The two commonly applied methods to assess dinitrogen (N2) fixation rates are the 15N2-tracer addition and the acetylene reduction assay (ARA). Discrepancies between the two methods as well as inconsistencies between N2 fixation rates and biomass/growth rates in culture experiments have been attributed to variable excretion of recently fixed N2. Here we demonstrate that the 15N2-tracer addition method underestimates N2 fixation rates significantly when the 15N2 tracer is introduced as a gas bubble. The injected 15N2 gas bubble does not attain equilibrium with the surrounding water leading to a 15N2 concentration lower than assumed by the method used to calculate 15N2-fixation rates. The resulting magnitude of underestimation varies with the incubation time, to a lesser extent on the amount of injected gas and is sensitive to the timing of the bubble injection relative to diel N2 fixation patterns. Here, we propose and test a modified 15N2 tracer method based on the addition of 15N2-enriched seawater that provides an instantaneous, constant enrichment and allows more accurate calculation of N2 fixation rates for both field and laboratory studies. We hypothesise that application of N2 fixation measurements using this modified method will significantly reduce the apparent imbalances in the oceanic fixed-nitrogen budget.

SamplingIn order to compare the commonly used 15 N 2 tracer addition method to measure N 2 fixation 1 with the addition of 15 N 2 -enriched water as suggested by Mohr et al. (2010) 2 , seawater was sampled on two cruises in the Atlantic Ocean, the first on board R/V Meteor (M80/1) on a longitudinal transect (23°W) between 15°N and 5°S, the second on board R/V Polarstern (ANT-XXVI/1) on a transect between 54°N and 54°S (Bremerhaven, Germany to Punta Arenas, Chile). In total 39 triplicate incubations were conducted with both methods in parallel. On the M80/1 cruise, seawater was sampled at 11 stations from the surface (bucket), 20 m depth and the chlorophyll maximum (CTD rosette sampler) at 7:00 in the morning, whereas on the ANT-XXVI/1 cruise, seawater was sampled at 6 stations at 16:00 from the ship's clean seawater supply which is installed at 11 m depth (keel of the ship). IncubationsSeawater samples were filled headspace-free (bubble addition method) or with a 100-150 ml headspace (dissolved method) into 4.5 L polycarbonate bottles and closed with Teflon ® -coated butyl rubber septum caps. To determine N 2 fixation rates with the bubble-addition method, a 4.5 mL 15 N 2 gas bubble (Sigma-Aldrich, ≥98 atom%) was injected through the septa into each of triplicate bottles (yielding a theoretical enrichment of ~12 atom% assuming a rapid isotopic equilibration between the added 15 N 2 gas and the ambient dissolved N 2 of the water sample). After injection, bottles were gently inverted one hundred times. For comparison of N 2 fixation rates, we added 15 N 2 -enriched seawater to a second set of triplicate bottles (dissolution method). In detail, the preparation of the 15 N 2 -enriched seawater was started by degassing filtered seawater (0.2 µm filtered, Durapore) using a membrane flowthrough system (Mini-Module, Membrana) in which the seawater flowed on the inside of the membrane and a vacuum (-960 mbar, water jet pump) was applied to the outer side of the membrane. The seawater flow rate was about 400 -500 mL min -1 and seawater was recirculated for the first 10-15 min of the degassing step. Degassed seawater was then filled directly from the flow-through system into evacuated gas-tight 3L Tedlar® bags without a headspace. Addition of 15 N 2 gas was dependent on the amount of seawater in the Tedlar® bag and was added at a ratio of 10 ml 15 N 2 per 1L seawater. The volume of degassed seawater in SUPPLEMENTARY INFORMATION

Microbial dinitrogen (N) fixation, the nitrogenase enzyme-catalysed reduction of N gas into biologically available ammonia, is the main source of new nitrogen (N) in the ocean. For more than 50 years, oceanic N fixation has mainly been attributed to the activity of the colonial cyanobacterium Trichodesmium. Other smaller N-fixing microorganisms (diazotrophs)-in particular the unicellular cyanobacteria group A (UCYN-A)-are, however, abundant enough to potentially contribute significantly to N fixation in the surface waters of the oceans. Despite their abundance, the contribution of UCYN-A to oceanic N fixation has so far not been directly quantified. Here, we show that in one of the main areas of oceanic N fixation, the tropical North Atlantic, the symbiotic cyanobacterium UCYN-A contributed to N fixation similarly to Trichodesmium. Two types of UCYN-A, UCYN-A1 and -A2, were observed to live in symbioses with specific eukaryotic algae. Single-cell analyses showed that both algae-UCYN-A symbioses actively fixed N, contributing ∼20% to N fixation in the tropical North Atlantic, revealing their significance in this region. These symbioses had growth rates five to ten times higher than Trichodesmium, implying a rapid transfer of UCYN-A-fixed N into the food web that might significantly raise their actual contribution to N fixation. Our analysis of global 16S rRNA gene databases showed that UCYN-A occurs in surface waters from the Arctic to the Antarctic Circle and thus probably contributes to N fixation in a much larger oceanic area than previously thought. Based on their high rates of N fixation and cosmopolitan distribution, we hypothesize that UCYN-A plays a major, but currently overlooked role in the oceanic N cycle.

