3012
Ind. Eng. Chem. Res. 2002, 41, 3012-3018
Supercritical Fluid Extraction of Fatty Acids and Carotenoids from the Microalgae Spirulina maxima A. Paula R. F. Canela,† Paulo T. V. Rosa,† Marcia O. M. Marques,‡ and M. Angela A. Meireles*,† LASEFI, DEA/FEA, Unicamp, Cx.P. 6121, 13083-970 Campinas, Sa˜ o Paulo, Brazil, and Centro de Gene´ tica, Biologia Molecular e Fitoquı´mica, IAC, Cx.P. 28, 13001-970 Campinas, Sa˜ o Paulo, Brazil
The supercritical fluid extraction of fatty acids and carotenoids from the microalgae Spirulina maxima with carbon dioxide was studied. The effects of pressure and temperature on the yield and chemical composition of the extracts were assessed. The experiments were conducted at temperatures of 20-70 °C and pressures of 15-180 bar. The solvent mass flow rate was 3.33 × 10-5 kg/s. Statistical analysis showed that neither the temperature nor the pressure significantly affected the total yield, but both the temperature and the pressure affected the extraction rate, and the effect of the temperature prevailed over that of the pressure. The extracts were rich in essential fatty acids and carotenes, and at 100 bar and 45 °C the extract contained no carotenes. Temperatures larger than 50 °C degraded the carotenes, as expected. The model of Goto et al. described the overall experimental extraction curves quite well. Introduction The microalgae Spirulina maxima is a blue-green cyanobacterium that is a source of the blue color pigment phycocyanin, which is not soluble in CO2 but is soluble in water. The direct use of water as a solvent medium results in a mixture that is not easily fractionated because of the presence of polar and nonpolar substances. The alga is of great interest for food, cosmetics, and pharmaceutical industries because of its chemical composition. Among other desirable components we may mention the carotenoids and the essential fatty acids such as γ-linolenic acid. There are several commercial large-scale facilities that produced 2500 tons of S. maxima in 1998.1 The carotenes are usually added to food to impart colors ranging from yellow to red. They are also used because of their pro-Vitamin A action and antioxidant activity.2 This activity comes from carotenes’ ability to deactivate the free radicals responsible for the cell degeneration that promotes cancer. Among the essential free fatty acids from S. maxima, γ-linolenic acid plays an important role as a precursor of prostaglandin (PG1), a substance similar to the hormones that control several functions of the human body.3 Many authors studied the nutritional value of Spirulina. Sarada et al.4 verified the effects of drying and extraction methods on the functional properties of Spirulina platensis. Despite its application as a food supplement, some blue-green algae can cause several toxic effects when ingested by human and animals. Salazar et al.5 verified that even high feeding levels of S. maxima did not produce adverse effects in mice. The cholesterol level in the blood decreased for high amounts of S. maxima. Paredes* To whom correspondence should be addressed. Phone: 55 19 3788-4033. Fax: 55 19 37884027. E-mail: meireles@ fea.unicamp.br. † LASEFI, DEA/FEA. ‡ IAC.
Carbajal et al.6 determined that S. maxima is able to prevent the adverse effects of a high fructose content diet, and Paredes-Carbajal et al.7 analyzed, in vitro, the effects of raw ethanolic extracts of S. maxima on the vasomotor responses of rat aortic rings. Torres-Dura´n et al.8 determined that the oil extract from S. maxima or its defatted fraction is capable of preventing fatty liver development, and so their results support the potential hepatoprotective role of Spirulina. Kim et al.9 showed that Spirulina inhibited the systemic allergic reaction in rats. Very recently, Careri et al.10 reported the effects of process parameters on the supercritical fluid extraction (SFE) of carotenoids β-carotene, β-cryptoxanthin, and zeaxanthin from Spirulina pacifica. They used the surface response methodology to determine that the best extraction conditions were the pressure of 350 bar and 15% of ethanol used as the cosolvent. The best processing temperature was specific for each target component, i.e., 40, 60, and 80 °C for β-carotene, β-cryptoxanthin, and zeaxanthin, respectively. Fatty acids and carotenoids are soluble in carbon dioxide at reasonably moderate conditions.2,11 Monteiro et al.12 obtained the fatty acids from the shells of Platonia insignis at a pressure of 63 bar and a temperature of 16 °C, and the extracts were rich in γ-linolenic acid. Sovova´ et al.13 reported in a survey of literature data that the solubilities of vegetable oils in carbon dioxide have been measured for temperatures of 40-70 °C and pressures of 90-1400 bar. Sakaki14 determined the solubility of β-carotene in carbon dioxide and nitrous oxide in temperatures ranging from 35 to 50 °C and pressures ranging from 96 to 300 bar. Maheshwari et al.11 reported the solubility of fatty acids in carbon dioxide in the ranges of 35-60 °C and 140-400 bar. Markon et al.15 and Franc¸ a and Meireles16 reported that β-carotene prefers the triglyceride-rich phase instead of the CO2 phase for the system palm oil + CO2. Markon et al.15 also observed that the pressure effect predomi-
