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\begin{document}

\title{Factors that influence the accumulation of fatty acids in Prorocentrum
micans}



\author[1]{Carla Aguilar}%
\author[2]{Hanna Hernandéz}%
\author[2]{Lennin Ramos}%
\author[2]{Anthony  Soto}%
\author[3]{Roberta Soares}%
\author[4]{Carlos Yure B. Oliveira}%
\author[3]{Jéssika Lima de Abreu Abreu}%
\author[5]{Alfredo Gálvez}%
\affil[1]{Instituto del Mar del Perú}%
\affil[2]{Instituto del Mar del Peru}%
\affil[3]{Universidade Federal Rural de Pernambuco}%
\affil[4]{Universidade Federal de Santa Catarina}%
\affil[5]{UFRPE}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Microalgae, historically relevant to aquaculture sector, can effectively
contribute to the sustainability in several production chain. The
physical and chemical environmental factors play a direct or indirect
role in shaping the composition of their intracellular compounds of
these organisms. Among these compounds, fatty acids are particularly
noteworthy, with various species harboring an extensive array of them.
Notably, the marine dinoflagellate Prorocentrum micans excels in the
production of essential polyunsaturated fatty acids (PUFA), including
DHA and EPA. The objective was to determine which factors contribute to
the accumulation of the PUFA in a P. micans strain. A factorial design
of 3x3 composed by the following factors: illumination (5, 12.5, 20 \selectlanguage{greek}µ\selectlanguage{english}mol
photons m-2 s-1), nitrate dose (0, 75, 150 mg ml-1) and inoculum volume
(50, 125, 200 ml) was examined to determine the better growth conditions
for P. micans. Furthermore, lipid profile and the fatty acids profile
were analyzed at the end of the cultivation. The results determined
that, maximum averages of DHA (20\%) and EPA (1\%), in the biomass, were
registered in cultures without nitrate, low irradiance (5 \selectlanguage{greek}µ\selectlanguage{english}mol photons
m-2 s-1) and high volume of inoculum (200 ml). The maximum cell
concentration recorded was 3.1\selectlanguage{ngerman}×104 cells ml-1, at the end of the
culture. So, it can be concluded that the evaluated strain is easily
adapted to the culture flow and, the culture conditions to which it was
subjected allows its biomass to be considered an interesting alternative
as lipid input in aquaculture activity.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Factors that contribute to the accumulation of fatty acids
in\emph{Prorocentrum micans}: a novel source of lipids for aquaculture}

Carla Aguilar S\textsuperscript{1}, Hanna Hern\selectlanguage{ngerman}ández
A\textsuperscript{1}, Lennin Flores R\textsuperscript{2}, Anthony Ruiz
S\textsuperscript{2}, Roberta Borda Soares\textsuperscript{3}, Carlos
Yure B. Oliveira\textsuperscript{4}, Jéssika Lima de
Abreu\textsuperscript{3*} and Alfredo Olivera Gálvez\textsuperscript{3}

1 Germplasm Bank of Aquatic Organisms, Directorate of Aquaculture
Research, Instituto del Mar del Perú, Peru

2 Instrumental Analysis Laboratory, Directorate of Aquaculture Research,
Instituto del Mar del Perú, Peru

3 Fisheries and Aquaculture Department, Federal Rural University of
Pernambuco, Brazil

4 Botany Department, Center for Biological Sciences, Federal University
of Santa Catarina, Brazil

\emph{List of abbreviations}

PUFA - polyunsaturated fatty acids

DHA - C22:6n\selectlanguage{english}-3 docosahexaenoic acid

EPA- C20:5n-3 eicosapentaenoic acid

FAMEs - fatty acid methyl ester

BGOA - Aquatic Organisms Germplasm Bank

mg ml\textsuperscript{-1} - milligram per milliliter

ng ml\textsuperscript{-1} - nanogram per milliliter

pg ml\textsuperscript{-1} - picogram per milliliter

\selectlanguage{greek}µ\selectlanguage{english}mol -- micromole

*Corresponding author: J\selectlanguage{ngerman}éssika Lima de Abreu

Federal Rural University of Pernambuco, Department of Fisheries and
Aquaculture, St. Rua Dom Manuel de Medeiros, s/n - Dois Irmãos, Recife -
PE, Zip code: 52171-900

