Algal Research 8 (2015) 128–134

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Pulsed electric field and pH assisted selective extraction of intracellular

components from microalgae Nannochloropsis
Oleksii Parniakov a,b, Francisco J. Barba c, Nabil Grimi a, Luc Marchal d, Sébastien Jubeau e,
Nikolai Lebovka a,b,⁎, Eugene Vorobiev a
a
Sorbonne Universités, Université de Technologie de Compiègne, Laboratoire Transformations Intégrées de la Matière Renouvelable (UTC/ESCOM, EA 4297 TIMR), Centre de Recherche Royallieu, CS
60319, 60203 Compiègne Cedex, France
b
Institute of Biocolloidal Chemistry named after F. D. Ovcharenko, NAS of Ukraine, 42, blvr. Vernadskogo, Kyiv 03142, Ukraine
c
Universitat de València, Nutrition and Food Science Area, Avda. Vicent Andrés Estellés, s/n 46100 Burjassot, València, Spain
d
LUNAM Université, CNRS, GEPEA, Université de Nantes, UMR6144, CRTT, Boulevard de l'Université, BP 406, 44602 Saint-Nazaire Cedex, France
e
AlgoSource Technologies, 37 Bd de l'Université, 44600 Saint-Nazaire, France

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

Article history: The study was aimed at investigation of the potential of pulsed electric field (PEF) pre-treatment as a preliminary
Received 6 August 2014 step of pH-assisted aqueous extraction of algae components from microalgae Nannochloropsis suspensions. The
Received in revised form 9 December 2014 PEF and sonication (S) were compared as pretreatment methods. They were applied at normal (pH = 8.5) and
Accepted 31 January 2015
basic (pH = 11) conditions, and supplementary basic extraction (at pH = 11) was done. The extracts were
Available online xxxx
analyzed for content of pigments, proteins, carbohydrates, total phenolic compounds and antioxidant capacity.
Keywords:
The colloidal stability of PEF- and S-pretreated suspensions was also evaluated. The data evidence that PEF tech-
Electroporation nique allows selective extraction of a portion of pure proteins that are different from proteins extracted from S-
Pulsed electric field pretreated suspensions. The discovered effects have shown the advantages of PEF-pretreatment at normal
Extraction conditions (pH = 8.5) and supplementary extraction at basic conditions (pH = 11) for selective extraction of
pH different intracellular components.
Microalgae © 2015 Elsevier B.V. All rights reserved.
Nannochloropsis

1. Introduction
Microalgae have high content of lipids, proteins, polyunsaturated
fatty acids, carotenoids, valuable pigments and vitamins, and can be
used in the food, feed, cosmetics, and pharmaceutical and bio-fuel in-
dustries [1–5]. Nowadays, the large scale photo-bioreactors for cultiva-
tion of algae are commercially available [6]. Typically, the extraction
efficiency from different strains of microalgae is highly dependent on
the size, wall composition and structure of their cells. Moreover, current
oil extraction from microalgae requires using of solvents, such as chloro-
form, methanol, acetone or hexane.
Recent efforts have shown a high potential of application of pulsed
electric fields (PEFs) for enhancement of extraction of valuable compo-
nents from different biological objects [7–9]. PEF-assisted techniques are
based on the phenomenon of electroporation, or electropermeabilization,
that reflects formation of pores in the cell membranes under the effect of
electric pulses with short duration (from several nanoseconds to several
⁎ Corresponding author at: Institute of Biocolloidal Chemistry, National Academy of
Sciences of Ukraine, 42, Vernadsky av., 03142 Kyiv, Ukraine and Université de Technologie
de Compiègne, TIMR, EA 4297, UTC/ESCOM, Centre de Recherche de Royallieu, B.P. 20529-
60205 Compiègne Cedex, France.
E-mail addresses: lebovka@gmail.com, Nikolai.Lebovka@utc.fr (N. Lebovka).
milliseconds), and high electric field strength (from 100–300 V/cm up
to 300 kV/cm) [10]. During the last decades electroporation was frequent-
ly used as an effective tool for gene transformation of algae protoplasts
(for a review, see [11]). However, small attention was paid to applications
of PEF treatment for purposes of extraction from microalgae species. It
was noted that PEF treatment of the chlorophycean microalgae
(Scendesmus sp. and Pseudochlorococcum sp.) can cause the increase of
distance between the cell wall and cytoplasmic membrane, as well as dis-
tortion and fusion of intracellular membranes [12].
Application of PEF allowed significant enhancement of the rate of lipid
recovery. It was also demonstrated that the increase in lipid recovery was
due to the electroporation and not due to temperature effects [13]. The
opportunity of using PEF treatment at the first step of extraction and sol-
vents at the second step of extraction was demonstrated [14]. Extraction
of intracellular components from microalgae Nannochloropsis sp. with
application of different cell disruption techniques, including PEF, high
voltage electrical discharge (HVED), sonication (S), and high pressure ho-
mogenization (HPH), was compared [15]. The PEF treatment allowed ex-
traction of ≈5.2% (w/w DW biomass) proteins, the supplementary
contributions of HVED (≈1.15%) or sonication (≈1.8%) were rather low
and the most fundamental contribution (≈91%) gave the HPH treatment.
PEF treatment was also proposed as an effective tool for industrial scale
control of predators in microalgae cultures [16]. Industrial attractiveness
 O. Parniakov et al. / Algal Research 8 (2015) 128–134 129

