Nitrogen Kinetic Isotope Effects of Nitrification by the Complete Ammonia Oxidizer Nitrospira inopinata.

Analysis of nitrogen isotope fractionation effects is useful for tracing biogeochemical nitrogen cycle processes. Nitrification can cause large nitrogen isotope effects through the enzymatic oxidation of ammonia (NH3) via nitrite (NO2-) to nitrate (NO3-) (15εNH4+→NO2- and 15εNO2-→NO3-). The isotope effects of ammonia-oxidizing bacteria (AOB) and archaea (AOA) and of nitrite-oxidizing bacteria (NOB) have been analyzed previously. Here, we studied the nitrogen isotope effects of the complete ammonia oxidizer (comammox) Nitrospira inopinata that oxidizes NH3 to NO3-. At high ammonium (NH4+) availability (1 mM) and pH between 6.5 and 8.5, its 15εNH4+→NO2- ranged from -33.1 to -27.1‰ based on substrate consumption (residual substrate isotopic composition) and -35.5 to -31.2‰ based on product formation (cumulative product isotopic composition), while the 15εNO2-→NO3- ranged from 6.5 to 11.1‰ based on substrate consumption. These values resemble isotope effects of AOB and AOA and of NOB in the genus Nitrospira, suggesting the absence of fundamental mechanistic differences between key enzymes for ammonia and nitrite oxidation in comammox and canonical nitrifiers. However, ambient pH and initial NH4+ concentrations influenced the isotope effects in N. inopinata. The 15εNH4+→NO2- based on product formation was smaller at pH 6.5 (-31.2‰) compared to pH 7.5 (-35.5‰) and pH 8.5 (-34.9‰), while 15εNO2-→NO3- was smaller at pH 8.5 (6.5‰) compared to pH 7.5 (8.8‰) and pH 6.5 (11.1‰). Isotopic fractionation via 15εNH4+→NO2- and 15εNO2-→NO3- was smaller at 0.1 mM NH4+ compared to 0.5 to 1.0 mM NH4+. Environmental factors, such as pH and NH4+ availability, therefore need to be considered when using isotope effects in 15N isotope fractionation models of nitrification. IMPORTANCE Nitrification is an important nitrogen cycle process in terrestrial and aquatic environments. The discovery of comammox has changed the view that canonical AOA, AOB, and NOB are the only chemolithoautotrophic organisms catalyzing nitrification. However, the contribution of comammox to nitrification in environmental and technical systems is far from being completely understood. This study revealed that, despite a phylogenetically distinct enzymatic repertoire for ammonia oxidation, nitrogen isotope effects of 15εNH4+→NO2- and 15εNO2-→NO3- in comammox do not differ significantly from those of canonical nitrifiers. Thus, nitrogen isotope effects are not suitable indicators to decipher the contribution of comammox to nitrification in environmental samples. Moreover, this is the first systematic study showing that the ambient pH and NH4+ concentration influence the isotope effects of nitrifiers. Hence, these key parameters should be considered in comparative analyses of isotope effects of nitrifiers across different growth conditions and environmental samples.

INTRODUCTION

Natural abundance isotope techniques have proven useful for studying nitrogen (N) transformation processes in aquatic and terrestrial ecosystems (1–3). Several process-oriented models that integrate isotope effects have been reported (4, 5). However, the successful integration of N isotopic composition into N cycle models requires the knowledge of the accurate isotope effects of each N transformation process. Until now, multiple N isotope effects have been reported based on soil and groundwater studies and for microbial isolates (3).

Nitrification represents a two-step N cycle process where ammonia (NH3) is first oxidized to nitrite (NO2−) by ammonia-oxidizing bacteria (AOB) and archaea (AOA), followed by the oxidation of NO2− to nitrate (NO3−) by nitrite-oxidizing bacteria (NOB). Intriguingly, nitrification has shown much larger isotope effects than other N cycle processes, including biological N2 fixation, mineral N uptake, ammonification, and denitrification (3). Isotope effects (15εNH4+→NO2-) for ammonia oxidation to NO2− have been determined in enriched and pure cultures of AOB and AOA (6–9). The AOB strain Nitrosomonas europaea exhibited an isotope effect of −38 to –32‰, whereas Nitrosomonas marina, Nitrosomonas sp. C-113a, and Nitrosospira tenuis showed smaller isotope effects in the range of −25 to −14‰ (6, 7). Measured isotope effects of AOA were in the same range as those of AOB, with a large variation of −21‰ ± 10‰ (8), regardless of whether the AOA had been cultured from marine or geothermal sources (9). The factors causing the large variations of the measured isotope effects in AOA and AOB have remained largely unknown. Possible causes include differences in the pH and in initial NH4+ concentrations between studies, amino acid substitutions in ammonia monooxygenase (AMO; the key enzyme for ammonia oxidation in AOB and AOA), and any isotope effect possibly involved in the subsequent oxidation of the AMO product hydroxylamine (NH2OH) to NO2−, and with gaseous N losses via nitric oxide (NO) or nitrous oxide (N2O) (7, 9).

