Nitrogen and Oxygen Isotope Signatures of Nitrogen Compounds during Anammox in the Laboratory and a Wastewater Treatment Plant

Abstract

Isotopic fractionation factors against ¹⁵N and ¹⁸O during anammox (anaerobic ammonia oxidization by nitrite) are critical for evaluating the importance of this process in natural environments. We performed batch incubation experiments with an anammox-dominated biomass to investigate nitrogen (N) and oxygen (O) isotopic fractionation factors during anammox and also examined apparent isotope fractionation factors during anammox in an actual wastewater treatment plant. We conducted one incubation experiment with high δ¹⁸O of water to investigate the effects of water δ¹⁸O. The N isotopic fractionation factors estimated from incubation experiments and the wastewater treatment plant were similar to previous values. We also found that the N isotopic effect (¹⁵ε_NXR of –77.8 to –65.9‰ and ¹⁵Δ_NXR of –31.3 to –30.4‰) and possibly O isotopic effect (¹⁸ε_NXR of –20.6‰) for anaerobic nitrite oxidation to nitrate were inverse. We applied the estimated isotopic fractionation factors to the ordinary differential equation model to clarify whether anammox induces deviations in the δ¹⁸O vs δ¹⁵N of nitrate from a linear trajectory of 1, similar to heterotrophic denitrification. Although this deviation has been attributed to nitrite oxidation, the O isotopic fractionation factor for anammox is crucial for obtaining a more detailed understanding of the mechanisms controlling this deviation. In our model, anammox induced the trajectory of the δ¹⁸O vs δ¹⁵N of nitrate during denitrification to less than one, which strongly indicates that this deviation is evidence of nitrite oxidation by anammox under denitrifying conditions.

Anammox (anaerobic ammonia oxidization by nitrite) has been intensively investigated since the discovery of its importance as a N removal process in natural ecosystems (Dalsgaard et al., 2003; Kuypers et al., 2003). The rates of anammox and denitrification are frequently similar (Kuypers et al., 2003; Hamersley et al., 2007; Lam et al., 2009). The detection of anammox in ecosystems is key for further investigations on the relative (quantitative) importance of anammox and denitrification to N losses. Molecular techniques, such as qPCR (Hamasaki et al., 2018) and biomarker analyses (ladderane lipids; Jaeschke et al., 2007), have generally been applied to detect anammox bacteria, followed by ¹⁵N tracer experiments (Amano et al., 2007) to assess anammox activities (rates) in the laboratory. Although this approach is promising, it only estimates potential anammox rates. Thus, it is crucial to develop screening techniques that estimate anammox in the field.

The naturally occurring stable isotope ratios of N (¹⁵N / ¹⁴N, expressed as δ¹⁵N) and O (¹⁸O / ¹⁶O, expressed as δ¹⁸O) are useful tracers for investigating the origins, transport, and biogeochemical processes of dissolved inorganic N (DIN), such as nitrate (NO₃^–), nitrite (NO₂^–), and ammonium (NH₄⁺), in ecosystems (Casciotti, 2016a, 2016b; Denk et al., 2017; Thuan et al., 2018). Regarding the use of δ¹⁵N and δ¹⁸O to interpret the complex dynamics of DIN, it is essential to apply the isotopic fractionation factors of specific reactions of DIN production and consumption. Previous studies on heterotrophic denitrification estimated ¹⁵N (Blackmer and Bremner, 1977; Chien et al., 1977; Mariotti et al., 1981, 1982; Bryan et al., 1983; Kawanishi et al., 1993; Barford et al., 2017) and ¹⁸O fractionation factors (Böttcher et al., 1990; Granger et al., 2008; Kritee et al., 2012; Frey et al., 2014; Martin and Casciotti, 2016; Osaka et al., 2018; Wang et al., 2018). Detailed information on isotopic fractionation during denitrification has encouraged the use of the δ¹⁵N and δ¹⁸O of NO₃^– in investigations on the occurrence and magnitude of denitrification in many ecosystems (Mariotti et al., 1988; Koba et al., 1997; Ostrom et al., 2002; Lehmann et al., 2003; Sigman et al., 2003; Houlton et al., 2006; Houlton and Bai, 2009; Miyajima et al., 2009; Fang et al., 2015; Lennon and Houlton, 2017).

In contrast to denitrification, few studies have used the δ¹⁵N and δ¹⁸O of DIN to examine anammox (Prokopenko et al., 2006; Prokopenko et al., 2013; Wenk et al., 2014; Dähnke and Thamdrup, 2016), and only two studies (Brunner et al., 2013; Kobayashi et al., 2019) have reported isotopic fractionation factors for the anammox reaction. These factors must be known in order to estimate the importance of anammox in a studied ecosystem with the δ¹⁵N and δ¹⁸O of DIN. Brunner et al. (2013) estimated a large inverse N isotope effect (i.e., the heavier isotope, ¹⁵N, reacts faster than the lighter isotope, ¹⁴N) during NO₃^– production (by anaerobic nitrite oxidation) in anammox as well as a large normal (i.e., the lighter ¹⁴N reacts faster than the heavier ¹⁵N) isotope effect for ammonium oxidation, which was confirmed in a later study by Kobayashi et al. (2019). However, they only reported the combined ¹⁸O isotope fractionation factors and do not provide the isotope fractionation factors for the NO₂^– oxidation and its relevant oxygen atom incorporation from water, involved in the combined factors.

Studies on ¹⁵N and ¹⁸O fractionation factors revealed that NO₃^– consumption (the assimilatory and dissimilatory reduction of NO₃^–) generally induced a 1:1 increase in the δ¹⁸O and δ¹⁵N of NO₃^– (Granger et al., 2008; Granger et al., 2010; Karsh et al., 2012; Rohde et al., 2015; Osaka et al., 2018). This finding prompted the use of the δ¹⁸O and δ¹⁵N of NO₃^– to detect NO₃^– consumption in the actual ecosystem as well as investigations on NO₃^– isotope anomalies, specifically isotopic deviations from a slope of 1 in the δ¹⁸O vs δ¹⁵N of NO₃^– (Δ[15, 18]; Sigman et al., 2005), in order to deepen insights into NO₃^– dynamics (Casciotti et al., 2008; Casciotti and Buchwald, 2012; Bourbonnais et al., 2013; Peters et al., 2018; White et al., 2019). Granger and Wankel (2016) proposed that widely observed deviations in the δ¹⁸O vs δ¹⁵N of NO₃^– from the denitrification slope of 1 in freshwater systems (Sigman et al., 2005; Granger et al., 2008; Kritee et al., 2012) must result from concurrent NO₃^– production (nitrification or anammox) in the denitrifying system that has been largely overlooked. However, they lacked information on the ¹⁸O fractionation factor for anammox, and assumed that the ¹⁸O fractionation factor during NO₃^– production by anammox was similar to that for aerobic nitrite oxidation to NO₃^– by nitrifiers (nitrification). Thus, it is essential to investigate the ¹⁵N and ¹⁸O fractionation factors during anammox not only for the better use of the δ¹⁸O and δ¹⁵N of NO₃^– in anammox studies, but also to obtain a more detailed understanding of ¹⁵N and ¹⁸O fractionation.

