Enzyme-Assisted Aqueous Extraction of Lipid From Microalgae

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Article
Enzyme-assisted aqueous extraction of lipid from microalgae
Liang Ke Hong, qinghua zhang, and Wei Cong
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 16 Oct 2012
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Page 1 of 28 Journal of Agricultural and Food Chemistry
1 Enzyme-assisted aqueous extraction of lipid from microalgae
2 Kehong Liang†‡, Qinghua Zhang†, Wei Cong†, *
†
3 National Key Laboratory of Biochemical Engineering, Institute of Process
4 Engineering, Chinese Academy of Sciences, Beijing 100080, PR China
‡
5 Graduate School of the Chinese Academy of Sciences, Beijing 100049, PR China
10
11
12
13 Corresponding author
14 Dr. Wei Cong
15 Professor of Biochemical Engineering
16 National Key Laboratory of Biochemical Engineering,
17 Institute of Process Engineering, Chinese Academy of Sciences,
18 Beijing 100190, P.R. China
19 Tel& Fax: +86-10-82627074
20 E-mail: weicong@home.ipe.ac.cn
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Journal of Agricultural and Food Chemistry Page 2 of 28
21 ABSTRACT
22 An improved lipid extraction process has been established for microalgal using
23 enzyme-assisted aqueous extraction processing (EAEP), which mainly involved in
24 sonication and enzyme treatment. As compared to cellulase, neutral protease and
25 alkaline protease, significantly higher lipid recovery was achieved by snailase and
26 trypsin. The highest lipid recovery of 49.82% was obtained by a combined
27 sonication-enzyme treatment at pH 4. The enhancement mechanism of the EAEP was
28 analyzed in terms of the particle size of cream and zeta potential. In addition,
29 microalgal lipid recovery was also affected by lipid class composition and the type of
30 algae. The present study demonstrates a promising alternative to conventional lipid
31 extraction of microalgae and the quantitative information on EAEP of oleaginous alga
32 can provide valuable data for process design at pilot and industrial scale.
33 KEYWORDS: Microalgae, Enzyme-assisted aqueous extraction processing, Lipid
34 extraction, Lipid recovery, Biodiesel
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43 ■ INTRODUCTION
44 Microalgae are sunlight-driven cell factories that convert carbon dioxide to
45 potential biofuels such as methane, biodiesel and biohydrogen.1 Although some of
46 microalgae have been attempted for oil and biofuels production on a commercial
47 scale,2 the lipid extraction is still quite restricted as compared with other processes. By
48 far, several extraction methods have been reported, including organic solvent
49 extraction, subcritical water extraction, supercritical fluid extraction and aqueous
50 extraction processing.3-6 However, none of them are satisfactory. For example, organic
51 solvent extraction is a simple method for extraction of lipids from microalgae,
52 however, this method is gradually abandoned from the environment and safety point
53 of view, since a great amount of organic solvent is used. As alternatives to organic
54 solvent extraction, both subcritical water extraction and supercritical fluid extraction
55 achieve higher selectivity and consume shorter extraction time. However, these
56 methods are impractical for industrial process because of their higher energy
57 consumption and cost.7, 8 Aqueous extraction processing (AEP) is an
58 environmental-friendly approach using water to extract oil from oilseeds, but its low
59 oil recovery is of great concern.5
60 Several assistant treatments have been attempted to increase the oil recovery of
61 AEP. For example, flaking and extruding are adopted to rupture cell walls and
62 facilitate flushing action of water, which is only suitable for dry seeds. In another way,
63 enzyme-assisted aqueous extraction processing (EAEP) together with sonication

