N2O Site Preference Research Articles

Acidification Offset Warming-Induced Increase in N2O Production in Estuarine and Coastal Sediments.

Global warming and acidification, induced by a substantial increase in anthropogenic CO2 emissions, are expected to have profound impacts on biogeochemical cycles. However, underlying mechanisms of nitrous oxide (N2O) production in estuarine and coastal sediments remain rarely constrained under warming and acidification. Here, the responses of sediment N2O production pathways to warming and acidification were examined using a series of anoxic incubation experiments. Denitrification and N2O production were largely stimulated by the warming, while N2O production decreased under the acidification as well as the denitrification rate and electron transfer efficiency. Compared to warming alone, the combination of warming and acidification decreased N2O production by 26 ± 4%, which was mainly attributed to the decline of the N2O yield by fungal denitrification. Fungal denitrification was mainly responsible for N2O production under the warming condition, while bacterial denitrification predominated N2O production under the acidification condition. The reduced site preference of N2O under acidification reflects that the dominant pathways of N2O production were likely shifted from fungal to bacterial denitrification. In addition, acidification decreased the diversity and abundance of nirS-type denitrifiers, which were the keystone taxa mediating the low N2O production. Collectively, acidification can decrease sediment N2O yield through shifting the responsible production pathways, partly counteracting the warming-induced increase in N2O emissions, further reducing the positive climate warming feedback loop.

Isotopic and geochemical modeling approach to evaluate abiotic nitrite reduction by ferrous iron

Nitrite reduction has often been treated as a biotic process in water treatment systems, but it also occurs abiotically, and it is difficult to distinguish both reactions as they can co-occur at field-scale. The potential reduction of NO2− was tested in 3 anaerobic experimental scenarios using a NO2− bearing solution amended with: i) siderite (FeCO3) (Sid experiment); ii) Fe(II) solution from FeCl2.4H2O(s) (DFe experiment); and iii) siderite mixed with Fe(II) solution (Sid+DFe experiment). Non-sterilized batch experiments were carried out in anaerobic conditions with an initial ratio of nitrogen to dissolved iron of 5 in DFe and Sid+DFe (Sid had no initially dissolved Fe(II)) and 1000 mg L−1 of siderite in Sid and Sid+DFe experiments. At the end of the experiments, the NO2− removed was 3% for Sid, 54% for DFe and 84% for Sid+DFe. The NO2− concentration decrease over time was characterized by an enrichment in the δ15NNO2 of the unreacted NO2−, increasing from −26.9 ‰ to −26.4 ‰ (Sid), −18.0 ‰ (DFe) and −15.9 ‰ (Sid+DFe). The calculated ε15NNO2 for Sid was −11.8 ‰, whereas for DFe was −12.0 ‰ and Sid+DFe was −13.0 ‰, suggesting a common NO2− degradation mechanism in all experiments. The Rayleigh distillation equation showed that the generated N2O was the final product of the abiotic nitrite reduction reaction, and the calculated N2O site preference (SP) was 22.5 ± 0.7 ‰ for DFe and 23.5 ± 0.5 ‰ for Sid+DFe. The continuous N2O measurement showed that only 25.3 ± 5.1% in DFe and 31.0 ± 6.3% in Sid+DFe of the generated N2O in water was recovered in the headspace vials, suggesting that a large portion of the produced N2O(aq) in solution did not diffuse from water. The coupled NO2− reduction and Fe(II) oxidation followed a second-order kinetic reaction with a rate equal to (9.39 ± 0.36)·10−4·[NO2−]·[Fe(II)] (mol L−1 s−1) in all experiments. The experimental conditions supported by the Rayleigh distillation equation using the experimentally calculated ε15N values, coupled with NO2− isotopic data and N2O SP values, showed that biological denitrification had a negligible influence on nitrite reduction and that chemodenitrification was the main NO2− attenuation pathway. A geochemical model coupling the kinetic chemodenitrification, isotope fractionation, aqueous speciation in equilibrium and precipitation and dissolution of calcite has been implemented and reproduced the experimental results. The geochemical model developed in our study can be applied to similar experimental studies and to field-scale studies to predict the efficiency of abiotic nitrite reduction treatments using Fe(II).