We herein report unique data on ¹⁸O fractionation factors during anammox. We calculated apparent ¹⁵N and ¹⁸O fractionation factors with data collected from a wastewater treatment plant (WWTP) at which anammox reactors were installed at the final stage of treatment (Isaka et al., 2017). We also performed anaerobic laboratory incubations with an anammox-dominated biomass to obtain more information on isotopic fractionation during anammox. We conducted one incubation experiment with high δ¹⁸O of water to investigate the effects of water δ¹⁸O. We then simulated system behavior with the observed isotopic fractionation factors to establish whether deviations in the δ¹⁸O vs δ¹⁵N of NO₃^– from the denitrification slope of 1 may be used to detect anammox activity.

Materials and Methods

Full-scale anammox wastewater treatment plant

Influent and treated water in the full-scale anammox wastewater treatment plant (Isaka et al., 2017) were sampled three times (28th April, 7th and 12th May 2015). Detailed information on water chemistry and plant performance have been reported by Isaka et al. (2017). The anammox plant consists of a denitrifier reactor (DN), biochemical oxygen demand (BOD) oxidation reactor (BD), nitrite-nitrification reactor (NT), and anammox reactor (ANX). We collected water samples from each reactor (Fig. S1). The wastewater introduced into this anammox plant was effluent from an ammonia plant, which was pure water containing mainly NH₄⁺ and methanol. Average NH₄⁺ and total organic concentrations were 658 and 37‍ ‍mg L^–1, respectively (Isaka et al., 2017). Solutions were sampled from these reactors (Fig. S1) to measure the concentrations and isotope ratios of DIN. Samples (10‍ ‍mL each) were immediately filtered through 0.45-μm disk filters (25CS045AN; ADVANTEC Toyo Kaisha) and then collected in plastic centrifuge tubes. Samples were frozen until further measurements.

Biomass incubation experiments

We performed batch incubations with anammox bacteria. Details on a small-scale anammox reactor with activated sludge, including start-up information, maintenance, performance, input solutions, and microbial communities of the reactor, are provided in Supplemental Information (SI Text 1.1).

In the first experiment (Experiment A), the biomass in the reactor was sampled and incubated with the media used for the reactor, while the sampled biomass was re-suspended in fresh, chemically defined media in the second (Experiment B) and third (Experiment C) experiments. Difficulties were associated with performing incubation experiments with the anammox biomass for isotopic measurements, and, thus, we employed slightly different settings and operations to facilitate constant and active anammox reactions. In each experiment, 15‍ ‍mL of the biomass suspension in media solution was filtered with filter paper (Reeve Angel, Whatman) and differences in filter weights before and after filtration were used to calculate the suspended solid (S.S.) concentration after the filter had been oven-dried (at 105°C).

Experiment A

The biofilm and incubation media solution (500‍ ‍mL in total) were sampled from the incubation membrane in the anammox reactor (SI Text 1.1). The incubation was performed anaerobically in the glovebox at room temperature (25–30°C) after the purging of media by N₂ gas to remove dissolved oxygen (DO). pH, DO, and the concentrations of NH₄⁺ and NO₂^– were regularly measured to confirm the anammox activity of the biofilm, and pH (8.0) was maintained by adding KH₂PO₄ and Na₂HPO₄·12H₂O solution. After the addition of NaNO₂, (NH₄)₂SO₄, and NaHCO₃, we started the incubation and sampled 10‍ ‍mL of media. Sampled media were filtered with a 0.20-μm syringe filter and then split into three; one for NO₃^– followed by the removal of NO₂^– (Granger and Sigman, 2009), another one for NO₂^– with high pH by the addition of 2M NaOH solution to prevent oxygen atom exchange between NO₂^– and water (Bourbonnais et al., 2017), and one for NH₄⁺ with low pH by the addition of 4.8 M H₂SO₄ to prevent NH₄⁺ from volatilizing. These subsamples were frozen (–30°C) until further analyses.

Experiment B

The granule biomass that accumulated at the bottom of the anammox reactor was sampled. Granules were rinsed anaerobically with new media (N₂ purged) in the glovebox. Media consisted of NaHCO₃ 502‍ ‍mg L^–1; MgSO₄·7H₂O 603‍ ‍mg L^–1; CaCl₂ 180.5‍ ‍mg L^–1; KH₂PO₄ 169‍ ‍mg L^–1; Na₂HPO₄·12H₂O 282‍ ‍mg L^–1; trace elements of solution I (containing EDTA 6.369‍ ‍g L^–1; FeSO₄·7H₂O 9.14‍ ‍g L^–1) 0.5‍ ‍mL and solution II (containing EDTA 19.106‍ ‍g L^–1; ZnSO₄·7H₂O 0.43‍ ‍g L^–1; CoCl₂·6H₂O 0.24‍ ‍g L^–1; MnCl₂·4H₂O 0.99‍ ‍g L^–1; CuSO₄·5H₂O 0.25‍ ‍g L^–1; Na₂MoO₄·2H₂O 0.22‍ ‍g L^–1; NiCl₂·6H₂O 0.19‍ ‍g L^–1; Na₂SeO₄·10H₂O 0.21‍ ‍g L^–1; H₃BO₃ 0.014‍ ‍g L^–1) 0.5‍ ‍mL. We added NaNO₂ and (NH₄)₂SO₄ to media (500‍ ‍mL) with the anammox granules and then started the incubation at room temperature (25–30°C). We monitored pH (7.9 to 8.8) and NO₂^– to assess the progress of anammox. Sampling was performed as described in Experiment A.

Experiment C

We incubated the biofilm collected from the incubation membrane in the anammox reactor with the same media used in Experiment B; however, the δ¹⁸O of water (δ¹⁸O_H2O) was markedly higher (229‰) than that in Experiments A and B (–8‰). This “heavy” water was prepared by mixing ¹⁸O-labeled water (10% atom ¹⁸O) with Milli-Q water. During the incubation, media with biofilms were shaken at a constant temperature (30°C) and continuously purged with a gas mixture (95% Ar + 5% CO₂) to maintain low DO levels. pH ranged between 7.1 and 7.5 and the monitoring and sampling scheme was identical to Experiment B

Chemical analysis

DIN concentrations in water samples from the anammox plant and incubation experiments were measured using colorimetric methods with an autoanalyzer (Quatro, BL-Tec) (Thuan et al., 2018) after appropriate dilutions. In Experiment A, NH₄⁺ concentrations were measured during the incubation by the o-phthaldialdehyde (OPA) method (Holmes et al., 1999). The DO and pH of the incubation media were monitored during the incubation with a DO meter (HQ30d; Hach) and pH meter (D-71; Horiba).