64 provides improved efficiency of AEP by the combination of biochemical and
65 mechanical treatments. In one hand, sonication employs high-frequency sound waves
66 to destroy cell walls within a short period of time,9 while in the other hand, the
67 destroyed cell walls and lipid bodies can be further decomposed. EAEP has been
68 employed to extract different compounds from plants, and has been proved to be
69 effective in improving the yield of the target component.10, 11 By using EAEP,
70 improved lipid extraction was observed in many different oil-bearing plant materials
71 including soybean,12 sunflower seeds13 and sesame.14 In addition, EAEP will make it
72 possible to extract and separate oil directly from algae in the natural aqueous
73 environment of algae cultures, which avoids the collection and drying process of algae
74 biomass. Although the cell wall of algae has similar structure and components as
75 those of land plants, differences to exist. The cell wall of most algae is composed of
76 cellulose, hemicellulose and polysaccharides.15 It has been shown that insoluble
77 non-hydrolysable biopolymers termed algaenans was detected in cell wall of some
78 algae,16-18 and made cell breakage a challenge. Thus, the EAEP methods established
79 for common terrestrial plants can not be applied directly to the lipid extraction from
80 microalgae and the detailed process should be carefully investigated. Unfortunately,
81 there have been few reports on the lipid extraction from microalgae using EAEP
82 combined with sonication up to now.
83 In this study, an efficient EAEP technique was developed for microalgae by
84 using Chlorella vulgaris as a model system. The process was investigated by
85 considering both the enzyme types and pH values. The underlying mechanism of

86 EAEP was elucidated by cell microstructure and the particle size of cream. Finally,
87 the established EAEP procedure was also verified in some other typical microalgae
88 including Scenedesmus dimorphus and Nannochloropsis sp.
89 ■ MATERIALS AND METHODS
90 Microalgae and growth.
91 Fresh cell of Chlorella vulgaris, Scenedesmus dimorphus and Nannochloropsis
92 sp. in paste form with 18% solid content were used in this study. All algae strains
93 were kindly provided by Qingdao Institute of Bioenergy and Bioprocess Technology,
94 Chinese Academy of Sciences (Qingdao, China). Scenedesmus dimorphus was
95 cultivated in BG11 medium,19 while Chlorella vulgaris and Nannochloropsis sp. were
96 incubated in F/2 medium,20 in a 2 L jar with a mixed air/CO2 (99:1, v/v) gas aerated at
97 constant rate of 120 µmol m-2 s-1.
98 Enzymes.
99 All the enzymes used in EAEP, including cellulose, snailase (a complex of more
100 than 30 enzymes, including cellulose, hemicellulase, galactase, proteolytic enzyme,
101 pectinase, β-glucuronidase, etc.), neutral protease, alkaline protease and trypsin, were
102 purchased from Xin-jing-ke biotechnology Co. (Beijing, China). The available
103 information on the enzyme formulations used in the experiments was summarized in
104 Table 1.
105 Quantification of total lipid in microalgae.
106 Algal cells were harvested by centrifugation at 8000 rpm for 15 min and washed
107 three times with distilled water. After dried in a freeze drier , the samples were

108 ultrasonicated for 30 min in 5 mL solution of chloroform/methanol (2:1, v/v).
109 subsequently, 1 mL chloroform and 1.5 mL water were added to the sample.21 The
110 mixture was centrifuged at 4500 rpm for 10 min, the lower solvent phase was
111 collected and evaporated in a rotary evaporator under vacuum at 60°C. The resulting
112 lipid was weighed and regarded as total lipid content. Thus, the lipid recovery in the
113 EAEP procedures can be calculated on the basis of the total lipid content determined.
114 Lipid fractionation.
115 The lipid extract was fractionated into neutral lipid, glycolipids and
116 phospholipids using a silica cartridges (Waters, Milford, MA ), according to Damiani
117 et al.22 In this method, silica Sep-Pak cartridges (500 mg) were initially equilibrated
118 with 10 mL methanol followed by 30 mL chloroform. Subsequently, 1 mL chloroform
119 solution containing 20 mg lipid was applied to a Sep-Pak cartridge. The cartridge was
120 eluted by 15 mL solution of chloroform/acetic acid (9:1, v: v) to collect neutral lipid,
121 by 20 mL solution of acetone/methanol (9:1, v: v) to collect glycolipids and by 20mL
122 methanol to collect phospholipids. Each content of neutral lipid, glycolipids and
123 phospholipids was determined by weighing after dried in a rotary evaporator under
124 vacuum at 60 °C.
125 Aqueous enzyme-assisted processing.
126 Each microalgae paste (~10 g fresh weight) was exactly weighed and placed in a
127 50 mL centrifuge tube. The sample was ultrasonicated with an ultrasonic cell
128 disintegrator (JY92-2D, Ningbo Scientz Co,.Ltd, Zhejiang Province, China) at 600 W
129 ultrasonic power, 4 s interval time/2 s ultrasonic time and 15 min total working time. The

