http://lod.bco-dmo.org/id/dataset/921924
eng; USA
utf8
dataset
Highest level of data collection, from a common set of sensors or instrumentation, usually within the same research project
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
2024-03-11
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Hemiaulus-Richelia physiological response to different nitrogen sources
2024-03-11
publication
2024-03-11
revision
Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)
2024-06-13
publication
https://doi.org/10.26008/1912/bco-dmo.921924.1
Tatiana A. Rynearson
University of Rhode Island
principalInvestigator
Kei Inomura
University of Rhode Island
principalInvestigator
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
publisher
Cite this dataset as: Setta, S. P., Rynearson, T. A. (2024) Hemiaulus-Richelia physiological response to different nitrogen sources. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2024-03-11 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.921924.1 [access date]
DDA nutrient acquisition Dataset Description: Methods and Sampling: <p>Samples were collected from culture flasks of a <em>Hemiaulus</em>-<em>Richelia</em> diatom diazotroph association (DDA) over the course of 2.5 weeks. Sargasso Seawater collected on the R/V Atlantic Explorer AE1812 cruise in May 2018 (at 32.42°N, 63.48°W) was used for media and filtered via peristaltic pump over 0.2 micrometers (µm) 47-millimeter (mm) filters (Polyethersulfone filters Millipore Express PLUS #GPWP04700). All flasks were grown in f/5 media without nitrogen, one set of flasks had no added nitrogen (Only N2), one set 10 µM added ammonium treatment (+NH4), and one set 10 µM added nitrate treatment (+NO3). Flasks were grown at 22 degrees Celsius (°C), ~130 micromoles per square meter per second (µmol m-2 s-1) in a 12:12 light:dark cycle. Two time points were used, an initial (T=0) and a final (T=15,17,18,19, depending on flask growth). Samples from the culture innoculum (Innoc1, Innoc2, Innoc3) were collected before all three innoculum flasks were thoroughly mixed and added to the experiment flasks.</p>
<p>Total chlorophyll a fluorescence was sampled by filtering over GF/F (~0.7 µm Whatmanfilters) and immediately extracted using 90% acetone over 24 hours (Strickland &amp; Parsons, 1968). Total chlorophyll a, phaeophytin, and relative fluorescence were measured daily using a 10 AU fluorometer (Turner). FRR (Fv_Fm and sigma) was measured using a FIRe Fluorometer System (Satlantic) (Kolber et al., 1998) with settings of 100 microseconds (µs) Single Turnover Flash (STF), 80 µs STRI, 20 µs MTF, 40 µs MTRP, 100 µs MTRI, and with gain adjusted based on the fluorescence yield. Samples were preserved for cell counts during the experiment by preserving with 0.125% gluteraldehyde, flash freezing, and storing at -80°C. Samples were then thawed and aliquoted onto a Sedgewick Rafter (PYSER-SGI) and counted. Cells<em> </em>were counted on an Eclipse E800 (Nikon) light microscope, with phycoerythrin emission (565 nanometers (nm) ± 40 nm) and excitation (530 nm ± 30 nm) wavelength filters to count the diazotroph symbionts. Cell size was averaged using at least 30 pictures each of the host and symbiont per flask, and measured using ImageJ and an image of a stage micrometer (OMAX A36CALM1 0.01 mm) at the same magnification of cell pictures. Cell volume and surface area were calculated using 29-H (<em>Hemiaulus</em> <em>hauckii</em>) and 1-H shaped cells (<em>Richelia</em> <em>euintracellularis</em>) equations from Sun and Liu (2003). Dissolved and particulate nutrients were sampled from the same ~100 milliliters (mL) aliqout (Part_vol_mL). Particulate nutrients were filtered out using pre-combusted GF/F (~0.7 µm) Whatman filters. Dissolved nutrients (NO3_uM, NO2_uM, NH4_uM, SiOH4_uM, and PO4_uM) were immediately frozen before measuring on a Seal Analytical AA3 nutrient autoanalyzer and total reduced nitrogen (TDN_uM) was analyzed by oxidizing all nitrogen to nitrate following a persulfate odixation method (Knapp et al., 2005). Particulate nutrients (PC and PN) were immediately frozen until processing, during which samples were dried at 60 °C for 24 hours, packed in 9x10mm tin capsules (Costech) and sent for analysis on the Carlo Erba NC 2500 Elemental Analyzer (with a Costech zero-blank autosampler) at the Central Appalachians Stable Isotope Facility (CASIF) at the University of Maryland.</p>
