Nitrogen and oxygen isotope enrichment factors of nitrate at different denitrification rates in an agricultural soil

ABSTRACT

Quantitative evaluation of denitrification by the dual isotope approach, which measures the stable isotope ratios of nitrogen (δ¹⁵N) and oxygen (δ¹⁸O) in nitrate, has been hampered by the wide range of values reported for the ratio of enrichment factors for ¹⁵N and ¹⁸O (¹⁵ε and ¹⁸ε, respectively) during denitrification. The objectives of this study were to determine ¹⁵ε and ¹⁸ε values at different denitrification rates under controlled conditions, and to infer possible mechanisms by which the ¹⁸ε/¹⁵ε ratio is influenced under different conditions. Column experiments were conducted at 25, 15, and 10°C, which enabled determination of ¹⁵ε and ¹⁸ε at different denitrification rates, in the absence of nitrate replenishment from ammonium oxidation and other sources. The values of ¹⁵ε and ¹⁸ε ranged from −11.8 to −14.9‰ and from −8.4 to −15.9‰, respectively, with ¹⁵ε less sensitive to changes in the denitrification rates. The resultant ¹⁸ε/¹⁵ε ratio, ranging from 0.70 to 1.17, was close to the values reported for sediment incubations, and larger than those for groundwater systems. These results are consistent with the explanations that ¹⁸ε/¹⁵ε value itself is close to unity during denitrification, and that at smaller denitrification rates, concurrent reactions including re-oxidation of nitrite to nitrate lead to smaller apparent fractionation of ¹⁸O and smaller ¹⁸ε/¹⁵ε ratios. This suggests that while linear relationships between δ¹⁸O and δ¹⁵N give a strong evidence of denitrification, apparent ¹⁸ε/¹⁵ε values are site specific and depend on the ambient conditions. In evaluating denitrification in such systems, we suggest the use of ¹⁵ε in preference to ¹⁸ε because ¹⁵ε is less sensitive to denitrification rates.

KEY WORDS:

• Denitrification
• Isotope
• nitrogen cycle
• dual isotope method

1. Introduction

Nitrate (NO₃⁻) levels in the biosphere have been increasing in recent years due to the use of fertilizers and combustion of fossil fuels (Galloway et al. 2004, 2008). Excess NO₃⁻ in terrestrial ecosystems can contribute to various environmental problems, such as nitrogen saturation in forests (Stoddard 1994), eutrophication in aquatic ecosystems (Vitousek and Howarth 1991), and the deterioration of drinking water quality, and has prompted many studies of NO₃⁻ dynamics in terrestrial ecosystems.

Denitrification is a microbial process that can decrease terrestrial NO₃⁻ levels by reduction via nitrite (NO₂⁻) and nitrous oxide (N₂O) to dinitrogen (N₂) under anaerobic conditions. The identification and quantitative evaluation of denitrification processes is crucial for predicting the fate of NO₃⁻ in terrestrial ecosystems. In the last few decades, the dual isotope approach, which measures stable isotope ratios of nitrogen and oxygen in NO₃⁻ (designated by δ¹⁵N_NO3 and δ¹⁸O_NO3, respectively), has been used to investigate denitrification processes and their role in nitrogen cycles in terrestrial ecosystems (e.g., Campbell et al. 2002; Ohte et al. 2004; Panno et al. 2006; Kendall et al. 2007; Osaka et al. 2010). The approach is based on the observations that NO₃⁻ derived from different sources have different values of δ¹⁵N_NO3 and δ¹⁸O_NO3, and that there is a simultaneous increase in δ¹⁵N_NO3 and δ¹⁸O_NO3 values during denitrification process (Kendall et al. 2007), as expressed by the Rayleigh equations:

(1)

(2)

where C is the NO₃⁻ concentration in the solution (g N m⁻³), Δδ¹⁵N_NO3 and Δδ¹⁸O_NO3 are respective changes in δ¹⁵N_NO3 and δ¹⁸O_NO3 from the values at an arbitrarily chosen reference point where C = C₀. The enrichment factors for ¹⁵N and ¹⁸O during denitrification are designated by ¹⁵ε and ¹⁸ε, respectively. A positive constant Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 ratio observed in a spatial or temporal sequence is indicative of the presence of denitrification, with Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 = ¹⁸ε/¹⁵ε if Δδ¹⁸O_NO3 and Δδ¹⁵N_NO3 follow Eq. (1) and (2). The method has enabled the identification of denitrification as well as NO₃⁻ sources in the environment, where the mixing of NO₃⁻ from different sources with different δ¹⁵N_NO3 values could also be responsible for the spatial variation of δ¹⁵N_NO3.

