Growth of the diatoms
\nThe 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 \u00b0C with 100 \u00b5M\u00a0 NaNO3, 200 \u00b5M of NaH2PO4\u00b7H2O, and 200 \u00b5M of Na2SiO3\u00b79H2O. Illumination was on a 14 h:10 h light:dark cycle at a photon flux density of 160 \u00b5mol 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
\nCounts 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\u2019s 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.
\nChlorophyll 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.
\nBacteria abundance
\nBacteria (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 \u00b5g ml-1.
Carbohydrate analysis
\nTwo 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
\nUptake 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
\nTransparent 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
\nCoomassie 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).
References cited
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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.
\nBerges, 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
\nDubois, 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\u2013356. doi:10.1021/ac60111a017
\nEngel, A. 2009. Determination of Marine Gel Particles. In Wurl, O. [Ed.] Practical Guidelines for the Analysis of Seawater. CRC Press, Taylor & Francis Group, Boca Raton, Florida, pp.125-142.
\nFranklin, 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\u2013317. doi:10.4319/lo.2012.57.1.0305
\nGuillard, 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.
\nLogan, 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
\nMenden-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
\nMyklestad, S. M., Skanoy, E., Hestmann S. 1997. A sensitive and rapid method for analysis of dissolved mono- and polysaccharides in seawater. Marine Chemistry 56: 279-286. doi:10.1016/S0304-4203(96)00074-6
\nParsons, T. R., Maita, Y. & Lalli, C. M. 1984. A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, Oxford, UK.
\nPassow, 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
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\nUnderwood, G. J. C., Paterson D. M., Parkes R. J. 1995. The measurement of microbial carbohydrate exopolymers from intertidal sediments. Limnol. Oceanogr. 40: 1243-1253. doi:10.4319/lo.1995.40.7.1243
\nUnderwood, G. J. C., Boulcott, M., Raines, C. A., Waldron K. 2004. Environmental effects on exopolymer production by marine benthic diatoms: Dynamics, changes in composition, and pathways of production. J. Phycol. 40: 293-304. doi:10.1111/j.1529-8817.2004.03076.x
\nVeldhuis, 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\u2013177. doi:10.1080/09670260110001735318
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Data from laboratory experiment on exopolymer and carbohydrate production by the diatoms Thalassiosira weissflogii (CCMP 1051), Skeletonema marinoi (CCMP 1332), and Cylindrotheca closterium (CCMP 339) during the growth to death phases of the cultures.
\nRelated references:
\nChen, J. 2014. Factors affecting carbohydrate production and the formation of transparent exopolymer particles (TEP) by diatoms. Ph.D. dissertation, Texas A&M University, College Station, TX.
Limited processing was necessary with this dataset. As this was a laboratory experiment it was designed in such a way to ensure that the parameters we measured were likely to be within a measurable range and therefore there were no measurements below detection limits. Chlorophyll concentrations were frequently too high; this was resolved by diluting the sample into the measurable range. Measured parameters were normalized to volume as most of the parameters were expressed as concentrations.