Only a few studies have addressed isotopic fractionation during NO2− oxidation. The isotope effect of the key nitrite-oxidizing enzyme, nitrite oxidoreductase (NXR) (15εNO2→NO3-), became a focus of attention when the difference of δ15N between NO3− and NO2− in marine samples turned out to be surprisingly large (10, 11). This was unexpected, because processes consuming NO2− (such as NO2− oxidation) or NO3− (denitrification) should increase the δ15 N residual NO2− and NO3−, respectively, and lead to a smaller offset in the δ15N than observed. Hence, these results were taken as an indication for a process that actually decreased the δ15NO2− relative to δ15NO3− (12). Indeed, a subsequent study revealed an inverse isotope effect of NXR, where the substrate (NO2−) became more depleted in 15N compared to the product (NO3−) in the marine NOB Nitrococcus mobilis (10). This contrasts with most other enzymatic processes of the N cycle, where the substrate becomes 15N enriched and the product 15N depleted. Three explanations for such inverse kinetic isotope effects were studied, i.e., equilibrium isotope effects between NO2− and nitrous acid before reaction, reaction reversibility at the enzyme level, and real inverse kinetic isotope fractionation (10). The inverse isotope effect of NXR most likely originates at the enzyme level, where larger force constants in the transition state explain the inverse kinetic isotope effect when stretching vibrational contributions dominate the kinetic isotope effect (13). Follow-up studies demonstrated inverse isotope effects of the NOB Nitrococcus and Nitrobacter to be similar and around 20.5‰, whereas the inverse isotope effects of the NOB Nitrospira and Nitrospina were less pronounced and close to 9.5‰ (11, 14), and those of anammox bacteria were larger with 30.1 to 45.3‰ (15). The cause for the quantitatively different isotope effects of 15εNO2-→NO3- in the diverse NOB remained unclear. Possibly responsible factors include the orientation of the membrane-attached NXR toward either the cytoplasm or periplasm, kinetic differences among the known forms of NXR, and the reversibility of NO2− oxidation by this enzyme (i.e., its capability to also catalyze NO3− reduction) (14).

Complete ammonia oxidizers (comammox organisms), which oxidize NH3 to NO3− on their own, were recently discovered in the genus Nitrospira (16, 17). Comammox organisms are widespread in natural and engineered ecosystems (18). They possess a distinct form of AMO, which is phylogenetically moderately related to the AMO of betaproteobacterial AOB (16, 17). Generally, it is assumed that NH3 and not NH4+ is the substrate for AMO in AOB, AOA, and comammox (19, 20). The only available comammox isolate, Nitrospira inopinata, has a very high substrate affinity for ammonia that exceeds the affinities of all characterized AOB and several AOA (21). Comammox bacteria share a highly similar NXR with canonical (only NO2-oxidizing) Nitrospira, but the affinity of N. inopinata for NO2− is much lower than that reported for canonical Nitrospira (21, 22). The unique kinetic properties of comammox, and the distinct AMO, raise the question of whether NH3 and NO2− oxidation by comammox might show comparable or different isotope effects than found in the canonical nitrifiers. So far, however, no isotope fractionation data from comammox have been available. In this study, we analyzed the N kinetic isotope effects of ammonia oxidation (15εNH4+→NO2-) and nitrite oxidation (15εNO2-→NO3-) of a pure culture of N. inopinata. In addition, we explored whether the isotope effect is influenced by selected environmental factors (medium pH and initial NH4+ concentration). The results provide important constraints for the interpretation of natural abundance stable isotope ratios for N compounds in systems where comammox Nitrospira are prevalent.

RESULTS

Experiment 1: ammonia oxidation with an initial concentration of 1 mM NH4.

Within 2 weeks of this incubation experiment, N. inopinata oxidized the initially provided 1 mM NH4+ to approximately 90% NO3− and 10% NO2− (Fig. 1A). A transient accumulation of NO2− was also observed in previous studies with N. inopinata, where the residual NO2− was finally converted to NO3− during prolonged incubations after NH4+ depletion (16, 21). The ratio of NH4+ consumption to NO2− plus NO3− formation was close to 1.0, indicating that ammonia was almost stoichiometrically oxidized to NO2− and NO3− (Fig. 1A). The initial δ15N of NH4+ was −0.6‰. The δ15N of NH4+ increased exponentially with incubation time, along with an increase of δ15N of NO2− and NO3−. Moreover, the δ15N of NO2− was depleted compared to the δ15N of NH4+ and NO3− during the incubation (Fig. 1B). The 15εNH4+→NO2- was −27.1‰ ± 0.8‰ based on the residual substrate (equation 2, Fig. 1C, and Table 1) and −32.2‰ ± 1.4‰ based on the cumulative product (Table 1), which was in agreement with the 15εNH4+→NO2- (−38 to −14‰) of canonical AOB and AOA (7, 8). The 15εNO2-→NO3- was 7.6‰ ± 0.2‰ based on the residual substrate from the Solver model (Table 1), which was close to the canonical Nitrospira NOB (9‰) (11, 14).

FIG 1

Kinetic isotope effect of N. inopinata cultivated in CaCO3-buffered medium with 1 mM ammonium (NH4+) initial concentration. (A) Concentrations of NH4+, nitrite (NO2−), and nitrate (NO3−). (B) Isotopic signatures of NH4+, NO2−, and NO3−. (C) 15εNH4+→NO2- based on the residual substrate (εAMO-RS).

TABLE 1

Modeled kinetic isotope effects (mean ± s.d., n = 3) of AMO and NXR of Nitrospira inopinata

NH4+ (mM)	pH	NH4+/NO2− oxidation rate (μM/h)	NH4+ oxidation rate [μmol N (mg protein)−1 h−1]	εAMO-RS (‰)	εAMO-CP (‰)	εNXR-RS (‰)
1	6.5	15.8 ± 2.6	Vmax (12.8)	−30.1 ± 0.5	−31.2 ± 0.3	11.1 ± 0.6
1	7.5	18.6 ± 0.7	Vmax (12.8)	−31.6 ± 0.5	−35.5 ± 0.2	8.8 ± 0.6
1	8.5	14.0 ± 1.7	Vmax (12.8)	−33.1 ± 0.8	−34.9 ± 1.6	6.5 ± 1.0
0.1	8.2	5.8 ± 0.3b	2.1 ± 0.1	−19.7	−17.3	6.2
0.25	8.2	8.6 ± 0.4	3.1 ± 0.1	−21.1 ± 1.3	−21.2 ± 2.3	10.8 ± 1.1
0.5	8.2	10.7 ± 0.5	3.8 ± 0.2	−24.8 ± 0.2	−24.3 ± 1.9	10.5 ± 2.3
1	8.2	6.2 ± 0.4c		−27.1 ± 0.8	−32.2 ± 1.4	7.6 ± 0.2
1	8.2	39.5 ± 4.6				9.2 ± 0.5

Modeled kinetic isotope effects (mean ± standard deviation, n = 3) of AMO and NXR of Nitrospira inopinata based on the Solver model at pH 6.5 to 8.5 with initial NH4+ concentrations of 0.1 to 1 mM.