A challenge in the quantitative evaluation of denitrification by the dual isotope method is that a wide range of values, ranging from 0.48 to 0.76, have been reported for the Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 (and hence ¹⁸ε/¹⁵ε) ratio for denitrification (Böttcher et al. 1990; Aravena and Robertson, 1998; Mengis et al. 1999; Fukada et al. 2003; Panno et al. 2006). Because of the large volume of sample solutions required for δ¹⁵N_NO3 and δ¹⁸O_NO3 analyses in earlier studies, the values cited above were based on watershed-scale field observations. In addition, δ¹⁸O_NO3 values determined in the earlier studies were potentially biased because of the exchange of oxygen with glassware or contamination by oxygen-bearing contaminants in silver nitrate (Revesz and BöHlke 2002; Kendall et al. 2007).

The development of the denitrifier method for measuring isotopic compositions of NO₃⁻ (Sigman et al. 2001; Casciotti et al. 2002) has enabled accurate determination of δ¹⁵N_NO3 and δ¹⁸O_NO3 in much smaller samples than before. Using the denitrifier method, Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 values for denitrification at watershed scales were reported to range from 0.50 to 0.76 (Lohse et al. 2013; Wexler et al. 2014; Ji et al. 2017). Granger et al. (2008) reported that most of the denitrifiers they studied had Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 values close to unity during pure incubation in vitro. Using aquifer, lake and marine sediment materials, Carrey et al. (2013), Vidal-Gavilan et al. (2013), Carrey et al. (2014), and Kessler et al. (2014) investigated isotopic fractionations during denitrification in laboratory columns, and reported ¹⁸ε/¹⁵ε values of 0.88–1.04, 0.68–0.88, 0.99, and 0.78, respectively. These values were larger than those reported from field observations, and reinforced the necessity to determine the ¹⁸ε/¹⁵ε values under a wider range of experimental conditions and to identify the factors affecting the apparent fractionation.

A plausible explanation for the different ¹⁸ε/¹⁵ε values between field observation and laboratory incubation is that isotope ratios at watershed scales are likely to be affected by N transformation processes other than denitrification, and could lead to an erroneous estimate of ¹⁸ε/¹⁵ε for denitrification. Osaka et al. (2010) suggested that the concurrence of nitrification and denitrification led to Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 values in the groundwater of forested watersheds lower than those expected when denitrification alone was occurring. Another plausible explanation is the differences in the denitrification rate in the field and laboratory incubation. Mariotti et al. (1988) reported that ¹⁵ε in denitrification is influenced by the denitrification rate, with a larger fractionation (i.e., more negative ¹⁵ε) expected for a smaller denitrification rate.

Despite the importance of accurate measurements of ¹⁸ε/¹⁵ε values in quantitative evaluation of denitrification at field scales, there have been a limited number of studies which investigate how the ¹⁸ε/¹⁵ε value varies under different conditions. In the present study, we hypothesized that denitrification rates could exert influences of different magnitudes on ¹⁵ε and ¹⁸ε, and that this could partly explain variations of ¹⁸ε/¹⁵ε values under different conditions. The objectives of the study were (i) to determine the enrichment factors ¹⁵ε and ¹⁸ε at different denitrification rates in soil column experiments in the absence of nitrification or mixing of NO₃^– from different sources, and (ii) to infer possible mechanisms by which the Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 ratio are influenced by denitrification rates. Implication of the results in the interpretation of field-measured ¹⁸ε/¹⁵ε values is also discussed.

