Impact of nitrogen compounds on fungal and bacterial contributions to codenitrification in a pasture soil

Ruminant urine patches on grazed grassland are a significant source of agricultural nitrous oxide (N2O) emissions. Of the many biotic and abiotic N2O production mechanisms initiated following urine-urea deposition, codenitrification resulting in the formation of hybrid N2O, is one of the least understood. Codenitrification forms hybrid N2O via biotic N-nitrosation, co-metabolising organic and inorganic N compounds (N substrates) to produce N2O. The objective of this study was to assess the relative significance of different N substrates on codenitrification and to determine the contributions of fungi and bacteria to codenitrification. 15N-labelled ammonium, hydroxylamine (NH2OH) and two amino acids (phenylalanine or glycine) were applied, separately, to sieved soil mesocosms eight days after a simulated urine event, in the absence or presence of bacterial and fungal inhibitors. Soil chemical variables and N2O fluxes were monitored and the codenitrified N2O fluxes determined. Fungal inhibition decreased N2O fluxes by ca. 40% for both amino acid treatments, while bacterial inhibition only decreased the N2O flux of the glycine treatment, by 14%. Hydroxylamine (NH2OH) generated the highest N2O fluxes which declined with either fungal or bacterial inhibition alone, while combined inhibition resulted in a 60% decrease in the N2O flux. All the N substrates examined participated to some extent in codenitrification. Trends for codenitrification under the NH2OH substrate treatment followed those of total N2O fluxes (85.7% of total N2O flux). Codenitrification fluxes under non-NH2OH substrate treatments (0.7–1.2% of total N2O flux) were two orders of magnitude lower, and significant decreases in these treatments only occurred with fungal inhibition in the amino acid substrate treatments. These results demonstrate that in situ studies are required to better understand the dynamics of codenitrification substrates in grazed pasture soils and the associated role that fungi have with respect to codenitrification.

, NO and N 2 O, which are all obligate intermediaries of the denitrification pathway 12,[18][19][20] to finally create dinitrogen (N 2 ). In order to conserve both energy and oxygen, nitrifier-denitrification may occur in response to limited soil oxygen conditions 21 , whereupon nitrifiers convert NO 2 − to NO, N 2 O and N 2 12 although the significance of this process may have been overestimated in some studies 22 . In addition to these N 2 O production pathways, N 2 O may also be produced as 'hybrid' N 2 O via codenitrification, a process involving two different N pools 20,23 . Spott et al. 20 reviewed possible biotic and abiotic reactions that may be included under the term 'codenitrification' . For example, abiotic reactions involving reduced iron (Fe 2+ ) and NO 2 − , may occur at the interface between an aerobic zone overlying an anaerobic zone when NO 2 − diffusing downwards meets Fe 2+24, 25 . However, this process is unlikely to contribute significantly to N 2 O emissions due to insufficient Fe 2+ ion concentrations in most soils 26,27 . A more common abiotic reaction that occurs in acidic soil (pH < 5.0) is that of chemodenitrification (abiotic-nitrosation), whereby NO 2 − and H + react to form nitrous acid (HNO 2 ), which can then react with amino compounds, NH 2 OH, NH 4 + or other organic N compounds resulting in the formation of N 2 O 28,29 . However, under alkaline conditions when oxygen is depleted codenitrification may occur via biologically mediated nitrosation 20,30 . Under such conditions the hydrogen atom in an organic compound is replaced with a nitroso group (−N=O). Enzymatic nitrosyl compounds attract nucleophile compounds (e.g. NH 2 OH, NH 4 + , hydrazine (N 2 H 2 ), amino compounds and NH 3 ) resulting in hybrid N 2 O or N 2 species, containing one N atom derived from the nucleophile and one N atom derived from the nitrosyl compound 20 . Recent studies have revealed the significant contribution of codenitrification to gaseous N losses from grassland soils [30][31][32] . Using a 15 N tracer approach, Laughlin and Stevens 