Tel.: +55 87 98581 3442

E-mail address: jessik.labreu@gmail.com

\textbf{Abstract}

Microalgae, historically relevant to aquaculture sector, can effectively
contribute to the sustainability in several production chain. The
physical and chemical environmental factors play a direct or indirect
role in shaping the composition of their intracellular compounds of
these organisms. Among these compounds, fatty acids are particularly
noteworthy, with various species harboring an extensive array of them.
Notably, the marine dinoflagellate \emph{Prorocentrum micans} excels in
the production of essential polyunsaturated fatty acids (PUFA),
including DHA and EPA. The objective was to determine which factors
contribute to the accumulation of the PUFA in a \emph{P. micans} strain.
A factorial design of 3x3 composed by the following factors:
illumination (5, 12.5, 20 \selectlanguage{greek}µ\selectlanguage{english}mol photons
m\textsuperscript{-2}s\textsuperscript{-1}), nitrate dose (0, 75, 150 mg
ml\textsuperscript{-1}) and inoculum volume (50, 125, 200 ml) was
examined to determine the better growth conditions for \emph{P. micans}
. Furthermore, lipid profile and the fatty acids profile were analyzed
at the end of the cultivation. The results determined that, maximum
averages of DHA (20\%) and EPA (1\%), in the biomass, were registered in
cultures without nitrate, low irradiance (5 \selectlanguage{greek}µ\selectlanguage{english}mol photons
m\textsuperscript{-2} s\textsuperscript{-1}) and high volume of inoculum
(200 ml). The maximum cell concentration recorded was
3.1\selectlanguage{ngerman}×10\textsuperscript{4} cells ml\textsuperscript{-1}, at the end of
the culture. So, it can be concluded that the evaluated strain is easily
adapted to the culture flow and, the culture conditions to which it was
subjected allows its biomass to be considered an interesting alternative
as lipid input in aquaculture activity.

\textbf{Keyword} : \emph{P. micans} DHA, EPA, nitrate

\textbf{Introduction}

Due to the global changes that humanity faces to maintain, protect and
improve food security, aquaculture has several challenges to overcome,
mainly sustainability, biosecurity, regulatory structures in accordance
with ocean ecosystems and food supplies, since these charge greater
relevance due to the nutritional level they perform, especially as
unique and extremely diverse providers of omega-3 fatty acids and
essential bioavailable micronutrients (MUSTAFA and SHAPAWI, 2015; FAO
2022). In the aquatic environment, microalgae are the base of the
trophic chain, capable of accumulating a series of active compounds,
among which fatty acids stand out, especially polyunsaturated fatty
acids (PUFAs). important for the development and reproduction of
zooplankton organisms, seeds and other secondary consumers in their
initial levels of development (IKAWA, 2004; HERNÁNDEZ, 2016; GONÇALVEZ
et al., 2017; REMMENS et al., 2018; PELTOMA et al., 2019; OLIVEIRA et
al., 2022).

The importance of microalgae in the diet of the larval stages of various
aquatic organisms is mainly due to the presence of polyunsaturated fatty
acids such as; C22:6n\selectlanguage{english}-3 docosahexaenoic acid (DHA) and C20:5n\selectlanguage{english}-3
eicosapentaenoic acid (EPA) that are synthesized and transferred through
the food chain ingested by secondary consumers, as they play a crucial
role in development neural, tissue formation, physiological functions,
cell walls and energy storage of the latter, it is due to this, that
various studies refer to a connection between the nutritional value of
microalgae and their lipid profile (ADAME et al., 2012; GONCALVEZ et
al., 2017; RAJA et al., 2018; PELTOMA et al., 2019).

With the development of the aquaculture industry and the demand that
this implies, in terms of quality and quantity of organisms produced in
closed or open spaces, inputs rich in lipid compounds are increasingly
scarce or are replaced by alternative products that lack the necessary
nutrients for a balanced feed of aquatic organisms (PELTOMA et al.,
2019). In this context and given that microalgae contain interesting
lipid profiles, they are positioned as a potential resource so that,
through mass cultivation processes, biomass or extracts can be obtained,
as raw material or biochemically adequate input for feeding cultivable
organisms, also considering that microalgal productivity does not
interfere with food safety and favors the environment (ATKINSON, 2013;
FU et al., 2017; LORENZEN et al., 2017; REMMENS et al., 2018; PELTOMA,
2019).

