Soil Formate Regulates the Fungal Nitrous Oxide Emission Pathway

Fungal activity is a major driver in the global nitrogen cycle, and mounting evidence suggests that fungal denitrification activity contributes significantly to soil emissions of the greenhouse gas nitrous oxide (N₂O). The metabolic pathway and oxygen requirement for fungal denitrification are different from those for bacterial denitrification. We hypothesized that the soil N₂O emission from fungi is formate and O₂ dependent and that land use and landforms could influence the proportion of N₂O coming from fungi. Using substrate-induced respiration inhibition under anaerobic and aerobic conditions in combination with ¹⁵N gas analysis, we found that formate and hypoxia (versus anaerobiosis) were essential for the fungal reduction of ¹⁵N-labeled nitrate to ¹⁵N₂O. As much as 65% of soil-emitted N₂O was attributable to fungi; however, this was found only in soils from water-accumulating landforms. From these results, we hypothesize that plant root exudates could affect N₂O production from fungi via the proposed formate-dependent pathway.     

Corn Yield and Soil Nitrous Oxide Emission under Different Fertilizer and Soil Management: A Three-Year Field Experiment in Middle Tennessee

Background

A three-year field experiment was conducted to examine the responses of corn yield and soil nitrous oxide (N₂O) emission to various management practices in middle Tennessee.

Methodology/Principal Findings

The management practices include no-tillage + regular applications of urea ammonium nitrate (NT-URAN); no-tillage + regular applications of URAN + denitrification inhibitor (NT-inhibitor); no-tillage + regular applications of URAN + biochar (NT-biochar); no-tillage + 20% applications of URAN + chicken litter (NT-litter), no-tillage + split applications of URAN (NT-split); and conventional tillage + regular applications of URAN as a control (CT-URAN). Fertilizer equivalent to 217 kg N ha^-1 was applied to each of the experimental plots. Results showed that no-tillage (NT-URAN) significantly increased corn yield by 28% over the conventional tillage (CT-URAN) due to soil water conservation. The management practices significantly altered soil N₂O emission, with the highest in the CT-URAN (0.48 mg N₂O m^-2 h^-1) and the lowest in the NT-inhibitor (0.20 mg N₂O m^-2 h^-1) and NT-biochar (0.16 mg N₂O m^-2 h^-1) treatments. Significant exponential relationships between soil N₂O emission and water filled pore space were revealed in all treatments. However, variations in soil N₂O emission among the treatments were positively correlated with the moisture sensitivity of soil N₂O emission that likely reflects an interactive effect between soil properties and WFPS.

Conclusion/Significance

Our results indicated that improved fertilizer and soil management have the potential to maintain highly productive corn yield while reducing greenhouse gas emissions.     

Investigation on Emission of Nitrous Oxide by Aseptic Soybean Plant

The emission of nitrous oxide is not only a way of nitrogen loss in ecological system of agriculture, it is also a contamination source of increasing nitrogen oxide concentration in the atmosphere. This paper reports the analysis of gas chromatographic determination of N2O on the soybean plant and its different four organs-root, stem, leaf and cotyledon under strict aseptic condition. The results showed ,hat they all are able to emit nitrous oxide.     