10.1021/ie010469i CCC: $22.00 © 2002 American Chemical Society Published on Web 05/14/2002
Ind. Eng. Chem. Res., Vol. 41, No. 12, 2002 3013
nated over the temperature’s effect in the fractionation of crude palm oil. Because of the commercial value of carotenoids and γ-linolenic acid and because the organic solvent extraction produces a crude extract that is hard to fractionate, the SFE of fatty acids and carotenoids may find its application as the first step in the fractionation of S. maxima compounds. For these reasons, the objective of this work was to investigate the selective extraction of fatty acids and carotenoids of S. maxima. To avoid an excessive coextraction of triglycerides, the experimental runs were carried out at temperatures in the range of 20-70 °C and pressures in the range of 150-180 bar, in a fixed-bed extractor. The extracts were analyzed using a UV spectrophotometer and a gas chromatograph (GC)-mass spectrometer (MS) system. The overall extraction curves (OECs) were modeled using the Goto et al.17 model. Materials and Methods S. maxima was from Chile. The alga was supplied dried by the company Minera Chan˜ar Blanco and by the University of La Serena (Chile). It was produced in artificial ponds and dried at ambient temperature (20 °C). The material was stored at 20 °C under vacuum in plastic bags containing aluminum foil, to prevent photodegradation. The raw material humidity was determined using the AOAC 925.09 method,18 the protein content was evaluated with the AOAC 979.09 method,18 the ash amount was determined using the AOAC 923.03 method,18 and the total amount of lipidsoluble substances was measured using the Bligh and Dyer method.19 Raw Material Preparation. The raw material was ground using the following procedure: 60 g of S. maxima was placed inside a large black plastic bag. The material was broken into small pieces using a cylindrical rod to reach the appropriate particle size distribution. The solid particle size distribution was determined using sieve trays under mechanical agitation (Granutest, series Tyler, Abrosinox), with sieve meshes of 16, 24, 32, and 48. Bed and Particles Characterization. The bed was formed using equal amounts of S. maxima and glass beads. The real or true density (Fs) of S. maxima was measured using a helium pycnometer (Micrometrics, model multivolume pycnometer 1305), at the Analytical Facilities of the Chemistry Institute (IQ-Unicamp). The apparent bed density (Fa) was determined using the bed volume and the mass of the feed (S. maxima + glass beads). The apparent density of the S. maxima particles (Fp) was estimated measuring the volume of the rectangular pieces. Extraction Procedure for the Total Amount of Soluble Material. The total amount of soluble material (X0) at a given temperature and pressure was determined using a Spe-ed SFE system (Applied Separations) equipped with a 3 or 5 cm3 extraction cell (Thar Designs). The bed density was kept at 0.8 kg of S. maxima/cm3 of bed. The CO2 was admitted into the system at a flow rate of 8.5 × 10-2 kg/s, up to the point where no solute was observed at the exit of the column. The amount of CO2-soluble material was calculated as the ratio of the total mass of extract and the total initial mass of S. maxima. The experiments were run at pressures of 180 and 150 bar, and because the SFE
system operates above room temperature (25 °C in our laboratory), the assays were performed at temperatures ranging from 30 to 70 °C. Extraction Procedure for Kinetic Experiments. Kinetic experimental runs were performed using a standard extraction unit containing an extraction cell of approximately 368.4 cm3 (Bergoht, model HB-500, maximum pressure 200 bar) described by Rodrigues et al.20 The bed was formed inside the extraction cell with 173 ( 2 g of microalgae and 173 ( 2 g of glass beads. The particles of the microalgae had sizes of 24, 32, and 48 mesh in proportions of 23, 34, and 43%, respectively, and the glass beads had sizes of 24 and 48 mesh (Potters Industrial Ltd., 3R). These particles were used to form more homogeneous fixed beds, thus having a better pressure control during the experiments. To minimize the bed heterogeneity, the particles (S. maxima + glass beads) were fed into the extraction cell in portions of 15 g of the mixture. Samples were collected every hour and the runs continued for 12 h. Content of Total Carotenes. The amount of carotenes was determined by a UV spectrophotometer (Hitachi, model U-3010). The samples were diluted in hexane (chromatographic grade, EM Science, 38211). The UV spectrum was scanned from 300 to 600 nm because the maximum absorbance was expected to be at 450 nm. The total amount of carotenes was calculated using Beer’s law with the coefficient of optical extinction equal to 259.2 mL/mg‚cm given by Mendes et al.2 Fatty Acids Quantification and Identification. The samples used to determine the total carotenes were analyzed with respect to the fatty acids