AOA/AOB medium buffered with CaCO3 (pH around 8.2) was used for this batch experiment with 0.1, 0.25, and 0.5 mM NH4+.

The batch experiment was performed with CaCO3-buffered medium but with much less biomass.

Experiment 2: nitrite oxidation with an initial concentration of 1 mM NO2.

In a subsequent experiment, the 15εNO2-→NO3- was directly measured from a batch incubation with NO2− as a substrate. Since N. inopinata is unable to utilize NO2− as an N source for assimilation (16), growth was not expected to occur during the incubation experiment with NO2−. Thus, we used an already highly concentrated cell suspension to analyze the 15εNO2-→NO3-. Within 2 days of incubation, the initially provided 1 mM NO2− was almost stoichiometrically oxidized to NO3− (Fig. 2A). The ratio of NO2− oxidation to NO3− production was 1.03 ± 0.03. The initial δ15N of NO2− was −25‰, and the δ15N of both NO2− and NO3− decreased along with NO2− oxidation. In agreement with previous studies of canonical NOB (see introduction), the δ15N of NO2− was depleted compared to the δ15N of NO3− during NO2− oxidation (Fig. 2B). The calculated, inverse isotope effect of 15εNO2-→NO3- was 9.2‰ ± 0.5‰ based on the Rayleigh models for the residual substrate (Fig. 2C), which was similar with the above-mentioned 15εNO2-→NO3- (7.6‰ ± 0.2‰) calculated from the Solver model during NH3 oxidation. Figure 2D shows the calculated 15εNO2-→NO3- based on the cumulative product.

FIG 2

Kinetic isotope effect of N. inopinata cultivated in CaCO3-buffered medium with 1 mM NO2− initial concentration. (A) Concentrations of NO2− and NO3−. (B) Isotopic signatures of NO2− and NO3−. (C and D) 15εNO2-→NO3- based on the residual substrate (εNXR-RS) (C) and cumulative product (εNXR-CP) (D).

Experiment 3: ammonia oxidation with an initial concentration of 0.1, 0.25, and 0.5 mM NH4.

Similar patterns of NH4+ oxidation, NO2− production and consumption, and NO3− production were observed for all tested initial NH4+ concentrations (Fig. 3A to C). However, the NH4+ oxidation rates increased with higher initial NH4 concentrations (Table 1). The concentration of transiently accumulated NO2− also increased with the initial NH4+ concentration (Fig. 3A to C). The δ15N of NH4+ increased with ongoing NH4+ oxidation, along with an increase in the δ15N of NO2− and NO3−. After about 93% of the NH4+ had been consumed, a pronounced decrease in the δ15N of NO2− was observed, which was consistent with the net consumption of NO2− (Fig. 3, especially Fig. 3D to F). The 15εNH4+→NO2- values based on the residual substrate were significantly (P < 0.05) larger for the 0.5 mM initial NH4+ concentration (−24.8‰) compared to that for the 0.25 mM initial NH4+ concentration (−21.1‰). The 15εNH4+→NO2- was smallest (−19.1‰) for the 0.1 mM initial NH4+ concentration among all the tested initial NH4+ concentrations (Fig. 3G to I). The calculated 15εNH4+→NO2- values based on the cumulative product were similar to those based on the residual substrate, with values of −17.3, −21.2, and −24.3‰ for the 0.1, 0.25, and 0.5 mM NH4+ addition, respectively (Table 1). As outlined below (see Discussion), the weaker 15εNH4+→NO2- at the lowest NH4+ concentration could be due to the low NH4+ oxidation rates with 0.1 mM NH4+ [2.1 μmol N (mg protein)−1 h−1; total protein content was ∼2.8 μg ml−1], which was significantly smaller than the Vmax [12.8 μmol N (mg protein)−1 h−1] of N. inopinata (21). The 15εNO2-→NO3- values based on the Solver model were 6.2, 10.8, and 10.5‰ for the 0.1, 0.25, and 0.5 mM NH4+ treatments, respectively (Table 1). No significant difference of 15εNO2-→NO3- was observed between the 0.25 and 0.5 mM initial NH4+ concentration treatments.

FIG 3

Kinetic isotope effect of N. inopinata cultivated in CaCO3-buffered medium with 0.1, 0.25, and 0.5 mM NH4+ initial concentration. (A to C) Concentrations of NH4+, NO2−, and NO3−. (D to F) Isotopic signatures of NH4+, NO2−, and NO3−. (G to I) 15εNH4+→NO2- based on the residual substrate (εAMO-RS).

Experiment 4: ammonia oxidation at different pH values with an initial concentration of 1 mM NH4.

In this experiment, the maximum NH4+ oxidation rates (day 3 to 4 for pH 6.5 and 7.5; day 4 to 5 for pH 8.5) were significantly (P < 0.05) lower at pH 8.5 than at pH 7.5 (Table 1 and Fig. 4A to C). The NO2− concentration was significantly (P < 0.05) higher at pH 6.5 (361 μM) than that at pH 8.5 (234 μM), which was consistent with the trend of maximum NO2− oxidation rate (this was calculated during the period of NO2− oxidation when NH4+ was almost completely consumed) that was significantly (P < 0.05) lower at pH 6.5 (9.7 μM h−1) than at pH 8.5 (13.5 μM h−1). The δ15N of NH4+, NO2−, and NO3− showed similar patterns as in the other experiments, i.e., the δ15N of NH4+, NO2−, and NO3− increased simultaneously with ammonia oxidation until more than 90% NH4+ was oxidized, followed by a decrease in the δ15N of NO2− until the end of the incubations (Fig. 4D to F). There was no significant difference for the 15εNH4+→NO2- calculated based on the residual substrate among the three pH levels (Table 1 and Fig. 4G to I), but the 15εNH4+→NO2- based on product formation was significantly (P < 0.05) weaker at pH 6.5 than at pH 7.5 and pH 8.5 (Table 1). Interestingly, the δ15N of NO2− was significantly (P < 0.05) lower at pH 6.5 than at pH 8.5 at the beginning of the experiment. Moreover, the pH affected the isotope effect of NO2− oxidation significantly, where the 15εNO2-→NO3- was significantly (P < 0.05) weaker at pH 8.5 than that at pH 7.5 and 6.5, based on the Solver model (Table 1).

