Methodology:

We performed these experiments to determine whether coccolithophores (CCMP289 and CCMP3337) can sustain themselves in darkness by using organic compounds as energy and/or carbon sources.

Sampling and analytical procedures:

First, we prepared 350 ml of L1 medium and log phase cells from each strain. The cell concentrations of CCMP289 and CCMP3337 were 5×104 cells L-1 and 1×104 cells L-1, respectively. We then poured 15 mL aliquots into 16 vials were kept in darkness. Our goal was to determine the effect of concentration on growth: one vial was the control with no organics added, and 5 vials with each organic compound in final concentrations at 10, 30, 100, 300, and 1000 µmol L-1. The experiment lasted for approximately 30 days; temperature and irradiance for the illuminated cultures were the same as for culture growth and maintenance, vials kept in darkness were also kept at the original growth temperature and additionally covered in black aluminum foil, to ensure complete darkness. We sampled the vials for cell counts every 2-3 days, and during sampling we kept light levels corresponding to experimental conditions. This time-course experiment was performed without replicates, however, we took repeated duplicate samples for cell counts (technical duplicates). Cell concentration was determined using a hemocytometer on an American Optical Microscope (Spencer Lens Company, Buffalo, NY, USA) with polarization optics for CCMP289, as well as a Moxi Z Cell Counter (Andwin Scientific, Simi Valley, CA, USA) for CCMP3337. The Moxi Z uses Gaussian curve-fitting with a coincidence correction algorithm of cell count (vs. diameter) histograms to extract precise (>95%) cell count metrics in a sample. The extracted raw data was further used for cellular carbon calculations of CCMP 3337. After the experiment, the vials, which were kept in the dark, were placed in the light, and after 10 days we were able to qualitatively confirm, under the microscope, renewed growth of coccolithophore cells.

batch_growth_experiment_new_phyto-1.csv