Bulk uptake 15N2

Data types › Nitrogen Fixation › Bulk uptake 15N2

• Page authors: PRIMO
• Responsible curator: Hagen Buck-Wiese

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N2 fixation (bulk 15N2)
Approach: 15N2 stable isotope tracer uptake
Context: incubation, in situ
Spatial scale: point sample
Temporal scale: hours to one day
Units: nmol N L-1 d-1; µmol N m-2 d-1 (depth-integrated)
Community captured: bulk, size-fractionated
Co-measurements: background δ15N (PON), 15N2 enrichment of dissolved phase; can couple with 13CO2 uptake

Method Overview

¹⁵N₂ gas is dissolved into seawater and added to incubation bottles, labelling the dissolved N₂ pool. Diazotrophic microorganisms fix the labelled N₂, incorporating ¹⁵N into their biomass. After incubation, the particulate fraction is collected on pre-combusted GF/F filters; the ¹⁵N enrichment in particulate nitrogen (PN) is measured by EA-IRMS. The N₂ fixation rate is calculated from the ¹⁵N excess in PN relative to the ¹⁵N enrichment of the dissolved N₂ pool, measured by MIMS or GC-IRMS.

Two labelling approaches are used: (1) direct injection of ¹⁵N₂ gas bubbles followed by vigorous shaking and equilibration; and (2) the dissolution method, in which pre-dissolved ¹⁵N₂-enriched water is injected, achieving more homogeneous labelling. The method can be coupled with ¹³CO₂ uptake in the same incubation to simultaneously measure N₂ fixation and carbon fixation by diazotrophs.

Scale of measurement

Each incubation provides a point measurement. Depth-integrated fixation rates are obtained by incubating bottles collected from multiple depths and integrating over the euphotic zone. Incubation duration is typically hours to one day.

Data generated

N₂ fixation rates in nmol N L^-1 d^-1 or µmol N m^-2 d^-1 (depth-integrated). Rates can be size-fractionated (e.g., 0.2–10 µm versus > 10 µm) to attribute fixation to different diazotroph size classes (unicellular vs. filamentous). Fixed nitrogen can also be tracked into the dissolved organic nitrogen (DON) pool.

Units & currency

Units are nmol N L^-1 d^-1 or µmol N m^-2 d^-1. The currency is nitrogen.

Sample size

Typical samples are 1–5 L in volume per incubation.

Limitations

The ¹⁵N₂ enrichment of the dissolved pool must be measured accurately; under-estimation of the ¹⁵N₂ enrichment (e.g., due to incomplete equilibration of bubbles) leads to overestimation of fixation rates. Bubble injection produces heterogeneous enrichment across replicate bottles; the dissolution method mitigates this. Diazotroph community composition affects the timing of fixation: Crocosphaera fixes N₂ primarily at night, UCYN-A primarily during the day. Bottle effects apply.

Example Applications & Protocols

Classic examples

• Montoya et al. (1996) A simple, high-precision, high-sensitivity tracer assay for N₂ fixation
• Wilson et al. (2012) Comparative assessment of nitrogen fixation methodologies, conducted in the oligotrophic North Pacific Ocean
• Großkopf et al. (2012) Doubling of marine dinitrogen-fixation rates based on direct measurements

Recent applications

• Tschitschko et al. (2024) Rhizobia–diatom symbiosis fixes missing nitrogen in the ocean

Common calculations/conversions

• N₂ fixation rate (nmol N L^-1 d^-1) = [(¹⁵N_PN,final − ¹⁵N_PN,initial) / (¹⁵N_N2,dissolved − ¹⁵N_natural)] × [PN] / incubation time.
• Depth-integrated rate (µmol N m^-2 d^-1) = ∫ rate(z) dz over the euphotic zone.