The microalgae with considerable lipid profiles are those of the group
of diatoms and dinoflagellates, the first of which is characterized by
accumulating EPA and the second DHA in a greater proportion compared to
other fatty acids (SEXTON and LOMAS 2018; ZULU et al., 2018; PELTOMA et
al., 2019), both groups are widely distributed in the aquatic
environment and even when some of them face certain factors such as;
temperature, irradiance, salinity tend to proliferate forming algal
blooms, only dinoflagellates can adapt very well due to their
mixotrophic or heterotrophic condition to sudden changes, losing their
inhibitory capacity on the development of other organisms (LEE et al.,
2017; ABD et al. ., 2022).

Within the group of dinoflagellates, \emph{Prorocentrum}
\emph{micans}turns out to adapt quite easily to culture conditions, it
stands out for its high content of DHA over other lipid components,
which makes it a potential resource whose biomass would be an
interesting input or raw material in the production of aquaculture
products (KATTNER and BROCKMANN, 1978; SUH et al., 2015; HERNANDEZ et
al., 2019).

In culture, \emph{P. micans} is a species whose growth is mainly
impacted by thermal and light variation, as well as by nutrient levels,
especially nitrates (LEE et al., 2017; REMMENS et al., 2018). The
variations of these factors determine the lipid profile of the species;
among the fatty acids that can be recorded and are related to algal
bloom events are considered; octadecapentaenoic acid C18:5n3 (OPA) and
octadecatetraenoic acid C18:4n3 (ODTA), the latter quite toxic (IKAWA,
2004).

Therefore, to explore and evaluate the factors that contribute to the
accumulation of fatty acids of interest in the aquaculture industry
in\emph{P. micans} and minimize or inhibit toxic substances, light
intensity and nitrate content should be taken into account, given which
are critical factors that affect the photosynthesis of these algal
organisms, also considering that the toxic activity of PUFAs may be
related to photooxidation (IKAWA, 2004; LEE et al., 2017; REMMENS et
al., 2018).

In this sense, the objective of the present study was to evaluate the
culture conditions in a controlled environment that contribute to the
accumulation of fatty acids with emphasis on DHA and EPA in the marine
dinoflagellate \emph{P. micans} strain IMP-BG-036, with the purpose of
that is considered a potential input for the preparation of enrichments
or other products that are required in the aquaculture activity. It is
necessary to mention that the afore mentioned strain was evaluated and
selected from a set of strains of the same species isolated from
different places on the Peruvian coast and was the one that best adapted
to the culture flow with high levels of various fatty acids, among which
stand out the DHA.

\textbf{Material and methods}

\emph{2.1 Culture conditions}

The biological material was the IMP-BG-036 strain, corresponding to the
marine dinoflagellate \emph{P. micans} , which is part of the strain of
the Aquatic Organisms Germplasm Bank (BGOA). The cultivation was of the
batch type, for 11 days, enriched with medium FL, arranged in 250ml
glass flaks and in triplicate, submitted to a temperature of 18degC, with
photoperiod 12 h, according to experimental design factors detailed in
Table 1.

{[}Table 1{]}

The data generated was subjected to a complete factorial experiment 23,
with 3 replicates and 3 central points, completely randomized, the
treatments were shown in table 2.

{[}Table 2{]}

\emph{2.2 Determination of growth characteristics}

The evaluation of cell density was carried out using cell counts, after
a period of 11 days of triplicate culture, in a Sedgwick-Rafter chamber,
extracting a 2 ml culture sample set in lugol at 4\%.

\emph{2.3 Determination of lipid content}

The lipid profile was determined using the Folch method (1957). 50 ml of
sample was centrifuged for analysis, then 3 ml of chloroform: methanol
(1:1) was added to perform lipid extraction. A Branson 2510 ultrasonic
bath was used to enhance lipid extraction, the chloroform:lipid phase
was separated with ultrapure water and evaporated with nitrogen gas. The
result was expressed as weight percentage.

\emph{2.4 Determination of fatty acids}

The derivatization of these compounds was carried out using the method
of Ichihara and Fukubayashi (2010). 0.2ml toluene, 1.5ml methanol and a
mixture of 1ml concentrated hydrochloric acid with 4.3ml methanol were
added to the extracted lipids and the mixture was incubated at 45degC,
then FAMEs were extracted with 1.5ml hexane and 1ml of water. Varian
CP-3800 Gas Chromatograph conditions were as follows (Table 3).