<p>Carbon and nitrogen fixation measurements were taken using ¹³C and ¹⁵N stable isotope incubations (Hama et al. 1983, Montoya et al. 1996) during the last 24 hours of the experiment (T=Final), following methods from White et al. (2020) and Klawonn et al. (2015). Sample aliqouts (~550 mL) were added to 630 mL polycarbonate bottles (Nalgene), with 214 mL of 0.05 grams per milliliter (g/mL) of H₁₃CO₃ (Sodium Bicarbonate, 13C 99% Cambridge Isotopes) for a final concentration of 200 µM and 63 mL ¹⁵N₂ solution using Cambridge Isotopes ¹⁵N₂, 98%+ (Lot No. I-24583/AR0483820) for a final ¹⁵N₂ dilution of 10% v/v. The ¹⁵N₂ (Cambridge Isotopes 15N2 98%+, Cat # NLM-363-1-LB, Lot # 1-24583/AR0483820) solution added to each flask was made using the dissolution method (Mohr et al. 2010; Klawonn et al. 2015) and letting serum bottles with dissolved gas sit for ~12 hours before the start of the incubation to allow more ¹⁵N₂ to move into solution. After 24 hours, 10 mL of incubation sample was collected through the bottle septum caps, added to helium-flushed 20 mL vials with 20 mm rubber butyl septa crimp caps (Sigma-Aldrich) for % ¹⁵N₂ dissolved gas analysis. A 50% solution of ZnCl₂ was added to each 20 mL vial for sample preservation and vials were stored upside down, submerged in DI water before sending for sample analysis at the UC Davis Stable Isotope Facility for analysis on the GasBench-Precon-IRMS for ¹⁵N₂/N₂ atom % measurements. After dissolved gas samples were collected from incubation bottles, 225-275 mL isotope incubation volume was filtered over pre-combusted GF/F (~0.7 µm) filters (Whatman), which were then immediately frozen and processed in the same way as particulate samples before sending to the CASIF at the University of Maryland (UMD) for isotope analysis (d¹⁵N and d¹³C) on a Thermo Fisher Delta V+ isotope ratio mass spectrometer interfaced with the Carlo Erba NC 2500 Elemental Analyzer. One sample (NegCon) was incubated with a diatom (<em>Thalassiosira pseudonana -</em> f/2 media) with no nitrogen fixers to test for contamination (¹⁵NO₃ or ¹⁵NH₄) in the ¹⁵N₂ gas stock, from which there was no indication of contamination.</p>
<p>The culture used in this experiment was isolated in 2018; these data were collected in 2022.</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-2227425 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=2227425
completed
Tatiana A. Rynearson
University of Rhode Island
401-874-6022
Graduate School of Oceanography, University of Rhode Island 215 South Ferry Rd.
Narragansett
RI
02882
USA
rynearson@uri.edu
pointOfContact
Kei Inomura
University of Rhode Island
215 S Ferry Rd.
Narragansett
RI
02882
USA
inomura@uri.edu
pointOfContact
asNeeded
Dataset Version: 1
Unknown
Flask
Time_gp
Timepoint
Treatment
RFU_Turner
chla
phaeophytin
Hemiaulus_cellspermL
Richelia_cellspermL
Asymb_cellspermL
Hemaiulus_mean_SA
Hemaiulus_mean_vol
Richelia_mean_SA
Richelia_mean_vol
NO3_uM
NO2_uM
NH4_uM
SiOH4_uM
PO4_uM
TDN_uM
Part_vol_mL
PC
PN
Fv_Fm
sigma
d15N
massN_mg
d13C
massC_mg
atperc_15N2_N2
vol_filt_L
inc_time_min
pp_rate_ugCperLperday
NFR_nmolNperLperday
Carlo Erba NC 2500 Elemental Analyzer
Thermo Fisher Delta V+ isotope ratio mass spectrometer
GasBench-Precon-IRMS
Costech zero-blank autosampler
Eclipse E800 (Nikon) light microscope
FIRe Fluorometer System (Satlantic)
Sedgewick Rafter (PYSER-SGI)
10 AU fluorometer (Turner)
theme
None, User defined
sample identification
time_point
treatment
fluorescence
chlorophyll a
total phaeopigment
abundance
surface_area
volume
Nitrate
Nitrite
Ammonium
Silicate, Si(OH)4, silicic acid
reactive phosphorus (PO4)
Total Dissolved Nitrogen
Elemental carbon (C)
Nitrogen
Fv to Fm ratio
No BCO-DMO term
d15N
mass
d13C
percent composition
time_elapsed
rate of 14C fixation
nitrogen fixation rate
featureType
BCO-DMO Standard Parameters
Carlo Erba NC 2500 elemental analyzer
Isotope-ratio Mass Spectrometer
Isotope-ratio Mass Spectrometer
Laboratory Autosampler
Microscope - Optical
Satlantic Fluorescence Induction and Relaxation of Emission Spectrometer
Sedgewick Rafter Counting Chamber
Turner Designs Fluorometer 10-AU
instrument
BCO-DMO Standard Instruments
otherRestrictions
otherRestrictions
Access Constraints: none. Use Constraints: Please follow guidelines at: http://www.bco-dmo.org/terms-use Distribution liability: Under no circumstances shall BCO-DMO be liable for any direct, incidental, special, consequential, indirect, or punitive damages that result from the use of, or the inability to use, the materials in this data submission. If you are dissatisfied with any materials in this data submission your sole and exclusive remedy is to discontinue use.
