b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5

Available online at www.sciencedirect.com

ScienceDirect

http://www.elsevier.com/locate/biombioe

Characterization of fatty acid and carotenoid

production in an Acutodesmus microalga isolated
from the Algerian Sahara

Borhane Samir Grama a,b, Samira Chader c, Douadi Khelifi a, Ben Stenuit b,
Clayton Jeffryes b,d, Spiros N. Agathos b,*
a
Laboratory of Genetic Biochemistry and Plant Biotechnology, Faculty of Nature and Life Sciences, Universite
Constantine 1, Constantine, Algeria
b
Earth & Life Institute e Bioengineering Laboratory, Universite Catholique de Louvain, Place Croix du Sud 2,
bte. L07.05.19, B-1348 Louvain-la-Neuve, Belgium
c
Algerian Center of Renewable Energy (Centre de Developpement des Energies Renouvelables), Unite de
Developpement des Equipements Solaires (UDES), Bou-Ismail, 42415 W. Tipaza, Algeria
d
Fonds de la Recherche e FNRS, Belgium

article info abstract

Article history: An Acutodesmus microalga of the Scenedesmaceae family isolated from the Algerian Sahara
Received 22 February 2014 was characterized for its ability to produce lipids, fatty acids (FA) and carotenoids under
Received in revised form nitrate starvation, osmotic stress and a varying level of oxidative stress. The FA produced
1 July 2014 by this strain isolated from an environment typified by extreme heat showed a lower de-
Accepted 22 July 2014 gree of unsaturation than those of other Scenedesmus which have been isolated from cold or
Available online temperate water environments, with no unsaturated C16 FA and more than 92% of the FA
profile being comprised of FA with less than three unsaturated bonds. According to the
Keywords: degree of unsaturation and the cetane number, the FA profile from this strain is highly
Acutodesmus favorable for biofuel production. Lipogenesis and carotenogenesis were easily reversible
Scenedesmus upon removal of the stress. These results constitute an advance in developing strains
Lipids suitable for industrial-scale production of biofuels.
Fatty acids © 2014 Elsevier Ltd. All rights reserved.
Canthaxanthin
Photosynthetic biorefinery

of microalgae have already demonstrated the capacity to

1. Introduction
Microalgae are emerging as important cell factories for the
production of various commercial products such as caroten-
oids [8,9] and fatty acids (FA) [32] and the potential still exists
to convert algal lipids into biofuels [7]. Indeed, several species
accumulate intracellular concentrations of over 50% lipids on
a dry cell weight (DCW) basis under species-specific stresses
such as nitrate [19] or silicon [27] limitation. Lipid accumula-
tion is often in the form of triacylglycerides (TAG) which are
deposited in cytoplasmic oil bodies (OB). TAG are the source of
medium- and long-chain FA, the raw material used to make

