The “dissolution” method to measure N2 fixation rates with 15N2 gas tracer involves the preparation of 15N2-enriched water that is then added to each incubation bottle. Investigators typically measure the 15N2 atom% of the 15N2-enriched inoculum by MIMS, and extrapolate the 15N2 atom% in the incubations based on the inoculum value. Here, we demonstrate that such extrapolation yields inaccurate estimates of the 15N2 atom% of incubations. The latter should be measured directly.

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Version Date	Archive	Persistent Identifier	Date Assigned
2019-10-02	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.1575/1912/bco-dmo.778158.1	2020-01-06

Description

Trial b test of the dissolution method

Methods & Sampling

Inocula of 15N2-enriched water were prepared according to either of two protocols outlined by Klawonn et al. (2015).
In a first Trial A, respective 1.9 mL of 15N2 gas aliquots (Cambridge Isotope Laboratories, Lot #I-21065) were injected into crimped-sealed 120 mL glass serum vials filled with deionized water. To dissolve the 15N2 bubble, each of the two serum vials was vortexed for 5 minutes. Two subsamples of each inoculum were dispensed into Exetainers™ with a peristaltic pump for analysis on the MIMS. An aliquot of each inoculum (5 % vol/vol) was then dispensed in replicate 160 mL serum incubation bottles containing air-equilibrated deionized water (Trials A1-A4), which were then crimped-sealed. Following homogenization, triplicate subsamples of each incubation were collected in Exetainers™ for MIMS analysis. The 15N atom % of the inocula and of the corresponding incubations were measured by MIMS at the University of Connecticut (Bay Instruments) and computed as follows:

Equation 4:

In Trial B, duplicate 6 mL, 12 mL, and 24 mL aliquots of enriched seawater, prepared as per Wilson et al. (2012; Cambridge Isotope Laboratories 15N2 gas aliquots, Lot #I-19168A), were added to 100 mL glass serum vials, filled with air equilibrated seawater, and crimp-sealed with no headspace using Teflon-lined septa. Triplicate subsamples of this dilution series and the enriched seawater were analyzed at the University of Hawaii on a MIMS (Bay Instruments; Eq. 4).

In both trials, the concentration of N2 isotopologues (m/z 28, 29, and 30) in each of the 15N2-enriched inocula was then extrapolated from the ionization efficiency of N isotopologues in air-equilibrated seawater. We define the ionization efficiency as the ratio of the isotopologue ion current measured by MIMS relative to its concentration in air-equilibrated seawater (ASW):

Equation S2:

For instance, at a temperature of 25ºC and salinity of 35 psu, the solubility coefficients of Hamme and Emerson (2004) predict a N2 concentration of 388.9 μmol kg-1. The fraction of 15N in N2 (i.e., 15N/(14N+15N)) for air-equilibrated seawater is 0.003663 (Mariotti, 1983), such that the expected fractions of 28N2, 29N2, and 30N2 derived from their binomial probability distributions are as follows:

= 99.2687 % Equation S3a

= 0.7299 % Equation S3b

= 0.0013 % Equation S3c

Accordingly, air-equilibrated concentrations of 28N2, 29N2, and 30N2 at this temperature and salinity are 386.0, 2.8, and 0.005 μmol kg-1, respectively. The ionization efficiency of the isotopologues is then equal to the ion current of m/z 28 recorded for ASW divided by the corresponding 28N2 concentration (Eq. S2). We used the ionization efficiency of m/z 28 in ASW to derive the N2 isotopologue concentrations in the inocula from their respective MIMS ion currents. We did not derive distinct ionization efficiencies from the ion current-to-concentration of m/z 29 and 30 in ASW, as these isotopologues are poorly resolved by the MIMS at natural abundance. Thus, we are assuming that the ionization efficiency of m/z 29 and 30 isotopologues is roughly similar to that of m/z 28 (i.e., that ionization isotope effects are negligible for our purposes). The initial expected AN2 of the “incubations” was then calculated using a linear mixing model with N2 isotopologue concentrations in ambient and enriched seawater

Data Processing Description

BCO-DMO Processing Notes:
- table was extracted from original spreadsheet.
- added conventional header with dataset name, PI name, version date
- modified parameter names to conform with BCO-DMO naming conventions

Data Files

File
trial_b.csv (Comma Separated Values (.csv), 3.67 KB) MD5:18132efe094760118290a877fd6b8fcf Primary data file for dataset ID 778158

Supplemental Files

File
equation 4 EAGER_NitFix filename: equation_4.jpg (JPEG Image (.jpg), 24.62 KB) MD5:25f19b478991407bd347bd9221c4857b The 15N atom % of the inocula and of the corresponding incubations were measured by MIMS at the University of Connecticut (Bay Instruments) and computed using this equation.
equation S2 Eager_NitFix filename: equation_s2.jpg (JPEG Image (.jpg), 23.66 KB) MD5:af88aa623a8e948669b26dbeb4dbaeaf Equation for the definition of the ionization efficiency as the ratio of the isotopologue ion current measured by MIMS relative to its concentration in air-equilibrated seawater (ASW).
equation s3a Eager_NitFix filename: equation_s3a.jpg (JPEG Image (.jpg), 9.58 KB) MD5:fdc41b085863b05134fbb25f6cc25c54 Equation for the binomial probability distribution for 28N2.
equation s3b Eager_NitFix filename: equation_s3b.jpg (JPEG Image (.jpg), 22.12 KB) MD5:d7200880be28043d090d28bebeda9d12 Equation for the binomial probability distribution for 29N2.
equation s3c Eager_NitFix filename: equation_s3c.jpg (JPEG Image (.jpg), 14.71 KB) MD5:04c7942d3273c43e9c10e3ed2b41b0fd Equation for the binomial probability distribution for 30N2.

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1732246

Deployments

None available yet

Instruments

Isotope-ratio Mass Spectrometer

Supplied Name: Isotope Ratio Mass Spectrometer

Supplied Description:

continuous flow Delta V Isotope Ratio Mass Spectrometer (Smith et al. 2015), and continuous flow-GV Isoprime IRMS (Charoenpong et al., 2014)

Instrument Type

Generic Name: Isotope-ratio Mass Spectrometer

Acronym: IR Mass Spec; IRMS

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB16/

Generic 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).

Membrane Inlet Mass Spectrometer

Supplied Description:

Membrane Inlet Mass Spectrometer (Bay Instruments)

Instrument Type

Generic Name: Membrane Inlet Mass Spectrometer

Acronym: MIMS

Generic Description:

Membrane-introduction mass spectrometry (MIMS) is a method of introducing analytes into the mass spectrometer's vacuum chamber via a semipermeable membrane.

Parameters

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

Supplied Name	Supplied description	Supplied Units	Standard Name
Time	time	unitless	no_bcodmo_term
Sample_ID	sample	unitless	no_bcodmo_term
ratio_30_28	ratio 30/28	unitless	no_bcodmo_term
O2_Ar	O2 to Ar ratio	unitless	no_bcodmo_term
Mass_z_40	mass-to-charge	unitless	no_bcodmo_term
Mass_z_32	mass-to-charge	unitless	no_bcodmo_term
Mass_z_30	mass-to-charge	unitless	no_bcodmo_term
Mass_z_29	mass-to-charge	unitless	no_bcodmo_term
Mass_z_28	mass-to-charge	unitless	no_bcodmo_term
atom_pcnt_15N	15N atom %	unitless	no_bcodmo_term

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