META-DDA: METabolic Activities of Diatom-Diazotroph Associations
https://osprey.bco-dmo.org/project/913989
META-DDA: METabolic Activities of Diatom-Diazotroph Associations
<p>Diatom diazotroph associations (DDAs) have broad geographic distributions, provide bioavailable nitrogen to the biosphere via nitrogen fixation, affect ecosystem functioning and influence biogeochemical cycling. Despite the importance of these symbioses, little is known about their basic physiology and metabolism because DDAs are rarely brought into and kept in culture. Without cultured strains, it is difficult to study experimentally how the partners interact, share and potentially compete for resources. Here we evaluate and model key physiological characteristics of a DDA (Hemiaulus hauckii-Richelia intracellularis) isolated from the Sargasso Sea to investigate how their physiology is altered by nitrogen sources and temperature. The isolated strain is growing well, fixing nitrogen and can be manipulated in the laboratory, allowing for an unprecedented view into the physiology and metabolism of a biogeochemically important ocean symbiosis. Empirical data informs the development of quantitative cell flux model of DDAs (CFM-DDA) embedded into a simple ecosystem model to test how different nitrogen sources and temperatures shape the niche of DDAs. The physiology of DDAs is compared to asymbiotic diatoms to examine the conditions where diazotrophic symbionts benefit the host diatoms and allow them to expand their ecological niche. This research addresses fundamental knowledge gaps that will lead to an enhanced understanding of DDA distributions and activities both in today's ocean and in a future ocean with altered temperature and nutrient fields. </p>
<p><em><strong>NSF Award Abstract:</strong></em><br />
Phytoplankton are photosynthetic microbes that inhabit the surface ocean, form the base of marine food webs, and drive the global cycling of elements like carbon and nitrogen. To survive in regions with limiting nutrients for growth, some phytoplankton have evolved symbiotic relationships. In many cases, it remains unknown how the symbioses influence the survival of each partner or their impacts on ecosystem function and cycling of nutrients. This project focuses on the symbiotic relationship between two phytoplankton -- a single-celled eukaryote diatom and a single-celled nitrogen fixing cyanobacteria called a diazotroph. These diatom-diazotroph associations (DDAs) have broad geographic distributions, provide bioavailable nitrogen to the biosphere via the fixation of nitrogen gas, affect marine food webs, and influence the cycling of carbon and nitrogen. Despite the importance of these symbioses, little is known about their basic physiology and metabolism because it has been difficult for researchers to grow DDAs in the laboratory. In this project, a team of investigators from the University of Rhode Island is applying a method they developed to grow DDAs in the laboratory and conducting experiments on the effects of temperature and nutrients on DDA cellular metabolism. The newly-generated laboratory data is informing the development of a computer model of DDA cellular functioning that embedded in a simple ecosystem model to test how different nitrogen sources and temperatures influence DDA ecology and ecosystem function. The project is addressing fundamental knowledge gaps, leading to an enhanced understanding of DDA geographic distributions and activities both in today's ocean and in a future ocean with altered temperature and nutrient fields. Broader impacts of this study include the provision of DDA cultures to the oceanographic community, graduate student training, computational model distribution, and outreach to the broader community. Graduate students supported by the project are being cross-trained in experimental and modeling approaches. Outreach to the broader community includes hosting a high school student intern in the lab each year and the development of educational videos for the general public and K-12 students. Collectively, these activities are designed to broaden the public understanding of DDAs, a globally significant symbiosis.</p>