to extraction [7,19,20]. Rather interesting is a combination of PEF treat-

Nomenclature
ment of microalgae with traditional solvent extraction and regulation of
extraction efficiency by changing the temperature and pH of the medium.
E is the electrical field strength, kV/cm
Note that a strongly acidic or basic pH can have a positive impact on the
N is the number of pulses
aqueous extraction efficiency. However, strong deviation from the normal
ti is the effective decay time, μs
pH commonly affects protein activity and may result in rapid denatur-
Δtp is the pause between pulses, s
ation of the proteins. Recent investigation has shown the good potential
tPEF is the pulse electric field duration, ms
of the aqueous extraction of algae proteins from Nannochloropsis biomass
W is the sonication power, W
at pH 11 and temperature of 333 K [21].
f is the frequency of sonication, kHz
The objectives of the present study were to investigate the potential
ts is the duration of sonication treatment, s
of PEF pre-treatment as a preliminary step of pH-assisted aqueous
tE is the total extraction time, s
extraction of algae components from Nannochloropsis suspensions
g is the acceleration, m/s2
(1 wt.%). The data for PEF-treated and sonication (S)-treated suspen-
g0 is the gravity of Earth, m/s2
sions were also compared. The extracts were analyzed for content of
R is the axis of rotation, m
pigments, proteins, carbohydrates, total phenolic compounds and anti-
Tr is the mean light transmission
oxidant capacity. In addition, the colloidal stability of PEF- and S-treated
tc is the time of centrifugation, s
suspensions was also evaluated.
Trb is the turbidity of the extract, NTU
A is the absorbance
2. Materials and methods
Aach is the maximum absorbance of chlorophyll a
Abch is the maximum absorbance of chlorophyll b
2.1. Cell growth
Acr is the maximum absorbance of total carotene
Ag is the absorbance of green pigments
Nannochloropsis spp. (provided by Alphabiotech, Asserac, France) is
Ao is the absorbance of orange pigments
a marine green algae belonging to the Eustigmataceae family. The
Ar is the absorbance of red pigments
microalgae were produced in two steps. The first step was done under
Rgo is the ratio of green/orange pigments
non-stressful conditions to promote the production of biomass and
Rgr is the ratio of green/red pigments
the second one was done under conditions of nitrogen starvation to
Cach is the concentration of chlorophyll a, mg/g
maximize the accumulation of lipids. Analysis of the valuable nutritional
Cbch is the concentration of chlorophyll b, mg/g
compounds was done using standard assay procedures. The concentra-
Cch is the concentration of total chlorophyll mg/g
tion of carbohydrates was determined by the phenol–sulfuric acid
Ccr is the concentration of total carotene, mg/g
method [22]. The concentration of proteins was determined by
Cc is the concentration of carbohydrates, mg/g
bicinchoninic acid microtiter plate assay [23]. The total lipid quantifica-
CTPC is the concentration of total phenolic compounds, mg/g
tion was carried out after chloroform/methanol extraction by gas chro-
Cp is the concentration of proteins, mg/g
matography coupled to flame ionization detector (GC-FID) as described
in [24]. As a result it was determined that the microalgae contained
Greek symbols
30.0% ± 1.5% (w/w DW biomass) of carbohydrates, 20.0% ± 0.8% of pro-
σw is the electrical conductivity of water, μS/cm
teins and 25% ± 1% of total lipids.
ω is the rotor speed, rad/s
The cells had approximately spherical shape, and the mean diam-
λ is the wavelength, nm
eter of the completely swelled cells was found to be about 2 μm.
Subscripts and superscript Nannochloropsis spp. was obtained as a frozen algae paste (12–15%
b is the basic condition at pH = 11 solid content). The biomass was thawed at ambient temperature
n is the normal condition at pH = 8.5 and then harvested by centrifugation and washed 3 times by deion-
ized water. The deionized water with electrical conductivity σ w of
Abbreviations ≈ 0.18 μS/cm was obtained using Elix advantage water purification
DW is the dry weight system E-POD (Merck Millipore, France). Finally, the 1 wt.% untreat-
E is the aqueous extraction ed suspension with initial electrical conductivity of 60 ± 2 μS/cm
HPH is the high pressure homogenization was prepared. The hydrochloric acid or sodium hydroxide was used
HVED is the high voltage electrical discharge for adjustment of pH values. The pH was controlled using a pH
PEF is the pulsed electric field meter Consort C931 (Bioblock Scientific, France) at temperature of
S is the sonication 293 K.
TPC is the total phenolic compounds
TEAC is the trolox equivalent antioxidant capacity, mM 2.2. Experimental operations

