulting from the increase of temperature and pressure is not significant as compared to the yield from the conventional methanol extraction method. In addition, the increase of temperature leads to the increased impurity observed in the HPLC profile. To further enhance the lutein recovery yield, the addition of a co-solvent in SFE is performed. Of the five solvent powders investigated, ethanol is regarded as the optimum co-solvent for use in lutein extraction. The optimum amount of ethanol to be added in the SFE operation is determined. The best lutein recovery yield obtained in this study of SFE operation was 76.7% (compared to the conventional methanol extraction method) under the conditions of 400 bar, 70 8C and ethanol as the co-solvent at a ratio of 30 mol%. ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Co-solvent Lutein Ethanol SFE Scenedesmus
1. Introduction Lutein is a xanthophyll compound which is now recommended as a health supplement for the prevention of cancer and diseases related to retinal degeneration or to ameliorate the effects of degenerative diseases such as age-related macular degeneration (AMD) [1]. The total commercial market value of lutein in the US of 2010 has been estimated at about $150 million per year [2], with the lutein being produced from marigold flowers by the solvent extraction method. However, the productivity of lutein is restricted due to the limitations of land usage and the slow growth of plants [2,3]. Therefore, a new process involving the production of lutein using microalgae instead of marigold has been seen as an attractive alternative. Several microalgae have been proposed as potentially adequate lutein sources, such as Muriellopsis sp. [4], Chlorella zofingensis [5], or Chlorella protothecoides [6]. In general, the lutein content in microalgal biomass is in the range of 3–5 mg per g of dry biomass, which is comparable to that in marigold. Of all microalgae tested, Muriellopsis sp., which is a chlorophycean microalgae, achieved a high lutein production concentration (up to 35 mg/L) under specific culture conditions, as well as a high growth rate and high cell density.
* Corresponding author at: 181, 3rd Section, Taichung Harbor Rd, Taichung, Taiwan. Fax: +886 4 23590009. E-mail address: [email protected] (H.-W. Yen).
Lutein from the microalgae Cholerella vulgaris was successfully extracted and purified using a conventional extraction method by Li et al. [7]. Crude lutein was obtained by extraction with dichloromethane from the microalgae after saponification. The reported final purity of the lutein thus obtained was 90–98%, and the yield was 85–91%. Nevertheless, using organic solvents for the extraction is not the preferred route in health food production due to the inherent toxicity of residual solvents contained in the foods, and also the issue of environmental pollution. Development of an efficient extraction technique is required for the separation and isolation of pharmaceutical compounds from natural resources. Supercritical fluid extraction, a ‘natural and green’ method of product extraction, is currently regarded as an important alternative to conventional separation methods, not only because it is simpler, faster, and more efficient, but also because it does not require the consumption of large amounts of organic solvents which are both expensive and potentially harmful. The extraction of compounds from natural sources is the most widely studied application of supercritical fluids (SCFs), with several hundreds of published scientific papers. Indeed, supercritical fluid extraction (SFE) has immediate advantages over traditional extraction techniques: it is a flexible process due to the possibility of continuous modulation of the solvent power/ selectivity of the SCF, and it allows the elimination of polluting organic solvents and of the expensive post-processing of the extracts for solvent elimination. Supercritical CO2 is the most commonly adopted extraction agent due to its non-toxicity,
1876-1070/$ – see front matter ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jtice.2011.07.010
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H.-W. Yen et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 53–57
method. As well as the pressure and temperature effects, the effect of each of the 5 different co-solvents added in the SFE process – methanol, ethanol, propanol, butanol and acetone – on lutein extraction has been discussed. The optimum amount of co-solvent to be added (as compared to the flow rate of CO2) for the maximum lutein recovery yield was then determined. The goals of this study were to discover a SFE method to replace the conventional extraction method for the recovery process of lutein from Scenedesmus powders. 2. Materials and methods 2.1. Microorganism and cultivation
Fig. 1. Scheme of 20 l photobioreactor.
