**2. Results and Discussion**

### *2.1. Adaptation of Autotrophic to Heterotrophic Culture of T. suecica*

The heterotrophic culture of *T. suecica* showed that cell density, cell concentration and biovolume were statistically higher in the heterotrophic culture (*p* < 0.05), while the cell volume was 17 times lower compared with the autotrophic culture (*p* < 0.05) (Table 1). The specific growth rate between autotrophic and heterotrophic cultures no showed statistic differences significantly (Table 1).


**Table 1.** Population parameter from autotrophic and heterotrophic cultures of *T. suecica* (mean ± SD; *n* = 3). Asterisks denote significant di fferences (*p* < 0.05, Student's *t*-test).

Azma et al. [12] obtained di fferences in the final cell concentration of *T. suecica* grown in autotrophy and heterotrophy. On the contrary, Day and Tsavalos [33] found no di fferences in the final cell concentration of *Tetraselmis* sp. between the two culture conditions. These variations could be due to growth in the absence of light, and the presence of organic substrates can change the metabolism and morphology of cells. In our investigation, glucose was used as the source of organic carbon, which generated high cellular concentrations due to the energy provided (2.8 kJ mol−1), compared to the 0.8 kJ mol−<sup>1</sup> for acetate used in Azma et al.'s [12] investigation.

The adaptation of autotrophic to heterotrophic culture was performed by the progressive reduction of the illumination times in the photoperiod, preserving the irradiance of the *T. suecica* cultures. However, Azma et al. [21] made a progressive decrease in lighting for *T. suecica* cultures with longer periods, adding a total of 1650 h compared to the present study, which was 1080 h for adaptation to heterotrophy, meaning 35% less hours of adaptation, which would be due to the di fferent media used in cultivation. The Walne medium [34] used in Azma et al.'s [21] investigation contained concentrations of nitrate, phosphate, ethylenediaminetetraacetic acid (EDTA), zinc, molybdenum and manganese higher than F/2 used in the present study. Therefore, *T. suecica* has the ability to regulate its metabolism to achieve balanced growth in heterotrophic culture; this capability can be used to increase the production of metabolites of biotechnological interest.

### *2.2. Elemental Analysis of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

Heterotrophic cultures of *T. suecica* showed higher carbon and nitrogen contents than those observed in autotrophy (*p* < 0.05; Table 2). The C/N ratio did not show statistical di fferences (*p* > 0.05) between both culture conditions (Table 2). The C/N ratio is a nutritional indicator of the microalgae. When N is low, the C/N ratio favors the biosynthesis and accumulation of carbohydrates [35]. However, Cheng et al. [36] mentioned that a C/N ratio higher than 10 allowed lipid accumulation in heterotrophic cultures. In our study, the high C/N ratio is favored for the accumulation of carbon, which will be used to increase the accumulation of lipids or carbohydrates.

**Table 2.** Total carbon (TC), total nitrogen (TN) content and C/N ratio in the biomass obtained from autotrophic and heterotrophic cultures of *T. suecica* (mean ± SD; *n* =3). Asterisks denote significant di fferences (*p* < 0.05, Student's *t*-test).


### *2.3. Biochemical Composition of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

Heterotrophic cultures eliminate the light limitations that autotrophic cultures require, generating metabolic changes in microalgae, thus presenting variations in the biochemical composition of the biomass [6,7]. In our study, except for the percentage of ash (*p* > 0.05; Table 3), statistical di fferences in the biochemical composition of the autotrophic and heterotrophic cultures of *T. suecica* were found (*p* < 0.05; Table 3).

**Table 3.** Content of proteins, carbohydrates, lipids, ash and moisture in the biomass from autotrophic and heterotrophic cultures of *T. suecica* (% of dry weight (DW); mean ± SD; *n* = 3). Asterisks denote significant differences (*p* < 0.05, Student's *t*-test).


