| Contributors | Affiliation | Role |
|---|---|---|
| Thornton, Daniel C.O. | Texas A&M University (TAMU) | Principal Investigator |
| Rauch, Shannon | Woods Hole Oceanographic Institution (WHOI BCO-DMO) | BCO-DMO Data Manager |
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.
Related references:
Chen, 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.
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 NaNO3, 200 µM of NaH2PO4·H2O, and 200 µM of Na2SiO3·9H2O. 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).
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
Engel, 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.
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
Myklestad, 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
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
Underwood, 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
Underwood, 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
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
Zack, G. W., Rogers, W.E., Latt S. A. 1977. Automatic-measurement of sister chromatid exchange frequency, J. Histochem. Cytochem., 25(7), 741-753. doi:10.1177/25.7.70454
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.
| File |
|---|
growth_phase_exopolymers.csv (Comma Separated Values (.csv), 18.94 KB) MD5:013dac5262adb9dd8c9b3a97328819ee Primary data file for dataset ID 511526 |
| Parameter | Description | Units |
| species | Species name. | dimensionless |
| growth_phase | Growth phase of the diatom (exponential, stationary, declining, death). | dimensionless |
| day | Day of the experiment. | dimensionless |
| culture | Identifier of the culture replicate. | dimensionless |
| cell_abundance | Cell count. Counts of 400 cells were made by transmitted light microscopy using a hemacytometer (Fuchs-Rosenthal ruling Hauser Scientific) (Guillard & Sieracki 2005). | cells per milliliter (mL-1) |
| cell_vol_mean | Mean cell volume calculated assuming 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. | cubic micrometers (um^3) |
| chla | Concentration of chlorophyll a measured by fluorescence (Arar & Collins 1997; Method 445.0. EPA). | micrograms per liter (ug L-1) |
| chla_per_cell | Concentration of chlorophyll a per cell. | picograms per cell (pg cell-1) |
| chla_per_cell_vol | Concentration of chlorophyll a per cell volume. | femtograms per cubic micrometer (fg um-3) |
| tot_carb | Total carbohydrate concentration measured using the PSA method (Dubois et al. 1956). | micrograms per milliliter (ug mL-1) |
| tot_carb_per_cell | Total carbohydrate concentration per cell. | picograms per cell (pg cell-1) |
| tot_carb_per_cell_vol | Total carbohydrate concentration per cell volume. | femtograms per cubic micrometer (fg um-3) |
| colloidal_carb | Colloidal carbohydrate concentration. Different fractions of carbohydrate were extracted from the cultures using methods described in Underwood et al. (1995) and Underwood et al. (2004). The colloidal carbohydrate fractions were measured using the PSA method (Dubois et al. 1956). | micrograms per milliliter (ug mL-1) |
| collodial_per_cell | Colloidal carbohydrate concentration per cell. | picograms per cell (pg cell-1) |
| colloidal_per_cell_vol | Colloidal carbohydrate concentration per cell volume. | femtograms per cubic micrometer (fg um-3) |
| EPS_carb | Exopolymer (EPS) carbohydrate concentration. Different fractions of carbohydrate were extracted from the cultures using methods described in Underwood et al. (1995) and Underwood et al. (2004). The EPS carbohydrate fractions were measured using the PSA method (Dubois et al. 1956). | micrograms per milliliter (ug mL-1) |
| EPS_carb_per_cell | Exopolymer (EPS) carbohydrate concentration per cell. | picograms per cell (pg cell-1) |
| EPS_carb_per_cell_vol | Exopolymer (EPS) carbohydrate concentration per cell volume. | femtograms per cubic micrometer (fg um-3) |
| HW_carb | Intracellular carbohydrate (hot water (HW) extraction) concentration. Different fractions of carbohydrate were extracted from the cultures using methods described in Underwood et al. (1995) and Underwood et al. (2004). The HW carbohydrate fractions were measured using the PSA method (Dubois et al. 1956). | micrograms per milliliter (ug mL-1) |
| HW_carb_per_cell | Intracellular carbohydrate (hot water (HW) extraction) concentration per cell. | picograms per cell (pg cell-1) |
| HW_carb_per_cell_vol | Intracellular carbohydrate (hot water (HW) extraction) concentration per cell volume. | femtograms per cubic micrometer (fg um-3) |
| HB_carb | Cell-wall associated carbohydrate (hot bicarbonate (HB) extraction) concentration. Different fractions of carbohydrate were extracted from the cultures using methods described in Underwood et al. (1995) and Underwood et al. (2004). The HB carbohydrate fractions were measured using the PSA method (Dubois et al. 1956). | micrograms per milliliter (ug mL-1) |
| HB_carb_per_cell | Cell-wall associated carbohydrate (hot bicarbonate (HB) extraction) concentration per cell. | picograms per cell (pg cell-1) |
