The mechanism of oxygen isotope fractionation during N2O production by denitrification

The isotopic composition of soil-derived N2O can help differentiate between N2O production pathways and estimate the fraction of N2O reduced to N2. Until now, δ18O of N2O has been rarely used in the interpretation of N2O isotopic signatures because of the rather complex oxygen isotope fractionations during N2O production by denitrification. The latter process involves nitrate reduction mediated through the following three enzymes: nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR). Each step removes one oxygen atom as water (H2O), which gives rise to a branching isotope effect. Moreover, denitrification intermediates may partially or fully exchange oxygen isotopes with ambient water, which is associated with an exchange isotope effect. The main objective of this study was to decipher the mechanism of oxygen isotope fractionation during N2O production by denitrification and, in particular, to investigate the relationship between the extent of oxygen isotope exchange with soil water and the δ18O values of the produced N2O. We performed several soil incubation experiments. For the first time, ∆17 O isotope tracing was applied to simultaneously determine the extent of oxygen isotope exchange and any associated oxygen isotope effect. We found bacterial denitrification to be typically associated with almost complete oxygen isotope exchange and a stable difference in δ18O between soil water and the produced N2O of δ18O(N2O / H2O) = (17.5±1.2) ‰. However, some experimental setups yielded oxygen isotope exchange as low as 56 % and a higher δ18O(N2O / H2O) of up to 37‰. The extent of isotope exchange and δ18O(N2O / H2O) showed a very significant correlation (R2 = 0.70, p < 0.00001). We hypothesise that this observation was due to the contribution of N2O from another production process, most probably fungal denitrification. An oxygen isotope fractionation model was used to test various scenarios with different magnitudes of branching isotope effects at different steps in the reduction process. The results suggest that during denitrification the isotope exchange occurs prior to the isotope branching and that the mechanism of this exchange is mostly associated with the enzymatic nitrite reduction mediated by NIR. For bacterial denitrification, the branching isotope effect can be surprisingly low, about (0.0±0.9) ‰; in contrast to fungal denitrification where higher values of up to 30‰ have been reported previously. This suggests that δ18O might be used as a tracer for differentiation between bacte- 5 rial and fungal denitrification, due to their different magnitudes of branching isotope effect

Oxygen isotope fractionation during N2O production by soil denitrification

The isotopic composition of soil-derived N₂O can help differentiate between N₂O production pathways and estimate the fraction of N₂O reduced to N₂. Until now, <i>δ</i>¹⁸O of N₂O has been rarely used in the interpretation of N₂O isotopic signatures because of the rather complex oxygen isotope fractionations during N₂O production by denitrification. The latter process involves nitrate reduction mediated through the following three enzymes: nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR). Each step removes one oxygen atom as water (H₂O), which gives rise to a branching isotope effect. Moreover, denitrification intermediates may partially or fully exchange oxygen isotopes with ambient water, which is associated with an exchange isotope effect. The main objective of this study was to decipher the mechanism of oxygen isotope fractionation during N₂O production by soil denitrification and, in particular, to investigate the relationship between the extent of oxygen isotope exchange with soil water and the <i>δ</i>¹⁸O values of the produced N₂O. <br><br> In our soil incubation experiments Δ¹⁷O isotope tracing was applied for the first time to simultaneously determine the extent of oxygen isotope exchange and any associated oxygen isotope effect. We found that N₂O formation in static anoxic incubation experiments was typically associated with oxygen isotope exchange close to 100 % and a stable difference between the ¹⁸O ∕ ¹⁶O ratio of soil water and the N₂O product of <i>δ</i>¹⁸O(N₂O ∕ H₂O)  =  (17.5 ± 1.2) ‰. However, flow-through experiments gave lower oxygen isotope exchange down to 56 % and a higher <i>δ</i>¹⁸O(N₂O ∕ H₂O) of up to 37 ‰. The extent of isotope exchange and <i>δ</i>¹⁸O(N₂O ∕ H₂O) showed a significant correlation (<i>R</i>² = 0.70, <i>p</i> &lt;  0.00001). We hypothesize that this observation was due to the contribution of N₂O from another production process, most probably fungal denitrification. <br><br> An oxygen isotope fractionation model was used to test various scenarios with different magnitudes of branching isotope effects at different steps in the reduction process. The results suggest that during denitrification, isotope exchange occurs prior to isotope branching and that this exchange is mostly associated with the enzymatic nitrite reduction mediated by NIR. For bacterial denitrification, the branching isotope effect can be surprisingly low, about (0.0 ± 0.9) ‰, in contrast to fungal denitrification where higher values of up to 30 ‰ have been reported previously. This suggests that <i>δ</i>¹⁸O might be used as a tracer for differentiation between bacterial and fungal denitrification, due to their different magnitudes of branching isotope effects