2. Materials and methods

2.1. Soil

The soil samples used in this study were collected from the topsoil layer (0–15 cm depth) of a paddy field at the Central Region Agricultural Research Center, National Agriculture and Food Research Organization, Joetsu, Japan (37°06′58′′ N, 138°15′30′′ E). The light clayey soil, having sand (>0.02 mm), silt (2–20 μm), and clay (<2 μm) contents of 0.265, 0.382, and 0.353 kg kg⁻¹, respectively, was classified as montmorillonitic, mesic, Typic Epiaquept (Soil Survey Staff 1992). The field had received 55 kg N ha⁻¹yr⁻¹ of ammonium chloride and 15 Mg ha⁻¹yr⁻¹ of rice straw compost for more than 10 years. The total nitrogen and carbon contents of the soil were 3.1 and 30.2 g kg⁻¹, respectively, and the δ¹⁵N value of the total nitrogen was 0.7‰. The soil was air dried at room temperature, sieved with a 2.0-mm screen, and used for subsequent experiments.

2.2. Column denitrification experiments

Laboratory denitrification experiments were conducted to determine the isotope enrichment factors (¹⁸ε and ¹⁵ε) from the measurement of δ¹⁵N_NO3, δ¹⁸O_NO3, and NO₃⁻ concentration profiles in the pore water in soil columns, to which a solution containing NO₃⁻ was continuously loaded. The experiments were conducted at different ambient temperatures to examine possible effects of denitrification rates on isotope fractionation.

Two series of experiments, Ex-1 and Ex-2, were conducted using soil columns of different lengths. In Ex-1, the ambient temperature was maintained at 25°C, and a 15-cm length column with an inside diameter of 5.0 cm (Column A in Fig. 1) was used. In Ex-2, the ambient temperature was lower, and was initially set at 15°C. After loading NO₃⁻-containing solution for 216 h, the temperature was lowered to 10°C. A longer, 24-cm column with the same inside diameter (Column B in Fig. 1) enabled a longer period of observation of slower denitrification processes. Both columns were equipped with sampling ports from which porous plastic water samplers (0.25 cm in diameter and 4.5 cm long; Rhizosphere Research Products, Wageningen, The Netherlands) were inserted to allow the collection of pore water at different distances from the column inlet using 5.0-mL plastic syringes. The columns were placed in a thermostatic chamber to control the ambient temperature.

Figure 1. Setup of laboratory soil columns used in denitrification experiments. The calcium nitrate solution was loaded from the inlet and pushed upward by a micro-tube pump. The columns were equipped with sampling ports for the collection of pore water with porous plastic tubes. Columns A and B were used in Experiments 1 and 2, respectively.

After packing air-dried soil into columns to a bulk density of 1.06–1.10 Mg m⁻³, a culture solution containing approximately 50 mg N L⁻¹ of calcium nitrate and 100 mg L⁻¹ of 2-amino-4-chloro-6-methylpyrimidine (AM), added as a nitrification inhibitor, was injected to the columns in the upward direction using a microtube pump (Fig. 1). The initial NO₃⁻ concentration of approximately 50 mg N L⁻¹ was within the range typically found in agricultural soils. The AM interferes with the oxidation of NH₄⁺ to NO₂⁻ by Nitrosomonas sp. (Prasad et al. 1971). The solution was supplied from a 20-L polyethylene tank through a Tygon® tube to the bottom of the columns at a constant flux of 0.60–0.66 cm h⁻¹ (Table 1). This water flux was chosen such that there was sufficient residence time in the columns for the changes in the NO₃⁻ concentration as well as δ¹⁵N_NO3 and δ¹⁸O_NO3 values to be easily detected while minimizing confounding effects of NO₃⁻ diffusion on the isotopic values (Lehmann et al. 2003). Near-saturated conditions developed in the columns approximately 24 and 36 h after commencing the loading of the solution in Ex-1 and Ex-2, respectively. About 5 mL of pore water was collected from the sampling ports of the columns, located at 3, 6, 9, and 12 cm (Ex-1), and 2, 7, 12, 17, and 22 cm (Ex-2) distance from the column inlet, at elapsed times of t = 118, 165, and 216 h in Ex-1 and t = 121, 192, and 314 h in Ex-2. A time interval of 98 h between the switch of temperature and the third sampling in Ex-2 was large enough to make the solution in the columns completely displaced so that the effects of the earlier temperature did not persist at the third sampling. The column experiments were conducted in triplicate (Columns 1–3 in Ex-1, and Columns 4–6 in Ex-2).