<p>This project is examining the cellular metabolism and physiology of diatom-diazotroph associations (DDAs) and evaluating their ecosystem and biogeochemical impacts by addressing the following critical, longstanding questions: 1) What is the cellular response of the Hemiaulus DDA to different nitrogen sources? 2) How does the thermal niche of the DDA influence nitrogen fixation, nutrient stoichiometries, and geographic distribution? 3) How does DDA physiology and metabolism differ from asymbiotic diatoms, and what are the ecosystem-level impacts of the symbiosis? Due to a scarcity of culture data, major ecological models assume DDAs gain 100% of their nitrogen from N2, although there is intriguing experimental evidence suggesting otherwise. If DDA physiology is affected by different N sources, key assumptions in modern ecosystem models will be altered, refining our understanding of the role DDAs play in ecosystem and biogeochemical functioning. In addition to N sources, temperature is an important regulator of cellular metabolism and a key variable in ecosystem models. The team of researchers is examining the roles of each partner in the symbiosis in setting the DDA thermal niche and examining ecosystem-level impacts via modeling both in the present day and future oceans. Finally, the impacts of the endosymbiont on the host genome, transcriptome, and resulting physiology are practically unknown. Comparison of DDAs with asymbiotic diatoms is providing new insights into the metabolic modifications of the host and providing new understanding of DDAs as a symbiosis. Addressing these three questions advances fundamental understanding of the impact of this widely-distributed symbiosis.</p>
META-DDA
largerWorkCitation
project
eng; USA
oceans
-63.48
-63.48
32.42
32.42
2018-05-01
2022-05-31
Oligotrophic and shelf waters
0
BCO-DMO catalogue of parameters from Hemiaulus-Richelia physiological response to different nitrogen sources
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
http://lod.bco-dmo.org/id/dataset-parameter/921945.rdf
Name: Flask
Units: unitless
Description: <p>Either flask (F1-F16, Innoc1-3) or sample id (NegCon)</p>
http://lod.bco-dmo.org/id/dataset-parameter/921946.rdf
Name: Time_gp
Units: unitless
Description: <p>Time point group (either initial or final)</p>
http://lod.bco-dmo.org/id/dataset-parameter/921947.rdf
Name: Timepoint
Units: days
Description: <p>Day each sample was collected</p>
http://lod.bco-dmo.org/id/dataset-parameter/921948.rdf
Name: Treatment
Units: unitless
Description: <p>Type of nutrient addition treatment: either no added nitrogen (Only N2, control), 10 uM added ammonium ( +NH4), or 10 uM added nitrate ( +NO3). Innoculum cultures are indicated with Innoc, and the isotope incubation negative control is indicated with NegCon.</p>
http://lod.bco-dmo.org/id/dataset-parameter/921949.rdf
Name: RFU_Turner
Units: relative fluorescence units
Description: <p>chlorophyll a relative fluorescence</p>
http://lod.bco-dmo.org/id/dataset-parameter/921950.rdf
Name: chla
Units: micrograms per cell (ug/cell)
Description: <p>total chlorophyll a concentration</p>
http://lod.bco-dmo.org/id/dataset-parameter/921951.rdf
Name: phaeophytin
Units: micrograms per cell (ug/cell)
Description: <p>phaeophytin concentration</p>
http://lod.bco-dmo.org/id/dataset-parameter/921952.rdf
Name: Hemiaulus_cellspermL
Units: cells per milliliter (cells/mL)
Description: <p>Cell counts of Hemiaulus hauckii</p>
http://lod.bco-dmo.org/id/dataset-parameter/921953.rdf
Name: Richelia_cellspermL
Units: cells per milliliter (cells/mL)
