Bioresource Technology 189 (2015) 162–168
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Continuous extraction of lipids from Schizochytrium sp. by CO2-expanded ethanol Hsin-Chih Wang, Worasaung Klinthong, Yi-Hung Yang, Chung-Sung Tan ⇑ Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC
h i g h l i g h t s CO2-expanded ethanol (CXE) is a promising solvent for lipid extraction. Continuous CXE extraction can be operated at low pressures and temperatures. CXE provides an extracted lipid yield of 87 wt% within only 30 min. CXE is superior to ethanol and pressurized ethanol for extraction. The advantages of using CXE include less amount of ethanol and less time required.
a r t i c l e
i n f o
Article history: Received 20 January 2015 Received in revised form 1 April 2015 Accepted 2 April 2015 Available online 8 April 2015 Keywords: Extraction Microalgae CO2-expanded ethanol Lipid Yield
a b s t r a c t CO2-expanded ethanol (CXE) was used to extract DHA-containing lipids from Schizochytrium sp. with a 35.7 wt% lipid content of dry biomass in a continuous mode. The effects of operation variables such as temperature, pressure, ethanol flow rate and CO2 flow rate on extraction performance were investigated. Based on a 24-central composite design and response surface methodology, the optimal operating conditions were determined to be a pressure of 6.9 MPa, a temperature of 313 K, an ethanol flow rate of 1 mL/ min and a CO2 flow rate of 6.0 mL/min, providing an extracted lipid yield of 87 wt% over an extraction period of 30 min. Not only the lipid yield obtained using CXE was observed to be significantly greater than those using ethanol and pressurized ethanol as the solvents, but also a lower amount of ethanol and less time were required to achieve the same extraction yield by using CXE. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are microscopic organisms that typically grow suspended in water and are driven by the same photosynthetic process adopted by higher plants (Hanelt et al., 2007). Microalgae convert solar energy to biomass more efficiently than terrestrial plants due to their simple cellular structure and easy access to basic nutrients. They can also produce a wide range of unique, high-value products used in pharmaceuticals, cosmetics and foods (Reyes et al., 2014). Schizochytrium sp. is one type of microalga that is commercially utilized to produce docosahexaenoic acid (DHA, C22:6, n-3) (Ashford et al., 2000; Wu and Lin, 2003; Gupta et al., 2012; Yao et al., 2013). In addition to DHA, the microalga also contains a high level of total fatty acids (50% of dry biomass), which may also serve as an ideal source for producing biodiesel as renewable energy (Johnson and Wen, 2009; Wang and Wang, 2012). ⇑ Corresponding author. Tel.: +886 3572 1189; fax: +886 3572 1684. E-mail address: [email protected] (C.-S. Tan). http://dx.doi.org/10.1016/j.biortech.2015.04.011 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Cost- and extraction-effective methods from which the extracted microalgal lipids can be optimally utilized as nutrients or as biofuel feedstock are highly required (Choi et al., 2014). Various methods can be used to extract lipids from microalgae, including solvent extraction, pressurized solvent extraction, and supercritical fluid extraction (Shin et al., 2011). Lipid-solvent extraction systems are governed by the principle of like-dissolves-like through which lipids are extracted by organic solvents, such as n-hexane, methanol, and ethanol, and mixed polar/non-polar solvents, such as methanol/chloroform (2:1 v/v, Bligh and Dyer method) or isopropanol/hexane (Geciova et al., 2002); however, the extraction efficiency is highly dependent on the microalgae strains used (Halim et al., 2011). Halim et al. (2011) found that using isopropanol as a co-solvent could increase the lipid yield extracted from Chlorococum sp. by up to 300% relative to that extracted using pure hexane. Ranjan et al. (2010) observed that a mixture of chloroform and methanol yielded the highest lipid yield extracted from Botryococcus braunii among the five organic solvents tested. Cho et al. (1996) also found that a methanol/
