Simple approach for the preparation of 15−15N2-enriched water for nitrogen fixation assessments: evaluation, application and recommendations

Recent findings revealed that the commonly used 15N2 tracer assay for the determination of dinitrogen (N2) fixation can underestimate the activity of aquatic N2-fixing organisms. Therefore, a modification to the method using pre-prepared 15−15N2-enriched water was proposed. Here, we present a rigorous assessment and outline a simple procedure for the preparation of 15−15N2-enriched water. We recommend to fill sterile-filtered water into serum bottles and to add 15−15N2 gas to the water in amounts exceeding the standard N2 solubility, followed by vigorous agitation (vortex mixing ≥ 5 min). Optionally, water can be degassed at low-pressure (≥950 mbar) for 10 min prior to the 15−15N2 gas addition to indirectly enhance the 15−15N2 concentration. This preparation of 15−15N2-enriched water can be done within 1 h using standard laboratory equipment. The final 15N-atom% excess was 5% after replacing 2–5% of the incubation volume with 15−15N2-enriched water. Notably, the addition of 15−15N2-enriched water can alter levels of trace elements in the incubation water due to the contact of 15−15N2-enriched water with glass, plastic and rubber ware. In our tests, levels of trace elements (Fe, P, Mn, Mo, Cu, Zn) increased by up to 0.1 nmol L−1 in the final incubation volume, which may bias rate measurements in regions where N2 fixation is limited by trace elements. For these regions, we tested an alternative way to enrich water with 15−15N2. The 15−15N2 was injected as a bubble directly to the incubation water, followed by gentle shaking. Immediately thereafter, the bubble was replaced with water to stop the 15−15N2 equilibration. This approach achieved a 15N-atom% excess of 6.6 ± 1.7% when adding 2 mL 15−15N2 per liter of incubation water. The herein presented methodological tests offer guidelines for the 15N2 tracer assay and thus, are crucial to circumvent methodological draw-backs for future N2 fixation assessments.


Introduction
The availability of fixed nitrogen (N) in N-limiting habitats is a proximal driver of aquatic productivity and the subsequent sequestration of carbon dioxide (CO 2 ) from the atmosphere to the sediment (Capone, 2001;Gruber and Galloway, 2008). Biological N 2 fixation is the largest source of fixed nitrogen to the marine biosphere  Tg N year −1 ) (Codispoti, 2007;Grosskopf et al., 2012). Rates of N 2 fixation can be estimated using a mass balance approach, for example, through deep-water nutrient stoichiometry (Gruber and Sarmiento, 1997;Deutsch et al., 2007) or natural signatures of stable isotopes (Montoya et al., 2002). Alternatively, N 2 fixation can be measured indirectly with the acetylene reduction assay or directly with the 15 N 2 tracer assay (Zehr and Montoya, 2007 and references therein).
Recently, it has been demonstrated that the commonly used 15 N 2 tracer assay leads to a significant underestimation of true N 2 fixation rates, which may explain, at least partially, the apparent imbalance of sources and sinks of N in the global oceans (Grosskopf et al., 2012). In the former protocol of the 15 N 2 tracer assay, 15−15 N 2 gas was added directly as a bubble to water, and an instantaneous equilibrium between the 15−15 N 2 gas bubble and the N 2 dissolved in water was assumed. Rates of N 2 fixation were then calculated from the incorporation of 15−15 N 2 gas into biomass assuming a constant 15 N-atom percent (atom%) in the dissolved N 2 pool from the time of the tracer addition until the end of incubations (Montoya et al., 1996). The dissolution of a 15−15 N 2 gas bubble in water, however, is not instantaneous but time-delayed (Mohr et al., 2010). Consequently, in the past, rates of N 2 fixation might have been underestimated depending on the incubation time, the timing of the injection of the 15−15 N 2 bubble relative to the diel cycle of organisms and the species composition of the diazotrophic community (Mohr et al., 2010;Grosskopf et al., 2012). To circumvent the potential underestimation of N 2 fixation rates, a simple but effective twist has been proposed: the 15 N tracer was added as an aliquot of 15−15 N 2 -enriched seawater, which is prepared prior to incubations. Thereby, a constant 15 N-atom% throughout incubations was ensured (Mohr et al., 2010).
These methodological advances stimulated a debate on how to prepare 15−15 N 2 -enriched seawater-bearing in mind the practicability and time efficiency of the overall protocol. We took this debate as an inspiration, and tested methodological options to facilitate the preparation of 15−15 N 2 -solutions and evaluated their benefits on the dissolution of 15−15 N 2 gas in water in relation to their time effort. We also estimated the risk of contaminations with trace elements (Fe, P, Mo, Mn, Zn, Cu) during the preparation of 15−15 N 2 -enriched water, which may bias N 2 fixation measurements in regions where N 2 fixation is limited by the availability of P or Fe. Finally, recommendations are given for a rapid, simple and reliable procedure to enrich water with 15−15 N 2 gas.

