Dataset: Nitrogen isotope fractionation for ammonium assimilation by marine phytoplankton

Name: Nitrogen isotope fractionation for ammonium assimilation by marine phytoplankton (Biological Nitrogen Isotope Fractionation project)
Published: 2021-11-15
Keywords: oceans

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Cite This Dataset

Temporal Extent: 2018-09-09 - 2020-01-03

Project:

CAREER: The biological nitrogen isotope systematics of ammonium consumption and production (Biological Nitrogen Isotope Fractionation)

Principal Investigator:

Julie Granger (University of Connecticut, UConn)

Contact:

Michael Mathuri (University of Connecticut, UConn)

Analyst:

Cesar Barbedo Rocha (Woods Hole Oceanographic Institution, WHOI)

BCO-DMO Data Manager:

Sawyer Newman (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2021-11-15

Restricted:

Validated:

Current State:

Data not available

Nitrogen isotope fractionation for ammonium assimilation by marine phytoplankton (Biological Nitrogen Isotope Fractionation project)

Abstract:

Results of batch cultures and short-term ammonium (NH4+) uptake experiments were conducted using marine phytoplankton to verify concentration dependence of nitrogen (N) isotope fractionation for NH4+ assimilation.

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Description

Model code description

To query the physiological mechanism of nitrogen (N) isotope fractionation during ammonium (NH4+) assimilation, we constructed a time-dependent finite-differencing box model that tracks discrete 14N and 15N in the different N pools during the growth of marine algae with NH4+ as the sole N source. The model entails three N reservoirs: external N (medium), intracellular reservoir, and the phytoplankton nitrogen (biomass). We prescribe a non-fractionating active NH4+ uptake by the cells via specialized NH4+ transport proteins (AMTs), that follows Michaelis-Menten kinetics with half-saturation constant of 50 nM. Inside the cell, NH4+ is condensed with glutamate by glutamine synthetase (GS) to biomass – the rate-determining and isotope-fractionating step for NH4+ assimilation. The isotope fractionation imparted internally is then communicated to the external medium largely by passive diffusion of ammonia (NH3). A full description of the physiological model and the different scenarios tested can be found in the article. Below is an outline of the various constants and variables used in the model.

Constants

0.00367 – initial ratio of 15N/14N for all the N pools other than external NH4+

0.003695 – initial ratio of 15N/14N for external NH4+

1.76 x 10-5 – equilibrium constant for protonation of NH3

1.58 x 10-6 – concentration of OH- in the external medium at pH 8.2 (mol L-1)

1.0 x 10-7 – concentration of OH- in the cytoplasm at pH 7.0 (mol L-1)

1.0 x 10-9 – concentration of OH- in the vacuole at pH 5.0 (mol L-1)

µmax – maximum specific growth rate (hr-1)

VGSmax – maximum GS rate for NH4+ condensation with glutamate (hr-1)

alphaGS – N isotope fractionation factor for GS

alphaCat – N isotope fractionation factor for catabolic NH4+ production

MultLA – multiplier of the maximum specific growth rate for the low-affinity uptake

MultHA – multiplier of the maximum specific growth rate for the high-affinity uptake

PC_NH3 – permeability coefficient for NH3 (cm hr-1)

PC_NH4 – permeability coefficient for NH4+ (cm hr-1)

BetaNH4 – a term in the constant field equation for NH4+ diffusion across the cellular membrane

expBetaNH4 – exponent of BetaNH4

BetaVac – a term in the constant field equation for NH4+ diffusion across the vacuolar membrane

expBetaVac – exponent of BetaVac

SA – surface area of the phytoplankton cell (cm-2)

SAVac – surface area of the cell vacuole (cm-2)

Cellular N quota – 1.66 x 10-6 µmol N cell-1

Phytoplankton cell volume – 1.15 x 10-12 L

Vacuole volume – 3.45 x 10-13 L

NH4+-NH3 equilibrium isotope effect – 15‰

Variables

CellN14 – 14N in the phytoplankton nitrogen in the culture (µmol L-1)

CellN15 – 15N in the phytoplankton nitrogen in the culture (µmol L-1)

Gln14 – 14N in the glutamine pool in the culture (µmol L-1)