H.-C. Wang et al. / Bioresource Technology 189 (2015) 162–168
chloroform (2:1) mixture was superior to the tested organic solvents in extracting DHA from lyophilized Thraustochytrium sp. The DHA-containing lipids of Schizochytrium sp. were extracted by ethanol and 70% aqueous isopropanol by refluxing at 353 K for 30 min; the extracted lipids were mainly composed of phospholipids, and a lipid yield in excess of 70% was achieved (Wang and Wang, 2012; Yao et al., 2013). However, because all extraction processes require large amounts of organic solvents, proper precautionary steps should be taken because of the flammable or toxic nature of these solvents and their negative effects on the nutritional and functional properties of the extracted compounds as well as on human health (Lam and Lee, 2012). Pressurized liquid extraction (also called accelerated solvent extraction) and supercritical fluid extraction have been proposed as alternative approaches to normal pressure solvent extraction. These approaches can reduce the amount of solvent used, increase the selectivity toward valuable bioactive compounds, and preserve the bioactivity of these compounds (Bahadar and Khan, 2013; Herrero et al., 2013). The physicochemical properties of pressurized solvents, supercritical fluids and fluid mixtures, such as their density, diffusivity, viscosity and dielectric constant, can be easily adjusted by varying the operating conditions to obtain versatile and effective solvents for extraction (del Valle et al., 2005; Herrero et al., 2013). Pressurized organic solvents, including ethanol, n-hexane, n-hexane/2-propanol (2:1), were applied to extract fatty acids from Nannochloropsis oculata (Pieber et al., 2012). For pressurized organic solvents, however, the properties such as density, viscosity, diffusivity and solubility, are not so significantly varied as supercritical fluids. A maximum yield of 16.7 wt% total fatty acids, on a dry input basis, was achieved using pressurized ethanol at a pressure of 11 MPa and a temperature of 333 K. Jaime et al. (2010) used pressurized ethanol and n-hexane to extract carotenoids from the microalga Haematococcus pluvialis. The best yields were obtained with ethanol at a pressure of 10.3 MPa and a temperature of 473 K. Iqbal and Theegala (2013) optimized a continuous lipid extraction system for the extraction of lipids from the microalga Nannochloropsis sp. using a ethanol/chloroform (2:1) mixture. The optimum temperature and pressure were observed to be 373 K and 0.3 MPa. The use of supercritical carbon dioxide (scCO2) to extract microalgae lipids for biodiesel production has recently been explored. Lipid from wet Chlorococum sp. paste was extracted using scCO2 with a yield of 7.1 wt% at a temperature of 333 K, a pressure of 30 MPa and an extraction time of 80 min (Halim et al., 2011). However, a comparison of the amount of lipids obtained from Crypthecodinium cohnii using scCO2 extraction with that obtained by organic solvent extraction showed that the latter was approximately twice as large (Couto et al., 2010). On the other hand, for the extraction of lipids from Tetraselmis sp. (strain M8), Li et al. (2014) observed that scCO2 extraction at a pressure of 15 MPa and a temperature of 313 K over 12 h of soaking at a flow rate of 5 mL/min, with 30 min of flushing, resulted in more effective extraction performance compared with Bligh and Dyer lipid extraction, organic solvent extraction and direct saponification using KOH in ethanol. In recent years, various principles and applications of CO2expanded liquids (CXLs), including extraction, reaction and separation, have been reported (Golmakani et al., 2012; Yang et al., 2012; Lin et al., 2013). CXLs can be continuously tuned from neat organic solvents to scCO2 by changing the CO2 composition in the solvent via pressure and temperature adjustments (Jessop and Subramaniam, 2007). A certain amount of CO2 in CXL generates a mixture with favorable transport properties, such as density, viscosity and diffusivity and solubility, resulting from volume expansion, whereas the presence of a suitable amount of polar organic solvent favors the solubility of solids and liquid solutes (Jessop and Subramaniam, 2007; Lin and Tan, 2008; Yang et al.,