Description: <p>Cell counts of Richelia intracellularis</p>
http://lod.bco-dmo.org/id/dataset-parameter/921954.rdf
Name: Asymb_cellspermL
Units: cells per milliliter (cells/mL)
Description: <p>Cell counts of Hemiaulus hauckii host cells without symbionts</p>
http://lod.bco-dmo.org/id/dataset-parameter/921955.rdf
Name: Hemaiulus_mean_SA
Units: square micrometers (um^2)
Description: <p>mean surface area of Hemiaulus hauckii cells</p>
http://lod.bco-dmo.org/id/dataset-parameter/921956.rdf
Name: Hemaiulus_mean_vol
Units: cubic micrometers (um^3)
Description: <p>mean volume of Hemiaulus hauckii cells</p>
http://lod.bco-dmo.org/id/dataset-parameter/921957.rdf
Name: Richelia_mean_SA
Units: square micrometers (um^2)
Description: <p>mean surface area of Richelia intracellularis cells</p>
http://lod.bco-dmo.org/id/dataset-parameter/921958.rdf
Name: Richelia_mean_vol
Units: cubic micrometers (um^3)
Description: <p>mean volume of Richelia intracellularis cells</p>
http://lod.bco-dmo.org/id/dataset-parameter/921959.rdf
Name: NO3_uM
Units: micromolar (uM)
Description: <p>dissolved nitrate concentration</p>
http://lod.bco-dmo.org/id/dataset-parameter/921960.rdf
Name: NO2_uM
Units: micromolar (uM)
Description: <p>dissolved nitrite concentration</p>
http://lod.bco-dmo.org/id/dataset-parameter/921961.rdf
Name: NH4_uM
Units: micromolar (uM)
Description: <p>dissolved ammonium concentration</p>
http://lod.bco-dmo.org/id/dataset-parameter/921962.rdf
Name: SiOH4_uM
Units: micromolar (uM)
Description: <p>dissolved silicate concentration</p>
http://lod.bco-dmo.org/id/dataset-parameter/921963.rdf
Name: PO4_uM
Units: micromolar (uM)
Description: <p>dissolved phosphate concentration</p>
http://lod.bco-dmo.org/id/dataset-parameter/921964.rdf
Name: TDN_uM
Units: micromolar (uM)
Description: <p>total dissolved nitrogen concentration</p>
http://lod.bco-dmo.org/id/dataset-parameter/921965.rdf
Name: Part_vol_mL
Units: milliliters (mL)
Description: <p>volume of sample filtered for particulate nutrients</p>
http://lod.bco-dmo.org/id/dataset-parameter/921966.rdf
Name: PC
Units: micromolar (uM)
Description: <p>particulate carbon</p>
http://lod.bco-dmo.org/id/dataset-parameter/921967.rdf
Name: PN
Units: micromolar (uM)
Description: <p>particulate nitrogen</p>
http://lod.bco-dmo.org/id/dataset-parameter/921968.rdf
Name: Fv_Fm
Units: unitless
Description: <p>FRR variable fluorescence (Fv/Fm) or PSII photosynthetic efficiency</p>
http://lod.bco-dmo.org/id/dataset-parameter/921969.rdf
Name: sigma
Units: square Angstroms per quanta (Å^2 (quanta)^-1)
Description: <p>FRR PSII Absorption cross-section</p>
http://lod.bco-dmo.org/id/dataset-parameter/921970.rdf
Name: d15N
Units: parts per thousand (ppt)
Description: <p>delta 15N</p>
http://lod.bco-dmo.org/id/dataset-parameter/921971.rdf
Name: massN_mg
Units: milligrams (mg)
Description: <p>mass of particulate N for delta 15N samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/921972.rdf
Name: d13C
Units: parts per thousand (ppt)
Description: <p>delta 13C</p>
http://lod.bco-dmo.org/id/dataset-parameter/921973.rdf
Name: massC_mg
Units: milligrams (mg)
Description: <p>mass of particulate C for delta 13C samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/921974.rdf
Name: atperc_15N2_N2
Units: percent
Description: <p>atom % of 15N2/N2 dissolved gasses</p>
http://lod.bco-dmo.org/id/dataset-parameter/921975.rdf
Name: vol_filt_L
Units: liters (L)
Description: <p>volume of isotope incubation filtered</p>
http://lod.bco-dmo.org/id/dataset-parameter/921976.rdf
Name: inc_time_min
Units: minutes
Description: <p>duration of the isotope incubation experiments</p>
http://lod.bco-dmo.org/id/dataset-parameter/921977.rdf
Name: pp_rate_ugCperLperday
Units: micrograms C per liter per day (ug C L^-1 d^-1)
Description: <p>carbon fixation rate</p>
http://lod.bco-dmo.org/id/dataset-parameter/921978.rdf
Name: NFR_nmolNperLperday
