Process Biochemistry 51 (2016) 1636–1643

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Carotenoid and lipid extraction from Heterochlorella luteoviridis using

moderate electric field and ethanol
Débora Pez Jaeschke a,∗ , Tania Menegol b , Rosane Rech b , Giovana Domeneghini Mercali b ,
Ligia Damasceno Ferreira Marczak a
a
Chemical Engineering Department, Federal University of Rio Grande do Sul (UFRGS), Engenheiro Luiz Englert Street, Porto Alegre, RS, 90040-040, Brazil
b
Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Bento Gonçalves Avenue, 9500, Porto Alegre, RS, 91501-970, Brazil

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

Article history: This study evaluated the effect of moderate electric field (MEF) and ethanol concentration on carotenoid
Received 23 March 2016 and lipid extraction from the microalgae Heterochlorella luteoviridis. The extraction was performed using
Received in revised form 10 June 2016 0–180 V and 25 mL/100 mL of ethanol/water solution during 10 min of pretreatment, followed by a diffu-
Accepted 15 July 2016
sive step using 25–75 mL/100 mL of ethanol/water solution during 50 min. Carotenoid and lipid content
Available online 21 July 2016
analyses were performed on the supernatant after each extraction process. Results showed that MEF
and ethanol concentration affected carotenoid extraction, while lipid extraction was only affected by
Keywords:
ethanol concentration. The application of MEF combined with ethanol as solvent (180 V, 75 mL/100 mL
MEF
Pigments
of ethanol solution) yielded up to 73% of carotenoid extraction, proving to be an attractive alternative
Fatty acids method to extract these compounds from the microalgae Heterochlorella luteoviridis biomass. The highest
Microalgae lipid extraction yield (83%) was achieved when 75 mL/100 mL of ethanol solution was used.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction function in immune and inflammatory cells, decreasing risk of car-

Microalgae are interesting sources of carotenoids and lipids
due to its cultivation characteristics, such as good adaptation to
changing environmental conditions and the fact that its cultiva-
tion does not compete with food production [1]. Green microalgae,
as Chlorellas, are considered a good source of lutein, ␤-carotene,
zeaxanthin and violaxanthin. Inbaraj, Chien, & Chen [2] evalu-
ated the carotenoid profile from Chlorella pyrenoidosa and reported
that lutein accounted for 93 % of the total carotenoid content
(125.03 ± 0.042 mg g−1 ), followed by 2.6 % of ␤-carotene, 2.6 % of
␣-carotene and 1.3 % of zeaxanthin. Carotenoids consumption has
been associated with health benefits, as cancer, cardiovascular and
chronic diseases prevention. These benefits are probably related to
their antioxidant capacity. Besides these nutraceutical properties,
carotenoids have potential as food colorants and pharmaceutical
and cosmetic usages [3,4].
Green microalgae are also rich in essential fatty acids, such as
linoleic (C:18:2n6) and ␣-linolenic (C18:3n3) acids. These fatty
acids, also known as ␻-3 and ␻-6 fatty acids, develop an important
∗ Corresponding author.
E-mail addresses: debora jaeschke@hotmail.com, deborapj@enq.ufrgs.br
(D.P. Jaeschke).
diovascular diseases [5,6]. These compounds can be usually found
at marine plants and animals [8]. It is well known that fish accu-
mulate polyunsaturated fatty acids by the plankton consumption,
such as algae and microalgae. Thus, it is important to highlight that
algae and microalgae are a primary source of polyunsaturated fatty
acids and they are considered a vegan source of these compounds
[9,10].
The conventional lipid and carotenoid extraction methods are
based on the usage of organic solvents, such as methanol and chlo-
roform [11,12], hexane and isopropanol [13], methylene chloride
and methanol [14], hexane, acetone and diethyl ether [1,15,16].
Furthermore, carotenoids extraction methods have the disadvan-
tage of being time consuming due to the maceration step [17].
Recently, novel technologies, such as pulsed electric field (PEF)
[18,19] and ultrasound [20,21] have shown to be attractive alterna-
tives to extract selectively intracellular compounds from different
microalgae species. However, to the best of our knowledge, mod-
erate electric field (MEF) has not been applied for microalgae
compounds extraction, although interesting studies were reported
on food compounds extraction. MEF is an electrical technology
that involves the application of electric fields typically under
1000 V cm−1 , with or without heating, to accomplish specific objec-
tives. It has been studied associated with extraction processes

