CHEMICAL ENGINEERING TRANSACTIONS

VOL. 79, 2020

A publication of

The Italian Association

of Chemical Engineering
Online at www.cetjournal.it

Guest Editors: Enrico Bardone, Antonio Marzocchella, Marco Bravi
Copyright © 2020, AIDIC Servizi S.r.l.
ISBN 978-88-95608-77-8; ISSN 2283-9216

Research into the Influence of Cultivation Conditions on the
Fatty Acid Composition of Lipids of Chlorella Vulgaris

Microalgae

Dmitry Dvoretsky*, Stanislav Dvoretsky, Mikhail Temnov, Evgeny Akulinin,
Ilya Markin, Yana Ustinskaya, Maria Yeskova, Kirill Meronyuk

Тambov State Technical University, ul. Sovetskaya 106, Tambov 392000, Russia
dvoretsky@yahoo.com

The effect of temperature and illumination intensity on the biomass growth of Chlorella vulgaris Beijer IPPAS
C-2 microalgae cells, lipid biosynthesis and their fatty acid composition was studied experimentally. It has
been established that temperature and photosynthetically active radiation (PAR) have a significant influence
on the kinetics of microalgae biomass growth, biosynthesis of lipids and their fatty acid composition.
Cultivation with increased levels of PAR and temperature leads to intensive cell division, which can be useful
for seed preparation. At lower PAR levels and temperatures, the strain accumulates the maximum amount of
biomass, lipids, saturated and unsaturated fatty acids. The analysis of fatty acid composition has shown that
this strain may be promising in the technology of producing raw materials for biofuel production and food
additives.

1. Introduction
Sustainable use of natural resources and production of minimal amount of environmentally harmful by-
products has been on the agenda of many researchers worldwide. Technologies of complex use of microalgae
for obtaining food ingredients, wastewater treatment, production of raw materials for biofuel, etc. have the
potential for wide dissemination in the closed-loop economy. One of the key advantages of microalgae is their
"flexibility" in terms of the composition and quality of the components they synthesize - the chemical
composition of the biomass and the productivity of the microalgae strain can vary widely depending on the
conditions of cultivation (Barghbani et al., 2012; Blair et al., 2014). Despite the plethora of studies (Ma et al.,
2018; Chinnasamy et al., 2009) on microalgae cultivation regimes, their diversity and the peculiarities of the
biosynthesis of external and internal metabolites existing in each strain, enables us to look for new
approaches to their sustainable production. At the same time, one of the problems of rapid and reliable
determination of optimal cultivation regimes is related to the fact that at the moment there is no clear
understanding of the links between process conditions of microalgae cultivation and the intensity of
biochemical and chemical reactions in the cell.
One of the promising microalgae species for industrial cultivation is Chlorella vulgaris, because it has a high
growth rate, the ability to respond to different conditions of cultivation by changing the content of nutrients, and
it is resistant to infection. Chlorella vulgaris microalgae cells contain up to 50 % of lipids - including saturated
and unsaturated fatty acids used in the production of biofuels and nutritional supplements (ω-3 fatty acids),
respectively (Araujo et al., 2013).
The analysis of the processes taking place in the Chlorella vulgaris microalgae cell (Figure 1) (Salati and
Goodridge, 1996) shows that the main parameters in microalgae cultivation are the intensity of illumination
and culturing temperature (Mayo, 1997; Gong et al., 2014). The temperature and illumination have a critical
influence on the amount of the intermediate product, acetyl-CoA, which is the starting substance for the
biosynthesis of lipids and fatty acids in the microalgae cell. In addition, the temperature influences membrane
fluidity, cytoplasm viscosity and, as a consequence, the rate of reactions taking place in the cell. As a result of
biochemical reactions from acetyl-CoA, saturated and unsaturated fatty acids are synthesized. The resulting

DOI: 10.3303/CET2079006

Paper Received: 11 October 2019; Revised: 19 January 2020; Accepted: 17 February 2020
Please cite this article as: Dvoretsky D., Dvoretsky S., Temnov M., Akulinin E., Markin I., Ustinskaya Y., Yeskova M., Meronyuk K., 2020,
Research Into the Influence of Cultivation Conditions on the Fatty Acid Composition of Lipids of Chlorella Vulgaris Microalgae, Chemical
Engineering Transactions, 79, 31-36 DOI:10.3303/CET2079006

acids form molecules of saturated and unsaturated triglycerides. At a higher temperature, there is a decrease
in the proportion of unsaturated fatty acids as a result of their predominant oxidation and detachment. Under
these conditions there is a decrease in the functional activity of photosystems and ATP-synthesizing complex,
which leads to a slowdown in cell growth. At the same time, desaturase genes, which catalyze the formation of
double bonds in fatty acids, are also expressed (Los, 2014).