chemical inertia, low cost and the most important thing, carbon dioxide owned a low critical temperature value (Tc = 31.8 8C). The characteristic of low critical temperature makes CO2 ideal for the extraction of thermally labile compounds. For these reasons, using CO2 as the extraction fluid has been successfully reported in literatures for isolation of many compounds from various resources [8,9]. This study investigated the application of supercritical CO2 to extract lutein from Scenedesmus powders cultured in an autotrophically inner-illuminated photobioreactor. The effects of temperature and pressure on lutein recovery were examined as compared to those obtained by the conventional organic solvent extraction
The microalgae Scenedesmus sp. used in this study was generously provided by Prof. Jo-Shu Chang (National Cheng Kung University, Taiwan). S. sp was autotrophically cultured in a 20 l photobioreactor (the dimensions of the photobioreactor are shown in Fig. 1) with 0.5 vvm of mixing gas (98% of air and balanced with CO2) and inner-illuminated with T8 LED lamps at 350–400 mmol/m2 s. The algal biomass was collected after 7–10 days of cultivation. The maximum biomass was 1.1 g/l after 8 days of cultivation. The freeze-dried algal biomass was milled to obtain homogenized powders that were then stored in the dark at 4 8C for the following experiments. The average lutein content of the collected freeze-dried S. sp powders was about 0.3% (g/g), which was analyzed by the conventional solvent (methanol) extraction method. 2.2. Supercritical CO2 extraction The experiments were carried out in a Speed SFE model 7070 (Applied Separation Co. Ltd., USA) equipped with a CO2 pump, an oven extractor, a 32 ml extractor cell and a wet gas meter. The scheme structure of SFE was shown in Fig. 2. The operating methodology involved loading approximately 1.0 g of the freeze-dried biomass, which had previously been homogenized to maintain a constant apparent density in all experiments. Then, the extractor cell was installed in the oven
Fig. 2. Scheme of SFE structure.
H.-W. Yen et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 53–57
55
extractor and left for 15 min to reach the operating pressure and temperature. When a balanced state had been attained, the thermostatically controlled micrometric valve was opened up (at 170 8C) until a constant flow of 750–800 ml/min was achieved. The operation of supercritical extraction was then carried out for 1 h. The extracts were collected in glass tubes containing methanol to prevent the extract being lost in the gas flow of CO2. After the extraction process was complete, the solvent was removed with a rotary evaporator at a temperature of 50 8C. The residual extracted powders were re-dissolved in a defined amount of methanol (30 ml) for the subsequent HPLC analysis. 2.3. Analytical methods For the conventional organic extraction method, methanol was adopted as the extraction solvent for the comparison to the lutein recovery yield of the SFE operation. Firstly, five milliliters of thoroughly mixed culture fluid from the fermentor was transferred to a 15-ml conical tube. The culture was then centrifuged at 8000 rpm for 15 min, and the supernatant was discarded. Five milliliters of methanol were subsequently added to the mixture for the extraction of lutein ultrasonically. The mixture was then centrifuged at 8000 rpm for 15 min at 4 8C. The lutein was analyzed using an HPLC system under the following conditions: column, N 5ODS (250 4.68 mm); temperature, ambient (27 1 8C); and a mobile phase, initially consisting of 60% solvent A (acetonitrile/ methanol, 20/80, v/v) and 40% solvent B (methanol/acetone, 80/20, v/v), which was finally brought to 30% solvent A and 70% solvent B over a period of 15 min. The column was subsequently returned to its original solvent composition of 60% solvent A and 40% solvent B over the next 6 min prior to the injection of a new sample; flow rate, 1.0 ml/min; and UV detector (Hitachi) at 450 nm. The biomass concentration was estimated by absorbance at 560 nm, with the relationship between optical density (OD560) and dry cell weight (DCW) given as DCW (g/l) = 0.8939 OD560 0.0351. 3. Results and discussion 3.1. Effects of temperature on lutein extraction The results of temperatures in the range of 35–80 8C at a pressure of 400 bar on lutein recovery in the SFE operation, as shown in Table 1, revealed that the increase of temperature led to an increase in lutein recovery. Macı´as-Sa´nchez et al. reported that an SFE temperature of 60 8C resulted in a higher lutein recovery than for 32 8C and 46 8C at a pressure of 400 bar [10]. The best lutein recovered ratio (lutein content by SFE/reference data) was 2% at 400 bar and 60 8C in the study by Macı´as-Sa´nchez and his colleagues, which was lower than the value of 5.27% obtained in this study at the same conditions [10]. A longer SFE operation of 1 h used in this study than that of 30 min used in the study by Macı´asSa´nchez and his colleagues probably led to a higher lutein recovery ratio observed. However, the increase in the operational temperature raised another problem, which was the increase of impurity
Table 1 Effects of temperature on lutein recovery by SFE at the pressure of 400 bar. Pressure (bar)
Temperature (8C)
Lutein (mg/g)
Recovery yielda (%)
400
35.0 47.5 60.0 70.0 80.0
0.039 0.085 0.152 0.206 0.254
1.34 2.84 5.27 7.15 8.82
a
As compared to the solvents extraction method.
