Nitrogen Isotope Fractionation During Archaeal Ammonia Oxidation: Coupled Estimates From Measurements of Residual Ammonium and Accumulated Nitrite

Abstract

The naturally occurring nitrogen (N) isotopes, ¹⁵N and ¹⁴N, exhibit different reaction rates during many microbial N transformation processes, which results in N isotope fractionation. Such isotope effects are critical parameters for interpreting natural stable isotope abundances as proxies for biological process rates in the environment across scales. The kinetic isotope effect of ammonia oxidation (AO) to nitrite (NO₂^–), performed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), is generally ascribed to the enzyme ammonia monooxygenase (AMO), which catalyzes the first step in this process. However, the kinetic isotope effect of AMO, or ε_AMO, has been typically determined based on isotope kinetics during product formation (cumulative product, NO₂^–) alone, which may have overestimated ε_AMO due to possible accumulation of chemical intermediates and alternative sinks of ammonia/ammonium (NH₃/NH₄⁺). Here, we analyzed ¹⁵N isotope fractionation during archaeal ammonia oxidation based on both isotopic changes in residual substrate (RS, NH₄⁺) and cumulative product (CP, NO₂^–) pools in pure cultures of the soil strain Nitrososphaera viennensis EN76 and in highly enriched cultures of the marine strain Nitrosopumilus adriaticus NF5, under non-limiting substrate conditions. We obtained ε_AMO values of 31.9–33.1‰ for both strains based on RS (δ¹⁵NH₄⁺) and showed that estimates based on CP (δ¹⁵NO₂^–) give larger isotope fractionation factors by 6–8‰. Complementary analyses showed that, at the end of the growth period, microbial biomass was ¹⁵N-enriched (10.1‰), whereas nitrous oxide (N₂O) was highly ¹⁵N depleted (−38.1‰) relative to the initial substrate. Although we did not determine the isotope effect of NH₄⁺ assimilation (biomass formation) and N₂O production by AOA, our results nevertheless show that the discrepancy between ε_AMO estimates based on RS and CP might have derived from the incorporation of ¹⁵N-enriched residual NH₄⁺ after AMO reaction into microbial biomass and that N₂O production did not affect isotope fractionation estimates significantly.

Introduction

Knowledge of natural ¹⁵N abundances and of nitrogen (N) isotope fractionation effects associated with key microbial N transformation processes has contributed greatly to our understanding of the marine N cycle (Casciotti and Buchwald, 2012; Buchwald and Casciotti, 2013) and of terrestrial gaseous N emissions (Houlton and Bai, 2009), namely atmospheric N₂O sources and sinks (Yoshida and Toyoda, 2000), and biological N fixation (Vitousek et al., 2013). The oxidation of NH₄⁺ to NO₂^–—the first and rate-limiting step in nitrification—is a central process in the marine and terrestrial N cycles, as well as the major driver of a large N isotope effect that leads to formation of ¹⁵N-depleted products such as NO, N₂O, NO₂^–, and NO₃^–, while residual NH₄⁺ becomes ¹⁵N-enriched during that process (Mariotti et al., 1981; Sigman and Casciotti, 2001). A detailed understanding of N isotope effects of the range of N transformation processes is thus critical for adequate biological interpretation of natural ¹⁵N isotope patterns in the environment (Casciotti, 2016).

