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1 Extraction of carotenoids and lipids from algae by supercritical CO2 and

2 subcritical dimethyl ether

4 Motonobu Gotoa, *, Hideki Kandaa, b, Wahyudionoc, Siti Machmudahd

a
5 Department of Chemical Engineering, Nagoya University, Furo-cho, Chikusa-ku,

6 Nagoya 464-8603, Japan

b
7 Japan Science and Technology Agency, Chiyoda, Tokyo 102-0076, Japan
c
8 Department of Chemical Engineering, University of Surabaya, Raya Kalirungkut,

9 Surabaya 60293, Indonesia

d
10 Sepuluh Nopember Institute of Technology, Kampus ITS, Sukolilo Surabaya 60111,

11 Indonesia

12

13

14 * Corresponding author. Tel.: +81-52-789-3392; fax: +81-52-789-3389.

15 E-mail address: mgoto@nuce.nagoya-u.ac.jp (M. Goto)

16

17 Abstract

18 Algae contain lipids and functional compounds such as carotenoids. Especially,

19 micoalgae are recently focused as a source of biofuel. To extract these components,

20 organic solvent or supercritical carbon dioxide have been used. We have been

21 developing wet extraction process using liquefied (subcritical) dimethyl ether (DME) as

22 solvent at around 0.59 MPa. The extraction process usually requires energy consuming

23 drying and grinding process as in the case of supercritical CO2. We have applied

24 liquefied DME for the extraction of lipids and functional compounds from various kinds

© 2014 published by Elsevier. This manuscript is made available under the Elsevier user license
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25 of algae. Since the liquefied DME extraction process can eliminate the process for

26 drying, cell disruption, and solvent evaporation, it can realize simpler and low energy

27 consumption system. In this paper, our studies on the extraction from algae by using

28 supercritical CO2 or subcritical DME are reviewed.

29

30 Keywords: Algae, lipid, carotenoid, extraction, carbon dioxide, dimethyl ether

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31 1. Introduction

32 Algae are a diverse group of eukaryotic organisms, ranging from unicellular to

33 multicellular forms, and various species contain valuable organic compounds. Algae are

34 known to possess several functional properties such as antioxidative, anticancer,

35 antiviral, and antiobesity activities. They contain lipids, sulfated cell-wall

36 polysaccharides such as fucoidans, and several functional compounds. Recently,

37 microalgae have become the focus of several research topics owing to their utility as

38 sources of biofuel. A well-known species is the brown alga Undaria pinnatifida,

39 commonly known as “wakame” in Japan. Wakame is an edible seaweed that is widely

40 consumed and is a part of the diet in several countries, especially Korea and Japan. The

41 microalga Haematococcus pluvialis is the richest known natural source of the

42 ketocarotenoid astaxanthin. Astaxanthin (3,3’-dihydroxy-β,β’-carotene-4,4’-dione) is

43 vibrantly red pigmented; therefore, it is used as a source of pigments in poultry and

44 aquaculture industries. Furthermore, astaxanthin also has several key biological

45 functions in poultry animals; it serves as a precursor of vitamin A and is associated with

46 cell reproduction, embryo development, as well as protection against oxidative damage.

47 Astaxanthin has been gaining widespread popularity as a human dietary supplement

48 owing to its superior antioxidant properties compared to those of β-carotene, α-carotene,

49 lutein, lycopene, canthaxanthin, and vitamin E [1-4]. Several astaxanthin-based

50 products are now commercially available and are being promoted as anticancer,

51 anti-inflammatory, and immunostimulating agents. Another important microalgal

52 species is Chlorella vulgaris, which is known to contain very high concentrations of

53 chlorophyll and a large amount of carotenoids like astaxanthin and cantaxanthin and

54 minor amounts of β-carotene and lutein. Carotenoids derived from C. vulgaris have

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55 already been successfully tested as sources of pigments for poultry products, along with

56 their ability to impart functional activity (e.g., antioxidative activity) [5-8]. Lutein is one

57 of the most abundant carotenoids acquired through diet. Lutein is a desirable carotenoid

58 since it is inexpensive and possesses antioxidant and anticarcinogenic properties. It can

59 be obtained from animal sources such as egg yolk and plant sources such as marigold.

60 However, the production of lutein from these sources is limited by a number of reasons.

61 For example, homogeneous biomass cannot be produced at a constant rate at all times

62 and under various weather conditions because marigold is a plant.