Nitrite induced transcription of p450nor during denitrification by Fusarium oxysporum correlates with the production of N2O with a high 15N site preference

The greenhouse gas nitrous oxide (N2O) is produced in soil as a consequence of complex co-occurring processes conducted by diverse microbial species, including fungi. The fungal p450nor gene encodes a nitric oxide reductase associated with fungal denitrification. We thus hypothesized that p450nor gene expression is a marker for ongoing fungal denitrification. Specific PCR primers and quantitative PCR (qPCR) assays were developed targeting p450nor genes and transcripts. The novel PCR primers successfully amplified p450nor from pure cultures, and were used in an mRNA targeted qPCR to quantify p450nor gene transcription (i.e., gene expression) during denitrification activity in cultures of the fungal model denitrifier Fusarium oxysporum. Gene expression was induced by high (5 mM) and low (0.25 mM) nitrite concentrations. Nitrite stimulated N2O production rates by F. oxysporum, which correlated well with an up to 70-fold increase in p450nor gene expression during the first 12–24 h of anoxic incubation. The relative p450nor gene peak expression and peak N2O production rates declined 20- and 2-fold on average, respectively, towards the later phase of incubation (48–120 h). The 15N site preference of N2O (SP(N2O)) was high for F. oxysporum and independent of reaction progress, confirming the fungal origin of N2O produced. In conclusion, the developed fungal p450nor gene expression assay together with the analysis of SP(N2O) values provide a basis to improve current tools for the identification of fungal denitrification and/or N2O production in natural systems like soils

Development of a method for in situ measurement of denitrification in aquifers using 15N tracer tests and membrane inlet mass spectrometry

We present a new approach for in situ-measurement of denitrification using a combination of 15N-tracer push-pull experiments with in situ analysis of 15N-labled N2 and N2O using membrane inlet mass spectrometry (MIMS). In the 15N-tracer experiment we present here we supplemented Aquifer material of two depths with 15N labeled nitrate. The results of our laboratory 15N-tracer test showed a linear increase of denitrification products (15(N2O+N2)) over time. At the end of our experiment we measured up to 1500 and 3700 µg/L 15(N2O+N2) in the water samples from the supplemented aquifer material. The online measurement with MIMS enabled us to see during the experiment if and when the production of the labeled denitrification products started. We took also parallel samples for isotope ratio mass spectrometry (IRMS) analysis to check our MIMS measurements. The measured 15(N2O+N2) values for IRMS matches the MIMS measurements very well. With the MIMS-method there is no need for sample preparation and so we were able to run the MIMS part of the 15N-tracer test automatically. Later-on this approach will be used in the field

Interaction of straw amendment and soil NO3- content controls fungal denitrification and denitrification product stoichiometry in a sandy soil

The return of agricultural crop residues are vital to maintain or even enhance soil fertility. However, the influence of application rate of crop residues on denitrification and its related gaseous N emissions is not fully understood. We conducted a fully robotized continuous flow incubation experiment using a Helium/Oxygen atmosphere over 30 days to examine the effect of maize straw application rate on: i) the rate of denitrification, ii) denitrification product stoichiometry N2O/(N2O+N2), and iii) the contribution of fungal denitrification to N2O fluxes. Five treatments were established using sieved, repacked sandy textured soil; i) non-amended control, ii) nitrate only, iii) low rate of straw + nitrate, iv) medium rate of straw + nitrate, and iv) high rate of straw + nitrate (n = 3). We simultaneously measured NO, N2O as well as direct N2 emissions and used the N2O 15N site preference signatures of soil-emitted N2O to distinguish N2O production from fungal and bacterial denitrification. Uniquely, soil NO3− measurements were also made throughout the incubation. Emissions of N2O during the initial phase of the experiment (0–13 days) increased almost linearly with increasing rate of straw incorporation and with (almost) no N2 production. However, the rate of straw amendment was negatively correlated with N2O, but positively correlated with N2 fluxes later in the experimental period (13–30 days). Soil NO3− content, in all treatments, was identified as the main factor responsible for the shift from N2O production to N2O reduction. Straw amendment immediately lowered the proportion of N2O from bacterial denitrification, thus implying that more of the N2O emitted was derived from fungi (18 ± 0.7% in control and up to 40 ± 3.0% in high straw treatments during the first 13 days). However, after day 15 when soil NO3− content decreased to <40 mg NO3−-N kg−1 soil, the N2O 15N site preference values of the N2O produced in the medium straw rate treatment showed a sharp declining trend 15 days after onset of experiment thereby indicating a clear shift towards a more dominant bacterial source of N2O. Our study singularly highlights the complex interrelationship between soil NO3− kinetics, crop residue incorporation, fungal denitrification and N2O/(N2O + N2) ratio. Overall we found that the effect of crop residue applications on soil N2O and N2 emissions depends mainly on soil NO3− content, as NO3− was the primary regulator of the N2O/(N2O + N2) product ratio of denitrification. Furthermore, the application of straw residue enhanced fungal denitrification, but only when the soil NO3− content was sufficient to supply enough electron acceptors to the denitrifiers