Acetyl-CoA

Malonyl-CoA

Malonyl-ACP

3 Ketoacyl-ACP

Acyl-ACP 3 Hydroxyacyl-
ACP

Trans Enoyl-ACP

Saturated fatty acid

Unsaturated fatty acid

Glycerol-3-phospate

Lysophosphatidic acid

Phosphatidic acid

Diacylglycerol

Triacylglycerol

Fatty acid-CoA Acyl-CoA

Triacylglycerides

Fatty acid
synthesis

PLASTID

CYTOSOL

Temperature
Illuminance

Endoplasmic reticulumСhloroplast

Figure 1: Biosynthesis of lipids in a Chlorella vulgaris cell

The increase in the level of illumination and temperature to some critical level has a positive effect on biomass
accumulation (Takeshita et al., 2014). At low cultivation temperatures and illumination levels, metabolic
pathways shift towards lipid accumulation (in particular, with unsaturated fatty acids). At the same time, the
mass fraction of lipids in the cell is less affected by temperature than the illumination factor (Villarruel-López et
al., 2017).
Converti et al. (2009) and Khoeyi et al. (2012) have found that the metabolism shifted to the synthesis of
saturated fatty acids with a threefold increase in the intensity of illumination, while the amount of
monounsaturated and polyunsaturated fatty acids decreased with an increase in illumination and the duration
of the light phase. The maximum percentage of monounsaturated and polyunsaturated fatty acids (of the total
amount of fatty acids): 15.93 - 27.40 % - was recorded with a 1.5 times decrease in the intensity of
illumination.
The effect of light on the fatty acid content of Chlorella vulgaris biomass was studied in Seyfabadi et al.
(2011), Dickinson et al. (2017), Kanokwan Chankhong et al. (2018). It was found that the total content of
saturated fatty acids increases, while monounsaturated and polyunsaturated fatty acids decrease with
increasing light intensity and duration of the light phase of the lighting period.
Changes in the composition of fatty acids are observed when the culturing temperature changes: as the
temperature decreases, the amount of unsaturated fatty acids in lipids increases. Rohit and Venkata Rohan
(2018) established that lipid content in microalgae depends on temperature. With a 5 °C increase in
temperature, the lipid content in the Chlorella vulgaris biomass decreases by 40 %.
The analysis of the sources allows to conclude that the peculiarities of the microalgae lipid metabolism
pathway of the Chlorella vulgaris species, as well as the influence of cultivation temperature and the level of
illumination on the fatty acid composition of microalgae lipids, have not been fully studied.
Therefore, the aim of the study was to determine the regularities of the influence of Chlorella vulgaris
cultivation conditions on the fatty acid composition of biomass.

2. Methods and materials
2.1 Biomass cultivation

For the experimental study, the strain Chlorella vulgaris Beijer IPPAS C-2 from the Timiryazev Institute of
Plant Physiology of the Russian Academy of Sciences was used.

Microalgae cultivation was carried out on the Tamiya nutrient medium (Tamiya, 1957). On the fourth day of
cultivation, mineral salts were added to the microalgae suspension, which are part of the Tamiya nutrient
medium (Dvoretsky et al., 2015). The process of cultivation was carried out in a cylindrical photobioreactor of
0.3 m height and 0.1 m diameter under the following conditions: 1) the amount of the introduced seed selected
at the stationary growth stage was 10 % of the total suspension volume (cell titre - 13.5 million cells / mL) -

400 mL. The stock culture was grown at the temperature of 25 °С and the level of PAR of 105 µmol of photons
/(m2·s); 2) the pH level changed in the range of 6.2 - 8.0; 3) aeration of the suspension was carried out by a
gas-air mixture (70 - 80 L/h) with the content of carbon dioxide 0.03 %. Microalgae were cultivated under the
conditions presented in Table 1. The concentration of microalgae cells in the suspension was determined by
direct count in the Goryayev chamber. Cells were counted in 25 large squares. The concentration was
determined by the formula: n = a / 100⋅106, where n is concentration of microalgae cells, million cells / mL; a is
number of cells in 25 large squares, pcs.

