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Supercritical Carbon Dioxide Extraction of Molecules of Interest from Microalgae and Seaweeds Christelle Crampon,* Olivier Boutin, and Elisabeth Badens Universite Paul Cezanne Aix-Marseille III, Mecanique, Modelisation et Procedes Propres, UMR CNRS 6181, Europole de l'Arbois, BP80, Pavillon Laennec, Hall C, Aix en Provence Cedex 04, France 13545 ABSTRACT: The purpose of this paper is to guide lectors in the extraction of algal (microalgae and seaweeds) compounds using supercritical carbon dioxide (SC-CO2) from dry biomass. It proposes a review of ∼30 articles dealing with the SC-CO2 extraction of molecules of interest from microalgae and seaweeds. Among these papers, ∼20 are devoted to microalgae. The most extracted compounds are neutral lipids and antioxidants. Several operating conditions have been tested: pressures from 7.8 to 70 MPa, temperatures from 313.15 to 349.15 K, and CO2/algae mass ratio from 6 to 500. All extraction studies were performed at laboratory scale, with the masses of dry algae powder never exceeding 180 g. Extraction yields vary significantly with operating conditions: pressure seems to be the most influential parameter. The higher the pressure, the higher the yields and/or the faster the extraction kinetics. Temperature also has an influence, but its effect is dependent on pressure (retrograde behavior). Moreover, as expected, it is advised to work with a high CO2/algae mass ratio. From these works, it appears that, to perform an efficient extraction with SC CO2, the influence of the algae pretreatment is highly significant. The first step is a centrifugation. The resulting concentrated algal suspension must then undergo a drying operation, which is generally freeze-drying or low-temperature drying. Finally, the algae are crushed. Concerning the influence of crushing, the reported results show that, as expected, the smaller the particles, the more rapid the extraction kinetics and/or the higher the yields.
1. INTRODUCTION Seaweeds and microalgae are photosynthetic organisms present in multitudes of species on the surface of the globe. They can develop in a marine environment and in fresh or brackish water and are ubiquitous in many environments, from polar ices to deserts or other extreme milieu. This adaptability and their biological diversity allow the prediction of the presence of a multitude of molecules of interest for many application fields, such as human health or energy production. Microalgae are able to accumulate oil up to 80% of their dry weight1 and particularly when submitted to nitrogen defaults. The oil quality, because of the presence of polyunsaturated fatty acids (such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or γ-linolenic acid (GLA))2 13 and antioxidants (i.e., β-carotene),4,14 16 allows their use in the food and pharmaceutical industries. Many microalgae are even able to synthesize antimicrobial and antiviral molecules, which increase the interest of pharmaceutical industries.13,17 19 Over the last two decades, microalga properties have also been explored in the energy field for the production of third-generation biofuels.17,20 34 The production of first-generation biofuels has the drawback of being in competition with the use of land for food crops; moreover, only a small fraction of the plant is used. With regard to the second-generation biofuels, they are produced from biomass using a high fraction or the totality of the plant; however, the high demand for biofuels leads to significant deforestation in certain regions of the world, which affects biotopes rich in biodiversity. In this context, microalgae would be an attractive alternative, since the depletion of land resources can be avoided. r 2011 American Chemical Society