| HB_carb_per_cell_vol | Cell-wall associated carbohydrate (hot bicarbonate (HB) extraction) concentration per cell volume. | femtograms per cubic micrometer (fg um-3) |
| residual_carb | Residual carbohydrate concentration. Different fractions of carbohydrate were extracted from the cultures using methods described in Underwood et al. (1995) and Underwood et al. (2004). The residual carbohydrate fractions were measured using the PSA method (Dubois et al. 1956). | micrograms per milliliter (ug mL-1) |
| residual_carb_per_cell | Residual carbohydrate concentration per cell. | picograms per cell (pg cell-1) |
| residual_carb_per_cell_vol | Residual carbohydrate concentration per cell volume. | femtograms per cubic micrometer (fg um-3) |
| TPTZ_intracell_mono | Intracellular monosaccharide concentration determined using the TPTZ method (Myklestad et al. 1997). | micrograms per milliliter (ug mL-1) |
| TPTZ_extracell_mono | Extracellular monosaccharide concentration determined using the TPTZ method (Myklestad et al. 1997). | micrograms per milliliter (ug mL-1) |
| TPTZ_intracell_polysacc | Intracellular polysaccharide concentration determined using the TPTZ method (Myklestad et al. 1997). | micrograms per milliliter (ug mL-1) |
| TPTZ_extracell_polysacc | Extracellular polysaccharide concentration determined using the TPTZ method (Myklestad et al. 1997). | micrograms per milliliter (ug mL-1) |
| TEP_conc_mean | Mean transparent exopolymer particle (TEP) concentration. TEP retained on 0.4 polycarbonate filters and stained with Alcian blue (Alldredge et al. 1993). | TEP per milliliter (TEP mL-1) |
| TEP_mean_size | Mean size of Transparent exopolymer particles (TEP). | square micrometers (um^2) |
| tot_TEP_area | Total TEP area. | square millimeters per milliliter (mm^2 mL-1) |
| CSP_conc_mean | Mean coomassie staining particle (CSP) concentration. CSP retained on 0.4 polycarbonate filters and stained with Coomassie briliant blue blue (Long & Azam 1996). | CSP per milliliter (mL-1) |
| CSP_mean_size | Mean size of coomassie staining particle (CSP). | square micrometers (um^2) |
| tot_CSP_area | Total CSP area. | square millimeters per milliliter (mm^2 mL-1) |
| stained_cells_pcnt | % of SYTOX Green stained cells. Cell permeability was determined by SYTOX Green staining (Veldhuis et al. 1997). Four hundred cells were examined using an epifluorescence microscope and the number of cells that stained with SYTOX Green was enumerated. | percent (%) |
| bacteria | Bacteria abundance determined by DAPI staining and counts using an epifluorescence microscope (Porter & Feig 1980). | cells per milliliter (mL-1) |
| bact_per_diatom | Bacteria abundance per diatom. | dimensionless |
| Dataset-specific Instrument Name | Epifluorescence Microscope |
| Generic Instrument Name | Fluorescence Microscope |
| Dataset-specific Description | Bacteria were counted and cell permeability was determined using an epifluorescence microscope (Axioplan 2, Carl Zeiss MicroImaging). |
| Generic Instrument Description | Instruments that generate enlarged images of samples using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption of visible light. Includes conventional and inverted instruments. |
| Dataset-specific Instrument Name | Hemocytometer |
| Generic Instrument Name | Hemocytometer |
| Dataset-specific Description | Counts of 400 cells from each culture were made using a hemocytometer (Fuchs-Rosenthal ruling, Hauser Scientific) (Guillard and Sieracki 2005) from samples preserved in Lugol’s iodine (Parsons et al. 1984) using a light microscope. |
| Generic Instrument Description | A hemocytometer is a small glass chamber, resembling a thick microscope slide, used for determining the number of cells per unit volume of a suspension. Originally used for performing blood cell counts, a hemocytometer can be used to count a variety of cell types in the laboratory. Also spelled as "haemocytometer". Description from:
http://hlsweb.dmu.ac.uk/ahs/elearning/RITA/Haem1/Haem1.html. |
| Dataset-specific Instrument Name | Light Microscope |
| Generic Instrument Name | Microscope - Optical |
| Dataset-specific Description | 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). A light microscope was also used to determine cell volume and to enumerate TEP and CSP by image analysis. |
| Generic Instrument Description | Instruments that generate enlarged images of samples using the phenomena of reflection and absorption of visible light. Includes conventional and inverted instruments. Also called a "light microscope". |
| Dataset-specific Instrument Name | Turner Designs 700 Fluorometer |
| Generic Instrument Name | Turner Designs 700 Laboratory Fluorometer |
| Dataset-specific Description | 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). |