Emission of Methane and Nitrous Oxide by Australian Mangrove Ecosystems

Abstract: The fluxes of the greenhouse gases methane (CH₄) and nitrous oxide (N₂O) were measured in mangrove wetlands in Queensland, Australia, using the closed chamber technique. Large differences in the fluxes of both gases from different study sites were observed, which presumably depended on differences in substrate availability. CH₄ emission rates were in the range of 20 to 350 μg m^‐2 h^‐1, whereas N₂O fluxes were lower, amounting to ‐ 2 to 14 μg m^‐2 h^‐1. In general, the field sites with high substrate availability showed higher emissions than sites with poor nutrient supply. This assumption is supported by the observation of dramatically increased N₂O emissions (150 ‐ 400 μg m^‐2 h^‐1) if study sites were artificially fertilised with additional N. As expected, N fertilisation did not alter CH₄ fluxes during the period of investigation. In the present study, it was confirmed that the mangrove vegetation may play a role as a transport path for CH₄ and N₂O by facilitating diffusion out of the soil. Prop roots from Rhizophora stylosa emitted CH₄ and N₂O at rates of 2.6 and 3.3 μg m^‐2 root surface h^‐1, respectively, whereas the soil of this stand acted as a sink for CH₄. As a consequence, the ecosystem as a whole could constitute a CH₄ source despite CH₄ uptake by the soil. In contrast to prop roots, the presence of pneumatophores in Avicennia marina led to a significant increase in CH₄ emissions from mangrove soils, but did not enhance N₂O emissions. These findings indicate that mangrove ecosystems may be considered a significant source of N₂O and that anthropogenic nutrient input into these ecosystems will lead to enhanced source strengths. For an up‐scaling of greenhouse gas emissions from mangrove forests to a global scale, more information is needed, particularly on the significance of vegetation.     

Role of Microorganisms in Emission of Nitrous Oxide and Methane in Pulse Cultivated Soil Under Laboratory Incubation Condition

Soil from a pulse cultivated farmers land of Odisha, India, have been subjected to incubation studies for 40 consecutive days, to establish the impact of various nitrogenous fertilizers and water filled pore space (WFPS) on green house gas emission (N₂O & CH₄). C₂H₂ inhibition technique was followed to have a comprehensive understanding about the individual contribution of nitrifiers and denitrifiers towards the emission of N₂O. Nevertheless, low concentration of C₂H₂ (5 ml: flow rate 0.1 kg/cm²) is hypothesized to partially impede the metabolic pathways of denitrifying bacterial population, thus reducing the overall N₂O emission rate. Different soil parameters of the experimental soil such as moisture, total organic carbon, ammonium content and nitrate–nitrogen contents were measured at regular intervals. Application of external N-sources under different WFPS conditions revealed the diverse role played by the indigenous soil microorganism towards green house gas emission. Isolation of heterotrophic microorganisms (Pseudomonas) from the soil samples, further supported the fact that denitrification might be prevailing during specific conditions thus contributing to N₂O emission. Statistical analysis showed that WFPS was the most influential parameter affecting N₂O formation in soil in absence of an inhibitor like C₂H₂.     

Effects of Climate Change Drivers on Nitrous Oxide Fluxes in an Upland Temperate Grassland

Despite increasing interest in the patterns of trace gas emissions in terrestrial ecosystems, little is known about the impacts of climate change on nitrous oxide (N₂O) fluxes. The aim of this study was to determine the importance of the three main drivers of climate change (warming, summer drought, and elevated CO₂ concentrations) on N₂O fluxes from an extensively managed, upland grassland. Over a 2-year period, we monitored N₂O fluxes in an in situ ecosystem manipulation experiment simulating the climate predicted for the study area in 2080 (3.5°C temperature increase, 20% reduction in summer rainfall and atmospheric CO₂ levels of 600 ppm). N₂O fluxes showed significant seasonal and interannual variation irrespective of climate treatment, and were higher in summer and autumn compared with winter and spring. Overall, N₂O emissions showed a positive correlation with soil temperature and rainfall. Elevated temperature had a positive impact on mean annual N₂O fluxes but effects were only significant in 2007. Contrary to expectations, neither combined summer drought and warming nor the simultaneous application of elevated atmospheric CO₂ concentrations, summer drought and warming had any significant effect on annual N₂O fluxes. However, the maximum N₂O flux rates observed during the study occurred when elevated CO₂ was combined with warming and drought, suggesting the potential for important, short-term N₂O–N losses in enriched CO₂ environments. Taken together, our results suggest that the N₂O responses of temperate, extensively managed grasslands to future climate change scenarios may be primarily driven by temperature effects.     