δ¹⁵N and δ¹⁸O values were assessed by GC-IRMS (Sercon 20–22 with Cryoprep) (Thuan et al., 2018) with the denitrifier method (Sigman et al., 2001; Casciotti et al., 2002) for NO₃^– (δ¹⁵N_NO3– and δ¹⁸O_NO3–, respectively) with USGS 32, 34, 35, and IAEA-2 as standards, and with the azide method (McIlvin and Altabet, 2005) for NO₂^– (δ¹⁵N_NO2– and δ¹⁸O_NO2–, respectively) with TUAT-NO2-1 to TUAT-NO2-5 (Thuan et al., 2018) calibrated against N-23, N-7373, and N-10219 (Casciotti et al., 2007) as the standards. Analytical precision (expressed as the standard deviation of repeatedly measured samples) was ±0.2‰ for δ¹⁵N_NO2– and δ¹⁵N_NO3–, and ±0.5‰ for δ¹⁸O_NO2– and δ¹⁸O_NO3–. The δ¹⁵N values of NH₄⁺ (δ¹⁵N_NH4+) were evaluated using GC-IRMS with the denitrifier method after the conversion of NH₄⁺ to NO₃^– by persulfate oxidation (Koba et al., 2012; Thuan et al., 2018) with USGS 25, 26, and IAEA-N-2 as the standards. Analytical precision was ±0.5‰ for the δ¹⁵N of NH₄⁺. Water with high δ¹⁸O (229‰; measured by GC-IRMS with the modified azide method; McIlvin and Casciotti, 2006; Thuan et al., 2018) from Experiment C was used to prepare NO₃^– and NO₂^– isotope standards for NO₃^– and NO₂^– measurements in order to correct for the effects of oxygen atom incorporation during the analysis. δ¹⁵N and δ¹⁸O are expressed as (R_SampleN/R_Nitrogen)–1 and (R_SampleO/R_Oxygen)–1 where R_SampleN and R_SampleO are [¹⁵N/¹⁴N] and [¹⁸O/¹⁶O] of the sample, respectively, R_Nitrogen is [¹⁵N/¹⁴N] of atmospheric N₂ and R_Oxygen is [¹⁸O/¹⁶O] of Vienna Standard Mean Ocean Water (Table S1).

Calculation of apparent isotopic fractionation factors for the anammox plant

Apparent isotopic fractionation factors regarding anammox in the anammox plant were calculated as described by Kobayashi et al. (2019) based on steady-state, open-system isotope systematics reported by Fry (2006).

Apparent N isotope effects of the ammonium oxidation to N₂, and nitrite reduction and oxidation for the anammox plant

The ammonium oxidation to N₂ by NO₂^– has isotope fractionation defined as ¹⁵Δ_AMX. The δ¹⁵N of influx NH₄⁺ (δ¹⁵N_{NH4+_NT}), residual NH₄⁺ (δ¹⁵N_{NH4+_ANX}), and the fraction of NH₄⁺ reacting (f_NH4+) in ANX reactor (Fig. S1) at a steady state are used to estimate ¹⁵Δ_AMX (Fry, 2006; Kobayashi et al., 2019):

¹⁵Δ_AMX = (δ¹⁵N_{NH4+_ANX} – δ¹⁵N_{NH4+_NT}) / f_NH4+ --- eq. (1)

f_NH4+ = ([NH₄⁺]_NT – [NH₄⁺]_ANX) / [NH₄⁺]_NT

where [NH₄⁺]_NT and [NH₄⁺]_ANX are the NH₄⁺ concentrations in NT and ANX reactors, respectively.

The δ¹⁵N of NO₂^–, NO₃^–, and N₂ in ANX reactor (δ¹⁵N_{NO2–_ANX}, δ¹⁵N_{NO3–_ANX}, and δ¹⁵N_{N2_ANX}, respectively) at the steady state are given as follows (Fry, 2006; Kobayashi et al., 2019):

¹⁵Δ_AMXNIR = δ¹⁵N_{NO2–_ANX} – δ¹⁵N_{N2_ANX} --- eq. (2)

¹⁵Δ_NXR = δ¹⁵N_{NO2–_ANX} – δ¹⁵N_{NO3–_ANX} --- eq. (3)

where ¹⁵N fractionation for nitrite reduction in anammox and nitrite oxidation are ¹⁵Δ_AMXNIR and ¹⁵Δ_NXR, respectively.

δ¹⁵N_{NO2–_NT} is defined as:

δ¹⁵N_{NO2–_NT} = (1 – a – b) × δ¹⁵N_{N2_ANX} + a × δ¹⁵N_{NO2–_ANX} + b × δ¹⁵N_{NO3–_ANX} --- eq. (4)

with

a = [NO₂^–]_ANX / ([NO₂ ^– ]_NT – [NO₂^–]_ANX)

b = ([NO₃^–]_ANX – [NO₃^–]_NT) / ([NO₂^–]_NT – [NO₂^–]_ANX)

where δ¹⁵N_{NO2–_NT} is δ¹⁵N_NO2– in NT reactor and the concentrations of NO₂^– and NO₃^– in NT and ANX reactors are [NO₂^–]_NT, [NO₂^–]_ANX, [NO₃^–]_NT, and [NO₃^–]_ANX, respectively.

