Two agricultural soils were selected for the experiments (Table 1). Soil HP was used as a reference soil, since it had been previously used in numerous studies that proved that denitrification was the major process responsible for N₂O emissions (Čuhel et al., 2010). Soil HC was selected from a series of agricultural soils because it was the only one where (under identical optimal denitrifying conditions) the addition of greenwaste biochar increased N₂O emissions. The soils were sampled from a depth of 0–0.25 m, air-dried and sieved (<2 mm).

We used a biochar produced by continuous slow pyrolysis of greenwaste at 550°C provided by Pacific Pyrolysis Pty. Ltd. (Australia) (Table 1). Herbaceous and woody biochars have been found to be the most promising for mitigating N₂O emissions from soil (Cayuela et al., 2014). Therefore, this biochar was selected for its mitigation potential and as a representative standard biochar commonly used in other studies. The biochar was ground to a particle size <1 mm before soil application.

The incubation experiments were performed in 250 ml polypropylene jars at optimum conditions for denitrification: 25°C and moisture content of 90% water filled pore space (WFPS). The control treatments consisted of 100 g dry soil and the biochar treatments of 98 g dry soil and 2 g biochar (2% w:w). The biochar was thoroughly mixed with the dry soil to obtain a completely homogeneous mixture. Subsequently deionized water (or a solution containing the appropriate concentration of N fertilizer) was added to reach 90% WFPS (and the required N concentration in the fertilized treatments). The jars were incubated aerobically, covered with a polyethylene sheet that allows gas exchange but minimizes evaporation. Moisture was gravimetrically adjusted every other day with the addition of deionised water for each individual jar. The experiments were laid out as randomized block designs with four replicates per treatment.

For N₂O sampling each unit was sealed with gas-tight polypropylene screw caps for an accumulation period of 60 min. The headspace gas was then sampled directly with a membrane air pump (Optimal 250, Schego, Offenbach am Main, Germany), attached to a gas chromatograph (VARIAN CP-4900 Micro-GC, Palo Alto, CA, USA) (Mondini et al., 2010).

N₂O fluxes were calculated assuming a linear increase during the accumulation (closed) period, an approach which was verified prior to the experiments. Cumulative N₂O was calculated assuming linear changes in fluxes between adjacent measurement points (Velthof et al., 2003).

The ¹⁵N atomic fraction in N₂O was calculated from the 45/44 and 46/44 ratios of N₂O. The ¹⁵N gas-flux method (Mulvaney and Boast, 1986; Stevens et al., 1993; Stevens and Laughlin, 2001) was used to quantify N₂O and N₂ emissions from denitrification in soil HP. The molar fraction of ¹⁵N-NO⁻₃ (¹⁵X_N) in the soil pool was calculated from Δ45R and Δ46R according to Stevens and Laughlin (2001). The flux of N₂ and N₂O was then calculated by the equations given by Mulvaney and Boast (1986). The presence of hybrid nitrous oxide (⁴⁵N₂O) co-metabolically introduced into the reaction pathway of denitrification was tested by the model developed by Spott and Florian Stange (2011). This model considers two different N sources, where each source generates non-hybrid N₂O (⁴⁶N₂O and ⁴⁴N₂O) and, simultaneously, both N sources can be combined to form hybrid N₂O (⁴⁵N₂O). According to this model, the contribution of each pathway to the total N₂O formation can be calculated from the mass distribution of the released N₂O and the ¹⁵N mole fraction of the labeled N source (Spott and Florian Stange, 2011).

Univariate analysis of variance was used to investigate the significant differences in N₂O emissions and mineral N concentrations between biochar and control treatments with IBM SPSS Statistics 21, Sommers, USA.

Soil HP emitted N₂O when NO⁻₃ was added but not in the absence of fertilizer or after the addition of urea. In this soil, biochar significantly reduced N₂O emissions, by an average of 76% (Figures 1A1–A3).

Soil HC emitted N₂O in all treatments: without N fertilization, after the addition of NO⁻₃ and urea. In this soil, biochar consistently increased total cumulative N₂O emissions and the average increase was larger in the non-fertilized (95%) and urea (129%) treatments than in the NO⁻₃ treatment (54%), (Figures 1B1–B3).

Comparing treatments without biochar, the addition of NO⁻₃ increased total N₂O emissions in soil HP (from 54 to 11580 μg N₂O-N kg⁻¹ soil), whereas it increased N₂O emissions slightly in soil HC (from 3443 to 4546 μg N₂O-N kg⁻¹ soil). The addition of urea had no impact on soil HP, and increased emissions in soil HC (from 3443 to 5799 μg N₂O-N kg⁻¹ soil).