Whatever the application fields, the extraction of molecules of interest from dry seaweeds or microalgae is usually performed using organic solvents such as n-hexane24,25 but such widely used solvents have major drawbacks: they are toxic, generally flammable, and low-selectivity solvents. This toxicity has a great impact, because it is problematic for the people who handle them during processing and some residual traces remain in the extracted end-product and in the residue. The new Regulation on Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH) came into force in Europe in 2007; thus, additional measures are being taken to improve human and environmental protection from exposure to highly dangerous chemical substances. Most of the organic solvents used for extraction are concerned by this legislation. Flammability is also an obvious problem during processing but also storage of residues due to the risk of explosion. Finally, for a liquid solvent, a separation step, generally energy-consuming, is necessary to remove the solvent from the extracted phase and because of their low selectivity, an additional separation step is sometimes necessary to recover a rich fraction of the molecule of interest. One alternative to avoid the use of toxic solvents to extract biooil or other molecules is to carry out extraction using supercritical carbon dioxide (SC-CO2) as a solvent. This safe and nonflammable solvent is selective. Moreover, the separation step to recover the target product may be avoided, since CO2 is gaseous at ambient pressure. This selectivity is obtained by varying pressure Received: November 14, 2010 Accepted: June 20, 2011 Revised: June 19, 2011 Published: June 20, 2011 8941
dx.doi.org/10.1021/ie102297d | Ind. Eng. Chem. Res. 2011, 50, 8941–8953
Industrial & Engineering Chemistry Research
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Table 1. Extracted Compounds from Algae and Microalgae Using Supercritical CO2 name
extracted compounds
applications
reference(s)
Seaweed Bangia atropurpurea
lipids
food
67
Chaetomorpha linum
lipids
energy
21, 22
Dictyopteris membranacea
volatile metabolites
health
64
Dilophus ligulatus
pigments, biological materials
health
50, 51
Galaxaura cylindrica
lipids
food
67
Grateloupia filicina
lipids
food
67
Helmintocladia australis
lipids
food
67
Hypnea charoides Liagora boergesenii
lipids lipids
food food
35 67
Liagora orientalis
lipids
food
67
Plocamium cartilagineum
halogenated monoterpenes
health
72
Porphyra angusta
lipids
food
67
Porphyra dentata
lipids
food
67
Sargassum hemiphyllum
lipids (5.39%(wt dry) algae)
food
36
Scenedesmus obliqqus
lipids
food
37
Scinaia monoliformis Undaria pinnatifida
lipids aliphatic hydrocarbons, polychlorinated biphenyls
food energy
67 45, 61
Botryococcus braunii
alkadiens (85%)
energy
46, 52
Chaetoceros muelleri
antioxidant extracts
health
74
Chlorella pyrenoidosa
antioxidant extracts, lutein
health
48, 68
Chlorella vulgaris
lipids (22 25%), carotenoids
food
39, 42, 46, 52
Dunaliella salina
β-caroten, antioxidant extracts
food, energy
52, 69, 77
Haematococcus pluvialis Nannochloropsis sp.
astaxantin (5%), carotenoids lipids (25%)
food health
53, 54, 62, 63 43
Ochromonas danica
lipids (28%)
food
55
Skeletonema costatum
lipids (8.6%), β-carotene
food
55
Microalgae
Spirulina maxima
lipids, γ-linolenic acid
food
40, 52
Spirulina pacifica
carotenoids, β-carotene, β-cryptoxanthin, zeaxanthin
food, health
60
Spirulina platensis
lipids (7.8%), carotenoids, and chlorophylls; antioxidant and antimicrobial extracts, vitamin E
food, health
6, 38, 41, 47, 49, 59
Synechococcus sp.
carotenoids and chlorophylls, β-carotene, β-cryptoxanthin, zeaxanthin
food, health
57, 58
and temperature. In addition, the depressurization can be done step by step, allowing a fractionating of the extracted compounds, based on their solubility variation with density. The extract yields, which depend on experimental conditions, can be the same as (or even higher than) those obtained with extraction processes using organic solvents, for shorter extraction durations. The environmental benefit in using microalgae is also more significant if extraction is done using a nonpolluting solvent such as SC-CO2. Moreover, the very low critical temperature of CO2 allows its use for thermolabile compound extractions. SC-CO2 solubilizes nonpolar compounds; when the molecule of interest is not soluble, the solvent power can be increased using a safe and polar modifier, such as ethanol. This technology is well-known today and is considered as a green process. Indeed, the use of organic solvent can be totally avoided, depending on the chemical nature of the extracted molecules. The major part of CO2 is recycled, therefore decreasing the consumption per extracted mass. For the last three decades, a large number of industrial extraction plants using supercritical fluids have been constructed, attesting that this technology is economically viable for a large number of applications.
