4.1. Microalgal strain and culture conditions
The microalgal strain used in this work was S. obliquus C1S was isolated from an artificial freshwater reservoir in Southeastern Buenos Aires, Argentina (38°0’0”S 57°33’0”W), in 2010[38].
S. obliquus C1S was routinely maintained in BG11 medium[39] solidified with 1% (w/v) agar-agar. For the preparation of inoculums, S. obliquus cells were cultivated in 500 mL sterilized glass bottles containing 300 mL of modified BG11 medium supplemented with 6mM NaNO3 as a N source and 0.42 g · L-1 NaHCO3 to buffer CO2 supplementation. Cultures were sparged with filtered air containing 2% (v/v) CO2 at 0.3 – 0.5 L · min−1 at 29 ± 1 °C under constant white light at 310 µmol photons · m-2 · s-1.
For cultivation in environmental photobioreactors, two Phenometrics™ ePBR laboratory benchtop photobioreactors [24]were used. Initially, the ePBRs were inoculated with S. obliquusC1S at an OD750 of 0.3 and cells were acclimated at the testing weather-simulated conditions for one to two weeks depending on the apparent doubling time at the stated conditions. Then, cells were collected by centrifugation at 3,900 x g for 10 min and fresh medium was inoculated with acclimated cells at an initial OD750 of 0.3 for data collection. In addition to the magnetic bar of the ePBRs, sterile air was moistened in a water trap, to minimize evaporation, and bubbled at 0.3 – 0.5 L · min-1 to help achieving homogeneous mixing. When supplemented with CO2, BG11 medium additionally contained 0.42 g · L-1 NaHCO3 to avoid sudden shifts in pH, and the ePBRs were set to inject pure CO2 into the water trap for its dilution in air at an output concentration of 2% (v/v) from 1 h after to 1 h before the light was switched on and off, respectively.
The simulated weather conditions are sown in Table 1. Raw data were obtained from the NASA Langley Research Center (LaRC) POWER Project funded through the NASA Earth Science/Applied Science Program (https://power.larc.nasa.gov/data-access-viewer/). The average maximum and minimum temperatures and mid-day irradiation in the Southern Hemisphere were calculated considering the mean values of each month and by pondering the number of days of that month in the season. Thus, mean values corresponded to 10 days of month 12, 31 days of month 1, 28 days of month 2, and 21 days of month 3 for summer; 10 days of month 3, 30 days of month 4, 31 days of month 5, and 21 days of month 6 for autumn; 9 days of month 6, 31 days of month 7, 30 days of month 8, and 21 days of month 9 for winter; and 9 days of month 9, 31 days of month 10, 30 days of month 11, and 21 days of month 12 for spring. Maximum irradiation at mid-day was estimated from daily total irradiation and average day length in each location and season.
When ponds’ depth was simulated at 5; 10; or 20 cm, the corresponding volume and area exposed to light were 90; 200; or 460 mL or 25.78; 32.47; or 36.78 cm2, respectively. In each case, the light irradiation on the culture’s surface was calibrated according to the ePBR manufacturer’s instructions.