Table 1. Summary of column denitrification experiments.

Column number	Elapsed time (h)	Temperature (°C)	Denitrification rate (mg N kg^−1 h^−1)	First-order rate constant k1 (h^−1)	Enrichment factor ^15ε (‰)	r^2	Enrichment factor ^18ε (‰)	r^2	^18ε/^15ε	Water flux (cm h^−1)
1	118	25	1.07	0.072	−14.9	1.00	−14.1	0.98	0.95	0.63
	165	25	1.15	0.083	−14.3	1.00	−13.7	1.00	0.96	0.62
	216	25	1.10	0.074	−13.5	1.00	−15.9	0.99	1.17	0.63
2	118	25	1.00	0.063	−14.4	1.00	−13.8	0.99	0.96	0.66
	165	25	1.08	0.072	−14.0	1.00	−13.3	0.99	0.95	0.64
	216	25	1.01	0.065	−14.5	1.00	−14.8	0.99	1.01	0.64
3	118	25	1.00	0.063	−12.9	1.00	−12.7	0.98	0.98	0.62
	165	25	1.10	0.073	−13.6	1.00	−13.4	1.00	0.98	0.64
	216	25	1.03	0.064	−14.1	0.99	−14.6	1.00	1.01	0.65
4	121	15	0.43	0.022	−11.8	0.99	−8.4	0.98	0.71	0.60
	192	15	0.45	0.022	−13.2	1.00	−10.9	0.96	0.83	0.63
	314	10	0.28	0.013	−13.0	1.00	−9.0	0.95	0.70	0.66
5	121	15	0.48	0.025	−12.2	1.00	−10.4	0.97	0.85	0.65
	192	15	0.49	0.025	−13.3	1.00	−9.9	0.97	0.75	0.65
	314	10	0.39	0.018	−13.4	1.00	−11.4	0.99	0.84	0.65
6	121	15	0.52	0.025	−12.5	1.00	−9.5	1.00	0.76	0.62
	192	15	0.52	0.027	−12.9	0.99	−10.2	0.97	0.80	0.64
	314	10	0.41	0.019	−12.6	0.99	−11.3	0.97	0.86	0.65

The collected pore water was filtered through a 0.20-μm cellulose acetate filter (25CS020AS; Advantec, Tokyo, Japan) and stored in polypropylene tubes at −25°C until analysis for dissolved ion concentrations and stable isotope ratios. The δ¹⁵N_NO3 and δ¹⁸O_NO3 values were determined by the denitrifier method (Sigman et al. 2001; Casciotti et al. 2002) using isotope ratio mass spectrometry (Delta V, Thermo Fisher Scientific, Waltham, MA, U.S.A). The dissolved NO₃⁻ concentration was determined with an ion chromatograph (Dionex DXi-500, Thermo Fisher Scientific).

2.3. Determination of NO₃⁻ consumption rate and nitrogen and oxygen enrichment factors

The average NO₃⁻ consumption rate per unit mass of soil, J (g N kg⁻¹ h⁻¹), during steady-state water flow was calculated for each column from

(3)

where C_in and C_out are NO₃⁻ concentrations (g N m⁻³) at the inlet and outlet of the column, respectively, q is the water flux density (m h⁻¹), A is the cross-sectional area of the column (m²), and m is the mass of dry soil (kg) in the column. Assuming a first-order reaction, the rate constant for denitrification k₁ (h⁻¹) is given by:

(4)

where t_L is the travel time of the solution through the column (h), calculated from the relationship

(5)

where L is the length of the column (m), and θ is the average volumetric soil water content (m³ m⁻³) in the column. In a saturated soil, θ is equal to the total porosity of soil, and calculated from

(6)

where ρ_s is the particle density of soil, taken as 2.60 × 10³ kg m⁻³ in the present study.

