Ultrasonics Sonochemistry 20 (2013) 9598

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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Extraction of lipids from microalgae by ultrasound application: Prospection

of the optimal extraction method
Glacio S. Araujo a, Leonardo J.B.L. Matos b, Jader O. Fernandes b, Samuel J.M. Cartaxo b,
Luciana R.B. Gonalves b, Fabiano A.N. Fernandes b,, Wladimir R.L. Farias a
a
Universidade Federal do Cear, Departamento de Engenharia de Pesca, Campus do Pici, Bloco 827, 60455-760 Fortaleza CE, Brazil
b
Universidade Federal do Cear, Departamento de Engenharia Qumica, Campus do Pici, Bloco 709, 60455-760 Fortaleza CE, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately
Received 7 December 2010 2050% of dry weight) of lipids. A successful and economically viable algae based oil industry will depend
Received in revised form 19 July 2011 on the selection of appropriate microalgal strains and the selection of the most suitable lipid extraction
Accepted 31 July 2012
method. In this paper, ve extraction methods were evaluated regarding the extraction of lipids from
Available online 12 August 2012
Chlorella vulgaris: Bligh and Dyer, Chen, Folch, Hara and Radin, and Soxhlet. Furthermore, the addition
of silica powder was studied to evaluate the introduction of more shear stress to the system as to increase
Keywords:
the disruption of cell walls. Among the studied methods, the Bligh and Dyer method assisted by ultra-
Microalgae
Lipid
sound resulted in the highest extraction of oil from C. vulgaris (52.5% w/w). Addition of powder silica
Oil did not improve the extraction of oil.
Ultrasound 2012 Elsevier B.V. All rights reserved.
Silica
Extraction

1. Introduction The difculty is in releasing the lipids from their intracellular

In the industry, microalgae have been used as source for a wide
variety of practical and potential metabolic products, such as food
supplements, pharmacological substances, lipids, polymers, toxins,
pigments, enzymes, biomass, wastewater treatment, and green
energy. Microalgae are also important in aquiculture as a source
of nutrients, in production of oxygen, in consumption of carbon
dioxide, and in consumption of nitrogen-based compounds [1,2].
The main driving force to grow microalgae commercially is har-
vesting metabolic products, feed for marine and terrestrial organ-
isms, food supplements for humans, or to use the microalgae for
environmental processes, such as wastewater treatment, fertiliza-
tion of soils, biofuels, and phytoremediation of toxic wastes [1].
Microalgae have been recognized as a promising alternative
source for lipid production [35,1]. Several species of microalgae
can be induced to produce specic lipids and fatty acids through
relative simple manipulations of the physical and chemical proper-
ties of their culture medium. Microalgae can accumulate substan-
tial amounts of lipids (approximately 2050% of dry weight) [6,7].
The accumulation of lipids in microalgae is attributed to consump-
tion of sugars at a rate higher than the rate of cell generation,
which promotes the conversion of excess sugar into lipids [8].
location in the most energy-efcient and economical way possible,
avoiding the use of large amounts of solvent. A key requirement is
that the oil be released and extracted without signicant contam-
ination by other cellular components, such as DNA or chlorophyll
[9]. There is much scope for approaches based on selective decom-
position of the cell wall, using ultrasound, microwave, enzymes,
pressurized uid extraction and abrasives, such as silica powder
[10,11]. The mechanism of each of these techniques is different
but most of the techniques involve disruption of cells to release
the lipids present in cytoplasm.
In this work, ve methods for lipid extraction were evaluated for
the extraction of lipid from microalgae: Bligh and Dyer, Chen, Folch,
Hara and Radin, and Soxhlet. The rst four methods were adapted
to be used subjected to ultrasonic waves. The study was carried
out with Chlorella vulgaris, microalgae which presents high content
of lipids (approximately 50% w/w). The extraction process was also
studied with and without the addition of silica powder to evaluate
the introduction of an abrasive into the extraction process.
