Dataset: growth phase exopolymers
Deployment: lab_Thornton

Exopolymer and carbohydrate production by diatoms with growth.

Principal Investigator:

Daniel C.O. Thornton (Texas A&M University, TAMU)

BCO-DMO Data Manager:

Shannon Rauch (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Project:

Effect of Temperature on Extracellular Polymeric Substance Production (EPS) by Diatoms (Diatom EPS Production)

Current State:

Final no updates expected

Version:

16 April 2014

Version Date:

2014-04-16

Data URL:

https://www.bco-dmo.org/dataset-deployment/511578/data

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Description

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.

Methods & Sampling

Dataset 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 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

Data Processing Description

Dataset Processing Description

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.

More information about this dataset deployment

Funding

Award Number	Funding Source
OCE-0726369	NSF Division of Ocean Sciences

Instruments

Fluorescence Microscope

Supplied Name: Epifluorescence Microscope

Supplied Description:

Bacteria were counted and cell permeability was determined using an epifluorescence microscope (Axioplan 2, Carl Zeiss MicroImaging).

Instrument Type

Generic Name: Fluorescence Microscope

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB06/

Generic 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.

Hemocytometer

Supplied 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.

Instrument Type

Generic Name: Hemocytometer

Generic 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.

Microscope - Optical

Supplied Name: Light Microscope

Supplied 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.

Instrument Type

Generic Name: Microscope - Optical

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/

Generic 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".

Turner Designs 700 Laboratory Fluorometer

Supplied Name: Turner Designs 700 Fluorometer

Supplied Description:

Instrument Type

Generic Name: Turner Designs 700 Laboratory Fluorometer

Acronym: TD-700

Community Identifier: http://vocab.nerc.ac.uk/collection/L22/current/TOOL0510/

Generic 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.

UV Spectrophotometer-Shimadzu

Supplied Name: UV-Mini 1240 Spectrophotometer

Supplied 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).

Instrument Type

Generic Name: UV Spectrophotometer-Shimadzu

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB20/

Generic 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.

Parameters

Supplied Name	Supplied description	Supplied Units	Standard Name
species	Species name.	dimensionless	species
growth_phase	Growth phase of the diatom (exponential, stationary, declining, death).	dimensionless	no_bcodmo_term
day	Day of the experiment.	dimensionless	no_bcodmo_term
culture	Identifier of the culture replicate.	dimensionless	replicate
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)	diatom
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)	no_bcodmo_term
chla	Concentration of chlorophyll a measured by fluorescence (Arar & Collins 1997; Method 445.0. EPA).	micrograms per liter (ug L-1)	chl_a
chla_per_cell	Concentration of chlorophyll a per cell.	picograms per cell (pg cell-1)	no_bcodmo_term
chla_per_cell_vol	Concentration of chlorophyll a per cell volume.	femtograms per cubic micrometer (fg um-3)	no_bcodmo_term
tot_carb	Total carbohydrate concentration measured using the PSA method (Dubois et al. 1956).	micrograms per milliliter (ug mL-1)	no_bcodmo_term
tot_carb_per_cell	Total carbohydrate concentration per cell.	picograms per cell (pg cell-1)	no_bcodmo_term
tot_carb_per_cell_vol	Total carbohydrate concentration per cell volume.	femtograms per cubic micrometer (fg um-3)	no_bcodmo_term
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)	no_bcodmo_term
collodial_per_cell	Colloidal carbohydrate concentration per cell.	picograms per cell (pg cell-1)	no_bcodmo_term
colloidal_per_cell_vol	Colloidal carbohydrate concentration per cell volume.	femtograms per cubic micrometer (fg um-3)	no_bcodmo_term
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)	no_bcodmo_term
EPS_carb_per_cell	Exopolymer (EPS) carbohydrate concentration per cell.	picograms per cell (pg cell-1)	no_bcodmo_term
EPS_carb_per_cell_vol	Exopolymer (EPS) carbohydrate concentration per cell volume.	femtograms per cubic micrometer (fg um-3)	no_bcodmo_term
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)	no_bcodmo_term
HW_carb_per_cell	Intracellular carbohydrate (hot water (HW) extraction) concentration per cell.	picograms per cell (pg cell-1)	no_bcodmo_term
HW_carb_per_cell_vol	Intracellular carbohydrate (hot water (HW) extraction) concentration per cell volume.	femtograms per cubic micrometer (fg um-3)	no_bcodmo_term
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)	no_bcodmo_term
HB_carb_per_cell	Cell-wall associated carbohydrate (hot bicarbonate (HB) extraction) concentration per cell.	picograms per cell (pg cell-1)	no_bcodmo_term
HB_carb_per_cell_vol	Cell-wall associated carbohydrate (hot bicarbonate (HB) extraction) concentration per cell volume.	femtograms per cubic micrometer (fg um-3)	no_bcodmo_term
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)	no_bcodmo_term
residual_carb_per_cell	Residual carbohydrate concentration per cell.	picograms per cell (pg cell-1)	no_bcodmo_term
residual_carb_per_cell_vol	Residual carbohydrate concentration per cell volume.	femtograms per cubic micrometer (fg um-3)	no_bcodmo_term
TPTZ_intracell_mono	Intracellular monosaccharide concentration determined using the TPTZ method (Myklestad et al. 1997).	micrograms per milliliter (ug mL-1)	no_bcodmo_term
TPTZ_extracell_mono	Extracellular monosaccharide concentration determined using the TPTZ method (Myklestad et al. 1997).	micrograms per milliliter (ug mL-1)	no_bcodmo_term
TPTZ_intracell_polysacc	Intracellular polysaccharide concentration determined using the TPTZ method (Myklestad et al. 1997).	micrograms per milliliter (ug mL-1)	no_bcodmo_term
TPTZ_extracell_polysacc	Extracellular polysaccharide concentration determined using the TPTZ method (Myklestad et al. 1997).	micrograms per milliliter (ug mL-1)	no_bcodmo_term
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)	no_bcodmo_term
TEP_mean_size	Mean size of Transparent exopolymer particles (TEP).	square micrometers (um^2)	no_bcodmo_term
tot_TEP_area	Total TEP area.	square millimeters per milliliter (mm^2 mL-1)	no_bcodmo_term
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)	no_bcodmo_term
CSP_mean_size	Mean size of coomassie staining particle (CSP).	square micrometers (um^2)	no_bcodmo_term
tot_CSP_area	Total CSP area.	square millimeters per milliliter (mm^2 mL-1)	no_bcodmo_term
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 (%)	no_bcodmo_term
bacteria	Bacteria abundance determined by DAPI staining and counts using an epifluorescence microscope (Porter & Feig 1980).	cells per milliliter (mL-1)	bact_abund
bact_per_diatom	Bacteria abundance per diatom.	dimensionless	no_bcodmo_term

Database

Contribute Data

Dataset: growth phase exopolymers
Deployment: lab_Thornton

Dataset acquisition description

Dataset Processing Description

Database

Contribute Data

Dataset: growth phase exopolymersDeployment: lab_Thornton

Dataset acquisition description

Dataset Processing Description

Dataset: growth phase exopolymers
Deployment: lab_Thornton