32 found evidence for fungal dominated 15 NO 3 − depletion leading to hybrid N 2 emissions where 92% of the N 2 evolved was derived from codenitrification. Selbie et al. 30 confirmed, in-situ, the dominance of codenitrification derived N 2 under urine patch conditions when 56% of applied urine was codenitrified. Recently, studies have found further evidence for N 2 O production via codenitrification under simulated ruminant urine patch conditions 31,33 . However, knowledge about the nucleophile species that potentially partake in codenitrification under ruminant urine patch conditions is still lacking. Different N substrates (as potential nucleophiles) such as amino acids, NH 4 + and NH 2 OH have previously been proven to be capable of generating hybrid N 2 O/N 2 in vitro when utilized by one microbial species in combination with either NO 3 − or NO 2 −34-37 . Amino acids have been reported to be freely available within the soil solution, for example, phenylalanine  µg N g −1 soil) and glycine  µg N g −1 soil) were measured in long-term agricultural land on a Stagni-Haplic Luvisol 38 and in different cattle manure treated crop fields on a sandy Orthic Luvisol 39 . Reported concentrations of NH 2 OH are orders of magnitude lower, for example, Liu et al. 40 reported concentrations of <0.0348 µg N g −1 in a forest soil, while NH 4 + and NH 3 are routinely reported following ruminant urine deposition events 41 . Therefore, we hypothesise that in a soil matrix under simulated ruminant urine deposition the N substrates applied in this study will be utilized for codenitrification reactions, with a microbial preference for NH 2 OH and that these reactions would be mainly fungi driven.

Results
Soil pH, and mineral N. Within 6 h of applying the urea solution to the soil surface pH values increased uniformly in all treatments from an average of 5.6 ± 0.2 on Day −2 to >7.6 on Day 0. The surface soil pH peaked 30 h after the urea application, at 7.9, followed by a steady decline to 4.8 ± 0.1 on Day 9 ( Fig. 1) in the positive control and all treatments. The surface pH in the negative control ranged from 5.4 ± 0.05 to 5.6 ± 0.06 over the course of the experiment (Fig. 1).
Soil NO 2 − concentrations were significantly elevated within the first 4 days following urea application (p < 0.05). Soil NO 2 − concentrations peaked at 1.5 ± 0.2 µg NO 2 − -N g −1 soil on Day 9, subsequent to the physical mixing and then decreased to 0.6 ± 0.1 µg NO 2 − -N g −1 soil on Day 11 (Fig. 1b The magnitude of the decrease in the N 2 O fluxes, following inhibition treatment, varied due to inhibitor type and N substrate applied ( Table 1). The N 2 O emissions were lower under fungal inhibition by 46, 34 and 21% in the glycine, phenylalanine, and NH 2 OH treatments, respectively, while fungal inhibition did not affect fluxes from the NH 4 + treatment. Bacterial inhibition decreased N 2 O fluxes by 14, and 26% in the glycine and NH 2 OH treatments, respectively, while fluxes from the phenylalanine and NH 4 + treatments were unaffected by bacterial inhibition (Table 1). Applying both inhibitors simultaneously (combined inhibition) resulted in N 2 O fluxes decreasing by 29-41% in all N substrate treatments (Table 1). In the glycine treatment fungal inhibition decreased N 2 O fluxes more than bacterial inhibition, but this decrease was not enhanced when the two inhibitors were combined (Table 1). While bacterial inhibition did not significantly lower N 2 O fluxes in the phenylalanine treatment, www.nature.com/scientificreports www.nature.com/scientificreports/ the fungal inhibition either alone or within the combined inhibition did decrease N 2 O fluxes (Table 1). Sterilizing effectively eliminated N 2 O fluxes in both the amino acid treatments, and the NH 4 + treatment (Table 1). However, this was not the case when NH 2 OH was applied, where emissions decreased by 72% (Table 1).  