During a two year incubation of a subantarctic E. huxleyi culture (2015-11-20 to 2017-12-01), at the final cross-over point (Day 670) an additional suite of measurements were performed with cells from Now (11°C and pH 8.1) and Future (14°C and pH 7.8) cultures, and also the crossover experiments - Future cells inoculated into Now medium and incubated under Now conditions (Future in Now), and Now cells incubated in Future medium under Future conditions (Now in Future). For in vitro chlorophyll a (chl-a) measurement, chl-a was extracted from the cells for 20 hours in 90 % acetone and the fluorescence, before and after acidification, measured using a Turner 10-AU fluorometer (Parsons et al. 1984; Welschmeyer 1994). Particulate organic carbon (POC) and particulate organic nitrogen (PON) cell content was determined in cells collected on pre-ashed GF/F (Whatman) filters with particulate inorganic carbon (PIC) removed by flooding the filter with 0.1N sulphuric acid on the filtration manifold (Kennedy et al. 2005). Dried filters were packed in tin capsules before analysis using a ThermoFlash 2000 CHN Elemental Analyser by the Campbell Microanalytical Laboratory, University of Otago. PIC was determined by measuring total carbon in a similar way to POC, and then subtracting the POC. Particulate organic phosphorus (POP) content was measured according to the methods of Solorzano and Sharp (1980). Cell counts of 1% glutaraldehyde subsamples were made under an Olympus IX70 inverted microscope after mounting in a nanoplankton chamber (Phycotech). Uptake rates of dissolved inorganic carbon (DIC) into POC (photosynthetic rates) were measured using C-14 labelled sodium bicarbonate (Perkin Elmer; Parsons et al. 1984; Knap et al. 1996). Unlabelled DIC was measured in subsamples of culture fixed with saturated mercury dichloride (HgCl2) that had been stored in glass vials with rubber sealed caps until analysis using an Airica micro DIC system (Marianda, Germany). Calcium (Ca) uptake rate was measured using Ca-45 (Perkin Elmer) as a tracer for total Ca uptake. Briefly, Ca-45 (0.5 – 1 x 10-4 mg) was added to 10 ml culture subsamples and incubated for 6 to 7 hours in the light. Cells were then collected on GF/F filters and the radioactive label counted by scintillation counter (Perkin Elmer) after the addition of Hi-Safe II scintillant (Perkin Elmer). The total calcium uptake was calculated in a similar manner to that for inorganic carbon uptake using C-14 tracer (Knap et al. 1996). The unlabelled calcium content in the culture medium required for this calculation was measured by inductively coupled plasma mass spectrometry in the Trace Element Centre, University of Otago. In a previous experiment measuring Ca-45 only, it was determined that 97% of the Ca-45 taken up by the cells was incorporated into coccoliths (Satoh et al. 2009). Assuming there is no fractionation within the cell then 97% of the total Ca uptake would be used for calcification.
The study was supported by Coasts and Oceans Centre (Strategic Science Investment Fund of the National Institute of Water and Atmospheric Research).