The combination of eqs. (2) and (4) gives

δ¹⁵N_{NO2–_NT} = (1 – a – b) × δ¹⁵N_{N2_ANX} + a × δ¹⁵N_{NO2–_ANX} + b × δ¹⁵N_{NO3–_ANX}

= (1 – a – b) × (δ¹⁵N_{NO2–_ANX} – ¹⁵Δ_AMXNIR) + a × δ¹⁵N_{NO2–_ANX} + b × δ¹⁵N_{NO3–_ANX}

= δ¹⁵N_{NO2–_ANX} – ¹⁵Δ_AMXNIR – a × δ¹⁵N_{NO2–_ANX}+ a × ¹⁵Δ_AMXNIR – b × δ¹⁵N_{NO2–_ANX} + b × ¹⁵Δ_AMXNIR+ a × δ¹⁵N_{NO2–_ANX} + b × δ¹⁵N_{NO3–_ANX}

= b × (δ¹⁵N_{NO3–_ANX} – δ¹⁵N_{NO2–_ANX}) + δ¹⁵N_{NO2–_ANX}+ ¹⁵Δ_AMXNIR × (a + b – 1)

= – (b × ¹⁵Δ_NXR) + δ¹⁵N_{NO2–_ANX} + ¹⁵Δ_AMXNIR × (a + b – 1)

¹⁵Δ_AMXNIR = [δ¹⁵N_{NO2–_ANX} – δ¹⁵N_{NO2–_NT} – b × ¹⁵Δ_NXR] / (a + b – 1) --- eq. (5)

Apparent combined O isotope effect of nitrite oxidation for the anammox plant

To calculate ¹⁸O fractionation during nitrite oxidation to nitrate, we followed the approach described by Kobayashi et al. (2019) to calculate combined isotope fractionation (¹⁸E_AMXcombined) because of the lack of detailed information on isotopic fractionation for the nitrite oxidation and oxygen atom incorporation during nitrite oxidation. Thus, we calculated ¹⁸E_AMXcombined as follows:

¹⁸E_AMXcombined = 2/3 δ¹⁸O_{NO2–_ANX} + 1/3 δ¹⁸O_H2O – δ¹⁸O_{NO3–_ANX} --- eq. (6)

where δ¹⁸O_{NO2–_ANX}, δ¹⁸O_H2O, and δ¹⁸O_{NO3–_ANX} are the ¹⁸O ratios of NO₂^–, water, and NO₃^– in ANX reactor, respectively.

Calculation of isotopic fractionation factors for incubations and a simulation with the dynamic model (the anammox model)

We developed an ordinary differential equation model as described by Casciotti and Buchwald (2012), Granger and Wankel (2016), and He and Bao (2019). We prepared the model (the anammox model) to calculate the isotopic fractionation factors for Experiments A, B, and C. The N transformations and associated isotopic fractionation (Fig. 1) were implemented in the anammox model with Berkeley Madonna (BM) software (Macey et al., 2000), with a 4th-order Runge–Kutta method for integration. We initially used the curve-fitting function in BM software (least squares fitting) to calculate the rate constant of the ammonium oxidation based on concentration data in each experiment. Isotopic fractionation factors and the exchange rate of oxygen atoms between water and NO₂^– were then estimated from isotopic data.

Fig. 1.

Schematic of the anammox and denitrification system. Dotted arrows indicate denitrification processes that were not included in the anammox model.

Fluxes regarding the anammox process (Fig. 1) are defined as

AMX = AMXNIR = k_AMO14N × [¹⁴NH₄⁺] --- eq. (7)

NXR = AMXNIR × (x / (1 – x)) --- eq. (8)

where AMX, NXR, and AMXNIR are the (¹⁴N) fluxes of ammonium oxidation, nitrite oxidation, and reduction by anammox (Fig. 1), k_AMO14N is the rate constant for AMX, and x is a stoichiometric ratio (increase in [NO₃^–]/decrease in [NO₂^–]) (Brunner et al., 2013). We omitted the two N transformation processes regarding denitrification (nitrate and nitrite reduction by denitrification, NAR, and DENNIR, respectively; Fig. 1) in the anammox model because of the small contributions of denitrifying bacteria to the total microbial community (Fig. S2) and the small contribution of denitrification of only 5–10% at most to the total N removal rate in this study (estimated by ¹⁵N tracer measurements; D. Ikeda, personal communications).

Regarding NO₂^–;

d/dt [¹⁴NO₂^–] = – NXR – AMXNIR --- eq. (9)

d/dt [¹⁵NO₂^–] = – (R_NitriteN × NXR / ¹⁵ε_NXR)– (R_NitriteN × AMXNIR / ¹⁵ε_AMXNIR) --- eq. (10)

d/dt [N¹⁶O₂^–] = – 2NXR – 2AMXNIR --- eq. (11)

d/dt [N¹⁶O¹⁸O^–] = – (R_NitriteO × 2NXR / ¹⁸ε_NXR)– (R_NitriteO × 2AMXNIR / ¹⁸ε_AMXNIR)– N¹⁶O¹⁸O^–_{exch_OUT} + N¹⁶O¹⁸O^–_{exch_IN}

= – (R_NitriteO × 2NXR / ¹⁸ε_NXR)– (R_NitriteO × 2AMXNIR / ¹⁸ε_AMXNIR)– k_exch × [N¹⁶O¹⁸O^–] + k_exch×R_WaterO / ¹⁸ε_EQ --- eq. (12)

where R_NitriteO, R_NitrateO, R_NitriteN, and R_NitrateN are the ¹⁸O/¹⁶O and ¹⁵N/¹⁴N of [NO₂^–] and [NO₃^–], respectively. R_WaterO is the [¹⁸O/¹⁶O] of H₂O. ¹⁵ε_NXR and ¹⁵ε_AMXNIR are the ¹⁵N fractionation factors of NXR and AMXNIR. ¹⁸ε_NXR and ¹⁸ε_AMXNIR are the ¹⁸O fractionation factors of NXR and AMXNIR, respectively. ¹⁸ε_EQ, the ¹⁸O fractionation factor of the equilibration between NO₂^– and H₂O, was set at 13‰ in the present study based on the incubation temperature and pH (Table S1; Buchwald and Casciotti, 2013). We applied k_exch (rate coefficient for oxygen atom exchange), N¹⁶O¹⁸O^–_{exch_OUT}, and N¹⁶O¹⁸O^–_{exch_IN} (N¹⁶O¹⁸O^– efflux and influx regarding the N¹⁶O¹⁸O^– pool, respectively) as described by He and Bao (2019) to implement oxygen atom exchange rates between NO₂^– and H₂O.

Regarding NO₃^–;

d/dt [¹⁴NO₃^–] = NXR --- eq. (13)

d/dt [¹⁵NO₃^–] = (R_NitriteN × NXR / ¹⁵ε_NXR) --- eq. (14)

d/dt [N¹⁶O₃^–] = 3 NXR --- eq. (15)

d/dt [N¹⁸O¹⁶O₂^–] = (R_NitriteO × 2 NXR / ¹⁸ε_NXR)+ (R_WaterO × NXR) / ¹⁸ε_H2ONXR) --- eq. (16)

where ¹⁸ε_H2ONXR (assigned as 10.0‰; Table S1; Buchwald and Casciotti, 2010; Casciotti and Buchwald, 2012) is the ¹⁸O fractionation factor for the incorporation of oxygen from H₂O into NO₃^– during the NXR reaction (Fig. 1).