For each column, Δδ¹⁵N_NO3 and Δδ¹⁸O_NO3 of the pore water samples (i.e., changes in δ¹⁵N_NO3 and δ¹⁸O_NO3 from those in the incoming solution) were plotted against ln ([NO₃⁻]/[NO₃⁻]_ini), where [NO₃⁻] and [NO₃⁻]_ini are NO₃⁻ concentrations in the pore water and the incoming solution, respectively. The enrichment factors ¹⁵ε and ¹⁸ε were obtained as the slopes of the linear regression lines for Δδ¹⁵N_NO3 and Δδ¹⁸O_NO3, respectively, with C/C₀ = [NO₃⁻]/[NO₃⁻]_ini in Eq. (1) and (2). In calculating ¹⁵ε and ¹⁸ε, we assumed that NO₃⁻ diffusion had negligible effects on the distribution of NO₃⁻ concentration and isotope compositions in the columns. In our column experiments, the Peclet number (Smedt and Wierenga 1979), defined as Lu/D where L is the column length, u (= q/θ) is the local flow velocity, and D is the NO₃⁻ diffusion coefficient (= 1.90 × 10⁻⁵ cm² s⁻¹), was as high as 233–394, showing the predominance of convective transport in the NO₃⁻ transport in the columns.

3. Results

3.1. Nitrate consumption process in the columns

With increasing distance from the inlet of the columns, there were gradual decreases in NO₃⁻ concentration, accompanied by simultaneous increases in δ¹⁵N_NO3 and δ¹⁸O_NO3 (Fig. 2). Concentration of NO₂⁻ was below the limit of quantitation. The NO₃⁻ consumption rates calculated with Eq. (3) were in the range of 1.00–1.15, 0.43–0.52, and 0.28–0.41 mg N kg⁻¹ h⁻¹ at the ambient temperatures of 25°C, 15°C, and 10°C, respectively. Steady-state NO₃⁻ consumption was established before t = 118 h in Ex-1, and t = 121 h in Ex-2 (at 15°C). With further passage of time, only small changes in the NO₃⁻ concentration and δ¹⁵N_NO3 and δ¹⁸O_NO3 values were observed, and the resultant NO₃⁻ consumption depended on the temperature only. We presume that steady-state NO₃⁻ consumption had also been established in Ex-2 before the solution sampling at t = 314 h, which was conducted 98 h after resetting the temperature from 15°C to 10°C.

Figure 2. Profiles of (a) NO₃⁻ concentration and (b) δ¹⁵N_NO3 and (c) δ¹⁸O_NO3 values at different elapsed times in the columns.

3.2. Nitrogen and oxygen enrichment factors and Δδ¹⁸o_No3/Δδ¹⁵n_No3 in denitrification

Fig. 3 shows plots of Δδ¹⁵N_NO3 and Δδ¹⁸O_NO3 against ln ([NO₃⁻]/[NO₃⁻]_ini) at different locations in the columns. The values of ¹⁵ε and ¹⁸ε for each column, obtained from linear regression analyses using Eq. (1) and (2), ranged from −11.8 to −14.9‰ and from −8.4 to −15.9‰, respectively (Table 1). The ¹⁸ε values were less negative (i.e., smaller fractionation) at lower ambient temperatures with a smaller denitrification rate, while ¹⁵ε values were relatively insensitive to the ambient temperature (Table 1, Fig. 3). Fig. 4 presents the relationships between Δδ¹⁵N_NO3 and Δδ ¹⁸O_NO3 at different ambient temperatures. The difference in the temperature sensitivity between ¹⁸ε and ¹⁵ε resulted in a smaller Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 at low ambient temperatures with lower denitrification rates. In Fig. 5, the isotopic enrichment factors ¹⁵ε and ¹⁸ε and the ratio ¹⁸ε/¹⁵ε are plotted against the denitrification rate constant k₁ for different ambient temperatures. At ambient temperatures of 25, 15, and 10°C, with the average k₁ of 0.066, 0.025, and 0.016, the average ¹⁸ε/¹⁵ε was 1.00, 0.78, and 0.80, respectively.