Figure 2 shows NH⁺₄-N concentration in soils HP and HC at the end of the experiment. The original concentration of NH⁺₄ in soil HP was 19.3 mg N kg⁻¹ soil. After 14 days of incubation, soil HP underwent a significant increase in NH⁺₄ content for all fertilization treatments (74.5–110.4 mg N kg⁻¹ soil). The highest increase was observed when soil HP was fertilized with urea. Biochar addition did not have a significant impact on the final NH⁺₄ concentration in this soil.

Soil HC similarly increased its NH⁺₄ concentration throughout the incubation (initial concentration: 2.8 mg kg⁻¹ soil), excluding the KNO₃ treatment. In this soil biochar significantly decreased the amount of NH⁺₄ by the end of the incubation for the non-fertilized soil. Biochar also decreased mean NH⁺₄ concentration in the urea treatment, although not significantly due to the high variability in the biochar samples.

Figure 3 shows (NO⁻₃ + NO⁻₂)-N concentrations in soils HP and HC. The concentrations of (NO⁻₃ + NO⁻₂)-N in soil HP were very low (<2.0 mg kg⁻¹) for all fertilization treatments and biochar did not have a significant impact. However, NO⁻₂ was detected in biochar amended soils and not in the control. Soil HC had low (NO⁻₃ + NO⁻₂)-N concentrations when no fertilizer was added or after the addition of urea. In contrast, 33.3 mg of NO⁻₃-N kg⁻¹ were found in the KNO₃ treatment irrespective of the biochar addition.

Figure 4 shows the ¹⁵N atomic fraction in N₂O emitted from soils HP and HC in Experiment 2. When ¹⁵NO⁻₃ was added, the initial ¹⁵N atomic fraction in N₂O emitted from soil HP was 0.74, decreasing gradually to reach 0.04 at day 10 (Figure 4A). In contrast, the ¹⁵N isotopic composition in soil HC followed totally different dynamics: the initial ¹⁵N atomic fraction in N₂O was only 0.18; it increased slightly to 0.33 by day three, and reached a final value of 0.10 by day 10 (Figure 4B1). Biochar altered the isotopic composition of N₂O emitted in both soils.

When urea was added, soil HP did not emit N₂O (Figure 1A3). In soil HC (even when emissions were high) the initial ¹⁵N atomic fraction in N₂O was zero (Figure 4B2), it successively increased, but always remained beneath 0.15. The biochar and control treatments showed identical ¹⁵N-N₂O concentration dynamics.

Table 2 shows the molar fraction of ¹⁵N-NO⁻₃ and the ratio N₂O/(N₂ + N₂O) calculated by the ¹⁵N gas flux method (Mulvaney and Boast, 1986) and the contribution of codenitrification to N₂O formation according to Spott and Florian Stange (2011) in soil HP. The ratio N₂O/(N₂ + N₂O) was very high during the first 3 days, which demonstrates that most N was lost as N₂O. Biochar decreased the N₂O/N₂ ratio, particularly at day three (the peak of emissions in the control soil). The contribution of codenitrification was zero (see C in Table 2). This method of calculation could not be applied to soil HC, since other mechanisms than denitrification were operating in this soil and we could not calculate the enrichment of the source [¹⁵NO⁻₃ in soil (¹⁵X_N)] (Mulvaney and Boast, 1986). Nonetheless, we found a high proportion of N₂O with a hybrid bond (⁴⁵N₂O) in soil HC.

Addition of NO⁻₂ to soil HC produced the highest N₂O emissions peak monitored in this soil (Figure 5B1); fourfold higher than that of the non-fertilized soil (Figure 1B1). Under these conditions, the biochar amendment did not modify cumulative N₂O emissions.

The ¹⁵N atomic fraction in N₂O (Figure 5B2) followed a different pattern than with ¹⁵NO⁻₃ (Experiment 2; Figure 4B1). The initial ¹⁵N atomic fraction in the N₂O emitted was 0.30, decreasing gradually to reach 0.06 at day 10 (Figure 5B2). Biochar did not significantly modify this pattern.

The biochar amended soil had a significantly lower concentration of NH⁺₄ at the end of the incubation (Figure 5B3). The concentration of NO⁻₃ was low (below 5 mg kg⁻¹ soil) and not affected by biochar addition.

