Mc 1000 multicultivator

Cultures of each strain were harvested in exponential growth phase by centrifugation (6 min, 3700g), washed repeatedly with the treated wastewater medium, and resuspended in it. Initial cultures for the experiments were then prepared by adjusting the optical density (OD₆₃₀) of the suspensions to 0.06 and placing 60 mL aliquots in tubes designed to fit slots in a MC 1000 multi-cultivator (Photon System Instruments, Drásov, Czech Republic). Each species was grown in triplicates at 50, 150 and 300 μE m⁻² s⁻¹ (16 h light: 8 h dark photoperiod). The temperature was 25 ± 2 °C and the tubes were aerated (0.1 L min⁻¹). The algae were grown under these conditions for either 8 or 15 days, and OD₆₃₀ was measured daily to monitor their growth. Samples (50 mL) were then harvested by centrifugation at 3700 g for 6 min and freeze-dried for 3 days. Freeze-dried algae were used for lipid extraction and FTIRS analysis. The amounts of nitrogen and phosphorus present in the wastewater were determined before and after each experiment using LCK 138 and LCK349 kits, respectively, and a DR 3900 spectrophotometer, operated according to the manufacturer’s manual (Hach Lange, Germany).

The wild-type Synechococcus elongatus UTEX 2973 and constructed mutant strains in this study (listed in Table S1) were grown as described in Ungerer et al. (20 (link)) unless otherwise indicated. Cultures were maintained on BG11 agar plates at 38°C, 150 μmol photons m⁻² s⁻¹. To determine the effect of high light stress conditions on gene expression, cells were grown to midlogarithmic growth phase (optical density at 730 nm of 0.6–0.8) in an MC-1000 multicultivator (Photon Systems Instruments, Czech Republic), and diluted with fresh medium to an OD 730 of ~0.3 before exposure to high light (1,500 μmol photons m⁻² s⁻¹) for 30 min, 1 h, and 2 h, when cells were harvested. Growth experiments were repeated at least three times to confirm the growth patterns. Doubling times were calculated by fitting an exponential curve to the logarithmic section of the growth data (typically OD₇₂₀ of <0.3) and using the slope, m, as K′ (y = ke^mx). Doubling times were then calculated as ln(2)/K′. Mean doubling times were compared to Synechococcus 2973 using a one-way ANOVA and Dunnett’s multiple-comparison test.