FIG 4

Kinetic isotope effect of N. inopinata cultivated with 1 mM NH4+ (initial concentration) at pH 6.5, 7.5, and 8.5. (A to C) Concentrations of NH4+, NO2− and NO3−. (D to F) Isotopic signatures of NH4+, NO2−, and NO3−. (G to I) 15εNH4+→NO2- based on the residual substrate (εAMO-RS).

DISCUSSION

Isotope effects of ammonia and nitrite oxidation by N. inopinata.

The measured N isotope effect for ammonia oxidation (15εNH4+→NO2-) by N. inopinata (residual substrate [RS], −33.0 to −30.7‰; cumulative product [CP], −35.5 to −31.2‰) with an initial substrate concentration of 1 mM NH4+ fell into the range of 15εNH4+→NO2- values determined previously for AOB (−38.2 to −14.2‰) and AOA (−41 to −13‰) (7–9) (Table 2), as well as the measured N isotope effect of the two AOA species Nitrososphaera gargensis (RS, −22.3‰; CP, −32.8‰) and Nitrosocosmicus oleophilus that were also determined in this study (RS, −36.1‰; CP, −36.3‰) (see Fig. S1 and S2 in the supplemental material). However, isotope fractionation data are currently still lacking for many phylogenetic lineages of AOB and AOA. The most similar isotope effects, compared with N. inopinata, have been reported for Nitrosomonas europaea (−38.2‰) and Nitrosomonas eutropha (−32.8‰) (6, 7) (Table 2). In this context, it is noteworthy that comammox Nitrospira, betaproteobacterial AOB, and AOA possess three phylogenetically different types of AMO (16, 17, 23). Moreover, the characterized comammox Nitrospira and many AOA have a much higher substrate affinity for NH3 than AOB (21, 24, 25). Despite the distinct phylogenetic and kinetic properties of the AMO forms, no difference in the magnitude of 15εNH4+→NO2- between comammox, AOB, and AOA became apparent. This result may indicate that the enzymatic mechanism and transition states of the NH3 oxidation step catalyzed by AMO are similar across all ammonia oxidizers. However, the reported 15εNH4+→NO2- values varied strongly, even within one species or strain, in previous research (7–9). Hence, the kinetic isotope effects of ammonia oxidizers may be modulated by environmental factors, some of which have been investigated in our study (see below).

TABLE 2

Compilation of kinetic isotope effects of canonical AOA, AOB, and NOB

Nitrifiergroup	Strain	Initialsubstrate (mM)	pH	εRS (‰)	εCP (‰)	Reference
AOA	Nitrosopumilus adriaticus	1	7.6	−32 ± 1	−40 ± 1	Mooshammer et al. (33)
	Nitrososphaera viennensis	1−2	7.5	−32 ± 1	−39	Mooshammer et al. (33)
	Nitrososphaera gargensis	0.25	8.2	−22 ± 0	−33 ± 2	This study (Fig. S1 and S2)
	Nitrosocosmicus oleophilus	1	7.5	−36 ± 5	−36 ± 5	This study (Fig. S1 and S2)
	AOA enrichment CN25				−22 ± 5	Santoro and Casciotti (8)
	AOA enrichment CN75	0.01−0.075			−21 ± 10	Santoro and Casciotti (8)
	AOA enrichment CN150				−22 ± 5	Santoro and Casciotti (8)
	“Candidatus Nitrosocaldus”	0.2	8.2−8.6		−25 ± 2	Nishizawa et al. (9)
	“Candidatus Nitrosocaldus”	14	8.0		−32 ± 1	Nishizawa et al. (9)
AOB	Nitrosomonas europaea	4.7−25	7.5	−35 ± 3	−32 ± 6	Mariotti et al. (6)
	Nitrosomonas europaea	38			−32 to −25	Yoshida (30)
	Nitrosomonas europaea	1			−38 ± 2	Casciotti et al. (7)
	Nitrosomonas marina	2			−14 ± 4	Casciotti et al. (7)
	Nitrosomonas sp. C-113a	2	8.0		−19 ± 1	Casciotti et al. (7)
	Nitrosospira tenuis	1			−25 ± 1	Casciotti et al. (7)
	Nitrosomonas eutropha	1			−33 ± 2	Casciotti et al. (7)
	Nitrosomonas sp. C-113a					Casciotti et al. (43)
	Nitrosococcus oceani	0.005−0.05	8.2		−46 to −30	Casciotti et al. (43)
	Nitrosospira briensis					Casciotti et al. (43)
NOB	Nitrococcus mobilis			20 ± 3		Buchwald and Casciotti (11)
	Nitrobacter sp. Nb 355	0.05	8.2	21 ± 3		Buchwald and Casciotti (11)
	Nitrospira marina			9 ± 2		Buchwald and Casciotti (11)
	Nitrospira sp. Ecomares 2.1	0.5−1	7.5	10 ± 1		Jacob et al. (14)
	Nitrospina watsonii 347	0.6−1.6		10 ± 1		Jacob et al. (14)
	Nitrospira moscoviensis	1	7.5	9 ± 1		This study (Fig. S3)

Ammonia oxidation (A), isotopic signature (B) of NH4+ and NO2−, and 15εNH4+→NO2- based on substrate (εAMO-RS) (C) and product (εAMO-CP) (D) of Nitrososphaera gargensis cultivated in CaCO3-buffered medium with 0.25 mM NH4+ addition. Download FIG S1, TIF file, 0.06 MB.