The experimental operations used for microalgae cell treatment,

of PEF-assisted extraction technique was supported also by Eltron
Research & Development Inc. that has designed method for treatment of
a highly conductive marine algae sample [17].
However, there still exist a lot of problems related with development
of the effective extraction protocols for different microalgae species. The
state of cell walls in strain of microalgae [18] and the composition of a
liquid medium used for electroporation [11] may play also an extremely
important role. Moreover, even better extraction efficiency may be ex-
pected by application of combination of conventional and electrically
based techniques. For example, PEF processing can be simultaneous
with solvent extraction or can be applied as a pretreatment step prior
characterization of extracts and analysis of suspension stability, are pre-
sented in Fig. 1. These operations involve PEF-treatment, S-treatment or
aqueous basic extraction (Eb).
2.2.1. PEF treatment
PEF treatment was done using high voltage pulsed power 40 kV–
10 kA generator (Tomsk Polytechnic University, Tomsk, Russia). PEF
treatment was carried out in a cylindrical batch chamber (with diame-
ter of 11 cm) between two plate electrodes. The distance between the
electrodes was fixed at 2 cm and corresponding electric field strength
E was 20 kV/cm. PEF treatment comprised application of N successive
pulses [15]. The exponential decay of voltage U ∝ exp (−t/ti) with
130 O. Parniakov et al. / Algal Research 8 (2015) 128–134

Δ tt 2.2.4. Other operations

40
PEF

Voltage, kV
ti
+Eb For eliminating the influence of microalgae cells and cell debris on
Washing in distilled water

Washing in distilled water

20
the results of spectroscopic measurement after PEF treatment, S treat-

aqueous extraction
0
0 10 20 0 10 20 ment or E extraction (denoted as Eb at pH = 11), the suspension was
Centrifugation

Centrifugation

Supplementary
Time, μs
centrifuged during 10 min at the fixed rotor speed ω = 1518 rad/s
S (14,500 rpm) using 3-16P centrifuge (Sigma, Germany). The centrifugal
acceleration at the bottom of the cell was g = 21,475 g0, where g0 =
9.807 m/s2 is the gravity of Earth. The supernatant was decanted from
microalgae cells and analyzed.
E For supplementary extraction, the biomass was washed with deion-
ized water and harvested by centrifugation for 3 times for elimination of
all extracted components. The microalgae residue, obtained after PEF
Aqueous extraction and S experiments, was re-suspended, 1 wt.% suspension was prepared
and exposed to supplementary aqueous extraction (+Eb). This supple-
Analyses of supernatants: Colloidal stability mentary basic extraction (+Eb) was done under the same conditions as
UV-VIS, proteins, polyphenols, LUM for the Eb method (pH = 11, T = 323 K, tE = 10,800 s). Then after +Eb
carbohydrates, pigments, analysis extraction suspension was centrifuged and supernatant was used for
antioxidants further analysis.

Fig. 1. Experimental operations for microalgae cell treatment (pulsed electric field (PEF), 2.3. Sedimentation stability of suspensions
sonication (S), or aqueous basic extraction (Eb) and characterization of extracts.