Fig. 3. Effects of different temperature on HPLC profiles (the first peak is lutein and the second one is the impurity).
observed in the analysis profile of HPLC (Fig. 3). As shown in Fig. 3, the peak of lutein standard was presented at a retention time of around 6 min. After the peak of lutein standard, there was a small peak shown at around 8.5 min. The peak area increased with the increase in the SFE temperature, which suggested that the impurity was probably separated from the lutein compound under high temperature of SFE operation. Besides the impurity problem, while the SFE temperature was higher than 70 8C, the collected lutein samples presented slightly black in color. To avoid potential purification problems, a temperature of less than 70 8C was determined for the SFE extraction of lutein from the Scenedesmus biomass.
Table 2 Effects of pressure on lutein recovery by SFE at 47.5 8C. Pressure (bar)
Temperature (8C)
Lutein (mg/g)
Recovery yield (%)
200 300 400
47.5
0.011 0.031 0.089
0.39 1.07 3.07
Table 3 Effects of different solvents on lutein recovery by the conventional organic solvent extraction method. Solvent
Sample weight (g)
Lutein (mg/g)
Methanol Ethanol Propanol Butanol Acetone
0.1000 0.1001 0.1001 0.1002 0.0999
0.388 0.345 0.291 0.269 0.357
H.-W. Yen et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 53–57
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Table 4 Effects of co-solvent addition on lutein recovery in SFE operation at 400 bar and 70 8C. Co-solvent
Co-solvent/CO2 (mol%)
Pressure (bar)
Temperature (8C)
Lutein (mg/g)
Recovery yield (%)
Methanol Ethanol Propanol Butanol Acetone
40
400
70
1.464 1.794 1.487 1.116 0.488
50.75 62.20 51.55 38.68 16.91
Table 5 Effects of ethanol addition rate on lutein recovery by SFE.
Ethanol
Ethanol/CO2 (mol%)
Pressure (bar)
Temperature (8C)
Lutein (mg/g)
Recovery yield (%)
10 20 30 40
400
70
0.037 0.174 2.210 1.783
1.28 6.04 76.65 62.20
3.2. Effects of pressure on lutein extraction The results of the tests for the effect of pressure on lutein recovery are shown in Table 2. It was clear that an increase in pressure in the range of 200–400 bar at 47.5 8C led to an increase in lutein recovery. However, due to safety considerations for the SFE equipment used in this study, we did not perform tests at pressures higher than 400 bar. The increase in lutein recovery with an increase in pressure from 200 to 600 bar was as noted in the literature [10]. Even at the highest pressure of 400 bar, the lutein recovery yield was still far lower than with the conventional solvent extraction method, with only 3.07% obtained with the SFE operation. Obviously, it was unrealistic to use SFE for lutein extraction with such a low recovery yield obtained thus far by adjusting the temperature and the pressure. To further enhance the recovery yield, the addition of different co-solvents in the SFE operation was performed. 3.3. Effects of co-solvents on lutein extraction It was thought that the addition of a co-solvent would modify the interaction between the supercritical fluid and the compounds and serve to enhance the extraction efficiency of SFE. To screen the potential co-solvents used in the SFE operation, 5 different common extraction solvents: methanol, ethanol, propanol, butanol and acetone were investigated regarding their performance in lutein recovery by the simple conventional extraction method (Table 3). As shown in the table, methanol had the maximum lutein content obtained of all solvents investigated. With the increase of the carbon number from one to four carbons of the alcohol compounds (methanol, ethanol, propanol and butanol), a decrease in lutein extraction efficiency was observed. As well as methanol, acetone was regarded as a suitable solvent for lutein recovery by the conventional solvent extraction method. For comparison with the conventional solvent extraction method, the same five solvents were used as the co-solvent added in the SFE operation for the investigation of lutein recovery. The results of using the different solvents as the co-solvent added in the SFE operation are shown in Table 4. As seen in the table, with ethanol as the co-solvent the lutein recovery performance was superior to that using the other solvents. At the conditions of 400 bar and 70 8C, ethanol as the co-solvent at a ratio of 40 mol% (the ratio of co-solvent flow vs. CO2 flow rate, which was equal to 0.786 ml ethanol/min) had a lutein recovery yield of 62.2% as compared to that of the conventional methanol extraction