Regarding NH₄⁺;

d/dt [¹⁴NH₄⁺] = – AMX --- eq. (17)

d/dt [¹⁵NH₄⁺] = – (R_AmmoniumN × AMX/ ¹⁵ε_AMX) --- eq. (18)

where R_AmmoniumN is the ¹⁵N / ¹⁴N of [NH₄⁺] and ¹⁵ε_AMX is the N isotopic fractionation factor for NH₄⁺ consumption by anammox (Fig. 1).

The approximate stoichiometry of the anammox process converting NO₂^– and NH₄⁺ to N₂ and NO₃^– is as follows (Brunner et al., 2013):

1.3NO₂^– + 1NH₄⁺ → 1N₂ + 0.3NO₃^– + 2H₂O --- eq. (19)

However, this stoichiometry between nitrite removal and nitrate production has been reported to vary (Brunner et al., 2013). Thus, we estimated this stoichiometry (x) together with k_AMO14N with concentration data, which provided the AMX, AMXNIR, and NXR fluxes used in the calculation above (eq. [7] and [8]; Table S1). After estimating x and k_AMO14N, we estimated the k_exch of oxygen atoms between H₂O and NO₂^– (Table S1) using the curve-fitting functions for Experiments A and B. In Experiment C with high δ¹⁸O_H2O, we performed another incubation without the anammox biofilm (Fig. S3) to measure k_exch. At the same time, we estimated other isotopic fractionation factors (¹⁵ε_AMXNIR, ¹⁵ε_NXR, ¹⁵ε_AMX, ¹⁸ε_AMXNIR, and ¹⁸ε_NXR). We assigned the range from 0 to 60‰ (with 5 and 10‰ as the initial values for the curve-fitting function of BM software) to estimate isotopic fractionation factors. We considered this 60‰ range for the curve-fitting estimate to be reasonable because isotopic fractionation factors larger than 60‰ are rarely observed (Denk et al., 2017). It is important to note that curve-fitting for Experiments B and C was not successfully achieved for ¹⁸ε_AMXNIR, resulting in extremely high or low estimated values (calculated ¹⁸ε_AMXNIR values were 0 and 60‰ for Experiments B and C, respectively. In addition, ¹⁸ε_NXR (calculated as –11.2 and –84.3‰ for Experiments B and C, respectively) and consequently ¹⁸E_AMXcombined (calculated as –4.2 and –52.9‰ for Experiments B and C, respectively), were not all successfully estimated for Experiments B and C. Based on these uncertainties in parameter estimations, we did not report these calculated values for Experiments B and C; however, we speculate that these calculated parameter sets support ¹⁸ε_AMXNIR as normal and ¹⁸ε_NXR being inverse isotope fractionation, as discussed below for Experiment A. The curve-fitting function (“multiple-fit” in BM software) (Macey et al., 2000) was applied with a tolerance of 1 × 10^–6. BM codes for the anammox model for curve fittings with concentrations and isotopic data are provided in the Zenodo website (https://doi.org/10.5281/zenodo.3895346) and Table S2 showed the root mean square errors (RMSE) for concentrations and isotope values for the fitted model.

Simulation exercise for denitrification and anammox (the anammox-denitrification model)

We added the fluxes of denitrification (NAR and DENNIR; Fig. 3) to the anammox model in order for the anammox-denitrification model to simulate anammox and denitrification as follows:

Fig. 3.

Isotopic fractionation factors applied in the anammox-denitrification simulation model. These factors were from Experiment A or previous studies; (A) Buchwald and Casciotti, 2010; (B) Granger and Wankel, 2016; (C) Casciotti et al., 2002; (D) Buchwald and Casciotti, 2013.

Regarding NO₂^–;

d/dt [¹⁴NO₂^–] = – NXR + NAR – DENNIR – AMXNIR --- eq. (20)

d/dt [¹⁵NO₂^–] = – (R_NitriteN × NXR / ¹⁵ε_NXR)+ (R_NitrateN × NAR / ¹⁵¹⁸ε_NAR)– (R_NitriteN × DENNIR / ¹⁵ε_DENNIR)– (R_NitriteN × AMXNIR / ¹⁵ε_AMXNIR) --- eq. (21)

d/dt [N¹⁶O₂^–] = – 2 NXR + 2 NAR – 2 DENNIR – 2 AMXNIR --- eq. (22)

where ¹⁵¹⁸ε_NAR (assigned as 15‰, Granger et al., 2008; Table S1) is the N and O isotopic fractionation factor of NAR (i.e., ¹⁵ε_NAR = ¹⁸ε_NAR, Sigman et al., 2005; Granger et al., 2008; Granger et al., 2010; Rohde et al., 2015; Osaka et al., 2018) and ¹⁵ε_DENNIR (assigned as 5‰, Granger and Wankel, 2016; Table S1) is the ¹⁵N fractionation factor of DENNIR.

In the case of no exchange of oxygen atoms between NO₂^– and H₂O,

d/dt [N¹⁶O¹⁸O^–] = – (R_NitriteO × 2 NXR / ¹⁸ε_NXR)+ (R_NitrateO × 2 NAR / ¹⁵¹⁸ε_NAR) / ¹⁸ε_H2OBRNAR– (R_NitriteO × 2 DENNIR / ¹⁸ε_DENNIR)– (R_NitriteO × 2 AMXNIR / ¹⁸ε_AMXNIR) --- eq. (23a)

where ¹⁸ε_H2OBRNAR is the ¹⁸O fractionation factor for the “branching effect” (assigned as 25‰, Casciotti and McIlvin, 2007; Table S1) during NAR (Fig. 1).

In the case of full exchange between NO₂^– and H₂O,

d/dt [N¹⁶O¹⁸O^–] = d/dt [N¹⁶O₂^–] × R_Oxygen × [(δ¹⁸O_{NO2–_EQ} / 1000) + 1] --- eq. (23b)

where δ¹⁸O_{NO2–_EQ} is δ¹⁸O_NO2– at the equilibrium with H₂O (= δ¹⁸O_H2O + ¹⁸ε_EQ) and δ¹⁸O_NO2– is always set to δ¹⁸O_{NO2–_EQ}.

Regarding NO₃^–;

d/dt [¹⁴NO₃^–] = NXR – NAR --- eq. (24)

d/dt [¹⁵NO₃^–] = (R_NitriteN × NXR / ¹⁵ε_NXR) – (R_NitrateN × NAR / ¹⁵¹⁸ε_NAR) --- eq. (25)

d/dt [N¹⁸O¹⁶O₂^–] = 3 NXR – 3 NAR --- eq. (26)

d/dt [N¹⁸O¹⁶O₂^–] = (R_NitriteO × 2 NXR / ¹⁸ε_NXR)+ (R_WaterO × NXR / ¹⁸ε_H2ONXR)– (R_NitrateO × 3 NAR / ¹⁵¹⁸ε_NAR) --- eq. (27)

We applied the estimated isotopic fractionation factors from Experiment A (Table 2) together with the reported values for fractionation factors (Fig. 3) to simulate whether the stronger contribution of anammox to denitrification alters the slope of the δ¹⁸O vs δ¹⁵N of NO₃^– from the denitrification slope of 1 with or without oxygen atom exchange between H₂O and NO₂^– in freshwater (δ¹⁸O_H2O = –8‰) or seawater (0‰) environments. The BM code for the anammox and anammox-denitrification models is provided on the Zenodo website (https://doi.org/10.5281/zenodo.3895346).