The sedimentation stability of 1 wt.% microalgae suspensions was

effective decay time ti ≈ 10.0 ± 0.1 μs (it is RC-time constant of the ex- analyzed using analytical photocentrifuge LUMiSizer 610.0-135
ponential pulse generator) was observed in PEF treatment mode. The (L.U.M. GmbH, Germany). The analysis used a centrifugal rotor, a light
energy per pulse delivered to the treated suspension was 160 J. There source (pulsed near-infrared light-emitting 880 nm diode) and a light
was a pause of Δtp = 2 s between pulses. PEF treatment duration sensor. The 0.4 g weights of aqueous suspensions (1 wt.%) were subject-
tPEF = Nti was varied within 0.01–6 ms (N = 1–600). ed to centrifugation in the rectangular polycarbonate optical cells, sup-
Disrupted algae suspension characteristics were measured between plied by the photocentrifuge manufacturer. The operating principle of
successive applications of the PEF pulses. The initial temperature before the analytical photocentrifuge is based on the measurement of light
PEF treatment was 293 K and the final temperature after the PEF treat- transmission though the cell filled by the studied sample [25]. The radial
ment never exceeded 303 K. The temperature was controlled by K-type distance of the centrifugal cell bottom from the axis of rotation R was
thermocouple (± 0.1 K), connected to the data logger thermometer equal to 0.13 m. Centrifugation experiments were carried out at the
Center 305/306 (JDC Electronic SA, Yverdon-les-Bains, Switzerland). fixed rotor speed ω = 418.7 rad/s (4000 rpm). The centrifugal acceler-
ation at the bottom of the cell was g = R ∗ ω2 = 2324 g0. Mean light
2.2.2. Sonication treatment transmission, averaged over the height of the sample, Tr, versus the
Sonication (S) treatment (power W of 400 W and frequency f of time of centrifugation, tc, was measured.
24 kHz) was done using ultrasound processor UP 400S (Hielscher
GmbH, Germany). Sonication probe, acting as a wave amplifier, was 2.4. Characterization of microalgae extracts
plunged into a flask, containing 250 ± 5 g of microalgae suspension.
Treatment duration tS was varied within 0–600 s and the amplitude 2.4.1. Absorption spectra and turbidity
was fixed at 50%, which corresponded to the power W of 200 W [15]. Absorption spectra of the extracts were measured by UV-
The flask with the sample was immersed into a cooling bath with ice spectrophotometer Libra S32 (Biochrom, Lagny-sur-Marne, France).
for avoiding the heating induced by S treatment. The temperature was The wavelength range was within 350–800 nm against blank (with
measured by thermocouple during treatment and it never exceeded the precision of ±1 nm). The path length of the SUPRASIL quartz cu-
303 K. vette was 10 mm (Hellma, Müllheim, Germany).
Kinetics of extraction during the PEF and S treatments was con- The PeakFit program (Version 4.12, SeaSolve Software Inc.) was used
trolled by measuring the absorbance A of the aqueous supernatant. for analysis of the spectral shape of absorption bands and for their
The PEF and S treatments were done in neutral (pH = 8.5) and in graphical deconvolution. The autofit baseline option was used to re-
basic (pH = 11) media. The treatments at normal (pH = 8.5) and move the baseline trend prior to deconvolution of peaks, their fitting
basic (pH = 11) conditions are referred further on as PEFn, Sn and and estimation of their intensity. XR turbidimeter (HACH Company,
PEFb, and Sb, respectively. The hydrochloric acid or sodium hydroxide Loveland, CO, USA) was used for turbidity measurements of the extracts.
was used for adjustment of pH values. The pH was controlled using a Turbidity Trb was expressed in nephelometric turbidity units (NTU).
pH meter Consor C931 (Bioblock Scientific, France) at temperature of