content. The hexane was eliminated under a nitrogen atmosphere. Afterward, the methyl esters were prepared by the method of Maia.21 The extracts were saponified with a 0.5 N solution of sodium hydroxide in methanol, followed by esterification using solutions of ammonium chloride, sulfuric acid, and methanol in the proportion 1:1.5:30 (v/v). A saturated solution of sodium chloride and hexane was then added to the mixture. The composition of the extracts was determined using a gas chromatograph (GC-FID; Shimadzu, model 17A, Kyoto, Japan) equipped with a fused silica capillary column DB5 (30 m × 0.25 mm × 0.25 µm; J & W Scientific). The carrier gas was helium (1.0 mL/min), and a split ratio of 1:5 was used. The temperatures of the injector and of the detector were 250 and 280 °C, respectively. The column was heated to 50 °C for 6 min and programmed at 40 °C/min to 170 °C, at 1 °C/min to 205 °C, and at 10 °C/min to 230 °C. One microliter of the sample was injected. The identification of the substances was done by GC-MS (Shimadzu, model QP-5000, Kyoto, Japan) using the GC conditions, except for the detector temperature (230 °C), and was based on (i) comparison of the substance mass spectrum with the GC-MS system data bank (Nist 62 Library), (ii) comparison of the mass spectra with the data in the literature,22 and (iii) coinjection of the following fatty acids: palmitic, stearic, oleic, linoleic, and R-linoleic (Supelco Fame Mix GLC-10, 1891-1AM, LA-84222) and γ-linolenic (Sigma, L-6503). Experimental Planning and Kinetics Parameters Evaluation. The effects of temperature and pressure were quantified using a factorial experimental design without replication: temperatures of 20 and 30 °C, pressures of 150 and 180 bar, and a central point set at 165 bar and 25 °C (with replication). The
3014
Ind. Eng. Chem. Res., Vol. 41, No. 12, 2002 Table 2. Operational Conditions and Kinetic Parameters (QCO2 ) 3.33 × 10-5 kg/s) T ( 1 P ( 2 MCER × 108 tCER/60 RCER RTOTAL Rdesp RCER/ (°C) (bar) (s) (kg/s) (%) (%) (%) Rdesp
Figure 1. CO2-soluble material from S. maxima as a function of temperature. Table 1. S. maxima Composition content (% mass) humidity proteins lipids
9.1 54.5 3.1
content (% mass) ashes nonanalyzed
12.2 21.1
experiments were conducted at a fixed solvent flow rate (3.33 × 10-5 kg/s). To assess the influences of pressure and temperature, the kinetic parameter mass-transfer ratio (MCER) and yield (RCER) corresponding to the constant extraction rate (CER) period were calculated for the CER period using a spline algorithm.23 The yield was calculated with respect to the amount of the feed. The analysis of variance (ANOVA) and the spline fitting were performed using the software SAS 6.12. Results and Discussion Table 1 shows the composition of the microalgae used in the experiments. The values are within the range of variation found in the literature.24 The apparent bed density was 940 kg/m3, the apparent particle density was 665 kg/m3, and the particle true density was 1684 kg/m3. Based on preliminary experiments, the appropriate conditions to selectively extract fatty acids and carotenes were found to be pressures in the range of 150-200 bar and temperatures in the range of 20-70 °C. These assays also indicated that no carotenes were extracted at 100 bar and 45 °C. Figure 1 shows the amount of CO2-soluble material (X0) at 150 and 180 bar. The amount of soluble material at 150 bar remains approximately constant from 30 to 50 °C, increases at 60 °C to reach its maximum value, and sharply decreases at 70 °C. The parameter X0 is closely related to the solubility of the mixture of fatty acids and carotenes, measured in the system S. maxima + CO2, so it can be expected that the temperature and pressure will affect the behavior of X0 with the same trend as they have on the solubility. For the system S. maxima + CO2, this behavior can be quite complicated because of the nature of the solid substrate. This was indeed observed. At 150 bar, the CO2 density25 for the range of pressure tested decreased from 904 to 507 kg/ m3, but X0 had a maximum at 60 °C; i.e., the effect of the component vapor pressure prevailed. At 180 bar, the values of X0 were lower as compared to the corresponding values at 150 bar, a result that is related to the smaller solvent densities, which means that at this pressure the density effect (solvent power) preponderated. The effects of the temperature and pressure were quantified using a factorial experimental design. The experimental OECs, mass of extract against time, had the typical shape of the CER period, falling extraction rate (FER) period, and diffusion-controlled (DC) extrac-
30 30 20 20 25a
180 150 180 150 165
First Set of Experiments 2.32 ( 0.09 339 0.28 0.43 2.36 ( 0.08 315 0.26 0.42 1.82 ( 0.03 398 0.25 0.36 1.91 ( 0.05 245 0.17 0.32 1.42 ( 0.04 409 0.21 0.30
0.45 0.45 0.41 0.35 0.34
0.62 0.58 0.61 0.49 0.62
40 50 60 70
150 150 150 150
Second Set of Experiments 2.55 ( 0.04 351 0.29 0.44 2.38 ( 0.1 325 0.26 0.45 1.43 ( 0.08 366 0.21 0.34 1.35 ( 0.06 476 0.27 0.36
0.49 0.37 0.40
0.58 0.57 0.68
a
Mean of the two experiments.