Besides the recently discovered comammox bacteria (Daims et al., 2015; van Kessel et al., 2015), ammonia oxidation is catalyzed by both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), with different relative contributions across ecosystems and environmental conditions (Prosser and Nicol, 2012; Prosser et al., 2019). On a cellular level, ammonia oxidation is a multi-step process that comprises different enzymatic reactions and chemical equilibrium processes, which can all contribute to the N isotope fractionation effects inferred from extracellular N pools (Casciotti et al., 2003; Santoro and Casciotti, 2011). The isotopic fractionation effect (ε) of ammonia oxidizers has been typically inferred based on changes in δ¹⁵N of the cumulative product (CP) NO₂^– (ε_CP), and attributed to the initial enzymatic step catalyzed by the ammonia monooxygenase (AMO) enzyme, defined as ε_AMO. However, ε_CP estimates reflect the combined fractionation effects of the isotope equilibrium between NH₄⁺ and NH₃ [NH₃, the proposed substrate for ammonia oxidation, is depleted in ¹⁵N relative to NH₄⁺ (Hermes et al., 1985)], the AMO-catalyzed reaction, and accumulation of several intermediates derived from subsequent enzymatic processes (Casciotti et al., 2003). For instance, ε_CP estimates may be affected by the accumulation of essential intermediates, such as hydroxylamine (NH₂OH) and by the production of gaseous N by-products (nitric oxide, NO; and nitrous oxide, N₂O), which may represent further ¹⁵N fractionation steps. Consequently, this could result in a difference of kinetic isotope effect estimates derived from residual substrate (RS) and CP (Casciotti et al., 2003). Not only could these “leakage” processes alter CP-based estimates of ε_AMO, but their different contributions to ammonia utilization and to ε_CP may also underlie the large differences observed in ε_AMObetween ammonia-oxidizing organisms (Mariotti et al., 1981; Yoshida, 1988; Casciotti et al., 2003; Santoro and Casciotti, 2011).

Estimates of isotope effects based on the change in δ¹⁵NH₄⁺ (ε_RS) can circumvent many of the expected biases associated with ε_CP, as they are not affected by the multiple subsequent equilibria, enzymatic transformations, and intermediate N pools, as discussed but not quantified previously (Casciotti et al., 2003; Santoro and Casciotti, 2011). However, to our knowledge, only one study has determined the isotope fractionation factors based on concurrent measurements of changes in isotopic composition of RS and CP of ammonia oxidation, namely in cultures of the AOB Nitrosomonas europaea (Mariotti et al., 1981). This study found no difference between ε_RS and ε_CP, suggesting that ammonia oxidation can be effectively regarded as a “one-step process,” where the AMO-catalyzed reaction constitutes the rate-limiting and sole isotope fractionation step. On the other hand, AOB and AOA seem to harbor fundamentally distinct ammonia oxidation pathways and exhibit different yields of gaseous N compounds per mole of NH₄⁺ consumed (Walker et al., 2010; Kozlowski et al., 2016). Importantly, the enzyme hydroxylamine dehydrogenase (HAO), which performs the second step in ammonia oxidation of AOB, has not been identified in AOA, and thus it remains unclear how NH₂OH is converted to NO₂^– in AOA (Walker et al., 2010; Kerou et al., 2016). Moreover, a recent study has provided evidence that the bacterial HAO oxidizes NH₂OH to NO rather than to NO₂^–, as generally assumed, with the latter resulting from non-enzymatic oxidation of NO by oxygen (Caranto and Lancaster, 2017). Previous studies have shown that NO is also an essential intermediate in ammonia oxidation by AOA, as their growth and activity is highly sensitive to exposure to an NO scavenger (Shen et al., 2013; Kozlowski et al., 2016).

Here, we tested whether the kinetic isotope effect of archaeal ammonia oxidation based on CP (δ¹⁵NO₂^–) alone might be biased, by comparing the isotope fractionation factors inferred from both RS and CP pools. For this, we determined the kinetic isotope effects during growth of two phylogenetically and ecologically distinct AOA: the axenic strain Nitrososphaera viennensis EN76 (Stieglmeier et al., 2014a), isolated from soil, and the highly enriched marine strain Nitrosopumilus adriaticus NF5 (Bayer et al., 2016). This is also the first study of ¹⁵N isotope fractionation of ammonia oxidation by an AOA strain in pure culture. All previous studies of kinetic isotope effects of AOA have been performed with enrichment cultures with varying degrees of enrichment (Santoro and Casciotti, 2011; Nishizawa et al., 2016) and bacterial contaminants that may have contributed to the variation in isotope effects through consumption of and inputs to the same N pools.