Gln15 – 15N in the glutamine pool in the culture (µmol L-1)

NH4cyt14 – 14N in the cytoplasmic NH4+ in the culture (µmol L-1)

NH4cyt15 – 15N in the cytoplasmic NH4+ in the culture (µmol L-1)

NH4out14 – 14N in the external NH4+ pool (µmol L-1)

NH4out15 – 15N in the external NH4+ pool (µmol L-1)

Vacuole14 – 14N in the vacuolar NH4+ in the culture (µmol L-1)

Vacuole15 – 15N in the vacuolar NH4+ in the culture (µmol L-1)

MMLAU – Michaelis-Menten term for low-affinity NH4+ uptake with half-saturation constant of 30 µmol L-1

MMHAU – Michaelis-Menten term for high-affinity NH4+ uptake with half-saturation constant of 50 nmol L-1

Cell_N_total – phytoplankton nitrogen in the culture (µmol L-1)

"Cells_L-1" – cell density per liter of the culture medium (cells L-1)

CytoplasmNH4conc – cellular concentration of NH4+ in the cytoplasm (µmol L-1)

CytoplasmNH3conc – cellular concentration of NH3 in the cytoplasm (µmol L-1)

NH4concOut – external NH4+ pool (µmol L-1)

F15NH4out – fraction of 15N in the external NH4+

NH3concOut – external NH3 pool (µmol L-1)

Delta_NH3in – difference in NH3 concentration between the cytoplasm and external medium (µmol L-1)

EffluxNH3 – rate of cellular NH3 efflux from the cytoplasm to the external medium (µmol cell-1 hr-1)

d15NH4cyt – N isotope composition of NH4+ in the cytoplasm (‰ vs. air)

d15NH3cyt – N isotope composition of NH3 in the cytoplasm (‰ vs. air)

R15NH3cyt – ratio of 15N in the cytoplasmic NH3

F15NH3cyt – fraction of 15N in the cytoplasmic NH3

VENH314 – rate of 14N NH3 efflux from the cytoplasm to the external medium in the culture (µmol hr-1)

Delta_NH4in – difference in NH4+ concentration between the cytoplasm and external medium (µmol L-1)

EffluxNH4 – rate of cellular NH4+ efflux from the cytoplasm to the external medium (µmol cell-1 hr-1)

F15NH4cyt – fraction of 15N in the cytoplasmic NH4+

VENH414 – rate of 14N NH4+ efflux from the cytoplasm to the external medium in the culture (µmol hr-1)

VINH3Vac14 – rate of 14N NH3 influx from the cytoplasm to the vacuole in the culture (µmol hr-1)

MMGS – Michaelis-Menten term for GS with half-saturation constant of 10 µmol L-1

GS14 – rate of 14N NH4+ condensation with glutamate by GS in the culture (µmol hr-1)

GlnConc – cellular concentration of glutamine (µmol L-1)

MMGln – Michaelis-Menten term for glutamate synthase (GOGAT) with half-saturation constant of 700 µmol L-1

F15Gln – fraction of 15N in the glutamine pool

GOGAT14 – rate of assimilation of 14N glutamine into phytoplankton nitrogen by GOGAT in the culture (µmol hr-1)

VENH315 – rate of 15N NH3 efflux from the cytoplasm to the external medium in the culture (µmol hr-1)

VENH415 – rate of 15N NH4+ efflux from the cytoplasm to the external medium in the culture (µmol hr-1)

VINH3Vac15 – rate of 15N NH3 influx from the cytoplasm to the vacuole in the culture (µmol hr-1)

GS15 – rate of 15N NH4+ condensation with glutamate by GS in the culture (µmol hr-1)

GOGAT15 – rate of assimilation of 15N glutamine into phytoplankton nitrogen by GOGAT in the culture (µmol hr-1)

VENH4Vac14 – rate of 14N NH4+ efflux from the vacuole to the cytoplasm in the culture (µmol hr-1)

VENH4Vac15 – rate of 15N NH4+ efflux from the vacuole to the cytoplasm in the culture (µmol hr-1)

VU14 – rate of 14N NH4+ uptake into the cytoplasm via AMTs in the culture (µmol hr-1)

alphaTR – sigmoidal parametrization of N isotope fractionation factor for NH4+ transport via AMTs