Table 1, for simplicity only the non-inhibition treatments are depicted in Fig. 1 to visualize the range of increase. Below the NO 2 − concentration in the soils as measured in the NO 2 − control. (b) These partially destructive analysis was not performed within the treatment soils and the positive controls, but depicts the assumed NO 2 − concentration development within these soils. The soil surface pH was measured in all jars, however, all treatment soil surface pH values did not differ from the depicted positive control, in contrast to the negative control. (c) Each symbol represents a mean of n = 3, all error bars are ± SD.   (Table 2): the phenylalanine treatment with either no inhibition or bacterial inhibition, and the NH 4 + treatment with bacterial inhibition (Table 2). Within a given N substrate treatment, when comparing the N 2 O-15 N enrichment of the no inhibition treatment and a specific inhibitor treatment, few treatment differences occurred. Under glycine only the sterilized soil treatment varied, with a higher N 2 O-15 N enrichment relative to the no inhibition treatment ( Table 2). Applying phenylalanine also resulted in enhanced N 2 O-15 N enrichment, mostly when applied to the sterilized soil but this was not statistically different from the no inhibition treatment (Table 2). With NH 4 + as the N substrate the N 2 O-15 N enrichment was again highest in the sterilized soil treatment, but none of the inhibitor treatments caused N 2 O-15 N enrichment to differ from the no inhibitor treatment ( Table 2). The biggest shifts in N 2 O-15 N enrichment with inhibition treatments occurred in the NH 2 OH treatment where applying bacterial inhibition, either alone or within the combined inhibition treatment, caused significant decreases in N 2 O-15 N enrichment relative to the no inhibition treatment ( Table 2).  (Table 3). With sterilized soil under these N substrate treatments codenitrification fluxes ceased ( Table 3). The N 2 O co fluxes from the NH 2 OH treatment decreased significantly in the presence of the combined inhibition (>46%, Table 3) but not when applied individually. Under NH 2 OH, hybrid N 2 O fluxes equalled 3851 µg N 2 O co -N m −2 h −1 with no inhibition present. Sterilizing the soil significantly lowered NH 2 OH derived codenitrification fluxes to 617 µg N 2 O co -N m −2 h −1 . This corresponded to a decrease of >83%, compared to the no inhibition treatment; or a decrease of >71%, compared to the combined inhibitor treatment (Table 3).

Discussion
The hydrolysis of urea and its resulting products increases NH 4 + and OH − concentrations in the soil 5 with the latter responsible for the elevated soil surface pH observed in treatments containing urea. Urea application elevated soil NH 4 + -N concentrations, as evidenced by the higher concentrations in the positive control when compared with the negative control. Elevated soil pH will have resulted in the NH 4 + /NH 3 equilibrium shifting towards NH 3 5 . However, by Day 8 the concentration of NH 3 will have been relatively low based on soil pH values at this time 5 . While NH 3 can inhibit NO 2 − oxidisers under urea-affected soil 9,10 the elevated soil NO 3 − -N concentrations at the end of the experiment and the decline in NO 2 − from Day 1 to 7 demonstrates NO 2 − oxidisers were functioning. The soil NO 3 − -N concentration on Day 9 was higher when compared to a previous study by Rex et al. 33     www.nature.com/scientificreports www.nature.com/scientificreports/ the reduced potential for nitrifier inhibition 9,10 , a consequence of the lower urea-N rate used in the current study. Considering the soil pH and inorganic-N dynamics it can be concluded that the application of urea was representative of conditions under a typical urine patch 41,42 , and that the N substrate treatments were applied during a period of relatively rapid inorganic-N transformation.
The rapid increase in N 2 O fluxes following inhibitor application was partially the result of physically mixing the soil in order to distribute the inhibitors, which resulted in entrapped N 2 O, in the soil, being released 43 . Furthermore, soil, not previously exposed to oxygen, would have become exposed and thus there is also the possibility that inhibition of N 2 O reductase occurred, preventing complete denitrification 44 . However, the application of substrate-N also contributed to the N 2 O flux as demonstrated by the increased N 2 O-15 N enrichments, particularly in the case of the NH 2 OH treatment (Fig. 1a).
