Dataset: Thalassiosira wessiflogii growth rate and TEP

Name: Results from growth rate experiment with the diatom Thalassiosira wessiflogii in semi-continuous culture; conducted at the Thornton lab, TAMU from 2007-2012 (Diatom EPS Production project)
Published: 2014-04-07
Keywords: oceans

Cite This Dataset

DOI:10.1575/1912/bco-dmo.506135.1

Project:

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

Principal Investigator:

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

BCO-DMO Data Manager:

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

Version:

Version Date:

2014-04-07

Restricted:

Validated:

Yes

Current State:

Final no updates expected

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

Abstract:

Data from laboratory experiment on growth rate and transparent exopolymer particles (TEP) in the diatom Thalassiosira wessiflogii (CCMP 1051) in a semi-continuous culture (four replicate cultures).

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Archival Copy

Version Date	Archive	Persistent Identifier	Date Assigned
2014-04-07	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.1575/1912/bco-dmo.506135.1	2019-11-21

Description

Data from laboratory experiment on growth rate and transparent exopolymer particles (TEP) in the diatom Thalassiosira wessiflogii (CCMP 1051) in a semi-continuous culture (four replicate 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.

Chen, J., Thornton, D.C.O. (in revision). Effect of growth rate on TEP production and aggregation of Thalassiosira weissflogii. Journal of Phycology.

Methods & Sampling

Growth of the diatom
Thalassiosira wessiflogii (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 NaNO3), 100 µM (NaH2PO4), and 100 µM (Na2SiO3), 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. wessiflogii 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. weissiflogii 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

Data 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 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.

Data Files

File
growth_rate_TEP.csv (Comma Separated Values (.csv), 7.79 KB) MD5:397732374bb0fce3fd14cc930d9ca2e7 Primary data file for dataset ID 506135

More Information about this dataset

Funding Sources

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

Deployments

Deployment	Synonyms	Start Date	Platform	Investigator
lab_Thornton		2007-09-01	TAMU	Daniel C.O. Thornton (Principal Investigator)	data info

Instruments

Carlo-Erba NA-1500 Elemental Analyzer

Supplied Name: Carlo Erba NA1500 Elemental Analyzer

Supplied Description:

The carbon and nitrogen content of particulate organic matter in the cultures was determined by elemental analysis using a Carlo Erba NA1500 Elemental Analyzer.

Instrument Type

Generic Name: Carlo-Erba NA-1500 Elemental Analyzer

Acronym: Carlo-Erba NA-1500

Community Identifier: http://vocab.nerc.ac.uk/collection/L22/current/TOOL0470/

Generic Description:

A laboratory instrument that simultaneously determines total nitrogen and total carbon from a wide range of organic and inorganic sediment samples. The sample is completely and instantaneously oxidised by flash combustion, which converts all organic and inorganic substances into combustion products. The resulting combustion gases pass through a reduction furnace and are swept into the chromatographic column by the carrier gas which is helium. The gases are separated in the column and detected by the thermal conductivity detector which gives an output signal proportional to the concentration of the individual components of the mixture. The instrument was originally manufactured by Carlo-Erba, which has since been replaced by Thermo Scientific (part of Thermo Fisher Scientific). This model is no longer in production.

Fluorescence Microscope

Supplied Name: Epifluorescence Microscope

Supplied Description:

Bacterial abunance and cell permeability were 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 replicate culture were made by light microscopy using a hemocytometer (Fuchs-Rosenthal ruling, Hauser Scientific).

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:

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).

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".

Sequoia Scientific Laser In-Situ Sediment Size Transmissometer

Supplied Name: LISST-100X Type C Sequoia Scientific

Supplied Description:

The particle size distribution (PSD) and volume concentration of particles in the T. weissiflogii 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).

Instrument Type

Generic Name: Sequoia Scientific Laser In-Situ Sediment Size Transmissometer

Acronym: Sequoia LISST

Community Identifier: http://vocab.nerc.ac.uk/collection/L22/current/TOOL0044/

Generic Description:

A self-contained unit which measures the scattering of LASER light at a number of angles. This primary measurement is mathematically inverted to give a grain size distribution, and also scaled to obtain the volume scattering function. The size distribution is presented as concentration in each of the grain-size class bins. Optical transmission, water depth and temperature are recorded as supporting measurements.

The Sequoia LISST 100-X series instruments are available in two range sizes: 1.25-250 microns (Type B) and 2.5-500 microns (Type C).

Turner Designs 700 Laboratory Fluorometer

Supplied Name: Turner Designs 700 Fluorometer

Supplied 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).

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.

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
dilution_rate	Dilution rate.	per day (day-1)	no_bcodmo_term
culture	Identifier of the culture replicate.	dimensionless	replicate
day	Day of the experiment.	dimensionless	no_bcodmo_term
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	diatom
cell_vol_mean	Mean cell volume estimated assuming T. weissflogii (CCMP 1051) was a cyclinder using the method of Menden-Deuer & Lessard (2000).	cubic micrometers (um^3)	no_bcodmo_term
cell_vol_sd	Standard deviation of cell_vol_mean.	cubic micrometers (um^3)	std_dev
cell_vol_n	n (number of cells) used in determination of cell_vol_mean.	dimensionless	number
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
TEP	Transparent exopolymer particles (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
TEP_sd	Standard deviation of TEP_mean_size.	square micrometers (um^2)	std_dev
TEP_n	n used in determination of TEP_mean_size.	dimensionless	number
tot_TEP_area	Total TEP area.	square millimeters per milliliter (mm^2 mL-1)	no_bcodmo_term
TEP_prod_rate	TEP production rate.	square millimeters per milliliter per day (mm^2 mL-1 day-1)	no_bcodmo_term
vol_conc	Particulate volume concentration. Volume concentration and aggegation were measured using Laser in situ sacattering and transmissometry (LISST) (Rzadkowolski & Thornton 2012).	microliters per liter (uL L-1)	no_bcodmo_term
vol_conc_sd	Standard deviation of vol_conc.	microliters per liter (uL L-1)	std_dev
vol_conc_n	n used in determination of vol_conc.	dimensionless	number
agg_vol_conc	Aggregated volume concentration (particles > 63 um ESD). Particulate volume concentration and aggegation were measured using Laser in situ sacattering and transmissometry (LISST) (Rzadkowolski & Thornton 2012).	microliters per liter (uL L-1)	no_bcodmo_term
agg_vol_conc_sd	Standard deviation of agg_vol_conc.	microliters per liter (uL L-1)	std_dev
agg_vol_conc_n	n used in determination of agg_vol_conc.	dimensionless	number
stained_cells	Number 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.	cells per milliliter (cells 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 (cells mL-1)	bact_abund
bact_per_diatom	Bacteria abundance per diatom.	dimensionless	no_bcodmo_term
C_to_N	Ratio of carbon to nitrogen. C:N ratio was measured using a Carlo Erba NA1500 Elemental Analyzer. Standards were acetanilide, methionine, graphite (USGS 24, USGS 40, and USGS 41) (Verardo, Froelich, & McIntyre 1990).	dimensionless	C_to_N