Ammonia oxidation (A), isotopic signature (B) of NH4+ and NO2−, and 15εNH4+→NO2- based on substrate (εAMO-RS) (C) and product (εAMO-CP) (D) of Nitrosocosmicus oleophilus with 1 mM NH4+ addition. Download FIG S2, TIF file, 0.07 MB.

Like canonical NOB (10, 11, 14), N. inopinata displayed an inverse 15εNO2-→NO3-, meaning that 15NO2− was preferentially oxidized to NO3− during NO2− oxidation. The measured value of 15εNO2-→NO3- (9.2‰ ± 0.5‰ at pH 8.2 and 1 mM NO2−) was in line with previously determined 15εNO2-→NO3- values (9.1 to 10.2‰) of canonical Nitrospira NOB (11, 14) (Table 2 and Fig. S3). The NXR of N. inopinata clusters together with the NXR of canonical Nitrospira in phylogenetic analyses of the substrate-binding alpha subunit and the electron-channeling beta subunit of this enzyme (16). This close phylogenetic relationship is consistent with the highly similar kinetic isotope effects of comammox and canonical Nitrospira. Other NOB such as Nitrobacter (20.6‰) and Nitrococcus (12.8‰) showed remarkably stronger kinetic isotope effects than Nitrospira (10, 11). Interestingly, Nitrobacter and Nitrococcus have a lower whole-cell affinity (higher K(app)) for NO2− than Nitrospira (14, 22). Therefore, the differences in the kinetic isotope effect were suggested to be linked to the NO2− affinity of NOB, possibly caused by a different stability of the transition state in high- versus low-affinity NXR forms (14). However, this explanation turns out to be unlikely, considering that the isotope effect of the NXR of N. inopinata resembles that of other Nitrospira strains, whereas its whole-cell nitrite affinity is low and in the same range as the whole-cell affinity of Nitrobacter species (21). Instead, it may be more relevant that the NXR of Nitrospira (including comammox species) is located in the periplasmic space (where it may interact with the cytoplasmic membrane), whereas the membrane-attached NXR of Nitrobacter and Nitrococcus is oriented toward the cytoplasm (references 26 and 27 and references cited therein). The cellular localization of NXR determines whether transport of NO2− and NO3− over the cytoplasmic membrane is needed, which might also influence the kinetic isotope effect through the properties of the nitrite/nitrate transporter: it may limit the expression of the isotope effect of NXR if NO2− transport (has almost no isotope fractionation as a diffusional process) becomes limiting relative to NXR activity. The localization of NXR also affects the energy efficiency of nitrite oxidation, because only a periplasmic NXR contributes directly to proton motive force (26, 28). Moreover, the periplasmic and cytoplasmic NXR types represent phylogenetically unrelated lineages within the type II dimethyl sulfoxide (DMSO) reductase-like family of molybdopterin-containing enzymes (26, 29). The different magnitude of the kinetic isotope effect in NOB likely reflects the distinct functional properties and evolutionary history of the periplasmic and cytoplasmic NXR forms. This possibility was discussed previously (14) and gains further support from our results.

Nitrite oxidation (A), isotopic signature (B) of NO2−, and 15εNO2-→NO3- based on substrate (εNXR-RS) (C) of Nitrospira moscoviensis with 1 mM NO2− addition. Download FIG S3, TIF file, 0.03 MB.

Effects of substrate concentration and pH on the kinetic isotope effects of comammox bacteria.

Nitrogen isotope effects of ammonia oxidizers varied largely among previous studies, even within AOB and AOA (7–9). This variability might partly be caused by different enzyme (AMO) structures. However, a substantial variation in kinetic isotope effects can occur even within a single isolate, as reported for the AOB species Nitrosomonas europaea (6, 7, 30). Such variability indicates that the cultivation conditions and growth stage and specific factors, such as concentration-dependent diffusion limitations of substrate availability for the critical enzyme or the accumulation of intermediates in an N transformation pathway, can significantly affect the kinetic isotope effect (illustrated in Fig. 5). For comammox, possible effects of environmental conditions on the kinetic isotope effects of ammonia oxidation and nitrite oxidation, respectively, have not yet been studied. Here, we analyzed the effects of two conditions, the initial NH4+ concentration and culture pH, on the kinetic isotope effects of complete nitrification by N. inopinata.

FIG 5

Schematic overview of N processes and isotope fractionation effects involved in NH3 oxidation, NO2− oxidation, formation of intermediates, and growth of the comammox strain Nitrospira inopinata. Average kinetic isotope effects of NH3 and NO2− oxidation are presented for the residual substrate (NH4+, εRS1; NO2−, εRS2) and the cumulative product (NO2−, εCP1) with the addition of 1 mM NH4+ at pH 7.5. Isotope fractionation of NH3/NH4+ equilibration and NH3 uptake refer to reference 32. This schematic illustration is modified from reference 33.