{[}Table 3{]}

\emph{Statistical analysis}

For the evaluation of the data obtained, an analysis of variance (ANOVA)
and Tukey's post hoc test will be carried out to evaluate the value of
significance (\selectlanguage{greek}α\selectlanguage{english}=0.05). Likewise, for the relationship of the lipid
profile and fatty acids against the crops, a design of significant
effects and graphs (Pareto) will be carried out in the statistical
program Minitab 16.

\textbf{Results}

\emph{3.1 Growth characteristics of the P. micans strain IMP-BG-036}

Figure 1 shows that the cell concentration was affected by all the
factors as a whole: the volume of the inoculum, the lighting on the
culture and the dose of nitrate in the medium, without there being a
single selective factor. Although the standardized effects of the Pareto
Diagram show that the factor with the greatest impact was the volume of
the inoculum, the interaction between it and the lighting, as well as
the dose of nitrate and lighting allowed the recording of the maximum
cell concentrations at the end of the culture period on day 11. This
result (3.1x10\textsuperscript{4} cel.ml\textsuperscript{-1}), was
obtained with a lighting of 5
mol.m\textsuperscript{-2}.sec\textsuperscript{-1}, an inoculum volume of
200 ml and without nitrate dose. in the culture medium (Table 4).

{[}Figure 1{]}

{[}Table 4{]}

One aspect observed during the counting process and not considered in
the design was the number of empty shells found; with higher incidence
in crops whose inoculum was 200 mL. In those with a smaller volume of
inoculum (50 and 125 ml), the medium was more diluted, and the valves
found were very few.

\emph{3.2 Content and determination of fatty acids}

The experimental design allowed evaluating 27 combinations with the 3
factors considered: lighting, nitrate dose and inoculum volume. The
result allowed recording 12 fatty acids for this strain, among which are
distinguished; 3 saturated fatty acids (C14:0 myristic, C16:0 palmitic
and C18:0 stearic); 3 monounsaturated fatty acids (Palmitoleic C16:1n-7,
Oleic C18:1n-9 and Vaccenic C18:1n-7) and 6 polyunsaturated fatty acids
(Linoleic C18:2n-6, Linolenic C18:3n-3, Stearidonic C18:4n-3,
Docosahexanoic: DHA C22:6n-3, Eicosapentaenoic: EPA C20:5n-3 and
Octadecapentaenoic: OPA C18:5n-3).

The selective factor for the accumulation of DHA at the biomass level
(ng ml\textsuperscript{-1}) was mainly the volume of the inoculum,
followed by the lighting factor in interaction with the dose and the
volume separately, by themselves they did not have selective
representativeness (Figure 2a). On the contrary, at the cellular level
(pg.cel\textsuperscript{-1}), the factors related to illumination and
nitrate dose were selective on the accumulation of DHA, the first being
the most relevant. However, the inoculum volume interacts with the other
factors during the process (Figure 2b).

{[}Figure a, b{]}

Although, the interaction of the 3 factors (inoculum volume, lighting
and nitrate dose) contribute to the accumulation of DHA fatty acid at
the cellular level (pg.cel\textsuperscript{-1}). This condition is
evidenced when these factors interact at their maximum levels, that is,
when the cultures were exposed to a lighting of 20
\selectlanguage{greek}µ\selectlanguage{english}mol.m\textsuperscript{-2}.seg\textsuperscript{-1} and an inoculum
volume of 200 ml respectively (Fig 3).

{[}Figure 3{]}

Figure 4a shows that the selective factor in the accumulation of EPA at
the biomass level (ng ml\textsuperscript{-1}) was the volume of the
inoculum, followed by the interaction of the lighting factor with the
volume and dose of nitrate separately, realizing that, the selectivity
in this case, is evidenced through the interaction of these factors.
While, at the cellular level (pg ml\textsuperscript{-1}) there is not a
single selective value that contributes to the accumulation of EPA, but
all in parallel, only a joint interaction between the nitrate dose and
the volume of the inoculum is evidenced (Figure 4b).