Materials and Methods

Pure cultures of N. viennensis EN76 were cultivated in freshwater medium and incubated at 37°C, as described by Tourna et al. (2011). In a first experiment, quadruplicate cultures were supplemented with 1 mM NH₄⁺ and 0.1 mM pyruvate; in a second experiment, quadruplicate cultures were supplemented with 2 mM NH₄⁺ and 0.5 mM pyruvate to generate higher cell biomass and sufficient N₂O concentrations for isotopic analysis, in order to determine their potential effect on ε_AMO. Quadruplicate enrichment cultures of N. adriaticus NF5 were cultivated in Synthetic Crenarchaeota Medium (SCM) at 30°C as described by Bayer et al. (2016). The medium was supplemented with 1 mM NH₄⁺ and 5% (v/v) autoclaved seawater, which was sterile-filtered (0.22 μm GTTP, Millipore). Kanamycin at a final concentration of 100 μg ml^–1 was used to inhibit bacterial contaminants. At the time of the experiment (January 2013), the enrichment level of strain NF5 was ∼95%, as it contained a heterotrophic non-nitrifying/non-denitrifying contaminant of the alphaproteobacterial species Oceanicaulis alexandrii (Bayer et al., 2019).

Ammonia-oxidizing archaea growth was monitored by measuring nitrite production using the Griess method (Hood-Nowotny et al., 2010), coupled to NH₄⁺ consumption determined using the Berthelot method for N. viennensis cultures (Hood-Nowotny et al., 2010) and the o-Phthalaldehyde (OPA) method for N. adriaticus cultures (Goyal et al., 1988). δ¹⁵NH₄⁺ was quantified by microdiffusion (Sørensen and Jensen, 1991) with subsequent analysis on a continuous-flow isotope ratio mass spectrometer consisting of an elemental analyzer (EA1110, CE Instruments) coupled via a ConFlo III interface (Finnigan MAT, Thermo Fisher Scientific) to the isotope ratio mass spectrometer (IRMS; DeltaPLUS, Finnigan MAT, Thermo Fisher Scientific). δ¹⁵NO₂^– was determined based on the reduction of NO₂^– to N₂O by azide under acidified conditions (Lachouani et al., 2010). Concentrations and isotopic ratios of N₂O were determined using a purge-and-trap GC/IRMS system (PreCon - GasBench II headspace analyzer, Delta Advantage V IRMS; Thermo Fischer Scientific, Vienna, Austria). For NH₄⁺ and NO₂^– isotope measurements, we included blanks, concentration standards, and isotope standards varying in natural ¹⁵N abundance together with the samples through the full microdiffusion and azide procedures to allow corrections for blank contribution, incomplete reaction, and procedural isotope fractionation (Lachouani et al., 2010). Nitrogen content and δ¹⁵N signature of AOA biomass were determined by EA-IRMS as described above. δ¹⁵N signatures [‰ vs. AIR] were calculated relative to the ratio R (¹⁵N:¹⁴N) of the atmospheric N₂ standard (AIR), as δ¹⁵N = (R_sample/R_standard− 1) × 1000.

Isotope fractionation factors (ε) were calculated based on the Rayleigh closed system isotope fractionation, based on changes in the isotopic compositions of RS (i.e., NH₄⁺) and CP (i.e., NO₂^–) (Mariotti et al., 1981):

$${10}^3\text{ln}\frac{{10}^{-3}{\mathrm{\delta }}_{RS}+1}{{10}^{-3}{\mathrm{\delta }}_{S0}+1}=\mathrm{\epsilon }ln(f)$$ (1)

$${\mathrm{\delta }}_{CP}-{\mathrm{\delta }}_{S0}=-\mathrm{\epsilon }f\frac{\text{ln}(f)}{(1-f)},$$ (2)