63 In this study, subcritical dimethyl ether (DME; critical temperature, 126.85 °C;

64 critical pressure, 5.37 MPa) [9-12] and supercritical carbon dioxide (CO2) were used

65 [12-15] to extract lipids and the functional compounds (carotenoids) from various algae.

66 Supercritical fluids are widely accepted for extraction, purification, recrystallization,

67 and fractionation operations in many industries and are known to be more efficient

68 extraction fluids than traditional liquid solvents. Supercritical fluids act as selectively

69 dissolving liquid solvents when pressure and temperature are adjusted. Supercritical

70 CO2 is by far the most common supercritical fluid used in extraction of natural

71 compounds and in food processing. Since CO2 can be readily separated by process

72 depressurization, the extracts obtained after using supercritical CO2 are highly

73 concentrated. This leaves no trace of harsh organic chemicals or residues in the product.

74 Furthermore, since the CO2 gas stream can be recycled, supercritical CO2 extraction can

75 be regarded as an environmentally friendly process. Liquefied (subcritical) DME has

76 also used as an extractant in place of CO2 to extract fucoxanthin from Undaria

77 pinnatifida [12, 16]. DME is the simplest form of ether, possessing the following

78 characteristics: (i) DME has a low normal boiling point (−24.8 °C); therefore, it is not

4
 79 present in the final products at normal temperatures [17]; (ii) Relative permittivity of

80 DME is 1.08 and 5.34 at 30.5 °C, in gaseous and liquid states, respectively. Liquefied

81 DME has high affinity for oily substances [18] and partial miscibility with water [19];

82 (iii) DME has been approved as a safe extraction solvent for the production of

83 foodstuffs and food ingredients by the European Food Safety Authority (EFSA) [20], by

84 the Food Standards Australia New Zealand, and by the United States [21]. The EFSA

85 panels also consider the intended use of dimethyl ether as an extraction solvent to

86 remove fat from animal protein raw materials provided: (a) the defatted animal protein

87 is submitted to vacuum, which assures that most of the volatile dimethyl ether is

88 eliminated from the final animal protein products; (b) the maximum residual limit of

89 dimethyl ether is 9 μg kg-1 of extracted animal proteins and; (c) the extracted proteins

90 are used at a level of up to 2% in the final food product, at which the Panel considers

91 that there is no safety concern; (iv) DME exhibits resistance to autoxidation, unlike

92 other alkyl ethers [22].

93 Based on the characteristics of DME mentioned above, Kanda reported that

94 liquefied DME can be used as a solvent to extract water from high-water content solids

95 [23, 24], including low rank coals [23, 25-27], plants, foods [24, 28], sewage sludge

96 [29], and polymers [30]. Similarly, Catchpole et al. reported the extraction of pungent

97 components from low-water content chili, black pepper, and ginger by liquefied DME

98 [31]. Sato and Matsumura reported extraction of phenol in water phase by liquefied

99 DME [32]. Thereafter, Kanda [33] and Catchpole et al. [34] applied the water extraction

100 method for the extraction of oily substances from high-water content solids. Kanda et al.

101 reported extraction of odorous substances from sewage sludge [29, 35, 36], extraction of

102 heavy oil from oil-polluted soil [37], extraction of polychlorinated biphenyls from river

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103 sediment [38, 39], extraction of caffeine and catechins from green tea leaves [40], and

104 extraction of vegetal oil from plants [28]. Catchpole et al. further reported the use of

105 DME as an effective antisolvent for proteins in an aqueous solution and for

106 water-soluble coating compounds. Lipids were extracted from aqueous protein/lipid

107 mixtures, and this method was demonstrated on fresh and reconstituted egg yolks and

108 on selected dairy by-product streams [41, 42]. Catchpole et al. have also reported the

109 extraction of lipids from yeast and soil fungi [43]. Kanda et al. and Billakanti et al. have

110 also reported on the extraction of carotenoids and lipids from the macroalga U.

111 pinnatifida [12, 44] and microalgae Botryococcus braunii, Oscillatoria agardhii,

112 Microcystis aeruginosa, and several more by using liquefied DME [8-12].

113

114 2. Materials and Methods

115 2.1 Materials

116 All algae used in this study as starting materials were commercially available

117 in Japan. Samples were stored at 0 °C in tightly sealed aluminum bags until use to

118 prevent degradation of labile compounds. Standard lutein (70% purity), β-carotene

119 (80% purity), chlorophyll a (90% purity), and chlorophyll b (80% purity) and HPLC

120 grade ethanol, acetone, methanol, THF, and chloroform used for analysis were

121 purchased from Wako Pure Chemical Industries, Ltd., Japan. CO2 and DME were

122 obtained from Uchimura Co. Japan, Sogo Kariya Sanso, Inc. Japan and Tamiya, Inc.