* Corresponding author. Tel.: þ32 1047 3644; fax: þ32 1047 3062.
E-mail address: spiros.agathos@uclouvain.be (S.N. Agathos).
http://dx.doi.org/10.1016/j.biombioe.2014.07.023
0961-9534/© 2014 Elsevier Ltd. All rights reserved.
266 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5
biodiesel, and their synthesis is often associated with the
concomitant production of photoprotective, extraplastidic
secondary carotenoids [47]. These storage lipids are thought to
be an energy depot for the excessive photonic energy that
could not be utilized under unfavorable conditions [5,47].
By some estimates, there are hundreds of thousands to
millions of microalgal species, most of which have not been
isolated or characterized [33,46] and organisms isolated from
extreme environments (extremophiles) often possess unique
physiological responses that enable their survival in stressful
conditions such as extreme light, temperatures or pH,
nutrient limitation or high salinity [16,48] [48]. The molecules
which are overproduced in the presence of different types of
environmental stressors are often suitable for commercial
applications, such as astaxanthin produced in Haematococcus
pluvialis [10] or b-carotene from Dunaliella salina [4]. Addition-
ally, microalgal strains within the same species can exhibit
different physiological activities and metabolite profiles
depending on habitat and physicochemical condition range,
i.e., phenotypic plasticity within a single genotype [34]. It is
expected that strains belonging to the same, or closely related,
species but isolated from very different environments will
demonstrate different physiological responses or produce
different levels of biomolecules. Therefore, the diversity of
phenotypes is even greater than the diversity of genotypes,
which further enhances the interest to isolate species from
new biotopes. Consequently, the unmined richness of algal
resources is still largely unexplored and there is a high po-
tential to discover new strains of algae for the production of
industrially attractive compounds, such as secondary carot-
enoids and FA [2,14,20,38]. To this end, we have isolated
microalgal strains from the Sahara Desert of Algeria, where
the great diversity of microalgae remains almost completely
undiscovered. This region is an extreme environment that
covers nearly 2  106 km2 and experiences intense solar ra-
diation and high temperatures.
In this study, we show that an Acutodesmus sp. strain SG-1
from the Scenedesmaceae family isolated from the Sahara
Desert of Algeria has an FA profile with a low degree of
unsaturation with no unsaturated C16 FA, and minimal
amounts of both linolenic acid (C18:3, 7.1 ± 0.6%) and long-
chain fatty acids (>C18, 1.0 ± 0.3%). This finding differs from
previously characterized Acutodesmus obliquus strains
(formerly Scenedesmus obliquus) [17,23] which had higher levels
of C18:3 than C18:2 and significant levels of unsaturated C16
[1,11]. Under the highest lipid production conditions, the fatty
acid profile in the present study has a stationary phase degree
of unsaturation (DU, 97 ± 2) and predicted cetane number (CN,
55 ± 2), two measures of biodiesel quality, comparable to or
better than all readily available vegetable oil sources except
for palm [19].
The production of lipids and secondary carotenoids are
synchronous and the production of each enhances the pro-
duction of the other. Carotenoids are lipid soluble and there-
fore require a lipid body in order to accumulate at high
concentrations. Likewise, carotenoids protect against the ac-
tion of the lipid-peroxidizing reactive oxygen species (ROS) by
two mechanisms. The first mechanism is to act as a photo-
protectant which blocks photons from reaching the photo-
synthetic apparatus thereby reducing the production of ROS
in the electron transport chain [51]. The second mechanism is
to act as an antioxidant within the oil bodies which protects
the lipids from peroxidation [35]. Light is therefore a critical
environmental factor that affects the algal lipid metabolism
and composition [22] and was therefore selected to be the
critical variable in this study. We determined that the prin-
cipal carotenoid produced by our isolate was canthaxanthin.
The production of secondary carotenoids was proportional to
the lipid production suggesting that the primary role for sec-
ondary carotenogenesis in our isolate is to prevent peroxida-
tion of the lipids within OB.
The objective of the present work was to study the physi-
ological responses of the newly isolated strain under envi-
ronmental stress conditions that should induce the
production of carotenoids and fatty acids. Specifically, the
combination of osmotic, nutritional and oxidative stress and
the interactive effect of light intensity on lipid production and
composition during batch growth were investigated. We also
examined the recovery of the cell culture after the stress
conditions were removed in order to study the feasibility of a
reversible, and thus semi-continuous, cultivation process for
the production of algal bioproducts. This study is poised to
provide new insights into the commercial development for
fatty acid or carotenoids production by an algal strain isolated
from the Sahara.
2. Materials and methods
2.1. Microalgae strain
Acutodesmus sp. strain SG-1 was isolated from the Sid Ahmed
Timmi oasis, altitude 252e282 m, 0 15’E longitude, 27 45’N
latitude in the Sahara Desert of Algeria near Tamentit in
Adrar.
The internal transcribed spacer (ITS-1 and ITS-2) se-
quences of the nuclear ribosomal RNA operon were used for
phylogenetic analysis. A DNeasy® Plant Mini Kit (Qiagen
GmbH, Hilden, Germany) was employed to extract and purify
genomic DNA in accordance with the manufacturer's in-
structions. The purified genomic DNA was used as a template
for the PCR amplification reaction using the primer pair ITS5/
ITS4 (forward ITS5 primer: 5’-GGAAGTAAAAGTCGTAA-
CAAGG-3’; reverse ITS4 primer: 5’-TCCTCCGCTTATTGA-
TATGC-3’) [50]. The primer pair ITS5/ITS4 generates a 722-bp
PCR product (based on the sequence of A. obliquus CCAP 276/
48, Genbank accession number JQ082318). The region
including the ITS-1, 5.8S rDNA and ITS-2 sequences was
amplified in a 50-mL PCR mix containing 24.4 ng genomic DNA
with a GoTaq® Flexi DNA polymerase (Promega, Madison,
Wisconsin, USA) according to the manufacturer's protocol.
PCR amplification was performed in a Biometra TGradient
DNA Thermocycler (Westburg BV, Leusden, The Netherlands)
under the following conditions: thermocycler set at 95  C
