Algal Research 12 (2015) 368–376

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Algal Research

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

Isolation of prospective microalgal strains with high saturated fatty acid

content for biofuel production
A.V. Piligaev a,⁎, K.N. Sorokina a,b,c, A.V. Bryanskaya b, S.E. Peltek b, N.A. Kolchanov b,c, V.N. Parmon a,c
a
Boreskov Institute of Catalysis, pr. Lavrentieva 5, Novosibirsk 630090, Russia
b
Federal Research Center Institute of Cytology and Genetics of the Siberian Branch of the RAS, pr. Lavrentieva 10, Novosibirsk 630090, Russia
c
Novosibirsk State University, Pirogova str. 2, Novosibirsk 630090, Russia

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

Article history: Isolation of new microalgal strains with unique and valuable properties from diverse environmental conditions is
Received 3 March 2015 an important starting point for the establishment of high quality feedstock for biofuel production. In this study,
Received in revised form 20 July 2015 we have isolated twelve strains of microalgae, belonging to the genera Chlorella, Botryococcus, Scenedesmus,
Accepted 31 August 2015
Nannochloris and Bracteacoccus from samples obtained from various natural sources by flow cytometry. Five of
Available online xxxx
them were selected for further evaluation, as lipid producers on the basis of Nile Red fluorescent microscopy
Keywords:
screening. Analysis of the biomass in the exponential and stationary phases of growth showed that the strain
Microalgae Scenedesmus abundans A1175 has the highest specific growth rate (0.20 ± 0.01 d−1), biomass productivity
Biofuel (73.82 ± 4.53 mg L−1 d−1) and lipid content (44.4 ± 2.7%) in nitrogen depleted conditions. Strains Chlorella
Isolation of strains vulgaris A1123 and S. abundans A1175 have a high total content of saturated (SFA) and monounsaturated
Identification (MFA) fatty acids (67.0% and 72.8% respectively). S. abundans A1175 also had low polyunsaturated fatty acid
Fatty acid (PUFA) content that would allow for its use as a source of high quality biofuels.
Lipid © 2015 Elsevier B.V. All rights reserved.

1. Introduction standards EN 14214:2003 and ASTM D6751 is a low content of polyun-

Microalgae are widespread in nature, including soil, freshwater and
marine environments [1]. Microalgae biomass has been considered as a
promising source for production of motor fuels in comparison with
other sources of renewable biomass [2]. It was shown that oil produc-
tivity of microalgae is 100 times higher than that of some agricultural
crops, such as canola, soy and oil palm [3,4]. Currently, microalgal
biomass is used to produce third generation biofuels, biogas and
bioethanol, but is also considered as a source of other valuable compo-
nents: protein, polyunsaturated fatty acids, pigments, sugars and anti-
biotics [5].
High oil content of some microalgal strains makes them suitable for
biodiesel production. Microalgae belonging to such genera as Chlorella,
Dunaliella, Nannochloris, Nannochloropsis, Neochloris, Porphyridium, and
Scenedesmus contain 20–50% of lipids by weight, making them suitable
for biodiesel production [4]. Nevertheless, it was shown that strains
with high lipid content have a low biomass productivity that significant-
ly reduces their effectiveness as lipid producers [6]. In addition to high
lipid content, prospective microalgal strains need to have high growth
rates and a specific composition of produced lipids. An important cri-
terion for production of high quality biodiesel that meets biodiesel
⁎ Corresponding author.
E-mail address: piligaev@catalysis.ru (A.V. Piligaev).
saturated fatty acids in raw oils used a feedstock. Biofuels that contain
components with more saturated bonds are more resistant to oxidation,
and at combustion, glycerides remaining in the fuel are not polymer-
ized, which increases the reliability of diesel engines [7]. Thus, to find
a prospective microalgae strain for the production of high-quality biofu-
el, it is necessary to screen for natural habitat strains with a high content
of SFA and MFA with no additional requirements for expensive supple-
ments for fast growth (e.g. vitamins). A number of studies have shown
that the total content of SFA and MFA can reach high values (60–68% in
some strains like in Chlorella vulgaris ESP-31 [8]). However, features of
such strains remain poorly understood in terms of the physiology of
the accumulation and composition of lipids, and their applicability to
the production of valuable by-products, mainly protein and
carbohydrates.
In this study, we have isolated a number of microalgal strains from
various natural sources. The biomass accumulation, productivity, lipid
content and composition of the isolated strains were studied during
the exponential and stationary phases of autotrophic growth. Using
flow cytometry, a number of microalgal strains were isolated and iden-
tified. Five of them were selected through screening with Nile Red mi-
croscopy as lipid producers. Analysis of their productivity for biomass
and lipids, lipid accumulation and biochemical composition revealed
that two strains, S. abundans A1175 and C. vulgaris A-1123, have optimal
characteristics for production of high quality oil with high total content
of MSA and SFA.

