Nitrogen is essential for life but is often a major limiting nutrient for growth in the ocean. Biological dinitrogen fixation is a major source of new nitrogen to surface waters and promotes marine productivity. Yet the fate of diazotroph‐derived nitrogen (DDN) in marine ecosystems has been poorly studied, and its transfer to auto‐ and heterotrophic plankton has not been measured. Here, we use high‐resolution nanometer scale secondary ion mass spectrometry (nanoSIMS) coupled with ¹⁵N₂ isotopic labelling and flow cytometry cell sorting to examine the DDN transfer to specific groups of natural phytoplankton and bacteria during three diazotroph blooms dominated by the cyanobacterium Trichodesmium spp. in the South West Pacific. During these experiments, 13% ± 2% to 48% ± 5% of the fixed ¹⁵N₂ was released into the dissolved pool and 6% ± 1% to 8% ± 2% of this DDN was transferred to non‐diazotrophic plankton after 48 h. The primary beneficiaries of this DDN were diatoms (45% ± 4% to 61% ± 38%) and bacteria (22% ± 27% to 38% ± 12%), followed by pico‐phytoplankton (3% ± 1% to 21% ± 14%). The DDN was quickly converted to non‐diazotrophic plankton biomass, in particular that of diatoms, which increased in abundance by a factor of 1.4–15 over the course of the three experiments. The single‐cell approach we used enabled quantification of the actual transfer of DDN to specific groups of autotrophic and heterotrophic plankton in the surface ocean, revealing a previously unseen level of complexity in the pathways that occur between N₂ fixation and the eventual export of DDN from the photic zone.

Dinitrogen (N₂) fixation is an important source of biologically reactive nitrogen (N) to the global ocean. The magnitude of this flux, however, remains uncertain, in part because N₂ fixation rates have been estimated following divergent protocols and because associated levels of uncertainty are seldom reported—confounding comparison and extrapolation of rate measurements. A growing number of reports of relatively low but potentially significant rates of N₂ fixation in regions such as oxygen minimum zones, the mesopelagic water column of the tropical and subtropical oceans, and polar waters further highlights the need for standardized methodological protocols for measurements of N₂ fixation rates and for calculations of detection limits and propagated error terms. To this end, we examine current protocols of the ¹⁵N₂ tracer method used for estimating diazotrophic rates, present results of experiments testing the validity of specific practices, and describe established metrics for reporting detection limits. We put forth a set of recommendations for best practices to estimate N₂ fixation rates using ¹⁵N₂ tracer, with the goal of fostering transparency in reporting sources of uncertainty in estimates, and to render N₂ fixation rate estimates intercomparable among studies.

Traditionally, cyanobacterial activity in oceanic photic layers was considered responsible for the marine pelagic dinitrogen (N2) fixation. Other potentially N2-fixing bacteria and archaea have also been detected in the pelagic water column, however, the activity and importance of these non-cyanobacterial diazotrophs (NCDs) remain poorly constrained. In this perspective we summarize the N2 fixation rates from recently published studies on photic and aphotic layers that have been attributed to NCD activity via parallel molecular measurements, and discuss the status, challenges, and data gaps in estimating NCNF in the ocean. Rates attributed to NCNF have generally been near the detection limit thus far (<1 nmol N L-1 d-1). Yet, if considering the large volume of the dark ocean, even low rates of non-cyanobacterial N2 fixation (NCNF) could make a significant contribution to the new nitrogen input to the ocean. The synthesis here shows that nifH transcription data for NCDs have been reported in only very few studies where N2 fixation rates were reported in the absence of diazotrophic cyanobacteria. In addition, high apparent diversity and regional variability in the NCDs complicate investigations of these communities. Future studies should focus on further investigating impacts of environmental drivers including oxygen, dissolved organic matter, and dissolved inorganic nitrogen on NCNF. Describing the ecology of NCDs and accurately measuring NCNF rates, are critical for a future evaluation of the contribution of NCNF to the marine nitrogen budget.