method. Generally speaking, adding a co-solvent in the SFE operation enhanced the performance of lutein recovery as compared to the values obtained when a co-solvent was not added. The reasons for ethanol being the co-solvent having the maximum lutein recovery were not clear. However, it was believed that ethanol played a role as an ‘‘entrainer’’ in the phase diagram compositing of CO2, lutein and ethanol, which led to the increase of solubility of lutein in CO2 and resulted in the increase in the lutein recovery yield. Many research reports have suggested that ethanol would be a good co-solvent to enhance the extraction efficiency in the SFE operation [10,11]. Tzeng and his colleagues have also suggested that ethanol could modify SC-CO2 extraction investigated at the pressures and temperatures produced more pure artemisinin and copoletin than Soxhlet solvent extraction did [12]. However, while ethanol was the best co-solvent in the SFE operation, methanol was the best solvent used in the conventional extraction method. To further determine the optimum amount of ethanol to be added in the SFE operation, ratios in the range of 10–40 mol% (as compared to the flow rate of CO2 at 750 ml/min) were investigated. The results, as shown in Table 5, indicated that a ratio of 30 mol% obtained the maximum lutein recovery yield. The best lutein recovery yield obtained in this study of SFE operation was 76.7% under the conditions of 400 bar, 70 8C and ethanol as the co-solvent at a ratio of 30 mol%. 4. Conclusions The application of SFE in lutein extraction is regarded as a green process as lower amounts of solvents are used. Increases in temperature and pressure would be beneficial to the increase of lutein recovery. However, a higher temperature can lead to increased impurity as observed in the HPLC analysis profile. To further improve the recovery yield, the addition of a co-solvent in the SFE operation was investigated. Ethanol was regarded as the optimum co-solvent for lutein extraction from Scenedesmus powder. The best lutein recovery yield obtained in this study of the SFE operation was 76.7% (400 bar, 70 8C and ethanol as the cosolvent at a ratio of 30 mol%) as compared to the conventional methanol extraction method. Acknowledgements The authors wish to thank the National Science Council of the R.O.C. for financial supports.
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References [1] Ziegler R, Colavito E, Hartge P, McAdams M, Schoenberg J, Mason T, et al. Importance of alpha-carotene, beta-carotene and other phytochemicals in the etiology of lung cancer. J Natl Cancer Inst 1996;88:612. [2] Ferna´ndez-Sevilla J, Acie´n FF, Molina GE. Biotechnological production of lutein and its applications. Appl Microbiol Biotechnol 2010;86:27. [3] Piccaglia R, Marotti M, Grandi S. Lutein and lutein ester content in different types of Tagetes patula and T. erecta. Ind Crop Prod 1998;8:45. [4] Del Campo JA, Rodrı´guez H, Moreno J, Vargas MA, Rivas J, Guerrero MG. Lutein production by Muriellopsis sp. in an outdoor tubular photobioreactor. J Biotechnol 2001;85:289. [5] Del Campo JA, Rodrı´guez H, Moreno J, Vargas MA, Rivas J, Guerrero MG. Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta). J Biotechnol 2000; 76:51. [6] Shi XM, Zhang XW, Chen F. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme Microb Technol 2000;27:312.
