scholarly journals Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria

2014 ◽  
Vol 11 (10) ◽  
pp. 2679-2689 ◽  
Author(s):  
T. Yamazaki ◽  
T. Hozuki ◽  
K. Arai ◽  
S. Toyoda ◽  
K. Koba ◽  
...  
Keyword(s):  
Nitrous Oxide ◽  
Pure Culture ◽  
Paracoccus Denitrificans ◽  
In Vivo Studies ◽  
Site Preference ◽  
Ammonia Oxidizing Bacteria ◽  
Small Contribution ◽  
N2o Production ◽  
No2 Reduction

Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification by various microorganisms. Site preference (SP) of 15N in N2O, which is defined as the difference in the natural abundance of isotopomers 14N15NO and 15N14NO relative to 14N14NO, has been reported to be a useful tool to quantitatively distinguish N2O production pathways. To determine representative SP values for each microbial process, we firstly measured SP of N2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans. The SP value for NOR reaction (−5.9 ± 2.1‰) showed nearly the same value as that reported for N2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 ± 2.3‰) was a little higher than the values reported for N2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO2–) reduction (which is followed by NO reduction) to N2O production by N. oceani incubated under different O2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for the NH2OH oxidation pathway possibly due to a small contribution of NO2– reduction pathway. Further evaluation of isotopomer signatures of N2O using common enzymes of other processes related to N2O would improve the isotopomer analysis of N2O in various environments.

scholarly journals Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria

2013 ◽  
Vol 10 (10) ◽  
pp. 16615-16643 ◽  
Author(s):  
T. Yamazaki ◽  
T. Hozuki ◽  
K. Arai ◽  
S. Toyoda ◽  
K. Koba ◽  
...  
Keyword(s):  
Nitrous Oxide ◽  
Pure Culture ◽  
Paracoccus Denitrificans ◽  
In Vivo Studies ◽  
Site Preference ◽  
Ammonia Oxidizing Bacteria ◽  
Small Contribution ◽  
N2o Production ◽  
No2 Reduction

Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification in environmental nitrogen cycle by various microorganism. Site preference (SP) of 15N in N2O, which is defined as the difference in the natural abundance of isotopomers 14N15NO and 15N14NO relative to 14N14NO, has been reported to be a useful tool to quantitatively distinguish N2O production pathway. To determine representative SP value for each microbial process, we firstly measured SP of N2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans, respectively. The SP value for NOR reaction (−5.9 ± 2.1‰) showed nearly the same value as that reported for N2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 ± 2.3‰) was a little higher than the values reported for N2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO2–) reduction (which is followed by NO reduction) to N2O production by N. oceani incubated under different O2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for NH2OH oxidation pathway possibly due to a small contribution of NO2– reduction pathway. Further evaluation of isotopomer signatures of N2O using common enzymes of other processes related to N2O would improve the isotopomer analysis of N2O in various environments.

scholarly journals Biogeochemical controls and isotopic signatures of nitrous oxide production by a marine ammonia-oxidizing bacterium

2010 ◽  
Vol 7 (9) ◽  
pp. 2695-2709 ◽  
Author(s):  
C. H. Frame ◽  
K. L. Casciotti
Keyword(s):  
Nitrous Oxide ◽  
Stratospheric Ozone ◽  
Nitrifying Bacteria ◽  
Site Preference ◽  
Ammonia Oxidizing Bacteria ◽  
N2o Production ◽  
O2 Concentration ◽  
Cell Densities ◽  
Dissolved O2