The percentage of proteins in *T. suecica* statistically increased from 16.76% in autotrophy to 20.78% in heterotrophy (*p* < 0.05; Table 3). To the best of our knowledge, no studies regarding protein increase with respect to heterotrophic cultures has been reported; however, Cid et al. [37] showed that the addition of organic compounds to the culture medium increased the protein fraction in *T. suecica* mixotrophic cultures. El-Sheekh et al. [16] reported a significant increase in the percentage of proteins in mixotrophic cultures of *Chlorella vulgaris* and *Scenedesmus obliquus* with the addition of hydrolyzed wheat bran with respect to autotrophy. Canelli et al. [38] mentioned that heterotrophic cells convert the storage of cellular nitrogen into proteins, and when this nitrogen reserve is depleted, the consumption of intracellular carbon begins to increase the protein fraction. However, it is important to consider that a low C/N ratio induces protein accumulation [39].

In the case of lipids and carbohydrates, an increase in heterotrophic cultures of *T. suecica* is observed with respect to autotrophic cultures (*p* < 0.05; Table 3). In this sense, Azma et al. [12] observed that the heterotrophic culture of T. suecica presented a higher percentage of lipids with respect to the autotrophic culture. Furthermore, similar results were observed in heterotrophic cultures of *Chlorella protothecoides* and *C. vulgaris*, with lipid accumulations between 50–60% in the biomass [11,13]. Similar results of carbohydrate accumulation (>45%) in dry weight were observed for these microalgae species [15]. In our study, *T. suecica* increased in a greater percentage the carbohydrate content in relation to the lipids (Table 3). This could be because there was probably a depletion of N, observed by the high C/N index (Table 2). High growth rates in heterotrophic cultures led to nutrient depletion, decreasing cell division and allow them to accumulate carbon for the synthesis of lipids or carbohydrates [40]. Another factor could be the nitrogen deficiency, which induced the increase of lipids in the biomass [41]. Furthermore, Garcia-Ferris et al. [41] observed that, in periods of nitrogen starvation and heterotrophy, there was a decrease in the size of the chloroplast of *Euglena gracilis*. Additionally, in our study was observed a reduction in chloroplast size in the heterotrophic culture (data not shown). Similar results were observed by Gladue and Maxey [14] for *Tetraselmis* sp. in a heterotrophic culture.

### *2.4. Phenolic Compounds and Antioxidant Activity of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

The phenol content of heterotrophic cultures of *T. suecica* was higher than autotrophic (*p* < 0.05; Table 4). To the best of our knowledge, our results are the first report of phenol content in heterotrophic cultures for the species under study. In heterotrophy, the mechanisms of accumulation and antioxidant response are related to nutritional stress [42]. Phenolic compounds are a defense mechanism against the excess of oxygen produced in photosynthesis, and the depletion of nutrients causes their accumulation [42]. Quiñones-Galvez et al. [43] showed that the heterotrophic cultivation of calluses of *Theobroma cacao* increased the phenolic content, due to the osmotic stress caused by the addition of glucose, favoring their synthesis and accumulation. This increase in phenolic compounds in heterotrophy was also observed for *C. vulgaris* and *S. obliquus* [44].

**Table 4.** Total phenolic content and antioxidant capacity measured by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay in the biomass from autotrophic and heterotrophic cultures of *T. suecica* (mean ± SD; *n* = 3). Asterisks denote significant differences (*p* < 0.05, Student's *t*-test). TE: Trolox equivalents.


The increase in antioxidant activity is due to the fact that the photosystem II of the cells produces reactive oxygen species (ROS), caused by the photosynthetic process [45]. However, in the adaptation of autotrophy to heterotrophy, photosystem II is reduced due to its low photosynthetic activity [46], decreasing the chlorophyll, carotene and phycobiliprotein contents, which are related to the nitrogen availability, and causing alterations in the electron transport system, leading to an increase in antioxidant activity [47,48].

