New Perspectives on Nitrogen Fixation Measurements Using 15N2 Gas

Nicola Wannicke,Mar Benavides,Joseph P Montoya,Joachim W Dippner,Maren Voss,Tage Dalsgaard

doi:10.3389/fmars.2018.00120

Abstract

Recently, the method widely used to determine 15N2 fixation rates in marine and freshwater environments was found to underestimate rates because the dissolution of the added 15N2 gas bubble in seawater takes longer than theoretically calculated. As a solution to the potential underestimate of rate measurements, the usage of the enriched water method was proposed to provide constant 15N2 enrichment. Still, the superiority of enriched water method over the previously used bubble injection remains inconclusive. To clarify this issue, we performed laboratory based experiments and implemented the results into an error analysis of 15N2 fixation rates. Moreover, we conducted a literature search on the comparison of the two methods to calculate a mean effect size using a meta-analysis approach. Our results indicate that the error potentially introduced by an equilibrium phase of the 15N2 gas is -72% at maximum for experiments with very short incubation times of 1 hour. In contrast, the underestimation was negligible for incubations lasting 12 to 24 hours (error is -0.2%). Our meta-analysis indicates that 84 % of the measurements in the two groups will overlap and there is a 61% chance that a sample picked at random from the enriched water group will have a higher value than one picked at random from the bubble group. Overall, the underestimation of N2 fixation rates when using the bubble method relative to the enriched water method is highly dependent on incubation time and other experimental conditions and cannot be generalized.

Highlights

Over the last few decades, the stable isotopic tracer 15N2 was used to measure the production of diazotroph (N2-fixer) biomass directly
We combined a theoretical examination of the error associated with the equilibration time of the bubble of 15N2 gas and a meta-analysis of published and unpublished sets of N2-fixation measurements comparing both methods
Our error estimation of the bubble method during a 24 h experiment reveals a negligible error of −0.2% assuming a diazotroph community that fixes only during 12 h daytime and starting of nitrogen fixation 6 h after the injection of 15N2 gas i.e., the addition was done 6 h before sunrise

Summary

Introduction

Over the last few decades, the stable isotopic tracer 15N2 was used to measure the production of diazotroph (N2-fixer) biomass directly. This isotopic approach was first introduced by Burris and Miller (1941), but it was not until the 1990s that isotope ratio mass spectrometers (IRMS) were sensitive enough to measure low, open ocean low N2 fixation rates. The method consists of adding a volume of 15N2 gas into a seawater sample, Comparing 15N2 Fixation Measurements which is incubated for a given period (on deck or in situ), and terminated by filtration through glass fiber filters. The filters are later analyzed by IRMS to determine the amount of 15N2 transferred from the aqueous phase to the particulate cell material. Montoya et al (1996) introduced the calculation of the

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