http://dx.doi.org/10.1016/j.algal.2015.08.026
2211-9264/© 2015 Elsevier B.V. All rights reserved.
 A.V. Piligaev et al. / Algal Research 12 (2015) 368–376
2. Materials and methods
2.1. Isolation of microalgae by cell sorting with flow cytometry
Samples of soil and water were inoculated in 50 cm3 Bold's Basal Me-
dium (BBM) [9] and cultivated for two weeks at a temperature of 28 °C,
under a 16 h-light/8 h-dark cycle, and illumination of 60 μmol m−2 s−1.
Microalgae cells were separated from the contaminating microorgan-
isms by flow cytometry. Cell sorting was performed on BD FACSAria
flow cytometer, and a sapphire laser with a wavelength of 488 nm
was used for excitation of chlorophyll fluorescence. The fluorescence in-
tensity was determined by photodetector with a dichroic mirror 655LP
and a narrowband optical filter 695/40 nm. FACSFlow™ was used as a
compressing fluid in the sorting process. Before sorting, suspension
was purified from large aggregates, using cell strainers (35 μm, BD Fal-
con). Microalgae cell fraction had at least 6 × 104 cells that were plated
onto BBM agar and cultured as described until visible colonies were ob-
served. The purity of microalgal cultures was verified by microscopy
and by the absence of bacterial and fungal growth on LB and PDA agar
layered with diluted microalgae cultures from BBM agar plates.
2.2. Identification of the isolated strains of microalgae and
phylogenetic analysis
Genomic DNA from microalgal cultures was isolated using the Wiz-
ard SV Genomic DNA Purification System (Promega, USA) according to
the manufacturer's instructions. The partial sequence of the 18S rRNA
gene was amplified from the 50 ng genomic DNA by PCR by Taq poly-
merase using universal primers 5′-ACCTGGTTGATCCTGCCAGT-3′
(forward) and 5′-TCAGCCTTGCGACCATAC-3′ (reverse) [10], PCR was
performed under the following conditions: 1 cycle 95 °C — 3 min, 40 cy-
cles (95 °C — 30 s, 54 °C — 20 s, and 72 °C —1 min 30 s), 1 cycle — 72 °C
for 10 min and products were analyzed by electrophoresis. DNA se-
quencing was performed with BigDye® Terminator v3.1 Kit (Applied
Biosystems) and the nucleotide sequence was determined on ABI
3730XL sequencer (Applied Biosystems). The obtained sequences
were aligned with BLAST to sequences of microalgae 18 s rRNA from
GenBank database [11]. Multiple sequence alignment and phylogenetic
tree construction were performed using MEGA 5 [12]. The phylogenetic
tree was built using the Neighbor-Joining algorithm [13], and the statis-
tical reliability of its topology was determined by the Bootstrap test [14].
2.3. Preliminary screening for lipid production in microalgae
Equal amounts of cells from each microalgae isolates obtained dur-
ing the isolation were cultured in 24 well plates in 1.5 cm3 BBM-3 N me-
dium supplemented with 0.75 g L−1 NaNO3 under a 16 h-light/8 h-dark
cycle illumination at 60 μmol m−2 s−1 in 2% CO2 atmosphere for 7 days
and were agitated in an orbital shaker at 150 rpm. After incubation, cells
were harvested by centrifugation at 1000 g for 5 min, transferred to the
BBM medium without NaNO3, and similarly cultured in 24 well plates
for up to 10 days to trigger lipid accumulation in cells. During cultivation
in the BBM medium, the pattern of accumulation of lipids was observed
by fluorescent microscopy with Nile Red cell staining.
2.4. Cultivation of microalgae
A single colony of each strain cultivated on BBM agar was picked up
and inoculated into 50 cm3 Erlenmeyer flacks in 25 cm3 BBM media and
cultivated at 80 rpm agitation on an orbital shaker at 28 °C (without CO2
supplementation), at an illumination of 60 μmol m−2 s−1 under a 16 h-
light/8 h-dark cycle until the mid-exponential phase was reached. Cul-
ture growth was monitored by measuring the optical density (OD) at
680 nm using a UV–VIS spectrophotometer (Epoch, Biotek). Cells were
inoculated in 250 cm3 conical flasks, containing 100 cm3 of BBM at OD
369
0.08, and cultured at 150 rpm in an orbital shaker at 28 °C as described
until the stationary phase of growth was reached.
2.5. Determination of dry cell weight
The relationship between OD and dry cell weight (DW) was deter-
mined for each strain. During the exponential phase of growth, the OD
was measured at 680 nm, and 10 cm3 of liquid was withdrawn from cul-
ture (not less than 5 times at different time points in triplicate) and
transferred on a glass fiber filter, dried at 105 ºС overnight and weighed.
The relationship between OD and DW was determined through linear
regression and converted to g L−1 with R2 N 0.98.
2.6. Evaluation of growth rates and productivity parameters
Biomass productivity (BP) was calculated through the equation BP
(mg L−1 d−1) = (X2 − X1) · (t2 − t1)−1 where X1 and X2 were the bio-
mass dry weight concentrations (mg L−1) on days t1 (start of the expo-
nential phase) and t2 (end of the exponential phase), respectively.
Specific biomass growth rate (μ) was calculated in the exponential
growth phase by μ (d−1) = (lnX2 − lnX1) · (t2 − t1)−1 where X1 and
X2 are the lipid dry weight concentrations (mg L−1) on days t1 (start
of exponential growth) and t2 (end of exponential growth),
respectively.
Doubling time (DT) is defined as DT = ln2 · (μ) −1.
All experiments were carried out in triplicate, and data are expressed
as mean ± SD.
2.7. Fluorescent microscopy of cells after Nile Red staining
Fluorescent staining of 1 cm3 of microalgal cell suspension (105–106
cells) was performed using 0.05 cm3 of the Nile Red solution in acetone
(0.1 mg cm−3). The mixture was incubated for 10 min at 37 °C in dark-
ness. The fluorescence of the cells of stained and control samples of
microalgae were observed on Axioskop 2 Plus (Carl Zeiss) in the Inter-
institutional Shared Center for Microscopic Analysis of Biological
Objects SB RAS. Zeiss filter sets FS10 (excitation bandpass, 450 to
490 nm; emission bandpass, 515 to 565 nm) and FS20 (excitation
bandpass, 546/12 nm; emission bandpass, 575 to 640 nm) were used
for fluorescence detection. Images were taken with Axiovision imaging
v4.6. Acquisition and processing of data was done using the Axiovision
v4.6 software.
2.8. Determination of Nile Red fluorescence
Fluorescent staining of neutral lipids of microalgae was performed
using the Nile Red dye. For this purpose, 0.025 cm3 of Nile Red in ace-