Grazing exclusion alters denitrification N2O/(N2O + N2) ratio in alpine meadow of Qinghai–Tibet Plateau

Grazing exclusion has been implemented worldwide as a nature-based solution for restoring degraded grassland ecosystems that arise from overgrazing. However, the effect of grazing exclusion on soil nitrogen cycle processes, subsequent greenhouse gas emissions and underlying mechanisms remain unclear. Here, we investigated the effect of four-year grazing exclusion on plant communities, soil properties, and soil nitrogen cycle-related functional gene abundance in an alpine meadow on the Qinghai–Tibet Plateau. Using an automated continuous-flow incubation system, we performed an incubation experiment and measured soil-borne N2O, N2, and CO2 fluxes to three successive “hot moment” events (precipitation, N deposition, and oxic-to-anoxic transition) between grazing-excluded and grazing soil. Higher soil N contents (total nitrogen, NH4+, NO3−) and extracellular enzyme activities (β-1,4-glucosidase, β-1,4-N-acetyl-glucosaminidase, cellobiohydrolase) are observed under grazing exclusion. The aboveground and litter biomass of plant community was significantly increased by grazing exclusion, but grazing exclusion decreased the average number of plant species and microbial diversity. The N2O + N2 fluxes observed under grazing exclusion were higher than those observed under free grazing. The N2 emissions and N2O/(N2O + N2) ratios observed under grazing exclusion were higher than those observed under free grazing in oxic conditions. Instead, higher N2O fluxes and lower denitrification functional gene abundances (nirS, nirK, nosZ, and nirK + nirS) under anoxia were found under grazing exclusion than under free grazing. The N2O site-preference value indicates that under grazing exclusion, bacterial denitrification contributes more to higher N2O production compared with under free grazing (81.6 % vs. 59.9 %). We conclude that grazing exclusion could improve soil fertility and plant biomass, nevertheless it may lower plant and microbial diversity and increase potential N2O emission risk via the alteration of the denitrification end-product ratio. This indicates that not all grassland management options result in a mutually beneficial situation among wider environmental goals such as greenhouse gas mitigation, biodiversity, and social welfare.

Tracing Microbial Production and Consumption Sources of N2O in Rivers on the Qinghai-Tibet Plateau via Isotopocule and Functional Microbe Analyses

Nitrous oxide (N2O), a potent greenhouse gas, is produced in rivers through a series of microbial metabolic pathways. However, the microbial source of N2O production and the degree of N2O reduction in river systems are not well understood and quantified. This work investigated isotopic compositions (δ15N-N2O and δ18O-N2O) and N2O site preference as well as N2O-related microbial features, thereby differentiating the importance of nitrification, denitrification, and N2O reduction in controlling N2O emissions from five rivers on the eastern Qinghai-Tibet Plateau (EQTP). The average N2O concentration in overlying water (15.2 nmol L-1) was close to that in porewater (17.5 nmol L-1), suggesting that both overlying water and sediment are potentially important sources of N2O. Canonical and nitrifier denitrification dominated riverine N2O production, with contribution being approximately 90%. Nitrification is a non-negligible source of N2O production, and N2O concentration was positively correlated with nitrification genetic potential. The degree of N2O reduction ranged from 78.1 to 94.1% (averaging 90%), significantly exceeding the reported values (averaging 70%) in other freshwaters, which was attributed to the higher ratios of organic carbon to nitrogen and lower ratio of (nirS + nirK)/nosZ in EQTP rivers. This study indicates that a combination of isotopic and isotopocule values with functional microbe analysis is useful for quantifying the microbial sources of N2O in rivers, and the intense microbial reduction of N2O significantly accounts for the low N2O emissions observed in EQTP rivers, suggesting that both the production and consumption of N2O in rivers should be considered in the future.