| Generic Instrument Description | The TD-700 Laboratory Fluorometer is a benchtop fluorometer designed to detect fluorescence over the UV to red range. The instrument can measure concentrations of a variety of compounds, including chlorophyll-a and fluorescent dyes, and is thus suitable for a range of applications, including chlorophyll, water quality monitoring and fluorescent tracer studies. Data can be output as concentrations or raw fluorescence measurements. |
| Dataset-specific Instrument Name | UV-Mini 1240 Spectrophotometer |
| Generic Instrument Name | UV Spectrophotometer-Shimadzu |
| Dataset-specific Description | Turbidity of the cultures was measured by absorbance at 750 nm in a 1 cm path cuvette using a UV-Mini 1240 spectrophotometer (Shimadzu Corporation). 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). |
| Generic Instrument Description | The Shimadzu UV Spectrophotometer is manufactured by Shimadzu Scientific Instruments (ssi.shimadzu.com). Shimadzu manufacturers several models of spectrophotometer; refer to dataset for make/model information. |
| Website | |
| Platform | TAMU |
| Start Date | 2007-09-01 |
| End Date | 2012-08-01 |
| Description | Experiments conducted in the lab of Daniel C.O. Thornton located at:
Department of Oceanography
Texas A&M University
College Station, Texas, 77843
United States |
Description from NSF Propsoal:
It is necessary to determine the fate of organic matter in the ocean to understand marine food webs, biogeochemical cycles, and climate change. Diatoms fix approximately a quarter of the net global primary production each year, and a significant proportion of this production is excreted as extracellular polymeric substances (EPS). EPS have a profound impact on pelagic ecosystems by affecting the formation of aggregates. Diatoms and other particulate organic carbon (POC) sink rapidly as aggregates, affecting the biological carbon pump, which plays a pivotal role in the sequestration of carbon in the ocean. The proposed research will test the central hypothesis: Temperature increase affects diatom release of EPS, which act as a glue, increasing aggregation. Previous work by the investigator showed that increased temperatures affected the aggregation of Skeletonema costatum. Four specific hypotheses will be tested:
H1: Diatoms produce more EPS with increasing temperature.
H2: Diatoms produce more transparent exopolymer particles (TEP) with increasing temperature.
H3: The quantity or composition of cell-surface carbohydrates in diatoms changes with temperature.
H4: Aggregation of diatom cultures and natural plankton increases with temperature.
Laboratory experiments (years 1 - 2) will be conducted with three model diatom species grown at controlled growth rates and defined limitation (nitrogen or light) in continuous culture. Culture temperature will be stepped up or down in small increments to determine the effect of the temperature change on EPS production, aggregation, and partitioning of carbon in intra- and extracellular pools. Similar experiments in year 3 will be carried out using natural plankton populations from a coastal site where diatoms contribute a significant proportion to the biomass.
The proposed research will increase our understanding of the ecology and physiology of one of the dominant groups of primary producers on Earth. EPS are a central aspect of diatom biology, though the physiology, function and broader ecosystem impacts of EPS production remain unknown. This research will determine how temperature, light limitation, and nutrient limitation affect the partitioning of production between dissolved, gel, and particulate phases in the ocean. Measurements of plankton stickiness (alpha) under different conditions will be important to model aggregation processes in the ocean as alpha is an important (and variable) term in coagulation models. Determining how carbon is cycled between the ocean, atmosphere and lithosphere is key to understanding climate change on both geological and human time scales. This is a major societal issue as atmospheric CO2 concentrations are steadily increasing, correlating with a 0.6 C rise in global average temperature during the last century. This research will address potential feedbacks between warming of the surface ocean, diatom ecophysiology and the biological carbon pump.
Related Publications:
Rzadkowolski, Charles E. and Thornton, Daniel C. O. (2012) Using laser scattering to identify diatoms and conduct aggregation experiments. Eur. J. Phycol., 47(1): 30-41. DOI: 10.1080/09670262.2011.646314
Thornton, Daniel C. O. (2009) Effect of Low pH on Carbohydrate Production by a Marine Planktonic Diatom (Chaetoceros muelleri). Research Letters in Ecology, vol. 2009, Article ID 105901, 4 pages. DOI: 10.1155/2009/105901
Thornton, D.C.O. (2014) Dissolved organic matter (DOM) release by phytoplankton in the contemporary and future ocean. European Journal of Phycology 49: 20-46. DOI: 10.1080/09670262.2013.875596
Thornton, D.C.O., Visser, L.A. (2009) Measurement of acid polysaccharides (APS) associated with microphytobenthos in salt marsh sediments. Aquat Microb Ecol 54:185-198. DOI: 10.3354/ame01265
| Funding Source | Award |
|---|---|
| NSF Division of Ocean Sciences (NSF OCE) |