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.