http://dx.doi.org/10.1016/j.procbio.2016.07.016
1359-5113/© 2016 Elsevier Ltd. All rights reserved.
 D.P. Jaeschke et al. / Process Biochemistry 51 (2016) 1636–1643
because it can promote electroporation of the cell membranes,
increasing its permeability [22].
Kulshrestha & Sastry [23] evaluated the diffusion of beet dye
using MEF (0–23.9 V cm−1 and 0–5000 Hz) and found that diffu-
sion increased when higher voltages and lower frequencies were
applied. Kulshrestha & Sastry [24] studied potato and beet cells per-
meabilization via MEF; the researchers observed that, even when
low voltages were applied to the food, there was an increase on
diffusion rates, suggesting that low energy consuming treatments,
as MEF, could be used to intensify the compounds diffusion from
inside vegetable cells. The MEF application on the extraction would
be quite interesting once this electrical technology is considered
less expensive than PEF or high pressure processes considered for
the same purpose [25].
Lipid quantification methods, such as Bligh & Dyer [11] and
Folch, Lees, & Stanley [12], have the disadvantage of using rela-
tively large amount of dry biomass (approximately 1 g); beyond
that, the drying and extraction processes are time and energy con-
suming. Sulfo-phospho-vanillin method (SPV) is a well-established
lipid quantification methodology at medical field and was originally
developed using human serum [26]. Recently, this methodology
was adjusted and employed for lipid quantification from microal-
gae [27]. It has the advantages of being fast and simple, requiring
wet and lower amounts of biomass.
Thereby, the aim of this work was to evaluate MEF application
in the presence of ethanol as a pretreatment to carotenoids and
lipids extraction from the microalgae Heterochlorella luteoviridis.
The influence of different ethanol concentrations on the diffusive
step of the extraction process was also evaluated. Moreover, the
SPV method was compared to the traditional Bligh & Dyer method
to evaluate its application as an alternative lipid quantification
methodology.
2. Materials and methods
2.1. Chemicals
The standards all-trans-␤-carotene, Supelco 37 component
FAME mix and olive oil were purchase from Sigma Aldrich (St. Louis,
USA). HPLC-grade solvents methanol and methyl tert-butyl ether
were obtained from Vetec (Duque de Caxias, Brazil) and hexane
were obtained from Dinâmica (Diadema, Brazil).
2.2. Microalgae cultivation
The microalgae Heterochlorella luteoviridis was obtained from
the culture collection of the Department of Marine Biology
at Fluminense Federal University (Niteroi, Brazil). Microalgae
cultivation was performed in flat-plate airlift photobioreactors
as described elsewhere [28]. The cultivation was performed
using modified f1/2 medium [29] with the NaNO3 concentration
increased to 300 mg L−1 . To avoid nutrients starvation, 1 mL L−1
of phosphate solution (5 g L−1 of NaH2 PO4 ·H2 O) and 1 mL L−1 of
trace metals solution (9.8 mg L−1 of CuSO4 ·5H2 O, 22 mg L−1 of
ZnSO4 ·7H2 O,1 mg L−1 of CoCl2 ·6H2 O, 180 mg L−1 of MnCl2 ·4H2 O,
6.3 mg L−1 of Na2 MoO4 ·2H2 O, 4.36 g L−1 of Na2 EDTA, and 3.15 g of
FeCl3 ·6H2 O) were daily added to the photobioreactors. The culti-
vation was conducted under a semi-continuous mode: four days
after the beginning of the cultivation, half of the bioreactors vol-
ume, corresponding to 1.2 L, was collected and centrifuged (Hitachi,
CR 21GIII, 10,000g, 10 ◦ C, 10 min); then, the bioreactors volume was
topped up with cultivation medium. This procedure was repeated
every 48 h during 38 days. After centrifugation, the biomass was
frozen with liquid nitrogen, freeze-dried (LIOTOP, Model L101,
Brazil) and homogenized.
1637
2.3. Extraction experiments
2.3.1. Extraction assisted by MEF
The MEF apparatus used to performed the experiments com-
prised a data acquisition system, a power supply, a variable
transformer (Sociedade Técnica Paulista LTDA, model Varivolt, São
Paulo, SP, Brazil), a stabilizer (Forceline, model EV 1000 T/2-2, São
Paulo, SP, Brazil), a computer and two extraction cells. The extrac-
tion cells, EC1 and EC2, are vessels, made of Pyrex glass, with a
water jacket and a working volume of 100 mL and 300 mL, respec-
tively. EC1 was used during the pretreatment step, and EC2 during
the diffusive step. The electrodes were made of titanium (3.5 cm
of height) and were curved to conform EC1 dimensions. The inter-
electrode gap of EC1 was between 4.5 cm and 5.0 cm. More details
of the apparatus can be found elsewhere [30].
Before the extraction experiments, the electrical conductivity
of the samples was adjusted to 500 ␮S cm−1 . According to Goul-
lieux & Pain [31], electrical conductivities above 500 ␮S cm−1 are
consider ideal to conduct electricity. This procedure allowed the
application of relatively high voltages without increasing the tem-
perature above 35 ◦ C. This adjustment was necessary because the
biomass was cultivated in 34 g L−1 salt solution, resulting in a high
electrical conductivity of the medium (6 mS cm−1 ). The decrease of
the electrical conductivity was achieved by washing the biomass
with distillated water, centrifuging and adjusting to the target value
using NaCl solution (2 g L−1 ).
The extraction experiments were performed using 4 g of dry
biomass and comprised 10 min of a pretreatment and 50 min of
a diffusive step. For the pre-treatment, 80 mL of a 25 mL/100 mL
of ethanol solution (volume fraction) was added to the biomass
in EC1; the voltage was set at values between 0 and 180 V (60 Hz
of frequency), according to an experimental design. Temperature
inside the vessel was controlled by means of a water jacket. This
procedure decreased the heating rate in order to maintain the tem-
perature below 35 ◦ C. For the diffusive step, ethanol concentrations
varied from 25 to 75 mL/100 mL of ethanol solution, according to
the experimental design. This step was performed using EC2 cell,
mixing the EC1 content with ethanol solution until a final volume
of 300 mL. During this diffusive step, temperature in the cell was
maintained at 30 ◦ C. A magnetic stirrer plate (IKA C-MAG, Model HS
10, Brazil) provided mixing inside the vessel during both steps.
After the extraction process, samples were centrifuged (4757g,
20 ◦ C, 10 min, Cientec, Model CT 5000R, Brazil) and filtered (Allcrom,
PTFE, 0.45 ␮m). Carotenoids and lipid analyses were performed