Nitrous oxide production and isotopomer composition by fungi isolated from salt marsh sediments

The emissions of nitrous oxide (N2O), a potent greenhouse gas and ozone-depleting agent, have been steadily increasing from coastal environments, such as salt marsh sediments, as a result of anthropogenic nutrient loading. Biotic processes, including nitrification and denitrification, are the largest sources of N2O from salt marsh sediments. While it is assumed that the bulk of N2O from salt marsh sediment is produced by nitrification and bacterial denitrification, recent reports suggest fungal denitrification may contribute significantly. In this study, four fungi capable of growth under sulfidic conditions were isolated from salt marsh sediments in North Inlet, South Carolina, USA. Fungal species included Purpureocillium lilacinum, Trichoderma harzianum, Trichoderma virens, and Rhodotorula glutinis, as determined by sequencing the18S and 28S rRNA genes. The isotopomer signatures of N2O produced by these fungi were measured using isotope ratio mass spectrometry, which can be used to estimate the contribution of different sources of N2O. Up to 22.8% of nitrite provided in growth media was converted to N2O by fungal strains isolated from salt marsh sediments. The site preference (SP) of N2O produced by salt marsh sediment fungi ranged from 7.5 ± 1.6‰ to 33.4 ± 1.2‰. These values are lower than the SP of N2O from the model fungal denitrifier Fusarium oxysporum (37.1 ± 2.5‰), which is the SP typically used as an endmember in isotope mass balance considerations. The N2O SP values we measured expand the range of N2O SP used for isotope mass balances calculations to determine the relative contribution of fungi to N2O production in salt marsh sediments.

Versatile soil gas concentration and isotope monitoring: optimization and integration of novel soil gas probes with online trace gas detection

Abstract. Gas concentrations and isotopic signatures can unveil microbial metabolisms and their responses to environmental changes in soil. Currently, few methods measure in situ soil trace gases such as the products of nitrogen and carbon cycling or volatile organic compounds (VOCs) that constrain microbial biochemical processes like nitrification, methanogenesis, respiration, and microbial communication. Versatile trace gas sampling systems that integrate soil probes with sensitive trace gas analyzers could fill this gap with in situ soil gas measurements that resolve spatial (centimeters) and temporal (minutes) patterns. We developed a system that integrates new porous and hydrophobic sintered polytetrafluoroethylene (sPTFE) diffusive soil gas probes that non-disruptively collect soil gas samples with a transfer system to direct gas from multiple probes to one or more central gas analyzer(s) such as laser and mass spectrometers. Here, we demonstrate the feasibility and versatility of this automated multiprobe system for soil gas measurements of isotopic ratios of nitrous oxide (δ18O, δ15N, and the 15N site preference of N2O), methane, carbon dioxide (δ13C), and VOCs. First, we used an inert silica matrix to challenge probe measurements under controlled gas conditions. By changing and controlling system flow parameters, including the probe flow rate, we optimized recovery of representative soil gas samples while reducing sampling artifacts on subsurface concentrations. Second, we used this system to provide a real-time window into the impact of environmental manipulation of irrigation and soil redox conditions on in situ N2O and VOC concentrations. Moreover, to reveal the dynamics in the stable isotope ratios of N2O (i.e., 14N14N16O, 14N15N16O, 15N14N16O, and 14N14N18O), we developed a new high-precision laser spectrometer with a reduced sample volume demand. Our integrated system – a tunable infrared laser direct absorption spectrometry (TILDAS) in parallel with Vocus proton transfer reaction mass spectrometry (PTR-MS), in line with sPTFE soil gas probes – successfully quantified isotopic signatures for N2O, CO2, and VOCs in real time as responses to changes in the dry–wetting cycle and redox conditions. Broadening the collection of trace gases that can be monitored in the subsurface is critical for monitoring biogeochemical cycles, ecosystem health, and management practices at scales relevant to the soil system.