2. Materials and methods
2.1. Microalgae strain and materials

Corresponding author. Tel.: +55 85 33669611; fax: +55 85 33669610. C. vulgaris was obtained from the Aquiculture Technology Cen-
E-mail address: fabiano@ufc.br (F.A.N. Fernandes). ter (Fortaleza CE) bank. C. vulgaris belongs to the phylum Chloro-

1350-4177/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ultsonch.2012.07.027
96 G.S. Araujo et al. / Ultrasonics Sonochemistry 20 (2013) 9598
phyta and the family Chlorellaceae. This alga has a spherical shape
and its diameter ranges from 2 to 10 lm. It has a characteristic
green color given by the presence of chlorophyll in its chloroplast.
All reagents were of analytical grade and were obtained from
Synth (So Paulo SP).
2.2. Microalgae cultivation
Prior to use, the stock cultures were maintained at 22 2 C in
tubes under articial light with a photoperiod consisting of an
18:6 h lightdark cycle. The f/2 culture media was used to main-
tain the inoculums and to carry out the experiments [12,13].
The production started from a volume of 20 mL of stock culture
in a 250 mL Erlenmeyer. Every two days more 20 mL of culture
media was added to the culture ask. The culture was transferred
to a 1 L ask when the culture volume reached 200 mL, and after-
wards was transferred to a 3 L ask when the culture volume
reached 350 mL. At this point, 2650 mL of culture media was added
to the ask. The culture in the 3 L glass ask was subjected to con-
stant aeration at an air ow rate of 2 L/min. Illumination was set at
20 lE/cm2 s and was provided by 40 W uorescent lamps. The
room temperature was maintained at 22 2 C. The cultivation
was carried out at constant volume.
2.3. Harvesting
Chemical occulation was applied to separate the microalgae
from the culture media. Evaluation of several harvesting methods
showed that occulation combined with otation or sedimentation
and subsequent further dewatering by centrifugation or ltration
is the most promising cost and energy efcient alternative [14].
During occulation, the dispersed microalgal cells aggregate and
form larger particles with higher sedimentation rate.
A solution of 2 mol/L NaOH was added to the culture for occu-
lation of the biomass. The supernatant consisting of the culture
medium was removed. The biomass, rich in salt, was washed with
distilled water till total reduction of the salinity. The salinity of the
washing water was monitored using a manual refractometer.
The biomass was dried for 24 h in a drying oven with forced air
circulation set at 60 C. After drying, the biomass was milled and
weighted to determine the yield into biomass (mass of dried bio-
mass per culture volume).
2.4. Lipid extraction
Five methods of extraction were studied: Bligh and Dyer [15]
Chen et al. [17], Folch et al. [16], Hara and Radin [18] (cold meth-
ods) and Soxhlet [19]. The rst four methods were adapted to be
carried out under ultrasound application. The Soxhlet method is
a conventional method used to extract natural products from or-
ganic matrixes and was carried out to compare the results of the
four ultrasonic-assisted methods with a well established conven-
tional method.
A mass of 5 g of dried microalgae was used in each experiment.
All experiments with ultrasound application were carried out in an
ultrasonic bath working at 40 kHz and producing an ultrasonic
intensity of 29.7 W/L or 2.68 W/m2 (Unique model 40USC Indaia-
tuba, Brazil, 2.7 L, internal dimensions: 24  14  9 cm). All exper-
iments were carried out in triplicate.
2.4.1. Bligh and Dyer method
The biomass (5 g) was mixed and homogenized with 25 mL of
methanol, 12.5 mL of chloroform and 5 mL of water. The mixture
was subjected to ultrasonic energy during 40 min. Chloroform
(12.5 mL) and a solution of 1.5% w/v sodium sulfate (12.5 mL)
was added to the mixture and sonicated for 20 min. The extraction
was carried out at ambient temperature (25 C).
2.4.2. Chen et al. method
The biomass (5 g) was mixed and homogenized with 25 mL of
methanol and was sonicated for 3 min. Dichlorometane (50 mL)
was added and the mixture was sonicated for 27 min. The extrac-
tion was carried out at ambient temperature (25 C).
2.4.3. Folch et al. method
The biomass (5 g) was mixed and homogenized with 25 mL of
methanol. The mixture was subjected to ultrasonic energy for
3 min. Chloroform (50 mL) was added and the mixture was soni-
cated for 27 min. The extraction was carried out at ambient tem-
perature (25 C).