where δ_S0 is δ¹⁵N of initial NH₄⁺, δ_RS is δ¹⁵NH₄⁺, δ_CP is δ¹⁵NO₂^– and f is the fraction of the initial [NH₄⁺] remaining in the culture. Plots of ${10}^3\frac{{10}^{-3}{\mathrm{\delta }}_{RS}+1}{{10}^{-3}{\mathrm{\delta }}_{S0}+1}$ versus ln⁡(f) and of δ_CP−δ_S0 versus$f\frac{\text{ln}(f)}{(1-f)}$ yield linear relations, with the slope representing the kinetic isotope effect based on the isotopic change in substrate (ε_RS) and product (ε_AP), respectively. Uncertainties of ε are expressed as standard error of the slope. Differences in isotope fractionation effects between cultures were assessed by testing significant differences between their regression plots, using R (R Development Core Team, 2012).

Results and Discussion

Based on the oxidation of NH₃/NH₄⁺ to NO₂^–—a typical proxy for ammonia oxidizer growth, as it strongly correlates with growth rates (Stieglmeier et al., 2014a; Bayer et al., 2016)—all cultures showed growth curves typical for batch cultures of AOA, reaching stationary phase after 7 days for N. adriaticus, and after 3–4 days for N. viennensis cultures (Figures 1A–C). Nitrogen isotope fractionation was reflected in both the substrate (i.e., NH₄⁺) and the product (i.e., NO₂^–) of ammonia oxidation, and followed typical Rayleigh isotope fractionation kinetics for closed systems (Figures 1D–F): NH₄⁺ became increasingly ¹⁵N-enriched with the fraction of NH₄⁺ oxidized, while NO₂^– was strongly ¹⁵N-depleted after correction for NO₂^– deriving from the inoculum. With an increasing fraction of NH₄⁺ oxidized, δ¹⁵NO₂^– converged toward the isotopic signature of the initial NH₄⁺. Both N. adriaticus and N. viennensis (including cultures grown on 1 and 2 mM NH₄⁺) exhibited ¹⁵N isotope fractionation factors based on substrate (ε_RS) between 31.9 and 33.1‰, and based on product (ε_CP) between 37.7 and 49.1‰ (Figures 2A–F). We found no significant difference between the isotope fractionation factors of the different AOA cultures studied here based on δ¹⁵N evolution of the substrate (ε_RS; comparison of slopes, df = 2, F = 0.519, p = 0.598) or the product (ε_CP; comparison of slopes, df = 2, F = 2.380, p = 0.102). The N isotope fractionation factors based on δ¹⁵NO₂^– (ε_CP) were larger than those based on δ¹⁵NH₄⁺ (ε_RS) by 8.0, 5.8, and 5.9‰ for N. adriaticus, and for N. viennensis grown on 1 mM or 2 mM NH₄⁺, respectively.

FIGURE 1.

Ammonia oxidation and isotopic signature of NH₄⁺ and NO₂^– of a marine (N. adriaticus NF5 enrichment culture) and a soil (N. viennensis EN76) AOA. (A–C) Time course of NH₄⁺ oxidation to NO₂^–. (D–F) Isotopic composition of NH₄⁺ and NO₂^– as a function of initial NH₄⁺ oxidized. Some error bars are smaller than the symbol.

FIGURE 2.

Kinetic isotope effects of the marine AOA N. adriaticus and the soil AOA N. viennensis. Isotope fractionation factors (ε_RS and ε_CP) were calculated based on changes in (A–C) δ¹⁵N-NH₄⁺ (δ_RS) and (D–F) δ¹⁵N-NO₂^– (δ_CP), using linear regressions of ${10}^3\frac{{10}^{-3}{\mathrm{\delta }}_{RS}+1}{{10}^{-3}{\mathrm{\delta }}_{S0}+1}$ versus ln(f) and δ_CP−δ_S0 versus $f\frac{\text{ln}(f)}{(1-f)}$, respectively, as described in Mariotti et al. (1981). δ_S0 is δ¹⁵N of initial NH₄⁺ and f is the fraction of the initial [NH₄⁺] remaining in the culture. Uncertainties of ε_RS and ε_CP are expressed as SE of the slope. We used multiple-point estimates because they have lower uncertainty than single-point analyses.