130 sample was kept in an ice-water bath to avoid overheating. Before the specific enzyme
131 was added, the mixture was pre-adjusted to the optimum pH value and pre-heated in a
132 water bath to the optimum temperature, as indicated in Table 1. Various amounts of
133 enzyme were added and incubated for a specific period of time. Subsequently, the
134 reaction was stopped by heating in a water bath at 95 °C for 10 min to make enzyme
135 deactivated. The hydrolysate was then centrifuged (4500 rpm, 15 min) to separate
136 three distinct phases: oil phase, cream phase and aqueous phase. Subsequently, 5 mL
137 hexane was added to the supernatant and the upper layer was collected. Finally, the
138 weight of lipid was determined gravimetrically after the solvent was evaporated under
139 vacuum at 60 °C.
140 pH treatments.
141 Initially, the pH of the cream (cream sequently treated by snailase and trypsin
142 during the extraction step using the condition described above) was adjusted to the
143 desired value by adding 2 mol/L NaOH or 2 mol/L HCl. Subsequently, the resulted
144 cream was incubated at room temperature for 30 min with constant stirring. Following
145 concentration at 4500 rpm for 15 min, free oil was collected.
146 Scanning electron micrographs
147 The samples obtained from different treatment methods were observed by
148 scanning electron microscrope (SEM; S-4800, Hitachi High-technologies Corporation,
149 Japan) after freeze-dried, fixed to a specimen holder and sputtered with gold.
150 Particle Size Distributions and Zeta potential measurements.
151 Samples obtained after treatments and before centrifugation were used for

152 droplet size distribution and zeta potential measurements. Both particle size
153 distribution and zeta potential were analyzed by a DelsaNano C instrument (Beckman
154 Coulter, Inc. USA). The samples were diluted at a 1: 50 ratio in distilled water before
155 the analysis. Each measurement was repeated three times at 25 °C. All samples were
156 tested for at least three times and the obtained results were expressed as the average
157 and standard deviation.
158 Statistical Analysis.
159 All experiments were conducted with triplicate treatments. The data were
160 analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s
161 Honeatly Significant Differences Test. P value ≤ 0.05 was regarded as significant, and
162 data were presented as mean ± standard deviation (SD).
163 ■ RESULTS AND DISCUSSION
164 Enzyme-assisted extraction of lipid from Chlorella vulgaris.
165 Microalgal cell wall is predominantly composed of cellulose, hemicellulose and
166 saccharides which hinder the release of intracellular lipids.23 Microalgal lipid is stored
167 in subcellular compartments called lipid bodies, lipid droplets or oleosomes. Lipid
168 bodies are spherical organelles consisting of a neutral lipid core enclosed by a
169 monolayer lipid membrane coated with proteins.24 Thus, the extraction of lipid, to
170 some extent, depends on the destruction of cellular and subcellular structure. However,
171 pretreatment using sonication can only achieve 3.97% of the total algal lipid (data not
172 show), indicating that this kind of physical treatment is insufficient. Based on this
173 result, cellulase, snailase, neutral protease, alkaline protease and trypsin were selected

