Re-examining extreme carbon isotope fractionation in the coccolithophore Ochrosphaera neapolitana

Laboratory culture

Ochrosphaera neapolitana (RCC1357) was precultured in K/2 medium without Tris buffer⁸ using artificial seawater (ASW) supplemented with NaHCO₃ and HCl to yield an initial DIC of 2050 µM. In triplicate, 1-L bottles were filled with 150 mL of seawater medium with air in the bottle headspace and inoculated with a mid-log phase preculture at an initial cell concentration of 10⁴ cells mL⁻¹. Cultures were grown at 18 °C under a warm white LED light at 100 ± 20 µE on a 16h-light/8h-dark cycle. Bottles were orbitally shaken at 60 rpm to keep cells in suspension. Cell growth was monitored with a Multisizer 4e particle counter and sizer (Beckman Coulter). At ~1.4 × 10⁵ cells mL⁻¹, cells were diluted up to 300 mL to 2–3 × 10⁴ cells mL⁻¹ and harvested after 2 days of more exponential growth up to 7.9 ± 0.6 × 10⁴ cells mL⁻¹. More detailed culture results are listed in the Supplementary Note 1.

Immediately after harvesting, pH was measured using a pH probe calibrated with Mettler Toledo NBS standards (it should be noted here that high ionic strength calibration standards would be optimal for pH measurement of liquids like seawater). There was a carbonate system shift during the batch culture and more details are shown in Supplementary Fig. S1. Cells in 50 mL were pelleted by centrifuging at ~1650 × g for 5 min. Seawater supernatant was analyzed for DIC and δ¹³C_DIC by injecting 3.5 mL into an Apollo analyzer and injecting 1 mL into He-flushed glass vials containing H₃PO₄ for the Gas Bench.

For seawater DIC, an Apollo SciTech DIC-C13 Analyzer coupled to a Picarro CO₂ analyzer was calibrated with in-house NaHCO₃ standards dissolved in deionized water at different known concentrations and δ¹³C values from −4.66 to −7.94‰. δ¹³C_DIC in media were measured with a Gas Bench II with an autosampler (CTC Analytics AG, Switzerland) coupled to ConFlow IV Interface and a Delta V Plus mass spectrometer (Thermo Fischer Scientific). Pelleted cells were snap-frozen with N₂ (l) and stored at −80 °C. For PIC analysis, pellet was resuspended in 1 mL methanol and vortexed. After centrifugation, the methanol phase with extracted organics was removed and the pellet containing the coccoliths was dried at 60 °C overnight. About 300 mg of dried coccolith powder were placed in air-tight glass vials, flushed with He and reacted with five drops of phosphoric acid at 70 °C. PIC δ¹³C and δ¹⁸O were measured by the same Gas Bench system. The system and abovementioned in-house standards were calibrated using international standards NBS 18 (δ¹³C = −5.01‰, δ¹⁸O = +23.00‰) and NBS 19 (δ¹³C = +1.95‰, δ¹⁸O = +2.2‰). The analytical error for DIC concentration and δ¹³C is <10 μM and 0.1‰, respectively.

POC and PON were determined from cells harvested on pre-combusted QFF filters and deep-frozen until analysis. Inorganic carbon from cells on filters was removed by fuming sulfurous acid during 24 h. Filters were placed inside a desiccator on a porous tray and 50 mL sulfurous acid below was fumed with a vacuum pump. Gases were evacuated and filters were further dried at 60 °C overnight. Right before Elemental Analysis (EA), filters were compacted and wrapped into tin cups with the help of tweezers and a press. Samples loaded on a 96-well plate were combusted in the oxidation column at 1020 °C of a Thermo Fisher Flash-EA 1112 coupled with a Conflo IV interface to a Thermo Fisher Delta V-IRMS (isotope ratio mass spectrometer). Combustion gas passed through a reduction column at 650 °C producing N₂ and CO₂ which were separated by chromatography and into a split to the IRMS for an on-line isotope measurement.

Simulations of carbon isotope evolution during aeration

The DIC carbon isotope evolution model is simplified from the model in Zhang et al.⁷ The exchanging rate (with a unit of mol s⁻¹) between CO₂(g) and CO₂(aq) depends on the CO₂ gradient and exchanging rate constant (k_E, with a unit of ppm s⁻¹): ER =  k_E(CO_2(g)–k_HCO_2(aq)), where the k_H is Henry’s law constant with a unit of ppm µM⁻¹. The evolutions of DIC and DIC carbon isotope ratios during CO₂ aeration can be calculated by four differential equations:

where capital letters G, C, B, H, and OH represent CO₂(g), CO₂(aq), HCO₃⁻ + CO₃²⁻, H⁺, and OH⁻, respectively. The V stands for volume. The α is the isotopic fractionation, e.g. α_g2aq represents the carbon isotope fractionation of CO₂ gas diffusion into liquid phase. The XB1 and X¹³B1 are the fraction of HCO₃⁻ in (HCO₃⁻ + CO₃²⁻) and the fraction of H¹³CO₃⁻ in (H¹³CO₃⁻ + ¹³CO₃²⁻). The XB1 can be calculated by ({{{{{rm{XB}}}}}}1=frac{1}{1+frac{K2}{left[{{{{{{mathrm{H}}}}}}}^{+}right]}}) and the X¹³B1 can be calculated by ({{{{{{rm{X}}}}}}}^{13}{{{{{mathrm{B}}}}}}1=frac{1}{1+frac{K2}{left[{{{{{{mathrm{H}}}}}}}^{+}right]}{alpha }_{{{{{{{mathrm{CO}}}}}}_{3}}-{{{{{{mathrm{HCO}}}}}}_{3}}}}), where the ({alpha }_{{{{{{{mathrm{CO}}}}}}}3-{{{{{{mathrm{HCO}}}}}}}3}) is the carbon isotope fractionation between CO₃²⁻ and HCO₃⁻⁹. The k₊₁ is the reaction rate constant of CO₂ hydration, which can be calculated by ({{{{{rm{ln}}}}}}{k}_{!+1}=1246.98-,frac{61900}{{T}_{k}}-183{{{{{rm{ln}}}}}}{T}_{k})¹⁰. The k₋₁, the reaction rate constant of HCO₃⁻ dehydration, can be calculated from k₊₁, ({k}_{-1}=,frac{{k}_{+1}}{K1}) . The k₋₄ and k₊₄ are the reaction rate constants of CO₂ hydroxylation and HCO₃⁻ dihydroxylation. The k₊₄ is calculated by(,{{{{{rm{ln}}}}}}{k}_{+4}=17.67- ,frac{2790.47}{{T}_{k}})¹⁰ and ({k}_{-4}={k}_{+4}frac{{K}_{{{{{{mathrm{w}}}}}}}}{K1}), where K_w is the stoichiometric ion product of water. The K1 and K2 are the first and second dissociation constants of carbonic acid and in this study, we employed equations from¹¹, in which the K1 and K2 were calculated for pH in NBS scale. The reaction rate constants for ¹³C (({k}_{-1}^{13}), ({k}_{+1}^{13}), ({k}_{-4}^{13}) and ({k}_{+4}^{13})). The initial values for these differential equations are described in the Supplementary Note 2.

Source: Ecology - nature.com