Fig. 3: Cell performance of the transgenic strains compared with the WT strain.

a Spot culture with serial dilution on agar medium (supplemented with acetic acid) at pH 5.5. b The pH tolerance during liquid cultivation (pH 5.5; supplemented with acetic acid) is markedly enhanced via PMA4∆Cter expression. Shown are data from duplicate cell culture. The cell states at Day 2 of WT, PMA4∆Cter-V4, and PMA4∆Cter-V10 are displayed from the far left of the top panel. c The photoautotrophic biomass production (upper panel) and CO₂ fixation rate (R_CO2; lower panel) are improved under extremely high CO₂ conditions (20% CO₂). The two graphs share the same timescale. The arrows indicate the time points (distinguished by the colors) that the cell lines reached their maximum concentrations. Data from duplicate cell cultures are shown. Each dot in the lower panel depicts two biological replicates. d Per-cell gross photosynthetic rate of the strains exposed to high CO₂. The parameter was estimated as light-dependent O₂ evolution plus dark respiration. The double asterisks indicate statistically significant differences in the gross photosynthetic rate of each strain under the high CO₂ environment compared with those under the ambient conditions (p < 0.05). Each bar represents the mean ± SD of three independent experiments. e Box and whisker plots demonstrating the pH_i of the microalgal strains exposed to high CO₂ conditions derived from four biological replicates. The median (the center line) ± whiskers (1.5× the interquartile range from the lower and upper quartiles) with the interquartile range (the boundaries) are provided. The double asterisks indicate statistically significant differences compared with the pH_i of the WT strain after 2 h of exposure to CO₂ (p < 0.05). N.S. represents not significant. f In vitro ATPase activity assay using isolated PM-enriched fraction samples. The double (p < 0.05) and triple (p < 0.01) asterisks denote statistically significant differences compared with the ATPase activity of the WT strain, respectively. Each bar represents the mean ± SD of four independent experiments. In d, e, and f, the statistically significant differences were determined by a two-tailed Student’s t test. g Representative image of bromocresol purple-mediated in vivo ATPase activity test. Source data are provided as a Source Data file.