using the filtrated supernatant.
2.3.2. Exhaustive extractions
In order to determine the total carotenoid and lipid content
of the freeze-dried Heterochlorella luteoviridis biomass, exhaus-
tive extractions were performed. The carotenoid extraction from
biomass was carried out according to the methodology described
by Mandelli, Yamashita, Pereira, & Mercadante [32] with some
modifications. Dry biomass was macerated with ethyl acetate fol-
lowed by methanol and centrifuged. This step was performed
until the biomass became colorless. The subsequent steps (saponi-
fication, washing and drying) were performed with the same
methodology used for spectrophotometric analysis. The results
were expressed in milligrams of lutein per milligram of dried
biomass (mg g−1 ).
The total lipid content of the freeze-dried biomass was deter-
mined by Bligh & Dyer [11] and SPV method [27]. The analyses
were performed using 0.05 g of freeze-dried biomass; for the SPV
method, samples were previously hydrated with 2 mL of water, and
100 ␮L of this hydrated sample was used to perform the analysis.
1638 D.P. Jaeschke et al. / Process Biochemistry 51 (2016) 1636–1643
2.4. Quantification of the carotenoid content of the extracts
The carotenoid content of the ethanolic extracts was determined
using the methodology described by Rodriguez-Amaya [3] with
some modifications. Five grams of ethanolic extract was added
to 20 mL of acetone. The samples were homogenized in a vortex
(Phoenix Luferco, Model AP 56, Brazil) for 30 s and kept in the dark for
10 min. The partition step was made using ethylic and petroleum
ether (1:1). The saponification step was performed using methanol-
KOH solution (10 %, mass fraction) overnight. After saponification,
samples were washed with distilled water, dried using a rotatory
evaporator (Fisatom, Model 450-5, Brazil), diluted with ethanol and
analyzed on a spectrophotometer (PG Instruments Ltd., Model T80
UV–vis, United Kingdom) set at 450 nm.
2.5. Analysis of the carotenoid profile
The carotenoid profile of the biomass (exhaustive extraction)
and the ethanolic extract with the best extraction yield were ana-
lyzed by HPLC (Waters, Alliance e2695 Separations Module, USA),
according to the methodology described by Rodrigues et al. [16].
It was used a chromatography system equipped with quaternary
pump and a UV–vis detector (Rheodyne, Rohnert Park – CA, USA).
The UV–vis spectra were obtained between 250 nm and 600 nm
and the chromatograms were analyzed at 450 nm. Separation was
conducted on a C30 YMC column (5 ␮m, 250 × 4.6 mm id., Waters,
Wilmington − DE, USA). The mobile phases comprised eluents A
(methanol) and B (methyl tert-butyl ether). A linear gradient was
applied from 95:5 to 70:30 in the first 30 min, from 70:30 to 50:50
during the subsequent 20 min, maintaining this proportion for the
last 15 min. The identification was performed according to the elu-
tion order on C30 column, UV–vis spectrum characteristics and the
injection of all-trans-ˇ-carotene standard (0.125–15 ␮g.mL−1 ).
2.6. Quantification of the lipid content of the extracts
The lipid content of the extracts was determined by two meth-
ods: Bligh & Dyer [11] and SPV (Sulfo-phospho-vanillin method)
[27]. For the Bligh & Dyer method, 20 g of ethanolic extract was
added to 20 mL of ethanol and 10 mL of chloroform. Then, the sam-
ples were mixed during 30 min using a magnetic stirring plate (IKA
C-MAG, Model HS 10, Germany); after this step, 10 mL of chloroform
and 15 mL of a 10 % Na2 SO4 solution were added to the samples.
The chloroform phase was separated and filtered in the presence of
Na2 SO4 . Lipid content was determined by gravimetric difference.
The SPV method was performed using 100 ␮L of the extracts.
Two milliliters of sulfuric acid were added to the samples, main-
taining the temperature at 100 ◦ C with a hot plate (Fisatom, 752A,
Brazil). After, the samples were cooled and 5 mL of the vanillin
solution was added. Samples were kept in the dark for 15 min and
the absorbance was read at 530 nm using a spectrophotomer (PG
Instruments Ltd., Model T80 UV–vis, United Kingdom). The lipid con-
centration of each sample was determined using a calibration curve
[27] performed with olive oil standard (0.4–2 mg of olive oil/mL of
chloroform).
2.7. Analysis of fatty acid methyl esters (FAMEs)
The fatty acid profile of the biomass and the ethanolic extract
obtained under the conditions that promoted the best yield were
evaluated. Prior to the analysis, transesterification was performed
according to the methodology described by Joseph & Ackman [33].
This step was performed adding 1.5 mL of a 0.5 N NaOH in methanol
solution to the samples (chloroform phase of the Bligh & Dyer
method, dried using nitrogen gas); then, samples were heated in
a water bath at 100 ◦ C for 5 min and subsequently cooled at room
temperature. Next, 2 mL of BF3 (boron trifluoride) were added to the
samples, which were heated again for 30 min at 100 ◦ C and cooled.
Later, 5 mL of a saturated NaCl solution (360 g L−1 ) was added to
the tubes and the samples were stirred. Then, 1 mL of isooctane
was added and, when the separation of phases occurred, isooctane
was withdrawn to another tube. Samples were dried using nitrogen
gas and diluted with a BHT/hexane solution right before injection
into the gas chromatographer. During all this procedure, tubes were
kept closed with nitrogen inert atmosphere.
Fatty acid esters were separated in a gas chromatograph (Shi-
madzu, GC – 2010 Plus, Japan) equipped with a capillary column
of fused silica (Shimadzu, SBL 100, 30 m x 0.25 mm and 0.25 ␮m
of film thickness). The flame ionization detector (FID) and the
injection port temperature were 260 ◦ C and 240 ◦ C, respectively.
Column temperature was maintained at 50 ◦ C for 1 min and then
was increased at 10 ◦ C min−1 to 250 ◦ C, with a final holding time
of 74 min. The carrier gas was hydrogen with a constant injection
flow rate of 1.0 mL min−1 . The samples were injected in triplicate.
For fatty acid identification, the retention times were compared
to methyl-esters standard (Supelco, 37 component FAME mix)
previously analyzed by gas chromatograhy–mass spectrometry
(GC–MS).
2.8. Scanning electron microscope (SEM) analysis
SEM analysis of the biomass treated by MEF (experiments 0 V
and 25 mL/100 mL of ethanol solution, 180 V and 25 mL/100 mL of