163
2012). Because CXLs can be operated at mild pressures and temperatures, this tunability leads to a reduction in energy consumption and process costs (Sih et al., 2008). Similarly to supercritical fluids, CXLs have been shown to improve mass transfer rates by decreasing interfacial tension and viscosity as well as increasing diffusivity (Eckert et al., 2007; Lin and Tan, 2008; Herrero et al., 2013). Recently, CO2-expanded ethanol (CXE) has been applied for the extraction of valuable bioactive compounds from natural sources because ethanol is regarded as a greener solvent than other organic solvents. Reyes et al. (2014) noted that temperature and ethanol content had significant effects on the amount of astaxanthin extracted from H. pluvialis and antioxidant activity using CXE as the solvent. The greatest yield of astaxanthin (62.57 mg/g) was obtained with CXE at a pressure of 7 MPa, a temperature of 318 K and an ethanol content of 50%. Although CXE has been proposed as a promising solvent for chemical reactions and extraction of valuable bioactives from natural sources, only one study has been reported in the open literature regarding the application of CXE for extraction of lipids from microalgae in a continuous operation mode. Golmakani et al. (2012) used CXE to extract gamma-linolenic acid from Arthrospira platensis (Spirulina) and observed the extraction yield to be similar to that achieved by pressurized ethanol extraction but greater than that by scCO2 extraction; however, the CXE was operated at relatively high pressures in a range of 10–30 MPa. Until now, there are few reports for the use of CXE to extract DHA-containing lipids from microalgae. Therefore, the objective of this work was to assess and verify CXE as a promising solvent for extraction of DHA-containing lipids from Schizochytrium sp. under mild operating conditions in a continuous extraction mode. In the operation, microalgae were loaded in a vessel and CXE flowed continuously through the vessel. In this study, the pressure and temperature were varied from 2.8 to 9.0 MPa and from 313 to 333 K, respectively. A 24-central composite design (CCD) and response surface methodology were adopted to examine the effects of operation variables such as pressure, temperature, ethanol flow rate and CO2 flow rate on the extracted lipid yield and to determine the optimal operating conditions. The resulting yield at the optimal operation conditions using CXE was compared with the yields obtained using conventional ethanol extraction and pressurized ethanol extraction to verify the feasibility of the use of CXE as the solvent. 2. Methods 2.1. Materials The spray-dried microalgae strain Schizochytrium sp. was supplied by Far East Microalgae Ind. Ltd. (Taiwan) cultivated in a large scale of circular pound for commercial production of DHA-containing health care products and used for lipid extraction. The microalgae were kept in a chiller at 277 K to avoid oxidative degradation. CO2 with a purity of 99.5% was purchased from Boclh Industrial Gases Co. (Taiwan). The chemical reagents methyl undecanoate (P99%, Sigma–Aldrich), methyl tetradecanoate (P99%, Sigma– Aldrich), methyl hexadecanoate (P99%, Sigma–Aldrich), methyl oleate (P99%, Sigma–Aldrich), methyl docosahexaenoate (P99%, Sigma–Aldrich), ethanol (99.9%, Sigma–Aldrich), methanol (99.9%, J.T. Baker), n-hexane (99.9%, J.T. Baker), sulfuric acid (H2SO4, 95– 97 wt%, Sigma–Aldrich) and potassium hydroxide (KOH, 99%, Sigma–Aldrich) were used as received. 2.2. Soxhlet extraction Soxhlet extraction over a long period of operation was assumed to be the method that could completely extract all the lipids from
164
H.-C. Wang et al. / Bioresource Technology 189 (2015) 162–168
1 3
5
10 6 2
11
9
8
4 13 12
7
14
Fig. 1. Continuous extraction apparatus (1: CO2 cylinder; 2: ethanol vessel; 3: syringe pump; 4: liquid chromatography pump; 5: check valve; 6: coil; 7: oven; 8: extraction column; 9: valve; 10: pressure sensor; 11: back-pressure control valve; 12: water bath; 13: valve; 14: collector).