tion rate period. The OEC were fitted to a linear spline as explained by Povh et al.26 in order to calculate the kinetic parameters MCER, RCER, and RTOTAL. Because the spline quantitatively described the experimental OECs, the effects of temperature and pressure were assessed using the kinetic parameters (response variables) shown in Table 2. In Table 2, Rdesp refers to the increase in yield obtained during the depressurization of the system, which took 1.5 h. The last column in Table 2 shows the ratio of RCER to Rdesp. The results show that more than 49% of the CO2-soluble materials is removed from the S. maxima solid matrix during the CER period, and removing the remaining CO2-soluble material required an additional period of 6 h. The statistical analysis (ANOVA) performed for the response variable RTOTAL showed that the effects of both the temperature (p ) 0.1206) and the pressure (p ) 0.4117) were negligible, but the effect of the temperature was slightly more significant than that of the pressure. When the ANOVA was performed for the response variable MCER, it was observed that the effects of temperature and pressure were significant and that the effect of temperature was more significant (p ) 0.0014) than that of the pressure (p ) 0.0667). Therefore, considering the effects of the operating variables temperature and pressure on RTOTAL and MCER, it was decided to keep the pressure constant at 150 bar and search for the best operating temperature, and so the additional experiments were conducted at temperatures of 40, 50, 60, and 70 °C (Table 2). Data from Figure 1 and Table 2 show the same general trend, but the behavior of MCER was slightly different: MCER increased from 20 to 30 °C, remained almost constant up to 50 °C, and decreased sharply at 60 °C. From the information given above the best values of the operating variables pressure and temperature are 150 bar and 60 °C. However, to make a final decision, it is necessary to consider the composition of the extract with respect to the target components carotenes and fatty acids. These contents were determined in samples collected after 2, 5, 8, and 11 h of extraction. The samples were denoted by fractions F2, F5, F8, and F11, respectively, and the fractions correspond to the CER (F2 and F5), FER (F8), and DC (F11) periods. Table 3 shows the amount of carotenes obtained in each fraction, and the last column (total) refers to the total amount of carotene in the four fractions analyzed. The maximum amount of carotene was obtained at 180 bar and 30 °C and the smallest at 150 bar and 70 °C. This can be observed in the UV spectra of fraction F2 (Figure 2). The shoulder that appears at about 328 nm increased as the temperature rose from 20 to 70 °C, accompanied
Ind. Eng. Chem. Res., Vol. 41, No. 12, 2002 3015
Figure 2. UV spectra for sample F2 from experiments performed at 150 bar and temperatures of 20, 50, 60, and 70 °C. Table 3. Total Carotenoids for Selected Fractions of SFE Extracts of S. maxima total carotenoids (mg) 20 °C
25 °C
Figure 3. Effect of heating on total carotenes of samples F3 for pressures of 150 and 180 bar.