Soil N 2 O emissions are strongly driven by the presence and turn-over of NO 2 − which is the 'gate-way molecule' for N 2 O production 9,45 . In the current study soil NO 2 − concentrations were elevated on Day 9 but at concentrations lower than previously observed (e.g. Clough et al. 31 ) due to the lower urea application rate in the current study preventing NH 3 inhibition of NO 2 − oxidation 45 . Hence, the ensuing N 2 O emissions most likely result from the net effects of microbial processes utilising NO 2 − and/or the N substrate added. The effects of the microbial inhibitors, cycloheximide, streptomycin and heat sterilization on N 2 O production were assessed 12 h after inhibitor application since maximum efficacy is reported within 24 h of application 46  , as was the case in the NH 2 OH treatment of the current study. Thus, the decline in N 2 O emissions in the NH 2 OH treatment, with fungal inhibition, implies a fungal mechanism was partially responsible for the N 2 O flux, via NH 2 OH utilisation.
With bacterial inhibition, the decline in the N 2 O flux under the NH 2 OH treatment likely occurred due to the bacterial inhibitor preventing the function of the ammonia oxidising bacteria (AOB), which utilise NH 2 OH to gain energy 50 . Increased mRNA transcription levels of the functional genes present in AOB that encode for NH 2 OH oxidoreductase (haoA), and the reductases for NO 2 − and NO, which are nirK and norB, respectively, become elevated following NH 2 OH application 50 . A similar result and explanation might have been expected following bacterial inhibition in the NH 4 + treatment, given that NH 2 OH is an intermediate in the nitrification pathway, however the result was not statistically significant (Table 1). Lower N 2 O fluxes from the glycine treatment under bacterial inhibition may have also resulted from a diminished nitrification rate of the NH 4 + derived from the mineralized glycine-N, and thus delivering less NO 2 − to the soil pool. However, this did not occur under the phenylalanine treatment possibly because it is a more complex molecule and potentially slower to be mineralized, and thus potentially bacteria played less of a role in the N 2 O fluxes derived from phenylalanine. Again, with glycine the combined inhibition treatment demonstrated the role of fungi in generating N 2 O. This was also the case with phenylalanine where the combined inhibition cut N 2 O emissions to a level comparable to fungal inhibition alone.
The near complete suppression of N 2 O emissions in the amino acid and NH 4 + treatments, under the combined inhibition treatment, demonstrates that the observed N 2 O fluxes were almost entirely from biologically driven processes. As previously shown, from the δ 13 C signatures of respired amino acid-CO 2 -C, amino acids are readily mineralized, forming NH 4 +51 . Consequently, amino acids will contribute to N 2 O fluxes if this NH 4 + is nitrified, or via the denitrification of the nitrification products 51 . The residence time of amino acids in soils is generally reported in hours and depends on soil type [51][52][53] . However, the lack of a significant N 2 O flux response to amino acid and NH 4 + substrate additions at Day 9, relative to the positive control ( Fig. 1), is most likely due to the large background NH 4 + pool present at the time of N substrate addition, derived from the urea addition. Hence, the NH 4 + formed from either amino acid mineralization or direct NH 4 + addition will have been diluted by at least 10-fold, assuming all substrate-N was immediately available. Furthermore, it is likely other amino acids were also present to further dilute the amino acid additions. For example, after extracting three soils McLain and Martens 51 found the sum of 18 amino acids to range from 9 to 20 g kg −1 of soil, when examining an arid grassland (Well-drained Typic Torrifluvents of the Pima series). In contrast to the soil used in this study, these amino acid concentrations referred to a non-irrigated soil with an expected lower microbial abundance.
With the exception of NH 2 OH, the near-zero N 2 O emissions after applying the N substrates to the sterilized soils indicated that the N 2 O fluxes were dominated by biotic processes. This was not the case for NH 2 OH where the N 2 O flux from the sterilized soil was ∼28% that of the no inhibition treatment. It has previously been shown that the NH 2 OH molecule may decompose abiotically to produce N 2 O 50,54-56 .
The lack of any corresponding shifts in the relatively low 15 N enrichments of the N 2 O evolved from the amino acid treatments, under the various inhibition treatments, suggests fungi were not directly utilising the amino acids for N 2 O production. The codenitrification product depends on the redox state of the N-donor, and prior studies have shown amines (-R-NH 2 ) to be codenitrified to N 2 47 . Thus, the lack of any corresponding shifts in the relatively low 15 N enrichments of the N 2 O evolved from the amino acid treatments may have also been the result of N 2 being produced. Despite this, fungal inhibition lowered amino acid derived codenitrified N 2 O (Table 3), indicating that products derived from the amino acid mineralization are involved in fungal codenitrification. The lack of any bacterial inhibition effect on the codenitrification flux demonstrates the dominant role of fungi in codenitrification 33 .