Efficiency of Water Degassing
Two liters of deionized water were filled into a 4 L vacuum filtration flask suitable for low pressures. The flask was closed gastight, connected to a vacuum pump (Diaphragm Vacuum Pump N 026.3AN.18,KNF Neuberger GmbH,Freiburg,Germany) via gas-tight tubing (5 mm i.d.) and placed on a magnetic stirring block (Heidolph MR Hei-Mix L) (Figure 1). The maximum vacuum was 950 mbar below atmospheric pressure (atm. pressure) and water was mixed vigorously while degassing (magnetic stirring bar 40 × 8 mm, 1400 rpm). Degassing was conducted at 0, 200, 600, and 950 mbar below atm. pressure for up to 30 min. The stirring was stopped after the indicated degassing time and the pump switched off as soon as the water turbulence ceased. The water was transferred from the filtration flask into 160 mL borosilicate glass serum bottles using gas-tight tubing (transparent Tygon R , 8 mm o.d., 5 mm i.d.) via siphoning, i.e., atm. pressure was used to force water to flow from the filtration flask into a lower placed serum bottle. The tubing ends were positioned at the bottom of the serum bottle and filtration flask to limit the contact of water with air. As a measure of degassing efficiency, we determined the O 2 concentration using a calibrated Clark-type microelectrode (tip diameter <100 µm, Unisense A/S, Denmark) after the water was transferred into the serum bottles. The theoretical O 2 concentration was calculated as where TGP is the total gas pressure (here equivalent to the absolute degassing pressure = standard atm. pressure-degassing pressure), χ O2 the mole fraction of O 2 (= 0.2095) and k H the Henry law constant (772.55 L atm mol −1 at 24 • C, salinity 0).

Preparation of 15−15 N 2 -enriched Seawater
The common procedure for all tests on the 15−15 N 2 dissolution was as follows: About 1.5 L of deionized water was degassed at 0 or 950 mbar low-pressure for 15 min as described above.
The water was siphoned into 160 mL serum bottles and the bottles were crimp-sealed headspace-free with thick rubber stoppers (h = 1 cm) which can withstand over-pressure in the serum bottles (Figure 1)  Water was filled into a filtration flask, which was closed air-tight, placed on a magnetic stirring block and connected to a vacuum pump. During degassing the stirring was set to its maximum (1400 rpm) causing a turbulent vortex. (B) Water was transferred into borosilicate serum bottles via siphoning through gas-tight tubing so that atmospheric pressure forced the water to flow from the filtration flask into the serum bottles. Siphoning was initiated by air suctioning using a syringe. The tubing ends were kept at the bottoms of both flasks to avoid any water dripping and thus to minimize the gas-water interface. (C) The serum bottles were crimp-sealed headspace-free. A needle attached to a syringe without plunger was used as an outlet to not introduce air bubbles into the bottle while closing the bottle with a rubber stopper.
(D) Compression of injected gas bubble: A fraction of 15−15 N 2 will not dissolve in water but remain in the gaseous phase due to vapor-liquid equilibrium. Consequently, more 15−15 N 2 gas can be forced into the aqueous phase by reducing the bubble volume. We compressed the bubble volume by pressing 0.5 mL of water into half of the serum bottles after 15−15 N 2 gas had been injected. The subsequently added water was degassed at low-pressure when added to degassed water in the serum bottles, and not degassed when added to non-degassed water. The bubble volume was estimated by determining the bubble diameter with a ruler through the glass wall of the serum bottles. (E) Storage time and temperature of the 15−15 N 2 -enriched water: The solutions were stored for 1 or 24 h at 4 • C (for 2.5 mL 15−15 N 2 ) or 24 • C (for 2.5 and 5.0 mL 15−15 N 2 addition) after the 15−15 N 2 gas injection. In practice, the temperature of the 15−15 N 2 aliquot should equal the temperature of the incubation water. Thus, the water temperature was raised from 4 • C back to 24 • C in a water bath after the indicated storage time and before analyzing the 15−15 N 2 concentration.
Isotope ratios of dissolved N 2 and concentrations of 15−15 N 2 were analyzed using either a membrane-inlet mass spectrometer (MIMS; GAM200, IPI) at the Max Planck Institute for Marine Microbiology in Bremen, Germany or a gas chromatography isotope ratio mass spectrometer (GC-IRMS; Thermo Delta V, Thermo Fisher Scientific Inc.) at the Stable Isotope Laboratories at the Department of Geology, Stockholm University, Sweden. For MIMS measurements, water was analyzed immediately and directly in each 160 mL serum bottle. For GC-IRMS measurements, subsamples from serum bottles were transferred to 12 mL Exetainer R using a wide needle (2 × 80 mm) and a 50 mL disposable syringe. All tests on the 15−15 N 2 dissolution were conducted with deionized water. In order to test the validity of our results over a range of salinities, artificial seawater with a salinity of 0, 5, 20, and 35 was enriched with 5 mL 15−15 N 2 per 160 mL water. Generally, the solubility of gases decreases with elevated salinity. In our tests, this was confirmed by a negative linear correlation between the absolute 15−15 N 2 concentration and salinity (R 2 = 0.9809). However, the final 15 N-atom% excess was identical in saline and non-saline solutions because the relative solubility of 14−14 N 2 , 14−15 N 2 and 15−15 N 2 decreased by the same magnitude at increasing salinity. Hence, our tests with deionized water are applicable for fresh-and seawater.

