Abstract
Lower pH and elevated temperature alter phytoplankton growth and biomass in short-term incubations, but longer-term responses and adaptation potential are less well-studied. To determine the future of the coccolithophore Emiliania huxleyi, in subantarctic waters, a mixed genotype culture was incubated for two years. E. huxleyi was isolated from subantarctic waters at the end of the Munida Transect (Currie et al. 2011) east of New Zealand (-45.829 171.532) on 3rd June 2014. The coccolithophore was isolated in Aquil medium but then maintained in a 10-fold dilution of the recommended addition of Guillards f/2 (Sigma G0154) to natural seawater supplemented with additional nutrients to nitrate 96 μM and phosphate 6 μM (f/20). In this medium, the strain continued to calcify throughout the experiment after treatment with the antibiotics penicillin, streptomycin and neomycin (Sigma P4083) at the recommended dosage to remove bacteria prior to the start of the incubation (20th November 2015). E. huxleyi was incubated for two years under present-day summer temperature and pH (11°C and pH 8.1; Now), and also projected future conditions by the year 2100 (14°C and pH 7.8; Future; Law et al. 2018). The pH of the medium was amended by bubbling with 10% CO₂ prior to cells being added. Special air mixes (21% oxygen in nitrogen, with 380 ppm CO₂ for Now cultures and 750 ppm CO₂ for Future cultures; BOC Gas NZ) were passed through an inlet into the culture bottle headspace via a 0.22 μm syringe filter (Millipore) to maintain target pH with an exhaust with 0.22μm filter attached to relieve pressure. The cultures were maintained at 60 µmol m⁻² s⁻¹ in a 12 hour: 12 hour light: dark cycle. Cultures were grown in a semi-batch style to ensure the cells remained in exponential growth, with cell concentration maintained at low levels (<80000 cells ml⁻¹) to maintain pH at the target value. Growth rates of each population were measured throughout the experiment (20th November 2015 - 1st December 2017) and calculated by least squares regression of the natural logarithm of in vivo fluorescence versus time during exponential growth. At five time points, some cells from the Now culture were moved to Future conditions (Now in Future) and vice versa (Future in Now) with growth rates and cell sizes being determined in the Now, Future, Now in Future and Future in Now cultures. At the final cross-over, additional measurements were made - cell chlorophyll, inorganic carbon and organic carbon, nitrogen and phosphorus contents. From these measurements cell ratios were calculated. In addition, the uptake of inorganic carbon (C-14) and calcium (Ca-45) were measured to indicate primary production and calcification rates. At the end of the incubation a temperature response experiment was performed with the Now and Future populations measuring the cell growth rates at a range of temperatures covering current and projected conditions in the subantarctic waters. The experiments were carried out in the Department of Chemistry, University of Otago, New Zealand.