Results and Discussion

Anammox plant data

Throughout the 17-day span of the three sampling times, the DIN concentrations and their isotopic signatures were stable (Table 1) for each reactor. Stoichiometries for the anammox process were calculated by changes in DIN concentrations between NT and ANX reactors (decreases in the concentrations of NO₂^– and NH₄⁺ ΔNO₂^– / ΔNH₄⁺, for NO₂^– consumption, and an increase in NO₃^– with a decrease in NO₂^–, ΔNO₃^– / ΔNH₄⁺, for NO₃^– production). ΔNO₂^– / ΔNH₄⁺ ranged 1.22 ~ 1.26 and ΔNO₃^– / ΔNH₄⁺ was 0.14 ~ 0.15, both of which were within the range for anammox reactions (1.03 to 1.32 for ΔNO₂^– / ΔNH₄⁺ and 0.14 to 0.35 for ΔNO₃^– / ΔNH₄⁺) (Yao et al., 2015). Isaka et al. (2017) also reported that ΔNO₂^– / ΔNH₄⁺ was 1.23 from this anammox plant, which indicates the appropriate performance of anammox in ANX reactor. The ammonium in the influent with a high concentration (44.5‍ ‍mM) and low δ¹⁵N_NH4+ (–10.4‰) was gradually consumed in the reactors with normal isotopic fractionation (i.e., with increasing δ¹⁵N), resulting in a low concentration (1.9‍ ‍mM) with high δ¹⁵N_NH4+ (50.2‰) at final ANX reactor (Table 1). Nitrite produced in DN, BD, and NT reactors and consumed in ANX reactor had low δ¹⁵N_NO2– values (–38.8 to –21.6‰) and relatively stable δ¹⁸O_NO2– values (3.3 to 4.7‰; Table 1). Nitrate was not produced before ANX reactor and δ¹⁵N_NO3– (9.3‰) was higher than δ¹⁵N_NO2– (–21.6‰), with no significant difference between δ¹⁸O_NO3– and δ¹⁸O_NO2– in ANX (Table 1). In comparisons with the isotopic data for other types of WWTP (Table 1), we found that the lower δ¹⁸O_NO3– and higher δ¹⁵N_NH4+ from the anammox plant was useful for tracking the fate of N derived from the anammox wastewater plant.

Table 1. Average concentrations and isotopic compositions of DIN in the anammox plant and isotopic data from different types of WWTP

Reactor	[NH4+] (mM)	[NO2–] (mM)	[NO3–] (mM)	δ15NNH4+ (‰)	δ15NNO2– (‰)	δ18ONO2– (‰)	δ15NNO3– (‰)	δ18ONO3– (‰)
Influent	44.5 (0.1)	0 (0)	0 (0)	–10.4 (0.2)	n.d.	n.d.	n.d.	n.d.
DN	40.7 (0.2)	0.2 (0)	0 (0)	–8.4 (0.2)	–27.3 (0.4)	4.7 (0.2)	n.d.	n.d.
BD	32.8 (0.7)	7.7 (0.6)	0 (0)	0.6 (0.9)	–38.8 (0.8)	4.3 (0.2)	n.d.	n.d.
NT	18.7 (0.4)	21.2 (0.3)	0 (0)	19.2 (0.7)	–28.2 (0.4)	4.5 (0.1)	n.d.	n.d.
ANX	1.9 (0.5)	0.4 (0)	2.5 (0)	50.2 (1.4)	–21.6 (0.4)	3.3 (0.3)	9.3 (0.4)	2.7 (0.2)
WWTP type#									Reference
CAS				0 to 36			13 to 15	–1 to 0	Tumendelger et al., 2014
A2O							8.1*	–4.5*	Toyoda et al., 2011
Preliminary							11.5 (3.1)**	4.9 (4.2)**	Archana et al., 2016
Primary							14.8 (3.9)**	8.6 (3.4)**	Archana et al., 2016
CEPT							10.6 (4.9)**	–2.1 (3.6)**	Archana et al., 2016
Secondary							12.5 (4.3)**	3.8 (2.4)**	Archana et al., 2016
Tertiary							90.7 (83.9)**	87.7 (90.6)**	Archana et al., 2016

Means from three sampling times with standard errors (in parentheses) are shown. n.d.: not determined

* Data from the sampling point closest to the outlet to the river

** Means from several WWTP with standard deviations (in parentheses) are shown.

^# CAS: Conventional activated sludge, A₂O: Anaerobic-Anoxic-Oxic treatment, CEPT: Chemically Enhanced Primary Treatment

The calculated ¹⁵Δ_AMX was large (34.0 ~ 34.8‰; Table 2), which was similar to the reported value for anammox (30.9 ~ 32.7‰; Kobayashi et al., 2019) and to the isotope effect for aerobic ammonia oxidization (29.6 ± 4.9‰; Denk et al., 2017). The two NO₂^– consumption pathways in the ANX reactor had different ¹⁵N fractionation; normal (positive), large ¹⁵Δ_AMXNIR (11.8 ~ 12.4‰), and inverse (negative) ¹⁵Δ_NXR (–30.4 ~ –31.3‰), which fell within reported values (Kobayashi et al., 2019) (Table 2). The present results confirmed an inverse ¹⁵N effect during anaerobic NO₂^– oxidation to NO₃^–, as previously reported (Brunner et al., 2013; Kobayashi et al., 2019) for aerobic NO₂^– oxidation to NO₃^– (Casciotti, 2009; Buchwald and Casciotti, 2010). Similarly, the small and negative apparent “combined” ¹⁸O fractionation for ammonium oxidization by NO₂^– (–2.5 ~ –3.8‰; ¹⁸E_AMXcombined) also fell within the reported range of –1.5 to –‍12‰ (Kobayashi et al., 2019) (Table 2). The negative ¹⁸E_AMXcombined values reported here and by Kobayashi et al. (2019) during NO₃^– production in anammox agree with the inverse ¹⁸O fractionation for aerobic nitrite oxidation to NO₃^– (Casciotti, 2009; Buchwald and Casciotti, 2010).