Trace Elements
Contaminations with trace elements can occur due to the contact of water with glass, plastic and rubber ware (Heinrichs and Hermann, 1990) or due to the use of artificial seawater as a solvent for 15−15 N 2 gas. We investigated whether trace elements accumulated during the preparation of 15−15 N 2enriched water. All glass and plastic ware used in the above described experiments were washed with ultrapure HCl (10%, vol/vol), and rinsed and soaked in ultrapure water (Milli Q). HDPE-vials for trace element subsamples were preconditioned in 10% HCl overnight and thereafter rinsed with Milli Q water. During the preparation of 15−15 N 2 -enriched water, 25 mL-subsamples were transferred into HDPE-vials (triplicates) after the following steps in the protocol: (step 1) vacuum degassing, (step 2) water transfer to serum bottles and 5 min of vortex-mixing, and (step 3) 15−15 N 2 gas injection and subsequent transfer of a 15−15 N 2 aliquot to the incubation volume. In addition, we prepared 15−15 N 2 -enriched artificial seawater (S9883 Sigma) with a salinity of 35 to test whether artificial seawater can be an alternative to sterile filtered natural seawater, or whether it constitutes a substantial source of trace elements.
Trace element sub-samples were acidified with ultrapure HNO 3 (Fisher Scientific) to a final concentration of 2% (vol/vol). Samples were analyzed for phosphorus (P), iron (Fe), molybdenum (Mo), manganese (Mn), zinc (Zn), and copper (Cu) using high-resolution inductively coupled plasma   mass spectrometry (ICP-MS; Thermo Fisher Element II) at the ICBM, University of Oldenburg. ICP-MS measurements were done in medium resolution mode to separate molecular interferences from the analytes. The trace element concentrations were assessed with one-point calibration using single-element standards (Roth R or Sigma-Aldrich R ) in the ppt (10 −12 ) to lower ppb (10 −9 ) range. Ultrapure 2% HNO 3 (vol/vol) served as blank. The relative standard deviation was 2% for concentrations in the upper ppt to ppb range, and 15% (Fe, Cu, Mn, Zn) and 30% (Mo, P) for concentrations ≤ 10 ppt.

Modified Bubble injection of 15−15 N 2 assay
The addition of pre-prepared 15−15 N 2 -enriched water may alter the trace element composition in the incubation water. To test an alternative and less invasive addition of 15−15 N 2 to an incubation volume, we combined the previously used bubble approach (Montoya et al., 1996) and the recently proposed dissolution approach (Mohr et al., 2010). Natural seawater samples were taken in the North Sea at different sampling stations and days. The seawater was filled headspace-free in 2 L polycarbonate bottles fitted with septum caps and the 15 N tracer (98% + 15−15 N 2 , Cambridge Isotope Laboratories, lot#I-17229) was added at a ratio of 2 mL 15−15 N 2 per liter of seawater. Bottles were gently mixed by hand for 15 min. Subsequently, the remaining gas bubble was removed in order to stop equilibration of N 2 between the gas and aqueous phase. A water subsample was transferred into 12 mL Exetainer R and preserved with 100 µL of saturated HgCl 2 solution for later analysis of the 15−15 N 2 concentration. After sub-sampling, the incubation bottles were refilled headspace-free with seawater to prevent any loss of 15−15 N 2 gas during the incubation.