Nitrogen isotope fractionation has been studied in several AOB strains, but only in three marine and one thermophilic AOA enrichment cultures. These AOA enrichment cultures showed average N isotope fractionation factors between 22 and 25‰ at low substrate concentrations, and up to 32.0‰ at higher ammonium concentrations (Santoro and Casciotti, 2011; Nishizawa et al., 2016, measured via the isotopic composition of the product nitrite; see Table 1). These estimates are in the same range as the reported average isotope effects for different AOB strains, i.e., 14–38‰ (Delwiche and Steyn, 1970; Mariotti et al., 1981; Casciotti et al., 2003). ¹⁵N isotope fractionation factors of N. viennensis and N. adriaticus are in the upper range, or higher, than those previously reported for AOA, which might be due to the higher ammonia concentrations applied in our study (1–2 mM in our study vs. 200 μM in Nishizawa et al., 2016; 10–75 μM in Santoro and Casciotti, 2011). Previous studies have indicated that higher initial ammonia concentrations lead to more stable and higher ¹⁵N isotope fractionation (Casciotti et al., 2003; Santoro and Casciotti, 2011).

TABLE 1.

Compilation of published kinetic isotope effects of AOA and AOB.

Source	AOA/AOB	Strain	Initial [NH4+] (mM)	Other conditions	εRS		εCP
					Mean	SD	Mean	SD
This study	AOA	Nitrosopumilus adriaticus NF5	1		32.1	1.0	40.1	0.7
	AOA	Nitrososphaera viennensis EN76	1		31.9	1.2	37.7	0.8
	AOA	Nitrososphaera viennensis EN76	2		33.1	0.7	39.0	1.2
Santoro and Casciotti (2011)	AOA	Marine AOA enrichment CN25†	0.01–0.075				22	5
		Marine AOA enrichment CN75					21	10
		Marine AOA enrichment CN150					22	5
Nishizawa et al. (2016)	AOA	Candidatus Nitrosocaldus sp.	0.2				22.0	5.0
		Candidatus Nitrosocaldus sp.	14				24.7	2.1
Mariotti et al. (1981)	AOB	Nitrosomonas europaea	4.7–25		34.7	2.5	31.9	6.4
Delwiche and Steyn (1970)	AOB	Nitrosomonas europaea					26.0	5.6
Yoshida (1988)	AOB	Nitrosomonas europaea	38	pO2 low			24.6
	AOB	Nitrosomonas europaea	38	pO2 medium			29.0
	AOB	Nitrosomonas europaea	38	pO2 high			32.0
Casciotti et al. (2003)	AOB	Nitrosomonas marina	2				14.2	3.6
		Nitrosomonas sp. C-113a	2				19.1	1.2
		Nitrosospira tenuis	1				24.6	1.4
		Nitrosomonas eutropha	1				32.8	1.7
		Nitrosomonas europaea	1				38.2	1.6
Casciotti et al. (2010)	AOB	Nitrosomonas sp. C-113a	0.005–0.05				30–46
	AOB	Nitrosococcus oceani	0.005–0.05				30–46
	AOB	Nitrosospira briensis	0.005–0.05				30–46

^†Currently designated as Candidatus Nitrosopelagicus brevis CN25 (Santoro et al., 2015).