Units: nanomoles N per liter per day (nM N L^-1 d^-1)
Description: <p>nitrogen fixation rate</p>
GB/NERC/BODC > British Oceanographic Data Centre, Natural Environment Research Council, United Kingdom
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
8755
https://darchive.mblwhoilibrary.org/server/api/core/bitstreams/17e0faf6-1934-41a7-bf98-268ac8339bcf/content
download
https://doi.org/10.26008/1912/bco-dmo.921924.1
download
onLine
dataset
<p>Samples were collected from culture flasks of a <em>Hemiaulus</em>-<em>Richelia</em> diatom diazotroph association (DDA) over the course of 2.5 weeks. Sargasso Seawater collected on the R/V Atlantic Explorer AE1812 cruise in May 2018 (at 32.42°N, 63.48°W) was used for media and filtered via peristaltic pump over 0.2 micrometers (µm) 47-millimeter (mm) filters (Polyethersulfone filters Millipore Express PLUS #GPWP04700). All flasks were grown in f/5 media without nitrogen, one set of flasks had no added nitrogen (Only N2), one set 10 µM added ammonium treatment (+NH4), and one set 10 µM added nitrate treatment (+NO3). Flasks were grown at 22 degrees Celsius (°C), ~130 micromoles per square meter per second (µmol m-2 s-1) in a 12:12 light:dark cycle. Two time points were used, an initial (T=0) and a final (T=15,17,18,19, depending on flask growth). Samples from the culture innoculum (Innoc1, Innoc2, Innoc3) were collected before all three innoculum flasks were thoroughly mixed and added to the experiment flasks.</p>
<p>Total chlorophyll a fluorescence was sampled by filtering over GF/F (~0.7 µm Whatmanfilters) and immediately extracted using 90% acetone over 24 hours (Strickland &amp; Parsons, 1968). Total chlorophyll a, phaeophytin, and relative fluorescence were measured daily using a 10 AU fluorometer (Turner). FRR (Fv_Fm and sigma) was measured using a FIRe Fluorometer System (Satlantic) (Kolber et al., 1998) with settings of 100 microseconds (µs) Single Turnover Flash (STF), 80 µs STRI, 20 µs MTF, 40 µs MTRP, 100 µs MTRI, and with gain adjusted based on the fluorescence yield. Samples were preserved for cell counts during the experiment by preserving with 0.125% gluteraldehyde, flash freezing, and storing at -80°C. Samples were then thawed and aliquoted onto a Sedgewick Rafter (PYSER-SGI) and counted. Cells<em> </em>were counted on an Eclipse E800 (Nikon) light microscope, with phycoerythrin emission (565 nanometers (nm) ± 40 nm) and excitation (530 nm ± 30 nm) wavelength filters to count the diazotroph symbionts. Cell size was averaged using at least 30 pictures each of the host and symbiont per flask, and measured using ImageJ and an image of a stage micrometer (OMAX A36CALM1 0.01 mm) at the same magnification of cell pictures. Cell volume and surface area were calculated using 29-H (<em>Hemiaulus</em> <em>hauckii</em>) and 1-H shaped cells (<em>Richelia</em> <em>euintracellularis</em>) equations from Sun and Liu (2003). Dissolved and particulate nutrients were sampled from the same ~100 milliliters (mL) aliqout (Part_vol_mL). Particulate nutrients were filtered out using pre-combusted GF/F (~0.7 µm) Whatman filters. Dissolved nutrients (NO3_uM, NO2_uM, NH4_uM, SiOH4_uM, and PO4_uM) were immediately frozen before measuring on a Seal Analytical AA3 nutrient autoanalyzer and total reduced nitrogen (TDN_uM) was analyzed by oxidizing all nitrogen to nitrate following a persulfate odixation method (Knapp et al., 2005). Particulate nutrients (PC and PN) were immediately frozen until processing, during which samples were dried at 60 °C for 24 hours, packed in 9x10mm tin capsules (Costech) and sent for analysis on the Carlo Erba NC 2500 Elemental Analyzer (with a Costech zero-blank autosampler) at the Central Appalachians Stable Isotope Facility (CASIF) at the University of Maryland.</p>