This bibliographic study focused on the extraction from microalgae and seaweeds with SC-CO2. Seaweeds are included in the study because they were studied before microalgae for similar applications. The interest for the extraction of compounds from seaweeds or microalgae drastically increases, and SC-CO2 extraction should play an important role in the future. Among the identified papers dealing with SC-CO2 extraction of molecules of interest from microalgae and seaweeds, the main application fields are related to food and health, since one-quarter relates to the energy. The most extracted compounds are neutral lipids and antioxidants. This work proposes a critical overview on what has been done in this field, in order to highlight the experimental conditions leading to good extraction yields. The different algae used in supercritical extraction studies and the commercial applications of the extracted compounds are specified.6 8,21,22,35 78 Since algae pretreatment is very important for the success of the extraction operation, a review of the different pretreatments used is proposed. The oil extraction results obtained in the different studies reported in the literature are presented in a synthetic way. Finally, the last part is dedicated to the extraction of other compounds from algae. 8942
dx.doi.org/10.1021/ie102297d |Ind. Eng. Chem. Res. 2011, 50, 8941–8953
Industrial & Engineering Chemistry Research
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Table 2. Supercritical CO2 Extraction of Oil from Algae pretreatment
solvent
pressure,
temperature,
duration
lipid extracts
extraction
P (MPa)
T (K)
(h)
(%/wet g)
yield (%)
7
4.5
21, 22
reference(s)
Chaetomorpha linum drying at low temperature, mechanical crushing
CO2
freeze-drying, mechanical crushing (1 mm)
CO2
24.1
313.15
1
4.1
35
CO2
31.0
313.15
1
5.1
35
CO2
37.9
313.15
1
5.8
35
CO2
24.1
323.15
1
3.4
35
CO2 CO2
31.0 37.9
323.15 323.15
1 1
6.4 6.7
35 35
26.0
323.15
Hypnea charoides
Sargassum hemiphyllum freeze-drying, mechanical crushing (1 mm)
CO2
24.1
313.15
1
3.9
71.4
36
CO2
31.0
313.15
1
4.4
80.7
36
CO2
37.9
313.15
1
5.0
92.9
36
CO2
24.1
323.15
1
2.8
51.0
36
CO2
31.0
323.15
1
5.2
97.0
36
CO2
37.9
323.15
1
5.6
100
36
Scenedesmus obliqqus freeze-drying, manual crushing
CO2
37.9
313.15
4.2
37
CO2 + 15 mol %
37.9
313.15
13.9
37
3
21.5
67
3
13.6
67
3
17.0
67
3
19.7
67
3
19.8
67
3
18.8
67
3
17.6
67
3
12.4
67
3
11.2
67
3
13.3
67
ethanol Liagora boergesenii freeze-drying, manual crushing
CO2
freeze-drying, manual crushing
CO2
freeze-drying, manual crushing
CO2
freeze-drying, manual crushing
CO2
freeze-drying, manual crushing
CO2
freeze-drying, manual crushing
CO2
34.5
328.15
Grateloupia filicina 34.5
328.15
Scinaia monoliformis 34.5
328.15
Helmintthocladia australis 34.5
328.15
Galaxaura cylindra 34.5
328.15
Halymenia ceylanica 34.5
328.15
Liagora orientalis freeze-drying, manual crushing
CO2
freeze-drying, manual crushing
CO2
freeze-drying, manual crushing
CO2
freeze-drying, manual crushing
CO2
34.5
328.15
Porphyra angusta 34.5
328.15
Porphyra dentata 34.5
328.15
Bangia atropurpurea 34.5
2. LIST OF ALGAE AND MICROALGAE TREATED BY SUPERCRITICAL FLUID AND THEIR APPLICATIONS A synthesis of results from SC-CO2 extraction from seaweeds and microalgae is proposed. Approximately 30 different algae have been used for the SC-CO2 extraction of compounds of interest. Table 1 reports the extracted compounds for each alga or microalga studied.