Nitrous Oxide Production and Methane Oxidation by Different Ammonia-Oxidizing Bacteria

Ammonia-oxidizing bacteria (AOB) are thought to contribute significantly to N₂O production and methane oxidation in soils. Most of our knowledge derives from experiments with Nitrosomonas europaea, which appears to be of minor importance in most soils compared to Nitrosospira spp. We have conducted a comparative study of levels of aerobic N₂O production in six phylogenetically different Nitrosospira strains newly isolated from soils and in two N. europaea and Nitrosospira multiformis type strains. The fraction of oxidized ammonium released as N₂O during aerobic growth was remarkably constant (0.07 to 0.1%) for all the Nitrosospira strains, irrespective of the substrate supply (urea versus ammonium), the pH, or substrate limitation. N. europaea and Nitrosospira multiformis released similar fractions of N₂O when they were supplied with ample amounts of substrates, but the fractions rose sharply (to 1 to 5%) when they were restricted by a low pH or substrate limitation. Phosphate buffer (versus HEPES) doubled the N₂O release for all types of AOB. No detectable oxidation of atmospheric methane was detected. Calculations based on detection limits as well as data in the literature on CH₄ oxidation by AOB bacteria prove that none of the tested strains contribute significantly to the oxidation of atmospheric CH₄ in soils.     

Nitrous Oxide Emission Associated with Autotrophic Ammonium Oxidation in Acid Coniferous Forest Soil

Aerobic N₂O production was studied in nitrifying humus from urea-fertilized pine forest soil. Acetylene and nitrapyrin inhibited both NH₄⁺ oxidation and N₂O production, indicating that N₂O production was closely associated with autotrophic NH₄⁺ oxidation. N₂O production was enhanced by low soil pH; it was negligible above pH 4.7. When soil pH decreased from 4.7 to 4.1, the relative amount of N₂O-N produced from NH₄⁺-N oxidized increased exponentially to 20%. There was also some evidence that N₂O formation was stimulated by salts (potassium sulfate and sodium phosphates). The maximum rate of N₂O-N production was 0.17 μg of N₂O-N per g of soil per h. When humus was treated with NO₂⁻, N₂O evolved immediately, indicating chemical formation, but no N₂O was formed on the addition of NO₃⁻. The amount of N₂O-N evolved was 0.6 to 4.6% of NO₂⁻-N added. A high concentration of NO₂⁻ and low soil pH enhanced chemical production of N₂O. There was no accumulation of NO₂⁻ during nitrification. The calculations indicated that chemical formation of N₂O was not the main source of N₂O during NH₄⁺ oxidation. After the addition of inhibitors of NH₄⁺ oxidation the soils contained NO₃⁻, but no N₂O was produced. The results suggest that enhanced autotrophic NH₄⁺ oxidation is a potential source of N₂O in fertilized acid forest soil.     

Separate Nitrite, Nitric Oxide, and Nitrous Oxide Reducing Fractions from Pseudomonas perfectomarinus

Pseudomonas perfectomarinus was found to grow anaerobically at the expense of nitrate, nitrite, or nitrous oxide but not chlorate or nitric oxide. In several repetitive experiments, anaerobic incubation in culture media containing nitrate revealed that an average of 82% of the cells in aerobically grown populations were converted to the capacity for respiration of nitrate. Although they did not form colonies under these conditions, the bacteria synthesized the denitrifying enzymes within 3 hr in the absence of oxygen or another acceptable inorganic oxidant. This was demonstrated by the ability, after anaerobic incubation, of cells and of extracts to reduce nitrite, nitric oxide, and nitrous oxide to nitrogen. From crude extracts of cells grown on nitrate, nitrite, or nitrous oxide, separate complex fractions were obtained that utilized reduced nicotinamide adenine dinucleotide as the source of electrons for the reduction of (i) nitrite to nitric oxide, (ii) nitric oxide to nitrous oxide, and (iii) nitrous oxide to nitrogen. Gas chromatographic analyses revealed that each of these fractions reduced only one of the nitrogenous oxides.     