174 to facilitate the release of lipid from lipid bodies. As shown in Figure 1, when the alga
175 was treated with 0.5% alkaline protease, the lipid recovery increased to 5.27%. Higher
176 lipid recovery was obtained by snailase and trypsin as compared to the other three
177 types of enzymes. Snailase is a complex of cellulase, hemicellulase, pectinase and
178 β-glucuronidase, which makes the the cellulosic component of cell wall degraded
179 more effectively. While, proteolytic enzymes could hydrolyze the proteins of
180 membranes and cytoplasmic and modify the emulsifying capacity of some proteins
181 that is related to the aqueous efficiency.25 Trypsin, that attack basic residues (i.e.,
182 arginine and lysine),26 is very effective in the hydrolysis of protein because these
183 ionizable groups typically exist on the protein surface. As a result, when treated by
184 snailase and trypsin, more lipid was released from microalgae.
185 Besides the enzyme types, the enzyme dosage and incubation time could also
186 influence the lipid recovery. As shown in Figure 2, the lipid recovery was low when
187 enzyme dosage is lower than 4%. When the enzyme dosage further increased, no
188 remarkable improvement for lipid recovery could be observed. The lipid recovery also
189 increased with reaction time, and reached a maximum value at 12 h. in another aspect,
190 when the enzyme dosage was lower than 2%, significant reduction of lipid recovery
191 was observed irrespective of the incubation time.
192 In order to get an insight to the EAEP process for algal lipid recovery, the algae
193 materials were analyzed by SEM. Untreated Chlorella vulgaris was used as a control,
194 in which fully intact cell wall was observed with no signs of pitting or damage (Figure
195 3a). The sonicated cells showed breakdown of cell walls (Figure 3b) since the cells

196 were believed to be mechnically broken by the oscillation and collapse of cavitations
197 bubbles created by ultrasound.27 In previous studies, different cell disruption methods,
198 such as sonication, autoclaving, bead beating (bead diameter 0.1 mm), microwave and
199 10% NaCl solution treatments, have been tested for lipid extraction from
200 microalgae.28 It was found that sonication was the best for lipid extraction from
201 microalgae. With the aid of sonication, the algal cells were further disrupted into
202 pieces by snailase treatment (Figure 3c), indicating that the combination of sonication
203 and snailase treatment was more efficient for the degradation of algal cells. Therefore,
204 the effective cracking of the cell wall was a key to increasing the lipid extraction
205 efficiency.
206 Influence of pH on algal lipid recovery
207 Lipid recovery was found to be strongly dependent on pH value of the cream
208 layer (Figure 4). By adjustment of pH value of the system, the lipid recovery can be
209 increased from 39.82% at the initial pH of cream (~ 6.0) to 49.82% at pH 4.0. While
210 further decreasing the pH value, slight decrease of lipid recovery was observed. This
211 phenomenon can be interpreted by the destabilization effect of pH on the emulsion, 29
212 since electrostatic attraction between protein-stabilized emulsion droplets is
213 particularly sensitive to pH value. The isoelectric point of algal protein was between
214 pH 4.0 and 5.0 (data not shown). At pH near the isoelectric point of proteins the
215 charge on the droplets is low and thus the electrostatic repulsion is weak, so droplets
216 tend to aggregate.30
217 Particle size distribution measurements indicated that the majority of particles in
10