the microalgae; thus, the extracted amount was used as the bases for comparison. In Soxhlet extraction, 10 g of microalgae was exposed to 200 mL of ethanol at 363 K for 48 h. After extraction, liquid ethanol was removed by a rotary evaporator, and lipids were then obtained. 2.3. Continuous CO2-expanded ethanol extraction The lipids in the microalgae were extracted by CXE in a continuous operation mode as follows. First, pressurized CO2 was dissolved in ethanol to form CXE. Second, the CXE diffused through the cell membrane to the cytoplasm of the microalgae. Third, CXE interacted with lipids to form a CXE-lipid complex, which then diffused across the cell membrane to a bulk flowing CXE stream. Finally, the extracted lipids were collected. The continuous CXE extraction system is illustrated in Fig. 1. A syringe pump and a liquid chromatography pump were used to feed CO2 and ethanol, respectively, at the desired pressures and flow rates. The temperature of the extraction column was controlled by an oven, and the pressure of the system was controlled by a back-pressure control valve. The extracted liquid fraction was collected in a glass-chamber collector. Ten grams of microalgae was added to a 25 mL extraction column. After the desired temperature was reached, CO2 and ethanol were fed into the column. The temperature and pressure were varied over the ranges of 313–333 K and 2.8– 9.0 MPa, respectively. The ethanol flow rate was varied from 0.5 to 1.0 mL/min. In a preliminary study, the extracted lipid yields for one-phase and two-phase CXE operations in continuous mode were compared. The results showed that the extracted lipid yield obtained from the former was up to 68% greater than that obtained from the latter; thus, the one-phase continuous CXE process was chosen in this study. Because the amount of CO2 dissolved in ethanol was limited by the equilibrium solubility at each temperature and pressure, the molar ratio of CO2 to ethanol was controlled to be less than the equilibrium solubility. In all experiments involving one-phase CXE operation, the CO2 flow rates were controlled to reach 30–90% of the equilibrium solubility of CO2, which are listed in Table S1. The extraction was performed continuously, and the effluent liquid was collected every 10, 30, 60, 90 and 120 min. Ethanol in the collected liquid was removed by a rotary evaporator. 2.4. Transesterification of lipids Fatty acid methyl esters were prepared by the transesterification of microalgae lipids using acid and alkaline catalysts. The extracted lipids obtained from the Soxhlet extraction and continuous CXE were mixed with 20 mL of methanol. Five milliliters of 10 wt% H2SO4 in methanol was then added to the mixture. The mixture was heated to 333 K and held at that temperature for 2 h. Afterward, KOH was added until a pH level of 13 was achieved.
Table 1 Operation variables and their values used in the response surface design. Independent variable
1
0
+1
A B
0.5 30
0.75 60
1.0 90
2.8 313
4.8 323
6.9 333
C D
Ethanol flow rate (mL/min) CO2 flow rate resulting in a percentage of CO2 equilibrium solubility (%)a Pressure (MPa) Temperature (K)
a CO2 flow rates to reach equilibrium solubility in ethanol for an ethanol flow rate of 1 mL/min at various temperatures and pressures are shown in Table S1.
The mixture was then heated to 333 K and again held for 2 h to complete the transesterification of the lipids. The solvent in the mixture was then removed by oven drying at 333 K for 24 h. Twenty milliliters of n-hexane was then added to the mixture to extract fatty acid methyl esters (FAME). The n-hexane solution was used for the subsequent gas chromatography analysis. 2.5. Gas chromatography analysis and extraction yield The fatty acid compositions were analyzed using a gas chromatography-flame ionization detector (GC-FID, Agilent Technologies 6890N). The FAME-hexane solution was injected into the GC to analyze the composition of the FAME. The compositions were identified using the standard methyl undecanoate. The extracted lipid yield was calculated as follows:
Extracted lipid yield ð%Þ ¼ 100 extracted FAME ðgÞ= total lipid content ðgÞ
ð1Þ
Reproducibility tests at several operating conditions were performed and the measured extracted lipid yields were found to be reproduced with a deviation of less than 5%, indicating reliability of the experimental data. 2.6. Experimental design and analysis A statistical 24-CCD was used as the experimental design tool. The CCD was a full 19 factorial design (4 operation variables with 2 levels and 3 central points). The response y was the extracted lipid yield over a 30 min period. The four variables chosen were denoted as A (ethanol flow rate, mL/min), B (CO2 flow rate resulting in a percentage of CO2 equilibrium solubility in ethanol, %), C (pressure, MPa) and D (temperature, K). The ranges and center point values for the four independent variables (Table 1) were determined from the results of the preliminary study. Analysis of variance (ANOVA) was performed to determine significant differences between independent variables (p-values
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Continuous extraction of lipids from Schizochytrium sp. by CO2-expanded ethanol Hsin-Chih Wang, Worasaung Klinthong, Yi-Hung Yang, Chung-Sung Tan ⇑ Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC
h i g h l i g h t s CO2-expanded ethanol (CXE) is a promising solvent for lipid extraction. Continuous CXE extraction can be operated at low pressures and temperatures. CXE provides an extracted lipid yield of 87 wt% within only 30 min. CXE is superior to ethanol and pressurized ethanol for extraction. The advantages of using CXE include less amount of ethanol and less time required.