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[7] Li HB, Jiang Y, Chen F. Isolation and purification of lutein from the microalga Chlorella vulgaris by extraction after saponification. J Agric Food Chem 2002;50:1070. [8] Kitada K, Machmudah S, Sasaki M, Goto M, Nakashima Y, Kumamotod S, et al. Supercritical CO2 extraction of pigment components with pharmaceutical importance from Chlorella vulgaris. J Chem Technol Biotechnol 2009;84:657. [9] Careri M, Furlattini L, Mangia A, Musci M, Anklam E, Theobald A, et al. Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina Pacifica algae: a chemometric approach. J Chromatogr A 2001;912:61. [10] Macı´as-Sa´nchez MD, Fernandez-Sevilla JM, Acie´n Ferna´ndez FG, Cero´n Garcı´a MC, Molina Grima E. Supercritical fluid extraction of carotenoids from Scenedesmus almeriensis. Food Chem 2010;123:928. [11] Wu Z, Wu S, Shi X. Supercritical fluid extraction and dterminationof lutein in heterotrophically cultivated Chlorella pyrenoidosa. J Food Process Eng 2007; 30:174. [12] Tzeng TC, Lin YL, Jong TT, Chang CJ. Ethanol modified supercritical fluids extraction of scopoletin and artemisinin from Artemisia annua L. Sep Purif Technol 2007;56:18.
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Supercritical fluid extraction of lutein from Scenedesmus cultured in an autotrophical photobioreactor Hong-Wei Yen *, Wei-Cheng Chiang, Cheng-Hsiung Sun Department of Chemical Engineering, Tunghai University, Taichung, Taiwan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 May 2011 Received in revised form 14 July 2011 Accepted 31 July 2011 Available online 19 October 2011
Lutein is a known health food supplement which is mainly extracted from marigold through the conventional solvent extraction route. The results of lutein recovery by the supercritical fluid extraction (SFE) of CO2 from Scenedesmus powder are reported here. The collected Scenedesmus biomass is cultured in an inner-illuminated photobioreactor at a light intensity of 350–400 mmol/m2 s. The increase of pressure and temperature in the SFE operation enhances the lutein recovery yield. However, the enhancement resulting from the increase of temperature and pressure is not significant as compared to the yield from the conventional methanol extraction method. In addition, the increase of temperature leads to the increased impurity observed in the HPLC profile. To further enhance the lutein recovery yield, the addition of a co-solvent in SFE is performed. Of the five solvent powders investigated, ethanol is regarded as the optimum co-solvent for use in lutein extraction. The optimum amount of ethanol to be added in the SFE operation is determined. The best lutein recovery yield obtained in this study of SFE operation was 76.7% (compared to the conventional methanol extraction method) under the conditions of 400 bar, 70 8C and ethanol as the co-solvent at a ratio of 30 mol%. ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Co-solvent Lutein Ethanol SFE Scenedesmus
1. Introduction Lutein is a xanthophyll compound which is now recommended as a health supplement for the prevention of cancer and diseases related to retinal degeneration or to ameliorate the effects of degenerative diseases such as age-related macular degeneration (AMD) [1]. The total commercial market value of lutein in the US of 2010 has been estimated at about $150 million per year [2], with the lutein being produced from marigold flowers by the solvent extraction method. However, the productivity of lutein is restricted due to the limitations of land usage and the slow growth of plants [2,3]. Therefore, a new process involving the production of lutein using microalgae instead of marigold has been seen as an attractive alternative. Several microalgae have been proposed as potentially adequate lutein sources, such as Muriellopsis sp. [4], Chlorella zofingensis [5], or Chlorella protothecoides [6]. In general, the lutein content in microalgal biomass is in the range of 3–5 mg per g of dry biomass, which is comparable to that in marigold. Of all microalgae tested, Muriellopsis sp., which is a chlorophycean microalgae, achieved a high lutein production concentration (up to 35 mg/L) under specific culture conditions, as well as a high growth rate and high cell density.
* Corresponding author at: 181, 3rd Section, Taichung Harbor Rd, Taichung, Taiwan. Fax: +886 4 23590009. E-mail address: [email protected] (H.-W. Yen).