The 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) method measures hydrophilic and lipophilic antioxidants [49]. *T. suecica* increased the antioxidant activity measured by the ABTS method in heterotrophic cultures with respect to autotrophic (*p* < 0.05; Table 4). A positive correlation was found in the heterotrophic culture of *T. suecica* between ABTS and proteins, lipids, carbohydrates, total carbon (TC) and total nitrogen (TN) (Supplementary Table S1). This could be because the method also measures fat-soluble antioxidants (carotenoid, chlorophylls, vitamin E or tocopherols, PUFAs and polysaccharides) that are part of the biomass [49]. Although, in this study, no analysis of fatty acid composition was performed, an increase in the content of PUFAs in an heterotrophic cultivation of *T. suecica* has been reported [14,20], which would indicate that the increase in fatty acid composition is related to higher antioxidant activity [50].

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method measures the reducing capacity of the hydrophilic fraction of the compound [51]. In our study, *T. suecica* increased the antioxidant capacity in heterotrophic cultures with respect to autotrophic measured by the DPPH method (*p* < 0.05; Table 4). A positive correlation was found in the heterotrophic culture of *T. suecica* between the DPPH and phenolic content, lipids and TC (Supplementary Table S1). Therefore, it is attributed that the increase in antioxidant activity by the DPPH method is related to the content of phenols, because this assay performs a better measurement of hydrophilic compounds. Significant correlations have been observed in macroalgae [52]; however, in microalgae so far has not been found a correlation between the phenol content and DPPH, so this study shows the first evidence for a heterotrophic culture of *T. suecica*. Other authors di ffer from this relation, indicating that the variety of specific phenolic compounds in microalgae must be understood to which these correlation di fferences are attributed [48,53]. However, a synergistic e ffect among other compounds or substances could be involved in the antioxidant activity of microalgae, so future research would focus on correlating the increase in antioxidant activity in heterotrophic cultures with other variables involved in this type of condition.

### *2.5. Pigment Content of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

The heterotrophic culture of *T. suecica* reduced chlorophyll and carotenoid levels to 1% and 12%, respectively (*p* < 0.05), with respect to that observed in autotrophy (Figure 1). Our results are in-line with those by Day and Tsavalos [33], who reported a reduction of chlorophyll levels to 1% and carotenes to 50% of *T. suecica* in heterotrophic culture, changing the cells from green to bright yellow, an adaptation caused by the absence of light and observed in higher plants [54].

**Figure 1.** Pigment content extracted of the biomass from autotrophic and heterotrophic cultures of *Tetraselmis suecica* (mean ± SD; *n* = 3). Asterisks denote significant differences (*p* < 0.05, Student's *t*-test). DW: dry weight, Chl a and Chl b: chlorophyll-a and chlorophyll-b.

The reduction of photosynthetic and auxiliary pigments is related to the absence of light in heterotrophic cultures and to nitrogen depletion [41,55]. The stress generated by the changes in the trophic conditions and nutrients must be evaluated to identify potential microalgae that may produce some pigment of interest under dark conditions.

### *2.6. Production and Extraction of Exopolysaccharides (EPS) of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

The maximum concentration of total and acid exopolysaccharides (EPS) extracted from the heterotrophic culture of *T. suecica* was 4.2 and 8 times higher, respectively, with respect to that obtained in the autotrophic culture (*p* < 0.05; Figure 2).

**Figure 2.** Total and acid exopolysaccharides (EPS) production from autotrophic and heterotrophic cultures of *T. suecica* (mean ± SD; *n* = 3). Different letters indicate significant differences (ANOVA, Tukey's test, *p* < 0.05).

