3.3.2. Exopolysaccharides Extraction

Once the autotrophic and heterotrophic cultures reached the stationary phase, they were centrifuged at 3500 rpm for 8 min at 4 ◦C, and the supernatant was used for the extraction of total and acid EPS from *T. suecica*. Before extraction, phenols were removed by precipitation with polyvinylpyrrolidone (Sigma-Aldrich, St. Louis, MO, USA) and centrifugation 4500 rpm, 5 min, 4 ◦C. For this, total EPS were precipitated with the addition of ethanol (*v*/*v*) [82] and acid EPS with N-cetylpyridinium bromide (Cetavlon) (Sigma-Aldrich, St. Louis, MO, USA) 2% ( *w*/*v*) for 24 h [83]. Then, the EPS were centrifuged at 4500 rpm for 5 min, 4 ◦C. The supernatant was discarded, and 10 mL of 4-M NaCl (Sigma-Aldrich, St. Louis, MO, USA) was added. This mix was stirred until completely dissolved. Once cooled, ethanol was placed in a ratio (*v*/*v*) and kept at 4 ◦C for 24 h. After centrifugation at 4500 rpm, 5 min, 4 ◦C, the pellet containing the polysaccharides and salts was placed on a dialysis membrane (Sigma-Aldrich, St. Louis, MO, USA) in a 0.5-M NaCl solution overnight at 4 ◦C. Then, the dialyzed EPS was centrifuged at 4500 rpm for 5 min, 4 ◦C, and washed with absolute ethanol. Finally, acid and total EPS from both culture conditions (per triplicate, *n* = 3) were stored at −80 ◦C and subsequently freeze-dried at −50 ◦C. The EPS were quantified on an analytical balance, and the maximum concentrations obtained in the autotrophic and heterotrophic cultures were compared according to the culture volume and cell density.

### *3.4. Population Parameters (Cell Density, Cell Concentration, Specific Growth Rate, Biovolume and Cell Volume)*

Cell concentration was calculated according to Equation 1 [84]:

$$\text{Cell concentration} = \left(\frac{\mathcal{W}\_1 - \mathcal{W}\_0}{\mathcal{Volume}}\right) \tag{1}$$

where W1 and W0 are the di fferences in total weight of cells g L−<sup>1</sup> at volume filtration.

Through cell quantification in a 100-μm cuvette (Beckman Coulter ™ AccuComp Z2, Indianapolis, IN, USA), at a pre-established dilution of 1/20, the specific growth rate μ (day−1) and the daily doubling of biomass was calculated, and the results were shown as the sum of these with the algorithm of Arredondo and Voltolina [84], summarized in Equation (2):

$$\mu = \frac{\ln(N\_2 - N\_1)}{t\_2 - t\_1} \tag{2}$$

where *N*2 and *N*1 are the density of cells mL−<sup>1</sup> at times *t*2 and *t*1, respectively.

The biovolume was calculated from the mean of the population (μm<sup>3</sup> cell−1) provided by cell quantification and the value of the cell density (cell mL−1) to obtain its final value in μm<sup>3</sup> mL−1.

### *3.5. Total Carbon (C), Hydrogen (H), Nitrogen (N) and Sulfur (S) of Dry Biomass and Exopolysaccharides*

Total carbon (C), nitrogen (N) and sulfur (S) were determined from dry biomass and the extracted polysaccharides using the total combustion technique used in the LECO TruSppec Micro CHNSO-Elemental Analyzer (St. Joseph, MI, USA). This technique is based on the complete and instantaneous oxidation of the sample by pure combustion with controlled oxygen at a temperature of up to 1050 ◦C (C, H, N and S) and pyrolysis at 1300 ◦C (O) for decomposition of O as CO and oxidation to CO2. The resulting combustion products, CO2, H2O, SO2 and N2 are subsequently quantified by selective IR Pleabsorption detector (C, H and S) and TCD (N) di fferential thermoconductivity sensor. The result of each element (C, H, N and S) is expressed in % with respect to the weight of the sample.

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

For the biochemical composition (*n* = 3), total proteins were calculated from the elemental N determination using the N-protein conversion factor of 4.80 reported by Lourenço et al. [85]; lipids were extracted according to the Folch method [86] and carbohydrates according to the phenol-sulfuric procedure [87]. Moisture and ash levels were determined gravimetrically by drying in an oven at 105 ◦C and after incineration in a mu ffle furnace at 550 ◦C, respectively, until constant weight.
