Coupled nitrification and N₂ gas production as a cryptic process in oxic riverbeds

Abstract

The coupling between nitrification and N₂ gas production to recycle ammonia back to the atmosphere is a key step in the nitrogen cycle that has been researched widely. An assumption for such research is that the products of nitrification (nitrite or nitrate) mix freely in the environment before reduction to N₂ gas. Here we show, in oxic riverbeds, that the pattern of N₂ gas production from ammonia deviates by ~3- to 16-fold from that predicted for denitrification or anammox involving nitrite or nitrate as free porewater intermediates. Rather, the patterns match that for a coupling through a cryptic pool, isolated from the porewater. A cryptic pool challenges our understanding of a key step in the nitrogen cycle and masks our ability to distinguish between sources of N₂ gas that 20 years’ research has sought to identify. Our reasoning suggests a new pathway or a new type of coupling between known pathways in the nitrogen cycle.

The N cycle involves complex, microbially-mediated shuttling between ammonium, nitrite and nitrate, with climatically important greenhouse gas byproducts. Here the authors use isotope labeling experiments in river sediments and find a cryptic new step in the N cycle between nitrification and the removal of fixed N through N₂ gas production.

Introduction

Nitrogen is a key bio-element for life on Earth, integral to proteins and the very DNA that tells life what to do. A vast reservoir of nitrogen resides in the atmosphere as N₂ gas, unavailable to the majority of life until being fixed by either biological or anthropogenic nitrogen fixation. Life’s organically-bound nitrogen in turn decays to ammonia following excretion or death. To complete the cycle, first nitrogen must be oxidised to nitrite or nitrate which can then be reduced back to atmospheric N₂ gas. This process of ammonia oxidation—known as nitrification—typically occurs in two stages carried out by specialised aerobic chemoautotrophic ammonia- and nitrite-oxidising microbes, for example, in soils, sediments, freshwater, or marine ecosystems (Eqs. 1 and 2, respectively):

$$2{\mathrm{NH}}_4^{+}+3{\mathrm{O}}_2\to 2{\mathrm{NO}}_2^{-}+2{\mathrm{H}}_2\mathrm{O}+4{\mathrm{H}}^{+}\mathrm{\Delta }{G}^{\circ \prime }=-270\mathrm{kJ}\left(\mathrm{per}{\mathrm{NH}}_4^{+}\right)$$ 1

$$2{\mathrm{NO}}_2^{-}+{\mathrm{O}}_2\to 2{\mathrm{NO}}_3^{-}\mathrm{\Delta }{G}^{\circ \prime }=-79\mathrm{kJ}\left(\mathrm{per}{\mathrm{NO}}_2^{-}\right)$$ 2

Nitrite and nitrate can then be reduced to N₂ gas either alone, in a phylogenetically widespread form of microbial anaerobic respiration termed denitrification¹ (Eq. 3a, b) or, in combination with ammonia, in a phylogenetically narrow respiratory pathway termed anaerobic ammonia oxidation, namely anammox² (Eq. 4).

$$2{\mathrm{NO}}_3^{-}+10{\mathrm{e}}^{-}+12{\mathrm{H}}^{+}\to {\mathrm{N}}_2+6{\mathrm{H}}_2\mathrm{O}\mathrm{\Delta }{G}^{\circ \prime }=-360\mathrm{kJ}\left(\mathrm{per}{\mathrm{NO}}_3^{-}\right)$$ 3a

$$2{\mathrm{NO}}_2^{-}+6{\mathrm{e}}^{-}+8{\mathrm{H}}^{+}\to {\mathrm{N}}_2+4{\mathrm{H}}_2\mathrm{O}\mathrm{\Delta }{G}^{\circ \prime }=-282\mathrm{kJ}\left(\mathrm{per}{\mathrm{NO}}_2^{-}\right)$$ 3b

$${\mathrm{NH}}_4^{+}+{\mathrm{NO}}_2^{-}\to {\mathrm{N}}_2+2{\mathrm{H}}_2\mathrm{O}\mathrm{\Delta }{G}^{\circ \prime }=-358\mathrm{kJ}$$ 4

In addition, smaller amounts of N can be returned to the atmosphere as nitrous oxide (N₂O) but we do not consider those further here^3–5. Combinations of Eqs. (1) to (4) recycle ammonia back into atmospheric N₂ gas and this coupling between aerobic nitrification and anaerobic N₂ gas production is a key concept in the nitrogen cycle, controlling ecosystem production and the abundance of life on Earth^6,7.

Besides the now accepted reactions described in Eqs. (1) to (4), Broda’s original thermodynamic predictions that drove the quest for anammox^8,9 also included the potential for complete aerobic ammonia oxidation to N₂ gas—that, to the best of our knowledge—has yet to be observed in nature:

$$4{\mathrm{NH}}_4^{+}+3{\mathrm{O}}_2\to 2{\mathrm{N}}_2+6{\mathrm{H}}_2\mathrm{O}+4{\mathrm{H}}^{+}\mathrm{\Delta }{G}^{\circ \prime }=-316\mathrm{kJ}\left(\mathrm{per}{\mathrm{NH}}_4^{+}\right)$$ 5