{[}Figure 4{]}

The principal components graph showed that, at the cellular level (pg
cel\textsuperscript{-1}), the accumulation of EPA fatty acid requires a
minimum inoculum volume, without nitrate doses and lighting of 20 \selectlanguage{greek}µ\selectlanguage{english}mol
photons m\textsuperscript{-2} s\textsuperscript{-1}, (Figure 5a). The
interaction of factors that was recorded for the accumulation of EPA
fatty acid at the cellular level was between the volume of the inoculum
(50 ml) and the dose of nitrate (without nitrate), even though the
average values were of the order of 2.5 pg
cel\textsuperscript{-1}(Figure 5b).

{[}Figure 5{]}

A fatty acid whose presence called attention because it is related to
HABs was OPA and for this reason the Pareto Diagram is presented for the
factors involved in its accumulation. At the biomass level (mg
ml\textsuperscript{-1}) two selective factors are evident, lighting and
the volume of the inoculum, the other two factors are an interaction of
the three: Lighting and inoculum volume and lighting and nitrate dose.
At the cellular level (pg cel\textsuperscript{-1}) the selective factor
was lighting, no factor interaction was recorded (Figure 6). Both the
principal component plot and the interaction plot were not determinative
for this fatty acid.

{[}Figure 6{]}

During the cell count, a high incidence of empty valves was recorded in
the cultures whose inoculum was 200 ml. This condition may have been
related to the low levels of oil concentration at the cell level and
high concentrations at the biomass level. On the other hand, the
cultures with 50 ml inoculum did not present the presence of empty
valves, they were more diluted and exposed to a maximum illumination of
20 \selectlanguage{greek}µ\selectlanguage{english}mol photons m\textsuperscript{-2} s\textsuperscript{-1} and a double
dose of nitrate.

Of the 12 fatty acids registered for the IMP-BG-036 strain of \emph{P.
micans} , 11 reached their maximum average values, in the nitrate-free
medium, independent of lighting and inoculum volume, these were in
descending order; OPA (1783 ng ml\textsuperscript{-1}), Palmitic (1367
ng ml\textsuperscript{-1}), DHA (1162 ng ml\textsuperscript{-1}),
Steridonic (647 ng ml\textsuperscript{-1}), Vaccenic (472 ng
ml\textsuperscript{-1}), Myristic (133 ng ml\textsuperscript{-1}),
Linolenic (95 ng ml\textsuperscript{-1}), Stearic (84 mg
ml\textsuperscript{-1}), Palmitoleic (73 ng ml\textsuperscript{-1}), EPA
(71 ng ml\textsuperscript{-1}) and Linoleic (70 ng
ml\textsuperscript{-1}). The only one that registered its highest
average value in the medium with double dose of nitrate, minimum
lighting factor and inoculum volume was the Oleic (103 ng
ml\textsuperscript{-1}).

Given the interest in relevant fatty acids in aquaculture products, the
biomass level is compared with the content at the cellular level only of
DHA and EPA; where it is observed that, in the medium lacking nitrate,
the maximum average values at the biomass level are recorded when;
lighting was minimal and the inoculum volume was maximum, while the
maximum mean values at the level of cell content were recorded when the
lighting factor was maximum independent of the inoculum volume factor
(Table 5).

{[}Table 5{]}

The lipid variety for the \emph{P. micans} strain IMP-BG-036 recorded in
table 6 shows that, when the nitrate dose was doubled, it did not exceed
the average concentrations recorded in Table 5, in neither of the two
levels evaluated. biomass and cell. However, the pattern that the
highest values are shown in low lighting conditions is maintained,
despite the fact that the nitrate dose was double, taking the L1 culture
medium as reference.

{[}Table 6{]}

The results recorded in Table 7, where the factors were intermediate,
did not exceed the values recorded in Table 6.

{[}Table 7{]}

\textbf{Discussion}

The dinoflagellate \emph{P. micans} is one of the many microalgae that,
due to its vertical movement in the water column, can adapt to fairly
wide light intensities and thus experience the absorption of a variation
of nutrients in that path (JEON et al., 2010). Also, during the
photosynthetic process two important factors are the light intensity and
the nutrients available in the medium, which are related to the growth
of all photosynthetic organisms (LEE et al., 2017). In this sense, the
present study considered three factors at three levels each for the
cultivation of the strain; illumination of 5, 12.5 and 20
\selectlanguage{greek}µ\selectlanguage{english}mol.m\textsuperscript{-2}.seg\textsuperscript{-1}; the dose of nitrate
as part of the nutrients of the culture medium of 0, 75 and 150
mg.ml\textsuperscript{-1} and the volume of the inoculum (50, 125 and
200 ml), so that some of these combinations of variables allow cells
carry out the synthesis of active substances such as fatty acids more
easily.