N₂O emissions almost ceased when DCD was added to soil HC (Figure 6). The highest emissions were observed when the soil was fertilized with NO⁻₃ (Figure 6B2), but still represented less than 0.4% of the added N (compared to 12.7% without DCD (Figure 1B2).

The highest NH⁺₄ concentrations were found in the soil amended with urea, followed by the non-fertilized soil and the soil amended with KNO₃. Biochar (compared to the control) systematically decreased the concentration of NH⁺₄ by the end of the incubation for all treatments (non-fertilized soil, KNO₃, and urea). NO⁻₃ concentration was lower than the original in soil (16.9 mg NO⁻₃-N kg⁻¹ soil).

Nitrous oxide emissions patterns and their response to the addition of different N fertilizers were different in soils HP and HC, which clearly reflected the different N₂O production pathways involved.

Figure 7 illustrates the main pathways for N₂O formation in soil. Ammonia oxidation takes place in two steps: first NH₃ is oxidized to NH₂OH, which is then oxidized to NO⁻₂. N₂O may be directly released as a by-product of ammonia oxidation (nitrifier-nitrification) (Hooper and Terry, 1979) or it can be produced through a denitrification pathway where NO⁻₂ is reduced to N₂O (nitrifier-denitrification) (Kool et al., 2011). The ability to denitrify is a widespread, if not ubiquitous, attribute in ammonia oxidizers (Shaw et al., 2006). Classically, denitrification (from NO⁻₃) has been considered the main N₂O formation pathway in soils. However, other pathways that have been systematically overlooked in soil studies could play a more important role than originally estimated (Baggs, 2011; Spott et al., 2011). This is the case for codenitrification, which is potentially a widespread pathway of microbial N transformation in terrestrial environments (Spott et al., 2011) and dissimilatory nitrate reduction to ammonia (DNRA) (Giles et al., 2012). Although our knowledge of microbial N transformation in soil has evolved significantly over the last decades, recent findings show that, even today, our understanding of N₂O formation and consumption in soil is still very limited (Sanford et al., 2012; Long et al., 2013).

In the nearly water-saturated soil conditions used in our experiments (90% WFPS), N₂O production is expected to be dominated by denitrification of NO⁻₃. This was the case in soil HP, where emissions were clearly controlled by the conventional denitrification pathway. This can be deduced from the following facts: (i) This soil only emitted N₂O after the addition of NO⁻₃ (Figure 1A2); (ii) the ¹⁵N atomic fraction of the N₂O emitted at day one was 0.74 (Figure 4A), which shows that N₂O was primarily produced from the added ¹⁵NO⁻₃. The ¹⁵N atomic fraction decreased over time, showing the depletion of the labeled source; (iii) given the limited nitrification activity detected in this soil, addition of NO⁻₃ did not increase the final NH⁺₄ concentration (with respect to the non-fertilized soil), which suggests that DNRA was not a relevant pathway, and (iv) applying the equations developed by Spott and Florian Stange (2011), codenitrification was found to be null (Table 2).

As previously found in other soils under analogous optimal denitrifying conditions (Cayuela et al., 2013), biochar significantly decreased total N₂O emissions in this soil.

In soil HC, the weak response of N₂O emissions to NO⁻₃ addition pointed out to a low contribution of denitrification or DNRA in this soil. Given that the original NO⁻₃ concentration in the soil was 16.9 mg N kg⁻¹ at a natural abundance of 0.364% ¹⁵N, and that we added 55 mg N kg⁻¹ of ¹⁵NO⁻₃ (>99% enrichment), the ¹⁵N-NO⁻₃ enrichment in the soil at the beginning of the incubation was 75.8%. Yet, the ¹⁵N atomic fraction in the N₂O emitted at day one (Figure 4B1) was only 0.18, which demonstrates that some N₂O originated from denitrification, but also that NO⁻₃ was not the only source of N₂O. Moreover, the low C:N ratio of this soil and the NH⁺₄ concentration at the end of the incubation in the KNO₃ treatment (Figure 2B2) indicates that DNRA was not a major N₂O formation route in this soil (Giles et al., 2012). Instead, we hypothesize that N₂O formation in soil HC was mainly the result of nitrification-mediated processes. The results supporting this hypothesis can be summarized: (i) The addition of extra NO⁻₃ did not increase N₂O emissions in this soil, whereas the addition of extra urea did; (ii) the ¹⁵N atomic fraction of the N₂O emitted at day one was 17.7% (Figure 4B1), which shows that N₂O was not pre-dominantly formed from the added ¹⁵NO⁻₃. (iii) The concentration of dissolved organic N in this soil was very high (35.9 mg N kg⁻¹soil), which can explain the low contribution of the labeled urea to the emitted ¹⁵N₂O (Figure 4B2). However, significant hybrid N₂O (⁴⁵N₂O) was produced (data not shown) and we cannot discard the contribution of codenitrification to N₂O formation in soil HC.