Increasing 15  Using soil suspensions Spott and Stange 57 concluded N 2 O production from NH 2 OH in soil was complex due to the interaction of production pathways involving both abiotic formation and biogenic formation, resulting from both codenitrification and denitrification. Adding the NH 2 OH substrate to the sterilized soil (abiotic conditions) the 15 N enrichment of the N 2 O (∼44 atom%) aligned closely with the calculated 15 N enrichment of 49 atom% that indicates hybrid N 2 O production via abiotic N-nitrosation. The formation of N 2 O via NH 2 OH reacting with NO 2 − occurs due to abiotic nitrosation processes 58 , and has been previously observed in sterilized soils 56 . The NH 2 OH compound has also been reported to decay abiotically to form N 2 O with the process slowed down when NO 2 − is preesent 58 . However, had this been the main process for N 2 O formation the 15 N enrichment of the N 2 O evolved would have aligned more with the applied NH 2 OH-15 N enrichment. The combined inhibition treatment significantly decreased the N 2 O codenitrification flux by 50% (Table 3) compared to the no inhibition treatment ( Table 2) indicating abiotic reactions were also contributing substantially to the observed N 2 O flux.
Fungi contributed to N 2 O production when NH 2 OH was applied, as indicated by the flux decrease under the fungal inhibition treatment, however, the lack of any change in the N 2 O-15 N enrichment indicates fungal inhibition was not affecting the process generating 15 N enriched N 2 O. Conversely, the further decrease in both the N 2 O flux and N 2 O-15 N enrichment in the bacterial inhibition and the combined inhibition treatments, showed that the N 2 O production process was inhibited, and that less 15 N enriched NH 2 OH contributed to the N 2 O flux produced. Therefore, the codenitrification flux also tended to decline in the presence of the bacterial inhibitor. Bacterial inhibition diminishes, amongst others, the activity of AOB and thus (i) lowers the consumption of NH 2 OH via bacterial nitrification, (ii) lowers the enrichment of the nitrification products derived from 15

conclusions
Codenitrification occurs when N-donors, such as those studied here (NH 4 + , glycine, phenylalanine and NH 2 OH) react with a nitrosyl compound, to form hybrid N 2 O. Using selective microbial inhibition treatments, and simulating a ruminant urine patch environment, we demonstrated that all the used 15 N-labelled N substrates contributed to codenitrification in a soil matrix. Hydroxylamine was the most important N substrate with respect to increasing the N 2 O flux and contributing to codenitrification (85.7% of total flux), likely because of its more reactive character compared to the other N substrates. The codenitrification N 2 O fluxes following amino acid-15 N addition were orders of magnitude lower (0.7-1.2% of total flux), potentially due to dilution from antecedent amino acids or their break down products, which in turn means that a contribution of these natural amino acids could be assumed under the experimental conditions. Fungal inhibition resulted in a significant decline in the formation of amino acid derived codenitrification fluxes, underlining once more the importance of fungal codenitrification vs. bacterial codenitrification. The relatively lower codenitrification N 2 O fluxes with amino acids may also be a result of the microbial community structure that is present 20 . Alternatively, codenitrification of NH 2 OH to form N 2 O has been reported in the absence of organic electron donors 59 hence, given that codenitrification is in principle dependent on organic carbon respiration a lack of organic substrate or variations in its form may have favoured codenitrification of NH 2 OH 20 . The results of this study, demonstrated that codenitrification occurs via multiple pathways in a pasture soil following a simulated bovine urine event. Codenitrification resulting from the presence of NH 2 OH is likely to be the dominant process, in the short-term following the deposition of ruminant urine with its relatively high urea-N loading. The results warrant further in situ investigation of the dynamics of potential N-donors, in conjunction with N 2 O fluxes, under ruminant urine patches.