tone (0.1 mg cm−3) was added to 0.5 cm3 of the cell suspension con-
taining 20% DMSO, with the concentration of cells normalized at OD
0.2 (680 nm). The mixture was incubated for 10 min at 37 °C in dark-
ness. Analysis was performed with Perkin-Elmer EnVision2103
Multilabel Reader using 525/579 excitation/emission filters. The relative
fluorescence intensities were obtained by subtracting of both the auto-
fluorescence of unstained algal cells and fluorescence intensity of Nile
Red of blank medium containing DMSO and Nile Red Dye from the fluo-
rescence intensities measured in the stained cells.
2.9. Analysis of nitrate concentration in medium
Nitrate concentration during the cultivation of microalgal strains in
medium was measured as described in [15]. Samples of cultural liquid
were centrifuged at 3000 g for 5 min. The supernatant was collected
and the absorbance was measured at 220 nm. Sodium nitrate solution
with concentrations of up to 500 μM were used as a standard in all
measurements.
370 A.V. Piligaev et al. / Algal Research 12 (2015) 368–376
2.10. Analysis of total lipid content and productivity
For lipid analysis, cells were pelleted by centrifugation at 3000 g for
10 min, washed twice with milli-Q water and then lyophilized in a
freeze drier (FreeZone, Labconco) and weighed. Dry biomass was used
for experiments and all measurements were done in triplicate. For
lipid extraction up to 100 mg lyophilized algae biomass was dissolved
in 2 cm3 of chloroform:methanol (2:1), and sonicated for 2 min at
130 W with CPX 130 Ultrasonic Processor (Cole Parmer, USA). The mix-
ture was further added with 0.25 volume of 0.9% NaCl solution and
stirred vigorously. After phase layering, the organic phase was evaporat-
ed in a stream of nitrogen, and the sample was weighed. Lipid content
was determined as a ratio of weight of lipid extract to weight of dry bio-
mass. Lipid extracts were also used for the analysis of fatty acids and
lipid composition.
Lipid productivity was calculated as (mg L−1 d− 1) = (X2 −
X1) · (t2 − t1)−1 where X1 and X2 were the lipid dry weight concentra-
tions (mg L−1) for days t1 (start of the exponential growth phase) and t2
(end of the exponential growth phase), respectively. Experiments were
performed in triplicate, data are shown as mean ± SD.
2.11. TLC of microalgal lipid extract
To analyze the lipid composition in microalgal biomass, thin layer
chromatography (TLC) was performed on TLC silica gel 60G plates
(Merck). 0.003 cm3 of extracted lipid solution (10 mg in 1 cm3 of chlo-
roform) was layered on 10 × 15 cm plates, air-dryed and separated
using hexane-diethyl ether-acetic acid (80:20:1) as an eluent mixture.
A mixture (10 mg cm−3) of lipids, including a mono-, di-, and triglycer-
ide mix (Sigma) and oleic acid (Sigma) was used as a standard. Spots
were detected after spraying the plate with 1% solution of phospho-
molybdic acid in ethanol and heating at 120 °C for 15 min.
2.12. GC–MS analysis of FAME
To analyze the composition of fatty acids in the microalgal oil, the
lipids were esterified with methanol to obtain fatty acid methyl esters
(FAME) [16]. To avoid the oxidation of unsaturated fatty acids, all sam-
ples were processed under a nitrogen atmosphere. For this reaction, up
to 2 mg of lipids was added to 0.3 cm3 of a 2% H2SO4 in methanol solu-
tion and incubated for 1.5 h at 80 °C. 50 μg of heptadecanoic acid C17:0
(Sigma) was used as an internal standard for esterification and added to
the mixture prior to reaction. 0.3 cm3 of 0.9% (w/v) NaCl and 0.3 cm3 of
hexane were then added, vortexed and separated by centrifugation at
3000 g for 5 min. The upper hexane layer was used for GC–MS analysis.
The composition of fatty acid methyl esters was analyzed by GC–MS
on Agilent 7000B with electron impact ionization (70 eV) on a ZB-WAX
column (30 m × 0.25 mm ∗ 0.25 μm), with a temperature gradient from
100 °C to 260 °C at an increase rate of 12 °C min−1. Injector temperature
was set at 260 °C. Carrier gas (helium) flow was set to 1.2 cm3 min−1.
Peaks of fatty acids were identified by using the NIST'11 library, and
the relative amount of individual fatty acid was calculated by the inte-
grated area percentage from the total amount of fatty acids. Experi-
ments were carried out in triplicate, and the data are expressed as
mean ± SD.
2.13. Determination of protein content
For protein extraction, 10 mg of lyophilized biomass was added to
10 cm3 of 0.5 M solution of NaOH and incubated for 10 min at 80 °C,
and then centrifuged for 5 min at 14,000 g. Protein content in the ob-
tained extract was examined with the Quick Start kit (Bio-Rad) accord-
ing to the manufacturer instructions and using BSA as a standard.
Experiments were performed in triplicate, and the data are expressed
as mean ± SD.
2.14. Determination of carbohydrate content
The carbohydrate content of the microalgal biomass was deter-
mined with the phenol-sulfuric acid method [17]. For this purpose,
10 mg of lyophilized algae was added to 10 cm 3 of water. After
this, a 1 cm3 aliquot of sample was added to 3 cm3 of sulfuric acid
and 1 cm 3 of 5% aqueous solution of phenol, and the mixture was
stirred and incubated for 5 min at 90 °C. The amount of carbo-
hydrates was determined using a wavelength of 488 nm against a
calibration curve based off of a known concentration of glucose. Ex-
periments were carried out in triplicate, and the data are expressed
as mean ± SD.
3. Results and discussion
3.1. Isolation of microalgae with flow cytometry
In this study, more than 100 environmental samples were cultivated
from different sources (the salt lakes of Novosibirsk and Altai regions,
the anthropogenic ecosystems of Kemerovo oblast (Western Siberia),
and the terrestrial hot springs in the Uzon Caldera and Valley of Geysers
(Kamchatka) for the isolation and screening of microalgae strains. Only
49 of the samples revealed growth of microalgaе and were used in our
work to obtain microalgae cultures.
In previous studies, it was shown [18–20] that flow cytometry can be
successfully applied to reduce contamination of the isolated microalgae
cultures by extraneous microflora through the separation of subpopula-
tions of cells in the suspension. In our study, for the detection of
microalgae cells during sorting, we measured the following parameters:
the red fluorescence of chlorophyll а (N650 nm) and forward light scat-