a r t i c l e
i n f o
Article history: Received 20 January 2015 Received in revised form 1 April 2015 Accepted 2 April 2015 Available online 8 April 2015 Keywords: Extraction Microalgae CO2-expanded ethanol Lipid Yield
a b s t r a c t CO2-expanded ethanol (CXE) was used to extract DHA-containing lipids from Schizochytrium sp. with a 35.7 wt% lipid content of dry biomass in a continuous mode. The effects of operation variables such as temperature, pressure, ethanol flow rate and CO2 flow rate on extraction performance were investigated. Based on a 24-central composite design and response surface methodology, the optimal operating conditions were determined to be a pressure of 6.9 MPa, a temperature of 313 K, an ethanol flow rate of 1 mL/ min and a CO2 flow rate of 6.0 mL/min, providing an extracted lipid yield of 87 wt% over an extraction period of 30 min. Not only the lipid yield obtained using CXE was observed to be significantly greater than those using ethanol and pressurized ethanol as the solvents, but also a lower amount of ethanol and less time were required to achieve the same extraction yield by using CXE. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are microscopic organisms that typically grow suspended in water and are driven by the same photosynthetic process adopted by higher plants (Hanelt et al., 2007). Microalgae convert solar energy to biomass more efficiently than terrestrial plants due to their simple cellular structure and easy access to basic nutrients. They can also produce a wide range of unique, high-value products used in pharmaceuticals, cosmetics and foods (Reyes et al., 2014). Schizochytrium sp. is one type of microalga that is commercially utilized to produce docosahexaenoic acid (DHA, C22:6, n-3) (Ashford et al., 2000; Wu and Lin, 2003; Gupta et al., 2012; Yao et al., 2013). In addition to DHA, the microalga also contains a high level of total fatty acids (50% of dry biomass), which may also serve as an ideal source for producing biodiesel as renewable energy (Johnson and Wen, 2009; Wang and Wang, 2012). ⇑ Corresponding author. Tel.: +886 3572 1189; fax: +886 3572 1684. E-mail address: [email protected] (C.-S. Tan). http://dx.doi.org/10.1016/j.biortech.2015.04.011 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Cost- and extraction-effective methods from which the extracted microalgal lipids can be optimally utilized as nutrients or as biofuel feedstock are highly required (Choi et al., 2014). Various methods can be used to extract lipids from microalgae, including solvent extraction, pressurized solvent extraction, and supercritical fluid extraction (Shin et al., 2011). Lipid-solvent extraction systems are governed by the principle of like-dissolves-like through which lipids are extracted by organic solvents, such as n-hexane, methanol, and ethanol, and mixed polar/non-polar solvents, such as methanol/chloroform (2:1 v/v, Bligh and Dyer method) or isopropanol/hexane (Geciova et al., 2002); however, the extraction efficiency is highly dependent on the microalgae strains used (Halim et al., 2011). Halim et al. (2011) found that using isopropanol as a co-solvent could increase the lipid yield extracted from Chlorococum sp. by up to 300% relative to that extracted using pure hexane. Ranjan et al. (2010) observed that a mixture of chloroform and methanol yielded the highest lipid yield extracted from Botryococcus braunii among the five organic solvents tested. Cho et al. (1996) also found that a methanol/
H.-C. Wang et al. / Bioresource Technology 189 (2015) 162–168