ethanol solution, 180 V and 75 mL/100 mL of ethanol solution) were
performed to detect cell permeabilization. Previously to SEM analy-
sis, conducted in a scanning electron microscope (Hitachi, Tabletop
Microscope TM 3000, Japan), the biomass was dried at 30 ◦ C during
24 h (Solab, SL 102/100, Brazil) and fixed in stubs (1 cm of diameter)
with a metallic strip.
2.9. Experimental design and statistical data analyses
The experiments were planned to identify the effect of the volt-
age (V) and the ethanol concentration (mL/100 mL) on carotenoid
and lipid extraction from microalgae. Voltage ranged from 0 V to
180 V, and ethanol concentration varied from 25 mL/100 mL to
75 mL/100 mL; the voltage range was selected based on the lim-
itations of the MEF equipment. All extraction conditions were
performed in duplicate. Statistical analyses were performed using
the software Statistica® (7.0, Statsoft Inc., Tulsa, USA). Statistical
significance was determined by ANOVA and Tukey tests with a
95% of confidence level. Experimental data were analyzed using
a polynomial regression model, presented in Equation (1), where
Y represents the carotenoid or lipid content extracted from the
dried biomass (mg g−1 ), X1 is the coded variable for voltage (V)
(Equation (2)), X2 is the coded variable for ethanol concentration
(CEtOH ) (Equation (3)) and ˇ0 , ˇ1 , ˇ2 , ˇ11 , ˇ22 and ˇ12 represent the
equation coefficients.
Y = ˇ0 + ˇ1 X1 + ˇ2 X2 + ˇ11 X1 2 + ˇ22 X2 2 + ˇ12 X1 X2 (1)
where
V − 90
X1 = (2)
90
CEtOH − 50
X2 = (3)
25
 D.P. Jaeschke et al. / Process Biochemistry 51 (2016) 1636–1643
Table 1
Regression coefficients of the coded variables for carotenoid and lipid extraction.
Carotenoids
Regression Coefficients
coefficient p-value
ˇ0 0.068 0.027
ˇ1 0.236 <0.001
ˇ2 −0.104 0.021
ˇ11 0.340 <0.001
ˇ22 0.389 <0.001
ˇ12 0.280 <0.001
Blocks – 0.004
Regression R2 0.987
Regression p-value 1.3 × 10−11
Fig. 1. Contour lines of carotenoid extraction from algal biomass (mg g−1 ) under
different ethanol concentration (%) and voltage (V) conditions.
3. Results and discussion
3.1. Moderate electric field and ethanol effect
The effect of MEF and ethanol concentration on carotenoid and
lipid extraction from Heterochlorella luteoviridis was evaluated and
the experimental data were used to determine the polynomial
equation coefficients, which are presented in Table 1. The models
obtained adequately represent experimental data with a determi-
nation coefficient (R2 ) of 0.987, 0.959 and 0.956 for carotenoids,
Bligh & Dyer and SPV results, respectively. The experimental
design showed that ethanol concentration significantly affected
carotenoid and lipid extraction (p < 0.05), while MEF showed no
effect on the lipid extraction. A discussion of the extraction yield
for these two compounds in the different conditions evaluated is
presented separately in the following section.
The effect of ethanol concentration and MEF on carotenoid
extraction is presented as a contour plot shown in Fig. 1. It was
observed that carotenoid extraction increased as electrical field
strength and ethanol concentration increased. It can also be seen a
combined effect of both variables studied with the highest extrac-
tion yield at the maximum values of the two variables (180 V
and 75 mL/100 mL of ethanol concentration). It is important to
point out that only the presence of ethanol 75 mL/100 mL was
not sufficient for carotenoid extraction, being necessary the pres-
ence of the electric field. In microalgae, carotenoids are stored
within the chloroplasts that are surrounded by two phospholipidic
membranes; MEF probably modified the chloroplasts membrane
structure, allowing carotenoids extraction. Sensoy & Sastry [22]
evaluated solutes extraction from black tea leaves using MEF and
compared with an extraction using water (control). The authors
1639
Bligh & Dyer SPV
coefficient p-value coefficient p-value
3.57 <0.001 3.38 <0.001
– – – –
5.24 <0.001 5.21 <0.001
– – – –
2.75 <0.001 2.94 <0.001
– – – –
– – – –
0.959 0.956
1.4 × 10−12 1.9 × 10−12
Fig. 2. Lipid content at different extraction conditions (ethanol concentration from
25 to 75 mL/100 mL, v/v). The lipid quantification of the ethanol extracts were per-
formed using the Bligh & Dyer and the SPV methods.
observed that MEF enhanced the extraction from fresh tea leaves
and reported an elongation (compression) on the chloroplasts
structure on MEF treated samples. This result is in agreement with
the ones observed in the present work. As can be seen in Fig. 1, even
at the highest ethanol concentration (75 mL/100 mL), the solvent
itself was not able to disorder the chloroplast structure, resulting
in low carotenoid extraction yield.
Luengo et al. [34] evaluated pigments extraction from microal-
gae Chlorella vulgaris using PEF. The electrical field strength varied
from 10 to 25 kV cm−1 and the frequency of each pulse was 0.5 Hz.
The authors evaluated pigment extraction right after PEF treat-
ment and 1 h after the treatment. They observed that 1 h after the
extraction process the content of chlorophyll a, chlorophyll b and
carotenoid was higher comparing to the control (no PEF treatment).
Moreover, no pigments were observed when the samples were
analyzed right after applying PEF. This was attributed to chloro-
plasts plasmolysis that occurs by an osmotic imbalance once the
cell membrane is permeabilized by PEF treatment, indicating that
the extraction depends on a diffusion step. In the same study, the
authors did not attribute pigments extraction to chloroplasts com-
pression, but to the osmotic imbalance of the medium. Therefore,
more studies are needed to elucidate the influence of electric field
on chloroplast structure.
The effect of ethanol concentration on lipid extraction (for SPV
and Bligh & Dyer methods) can be seen in Fig. 2. It was observed
that lipid extraction increased as ethanol concentrations increased.
This result is explained by the interaction between ethanol and
the phospholipid layer of the cell membrane, leading to a disor-
der of the membrane physical structure [35,36]. Ethanol is largely
used as sanitizing agent to eliminate microorganisms. Although
its ideal concentration depends on the microorganism species,
a 70 mL/100 mL ethanol/water solution is usually used. A high
carotenoid and lipid extraction yield was obtained with this con-
centration in the present work. Gurtovenko & Anwar [37] evaluated