Previous studies with AOB and AOA mostly found 15ε to be below −20‰, which is the range seen in our experiments with comammox at high initial NH4+ concentrations (Tables 1 and 2). However, we observed salient decreasing trends of 15εNH4+→NO2- with decreasing NH4+ concentrations between 1 and 0.1 mM. This result is consistent with previous data from an enrichment of thermophilic canonical AOA, where smaller isotope effects were found at lower NH4+ concentrations (0.25 mM) compared to those in experiments with high NH4+ concentrations (10 mM) (9). Also for denitrifiers, a decrease of the N isotope effect was observed along with decreasing NO3− concentrations in the medium (31). In our study, the NH3 oxidation rates were lower at 0.1 mM NH4+ [2.1 μmol N (mg protein)−1 h−1] than at 0.25 mM [3.1 μmol N (mg protein)−1 h−1] and 0.5 mM NH4+ [3.8 μmol N (mg protein)−1 h−1], which were much lower than the Vmax [12.8 μmol N (mg protein)−1 h−1] of N. inopinata (21). Thus, the rate-limiting step for NH3 oxidation at low NH4+ concentrations was probably more dependent on NH3 diffusion/transport from the extracellular space into the periplasm, where the active sites of the enzymes involved in ammonia oxidation are likely located. Therefore, under these conditions, 15εNH4+→NO2- did not reflect the enzymatic isotope effect, but rather the equilibrium isotope effect 15εNH4+→NH3 at low NH4+ concentrations, which is around −19‰ (32). However, when NH4+ concentrations are higher and transport does not limit the AMO activity, enzymatically catalyzed NH3 oxidation will become the limiting step, and 15εNH4+→NO2- based on the residual substrate (εRS1) will converge to the enzymatic isotope effect of AMO. Moreover, the isotope effect based on εRS1 is also influenced by NH4+ assimilation for biomass formation and can therefore diverge from that based on residual substrate of ammonia oxidation. Compared to εRS1, 15εNH4+→NO2- based on product formation (εCP1) is affected by other factors, including N intermediate (NH2OH and NO) accumulation and N gas (N2O and NO) loss. Mooshammer et al. (33) demonstrated N assimilation to be the main factor responsible for the difference between εRS1 and εCP1 in the AOA species Nitrososphaera viennensis. In our experiment, we did not observe any significant change in the total protein content of the N. inopinata cultures at 0.1, 0.25, and 0.5 mM NH4+ during the incubation. The absence of detectable growth may explain the similar isotope effects of εRS1 and εCP1 at all three NH4+ concentrations. It appears that the NH4+ concentration also influenced the isotope effect of NO2− oxidation, as the modeled 15εNO2-→NO3- was considerably smaller at the lowest tested initial NH4+ concentration (0.1 mM; Table 1). The reason could be the low maximal NO2− concentration (around 10 μM) during NH3 and NO2− oxidation with 0.1 mM NH4+ (Fig. 3A). N. inopinata has quite a poor affinity (372 ± 55 μM) for NO2− during NO2− oxidation (21), and NO2− accumulated to higher concentrations in the experiments with 0.25 and 0.5 mM NH4+ (Fig. 3B and C). Accordingly, the kinetic isotope effect of NO2− oxidation was more pronounced at these higher concentrations (Table 1).

Medium pH is another factor potentially affecting kinetic isotope effects. N. inopinata has strong activities of NH3 and NO2− oxidation in the pH range of 6.5 to 8.5, with lower NH3 oxidation rates at pH 8.5 than those at pH 7.5 (Table 1). The NH3 oxidation rates were not significantly different between pH 6.5 and 7.5, while the NO2− oxidation rates (which were calculated in the later period of NH3 oxidation when NH4+ was almost completely consumed) were significantly lower at pH 6.5 than that at pH 8.5. The pH also influenced the 15εNH4+→NO2- and 15εNO2-→NO3-. The εRS1 did not change significantly among different pH values, while the εCP1 was much lower at pH 6.5 than that at pH 7.5 and pH 8.5 (Table 1). As discussed before, εCP1 was probably affected by the isotopic fractionation during intermediate formation (33). With N. inopinata cultures, release of small amounts of NH2OH, NO, and N2O from cells has been observed during the oxidation of NH3 to NO2− (34, 35). Any pH-dependent shifts in the amounts of these released compounds could lead to changes of 15εNH4+→NO2- (Fig. 5).

A pH-dependent shift was also observed for the 15εNO2-→NO3-, which decreased significantly from 11.1‰ to 6.0‰ when the pH increased from 6.5 to 8.5 (Table 1). As stated above, we assume that a limited NO2− availability caused the observed decrease of 15εNO2-→NO3- in the experiment with an initial NH4+ concentration of only 0.1 mM (Table 1). We observed that the concentration of transiently accumulated NO2− was lowest at pH 8.5 (compared to pH 6.5 and 7.5) during the whole NH3 oxidation period (Fig. 4A to C), which was in agreement with the lower rate of NH3 oxidation at pH 8.5 (Table 1). In addition, the maximum NO2− oxidation rate was higher at pH 8.5 than that at pH 6.5 and 7.5. Thus, the relatively high rate of NO2− oxidation and low rate of NH3 oxidation together led to the lower NO2− concentrations and thus can make NO2− diffusion the limiting step for NO2− oxidation at pH 8.5, especially for N. inopinata that has a low affinity for NO2− during NO2− oxidation. The effect of pH on NO2− self-decomposition was unlikely the cause of the different isotope effect in the pH range of 6.5 to 8.5. In our experiments, we found no significant change of the N balance and of δ15NNH3+NO2+NO3 at pH 6.5 to 8.5 (Fig. S4). This is consistent with the findings of Casciotti et al. (10), where no change of δ15N was observed in the δ15NNO2+NO3 in incubations of nitrite oxidizers and in control flasks in the pH range of 7.8 to 8.8. Until now, there has been no systematic investigation of the pH effect on 15εNO2-→NO3- of canonical NOB. Recently, a new NOB from the genus Nitrospira has been cultivated from an alkaline lake (36). It would be worthy to explore the 15εNO2-→NO3- of alkali-tolerant NOB and the mechanisms of NO2− oxidation at alkaline conditions in further studies.

Stability of the δ15NNH3+NO2+NO3 (A) and the total nitrogen balance (B), at pH values ranging from 6.5 to 8.5, in experiment 4 (see main text). Data points show the means of three replicates (error bars depict standard deviations). Download FIG S4, TIF file, 0.04 MB.

Conclusions.