chloroform (2:1) mixture was superior to the tested organic solvents in extracting DHA from lyophilized Thraustochytrium sp. The DHA-containing lipids of Schizochytrium sp. were extracted by ethanol and 70% aqueous isopropanol by refluxing at 353 K for 30 min; the extracted lipids were mainly composed of phospholipids, and a lipid yield in excess of 70% was achieved (Wang and Wang, 2012; Yao et al., 2013). However, because all extraction processes require large amounts of organic solvents, proper precautionary steps should be taken because of the flammable or toxic nature of these solvents and their negative effects on the nutritional and functional properties of the extracted compounds as well as on human health (Lam and Lee, 2012). Pressurized liquid extraction (also called accelerated solvent extraction) and supercritical fluid extraction have been proposed as alternative approaches to normal pressure solvent extraction. These approaches can reduce the amount of solvent used, increase the selectivity toward valuable bioactive compounds, and preserve the bioactivity of these compounds (Bahadar and Khan, 2013; Herrero et al., 2013). The physicochemical properties of pressurized solvents, supercritical fluids and fluid mixtures, such as their density, diffusivity, viscosity and dielectric constant, can be easily adjusted by varying the operating conditions to obtain versatile and effective solvents for extraction (del Valle et al., 2005; Herrero et al., 2013). Pressurized organic solvents, including ethanol, n-hexane, n-hexane/2-propanol (2:1), were applied to extract fatty acids from Nannochloropsis oculata (Pieber et al., 2012). For pressurized organic solvents, however, the properties such as density, viscosity, diffusivity and solubility, are not so significantly varied as supercritical fluids. A maximum yield of 16.7 wt% total fatty acids, on a dry input basis, was achieved using pressurized ethanol at a pressure of 11 MPa and a temperature of 333 K. Jaime et al. (2010) used pressurized ethanol and n-hexane to extract carotenoids from the microalga Haematococcus pluvialis. The best yields were obtained with ethanol at a pressure of 10.3 MPa and a temperature of 473 K. Iqbal and Theegala (2013) optimized a continuous lipid extraction system for the extraction of lipids from the microalga Nannochloropsis sp. using a ethanol/chloroform (2:1) mixture. The optimum temperature and pressure were observed to be 373 K and 0.3 MPa. The use of supercritical carbon dioxide (scCO2) to extract microalgae lipids for biodiesel production has recently been explored. Lipid from wet Chlorococum sp. paste was extracted using scCO2 with a yield of 7.1 wt% at a temperature of 333 K, a pressure of 30 MPa and an extraction time of 80 min (Halim et al., 2011). However, a comparison of the amount of lipids obtained from Crypthecodinium cohnii using scCO2 extraction with that obtained by organic solvent extraction showed that the latter was approximately twice as large (Couto et al., 2010). On the other hand, for the extraction of lipids from Tetraselmis sp. (strain M8), Li et al. (2014) observed that scCO2 extraction at a pressure of 15 MPa and a temperature of 313 K over 12 h of soaking at a flow rate of 5 mL/min, with 30 min of flushing, resulted in more effective extraction performance compared with Bligh and Dyer lipid extraction, organic solvent extraction and direct saponification using KOH in ethanol. In recent years, various principles and applications of CO2expanded liquids (CXLs), including extraction, reaction and separation, have been reported (Golmakani et al., 2012; Yang et al., 2012; Lin et al., 2013). CXLs can be continuously tuned from neat organic solvents to scCO2 by changing the CO2 composition in the solvent via pressure and temperature adjustments (Jessop and Subramaniam, 2007). A certain amount of CO2 in CXL generates a mixture with favorable transport properties, such as density, viscosity and diffusivity and solubility, resulting from volume expansion, whereas the presence of a suitable amount of polar organic solvent favors the solubility of solids and liquid solutes (Jessop and Subramaniam, 2007; Lin and Tan, 2008; Yang et al.,
163