VU15 – rate of 15N NH4+ uptake into the cytoplasm via AMTs in the culture (µmol hr-1)

d15NCell – N isotope composition of cellular N (‰ vs. air)

d15NH4Vac – N isotope composition of NH4+ in the vacuole (‰ vs. air)

d15NH3Vac – N isotope composition of NH3 in the vacuole (‰ vs. air)

d15NH4out – N isotope composition of NH4+ in the external pool (‰ vs. air)

NH4concVac – vacuolar concentration of NH4+ (µmol L-1)

NH3concVac – vacuolar concentration of NH3 (µmol L-1)

Delta_NH3Vac – difference in NH3 concentration between the cytoplasm and vacuole (µmol L-1)

Delta_NH4Vac – difference in NH4+ concentration between the cytoplasm and vacuole (µmol L-1)

EffluxNH4Vac – rate of cellular NH4+ efflux from the vacuole to the cytoplasm (µmol cell-1 hr-1)

F15NH4Vac – fraction of 15N in the vacuolar NH4+

InfluxNH3Vac – rate of cellular NH3 influx from the cytoplasm to the vacuole (µmol cell-1 hr-1)

Methods & Sampling

Phytoplankton cultures

Two strains of marine algae, the diatom Thalassiosira weissflogii (actin) and the prasinophyte Tetraselmis sp. were grown in batch cultures in sterile, acid-washed borosilicate glass or polycarbonate bottles using artificial seawater medium in an environmental chamber at 180C, illuminated with fluorescent light (40 μmol photons m-2 s-1 photosynthetically available radiation) on a 12-hour light and 12-hour dark cycle. The artificial seawater medium was prepared from low nutrient Instant Ocean™ salt dissolved in Milli-Q water and filtered through a 47 mm Whatman GF/F glass microfiber filter (0.7 μm nominal pore-size) and sterilized by autoclaving at 1000C via Pasteurization cycle in PRIMUS steam sterilizer, then supplemented with filter-sterilized 100 – 250 μM NH4+, 10 μM phosphate, 100 μM silicic acid (only for T. weissflogii cultures), and f/2 trace metals and vitamins (Guillard and Ryther 1962). The batch cultures were initiated from inocula of approximately 1,000 cells mL-1, and cell densities monitored daily using Multisizer 4 Beckman Coulter counter. Media subsamples were collected during exponential growth for analyses of NH4+ concentration and N isotope ratios. Subsamples were filtered through a 0.45 μm pore-size polyether-sulfone (PES) syringe filters and collected into acid-washed High Density Poly-Ethylene (HDPE) bottles, solution pH adjusted to ca. 4.5 with dilute hydrochloric acid in order to minimize ammonia volatilization during storage, and samples stored at –200C pending analysis. Particulate organic nitrogen (PON) was also sampled during the exponential growth phase by filtering aqueous subsamples onto a pre-combusted 25 mm Whatman GF/F glass microfiber filters (0.7 μm pore-size) and stored in pre-combusted aluminum foils at –200C pending analysis of N isotope ratio analysis. To capture lower NH4+ concentrations, short-term NH4+ uptake experiments with T. weissflogii and Tetraselmis sp. were conducted. A first set of experiments was conducted with T. weissflogii and Tetraselmis sp. cells in early stationary phase, wherein NH4+ was exhausted from the medium. The cells were collected by gentle filtration onto a 5 μm pore-size 47 mm IsoporeTM polycarbonate membrane filter and resuspended into fresh medium containing ~60 μM NH4+ for T. weissflogii and 20 μM NH4+ for Tetraselmis sp. Aqueous subsamples were collected at regular time intervals until NH4+ in the medium was exhausted. A second set of experiments was conducted with N-replete (cells in exponential growth phase) and N-starved cells (cells two days into stationary phase) of T. weissflogii and Tetraselmis sp. Cell cultures were either diluted into fresh medium containing ~20 µM NH4+, or gently filtered onto a polycarbonate membrane filter and resuspended into said medium. Short-term incubations occurred largely under constant illumination, although some Tetraselmis sp. uptake experiments were inadvertently subject to light-dark conditions.