Initially the jars, with soil, were placed in an incubator, in the dark, at 23 °C and wetted-up daily to preincubation weight. After four days, any germinated weed seedlings were removed and the experimental period of 14 days commenced (Day −2 to Day 11). An aqueous urea solution (500 µg urea-N g dry soil −1 ) was applied on Day 0 in order to simulate a bovine urine deposition event 31,60 . On Day 8, microbial inhibition treatments were applied with the N substrate treatments applied immediately after this in an aqueous solution (4 mL) as noted below.
Treatments consisted of 15 N enriched N substrate species (glycine (98), L-phenylalanine (98), NH 4 + (99) and NH 2 OH (98); atom% 15 N enrichment in bracket) with each N substrate treatment further split into five microbial inhibition treatments (no inhibition, fungal inhibition, bacterial inhibition, fungal and bacterial inhibition ('combined inhibition') and soil total microbial inhibition (heat sterilised soil)). Treatments were replicated thrice. The amino acid-N concentrations were based on the findings of Scheller and Raupp 39 , and in order to apply a realistic concentration, these were applied at a rate of 20 µg N g −1 dry soil. Hydroxylamine and NH 4 + were applied at equal N rates for comparative purposes.
According to Anderson and Domsch 61 cycloheximide, a fungal inhibitor, was applied at a rate of 8 mg g −1 soil and streptomycin, a bacterial inhibitor, at a rate of 5 mg g −1 soil. Both chemicals were applied as a dry powder on to the soil surface and subsequently mixed into the soil with a spatula for 1 min. The combined inhibition (2019) 9:13371 | https://doi.org/10.1038/s41598-019-49989-y www.nature.com/scientificreports www.nature.com/scientificreports/ included the simultaneous application of cycloheximide and streptomycin and was designed to inhibit both bacteria and fungi. Sterilizing (as complete microbial inhibition) was performed by heating the soil. This was achieved by microwaving the soil in the jars for 4 minutes, remoistening the dry soil, and then microwaving the jars for another 3 minutes, as microwave heating is a proven method to stop microbial activities 62,63 . Thereafter, the microwaved soils were readjusted to 50% water-holding-capacity and also mixed for 1 minute. The control treatment contained urea, but no inhibitors were applied, and the soil was mixed to replicate the physical disturbance of the other treatments. Immediately after application of the inhibitor treatments the N substrate treatments were applied according to treatment at a rate of 20 µg N g −1 dry soil, without subsequent mixing.
In addition, three further control treatments were set up; a positive control (soil with urea but no N substrate or inhibitor addition (n = 3), also physically mixed on Day 8; a negative control (n = 3) consisting of soil without urea, inhibitors, or N substrates, also physically mixed on Day 8; and a separate NO 2 − control (soil with urea but no N substrate addition, physically mixed on Day 8) for soil NO 2 − -N sampling at 4 different times over the duration of the experiment.
Gas sampling and analysis. On Day −2, −1, 0, 1, 2, 4, 6, 7, 8 (before inhibitor application), 9, 10 and 11, the jars were sealed with lids equipped with rubber septa. Jar headspace gas samples were taken with a plastic syringe, fitted with a three-way-stop cock and a 25G hypodermic needle, and injected into a previously evacuated Exetainer ® vials (Labco Ltd., High Wycombe, UK). The first gas sample (12 mL) was taken immediately after sealing the jar headspace. The second gas sample was taken after 1 h, only from the positive control to verify the linearity of the increase in the headspace gas concentration, and the third gas sample was taken after a 2 h incubation time (12 mL, all jars). On Days 8,9,10 and 11, the third gas sample (30 mL), was split between a 6 mL Exetainer ® that received 12 mL, and an evacuated and helium flushed 12 mL Exetainer ® that received 18 mL for 15 N-N 2 O determination.