Assessing the Soil Carbon,Biomass Production,and Nitrous Oxide Emission Impact of Corn Stover Management for Bioenergy Feedstock Production Using DAYCENT

Harvesting crop residue needs to be managed to protect agroecosystem health and productivity. DAYCENT, a process-based modeling tool, may be suited to accommodate region-specific factors and provide regional predictions for a broad array of agroecosystem impacts associated with corn stover harvest. Grain yield, soil C, and N₂O emission data collected at Corn Stover Regional Partnership experimental sites were used to test DAYCENT performance modeling the impacts of corn stover removal. DAYCENT estimations of stover yields were correlated and reasonably accurate (adjusted r ²?=?0.53, slope?=?1.18, p?<<?0.001, intercept?=?0.36, p?=?0.11). Measured and simulated average grain yields across sites did not differ as a function of residue removal, but the model tended to underestimate average measured grain yields. Modeled and measured soil organic carbon (SOC) change for all sites were correlated (adjusted r ²?=?0.54, p?<<?0.001), but DAYCENT overestimated SOC loss with conventional tillage. Simulated and measured SOC change did not vary by residue removal rate. DAYCENT simulated annual N₂O flux more accurately at low rates (≤2-kg N₂O-N ha^?1 year^?1) but underestimated when emission rates were >3-kg N₂O-N ha^?1 year^?1. Overall, DAYCENT performed well at simulating stover yields and low N₂O emission rates, reasonably well when simulating the effects of management practices on average grain yields and SOC change, and poorly when estimating high N₂O emissions. These biases should be considered when DAYCENT is used as a decision support tool for recommending sustainable corn stover removal practices to advance bioenergy industry based on corn stover feedstock material.     

Nitrate Reductase and Nitrous Oxide Production by Fusarium oxysporum 11dn1 Under Aerobic and Anaerobic Conditions

The fungus Fusarium oxysporum 11dn1 was found to be able to grow and produce nitrous oxide on nitrate-containing medium in anaerobic conditions. The rate of nitrous oxide formation was three to six orders of magnitude lower than the rates of molecular nitrogen production by common denitrifying bacteria. Acetylene and ammonia did not affect the release of nitrous oxide release. It was shown that under anaerobic conditions fast increase of nitrate reductase activity occurred, caused by the synthesis of enzyme de novo and protein dephosphorylation. Reverse transfer of the mycelium to aerobic conditions led to a decline in nitrate reductase activity and stopped nitrous oxide production. The presence of two nitrate reductases was shown, which differed in molecular mass, location, temperature optima, and activity in nitrate- and ammonium-containing media. Two enzymes represent assimilatory and dissimilatory nitrate reductases, which are active in aerobic and anaerobic conditions, respectively. Received: 2 February 2000 / Accepted: 28 February 2000     

Gut-Associated Denitrification and In Vivo Emission of Nitrous Oxide by the Earthworm Families Megascolecidae and Lumbricidae in New Zealand