In estuarine or coastal sea sediments, combinations of recognised aerobic and anaerobic metabolisms (Eqs. 1 to 4) buffer the flux of terrestrial nitrogen out to sea and are considered to be physically divided between the oxic and anoxic sediment layers—albeit by only a few tenths of millimetres¹⁰. In rivers, nitrite and nitrate borne from aerobic nitrification (Eqs. 1 and 2), in either the surrounding catchment soils or the riverbed itself, can be transported over large distances (1–100 km) before some 47 Tg N per year is removed from the fluvial network as N₂ gas^11–13. Regardless of the setting, the important point to appreciate here is that the products of aerobic nitrification (e.g., nitrate and nitrite) are assumed to be free to mix with any existing nitrate and nitrite in the surrounding porewater before they are subsequently metabolised, anaerobically, to N₂ gas. That is, there is—in effect—only one pool of nitrate and nitrite awaiting reduction to N₂ gas regardless of their origins. Indeed, this concept of free mixing between substrates lies at the very heart of the common ¹⁵N isotope pairing techniques used to disentangle and quantify the cycling of nitrogen in sediments that are major sources of N₂ gas on Earth^11,14,15.

Most research into the coupling between aerobic nitrification and anaerobic N₂ gas production in sediments has studied the two separately using either oxic or anoxic incubations, respectively¹⁶, but now work including oxygen is increasing¹⁷. Previously we demonstrated¹⁸ that oxic (~30% to 100% of air-saturation for oxygen) gravel and sandy riverbed sediments harbour a coupling between aerobic nitrification and, seemingly, anaerobic N₂ gas production with that production being attributed to a combination of denitrification and anammox¹⁸. We now show that the pattern of N₂ gas production from ammonia in these oxic riverbeds violates the prevailing concept that coupled nitrification and N₂ gas production is a two-step process with free nitrite or nitrate as intermediates. Not only does this challenge our understanding of a key coupling in the nitrogen cycle but it also masks our ability to distinguish between denitrification and anammox as sources of N₂ gas. Indeed, it may actually suggest a new pathway or at least a new type of coupling between known pathways in the nitrogen cycle.

Results and discussion

N₂ gas production is independent from porewater nitrite or nitrate

Following on from our original work¹⁸ on nitrification and putative anaerobic N₂ gas production in oxic riverbeds, we wanted to explore further how these two processes are coupled. We began by collecting sediment from four rivers—two each of predominantly gravel and sand and then extended our sampling to a total of twelve rivers (Supplementary Figure 1 and Supplementary Table 1). We added ¹⁵N-ammonia to oxic sediment microcosms (see Methods) to trace the coupling between nitrification and N₂ gas production both with and without the inhibitor of aerobic nitrification, allylthiourea¹⁹ (~80 µM ATU in the porewater, Treatments 1 & 2, Table 1 and Methods) that does not inhibit denitrification or anammox^2,20. As before¹⁸, we measured the immediate production of ¹⁵N-N₂-gas that was stopped by inhibiting the first step (Eq. 1) of aerobic ¹⁵N-ammonia oxidation with ATU (Fig. 1a, Table 1). The coupling between aerobic ammonia oxidation and N₂ gas production was clearly strong, however it was not complete. For example, across the twelve rivers, approximately 60% (Fig. 1b) of the oxidised ¹⁵N-ammonia tracer was recovered from the porewater as ¹⁵NO_x⁻, i.e., as either ¹⁵N-nitrite (Eq. 1) or the final product of nitrification, ¹⁵N-nitrate (Eq. 2) e.g., ¹⁵NO_x⁻ is the sum of ¹⁵NO₂⁻ and ¹⁵NO₃⁻.

Table 1.

Summary of total ¹⁵N-N₂ production in oxic incubations with ¹⁵NH₄⁺ or ¹⁵NO₂⁻. Mixed-effects models were used to estimate overall rates of total ¹⁵N-N₂ production for the incubations in Fig. 1a. Treatments 1 to 6 were applied to sediments from the first set of 4 rivers, and then just treatments 1 and 2 for the subsequent set of 12 rivers. Model fitting was carried out in the lme4 package in R⁴⁵ and rate estimates, standard errors (s.e.) and 95% confidence intervals derived using emtrends from the emmeans package (see Methods). Significant production (bold) of ¹⁵N-N₂ was only measured with treatments 1 and 3.

Code, Treatment	Rivers (replicates)	Total 15N-N2 (nmol N g−1 h−1)	s.e.	Lower 95% C.I.	Upper 95% C.I.
1, 15NH4+ + ATU	4 (5)	0.110	0.337	−0.667	0.886
2, 15NH4+	4 (5)	1.855	0.326	1.078	2.631
3, 15NH4+ + 14NO2− + ATU	4 (5)	0.152	0.337	−0.625	0.929
4, 15NH4+ + 14NO2−	4 (5)	1.941	0.326	1.165	2.717
5, 14NH4+ + 15NO2− + ATU	4 (5)	0.314	0.326	−0.462	1.091
6, 14NH4+ + 15NO2−	4 (5)	0.279	0.326	−0.497	1.055
1, 15NH4+ + ATU	12 (5)	0.129	0.178	−0.249	0.506
2, 15NH4+	12 (5)	1.465	0.176	1.091	1.839

Fig. 1. Oxic incubations with ¹⁵N-ammonia tracer produce both ¹⁵N₂ gas and ¹⁵NO_x⁻.