The studies by Lee et al. (2017) also recorded that, with the increase
in light intensity from 10 to 200
\selectlanguage{greek}µ\selectlanguage{english}E.m\textsuperscript{-2}.seg\textsuperscript{-1}, in Guillard medium
with silicate, the cell concentration of \emph{P. micans} increases
between 10,000 to 12,000 cel.ml\textsuperscript{-1} and the nitrate
concentration decreases from 100 uM to 1.5uM over time (between 6 to 20
days). In the present study, cell concentrations greater than 30,000
cel.ml\textsuperscript{-1} were recorded in the medium without nitrate
dose and with a light intensity of 5
\selectlanguage{greek}µ\selectlanguage{english}mol.m\textsuperscript{-2}.seg\textsuperscript{-1}, after 11 days of
culture. Even when the light intensity was 20
\selectlanguage{greek}µ\selectlanguage{english}mol.m\textsuperscript{-2}.seg\textsuperscript{-1}, the average cell
concentration was higher than 12,000 cel.ml\textsuperscript{-1}. From
these results it can be concluded that it is necessary to evaluate the
components of the medium in order to strengthen cell growth.

A relevant aspect of microalgal diversity, including dinoflagellates, is
the fatty acid content that characterizes them, since they play an
important role in their nutritional quality and growth, as well as in
the organisms that consume them; Thus, a correlation of the lipid
profile of microalgae with its nutritional value is established and,
from this, its usefulness as food can be determined (LEE et al., 2017),
indirectly, its use as an input for various products. During the present
study, the IMP-BG-036 strain of \emph{P. micans} recorded the presence
of 12 types of fatty acids, including saturated, monounsaturated and
polyunsaturated, of the latter the presence of DHA and EPA becomes
important due to their relevance in aquaculture activity.

An important aspect for the accumulation of fatty acids is the lack of
nitrate in the medium (REMMENS et al., 2018). Of the 12 fatty acids
registered for the strain IMP-BG-036 of \emph{P. micans} , 11 reached
their maximum average values, in the nitrate-free medium, independent of
lighting and inoculum volume, these were in descending order; OPA (1783
mg.ml\textsuperscript{-1}), Palmitic (1367 mg.ml\textsuperscript{-1}),
DHA (1162 ng.ml\textsuperscript{-1}), Stearidonic (647
mg.ml\textsuperscript{-1}), Vaccenic (472 mg.ml\textsuperscript{-1}),
Myristic (133 mg.ml-1), Linolenic (95 mg.ml\textsuperscript{-1}),
Stearic (84 mg.ml\textsuperscript{-1}), Palmitoleic (73
mg.ml\textsuperscript{-1}), EPA (71 mg.ml\textsuperscript{-1}) and
Linoleic (70 mg.ml\textsuperscript{-1}), the only fatty acid that was
recorded in the medium with a double dose of nitrate was Oleic (103
mg.ml\textsuperscript{-1}).

There are several biomolecules that various groups of microalgae
accumulate in response to the environment where they develop and the
strategies to which they can be exposed (FU et al., 2017; SUN et al.,
2019). After an exhaustive study on the importance of fatty acids
extracted from microalgae strains for aquaculture purposes, one of the
interesting species due to its lipid profiles, especially its DHA and
EPA content, was \emph{P. micans} (HERN\selectlanguage{ngerman}ÁNDEZ et al., 2019). Although it
is an organism related to the proliferation of Harmful Algal Blooms
(HABs), it is necessary to mention that its inhibitory capacity for the
development of other organisms can be affected by the variation of
certain factors such as lighting or some nutritional components in the
environment. medium as the source of nitrates and phosphates mainly
(IKAWA, 2004; LEE et al., 2017; ABD et al., 2022).