Lutein from the microalgae Cholerella vulgaris was successfully extracted and purified using a conventional extraction method by Li et al. [7]. Crude lutein was obtained by extraction with dichloromethane from the microalgae after saponification. The reported final purity of the lutein thus obtained was 90–98%, and the yield was 85–91%. Nevertheless, using organic solvents for the extraction is not the preferred route in health food production due to the inherent toxicity of residual solvents contained in the foods, and also the issue of environmental pollution. Development of an efficient extraction technique is required for the separation and isolation of pharmaceutical compounds from natural resources. Supercritical fluid extraction, a ‘natural and green’ method of product extraction, is currently regarded as an important alternative to conventional separation methods, not only because it is simpler, faster, and more efficient, but also because it does not require the consumption of large amounts of organic solvents which are both expensive and potentially harmful. The extraction of compounds from natural sources is the most widely studied application of supercritical fluids (SCFs), with several hundreds of published scientific papers. Indeed, supercritical fluid extraction (SFE) has immediate advantages over traditional extraction techniques: it is a flexible process due to the possibility of continuous modulation of the solvent power/ selectivity of the SCF, and it allows the elimination of polluting organic solvents and of the expensive post-processing of the extracts for solvent elimination. Supercritical CO2 is the most commonly adopted extraction agent due to its non-toxicity,
1876-1070/$ – see front matter ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jtice.2011.07.010
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H.-W. Yen et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 53–57
method. As well as the pressure and temperature effects, the effect of each of the 5 different co-solvents added in the SFE process – methanol, ethanol, propanol, butanol and acetone – on lutein extraction has been discussed. The optimum amount of co-solvent to be added (as compared to the flow rate of CO2) for the maximum lutein recovery yield was then determined. The goals of this study were to discover a SFE method to replace the conventional extraction method for the recovery process of lutein from Scenedesmus powders. 2. Materials and methods 2.1. Microorganism and cultivation
Fig. 1. Scheme of 20 l photobioreactor.
chemical inertia, low cost and the most important thing, carbon dioxide owned a low critical temperature value (Tc = 31.8 8C). The characteristic of low critical temperature makes CO2 ideal for the extraction of thermally labile compounds. For these reasons, using CO2 as the extraction fluid has been successfully reported in literatures for isolation of many compounds from various resources [8,9]. This study investigated the application of supercritical CO2 to extract lutein from Scenedesmus powders cultured in an autotrophically inner-illuminated photobioreactor. The effects of temperature and pressure on lutein recovery were examined as compared to those obtained by the conventional organic solvent extraction
The microalgae Scenedesmus sp. used in this study was generously provided by Prof. Jo-Shu Chang (National Cheng Kung University, Taiwan). S. sp was autotrophically cultured in a 20 l photobioreactor (the dimensions of the photobioreactor are shown in Fig. 1) with 0.5 vvm of mixing gas (98% of air and balanced with CO2) and inner-illuminated with T8 LED lamps at 350–400 mmol/m2 s. The algal biomass was collected after 7–10 days of cultivation. The maximum biomass was 1.1 g/l after 8 days of cultivation. The freeze-dried algal biomass was milled to obtain homogenized powders that were then stored in the dark at 4 8C for the following experiments. The average lutein content of the collected freeze-dried S. sp powders was about 0.3% (g/g), which was analyzed by the conventional solvent (methanol) extraction method. 2.2. Supercritical CO2 extraction The experiments were carried out in a Speed SFE model 7070 (Applied Separation Co. Ltd., USA) equipped with a CO2 pump, an oven extractor, a 32 ml extractor cell and a wet gas meter. The scheme structure of SFE was shown in Fig. 2. The operating methodology involved loading approximately 1.0 g of the freeze-dried biomass, which had previously been homogenized to maintain a constant apparent density in all experiments. Then, the extractor cell was installed in the oven
Fig. 2. Scheme of SFE structure.