We also measured ε_AMO based on changes in δ¹⁵NH₄⁺ (i.e., the residual substrate) to both circumvent and assess potential biases associated with estimates based on δ¹⁵NO₂^– (i.e., the cumulative product). It should be noted, however, that different apparent isotope effects in whole cells may also be observed in the NH₄⁺ pool, despite constant AMO enzyme-level isotope effects, depending, for example, on the balance between ammonia oxidation rates and ammonia diffusion across the S-layer (i.e., outermost cell envelope component in AOA) (Casciotti et al., 2003; Li et al., 2018). Published models of AOA and AOB metabolism favor the hypothesis of a (pseudo-)periplasmic location of the ammonia oxidation process (Arp and Stein, 2003; Walker et al., 2010; Simon and Klotz, 2013). However, AOA and AOB harbor very distinct NH₃/NH₄⁺ transport systems (Offre et al., 2014), whose role in ammonia oxidation and contribution to observed differences in ¹⁵N isotope fractionation remain unclear (Arp and Stein, 2003). At low ammonia concentrations, ammonia oxidation rates are expected to become limited by NH₄⁺ transport/NH₃ diffusion, resulting in the convergence of the isotope effect toward that of NH₄⁺/NH₃ equilibrium (if NH₃ is mainly taken up by the cells) or NH₄⁺/NH₃ transport. The NH₄⁺/NH₃ equilibrium isotope effect has been estimated to be 19.2‰ in aqueous solution (Hermes et al., 1985), whereas secondary active ammonium (AMT) transporters, which are highly expressed in AOA (Carini et al., 2017), have been shown to exert isotope fractionation of around 13–15‰, due to deprotonation of NH₄⁺ during transport (Ariz et al., 2018). It is unlikely that ammonia oxidation has been limited by NH₃ availability in our study, because of the high substrate concentrations used, which are well above the K_m of the AMO of N. viennensis (5.4 μM NH₃ + NH₄⁺; Kits et al., 2017) and that of the marine strain Nitrosopumilus maritimus strain SCM1 (0.13 μM NH₃ + NH₄⁺; Martens-Habbena et al., 2009), which is closely related to N. adriaticus. Furthermore, Nishizawa et al. (2016) estimated that, when NH₃ concentrations in the pseudo-periplasm are lower than in the medium under laboratory conditions, cell-specific NH₃ diffusion rates into the pseudo-periplasm are higher than cell-specific ammonia oxidation rates. It has also been proposed that the charged S-layer proteins of AOA enhance the diffusion of charged solutes, such as NH₄⁺, which concentrates NH₄⁺ in the pseudo-periplasmic space near the active site of the AMO (Li et al., 2018), where then the equilibrium reaction between NH₄⁺ and NH₃ is relatively fast and considered not to be rate-limiting.

Even if ammonia oxidation was not limited by periplasmatic NH₃ availability, the apparent isotope effect of the AMO can also be underestimated due to concurrent NH₄⁺ assimilation, which has a smaller isotope effect. This process would alter observed ε_RS estimates in proportion to the amount of NH₄⁺ assimilated and the isotope effect for NH₄⁺ assimilation (4–27‰; Hoch et al., 1992). Therefore, we also measured δ¹⁵N of the cell biomass at the end of incubation of N. viennensis grown on 2 mM NH₄⁺ (Figures 1F, 3). Although it is impossible to infer directly the contribution of N assimilation to ε_RS from just one end-point measurement, we propose that N assimilation substantially contributed to the decrease of ε_RS relative to ε_CP in our study, as biomass was ¹⁵N-enriched by ∼10‰ compared to initial NH₄⁺. Biomass N represented 3.1% (±0.3 SE) of ammonia oxidized by N. viennensis grown on 2 mM NH₄⁺. Although dissolved inorganic N (DIN) concentrations (sum of [NH₄⁺] and [NO₂^–]) were relatively constant over the course of ammonia oxidation, we recovered only 81.9% (±1.5 SE) of the initial DIN by the end of incubation of N. adriaticus, and 94.7% (±3.4 SE) and 90.7% (±1.1 SE) of N. viennensis grown on 1 mM NH₄⁺ or 2 mM NH₄⁺, respectively. In N. adriaticus cultures, assimilation of N by contaminant bacteria likely did not contribute substantially to the lower ε_RS relative to ε_CP, due to the high enrichment level of the culture (95%) at the time of the experiment, and the fact that ε_RS of N. adriaticus was similar to that of N. viennensis in pure culture. In addition, the ¹⁵N-enrichment of N. viennensis’ biomass shows that AMO preferentially, and primarily, reacts on pseudo-periplasmatic NH₃, causing ¹⁵N-enrichment of the residual ammonia, which is subsequently assimilated into biomass. We thus propose that under substrate replete conditions, the observed isotope effects of ε_RS of 31.9–33.1‰ primarily reflect the kinetic isotope effect of the AMO-catalyzed reaction, modified by the NH₄⁺/NH₃ equilibrium isotope effect (19.2‰; Hermes et al., 1985) and decreased by the contribution of the lower kinetic isotope effect of NH₄⁺ assimilation for anabolic purposes (4–27‰; Hoch et al., 1992). Moreover, it should be noted that some ammonia oxidizers use distinct pathways of NH₄⁺ assimilation, even among just AOA, which may contribute to different kinetic isotope effects. For instance, some members of the AOA genus Candidatus Nitrosocosmicus appear to assimilate NH₄⁺ via glutamate synthase (GOGAT), whereas all other known AOA use the glutamate dehydrogenase (GDH) pathway (Alves et al., 2019).