Application of the 15−15 N 2 Dissolution Assay
We incubated a culture of Nodularia spumigena KAC 12 (Karlberg and Wulff, 2013) by applying the dissolution (Mohr et al., 2010) and the bubble assay (Montoya et al., 1996). For the dissolution assay, water which was 0.2 µm-filtered (Isopore ™ Membrane Filters, GTTP, Merck Millipore Ltd. Ireland) and degassed at 950 mbar low-pressure, was filled into 160 mL serum bottles, enriched with 2.5 mL 15−15 N 2 gas and vortex-mixed for 1 min. The seawater was taken in the Baltic Sea at station B1 (N 58 • 48 ′ 18, E 17 • 37 ′ 52). Triplicate incubations were initiated by adding 20 mL of the 15−15 N 2 -enriched stock solution to 250 mL of N. spumigena suspension. Thereafter, the 250 mL serum bottles were crimp-sealed headspace-free. For the bubble assay, 300 µL of 15−15 N 2 were directly injected as a gas bubble to 250 mL N. spumigena suspension through the rubber stopper. The amount of 15−15 N 2 was calculated to give a similar 15 N-atom% for both the bubble and the dissolution assay. All bottles were gently inverted 50 times by hand after the 15−15 N 2 addition, and incubated at 150 µE m −2 s −1 and 18 • C for 0, 3, 6, 12, and 24 h. At the end of incubations, the following sub-samples were taken from each serum bottle: (1) Triplicate sub-samples were filled headspace-free into 12 mL Exetainer R vials to determine the 15 N-atom% in the N 2 . These samples were preserved with 100 µL of saturated ZnCl 2 solution.
spumigena. The cell counting resulted in 7.4 ± 0.3 × 10 7 cells L −1 (mean ± s.d., n = 9). (3) 150 mL were filtered onto pre-combusted GF/F filters and frozen at −80 • C to quantify the amount of 15−15 N 2 incorporated into biomass. The GF/F filters were dried at 50 • C overnight, pelletized into tin cups and analyzed on a Thermo Flash EA 1112 elemental analyzer coupled to an isotopic ratio mass spectrometer (Finnigan Delta Plus XP, Thermo Fisher Scientific) at the MPI, Bremen. Gases (calibrated against IAEA references) and caffeine were used as standards for the isotope ratios and the quantification of particulate organic carbon and nitrogen (POC, PON), respectively. N 2 fixation rates were calculated as where A is the 15 N-atom% in the dissolved N 2 pool (A N2 ) and particulate material (A PN ), and [PN] is the concentration of particulate material. A significant 15 N-uptake other than 15 N 2 fixation due to the impurity of 15 N 2 gas (Dabundo et al., 2014) could be excluded for our studies since traces of 15 NH + 4 , 15 NO − 3 , or 15 NO − 2 in the 15−15 N 2 -enriched water were not detectable using GC-IRMS.
The parameter A N2 , as given in Equation (2), is defined as 15 N-atom% in the dissolved N 2 pool, that is, the sum of naturally abundant and added 15 N. However, in the literature, A N2 is occasionally denoted as 15 N-enrichment or 15 N-excess enrichment. This misleading use of terms may lead to miscomprehension and we recommend a clear distinction between 15 N-atom% and 15 N-atom% excess in future studies.

Efficiency of Degassing
Recent studies applied water degassing at 750-960 mbar below atm. pressure for approximately 1 h, and degassing set-ups were similar to the one shown here (Figure 1) or a membrane flowthrough system was used (Mohr et al., 2010;Grosskopf et al., 2012;Rahav et al., 2013). Alternatively, ambient air was removed by purging with helium (Wilson et al., 2012). Nevertheless, the benefit of the degassing pressure/duration on the degassing efficiency and finally on the actual 15−15 N 2 dissolution has not been assessed systematically.
The solubility of gas follows Henry's law, i.e., the amount of gas dissolved in a liquid is proportional to its partial pressure.
where p is the partial pressure, c the concentration of the solute and k H the Henry law constant. In agreement, in our tests the degree of degassing and the degassing pressure were positively correlated (Figure 2A, R 2 = 0.9985). The efficiency of the degassing set-up to remove dissolved gas from water was high and met theoretical assumptions. Following Equation (1), water degassing at 200, 600, and 950 mbar low-pressure reduces the O 2 air-saturation to 83.0, 42.2, and 6.5%, respectively. By applying the set-up as shown in Figure 1, dissolved gas was removed from water to 84.2 ± 0.9% O 2 air-saturation (mean ± s.d., n = 6) at 200 mbar, 43.0 ± 1.9% (n = 6) at 600 mbar and 8.0 ± 0.7% (n = 6) at 950 mbar low-pressure. The minor difference between the measured and theoretical O 2 air-saturation can be explained either by a deviation of the actual atm. pressure from the standard atm. pressure or by O 2 contamination as the water was transferred from the filtration flask to the serum bottles. Degassing was accelerated by vigorous stirring and the maximum gas removal achieved after 5-10 min ( Figure 2B).
Hence, degassing at 950 mbar low-pressure in a simple set-up as applied here is an efficient and fast way of gas removal yielding a high degree of gas removal to ≤10% air-saturation within minutes ( Figure 2B).