before insertion of PCR tubes (simplified hot start method),
95  C for 5 min, 30 cycles of 95  C for 1 min, 53  C for 30 s, 72  C
for 1 min, and a final extension at 72  C for 5 min.
After staining with GelRed and visualization under UV
light, the resulting PCR amplicon was excised from the
agarose gel, extracted and purified by a Sigma GenElute™ Gel
 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5
Extraction kit (Sigma, USA) in accordance with the manufac-
turer's instructions. The purified PCR product was then
sequenced at Macrogen Europe (Amsterdam, The
Netherlands) using an ABI PRISM® 3100 Genetic Analyzer
(Applied Biosystems, Foster City, California, USA) with the
primers ITS5 and ITS4. The sequencing data were edited with
the Geneious Pro R6 software (version 6.1.4, Biomatters Ltd.,
Auckland, New Zealand) [29]. A BLASTN search against the nr
database was carried out in Geneious 6.1.4 using the default
parameters. The phylogenetic tree was generated from a
CLUSTALW alignment [31] using Tamura-Nei distance pa-
rameters and the neighbor-joining (NJ) tree build method
(Geneious Tree Builder). NJ bootstrapping was performed with
1000 replicates.
The strain was maintained as a pure culture on solid me-
dium consisting of modified 3N-BBM þ V (Culture Collection of
Algae and Protozoa, http://www.ccap.ac.uk/) plus 1.0 g L1
sodium acetate trihydrate, 2.0 g L1 Bacto™ tryptone, 2.0 g L1
Bacto™ yeast extract and 15 g L1 agar. The strain was sub-
cultured to fresh solid medium on a monthly basis. The
composition of the modified 3N-BBM þ V medium was 8.8 mM
NaNO3, 1.3 mM KH2PO4, 330 mM K2HPO4, 430 mM NaCl, 300 mM
MgSO4, 170 mM CaCl2, 12 mM EDTA, 2.2 mM Fe(III)Cl3, 2.0 mM
MnCl2, 220 nM ZnCl2, 99 nM Na2MoO4, and 51 nm CoCl2, 3.6 mM
vitamin B1 and 7.4 nM vitamin B12. All culture media were
sterilized by autoclaving for 20 min at 121  C and 1.6 bar,
except for the addition of the vitamin stocks, which were
0.2 mm filtered and maintained at room temperature before
addition.
Suspension cultures of Acutodesmus sp. strain SG-1 were
made up by carrying one loopful of cell mass from the agar-
based medium into 250 mL Cellstar® flasks (Greiner Bio-One
#658195, Monroe, NC) containing 50 mL 3N-BBM þ V me-
dium which were kept on an orbital shaker (Lab-Shaker,
Kühner, Switzerland) at 90 rpm, 20  C, and 30 mE m2 s1 using
a light:dark photoperiod cycle of 12:12 h. These axenic, sus-
pension cultures served to seed the growth phase precultures.
2.2. Lipogenesis process
2.2.1. Growth phase cultures
Green, growth phase Acutodesmus sp. strain SG-1 precultures
were set up to generate nitrate-starved seed cultures for the
lipogenesis experimental runs. These cell suspensions were
kept in an incubator (Lovibond Model ET 650-8) in 500 mL
Duran glass bottles with a working volume of 400 mL. The
cultures were gassed by introducing filtered air through sili-
cone tubing (I.D. 6 mm) at a flow rate of 200 mL min1. The
temperature was maintained at 25 ± 0.1  C and a light in-
tensity of 80 mE m2 s1 (measured at the outside surface of the
bottle) was supplied through 18 W cool white lamps. When the
NO  1
3 was at a measured level below 6.5 mg NO3 L , the green
culture was employed as seed for the lipogenesis experi-
ments. To begin lipogenesis, the nitrate-starved precultures
were transferred to the stress medium in the Multicultivator
MC 1000-OD (see below) by the process indicated below.
2.2.2. Stress phase
At the end of the growth phase, the nitrogen-limited algae
were introduced into a Multicultivator MC 1000-OD (Photon
267
Systems Instruments, Drasov, Czech Republic) for stress-
phase culturing. This cultivation device encompassed eight
bubble-column chambers containing 80 mL of suspension
culture under conditions of controlled aeration, light and
temperature. Each chamber was lit independently by a set of
warm white LEDs whose light intensity was adjustable. Air
was supplied to each chamber at a rate of 125 mL min1 by a
glass tube of 2 mm I.D.
The bubble columns of the Multicultivator were inoculated
by combining 20 mL of nitrate-starved growth phase cells
which had just become nitrogen limited (<6.5 mg NO3 L1)
with 60 mL of 3N-BBM þ V stress medium. The latter was
supplemented with 10 g L1 NaCl and contained no nitrate, but
otherwise was identical to 3N-BBM þ V. Thus the final con-
centration in the stress medium was 7.5 g L1 NaCl after
mixing with the cell culture and <1.6 mg NO3 L1. This resul-
ted in nutritional and osmotic stresses. A total of eight cul-
tures (four illumination conditions in duplicate: 100, 300, 600
and 900 mE m2 s1 at the light source as determined by the
device's software) were grown at 26  C. The source of illumi-
nation was situated at a distance of 2.0 cm from the external
wall of the vessel, whose I.D. was 15 mm. The mean light in-
tensities prevailing at the point when the final cell densities
had been reached were measured directly (US-SQS/L Spherical
Micro Quantum Sensor coupled to a ULM-500 Universal Light
Meter, Heinz Walz GmbH, Eichenring, Germany) and attained,
respectively, 40, 140, 280 and 380 mE m2 s1 for each duplicate
cultivation under nominal intensities of: 100, 300, 600 and
900 mE m2 s1. Given that the mean light intensity within the
culture varies with cell density and cell pigmentation, from
here onwards each experiment will be cited by the constant
light intensities at the source. An additional light-induced
stress arose as a consequence of photosystem over-
saturation at light intensities above the light half-saturation
value Ek, evaluated as in Section 2.4. The experiments were
performed for 144 h (six days).
To differentiate the influences of nitrate limitation and
salinity stress on the formation of lipids under conditions of
high illumination, two kinds of control cultivations were
carried out. One control cultivation substituted the stress
medium with 3N-BBM þ V without nitrate (N  NaCl control,
nitrate starvation without salinity stress) and the other
substituted the stress medium with 3N-BBM þ V containing
NaCl which resulted in a final level of 7.5 g L1 NaCl (þN þ NaCl
control, salinity stress in a nitrate replete medium) to inde-
pendently impose nitrate deficiency stress and osmotic stress,
respectively.
Both the þN þ NaCl and N  NaCl controls were grown in
duplicate at a 600 mE m2 s1 source illumination intensity for
144 h (six days). This corresponds to the light intensity which
maximized lipid production in the fully stressed cultures. The
initial light intensity within the cell cultures was
290 mE m2 s1.