Previous studies have documented the capacity of European earthworms belonging to the family Lumbricidae to emit the greenhouse gas nitrous oxide (N₂O), an activity attributed primarily to the activation of ingested soil denitrifiers. To extend the information base to earthworms in the Southern Hemisphere, four species of earthworms in New Zealand were examined for gut-associated denitrification. Lumbricus rubellus and Aporrectodea rosea (introduced species of Lumbricidae) emitted N₂O, whereas emission of N₂O by Octolasion cyaneum (an introduced species of Lumbricidae) and emission of N₂O by Octochaetus multiporus (a native species of Megascolecidae) were variable and negligible, respectively. Exposing earthworms to nitrite or nitrate and acetylene significantly increased the amount of N₂O emitted, implicating denitrification as the primary source of N₂O and indicating that earthworms emitted dinitrogen (N₂) in addition to N₂O. The alimentary canal displayed a high capacity to produce N₂O when it was supplemented with nitrite, and alimentary canal contents contained large amounts of carbohydrates and organic acids indicative of fermentation (e.g., succinate, acetate, and formate) that could serve as sources of reductant for denitrification. nosZ encodes a portion of the terminal oxidoreductase used in denitrification. The nosZ sequences detected in the alimentary canals of L. rubellus and O. multiporus were similar to those retrieved from soil and were distantly related to sequences of uncultured soil bacteria and genera common in soils (i.e., Bradyrhizobium, Azospirillum, Rhodopseudomonas, Rhodospirillum, Pseudomonas, Oligotropha, and Sinorhizobium). These findings (i) suggest that the capacity to emit N₂O and N₂ is a general trait of earthworms and not geographically restricted, (ii) indicate that species belonging to different earthworm families (i.e., Megascolecidae and Lumbricidae) may not have equal capacities to emit N₂O, and (iii) also corroborate previous findings that link this capacity to denitrification in the alimentary canal.Earthworms are dominant members of the soil fauna and affect the structure and fertility of soils (5, 20, 22, 23). Various species of European earthworms belonging to the family Lumbricidae (e.g., Aporrectodea caliginosa, Lumbricus rubellus, and Octolasion lacteum) emit dinitrogen (N₂) and the greenhouse gas nitrous oxide (N₂O), and their burrowing activities and feeding habits in combination with in situ conditions can influence the emission of nitrogenous gases from soils that they inhabit (1, 2, 13, 17, 25, 27, 39).The microbiology of the earthworm alimentary canal has been addressed in numerous studies (3, 4, 6, 9, 14, 16, 32). The alimentary canal of the earthworm is anoxic, in marked contrast to the aerated material that earthworms ingest (14, 39). Anoxia and other in situ conditions of the alimentary canal appear to stimulate soil microbes capable of surviving under anaerobic conditions during passage through the gut (3, 4). Soils are rich in denitrifying bacteria (37), and the capacity of European earthworms to emit nitrogenous gases has been attributed primarily to the in situ activity of ingested denitrifying bacteria that appear to be highly active under the anoxic conditions of the earthworm alimentary canal (12, 15, 17, 25, 39). However, it is not known if the capacity to emit nitrogenous gases is a general trait of earthworms independent of their taxonomic family or geographic location. The main objectives of this study were to examine the capacity of Southern Hemisphere earthworms in New Zealand to emit N₂O and to determine if this capacity was linked to denitrifying bacteria in the alimentary canal.     

Temporal Change in Nitrous Oxide and Dinitrogen from Denitrification Following Onset of Anaerobiosis

Similar temporal patterns were found in three mineral soils for the composition of the gaseous products of denitrification following the onset of anaerobic conditions. During the early period of anaerobiosis (0 up to 1 to 3 h), N₂ was the dominant product of denitrification. The NO₃⁻ → N₂O activity then increased, but was not accompanied by a corresponding increase in N₂O-reducing activity. This resulted in a relatively extended period of time (1 to 3 up to 16 to 33 h) during which N₂O was a major product. Eventually (after 16 to 33 h), an increase in N₂O-reducing activity occurred without a comparable increase in the N₂O-producing activity. The increase in the rate of N₂O reduction did not occur in the presence of chloramphenicol and required the presence of N₂O or NO₃⁻ during the preceding anaerobic incubation. During the final period (16 to 33, up to 48 h), N₂ was generally the sole product of denitrification, since the rate of N₂O reduction exceeded the rate of N₂O production. A similar sequential pattern was also found for a culture of a denitrifying Flavobacterium sp. shifted to anaerobic growth. A staggered synthesis of the enzymes in the denitrification sequence apparently occurred in response to anoxia, which caused first a net production of N₂O followed by consumption of N₂O.     

Clinical Trials of Different Concentrations of Oxygen and Nitrous Oxide for Obstetric Analgesia: Report to the Medical Research Council of the Committee on Nitrous Oxide and Oxygen Analgesia in Midwifery

Trials have been organized by a Medical Research Council committee to assess the effectiveness and safety for analgesia in labour of oxygen and nitrous oxide mixtures in different proportions. In a preliminary trial concentrations of 50% and 60% v/v nitrous oxide were compared, but, as the replies of 409 mothers revealed little difference between the two, the results of administering either 50% or 70% nitrous oxide to 778 mothers were then compared. The data relating to normal labour, obtained on 501 of the mothers in this main trial, showed that the relief of pain given was much the same. There was a suggestion, however, that the higher concentration of nitrous oxide might be useful in abnormal labour. The proportion of mothers with normal deliveries who lost consciousness, though very small, was significantly higher with 70% nitrous oxide than with the lower concentration. Ninety-two per cent. of mothers found the gas and oxygen machine helpful, and midwives reported complete or good co-operation by 77% of those using it. It is concluded that the 50% oxygen and 50% nitrous oxide mixture can safely be used by unsupervised midwives.     

