Experimental results: Exopolymer and carbohydrate production by diatoms with growth; conducted at the Thornton lab, TAMU from 2007-2012 (Diatom EPS Production project) (NCEI Accession 0278193)

Acquisition Description:
Growth of the diatoms
The diatoms Thalassiosira weissflogii (CCMP 1051), Skeletonema marinoi (CCMP 1332), and Cylindrotheca closterium (CCMP 339) were grown in artificial seawater (Berges et al. 2001) in batch culture at 20 °C with 100 µM NaNO 3 , 200 µM of NaH 2 PO 4 ·H 2 O, and 200 µM of Na 2 SiO 3 ·9H 2 O. Illumination was on a 14 h:10 h light:dark cycle at a photon flux density of 160 µmol m -2 s -1 . There were three replicate cultures. Cultures were sampled during both the growth and death of the cultures over several weeks.

Measures of diatom abundance and biomass
Counts of 400 cells from each culture were made using a hemacytometer (Fuchs-Rosenthal ruling, Hauser Scientific) (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).

Cell volume was determined using live cells (Menden-Deuer and Lessard 2000). The volume of 25 diatoms from each replicate culture was determined by measuring cell length (pervalver length) and width (valver length) at 400x magnification using a light microscope (Axioplan 2, Carl Zeiss MicroImaging). Cell volume was calculated based on the assumption that both T. wessiflogii and S. marinoi were cylinders. The volume of Cylindrotheca closterium was estimated assuming that its shape was equivalent to two cones.

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.

Bacteria abundance
Bacteria (400 cells) were counted using an epifluorescence microscope (Axioplan 2, Carl Zeiss MicroImaging) after staining with 4'6-diamidino-2-phenylindole dihydrochloride (DAPI) (Porter and Feig 1980) at a final concentration of 0.25 µg ml -1 .

Carbohydrate analysis
Two spectrophotometric methods were used to measure carbohydrates, the phenol sulfuric acid (PSA) method (Dubois et al. 1956) and the 2, 4, 6-tripyridyl-s-triazine (TPTZ) method (Myklestad et al. 1997). The color produced by both methods was measured in 1 cm path length cuvette using UV-Mini 1240 spectrophotometer (Shimadzu Corporation). Both methods were calibrated using D-glucose and the results are expressed as D-glucose equivalents. Different fractions of carbohydrate were extracted from the cultures using methods described in Underwood et al. (1995) and Underwood et al. (2004): total, colloidal, exopolymers (EPS), intracellular carbohydrate (hot water (HW) extraction), cell-wall associated carbohydrates (hot bicarbonate (HB) extraction), and residual. These carbohydrate fractions were measured using the PSA method. The TPTZ method was used to measure the intracellular and extracellular monosaccharide pools and the intracellular and extracellular polysaccharide pools after acid hydrolysis of the sample.

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).

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