328.15
Some algae and microalgae are known to have exceptional nutritional value, because of their content of antioxidants, fibers, phytosterols, nutriments, ... It is not surprising that the main application field is the health sector with interest for the food and pharmaceutical industries35 43,47 55,57 60,62 65,67 69,72,77 with supercritical extraction of lipids or molecules devoted to human or animal health. Indeed, algal lipids show much nutritional 8943
dx.doi.org/10.1021/ie102297d |Ind. Eng. Chem. Res. 2011, 50, 8941–8953
Industrial & Engineering Chemistry Research
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Table 3. Supercritical CO2 Extraction of Oil from Microalgae CO2/dry pressure, P (MPa)
temperature, T (K)
CO2flow rate (kg/h)
CO2
20.0
313.15
20.2
25
CO2 CO2
20.0 20.0
313.15 313.15
20.2 20.2
CO2
20.0
328.15
CO2
35.0
313.15
CO2
35.0
CO2
amount of CO2 (kg)
lipid duration (h)
extracts (%/wet g)
1.25
1.4
42
40 141
2 7
2.0 2.5
42 42
18.4
110
6
3.6
42
22.4
157
7
3.2
42
328.15
21.4
148
7
4.8
42
20.0
313.15
20.2
25
1.25
4.0
42
CO2
20.0
313.15
20.2
40
2
7.0
42
CO2 CO2
20.0 20.0
313.15 328.15
20.2 18.4
141 110
7 6
8.0 4.2
42 42
CO2
35.0
313.15
22.4
157
7
12.2
42
CO2
35.0
328.15
21.4
148
7
13.0
CO2
35.0
328.15
20
17
52
CO2
35.0
328.15
40
18
52
CO2
35.0
328.15
20
42
52
CO2
35.0
328.15
40
48
52
CO2
35.0
328.15
20
50
52
CO2
35.0
328.15
40
>60
52
CO2
25.0
323.15
0.5
100
4
0.9
40
CO2
25.0
323.15
1.5
300
12.5
1.5
40
CO2 + 10 mol %
25.0
323.15
1.5
300
12.5
2.0
40
CO2
25.0
323.15
1.4
280
2.5
52
CO2 + 10 mol %
25.0
323.15
1.3
260
3.1
52
ethanol CO2 + 10 mol %
25.0
333.15
1.2
240
2.2
52
CO2
25.0
313.15
10.00
40
500
4
7.5
96.1
40
CO2
25.0
315.15
10.00
10
125
1
3.2
41.0
40
CO2
40.0
315.15
10.00
10
125
1
5.6
71.7
40
CO2
55.0
315.15
10.00
10
125
1
7.0
89.9
40
CO2
70.0
315.15
10.00
10
125
1
7.5
96.4
40
CO2
25.0
328.15
10.00
10
125
1
2.0
25.3
40
CO2
40.0
328.15
10.00
10
125
1
4.8
61.2
40
CO2
55.0
328.15
10.00
10
125
1
7.8
99.7
40
CO2 CO2
70.0 40.0
328.15 313.15
10.00 24.00
10
125
1 4
7.8 7.0
pretreatment
solvent
algae (kg/kg)
extraction yield (%)
reference
Chlorella vulgaris freeze-drying, no crushing
freeze-drying, crushing
freeze-drying,
42
entire cells slightly crushed cells well-crushed cells
Spirulina maxima freeze-drying, crushing (0.2 mm)
ethanol freeze-drying, crushing
ethanol Spirulina platensis freeze-drying, crushing (0.37 mm)
no pretreatment
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100
40 47
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Table 3. Continued CO2/dry pressure, P (MPa)
temperature, T (K)
CO2flow rate (kg/h)
CO2
40.0
313.15
10.00
CO2
55.0
313.15
CO2
70.0
313.15
CO2
40.0
CO2
amount of CO2 (kg)
lipid
algae (kg/kg)
duration (h)
extracts (%/wet g)
extraction yield (%)
reference
14.00
77.8
1.4
13.5
54
43
10.00
14.00
77.8
1.4
19
76
43
10.00
14.00
77.8
1.4
22
88
43
328.15
10.00
14.00
77.8
1.4
15
60
43
55.0
328.15
10.00
14.00
77.8
1.4
20
80
43
CO2
70.0
328.15
10.00
14.00
77.8
1.4
23
92
43
CO2
40.0
313.15
10.00
28.00
155.6
2.8
20
80
43
CO2
55.0
313.15
10.00
28.00
155.6
2.8
23
92
43
CO2 CO2
70.0 40.0
313.15 328.15
10.00 10.00
28.00 28.00
155.6 155.6
2.8 2.8
25 21
100 84
43 43
CO2
55.0
328.15
10.00
28.00
155.6
2.8
24
96
43
CO2
70.0
328.15
10.00
28.00
155.6
2.8
25
100
43
CO2
17.2
313.15
0.279
46.5
4
3.3
CO2
24.0
313.15
0.279
46.5
4
CO2
31.0
313.15
0.279
46.5
4
CO2
17.2
313.15
0.279
46.5
CO2
24.0
313.15
0.279
CO2
31.0
313.15
0.279
pretreatment
solvent
Nannochloropsis sp. freeze-drying, crushing (0.37 mm)
Ochromonas danica freeze-drying,
11.8
55
2.5
8.9
55
6.0
21.4
55
4
0.6
3.0
55
46.5
4
2.5
29.1
55
46.5
4
1.7
19.8
55
crushing
Skeletonema costatum freeze-drying, crushing
interest, thanks to their high omega-3 fatty acid content. Marine algae are particularly rich in R-linolenic and eicosapentaenoic fatty acids. They also moderately contain docosapentaenoic and docosahexaenoic fatty acids.36 The second field is the use of algae and microalgae as a potential renewable energy source,21,22,45,46,52,61 since their content in neutral lipids can be high. Table 1 shows that, among microalgae, the microalgae Spirulina platensis and Chlorella vulgaris are the ones cited most often, since they offer the best compromise between an easy algae culture as well as the presence of co-products which may offer health benefits. At this point, note that studies of SC CO2 extractions from microalgae have been conducted at laboratory scale. The mass of processed dry biomass is generally in the range of tens of grams and never exceeds 180 g.43 Such data are therefore insufficient to consider the scale-up issues.