a Overall average production of total ¹⁵N-N₂ (i.e., ²⁹N₂ and ³⁰N₂) over time in the presence or absence of the inhibitor of ammonia mono-oxygenase, allylthiourea (ATU). The first 4 rivers (cyan circles, n = 40, 4 rivers x 5 replicates x 2 treatments at each time point, ± 1 s.e.) and the follow-up across 12 rivers (purple triangles, n = 60, 12 rivers x 5 replicates at each time point, ± 1 s.e.); open coloured symbols are the same plus ATU (see Table 1). b Proportions of oxidised ¹⁵N-ammonia tracer from a, recovered as either ¹⁵NO_x⁻ or ¹⁵N₂ across the 12 rivers (n = 60 as for a). Upper and lower box boundaries are 75^th and 25^th percentiles, respectively, upper and lower whiskers are 90^th and 10^th percentiles, respectively, the extreme outliers the maxima and minima and the horizontal line the centre, median value.

The presence of ¹⁵N-ammonia and ¹⁵N-NO_x⁻ together in the porewater generates two ¹⁵N-labelled substrate pools. The fraction of the pool labelled with ¹⁵N is termed F_A for ammonia (NH₃) and F_N for NO_x⁻ (Eqs. 10 and 11 in Methods). Theoretically, combinations of Eqs. (1) to (4) can draw on these two substrate pools (F_A and F_N) to produce both the single-¹⁵N-labelled, ²⁹N₂ gas (e.g., ¹⁴N, ¹⁵N) and the double-¹⁵N-labelled, ³⁰N₂ gas (e.g., ¹⁵N, ¹⁵N) which we illustrate schematically in Fig. 2a. Note that denitrification can draw on NO_x⁻ as either NO₂⁻ or NO₃⁻ but anammox is solely fuelled by NO₂⁻. The published and accepted mathematical framework²¹ (See derivation of equations in Supplementary Note 1) tells us that the fraction of ¹⁵N-labelling in each of the substrate pools (F_A and F_N) must influence the ratio of ²⁹N₂ to ³⁰N₂ (here termed R) and the overall fraction of ¹⁵N in the N₂ gas produced e.g., the overall blend of ²⁸N₂, ²⁹N₂ and ³⁰N₂ (here termed F_N2)^21,22. While complex, the accepted framework also tells us that so long as we know what fraction of each component part (F_A, F_N and F_N2) is labelled with ¹⁵N, then we can still calculate how the N₂ gas is produced e.g., by anammox or denitrification and understand the nature of this key coupling in the nitrogen cycle^21,22.

Fig. 2. Accepted and proposed cryptic couplings in oxic N cycling.

a ¹⁵NH₄⁺ tracer is added to oxic sediments to mix with ¹⁴NH₄⁺ in the porewater, with the fraction of ¹⁵N labelling known as F_A. Through reactions 1 and 2, ¹⁴NH₄⁺ and ¹⁵NH₄⁺ are oxidised aerobically to ^14,15NO₂⁻ and ^14,15NO₃⁻ to generate a ^14,15NO_x⁻ pool with ¹⁵N labelling known as F_N. NO₃⁻ and/or NO₂⁻ can be denitrified to N₂ gas (reactions 3a, 3b), or NO₂⁻ can oxidise NH₄⁺ anaerobically through anammox to N₂ gas (reaction 4). Regardless of the precise setting and combination of reactions, all substrates and products are free to mix and the measured ratio of ²⁹N₂ to ³⁰N₂ produced (R) can be predicted from the measured ¹⁵N labelling in the porewater. The downwards pointing orange arrow indicates NO₃⁻ respiration to NO₂⁻ that we do not consider further here. b In contrast, our measured values for R cannot be predicted using the measured fraction of ¹⁵N labelling in the porewater (F_A and F_N) and known combinations of reactions 1 to 4 but can only be approximated assuming a cryptic element (F_Ncry). A cryptic element could be a hidden substrate pool (6, novel or known) or novel parts of existing processes (7, e.g., complete nitrifier-denitrification beyond N₂O to N₂) and/or a completely new pathway (reaction 5 e.g., complete aerobic ammonia oxidation to N₂) or cryptic combinations of known pathways after partial aerobic ammonia oxidation to nitrite (reactions, 1, 3b, 4).

We tested the validity of this accepted mathematical framework by changing the fraction of porewater NO_x⁻ labelled with ¹⁵N (F_N) and looking for how this influenced the ratio of ²⁹N₂ to ³⁰N₂ produced (R). First we directly decreased F_N by adding ¹⁴N-nitrite to dilute the ¹⁵N-nitrite accumulating in the porewater from the oxidation of ¹⁵N-ammonia (Treatments 3 and 4, Table 1). Surprisingly, diluting F_N had no discernible effect on the values for R produced in the two sets of incubations (Fig. 3b. 2.32, 95% CI 2.01 to 2.64 versus 2.43, 95% CI 2.12 to 2.74, see Table 2 and Supplementary Table 2 for ²⁹N₂ and ³⁰N₂ production). We then repeated our incubations with just ¹⁵NH₄⁺ (with and without ATU, Treatments 1 and 2) across twelve rivers and measured a similar value for R of 1.8 (95% CI, 1.41 to 2.20, Fig. 3c) at an even lower value for F_N (see Table 1). Note, we might have expected R to increase steeply as an inverse function of F_N (Supplementary Figure 3). We can predict what values for R we might have expected if our N₂ gas had been produced by either denitrification or anammox fuelled by porewater nitrite and/or ammonia, respectively (Fig. 2a) and compare them to our measured R values to highlight the disparity between the two (Fig. 3b, c and Table 2):

$$\mathrm{Predicted}R\mathrm{for}\mathrm{denitrification},R=\frac{2\times F_N\times \left(1-F_N\right)}{F_N^2}$$ 6

$$\mathrm{Predicted}R\mathrm{for}\mathrm{anammox},R=\left(\frac{1}{F_N}-1\right)+\left(\frac{1}{F_A}-1\right)$$ 7