Determination of NH4+ concentration

Ammonium concentrations at or above 50 μM were measured fluorometrically following derivatization with o-phthalaldehyde (OPA; Holmes et al. 1999) while concentrations < 50 μM were analyzed with the indophenol method (Solórzano 1969).

Analyses of N isotopes of NH4+ and PON

Ammonium samples were diluted to 5 µM or 1 µM with deep Atlantic seawater and N isotope ratios determined using the hypobromite-azide method (Zhang et al. 2007), wherein NH4+ is first oxidized to nitrite by hypobromite, after which nitrite is converted to a nitrous oxide gas analyte by reacting with azide. The N isotopic composition of the nitrous oxide product was analyzed using a continuous flow purge and dual cryogenic trap system coupled to a custom-modified Gas Bench II device interfaced with a Thermo Delta V gas chromatograph isotope ratio mass spectrometer (GC-IRMS; see Casciotti et al. 2002; McIlvin and Casciotti 2011). Calibration to reference (dinitrogen gas in air) was achieved from parallel reactions of NH4+ reference materials IAEA-N1 and IAEA-N2 diluted in deep Atlantic seawater (5 µM or 1 µM solutions), with respective assigned δ15N values of 0.43‰ and 20.3‰ vs. air (Böhlke et al. 1993). To analyze N isotopes of PON, frozen glass microfiber filters were lyophilized for 24 hours using an Edwards Super Modulyo freeze-dryer. The filters were packed into tin capsules and analyzed by combustion to dinitrogen gas on a Costech ECS 4010 Elemental Analyzer followed by N isotope ratio analysis of the resulting dinitrogen gas on a Thermo Delta V isotope ratio mass spectrometer. Samples were calibrated with corresponding aliquots of L-glutamic acid reference materials USGS-40 and USGS-41, with δ15N values of –4.52 and 47.57‰ vs. air, respectively (Qi et al. 2003).

Data Processing Description

Processing notes from data submitter:

Data table generated from laboratory experiments were processed using Microsoft Excel
Model code written in MatLab

BCO-DMO Processing

- Dates converted from mm-dd-yy format to yyyy-mm-dd format
- UTC date time column added
- Column names changed to meet FAIR standards (spaces were replaced with underscores and special characters were removed).

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1554474

Deployments

None available yet

Instruments

Coulter Counter

Supplied Name: Multisizer 4 Beckman Coulter Counter

Supplied Description:

Used to determine phytoplankton cell density.

Instrument Type

Generic Name: Coulter Counter

Generic Description:

An apparatus for counting and sizing particles suspended in electrolytes. It is used for cells, bacteria, prokaryotic cells and virus particles. A typical Coulter counter has one or more microchannels that separate two chambers containing electrolyte solutions.

from https://en.wikipedia.org/wiki/Coulter_counter

Elemental Analyzer

Supplied Name: Costech ECS 4010 Elemental Analyzer coupled with Thermo Delta V isotope ratio mass spectrometer

Supplied Description:

Combustion of PON samples to produce dinitrogen gas for N isotope analyses, with calibration achieved using L-glutamic acid references USGS-40 and USGS-41.

Instrument Type

Generic Name: Elemental Analyzer

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB01/

Generic Description:

Instruments that quantify carbon, nitrogen and sometimes other elements by combusting the sample at very high temperature and assaying the resulting gaseous oxides. Usually used for samples including organic material.

Gas Chromatograph Mass Spectrometer

Supplied Name: Thermo Delta V gas chromatograph isotope ratio mass spectrometer (GC-IRMS)

Supplied Description:

Used to analyze N isotopes of nitrous oxide gas derived from chemical oxidation of NH4+ using the hypobromite-azide method (Zhang et al. 2007), with calibrations achieved using NH4+ reference materials IAEA-N1 and IAEA-N2.

Instrument Type

Generic Name: Gas Chromatograph Mass Spectrometer

Generic Description:

Instruments separating gases, volatile substances or substances dissolved in a volatile solvent by transporting an inert gas through a column packed with a sorbent to a detector for assay by a mass spectrometer.

Spectrophotometer

Supplied Name: U-3010 VIS Spectrophotometer

Supplied Description:

Used to measure NH4+ concentration < 50 µM.