30 °C 150 bar
fraction
150 bar
180 bar
165 bar
165 bar
150 bar
180 bar
F2 F5 F8 F11
0.353 0.275 0.158 0.213
0.549 0.562 0.211 0.123
0.316 0.252 0.152 0.116
0.541 0.540 0.231 0.122
0.359 0.454 0.288 0.231
0.896 0.660 0.381 0.333
0.544 0.447 0.364 0.280
0.192 0.129 0.189 0.129
0.069 0.070 0.079 0.047
total
1.00
1.45
0.84
1.43
1.33
2.27
1.64
0.64
0.27
50 °C 60 °C 70 °C
by a decrease in the absorbance at 445 nm, the characteristic wavelength of carotenes. To establish the occurrence of carotene thermal degradation during the SFE process, the following experiment was done: Samples collected after 3 h of extraction, for the two runs performed at 20 °C (150 and 180 bar), were diluted with hexane, were divided into five samples, and were kept for 2 h at temperatures of 20, 40, 50, 60, and 70 °C. Afterward, the absorbances of all samples were read, as previously described. Figure 3 shows that the heating treatment promoted a small increase in the absorbance read at 328 nm and a decrease of the absorbance read at 445 nm. Therefore, it is very possible that at least partially the increase in the shoulder at 328 nm observed in Figure 2 is due to the thermal degradation of the carotenes as the operating temperature increased. The fatty acids content of fraction F2, for the assay performed at 180 bar and 30 °C, is shown in Table 4, and Figure 4 shows its chromatogram. It can be observed that, besides fatty acids, some hydrocarbons were also identified. A similar profile was obtained for the other samples from the various runs. Table 5 shows the fatty acid content of fractions F2, F5, F8, and F11 for all runs. γ-Linolenic acid was the most abundant fatty acid in the samples. Its mass was approximately 2, 7, 3, and 25 times larger than the masses of palmitic acid, palmitoleic acid, linoleic acid, and stearic acid, respectively. These proportions remained about the same during the entire extraction process. These results are in accordance with the reported solubility of fatty acids.11 The experimental condition that provided a higher yield of fatty acids was 150 bar and 50 °C. At this condition, there was 16.2% of fatty acids in the extract.
Figure 4. Chromatogram of fraction F2 for SFE at 180 bar and 30 °C. Table 4. Main Compound Profile of Fraction F2 S. maxima Extract Obtained at 180 bar and 30 °C peak % area 1 2 5 9 11
2.19 1.34 30.09 6.25 25.12
compound n-pentadecane n-hexadecane n-heptadecane palmitoleic acid palmitic acid
peak % area 15 16 19
14.18 12.74 0.80
total
92.71
compound γ-linolenic acid linoleic acid stearic acid
Considering the previous discussion for the range of conditions studied, the conclusion is that in order to compromise the maximization of yield (RTOTAL) and the minimization of processing time (maximization of MCER) with respect to the amounts of fatty acids and carotenoids, the operating pressure should be 150 bar and the temperature kept at or lower than 50 °C, to avoid carotenoid thermal degradation. Mathematical Modeling. To transform SFE laboratory data in process design data, a mathematical model should be applied to describe the overall experimental curves to provide a tool for scale-up, but up to now no model is entirely accepted among the several ones available in the literature to describe the OECs.27-29 The
3016
Ind. Eng. Chem. Res., Vol. 41, No. 12, 2002
Table 5. Fatty Acid Content (mg) in Fractions F2, F5, F8, and F11 for Various Operating Conditions 20 °C fatty
acida
25 °C
30 °C
150 bar
fraction
150 bar
180 bar
165 bar
165 bar
150 bar
180 bar
50 °C
60 °C
70 °C
palmitic (C16:0)
F2 F5 F8 F11
1.94 0.85 0.51 1.35
1.70 2.19 0.82 0.44
3.70 1.06 0.38 0.16
1.38 1.73 0.51 0.29
2.15 1.27 1.16 0.72
2.54 3.03 1.38 0.36
3.54 2.41 1.64 0.51
2.00 1.20 1.23 1.19
1.83 2.17 1.78 1.28
palmitoleic (C16:1)
F2 F5 F8 F11
0.72 0.44 0.27 0.32
0.52 0.50 0.26 0.16
0.94 0.42 0.28 0.11
0.44 0.46 0.20 0.12
0.55 0.30 0.25 0.17
0.63 0.58 0.28 0.09
1.02 0.57 0.37 0.18
0.65 0.44 0.52 0.35
0.45 0.57 0.33 0.42
γ-linolenic (C18:3)
F2 F5 F8 F11
5.04 3.03 1.85 2.19
3.68 3.56 1.82 1.12
6.61 2.95 1.93 0.76
3.21 3.22 1.40 0.79
4.00 2.12 1.80 1.23
4.36 4.03 1.97 0.63
6.76 3.83 2.59 1.33
4.04 2.74 3.30 2.29
2.42 3.14 1.88 2.40
linoleic (C18:2)
F2 F5 F8 F11
1.46 0.89 0.54 0.64
1.10 1.05 0.54 0.34
1.96 0.83 0.55 0.22
0.89 0.96 0.40 0.24
1.15 0.60 0.54 0.37
1.29 1.20 0.59 0.18
2.04 1.16 0.78 0.40
1.24 0.85 1.01 0.72
0.76 0.97 0.58 0.76
stearic (C18:0)
F2 F5 F8 F11
0.15 0.08 0.06 0.07
0.13 0.12 0.05 0.04
0.23 0.07 0.00 0.00
0.12 0.12 0.00 0.00
0.20 0.11 0.06 0.05
0.18 0.22 0.09 0.00
0.35 0.19 0.14 0.09
0.24 0.19 0.22 0.13
0.18 0.14 0.12 0.18
22.37
20.16
23.16
16.48
18.80
23.64
29.91
24.57
22.35
totalb a
b
Cm:n: m is the number of carbons, and n is the number of double bonds. Total amount of analyzed fatty acids in fractions F2, F5, F8, and F11.