Preparation and Evaluation of 15−15 N 2 -enriched Water
The Effect of Water Degassing on the Dissolution of 15−15 N 2 Gas Gases and their isotopes have a similar solubility (Klots and Benson, 1963), and the N 2 solubility is linearly correlated to the partial pressure (Equation 3). Correspondingly, our data showed that water degassing had no direct effect on the amount of 15−15 N 2 which dissolved in water after 15−15 N 2 had been added ( Figure 3A). Nonetheless, water degassing had two indirect effects increasing the 15 N-atom% excess in the final incubation volume. Firstly, degassing at 950 mbar low-pressure lowered the 14−14 N 2 and 14−15 N 2 background in the 15−15 N 2 -enriched water to ≤10% (compare with Figure 2). Thereby, less 14 N is added together with 15−15 N 2 -enriched water to the incubation volume and the final 15 N-atom% excess in the incubation volume would increase by 4.5% (e.g., from 5.0 to 5.2% assuming that the aliquot of 15−15 N 2 -enriched water equals 5% of the final incubation volume). Secondly, water degassing lowered the initial total gas pressure and thus more 15−15 N 2 could be added to under-saturated water without risking the borosilicate serum bottles to explode. Roughly, the serum bottles could withstand the overpressure of a maximum of 5 mL 15−15 N 2 per 160 mL non-degassed water and 7 mL 15−15 N 2 per 160 mL of 950 mbar-degassed water. This can yield an additional increase in the final 15 N-atom excess of 30% (e.g., from 5.2 to 6.8% 15 N-atom% excess).
The Effect of the Volume of Injected 15−15 N 2 Gas on the 15−15 N 2 Concentration In agreement with Equation 3, we found a strong positive correlation between the amount of injected 15−15 N 2 gas and the measured 15−15 N 2 concentration in water even when exceeding the standard solubility of N 2 in water (R 2 = 0.9998, Figure 3B). The maximum 15−15 N 2 concentration was 1200 µmol L −1 , that is, adding a 20 mL aliquot of this solution to 1 L of incubation volume (24 • C, 0 PSU) would be sufficient to achieve a final 15 Natom excess of 5%. Further, instead of serum bottles, we tested the usage of Exetainer R vials as water containers. We injected 2 mL of 15−15 N 2 gas into a 12 mL Exetainer R filled with nondegassed water and vortex-mixed the solution for 1 min. The 15−15 N 2 concentration was 782 ± 48 µmol L −1 (n = 3), i.e., an aliquot of 30 mL added to 1 L of incubation volume (24 • C, 0 PSU) would be sufficient to achieve a final 15 N-atom excess of 5%. The preparation of 15−15 N 2 -enriched water in the Exetainer R vials was fast but the overpressure in the vials was high which made it difficult to press the total volume of 15−15 N 2 gas into the vials. Moreover, the 15−15 N 2 recovery was low (around 11%), presumably because a large gas bubble remained even after vortex-mixing.

The Effect of Agitation on the Dissolution of 15−15 N 2 Gas
In general, agitation promotes turbulent diffusion of gases across the gas-water interface. Hand shaking and vortex mixing had the same efficiency on the 15−15 N 2 dissolution relative to the mixing time; however, due to practical reasons we applied vortex-mixing  in cases when the mixing exceeded 30 s. Vortex mixing yielded the maximum 15−15 N 2 concentration within 5 min. The amount of 15−15 N 2 which dissolved in water could therefore be increased by 65% after 5 min of vortex mixing compared to the 15−15 N 2 dissolution after 30 s of hand-shaking ( Figure 3C).