the effect of ethanol concentration (2.5–30 %, mol/mol) on phos-
pholipid membranes and reported that concentrations lower than
12% (mol/mol, 30.5 mL/100 mL, v/v) promoted membrane struc-
1640 D.P. Jaeschke et al. / Process Biochemistry 51 (2016) 1636–1643
Fig. 3. SEM analysis of Heterochlorella luteoviridis biomass samples after the extraction treatment: (a) 0 V, 25 mL/100 mL of ethanol solution; (b) 180 V, 25 mL/100 mL of
ethanol solution; and (c) 180 V, 75 mL/100 mL of ethanol solution.
ture disorder, as well as a decrease of the membrane thickness.
Above this concentration, irregularities in the membrane could
be observed, and the bilayer structure started to be affected.
Grima et al. [38] evaluated seven solvent mixtures, including
ethanol (96 mL/100 mL, v/v), on lipid extraction from the microal-
gae Isochrysis galbana. The authors reported that 96 mL/100 mL
ethanol solution extracted approximately 85 mL/100 mL of the total
lipid content obtained by Bligh & Dyer method. This result is in
agreement to those obtained in the present work.
The MEF effect did not influenced lipid extraction. MEF was
supposed to act on cell membrane promoting permeabilization.
However, this effect was not observed in the present work as can
be seen in the micrographic images of biomass samples submitted
to 0 V, 25 mL/100 mL of ethanol (Fig. 3a) and 180 V, 25 mL/100 mL
of ethanol (Fig. 3b). After the treatments, biomass appeared to be
agglomerated and most of the cells presented a regular spherical
geometry, with no detectable visual differences between treat-
ments of 0 V and 180 V. These results suggest that irreversible
electroporation did not occur, once visible damage caused by
MEF was not observed. On the other hand, cells submitted to the
extraction treatment 180 V, 75 mL/100 mL of ethanol (Fig. 3c) were
apparently damaged (cells are compressed and elongated), proba-
bly due to ethanol effect on cellular membrane.
The voltage intensity (0–180 V) used in the present work may
not be enough to promote structural changes on the cell membrane
that could enhance the extraction; some studies have reported pore
formation when voltages higher than 180 V were used [18,39–41].
Zbinden et al. [42] evaluated PEF as a pre-treatment on lipid extrac-
tion from microalgae and reported that approximately 90% of the
cells were lysed, increasing the lipid extraction. Recently, stud-
ies have shown that electrical permeabilization depends on the
field strength and pulse parameters (type of pulse and pulse dura-
tion) [43]. In the present work, the alternating electric field with
a senoidal waveform was probably not enough to promote defini-
tive pore formation. It is presumable that reversible electroporation
could be occurred but this phenomenon was not sufficient to
increase lipid extraction.
3.2. Extraction efficiency
3.2.1. Carotenoid extraction efficiency
The extraction using MEF and ethanol was compared to the
carotenoid exhaustive extraction from biomass. The carotenoid
content of the biomass and extracts, shown in Table 2, ranged
from 0.04 ± 0.001 to 1.21 ± 0.080 mg g−1 . The standard devia-
tions between duplicate experiments were lower than 10%. The
results showed that, using a pre-treatment of MEF and ethanol,
an extraction up to 73% of total carotenoids from the microalgae
Chlorella sp. was achieved. Furthermore, experiments performed
with 180 V evidenced that ethanol concentrations of 25 mL/100 mL
and 50 mL/100 mL resulted in low extraction yields of 2% and
7%, respectively. These two experimental points statically dif-
fer (p < 0.05) from the experiment conducted using 180 V and
75 mL/100 mL of ethanol concentration. Moreover, the experimen-
tal condition of 135 V and 62.5 mL/100 mL of ethanol concentration
showed 20% of carotenoid extraction, which means that a high
ethanol concentration is required for the extraction. In addition,
analyzing the experimental conditions using ethanol concentra-
tion of 75 mL/100 mL and voltages varying from 0 V to 180 V, the
influence of the voltage also becomes evidenced, since 0 V and 90 V
yielded 14% and 41% of extraction, respectively.
It is important to point out that conventional extraction meth-
ods have the disadvantages of using toxic organic solvents, such
as acetone, hexane and ethyl acetate, and being time consuming
since they are based on sample maceration. On the other hand, the
methodology applied in this work uses an environmentally friendly
solvent, with no need of maceration step.
3.2.2. Lipid extraction efficiency
Lipid content and the extraction yield of all extracts analyzed by
Bligh & Dyer and SPV methods are shown in Table 2. The extraction
yield was determined by the ratio between the lipid contents of the
extract and the biomass, which was determined by an exhaustive
extraction.
Comparing the experimental results using the same voltage and
different ethanol concentrations, an increase of the extraction yield
was observed as ethanol concentration increased. Results showed
that up to 83% of the total lipid content of the biomass can be
extracted when a solution with 75 mL/100 mL of ethanol concen-
tration is used. Results obtained by SPV method did not statistically
differ (p < 0.05) from those obtained by Bligh & Dyer method, which
indicates that SPV method can be used as a simple and fast lipid
quantification method from Heterochlorella luteoviridis.
3.3. Carotenoid profile of the extracts
HPLC analyses were performed in the biomass and in the
extract with best yield (180 V, 75 mL/100 mL of ethanol con-
centration) in order to compare the carotenoid profiles. The
chromatograms obtained are presented in Fig. 4 for the biomass
(a) and the extract (b), respectively. Information regarding each
peak shown in the chromatograms is presented in Table 3. The
results showed that all-trans-lutein was the major carotenoid
(856 ± 56 ␮g g−1 ) found in Heterochlorella luteoviridis, repre-
senting 68.3% of the total carotenoid content, followed by
all-trans-zeaxanthin (244 ± 20 ␮g g−1 ) and all-trans-␤-carotene
(185 ± 2 ␮g g−1 ). Other carotenoids in lower concentrations were
identified on the biomass: all-trans-␣-carotene, 9–13-15-cis-␤-
carotene, cis-violaxanthin, all-trans-violaxanthin and 13–13 -cis-
lutein. This result is in agreement with the ones reported by
 D.P. Jaeschke et al. / Process Biochemistry 51 (2016) 1636–1643 1641