Table 1: Process conditions of the experiment

Sample # PAR, µmol of photons /(m
2·s) Temperature, °С

1 105 20
2 315 20
3 105 30
4 315 30

2.2 Processing and analysis of microalgae cells

Sampling of microalgae biomass (500 mL) for lipid extraction was performed prior to the experiment, as well
as on the 2nd, 4th, 6th, 8th and 10th day of culturing. Separation of fugate from microalgae biomass was
carried out using a Sigma 2-16 RK/2-16P centrifuge at a rotation speed of 4000 r/min for 5 minutes.
Microalgae cells were disintegrated in the form of paste with humidity of 98 - 99 % using an ultrasonic
disintegrator Scientz IID at ultrasound frequency of 25 kHz and power of 100 W within 20 minutes. Microalgae
cells were dried to determine the cell concentration in the suspension (g/L) in a dry-air oven "HS-121A" at
80 °C.
Extraction of lipids from microalgae biomass was carried out in the Soxhlet apparatus using petroleum ether
as a solvent. Lipids were extracted within 6 hours. The fat content was calculated by the formula:
x = (m1- m2)/ m3⋅100, where m1 is the weight of the sachet with the attachment and anhydrous sodium
sulphide before extraction, g; m2 is the weight of the sachet with the attachment and anhydrous sodium
sulphide after extraction, g; m3 is the weight of the attachment after drying, g.
The solvent was distilled using a rotary evaporator IR-1 M3 at a temperature of distillation 85 °C and the
speed of rotation of the flask 65 min-1.
The lipid content (mg) of 1 litre of microalgae suspension was calculated by the formula: m1 = M⋅x, where m1
is the mass of lipids in 1 L of microalgae suspension, mg/L; M is the mass of microalgae biomass, mg/L; x is
the mass fraction of lipids in microalgae biomass.
Analysis of lipid fatty acids contained in 'microalgae extract' was performed using a gas chromatograph
"Crystallux-4000M".

3. Results and discussion
3.1 Kinetics of biomass growth and lipid accumulation

Experimental studies have shown that the maximum growth rate of microalgae cells in all samples was
observed at the exponential growth phase (Figure 2a). And for samples 1, 2, 4 it equaled ≈ 0.4 days-1 on the
4th-6th day of cultivation (Table 2). The greatest number of cells was observed in sample 4, which was
cultivated at high temperatures and PAR: 2.1 million cells / mL on the 8th day of cultivation with an average
cell diameter of 7.4 µm. The highest mass concentration of cells (0.5 - 0.6 g/L) with the same number of cells
(0.5 - 0.75 million cells / mL) was observed at a lower level of PAR (105 µmol of photons/(m2·s) for samples 1,
3 on the 4th and 2nd day, respectively, see Figure 2b. At the same time, sample 1 reached the average cell
diameter of 9 - 9.4 µm in 4 days, and sample 3 - in 2 days. Thus, in terms of biomass, the best performance
was observed at lower levels of PAR.
The maximum amount of total lipids in microalgae cells was accumulated in samples 2, 4 and made up 45 %
of cell dry matter on the 4th day (Figure 2c), which is due to the fact that stock culture was cultivated with low
levels of PAR and samples were grown under stress conditions - with high levels of PAR
(315 µmol of photons/(m2·s)).
During the 4th to 6th day a decrease in the mass concentration of lipids in cells is observed, resulting from the
addition of a source of nitrogen to the nutrient medium. The increase of mass concentration of lipids in
samples 1 and 3 on 6 - 8 days is caused by nitrogen deficiency due to more intensive metabolism of the strain
at low levels of PAR. During this period of time a slight decrease in mass concentration of lipids was

registered in samples 2 and 4, which is due to the fact that acetyl-CoA is used for biosynthesis of antioxidant
substances necessary for the cell at higher levels of PAR (Salati and Goodridge, 1996). The greatest amount
of lipids (0.19 g/L) was observed at cultivation of sample 1 for 4 days (Figure 2d), at cell diameter ≈ 9.4 µm
and biomass concentration 0.58 g/L.

a) b)

c) d)

Figure 2: Kinetic characteristics of biomass with regard to cultivation conditions

3.2 Fatty acid composition

3.2.1 Saturated fatty acids

The maximum amount of saturated fatty acids in microalgae lipids (Figure 3a) was 181.5 mg/L on the 4th day
of cultivation in sample 1; the same sample had the highest rate of saturated fatty acid accumulation of
81.85 mg/(L·days). This is explained by the fact that during the period of cultivation (2 - 4 days) there was an
intensive growth of cells (Figure 2a), in which the active biosynthesis of fatty acids (primary product - saturated
palmitic acid) was carried out. Cultivation conditions of stock culture and of samples 1 and 3 (in terms of
illumination level) were identical, therefore these samples are assumed to have demonstrated the highest
rates of metabolism and amounts of lipids (Table 2) containing saturated fatty acids - energy reserves
necessary for cell growth and reproduction (Figure 3b). Chromatographic analysis of saturated fatty acids of
lipids in sample 1 (on the 4th day) showed the presence of the following fatty acids: myristic acid (C14:0) -
0.6 % (mass), pentadecanic acid (C15:0) - 30.3 % (mass), palmitic acid (C16:0) - 41.2 % (mass), margaric
acid (C17:0) - 18.4 % (mass), stearic acid (C18:0) - 2.4 % (mass). Thus, this cultivation regime can be
considered as promising for obtaining raw materials for biofuel production.