Figure 3. Relationships between (a) Δδ¹⁵N_NO3 and (b) Δδ¹⁸O_NO3 and the natural logarithms of NO₃⁻ concentration, normalized with respect to that in the incoming solution, in the column experiments. Δδ¹⁵N_NO3 and Δδ¹⁸O_NO3 are, respectively, the changes in δ¹⁵N_NO3 and δ¹⁸O _NO3 from their initial values in the solution fed to the columns. The broken and dotted lines represent the relationships expected for isotopic enrichment factors of nitrogen (¹⁵ε) and oxygen (¹⁸ε) of −5, −10, −15, and −20 ‰.

Figure 4. Relationships between Δδ¹⁵N_NO3 and Δδ ¹⁸O_NO3 at different ambient temperatures in the column experiments. Δδ¹⁵N_NO3 and Δδ¹⁸O_NO3 are, respectively, the changes in δ¹⁵N_NO3 and δ¹⁸O _NO3 from their initial values in the solution fed to the columns. The broken and dotted lines represent the relationships expected for enrichment factor ratio ¹⁸ε/¹⁵ε of 0.6, 0.8, 1.0, and 1.2.

Figure 5. Relationships between the denitrification rate constant k₁ and (a) isotopic enrichment factors for nitrogen (¹⁵ε) and oxygen (¹⁸ε) and (b) the ratio ¹⁸ε/¹⁵ε in the column experiments.

4. Discussion

The ¹⁵ε and ¹⁸ε values obtained in this study were in the middle range of the values reported in the literature. Their dependence on denitrification rates as revealed by the column experiments at different ambient temperatures suggests the reasons why a wide range of ¹⁸ε/¹⁵ε values have been reported in the literature. The reported ¹⁵ε values ranged from −4.7 to −30‰ in groundwater systems (Vogel et al. 1981; Mariotti et al. 1988; Böttcher et al. 1990; Mengis et al. 1999; Fukada et al. 2003; Wexler et al. 2014; Ji et al. 2017), from −11.65 to −34.2‰ in incubated soils and sediments (Blackmer and Bremner 1977; Mariotti et al. 1981; Grischek et al. 1998; Carrey et al. 2013; Vidal-Gavilan et al. 2013; Kessler et al. 2014), and from −4.7 to −39.8 ± 4.8‰ in pure microbe incubations (Barford et al. 1999; Granger et al. 2008; Frey et al. 2014; Hosono et al. 2015; Treibergs and Granger 2017).

Mariotti et al. (1988) argued that during denitrification, a larger fractionation (i.e., more negative ¹⁵ε) is expected for a smaller denitrification rate. In the present study, where the first-order rate constant of denitrification k₁ ranged from 0.013 to 0.083, ¹⁵ε values were in a narrow range of −11.8 to −14.9‰, and relatively insensitive to the denitrification rates. We note that the variation of ¹⁵ε values in the present study was smaller than those reported by Mariotti et al. (1988) for smaller denitrification rates with comparable values of k₁. The narrow range of ¹⁵ε values in the present study could be partly due to suppression of NH₄⁺ oxidation by the addition of the nitrification inhibitor AM, which should minimize confounding effects of NO₃⁻ replenished from mineralized soil N having various δ¹⁵N values different from that in the incoming NO₃⁻ solution (≈−2.9‰).