Comparing modified substrate-induced respiration with selective inhibition (SIRIN) and N<sub>2</sub>O isotope approaches to estimate fungal contribution to denitrification in three arable soils under anoxic conditions

Abstract. The coexistence of many N2O production pathways in soil hampers differentiation of microbial pathways. The question of whether fungi are significant contributors to soil emissions of the greenhouse gas nitrous oxide (N2O) from denitrification has not yet been resolved. Here, three approaches to independently investigate the fungal fraction contributing to N2O from denitrification were used simultaneously for, as far as we know, the first time (modified substrate-induced respiration with selective inhibition (SIRIN) approach and two isotopic approaches, i.e. end-member mixing approach (IEM) using the 15N site preference of N2O produced (SPN2O) and the SP/δ18O mapping approach (SP/δ18O Map)). This enabled a comparison of methods and a quantification of the importance of fungal denitrification in soil. Three soils were incubated in four treatments of the SIRIN approach under anaerobic conditions to promote denitrification. While one treatment without microbial inhibition served as a control, the other three treatments were amended with inhibitors to selectively inhibit bacterial, fungal, or bacterial and fungal growth. These treatments were performed in three variants. In one variant, the 15N tracer technique was used to estimate the effect of N2O reduction on the N2O produced, while two other variants were performed under natural isotopic conditions with and without acetylene. All three approaches revealed a small contribution of fungal denitrification to N2O fluxes (fFD) under anaerobic conditions in the soils tested. Quantifying the fungal fraction with modified SIRIN was not successful due to large amounts of uninhibited N2O production. In only one soil could fFD be estimated using modified SIRIN, and this resulted in 28 ± 9 %, which was possibly an overestimation, since results obtained by IEM and SP/δ18O Map for this soil resulted in fFD of below 15 % and 20 %, respectively. As a consequence of the unsuccessful SIRIN approach, estimation of fungal SPN2O values was impossible. While all successful methods consistently suggested a small or missing fungal contribution, further studies with stimulated fungal N2O fluxes by adding fungal C substrates and an improved modified SIRIN approach, including alternative inhibitors, are needed to better cross-validate the methods.

Can N Fertilizer Addition Affect N2O Isotopocule Signatures for Soil N2O Source Partitioning?

Isotopocule signatures of N2O (δ15Nbulk, δ18O and site preference) are useful for discerning soil N2O source, but sometimes, N fertilizer is needed to ensure that there is enough N2O flux for accurate isotopocule measurements. However, whether fertilizer affects these measurements is unknown. This study evaluated a gradient of NH4NO3 addition on N2O productions and isotopocule values in two acidic subtropical soils. The results showed that N2O production rates obviously amplified with increasing NH4NO3 (p < 0.01), although a lower N2O production rate and an increasing extent appeared in forest soil. The δ15Nbulk of N2O produced in forest soil was progressively enriched when more NH4NO3 was added, while becoming more depleted of agricultural soil. Moreover, the N2O site preference (SP) values collectively elevated with increasing NH4NO3 in both soils, indicating that N2O contributions changed. The increased N2O production in agricultural soil was predominantly due to the added NH4NO3 via autotrophic nitrification and fungal denitrification (beyond 50%), which significantly increased with added NH4NO3, whereas soil organic nitrogen contributed most to N2O production in forest soil, probably via heterotrophic nitrification. Lacking the characteristic SP of heterotrophic nitrification, its N2O contribution change cannot be accurately identified yet. Overall, N fertilizer should be applied strictly according to the field application rate or N deposition amount when using isotopocule signatures to estimate soil N2O processes.