To better understand which processes (within nitrification-mediated pathways) biochar might be modifying we performed Experiments 3 and 4.

In Experiment 3 the addition of NO⁻₂ to soil HC showed that, under high moisture conditions, this soil was able to rapidly reduce NO⁻₂ to N₂O, which was emitted in large quantities (38% of added NO⁻₂-N). It is very unlikely that the N₂O emitted was just the product of the chemical decomposition of NO⁻₂ (chemodenitrification), since this process, largely controlled by soil pH, only occurs in neutral and acidic soils (Bremner, 1997). Instead, NO⁻₂ was most probably used as electron acceptor for microbial respiration (nitrifier-denitrification). The high N₂O production in Experiment 3 (21.3 mg N kg⁻¹compared to 3.4 mg N kg⁻¹ in Experiment 1) may be related to enhanced nitrifier-denitrification for detoxifying NO⁻₂ (Jung et al., 2014).

The subsequent tracer experiment with application of ¹⁵NO⁻₂, demonstrated that significant nitrite reduction to N₂O occurs (the N₂O originating from the added ¹⁵NO⁻₂ at day one was 31.5%, see Figure 5B2), but also that it could not be the only process leading to N₂O emissions. This experiment demonstrated that biochar was not increasing N₂O emissions through the nitrifier-denitrification pathway, since N₂O emissions in the biochar and control treatments were not statistically different.

In our final experiment (Experiment 4), the high NH⁺₄ and low NO⁻₃ concentrations by the end of the experiment demonstrate the effectiveness of the DCD treatment to inhibit ammonia oxidation, which correlated with a large decrease in N₂O emissions for all treatments. We assumed that DCD did not inhibit other possible N₂O formation pathways. Although the impacts of DCD on other aspects of microbial N transformation in soil are largely unknown, Bremner and Yeomans (1986) demonstrated that DCD does not inhibit N₂O and N₂ emissions by denitrification when applied at similar rates to those used in this study. More recently, Wakelin et al. (2013) also demonstrated in a field study that the application of DCD had a minor impact on denitrifying bacteria activity (nirS).

Addition of biochar significantly and consistently decreased the NH⁺₄ concentration in soil HC. These results reinforce our conclusion that the production of N₂O in soil HC must be the consequence of nitrification processes (nitrifier-nitrification and associated nitrifier-denitrification). It seems that biochar does not promote the denitrification from NO⁻₂ (as was deduced from Experiment 3), but it does promote the oxidation of ammonia and concomitantly the formation of N₂O through nitrifier-nitrification. Clearly, if biochar raises the production of NO⁻₂ in soil, it will intrinsically enhance its denitrification (nitrifier-denitrification) when the soil is under low oxygen conditions (as in our experiments).

Our results are in agreement with recent findings by Prommer et al. (2014), who showed that biochar promotes soil ammonia-oxidizer populations and accelerates gross nitrification rates in a calcareous arable soil. The importance of nitrifier-nitrification and nitrifier-denitrification for N₂O production in calcareous soils has been recently documented by Huang et al. (2014), who demonstrated that these processes accounted for 35–53% and 44–58% of total N₂O emissions, respectively.

Here we present preliminary evidence that explains how biochar might affect N₂O emissions differently depending on the N₂O formation pathway operating in the soil. When denitrification was the main N₂O formation pathway (soil HP), biochar was found to decrease the N₂O/(N₂ + N₂O) ratio (Table 2), which is in agreement with previous findings (Cayuela et al., 2013). Recent studies have reported that biochar promotes an increase in the abundance of nitrous oxide reductase (nosZ) in soil (Harter et al., 2014), an enzyme that enhances the reduction of N₂O to N₂ (the last step in denitrification). In contrast, when N₂O was produced by nitrification (soil HC), biochar addition might have increased emissions by promoting gross nitrification. To our knowledge, there are not published studies explicitly relating to biochar and nitrification-N₂O production.

Another question that arises from this study is: why these two soils under identical experimental conditions follow different N₂O formation pathways, which we hypothesize might be linked to different soil microbial communities. In conclusion, predicting which N₂O formation pathway pre-dominates in a certain kind of soil will be necessary for guaranteeing the success of biochar as a N₂O mitigation strategy.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.