Our measured R values were too low to be explained by either denitrification or anammox fuelled by porewater F_N and/or F_A (Fig. 2a) and even a mixture of these two processes couldn’t produce such low values for R on average. This consistent disparity between our measured and predicted values for R, according to the accepted model, along with the constancy in R, despite differences in F_N (Table 2), strongly implies that porewater NO_x⁻ had little influence on the ¹⁵N-labelling of the N₂ gas produced from the oxidation of ¹⁵N-ammonia. Further, in an analogous set of incubations where we added ¹⁵N-nitrite instead of ¹⁵N-ammonia, we measured no consistent production of ¹⁵N-N₂ gas (Treatments 5 & 6 Table 1 and Methods). Hence, nitrogen for N₂ formation was not drawn primarily from the porewater NO_x⁻ pool (Fig. 2a). Instead, we propose that any N₂ producing pathways draw from a cryptic nitrogen pool (Fig. 2b) with ¹⁵N-labelled fraction, F_Ncry, instead of the familiar porewater pool with ¹⁵N-labelled fraction, F_Npw. Indeed, if we invoke a cryptic pool by making the ¹⁵N-labelling of F_N the same as ¹⁵N-ammonia in the porewater F_A in Eqs. (6) and (7) and thereby force denitrification and/or anammox to draw on that F_Ncry pool, then the predicted R values come closer to our measured R values (R cryptic, Fig. 3c and Table 2).

Fig. 3. Ratios of ²⁹N₂ and ³⁰N₂ production consistently below those predicted.

a Consistent ²⁹N₂ production (nmol g⁻¹ h⁻¹) from ¹⁵N-ammonia added to oxic sediments, against each corresponding measure of ³⁰N₂ production at each time-point (>0.5 h < 10 h) in each incubation in Fig. 1a presented here as the partial residuals from mixed-effects models (n = 100 and n = 300, for the 4- and 12-river datasets, respectively). b The corresponding measured values for R from a, for the first 4 rivers incubated with either ¹⁵NH₄⁺ (95% CI for R = 2.01 to 2.64) or ¹⁵NH₄⁺ and additional ¹⁴NO₂⁻ (95% CI for R = 2.12 to 2.74), against those predicted for denitrification of porewater NO₂⁻. c Measured R values for the 12 river sediments incubated with only ¹⁵NH₄⁺ (95% CI for R = 1.41 to 2.20), against predicted R values for denitrification, anammox, and a cryptic coupling. See main text and Table 2. Upper and lower box boundaries are 75th and 25th percentiles, respectively, upper and lower whiskers are 90th and 10th percentiles, respectively, the extreme outliers the maxima and minima and the horizontal line the centre, median value.

Table 2.

Summary of the overall measured and predicted ratios of ²⁹N₂ to ³⁰N₂ production (R) for treatments 2 and 4 and the fraction of ¹⁵N labelling in each substrate pool for F_N and F_A, on average. Overall measured and predicted R estimates, standard errors (s.e.) and 95% confidence intervals were derived with mixed-effects models using lme4 and emmeans (See Methods) and similarly for F_N and F_A. See Supplementary Table 3 for further details.

Code, Treatment	Rivers (replicates)	R (29N2/30N2)	Lower 95% C.I.	Upper 95% C.I.	P	FN‡		FA‡
		Measured				NO2−	NOx−
2, 15NH4+	4 (5)	2.32 (0.16)	2.0	2.6		0.32	0.41	0.57
4, 15NH4+ + 14NO2−	4 (5)	2.43 (0.16)	2.1	2.7		0.27	0.36	0.51
4 minus 2	4 (5)	0.11 (0.08)			0.20
2 & 4	4 (10)	2.38 (0.15)	2.1	2.7
2, 15NH4+	12 (5)	1.81 (0.20)	1.4	2.2		0.16	0.25	0.45
		Predicted†
2, 15NH4+	4 (5)	7.81 (1.36)D	5.1	10.5
4, 15NH4+ + 14NO2−	4 (5)	20.05 (1.49)D	16.9	22.3
2, 15NH4+	12 (5)	29.4 (2.28)D	24.8	33.9
2, 15NH4+	12 (5)	19.3 (2.28)A	14.8	23.9
2, 15NH4+	12 (5)	9.3 (2.28)C	4.8	13.8

^†Predicted R values, using Eqs. (6) and (7), for denitrification (D), anammox (A) and cryptic (C) processes fuelled by porewater NO₂⁻, see Supplementary Table 3 for NO_x⁻. Note also that the predicted values are derived using each individual measure of F_N and F_A in each vial, and that F_N^‡ are the overall mean values simply to illustrate the effect of adding ¹⁴NO₂⁻ to the incubations with sediments from the first 4 rivers and overall lower F_N value for the 12 river incubations.