<p>Carbon and nitrogen fixation measurements were taken using ¹³C and ¹⁵N stable isotope incubations (Hama et al. 1983, Montoya et al. 1996) during the last 24 hours of the experiment (T=Final), following methods from White et al. (2020) and Klawonn et al. (2015). Sample aliqouts (~550 mL) were added to 630 mL polycarbonate bottles (Nalgene), with 214 mL of 0.05 grams per milliliter (g/mL) of H₁₃CO₃ (Sodium Bicarbonate, 13C 99% Cambridge Isotopes) for a final concentration of 200 µM and 63 mL ¹⁵N₂ solution using Cambridge Isotopes ¹⁵N₂, 98%+ (Lot No. I-24583/AR0483820) for a final ¹⁵N₂ dilution of 10% v/v. The ¹⁵N₂ (Cambridge Isotopes 15N2 98%+, Cat # NLM-363-1-LB, Lot # 1-24583/AR0483820) solution added to each flask was made using the dissolution method (Mohr et al. 2010; Klawonn et al. 2015) and letting serum bottles with dissolved gas sit for ~12 hours before the start of the incubation to allow more ¹⁵N₂ to move into solution. After 24 hours, 10 mL of incubation sample was collected through the bottle septum caps, added to helium-flushed 20 mL vials with 20 mm rubber butyl septa crimp caps (Sigma-Aldrich) for % ¹⁵N₂ dissolved gas analysis. A 50% solution of ZnCl₂ was added to each 20 mL vial for sample preservation and vials were stored upside down, submerged in DI water before sending for sample analysis at the UC Davis Stable Isotope Facility for analysis on the GasBench-Precon-IRMS for ¹⁵N₂/N₂ atom % measurements. After dissolved gas samples were collected from incubation bottles, 225-275 mL isotope incubation volume was filtered over pre-combusted GF/F (~0.7 µm) filters (Whatman), which were then immediately frozen and processed in the same way as particulate samples before sending to the CASIF at the University of Maryland (UMD) for isotope analysis (d¹⁵N and d¹³C) on a Thermo Fisher Delta V+ isotope ratio mass spectrometer interfaced with the Carlo Erba NC 2500 Elemental Analyzer. One sample (NegCon) was incubated with a diatom (<em>Thalassiosira pseudonana -</em> f/2 media) with no nitrogen fixers to test for contamination (¹⁵NO₃ or ¹⁵NH₄) in the ¹⁵N₂ gas stock, from which there was no indication of contamination.</p>
<p>The culture used in this experiment was isolated in 2018; these data were collected in 2022.</p>
Specified by the Principal Investigator(s)
<p>R v4.3.0 was used for all isotope incubation calculations for carbon (pp_rate_ugCperLperday) and nitrogen fixation (NFR_nmolNperLperday).</p>
Specified by the Principal Investigator(s)
- Imported original file "Meta-DDA_NitAdd_2022.csv" into the BCO-DMO system.
- Marked "NA" as a missing data value; note that missing data are empty/blank in the final CSV file.
- Saved the final file as "921924_v1_dda_nutrient_acquisition.csv".
Specified by BCO-DMO Data Managers
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For questions regarding this resource, please contact BCO-DMO via the email address provided.
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Carlo Erba NC 2500 Elemental Analyzer
Carlo Erba NC 2500 Elemental Analyzer
PI Supplied Instrument Name: Carlo Erba NC 2500 Elemental Analyzer PI Supplied Instrument Description:Particulate nutrients were measured on a Carlo Erba NC 2500 Elemental Analyzer (with a Costech zero-blank autosampler). Instrument Name: Carlo Erba NC 2500 elemental analyzer Instrument Short Name:Carlo Erba NC 2500 EA Instrument Description: A laboratory instrument that simultaneously determines total nitrogen, total hydrogen, and total carbon in a solid sample. The sample is completely and instantaneously oxidized 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 helium carrier gas. The gases are separated in the column and quantified or they can be introduced into another instrument, such as an isotope ratio mass spectrometer, for further analysis. The instrument was originally manufactured by CE instruments (formerly Carlo Erba) and has since been replaced by Thermo Scientific (part of Thermo Fisher Scientific). This model is no longer in production.