Microbial potential for denitrification in the hyperarid Atacama Desert soils

The hyperarid soils of the Atacama Desert, Chile, contain the largest known nitrate deposits in the world. They also represent one of the most hostile environments for microbial life anywhere in the terrestrial biosphere. Despite known for its extreme dryness, several heavy rainfall events causing localised flash flooding have struck Atacama Desert core regions during the last five years. It remains unclear, however, whether these soils can support microbial denitrification. To answer this, we sampled soils along a hyperaridity gradient in the Atacama Desert and conducted incubation experiments using a robotized continuous flow system under a He/O2 atmosphere. The impacts of four successive extreme weather events on soil-borne N2O and N2 emissions were investigated, i) water addition, ii) NO3− addition, iii) labile carbon (C) addition, and iv) oxygen depletion. The 15N–N2O site-preference (SP) approach was further used to examine the source of N2O produced. Extremely low N2O fluxes were detected shortly after water and NO3− addition, whereas pronounced N2O and N2 emissions were recorded after labile-C (glucose) amendment in all soils. Under anoxia, N2 emissions increased drastically while N2O emissions decreased concomitantly, indicating the potential for complete denitrification at all sites. Although increasing aridity significantly reduced soil bacterial richness, microbial potential for denitrification and associated gene abundance (i.e., napA, narG, nirS, nirK, cnorB, qnorB and nosZ) was not affected. The N2O15N site preference values based on two end-member model suggested that fungal and bacterial denitrification co-contributed to N2O production in less arid sites, whereas bacterial denitrification dominated with increasing aridity. We conclude that soil denitrification functionality is preserved even with lowered microbial richness in the extreme hyperarid Atacama Desert. Future changes in land-use or extreme climate events therefore have a potential to destabilize the immense reserves of nitrate and induce significant N2O losses in the region.

Primings of soil organic matter and denitrification mediate the effects of moisture on nitrous oxide production

Nitrogen-fertilized agricultural soils are major emitters of the potent greenhouse gas nitrous oxide (N2O). Although soil moisture is known to drive N2O production, the influence of moisture on the priming effects of labile N additions, which alter the N2O production from native soil organic matter (SOM), remains understudied. We investigated how moisture impacts N2O primings, nitrification kinetics, and allocation of N2O sources between nitrification and denitrification. We evaluated urea additions at five water-filled pore spaces (WFPS): 31, 41, 53, 65, and 78%. Based on δ15N–N2O site preference (SP), the denitrification source (encompassing bacterial- and nitrifier-denitrification) amply dominated across all moistures. At moistures >60% WFPS, SP exhibited a narrow range of 1.2–1.6‰, whereas at 31% WFPS, SP rose to 9.4‰ (P < 0.001). These SPs translated into denitrification sourcing consistently four-fifth of the N2O production in the wettest soils. With drier soils, denitrification gradually decreased, but it still produced three-fifth of the N2O even at the driest soil (31% WFPS). Based on time courses of ammonium and nitrate concentrations, first-order kinetics (k1) modelling showed much slower nitrification rates at 31% WFPS (0.1 day−1) than the peak nitrification witnessed consistently at both 65 and 78% WFPSs (1.0 day−1). By using isotopically-labelled urea, we further allocated N2O production into SOM-N and urea-N sources. In soils receiving urea, SOM-N contribution to N2O production changed from being 77% in the driest soils to 46% in the wettest soil. Along this aeration-to-moisture gradient, net N2O priming steadily switched from being negative (−20%) in the driest soils to become positive (+14%) at the wettest soil, and this shifting trajectory intercepted the neutral 0% priming at 60% WFPS. Results indicate that pulse N2O emissions commonly reported in recently N-fertilized fields are triggered by soil moisture surpassing this threshold, which collectively accelerates positive primings of SOM and denitrification.

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.

Sources and priming of soil N2O and CO2 production: Nitrogen and simulated exudate additions

