*2.1. Algae Physiology and Biochemistry during the 24 h Experiment*

Cell abundance for *Tisochrysis lutea* during the 24 h of experiment increased sharply from t0 to t24. The experiment allowed cell concentration to double for the three balloons (Figure 2A). Despite the attention given to homogenization at the time of subculture from inoculum, the second enriched balloon had a cell concentration twice higher than Tl1 and TlT. This difference remained constant during the entire experiment. Cell abundance varied from 4.3 × 106 cells·mL−<sup>1</sup> to 9.5 × 106 cells·mL−<sup>1</sup> for the most concentrated balloon TI2 and from, on average, 2.6 × 106 cells·mL−<sup>1</sup> to 6.4 × <sup>10</sup><sup>6</sup> cells·mL−<sup>1</sup> for the two others (Figure 2A). Despite the concentration differences, the general slopes for the three balloons were very similar (0.22 cells·mL−1·h−<sup>1</sup> for Tl2 and 0.15 cells·mL−1·h−<sup>1</sup> for Tl1 + TlT) (Figure 2A). Bacteria were also found in higher abundance Tl2 (Figure 2B), almost five times higher than in Tl1 and TlT. However, bacteria increased only by a factor of 1.2 for Tl2 between t0 and t24 versus a factor 3.5 in average for Tl1 and TlT. Bacteria concentration was around 6.6 times higher than algae concentration for Tl1 and TlT on average over the 24 h of the experiment. For Tl2, bacteria concentration was 16 times higher than algae concentration at the beginning of the experiment, but this ratio decreased progressively until t24 (8.7 times higher) (Figure 2B).

FSC and SSC were, respectively, considered a proxy of cell size and cell complexity, and FL3 was considered a proxy of chlorophyll content. SSC and red fluorescence (FL3) did not significantly vary during the entire experiment (Bartlett tests, *p* > 0.05 and ANOVA *p* > 0.05) (Table S1). FSC increased slightly with time for the three balloons (Bartlett test, *p* > 0.05 and ANOVA *p* = 0.03) (Table S1). The percentage of dead microalgae (as measured by SYTOX staining assay) remained below 7% for the 24h of the experiment (Table S1).

Particulate organic carbon concentration increased similarly to the cell abundance for the two labelled balloons from 3.6 to 9.0 mmolC·L−<sup>1</sup> for Tl1, from 4.5 to 11.6 mmolC·L−<sup>1</sup> for Tl2, and from 3.4 to 8.2 mmolC·L−<sup>1</sup> for TlT (Figure 3A). Increase in POC, as a function of experiment duration (*R*<sup>2</sup> = 0.81, *p* < 0.0001), occurred at a relatively constant rate of 0.2 mmolC·L−1·h−<sup>1</sup> for Tl1, Tl2, and TlT considered together. Total fatty acid (TFA) concentration also increased for the three balloons (between 0.26 mmolC·L−<sup>1</sup> to 0.60 mmolC·L−<sup>1</sup> for Tl1, from 0.25 mmolC·L−<sup>1</sup> to 0.80 mmolC·L−<sup>1</sup> for Tl2, and from 0.17 mmolC·L−<sup>1</sup>

to 0.55 mmolC·L−<sup>1</sup> for TlT) (Figure 3B). The slope was 15 <sup>μ</sup>molC·L−1·h−<sup>1</sup> (*R*<sup>2</sup> = 0.72, *p* < 0.0001). POC was significantly correlated to cell concentration (*R*<sup>2</sup> = 0.80 *p* < 0.0001). The slope of the relation between POC and cell abundance is a proxy of carbon content per cell for *T. lutea,* which was, on average for the three balloons, equal to 1.07 fmolC·cell−<sup>1</sup> (Figure 3C). TFA concentration was linearly and positively correlated with POC concentration (*R*<sup>2</sup> = 0.73, *p* < 0.0001). The slope of the regression between TFA and POC concentration indicates that TFA represent in average 7.7% of bulk POC (Figure 3D).

**Figure 2.** Temporal dynamics of cell concentrations of the two enriched balloons of *Tisochrysis lutea* (TI1 and TI2, filled black circles and filled gray triangles, respectively) and of the control balloon (TIT, empty squares) (**A**) and corresponding bacteria concentrations (**B**) during the 24 h experiment.

**Figure 3.** Particulate organic carbon (POC) concentration (**A**) of the two enriched balloons of *Tisochrysis lutea* (TI1 and TI2, filled black circles and filled gray triangles, respectively) and of the control balloon (TIT, empty squares) and total fatty acids (TFA) concentration according to culture age in hours (**B**). POC concentration according to algae concentration (**C**). Relation between total fatty acids and POC concentration (**D**). All regressions (dotted lines) have been calculated with data from the three balloons combined together.