H.-W. Yen et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 53–57
55
extractor and left for 15 min to reach the operating pressure and temperature. When a balanced state had been attained, the thermostatically controlled micrometric valve was opened up (at 170 8C) until a constant flow of 750–800 ml/min was achieved. The operation of supercritical extraction was then carried out for 1 h. The extracts were collected in glass tubes containing methanol to prevent the extract being lost in the gas flow of CO2. After the extraction process was complete, the solvent was removed with a rotary evaporator at a temperature of 50 8C. The residual extracted powders were re-dissolved in a defined amount of methanol (30 ml) for the subsequent HPLC analysis. 2.3. Analytical methods For the conventional organic extraction method, methanol was adopted as the extraction solvent for the comparison to the lutein recovery yield of the SFE operation. Firstly, five milliliters of thoroughly mixed culture fluid from the fermentor was transferred to a 15-ml conical tube. The culture was then centrifuged at 8000 rpm for 15 min, and the supernatant was discarded. Five milliliters of methanol were subsequently added to the mixture for the extraction of lutein ultrasonically. The mixture was then centrifuged at 8000 rpm for 15 min at 4 8C. The lutein was analyzed using an HPLC system under the following conditions: column, N 5ODS (250 4.68 mm); temperature, ambient (27 1 8C); and a mobile phase, initially consisting of 60% solvent A (acetonitrile/ methanol, 20/80, v/v) and 40% solvent B (methanol/acetone, 80/20, v/v), which was finally brought to 30% solvent A and 70% solvent B over a period of 15 min. The column was subsequently returned to its original solvent composition of 60% solvent A and 40% solvent B over the next 6 min prior to the injection of a new sample; flow rate, 1.0 ml/min; and UV detector (Hitachi) at 450 nm. The biomass concentration was estimated by absorbance at 560 nm, with the relationship between optical density (OD560) and dry cell weight (DCW) given as DCW (g/l) = 0.8939 OD560 0.0351. 3. Results and discussion 3.1. Effects of temperature on lutein extraction The results of temperatures in the range of 35–80 8C at a pressure of 400 bar on lutein recovery in the SFE operation, as shown in Table 1, revealed that the increase of temperature led to an increase in lutein recovery. Macı´as-Sa´nchez et al. reported that an SFE temperature of 60 8C resulted in a higher lutein recovery than for 32 8C and 46 8C at a pressure of 400 bar [10]. The best lutein recovered ratio (lutein content by SFE/reference data) was 2% at 400 bar and 60 8C in the study by Macı´as-Sa´nchez and his colleagues, which was lower than the value of 5.27% obtained in this study at the same conditions [10]. A longer SFE operation of 1 h used in this study than that of 30 min used in the study by Macı´asSa´nchez and his colleagues probably led to a higher lutein recovery ratio observed. However, the increase in the operational temperature raised another problem, which was the increase of impurity
Table 1 Effects of temperature on lutein recovery by SFE at the pressure of 400 bar. Pressure (bar)
Temperature (8C)
Lutein (mg/g)
Recovery yielda (%)
400
35.0 47.5 60.0 70.0 80.0
0.039 0.085 0.152 0.206 0.254
1.34 2.84 5.27 7.15 8.82
a
As compared to the solvents extraction method.
Fig. 3. Effects of different temperature on HPLC profiles (the first peak is lutein and the second one is the impurity).
observed in the analysis profile of HPLC (Fig. 3). As shown in Fig. 3, the peak of lutein standard was presented at a retention time of around 6 min. After the peak of lutein standard, there was a small peak shown at around 8.5 min. The peak area increased with the increase in the SFE temperature, which suggested that the impurity was probably separated from the lutein compound under high temperature of SFE operation. Besides the impurity problem, while the SFE temperature was higher than 70 8C, the collected lutein samples presented slightly black in color. To avoid potential purification problems, a temperature of less than 70 8C was determined for the SFE extraction of lutein from the Scenedesmus biomass.
Table 2 Effects of pressure on lutein recovery by SFE at 47.5 8C. Pressure (bar)
Temperature (8C)
Lutein (mg/g)
Recovery yield (%)
200 300 400
47.5
0.011 0.031 0.089
0.39 1.07 3.07
Table 3 Effects of different solvents on lutein recovery by the conventional organic solvent extraction method. Solvent
Sample weight (g)
Lutein (mg/g)
Methanol Ethanol Propanol Butanol Acetone
0.1000 0.1001 0.1001 0.1002 0.0999
0.388 0.345 0.291 0.269 0.357
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Table 4 Effects of co-solvent addition on lutein recovery in SFE operation at 400 bar and 70 8C. Co-solvent
Co-solvent/CO2 (mol%)
Pressure (bar)
Temperature (8C)
Lutein (mg/g)
Recovery yield (%)
Methanol Ethanol Propanol Butanol Acetone
40
400
70
1.464 1.794 1.487 1.116 0.488
50.75 62.20 51.55 38.68 16.91
Table 5 Effects of ethanol addition rate on lutein recovery by SFE.