123 Japan, respectively.

124

125 2.2. Analytical Methods

126 The major carotenoids were determined by using HPLC (high-performance

6
127 liquid chromatography). The organic components in algal extracts were recovered with

128 2 mL of analytical solvent. All solutions were filtered using a disposable 0.45-μm filter

129 prior to HPLC analysis. Calibration curves (5 points) were obtained by using pure

130 carotenoid standards (fucoxanthin, astaxanthin, lutein, chlorophyll a, chlorophyll b, and

131 β-carotene). The HPLC instrument was equipped with an ultraviolet–visible

132 spectroscopy detector (UV-970; JASCO, Tokyo, Japan) and a diode array detector

133 SPD-M10A (Shimadzu, Japan), equipped with an ODS-3 column (5μm; 250 mm × 4.6

134 mm; GL Sciences, Tokyo, Japan), STR ODS-II column (5 μm; 4.6 × 250 mm; Shinwa

135 Chemical Industries, Ltd., Japan), and 5C18-MS Waters column (5μm; 4.6 × 150 mm;

136 Nacalai Tesque, Inc., Japan). The analytical methods used have been previously

137 described in detail [12-14]. All extracts were analyzed in duplicate.

138

139 2.3. Experimental Apparatus

140 Figure 1 shows a schematic diagram of the supercritical CO2 extraction

141 apparatus. This apparatus includes a high-pressure pump for CO2 (PU-2086; Jasco,

142 Hachioji, Japan), a heating chamber (WFO-400; EYELA, Tokyo, Japan), a 25-mL

143 extraction cell (Thar Technologies, Inc., Pittsburgh, PA, USA), and a back pressure

144 regulator (AKICO, Tokyo, Japan). In this study, the extraction of carotenoids by

145 supercritical CO2 was conducted at temperatures of 40–80 °C and pressures of 20–40

146 MPa by using a semi-continuous flow-type system under various CO2 flow rates. In

147 each experiment, 3–7 g of dry algae sample was loaded into the extraction vessel, filled

148 with glass beads at the bottom and top. The extraction vessel was placed in the heating

149 chamber to maintain the operating temperature. Extracts were collected every 30–60

150 min for 5 h and were weighed and analyzed immediately after collection. Bottles used

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151 for extract collection were wrapped in aluminum foil, and in all experiments, including

152 DME extraction, products were stored at −60 °C without further treatment. Extracts

153 were maintained under the conditions mentioned above until further analysis.

154 Co-solvent effect of ethanol on supercritical CO2 extraction was also examined. Ethanol

155 flow rates supplied by using high-pressure pump (PU-2080; Jasco, Hachioji, Japan)

156 were between 0.05 and 0.5 mL min−1.

157 For liquefied DME extraction, a semi-continuous flow-type apparatus was used.

158 Figure 2 depicts the scheme of apparatus used to evaluate the extraction efficiency of

159 the process. The major components of this apparatus were an extractor (HPG-10-5;

160 Taiatsu Techno Corp., Saitama, Japan; volume, 10 cm3), a needle valve to control the

161 DME flow rate, and an extract storage tank (HPG-96-3; Taiatsu Techno Corp.; volume,

162 96 cm3). These components were connected in series. The extractor and DME storage

163 tank were made of pressure-resistant glass coated with polycarbonate. Raw algae (water

164 content: 93.2%) was loaded into the lower half of the extractor, and the upper half was

165 loaded with colorless glass beads. DME flow rate was maintained at 10 ± 2 cm3 min−1.

166 All experiments were carried out in a temperature-regulated room, and the column

167 temperature was adjusted to approximately 25 ± 1 °C and measured by an infrared

168 thermometer. The pressure was approximately 0.59 ± 0.02 MPa, which is the saturated

169 vapor pressure at 25 ± 1 °C. Liquefied DME was passed through the extractor at

170 different time intervals after which it was evaporated by opening the reducing valve of

171 the storage vessel. Following this, liquefied DME was passed through the lower half of

172 the extractor whereby the color of the glass beads in the upper half changed to

173 olive-green. Since the glass beads and liquefied DME were colorless, a change of the

174 glass bead color to olive-green indicated probable presence of extract. The extraction

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175 test was completed when the color of the glass beads was restored to colorless. The total

176 extraction time was less than 43 min.