The polysaccharide production for *T. suecica* has mainly focused on intracellular and cell wall polysaccharides [56]. Kashif et al. [57] showed that a treatment with 1-M NaOH in the biomass increased the yield and quality of polysaccharides of *Tetraselmis* sp. Dogra et al. [56] reported that the most efficient extraction of *T. suecica* polysaccharides was in the biomass treated with the Fenton reaction. This reaction increases the productivity of polysaccharides, because it generates oxidative stress to the microalgae biomass. Guzman-Murillo and Ascencio [26] extracted acid EPS from *T. suecica* and *Tetraselmis* sp. in which their maximum concentration was 409 mg L−<sup>1</sup> and 1819 mg <sup>L</sup>−1, respectively, values higher than those obtained in our study. The di fference between our results and the ones previously cited [26] could be due to the di fferent salinities used in the culture media. In our study, we used a salinity of 35 ‰, while the aforementioned authors used salinities of 3–6 ‰, which would indicate that *T. suecica* produces a greater amount of acidic EPS at low salinities. The osmotic adjustment and the regulation of the turgor pressure of the microalgae is a ffected by salinity, since, when it is low, the cellular ionic concentrations increase and their ionic relationships are constant. On the contrary, at salinities greater than 20 ‰, the ionic relationships are variable [58]. It is important to consider that these variations in ionic relationships play a fundamental role in the excretion of polysaccharides. Furthermore, these variations will also depend on the species and its adjustment mechanisms to osmotic stress. For example, in the case of *Botryococcus braunii*, the increase in salinity allowed a greater production of polysaccharides [59]. Therefore, the increase in EPS production in *T. suecica* will depend on the cultivation condition, abiotic factors such as salinity and optimization of EPS extraction methods.

### *2.7. Elemental Analysis of Exopolysaccharides (EPS) of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

The acid EPS obtained from the heterotrophic culture of *T. suecica* showed the highest content of carbon and nitrogen (*p* < 0.05; Table 5), while acid and total EPS obtained from the autotrophic culture of *T. suecica* presented the lowest content of carbon and nitrogen, respectively. Although no sulfur was found in the autotrophic EPS, this element was present in the heterotrophic EPS, being statistically higher in acid EPS (*p* < 0.05). Total autotrophic EPS had the highest C/N ratio (*p* < 0.05), while the lowest C/N ratio was found in acid autotrophic EPS (*p* < 0.05). The EPS C/N ratio of microalgae, including *T. suecica*, has been poorly studied until now; therefore, the study of these relationships should be increased and specified. The sulfur content was only detected in the EPS of heterotrophic cultures. Within these, the acid EPS have a higher sulfur content than the total EPS (*p* < 0.05), mainly due to the fact that the extraction method is aimed exclusively at sulfated EPS.

**Table 5.** Total carbon (TC), total nitrogen (TN), ratio C/N and sulfur (S) (%) obtained in the total and acid polysaccharides extracted from autotrophic and heterotrophic cultures of *T. suecica*. The data represent the average ± standard deviation (*n* = 3). Di fferent letters indicate significant di fferences among polysaccharide types (ANOVA, Tukey's test, *p* < 0.05).


*2.8. Antioxidant Activity of Exopolysaccharides (EPS) of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

The total and acid heterotrophic EPS were 1.8 and 2.2 times higher than the autotrophic ones, respectively (*p* < 0.05; Figure 3).

**Table 6.** Percentage

 of principal

**Figure 3.** Antioxidant activity of total and acid exopolysaccharides from autotrophic and heterotrophic cultures of *T. suecica*. The antioxidant activity is expressed as micromoles of Trolox equivalents per gram of dry weight (μmol TE g – 1 DW) (mean ± SD; *n* = 3). Different letters indicate significant differences (ANOVA, Tukey's test, *p* < 0.05).

The EPS from marine microalgae have shown the ability to protect oxidative stress, avoiding the accumulation of free radicals and reactive oxygen species (ROS) [28]. Dogra et al. [56] and Kashif et al. [57] reported the reducing capacity to eliminate radicals generated by ABTS, DPPH and FRAP methods for total EPS in the autotrophy of *Tetraselmis* sp. However, in the present study, it was only measured by the ABTS method, in which the total autotrophic EPS presented similar results. In the case of acid autotrophic EPS, and acid and total heterotrophic EPS, to the best of our knowledge, there are no previous references to this study, this being the first report of antioxidant activity for these types of *T. suecica* exopolysaccharides.