Table 2
Experimental conditions, extraction yield and carotenoid and lipid content for each experimental point.*

Carotenoids Lipids
Voltage (V) CEtOH (mL/100 mL)
Concentration mg of lutein Yield (%) Bligh & Dyer g Yield (%) SPV g 100 g of Yield (%)
equivalent g of sample−1 100 g of sample−1 sample−1

0 25.0 0.11 ± 0.00e 6% 1.57 ± 0.031Ac 11% 1.56 ± 0.089Ac 9%

0 75.0 0.25 ± 0.00de 14% 10.46 ± 0.97Aa 71% 11.27 ± 0.52Aa 64%
90 50.0 0.07 ± 0.00e 4% 4.29 ± 0.07Abc 29% 4.64 ± 0.11Abc 26%
90 75.0 0.71 ± 0.01c 41% 12.29 ± 0.98Aa 83% 12.20 ± 0.12Aa 69%
135 62.5 0.35 ± 0.02d 20% 5.99 ± 0.60Ab 41% 5.26 ± 0.52Ab 30%
180 25.0 0.04 ± 0.00e 2% 0.49 ± 0.02Ac 3% 0.48 ± 0.02Ac 3%
180 50.0 0.11 ± 0.01e 7% 4.47 ± 0.06Abc 30% 4.29 ± 0.24Abc 24%
180 75.0 1.21 ± 0.08b 73% 11.92 ± 0.24Aa 81% 11.60 ± 0.77Aa 66%
Total content 1.73 ± 0.09a – 14.76 ± 0.52Aa – 17.67 ± 0.42Aa –
*
Mean ± standard deviation; means with the same lowercase letter in the same column does not present significant difference (p > 0.05); means with the same capital
letter in the same line does not present significant difference (p > 0.05).

Table 4
Retention time of all fatty acids peaks observed in the chromatograms.

Peaka Retention time (min) Fatty acid

1 19.9 Standard
2 30.3 C:14
3 32.6 C:14:1
4 34.7 C16:0
5 35.3 C:16:1n9
6 35.5 C:16:1n7
7 38.4 C:18:0
8 39.4 C18:1n6c
9 39.6 C18:1n9c
10 40.8 C:18:2n6c
11 41.4 C18:3n6
12 42.5 C18:3n3
13 43.1 C:22:0
14 45.2 C:24:0
a
Numbered according to the chromatograms shown in Fig. 5.

Tibbetts et al. [44] and Inbaraj et al. [2]. Tibbetts et al. [44]
evaluated the carotenoid profile of various microalgae species,
including Chlorella vulgaris. The authors reported that lutein was
the major carotenoid (132 ± 8.7 ␮g g−1 ), followed by zeaxanthin
(20.8 ± 2.0 ␮g g−1 ) and fucixanthin (6.2 ± 0.6 ␮g g−1 ). In the extract,
the carotenoids all-trans-lutein and all-trans-zeaxanthin could be
identified and represented 68.3% and 18.7% of the total content,
respectively. Other carotenoids, such as all-trans-violaxanthin, cis-
violaxanthin, 13-13 -cis-lutein, accounted for 8.5% of the total
carotenoid content. The peaks of ␣- and ␤-carotene were identi-
fied but the intensities were very low. This result showed that the
extracted carotenoids were mainly all-trans-lutein and all-trans-
Fig. 4. Chromatograms showing carotenoid profile of the (a) freeze-dried biomassa zeaxanthin. These carotenoids interacted with ethanol due to the
after exhaustive extraction and (b) ethanolic extract (180 V, 75 mL/100 mL of ethanol presence of the hydroxyl groups in their structure. Therefore, as
solution).
all-trans-lutein and all-trans-zeaxanthin are the major carotenoids
on Heterochlorella luteoviridis, ethanol showed to be an adequate
solvent for carotenoids extraction.
Table 3
Retention time of carotenoid peaks observed in the chromatograms.
3.4. Fatty acid methyl esters (FAMEs) profile
Peaka Carotenoid Retention time (min)b

1 all-trans-violaxantin 7.3 A gas chromatography analysis was performed to evaluate fatty