The Effect of Compressing the Gas Bubble on the Dissolution of 15−15 N 2 Gas
Dinitrogen gas is rather insoluble in water whereby a major part of N 2 remains in the gas phase. Thus, a reduction of the bubble volume by increasing the pressure inside the serum bottle should enhance the amount of 15−15 N 2 which dissolves in water. We injected 5 mL 15−15 N 2 gas to 160 mL non-degassed (0 mbar) and degassed water (950 mbar) which created a bubble of 0.58 ± 0.10 cm 3 (mean ± s.d., n = 12). After pressing 0.5 mL of water into half of the serum bottles, the bubble size was compressed to 0.07 ± 0.00 cm 3 (n = 6) irrespective of the earlier applied degassing pressure. This bubble compression led to a rise in 15−15 N 2 by 21-22% ( Figure 3D). In fact, this rise in 15−15 N 2 corresponded to the amount of 15−15 N 2 which was initially trapped in the bubble but dissolved in the liquid as the bubble was compressed. Compressing the gas bubble was a quick process but occasionally the pressure increase within the serum bottle caused the bottle to shatter risking the applicants' health.
The Effect of Storage Time and Temperature on the Dissolution of 15−15 N 2 Gas The amount of 15−15 N 2 which dissolved in water increased by 20-25% after 24 h compared to 1 h storage ( Figure 3E). Although this was a substantial effect, this approach to increase the 15−15 N 2 concentration seemed inefficient considering its time effort. Decreasing the temperature during storage from 24 • C to 4 • C did not yield higher 15−15 N 2 concentrations. Although gases are more soluble at colder temperatures, the re-warming of the 15−15 N 2 solution from 4 • C to 24 • C before the 15−15 N 2 concentration analysis might have reversed the effect of the colder storage temperature; however, this step is inevitable to not alter the temperature in the incubation volume.

Final 15 N-atom% Excess Using the Dissolution Approach
For most field studies an incubation volume of 1-4 L and a final 15 N-atom excess of 2-5% is applied (Grosskopf et al., 2012 Supplementary information). We achieved a 15 N-atom excess of 5% in the final incubation volume by applying the following steps: 5 mL of 15−15 N 2 gas were added to 160 mL water (degassed at 950 mbar low-pressure for 15 min) in serum bottles. Thereafter, the 15−15 N 2 solution was vortex-mixed for 1 min. We transferred 50 mL of the 15−15 N 2 stock solution to a 1 L Schott glass bottle which was filled to the brim (1150 mL), i.e., the volume of the 15−15 N 2 aliquot equaled less than 5% of the total incubation volume. The final 15 N-atom% excess was 4.8 ± 0.1% (n = 10) with no significant difference after 0.5 and 12 h following the 15−15 N 2 aliquot addition (p > 0.05). The 15 N-atom% excess might have been further enhanced by increasing the initial 15−15 N 2 gas addition to 7 mL 15−15 N 2 per 160 mL water and prolonging vortex-mixing to 5 min. During our later tests, the addition of 7 mL 15−15 N 2 gas to 160 mL degassed water yielded a 15−15 N 2 concentration of 1200 µmol L −1 (Figure 3B). Based on these results, only 2% of the incubation volume (24 • C, salinity 0) would have to be replaced with 15−15 N 2 -enriched water to reach a final 15 N-atom excess of 5%. Importantly, the amount of 15−15 N 2 that is transferred to the incubation water has to be adjusted according to the standard N 2 solubility at a given water salinity and temperature. We validated whether the transfer of 15−15 N 2 -enriched water from the incubation bottle to an Exetainer R vial may cause a loss of 15−15 N 2 to the atmosphere. The 15−15 N 2 -enriched water from an incubation bottle was gently aspirated with a 50 mL syringe (Plastic Sterile Plastipak) and expelled through a 0.2-µm syringe filter to the bottom of an Exetainer R vial. The gas-water interface was minimized during the water transfer by not dripping the solution into the Exetainer R vial, but using a needle (2 × 80 mm) or small tubing as extension of the syringe to gently release the water to the bottom of the vial. The 15−15 N 2 concentration was measured directly in the incubation volume and after a sub-sample was transferred to an Exetainer R using MIMS. On average the relative 15−15 N 2 loss was 4 ± 1% (mean ± s.d., n = 6) after the above described sample transfer. The addition of 2 mL 15−15 N 2 gas per liter seawater and subsequent removal of the bubble in the incubation volume yielded a 15 N-atom excess of 6.6 ± 1.7% (mean ± s.d., n = 12) ranging from 3.9 to 10.1% for water with salinities from 25 to 0.4. This wide range was driven by three bottles with considerably lower (3.9%) or higher (10.1%) values, but in nine of the 12 bottles the 15 N-atom excess was 6.8 ± 0.9%. When using the modified bubble addition, a consistent agitation should be used to ensure a small variability of 15 N-atom% among experiments. One disadvantage of this approach is the less efficient use of 15−15 N 2 gas (utilizing 40% of the 15−15 N 2 gas compared to 70% when adding 15−15 N 2 -enriched water as a stock solution). Nonetheless, the modified bubble approach is a less invasive approach for N 2 fixation measurements compared to the addition of preprepared 15−15 N 2 -enriched stock solutions and may minimize the risk of trace element contaminations (see Section Trace Elements).