N₂ is produced from ammonia through a cryptic intermediate

We can use both the accepted²¹ and a new mathematical framework to more formally justify our proposal for a cryptic intermediate pool or process. First, we define the proportion of N₂ gas coming from anammox relative to denitrification that is conventionally known as ra¹⁵. ra has to lie between 0 and 1 and, in the accepted framework, is expressed as a function of porewater F_A and F_N and R according to²¹ (See Eq. (1) to (14) in Supplementary Note 1):

$$ra=\frac{\left(R+2\right)\times F_N^2-2\times F_N}{\left(F_N-F_A\right)\times \left[\left(R+2\right)\times F_N-1\right]}$$ 8

In the accepted framework, however, our measured values for R and porewater F_A and F_N generate nonsensical estimates for ra (e.g., −6.06 to 3.03, not > 0 < 1). Just as for Fig. 3c, we cannot apportion N₂ gas between anammox and denitrification drawing on porewater F_N and/or F_A – in the conventional sense – to produce our measured R values (Fig. 2a). Next, we define the ¹⁵N- labelling of the N₂ gas produced (F_N2), which, like ra (Eq. 6), also has to lie between 0 and 1 (See Eq. (1) to (14) in Supplementary Note 1).

$$F_{N2}=F_N-\frac{R\times F_N+2\times \left(F_N-1\right)}{2\times \left(R+2-\frac{1}{F_N}\right)}$$ 9

Unlike ra, which is expressed as a function of both porewater F_A and F_N, only F_N is required to parameterise F_N2 (Eq. 9cf. Eq. 8). That is not to say that F_A has no influence on F_N2, as F_N—be it either the F_Ncry or F_Npw pools—must result from ammonia oxidation drawing on F_A (Fig. 2).

We can then use solutions to Eqs. (8) and (9) between > 0 < 1 to define a solution space for any combination of F_N, F_A, and realistic values for R (See Supplementary Figure 3 for R as a function of ¹⁵N atom %) that we can visualise as a 3D ribbon (Fig. 4). The height of the ribbon is defined in terms of F_N2 and is depicted here for our average value for F_A of 0.51 (Table 1 and see Supplementary Fig. 4 for F_A at 0.1 and 0.9). Overall the ribbon is very narrow and where F_A = F_N there are no solutions and this singularity appears as a gap in the ribbon. If F_Ncry is isolated and derives solely from the oxidation of F_A (Fig. 2b), then F_Ncry has to equal F_A. Further, if F_N2 is only dependent on F_N (Eq. 9) and this F_N is equivalent to F_Ncry, then our calculated values for F_N2—plotted as functions of our measured values for R and F_A (where F_Ncry equal F_A)—should fall near the gap in the ribbon where F_N equals F_A. This is indeed what we observe and especially for the better parameterised 12 river estimate (Fig. 4). In contrast, if we again force denitrification to be the only source of N₂, and calculate F_N2 assuming that F_N = F_Npw (Fig. 2a), then the points fall away from our measured R values. Hence, in the presence of ¹⁵N-ammonia and oxygen, our measured R values only make sense if we assume F_Ncry = F_A (Fig. 2b) i.e., the porewater nitrite pool essentially represents the left-overs of the cryptic transformations during which N₂ is produced.

Fig. 4. Orientations of the solution space ribbon with both measured and predicted values for R.

Here we present all data in just one solution space for the average fraction of ¹⁵N in the ammonia pool (F_A) of 0.51 and combinations of Eq. (8) (F_N2) and 9 (ra) both yielding values between > 0 < 1. R is the ratio of ²⁹N₂ to ³⁰N₂ and F_N and F_N2 the fraction of ¹⁵N in the NO_x⁻ and N₂ gas pools, respectively. To plot F_N2 for each of our measured values of R we have to assume that F_N equals F_A measured in the porewater. In the solution space, there are no solutions where F_A = F_N (i.e., 0.51) and this singularity appears as a gap in the ribbon. Despite measurable changes in porewater F_N, the average values for both the 4-river and 12-river study appear near to each other and the gap where F_A = F_N. Note that the better parameterised 12-river average touches the gap and by inference, F_A ≈ F_Ncry (Fig. 2b). Denitrification fuelled by porewater NO_x⁻ predicts values away from our measured values for R. Note, for the single predicted denitrification R values we use the median F_N values.

Internal NO_x⁻ cycling or a novel pathway or organism

We propose that the coupling between ammonia oxidation and N₂ gas production in oxic, permeable riverbed sediments involves a cryptic intermediate pool derived solely from the oxidation of ammonia that remains isolated from the porewater prior to the production of N₂ gas. In one scenario, a cryptic pool, similar to the porewater NO_x⁻ pool, is fed by the oxidation of ammonia to NO_x⁻, or possibly NO (ref. ^3,23,24), through nitrification. The pathway from F_Ncry to the production of N₂ gas, however, branches off before that NO_x⁻ mixes with the ambient porewater NO_x⁻ (Fig. 2b) and would require internal NO_x⁻ cycling. Internal NO_x⁻ cycling is recognised as a potential source of interference for ¹⁵N isotope tracer studies in the ocean^25,26 and is known in the consortia of ammonia oxidisers and anammox bacteria in wastewater CANON²⁷ reactors (Complete Autotrophic Nitrogen removal Over Nitrite. Figure 2b, reactions 1 & 4) – though the actual mechanism in nature remains unknown.

