Conceived and designed the experiments: WM DWRW JL. Performed the experiments: WM. Analyzed the data: WM TG DWRW JL. Wrote the paper: WM. Contributed to figure preparation/schematic diagrams: TG.
The authors have declared that no competing interests exist.
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.
Biological dinitrogen (N2) fixation is the major source of fixed nitrogen (N) in the oceanic N budget
The comparison of N2 fixation rates measured simultaneously using the 15N2-tracer addition and the ARA shows that the 15N2-tracer addition generally yields lower rates (for a summary see
The apparent oceanic N imbalance, differences between geochemical estimates and measured rates of N2 fixation, and the difficulties in reconciling discrepancies between ARA and 15N2-based estimates of N2 fixation in the field and in culture experiments, led us to re-assess the 15N2-tracer addition method. This method is based on the direct injection of a 15N2 gas bubble into a seawater sample
In applications of the method, all parameters of the equation are measured except for the atom% 15N in the dissolved N2 pool (
Here we report results of experiments that were designed to assess the rate of equilibration of an introduced 15N2 gas bubble with the surrounding water. Based on results of these experiments, we developed a modified approach involving addition of 15N2-enriched seawater which assured a well-defined and constant 15N enrichment of the dissolved N2 gas at the beginning of the incubations. We propose the application of the modified approach for future assessments of N2 fixation rates in natural microbial communities and in laboratory cultures.
A first set of experiments (isotopic equilibration experiments) was carried out to assess the time required to attain isotopic equilibrium in the dissolved pool of N2 gas after injection of a known amount of 15N2 gas as a bubble into sterile filtered seawater. A gas bubble of pure 15N2 was injected directly into incubation bottles which were manually inverted fifty-times (∼3 min agitation) and left standing for up to 24 h. Concentration of dissolved 15N2 was followed over the 24 h period to assess the degree of equilibration of the 15N2 gas bubble with the surrounding water as a function of time. Dissolved 15N2 concentrations in the seawater increased steadily with the incubation time (
Results are presented as a function of the time after bubble injection (white symbols). (A) Measured dissolved 15N2 concentrations as percentage of calculated concentration assuming rapid and complete isotopic equilibrium. (B) N2 fixation rates by
Similar results were obtained in the incubation experiments with pure culture of
Continuous, vigorous shaking (50 rpm) greatly increased the concentration of 15N2 in the media (
Values are presented as a percentage of the calculated concentration. The manually-shaken (3 min) sample was added to the plot for comparison (grey symbol).
Increasing the size of the incubation bottles, increasing the amount of gas injected per liter of seawater and the addition of dissolved organic matter (DOM) led in all cases except one to slower equilibration of the 15N2 gas bubble with the surrounding water (
Values are presented as a percentage of the calculated concentration. Bottles were incubated for 1 hour. Black bars, 0.13 L bottle and white bars, 1.15 L bottle.
Values are presented as a percentage of the calculated concentration (A) after 1 hour incubation in manually (3 min shaking and 1 h subsequent incubation), and (B) in continuously (1 h) shaken samples.
Both the isotopic equilibration and the culture experiments demonstrated clearly that the equilibration of 15N2 gas injected as a bubble into N2-saturated seawater is time-dependent and incomplete, even after 24 hours. The lack of complete equilibration causes the resulting calculated N2 fixation rates to be variably and significantly underestimated (see Equation 1). The equilibration,
The experiments with variable bottle sizes and DOM additions (
This study was motivated partly by the mismatches between the ARA and 15N2-based measurements of N2 fixation as well as imbalances between 15N2-fixation rates and biomass-specific rates (∼growth rate) or C∶N fixation ratios (
Organism/area | C2H2∶15N2 | C∶N fixation ratio | biomass-specific rate [d−1] | Reference |
808 |
||||
cyanobacterial bloom/Baltic | 3–20 | |||
3–22 | 75–133 | |||
1.5–6.9 | 0.002–0.011 |
|||
10–107 |
||||
13–437 | 0.006–0.03 |
C∶N fixation ratio is based on 15N2-fixation measurements.
Ratio calculated from DI13C and 15N2 fixation rates.
Calculated from 15N2 fixation rate divided by PON.
Calculated from doubling time with biomass-specific rate = ln (2)/doubling time.