The antioxidant activity could be related to the percentage of galacturonic and glucuronic acids present in the constitution of the EPS of *T. suecica* (see Section 2.9). The heterotrophic EPS of *T. suecica* had sulfate in their constitution (Table 6), and, according to Mendiola et al. [30] and Sun et al. [60], the content of uronic acids and sulfate are related to an increase in the reducing capacity of free radicals [28]. The increase of these elements in the constitution of the heterotrophic EPS with respect to the autotrophic ones of *T. suecica* contributed to the increase of antioxidant activity. Possible phenols from ESPs were removed by precipitation with polyvinylpyrrolidone. It should be noted that it was not measured by the DPPH method, because its extraction is carried out in organic solvent, so the EPS immediately precipitated.

extracted from autotrophic and heterotrophic cultures of *T. suecica.* **Monosaccharide Autotrophic Total (%) Autotrophic Acid (%) Heterotrophic Total (%) Heterotrophic Acid** ---

 obtained in the total and acid

polysaccharides

monosaccharides


### *2.9. Fourier-Transform Infrared Spectroscopy (FTIR) of Exopolysaccharides (EPS) of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

FTIR spectroscopy of exopolysaccharides obtained from autotrophic and heterotrophic cultures of T. suecica showed the presence of various functional groups in all samples, such as hydroxyl or carbonyls groups (Figure 4). Although the autotrophic EPS of *T. suecica* did not present the sulfate group peak (Figure 4A,C), the heterotrophic ones did (Figure 4B,D). To the best of our knowledge, this is the first characterization of total and acid EPS extracted from autotrophic and heterotrophic cultures of *T. suecica*.

**Figure 4.** Fourier-transform infrared spectroscopy (FTIR) spectra of (**A**) total EPS obtained from the autotrophic culture of *T. suecica*, (**B**) total EPS obtained from the heterotrophic culture of *T. suecica*, (**C**) acid EPS obtained from the autotrophic culture of *T. suecica* and (**D**) acid EPS obtained from heterotrophic culture of *T. suecica.*

Different absorbance peaks were observed in the spectra of the autotrophic and heterotrophic EPS, indicating their functional groups (Figure 4). The strongest and widest signals were located between 3000 and 3500 cm<sup>−</sup>1, which were attributed to the vibration of the -OH and -NH2 groups, followed by -CH2-methyl residues between 2800–2950 cm<sup>−</sup><sup>1</sup> characteristic of polysaccharides [61–63]. The peaks located between 1500 and 1700 cm<sup>−</sup><sup>1</sup> were due to the vibrations of the C=O groups and the stretching of C-N and the bending of NH [56,62,63]. The peak corresponding to the sulfate groups (S=O) was found between 1370–1240 cm<sup>−</sup><sup>1</sup> [64,65], which is characteristic of sulfated polysaccharides in marine microalgae [66]. The polysaccharides presented in the fingerprint zone from 1400 cm-1 to 700 cm<sup>−</sup>1, presenting various stretching and deformations corresponding to the polysaccharides bonds (C-O-C, C-O-P, C-N and P=O) [67].

Dogra et al. [56] and Kashif et al. [57] carried out the FTIR analysis to the soluble fraction of polysaccharides of *Tetraselmis* sp. biomass in which they found the peak of 1650 cm<sup>−</sup><sup>1</sup> of vibrations of C=O in accordance with the present investigation. Furthermore, they found peaks between 1068–1079 cm<sup>−</sup><sup>1</sup> indicating -COOH with α helix amino acids (low molecular weight proteins) and 1049 cm<sup>−</sup><sup>1</sup> attributed to an aliphatic group with a possible increase in antioxidant activity. These two peaks were not shown in the present work. According to Meng et al. [68], the FTIR method is a validated spectroscopic method, which characterizes algae polysaccharides, determines variations of other primary metabolites and evaluates the physiology of microalgae. This characterization method could be used to temporarily observe the EPS excretion dynamics, and the comparison of the

spectra with digital libraries would allow the identification of fractions of biotechnological interest and industrial application.