Alternatively, some aerobic ammonia oxidising bacteria first produce nitrite (reaction 1) that they then reduce to N₂O gas in a process known as nitrifier-denitrification³. Known nitrifier-denitrifier bacteria, however, lack a canonical N₂O-reductase (NOS, nosZ) to reduce N₂O to N₂ gas, so are not currently recognised as complete denitrifiers (reaction 7, Fig. 2b). Nitrosocyanin, a soluble red Cu protein isolated from Nitrosomonas europaea²⁸, is recognised as a plausible substitute to canonical N₂O-reductase that could enable complete nitrifier-denitrification to N₂ gas³. Our data enable us to test this hypothesis. For example, we know that ¹⁵NO₂⁻ from the initial oxidation of ¹⁵NH₄⁺ exchanges with the porewater (reaction 1, Figs. 1b and 2a) and we would expect, therefore, that ¹⁵NO₂⁻ added to the porewater would be available to any nitrifying-denitrifying bacteria²⁹. We have, however, already shown that adding ¹⁵NO₂⁻ to the porewater resulted in no consistent production of N₂ gas (Treatments 5 & 6, Table 1) i.e., N₂ gas production is dependent on the initial oxidation of ¹⁵N-ammonia. This fact, along with the clear discrepancy between the measured and predicted scenarios involving porewater NO_x⁻ (Figs. 3b, 3c & 4) make it hard to reconcile our N₂ gas production with either nitrifier-denitrification or canonical denitrification (reactions 3a, 3b & 7, Fig. 2).

Finally, it is theoretically possible for ammonia to be completely oxidised by oxygen to N₂ gas (equation 5⁸) within a single, unknown organism. Such a reaction offers the simplest explanation for our results, with their strong dependency on aerobic ammonia oxidation and lack of influence from external porewater nitrite. Regardless of the actual pathway that produces the N₂ gas (Fig. 2b), an isolated cryptic intermediate pool has to have the same ¹⁵N-labelling of the ammonia pool (F_Ncry = F_A). As a consequence of this equality, we can no longer distinguish between sources of N₂ gas, be it a denitrification-like pathway reductively combining N from an oxidised cryptic pool, an anammox-like process drawing on ammonia and cryptic N, or complete ammonia oxidation, as they would all produce ²⁹N₂ and ³⁰N₂ at the same ratio (Fig. 2b where R is equal for each process).

Our observations challenge the current understanding of a key coupling in the nitrogen cycle in permeable, oxic riverbed sediments that may also apply to other biomes where the oxidation of ammonia is tightly coupled to the production of N₂ gas, such as continental shelf-sediments^30,31 and groundwater aquifers¹⁷. Whether it transpires that our cryptic coupling is mediated by a novel organism or, as of yet, a masked combination of known players in the nitrogen cycle remains to be resolved.

Methods

Study sites and sediment sampling

We began by collecting sediment samples from four rivers which we subsequently widened to a total of twelve rivers in southern England, UK, between October 2015 and May 2016 (Supplementary Figure 1 and Supplementary Table 1). Among them, the Rivers Lambourn, Darent, Wylye, Rib, Pant, Stour (1) and Stour (2) have chalk-based, permeable gravel-dominated riverbeds, while the Rivers Marden, Hammer, Medway, Broadstone, and Nadder have less permeable, sand-dominated riverbeds as described elsewhere^18,32,33. At each river, surface sediments (<5 cm) were collected from five different locations using Perspex corers (13-cm × 9-cm internal diameter, 827 mL and sealed at one end with an oil-seal stopper)) which were then transferred to plastic zip-lock bags (VWR International) and stored in a cool bag (Thermo) during transport back to the laboratory. Each sediment sample from each river was then homogenised in the laboratory for the experiments described below.

Aerobic ammonia oxidation in oxic sediment slurries

¹⁵N-NH₄⁺ oxidation experiments were carried out with sediments first from four rivers (the rivers Lambourn, Wylye, Marden, and Hammer) and then all twelve. In a standard anoxic application of ¹⁵N isotope pairing techniques^34–36, ambient porewater nitrite, nitrate, and any residual oxygen are removed by pre-incubating the anoxic sediment slurries for 12 h to 24 h before adding any ¹⁵N-tracers^35,36. Here this was not possible as we were measuring the aerobic oxidation of NH₄⁺ and so to avoid contamination from the high background ¹⁴NO_x⁻ (¹⁴NO₃⁻ + ¹⁴NO₂⁻), which is typical for these rivers²⁴, instead we used nitrite- and nitrate-free synthetic river water (0.12 g/l NaHCO₃, 0.04 g/l KHCO₃, 0.07 g/l MgSO₄^.7H₂O, 0.09 g/l CaCl₂ 2H₂O, pH = 7) to make the sediment slurries as before¹⁸.

Oxic slurries were prepared by adding approximately 3 g sediment (~0.75 ml of porewater) and 2.7 ml air-saturated synthetic river water into 12 ml gas-tight vials (Exetainer, Labco), leaving an approximate 6 ml headspace of air which is equivalent to ~58 µmol O₂ per prepared vial. We know from previous incubations with similar sediments from 28 rivers³⁷ respiration rates to be ~187 nmol O₂ g⁻¹ h⁻¹, on average (±64.3, 95%, C.I.), that would consume ~12% of the total oxygen during a 12 h incubation. In addition, we also checked oxygen over time using a microelectrode (50 µm, Unisense) in parallel sets of scaled-up slurries (120 mL with the same ratio of sediment to water to headspace) for two rivers and found comparatively little consumption as before¹⁸ and see example in Supplementary Figure 2.