When early studies suffering from possible uncertainty of δ¹⁸O_NO3 measurement were excluded, ¹⁸ε values reported in the literature range from −4.8 ± 0.1 to −31.8 ± 4.1‰ in pure microbe incubations (Granger et al. 2008; Frey et al. 2014; Hosono et al. 2015; Treibergs and Granger 2017). The ¹⁸ε values in the present study were in a range of −8.4 to −15.9‰, and in close agreement with the values of −8.9 to −15.1‰ in incubated sediments reported by Carrey et al. (2013), Vidal-Gavilan et al. (2013) and Kessler et al. (2014). The consequent ¹⁸ε/¹⁵ε values in this study, ranging from 0.70 to 1.17 (Table 1), were larger than the values of 0.50–0.76 reported for groundwater and soilwater systems (Lohse et al. 2013; Wexler et al. 2014; Ji et al. 2017), and closer to the values of 0.68–0.88 observed in sediment incubations (Carrey et al. 2013; Vidal-Gavilan et al. 2013; Kessler et al. 2014), and 0.57–1.02 reported for a pure microbe incubation (Granger et al. 2008).

A plausible explanation for the variations in ¹⁸ε/¹⁵ε values under different conditions, including different temperatures in this study, is that in systems with a low denitrification rate, other processes that could decrease δ¹⁸O_NO3 were operating concurrently with denitrification. Osaka et al. (2010) suggested that if some of NO₃⁻ present in the system was derived from re-oxidation of NO₂⁻ produced by denitrifying bacteria, the observed Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 would be smaller than if denitrification was the only process occurring. This is because bacterial oxidation of NO₂⁻ to NO₃⁻ involves the uptake of an additional oxygen atom from water (DiSpirito and Hooper 1986), which often has lower δ¹⁸O_NO3 value than the original NO₃⁻ and could result in a smaller apparent fractionation during the whole process. Conducting anoxic sediment incubation experiments with nitrate-reduce microorganisms using ¹⁸O-enriched water, Wunderlich et al. (2013) found that up to 5.7% of the oxygen atoms in residual dissolved NO₃⁻ was exchanged by oxygen atom from ambient water, showing simultaneous occurrence of NO₂⁻ re-oxidation and denitrification. In reviewing nitrogen and oxygen isotopic studies of marine nitrogen cycles, Casciotti (2016) and concluded that simultaneous occurrence of NO₂⁻ re-oxidation and denitrification can either increase or decrease δ¹⁸O_NO3 values, relative to those in the absence of NO₂⁻ re-oxidation, depending on the relative δ¹⁸O values of NO₂⁻ and NO₃⁻. In the present study, the nitrification inhibitor AM interferes with the oxidation of NH₄⁺ to NO₂⁻, but not the oxidation of NO₂⁻ to NO₃⁻. It is plausible that this allowed simultaneous occurrence of reduction of NO₃⁻ to NO₂⁻ and re-oxidation of NO₂⁻ to NO₃⁻.

Granger and Wankel (2016) conducted numerical modeling studies to explore the influence of the δ¹⁸O value of water (δ¹⁸O_H2O) and relative rates of NO₂⁻ re-oxidation and NO₃⁻ reduction on Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 values during denitrification. They reported that with high rates of NO₂⁻ re-oxidation relative to NO₃⁻ reduction together with high δ¹⁸O_H2O values typical of seawater, Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 values were higher than unity expected for denitrification in the absence of NO₂⁻ re-oxidation. In contrast, with lower NO₂⁻ re-oxidation rates and lower δ¹⁸O_H2O values typical of terrestrial freshwater systems, Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 values were lower than unity. In marine sediment incubation, Dähnke and Thamdrup (2016) found Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 values smaller than unity. The degree of deviation of Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 from unity was more pronounced in sediments in which the ratio of NO₂⁻ pool to the total of NO₂⁻ and NO₃⁻ pools was larger, indicating the influence of NO₂⁻ re-oxidation on the observed Δδ¹⁵N_NO3/Δδ¹⁸O_NO3. In the present study, δ¹⁸O_H2O value of the solution in column experiments was estimate to be within a range of −7 to −10‰, values reported for freshwater in the region where the experiments were conducted (Mizota and Kusakabe 1994). For δ¹⁸O_H2O values within this range, Granger and Wankel (2016) showed the possibility that Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 lower than unity can be expected from the re-oxidation of NO₂⁻ produced by denitrifying bacteria.