2.4.4. Hara and Radin method
The biomass (5 g) was mixed and homogenized with 20 mL of
isopropanol and sonicated for 4 min. Hexane (30 mL) was added
and the mixture was sonicated for 56 min. The extraction was car-
ried out at ambient temperature (25 C).
2.4.5. Soxhlet method
The biomass (5 g) was kept over a Whatmann lter paper. The
lipids were selectively extracted into the solvent (110 mL of ace-
tone) during percolation. The solvent was heated at 120180 C
in Soxhlet extractor equipment. After 8 h of evaporation/condensa-
tion/percolation of solvent through biomass, the round-bottom
ask (containing mixture of solvent and extracted lipids) was ta-
ken out and the solvent was vaporized using a rotary evaporator
to recover the lipids.
2.5. Separation of lipids
The mixture contained the solid biomass and the liquid fraction
consisting of lipids and solvent was ltered to remove the solid
biomass. The liquid fraction was mixed with 75 mL of a solution
of KCl (0.88 w/v) and let to settle for 24 h in a pear-shaped separat-
ing funnel. The oil phase (lower phase) was transferred to a rotary
evaporator (Tecnal model TE-211 Piracicaba, Brazil) to remove
the remaining solvent. The rotary evaporator was set at 45 C
and vacuum of 200 mmHg was applied. The lipids remaining in
the ask after complete vaporization of the solvent were weighed.
2.6. Extraction assisted by addition of silica powder
The methods of Bligh and Dyer [15], Chen et al. [16], Folch et al.
[17] and Hara and Radin [18] were carried out with and without
the addition of silica powder.
In the experiments with addition of silica powder, the dried bio-
mass was mixed with 10% w/w of silica powder (0.5 g). The exper-
iments were carried out following the same procedure as described
previously. The silica powder was provided by Davison Grace (Por-
to Alegre Brazil). The brand Syloid 72 consisting of silica with an
average diameter of 40 lm was used in the experiments.
3. Results and discussion
Table 1 presents the yield into lipid extracted from C. vulgaris
biomass for the ve methods studied herein. The Bligh and Dyer
method assisted by ultrasound resulted in the highest oil extrac-
tion. Also, all ultrasound-assisted methods have recovered more
lipids than the conventional Soxhlet method.
The extraction methods have presented high repeatability and
high reproducibility. The standard error calculated for the percent-
 G.S. Araujo et al. / Ultrasonics Sonochemistry 20 (2013) 9598
Table 1
Yield in oil based in dried microalgae biomass extracted from Chlorella vulgaris by ve
different methods.
Method Yield (% w/w)
Without silica addition With silica addition
Bligh and Dyer (1959) 52.5 2.3 41.4 1.8
Chen (1981) 10.9 1.2 9.5 0.9
Folch et al. (1957) 16.1 0.8 11.7 1.0
Hara and Radin (1978) 2.2 0.3 1.8 0.2
Soxhlet 1.8 0.3
age of recovered lipids was between 0.3% and 2.3%. Regarding the
amount of lipids extracted by each method the Bligh and Dyer
method and the Folch method have presented the lowest standard
error (5%), thus higher repeatability. The reproducibility of all
methods was very high. The average values calculated after three
runs with microalgae produced by different batches resulted in a
reproducibility of 97% for the Bligh and Dyer, Folch and Chen meth-
ods and of 96% for the Soxhlet and the Hara and Radin methods.
Reproducibility was calculated at the standard error between dif-
ferent batches divided by the average value between different
batches.
The result obtained herein corroborates with a related study
from Ranjan et al. [11], which observed that the ultrasound as-
sisted Bligh and Dyer method resulted in an extraction of lipids
from Scenedesmus sp. higher than the obtained by application of
the Soxhlet method.
Extraction of lipids from microalgae is basically a mass transfer
operation, which depends on the nature of the solute and solvent,
the selectivity of the solvent and the level of convection in the
medium. The Soxhlet method involves washing of solid mass with
a solvent that has high solubility and selectivity for the solute. The
Soxhlet extraction mechanism is mainly diffusion; and the proce-
dure does not involve application of any shear stress to the bio-
mass. The results show that relying simply on diffusion of lipids
through the cell membrane is a slow process and results in low
yield of oil. Even using a polar solvent, such as acetone, no
improvement was achieved.