2.2.3. Recovery process
Cultures that had already attained stationary phase and per-
formed lipogenesis for a duration of 14 days to ensure a high
degree of cellular stress were used to measure the transition
from lipogenesis back to the growth phase. To eliminate NaCl
the algal biomass was spun down and re-suspended in an
268 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5
equal volume of 3N-BBM þ V. Subsequently, 30 mL of this
suspension were mixed with 50 mL 3N-BBM þ V for cultivation
in the Multicultivator at 26  C. The light intensity levels within
the duplicate recovery cultures were measured to be
170 mE m2 s1 at the end of the cultivation, although the
experiment will be referred to by the constant source light
intensity of 600 mE m2 s1. The recovery cultivations were
carried out for 144 h (six days).
2.3. Analytical techniques
2.3.1. Carotenoid analysis
Cells were separated from 1.0 mL of culture by centrifugation
in 2.0 mL Eppendorf tubes at 5000g for 5 min. The supernatant
was decanted and 100 mL of fucoxanthin internal standard
(0.001% in methanol) was added to the pellet. To extract the
carotenoids, glass beads and 100 mL methanol (MeOH) were
added to the pellet which was subsequently sonicated for
5 min in a Branson 2200 ultrasonic cleaner. After the first
5 min, 900 mL of MeOH were added to the sample and cell
disruption in the ultrasonic cleaner continued for 10 min.
Next, the aliquot was spun down for 5 min at 5000g to separate
the MeOH extract from the cell debris. The MeOH supernatant
(approximately 800 mL) was removed and placed into a clean,
10 mL test tube. The MeOH/sonication extraction steps were
reiterated twice more until the microalgae turned white. The
extract was fully dried in the test tube under a stream of N2 at
40  C. Then, the residue was suspended in precisely 200 mL
MeOH, to permit an exact quantification, and transferred to
HPLC sample vials for HPLC analysis.
Carotenoid analyses were carried out using an Agilent 1200
series HPLC system fitted with a UV/VIS diode array detector.
Liquid chromatography was conducted and optimized on an
RP-18e Lichrospher® 100 (250  4 mm i.d., 5 mm particle size)
column. Absorbance measurements were carried out at
450 nm. The HPLC separation employed the following mobile
phases: (a) MeOH, (b) acetone, (c) H2O: MeOH (90:10). The ca-
rotenoids were eluted at a flow rate of 1 mL min1 and at room
temperature using the following gradient: 0e3 min (90:0:10);
3e6 min (90:5:5); 6e9 min (90:5:5); 9e15 min (70:30:0);
15e25 min (50:50:0); 25e35 min (90:0:10). The chromatographic
peaks were identified and quantified by comparing retention
time, spectra and peak area against each standard. Each
determination was carried out in quadruplicate, i.e. duplicate
HPLC analyses for duplicate cultivations.
2.3.2. Lipid analysis
A modified Folch extraction method [18] was used to extract
the lipids from the algal cells and was coupled to the sulfo-
phospho-vanillin (SPV) method [13] for their quantification.
Initially, a 1.0 mL aliquot of algal suspension was collected and
spun down for 3 min at 5000g and the supernatant was dec-
anted. Next, the pellet was re-suspended in 0.5 mL of MeOH
and the cells were disrupted for 5 min in the presence of glass
beads in the ultrasonic cleaner. Then, 1.000 mL of CHCl3 was
added to the cells which were disrupted again for 10 min and
centrifuged again for 3 min at 5000g before collection of the
supernatant. The extraction and cell breakage steps were
reiterated and the extracts were combined. The pooled ex-
tracts were supplemented with a 0.8% solution of NaCl
(0.6 mL), spun down for 3 min at 5000g, the upper layer dec-
anted and the lower layer fully dried under N2 at 40  C. Then,
0.2 mL of H2SO4 (95e97%) was added and heated at 90  C for
10 min. Upon cooling, 5.0 mL of PV-reagent (1.2 g L1 vanillin in
68% phosphoric acid stored in the dark) was added, the sam-
ples were vortexed and absorbance values of 200 mL volumes
were determined at 525 nm in a spectrophotometric micro-
plate reader (Bioteck-Power wave XS2). All analyses were
carried out in quadruplicate, i.e., duplicate lipid measure-
ments for duplicate cultivations. The lipid content was
quantified by comparison to a calibration curve generated
from the standard addition of samples of 5, 10, 20 and 50 mg of
high-purity soybean oil from a 100 mg mL1 soy oil solution
(Sigma #S7381).
2.3.3. Nitrate analysis
Cells were separated from 1.00 mL of culture by centrifugation
in 2.0 mL Eppendorf tubes at 5000g for 5 min. The supernatant
was decanted and the NO 3 concentration was quantified in
triplicate for each cultivation following an earlier protocol
centered on UV absorption [43].
2.3.4. Cell characterization
A Beckman Multisizer™ IV Coulter Counter was used for
determining the cell size distribution. The electrolyte solution
was Coulter® Isoton® II Diluent, while an aperture diameter of
100 mm and 10 mm latex beads (Coulter CC size standard L10)
served for the size calibration. At least 104 cells were used for
the analyses of cell size. In addition to these analyses, light
microscopy was used for cell measurement and direct obser-
vation of cell characteristics using a Leica DMRA 2 microscope
aided by the QWin software package (Leica DM/RXA).
2.3.5. Cell culture density
Cell number density (cells mL1) was quantified using a
Beckman Multisizer™ IV Coulter Counter. Cell culture Ali-
quots of 0.10 mL were diluted in 10.0 mL of Coulter® Isoton® II
Diluent (BeckmaneCoulter) and cells between 5.0 and 16.0 mm
were counted using a 100 mm aperture. Triplicate measure-
ments were conducted for each cultivation.
To measure the dry cell mass density in the algal suspen-
sion, 10 mL of the cell culture was first spun down in glass
centrifuge tubes at 2000g for 10 min to separate the microalgal
biomass from the medium. Upon decanting of the superna-
tant the cells were re-suspended in 30 mL of distilled H2O and
centrifuged once more for 10 min at 2000g. The pellet washing
procedure was carried out twice, and the pellets were next put
into pre-dried, pre-weighed aluminum dishes at 80  C for 24 h
before being reweighed. Triplicate determinations were used
throughout.
2.3.6. Transmethylation and fatty acid analysis
Samples for lipid analysis were subjected to conversion to
fatty acid methyl esters using an adaptation to a previously
reported protocol [6]. For each sample, 2 mL of MeOH and
0.8 mL of cell culture were mixed and sonicated for 10 min to
disrupt the cells. After cell disruption, 1 mL of distilled water