The Impact of Using Mature Compost on Nitrous Oxide Emission and the Denitrifier Community in the Cattle Manure Composting Process

The diversity and dynamics of the denitrifying genes (nirS, nirK, and nosZ) encoding nitrite reductase and nitrous oxide (N₂O) reductase in the dairy cattle manure composting process were investigated. A mixture of dried grass with a cattle manure compost pile and a mature compost-added pile were used, and denaturing gradient gel electrophoresis was used for denitrifier community analysis. The diversity of nirK and nosZ genes significantly changed in the initial stage of composting. These variations might have been induced by the high temperature. The diversity of nirK was constant after the initial variation. On the other hand, the diversity of nosZ changed in the latter half of the process, a change which might have been induced by the accumulation of nitrate and nitrite. The nirS gene fragments could not be detected. The use of mature compost that contains nitrate and nitrite promoted the N₂O emission and significantly affected the variation of nosZ diversity in the initial stage of composting, but did not affect the variation of nirK diversity. Many Pseudomonas-like nirK and nosZ gene fragments were detected in the stage in which N₂O was actively emitted.     

Evidence for Involvement of Gut-Associated Denitrifying Bacteria in Emission of Nitrous Oxide (N2O) by Earthworms Obtained from Garden and Forest Soils

Earthworms (Aporrectodea caliginosa, Lumbricus rubellus, and Octolasion lacteum) obtained from nitrous oxide (N₂O)-emitting garden soils emitted 0.14 to 0.87 nmol of N₂O h⁻¹ g (fresh weight)⁻¹ under in vivo conditions. L. rubellus obtained from N₂O-emitting forest soil also emitted N₂O, which confirmed previous observations (G. R. Karsten and H. L. Drake, Appl. Environ. Microbiol. 63:1878–1882, 1997). In contrast, commercially obtained Lumbricus terrestris did not emit N₂O; however, such worms emitted N₂O when they were fed (i.e., preincubated in) garden soils. A. caliginosa, L. rubellus, and O. lacteum substantially increased the rates of N₂O emission of garden soil columns and microcosms. Extrapolation of the data to in situ conditions indicated that N₂O emission by earthworms accounted for approximately 33% of the N₂O emitted by garden soils. In vivo emission of N₂O by earthworms obtained from both garden and forest soils was greatly stimulated when worms were moistened with sterile solutions of nitrate or nitrite; in contrast, ammonium did not stimulate in vivo emission of N₂O. In the presence of nitrate, acetylene increased the N₂O emission rates of earthworms; in contrast, in the presence of nitrite, acetylene had little or no effect on emission of N₂O. In vivo emission of N₂O decreased by 80% when earthworms were preincubated in soil supplemented with streptomycin and tetracycline. On a fresh weight basis, the rates of N₂O emission of dissected earthworm gut sections were substantially higher than the rates of N₂O emission of dissected worms lacking gut sections, indicating that N₂O production occurred in the gut rather than on the worm surface. In contrast to living earthworms and gut sections that produced N₂O under oxic conditions (i.e., in the presence of air), fresh casts (feces) from N₂O-emitting earthworms produced N₂O only under anoxic conditions. Collectively, these results indicate that gut-associated denitrifying bacteria are responsible for the in vivo emission of N₂O by earthworms and contribute to the N₂O that is emitted from certain terrestrial ecosystems.     

Nitrous Oxide (N2O) Emissions from Subsurface Soils of Agricultural Ecosystems

Ecosystems - Nitrous oxide (N2O) is a major greenhouse gas and cultivated soils are the most important anthropogenic source. N2O production and consumption are known to occur at depths below the A...     

Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances

The intramolecular distribution of nitrogen isotopes in N₂O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N₂O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N₂O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N₂O from ammonia oxidation by N. europaea (31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N₂O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ~33‰ and ~0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N₂O.