tering, which indicates cell size. Fig. 1 shows typical cytograms of the
microalgae culture obtained from natural samples before and after
sorting.
During cell sorting, we observed two or more different cell
clusters on the cytogram. Clusters with the highest intensity of fluo-
rescence and the highest forward scattering corresponed to
microalgae cells, while clusters with low intensity of fluorescence
and low scattering probably contained non-photosynthetic cells
that may be normally present in the natural samples. Red color (re-
gion P1) in the Fig. 1 (a and b) shows the fraction with the highest
forward scattering and absorption in the area of 695 nm, which is
characteristic of microalgae cells. After flow sorting, 12 unique
microalgal strains were isolated and maintained as the axenic
cultures.
3.2. Taxonomic identification of microalgae
According to 18S rRNA analysis, the isolated microalgal strains be-
long to the genera Chlorella, Botryococcus, Scenedesmus, Nannochloris
and Bracteacoccus with more than 99% of homology (Fig. 2).
It was found that the isolated strains A1123, A1176, A1182 and
A1135 formed a single closely related cluster and had 99% homology
with the strains of C. vulgaris FR865683.1, Chlorella sp. AB713411.1,
Chlorella sp. AY195981.1 and Chlorella sorokiniana AB731602.1, respec-
tively. Strains A1139 and A1144 had 99% homology with the strains
Nannochloris bacillaris AB080300.1 and Bracteacoccus sp. JQ259951.1,
respectively. Strains A1175 and A1167 showed 99% and 100% degree
of similarity of genetic sequences with the strains of S. abundans
X73995.1 and Scenedesmus obliquus FR865738.1 respectively. Strains
A1115, A1138, A1113 and A1162 form a single cluster, belonging to
the genus Botryococcus. Strains A1115 and A1138 had 100% and 99%
homology with the strain Botryococcus sp. GU250969.1, and strains
A1113 and A1162 had 99% homology with the strain of Botryococcus
sp. AJ581914.1.
 A.V. Piligaev et al. / Algal Research 12 (2015) 368–376
Fig. 1. Cytograms of a highly contaminated sample of microalgal cuture before (a) and after (b) cell sorting. The X axis (PerCP-A) corresponds to the fluorescence channel 695/40 nm, and
axis Y (FSC-H) — with forward light scattering. The region P1 corresponds to the signal from microalgae fraction.
3.3. Screening of microalgal stains with high lipid content
To screen for prospective microalgal strains with high lipid content,
the accumulation of lipids in the microalgae cells under nitrogen-de-
pleted conditions for up to 10 days was observed using staining with
Nile Red. All of the isolated strains under study produced lipids after in-
duced starvation, but had different patterns of accumulation of lipid
droplets. For example, the majority of the cells in the cultures of
Scenedesmus obliquus A-1167, Botryococcus sp. A-1162 and S. abundans
A-1175 had synchronic manners of accumulation of lipid droplets.
Other strains visually had fewer lipid-producing cells (approximately
25%), except for the strains C. vulgaris A-1123 and C. sorokiniana A-
1135. More than 50% of the cells in these cultures contained visible
droplets. Based on this observation, we have selected strains
S. abundans A-1175, S. obliquus A-1167, Botryococcus sp. A-1162,
C. sorokiniana A-1135 and C. vulgaris A-1123 for further analysis.
Fig. 2. Phylogenetic analysis of the isolated strains of microalgae. Numbers represent the
results of 500 bootstrap replicates.
371
3.4. Cultivation of the selected microalgal strains
The most important parameters in the screening of microalgal
strains for biodiesel production are high lipid content and biomass pro-
ductivity [21]. Selected strains were cultured up to 26 days on BBM, and
the following parameters were evaluated: accumulation of lipid drop-
lets in cells stained with Nile Red and analyzed with fluorescent micros-
copy (Fig. 3), dry weight of the biomass (Fig. 4), nitrogen concentration
in the medium (Fig. 5) and the dynamics of lipid accumulation shown
by Nile Red fluorescence (Fig. 6). Growth rates and productivity param-
eters of the selected strains are presented in Table 1.
As shown in Fig. 4, the stationary phase of growth is achieved by
strain S. abundans A1175 on 19th day, and this timepoint correlates
with the total depletion of nitrogen (Fig. 5). This strain differs from
the others because of its coordinated accumulation of biomass and
lipids, as was discovered through the growth dynamics of biomass
and Nile Red fluorescence intensity (Figs. 4 and 6). In addition, this
strain has the highest specific growth rate of 0.20 ± 0.01 d−1 and bio-
mass productivity 73.82 ± 4.53 mg L−1 d−1, and, thus, the shortest dou-
bling time of 3.6 ± 0.5. Fluorescent microscopy analysis revealed the
formation of large oil droplets in the stationary phase of growth for
this strain (Fig. 3, BF2) on the 19th day. Also, in stationary phase of
growth, this strain had a higher lipid content (44.4 ± 2.7%) than
Botryococcus sp. A1162 (33.4 ± 2.3%) on 20th day, C. vulgaris A1123
(38.3 ± 1.0%) on 26th day, and this strain also had a lipid productivity
of 32.8 ± 1.5 mg L− 1d− 1. It exceeds the similar values from other
strains significantly; specifically, lipid production in S. abundans A1175
is 2.4 times greater than that of C. vulgaris A1123 and 3.3 times greater
than that of Botryococcus sp. A1162. Thus, S. abundans A1175 has the
greatest ability to produce biomass and lipids in the stationary phase
of growth.
Strains C. vulgaris A1123 and S. obliquus A1167 exhibited a relatively
high lipid content (38.3 ± 1.0% and 41.2 ± 2.2% respectively), that was
also confirmed with fluorescent microscopy (Fig. 3, BF1 and BF4). Nev-
ertheless, compared to S. abundans A1175, both strains have delayed
lipid accumulation as compared to the growth of biomass in the expo-
nential phase (Fig. 6), C. sorokiniana A1135, among the other strains
under study, has the lowest biomass (10.60 ± 0.34 mg L−1 d−1) and
lipid productivity (3.3 ± 0.1 mg L−1 d−1), accompanied by a low lipid
content (31.2 ± 3.4%) and rare oil droplet formation in cells (Fig. 6).
Low lipid productivity for this strain is also correlated with high residual
nitrogen content in the medium (0.08 g L−1) in the stationary phase of
growth (Fig. 5).
372 A.V. Piligaev et al. / Algal Research 12 (2015) 368–376