Ethanol
Ethanol/CO2 (mol%)
Pressure (bar)
Temperature (8C)
Lutein (mg/g)
Recovery yield (%)
10 20 30 40
400
70
0.037 0.174 2.210 1.783
1.28 6.04 76.65 62.20
3.2. Effects of pressure on lutein extraction The results of the tests for the effect of pressure on lutein recovery are shown in Table 2. It was clear that an increase in pressure in the range of 200–400 bar at 47.5 8C led to an increase in lutein recovery. However, due to safety considerations for the SFE equipment used in this study, we did not perform tests at pressures higher than 400 bar. The increase in lutein recovery with an increase in pressure from 200 to 600 bar was as noted in the literature [10]. Even at the highest pressure of 400 bar, the lutein recovery yield was still far lower than with the conventional solvent extraction method, with only 3.07% obtained with the SFE operation. Obviously, it was unrealistic to use SFE for lutein extraction with such a low recovery yield obtained thus far by adjusting the temperature and the pressure. To further enhance the recovery yield, the addition of different co-solvents in the SFE operation was performed. 3.3. Effects of co-solvents on lutein extraction It was thought that the addition of a co-solvent would modify the interaction between the supercritical fluid and the compounds and serve to enhance the extraction efficiency of SFE. To screen the potential co-solvents used in the SFE operation, 5 different common extraction solvents: methanol, ethanol, propanol, butanol and acetone were investigated regarding their performance in lutein recovery by the simple conventional extraction method (Table 3). As shown in the table, methanol had the maximum lutein content obtained of all solvents investigated. With the increase of the carbon number from one to four carbons of the alcohol compounds (methanol, ethanol, propanol and butanol), a decrease in lutein extraction efficiency was observed. As well as methanol, acetone was regarded as a suitable solvent for lutein recovery by the conventional solvent extraction method. For comparison with the conventional solvent extraction method, the same five solvents were used as the co-solvent added in the SFE operation for the investigation of lutein recovery. The results of using the different solvents as the co-solvent added in the SFE operation are shown in Table 4. As seen in the table, with ethanol as the co-solvent the lutein recovery performance was superior to that using the other solvents. At the conditions of 400 bar and 70 8C, ethanol as the co-solvent at a ratio of 40 mol% (the ratio of co-solvent flow vs. CO2 flow rate, which was equal to 0.786 ml ethanol/min) had a lutein recovery yield of 62.2% as compared to that of the conventional methanol extraction
method. Generally speaking, adding a co-solvent in the SFE operation enhanced the performance of lutein recovery as compared to the values obtained when a co-solvent was not added. The reasons for ethanol being the co-solvent having the maximum lutein recovery were not clear. However, it was believed that ethanol played a role as an ‘‘entrainer’’ in the phase diagram compositing of CO2, lutein and ethanol, which led to the increase of solubility of lutein in CO2 and resulted in the increase in the lutein recovery yield. Many research reports have suggested that ethanol would be a good co-solvent to enhance the extraction efficiency in the SFE operation [10,11]. Tzeng and his colleagues have also suggested that ethanol could modify SC-CO2 extraction investigated at the pressures and temperatures produced more pure artemisinin and copoletin than Soxhlet solvent extraction did [12]. However, while ethanol was the best co-solvent in the SFE operation, methanol was the best solvent used in the conventional extraction method. To further determine the optimum amount of ethanol to be added in the SFE operation, ratios in the range of 10–40 mol% (as compared to the flow rate of CO2 at 750 ml/min) were investigated. The results, as shown in Table 5, indicated that a ratio of 30 mol% obtained the maximum lutein recovery yield. The best lutein recovery yield obtained in this study of SFE operation was 76.7% under the conditions of 400 bar, 70 8C and ethanol as the co-solvent at a ratio of 30 mol%. 4. Conclusions The application of SFE in lutein extraction is regarded as a green process as lower amounts of solvents are used. Increases in temperature and pressure would be beneficial to the increase of lutein recovery. However, a higher temperature can lead to increased impurity as observed in the HPLC analysis profile. To further improve the recovery yield, the addition of a co-solvent in the SFE operation was investigated. Ethanol was regarded as the optimum co-solvent for lutein extraction from Scenedesmus powder. The best lutein recovery yield obtained in this study of the SFE operation was 76.7% (400 bar, 70 8C and ethanol as the cosolvent at a ratio of 30 mol%) as compared to the conventional methanol extraction method. Acknowledgements The authors wish to thank the National Science Council of the R.O.C. for financial supports.
H.-W. Yen et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 53–57
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