293 K. 2.4.2. Pigments
The samples of algae supernatant were mixed with acetone (20 ml
per each gram). The samples were filtered using filter paper (Qualitative
2.2.3. Aqueous extraction filter paper 5–13 μm, 413, VWR). The absorbance was estimated spec-
For comparison purposes the efficiency of aqueous extraction trophotometrically by analyzing filtrate at the wavelengths within the
assisted by PEF or S treatments was compared also with data for con- 350–800 nm range. The maximum absorbencies of chlorophyll a (Aach),
ventional aqueous extraction (E) without treatment. The suspension chlorophyll b (Abch), and total carotene (Acr) were located at the wave-
(1 wt.%) was placed in a closed flask and continuously agitated using lengths of λ ≈ 662 nm, λ ≈ 646 nm and λ ≈ 470 nm, respectively
the magnetic stirrer Arex (VELP Scientifica, Usmate, Italy) at the fixed [26]. The concentrations of pigments in the extract were calculated
temperature T = 323 K. The total extraction time was tE = 10,800 s using the following equations [27]:
(3 h). Preliminary experiments have shown that this time was long
enough for high level of extraction of the main components (pigments,
a a b
proteins and carbohydrates) from microalgae. C ch ¼ 11:75Ach −2:35Ach ; ð1Þ
 O. Parniakov et al. / Algal Research 8 (2015) 128–134
b b a
C ch ¼ 18:61Ach −3:96Ach ; ð2Þ
a b
C ch ¼ 1000Acr −2:27C ch −0:35C ch ; ð3Þ
a b
C ch ¼ C ch þ C ch ð4Þ
a
where Cch , Cbch, Cch and Ccr are the concentrations (mg of pigment/g of
dry mater, mg/g) of chlorophyll a, chlorophyll b, total chlorophyll and
total carotene, respectively.
2.4.3. Carbohydrates
The concentration of carbohydrates, Cc (mg of glucose equivalent/g
of dry mater, mg/g), was determined by the phenol–sulfuric acid meth-
od [28] that was partially modified in order to reduce the consumption
of reagents [29]. The color reaction was initiated by mixing 2 ml of ex-
tract with 1 ml of 5% phenol solution and 5 ml of concentrated sulfuric
acid (Sigma-Aldrich, France). The reaction mixture was kept at room
temperature (T = 293 K) for 30 min. The absorbance, A, was measured
at the wavelength of λ ≈ 490 nm. D-Glucose (Sigma-Aldrich, France)
was used for the calibration.
2.4.4. Total phenolic compounds
The concentration of total phenolic compounds (TPC), CTPC (mg of
gallic acid equivalent/g of dry mater), was measured by the Folin–
Ciocalteu method that was based on the colorimetric reduction/oxida-
tion reaction of phenols [30]. The 0.2 ml of extract and 1 ml of Folin–
Ciocalteu reagent (Sigma-Aldrich, France) (diluted by water 1:10)
were mixed. Then 0.8 ml of Na2CO3 (75 g/L) (VWR, France) was
added. The sample was incubated for 10 min at 323 K and then cooled
at room temperature. The absorbance was measured at the wavelength
of λ ≈ 750 nm. The gallic acid (Sigma-Aldrich, France) was used for
calibration.
2.4.5. Trolox equivalent antioxidant capacity
The trolox equivalent antioxidant capacity (TEAC, millimolar Trolox
equivalents, mM) measures the antioxidant capacity of a given substance
as compared to the standard, Trolox (Sigma-Aldrich, Steinheim,
Germany). TEAC was measured using the method [31] based on applica-
tion of ABTS Decolorization Assay (Sigma-Aldrich, Steinheim, Germany).
The ABTS radicals (ABTS•+) were generated using 440 μl of potassium
persulfate (140 mM). The solution was diluted by ethanol (Baker, Deven-
ter, The Netherlands) until absorbance of 0.70 was reached at 734 nm.
Once the radical was formed, 2 ml of ABTS•+ was mixed with 100 μl of
extract and the sample was incubated for 20 min at 20 °C. The absorbance,
A, was measured at the wavelength of λ ≈ 734 nm [32].
2.4.6. Proteins
The concentration of proteins, Cp (mg of bovine serum albumin