To trace the oxidation of ammonia to N₂ gas, the prepared oxic slurry vials were then sealed and injected with 100 µl of 14 mM ¹⁵NH₄⁺ stock-solutions (98 atom% ¹⁵N, Sigma-Aldrich) to generate final porewater concentrations of ~390 µM ¹⁵NH₄⁺. This high ¹⁵N concentration ensured sufficient labelling of the ammonia pool (~50%) to enable quantifiable production of both single-labelled, ²⁹N₂, and dual-labelled, ³⁰N₂, in order to calculate R in Eqs. (6) to (9). To link the production of N₂ gas to the initial aerobic oxidation of ammonia, an additional set of slurries were injected with 100 µl of 14 mM ¹⁵NH₄⁺ (as above), along with 2.8 mM (stock-solution) of the ammonia mono-oxygenase inhibitor¹⁹, allylthiourea (ATU), to give final porewater concentrations of ~390 µM ¹⁵NH₄⁺ and ~80 µM ATU. While we have shown previously that 80 µM ATU inhibits aerobic ammonia oxidation in gravel and sandy riverbed sediments¹⁸, higher concentrations maybe required in other settings³⁸. All of the oxic slurry vials were then incubated on a shaker (120 rpm, Stuart SSL1) for up to 12 h (Table 1, Treatments 1 and 2) in a temperature-controlled room at 12 °C. Incubations amended with just ¹⁵NH₄⁺ were terminated at 0 h, 0.5 h, 1 h, 3 h, 4.5 h, 6 h, 9 h, and 12 h while those amended with both ¹⁵NH₄⁺ and ATU were terminated at 0 h, 3 h, 6 h, and 12 h by injecting 100 μl of formaldehyde (38%, w/v) through the vial septa. All vials were then stored upside down prior to quantification of ²⁹N₂ and ³⁰N₂ by mass-spectrometry and R is then simply ²⁹N₂/³⁰N₂ (see below).

In addition to measuring the production of ²⁹N₂ and ³⁰N₂ gases (R), the fraction of ¹⁵N in the inorganic nitrogen porewater pools (F_A for ammonia and F_N for NO_x⁻ e.g., NO₂⁻ plus NO₃⁻) needed to be quantified too (see Eqs. 6 to 9). To avoid any potential interference from formaldehyde, on the analysis of the inorganic nitrogen species, a parallel set of ¹⁵NH₄⁺ amended slurries was prepared solely for nutrient analyses. At each time point (as above for N₂ gas analysis), vials were injected with 20 µL of 1.6 M NaOH to preserve nitrite before being frozen at −20 °C³⁹. Samples were defrosted and centrifuged at 1200 rpm for 10 min and the collected supernatant analysed (see below).

Manipulating the degree of ¹⁵N-labelling in the porewater NO₂⁻ pool (F_N as F_Npw)

In typical anoxic sediment slurry incubations used to quantify N₂ gas production from denitrification and anammox^34,35, the fraction of porewater substrate labelled with ¹⁵N (F_A or F_N) influences the ratio of ²⁹N₂ to ³⁰N₂ produced. To characterise the influence of porewater NO₂⁻ on the coupling between ¹⁵N-NH₄⁺ oxidation and ¹⁵N-N₂ production in oxic sediment slurries, we manipulated the fraction of porewater NO₂⁻ labelled with ¹⁵N. Oxic sediment slurries from the first four riverbeds were injected (100 µl) with combinations of stock-solutions of 14 mM ¹⁵NH₄⁺ and 840 µM ¹⁴NO₂⁻ or just 14 mM ¹⁵NH₄⁺ and both with or without 2.8 mM ATU. This generated final porewater concentrations of ~390 µM ¹⁵NH₄⁺, ~24 µM ¹⁴NO₂⁻ or ~80 µM ATU and the prepared vials were then incubated on a shaker as above (see Table 1, Treatments 3 and 4). As above, oxic slurry vials were sacrificed at different time points for ¹⁵N₂ gas analysis and with a parallel set of ¹⁵NH₄⁺ or ¹⁵NH₄⁺ plus NO₂⁻ amended slurries solely for nutrient analyses.

To further test the dependency of N₂ gas production on the initial oxidation of ¹⁵N-ammonia, we also performed a set of analogous incubations with sediments from the first four rivers with ¹⁵NO₂⁻ (Table 1, Treatments 5 and 6). Here everything was the same (amount of sediment, with or without ATU, incubation times, oxygen etc.,) except the ¹⁵N-labelling was added with nitrite rather than ammonia (as above) to final concentrations of ~390 µM ¹⁴NH₄⁺ and ~24 µM ¹⁵NO₂⁻ (98 atom% ¹⁵N, Sigma-Aldrich). If active, we would have expected N₂ gas production from reactions 3b and 4.