A slower denitrification coupled with a slower decomposition of substrates in the columns should be accompanied by less vigorous consumption of dissolved O₂ in the incoming solution, leaving more time for the re-oxidation of NO₂⁻ to NO₃⁻. Although the accumulation of NO₂⁻ was not detected in the column experiment, this could be due to immediate re-oxidation of NO₂⁻. A smaller Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 (and ¹⁸ε/¹⁵ε) is thus expected with a decrease of the denitrification rate.

Concurrent reduction of NO₃⁻ to NO₂⁻ and re-oxidation of NO₂⁻ to NO₃⁻ may also explain why larger ¹⁸ε/¹⁵ε values have been reported in laboratory experiments as compared with those found in the fields. Denitrification rates in laboratory experiments are often much larger than those observed in field groundwater systems. This was the case in our column experiments, where the denitrification rates were in the range of 0.28–1.15 mg N kg⁻¹ h⁻¹ (with the first-order rate constant k₁ of 0.012–0.078). The ¹⁸ε/¹⁵ε values of 0.70 to 1.17 in the present study, larger than those observed in groundwater systems, were likely due to higher denitrification rates relative to re-oxidation of NO₂⁻ to NO₃⁻. As discussed above, the dependence of ¹⁸ε/¹⁵ε on the denitrification rates was likely the result of smaller apparent fractionation of oxygen when denitrification and re-oxidation of NO₂⁻ were occurring simultaneously. Oxidation of NH₄⁺ to NO₂⁻ by ammonium oxidizing bacteria occurring in field groundwater could also contribute to lowering the apparent δ¹⁸O_NO3 values, while it also affects δ¹⁵N_NO3 values and could have confounding effects on observed Δδ¹⁸O_NO3/Δδ¹⁵N_NO3.

Alternatively, different denitrifying bacteria could be responsible for the different ¹⁸ε/¹⁵ε values. Granger et al. (2008) observed different ¹⁸ε/¹⁵ε values for denitrification by different denitrifying bacteria, and hypothesized that ¹⁸ε/¹⁵ε depends on the type of nitrate reductase of individual strains. Treibergs and Granger (2017) determined enzymatic N and O isotope effects during NO₃⁻ reduction in vivo, and reported ¹⁸ε/¹⁵ε of 0.57 for Nap isotope effects and ¹⁸ε/¹⁵ε of ≈ 1 for Nar and eukNR. This may explain the apparent discrepancies of ¹⁸ε/¹⁵ε values among different studies, but the explanation is not applicable to our column experiments where the same soil was used.

From the results of our column experiments together with the discussion above, we conclude that the ¹⁸ε/¹⁵ε value itself is close to unity during denitrification. Lower ¹⁸ε/¹⁵ε values reported in earlier studies as well as those observed at low ambient temperatures in the current study were likely due to concurrent reactions including re-oxidation of NO₂⁻ to NO₃⁻, which lowered observed ¹⁸ε values. We expect high ¹⁸ε/¹⁵ε values when denitrification rates are large enough to negate the influence of other reactions on the isotope ratios as well as in the systems with a negligible supply of O₂. Linear relationships between δ¹⁸O_NO3 and δ¹⁵N_NO3 frequently observed in the fields are a strong evidence of denitrification, but the Δδ¹⁸O_NO3/Δδ¹⁵N_NO3 ratio should depend on the ambient conditions which exert influences on the observed ¹⁸ε and ¹⁵ε values. The apparent ¹⁸ε/¹⁵ε values are thus site-specific. In a system where mixing effects on δ¹⁵N_NO3 from multiple NO₃⁻ sources are negligible, we suggest the use of ¹⁵ε for estimating denitrification because ¹⁵ε is much less sensitive to denitrification rates than ¹⁸ε.

Acknowledgments

We thank Drs. Satoshi Ohno and Tetsuo Sekiguchi of the Central Region Agricultural Research Center, who kindly provided the soil samples used in this study, and Mrs. Hiromi Gouhara and Mrs. Emiko Sakurai for their assistance with the chemical analysis. We also acknowledge the support and suggestions provided by the members of the Institute for Agro-Environmental Sciences, National Agriculture and Food Research Organization.