In summary, our results demonstrate that the 15εNH4+→NO2- and 15εNO2-→NO3- of comammox N. inopinata ranged from −33‰ to −27‰ and 6.5‰ to 9‰, respectively, with nonlimiting NH4+ and NO2− supply as the substrates at pH 7.5 to 8.5. Both substrate concentration and pH affected the 15εNH4+→NO2- and 15εNO2-→NO3- of N. inopinata during NH3 oxidation. At low NH4+ concentrations, especially when NH3 oxidation rates were much smaller than the Vmax of N. inopinata, the 15εNH4+→NO2- was closer to 15εNH4+→NH3 and did not reflect the enzymatic isotope effect of N. inopinata. Medium pH affected the 15εNH4+→NO2- of N. inopinata based on the cumulative product, due to the effect of pH on intermediate formation of N. inopinata. The exact reasons responsible for the weaker isotope effects of 15εNO2-→NO3- at higher pH remained elusive. Further studies should target the effects of pH and substrate concentration on the kinetic isotope effects of canonical AOB, AOA, and NOB to investigate the underlying mechanisms.

MATERIALS AND METHODS

Cultivation of Nitrospira inopinata.

Cultures of N. inopinata were maintained at 37°C with 1 mM NH4Cl in a CaCO3-buffered AOM medium containing (per liter) (37): 50 mg KH2PO4, 50 mg MgSO4·7H2O, 75 mg KCl, 584 mg NaCl, 4 g CaCO3 (solid buffer), 1 ml selenium-tungstate solution (SWS), and 1 ml trace element solution (TES). For the composition of TES and SWS, please refer to Widdel et al. (38). The pH of the medium was maintained at approximately 8.2. The cultivation conditions and experiments for Nitrososphaera gargensis, Nitrosocosmicus oleophilus, and Nitrospira moscoviensis are described in Text S1 in the supplemental material.

Culture conditions and description of the experiments performed with Nitrososphaera gargensis, Nitrosocosmicus oleophilus, and Nitrospira moscoviensis. Download Text S1, PDF file, 0.1 MB.

Incubation experiments. (i) Experiment 1: ammonia oxidation with an initial concentration of 1 mM NH4

Metabolically active (i.e., ammonia-oxidizing) N. inopinata cells (190 ml) were harvested by centrifugation (5,000 × g, 30 min), washed twice with CaCO3-buffered medium (pH ∼8.2), and resuspended in 300 ml of CaCO3-buffered medium containing 1 mM NH4+. Subsequently, the cell suspension was equally distributed into three autoclaved 250-ml glass bottles. On days 0, 7, 9, 13, and 14 after inoculation, 8-ml aliquots of each replicate were transferred into 15-ml plastic tubes and centrifuged (10,000 × g, 10 min). Aliquots (1 ml) of the supernatant were transferred into 1-ml Eppendorf tubes for NO2− and NO3− isotope analysis, respectively, and 5-ml aliquots of the supernatant were transferred into 15-ml plastic tubes for NH4+ isotope analysis. All aliquots of supernatants were frozen at –20°C immediately after sampling.

(ii) Experiment 2: nitrite oxidation with an initial concentration of 1 mM NO2−.

Metabolically active N. inopinata cells (2,000 ml) were harvested by centrifugation (5,000 × g, 30 min), washed once, and resuspended in 120 ml of CaCO3-buffered medium containing 1 mM NO2−. The cell suspension was equally distributed into three autoclaved 100-ml glass bottles. Samples were taken at 0, 11, 23, 35, and 47 h after inoculation and were centrifuged and stored as described above for experiment 1.

(iii) Experiment 3: ammonia oxidation with an initial concentration of 0.1, 0.25, and 0.5 mM NH4+.

Metabolically active N. inopinata cells (700 ml) were harvested by centrifugation (5,000 × g, 30 min), washed once, and resuspended in 1,000 ml of CaCO3-buffered medium without NH4+. Subsequently, 900 ml of the cell suspension was equally split into nine autoclaved 250-ml glass bottles. For the different NH4+ treatments, 0.02, 0.05, and 0.1 ml of a sterile 0.5 M NH4+ solution was added to three bottles, respectively, resulting in triplicates per NH4+ concentration. Samples were taken at 0, 4, 7.5, 16.5, 22, and 28 h (0.1 mM NH4+), at 0, 7.5, 16.5, 22, 28, and 46 h (0.25 mM NH4+), and at 0, 22, 31.5, 43, 54, and 66 h (0.5 mM NH4+) after inoculation. The samples were centrifuged and stored as described above for experiment 1.

(iv) Experiment 4: ammonia oxidation at different pH values with an initial concentration of 1 mM NH4+.

A stock culture of N. inopinata was transferred from CaCO3-buffered medium to medium buffered with 2 mM NaHCO3, followed by two rounds of growth and transfer in this medium, in order to remove solid CaCO3. Metabolically active cells (1,200 ml) were then harvested by centrifugation (8,000 × g, 15 min), washed with 2 mM NaHCO3-buffered medium, and resuspended in 10 ml of the same medium. This cell suspension was used to inoculate nine glass bottles containing 100 ml medium. During the incubations, 10 mM MES [2-(N-morpholino)ethanesulfonic acid], 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and 10 mM TAPS ([tris(hydroxymethyl)methylamino] propanesulfonic acid) were used as buffers to adjust the pH to 6.5, 7.5, and 8.5, respectively. The MES stock buffer was prepared by dissolving 9.76 g MES in 100 ml of 136 mM NaOH. The HEPES stock buffer was prepared by dissolving 23.83 g HEPES in 100 ml of 600 mM NaOH. The TAPS stock buffer was prepared by dissolving 24.3 g TAPS in 100 ml of 570 mM NaOH. Buffer stock solutions were diluted to 10 mM MES, HEPES, and TAPS, respectively, and sterilized. 1 M HCl and 1 M NaOH were used to adjust the pH to 6.5, 7.5, and 8.5. For each pH treatment, samples were taken on 0, 0.3, 1.3, 2.3, 3.3, 4.3, 5.3, 6.5, and 8.5 days after inoculation. The samples were centrifuged and stored as described above for experiment 1.