3. PRETREATMENT OF RAW MATERIALS When an alga is harvested, a suspension with a low concentration of dry matter is produced (a few grams per liter). This suspension must first be preconcentrated. Concentrated algae are then dried and crushed. The preconcentration step can be realized by centrifugation, followed by pressing or filtration. Nevertheless, this first step of pretreatment is not detailed in the different cited works.
After the preconcentration step, the algae are in the form of a pasty fluid, ∼25% of dry biomass. A drying step is then necessary, because of the presence of water in large quantities. For SC-CO2 extraction, the humidity rate should be 35 MPa may be economically advantageous, since as the pressure increases, the duration of the experiment decreases and the amount of CO2 may also decrease significantly. With regard to the CO2/algae mass ratio, values up to 500 were studied at laboratory scale. Rather, the ratio used at the industrial scale should be ∼50. Such mass ratio ranges may seem large, but it should be noted that the CO2 is recycled at the industrial scale. Lastly, the CO2 flow rate is also an important parameter and will influence the equipment cost. 5.2. Neutral Lipid Oil Composition. Concerning neutral lipids, when extraction yields obtained with pure SC-CO2 are at a maximum level (100%), the extract composition is obviously the same as in the oil initially contained in the algae. Each algae contains an oil with a given composition in fatty acids, but such oil is generally composed of fatty acids from 14:0 to 20:0 with strong fractions of 16:0, 18:1(omega-9), 20:4(omega-6), and 20:5(omega-3). However, thanks to the selectivity of SC-CO2, it is possible to slightly modify the composition of the oil, provided that lower yields are accepted. Cheung35 has shown that, under low pressures, more saturated fatty acids are extracted. In contrast, as the pressure is increased, the proportion of unsaturated fatty acids increases in the extracted phase. Concerning omega-3 fatty acids, Cheung et al.36 showed that the best yields occurred at low temperatures and high pressures. Little information is given about the coloration of extracted oil while algae and microalgae are known to contain significant amounts of red (carotenoids) and/or green (chlorophylls) pigments. Only Subra et al.,50,51 Choi,37 Gouveia et al.,39 and Mendes et al.42 reported that such pigments were extracted with the oil. Since they are generally liposoluble, it appears difficult to avoid their extraction (even by changing pressure and temperature conditions).
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5.3. Comparison with Extraction Using Organic Solvents. Some authors38,40,42,43,52 have compared SC-CO2 extraction yields and duration to those obtained using organic solvents: n-hexane, ethanol, acetone, or mixtures of chloroform, methanol, and water (following the method of Bligh and Dyer79). In addition to the several advantages of using SC-CO2 mentioned in the Introduction, the duration of the extraction can be radically decreased with SC-CO2, compared to the extraction with an organic solvent (from 72 h42 to 2 h52). Cheung et al.36 have shown for the alga Sargassum hemiphyllum that SCCO2 extraction under 37.9 MPa and at 323.15 K for 1 h resulted in maximum yields of lipids and was as effective as Soxhlet extraction (chloroform:methanol ratio of 2:1). Andrich et al.38 have compared their yields to those obtained with an extraction using n-hexane, using a Soxhlet apparatus. Table 4 reports their experiments: n-hexane extraction needed 8 h to extract the total amount of lipids, whereas SC-CO2 extraction needed