On the other hand, the studies by Peltoma et al., (2019), determined
that dinoflagellates, regardless of their habitat, register
concentrations between 10 to 15\% of DHA and less than 5\% of EPA. In
the present study, even when the results did not show statistically
significant differences, the average concentrations of EPA did not
exceed 3\% and for DHA they did not exceed 30\%.

When evaluating the influence of luminosity, nitrate doses and initial
concentration of the inoculum, it can be observed that the fatty acid
profiles of the produced biomass did not show differences among
themselves. The results indicate that the species has great potential
for its use in obtaining fatty acids, regardless of the culture
conditions that are subjected to it.

\textbf{Acknowledgments}

The authors would like to thank the National Council for Scientific and
Technological Development - CNPq for the financial support (Process nº
167938/2022-3, PQ 309669/2021-9 and PQ 308063/2019-8), the Fundação de
Amparo a Ciência e Tecnologia of PE - FACEPE (Process nº BFP - 0131 -
5.06/22) and finally the Foundation Coordination for the Improvement of
Higher Education Personnel - Capes (Process nº 88882.436231/2019-01).

\textbf{Authors' contributions}

CAS: Investigation, Conceptualization, Data curation, Formal analysis,
Visualization, Writing - original draft, Writing- review \& editing.
HHA: Investigation, Data curation, Formal analysis, Writing- review \&
editing. LFR: Investigation, Data curation, Formal analysis,
Visualization, Writing - original draft. ARS: Formal analysis, Data
Curation, Writing- review \& editing; RBS: Formal analysis, Writing-
review \& editing. CYBO: Conceptualization, Data curation, Writing-
review \& editing. JLA: Data curation, Formal analysis, Writing- review
\& editing. AOG: Supervision, Investigation, Project administration,
Visualization, Writing -review \& editing.

\textbf{Conflict of interest}

Authors declare that there is no conflict of interest regarding the
publication of this article.

\textbf{Data availability}

The datasets generated during and/or analyzed during the current study
are available from the corresponding author on reasonable request.

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\textbf{Tables}

Table 1: Factors and levels of factorial design\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Factors} & \textbf{Units} & \textbf{Levels} & \textbf{Levels} &
\textbf{Levels}\tabularnewline
\midrule
\endhead
& & -1 & 0 & +1\tabularnewline
\textbf{Inoculum volume} & ml & 50 & 125 & 200\tabularnewline
\textbf{Luminous intensity} & & 5 & 12.5 & 20\tabularnewline
\textbf{Nitrate dosage(*)} & g.ml\textsuperscript{-1} & 0 & 1 &
2\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{(*) Level 0 = no dose, level 1 = 75mg.ml-1, level 2 =
150mg.ml-1.}

