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-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-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-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
µmol.m-2.seg-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-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-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-1), the accumulation of EPA fatty acid requires a
minimum inoculum volume, without nitrate doses and lighting of 20 µmol
photons m-2 s-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-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-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-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 µmol photons m-2 s-1 and a double
dose of nitrate.
Of the 12 fatty acids registered for the IMP-BG-036 strain of 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-1), Palmitic (1367
ng ml-1), DHA (1162 ng ml-1),
Steridonic (647 ng ml-1), Vaccenic (472 ng
ml-1), Myristic (133 ng ml-1),
Linolenic (95 ng ml-1), Stearic (84 mg
ml-1), Palmitoleic (73 ng ml-1), EPA
(71 ng ml-1) and Linoleic (70 ng
ml-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-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 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]
Discussion
The dinoflagellate 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
µmol.m-2.seg-1; the dose of nitrate
as part of the nutrients of the culture medium of 0, 75 and 150
mg.ml-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
µE.m-2.seg-1, in Guillard medium
with silicate, the cell concentration of P. micans increases
between 10,000 to 12,000 cel.ml-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-1 were recorded in the medium without nitrate
dose and with a light intensity of 5
µmol.m-2.seg-1, after 11 days of
culture. Even when the light intensity was 20
µmol.m-2.seg-1, the average cell
concentration was higher than 12,000 cel.ml-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 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 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-1), Palmitic (1367 mg.ml-1),
DHA (1162 ng.ml-1), Stearidonic (647
mg.ml-1), Vaccenic (472 mg.ml-1),
Myristic (133 mg.ml-1), Linolenic (95 mg.ml-1),
Stearic (84 mg.ml-1), Palmitoleic (73
mg.ml-1), EPA (71 mg.ml-1) and
Linoleic (70 mg.ml-1), the only fatty acid that was
recorded in the medium with a double dose of nitrate was Oleic (103
mg.ml-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 P. micans (HERNÁNDEZ 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.