Abstract. Nitrous oxide (N2O) is a trace gas that contributes to the greenhouse effect and stratospheric ozone depletion. The N2O yield from nitrification (moles N2O-N produced per mole ammonium-N consumed) has been used to estimate marine N2O production rates from measured nitrification rates and global estimates of oceanic export production. However, the N2O yield from nitrification is not constant. Previous culture-based measurements indicate that N2O yield increases as oxygen (O2) concentration decreases and as nitrite (NO2−) concentration increases. Here, we have measured yields of N2O from cultures of the marine β-proteobacterium Nitrosomonas marina C-113a as they grew on low-ammonium (50 μM) media. These yields, which were typically between 4 × 10−4 and 7 × 10−4 for cultures with cell densities between 2 × 102 and 2.1 × 104 cells ml−1, were lower than previous reports for ammonia-oxidizing bacteria. The observed impact of O2 concentration on yield was also smaller than previously reported under all conditions except at high starting cell densities (1.5 × 106 cells ml−1), where 160-fold higher yields were observed at 0.5% O2 (5.1 μM dissolved O2) compared with 20% O2 (203 μM dissolved O2). At lower cell densities (2 × 102 and 2.1 × 104 cells ml−1), cultures grown under 0.5% O2 had yields that were only 1.25- to 1.73-fold higher than cultures grown under 20% O2. Thus, previously reported many-fold increases in N2O yield with dropping O2 could be reproduced only at cell densities that far exceeded those of ammonia oxidizers in the ocean. The presence of excess NO2− (up to 1 mM) in the growth medium also increased N2O yields by an average of 70% to 87% depending on O2 concentration. We made stable isotopic measurements on N2O from these cultures to identify the biochemical mechanisms behind variations in N2O yield. Based on measurements of δ15Nbulk, site preference (SP = δ15Nα−δ15Nβ), and δ18O of N2O (δ18O-N2O), we estimate that nitrifier-denitrification produced between 11% and 26% of N2O from cultures grown under 20% O2 and 43% to 87% under 0.5% O2. We also demonstrate that a positive correlation between SP and δ18O-N2O is expected when nitrifying bacteria produce N2O. A positive relationship between SP and δ18O-N2O has been observed in environmental N2O datasets, but until now, explanations for the observation invoked only denitrification. Such interpretations may overestimate the role of heterotrophic denitrification and underestimate the role of ammonia oxidation in environmental N2O production.

scholarly journals Impact of reactive surfaces on the abiotic reaction between nitrite and ferrous iron and associated nitrogen and oxygen isotope dynamics

2020 ◽  
Vol 17 (16) ◽  
pp. 4355-4374
Author(s):  
Anna-Neva Visser ◽  
Scott D. Wankel ◽  
Pascal A. Niklaus ◽  
James M. Byrne ◽  
Andreas A. Kappler ◽  
...  
Keyword(s):  
Isotope Effect ◽  
Ex Vivo ◽  
Reduction Rate ◽  
Site Preference ◽  
Experimental Conditions ◽  
N2o Production ◽  
O Isotope ◽  
No2 Reduction ◽  
The Impact ◽  
Reactive Surfaces

Abstract. Anaerobic nitrate-dependent Fe(II) oxidation (NDFeO) is widespread in various aquatic environments and plays a major role in iron and nitrogen redox dynamics. However, evidence for truly enzymatic, autotrophic NDFeO remains limited, with alternative explanations involving the coupling of heterotrophic denitrification with the abiotic oxidation of structurally bound or aqueous Fe(II) by reactive intermediate nitrogen (N) species (chemodenitrification). The extent to which chemodenitrification is caused (or enhanced) by ex vivo surface catalytic effects has not been directly tested to date. To determine whether the presence of either an Fe(II)-bearing mineral or dead biomass (DB) catalyses chemodenitrification, two different sets of anoxic batch experiments were conducted: 2 mM Fe(II) was added to a low-phosphate medium, resulting in the precipitation of vivianite (Fe3(PO4)2), to which 2 mM nitrite (NO2-) was later added, with or without an autoclaved cell suspension (∼1.96×108 cells mL−1) of Shewanella oneidensis MR-1. Concentrations of nitrite (NO2-), nitrous oxide (N2O), and iron (Fe2+, Fetot) were monitored over time in both set-ups to assess the impact of Fe(II) minerals and/or DB as catalysts of chemodenitrification. In addition, the natural-abundance isotope ratios of NO2- and N2O (δ15N and δ18O) were analysed to constrain the associated isotope effects. Up to 90 % of the Fe(II) was oxidized in the presence of DB, whereas only ∼65 % of the Fe(II) was oxidized under mineral-only conditions, suggesting an overall lower reactivity of the mineral-only set-up. Similarly, the average NO2- reduction rate in the mineral-only experiments (0.004±0.003 mmol L−1 d−1) was much lower than in the experiments with both mineral and DB (0.053±0.013 mmol L−1 d−1), as was N2O production (204.02±60.29 nmol L−1 d−1). The N2O yield per mole NO2- reduced was higher in the mineral-only set-ups (4 %) than in the experiments with DB (1 %), suggesting the catalysis-dependent differential formation of NO. N-NO2- isotope ratio measurements indicated a clear difference between both experimental conditions: in contrast to the marked 15N isotope enrichment during active NO2- reduction (15εNO2=+10.3 ‰) observed in the presence of DB, NO2- loss in the mineral-only experiments exhibited only a small N isotope effect (<+1 ‰). The NO2--O isotope effect was very low in both set-ups (18εNO2 <1 ‰), which was most likely due to substantial O isotope exchange with ambient water. Moreover, under low-turnover conditions (i.e. in the mineral-only experiments as well as initially in experiments with DB), the observed NO2- isotope systematics suggest, transiently, a small inverse isotope effect (i.e. decreasing NO2- δ15N and δ18O with decreasing concentrations), which was possibly related to transitory surface complexation mechanisms. Site preference (SP) of the 15N isotopes in the linear N2O molecule for both set-ups ranged between 0 ‰ and 14 ‰, which was notably lower than the values previously reported for chemodenitrification. Our results imply that chemodenitrification is dependent on the available reactive surfaces and that the NO2- (rather than the N2O) isotope signatures may be useful for distinguishing between chemodenitrification catalysed by minerals, chemodenitrification catalysed by dead microbial biomass, and possibly true enzymatic NDFeO.