Table 2: Experimental design with the strain selected to enhance its
lipid profile, subjected to nitrogen stress, according to possible
combinations at chance between levels and variables.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
N\selectlanguage{ngerman}° experiments\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Inoculum volume (ml)\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Luminous intensity
(umol.m\textsuperscript{2}.seg\textsuperscript{-1})\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Nitrate dosage (g.ml\textsuperscript{-1})\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Fatty acid (pg.cel\textsuperscript{-1})\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Final cell conc (cel.ml\textsuperscript{-1})\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
average\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
200\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
200\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
200\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
200\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
125\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
12.5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3} . Instrumental parameters and conditions of the
chromatograph used (Varian CP-3800)\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Instrumental parameters & Conditions\tabularnewline
\midrule
\endhead
Injection volume & 1 \selectlanguage{greek}µ\selectlanguage{english}L\tabularnewline
Injection mode & spitless\tabularnewline
Injection time & 0.5 min\tabularnewline
Injector temperature & 250 \selectlanguage{ngerman}ºC\tabularnewline
Capillary column & Restek, FAMEWAS \selectlanguage{ngerman}® WCOT fused silica 30m x 0.25 mm x
0.25 \selectlanguage{greek}µ\selectlanguage{english}m\tabularnewline
Carrier gas & Helium\tabularnewline
Gas flow & 1 mL min\textsuperscript{-1}\tabularnewline
Temperature program & 120 \selectlanguage{ngerman}ºC for 1 min, 30 ºC min\textsuperscript{-1} up
to 160 ºC, 160 ºC for 1 min, 4 ºC min\textsuperscript{-1} up to 240 ºC,
and 240 ºC for 7 min\tabularnewline
Total time of temperature program & 30.33 min\tabularnewline
Detector temperature & 260 \selectlanguage{ngerman}ºC\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Table 4: Cellular concentration of \emph{P. micans} , strain IMP-BG-036
at the end of culture\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
N\selectlanguage{ngerman}° experiments\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Vol., inoculum (ml)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Vol., inoculum (ml)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Luminous intensity
(\selectlanguage{greek}µ\selectlanguage{english}mol/m\textsuperscript{2}/s\textsuperscript{1})\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Nitrato dosage (g/ml)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Cell Concentration (cel/mL)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Cell Concentration (cel/mL)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
50\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.5E+04\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(\selectlanguage{ngerman}±334.33)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
200\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
2.91E+04\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(\selectlanguage{ngerman}±216.76)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
50\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.29E+04\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(\selectlanguage{ngerman}±242.53)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
200\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
2.77E+04\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(\selectlanguage{ngerman}±257.26)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
50\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.26E+04\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(\selectlanguage{ngerman}±161.11)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
200\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.52E+04\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(\selectlanguage{ngerman}±1346.01)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
50\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.71E+04\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(\selectlanguage{ngerman}±525.73)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
200\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.96E+04\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(\selectlanguage{ngerman}±300.04)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
125\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
12.5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
2.62E+04\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(\selectlanguage{ngerman}±391.64)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Table 5: Variety of the lipid content of the strain IMP-BG-036 of
\emph{P. micans} , under controlled culture conditions, induced by three
abiotic factors; Inoculum volume (ml), Illumination
(\selectlanguage{greek}µ\selectlanguage{english}mol.m\textsuperscript{-2}.sec\textsuperscript{-1}) and nitrate dose.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Table 6: Variety of the lipid content of the strain IMP-BG-036
of\emph{P. micans} , under controlled culture conditions, induced by
three abiotic factors; Inoculum volume (ml), Illumination
(\selectlanguage{greek}µ\selectlanguage{english}mol.m\textsuperscript{-2}.sec\textsuperscript{-1}), with a double dose
of nitrate.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Table 7: Variety of the lipid content of the strain IMP-BG-036
of\emph{P. micans} , under controlled culture conditions, induced by
three abiotic factors; Inoculum volume (ml), Illumination
(\selectlanguage{greek}µ\selectlanguage{english}mol.m\textsuperscript{-2}.seg\textsuperscript{-1}), with a dose of
nitrate (According to culture medium L1)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure capitions}

Figure 1: Factors involved in cell concentration during the cultivation
of the \emph{P. micans} strain IMP-BG-036 under laboratory conditions,
using the Pareto Diagram

Figure 2: Factors involved in the accumulation of DHA polyunsaturated
fatty acid, a) biomass level (ng.ml\textsuperscript{-1}), b) cellular
level (pg.cel\textsuperscript{-1}) of the IMP-BG-036 strain of \emph{P.
micans} under laboratory conditions, using the Pareto Diagram.

Figure 3: Graph of interaction of factors involved in the accumulation
of the fatty acid DHA of the strain IMP-Bg-036 of \emph{P. micans} , in
culture.

Figure 4: Factors involved in the accumulation of EPA polyunsaturated
fatty acid, a) biomass level (ng.ml\textsuperscript{-1}), b) cellular
level (pg.cel\textsuperscript{-1}) of the IMP-BG-036 strain of \emph{P.
micans} under laboratory conditions, using the Pareto Diagram.

Figure 5: a) Graph of principal components that determines the impact of
the factors volume of the inoculum, illumination and dose of nitrate on
the accumulation of EPA at the cellular level, b) Graph of interaction
of factors that intervene in the accumulation of the fatty acid EPA of
the strain IMP-Bg-036 of \emph{P. micans} , in culture under controlled
conditions.

Figure 6: Factors involved in the accumulation of the polyunsaturated
fatty acid OPA, a) biomass level (ng.ml\textsuperscript{-1}), b)
cellular level (pg.cel\textsuperscript{-1}) in the culture of the strain
IMP-BG-036 of \emph{P. micans} under laboratory conditions, using the
Pareto Diagram.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

A\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

B\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

A\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

B\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}

A\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}

B\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

\end{center}
\end{figure}

A\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}

B

\selectlanguage{english}
\FloatBarrier
\end{document}