Identifying the sources of nitrous oxide (N2O) and carbon dioxide (CO2) production from soil is central to enhancing the understanding and prediction of these emissions to the atmosphere. The magnitude of N2O and CO2 production derived from soil organic matter (SOM) can asymmetrically change due to stimuli from root exudation and nitrogen additions ‒ a response termed the priming effect. We conducted an incubation to examine the effects of N and artificial root exudate (ARE) additions on the priming of SOM. We also evaluated the changes in N2O production from nitrification and denitrification by measuring 15N–N2O site preference (SP). ARE consisted of a mixture of 99 atom% 13C labelled compounds at three rates (0, 6.2, 12.5 mg C kg−1 soil day−1) applied daily to microcosms with or without urea, a subset of which was also labelled with 5 atom% 15N. Additions of ARE or urea alone caused positive priming effects; however, addition of ARE and urea concurrently resulted in an antagonistic interactive effect that diminished the N2O production derived from SOM mineralization (P < 0.05). Moreover, CO2 production from SOM decreased in urea-treated microcosms (P < 0.01) such that all soils receiving ARE and urea exhibited reduced positive priming relative to their unfertilized counterparts. Based on SP results, the contributions of denitrification and nitrification to total N2O production were both amplified due to the combined inputs of ARE and urea compared to the untreated control (49.9 ± 10.1 and 28.3 ± 8.0 μg N2O–N kg−1, respectively). In soils receiving only ARE, N2O derived from denitrification decreased relative to a control, thus reducing overall N2O production (−9.5 ± 12.3 μg N2O–N kg−1); conversely, nitrification-derived N2O was differentially augmented (+17.2 ± 9.0 μg N2O–N kg−1). Results indicate that a combination of elevated root exudation with N fertilization has the potential to asymmetrically amplify N2O emissions due to increases in both nitrification and denitrification sources.

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

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.

Straw amendment with nitrate-N decreased N2O/(N2O+N2) ratio but increased soil N2O emission: A case study of direct soil-born N2 measurements

Straw application in combination with synthetic N fertilizer could increase crop yield and improve soil fertility, however, contradictory observations have been reported on the effects of straw addition on soil N2O emission. Straw application can affect both denitrification rate and its product stoichiometry (N2O/(N2O + N2) ratio), whereas the latter remains rather unclear since the ratio is strongly regulated by other soil parameters, e.g. nitrate and oxygen concentrations at denitrifying micro-sites. In this context, we conducted an incubation experiment with a robotized continuous flow incubation system using a He/O2 atmosphere and measured N2O and direct N2 fluxes over 22 days. Soil amended with and without rice straw (2.5 g kg−1 soil) in conjunction with nitrate fertilizer (10 mM KNO3) and non-amended control soil were incubated under 85% water-filled pore space. To simulate a short soil anoxic period, three different O2 partial pressures phases were set (20%, 5% and 10%). Additionally, N2O site preference signatures of soil-emitted N2O were analyzed to identify the processes contributing to N2O fluxes. Addition of nitrate increased cumulative N2O fluxes and decreased cumulative N2 fluxes compared with non-fertilized control, while rice straw amendment increased both N2O and N2 emissions drastically compared with the nitrate only treatment. The N2O SP values ranged from 0.4 to 2.7‰ among all treatments, indicating denitrification/nitrifier denitrification was the dominating source. The results suggest that straw amendment can trigger high denitrification rate, whereas the effect of straw amendment on the amount of emitted N2O and the N2O/(N2O + N2) product ratio strongly depends on soil NO3− concentration. As a conclusion, the present study suggests that straw amendment in conjunction with nitrate-N can increase soil N2O emissions under conditions favoring denitrification, even though it may decrease the overall N2O/(N2O + N2) product ratio.

Development of a field-deployable method for simultaneous, real-time measurements of the four most abundant N2O isotopocules

ABSTRACTUnderstanding and quantifying the biogeochemical cycle of N2O is essential to develop effective N2O emission mitigation strategies. This study presents a novel, fully automated measurement technique that allows simultaneous, high-precision quantification of the four main N2O isotopocules (14N14N16O, 14N15N16O, 15N14N16O and 14N14N18O) in ambient air. The instrumentation consists of a trace gas extractor (TREX) coupled to a quantum cascade laser absorption spectrometer, designed for autonomous operation at remote measurement sites. The main advantages this system has over its predecessors are a compact spectrometer design with improved temperature control and a more compact and powerful TREX device. The adopted TREX device enhances the flexibility of the preconcentration technique for higher adsorption volumes to target rare isotope species and lower adsorption temperatures for highly volatile substances. All system components have been integrated into a standardized instrument rack to improve portability and accessibility for maintenance. With an average sampling frequency of approximately 1 h–1, this instrumentation achieves a repeatability of 0.09, 0.13, 0.17 and 0.12 ‰ for δ15Nα, δ15Nβ, δ18O and site preference of N2O, respectively, for pressurized ambient air. The repeatability for N2O mole fraction measurements is better than 1 ppb (parts per billion, 10–9 moles per mole of dry air).