Nitrous oxide production by lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-removal systems

2011 ◽  
Vol 39 (6) ◽  
pp. 1832-1837 ◽  
Author(s):  
Kartik Chandran ◽  
Lisa Y. Stein ◽  
Martin G. Klotz ◽  
Mark C.M. van Loosdrecht
Keyword(s):  
Nitrous Oxide ◽  
Nitrogen Removal ◽  
Ammonia Oxidation ◽  
Wastewater Treatment Plants ◽  
N2o Emissions ◽  
Ammonia Oxidizing Bacteria ◽  
N2o Production ◽  
Nitrogen Emissions ◽  
Greenhouse Gas Inventories ◽  
Nitrogen Concentrations

Chemolithoautotrophic AOB (ammonia-oxidizing bacteria) form a crucial component in microbial nitrogen cycling in both natural and engineered systems. Under specific conditions, including transitions from anoxic to oxic conditions and/or excessive ammonia loading, and the presence of high nitrite (NO2−) concentrations, these bacteria are also documented to produce nitric oxide (NO) and nitrous oxide (N2O) gases. Essentially, ammonia oxidation in the presence of non-limiting substrate concentrations (ammonia and O2) is associated with N2O production. An exceptional scenario that leads to such conditions is the periodical switch between anoxic and oxic conditions, which is rather common in engineered nitrogen-removal systems. In particular, the recovery from, rather than imposition of, anoxic conditions has been demonstrated to result in N2O production. However, applied engineering perspectives, so far, have largely ignored the contribution of nitrification to N2O emissions in greenhouse gas inventories from wastewater-treatment plants. Recent field-scale measurements have revealed that nitrification-related N2O emissions are generally far higher than emissions assigned to heterotrophic denitrification. In the present paper, the metabolic pathways, which could potentially contribute to NO and N2O production by AOB have been conceptually reconstructed under conditions especially relevant to engineered nitrogen-removal systems. Taken together, the reconstructed pathways, field- and laboratory-scale results suggest that engineering designs that achieve low effluent aqueous nitrogen concentrations also minimize gaseous nitrogen emissions.

scholarly journals Comparison of different two-pathway models for describing the combined effect of DO and nitrite on the nitrous oxide production by ammonia-oxidizing bacteria

2016 ◽  
Vol 75 (3) ◽  
pp. 491-500 ◽  
Author(s):  
Longqi Lang ◽  
Mathieu Pocquet ◽  
Bing-Jie Ni ◽  
Zhiguo Yuan ◽  
Mathieu Spérandio
Keyword(s):  
Nitrous Oxide ◽  
Combined Effect ◽  
Ammonia Oxidizing Bacteria ◽  
N2o Production ◽  
Mathematical Structures ◽  
Nitrifier Denitrification ◽  
Nitrous Oxide Production ◽  
Pathway Models ◽  
Microbial Systems ◽  
Electron Carriers