Table 2. Isotopic fractionation factors during anammox (‰)

Open system	Anammox Plant (This study)				Reported values
	20150428	20150507	20150512		Kobayashi et al., (2019)
15ΔAMXNIR	11.8	12.0	12.4		5.9~29.5
15ΔNXR	–30.4	–31.1	–31.3		–30.1~ –45.3
15ΔAMX	34.0	34.8	34.4		30.9~32.7
18EAMXcombined*	–3.8	–2.5	–3.2		–1.5~ –12.1
Closed system	Batch incubations (This study)				Reported values
	Experiment A	Experiment B	Experiment C		Brunner et al., (2013)
15εAMXNIR	13.7	21.8	15.6		16.0
15εNXR	–77.8	–65.9	–71.1		–31.1
15εAMX	32.5	25.4	19.3		23.5~29.1
18EAMXcombined*	–10.4	n.d.	n.d.		n.d.
18εAMXNIR**	3.1	n.d.	n.d.		n.d.
18εNXR**	–20.6	n.d.	n.d.		n.d.

*: ¹⁸ε_NXR × 2 / 3 + ¹⁸ε_H2ONXR / 3 (Kobayashi et al., 2019)

**: assuming ¹⁸ε_EQ = 1.013, ¹⁸ε_H2ONXR = 1.010 (Table S2)

Incubation experiments

In all experiments, [NH₄⁺] and [NO₂^–] concurrently decreased as [NO₃^–] increased (Fig. 2a, b, and c). Averaged stoichiometries during anammox were 1.29, 1.51, and 1.48 for ΔNO₂^– / ΔNH₄⁺ and 0.16, 0.17, and 0.21 for ΔNO₃^– / ΔNH₄⁺ in Experiments A, B, and C, respectively (Fig. 2a, b, and c). These results were more consistent in their stoichiometry than previous findings with the same anammox bacterium (1.00 to 2.12 for ΔNO₂^– / ΔNH₄⁺ and 0.10 to 0.37 for ΔNO₃^– / ΔNH₄⁺; Ali et al., 2015). The estimated values of x, k_AMO14N, and k_exch were shown in Table S1. The estimated x values (0.13 to 0.21; Table S1) were similar to reported values (0.15 to 0.48; Brunner et al., 2013), and k_exch values were negligible (Table S1). The anammox rates based on NH₄⁺ consumption were 39.7, 61.7, and 12.4‍ ‍μM (g-S.S.)‍ ‍^–1‍ ‍h^–1 for Experiments A, B and C, respectively.

Fig. 2.

Concentrations and isotopic signatures of inorganic N in incubation experiments. The lines represent changes in the concentrations and isotopic signatures estimated by the curve-fitting of rate constants (for concentrations, upper panels) and ¹⁵N and ¹⁸O fractionation factors (for δ¹⁵N and δ¹⁸O values, middle and lower panels). The root mean square error (RMSE) for each fitting was shown in Table S1.

δ¹⁵N_NH4+, δ¹⁵N_NO2–, and δ¹⁵N_NO3– increased as [NH₄⁺] and [NO₂^–] decreased during anammox (Fig. 2d, e, and f). In contrast, δ¹⁸O_NO2– and δ¹⁸O_NO3– did not change in Experiment A (Fig. 2g), while δ¹⁸O_NO3– increased by ~2‰ and δ¹⁸O_NO2– decreased by ~3‰ in Experiment B (Fig. 2h). In Experiment C with the high δ¹⁸O of H₂O (229‰), δ¹⁸O_NO2– and δ¹⁸O_NO3– rapidly increased (Fig. 2i). δ¹⁸O_NO2– also rapidly increased in the negative control experiment without the biomass (Fig. S3). The isotope exchange between NO₂^– and NO₃^– needs to be taken into consideration (Brunner et al., 2013) when the rapid and large changes in δ¹⁵N and δ¹⁸O at the beginning of the incubation are observed. Since we did not observe such a marked change in δ¹⁵N and δ¹⁸O, indicating isotope exchange (Fig. 2), we did not include isotope exchange between NO₂^– and NO₃^– in the present study.

¹⁵ε_AMX values were calculated as 32.5, 25.4, and 19.3‰ for Experiments A, B, and C, respectively (Table 2, Fig. 2d, e, and f). These ¹⁵ε_AMX values were similar to previously reported values (23.5 ~ 29.1‰) for Kuenenia stuttgartiensis in batch incubation experiments (Brunner et al., 2013). ¹⁵ε_AMXNIR values were estimated to be 13.7, 21.8, and 15.6‰ (Table 2, Fig. 2d, e, and f), while ¹⁸ε_AMXNIR values were 3.1, 0 and 60.0‰ (Table 2, Fig. 2d, e, and f) for Experiments A, B and C, respectively. Although ¹⁸ε_AMXNIR values in Experiments B and C were not successfully measured (Table 2; see the Methods), estimated ¹⁵ε_AMXNIR values were consistent with the ¹⁵N values reported for NO₂^– reduction by Cu-NIR coded by the nirK gene (22 ± 2 and 2 ± 2‰) (Martin and Casciotti, 2016). The similarity in these values was attributed to the Cu-NIR of “Candidatus Jettenia” with the nirK gene (Hira et al., 2012; Ali et al., 2015), the dominant microbe in incubation experiments (Fig. S2).

In addition to normal isotopic fractionation, we estimated ¹⁵N and ¹⁸O fractionation factors during anaerobic nitrite oxidization to NO₃^– of –77.8‰ for ¹⁵ε_NXR and –20.6‰ for ¹⁸ε_NXR (Table 2, Fig. 2g, h, and i) in Experiment A. Although we also estimated ¹⁵N and ¹⁸O fractionation factors of –65.9 and –71.1‰ for ¹⁵ε_NXR and –11.2 and –84.3‰ for ¹⁸ε_NXR for Experiments B and C, respectively, ¹⁸ε_NXR values for these experiments were not precisely measured. The large inverse ¹⁵ε_NXR is consistent with the reported value with K. stuttgartiensis (–31.1 ± 3.9‰; Brunner et al., 2013), as well as aerobic nitrite oxidation to NO₃^– by nitrite-oxidizing bacteria (–12.8 ± 1.5‰; Casciotti, 2009). Regarding oxygen, although only ¹⁸ε_NXR in Experiment A was successfully assessed, the estimated ¹⁸ε_NXR value was negative and close to the inverse ¹⁸O fractionation factors for aerobic nitrite oxidization to NO₃^– by nitrite-oxidizing bacteria (–10 to –‍1‰; Buchwald and Casciotti, 2010).