177

178 3. Results and Discussion

179 3.1 Supercritical CO2 extraction

180 Figures 3a and 3b show the fucoxanthin extraction curves from U. pinnatifida

181 at different extraction times and under varying pressure (10–40 MPa; 60 °C) (Fig. 3a)

182 and varying temperature (40–70 °C; 40 MPa) (Fig. 3b). The physical appearance of the

183 extracts was oily and their amount increased with increase in pressure at constant

184 temperature. This tendency is attributed to a direct increase in density and hence the

185 solvating power of supercritical CO2. The yield of fucoxanthin increased significantly

186 up to an extraction time of 150 min. For example, the yield was about 47 µg g-1 dry

187 weight of U. pinnatifida after extraction for 30 min at 40 MPa and 60 °C. This increased

188 gradually to 58 µg g-1 dry weight at an extraction time of 150 min. Beyond an extraction

189 time of 150 min, the yield of fucoxanthin did not increase significantly. At the same

190 extraction temperature, a similar trend was observed at 10, 20, and 30 MPa. As

191 previously mentioned, the effect of pressure can be attributed to the increase in solvent

192 power due to the strengthening of intermolecular physical interactions. An increase in

193 pressure also affected the solubility of fucoxanthin in supercritical CO2. These results

194 are in agreement with the trends reported in other studies.

195 At constant pressure (40 MPa), the yield of fucoxanthin increased with

196 increasing extraction temperature. The yield increased rapidly until 150 min of

197 extraction and then remained constant after 200 min at each extraction temperature. The

198 extraction rate of fucoxanthin was highest when the extraction temperature was 60 °C

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199 (Fig. 3b). This indicated the dependence of fucoxanthin extraction on solute vapor

200 pressure which increased with an increase in temperature. Increasing the temperature

201 contributed to the decomposition of cell walls, and as a result the amount of fucoxanthin

202 and other extractable compounds increased. Figure 3b also showed that an increase in

203 temperature likely had a stronger effect on the solubility than an increase in pressure. At

204 70 °C, the yield of fucoxanthin is lower than at 60 °C. This might be possible due to the

205 degradation of fucoxanthin at higher temperature, considering that carotenoids are

206 heat-sensitive.

207 Figure 4 shows the amounts of astaxanthin extracted from Haematococcus

208 pluvialis by supercritical CO2 extraction with and without ethanol as an entrainer, under

209 various pressure conditions at 40 °C, when CO2 flow rate is maintained at 3 mL min−1.

210 In this work, the effect of addition of the entrainer was studied mainly at low

211 temperature, since the rate of extraction under this condition was low. Experiments were

212 conducted at up to 40 MPa, since the maximum capacity of the entrainer pump was 50

213 MPa. In this work, the use of ethanol at a concentration of 1.67% was found to enhance

214 the amount of extracted astaxanthin more than 2-fold. A significant increase in

215 extraction efficiency was due to ethanol that enhanced the solubility of specific

216 compounds in supercritical CO2. Ethanol functioned as polarity modifier and led to

217 swelling of the matrix [45-49], thus increasing the internal volume and the surface area

218 for the contact with supercritical CO2. The addition of ethanol in supercritical CO2 also

219 resulted in decomposition of the cell wall [49-51], resulting in increased astaxanthin

220 available for extraction. Under 40 MPa pressure and at 40 °C, the amount of extracted

221 astaxanthin also increased with increasing ethanol concentration (up to 5% (v/v)

222 ethanol). At this condition, more than 80% of astaxanthin could be extracted. However,

10
223 at a higher entrainer concentration of 7.5% (v/v), lower amount of astaxanthin was

224 extracted. This was probably due to the lowering of the density of the supercritical fluid.

225 Table 1 shows the main components extracted from C. vulgaris by supercritical

226 CO2 using entrainer (based on HPLC chromatogram). The yield of each pigment in the

227 extract, obtained by supercritical CO2 with ethanol as an entrainer, was higher than that

228 obtained with and without acetone as a co-solvent. This is because the solubility of

229 pigments, especially lutein and chlorophyll, is higher in ethanol than in acetone.

