Continuous measurements of nitrous oxide isotopomers during incubation experiments

Abstract

Nitrous oxide (N<sub>2</sub>O) is an important and strong greenhouse gas in the atmosphere and part of a feed-back loop with climate. N<sub>2</sub>O is produced by microbes during nitrification and denitrification in terrestrial and aquatic ecosystems. The main sinks for N<sub>2</sub>O are turnover by denitrification and photolysis and photo-oxidation in the stratosphere. The position of the isotope <sup>15</sup>N in the linear N = N = O molecule can be distinguished between the central or terminal position (isotopomers of N<sub>2</sub>O). It has been demonstrated that nitrifying and denitrifying microbes have a different relative preference for the terminal and central position. Therefore, measurements of the site preference in N<sub>2</sub>O can be used to determine the source of N<sub>2</sub>O i.e. nitrification or denitrification. Recent instrument development allows for continuous (on the order of days) position dependent <i>δ</i><sup>15</sup>N measurements at N<sub>2</sub>O concentrations relevant for studies of atmospheric chemistry. We present results from continuous incubation experiments with denitrifying bacteria, <i>Pseudomonas fluorescens</i> (producing and reducing N<sub>2</sub>O) and <i>P. chlororaphis</i> (only producing N<sub>2</sub>O). The continuous position dependent measurements reveal the transient pattern (KNO<sub>3</sub> to N<sub>2</sub>O and N<sub>2</sub>, respectively), which can be compared to previous reported site preference (SP) values. We find bulk isotope effects of −5.5 ‰ ± 0.9 for <i>P. chlororaphis</i>. For <i>P. fluorescens</i>, the bulk isotope effect during production of N<sub>2</sub>O is −50.4 ‰ ± 9.3 and 8.5 ‰ ± 3.7 during N<sub>2</sub>O reduction. The values for <i>P. fluorescens</i> are in line with earlier findings, whereas the values for <i>P. chlororaphis</i> are larger than previously published <i>δ</i><sup>15</sup>N<sub><i>bulk</i></sub> measurements from production. The calculations of the SP isotope effect from the measurements of <i>P. chlororaphis</i> result in values of −6.6 ‰ ± 1.8. For <i>P. fluorescens</i>, the calculations results in SP values of −5.7 ‰ ± 5.6 during production of N<sub>2</sub>O and 2.3 ‰ ± 3.2 during reduction of N<sub>2</sub>O. In summary, we implemented continuous measurements of N<sub>2</sub>O isotopomers during incubation of denitrifying bacteria and believe that similar experiments will lead to a better understanding of denitrifying bacteria and N<sub>2</sub>O turnover in soils and sediments and ultimately hands-on knowledge on the biotic mechanisms behind greenhouse gas exchange of the Globe.

An automated, laser‐based measurement system for nitrous oxide isotope and isotopomer ratios at nanomolar levels

“…Until recently, the main disadvantages of CRDS instruments have been the relatively large sample size requirement (20–130 nmol‐N 2 O) and the lack of automatic sample preparation. To this end, researchers who attempted to utilize laser‐based N 2 O systems for aquatic nitrogen and oxygen isotopic analyses were limited to (1) extraction of dissolved N 2 O using a gas equilibration device under continuous‐flow mode or (2) conversion of samples with high nitrite or nitrate concentrations to N 2 O via chemical or biological methods . We have now successfully coupled an automated purge‐and‐trap module to a CRDS system (referred to as PT‐CRDS), and we demonstrate the ability to conduct automated and high‐throughput measurements of N 2 O concentrations and isotope/isotopomer signatures from discrete seawater samples at much lower N 2 O concentrations, while maintaining accuracy and precision that are similar to those obtained by GC (N 2 O concentration measurements) and GC/IRMS (isotope/isotopomer measurements).…”

Isotopically characterised N2O reference materials for use as community standards

“…The informative value of N 2 O isotope data has been markedly increased by using the data to inform biogeochemical models, providing regional and global patterns of N 2 O losses and independent process information 9–12 . Advances in applications have been accompanied and accelerated by progress in analytics, complementing the traditional high‐precision isotope‐ratio mass‐spectrometry (IRMS) 1,13 by laser spectroscopic techniques, with the potential for field applicability and real‐time data coverage 14–19 …”

“…[9][10][11][12] Advances in applications have been accompanied and accelerated by progress in analytics, complementing the traditional high-precision isotope-ratio mass-spectrometry (IRMS) 1,13 by laser spectroscopic techniques, with the potential for field applicability and real-time data coverage. [14][15][16][17][18][19] The isotopic composition of a sample is reported using the delta (δ) notation, which is the relative difference in isotope ratio (R) between a sample P and a reference material, i.e. δ(P/ref) = R P / R ref À 1.…”

“…For nitrogen, the 15 N/ 14 N isotope ratio is used, R( 15 N/ 14 N) = x( 15 N)/x( 14 N), where x is the isotopic abundance and tropospheric N 2 is the international reference material for the Air-N 2 scale. For oxygen, the 18 O/ 16 O and 17 O/ 16 O ratios are used, which are related to the Vienna Standard Mean Ocean Water (VSMOW) scale. In addition, we adopt the following notation conventions: δ 15 N = δ( 15 N/ 14 N, P/Air-N 2 ) (average of both nitrogen atoms) and δ 18 O = δ( 18 O/ 16 O, P/VSMOW).…”

“…The 15 N site preference (SP) is defined by the predominance of 15 N substitution in the central (α) position as compared to the terminal (β) position, and calculated accordingly as δ 15 N SP = δ 15 N α À δ 15 N β . All δ values in this paper are reported against Air-N 2 (for 15 N/ 14 N ratios) and against VSMOW (for 18 O/ 16 O and 17 O/ 16 O ratios).…”