218 the cream were relatively small at pH values far away from the isoelectric point
219 (Figure 5). At pH 9.0, the lipid droplet size was in the range from about 170-360 nm,
220 and with the decrease of pH, the proportion of large lipid droplets increased. At pH
221 4.0, the majority of particles were distributed from 370 to 800 nm. By further
222 decreasing the pH to 3.0, the particle size fell to the range of 300-670 nm. These
223 results showed that larger particles size occurred at pH value closer to the pI of most
224 of the algal proteins.
225 Application of EAEP to other microalgal species
226 For Chlorella vulgaris, the lipid recovery by EAEP was 49.82% (Table 2), which
227 was lower than that of soybean (90%).31 This might be the result of different lipid
228 class compositions between Chlorella vulgaris and soybean. Chlorella vulgaris
229 contains only 66.88% neutral lipids (Table 2) whereas the soybean contains 88%.32
230 Neutral lipid bodies are composed of a hydrophobic core of lipids surrounded by a
231 monolayer of phospholipids,33 and are much more easily extracted by EAEP. In
232 contrast, glycolipids and phospholipids are polar hydrophilic molecules, which are
233 relatively easy to be dispersed in aqueous phases. The levels of polar lipids were
234 lower in the enzyme-extracted oil (Table 2). The lipid recovery from Scenedesmus
235 dimorphus was about 46.81%, which was similar to that of Chlorella vulgaris.
236 However, the lipid recovery of Nannochloropsis sp. was only 11.73% partly due to its
237 lower content of neutral lipid. The difference of the lipid droplet size distribution
238 during EAEP among different algal species was shown in Figure 6. The size of major
239 oil droplet fraction was around 405 nm, 336 nm and 194 nm for Chlorella vulgaris,
11

240 Scenedesmus dimorphus and Nannochloropsis sp., respectively. In general, smaller
241 emulsion droplets have greater kinetic stability and offer a more stable emulsion.34 As
242 per Stokes Law, larger emulsion droplets would have higher terminal velocities in
243 aqueous medium during settling, which could result in rapid formation of a cream
244 layer on the top part of EAEP derived mixture. Once the larger droplets are formed,
245 they are more likely to coalesce.35
246 The subcritical water extraction method has also been applied to lipid extraction
247 from the wet algal biomass. The yield of 100% was reported for wet algal biomass
248 (70% moisture) at 60 °C, 30 MPa.36 However, the application of high pressure in
249 SWE method makes it much more complicated for industrial production at higher
250 expense. Although the combination of chloroform and methanol has been reported as
251 the best solvent for lipid extraction from Synechosystis,6 the toxicity of chloroform
252 and methanol is of great concern due to environmental considerations, especially for
253 large scale production. Compared with these methods, less toxic solvent and mild
254 operation conditions such as room temperature and atmospheric pressure were
255 employed in current method. Furthermore, the procedures for ultrasonication prior to
256 EAEP showed high extraction efficiency. Similar results were obtained in other plant
257 materials (e.g Soybean and Jatropha curcas L.37, 38)
258 In this study, an effective method was developed by the combination of EAEP
259 with sonication for rupturing algae cell microstructure and extracting lipid from
260 microalgae. Microalgal lipid recovery was affected not only by the process conditions
261 such as enzyme type, enzyme dosage, incubation time and pH but also by the lipid
12

262 class composition and the type of algae. The highest lipid recovery of 49.82% was
263 achieved by using EAEP. As mild-extraction technique and promising alternative to
264 microalgal lipid extraction, the results obtained in this study are of great importance
265 for the future development. The major drawback in proposed process is the cost of the
266 enzyme. The enzymes may be used in an immobilized form to reuse them.
267 Immobilization can make enzymes more economical since it facilitates separation of
268 enzyme from product, allows reuse, and improves enzyme stability.39, 40 For example,
269 stabilization of multimeric enzymes may be easily achieved using CLEAs
270 (crosslinked aggregates) technology, and the rigidity of the enzyme will be usually
271 higher using multipoint covalent attachment.41 It should be better to immobilize
272 enzymes on the surface of non porous nanoparticles/ other carriers in order to be able
273 to attack no insoluble structures of microalgae. Further work using the immobilized
274 enzymes can be considered in enzyme-assisted aqueous extraction.
275 ■ ACKNOWLEDGMENTS
276 This work is supported by National Key Technologies R&D Program of China
277 (2011BAD14B02).
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390 2008, 42, 332–339.
391 (41) Garcia-Galan, C.; Berenguer-Murcia, A.; Fernandez-Lafuente, R.; Rodrigues,
392 R.C. Potential of different enzyme immobilization strategies to improve enzyme
393 performance. Adv. Synth. catal. 2011, 353, 2885-2904.
18

