Dataset: Si Depletion Experiment: Cell Counts

The methodology below describes all data collected from this experiment.  This dataset landing page serves the "Si Depletion Experiment: Cell Counts" data.  Other datasets can be found in the "Related Datasets" section of this page.

Experiment Overview: Phytoplankton Si utilization during an 8 d culture experiment was assessed. A variety of silicifying and non-silicifying species were grown in fully amended LH and F/2 media supplemented with 5 M Si. All species were grown in quadruplicates except for Thalassiosira weissflogii and the no cells control. Aliquots of cultures for cell counts, dissolved Si (DSi), and biogenic Si incorporated into the cells (BSi) were taken daily from T0 – T4, then on T6 and T8 and subsequently processed as described below. In addition, EDS analysis was used to determine if Si was present in biomineral structures.

Cell Counting: Cells were counted on each collection day using a hemocytometer or Sedgwick-Rafter chamber. A minimum of 300 cells were counted per sample. Growth curves were plotted and specific growth rates were calculated for each species throughout the 8 d sampling period.

DSi Method: Autoclaved and filtered Gulf Stream seawater was amended with LH or F/2 nutrients and [Si] measured prior to adding sufficient NaSiO3 to reach a starting [Si] of 5 M. Cells were harvested from cultures at early exponential phase and gently washed in Si-free media using a 0.4 m Nalgene polycarbonate filter unit. Replicate 200 mL cultures were seeded with washed cells for a starting density of 1-5 x 104 mL-1. Each sampling day, 15 mL culture aliquots were 0.2 m filtered (Merck Millipore Ltd.) and filtrate was stored at 4C prior to DSi analysis. The filters were frozen for later BSi analysis (see below). For AutoAnalyzer nutrient analysis, the molybdate method was used (modified from Brzezinski and Nelson (1995) and Brzezinski et al. (1997)). The system was washed with sodium dodecyl sulfate (SDS) to lubricate the Si lines. Oxalic acid concentrations were increased to saturated levels (143 g/L) to overcome any phosphate interference. Molybdate was made fresh for each run. The tubing diameter for the oxalic line was also increased to 0.035 in, with a flow rate of 0.41 at 40%.

BSi Method: The filters (See DSi explanation above) with cells were frozen at -20C prior to BSi analysis. For BSi determination, silicifying species were collected on 0.2 m polycarbonate filters, and processed using the alkaline digestion method as described by Brazinski and Nelson (1995), with modifications from Paasche (1973) and Krausse (1983). For coccolithophores, filters were first treated with 1mL 0.5 M HCl to fully dissolve coccoliths (Moheimani & Borowitzka, 2006), then treated with 4 mL 0.2 M NaOH to neutralize, before the alkaline digestion. For the alkaline digestion, each filter was placed in a 15 mL polymethylpentene tube (Diagenode, Inc.) with 4 mL of 0.2 M NaOH and brought to 100oC in a water bath for 20 min, cooled, and neutralized with 1 mL of 0.5 M HCl. The digest was centrifuged at 10,000 rpm for 9 minutes and aliquots of supernatant were removed and diluted for autoanalyzer analysis as appropriate. Analytical blanks with filter only (no cells) were included for each run. Autoanalyzer conditions are the same as described under the DSi Collection section.

EDS Method: For EDS analysis 1-3 mL of culture were filtered onto 13 mm 0.4 m isopore filters [Merck Millipore Ltd.] and rinsed with Nanopure water buffered to pH 8.0 with 1 mM HEPES to remove salts. Filters were air-dried and mounted onto a SEM stub with carbon adhesive tabs before coating with 10 nm Pt/Pd. EDS analysis was performed at the Joint School of Nanoscience and Nanoengineering (Zeiss Auriga SEM, with a Bruker Quantax detector and analysis software) or at North Carolina State University (FEI Verios 460L SEM, with an Oxford Xmax silicon drift EDS detector and AZtec acquisition and analysis software). A minimum of 500,000 counts were collected, between 2,000 – 8,000 cps with an average deadtime < 5%. Standardless quantification was used to determine atomic % and weight % for each element.