2 cis-violaxanthin
3 13-cis-lutein
4 13 -cis-lutein
5 all-trans-lutein
6 all-trans-zeaxanthin
7 15-cis-␤-caroteno
8 all- trans-␣-caroteno
9 13-cis-␤-caroteno
10 all-trans-␤-carotene
11 9-cis-␤-caroteno
a
Numbered according to the chromatogram shown in Fig. 4.
b
Retention time on the C30 column.
8.0
acid profile on the best extraction condition (75 mL/100 mL of
10.3
11.3
ethanol) and on the biomass (Fig. 5 and Table 4). The major fatty acid
12.3 found in Heterochlorella luteoviridis was the palmitic acid (C16:0),
14.5 representing 35% of the total fatty acid content. Among all fatty
27.5 acids identified, it was found 32% of linoleic acid (C:18:2n6), 10% of
29.9
␣-linoleic acid (C18:3n3), 7% of stearic acid (C:18:0) and 6% of oleic
31.1
34.1 acid (C18:1n9). This result is similar to other results found in the
36.3 literature [45–47]. Praveenkumar et al. [44] evaluated Chlorella sp.
productivity in various cultivation conditions. The authors reported
that, for all the conditions tested, the major fatty acids were linoleic
and palmitic acid, representing 21.1 ± 5.7 and 20.6 ± 4.9%, respec-
1642 D.P. Jaeschke et al. / Process Biochemistry 51 (2016) 1636–1643
Fig. 5. Chromatograms showing the fatty acid profile of the (a) biomass after
exhaustive extraction and the (b) ethanolic extract (180 V, 75 mL/100 mL of ethanol
solution).
tively. Ohse et al. [46] evaluated the lipid content and the fatty
acid profile of ten microalgae species, including Chlorella vulgaris.
The results obtained for C. vulgaris showed that the total lipid con-
tent was 16.1% and the major fatty acids were palmitic (C16:0) and
linolenic acid (C18:2n6), representing 21.17 and 22.71% of the total
fatty acid content, respectively. Stearic (C18:0), oleic (C18:1n9) and
linoleic (C18:3n3) acids were also identified and corresponded to
15.5, 13.5 and 7.4% of the total fatty acid content, respectively.
Comparing the FAMEs profile of the biomass and the extract,
similar results were found, since the main fatty acids were also
palmitic acid (39%), linoleic acid (25 %), ␣-linoleic acid (10%). This
result indicates that using a 75 mL/100 mL of ethanol/water solu-
tion it was possible to effectively extract the high added value fatty
acids, ␻-6 and ␻-3. These compounds, extracted using a less harm-
ful and non-toxic solvent, can be used in many applications, such
as in the food industry and as a lipid option for vegans.
4. Conclusion
The present work evaluated the application of MEF in the pres-
ence of ethanol as a pre-treatment followed by a diffusive step
with ethanol as solvent on carotenoid and lipid extraction from
the microalgae Heterochlorella luteoviridis. The results showed that
MEF combined with ethanol as solvent (180 V, 75 mL/100 mL of
ethanol concentration) yielded up to 73% of carotenoid extrac-
tion. For lipids, the highest lipid extraction obtained were 83 %
when 75 mL/100 mL of ethanol concentration was used. The pro-
posed methodology proved to be an attractive alternative method
to extract these compounds from the microalgae Heterochlorella
luteoviridis.
The carotenoid and FAMEs profile analyses of the extract
revealed that the xanthophylls all-trans-lutein and all-trans-
zeaxanthin were the major carotenoids extracted due to their
polarity. The high value added ␻-3 and ␻-6 fatty acids were also
effectively extracted by ethanol. Overall, the combination of MEF
and ethanol showed to be an interesting carotenoid and lipids
extraction method from the microalgae Heterochlorella luteoviridis
because it showed high-yield extraction and uses a less harmful
and non-toxic solvent.
Acknowledgements
The authors gratefully acknowledge the financial support
received from CAPES (Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior) as a scolarship for the first author and as the
PNPD (Programa Nacional de Pós Doutorado) grant.
References
[1] F. Ahmed, K. Fanning, M. Netzel, W. Turner, Y. Li, P.M. Schenk, Profiling of
carotenoids and antioxidant capacity of microalgae from subtropical coastal
and brackish waters, Food Chem. 165 (2014) 300–306.
[2] B.S. Inbaraj, J.T. Chien, B.H. Chen, Improved high performance liquid
chromatographic method for determination of carotenoids in the microalga
Chlorella pyrenoidosa, J. Chromatogr. 1102 (2006) 193–199.
[3] D.B. Rodriguez-Amaya, A Guide to Carotenoid Analysis in Foods, ILSI Press,
Washington DC, 2001.
[4] E. Ibañez, A. Cifuentes, Benefits of using algae as natural sources of functional
ingredients, J. Sci. Food Agric. 93 (2013) 703–709.
[5] C. Galli, P.C. Calder, Effects of fat and fatty acid intake on inflammatory and
immune responses: a critical review, Ann. Nutr. Metab. 55 (2009) 123–139.
[6] K.-H. Park, J.-Y. Kim, I. Choi, J.-R. Kim, K.-H. Cho, ␻-6 (18:2) and ␻-3 (18:3)
fatty acids in reconstituted high-density lipoproteins show different
functionality of anti-atherosclerotic properties and embryo toxicity, J. Nutr.
Biochem. 26 (2015) 1613–1621.
[8] M. Koller, A. Muhr, G. Braunegg, Microalgae as versatile cellular factories for
valued products, Algal Res. 6 (2014) 52–63.
[9] S. Pieber, S. Schober, M. Mittelbach, Pressurized fluid extraction of
polyunsaturated fatty acids from the microalga Nannochloropsis oculata,
Biomass Bioenergy 47 (2012) 474–482.
[10] E.G. Bligh, W.J. Dyer, A Rapid method of total lipid extraction and purification,
Can. J. Biochem. Physiol. 37 (1959) 911–917.
[11] J. Folch, M. Lees, G.H.S. Stanley, A simple method for the isolation and
purification of total lipides from animal tissues, J. Biol. Chem. 226 (1957)
497–509.
[12] A. Hara, N.S. Radin, Lipid extraction of tissues with a low-toxicity solvent,
Anal. Biochem. 90 (1978) 420–426.
[13] I.S. Chen, C.S.J. Shen, A.J. Sheppard, Comparison of methylene chloride and
chloroform for the extraction of fats from food products, J. Am. Oil Chem. Soc.
58 (1981) 599–601.
[14] J. Deli, S. Gonda, L.Z. Nagy, I. Szabó, G. Gulyás-Fekete, A. Agócs, K. Marton, G.
Vasas, Carotenoid composition of three bloom-forming algae species, Food
Res. Int. 65 (2014) 215–223.
[15] D.B. Rodrigues, É.M.M. Flores, J.S. Barin, A.Z. Mercadante, E. Jacob-Lopes, L.Q.
Zepka, Production of carotenoids from microalgae cultivated using
agroindustrial wastes, Food Res. Int. 65 (2014) 144–148.
[16] Y. Shao, J. Pan, C. Zhang, L. Jiang, Y. He, Detection in situ of carotenoid in
