Dataset: BioLog Eco-plates experiment - 14C-labeled uptake

We investigated the uptake of specific dissolved organic compounds, which showed high potential for osmotrophy. We selected five ¹⁴C-labeled-DOC compounds based on results of the BioLog Eco-plates survey as well as commercial availability of radiotracer-labeled compounds. The selected compounds included sugar alcohols (glycerol and mannitol), carbohydrate (xylose), and amino-acid (arginine). Additionally, we selected acetate due to its biochemical importance and availability in marine ecosystems (Ho et al. 2002; Wu et al. 1997). Specific activities of the radiotracers were: glycerol - 160 µCi µmol⁻¹, mannitol - 57 µCi µmol⁻¹, xylose - 200 µCi µmol⁻¹, arginine - 338 µCi µmol⁻¹, and acetate - 52 µCi µmol⁻¹ (acetic acid sodium salt) (PerkinElmer, Inc. Waltham, MA). As a reference uptake compound we used ¹⁴C-bicarbonate (56 µCi µmol) (MP Biomedicals, LLC, Santa Ana, CA, USA) incubations in photosaturated light conditions. We performed radiolabel uptake experiments on axenic coccolithophore strains, CCMP289 Cruciplacolithus neohelis and CCMP3337 Chrysotila carterae (NCMA lists the strain as Pleurochrysis carterae). We maintained the cultures in media and light conditions as described above, and at 22°C (CCMP289) and at 16°C (CCMP3337).

For the survey of arginine and xylose net uptake in darkness, we prepared two 70 mL master samples (concentration of 1×10⁵ cells L⁻¹) of CCMP289 and CCMP3337 cultures in log phase growth. We measured cell concentrations using a haemocytometer on an American Optical Microscope (Spencer Lens Company, Buffalo, N.Y.) with polarization optics. We added unlabeled arginine or xylose to each strain’s master sample up to a 20 µM final concentration. From each master sample, 10 mL were then removed into separate borosilicate vials that were kept in the dark for subsequent cell counts over the duration of the experiment. To the remaining 60 mL culture samples containing unlabeled arginine or xylose, we added ¹⁴C-arginine or ¹⁴C-xylose, to a final concentration (labeled and unlabeled) of 20.25 µM and 20.83 µM, respectively. We withdrew 45 mL of the 60 mL sample and divided that into three 15 mL replicate vials. We transferred the remaining 15 mL into a fourth vial with buffered formalin as a formalin-killed control. Due to logistical issues in sample manipulation, the actual time of addition of the first ¹⁴C-labeled compound was 10±5 min after addition of formalin to the labeled control. We then subsampled and filtered all 16 vials (12 samples (triplicates of the two ¹⁴C-labeled compounds x two strains) and 4 formalin samples (two compounds x two strains)). After the first time point, we placed samples in the dark incubators at 22°C for CCMP289 and 16°C for CCMP3337. Subsampling for each time course experiment was performed at 3 h, 6 h, 24 h, and 48 h. For subsampling, we performed filtration of each 2 mL of culture subsamples onto each 0.4 µm pore-size, 25 mm diameter polycarbonate filter. Following filtration, filters were carefully rinsed three times with ASW (including a careful rim rinse) to remove any ¹⁴C-labeled, dissolved compound left on the filter. Each filter was then placed in the bottom of a clean scintillation vial, and scintillation cocktail was added (Balch et al., 2000).

We also examined the net uptake of ¹⁴C-arginine and ¹⁴C-xylose uptake in illuminated cultures over 24 hours. We added these ¹⁴C- labeled compounds to axenic cultures (CCMP289 or CCMP3337) to a final concentration of 0.37 µM for ¹⁴C-arginine and 1 µM for ¹⁴C-xylose. We sampled at T15 min and T24 h, stopping the incubation by filtration, and measured the ¹⁴C uptake as described above.

Furthermore, we examined the net uptake of ¹⁴C-acetate, ¹⁴C-glycerol, and ¹⁴C-mannitol in darkness over 24 h and compared it with ¹⁴C-bicarbonate net uptake (in light). Prior to addition of radiolabeled compounds, axenic cultures (CCMP289 or CCMP3337) were divided into separate vials and 5 mL of log-phase culture were removed for the enumeration of cell concentration. To correct for any effects due to EtOH solvent in the ¹⁴C-acetate, in one 5 mL sample we added only 0.0125 mL of EtOH as a control. We added ¹⁴C- labeled compounds to each separate vial to a final concentration of 4.81 µM of ¹⁴C-acetate, 1.49 µM of ¹⁴C-glycerol, 4.18 µM of ¹⁴C-mannitol, and for comparison we used ¹⁴C-bicarbonate to a final concentration of 2.6 mM of labeled and unlabeled form. Triplicate samples for uptake measurements were filtered after 15 min and 24 h of darkness.