equivalent/g of dry mater, mgBSA/g), was determined using the Bradford
assay [33]. The supernatant samples were obtained by centrifugation at
3500 g for 10 min at 293 K, then 0.2 ml of the extract (diluted if it was
required) and 1.8 ml of three-fold diluted Bradford Reagent (Sigma-Al-
drich, St-Quentin Fallavier, France) were mixed.
All test tubes with the mixture were shaken for 10 s on the Vortex
and the sample was kept 5 min at room temperature. The absorbance
A was measured at the wavelength of λ ≈ 595 nm. Bovine Serum Albu-
min (Sigma-Aldrich, St-Quentin Fallavier, France) was used for the
calibration.
Fig. 2 presents examples of the kinetics of extraction of proteins at
different pH values. The similar curves were obtained for extraction of
carbohydrates and total phenolic compounds. The data shows that the
main compounds were extracted rather rapidly during initial period of
time, tE ≤ 3600 s, and later on the extraction process was slowed
down. For example, after a long period of time, tE ≈ 36,000 s, the
concentrations of extracted proteins were ≈ 6 mg/g, 12.2 mg/g and
131
22 tE=10800 s pH=12
20
18
16
14
pH=11
Cp, mg/g
12
10
8
pH=8.5
6
4
2
0
0 10000 20000 30000
tE, s
Fig. 2. Concentration of proteins, Cp, versus the time of aqueous extraction, tE, at different
pH values, T = 323 K.
20.5 mg/g at pH = 8.5, 11 and 12, respectively. It can be seen that ex-
traction during the period of tE = 10,800 s was sufficient for obtaining
high level of proteins in extracts, and further increase of extraction
time resulted in saturation of Cp(tE) dependencies.
The observed kinetics of extraction of proteins was in qualitative
correspondence with previously reported data for Nannochloropsis bio-
mass [21]. It was also noted that the extraction at pH = 11 was signifi-
cantly higher to that obtained at pH = 9.
2.5. Statistical analysis
Each experiment was repeated, at least, three times. One-way
analysis of variance was used for statistical analysis of the data with
the help of Statgraphics plus (version 5.1, Statpoint Technologies Inc.,
Warrenton, VA). Significance level of 5% was assumed for each analysis.
The error bars, presented on the figures, correspond to the standard
deviations.
3. Results and discussion
Fig. 3 presents examples of spectra of the extracts (supernatants),
obtained after different modes of treatment of microalgae suspensions
(Fig. 1).
PEFn (tPEF = 1–6 ms, Fig. 3a) and Sn (tS = 60–600 s, Fig. 3b) treat-
ments provoked increasing of peak intensities with maximums located
at ≈ 420–490 nm (yellow, orange and red pigments) and ≈ 620–
680 nm (green pigments), which can be attributed to the absorbance
of total carotene and the absorbance of chlorophylls, respectively [26].
Note that nucleic acid can display the absorption band in the vicinity
of ≈260 nm [34]. It should be noted that extraction efficiency treatment
was noticeably higher for Sn than for PEFn treatment and this result is in
correspondence with previously reported data, obtained for the similar
microalgae Nannochloropsis sp. [15]. However, power consumption was
smaller for PEFn (≈100 kJ/kg, 20 kV/cm, 6 ms) than for Sn (≈250 kJ/kg,
200 W, 10 min).
Supplementary extraction (+Eb) from microalgae treated by PEFn
(6 ms) or Sn (600 s) (Fig. 3c) allowed release of the certain quantity of
pigments. The total addition of + Eb operation was more appreciable
after PEFn treatment as compared with that obtained after Sn treatment.
Fig. 4 presents examples of extraction kinetics of pigments from
microalgae in the course of the PEFn and Sn treatments. The kinetics
was checked by measuring the absorbance of green pigments (chloro-
phylls) Ag versus the treatment times tPEF and tS. It can be seen that at
132 O. Parniakov et al. / Algal Research 8 (2015) 128–134