Goto et al.17 model was developed for the extraction of mint essential oil from peppermint leaves, and the shape of S. maxima resembles that of peppermint leaves, so this model was chosen to describe the extraction process. The model treats the solid substrate as a porous matrix. The solute is extracted after its desorption from the solid matrix. Diffusion occurs inside the particle pores, and there is a mass-transfer resistance in the film surrounding the particles. The mass balance equations for the interstitial space of the bed and particles are17
For the interstitial space:
(
)
φ(1 - l) Xs dX X X+ )dθ l l K
X) a1 )
Co
(
)
Xs φ XdXs K ) dθ p/K + (1 - p)
(
)
1 - l φ [p + (1 - p)K](a1 - a2) l
(2)
(3)
The solution for the set of differential equation is given by
X(θ) ) A[exp(a1θ) - exp(a2θ)]
(5)
)
(6) (7)
φ 1 φ(1 - l) ; + + l p + (1 - p)K l
(1)
X)0 K p + (1 - p)K
(
c)
Initial conditions:
Xs )
C hs t ; θ ) ; φ ) kpapτ Co τ
1 1 -b + xb2 - 4c ; a2 ) -b - xb2 - 4c 2 2 A)
b)
; Xs )
Co )
For the particle:
[
φ (8) [p + (1 - p)K]l
]
p + (1 - p) X0Fs K
(9)
where K is the solute partition coefficient, QCO2 is the volumetric solvent flow rate, t is time, Vc is the bed volume, X is the fraction of total solute present in the fluid phase, Xs is the solute mass ratio in the solid phase, X0 is the initial solute mass ratio in the solid phase, Y is the fluid-phase solute mass ratio, is the total bed porosity ( ) 1 - Fa/Fs), l is the interstitial bed porosity (l ) 1 - Fa/Fp), p is the particle porosity (p ) 1 - Fp/Fs), Fa is the apparent bed density, Fp is the particle apparent density, Fs is the solid true density, τ is the residence time of the supercritical fluid in the bed (τ ) Vc/QCO2), Kp is the combined mass-transfer coefficient, and ap is the specific surface area. The mass ratio of the solute in the fluid phase as a function of time can be obtained from
(4) Y(t) )
where
FCO2Y
[
] [ ( )
( )] (10)
p X0Fs t t A exp a1 - exp a2 + (1 - p) K FCO2 τ τ
Ind. Eng. Chem. Res., Vol. 41, No. 12, 2002 3017
Figure 5. Comparison between experimental (symbols) and fitted (lines) OECs at 180 bar.
Figure 6. Comparison between experimental (symbols) and fitted (lines) OECs at 150 bar.
Table 6. Parameters for the Goto et al.17 Model (QCO2 ) 3.33 × 10-5 kg/s)
Nomenclature
P (bar)
T (°C)
X0 × 103 (kg/kg)
K × 102
kpap × 103 (s-1)
180 150 150 150 150 150
30 30 40 50 60 70
6.3 6.7 7.7 7.1 9.3 4.7
1.53 2.02 1.87 1.74 10.10 1.65
3.15 3.72 2.28 1.77 4.25 1.17
The mass of extract at the bed outlet can be calculated from
m(t) )
∫0tY(t) QCO FCO 2
2
dt
(11)
Using eq 10 in eq 11, we obtain
m(t) )
[
]
p + (1 - p) X0FsQCO2Aτ × K 1 t t 1 exp a1 - 1 + 1 - exp a2 a1 τ a2 τ
{[ ( ) ] [
( )]} (12)
The parameters K and φ can be obtained from the leastsquares fitting of the experimental data to eq 12. Table 6 shows the model parameters for the Goto et al. model.17 Figures 5 and 6 compare the experimental overall extractions curves with the fitted curves and show that the model describes the experimental data quite well, regardless of the operating conditions. The data in Table 6 showed that the desorption constant for the assay at 150 bar and 60 °C was 1 order of magnitude greater than the others. The combined mass-transfer coefficient was approximately constant from 150 to 180 bar at 30 °C. At 150 bar, the behavior of the combined mass-transfer coefficient resembles that of the total maximum yield (Figure 1). Acknowledgment The authors thank Minera Chan˜ar Blanco S.A. (
[email protected]) and Dr. Jose´ Valderrama, Universidad de La Serena, both from La Serena, Chile, for providing the S. maxima used in the experiments. The authors are also grateful to FAPESP (1995/0562-3 and 1999/01962-1) for financial support. A.P.R.F.C. thanks FAPESP for the M.S. assistantship, and P.T.V.R. thanks CPP-Rhodia for the postdoc fellowship.