Nitrous oxide concentrations were determined using a gas chromatograph (SRI-8610, SRI Instruments, Torrance, CA) coupled to an autosampler (Gilson 222XL; Gilson, Middleton, WI) equipped with a 63 Ni electron capture detector 64 . PeakSimple 4.44 software (SRI Instruments, Torrance, CA) and several N 2 O standards (range 0-100 µL L −1 ,BOC, New Zealand) were used to determine the N 2 O concentrations. The N 2 O fluxes (µg N 2 O-N m −2 h −1 ) were determined using the following equation: The measured 15 N concentration of the headspace N 2 was close to natural abundance thus a determination of the N 2 flux was not possible, hence, the N 2 emissions were not considered further.

Codenitrification calculations.
As previously reported 20 conventional denitrification produces N 2 O (non-hybrid N 2 O) while N 2 O produced via codenitrification results in an N atom from NO 2 − and an N atom from a co-metabolised compound producing a hybrid N-N species, such as N 2 O. The following calculations determine the codenitrification flux, assuming that hybrid N 2 O only arises from codenitrification. We do not distinguish between the roles of biotic and abiotic reactions in this process. However, the use of biological inhibitors and soil sterilization indicate the relative roles of abiotic and biotic processes in producing hybrid N 2 O.
For the N 2 O evolved it was assumed that this was generated from one 15 N enriched pool-fraction (d′ D ) with 15 N enriched N ( 15 N atom fraction q′ D ), and a fraction (d′ N , equal to 1 − d′ D ) derived from a pool or pools at natural abundance ( 15 N atom fraction q′ N ).
The ratios r' 1 and r' 2 , were determined from the N 2 O m/z ion currents at m/z 44, 45 and 46 65 : www.nature.com/scientificreports www.nature.com/scientificreports/ When letting d′ N equal (1 − d′ D ) and a′ A equal the 15 N enrichment at natural abundance (0.003663) Eqs 3 and 4, when set to equal zero, become: Since a′ s and x′ s are known the values of d′ D and a′ D can be determined using the Solver function in Microsoft Excel TM , while setting the target value at zero, with the result accepted when the target value is <1 × 10 −5 .
Then the codenitrification flux was calculated according to Clough et al. (2001) as: were d CD is the fraction of N 2 O within the headspace derived from codenitrification and Δ 45 R is the 45 N 2 O/ 44 N 2 O ratio, while p 1 (0.9963) and q 1 (0.0037) are fractions of 14 N and 15 N in the natural abundance pool, and where q 2 equals a'D, derived above, with p 2 equal to 1 − q 2 . Finally the codenitrification flux was determined as:

CD C D 2
Surface pH and inorganic-N measurement. Surface pH was measured on Days −2, 0, 1, 3, 5, 7, 9 and 11, by adding one drop of deionised water to the soil surface and then placing a flat surface pH probe (Broadley James Corp., Irvine, California) onto the soil surface. The NO 2 − concentration in the unmixed NO 2 − control (soil + urea solution) was determined by subsampling soil with a corer (diameter 1.6 cm, depth 1.5 cm). The soil was then blended with 2 M potassium chloride (KCl), adjusted to pH 8 with potassium hydroxide 66 at a 1:6 ratio. This procedure was performed on Days 1, 4, 6 and 10.
Subsamples of moist soil (4 g dry weight) were taken after Day 11, from the positive and negative controls, and extracted with 2 M KCl in order to determine the NH 4 + and NO 3 − concentrations at the end of the experiment 67,68 . Inorganic-N concentrations in the extracts were determined using Flow Injection Analysis 67 .
Statistics. The single jars were defined as experimental units by the independent applications of treatments. The experiment focussed on achieving the most sensitive test of treatment differences and inference is not claimed for a population wider than the paddock, used for sampling. All statistical analyses were performed using SigmaPlot 13.0 (Systat Software Inc., Chicago). For each variable of interest a general linear model (ANOVA equivalent) was fitted with N substrate treatment or a factorial combination of N substrate treatment and inhibition method as explanatory variables. Using this method, the different inhibition treatments within each N substrate treatment were compared. Tests for normality (Shapiro-Wilk test) and variance (Brown-Forsythe test) were used to evaluate the residuals and define the most powerful test for each comparison of means. Hence, means comparisons were adjusted for multiplicity using Tukey, Holm-Sidak, Dunn's or Student's t-test adjustments to p values.