Chemical analyses.

Inorganic N concentrations were measured by using established protocols (39). Combined NH3 and NH4+ concentrations were determined by the indophenol blue method. NO2− concentrations were measured spectrophotometrically by the Griess reaction after reaction with sulfanilamide and N-1-naphthyl-ethylenediamine dihydrochloride. NO3− concentrations were measured by the Griess reaction after reduction to NO2− with vanadium chloride. Total protein concentrations were measured by the Bradford assay.

Nitrogen isotope analyses.

The δ15N values of NH4+ were analyzed by microdiffusion coupled to elemental analyzer-isotope ratio mass spectrometry (EA-IRMS [40]). For isotopic calibration, the following NH4+ standards were used: IAEA-N-2 (20.3‰ ± 0.2‰), IAEA-N-1 (0.4‰ ± 0.2‰), and USGS26 (53‰ ± 0.4‰). The δ15N signatures of NO2− and NO3− were measured by purge-and-trap isotope ratio mass spectrometry (PT-IRMS) after chemical conversion of NO2− and NO3− to N2O (40). In-house NO2− and NO3− standards, which ranged between −20.5 and 16.8‰, were used for isotopic calibration and were analyzed in parallel with the samples.

Natural 15N abundances are defined in the delta notation as follows: δ15N (‰) = [(15Nsample/14Nsample)/(15Nstd/14Nstd) − 1] × 1,000 where std stands for standard. Isotope ratios are reported relative to AIR (atmospheric dinitrogen).

Kinetic isotope effects were defined as follows: where kL is the first-order rate constant for the reaction of isotopically light molecules (e.g., 14N) and is the rate constant for the reaction of isotopically heavy molecules (e.g., 15N). The organism-level nitrogen isotope effect for ammonia oxidation (15ε ∑NH3/NO2−) was calculated using the Rayleigh residual substrate (RS) equation (equation 2) and the cumulative product (CP) equation (equation 3): where δRS is the N isotope signature of the residual substrate, i.e., NH3 (in the case of NH3 oxidation) or of NO2− (in the case of NO2− oxidation); δCP is the N isotope signature of the cumulative product, i.e., NO2− (in the case of NH3 oxidation) or NO3− (in the case of NO2− oxidation); δinput is the substrate isotope signal initially present in the medium, and f is the oxidizing fraction of substrate. δ15N at any time since the initial time point was calculated from the measured δ15N using the following isotope mass balance equation: where Ncon. is the measured concentration of a specific substrate or product, which includes the background from initial cultures plus the added substrates, and Ninitial is the concentration of substrates transferred with the initial cultures, i.e., the background concentrations of substrates. Initial measurements of the δ15N signatures of NH4+, NO2−, and NO3−, deriving from transferring the cultures to new media, when starting new experiments, were done for time zero, and corrected for from the following culture samples using mass and isotope balance equations (41).

Solver model.

An isotope fractionation model was constructed based on linearly connected, closed system isotope fractionation submodels depicting the coupled sequential processes of nitrification. We assumed that the processes in all incubations were operating under closed system conditions as shown elsewhere (6). Three processes were considered and incorporated in the model in sequential order: (i) NH3 oxidation (AO), (ii) NO2− production (NiP), and (iii) NO2− oxidation to NO3− (NiO). AO can also include isotope effects of equilibrium isotope fractionation between NH3 and NH4+ and of NH4+ uptake and is distinguished from NiP, because the kinetic isotope effect of RS and CP of NH3 oxidation can differ (33). The reason is that multiple processes consume NH3/NH4+, which is used for biomass formation and NH3 oxidation, and that the NH3 oxidation pathway in nitrifiers comprises several intermediates and by-products (hydroxylamine, nitric oxide, and nitrous oxide), which can also change the isotope effect as determined by RS and CP. The model is therefore composed of a system of nine equations (equations 5 to 13). It uses the measured N compound concentrations (NH4+initial, NH4+residual, NO2−residual, and NO3−cumulative) and their δ15N values (δ15NNH4+ initial, δ15NNH4+ residual, δ15NNO2- residual, and δ15NNO3- cumulative) to simulate the kinetic isotopic effects (ε) and fractions (f, 0 ≤ f ≤ 1) of N sources converted to N sinks in the three listed processes. Therefore, for the three coupled processes, we derive: where εAO, εNiP, and εNiO represent the kinetic isotopic effects of NH3 oxidation, NO2− production, and NO2− oxidation, respectively. The corresponding N fractions are denoted as fAO, fNiP, and fNiO. This system of equations was set up in Microsoft Excel and solved by the SOLVER macro in Microsoft Office Excel. The following setting was used: set objective (δ15NTDN as measured). The variable cells (all ε and f values of all processes) are solved so that all modeled RS and CP values (concentrations and δ15N signatures) conform to the measured values. The ranges of variable cells (ε and f values) for each process were subject to constraints (see Table S1 in the supplemental material) with reference to published synthesis studies (7–10, 14, 33). The model was run for each incubation experiment and replicate individually, based on 1,000 iterations and using the GRG nonlinear engine as the solving method. Model accuracy across all incubation experiments was examined by regressing the simulated N contents and δ15N values of individual N pools against the corresponding measured values. Model results with an adjusted R2 of >0.95 were accepted. Otherwise, variable constraints were adapted and outliers were deleted eventually.

Constraints for the Solver model. Download Table S1, PDF file, 0.1 MB.

Statistical analyses.

Analysis of variance (ANOVA) was used to test the effects of pH and concentration levels on the isotope effects of 15εNH4+→NO2- and 15εNO2-→NO3- using the R software package (version 3.4.3 [42]). Tukey’s tests (P < 0.05) were used to examine significant differences between the means of kinetic isotope effects at different pH or concentration levels.