microalgae by transmission spectroscopy, Comput. Electron. Agric. 112 (2015)
121–127.
[17] O. Parniakov, F.J. Barba, N. Grimi, L. Marchal, S. Jubeau, N. Lebovka, E. Vorobiev,
Pulsed electric field and pH assisted selective extraction of intracellular
components from microalgae Nannochloropsis, Algal Res. 8 (2015) 128–134.
[18] O. Parniakov, F.J. Barba, N. Grimi, L. Marchal, S. Jubeau, N. Lebovka, E. Vorobiev,
Pulsed electric field assisted extraction of nutritionally valuable compounds
from microalgae Nannochloropsis spp. using the binary mixture of organic
solvents and water, Innov. Food Sci. Emerg. Technol. 27 (2015) 79–85.
[19] O. Parniakov, E. Apicella, M. Koubaa, F.J. Barba, N. Grimi, N. Lebovka, G. Pataro,
G. Ferrari, E. Vorobiev, Ultrasound-assisted green solvent extraction of
high-added value compounds from microalgae Nannochloropsis spp,
Bioresour. Technol. 198 (2015) 262–267.
[20] J.A. Gerde, M. Montalbo-Lomboy, L. Yao, D. Grewell, T. Wang, Evaluation of
microalgae cell disruption by ultrasonic treatment, Bioresour. Technol. 125
(2012) 175–181.
[21] I. Sensoy, S.K. Sastry, Extraction using moderate electric fields, J. Food Sci. 69
(2004) 7–13.
[22] S. Kulshrestha, S. Sastry, Frequency and voltage effects on enhanced diffusion
during moderate electric field (MEF) treatment, Innov. Food Sci. Emerg.
Technol. 4 (2003) 189–194.
[23] S.A. Kulshrestha, S.K. Sastry, Changes in permeability of moderate electric
field (MEF) treated vegetable tissue over time, Innov. Food Sci. Emerg.
Technol. 11 (2010) 78–83.
[24] N. Lebovka, E. Vorobiev, Electrotechnologies for Extraction from Food Plants
and Biomaterials, Springer, 2008.
[25] G.T. Vatassery, M.A. Sheridan, A.M. Krezowski, A.S. Divine, H.L. Bach, Use of
the sulfo-phospo-vanillin reaction in a routine method for determining total
lipids in human cerebrospinal fluid, Clin. Biochem. 14 (1981) 21–24.
 D.P. Jaeschke et al. / Process Biochemistry 51 (2016) 1636–1643
[26] S.K. Mishra, W.I. Suh, W. Farooq, M. Moon, A. Shrivastav, M.S. Park, J.-W. Yang,
Rapid quantification of microalgal lipids in aqueous medium by a simple
colorimetric method, Bioresour. Technol. 155 (2014) 330–333.
[27] L.H. Kochem, N.C. Da Fré, C. Redaelli, R. Rech, N.R. Marcílio, Characterization of
a novel flat-panel airlift photobioreactor with an internal heat exchanger,
Chem. Eng. Technol. 37 (2014) 59–64.
[28] R.L. Guillard, Culture of phytoplankton for feeding marine invertebrates, in:
W. Smith, M. Chanley (Eds.), Culture of Marine Invertebrate Animals,
Springer, US, 1975, pp. 29–60.
[29] G.D. Mercali, D.P. Jaeschke, I.C. Tessaro, L.D.F. Marczak, Degradation kinetics
of anthocyanins in acerola pulp: comparison between ohmic and
conventional heat treatment, Food Chem. 136 (2013) 853–857.
[30] A. Goullieux, J.-P. Pain, 18 – Ohmic heating, in: D.-W. Sun (Ed.), Emerging
Technologies for Food Processing, Academic Press, London, 2005,
pp. 469–505.
[31] F. Mandelli, F. Yamashita, J.L. Pereira, A.Z. Mercadante, Evaluation of biomass
production, carotenoid level and antioxidant capacity produced by Thermus
filiformis using fractional factorial design, Braz. J. Microbiol. 43 (2012)
126–134.
[32] J.D. Joseph, R.G. Ackman, Capillary column gas chromatographic method for
analysis of encapsulated fish oils and fish oil ethyl esters: collaborative study,
J. AOAC Int. 75 (1992) 488–506.
[33] E. Luengo, S. Condón-Abanto, I. Álvarez, J. Raso, Effect of pulsed electric field
treatments on permeabilization and extraction of pigments from chlorella
vulgaris, J. Membr. Biol. 247 (2014) 1269–1277.
[34] D.B. Goldstein, Effect of alcohol on cellular membranes, Ann. Emerg. Med. 15
(1986) 1013–1018.
[35] M. Patra, E. Salonen, E. Terama, I. Vattulainen, R. Faller, B.W. Lee, J.
Holopainen, M. Karttunen, Under the influence of alcohol: the effect of
ethanol and methanol on lipid bilayers, Biophys. J. 90 (2006) 1121–1135.
[36] A.A. Gurtovenko, J. Anwar, Interaction of ethanol with biological membranes:
the formation of non-bilayer structures within the membrane interior and
their significance, J. Phys. Chem. 113 (2009) 1983–1992.
1643
[37] E.M. Grima, A.R. Medina, A.G. Giménez, J.A. Sánchez Pérez, F.G. Camacho, J.L.
García Sánchez, Comparison between extraction of lipids and fatty acids from
microalgal biomass, J. Am. Oil Chem. Soc. 71 (1994) 955–959.
[38] N.I. Lebovka, H. Mhemdi, N. Grimi, O. Bals, E. Vorobiev, Treatment of potato
tissue by pulsed electric fields with time-variable strength: theoretical and
experimental analysis, J. Food Eng. 137 (2014) 23–31.
[39] M. Goettel, C. Eing, C. Gusbeth, R. Straessner, W. Frey, Pulsed electric field
assisted extraction of intracellular valuables from microalgae, Algal Res. 2
(2013) 401–408.
[40] H. Bouzrara, E. Vorobiev, Solid–liquid expression of cellular materials
enhanced by pulsed electric field, Chem. Eng. Process.: Process Intensif. 42
(2003) 249–257.
[41] M.D.A. Zbinden, B.S.M. Sturm, R.D. Nord, W.J. Carey, D. Moore, H. Shinogle,
S.M. Stagg-Williams, Pulsed electric field (PEF) as an intensification
pretreatment for greener solvent lipid extraction from microalgae,
Biotechnol. Bioeng. 110 (2013) 1605–1615.
[42] E. Vorobiev, N.I. Lebovka, Extraction of intercellular components by pulsed
electric fields, in: J. Raso, V. Heinz (Eds.), Pulsed Electric Fields Technology for
the Food Industry, Springer, US, 2006, pp. 153–193.
[43] S.M. Tibbetts, C.G. Whitney, M.J. MacPherson, S. Bhatti, A.H. Banskota, R.
Stefanova, P.J. McGinn, Biochemical characterization of microalgal biomass
from freshwater species isolated in Alberta. Canada for animal feed
applications, Algal Res. 11 (2015) 435–447.
[44] R. Praveenkumar, B. Kim, E. Choi, K. Lee, J.-Y. Park, J.-S. Lee, Y.-C. Lee, Y.-K. Oh,
Improved biomass and lipid production in a mixotrophic culture of Chlorella
sp. KR-1 with addition of coal-fired flue-gas, Bioresour. Technol. 171 (2014)
500–505.
[45] S. Ohse, R. Bianchini Derner, R. Ávila Ozório, R. Gordo Corrêa, E. Badiale
Furlong, P.C. Roberto Cunha, Lipid content and fatty acid profiles in ten
species of microalgae, Idesia (Arica) 33 (2015) 93–101.
[46] I. Krzemińska, A. Piasecka, A. Nosalewicz, D. Simionato, J. Wawrzykowski,
Alterations of the lipid content and fatty acid profile of Chlorella
protothecoides under different light intensities, Bioresour. Technol. 196
(2015) 72–77.