The aim of this work is to compare the capability of two recently proposed two-pathway models for predicting nitrous oxide (N2O) production by ammonia-oxidizing bacteria (AOB) for varying ranges of dissolved oxygen (DO) and nitrite. The first model includes the electron carriers whereas the second model is based on direct coupling of electron donors and acceptors. Simulations are confronted to extensive sets of experiments (43 batches) from different studies with three different microbial systems. Despite their different mathematical structures, both models could well and similarly describe the combined effect of DO and nitrite on N2O production rate and emission factor. The model-predicted contributions for nitrifier denitrification pathway and hydroxylamine pathway also matched well with the available isotopic measurements. Based on sensitivity analysis, calibration procedures are described and discussed for facilitating the future use of those models.

scholarly journals Nitrous oxide distribution and its origin in the central and eastern South Pacific Subtropical Gyre

2007 ◽  
Vol 4 (5) ◽  
pp. 729-741 ◽  
Author(s):  
J. Charpentier ◽  
L. Farias ◽  
N. Yoshida ◽  
N. Boontanon ◽  
P. Raimbault
Keyword(s):  
Nitrous Oxide ◽  
Coastal Upwelling ◽  
South Pacific ◽  
Site Preference ◽  
Natural Environments ◽  
Intermediate Water ◽  
N2o Production ◽  
South Pacific Ocean ◽  
Nitrifier Denitrification ◽  
Organic Particles

Abstract. The mechanisms of microbial nitrous oxide (N2O) production in the ocean have been the subject of many discussions in recent years. New isotopomeric tools can further refine our knowledge of N2O sources in natural environments. This study compares hydrographic, N2O concentration, and N2O isotopic and isotopomeric data from three stations along a coast-perpendicular transect in the South Pacific Ocean, extending from the center (Sts. GYR and EGY) of the subtropical oligotrophic gyre (~26° S; 114° W) to the upwelling zone (St. UPX) off the central Chilean coast (~34° S). Although AOU/N2O and NO3− trends support the idea that most of the N2O (mainly from intermediate water (200–600 m)) comes from nitrification, N2O isotopomeric composition (intramolecular distribution of 15N isotopes) expressed as SP (site preference of 15N) shows low values (10 to 12\\permil) that could be attributed to the production through of microbial nitrifier denitrification (reduction of nitrite to N2O mediated by ammonium oxidizers). The coincidence of this SP signal with high – stability layer, where sinking organic particles can accumulate, suggests that N2O could be produced by nitrifier denitrification inside particles. It is postulated that deceleration of particles in the pycnocline can modify the advection - diffusion balance inside particles, allowing the accumulation of nitrite and O2 depletion suitable for nitrifier denitrication. As lateral advection seems to be relatively insignificant in the gyre, in situ nitrifier denitrification could account for 40–50% of the N2O produced in this layer. In contrast, coastal upwelling system is characterized by O2 deficient condition and some N deficit in a eutrophic system. Here, N2O accumulates up to 480% saturation, and isotopic and isotopomer signals show highly complex N2O production processes, which presumably reflect both the effect of nitrification and denitrification at low O2 levels on N2O production, but net N2O consumption by denitrification was not observed.

scholarly journals The isotopic composition of atmospheric nitrous oxide observed at the high-altitude research station Jungfraujoch, Switzerland

2020 ◽  
Vol 20 (11) ◽  
pp. 6495-6519 ◽  
Author(s):  
Longfei Yu ◽  
Eliza Harris ◽  
Stephan Henne ◽  
Sarah Eggleston ◽  
Martin Steinbacher ◽  
...  
Keyword(s):  
Nitrous Oxide ◽  
Isotopic Composition ◽  
High Altitude ◽  
High Precision ◽  
Large Scale ◽  
Late Summer ◽  
Site Preference ◽  
Research Station ◽  
N2o Production ◽  
Mixing Ratios