Most cold methods involve simultaneous extraction and parti-
tioning by mixing of the microalgal cell suspension in a mixture
of solvents. The mixture forms two phases after completion of
extraction. The cold methods also do not involve application of
shear stress to the algal biomass, thus, the predominant mecha-
nism is also diffusion.
To enhance the efciency of the cold methods, it is important to
disrupt the cell walls to release of lipids into the solvent mixture.
In this case, the extent of extraction should be independent of
the solvent properties. For this matter, sonication or addition of sil-
ica could provide the means to disrupt the cell walls of the
microalgae.
The association of the cold methods with ultrasound extraction
has contributed to the increase the amount of oil extracted from
the microalgal biomass. The chemical effect of cavitation is the
generation of highly reactive radicals due to dissociation of the en-
trapped vapor molecules in the cavitation bubble. Furthermore, the
implosion of the cavitation bubbles caused by ultrasound inside
and outside an organism may contribute to rupture of cell walls
[20].
Ranjan et al. [11] showed, through micrographs, that the use of
the Bligh and Dyer method without ultrasound application showed
some distorted clusters of biomass corresponding to disrupted
cells. The application of ultrasound increased the number of dis-
rupted cells. Similar result was obtained in our study. Fig. 1 shows
a normal cell and a disrupted cell. The micrograph was obtained
97
Fig. 1. Cells of Chlorella vulgaris after application of the Bligh and Dyer method,
showing a normal cell in the right and a disrupted cell in the left.
after application of the Bligh and Dyer method. After ultrasound-
assisted extraction most cells have been disrupted.
Solvents, such as chloroform and dichloromethane, may con-
tribute to weaken the cell wall, thus contributing toward a more
intense extraction of oil from the microalgae cells. The low recov-
ery observed for the Hara and Radin method may be attributed to
the use of n-hexane, which has a nonpolar character and low selec-
tivity toward microalgal lipids.
The higher extraction capability of the Bligh and Dyer method
assisted by ultrasound can be attributed to the disruption of the
cells wall provided by the cavitation mechanism, which releases
lipids to the solution; and to the higher selectivity of microalgal
lipids toward chloroformmethanolwater system, which has a
more polar nature.
Prabakaran and Ravindran [21] have studied the recovery of lip-
ids from C. vulgaris sonicating the microalgae in water and after-
wards extracting the lipids using the cold methods. The
methodology followed by Prabakaran and Ravindran [21] have re-
sulted a lower recovery of lipids (19%) than applying ultrasound-
assisted extraction.
The oil composition was not affected by the extraction methods.
The nal oil composition consisted of 39% of palmitic acid, 16% of
linoleic acid, 23% of oleic acid, 3% of linolenic acid and 19% of other
acids.
The recovery of lipids from the biomass in the ltration step
was also evaluated. Changes in the method described in Section
2 did not resulted in further recovery of lipids from the biomass.
Adding more solvent to wash the biomass in the lter and washing
the lter with the recovered mixture did not increase the nal
amount of lipids.
Silica powder was added to the extraction system to increase
the shear stress induced to the microalgae cells. The results
showed that the addition of silica powder did not increase the
extraction of oil. In fact, the addition of silica powder had a nega-
tive effect in the extraction process. It was expected that the in-
tense local turbulence in the medium created by ultrasound
would help mixing the silica powder, which in turn would increase
the shear stress induced to the microalgae cell wall. The results
suggest that the presence of silica powder reduced the effect of
ultrasound, leading to a lower extraction of oil.
4. Conclusions
The key step in extraction and recovery of lipids from microal-
gae is cell disruption. Sonication increased the efciency of the
extraction since it was partially responsible for cell disruption.
98 G.S. Araujo et al. / Ultrasonics Sonochemistry 20 (2013) 9598
The selection of the proper solvent system is still essential to the
extraction process because it may weaken the cell wall structure
facilitating cell disruption and therefore extraction of lipids. The
addition of powder silica did not improve the extraction of oil in
the cold methods assisted by ultrasound. The Bligh and Dyer meth-
od assisted by ultrasound resulted in the highest extraction of oil
from C. vulgaris (52.5% w/w).
Acknowledgements
The authors thank the Brazilian funding agency CNPq and
FUNCAP.
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