We reviewed published studies that have used the direct injection of a 15N2 gas bubble to assess N2 fixation rates in order to evaluate the magnitude of under-estimation. However, first attempts to assess the degree of underestimation of field and culture N2 fixation rates were obscured by a wide range of experimental conditions among the studies. Bottle sizes ranged from 14 ml to 10 L, the amount of 15N2 injected varied from 0.2 to 40.8 ml 15N2 per L seawater and incubation times ranged from 0.25 to 48 hours, with the majority of the field studies using 2–4 L bottles and 24 h incubations. In addition, information on agitation was, in general, not available. There were no obvious trends of reported N2 fixation rates with either bottle size, incubation time or the amount of injected 15N2 gas probably because of the large variability of geographic locations and environmental conditions prevailing in the individual studies, which would have a dominant effect on the local diazotrophic communities and their N2 fixation rates. An evaluation of the degree of possible underestimation of 15N2 fixation rates in environmental studies is further confounded by diel periodicity of N2 fixation
Schematic diagram illustrating the influence of diel N2 fixation patterns on N2 fixation rates when determined with the direct injection of a 15N2 gas bubble. A hypothetical diel N2 fixation pattern is shown (panel A) with a duration of the N2-fixing period of 12 h. Three possible time periods for 24 h incubations are indicated by the solid bars (A–F). The corresponding 15N enrichment in the dissolved N2 pool (panel B) is shown for the three incubation periods using the direct injection of a 15N2 gas bubble (solid lines; A, B and C) and the addition of 15N2-enriched seawater (dashed line; D, E and F). The resulting cumulative N2 fixation in each of the incubations (panel C) demonstrates that the timing of the incubation relative to diel N2 fixation patterns introduces a variable underestimation in the total N2 fixation rate measured during the incubation after a 15N2 gas bubble is injected (solid lines; A, B and C) as compared to the N2 fixation measured with the addition of 15N2-enriched seawater (dashed lines; D, E and F). The diagram is based on the observations made in the experiments described in this study.
The discrepancies and mismatches/imbalances observed in field and laboratory studies could, in part, be explained by the variable underestimation of the true N2 fixation rate due to the methodological uncertainty reported here. We propose the addition of 15N2-enriched seawater to incubations to assess N2 fixation rates in laboratory and field studies. We suggest that measurements using this approach are likely to increase measurements and estimates of N2 fixation at species, regional and global level and lead to a reduction in the apparent oceanic nitrogen imbalance.
The diazotrophic cyanobacterium
We first examined the rate of equilibration between an injected bubble of 15N2 gas and seawater. Two series of incubations were started by injecting 140 µl of 15N2 into 133 ml of an artificial seawater media (YBCII) contained in headspace-free, septum-capped glass bottles. In the first series (isotopic equilibration experiments), all bottles were inverted fifty times (∼3 min) after injection of the 15N2 gas bubble and left at room temperature in the laboratory. One bottle was sampled immediately after the agitation in order to determine how much 15N2 gas had dissolved initially. The other bottles were opened and sampled after standing for periods from 1 to 24 h. Upon opening of the bottles, samples to measure the dissolved 15N2 were taken and stored in gas-tight glass vials (Exetainer®) until analysis.
In the second series (culture experiments), the YBCII media was pre-heated to 28°C in a temperature-controlled chamber before being used to fill septum-capped glass bottles. As with the first series, samples were agitated and left standing for varying periods of time after the injection of a 15N2 gas bubble. Instead of taking subsamples for 15N2 analysis, 13 ml of media were replaced by
An alternative, modified 15N2 tracer addition method was developed, which involved addition of an aliquot of 15N2-enriched water to incubations. This alternative method was based on earlier approaches used to study oxygen cycling using 18O2
We assessed possible effects of varying bottle size, amounts of injected gas and different amounts of agitation on their contribution to the equilibration between a bubble of 15N2 gas and the surrounding seawater. For the bottle size comparison, incubations were performed in 0.13 L bottles and in 1.15 L bottles. The amount of injected gas varied between 1 ml 15N2 per 1 L seawater up to 8 ml 15N2 per 1 L seawater. The incubations were agitated either by inverting fifty times manually (∼3 min) or by continuous agitation on a rotating bench-top shaker (Biometra WT 17) at 50 rpm (rotations per minute). We also added marine broth (Difco 2216; 0.2 µm filter-sterilized; 230 mg DOM L−1 media) to some bottles to examine the effect of dissolved organic matter (DOM).
Subsamples taken during the equilibration experiments were analysed for 15N2 concentration with a membrane-inlet mass spectrometer (MIMS; GAM200, IPI) within one week of subsampling. Dried GF/F filters were pelletized in tin cups, and PON as well as isotope ratios were measured by means of flash combustion in an elemental analyser (Carlo Erba EA 1108) coupled to a mass spectrometer (Thermo Finnigan Delta S).
The expected concentration of 15N2 following bubble injections was calculated assuming rapid and complete isotopic equilibration between bubble and surrounding seawater and considering atmospheric equilibrium concentrations of dissolved N2
We thank Marcel Kuypers and Hannah Marchant (Max Planck Institute for Marine Microbiology Bremen) for providing access to and advice on the membrane-inlet mass spectrometry. The assistance and suggestions of Gert Petrick and Karen Stange (IFM-GEOMAR) concerning the early analyses of 15N2 is also gratefully acknowledged.