Culturing of Synechococcus clones:
Monocultures of six clones of Synechococcus were used to examine variation and controls on Si quotas and rates of Si accumulation. Cultures were procured from the National Center for Marine Algae and Microbiota (NCMA) at the Bigelow Laboratory for Ocean Sciences in Boothbay Harbor, Maine. Many of these clones are also available in other culture collections and have various strain names; here we will refer to each by their NCMA strain number (a.k.a. CCMP number).
All clones were maintained in aged surface Sargasso Sea water with f/2 media constituents with 10 – 100 uM Si depending on the experiment as detailed below. The temperature was 21C with low light 65 microeinsteins per second per square meter (uE/m2/s) on a 12 h light: 12 h dark photocycle. pH was regulated in all cultures by bubbling with humidified ambient air which was sterilized by passage through a bacterial filter prior to entering each culture vessel. Unless otherwise specified, all experiments were conducted under these temperature and light conditions. pH was monitored daily and remained below 8.5 in all experiments.
Water-soluble cellular silicon:
The water-soluble fraction of cellular silicon was measured for all six clones at external silicic acid concentrations of 1, 60 and 120 uM. Replicate 10-mL aliquots of each exponential culture were filtered onto separate 0.2 um polycarbonate membrane filters in parallel with the samples for total cellular Si measurement. Each filter was placed in a separate 15-mL polypropylene tube, immediately resuspended into 5 mL of dilute HCl (0.01 N) to avoid solubilization of any particle-bound Si which may be an amorphous solid, and flash frozen in liquid nitrogen. After the freeze, samples were thawed in a 35 C water bath, vortexed, and refrozen in liquid nitrogen. In total, four freeze/thaw cycles were conducted to lyse the cells. This method was determined to be the most effective for lysing cells, as the small volumes required for preserving the small analytical signal precluded the use of larger volumes that would enable the use of probe sonicators or pressure cells (e.g. French type). After the freeze/thaw cycles, cell suspensions were syringe-filtered through a 0.2 um filter and the filtrate analyzed for silicic acid in the same manner as for solubilized biogenic silica using NaOH–HF digestion in Teflon tubes as described in Krause et al. (2013).
Full methods are described in Brzezinski et al. (in review as of 05 Jan 2017).
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