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Phytoplankton form the basis of the marine food web and are responsible for approximately half of global carbon dioxide (CO2) fixation (∼ 50 Pg of carbon per year). Thus, these microscopic, photosynthetic organisms are vital in controlling the atmospheric CO2 concentration and Earth's climate. Phytoplankton are dependent on sunlight and their CO2-fixation activity is therefore restricted to the upper, sunlit surface ocean (that is, the euphotic zone). CO2 usually does not limit phytoplankton growth due to its high concentration in seawater. However, the vast majority of oceanic surface waters are depleted in inorganic nitrogen, phosphorus, iron and/or silica; nutrients that limit primary production in the ocean (Figure 1). Phytoplankton growth is mainly supported by either the recycling of nutrients or by reintroduction of nutrients from deeper waters by mixing. A small percentage of primary production, though, is fueled by 'external' or 'new' nutrients and it is these nutrients that determine the amount of carbon that can be sequestered long term in the deep ocean. For most nutrients such as phosphorus, iron, and silica, the external supply is limited to atmospheric deposition and/or coastal and riverine inputs, whereas their main sink is the sedimentation of particulate matter. Nitrogen, however, has an additional, biological source, the fixation of N2 gas, as well as biological sinks via the processes of denitrification and anammox. Despite the comparatively small contributions to the overall turnover of nutrients in the ocean, it is these biological processes that determine the ocean's capacity to sequester CO2 from the atmosphere on time scales of ocean circulation (∼ 1000 years). This primer will highlight shifts in the traditional paradigms of nutrient limitation in the ocean, with a focus on the uniqueness of the nitrogen cycling and its biological sources and sinks.

Dinitrogen (N 2 ) 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 N 2 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 N 2 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 N 2 fixation rates and for calculations of detection limits and propagated error terms. To this end, we examine current protocols of the 15 N 2 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 N 2 fixation rates using 15 N 2 tracer, with the goal of fostering transparency in reporting sources of uncertainty in estimates, and to render N 2 fixation rate estimates intercomparable among studies.

Mahoney Lake represents an extreme meromictic model system and is a valuable site for examining the organisms and processes that sustain photic zone euxinia (PZE). A single population of purple sulfur bacteria (PSB) living in a dense phototrophic plate in the chemocline is responsible for most of the primary production in Mahoney Lake. Here, we present metagenomic data from this phototrophic plate--including the genome of the major PSB, as obtained from both a highly enriched culture and from the metagenomic data--as well as evidence for multiple other taxa that contribute to the oxidative sulfur cycle and to sulfate reduction. The planktonic PSB is a member of the Chromatiaceae, here renamed Thiohalocapsa sp. strain ML1. It produces the carotenoid okenone, yet its closest relatives are benthic PSB isolates, a finding that may complicate the use of okenone (okenane) as a biomarker for ancient PZE. Favorable thermodynamics for non-phototrophic sulfide oxidation and sulfate reduction reactions also occur in the plate, and a suite of organisms capable of oxidizing and reducing sulfur is apparent in the metagenome. Fluctuating supplies of both reduced carbon and reduced sulfur to the chemocline may partly account for the diversity of both autotrophic and heterotrophic species. Collectively, the data demonstrate the physiological potential for maintaining complex sulfur and carbon cycles in an anoxic water column, driven by the input of exogenous organic matter. This is consistent with suggestions that high levels of oxygenic primary production maintain episodes of PZE in Earth's history and that such communities should support a diversity of sulfur cycle reactions.

Symbiotic relationships between phytoplankton and N 2 -fixing microorganisms play a crucial role in marine ecosystems. The abundant and widespread unicellular cyanobacteria group A (UCYN-A) has recently been found to live symbiotically with a haptophyte. Here, we investigated the effect of nitrogen (N), phosphorus (P), iron (Fe) and Saharan dust additions on nitrogen (N 2 ) fixation and primary production by the UCYN-A-haptophyte association in the subtropical eastern North Atlantic Ocean using nifH expression analysis and stable isotope incubations combined with single-cell measurements. N 2 fixation by UCYN-A was stimulated by the addition of Fe and Saharan dust, although this was not reflected in the nifH expression. CO 2 fixation by the haptophyte was stimulated by the addition of ammonium nitrate as well as Fe and Saharan dust. Intriguingly, the single-cell analysis using nanometer scale secondary ion mass spectrometry indicates that the increased CO 2 fixation by the haptophyte in treatments without added fixed N is likely an indirect result of the positive effect of Fe and/or P on UCYN-A N 2 fixation and the transfer of N 2 -derived N to the haptophyte. Our results reveal a direct linkage between the marine carbon and nitrogen cycles that is fuelled by the atmospheric deposition of dust. The comparison of single-cell rates suggests a tight coupling of nitrogen and carbon transfer that stays balanced even under changing nutrient regimes. However, it appears that the transfer of carbon from the haptophyte to UCYN-A requires a transfer of nitrogen from UCYN-A. This tight coupling indicates an obligate symbiosis of this globally important diazotrophic association.