a) a) 0 200
tS, s 400 600
0.01
0.008 680 nm -1 PEFn
tPEF, ms 10
0.006 Sn
A

A
440 nm 1
0.004 6
490 nm 10-2
0.002
0 0 2 4 6
400 500 600 700 tPEF, ms
 , nm
b) b) 0 200
tS, s 400 600
420 nm 30
0.3
Rgo
tS, s Un
20 Rgr
0.2

R
A

60 Rgo
PEFn Rgr
480
0.1 10
620 nm
490 nm

0 0
400 500 600 700 0 2 4 6
 , nm tPEF, ms
Fig. 4. Kinetics of extraction from microalgae in the course of PEFn and Sn treatments. Ab-
c) 425 nm sorbance of green pigments, Ag, (at λ = 680 nm) (a) and peak ratios, Rgo and Rgr, (b) versus
0.06 the treatment times tPEF and tS. Here, Rgo = Ag/Ao (green/orange) and Rgr = Ag/Ar (green/
675 nm red), where Ag, Ao, and Ar are the absorbance at 680 nm (green pigments), 440 nm (orange
PEFn +Eb pigments) and 490 nm (red pigments) wavelengths, respectively.
0.04
A

Sn +Eb

0.02 490 nm
0 with Sn treatment (pH = 8.5, normal conditions) and inverse situation
400 500 600 700
 , nm
Fig. 3. The absorption spectra of the supernatants, obtained after treatment of 1%
microalgae suspensions Nannochloropsis sp. by PEFn (a, 1–6 ms), Sn (b, 60–600 s) and sup-
plementary aqueous extraction +Eb (c, 10,800 s) from microalgae treated by PEFn (6 ms)
or Sn (600 s).
relatively long treatment times, tPEF N 6 ms, and tU N 600 s, the values of
Ag reached saturation (Fig. 4a). It is interesting that the ratio of green/or-
ange pigments Rgo = Ag/Ao and green/red pigments Rgr = Ag/Ar
displayed different behaviors (Fig. 4b). The most striking was the no-
ticeable increase of Rgr = Ag/Ar ratio in the course of Sn treatment
(Fig. 4b). This effect may be related to the damage of red pigments as
the result of Sn treatment. Besides, the red pigments may have good ad-
sorption affinity to the small cell debris originating from Sn treatment
and be removed from supernatant as a result of centrifugation.
Fig. 5 presents the final concentrations of total chlorophylls (a), ca-
rotenoids (b), proteins (c), carbohydrates (d), phenolic compounds
(e) and antioxidant capacity (f) for the extracts obtained using PEFn,
PEFb, Sn, Sb and Eb extraction procedures. The effects of supplementary
aqueous extraction +Eb are also shown.
In general, efficiency of extraction of various components, stimulat-
ed by PEFn treatment, was comparable with that of the aqueous extrac-
tion Eb in the basic medium. However, PEFn treatment was more
efficient than PEFb treatment. For example, the quantity of extracted
chlorophylls and carotenoids (Fig. 5a, b) was noticeably smaller at
PEFb treatment (pH = 11, basic conditions) as compared with PEFn
(pH = 8.5, normal conditions). Both PEFn and PEFb treatments gave ap-
proximately the same quantity of extracted total phenolic compounds
(Fig. 5e). In general, the larger quantity of extracted compounds was
obtained for Sn treatment as compared with PEFn and Eb procedures.
However, the differences between Sn and Sb treatments were not so
definitive as between PEFn and PEFb treatments. For example, the
quantity of extracted proteins and carbohydrates (Fig. 5c, d) was notice-
ably smaller at Sb treatment (pH = 11, basic conditions) as compared
was observed for carotenoids (Fig. 5b). However, in all the cases, the an-
tioxidant capacity (value of TEAC) was smaller for treatment at pH =
8.5 than for treatment at pH = 11 (Fig. 5f).
Supplementary basic extraction at pH = 11 (+Eb) allowed a notice-
able increase of the concentrations of all components in the extracts, as
well as the values of antioxidant capacity, TEAC. The best results demon-
strated the sonication assisted procedures followed by supplementary
basic extraction +Eb, e.g., Sb + Eb procedure for chlorophylls and carot-
enoids and Sn + Eb procedure for proteins, total phenolic compounds
and antioxidant capacity.
The sedimentation stability was rather different for PEFn and Sn
treated microalgae suspensions. Fig. 6 shows mean light transmission,
Tr, versus time of centrifugation tc for PEFn and Sn treated microalgae
suspensions (1 wt.%). In this test, the centrifugal acceleration was rather
moderate (g = 2324 g0). The increase of mean light transmission, Tr,
through the sample during centrifugation reflected continuous clarifi-
cation of suspension, caused by the settling of the microalgae cells in
the centrifugal field. The estimate has shown that the time of clarifica-
tion was significantly shorter after the PEFn treatment (tc ≈ 150 s) as
compared to that after the Sn-treatment (tc ≈ 11,000 s) (Fig. 6). High
sedimentation stability of the microalgae suspension after Sn-
treatment can be explained by production of small sized cell debris as
a result of sonication.
Finally, the supplementary analysis was done in order to reveal the
difference between turbidity of supernatants, obtained by intensive
centrifugation (during 10 min with centrifugal acceleration g =
21,475 g0) of PEFn and Sn treated microalgae suspensions (Fig. 7). In
these tests, the value of pH was adjusted before measurements of
turbidity.
The impact of pH value on the turbidity can reflect the effects of iso-
electric precipitation when the net protein charge is near zero (pH = 4–
5) and irreversible aggregation or denaturation driven by hydrophobic
interactions (at extremes of pH) [35]. Note that phenomenon of
 O. Parniakov et al. / Algal Research 8 (2015) 128–134 133

a) b) 0.4
0.8 Chlorophylls Carotenoids
+Eb
0.6 0.3

Ccr, mg/g
Cch, mg/g

0.4 0.2

0.2 0.1

0 0
PEFn PEFb Sn Sb Eb PEFn PEFb Sn Sb Eb

c) 50 Proteins
d)
+Eb 50 Carbohydrates
40
Cp, mg/g

40

Cc, mg/g
30
30
20 20
10 10

0 0
PEFn PEFb Sn Sb Eb PEFn PEFb Sn Sb Eb

e) f) 1.4
Total phenolic Antioxidant
5 +Eb
compounds 1.2
CTPC, mg/g

capacity
4 TEAC, mM 1
3 0.8
0.6
2
0.4
1 0.2
0 0
PEFn PEFb Sn Sb Eb PEFn PEFb Sn Sb Eb

Fig. 5. Concentration of total chlorophylls, Ccha, (a), carotenoids, Ccr, (b), proteins, Cp, (c), carbohydrates, Cc, (d), phenolic compounds, CTPC, (e) and antioxidant capacity, TEAC values, (f).
The data are presented for extracts, obtained using PEFn, PEFb, Sn, Sb and Eb extraction procedures. The effects of supplementary aqueous extraction +Eb are also shown. The time duration
was tPEF = 4 ms, tU = 600 s and tE = 10,800 s for PEF, U, and E extraction procedures, respectively.

