Macronutrient

Sampling was performed on board of the RSV Aurora Australis (AU1602, AA-V02 2016/17) between 8 December 2016 and 21 January 2017, in one station belonging to the Dalton polynya (D02) and two stations (M36 and M48) located within the Mertz polynya (East Antarctica; 67.2–66.8 ^°S and 119.5–145.8°E; Supplementary Figure S1A). These stations were chosen due to their contrasting biological, chemical, and physical characteristics, extensively described and discussed by Moreau et al. (2019) (link) and Ratnarajah et al. (2022) (link). For comparison with Ratnarajah et al. (2022) (link), D02 refers to St.2; M36 is EM03 and M48 is MG08. Briefly, both polynyas had similar sea surface temperatures ranging majorly between 0.0 and 1.5°C, but the Dalton polynya showed higher sea surface salinity than the Mertz polynya (34.0–34.3 g/kg vs. 32.5–33.5 g/kg, respectively), suggesting that the later could have experienced more sea ice melting than the Dalton polynya (Moreau et al., 2019 (link)). The Dalton polynya presented deeper euphotic depths (95 ± 56 m) and mixed layer depths (25 ± 12 m, excluding two stations where the mixed layer was down to 100 m and 154 m) than the Mertz polynya (40 ± 9 m and 13 ± 1 m, respectively) (Ratnarajah et al., 2022 (link)). In general, chlorophyll-a (Chl-a) concentrations in the surface were higher in the Dalton polynya compared to the Mertz polynya (max of 15 μg L⁻¹ vs. 8 μg L⁻¹), but Mertz presented a subsurface Chl-a maximum of ~10 μg L⁻¹ located between 20 and 70 m depth that was consistent along the whole polynya, whereas in the Dalton that layer was more variable (Moreau et al., 2019 (link)). Both polynyas also presented global differences in nutrient ratios (i.e., Si:N or N:P) suggesting different nutrient sources (i.e., water masses) and different phytoplanktonic communities, something which was also corroborated by microscope analyses (Moreau et al., 2019 (link)). During the time of sampling, the dominant phytoplankton groups differed between both polynyas, with Phaeocystis antarctica dominating in the Dalton station and diatoms dominating in the much more productive Mertz stations (Moreau et al., 2019 (link)).
The sampling and analyses of physicochemical and environmental parameters were performed as described in Moreau et al. (2019) (link) and Ratnarajah et al. (2022) (link). Temperature and salinity were obtained from the CTD (Rosenberg and Rintoul, 2017 ) and fluorescence values were obtained with a fluorometer (ECO-AFL/FL 756, Wetlabs, United States) that was installed also on the CTD rosette. Of particular interest for this study is the concentration of Chl-a, particulate organic carbon (POC) and inorganic nutrients. Chl-a concentrations were obtained from ~500 ml of filtered seawater, extracted with acetone and stored at −20°C for 24–48 h in the dark prior to analysis with a Turner Trilogy fluorometer. POC was determined following Knap et al. (1996) and analyzed using a Thermo Finnigan EA 1112 Series Flash Elemental Analyzer. Concentrations of inorganic macronutrients (nitrate, nitrite and silicic acid) were analyzed after the voyage at the CSIRO laboratory (Hobart, Australia) following the methods described in Murphy and Riley (1962) (link), Armstrong et al. (1967) (link), Wood et al. (1967) (link) and Kérouel and Aminot (1997) (link).

In support of the goal of determining whether cell growth will in fact occur within the AC- PBR and at what level of productivity, two runs with each of the three selected microalgal species were conducted in the respective media. An initial inoculum (O.D._680 nm = 0.6–0.8) of 10% (v/v) was used for all the species examined. Once suitable cellular O.D. was achieved in the shaker culture flasks, each algal suspension was harvested and added to freshly prepared medium, as described in “Microalgae cultivation” section. Prior to addition of the algae, the medium was enhanced by bubbling in filtered compressed CO₂ gas until solution saturation was reached (i.e., dropping pH stabilized) followed by addition of saturated sodium bicarbonate (NaHCO₃ = 48 g/L) until neutral pH was achieved. The final volume was increased stoichiometrically with macronutrient solution based on the amount of added NaHCO₃.
Before AC-PBR test commencement, each air-filled AC-PBR was placed under a UV-C germicidal lamp for 15 min to sterilize the interior volume and prevent contamination. Through a small opening in the AC, approximately 190 mL (working volume) of algal cell suspension were pipetted inside with any extra air removed, and the AC-PBR (total volume 380 mL) was resealed using a VacMaster Pro110 external vacuum sealer machine. The system was checked for leaks.
The AC-PBRs for each algal strain was kept under two rows of LED with light intensity of 180 µmol/m²s with a 16:8-h (light:dark) photoperiod, no shaking at 25 °C. The cultivation was carried till each alga reached early stationary phase (C. vulgaris- 6 days, N. oculata- 8 days and C. cryptica-7 days). Photos of the AC-PBR microalgal cultivation test set-up for each strain can be found in Supplementary Data.