2012). Because CXLs can be operated at mild pressures and temperatures, this tunability leads to a reduction in energy consumption and process costs (Sih et al., 2008). Similarly to supercritical fluids, CXLs have been shown to improve mass transfer rates by decreasing interfacial tension and viscosity as well as increasing diffusivity (Eckert et al., 2007; Lin and Tan, 2008; Herrero et al., 2013). Recently, CO2-expanded ethanol (CXE) has been applied for the extraction of valuable bioactive compounds from natural sources because ethanol is regarded as a greener solvent than other organic solvents. Reyes et al. (2014) noted that temperature and ethanol content had significant effects on the amount of astaxanthin extracted from H. pluvialis and antioxidant activity using CXE as the solvent. The greatest yield of astaxanthin (62.57 mg/g) was obtained with CXE at a pressure of 7 MPa, a temperature of 318 K and an ethanol content of 50%. Although CXE has been proposed as a promising solvent for chemical reactions and extraction of valuable bioactives from natural sources, only one study has been reported in the open literature regarding the application of CXE for extraction of lipids from microalgae in a continuous operation mode. Golmakani et al. (2012) used CXE to extract gamma-linolenic acid from Arthrospira platensis (Spirulina) and observed the extraction yield to be similar to that achieved by pressurized ethanol extraction but greater than that by scCO2 extraction; however, the CXE was operated at relatively high pressures in a range of 10–30 MPa. Until now, there are few reports for the use of CXE to extract DHA-containing lipids from microalgae. Therefore, the objective of this work was to assess and verify CXE as a promising solvent for extraction of DHA-containing lipids from Schizochytrium sp. under mild operating conditions in a continuous extraction mode. In the operation, microalgae were loaded in a vessel and CXE flowed continuously through the vessel. In this study, the pressure and temperature were varied from 2.8 to 9.0 MPa and from 313 to 333 K, respectively. A 24-central composite design (CCD) and response surface methodology were adopted to examine the effects of operation variables such as pressure, temperature, ethanol flow rate and CO2 flow rate on the extracted lipid yield and to determine the optimal operating conditions. The resulting yield at the optimal operation conditions using CXE was compared with the yields obtained using conventional ethanol extraction and pressurized ethanol extraction to verify the feasibility of the use of CXE as the solvent. 2. Methods 2.1. Materials The spray-dried microalgae strain Schizochytrium sp. was supplied by Far East Microalgae Ind. Ltd. (Taiwan) cultivated in a large scale of circular pound for commercial production of DHA-containing health care products and used for lipid extraction. The microalgae were kept in a chiller at 277 K to avoid oxidative degradation. CO2 with a purity of 99.5% was purchased from Boclh Industrial Gases Co. (Taiwan). The chemical reagents methyl undecanoate (P99%, Sigma–Aldrich), methyl tetradecanoate (P99%, Sigma– Aldrich), methyl hexadecanoate (P99%, Sigma–Aldrich), methyl oleate (P99%, Sigma–Aldrich), methyl docosahexaenoate (P99%, Sigma–Aldrich), ethanol (99.9%, Sigma–Aldrich), methanol (99.9%, J.T. Baker), n-hexane (99.9%, J.T. Baker), sulfuric acid (H2SO4, 95– 97 wt%, Sigma–Aldrich) and potassium hydroxide (KOH, 99%, Sigma–Aldrich) were used as received. 2.2. Soxhlet extraction Soxhlet extraction over a long period of operation was assumed to be the method that could completely extract all the lipids from
164
H.-C. Wang et al. / Bioresource Technology 189 (2015) 162–168
1 3
5
10 6 2
11
9
8
4 13 12
7
14
Fig. 1. Continuous extraction apparatus (1: CO2 cylinder; 2: ethanol vessel; 3: syringe pump; 4: liquid chromatography pump; 5: check valve; 6: coil; 7: oven; 8: extraction column; 9: valve; 10: pressure sensor; 11: back-pressure control valve; 12: water bath; 13: valve; 14: collector).