irreversible aggregation is rather difficult to predict on the basis of pro-
tein structure. The initial supernatant before adjustment of pH (i.e. at
pH = 8.5) was slightly more turbid for Sn treated suspension than for
PEF treated ones. It can be easily explained by the presence of small
sized cell debris as a result of sonication.
90
tc 150 s tc 11000 s
At basic pH, the turbidities of supernatants of Sn and PEFn treated sus-
pensions were rather similar. The turbidity of supernatants of PEFn treated
suspensions was practically constant within pH = 5–10, which evidences
stability of high molecular species against isoelectric precipitation and
1400

1200
80 Sn
PEFn Sn

1000
Trb
Tr

70

800

60 PEFn

600

6 8 10 12
50 1 pH
10 102 103 104
t, s
Fig. 7. Turbidity of supernatant, Trb, versus pH. The supernatants were obtained by inten-
Fig. 6. Mean light transmission, Tr, versus time of centrifugation, t, for 1% microalgae sus- sive centrifugation (for 10 min with centrifugal acceleration g = 21,475 g0) of PEFn and Sn
pensions obtained using PEFn and Sn treatments. The time duration was tPEF = 4 ms and treated microalgae suspensions. The time duration was tPEF = 4 ms and tS = 600 s for PEFn
tS = 600 s for PEFn and Sn treatments, respectively. Arrows show time of clarification, tc, and Sn treatments, respectively. The value of pH was adjusted before measurements of
for PEFn and Sn treated suspensions. turbidity.
134 O. Parniakov et al. / Algal Research 8 (2015) 128–134
denaturation [35]. However, at highly acidic pH, the turbidity of superna-
tants of Sn treated suspensions was very high. The clear difference between
PEFn and Sn and treated suspensions can be explained by the differences in
molecular weight, charge and structure of high molecular compounds and
their stability against denaturation. It surely evidences selectiveness of ex-
traction of some proteins at PEFn treatment as compared with Sn treatment.
4. Conclusions
This study was aimed for development of new method of processing
of algae biomass assisted by electroporation. PEF allowed selective extrac-
tion of ionic components and water soluble proteins. PEF pre-treatment
with parameters used in this work was ineffective for extraction of
pigments. For enhancement of efficiency of PEF pre-treatment the use
of the binary mixture of organic solvents and water or adjustment of ex-
tracellular media conductivity [36,37] is desirable. We shall look into
these experiments in our future work.
For extraction of pigments, the sonication pre-treatment was more effi-
cient. However, this mode of treatment required more power consumption
and supplementary purification of the output products. The different abili-
ties of extraction of different pigments (e.g., green or orange) were revealed
for sonication (S) pre-treatment, and it was explained by the possibility of
damage of some sorts of pigment under sonication. In general, efficiency of
extraction of various components, stimulated by PEF treatment, was com-
parable with that for aqueous extraction E in a basic medium. Supplemen-
tary basic extraction at pH = 11 (+Eb) allowed a noticeable increase of the
concentrations of all components in the extracts, as well as of the values of
antioxidant capacity, TEAC. The best results demonstrated the sonication
assisted procedures followed by the supplementary basic extraction +Eb,
e.g., Sb + Eb procedure for chlorophylls and carotenoids and Sn + Eb proce-
dure for proteins, total phenolic compounds and antioxidant capacity. The
sedimentation stability data have shown the better purity of extract obtain-
ed using PEF treatment.
Thus PEF pre-treatment has an excellent potential as a preliminary
step of aqueous extraction of algae components. Moreover, PEF tech-
nique allowed selective extraction of some pure proteins that were dif-
ferent from the proteins extracted after Sb treatment. The novelty of the
approach is based on combination PEF and pH-assisted extraction tech-
niques. The discovered effects have shown the advantages of PEF-
pretreatment application in a normal medium (pH = 8.5) and basic
medium supplementary extraction (pH = 11) for selective extraction
of different intracellular components.
Acknowledgment
The authors appreciate the support from the COST Action TD1104
(EP4Bio2Med — European network for development of electroporation-
based technologies and treatments). F.J. Barba thanks the Valencian
Autonomous Government (Consellería d'Educació, Cultura i Esport.
Generalitat Valenciana) for the postdoctoral fellowship of the VALi+d
program “Programa VALi+d per a investigadors en fase postdoctoral
2013” (APOSTD/2013/092). The authors also thank Dr. N. S. Pivovarova
for her help with the manuscript preparation.
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