### *2.10. Gas Chromatography—Mass Spectrometry (GC-MS) of Exopolysaccharides (EPS) of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

In the GC-MS spectrum of total EPS extracted from the autotrophic culture of *T. suecica*, the highest peak corresponds to galactopyranoside with a retention time of 27.37 min, followed by glucose, galactose and glucuronic acid (Supplementary Figure S1). Other minor monosaccharides (mannose, arabinose and ribose) were identified.

In the GC-MS spectrum of acid EPS extracted from the autotrophic culture of *T. suecica*, the highest peak corresponds to glucose with a retention time of 28.93 min, followed by galactopyranoside, glucuronic acid and galactose (Supplementary Figure S2). Other minor monosaccharides were identified as xylose, galacturonic acid, mannose and ribose.

In the GC-MS spectrum of total EPS extracted from the heterotrophic culture of *T. suecica*, the highest peak corresponds to mannose with a retention time of 26.05 min, followed by glucose, glucuronic acid and rhamnose (Supplementary Figure S3). Other minor monosaccharides (galactose, galacturonic acid, ribose, and fucose) were identified.

In the GC-MS spectrum of acidic EPS extracted from the heterotrophic culture of *T. suecica*, the highest peak corresponds to mannose with a retention time of 26.06 min, followed by glucose, glucuronic acid and galactopyranoside (Supplementary Figure S4). Other minor monosaccharides were identified as galacturonic acid, galactose, fucose, ribose and xylose.

According to our revision, to the best of our knowledge, the monosaccharides of the EPS of *T. suecica* have not been previously characterized. However, intracellular and cell wall polysaccharides of *T. suecica* have been characterized as having 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) (54%), 3-deoxy-lyxo-2-heptulosaric acid (Dha) (17%), galacturonic acid (21%) and galactose (6%) by GC-MS [69]. For *Tetraselmis striata*, similar cell wall monosaccharides were described by NMR spectroscopy [70–72]. Dogra et al. [56] carried out the high-performance anion exchange chromatography with a pulsed amperometric detector (HPAEC-PAD) analysis to the soluble fraction of polysaccharides of *Tetraselmis* sp. biomass, in which they found that the KCTC 12432 BP strain contained a higher percentage of galactose and glucose in a molar ratio (11.1:8.2) and the strain KCTC 12236 BP showed the peaks of rhamnose, galactose, glucose, mannose and xylose. Therefore, the constitution of the EPS of *T. suecica* is di fferent from the intracellular and cell wall polysaccharides. However, they have a similar monosaccharide composition with the soluble fraction of polysaccharides from *Tetraselmis* sp.

The EPS from autotrophic and heterotrophic cultures of *T. suecica* are composed in a higher percentage of glucose (23–37%), glucuronic acid (20–25%), mannose (2–36%), galactose (3–25%) and galactoryranoside (5–27%) and in lower percentages of galacturonic acid (0.1–3%); arabinose (5%); xylose (0.3–3%) and ribose, rhamnose and fucose (1%) (Table 6). Di fferences were found between the monosaccharide constitution of the EPS of autotrophic and heterotrophic cultures of this study. The highest percentage of mannose and fucose were the heterotrophic EPS, while the highest amounts of galactose and glucose were detected in autotrophic EPS. Xylose was present only in acid EPS from both culture conditions, while arabinose and rhamnose were found only in the total autotrophic and heterotrophic EPS.

According to Xiao and Zheng [73], the variations in EPS percentage are due to a nutritional stress of the culture conditions of origin of each one of them. These authors indicated that the di fferences and changes at the physiological level of the microalgae caused by di fferent culture conditions make the microalgae adapt and biosynthesize polysaccharides according to the environmental conditions. Despite the percentages of monosaccharides presented for each EPS, the particular weight of each of the monosaccharides must be analyzed, representing the same high values of uronic acids in heterotrophy (galacturonic and glucuronic acids). According to de Jesus Raposo [28], polysaccharides with high contents of uronic acids present high bioactivity.