Abstract. Atmospheric nitrous oxide (N2O) levels have been continuously growing since preindustrial times. Mitigation requires information about sources and sinks on the regional and global scales. Isotopic composition of N2O in the atmosphere could contribute valuable constraints. However, isotopic records of N2O in the unpolluted atmosphere remain too scarce for large-scale N2O models. Here, we report the results of discrete air samples collected weekly to biweekly over a 5-year period at the high-altitude research station Jungfraujoch, located in central Switzerland. High-precision N2O isotopic measurements were made using a recently developed preconcentration and laser spectroscopy technique. The measurements of discrete samples were accompanied by in situ continuous measurements of N2O mixing ratios. Our results indicate a pronounced seasonal pattern with minimum N2O mixing ratios in late summer, associated with a maximum in δ15Nbulk and a minimum in intramolecular 15N site preference (δ15NSP). This pattern is most likely due to stratosphere–troposphere exchange (STE), which delivers N2O-depleted but 15N-enriched air from the stratosphere into the troposphere. Variability in δ15NSP induced by changes in STE may be masked by biogeochemical N2O production processes in late summer, which are possibly dominated by a low-δ15NSP pathway of N2O production (denitrification), providing an explanation for the observed seasonality of δ15NSP. Footprint analyses and atmospheric transport simulations of N2O for Jungfraujoch suggest that regional emissions from the planetary boundary layer contribute to seasonal variations of atmospheric N2O isotopic composition at Jungfraujoch, albeit more clearly for δ15NSP and δ18O than for δ15Nbulk. With the time series of 5 years, we obtained a significant interannual trend for δ15Nbulk after deseasonalization (-0.052±0.012 ‰ a−1), indicating that the atmospheric N2O increase is due to isotopically depleted N2O sources. We estimated the average isotopic signature of anthropogenic N2O sources with a two-box model to be -8.6±0.6 ‰ for δ15Nbulk, 34.8±3 ‰ for δ18O and 10.7±4 ‰ for δ15NSP. Our study demonstrates that seasonal variation of N2O isotopic composition in the background atmosphere is important when determining interannual trends. More frequent, high-precision and interlaboratory-compatible measurements of atmospheric N2O isotopocules, especially for δ15NSP, are needed to better constrain anthropogenic N2O sources and thus the contribution of biogeochemical processes to N2O growth on the global scale.

scholarly journals Isotopic evidence for alteration of nitrous oxide emissions and producing pathways contribution under nitrifying conditions

2019 ◽  
Author(s):  
Guillaume Humbert ◽  
Mathieu Sébilo ◽  
Justine Fiat ◽  
Longqi Lang ◽  
Ahlem Filali ◽  
...  
Keyword(s):  
Nitrous Oxide ◽  
Biofilm Reactor ◽  
Nitrite Reduction ◽  
Nitrous Oxide Emissions ◽  
Site Preference ◽  
N2o Emissions ◽  
Ammonium Oxidation ◽  
N2o Production ◽  
Oxidation Rates ◽  
Nitrifier Denitrification

Abstract. Nitrous oxide (N2O) emissions by a nitrifying biofilm reactor were investigated with N2O isotopocules. The site preference of N2O (15N-SP) indicated the contribution of producing and consuming pathways in response to changes in oxygenation level (from 0 to 21 % O2 in the gas mix), temperature (from 13.5 to 22.3 °C), and ammonium concentrations (from 6.2 to 62.1 mg N L−1). Nitrite reduction, either nitrifier-denitrification or heterotrophic denitrification, was the main N2O producing pathway under the tested conditions. Nitrite oxidation rates decreased as compared to ammonium oxidation rates at temperatures above 20 °C and sub-optimal oxygen levels, increasing N2O production by the nitrite reduction pathway. Below 20 °C, a difference in temperature sensitivity between hydroxylamine and ammonium oxidation rates is most likely responsible for an increase in the N2O production via the hydroxylamine oxidation pathway (nitrification). A negative correlation between the reaction kinetics and the apparent isotope fractionation was additionally shown from the variations of δ15N and δ18O values of N2O produced from ammonium.

scholarly journals Isotopic evidence for alteration of nitrous oxide emissions and producing pathways' contribution under nitrifying conditions

2020 ◽  
Vol 17 (4) ◽  
pp. 979-993
Author(s):  
Guillaume Humbert ◽  
Mathieu Sébilo ◽  
Justine Fiat ◽  
Longqi Lang ◽  
Ahlem Filali ◽  
...  
Keyword(s):  
Nitrous Oxide ◽  
Biofilm Reactor ◽  
Nitrite Reduction ◽  
Nitrous Oxide Emissions ◽  
Site Preference ◽  
N2o Emissions ◽  
Ammonium Oxidation ◽  
N2o Production ◽  
Oxidation Rates ◽  
Nitrifier Denitrification