Trace Elements
Trace element concentrations in the 15−15 N 2 -enriched water were ≤2 nmol L −1 for Fe, P, Mn, Mo, Cu, Zn. The individual element concentrations in the final incubation volume increased by a maximum of 0.01 nmol P L −1 , 0.1 nmol Fe L −1 , 0.04 nmol Mn L −1 , 0.1 nmol Cu L −1 , and 0.09 nmol Cu L −1 . These concentrations were calculated by assuming that the volume of the 15−15 N 2 -enriched water which is transferred to the incubations equals 5% of the total incubation volume. Concentrations of Mo did not differ in the 15−15 N 2 -enriched water and blank solutions. These trace element contaminations should be seen as an estimate. We expect that contaminations vary depending on the specific material used during the preparation of 15−15 N 2 -enriched water and also the history of that material. In our tests, major contamination sources for Fe were the contact of water with glass ware and colored rubber stoppers. In contrast, the utilization of stainless steel needles (0.6 × 25 mm, Terumo Corporation, Leuven, Belgium), which were used for the 15−15 N 2 gas injection into the serum bottles, led to no substantial Fe-contamination because the cannulas were by default covered with silicone (personal communication with the supplier TERUMO BCT Europe).
The artificial seawater (salinity of 35) was highly enriched with trace metals, ranging from 23-70 nmol L −1 for Fe, Mn, Cu, Zn. Levels of Mo were low, 0.1 nmol L −1 , and levels of P were not different from blank levels. These concentrations confirmed the declaration of the manufacturer Sigma stating their sea salt S9883 may contain ≤500 nmol L −1 of trace elements (information received after written request). The usage of this specific artificial seawater would have led to trace metal concentrations in the incubation water of 3.5 nmol L −1 for Fe, 2.3 nmol L −1 for Mn, 2.9 nmol L −1 for Cu, 1.2 nmol for Zn and 5 pmol L −1 for Mo assuming that the volume of the artificial seawater added to the incubation volume equals 5 Vol%. Such concentration levels are in excess of those in wide regions of the marine environment. Artificial seawater prepared with sea salt of a different batch, supplied by manufacturers other than Sigma (S9883) or seawater prepared using ultrapure single element powder may yield lower (or higher) concentrations of trace elements.

Application of 15−15 N 2 Dissolution Assay
The dissolution approach attained a 15 N-atom% excess of 4.3 ± 0.3% (mean ± s.d., n = 35). The differences between the time points were low and are explained by differences of the initial 15−15 N 2 stock solution as we used a different serum bottle of the 15−15 N 2 stock solution for each set of triplicates of each time point. In contrast, the 15 N-atom% excess increased from FIGURE 4 | A Nodularia spumigena culture was incubated following the former bubble-addition and the modified dissolution assay. (A) The dissolution of a 15−15 N 2 gas bubble was time-dependent. The 15 N-atom% increased over time if the 15 N-tracer was added as a bubble directly to the incubation volume (see also Mohr et al., 2010). The 15 N-atom% was more consistent as an aliquot of 15−15 N 2 -enriched water was added to the N. spumigena solution (dissolution approach). (B) Rates of N 2 fixation were underestimated by 16-71% when using the bubble approach compared to the dissolution approach.
1.6 ± 0.1% (n = 9) after 15 min to 4.5 ± 0.3% (n = 9) after 24 h when the 15−15 N 2 gas was injected as a bubble ( Figure 4A). This led to an underestimation of N 2 fixation by 38% after 3 h and 16% after 24 h incubations if the 15 N-atom% was assumed to be 4.5% throughout the entire incubation period (Figure 4B). The decrease in underestimation of N 2 fixation with time was due to the fact that the 15−15 N 2 reached its vapor-liquid equilibrium after approximately 6 h ( Figure 4B). In addition, the positively buoyant colonies of Nodularia spumigena floated and assembled close to the gas bubble where they were exposed to a higher 15 N-atom% compared to organisms more distant from the gas bubble. This might have compensated for the initially lower 15−15 N 2 concentration in water (see also Grosskopf et al., 2012 SI;White, 2012).