394 Liang et al., Figure Captions
395 Figure 1. Effect of different enzymes on lipid recovery during enzyme-assisted
396 aqueous extraction processing. Values are represented as mean ± standard deviations
397 of three independent experiments. Identical superscripts indicate non-significant (p
398 ≤0.05) differences.
399 Figure 2. Effect of enzyme dosage on the total lipid recovered from Chlorella
400 vulgaris. Values are represented as mean ± standard deviations of three independent
401 experiments.
402 Figure 3. Scanning electron micrographs of Chlorella vulgaris. (A) untreated sample;
403 (B) sonication treatment sample; (C) combination of sonication and snaillase
404 treatment sample.
405 Figure 4. Effect of pH on the total lipid recovered from Chlorella vulgaris. Values are
406 represented as mean ± standard deviations of three independent experiments.
407 Figure 5. Particle size distribution profile of cream subjected to pH treatment.
408 Figure 6. Particle size distribution profile of cream suspensions from different
409 microalgal.
410
411
412
413
19

414 Table 1 Enzymes used in enzyme-assisted aqueous extraction processing.
Enzyme type Activity Source Optimum Optimum pH
temperature
Trichoderma
Cellulase 2.9 u/mg 55 °C 4.8
Snail
Snailase >90% a 37 °C 5.8
Bacillus subtilis
Neutral 200 u/mg 50 °C 7.0
protease
Baclicus
Alkaline 200 u/mg 55 °C 8.5
lincheniformis
protease
Trypsin 40 u/mg Porcine 37 °C 8.0
pancreas
a
415 Breakage rate of yeast cells per gram that are hydrolyzed by 30 mg snailase at
416 37 °C for 1 h.
417
418
419
420
421
422
20

423 Table 2 Lipid content, classes and recovery in different algal species. Values are
424 represented as means ± standard deviations of three replicates. Identical superscripts
425 indicate non-significant (p ≤ 0.05) differences.

426
Lipid Lipid class distribution (% total lipid) Lipid Lipid class distribution from EAEP
content recovery by (% enzyme-extracted lipid)
Species
(% dw of Neutral Glycolipids Phospholipids EAEP (% Neutral Glycolipids Phospholipids
cell) lipids total lipid ) lipids
Chlorella 15.11 66.88 26.30 6.82 49.82 a 72.29 25.28 2.43
vulgaris
Scenedesmus 10.62 65.09 31.75 3.16 46.81 a 71.51 27.47 1.02
dimorphus
Nannochloropsi 15.98 52.95 44.20 2.85 11.73 b 60.57 38.51 0.92
s sp.
427
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437
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438 Figure 1.
439
440
441
442
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444
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449 Figure 2.
450
451
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460 Figure 3.
461
462
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465
(A)
466
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470 (B)
471
472
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475 (C)
476
477
24

478 Figure 4.
479
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489 Figure 5.
490
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500 Figure 6.
501
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27

511 Table of Contents Graphic
512
513 An improved lipid extraction process has been established for microalgal using
514 enzyme-assisted aqueous extraction processing (EAEP), which mainly involved in
515 sonication and enzyme treatment. The enhancement mechanism of the EAEP was
516 analyzed in terms of the particle size of cream and zeta potential. In addition,
517 microalgal lipid recovery was also affected by lipid class composition and the type of
518 algae.
519
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Enzyme-Assisted Aqueous Extraction of Lipid From Microalgae

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Enzyme-Assisted Aqueous Extraction of Lipid From Microalgae