the microalgae; thus, the extracted amount was used as the bases for comparison. In Soxhlet extraction, 10 g of microalgae was exposed to 200 mL of ethanol at 363 K for 48 h. After extraction, liquid ethanol was removed by a rotary evaporator, and lipids were then obtained. 2.3. Continuous CO2-expanded ethanol extraction The lipids in the microalgae were extracted by CXE in a continuous operation mode as follows. First, pressurized CO2 was dissolved in ethanol to form CXE. Second, the CXE diffused through the cell membrane to the cytoplasm of the microalgae. Third, CXE interacted with lipids to form a CXE-lipid complex, which then diffused across the cell membrane to a bulk flowing CXE stream. Finally, the extracted lipids were collected. The continuous CXE extraction system is illustrated in Fig. 1. A syringe pump and a liquid chromatography pump were used to feed CO2 and ethanol, respectively, at the desired pressures and flow rates. The temperature of the extraction column was controlled by an oven, and the pressure of the system was controlled by a back-pressure control valve. The extracted liquid fraction was collected in a glass-chamber collector. Ten grams of microalgae was added to a 25 mL extraction column. After the desired temperature was reached, CO2 and ethanol were fed into the column. The temperature and pressure were varied over the ranges of 313–333 K and 2.8– 9.0 MPa, respectively. The ethanol flow rate was varied from 0.5 to 1.0 mL/min. In a preliminary study, the extracted lipid yields for one-phase and two-phase CXE operations in continuous mode were compared. The results showed that the extracted lipid yield obtained from the former was up to 68% greater than that obtained from the latter; thus, the one-phase continuous CXE process was chosen in this study. Because the amount of CO2 dissolved in ethanol was limited by the equilibrium solubility at each temperature and pressure, the molar ratio of CO2 to ethanol was controlled to be less than the equilibrium solubility. In all experiments involving one-phase CXE operation, the CO2 flow rates were controlled to reach 30–90% of the equilibrium solubility of CO2, which are listed in Table S1. The extraction was performed continuously, and the effluent liquid was collected every 10, 30, 60, 90 and 120 min. Ethanol in the collected liquid was removed by a rotary evaporator. 2.4. Transesterification of lipids Fatty acid methyl esters were prepared by the transesterification of microalgae lipids using acid and alkaline catalysts. The extracted lipids obtained from the Soxhlet extraction and continuous CXE were mixed with 20 mL of methanol. Five milliliters of 10 wt% H2SO4 in methanol was then added to the mixture. The mixture was heated to 333 K and held at that temperature for 2 h. Afterward, KOH was added until a pH level of 13 was achieved.
Table 1 Operation variables and their values used in the response surface design. Independent variable
1
0
+1
A B
0.5 30
0.75 60
1.0 90
2.8 313
4.8 323
6.9 333
C D
Ethanol flow rate (mL/min) CO2 flow rate resulting in a percentage of CO2 equilibrium solubility (%)a Pressure (MPa) Temperature (K)
a CO2 flow rates to reach equilibrium solubility in ethanol for an ethanol flow rate of 1 mL/min at various temperatures and pressures are shown in Table S1.
The mixture was then heated to 333 K and again held for 2 h to complete the transesterification of the lipids. The solvent in the mixture was then removed by oven drying at 333 K for 24 h. Twenty milliliters of n-hexane was then added to the mixture to extract fatty acid methyl esters (FAME). The n-hexane solution was used for the subsequent gas chromatography analysis. 2.5. Gas chromatography analysis and extraction yield The fatty acid compositions were analyzed using a gas chromatography-flame ionization detector (GC-FID, Agilent Technologies 6890N). The FAME-hexane solution was injected into the GC to analyze the composition of the FAME. The compositions were identified using the standard methyl undecanoate. The extracted lipid yield was calculated as follows:
Extracted lipid yield ð%Þ ¼ 100 extracted FAME ðgÞ= total lipid content ðgÞ
ð1Þ
Reproducibility tests at several operating conditions were performed and the measured extracted lipid yields were found to be reproduced with a deviation of less than 5%, indicating reliability of the experimental data. 2.6. Experimental design and analysis A statistical 24-CCD was used as the experimental design tool. The CCD was a full 19 factorial design (4 operation variables with 2 levels and 3 central points). The response y was the extracted lipid yield over a 30 min period. The four variables chosen were denoted as A (ethanol flow rate, mL/min), B (CO2 flow rate resulting in a percentage of CO2 equilibrium solubility in ethanol, %), C (pressure, MPa) and D (temperature, K). The ranges and center point values for the four independent variables (Table 1) were determined from the results of the preliminary study. Analysis of variance (ANOVA) was performed to determine significant differences between independent variables (p-values