Effects of grass species and grass growth on atmospheric nitrogen deposition to a bog ecosystem surrounded by intensive agricultural land use

We applied a N-15 dilution technique called Integrated Total Nitrogen Input (ITNI) to quantify annual atmospheric N input into a peatland surrounded by intensive agricultural practices over a 2-year period. Grass species and grass growth effects on atmospheric N deposition were investigated using Lolium multiflorum and Eriophorum vaginatum and different levels of added N resulting in increased biomass production. Plant biomass production was positively correlated with atmospheric N uptake (up to 102.7mg N pot(-1)) when using Lolium multiflorum. In contrast, atmospheric N deposition to Eriophorum vaginatum did not show a clear dependency to produced biomass and ranged from 81.9 to 138.2mgNpot(-1). Both species revealed a relationship between atmospheric N input and total biomass N contents. Airborne N deposition varied from about 24 to 55kgNha(-1)yr(-1). Partitioning of airborne N within the monitor system differed such that most of the deposited N was found in roots of Eriophorum vaginatum while the highest share was allocated in aboveground biomass of Lolium multiflorum. Compared to other approaches determining atmospheric N deposition, ITNI showed highest airborne N input and an up to fivefold exceedance of the ecosystem-specific critical load of 5-10kgNha(-1)yr(-1).Peer reviewe

Anteil von Pilzen und Bakterien an der Lachgasbildung in verschiedenen Böden

Welchen Anteil Pilze an den N2O-Emissionen aus der Denitrifikation haben, ist bisher noch nicht hinreichend untersucht worden. Während der pilzlichen Denitrifikation findet meistens keine N2O-Reduktion statt, so dass N2O das Endprodukt darstellt. Somit könnte der pilzliche N2O-Anteil aus der Denitrifikation höher ausfallen, als bisher vermutet wird. Reinkulturversuche ergaben, dass das N2O von Bakterien und Pilzen eine unterschiedliche 15N-Positionspräferenz aufweist. Erste Ergebnisse aus Inkubationsversuchen mit Bodenproben und selektiver Hemmung konnten die deutlich positive 15N-Positionspräferenz des pilzlichen N2O aus den Reinkulturen nicht zeigen. Die Ergebnisse verdeutlichen, dass weitere Versuche notwendig sind, um ein besseres Verständnis über die N2O-Bildungsprozesse zu erlangen

Emissionen von grundwasserbürtigem N2O in die Atmosphäre: Modellrechnungen zu einem 15N-Tracerversuch unter Feldbedingungen

In einem Tracerversuch mit isotopisch mar-kiertem Nitrat wurde unter Feldbedingungen (Gley-Podsol aus Talsand, darunter ein Lockergesteinsaquifer) die Denitrifikation im oberflächennahen Grundwasser und die daraus resultierende Emission von N2O in die Atmosphäre über einen Zeitraum von Juli bis September 2007 verfolgt. Die Messergebnisse dienten als Anfangs- und Randbedingungen und Vergleichswerte für Simulationsrechnungen zum Transport von N2O durch den Boden zur Atmosphäre mit einem numerischen Gasdiffusionsmodell. Die Ergebnisse der Simulationsrechnungen bestätigen, dass Diffusion der wesentliche Prozess für den N2O-Transport in dem untersuchten Boden war. Für die geringen grundwasserbürtigen N2O-Emissionen spielte N2O-Abbau im Boden keine Rolle. Wir schlussfolgern aus den Ergebnissen, dass die Simulationsrechnungen eine wertvolle Ergänzung zur experimentellen Datenbasis darstellen. Sie verdeutlichen u.a., dass die N2O-Emission am Versuchsstandort hauptsächlich durch N2O-Umsetzungen im Oberboden gesteuert wurde