Fig. 3. Transmitted light and fluorescent images of microalgae stained with Nile Red in exponential and stationary phase of growth. Scale bars show relative size of cells. F — fluorescent
image; T — transmitted light; A — exponential phase; B — stationary phase. 1 — Chlorella vulgaris A1123 (26th day); 2 — Scenedesmus abundans A1175 (19th day); 3 — Chlorella sorokiniana
A1135 (25th day); 4 — Scenedesmus obliquus A1167 (25th day); 5 — Botryococcus sp. A1162 (20th day).

3.5. Analysis of the composition of lipids by TLC
The composition lipid extracts from microalgae was studied in the
exponential and stationary growth phases through TLC (Fig. 7).
The stationary phase was indicated by the presence of triglycerides
that were mostly absent in the exponential phase in all strains except
for C. sorokiniana A1135, which has the lowest amount of lipids station-
ary phase (31.2 ± 3.4%) and the fewest lipid droplets in cells (Fig. 3,
BF3). It can also be noted that C. vulgaris A1123 and S. abundans
A1175 showed apparent increases in triglyceride content in the station-
ary phase compared with the other strains.
3.6. Analysis of fatty acid composition of the biomass of microalgae at
different stages of growth
An important parameter that determines the quality of biodiesel is
its fatty acid composition. The structure of the fatty acids affects key
properties of biodiesel, especially its cetane number, flash point, viscos-
ity, oxidation stability, and fluidity at low temperature [3,22]. In this re-
gard, a raw material for biofuel production has to meet a number of
requirements. For example, quality oil shall contain high levels of satu-
rated fatty acids and low levels of polyunsaturated ones (with the num-
ber of double bonds being greater than four) [23]. Polyunsaturated fatty
 A.V. Piligaev et al. / Algal Research 12 (2015) 368–376 373