Recommendations and Comments
The following guidelines are recommended as best practice for an efficient and reproducible preparation of 15−15 N 2 -enriched water and the reliable determination of N 2 fixation rates: (1) For the preparation of 15−15 N 2 -enriched water, we suggest to add 15−15 N 2 to sterile-filtered water in excess of the standard N 2 solubility and to mix the solution vigorously, preferably by vortex-mixing for at least 5 min. Prior to the addition of 15−15 N 2 , sterile-filtered water can be degassed at low-pressure (≥950 mbar) to indirectly increase the 15−15 N 2 -atom% excess in the final incubation volume (see Section The effect of water degassing on the dissolution of 15−15 N 2 gas). The protocol for the 15−15 N 2 enrichment, however, might be adjusted according to the experimental set-up, time plan and study area. A rating according to the benefits and drawbacks of the methodological steps for the preparation of 15−15 N 2 -enriched water is given in Table 2.
(2) Natural seawater from the specific sampling stations/depths should be used rather than artificial seawater in order to avoid trace element contaminations. In addition, to minimize possible contaminations during the preparation of 15−15 N 2 -enriched water, we recommend washing all equipment with 10% HCl (vol/vol) followed by several rinses with MilliQ, and to use old and worn glass ware which has lost its element impurities. Transparent rubber stoppers and tubing (for example, transparent PVC, PE or Tygon R , which are N-free) are preferable over colored rubber stoppers and tubing. In our tests, the mean concentration of trace elements in the incubation volume was estimated to increase by up to 0.1 nmol L −1 due to the addition of pre-prepared 15−15 N 2 -enriched water. We consider trace element contamination levels of up to 0.1 nmol L −1 as minor for many regions of the aquatic environment. Yet, marine pelagic (cyanobacterial) N 2 fixation can be limited by the availability of Fe and P, especially in the open ocean (e.g., Sanudo-Wilhelmy et al., 2001;Mills et al., 2004;Turk-Kubo et al., 2012). We therefore advise against the preparation of 15−15 N 2 enriched water as shown in Figure 1 when working in regions where concentrations of dissolved Fe are very low (≤0.1 nmol L −1 ), e.g., the South Atlantic Ocean or parts of the central North and South Pacific Gyres (Brown et al., 2005;Sohm et al., 2011). Here, the modified bubble addition approach is recommended due to a lower risk of trace element contamination.
(3) A sub-sample of the incubation water should be taken for MIMS or GC-IRMS analyses in order to determine the actual enrichment of 15−15 N 2 and to ensure the accurate calculation of N 2 fixation rates. When using the modified bubble method, every individual incubation bottle should be sub-sampled. We further recommend that 15 N 2 gas bottles are checked for potential impurities with 15 N-compounds other than 15 N 2 (Dabundo et al., 2014).
To highlight the importance of the determination of the 15 N-atom% in the incubation, we modeled the effect of under-/overestimating the 15 N-labeling on rates of N 2 fixation ( Figure 5). Rates of N 2 fixation are calculated according to Equation (2) and an incorrect assumption of the 15 N-atom% or 15 N-atom% excess leads to a significant bias in N 2 fixation rates. The percent deviation from true N 2 fixation rates is more pronounced if the 15 N-atom% or 15 N-atom% excess is underestimated compared to occasions when it is overestimated. Moreover, the magnitude of potential errors in N 2 fixation rates is lower when using a 15 N-atom% of ≥5% compared to a lower 15 N-atom% of ≤2% (Figure 5). A high 15 N-atom% should also be used in regions with high biomass and/or high productivity, but low N 2 -fixing activity. Here, a high 15 N-atom% increases the detection limit of N 2 fixation since the 15 N-PON signal from N 2 fixation can be attenuated by the presence of non-diazotroph PON. Currently, the 15 N 2 tracer assay is the only available method for a direct assessment of N 2 fixation by aquatic diazotrophs and its application has greatly advanced our understanding of the global N-cycle. Nevertheless, the systematic underestimation of Frontiers in Microbiology | www.frontiersin.org FIGURE 5 | Modeled deviation from true, actual N 2 fixation rates in correlation to the potential deviation from true values of 15 N-atom% (in black) or 15 N-atom% excess (in red). Following Equation 2, a deviation of 15 N-atom% or 15 N-atom% excess from the true value will lead to significant under-or overestimations of N 2 fixation. This error in N 2 fixation rates is especially pronounced when the 15 N-atom% or 15 N-atom% excess is underestimated. In addition, the variable 15 N-atom% is corrected for the naturally present 15 N (0.366%) for calculations of N 2 fixation rates. This correction has a more pronounced weighting on the calculated N 2 fixation when using a low 15 N-atom% of ≤2% compared to a higher 15 N-atom of ≥5%. The percent deviation was calculated as difference between the incorrect, i.e., falsely estimated value of 15 N-atom%/ 15 N-atom% excess and their true values.
actual N 2 fixation rates by the former bubble approach demanded the development and evaluation of a revised protocol. In the future, the application of a reliable 15 N 2 tracer assay will be of importance to advance our knowledge in diazotrophic activity and biogeography in the aquatic environment. Especially, the activity of unicellular and symbiotic diazotrophs is believed to have been underestimated up to 6-fold during past field campaigns (Grosskopf et al., 2012). A modified, standardized N 2 fixation methodology is therefore expected to yield higher rates of N 2 fixation which may, at least partially, resolve the discrepancy between N-gain and -loss processes in marine N budget calculations (Codispoti, 2007).