Simulation for denitrification and anammox

We developed an anammox-denitrification model with the estimated isotopic fractionation factors (from Experiment A; Table 2) and reported values (Fig. 3) to clarify whether anammox induces a deviation in δ¹⁸O_NO3– vs δ¹⁵N_NO3– from the denitrification slope of 1 (i.e., Δ(15, 18); defined as (δ¹⁵N – δ¹⁵N_initial) – (¹⁸ε / ¹⁵ε)(δ¹⁸O – δ¹⁸O_initial), where ¹⁸ε / ¹⁵ε is the ratio of isotopic fractionation for O and N during denitrification, respectively, and assigned as 1; see the inset in Fig. 4a; Sigman et al., 2005). Each simulation was run until more than 25% of NO₂^– was consumed. In the case of denitrification in which AMX / NAR is equal to 0 (indicating no anammox), δ¹⁸O_NO3– vs δ¹⁵N_NO3– was set to show a slope of 1 (the dotted lines in Fig. 4a, d, c, and d). As reported in previous studies (Casciotti and Buchwald, 2012; Granger and Wankel, 2016; He and Bao, 2019), larger anammox rates (larger AMX / NAR) induced greater offsets (larger Δ[15, 18]) from the 1:1 relationship between δ¹⁸O_NO3– and δ¹⁵N_NO3– in all cases (Fig. 4a, b, c, and d). The effect of oxygen atom exchange was small, but obvious (Fig. 4c for freshwater and Fig. 4d for seawater) with a larger offset with the exchange. Although the present results revealed that Δ(15, 18) is a sensitive parameter for the occurrence of anammox, its usefulness diminishes with smaller ¹⁵ε_NXR values (–31.1‰, Table 2 and Fig. S4), indicating the sensitivity of Δ(15, 18) against ¹⁵ε_NXR (i.e., the stronger ¹⁵ε_NXR, the larger Δ [15, 18]). To elucidate the relationship between Δ(15, 18), AMX / NAR, and isotopic fractionation factors, we simulated the δ¹⁸O_NO3– and δ¹⁵N_NO3– trajectories along with the different AMX / NAR ratios and ¹⁵ε_AMXNXR (Fig. 5 with ¹⁵ε_NXR = –77.8‰ and Fig. S5 with –31.1‰). Similar to ¹⁵ε_NXR, stronger ¹⁵ε_AMXNXR resulted in larger Δ(15, 18); however, Δ(15, 18) depended on AMX / NAR, ¹⁵ε_AMXNXR, and ¹⁵ε_NXR (Fig. 5 and S5). Therefore, a simple comparison of Δ(15, 18) data does not permit quantitative estimations of AMX / NAR because Δ(15, 18) increases with NO₂^– and NO₃^– consumption whenever anammox is active (AMX / NAR > 0; Fig. 4 and 5) and Δ(15, 18) levels strongly depend on many parameters, including ¹⁵ε_AMXNXR and ¹⁵ε_NXR. Although more information on isotopic fractionation factors is needed for quantitative interpretations due to the sensitivity of Δ(15, 18), our simulation exercise revealed that Δ(15, 18), the offset from the 1:1 relationship between δ¹⁵N and δ¹⁸O, may be useful for detecting NXR (nitrite oxidation) in denitrifying systems in both freshwater and seawater.

Fig. 4.

Results from the anammox-denitrification model for variable ratios of anammox (AMX) and denitrification (NAR), and with or without oxygen atom exchange between water and NO₂^–. The simulation was run with ¹⁵ε_NXR = –77.8‰ (Table 2) until more than 25% of the initial NO₂^– pool was consumed; however, NO₂^– consumption in simulations with the same run times varied according to the different AMX / NAR ratios. The end point of each simulation run was not important, whereas the slope of each run was. The dotted line in each panel illustrated the denitrification slope (1:1) and the inset in Fig. 4a shows Δ(15, 18) in the δ¹⁵N and δ¹⁸O space.

Fig. 5.

Results of the anammox-denitrification model for variable ANX / NAR ratios with variable ¹⁵ε_AMXNIR values with the full oxygen atom exchange between water (freshwater with δ¹⁸O_H2O = –8‰) and NO₂^–. The simulation was run with ¹⁵ε_NXR = –77.8‰ (Table 2) until more than 25% of the initial NO₂^– pool was consumed; however, NO₂^– consumption in simulations with the same run times varied according to the different AMX / NAR ratios. The end point of each simulation run was not important, whereas the slope of each run was crucial. The inset in Fig. 5c shows Δ(15, 18) in the δ¹⁵N and δ¹⁸O space.

Besides NXR, some chemolithoautotrophic (e.g., sulfide-dependent) denitrification with auxiliary Nap NO₃^– reductase may exhibit a 2:1 rather than 1:1 relationship between δ¹⁵N and δ¹⁸O, resulting in an offset from the 1:1 relationship (Frey et al., 2014). Although this non-respiratory pathway (i.e., Nap NO₃^– reduction) is not considered to be a major environmental sink for NO₃^– (Granger and Wankel, 2016), and, thus, was not included in our models, it is worthwhile considering this autotrophic denitrification as a driver of the offset in a sulfide-rich environment in which anammox may be inhibited (Jensen et al., 2008) and sulfide-dependent denitrification enhanced.

Conclusion

We estimated ¹⁵N and ¹⁸O fractionation factors during anammox. The inverse ¹⁵N effects for NXR (and possibly inverse O isotope effects) may induce an offset from the denitrification trajectory (1:1 relationship between δ¹⁵N and δ¹⁸O of NO₃^–, Δ[15, 18]). In practice, Δ(15, 18) may be evaluated with time-course samplings or short incubation studies to investigate the occurrence of anammox, similar to denitrification. This technique will be advantageous because of its potential in evaluations of the quantitative contribution in situ of anammox versus denitrification. Although the detection and quantification of functional genes in denitrification and anammox may be readily performed, difficulties are associated with detecting the in situ occurrence of denitrification and anammox. Although the isotopic fractionation factors used also need to be considered, Δ(15, 18) is a promising parameter to complement molecular data and the results from laboratory incubation experiments in the study of anammox.

Citation

Kotajima, S., Koba, K., Ikeda, D., Terada, A., Isaka, K., Nishina, K., et al. (2020) Nitrogen and Oxygen Isotope Signatures of Nitrogen Compounds during Anammox in the Laboratory and a Wastewater Treatment Plant. Microbes Environ 35: ME20031.

https://doi.org/10.1264/jsme2.ME20031

Acknowledgements

We thank T. Makita, A. Koba, and E. Murata for helping with sampling and the chemical analysis, and K. Casciotti and M. McIlvin for the calibration of our in-house NO₂^– standards. We also appreciate E. Hobbie for fruitful discussions and English editing. This study was financially supported by research funds from JSPS (26252020, 16H02524, 17H06297, and 18H04138).

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