Fig. 4. DW of microalgal strains during cultivation.

acids are susceptible to polymerization, and, as such, resins can form
through oxidation during storage or through oxidative and thermal po-
lymerization at higher temperatures and pressures during combustion.
In addition, biofuels must have a sufficiently high level of saturated fatty
acids to increase the cetane number and, at the same time, a sufficient
level of monounsaturated fatty acids. It is shown that palmitoleic, as
well as oleic acid, significantly enhances cold flow properties of biofuels
[24].
Compared to oils obtained from soybeans and canola, oil produced
by microalgae has a higher content of fully saturated fatty acids:
palmitic C16:0 and stearic C18:0. Soyabean oil and rapeseed oil are char-
acterized by a high content of linoleic С18:2 and oleic acid С18:1 [25].
For the accurate determination of unsaturated fatty acids in our
microalgae cultures, we used GC–MS. All manipulations with lipid ex-
tracts were conducted under a nitrogen atmosphere. All strains had a
tendency to change fatty acid composition during cultivation
(Table 2). For example, in the exponential and stationary phases of
growth. High levels of palmitic С16:0, stearic С18:0 and oleic С18:1
acids were observed. However, in the stationary phase, a significant de-
crease in the content of stearic С18:0 acid was observed in C. vulgaris
A1123 (27.8 ± 4.9%), S. obliquus A1167 (19.7 ± 0.7%) and Botryococcus
sp. A1162 (20.2 ± 1.5%).
The amount of oleic acid С18:1 remained stable in all strains in both
growth phases, except the strains C. vulgaris A1123 and Botryococcus sp.
A1162 showed a nearly twofold increase in C18:1 levels during the sta-
tionary phase. The amount of n-2 unsaturated fatty acid biomass was
significantly different among different strains: in exponential phase,
the linoleic acid С18:2 content of C. vulgaris A1123 was 9.5 ± 3.1%,
S. obliquus A1167, 3.9 ± 0.6% and Botryococcus sp. A1162, 13.1 ± 0.2%.
During the stationary phase, these values increased by almost two

Fig. 5. Nitrogen concentration during cultivation of microalgae.

374 A.V. Piligaev et al. / Algal Research 12 (2015) 368–376
Fig. 6. Nile Red fluorescence intensity of stained microalgae cells.
times, up to 21.5 ± 3.4%, 6.9 ± 0.8 and 20.2 ± 4.7%, respectively. The
highest content of n-3 PUFA, such as hexadecatrienoic acid С16:3, was
in C. sorokiniana A1135, which had levels of 7.8 ± 0.9% in the exponen-
tial phase and 6.8 ± 0.3% in the stationary phase. Linolenic acid С18:3
content was the highest in the exponential and stationary phases of
C. sorokiniana A1135 (14.0 ± 4.0% and 13.8 ± 0.8% respectively) and
S. obliquus A1167 (13.4 ± 3.2% and 20.5 ± 2.9 respectively). In addition,
high hexadecatetraenoic acid С16:4 levels were found in S. abundans
A1175 (3.6 ± 0.7%) and S. obliquus A1167 (7.5 ± 1.7%) in the exponen-
tial phases, which significantly increased during nitrogen depletion only
for S. obliquus A1167 (to 10.5 ± 0.6%). The latter strain also had a rela-
tively high content of octadecatetraenoic acid С18:4 (2.5 ± 0.4%) during
nitrogen depletion. Thus, the total content of PUFA was maximal in the
strain C. sorokiniana A1135 (33.4%) in the stationary phase of growth,
and minimal in Botryococcus sp. A1162 (22.1%).
The highest total content of SFA in all strains during nitrogen
starvation was found in C. vulgaris A1123, S. abundans A1175 and
C. sorokiniana A1135, which had levels of 47.6, 51.3 and 46.7% of the
total fatty acid content, respectively. These strains also had very similar
total amounts of unsaturated fatty acids (UFA): 52.4, 48.7 and 53.3%, re-
spectively. Nevertheless, during nitrogen depletion, the biggest total
amounts of SFA and MFA, accompanied by a high specific growth rate
in nitrogen replete conditions, were found in C. vulgaris A1123 (67.0%)
and S. abundans A1175 (72.8%). Another feature of the S. abundans
A1175 was a relatively constant level of production of SFA, MFA and
PUFA throughout the cultivation, while the majority of other strains
had significant differences in the number of saturated bonds in the
fatty acids, depending on growth phase. S. abundans A1175, when
Table 1
Analysis of the properties of microalgal strains under study.
Strain μ, d−1 DT
Chlorella vulgaris A1123 0.09 ± 0.01 8.0 ± 0.7
Scenedesmus abundans A1175 0.20 ± 0.01 3.6 ± 0.5
Chlorella sorokiniana A1135 0.05 ± 0.01 13.5 ± 0.1
Scenedesmus obliquus A1167 0.07 ± 0.01 9.5 ± 1.0
Botryococcus sp. A1162 0.11 ± 0.01 6.1 ± 0.5
compared with all other strains, showed lower levels of n − 2 and
n−3 PUFA.
These results are consistent with other studies. For example, C.
vulgaris A1123 also had a higher content of saturated fatty acids than
C. vulgaris YSL04 [26] and Chlorella sp. BR2 [27]. C. vulgaris A1123 also
had comparable biomass productivity to the values for strain C. vulgaris
259 that were published in the work [28]. S. abundans A1175, isolated in
this work, has a little bit smaller lipid productivity and content than the
strain S. abundans from [29]. This can be partly explained by its culti-
vation in another medium. Results on the total content of MFA and
SFA obtained for S. abundans A1175 in this study are comparable to
the data from [29], which also investigated the composition of
fatty acids of S. abundans, which showed comparable results for
total amounts of MFA and SFA (76%) and linolenic acid C18:3.
Nevertheless, it is worth noting the presence of hexadecatetraenoic
acid С16:4 (3.9 ± 0.5%) during nitrogen depletion in a
biomass of S. abundans A1175 and also some minor amounts of
octadecatetraenoic acid С18:4 (0.3 ± 0.0%), undetected by the re-
sults GC–MS analysis in study [29].
3.7. Protein and carbohydrate content
Microalgae are also considered as a valuable stock for the industry,
as some strains have high protein and carbohydrate content [30,31].
Therefore, to evaluate the applicability of the strains C. vulgaris A-1123
and S. abundans A1175 for the production of protein and carbohydrates
as a byproduct of their biomass processing, we have performed
Biomass productivity, Lipid productivity, mg L−1 d−1 Lipid content, %
mg L−1 d−1
35.8 ± 0.24 13.7 ± 0.2 38.3 ± 1.0
73.82 ± 4.53 32.8 ± 1.5 44.4 ± 2.7
10.60 ± 0.34 3.3 ± 0.1 31.2 ± 3.4
20.85 ± 0.71 8.6 ± 0.7 41.2 ± 2.2
29.61 ± 1.46 9.9 ± 0.3 33.4 ± 2.3
 A.V. Piligaev et al. / Algal Research 12 (2015) 368–376 375