Marine diazotrophs convert dinitrogen (N₂) gas into bioavailable nitrogen (N), supporting life in the global ocean. In 2012, the first version of the global oceanic diazotroph database (version 1) was published. Here, we present an updated version of the database (version 2), significantly increasing the number of in situ diazotrophic measurements from 13 565 to 55 286. Data points for N₂ fixation rates, diazotrophic cell abundance, and nifH gene copy abundance have increased by 184 %, 86 %, and 809 %, respectively. Version 2 includes two new data sheets for the nifH gene copy abundance of non-cyanobacterial diazotrophs and cell-specific N₂ fixation rates. The measurements of N₂ fixation rates approximately follow a log-normal distribution in both version 1 and version 2. However, version 2 considerably extends both the left and right tails of the distribution. Consequently, when estimating global oceanic N₂ fixation rates using the geometric means of different ocean basins, version 1 and version 2 yield similar rates (43–57 versus 45–63 Tg N yr⁻¹; ranges based on one geometric standard error). In contrast, when using arithmetic means, version 2 suggests a significantly higher rate of 223±30 Tg N yr⁻¹ (mean ± standard error; same hereafter) compared to version 1 (74±7 Tg N yr⁻¹). Specifically, substantial rate increases are estimated for the South Pacific Ocean (88±23 versus 20±2 Tg N yr⁻¹), primarily driven by measurements in the southwestern subtropics, and for the North Atlantic Ocean (40±9 versus 10±2 Tg N yr⁻¹). Moreover, version 2 estimates the N₂ fixation rate in the Indian Ocean to be 35±14 Tg N yr⁻¹, which could not be estimated using version 1 due to limited data availability. Furthermore, a comparison of N₂ fixation rates obtained through different measurement methods at the same months, locations, and depths reveals that the conventional ¹⁵N₂ bubble method yields lower rates in 69 % cases compared to the new ¹⁵N₂ dissolution method. This updated version of the database can facilitate future studies in marine ecology and biogeochemistry. The database is stored at the Figshare repository (https://doi.org/10.6084/m9.figshare.21677687; Shao et al., 2022).

Dinitrogen (N₂) fixation was investigated together with organic matter composition in the mesopelagic zone of the Bismarck (Transect 1) and Solomon (Transect 2) Seas (Southwest Pacific). Transparent exopolymer particles (TEP) and the presence of compounds sharing molecular formulae with saturated fatty acids and sugars, as well as dissolved organic matter (DOM) compounds containing nitrogen (N) and phosphorus (P) were higher on Transect 1 than on Transect 2, while oxygen concentrations showed an opposite pattern. N₂ fixation rates (up to ~1 nmol N L^-1 d^-1) were higher in Transect 1 than in Transect 2, and correlated positively with TEP, suggesting a dependence of diazotroph activity on organic matter. The scores of the multivariate ordination of DOM molecular formulae and their relative abundance correlated negatively with bacterial abundances and positively with N₂ fixation rates, suggesting an active bacterial exploitation of DOM and its use to sustain diazotrophic activity. Sequences of the nifH gene clustered with Alpha-, Beta-, Gamma- and Deltaproteobacteria, and included representatives from Clusters I, III and IV. A third of the clone library included sequences close to the potentially anaerobic Cluster III, suggesting that N₂ fixation was partially supported by presumably particle-attached diazotrophs. Quantitative polymerase chain reaction (qPCR) primer-probe sets were designed for three phylotypes and showed low abundances, with a phylotype within Cluster III at up to 10³ nifH gene copies L^-1. These results provide new insights into the ecology of non-cyanobacterial diazotrophs and suggest that organic matter sustains their activity in the mesopelagic ocean.

Here we report N ₂ fixation rates from a ∼ 4000 km transect in the western and central tropical South Pacific, a particularly undersampled region in the world ocean. Water samples were collected in the euphotic layer along a west to east transect from 160 ^∘ E to 160 ^∘ W that covered contrasting trophic regimes, from oligotrophy in the Melanesian archipelago (MA) waters to ultra-oligotrophy in the South Pacific Gyre (GY) waters. N ₂ fixation was detected at all 17 sampled stations with an average depth-integrated rate of 631 ± 286 µmolNm-2d-1 (range 196–1153 µmolNm-2d-1) in MA waters and of 85 ± 79 µmolNm-2d-1 (range 18–172 µmolNm-2d-1) in GY waters. Two cyanobacteria, the larger colonial filamentous Trichodesmium and the smaller UCYN-B, dominated the enumerated diazotroph community (> 80 %) and gene expression of the nifH gene (cDNA > 10 ⁵ nifH copies L ⁻¹ ) in MA waters. Single-cell isotopic analyses performed by nanoscale secondary ion mass spectrometry (nanoSIMS) at selected stations revealed that Trichodesmium was always the major contributor to N ₂ fixation in MA waters, accounting for 47.1–83.8 % of bulk N ₂ fixation. The most plausible environmental factors explaining such exceptionally high rates of N ₂ fixation in MA waters are discussed in detail, emphasizing the role of macro- and micro-nutrient (e.g., iron) availability, seawater temperature and currents.