### *2.11. Cytotoxic E*ff*ects on Tumor Cells of Exopolysaccharides (EPS) of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

The results obtained in this study showed that the EPS obtained from *T. suecica* in autotrophic and heterotrophic cultures have high cytotoxic effects on tumor cells (Figure 5). In the human leukemia cell line HL-60, inhibitory concentration (IC50) of 36 μg mL−<sup>1</sup> and 68 μg mL−<sup>1</sup> were determined for acidic autotrophic and heterotrophic EPS, respectively, and IC50 of 1784 μg mL−<sup>1</sup> and 5183 μg mL−<sup>1</sup> for total autotrophic and heterotrophic EPS, respectively (Figure 5A). In the breast cancer cell line (MCF-7), the acidic autotrophic and heterotrophic EPS showed IC50 of 60 μg mL−<sup>1</sup> and 141 μg mL−1, respectively, while the total autotrophic and heterotrophic EPS showed lower effects with IC50 of 9461 μg mL−<sup>1</sup> and 9135 μg mL−1, respectively (Figure 5B). In the case of the lung cancer cell line (NCI-H460), the acidic autotrophic and heterotrophic EPS showed high cytotoxic effects with IC50 of 118 μg mL−<sup>1</sup> and 110 μg mL−1, respectively, whilst the total autotrophic and heterotrophic EPS had lower activity with IC50 of 5160 μg mL−<sup>1</sup> and 8000 μg mL−1, respectively (Figure 5C).

**Figure 5.** (**A**). Survival (%) of the human leukemia cell line (HL-60) exposed to different concentrations of EPS from *T. suecica*. (**B**). Survival (%) of the human breast cancer cell line (MCF-7) exposed to different concentrations of EPS from *T. suecica*. (**C**). Survival (%) of the human lung cancer cell line (NCI-H460) exposed to different concentrations of EPS from *T. suecica*.

This is the first evidence that EPS from *T. suecica* have cytotoxic effects on tumor cells. Previously, it has only been demonstrated that acidic EPS from *Tetraselmis* sp. inhibited the adhesion of *Helicobacter pylori* to HeLa S3 cells, indicating a possible prophylactic treatment in microbial infections, although in vivo experimental models are necessary [26]. Microalgae polysaccharides are interesting candidates for antitumor therapies. Polysaccharides from *Tribonema* sp. and *Phaedactylum tricornum* induced apoptosis in the liver cancer cell line (HepG2) [74,75]. Polysaccharides from *Artrosphira platensis* reduced cell proliferation in HepG2 and the breast cancer cell line (MCF-7) [76]. Other studies have described the antiproliferative activity of EPS from *Porphyridium cruentum* in the human cervical cancer cell line (HeLa) [77], MCF-7 cell line [78] and the inhibition of tumor growth Gra ffi myeloids [32]. Therefore, EPS from marine microalgae can be used as functional ingredients in foods or possible nutraceuticals to decrease the likelihood of tumor formation and development in the human body.

### *2.12. Cytotoxic of Exopolysaccharides (EPS) of Autotrophic and Heterotrophic Biomass Cultures of T. suecica*

The autotrophic and heterotrophic total EPS did not reach the IC50 at the concentrations tested; therefore, they did not have a cytotoxic e ffect on the proliferation of the gingival fibroblast cell line (HGF-1) (Figure 6). However, the autotrophic and heterotrophic acid EPS showed cytotoxicity e ffects with IC50 of 165 μg mL−<sup>1</sup> and 61 μg mL−1, respectively (Figure 6). The elemental characteristic of cancer chemotherapeutics is that the compounds used do not a ffect the normal cell growth and have specific cytotoxicity [79]. Gingival fibroblast cell line (HGF-1) is a representative mammalian cell line that has been used for the investigation of anticancer activity [80]. Based on our results, the acids EPS showed high cytotoxicity; therefore, they are not suitable for therapeutic use. In contrast, the total EPS did not show cytotoxicity, so they could be a good candidate for anticancer investigation. However, it is important to deepen the studies and test other types of healthy cell lines.

**Figure 6.** Survival (%) of the human gingival fibroblast cell line (HGF-1) exposed to di fferent concentrations of EPS from *T. suecica***.**