3.2.2 Unsaturated fatty acids

Sample 1 also demonstrated the largest amount of unsaturated fatty acids (31.0 mg/L) and the rate of their
accumulation (11.25 mg/(L⋅days)) on the 6th day of cultivation (Figure 3c). This is explained by the fact that in
the process of intensive cell division a large number of unsaturated fatty acid molecules are required to form
cytoplasmic membranes. Lower content of unsaturated fatty acids in samples 2 and 4 is associated with the
process of photo-oxidative stress that may occur when microalgae cells are cultured at high levels of PAR.
With temperature rise, the content of unsaturated fatty acids in cells (sample 3, Figure 3d) decreased.

0 2 4 6 8 10
0

0.5

1.5

Time of cultivation, days

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t.)

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s
us

pe
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io
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g
/L

1
2
3
4

a) b)

c) d)

Figure 3: Kinetic characteristics of fatty acids in microalgae biomass

Table 2: Parameters of microalgae biomass during cultivation

Parameter Sample 1 Sample 2 Sample 3 Sample 4

Biomass

μcells, day
-1

0.38 (4 - 6 days) 0.41 (4 - 6 days) 0.31 (4 - 6 days) 0.38 (4 - 6 days)

xmax,
mln cells/mL

1.6 (8th day) 1.6 (8th day) 1.5 (8th day) 2.1 (8th day)

μbiomass,
g / L·day

-1

0.15 (2 - 4 days) 0.1 (2 - 4 days) 0.21 (0 - 2 days) 0.1 (8 - 10 days)

xmax, g/ L 0.58 (4th day) 0.45 (8th day) 0.51 (2nd day) 0.29 (6th, 8th days)

Lipids
μlipids,

g / L ·day
-1

0.085 (2 - 4 days) 0.05 (2 - 4 days) 0.07 (2 - 4 days) 0.04 (2 - 4 days)

xmax , g / L 0.19 (4th day) 0.16 (8th day) 0.18 (4th, 8th days) 0.11 (4th, 6th days)

Saturated
fatty
acids

μSFA,
mg / L·day

-1

81.85 (0 - 2 days) 42.7 (2 - 4 days) 62.51 (2 - 4 days) 37.97 (2 - 4 days)

xmax, mg / L 181.5 (4th day) 145.76 (8th day) 161.82 (4th day) 101.31 (6th days)

Unsaturat
ed fatty
acids

μUFA,
mg / L ·day

-1

11.25 (4 - 6 days) 8.3 (2 - 4 days) 7.47 (2 - 4 days) 5.35 (2 - 4 days)

xmax , mg / L 31.0 (6th day) 22.12 (4th day) 23.58 (8th day) 13.42 (4th day)

(SFA - saturated fatty acids, UFA - unsaturated fatty acids, μ – maximum growth rate, xmax – maximum concentration)

The analysis of unsaturated fatty acids of lipids of sample 1 (on the 6th day) showed the presence of the
following fatty acids: heptadecenoic (C17:1) - 21.5 % (mass), oleic (C18:1) - 3.9 % (mass), linoleic (C18:2) -
2.7 % (mass). That makes this cultivation regime a potentially attractive technology of obtaining raw materials
for the production of food additives. The results obtained are consistent with the trends described in the works
of Tsoglin and Pronina (2012) and Los (2014).

4. Conclusions
Experiments have shown that temperature and PAR have a significant influence on the kinetics of microalgae
biomass growth, lipid biosynthesis and their fatty acid composition. Cultivation at elevated levels of PAR and
temperature leads to intensive cell division, which may be useful for seed preparation. At lower PAR levels
and temperatures, the strain Chlorella vulgaris Beijer IPPAS C-2 accumulates the maximum amount of

0 2 4 6 8 10
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150

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4

biomass (0.58 g/L (4th day)), lipids (0.19 g/L (4th day)), saturated (181.5 mg/L (4th day)) and unsaturated fatty
acids (31.0 mg/L (6th day)). The analysis of fatty acid composition has shown that this strain may be
promising in the technology of production of raw materials for biofuel and food additives.

Acknowledgments

The work was commissioned and carried out with financial support of the Ministry of Education and Science of
the Russian Federation.

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