230 Pigments are soluble in alcohol but have low solubility in ketones. In addition, ethanol

231 present in supercritical CO2 led to modification of the characteristics of supercritical

232 CO2, and resulted in enhancement of its solvent properties, thus facilitating the

233 extraction of pigment components. In this case, the undesirable components were not

234 extracted together with the target component. Furthermore, also when food safety is

235 considered, ethanol is more appropriate as an entrainer than acetone owing to its low

236 toxicity compared with acetone.

237

238 3.2 Liquefied (Subcritical) DME Extraction

239 Figure 5 shows the yield of fucoxanthin extracted from U. pinnatifida with

240 liquefied DME. Increasing amount of DME led to an increase in the amount of

241 extracted fucoxanthin. When 286 g DME was used (at the last time point of extraction

242 process), the residue of the U. pinnatifida was almost completely dehydrated and almost

243 resembled a dried paper. At this point, the color of U. pinnatifida residue was light

244 green and the amount of fucoxanthin was approximately 390 µg g-1 dry weight of U.

245 pinnatifida. It is well known that liquefied DME can dissolve a wide range of polar and

246 non-polar substances. They are also good solvents for many hydrogen-bonded

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247 substances. To dissolve hydrogen-bonded substances, high solvation energy was needed.

248 In this case, DME has the ability to form hydrogen bonds with hydrogen-bonding

249 solutes. Therefore, liquefied DME can penetrate into U. pinnatifida cells and extract

250 components like fucoxanthin into the medium. The cumulative fucoxanthin extracted

251 seemed significantly enhanced with increasing DME consumption. Like previously

252 mentioned, despite possessing a high dissolving ability, liquefied DME could also

253 generate a less viscous matrix, and therefore allowed better diffusion of the solute from

254 the solid phase to the solvent. Consequently, the extraction of fucoxanthin in U.

255 pinnatifida was achieved easily.

256 Lipids can be extracted from various types of algae by liquefied DME owing to

257 its characteristic properties. A basic scheme of the process of extraction of lipids is

258 depicted in Fig. 6. The operational temperature shown in Fig. 6 can be set arbitrarily

259 close to the ordinary room temperature, and 30 °C, which is depicted in the figure, is a

260 representative example. The operational pressure is the saturated vapor pressure of

261 DME. After partial dewatering, microalgae are collected and packed in the extraction

262 column. Water and lipids are further extracted using liquefied DME and are discharged

263 from the extraction column in the form of a mixture. In case the lipid concentration is

264 not sufficiently high, the mixture is returned to the extraction column. The DME with

265 saturated extracted oil is heated and evaporated using hot water. The residual waste heat

266 from a high-grade heat source can be used for this purpose. However, sun-warmed hot

267 water or heat generated from small biomass electric generator with not much of

268 electricity generation efficiency is desirable. 45 °C setting of sun-warmed water as

269 depicted in the figure is representative, and it is advisable to increase the approach

270 temperature in order to reduce the size of the heat exchanger. The evaporated DME is in

12
271 a gaseous high-pressure state at ordinary temperature that can be condensed by using

272 seawater and reused as liquefied DME. In order to reduce the size of the heat exchanger,

273 it is advisable that the temperature of the cold source is low. In this context, the actual

274 temperature of circulating DME may be decided based on the temperature of the cold

275 source and the approach temperature of the heat exchanger.

276 Figure 7 shows a comparison of the yield of lipid extraction from various algal

277 sources using the liquefied DME and Bligh-Dyer method. Microalgae are initially

278 partially dried by centrifugation and filled in a column of inner volume of 5 cm3 in a

279 state of 70%–90% water content. The quantity of oil obtained after passing sufficient

280 amount of liquefied DME through the column is shown in Fig. 7. The proportion of

281 lipids obtained by means of liquefied DME, based on dry weight of microalgae, is

282 shown in white. Lipids obtained using the Bligh-Dyer method, wherein the microalgae

283 are dried and cell disrupted, are shown in black. The ratio of the weight of the lipid

284 obtained by DME to that extracted by Bligh-Dyer method is mentioned. The Bligh-Dyer

285 method makes use of a methanol-chloroform mixture and is regarded effective for the

286 extraction of lipids of various polarities. As seen in Figure 7, at least 97% lipid was

287 obtained from the different algal sources by using liquefied DME by considering

