Experimental results: Exopolymer production by phytoplankton under oxidative stress; conducted at the Thornton lab, TAMU from 2007-2012 (Diatom EPS Production project) (NCEI Accession 0278027)

Acquisition Description:
Growth of the phytoplankton
The diatom Thalassiosira weissflogii (CCMP 1051) and the cyanobacterium Synechococcus elongates_cf (CCMP 1379) were obtained from the National Center for Culture of Marine Algae and Microbiota (NCMA). Replicated (n = 3) Batch cultures were grown in artificial seawater (Berges et al. 2001) containing nitrogen, phosphorus and silicon at 400 µM (as NaNO 3 ), 25 µM (NaH 2 PO 4 ), and 400 µM (Na 2 SiO 3 ), respectively. Culture temperatures were maintained at 20 ± 1 °C. Photon flux density on the surface of the culture bottles was 40 to 45 µmol m -2 s -1 on a 14 hour light: 10 hour dark cycle. During exponential growth, each culture was split into three treatments in which oxidative stress was induced by the addition of hydrogen peroxide at final concentrations of 0 (control), 10 and 100 µM H 2 O 2 . The treatments were sampled once a day over the next three days.

Measures of phytoplankton abundance and biomass
Counts of 400 cells from each culture were made using hemocytometers (Guillard and Sieracki 2005) from samples preserved in Lugol’s iodine (Parsons et al. 1984) using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Turbidity of the cultures, used as an indicator of growth, was measured by absorbance at 750 nm in a 1 cm path cuvette using a UV-Mini 1240 spectrophotometer (Shimadzu Corporation).

Chlorophyll a concentration 90% acetone extractions from biomass retained on GF/C (Whatman) were measured using a Turner Designs 700 fluorometer, which was calibrated using chlorophyll a standards (Sigma) (Arar and Collins 1997). The extract was diluted with 90% acetone if the chl a concentration were too high.

Cell permeability
Uptake and staining with the membrane-impermeable SYTOX Green (Invitrogen) was used to determine what proportion of the diatom population had permeable cell membranes (Veldhuis et al. 2001, Franklin et al. 2012). Four hundred cells were examined using an epifluorescence microscope (Axioplan 2, Carl Zeiss MicroImaging) and the number of cells that stained with SYTOX Green was enumerated.

TEP staining and analysis
Transparent exopolymer particles (TEP) were sampled according to Alldredge et al. (1993) and TEP abundance was enumerated by image analysis (Logan et al. 1994, Engel 2009). Ten photomicrographs were taken of each slide using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Images were analyzed using ImageJ software (National Institutes of Health) based on the method of Engel (2009). Thresholding during image processing was done using the triangle method (Zack et al. 1977).

CSP staining and analysis
Coomassie staining particles (CSP) were sampled according to Long and Azam et al. (1996) and CSP abundance was enumerated by image analysis (Logan et al. 1994, Engel 2009). Ten photomicrographs were taken of each slide using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Images were analyzed using ImageJ software (National Institutes of Health) based on the method of Engel (2009). Thresholding during image processing was done using the triangle method (Zack et al. 1977).

Quantum yield of photosystem II
The quantum yield of photosystem II was used as an indicator of phytoplankton health and measured using the saturating pulse method (Genty et al. 1989) using a pulse amplitude modulated fluorometer (PAM-210, Heinz Walz GmbH) folowing a protocol based on Marwood et al. (1999).

Caspase-like activity
Caspase-like activity was measured based on the method of Bouchard & Purdie (2011). Phytoplankton were collected by centrifugation, then lysed in a buffer, and the caspase-3 like activity was measured in the extracted proteins using a Enzcheck Caspase-3 Assay Kit #1 (Invitrogen inc.). The fluorescent product was measured by fluorescence using a microplate reader (SPECTRAmax GeminiEM, Molecular Devices).

References cited
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