N2O and NOx emissions by reactions of nitrite with soil organic matter of a Norway spruce forest

Nitrite (NO2 −) as an important intermediate of the biological nitrogen cycle is particularly reactive in acidic soils and acts as a source of N2O and NOx (NO and NO2). However, abiotic and biotic pathways of NO2 −-driven N2O and NOx production in forest soil and the role of soil organic matter (SOM) in these processes are still unclear. In this study, NO2 − was applied to both unsterile and sterilized soil samples as well as to different SOM fractions from a Norway spruce forest. Biotic and abiotic N2O emission was measured with an infrared absorption analyzer and gas chromatography, while NOx emission was quantified with a chemiluminescence analyzer. Isotopic signatures of N2O (δ15Nbulk, δ18O, and 15N-N2O site preference) were analyzed with an isotope ratio mass spectrometer. After NO2 − addition, a large amount of NOx was emitted immediately, while N2O emission occurred 15–60 min later and was much lower compared to NOx. Sterilization of soil decreased N2O emission significantly, but not NOx emission. The 15N site preference of N2O ranged from 7.98 to 11.58‰ for abiotic and 4.69–7.42‰ for biotic sources. The fulvic acid fraction contributed the most to abiotic N2O emission, while the fastest NO and N2O emission occurred after NO2 −application to the humin fraction, followed by the humic acid fraction. These results are important for the future understanding of NOx and N2O sources, as well as the use of isotopic signatures for source-partitioning N2O emission from soil.

Ecosystem metabolism and greenhouse gas production in a mesotrophic northern temperate lake experiencing seasonal hypoxia

Many lacustrine systems, despite management efforts to control eutrophication, are hypoxic during stratified periods. Hypoxia is a major concern, not only for its impact on aquatic life but also for its potential to stimulate production of the greenhouse gases, methane (CH4) and nitrous oxide (N2O). We investigated the drivers of hypoxia in Muskegon Lake, a temperate dimictic freshwater estuary that experiences frequent hypolimnetic mixing due to atmospheric forces, riverine inputs, and intrusion of oxic water from coastal upwelling in Lake Michigan. Primary production and respiration (R) rates obtained from a δ18O mass balance model were similar to other mesotrophic environments (0.56–26.31 and 0.57–13.15 mmol O2 m−3 day−1, respectively), although high P/R (≥2 in mid-summer) indicated there is sufficient autochthonous production to support hypoxia development and persistence. The isotopic enrichment factor for respiration (eobs) varied markedly and was least negative in August of both sampling years, consistent with high R rates. Hypoxic conditions were associated with accumulation of N2O but not CH4, and emissions of N2O are among the highest reported from lakes. The average N2O site preference value of 25.4‰ indicates that the majority of N2O was produced by nitrification via hydroxylamine oxidation, despite the presence of resilient hypoxia. While it has been hypothesized that denitrification acts as a sink for N2O in hypoxic lakes, it is clear that Muskegon Lake functions as a strong source of N2O via nitrification. Further considerations of lakes as global sources of N2O thus warrant a closer evaluation of nitrification-fueled N2O production.