Analytical methods

Headspaces of the oxic slurry samples were analysed for ¹⁵N-N₂ using a continuous-flow isotope ratio mass spectrometer (Sercon 20–22, UK) as described elsewhere¹⁸. The mass spectrometer has a sensitivity of 0.1 ‰ ¹⁵N which here translates to approximately 0.1 nmol ¹⁵N-N₂ g⁻¹ dry sediment. To determine porewater F_N (NO₂⁻ or NO_x⁻, below) the concentration of ¹⁵NO₂⁻ in the ¹⁵NH₄⁺ treatments was measured, the preserved supernatants were diluted and 3 ml of sample transferred into a new 3 ml gas-tight vial (Exetainer, Labco), the vial capped and a 0.5 ml helium headspace (BOC) added. Samples were injected with 100 μl of sulfamic acid (4 mM in 4 M HCl) and placed on a shaker (120 rpm, Stuart SSL1) overnight to reduce ¹⁵NO₂⁻ to ¹⁵N-N₂ and the headspaces subsequently analysed for ¹⁵N-N₂ as above^18,40. For ¹⁵NO_x⁻ (¹⁵NO₂⁻ plus ¹⁵NO₃⁻) analysis, 0.3 g spongy cadmium and 200 µl of 1 M imidazole, along with 3.5 ml of sample were added to each gas-tight vial (12 ml, Exetainer, Labco) and the vials shaken (120 rpm, Stuart SSL1) for 2.5 h to reduce ¹⁵NO₃⁻ to ¹⁵NO₂⁻ and the samples then treated as above to convert ¹⁵NO₂⁻ to N₂^18,41. The sensitivity for ¹⁵NO_x⁻ was approximately 0.4 nmol ¹⁵N g⁻¹ dry sediment. F_N was then calculated for NO₂⁻ or NO_x⁻ as:

$$F_N=\frac{{}^{15}{\mathrm{NO}}_x^{-}}{\left({}^{15}{\mathrm{NO}}_x^{-}+{}^{14}{\mathrm{NO}}_x^{-}\right)}$$ 10

And similarly for F_A:

$$F_A=\frac{{}^{15}{\mathrm{NH}}_4^{+}}{\left({}^{15}{\mathrm{NH}}_4^{+}+{}^{14}{\mathrm{NH}}_4^{+}\right)}$$ 11

Where ¹⁵NH₄⁺ was determined by the increase in concentration, measured by standard indophenol-blue wet-chemistry, above ambient background in controls after the addition of ¹⁵NH₄⁺.

Sediment particle size was determined by sorting the dried sediments through a series of sieves (Endecotts Ltd, England) from 16 mm, 13.2, 8, 4, 1.4, 0.5, 0.25, 0.125, to 0.0625 mm and then weighing each size fraction. Grain size distributions were calculated and classified on the Wentworth scale as gravel (particles coarser than 2 mm), sand (particles between 0.0625 and 2 mm), mud (silt plus clay material finer than 0.0625 mm)⁴². For sediment organic C and N content, disaggregated samples were oven-dried, acidified by HCl (2 M) to remove inorganic carbonates⁴³ and re-dried to a constant weight. Then ~50 mg of sediments were transferred to tin-cups, reweighed, and combusted at 1000 °C in an integrated elemental analyser and mass-spectrometer (Sercon, Integra 2, UK).

Statistical analysis

We used mixed-effects models to estimate overall rates of total ¹⁵N-N₂ gas production during the incubations (Fig. 1a), treating each of either the first four or subsequent twelve rivers as genuine, independent replicates. Within each river, each of the 5 technical replicates were nested within each respective river and fitted as random effects on the slope and intercept in each case; though it was not always necessary to retain replicate or all the random effects in a model to get the best fit to the data – based on lowest AIC (Akaike Information Criterion). To visualise the consistent production of ²⁹N₂ to ³⁰N₂ across the incubations with ¹⁵N-ammonia, we regressed each measure of ²⁹N₂ on each measure of ³⁰N₂, at each time point, in each incubation and display (Fig. 3a) the partial residuals for the best fitting model⁴⁴. To estimate the overall average measured and predicted ratios of ²⁹N₂ to ³⁰N₂ (R) we only used the data for the time points >0.5 h < 10 h i.e., when there was measurable (~0.1 nmol N₂ g⁻¹ dry sediment), steady-production of both ¹⁵N labelled gases, divided each measure of ²⁹N₂ by each respective measure of ³⁰N₂ at each time point, in each incubation and treated river and replicate as above. For the first 4 rivers, the ratio R was estimated by fitting each time point as a random-effect, but for the larger, 12 river dataset, time was fitted as a fixed-effect and R estimated for the middle time point in the incubations and similarly for F_N (for both NO₂⁻ and NO_x⁻) and F_A. All statistical analyses were performed in R (version 3.6.3, 2020-02-29) under RStudio (version 1.2.5033). Model fitting was carried out in the “lme4” package (version 1.1-21) and parameter (marginal mean) estimates, standard errors, and confidence intervals derived using the “emmeans” package (version 1.4.5) with Kenwood-Roger degrees of freedom and Tukey correction where appropriate.

Source data

Source Data ^{(78.5KB, xlsx)}

Author contributions

M.T. and L.O. conceived the study and L.O. performed all of the experiments and B.T. formulated the mathematical framework. L.O. and M.T. analysed the data and M.T. and B.T. drafted the manuscript. All authors commented on and revised the manuscript.

Data availability

Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-021-21400-3.