288 Bligh-Dyer method as a reference. Maximum amount of lipids was obtained from the

289 blue-green microalgae collected from Hirosawa lake in Kyoto city, Japan, and it

290 accounted for 40.1% of dry weight in case of liquefied DME. In the Bligh-Dyer method,

291 it was 40.2%. In this case, if the cell-rupture treatment was not used in the Bligh-Dyer

292 method, the quantity of extracted oil was not more than 0.6%. Thus, it is clear that

293 microalgal cell disruption, which is a necessary step in the Bligh-Dyer method to

294 increase the amount of extracted lipids, can be skipped when the liquefied DME method

13
295 is used. Based on the analysis of lipid properties; such as molecular weight distribution

296 determined by gel permeation chromatography, elemental composition and calorific

297 value; it was concluded that the properties of lipids obtained by using liquefied DME

298 were almost the same as that obtained by the Bligh-Dyer method. Furthermore, the

299 extraction of hydrocarbons from Botryococcus braunii by using DME, without using

300 any pretreatment like cell-drying or cell-disruption, could be confirmed.

301

302 3.3 Development of Extraction Technology

303 The process of extraction of bioactive compounds from plants has usually been

304 accomplished by traditional methods such as solid-liquid extractions employing

305 methanol, ethanol, acetone, or hexane and through steam distillation or evaporation

306 processes for removal of solvents from extracts. Currently, the demand for natural

307 bioactive compounds is on the rise due to their application in the functional food and

308 pharmaceutical industry [52]. To cope with this demand, the development of an

309 appropriate extraction technology that is able to provide high-quality extracts is needed.

310 Development of an efficient extraction technology is also needed for the following

311 reasons: (a) tightening government regulations on toxic-chemical solvent residues and

312 pollution control, (b) consumer concerns over the use of toxic-chemical solvents during

313 processing, and (c) increased demand for higher quality products that traditional

314 processing techniques cannot meet [52]. In this study, the connection between

315 supercritical CO2 and liquefied DME as clean extraction processes for bioactive

316 compounds from algae has been demonstrated. This process could be operated safely

317 and is one of the major advantages associated with the development of new extraction

318 processes. As a non-polar substance, SCCO2 mainly solubilizes low molecular weight,

14
319 non-polar compounds and is a poor solvent for high molecular weight, polar compounds.

320 This makes extractions using SCCO2 very selective due to penetration of CO2 into

321 complex matrices. Further, the solubility of polar solutes in SCCO2 could be

322 significantly increased by adding a small amount of polar co-solvent. However, this

323 process might be difficult to apply due to potential issues concerning separation of the

324 co-solvent at a later time point. This problem was overcome by employing DME as an

325 extractant. In summary, we developed a very simple, versatile, and feasible technique

326 for extraction of bioactive compounds from microalgae.

327

328 4. Conclusions

329 This study provides an overview about the extraction of carotenoids and lipids

330 from algae by using supercritical carbon dioxide. This process employing subcritical

331 DME saved energy and was effective for extraction of bioactive compounds from wet

332 algae. Since the water content of biomaterials is very high, thorough drying is necessary.

333 However, drying of wet biomaterials is highly energy consuming. Subcritical (liquefied)

334 DME is unique and suitable for extraction of water and oily substances from high

335 water-content biomaterials and has potential to cut energy consumption of extraction

336 procedures.

337

338 Acknowledgment

339 A part of this research was supported by Industrial Technology Research Grant

340 Program (Project ID: 09B40009c, H. Kanda) from New Energy and Industrial

341 Technology Development Organization of Japan and a grant from the Precursory

342 Research for Embryonic Science and Technology Program (PRESTO; H. Kanda) of

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343 Japan Science and Technology Agency.

344

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496

22
497 Figure captions

498 Figure 1. Schematic diagram depicting supercritical CO2 extraction

499 Figure 2. Schematic diagram depicting liquefied DME extraction

500 Figure 3a. Effect of pressure on fucoxanthin extraction as a function of time at an

501 extraction temperature of 60 °C [12].

502 Figure 3b. Effect of temperature on fucoxanthin extraction as a function of time at an

503 extraction pressure of 40 MPa [12].

504 Figure 4. Comparison of supercritical CO2 extraction with and without 1.67% entrainer

505 at various pressure conditions, at 40 °C and a flow rate of 3 mL min-1 [13].

506 Figure 5. Fucoxanthin yield in the extract obtained from wet Undaria pinnatifida by

507 liquefied DME [12].