and 1 mL of chloroform were added. Next, the aliquot was
vortexed for 30 s and centrifuged at 1000g for 5 min to separate
the two layers. The organic phase was recovered and dried
 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5
under nitrogen. After extraction, samples were methylated
when 1.00 mL of 0.10 M KOH in MeOH was added to the sample
and heated at 70  C for 1 h. The samples were mixed at t ¼ 20
and 40 min. After cooling, 0.40 mL of 1.2 M HCl in MeOH was
added and the mixture was heated at 70  C for 15 min, then
the sample was cooled before 2.00 mL of n-hexane and 1.00 mL
of milliQ water (18.2  106 U) was added and then vortexed for
10 s. Finally, the mixture was centrifuged at 3000g for 5 min.
The n-hexane layer with fatty acid methyl esters (FAMEs) was
analyzed by gas chromatography.
Gas chromatography of the FAMEs was carried out by
injecting 1.5 mL of sample in column mode on a Trace GC
(ThermoQuest, ThermoFinnigan, Milan, Italy) equipped with a
flame ionization detector and a fused silica capillary column
(100 m length, 0.25 mm I.D.) coated with a 0.2 mm film of bis-
cyanopropyl polysiloxane (Rt-2560, Restek, Bellefonte, PA,
USA). Helium served as the carrier gas and the system was run
at a constant pressure of 200 kPa. The initial oven temperature
was 80  C with a 25  C min1 ramp to 175  C (maintained for
25 min), then raised with a ramp of 10  C min1 to 205  C (held
for 4 min), further augmented at 10  C min1 to 225  C
(maintained for 20 min) and finally diminished at 20  C min1
to 80  C. The flame ionization detector temperature was kept
at 255  C. The flow of helium to the detector was 35 mL min1
and airflow was 350 mL min1. A calibration injection with
authentic standards (SigmaeAldrich, MO, USA) was used to
determine the retention times and response factors of each
fatty acid. A hexane injection was used as the blank. The
sample injections used C11:0 and C17:0 as internal standards
which served to verify retention time alignment with the
calibration injection. The ChromQuest software automatically
identified the peaks and performed the integration. Each FA in
the sample injections was quantified by using the injected
quantity of the C17:0 internal standard and the ratio of its
response factor to the response factor of each FA from the
authentic standard. A representative FAME scan gas chro-
matogram is presented in supplementary information, Fig. S2.
2.4. Photosynthesiseillumination (PeE) curves
Photosynthesis versus light intensity (PeE) curves were ob-
tained for cell cultures in the growth phase at 26  C following
published protocols [24,25]. The measurements were carried
out in a DW1 electrode chamber connected to an Oxylab
control system and O2 View software, all purchased from
Hansatech Instruments Limited (Norfolk, UK). The light was
delivered to the electrode chamber by a Decostar Titan lamp
(12 V, 35 W, 60 reflector). The mean photosynthetically active
photon flux density (Em, mmol photons m2 s1) was quantified
by positioning a US-SQS/L Spherical Micro Quantum Sensor
connected to a ULM-500 Universal Light Meter (Heinz Walz
GmbH, Eichenring, Germany) directly inside algal suspensions
which were already put in the electrode chamber. Calibration
and operation was carried out according to the manufacturer's
instructions and the system was held isothermal by con-
necting the water jacket of the electrode chamber to a ther-
moregulating bath.
Measurements were carried out in duplicate at light in-
tensities of 25, 50, 100, 200, 300, 400, 600 and 900 mE m2 s1
and in the dark with cultures which had a dry cell density of
269
535 ± 12 mg DCW L1. The cell culture within the photosyn-
thetic measurement chamber had a small light path (1 cm),
was well mixed (60 rpm stir speed) and the PAR values were
measured directly within the culture, so it can be assumed
that the stated mean light intensities are accurate even
though a cell culture density of ~0.5 g L1 will experience some
self-shading. The rate of photosynthesis, rO2 (mmol O2 s1 g
DCW1), versus mean light intensity, Em (mE m2 s1), was
adjusted for respiration and fit to a Monod model in view of
quantifying the half-saturation constant, EK (mE m2 s1). The
maximum light intensity used in our PeE analysis
(900 mE m2 s1) was not enough to induce photoinhibition
because a reduction in photosynthetic rate was not observed,
so a PeE curve model which includes an inhibition term was
not considered. The oxygen evolution data were fit to the
following Monod form equation:
Em
rO2 ¼ rO2 ;max (1)
Ek þ Em
where rO2 and rO2 ;max are the measured and maximum specific
oxygen evolution rates (mmol O2 g DCW1 s1) and Em and Ek
are the mean photon flux within the test chamber and the
Monod half-saturation constant (mE m2 s1), respectively.
3. Results and discussion
3.1. Phylogenetic analysis
The BLASTN analysis indicates that the microalgal isolate
(named Acutodesmus sp. strain SG-1) showed the highest per-
centage identity (99.8%, query coverage 100%) with A. obliquus
strain CCAP 276/2 (GenBank accession number FR865715),
strain UTEX 1450 (AJ249506), strain CCAP 276/49 (FR865726)
and strain CCAP 279/46 (FR865738), and Acutodesmus bernardii
strain CCAP 276/38 (JQ082329). The NJ phylogenetic tree
inferred from the ITS rRNA gene sequences of selected
microalgae is shown in Fig. 1 and contains some members of
the Scenedesmaceae family and other selected families of the
Chlorophyceae class. As shown by Refs. [17] and [23]; the Sce-
nedesmus genus (sensu stricto) and the Acutodesmus genus
(sensu stricto) form separate clusters in the constructed NJ
phylogenetic tree (Fig. 1), even though they are generally quite
similar morphologically and physiologically.
3.2. PeE curves
The data from the PeE curve served to determine the level of
light intensity at which oxidative stress would start having a
significant influence. As light intensities approach and exceed
the value of Ek the quantum yield decreases and oxidative
stress begins to increase [49], which leads to the formation of
potentially damaging ROS. The values of Ek and rO2 ;max were
determined from the PeE curve at 26  C to be 36 ± 7 mE m2 s1
and 1.2 ± 0.1 mmol O2 g DCW1 h1. Therefore, light intensities
within the cell culture for the lipogenesis experiments were
selected to be approximately the Ek for the low light stress
condition and up to 10 the Ek within the cell culture at the
highest light intensity. The PeE curve is presented in the
Supplementary information, Fig. S1.
270 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5