used in [35], which was 51.0 ± 0.7% carbohydrate. At the same time,
S. abundans A1175 had a carbohydrate content of 43.3 ± 1.2%, which
exceeded the corresponding data for Scenedesmus sp. CCNM 1077 [34].
Thus, based on these data, C. vulgaris A-1123, during nitrogen starvation,
exceeds S. abundans A1175 in protein content but has a comparable
amounts of carbohydrates. Thus, both of these strains can be used as a
source of carbohydrates, which can then be converted into valuable
products, e.g., ethanol [36].

4. Conclusions

In this work, we have studied the properties of a number of strains

Fig. 7. TLC analysis of the lipid extracts of microalgal strains in exponential and stationary
growth phases. A — exponential phase of growth; B — stationary phase of growth; 1 and 6
— Chlorella vulgaris A1123; 2 and 7 — Scenedesmus abundans A1175; 3 and 8 — Chlorella
sorokiniana A1135; 4 and 9 —Scenedesmus obliquus A1167; 5 and 10 — Botryococcus sp.
A1162.
additional analysis of biomass composition, namely the content of car-
bohydrates and protein in nitrogen depleted conditions.
It has been shown that the protein content in the biomass of strain C.
vulgaris A-1123 in the stationary phase of growth was 26.0 ± 1.2%. This
is lower than the other strains. For example, C. vulgaris [32] contains
47.82 ± 0.05% of protein. Protein content of S. abundans A1175 was
15.7 ± 0.7%, which was significantly lower than other closely related
species, e.g. Scenedesmus sp. NT1d (33.08%) and Scenedesmus dimorphus
NT8c (22.66%) [33]. In addition, in the work [34], it has been shown that
the complete absence of nitrogen in the medium results in reduced pro-
duction of protein by microalgae Scenedesmus sp. CCNM 1077, from a
biomass percentage of 47.75 to 16.87. Thus, the low protein content
compared with other strains may be explained by their individual char-
acteristics under nitrogen starvation.
The carbohydrate content in the stationary phase for C. vulgaris A-
1123 was 37.8 ± 1.4%. This is higher than that in C. vulgaris [32],
which was 8.08 ± 0.09% carbohydrate, but less than C. vulgaris FSP-E
isolated from different climatic conditions, all of which were easily
maintained as a pure cultures in our laboratory conditions. Isolated
strains belonged to genera Chlorella, Botryococcus and Scenedesmus, all
of which are widespread in nature. It was shown that the microalgal
strains have different fatty acid compositions in various stages of
growth, and, generally, the amount of SFA decreased in the stationary
phase, excluding strain S. abundans A1175, which had a relatively con-
stant composition of SFA, MFA and PUFA. It was shown that the proper-
ties of S. abundans A1175 significantly surpassed the properties of other
strains of microalgae isolated in this work, with regard to biofuel appli-
cations. This strain has a high growth rate and high lipid (including tri-
glycerides) content by biomass (44.4 ± 2.7%) with 72.8% SFA and MFA.
It is worth noting that the use of S. abundans A1175 for biofuel produc-
tion is preferable because of its relatively low content of PUFA, which is
strongly regulated by biofuel standards. However, another strain, C.
vulgaris A1123, also had a high content of SFA and MFA (67.0%), but it
also had a specific growth rate of two times less than S. abundans
A1175. C. vulgaris A1123 had higher protein content during nitrogen
starvation than S. abundans A1175, while the amounts of carbohydrate
content was comparable in both strains. Thus, S. abundans A1175 is a
promising strain for producing high-quality biodiesel that meets mod-
ern industrial standards.
Acknowledgment
This work was done by the financial support of the Russian
Foundation for Basic Research (project no. 14-08-31589 mol_a).