Instrument Type

Generic Name: Spectrophotometer

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

Generic Description:

An instrument used to measure the relative absorption of electromagnetic radiation of different wavelengths in the near infra-red, visible and ultraviolet wavebands by samples.

Turner Designs Trilogy fluorometer

Supplied Name: Turner Trilogy Fluorometer

Supplied Description:

Used to measure NH4+ concentration ≥ 50 µM.

Instrument Type

Generic Name: Turner Designs Trilogy fluorometer

Acronym: Turner Trilogy

Generic Description:

The Trilogy Laboratory Fluorometer is a compact laboratory instrument for making fluorescence, absorbance, and turbidity measurements using the appropriate snap-in application module. Fluorescence modules are available for discrete sample measurements of various fluorescent materials including chlorophyll (in vivo and extracted), rhodamine, fluorescein, cyanobacteria pigments, ammonium, CDOM, optical brighteners, and other fluorescent compounds.

Parameters

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

Supplied Name	Supplied description	Supplied Units	Standard Name
NH4_plus	Ammonium concentration. Concentrations ≥ 50 µM measured fluorometrically on Turner Trilogy Fluorometer with the o-phthalaldehyde (OPA) method (Holmes et al. 1999). Concentrations < 50 _M analyzed on U-3010 VIS Specrophotometer with the indophenol method (Solorzano 1969)	umol L-1	NH4
Date_and_time_EST	Date and time of sampling in EST; %Y-%m-%d %H:%M Format: %Y-%m-%d %H:%M	unitless	ISO_DateTime_Local
Date_and_time_UTC	Date and time of sampling in UTC; %Y-%m-%dT%H:%MZ Format: %Y-%m-%dT%H:%MZ	unitless	ISO_DateTime_UTC
Experiment_number	Experiment identifier; a and b represent replicate trials for batch cultures	unitless	exp_id
Light_conditions	Experiments conducted under 12-hour light and 12-hour dark conditions. Most of the short-term experiments were conducted in light but a few with Tetraselmis sp. extended into the dark period	unitless	treatment
delta_15NNH4_plus	N isotope composition of NH4+ measured using hypobronite-azide method (Zhang et al. 2007) on Thermo Delta V GC-IRMS with modified Gas Bench II and a PAL autosampler	‰ vs. air	NH4
delta_15NPON	N isotope composition of PON measured by combustion on a Thermo Delta V IRMS linked through a Costech ECS 4010 Elementral Analyzer	‰ vs. air	pcent_composition
negative_natural_logarithm_of_NH4_plus	Negative natural logarithm of ammonium concentration as used in the Rayleigh substrate (NH4+) equation (Mariotti et al. 1981)	unitless	NH4
Notes	Notes	unitless	comment
Cell_density	Number of cells per one milliliter of the culture media at different times during the incubation period as determined using Beckman Coulter counter	cells mL-1	cell_concentration
Initial_cell_density	Number of cells per one milliliter of the culture media at start of short-term uptake experiments	cells mL-1	cell_concentration
Incubation_time_in_days	Number of days from the start of the experiment	days	time_inc
Growth_stage	Short-term uptake experiments were conducted with cells harvested in their exponential growth phase (N-replete), early stationary phase (stationary), and in late stationary phase (N-starved).	unitless	stage
stdev_sigma_NH4_plus	Standard deviation for [NH4+] derived from analytical replicates	unitless	NH4
stdev_sigma_delta_15NNH4_plus	Standard deviation for _15NNH4+ derived from analytical replicates using monte carlo error propagation	units	NH4
Experiment_type	The algal strains were incubated either under batch cultures or short-term ammonium uptake experiments	unitless	exp_type
Strain	The two marine algal strains used in the laboratory experiments; a diatom Thalassiosira weissflogii and a prasinophyte Tetraselmis sp.	unitless	species
f_lnf_1_f	Used in the Rayleigh product (PON) equation (Mariotti et al. 1981), where f represents the fraction of ammonium in the media at every sampling time relative to the initial concentration	units	NH4

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Dataset: Nitrogen isotope fractionation for ammonium assimilation by marine phytoplankton