Fig. 1 e Neighbor-joining (NJ) phylogenetic tree generated using the Geneious Pro R6 software, v 6.1.4 and phylogenetic
position of Acutodesmus sp. strain SG-1 based on ITS-1e5.8SeITS-2 rRNA gene sequence data. Acutodesmus sp. strain SG-1
(indicated in bold with an asterisk) belongs to the Acutodesmus genus (sensu stricto) [17,23].

3.3. Lipogenesis cultivation
Lipogenesis was initiated by transferring cells from the
growth phase to the Multicultivator where they were cultured
under conditions that were conducive to increased lipid for-
mation and accumulation. The formation and accumulation
of lipids in microalgae can emanate from different kinds of
stress [37,40,47]. In this study, we used nitrate starvation as it
has been documented to be the most reliable approach to
induce lipogenesis, while salinity and illumination were of
direct relevance to the ecosystem from which our strain
originated and, furthermore, were convenient to impose and
modulate in our present system.
In recent reports approaches similar to our study were
employed to characterize microalgal formation of lipids and
carotenoids [12,39]. Nonetheless, the strains used by Park et al.
[39] were saline-tolerant Dunaliellas so the effect of salinity
was not studied. Campenni et al. [12] examined production
levels at different salt concentrations and lower light in-
tensities. The maximum incident light intensity in their study
was 78.3 mE m2 s1 (there was no indication of light intensity
within the cultures) and the long term (15e49 days after the
start of the stress phase) production was reported in contrast
to this study which reports the short term trends.
The current study employed source light intensity levels of
100, 300, 600 and 900 mE m2 s1 (40, 140, 280 and
380 mE m2 s1 mean light intensity quantified within the
cultures), to illustrate the higher mean light intensities ex-
pected to prevail in large-scale cultures in Algeria and
measured the carotenoids and lipids every day over a shorter
 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5
period (144 h) to mimic the shorter production cycles of a cost-
effective, industrial-scale algal cultivation.
3.3.1. Biomass production and cell characterization
At the beginning of the culture, the cell density in terms of dry
cell weight was 1.39 ± 0.06 g L1, which increased to
1.45 ± 0.08, 1.65 ± 0.03, 2.09 ± 0.16 and 1.74 ± 0.12 g L1 for the
100, 300, 600 and 900 mE m2 s1 source light intensity culti-
vations, respectively, even though the cell number densities
slightly decreased (Fig. 2). The initial pH in the cell culture was
7.39 ± 0.01, and never exceeded 8.65 in any of the cultures
during lipogenesis. Similar to previous work [20], microscopic
observation showed an increase in cell size and a shift
Fig. 2 e (A) Cell number density and (B) cell mass density
during lipogenesis, recovery, and the control cultivations
which were carried out with both nitrate and NaCl
(þN þ NaCl), or with neither (¡N ¡ NaCl). The data
represent the initial conditions (t ¼ 0) and the final
measurements (t ¼ 144 h) for each cultivation. The values
on the x-axis represent the cultivation light intensities,
which were only at a source light intensity of
600 mE m¡2 s¡1 for the recovery and control cultivations.
271
towards orange pigmentation during the stress phase, but due
to the irregular shape of the individual cells the change in cell
diameter was difficult to quantify and the mean cell sizes are
therefore not reported.
The cell number density decreased slightly in all of the
lipogenesis cultivations while the cell number density for both
the þN þ NaCl and N  NaCl controls remained constant
(Fig. 2A). Therefore, nitrate starvation and osmotic stress both
arrested cell division in this strain, with slightly more cell
attrition when both stresses were present. However, all cul-
tivations increased in dry cell mass, with the increase in cell
mass following the increase in lipid content (Fig. 2B and 3). In
almost all cases, nutrient starvation arrests cell division, but
CO2 fixation can continue with lipids and starch being the
primary carbon sinks [47]. This is in agreement with other
work [30] which proposes that algal biomass productivity can
be described by a compartmental model that segregates the
biomass into functional and storage components and that
under nitrogen starvation an increase in biomass can be pri-
marily attributed to an increase in storage compounds.
3.3.2. Lipids production
As shown in Fig. 3, under all illumination intensity conditions
used the lipid content of the cells increased during lipogen-
esis. In the beginning of lipogenesis, the starting lipid content
was 190 ± 15 mg L1 and it reached up to 700 ± 54 mg L1 after
144 h at a light intensity of 600 mE m2 s1, even though the cell
number density did not increase. The lipid content in the
N  NaCl and þN þ NaCl control cell cultures at
600 mE m2 s1 (Fig. 3) increased from 160 ± 29 mg L1 at the
inoculum conditions to 480 ± 138 mg L1 and to
380 ± 53 mg L1 at shutdown, respectively. Therefore, even
though salinity stress induced a state of lipogenesis, nitrogen
starvation was more effective, but not as effective as a com-
bination of both stresses. Therefore, a bioprocess based on
Fig. 3 e The lipid composition during the lipogenesis and
control cultivations measured per culture volume, per cell
and by percent of dry weight for the inoculum condition
(t ¼ 0) and at the end of the cultivation at t ¼ 144 h. The
values 100, 300, 600 and 900 mE m¡2 s¡1 indicate the source
light intensity levels. C, t ¼ 0 indicates the inoculum
condition for the controls which were carried out with both
nitrate and NaCl (þþ), or with neither (¡¡). The controls
were conducted at a source light intensity of
600 mE m¡2 s¡1 and carried out for 144 h.
272 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5
salinity stress which is more readily applied than a nutrient
stress could be developed, although the process yield would
be lower than a combination of stresses.
3.3.3. Fatty acid profile
The fatty acid distribution in the cell mass at the time of
inoculation and at shutdown (t ¼ 144 h) for the source light
intensity with low oxidative stress (100 mE m2 s1) and at
the intensity which had the highest lipid production levels
(600 mE m2 s1) can be seen in Fig. 4. The fatty acid profiles
between inoculation and shutdown were minimally
changed, except for small reductions in medium chain (<16)
and C16:0 FA, which were shifted towards C18:1. It is
apparently not possible to significantly change the fatty acid
profile for this strain using the stresses applied in this
study.
The FA profile had a low degree of unsaturation [15,42];
when compared to strains of the closely related A. obliquus,
UTEX 393 and 417 [1,11,23], isolated from cold-water envi-
ronments. In this study at stationary phase and at the light
intensity with the highest lipid production (600 mE m2 s1),
the primary FA produced was oleic acid (C18:1, 49.7 ± 0.7%)
followed by palmitic acid (C16:0, 21 ± 1%) and linoleic acid
(C18:2, 15 ± 1%) with no unsaturated C16 fatty acids, and
minimal amounts of both linolenic acid (C18:3, 7.1 ± 0.6%) and
long-chain fatty acids (>C18, 1.0 ± 0.3%). The fatty acid profile
had a calculated degree of unsaturation (DU, 97 ± 2) and a
predicted cetane number (CN, 55 ± 2), two measures of bio-
diesel quality, comparable to or better than all readily avail-
able vegetable oil sources except for palm [19]. This differed
from the previously characterized A. obliquus strains which
have higher levels of C18:3 than C18:2 and significant levels of
unsaturated C16 [1,11]. Additionally, the measured content of
linolenic acid was well below the limit of 12% established by
Fig. 4 e Fatty acid distribution in the cell mass on the day of
inoculation and at shutdown for the source light intensity
with low oxidative stress (100 mE m¡2 s¡1) and at the
highest production level of lipids (600 mE m¡2 s¡1) at
shutdown (day 6). All FA with a carbon chain less than 16
carbons are represented by < C16 and all FA with a carbon
chain more than 18 carbons are represented by >18. All
other FA were less than 0.2% of the FA profile.
the European Committee for Standardization (regulation EN
14214), which describes the requirements and testing
methods for biodiesel.
It has been shown that the selective pressure of cold-
temperature environments results in algal strains with
higher ratios of unsaturated FA [3,21], that decreasing culti-
vation temperature increases the unsaturation of FA [28] and
that increasing the cultivation temperature can increase
saturation of FA [45]. Therefore, we postulate that microalgal
species with adequate levels of saturation and a fatty acid
profile amenable to biofuel production could be isolated
from the many yet to be exploited high-temperature
biotopes.
3.3.4. Content of carotenoids and photosynthetic pigments
At the start of lipogenesis, the photosynthetic pigments,
including chlorophyll and primary carotenoids, were pre-
dominant (Fig. 5A). However, due to the induction of car-
otenogenesis in response to the stress conditions, the
canthaxanthin had already doubled by the second day in the
600 and 900 mE m2 s1 lipogenesis experiments. The chloro-
phyll b content had already diminished between 72% and 84%
by the second day and had decreased from 3.45 ± 0.01 mg L1
at the start of the cultivation to 0.43 ± 0.01 mg L1 by day six for
the 900 mE m2 s1 cultivation (Fig. 5A and B). At the end of the
cultivation, there were considerable differences in the accu-
mulation level of canthaxanthin under the various light in-
tensities. After 144 h of stress culture (lipogenesis),
intracellular canthaxanthin evolution was least at a light in-
tensity of 100 mE m2 s1 and highest at both 600 and
900 mE m2 s1 with cell culture concentrations of 0.94 ± 0.04
and 1.44 ± 0.03 mg L1 of culture, respectively. The increase in
canthaxanthin content was most remarkable when normal-
ized to chlorophyll b content, from 0.13 ± 0.01 on day one to
3.4 ± 0.1 mg canthaxanthin mg1 chl b (>25-fold increase) after
144 h at a light intensity of 900 mE m2 s1 (Fig. 5C). This verifies
the effectiveness of the stress combination because the
canthaxanthin/chl b ratio can be used as a measure of cellular
stress, and the ratio in this study is on the same order and
higher than in other stress studies [36]; [44], indicating that the
photosystems of the cells were sufficiently, if not maximally,
stressed. However, the intracellular concentration of cantha-
xanthin could also be correlated to the intracellular concen-
tration of lipids (Fig. 5D), which implies that the production of
secondary carotenoids in this strain could be for the preven-
tion lipid peroxidation [35].
In the þN þ NaCl and N  NaCl control cultivations, it
was possible to differentiate the effects of both nitrate stress
and salinity stress on the production of canthaxanthin
(Fig. 5C and D). The þN þ NaCl control showed an increase in
canthaxanthin concentration from 0.20 ± 0.06 to
0.99 ± 0.02 mg L1 while the N  NaCl cultivation man-
ifested a more modest rise in canthaxanthin concentration
from the same initial value to 0.73 ± 0.08 mg L1. However,
the combination of the two stresses resulted in a greater in-
fluence (Fig. 5A). Consistent with the fully stressed lipogen-
esis experiments, the intracellular canthaxanthin
concentration remained proportional to the increase in lipid
content (Fig. 5D). This result is contrary to a previous study
using a Dactylococcus microalga isolated from the same oasis
 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5 273