Abstract. Nitrous oxide (N2O) emissions from a nitrifying biofilm reactor were investigated with N2O isotopocules. The nitrogen isotopomer site preference of N2O (15N-SP) indicated the contribution of producing and consuming pathways in response to changes in oxygenation level (from 0 % to 21 % O2 in the gas mix), temperature (from 13.5 to 22.3 ∘C) and ammonium concentrations (from 6.2 to 62.1 mg N L−1). Nitrite reduction, either nitrifier denitrification or heterotrophic denitrification, was the main N2O-producing pathway under the tested conditions. Difference between oxidative and reductive rates of nitrite consumption was discussed in relation to NO2- concentrations and N2O emissions. Hence, nitrite oxidation rates seem to decrease as compared to ammonium oxidation rates at temperatures above 20 ∘C and under oxygen-depleted atmosphere, increasing N2O production by the nitrite reduction pathway. Below 20 ∘C, a difference in temperature sensitivity between hydroxylamine and ammonium oxidation rates is most likely responsible for an increase in N2O production via the hydroxylamine oxidation pathway (nitrification). A negative correlation between the reaction kinetics and the apparent isotope fractionation was additionally shown from the variations of δ15N and δ18O values of N2O produced from ammonium. The approach and results obtained here, for a nitrifying biofilm reactor under variable environmental conditions, should allow for application and extrapolation of N2O emissions from other systems such as lakes, soils and sediments.

scholarly journals Estimation of isotope variation of N<sub>2</sub>O during denitrification by <i>Pseudomonas aureofaciens</i> and <i>Pseudomonas chlororaphis</i>: implications for N<sub>2</sub>O source apportionment

2018 ◽  
Vol 15 (12) ◽  
pp. 3873-3882 ◽  
Author(s):  
Joshua A. Haslun ◽  
Nathaniel E. Ostrom ◽  
Eric L. Hegg ◽  
Peggy H. Ostrom
Keyword(s):  
Nitrous Oxide ◽  
Electron Donor ◽  
Isotope Effects ◽  
Microbial Processes ◽  
Site Preference ◽  
N2o Emissions ◽  
Pseudomonas Chlororaphis ◽  
Fractionation Factor ◽  
N2o Production ◽  
Soil Microbial

Abstract. Soil microbial processes, stimulated by agricultural fertilization, account for 90 % of anthropogenic nitrous oxide (N2O), the leading source of ozone depletion and a potent greenhouse gas. Efforts to reduce N2O flux commonly focus on reducing fertilization rates. Management of microbial processes responsible for N2O production may also be used to reduce N2O emissions, but this requires knowledge of the prevailing process. To this end, stable isotopes of N2O have been applied to differentiate N2O produced by nitrification and denitrification. To better understand the factors contributing to isotopic variation during denitrification, we characterized the δ15N, δ18O and site preference (SP; the intramolecular distribution of 15N in N2O) of N2O produced during NO3- reduction by Pseudomonas chlororaphis subsp. aureofaciens and P. c. subsp. chlororaphis. The terminal product of denitrification for these two species is N2O because they lack the gene nitrous oxide reductase, which is responsible for the reduction of N2O to N2. In addition to species, treatments included electron donor (citrate and succinate) and electron donor concentration (0.01, 0.1, 1 and 10 mM) as factors. In contrast to the expectation of a Rayleigh model, all treatments exhibited curvilinear behaviour between δ15N or δ18O and the extent of the reaction. The curvilinear behaviour indicates that the fractionation factor changed over the course of the reaction, something that is not unexpected for a multi-step process such as denitrification. Using the derivative of the equation, we estimated that the net isotope effects (η) vary by as much as 100 ‰ over the course of a single reaction, presenting challenges for using δ15N and δ18O as apportionment tools. In contrast, SP for denitrification was not affected by the extent of the reaction, the electron donor source or concentration, although the mean SP of N2O produced by each species differed. Therefore, SP remains a robust indicator of the origin of N2O. To improve apportionment estimates with SP, future studies could evaluate other factors that contribute to the variation in SP.

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