508 Figure 6. Lipid extraction from wet microalgae by using liquefied DME.

509 Figure 7. Lipid extraction ratio for several species of natural microalgae by using the

510 DME method [10].

511

512

513

23
514 Table 1. Composition of Chlorella vulgaris extract at 60 °C, 30 MPa, and CO2 flow rate

515 of 2.5 mL min−1, with 7.5% ethanol for 120 min [13].
516
Retention time [min] Component Peak area [%]
2.22 Unidentified 16.91
2.84 Lutein 19.00
3.63 Unidentified 6.32
5.10 Chlorophyll b 2.83
7.17 Chlorophyll a 37.50
7.89 Unidentified 9.70
15.41 Unidentified 4.71
19.23 β-carotene 2.93
517
518

24
519
520
521
522 Back pressure valve
Flow meter
523 Solvent mixer
524
525
Extractor
526
527 CO2
pump
528
529
530 extract CO2
531
532
Water
533 Chiller Heating
bath
chamber Glass beads
534 Ethanol
CO2 cylinder
535 pump
536
Ethanol
537
538 Figure 1
539
540 DME vapor
Needle valve
541
DME liquid
542
Reducing valve
543
544
545 Glass beads
Extractor

546
547
Feed
548
549
550
551
552
Extract
Liquefied DME cylinder
553
554 Figure 2

25
555
556
557 40MPa
558 Fucoxanthin / dry U. pinnatifida [ µg/g ] 80 30MPa
559 20MPa
560 10MPa
60
561
562
563 40
564
565 20
566
567 0
568 0 100 200 300
569 Time [ min ]
570 Figure 3a
571
572 70 ºC
Fucoxanthin / dry U. pinnatifida [ µg/g ]

573 80 60 ºC
574 50 ºC
575 40 ºC
60
576
577
578 40
579
580 20
581
582 0
583 0 100 200 300
584 Time [ min ]
585 Figure 3b
586

26
587
588 P = 20 MPa P = 30 MPa
589 P = 35 MPa P = 40 MPa
590 25 20 MPa with entrainer 30 MPa with entrainer
591 35 MPa with entrainer 40 MPa with entrainer
Astaxanthin extracted (%)

592 20
593
594 15
595
596 10
597
598 5
599
600 0
601 0 60 120 180 240
602 Time (min)
603 Figure 4.
604
605
606 DME amount [ g ]
Fucoxanthin / dry U. pinnatifida [ µg/g ]

607 0 50 100 150 200 250 300

608 500
609
DME extraction.
400
610
611
300
612
613
200
614
615 100
616
617 0
618 0 10 20 30 40
619 Time [ min ]
620 Figure 5
621

27
622
623
624 Seawater,
Heat exchanger
Groundwater
625 at 15 ºC DME gas at 30 ºC, 0.68 MPa
626
627 DME condensation Sun-warmed
water
628 at 45 ºC
Liquefied DME
at 30 ºC, 0.68 MPa Heat exchanger
629
Extraction column
630 at 30 ºC, 0.68 MPa

631
DME evaporation
632
633 Cell
Lipids Water-lipids-DME
634 Water mixture Residue Water Lipids
Wet microalgae
635
636 Figure 6
637
638
639 50
50
Lipids / dry weight of microalgae [%]

DME method
640 99.7%
40
40 Bligh-Dyer method
641
642 30
30
643 97.0%
644 20
20 97.5%
98.8% 98.4%
645 98.7%
98.2%
10
10
646
647
00
648 NIES-595 NIES-1263 ONC01B10 GSK01B30 GK12 Kanogawa Hirosawa
649 Oscillatoria agardhii Microcystis aeruginosa Monoraphidium Mainly Mainly
chlorophyta Cymbella Microcystis
650
651 Figure 7
652
653

28
Graphical Abstract

Solvent cycle for lipid extraction process from wet microalgae by using subcritical

dimethyl ether (DME).

Heat exchanger
Seawater,
Groundwater
at 15 ºC DME gas at 30 ºC, 0.68 MPa

DME condensation Sun-warmed

water
at 45 ºC
Liquefied DME
at 30 ºC, 0.68 MPa Heat exchanger
Extraction column
at 30 ºC, 0.68 MPa

DME evaporation

Cell
Lipids Water-lipids-DME
Water mixture Residue Water Lipids
Wet microalgae