FIGURE 3.

Schematic overview of processes and isotope fractionation effects involved in ammonia oxidation, growth and intermediate formation of the soil AOA N. viennensis. δ¹⁵N values are given for endpoint measurements of N₂O and biomass, while average kinetic isotope effects of ammonia oxidation are presented for substrate (NH₄⁺, ε_RS) and product (NO₂^–, ε_CP). Literature values for isotope fractionation of NH₃/NH₄⁺ equilibration (ε_eq; causing ¹⁵N depletion of NH₃), for secondary active NH₄⁺ uptake (ε_upt) and ammonia assimilation (ε_aa) are presented as well. The identity of the enzyme oxidizing NH₂OH to NO₂^– and its inherent isotope fractionation are currently unknown for AOA.

Despite these potential isotope fractionation effects on the RS level, a higher ε_CP relative to ε_RS may also result from accumulation of metabolic intermediates, allowing for at least a second ¹⁵N isotope fractionation step to be observed. First, accumulation of NH₂OH, or any other intermediate, has not been observed in AOA cells, and the coupled activities of AMO and hydroxylamine oxidoreductase (yet unknown in AOA) are assumed to maintain NH₂OH at low steady-state concentrations. Therefore, isotope effects associated with NH₂OH oxidation should have a limited impact on δ¹⁵NO₂^–. Second, any process that adds an additional isotope fractionation step, either prior, or subsequent, to NO₂^– formation, such as the production of the gases NO and N₂O, may result in an under- or overestimation of the kinetic isotope fractionation factor. The effect of N₂O and NO production on δ¹⁵NO₂^– will depend on their formation pathways, and respective isotope effects and by-product yields. In the N. viennensis culture grown on 2 mM NH₄⁺, we measured cumulative N₂O at the end of the incubation, which represented 0.5% (±0.01 SE) of the NH₄⁺ oxidized, or 3.5% (±0.4 SE) of the “missing” N pool. N₂O yields of AOA are generally low. For example, N. viennensis has been shown to produce N₂O at rates of about 0.1% of those of ammonia oxidation when grown on 1 mM NH₄⁺/NH₃ (Stieglmeier et al., 2014b), whereas the marine AOA N. maritimus SCM1 produces even less (0.002–0.03%; Löscher et al., 2012). Here, the cumulative N₂O of the N. viennensis culture had a δ¹⁵N of −38.1‰ (±0.3 SE) (Figures 1F, 3), which was more ¹⁵N-depleted than previously observed for AOA enrichment cultures. AOA have been shown to produce N₂O with δ¹⁵N signatures ranging between −35 and −13‰ in soil enrichment cultures and −9‰ in marine enrichment cultures (Santoro et al., 2011; Jung et al., 2014), while N₂O produced by AOB tends to have lower δ¹⁵N, ranging between −66‰ (Nitrosomonas europaea; Yoshida, 1988) and −10‰ (Nitrosomonas marina; Frame and Casciotti, 2010). Furthermore, by-products that are more ¹⁵N-depleted than the main product of ammonia oxidation (i.e., NO₂^–) would decrease the apparent kinetic isotope effect of AMO (ε_CP), instead of increasing it. Using an isotopic mass balance approach, we calculated that the missing N pool (i.e., the NH₄⁺ taken up which was not oxidized to NO₂^–), would need to have a δ¹⁵N of −18.5‰ (±1.7 SE) in order to account for the difference in isotope fractionation between ε_RS and ε_CP (Table 2). Therefore, the δ¹⁵N₂O signature of −38.1‰ cannot explain the observed large isotope fractionation based on δ¹⁵NO₂^–, since the N₂O produced would need to be a larger contributor to the “missing” N pool, as well as to be ¹⁵N-enriched relative to NO₂^–.