Thermo Fisher Delta V+ isotope ratio mass spectrometer
Thermo Fisher Delta V+ isotope ratio mass spectrometer
PI Supplied Instrument Name: Thermo Fisher Delta V+ isotope ratio mass spectrometer PI Supplied Instrument Description:Particulate isotope associated with isotope incubations were measured on a Thermo Fisher Delta V+ isotope ratio mass spectrometer interfaced with the Carlo Erba NC 2500 Elemental Analyzer. Both instruments are at the CASIF at the University of Maryland (UMD). Instrument Name: Isotope-ratio Mass Spectrometer Instrument Short Name:IR Mass Spec; IRMS Instrument Description: The Isotope-ratio Mass Spectrometer is a particular type of mass spectrometer used to measure the relative abundance of isotopes in a given sample (e.g. VG Prism II Isotope Ratio Mass-Spectrometer). Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB16/
GasBench-Precon-IRMS
GasBench-Precon-IRMS
PI Supplied Instrument Name: GasBench-Precon-IRMS PI Supplied Instrument Description:Samples for 15N2/N2 atom % were analyzed on the GasBench-Precon-IRMS at the UC Davis Stable Isotope Facility. Instrument Name: Isotope-ratio Mass Spectrometer Instrument Short Name:IR Mass Spec; IRMS Instrument Description: The Isotope-ratio Mass Spectrometer is a particular type of mass spectrometer used to measure the relative abundance of isotopes in a given sample (e.g. VG Prism II Isotope Ratio Mass-Spectrometer). Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB16/
Costech zero-blank autosampler
Costech zero-blank autosampler
PI Supplied Instrument Name: Costech zero-blank autosampler PI Supplied Instrument Description:Particulate nutrients were measured on a Carlo Erba NC 2500 Elemental Analyzer (with a Costech zero-blank autosampler). Instrument Name: Laboratory Autosampler Instrument Short Name: Instrument Description: Laboratory apparatus that automatically introduces one or more samples with a predetermined volume or mass into an analytical instrument.
Eclipse E800 (Nikon) light microscope
Eclipse E800 (Nikon) light microscope
PI Supplied Instrument Name: Eclipse E800 (Nikon) light microscope PI Supplied Instrument Description:A Sedgewick Rafter (PYSER-SGI), Eclipse E800 (Nikon) light microscope, and stage micrometer (OMAX A36CALM1 0.01 mm) were used for cell size and count measurements. Instrument Name: Microscope - Optical Instrument Short Name: 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". Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/
FIRe Fluorometer System (Satlantic)
FIRe Fluorometer System (Satlantic)
PI Supplied Instrument Name: FIRe Fluorometer System (Satlantic) PI Supplied Instrument Description:The FIRe Fluorometer System (Satlantic) was used for FRR measurements. Instrument Name: Satlantic Fluorescence Induction and Relaxation of Emission Spectrometer Instrument Short Name:Satlantic FIRe Instrument Description: The Satlantic FIRe (Fluorescence Induction and Relaxation) System is a bio-optical technology used to measure variable chlorophyll fluorescence in photosynthetic organisms. Based on the Fast Repetition Rate Fluorometry (FRRF) technique, the FIRe was developed in collaboration with Dr. Maxim Gorbunov and Dr. Paul Falkowski from Rutgers University.
More information on FIRe (PDF).
Sedgewick Rafter (PYSER-SGI)
Sedgewick Rafter (PYSER-SGI)
PI Supplied Instrument Name: Sedgewick Rafter (PYSER-SGI) PI Supplied Instrument Description:A Sedgewick Rafter (PYSER-SGI), Eclipse E800 (Nikon) light microscope, and stage micrometer (OMAX A36CALM1 0.01 mm) were used for cell size and count measurements. Instrument Name: Sedgewick Rafter Counting Chamber Instrument Short Name: Instrument Description: Sedgewick Rafter Counting Chambers are transparent slides widely water analysis, culture inspection, and for other applications where particles per unit volume in fluid must be determined. The slide has a base that is ruled in one-thousand 1-millimeter squares. When a liquid is held in the cell by a coverglass, the grid subdivides 1 milliliter of liquid into 1 microliter volume.
10 AU fluorometer (Turner)
10 AU fluorometer (Turner)
PI Supplied Instrument Name: 10 AU fluorometer (Turner) PI Supplied Instrument Description:A 10 AU fluorometer (Turner) was used for fluorescence measurements. Instrument Name: Turner Designs Fluorometer 10-AU Instrument Short Name:Turner Fluorometer 10-AU Instrument Description: The Turner Designs 10-AU Field Fluorometer is used to measure Chlorophyll fluorescence. The 10AU Fluorometer can be set up for continuous-flow monitoring or discrete sample analyses. A variety of compounds can be measured using application-specific optical filters available from the manufacturer (read more from Turner Designs, turnerdesigns.com, Sunnyvale, CA, USA). Community Standard Description: http://vocab.nerc.ac.uk/collection/L22/current/TOOL0393/