Fig. 5 e Pigment compositions on a cell culture basis. The label t ¼ 0 represents the inoculum conditions immediately after
starting the stress phase and the labels 100, 300, 600, and 900 indicate the incident light intensities to the cell cultures for
the indicated measurement at shutdown (t ¼ 144 h). C, t ¼ 0 indicates the inoculum condition for the controls which were
carried out with both nitrate and NaCl (þþ), or with neither (¡¡). (A) The cell culture composition of canthaxanthin, lutein
and the chlorophylls. (B) Cell culture concentrations of canthaxanthin (can) and chlorophyll b (Chlb) with respect to time. (C)
Canthaxanthin composition based on dry cell mass, per cell and normalized to chlorophyll b content. (D) Intracellular
canthaxanthin per gram of intracellular lipids.

[20], which implies that secondary carotenogenesis in Acuto-
desmus sp. SG-1 is primarily a function of the lipid production,
not the type of stress, and that carotenogenesis was likely in
response to the cellular need to protect the oil bodies against
peroxidation. The present Acutodesmus strain is therefore of
primary interest for its lipogenesis of fatty acids with a degree
of saturation adequate for biofuel production, with cantha-
xanthin being produced in lower concentrations and in
response to lipogenesis.
3.4. Recovery process
Algal cultures which had been maintained under lipogenesis
conditions for 14 days were centrifuged to remove NaCl from
the medium and the cells were re-suspended in 3N-BBM þ V
medium in the Multicultivator at a source light intensity of
600 mE m2 s1. At this stage, the algal biomass was under
neither nitrate starvation nor osmotic stress, thus cell growth
restarted and the cell number density increased from
(1.74 ± 0.03)  107 to (3.42 ± 0.01)  107 cells mL1. Likewise, the
dry cell mass density increased from 0.96 ± 0.06 to
2.5 ± 0.3 g DCW L1 and the cellular lipid composition
decreased from 30 ± 2 to 11 ± 2 wt% lipids on a DCW basis,
approximately the lipid composition (14 ± 1 wt%) at the start
of the lipogenesis cultivation (Fig. 3). This corresponds to
starting and ending lipid contents of 290 ± 30 and
350 ± 50 mg L1 on a cell culture basis and a productivity of
10 ± 8 mg L1 d1, which is significantly less than the lipid
productivity during the lipogenesis cultivation under the
same illumination conditions (85 ± 8 mg L1 d1).
A reduction of carotenoids in the cell culture and the pro-
duction of photosynthetic pigments were also observed
(Fig. 6). The canthaxanthin concentration decreased from
1.05 ± 0.09 mg L1 in the initial cell culture to 0.27 ± 0.01 mg L1
by 144 h, while chlorophyll a and b were restored. The recov-
ery process also resulted in an increase of lutein from
0.71 ± 0.04 to 2.11 ± 0.28 mg L1 by the end of cultivation. The
increase in lutein was expected because its primary role is in
transferring excitation energy to chlorophyll and in quench-
ing 3Chl states [26], thus the restoration of the chlorophylls
restored the cellular requirement for lutein.
Given that the metabolic stress of this algal strain is easily
reversible, which has also been demonstrated in other mem-
bers of the Acutodesmus genus [41], it is a good candidate for
274 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5
Fig. 6 e Cell culture pigment composition at the start (R,
t ¼ 0) and at shutdown (R, t ¼ 144 h) of the recovery phase
cultivation.
the development of semi-batch or semi-continuous cultures
at the industrial scale.
4. Conclusion
The production of lipids, carotenoids and FA by Acutodesmus sp.
strain SG-1, a strain isolated from the Algerian Sahara, under
nitrate starvation, NaCl and oxidative stress, has been charac-
terized. The production of lipids was a function of the light in-
tensity and the principal secondary carotenoid was
canthaxanthin, which was believed to act as a lipid preserva-
tive. The FA profile was more saturated than previously char-
acterized strains of Acutodesmus, probably due to the selective
pressure of its high-temperature native environment. Further-
more, the FA profile was characteristic of a high quality biofuel.
The metabolic stress responses were readily reversible, thus a
semi-continuous bioprocess is possible at the industrial scale.
Acknowledgements
Support for this work was obtained from The Belgian National
Fund for Scientific Research (Fonds de la Recherche Scientifi-
que e FNRS) project PHOTOFUNDS, the Walloon Region proj-
ect FOTOBIOMAT (contract #1117323) and the Algerian
Ministry of Higher Education and Scientific Research (Minis-
 re de l'enseignement supe
te rieur et de la recherche scientifi-
que e Alge  rie).
The authors declare the absence of any conflict of interest
or financial involvement with any entity with a financial in-
terest, conflict, or motive which could influence the content of
this manuscript.
 le
 ne Dailly (Universite
 catho-
We gratefully appreciate He
lique de Louvain, Laboratory of Bioengineering) for her assis-
tance and skill with the analyses of lipids and
chromatography methodologies; Yvan Larondelle and Eric
Mignolet (Universite  catholique de Louvain) for access to their
GC equipment and help with fatty acid transesterification; and
Theocharis Efthymiopoulos (Universite  catholique de Lou-
vain, Laboratory of Bioengineering) for his assistance in the
phylogenetic characterization of the strain.
Supplementary information
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.biombioe.2014.07.023.
references
[1] Abomohra AEF, Wagner M, El-Sheekh M, Hanelt D. Lipid and
total fatty acid productivity in photoautotrophic fresh water
microalgae: Screening studies towards biodiesel production.
J Appl Phycol 2013;25(4):931e6.
[2] Aburai N, Ohkubo S, Miyashita H, Abe K. Composition of
carotenoids and identification of aerial microalgae isolated
from the surface of rocks in mountainous districts of Japan.
Algal Res 2013;2(3):237e43.
[3] An M, Mou S, Zhang X, Ye N, Zheng Z, Cao S, et al.
Temperature regulates fatty acid desaturases at a
transcriptional level and modulates the fatty acid profile in
the Antarctic microalga Chlamydomonas sp. ICE-L. Bioresour
Technol 2013;134:151e7.
[4] Ben Amotz A, Avron M. The biotechnology of mass culturing
Dunaliella for products of commercial interest. In:
Cresswell RC, Rees TAV, Shah N, editors. Algal and
cyanobacterial biotechnology. New York: Longman Scientific
& Technical; 1989. p. 91e114.
[5] Bigogno C, Khozin-Goldberg I, Cohen Z. Accumulation of
arachidonic acid-rich triacylglycerols in the microalga
Parietochloris incisa (trebuxiophyceae, chlorophyta).
Phytochemistry 2002;60(2):135e43.
[6] Bligh EG, Dyer WJ. A rapid method of total lipid extraction
and purification. Can J Biochem Physiol 1959;37(8):911e7.
[7] Borowitzka MA, Moheimani NR. Algae for biofuels and
energy. In: Borowitzka MA, editor. Developments in applied
phycology, vol. 5. New York: Springer; 2013.
[8] Borowitzka MA. Carotenoid production using
microorganisms. In: Ratledge C, Cohen Z, editors. Single cell
oils. Urbana: AOCS Publishing; 2010. p. 225e40.
[9] Borowitzka MA. High-value products from microalgae-their
development and commercialisation. J Appl Phycol
2013;25(3):743e56.
[10] Boussiba S, Vonshak A. Astaxanthin accumulation in the
green alga haematococcus pluvialis. Plant Cell Physiol
1991;32(7):1077e82.
[11] Breuer G, Lamers PP, Martens DE, Draaisma RB, Wijffels RH.
Effect of light intensity, pH, and temperature on
triacylglycerol (TAG) accumulation induced by nitrogen
starvation in Scenedesmus obliquus. Bioresour Technol
2013;143:1e9.
[12] Campenni L, Nobre BP, Santos CA, Oliveira AC, Aires-
Barros MR, Palavra AMF, et al. Carotenoid and lipid
production by the autotrophic microalga Chlorella
protothecoides under nutritional, salinity, and luminosity
stress conditions. Appl Microbiol Biotechnol
2013;97(3):1383e93.
[13] Chabrol E, Castellano A. SVP method for estimation of total
serum lipid. J Lab Clin Med 1961;57:300.
[14] Chan MC, Ho SH, Lee DJ, Chen CY, Huang CC, Chang JS.
Characterization, extraction and purification of lutein
 b i o m a s s a n d b i o e n e r g y 6 9 ( 2 0 1 4 ) 2 6 5 e2 7 5
produced by an indigenous microalga Scenedesmus obliquus
CNW-N. Biochem Eng J 2013;78:24e31.
[15] Clements LD. Blending rules for formulating biodiesel fuel.
Liquid fuel and industrial products from renewable
resources. In: Proceedings of the third liquid fuel conference.
September 15e17, Nashville; 1996. pp. 44e53.
[16] Czygan FC. Blooderain and bloodesnow: nitrogen-deficient
cells of Haematococcus pluvialis and Chlamydomonas nivalis.
Arch Mikrobiol 1970;74(1):69e76.
[17] 
Elia s M, Nemcova 
 Y, Skaloud P, Neustupa J, Kaufnerova  V,