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Journal of Agricultural and Food Chemistry is published by the American Chemical

1 Enzyme-assisted aqueous extraction of lipid from microalgae

2 Kehong Liang†‡, Qinghua Zhang†, Wei Cong†, *

4 Engineering, Chinese Academy of Sciences, Beijing 100080, PR China

14 Dr. Wei Cong

15 Professor of Biochemical Engineering

16 National Key Laboratory of Biochemical Engineering,

17 Institute of Process Engineering, Chinese Academy of Sciences,

18 Beijing 100190, P.R. China

19 Tel& Fax: +86-10-82627074

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23 enzyme-assisted aqueous extraction processing (EAEP), which mainly involved in

24 sonication and enzyme treatment. As compared to cellulase, neutral protease and

26 trypsin. The highest lipid recovery of 49.82% was obtained by a combined

27 sonication-enzyme treatment at pH 4. The enhancement mechanism of the EAEP was

30 algae. The present study demonstrates a promising alternative to conventional lipid

31 extraction of microalgae and the quantitative information on EAEP of oleaginous alga

33 KEYWORDS: Microalgae, Enzyme-assisted aqueous extraction processing, Lipid

34 extraction, Lipid recovery, Biodiesel

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44 Microalgae are sunlight-driven cell factories that convert carbon dioxide to

45 potential biofuels such as methane, biodiesel and biohydrogen.1 Although some of

49 extraction, subcritical water extraction, supercritical fluid extraction and aqueous

51 solvent extraction is a simple method for extraction of lipids from microalgae,

53 of view, since a great amount of organic solvent is used. As alternatives to organic

57 consumption and cost.7, 8 Aqueous extraction processing (AEP) is an

59 oil recovery is of great concern.5

63 enzyme-assisted aqueous extraction processing (EAEP) together with sonication

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64 provides improved efficiency of AEP by the combination of biochemical and

65 mechanical treatments. In one hand, sonication employs high-frequency sound waves

69 effective in improving the yield of the target component.10, 11 By using EAEP,

76 cellulose, hemicellulose and polysaccharides.15 It has been shown that insoluble

77 non-hydrolysable biopolymers termed algaenans was detected in cell wall of some

80 microalgae and the detailed process should be carefully investigated. Unfortunately,

82 combined with sonication up to now.

83 In this study, an efficient EAEP technique was developed for microalgae by

84 using Chlorella vulgaris as a model system. The process was investigated by

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88 including Scenedesmus dimorphus and Nannochloropsis sp.

89 ■ MATERIALS AND METHODS

90 Microalgae and growth.

91 Fresh cell of Chlorella vulgaris, Scenedesmus dimorphus and Nannochloropsis

93 were kindly provided by Qingdao Institute of Bioenergy and Bioprocess Technology,

94 Chinese Academy of Sciences (Qingdao, China). Scenedesmus dimorphus was

97 constant rate of 120 µmol m-2 s-1.

100 than 30 enzymes, including cellulose, hemicellulase, galactase, proteolytic enzyme,

105 Quantification of total lipid in microalgae.

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108 ultrasonicated for 30 min in 5 mL solution of chloroform/methanol (2:1, v/v).

114 Lipid fractionation.

116 phospholipids using a silica cartridges (Waters, Milford, MA ), according to Damiani

118 with 10 mL methanol followed by 30 mL chloroform. Subsequently, 1 mL chloroform

120 eluted by 15 mL solution of chloroform/acetic acid (9:1, v: v) to collect neutral lipid,

121 by 20 mL solution of acetone/methanol (9:1, v: v) to collect glycolipids and by 20mL

124 vacuum at 60 °C.

125 Aqueous enzyme-assisted processing.

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139 vacuum at 60 °C.

146 Scanning electron micrographs

148 scanning electron microscrope (SEM; S-4800, Hitachi High-technologies Corporation,

150 Particle Size Distributions and Zeta potential measurements.

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