a1, a2 ) constants defined in eq 6 ap ) specific surface area (1/L) A ) constant defined in eq 7 b, c ) constants defined in eq 8 CER ) constant extraction rate Co ) total solute concentration (M/L3) Cs ) average value of the solute concentration in the solid phase (M/L3) DC ) diffusion controlled FER ) falling extraction rate K ) partition coefficient of the solvent in the solute kp ) combined mass-transfer coefficient (L/T) MCER ) solute mass flow rate during the constant extraction rate period (M/T) OEC ) overall extraction curve P ) extraction pressure QCO2 ) volumetric CO2 flow rate (L3/T) RCER ) yield of solute during the constant extraction rate period Rdesp ) yield of solute during the depressurization period Rtotal ) total yield of solute during extraction t ) extraction time (T) tCER ) end of the constant extraction rate period (T) T ) extraction temperature Vc ) bed volume (L3) X ) fraction of the total solute present in the fluid phase X0 ) initial solute mass ratio in the solid phase Xs ) solute mass ratio in the solid phase (M/M) Y ) solute mass ratio in the fluid phase (M/M) Greek Letters ) total bed porosity l ) interstitial bed porosity p ) particle porosity φ ) dimensionless number defined in eq 5 θ ) dimensionless time Fa ) bed density (M/L3) FCO2 ) carbon dioxide density (M/L3) Fp ) particle density (M/L3) τ ) CO2 residence time (T)
Literature Cited (1) Borowitzka, M. A. Commercial production of microalgae: ponds, tanks, tubes and fermenters. J. Biotechnol. 1999, 70, 313321. (2) Mendes, R. L.; Nobre, B. P.; Coelho, J. P.; Palavra, A. F. Solubility of β-carotene in supercritical carbon dioxide and ethane. J. Supercrit. Fluids 1999, 16, 99-106.
3018
Ind. Eng. Chem. Res., Vol. 41, No. 12, 2002
(3) Henrikson, R. In Microalga Spirulina: Superalimento del futuro; Herce, A. C., Translator; Ediciones Urano: Barcelona, 1994; p 221. (4) Sarada, R.; Pillai, M. G.; Ravishankar, G. A. Phycocyanin from Spirulina sp: influence of processing of biomass on phycocyanin yield, analysis of efficacy of extraction methods and stability studies on phycocyanin. Process Biochem. 1999, 34, 795-801. (5) Salazar, M.; Martinez, E.; Madrigal, E.; Ruiz, L. E.; Chamorro, G. A. Subchronic toxicity study in mice fed Spirulina maxima. J. Ethnopharmacol. 1998, 62, 235-241. (6) Paredes-Carbajal, M. C.; Torres-Dura´n, P. V.; RivasArancibia, S.; Zamora-Gonza´lez, J.; Mascher, D.; Jua´rezOropeza, M. A. Effects of dietary Spirulina maxima on vasomotor responses of aorta rings from rats fed a fructose-rich diet. Nutr. Res. 1998, 18, 1769-1782. (7) Paredes-Carbajal, M. C.; Torres-Dura´n, P. V.; Diaz-Zagoya, J. C.; Mascher, D.; Jua´rez-Oropeza, M. A. Effects of the ethanolic extract of Spirulina maxima on endothelium dependent vasomotor responses of rat aortic rings. J. Ethnopharmacol. 2001, 75, 3744. (8) Torres-Dura´n, P. V.; Miranda-Zamora, R.; Paredes-Carbajal, M. C.; Mascher, D.; Ble´-Castillo, J.; Diaz-Zagoya, J. C.; Jua´rezOropeza, M. A. Studies on the preventive effect of Spirulina maxima on fatty liver development induced by carbon tetrachloride in rat. J. Ethnopharmacol. 1999, 64, 141-147. (9) Kim, H.-M.; Lee, E.-H.; Cho, H.-H.; Moon, Y.-H. Inhibitory effect of mast cell-mediated immediate-type allergic reactions in rats by Spirulina. Biochem. Pharmacol. 1998, 55, 1071-1076. (10) Careri, M.; Furlattini, L.; Mangia, A.; Musci, M.; Anklam, E.; Theobald, A.; von Holst, C. Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina pacifica algae: a chemometric approach. J. Chromatogr. A 2001, 912, 61-71. (11) Maheshwari, P.; Nikolov, Z. L.; White, T. M.; Hartel, R. Solubility of fatty acids in supercritical carbon dioxide. J. Am. Oil Chem. Soc. 1992, 69 (11), 1069-1076. (12) Monteiro, A. R.; Meireles, M. A. A.; Marques, M. O. M.; Petenate, A. J. J. Supercrit. Fluids 1997, 11 (1/2), 91-102. (13) Sovova´, H.; Zarevu´cka, M.; Vacek, M.; Stra´nsky, K. Solubility of two vegetable oils in supercritical CO2. J. Supercrit. Fluids 2001, 20, 15-28. (14) Sakaki, K. Solubility of β-carotene in dense carbon dioxide and nitrous oxide from 308 to 323 K and from 9.6 to 30 MPa. J. Chem. Eng. Data 1992, 37, 249-251. (15) Markon, M.; Singh, H.; Hasan, M. Supercritical CO2 fractionation of crude palm oil. J. Supercrit. Fluids 2001, 20, 4553. (16) Franc¸ a, L. F.; Meireles, M. A. A. Modeling the extraction of carotene and lipids from pressed palm oil (Elaes guuineensis) fibers using supercritical CO2. J. Supercrit. Fluids 2000, 18 (3), 35-47.
(17) Goto, M.; Sato, M.; Hirose, T. Extraction of peppermint oil by supercritical carbon dioxide. J. Chem. Eng. Jpn. 1993, 26 (4), 401-407. (18) Official Methods of Analysis of the Association of Agricultural Chemist, 16th ed.; AOAC: Gaithersburg, MD, 1995. (19) Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959, 37, 911-917. (20) Rodrigues, V. M.; Meireles, M. A. A.; Marques, M. O. M. Determination of solubility of clove essential oil in ScCO2: a standardization of the dynamic method. Proceedings of the 5th ISSF, Atlanta, GA, Apr 8-12, 2000. (21) Maia, E. L. Otimizac¸ a˜o da metodologia para caracterizac¸a˜o de constituintes lipı´dicos e determinac¸ a˜o da composic¸ a˜o em a´cidos graxos e aminoa´cidos de peixe em a´gua doce. Ph.D. Dissertation, Faculty of Food Engineering, Universidade Estadual de Campinas (UNICAMP), 1992; p 249. (22) McLaferty, F. W.; Stauffer, D. B. The Wiley/NBS Registry of Mass Spectral Data; John Wiley and Sons: New York, 1989; Vol. 1. (23) Freund, R. J.; Littell, R. C. SAS System for Regressions SAS Series in Statistical Applications, 2nd ed.; SAS Institute Inc.: Cary, NC, 1995; p 211. (24) Herrera, A.; Boussiba, S.; Napoleone, V.; Hohlberg, A. Recovery of c-phycocyanin from cyanobacterium Spirulina maxima. J. Appl. Phycol. 1989, 1, 325-331. (25) Angus, S., Armstrong, B., de Reuck, K. M., Eds. International Thermodynamic Tables of the Fluid StatesCarbon Dioxide; Pergamon Press: Oxford, U.K., 1976; p 385. (26) Povh, N. P.; Marques, M. O. M.; Meireles, M. A. A. Supercritical CO2 Extraction of essential oil and oleoresin from chamomile (Chamomilla recutita [L.] Rauschert). J. Supercrit. Fluids 2001, 21 (3), 245-256. (27) Esquı´vel, M. M.; Bernardo-Gil, M. G.; King, M. B. Mathematical Models for Supercritical Extraction of Olive Husk Oil. J. Supercrit. Fluids 1999, 16 (1), 43-58. (28) Sovova´, H. Rate of the vegetable oil extraction with supercritical CO2sI. Modeling of extraction curves. Chem. Eng. Sci. 1994, 49 (3), 409-414. (29) Reis-Vasco, E. M. C.; Coelho, J. A. P.; Palavra, A. M. F.; Marrone, C.; Reverchon, E. Mathematical Modeling and Simulation of Pennyroyal Essential Oil Supercritical Extraction. Chem. Eng. Sci. 2000, 55 (15), 2917-2922.
Received for review May 29, 2001 Revised manuscript received March 21, 2002 Accepted March 26, 2002 IE010469I