Sejnohov  L. Hylodesmus singaporensis gen. et sp. nov., a
a [37] Pal D, Khozin-Goldberg I, Cohen Z, Boussiba S. The effect of
new autosporic subaerial green alga (Scenedesmaceae,
Chlorophyta) from Singapore. Int J Syst Evol Microbiol
2010;60(5):1224e35.
[18] Folch J, Lees M, Sloane Stanley GH. A simple method for the
isolation and purification of total lipides from animal tissues.
J Biol Chem 1957;226(1):497e509.
[19] Gouveia L, Oliveira AC. Microalgae as a raw material for
biofuels production. J Ind Microbiol Biotechnol
2009;36(2):269e74.
[20] Grama BS, Chader S, Khelifi D, Agathos SN, Jeffryes C.
Induction of canthaxanthin production in a Dactylococcus
microalga isolated from the Algerian Sahara. Bioresour
Technol 2014;151:297e305.
[21] Guschina IA, Harwood JL. Lipids and lipid metabolism in
eukaryotic algae. Prog Lipid Res 2006;45(2):160e86.
[22] Harwood JL, Vı́gh L. Membranes in stress and adaptation.
Ann NY Acad Sci 1998;851:162e8.
[23] Hegewald E, Bock C, Krienitz L. A phylogenetic study on
Scenedesmaceae with the description of a new species of
Pectinodesmus and the new genera Verrucodesmus and
Chodatodesmus (Chlorophyta, Chlorophyceae). Fottea
2013;13(2):149e64.
[24] Huang Y-m, Rorrer GL. Dynamics of oxygen evolution and
biomass production during cultivation of Agardhiella
subulata microplantlets in a bubble-column photobioreactor
under medium perfusion. Biotechnol Prog 2002;18:62e71.
[25] Huang Y-m, Maliakal S, Cheney DP, Rorrer GL. Comparison of
development and photosynthetic growth for filament
clumps and regenerated microplantlet cultures of
Agardhiella subulata (Rhodophyta, Gigartinales). J Phycol
1998;34:893e901.
[26] Jahns P, Holzwarth AR. The role of the xanthophyll cycle and
of lutein in photoprotection of photosystem II. Biochim
Biophys Acta - Bioenerg 2012;1817(1):182e93.
[27] Jeffryes C, Rosenberger J, Rorrer GL. Fed-batch cultivation
and bioprocess modeling of Cyclotella sp. for enhanced fatty
acid production by controlled silicon limitation. Algal Res
2013;2(1):16e27.
[28] Jiang H, Gao K. Effects of lowering temperature during
culture on the production of polyunsaturated fatty acids in
the marine diatom Phaeodactylum tricornutum
(Bacillariophyceae). J Phycol 2004;40(4):651e4.
[29] Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M,
Sturrock S, et al. Geneious basic: an integrated and
extendable desktop software platform for the organization
and analysis of sequence data. Bioinformatics
2012;28(12):1647e9.
[30] Klok AJ, Verbaanderd JA, Lamers PP, Martens DE, Rinzema A,
Wijffels RH. A model for customising biomass composition
in continuous microalgae production. Bioresour Technol
2013;146:89e100.
[31] Larkin MA, Blackshields G, Brown NP, Chenna R,
McGettigan PA, McWilliam H, et al. Clustal W and Clustal X
version 2.0. Bioinformatics 2007;23(21):2947e8.
[32] Mendes A, Reis A, Vasconcelos R, Guerra P, Lopes Da Silva T.
Crypthecodinium cohnii with emphasis on DHA production: a
review. J Appl Phycol 2009;21(2):199e214.
275
[33] Metting FB. Biodiversity and application of microalgae. J Ind
Microbiol Biotechnol 1996;17(5e6):477e89.
[34] Miner BG, Sultan SE, Morgan SG, Padilla DK, Relyea RA.
Ecological consequences of phenotypic plasticity. Trends
Ecol Evol 2005;20(12):685e92.
[35] Niyogi KK. Photoprotection revisited: genetic and molecular
approaches. Annu Rev Plant Biol 1999;50:333e59.
[36] Orosa M, Franqueira D, Cid A, Abalde J. Analysis and
enhancement of astaxanthin accumulation in Haematococcus
pluvialis. Bioresour Technol 2005;96(3):373e8.
light, salinity, and nitrogen availability on lipid production
by Nannochloropsis sp. Appl Microbiol Biotechnol
2011;90(4):1429e41.
[38] Park KC, Whitney C, McNichol JC, Dickinson KE,
MacQuarrie S, Skrupski BP, et al. Mixotrophic and
photoautotrophic cultivation of 14 microalgae isolates from
Saskatchewan, Canada: potential applications for
wastewater remediation for biofuel production. J Appl Phycol
2012;24(3):339e48.
[39] Park S, Lee Y, Jin ES. Comparison of the responses of two
Dunaliella strains, Dunaliella salina CCAP 19/18 and
Dunaliella bardawil to light intensity with special emphasis
on carotenogenesis. Algae 2013;28(2):203e11.
[40] Patterson GW. Effect of culture temperature on fatty acid
composition of Chlorella sorokiniana. Lipids 1970;5(7):597e600.
[41] Qin S, Liu G-X, Hu Z-Y. The accumulation and metabolism of
astaxanthin in. Scenedesmus obliq (Chlorophyceae). Process
Biochem 2008;43:795e802.
[42] Ramos MJ, Ferna  ndez CM, Casas A, Rodrı́guez L, Perez A.
Influence of fatty acid composition of raw materials on
biodiesel properties. Bioresour Technol 2008;100(1):261e8.
[43] Rice EW, Baird RB, Eaton AD, Clesceri LS. In: Standard
methods for the examination of water and wastewater. 20th
ed. American Water Works Association; 1998.
[44] Saha SK, Moane S, Murray P. Effect of macro- and micro-
nutrient limitation on superoxide dismutase activities and
carotenoid levels in microalga Dunaliella salina CCAP 19/18.
Bioresour Technol 2013;147:23e8.
[45] Sato N, Sonoike K, Kawaguchi A, Tsuzuki M. Contribution of
lowered unsaturation levels of chloroplast lipids to high
temperature tolerance of photosynthesis in
Chlamydomonas reinhardtii. J Photochem Photobiol B Biol
1996;36(3):333e7.
[46] Sheehan J, Dunahay T, Benemann J, Roessler P. A look back at
the U.S. Department of Energy's aquatic species program:
biodiesel from algae (close-out report). National Renewable
Energy Laboratory aquatic species program closing report; 1998.
[47] Solovchenko AE. Physiological role of neutral lipid
accumulation in eukaryotic microalgae under stresses. Russ J
Plant Physiol 2012;59(2):167e76.
[48] Solovchenko AE. Physiology and adaptive significance of
secondary carotenogenesis in green microalgae. Russ J Plant
Physiol 2013;60(1):1e13.
[49] Takache H, Pruvost J, Cornet JF. Kinetic modeling of the
photosynthetic growth of Chlamydomonas reinhardtii in a
photobioreactor. Biotechnol Prog 2012;28(3):681e92.
[50] White TJ, Bruns TJ, Lee S, Taylor J. Amplification and direct
sequencing of fungal ribosomal RNA genes for 64 nuclear
sequence analysis of Skeletonema costalum phylogenetics. In:
Innis M, Gelfand M, Sainsky J, White TJ, editors. PCR
protocols: a guide to methods and applications. Orlando, FL:
Academic Press; 1990. p. 315e22.
[51] Zhekisheva M, Zarka A, Khozin-Goldberg I, Cohen Z,
Boussiba S. Inhibition of astaxanthin synthesis under high
irradiance does not abolish triacylglycerol accumulation in
the green alga Haematococcus pluvialis (Chlorophyceae). J
Phycol 2005;41(4):819e26.