Table 2
FAME analysis with GC–MSa. Given values are expressed in % as mean ± standard deviation.*a

Scenedesmus abundans
Chlorella vulgaris A1123 Chlorella sorokiniana Scenedesmus obliquus Botryococcus sp. A1162
FA A1175
A1135 A1167

Exp St Exp St Exp St Exp St Exp St

C14:0 1.2 ± 0.1 1.1 ± 0.4 1.0 ± 0.1 1.5 ± 0.2 1.2 ± 0 1.2 ± 0.2 1.2 ± 0.2 1.1 ± 0.1 1.5 ± 0.1 1.4 ± 0
C16:0 18.7 ± 1.6 18.6 ± 1.8 17.5 ± 0.5 22.0 ± 5.1 16.1 ± 0.6 16.0 ± 3.1 17.2 ± 0.5 14.3 ± 0.9 16.0 ± 0.6 14.4 ± 0.7
C16:1 1.0 ± 0.2 1.3 ± 0.1 1.5 ± 0.3 1.5 ± 0.3 2.1 ± 0.5 2.4 ± 0.2 1.8 ± 0.2 2.8 ± 0.2 3.8 ± 0.3 4.5 ± 0.9
C16:2 3.1 ± 0.7 5.6 ± 0.4 1.3 ± 0.1 2.2 ± 0.4 3.2 ± 0.8 4.0 ± 0.4 0.7 ± 0.1 0.7 ± 0.1 7.0 ± 0.5 8.9 ± 0.1
C16:3 1.1 ± 0.2 1.7 ± 0.3 1.6 ± 0.0 2.3 ± 0.4 7.8 ± 0.9 6.8 ± 0.3 0.8 ± 0.1 1.3 ± 0.0 0.9 ± 0.1 1.4 ± 0.3
C16:4 0.5 ± 0.6 0.5 ± 0.0 3.6 ± 0.7 3.9 ± 0.5 0.8 ± 0.2 0.6 ± 0.1 7.5 ± 1.7 10.5 ± 0.6 2.1 ± 0.1 2.5 ± 0.1
C18:0 42.3 ± 3.8 27.8 ± 4.9 36.5 ± 4.8 27.5 ± 0.1 35.6 ± 5.3 29.3 ± 4.6 33.8 ± 4.6 19.7 ± 0.7 32.1 ± 1.6 20.2 ± 1.5
C18:1 20.0 ± 4.6 17.7 ± 0.6 23 ± 3.5 18.2 ± 3.6 13.2 ± 0.5 16.8 ± 2.7 16.6 ± 1.4 18.3 ± 4.5 18.3 ± 3.9 18.4 ± 0.9
C18:2 9.5 ± 3.1 21.5 ± 3.4 8.0 ± 1.6 11.8 ± 2.4 6.0 ± 0.7 8.2 ± 1.8 3.9 ± 0.6 6.9 ± 0.8 13.1 ± 0.2 20.2 ± 4.7
C18:3 2.3 ± 0.5 3.3 ± 0.2 6.1 ± 1.4 6.6 ± 1.1 14.0 ± 4.0 13.8 ± 0.8 13.4 ± 3.2 20.5 ± 2.9 5.3 ± 1.2 6.6 ± 1.6
C18:4 – 0.4 ± 0.0 – 0.3 ± 0.0 – – 1.4 ± 0.8 2.5 ± 0.4 – 0.9 ± 0.2
C20:0 – 0.1 ± 0.0 – 0.3 ± 0.0 – 0.2 ± 0.0 – – – –
C20:1 0.3 ± 0.0 0.5 ± 0.1 – 1.8 ± 0.1 – 0.8 ± 0.1 1.7 ± 0.5 1.2 ± 0.2 – 0.7 ± 0.1
SFA 62.2 47.6 55.0 51.3 52.9 46.7 52.2 35.2 49.6 36.0
UFA 37.8 52.4 45.0 48.7 47.1 53.3 47.8 64.8 50.4 64.0
MFA 21.3 19.5 24.5 21.5 15.3 20.0 20.0 22.3 22.0 23.5
PUFA 23.1 33.0 25.0 27.1 26.4 33.4 23.7 23.7 42.4 22.1
SFA +
83.5 67.0 79.4 72.8 68.3 66.6 72.2 57.7 71.7 59.6
MFA
a
Exp — exponential phase of growth; St — stationary phase of growth; FA — fatty acids; UFA — unsaturated FA
376 A.V. Piligaev et al. / Algal Research 12 (2015) 368–376
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