Results from growth rate experiment with the diatom Thalassiosira weissflogii in semi-continuous culture; conducted at the Thornton lab, TAMU from 2007-2012 (Diatom EPS Production project) (NCEI Accession 0278830)

Acquisition Description:
Growth of the diatom
Thalassiosira weissflogii (CCMP 1051) was obtained from the National Center for Culture of Marine Algae and Microbiota (NCMA). The diatom was grown in artificial seawater (Berges et al. 2001) in nitrogen-limited 1000 ml semi-continuous cultures at a sequence of dilution rates. The macronutrient concentrations in the artificial seawater recipe were modified from Berges et al. (2001) to affect nitrogen limitation; concentrations of nitrogen, phosphorus and silicon were 60 µM (as NaNO 3 ), 100 µM (NaH 2 PO 4 ), and 100 µM (Na 2 SiO 3 ), respectively. Culture temperature was maintained at 20 ± 0.1 °C throughout the experiment. Photon flux density on the surface of the culture bottles was 150 µmol m -2 s -1 . The cultures were stirred with 2.5 cm long stir bars using magnetic stirrers at 120 revolutions per minute. The cultures were grown at a sequence of dilution rates (0.3, 0.5, 0.7, 0.9 and 0.3 day -1 ) affected by daily dilution at 10:00 am every day. To induce a dilution rate of 0.3 day -1 , 0.3 of the culture volume (300 ml) was removed and replaced with 300 ml of fresh medium to maintain a constant total culture volume (1000 ml).

Measures of phytoplankton abundance and biomass
Counts of 400 cells from each replicate culture were made by light microscopy using a hemocytometer (Fuchs-Rosenthal ruling, Hauser Scientific) (Guillard and Sieracki 2005) from samples preserved in Lugol’s iodine (Parsons et al. 1984). Cell volume was determined using live cells (Menden-Deuer and Lessard 2000). The volume of 100 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 T. weissflogii is a cylinder.

Chlorophyll a concentrations in the cultures was determined by fluorescence (Arar and Collins 1997). 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.

The carbon and nitrogen content of particulate organic matter in the cultures was determined by elemental analysis using a Carlo Erba NA1500 Elemental Analyzer. Standards were acetanilide, methionine, graphite (USGS 24, USGS 40, and USGS 41) (Verardo et al. 1990).

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 .

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 and the number of cells that stained with SYTOX Green was enumerated.

Total carbohydrate
Total carbohydrate concentrations were determined in unfiltered liquid samples from the cultures using the phenol-sulfuric acid (PSA) method (Dubois et al. 1956) calibrated with d-glucose. The concentration of total carbohydrate was expressed as glucose equivalents.

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 and the area of 100 TEP particles from each replicate culture was determined after manually drawing around each particle using Axio Vision 4.8 (Carl Zeiss MicroImaging ) image analysis software.

Particle size distribution and aggregation
The particle size distribution (PSD) and volume concentration of particles in the T. weissflogii cultures was measured using laser scattering following the method of Rzadkowolski and Thornton (2012) using a Laser In Situ Scattering and Transmissometry instrument (LISST-100X, Type C; Sequoia Scientific). Sample (150 ml) from each replicate culture was placed into a chamber attached to the LISST and the PSD was measured 100 times at a rate of 1 Hz. The PSD of the culture was blank corrected by subtracting the PSD of 0.2 µm filtered artificial seawater.

References cited
Alldredge, A. L., Passow, U. & Logan B. E. 1993. The abundance and significance of a class of large, transparent organic particles in the ocean. Deep-Sea Res . Oceanogr ., I. 40: 1131-1140. doi: 10.1016/0967-0637(93)90129-Q

Arar, E. J. & Collins, G. B. 1997. Method 445.0. In Vitro Determination of Chlorophyll a and Pheophytin a in Marine and Freshwater Algae by Fluorescence U.S. Environmental Protection Agency, Cincinnati, Ohio.

Berges, J. A., Franklin D. J. & Harrison, P. J. 2001. Evolution of an artificial seawater medium: Improvements in enriched seawater, artificial water over the last two decades. J. Phycol . 37:1138-1145. doi: 10.1046/j.1529-8817.2001.01052.x

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350–356. doi: 10.1021/ac60111a017

Franklin, D. J., Airs, R. L., Fernandes, M., Bell, T. G., Bongaerts, R. J., Berges, J. A. & Malin, G. 2012. Identification of senescence and death in Emiliania huxleyi and Thalassiosira pseudonana : Cell staining, chlorophyll alterations, and dimethylsulfoniopropionate (DMSP) metabolism. Limnol. Oceanogr. 57: 305–317. doi:10.4319/lo.2012.57.1.0305

Guillard, R. R. L. & Sieracki, M. S. 2005. Counting cells in cultures with the light microscope. In Andersen R. A. [Ed.] Algal Culturing Techniques . Elsevier Academic Press, Burlington, MA, pp. 239-252.

Logan, B. E., Grossart, H. P. & Simon, M. 1994. Direct observation of phytoplankton, TEP and aggregates on polycarbonate filters using brightfield microscopy. J. Plankton Res. 16: 1811-1815.doi: 10.1093/plankt/16.12.1811

Menden-Deuer S. & Lessard, E. J. 2000. Carbon to volume relationships for dinoflagellates, diatoms, and other protists plankton. Limnol. Oceanogr. 45: 569- 579. doi: 10.4319/lo.2000.45.3.0569

Parsons, T. R., Maita, Y. & Lalli, C. M. 1984. A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press , Oxford, UK.

Passow, U. & Alldredge, A. L. 1995. A dye-binding assay for the spectrophotometric measurement of transparent exopolymer particles (TEP). Limnol. Oceanogr. 40: 1326-1335. doi: 10.4319/lo.1995.40.7.1326

Porter, K. G. & Feig, Y. S. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25:943–948. doi: 10.4319/lo.1980.25.5.0943

Rzadkowlski, C. E. & Thornton, D. C. O. 2012. Using laser scattering to identify diatoms and conduct aggregation experiments. Eur. J. Phycol. 47:30-41. doi: 10.1080/09670262.2011.646314

Veldhuis, M. J. W., Kraay, G. W. & Timmermans, K. R. 2001. Cell death in phytoplankton: correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth. Eur. J. Phycol. 36: 167–177. doi: 10.1080/09670260110001735318

Verardo, D. J., Froelich, P. N. & McIntyre, A. 1990. Determination of organic carbon and nitrogen in marine sediments using the Carlo Erba NA-1500 analyzer. Deep-Sea Res.A 37:157-165. doi: 10.1016/0198-0149(90)90034-S