TABLE 2.

Nitrogen pools for N. viennensis culture grown on 2 mM NH₄⁺.

	N pool (μM)	δ15N of N pool (‰)	Percent of missing N pool	Percent of ammonia oxidized
Missing	204.4 (±25.2)	−7.6 (±5.2)
Biomass	38.8 (±3.4)	10.1 (±0.1)	22.8 (±4.1)	3.1 (±0.3)
N2O-N	6.5 (±0.2)	−38.1 (±0.3)	3.5 (±0.4)	0.5 (±0.1)
Unaccounted	139.2 (±27.1)	−18.5 (±1.7)	73.7 (±4.5)

The missing N pool is N that was not present at NH₄⁺ and NO₂^– at the last sampling time point when biomass and N₂O was collected for isotope analysis. We used a mass balance approach to calculate δ¹⁵N of the missing and unaccounted N pool. Standard errors are given in parentheses.

Nitric oxide is an important intermediate in the ammonia oxidation pathway, particularly in that of AOA. Unlike in AOB, NO is a necessary co-reactant for the oxidation of NH₂OH to NO₂^– in AOA, despite being produced in relatively small amounts (Kozlowski et al., 2016). Although the δ¹⁵N signature of NO produced by AOA has not yet been determined, Yoshida (1988) found that NO produced during nitrification by N. europaea had a δ¹⁵N between 0 and +20‰. The production of such ¹⁵N-enriched NO could significantly contribute to the observed overestimation of ε_CP in AOA.

Conclusion

In conclusion, our results show that, under non-limiting substrate conditions, the ε_AMO of two phylogenetically and ecologically distinct AOA strains was 31.9–33.1‰ based on δ¹⁵NH₄⁺, whereas the more commonly estimated ε_AMO based on δ¹⁵NO₂^– was higher (37.7–40.1‰). Thus, NH₄⁺ assimilation, but not N₂O production, significantly affected the isotope fractionation factor of AMO estimated for N. viennensis (Figure 3). Although the potential role of NO in this context remains to be tested, isotopic analysis of this molecule is difficult and therefore future measurements of ε_AMO may rely on coupled estimates from δ¹⁵NH₄⁺ and δ¹⁵NO₂^–.

Data Availability Statement

The raw data supporting the conclusion of this article have been deposited at DRYAD (doi: 10.5061/dryad.0gb5mkkz1).

Author Contributions

WW designed the study. MMo, RA, BB, MMe, MS, LJ, SR, and MW performed the experiments. MMo, RA, BB, MS, and LJ analyzed the data. GH and CS provided the resources and strains. MMo, RA, and WW wrote the manuscript with contributions from all co-authors.

Conflict of Interest

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.