Probing the biological sources of soil N2O emissions by quantum cascade laser-based 15N isotopocule analysis

Isotopocule analysis by quantum cascade laser spectrometry (QCL) is a promising approach for in situ, real-time tracking of the biological sources of soil N2O emissions. However, background atmospheric N2O is an important source of variability in the measurement of isotopocule ratios (i.e., 14N15N16O/14N14N16O and 15N14N16O/14N14N16O) of gas samples. Here, a method based on Keeling plot for determining the intramolecular 15N distribution in N2O is introduced. The sensitivity and reliability of this method are examined against N2O of known 15N site preference (SP), and N2O produced from fungal and bacterial isolates, soils with different moisture contents and organic amendments, and a soil chamber under field conditions. The isotopocules of N2O determined by standard gases supported that the Keeling plot method was more reliable than the averaging method. Using this method, SP of N2O was greater in fungal than bacterial denitrifiers, as well as in soil at 60% water filled pore space (WFPS) than 80–100% WFPS. This method also determined that the SP of N2O was distinct between soils of different substrate complexity. Further, we observed a N2O SP between −18.9‰ and 2.2% in a soil flux chamber deployed in a corn field after 2 d of rainfalls that is consistent with the SP of N2O produced from bacterial denitrification and nitrifier denitrification. Our data demonstrate that this Keeling plot method provides accurate discrimination of biological sources when N2O is analyzed by the QCL system.

N2O source partitioning in soils using (15)N site preference values corrected for the N2O reduction effect.

The aim of this study was to determine the impact of isotope fractionation associated with N2O reduction during soil denitrification on N2O site preference (SP) values and hence quantify the potential bias on SP-based N2O source partitioning. The N2O SP values (n = 431) were derived from six soil incubation studies in N2-free atmosphere, and determined by isotope ratio mass spectrometry (IRMS). The N2 and N2O concentrations were measured directly by gas chromatography. Net isotope effects (NIE) during N2O reduction to N2 were compensated for using three different approaches: a closed-system model, an open-system model and a dynamic apparent NIE function. The resulting SP values were used for N2O source partitioning based on a two end-member isotopic mass balance. The average SP0 value, i.e. the average SP values of N2O prior to N2O reduction, was recalculated with the closed-system model, resulting in -2.6 ‰ (±9.5), while the open-system model and the dynamic apparent NIE model gave average SP0 values of 2.9 ‰ (±6.3) and 1.7 ‰ (±6.3), respectively. The average source contribution of N2O from nitrification/fungal denitrification was 18.7% (±21.0) according to the closed-system model, while the open-system model and the dynamic apparent NIE function resulted in values of 31.0% (±14.0) and 28.3% (±14.0), respectively. Using a closed-system model with a fixed SP isotope effect may significantly overestimate the N2O reduction effect on SP values, especially when N2O reduction rates are high. This is probably due to soil inhomogeneity and can be compensated for by the application of a dynamic apparent NIE function, which takes the variable reduction rates in soil micropores into account.

Stable isotopes and iron oxide mineral products as markers of chemodenitrification.

When oxygen is limiting in soils and sediments, microorganisms utilize nitrate (NO3-) in respiration--through the process of denitrification--leading to the production of dinitrogen (N2) gas and trace amounts of nitrous (N2O) and nitric (NO) oxides. A chemical pathway involving reaction of ferrous iron (Fe2+) with nitrite (NO2-), an intermediate in the denitrification pathway, can also result in production of N2O. We examine the chemical reduction of NO2- by Fe(II)--chemodenitrification--in anoxic batch incubations at neutral pH. Aqueous Fe2+ and NO2- reacted rapidly, producing N2O and generating Fe(III) (hydr)oxide mineral products. Lepidocrotite and goethite, identified by synchrotron X-ray diffraction (XRD) and extended X-ray absorption fine structure (EXAFS) spectroscopy, were produced from initially aqueous reactants, with two-line ferrihydrite increasing in abundance later in the reaction sequence. Based on the similarity of apparent rate constants with different mineral catalysts, we propose that the chemodenitrification rate is insensitive to the type of Fe(III) (hydr)oxide. With stable isotope measurements, we reveal a narrow range of isotopic fractionation during NO2- reduction to N2O. The location of N isotopes in the linear N2O molecule, known as site preference, was also constrained to a signature range. The coexistence of Fe(III) (hydr)oxide, characteristic 15N and 18O fractionation, and N2O site preference may be used in combination to qualitatively distinguish between abiotic and biogenically emitted N2O--a finding important for determining N2O sources in natural systems.