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Isotopic overprinting of nitrification on denitrificationas a ubiquitous and unifying feature ofenvironmental nitrogen cyclingJulie Grangera,1 and Scott D. Wankelb,1
aDepartment of Marine Sciences, University of Connecticut, Groton, CT 06340; and bDepartment of Marine Chemistry and Geochemistry, Woods HoleOceanographic Institution, Woods Hole, MA 02540
Edited by Donald E. Canfield, Institute of Biology and Nordic Center for Earth Evolution (NordCEE), University of Southern Denmark, Odense M., Denmark,and approved August 26, 2016 (received for review January 25, 2016)
Natural abundance nitrogen and oxygen isotopes of nitrate (δ15NNO3
and δ18ONO3) provide an important tool for evaluating sources andtransformations of natural and contaminant nitrate (NO3
−) in theenvironment. Nevertheless, conventional interpretations of NO3
isotope distributions appear at odds with patterns emerging fromstudies of nitrifying and denitrifying bacterial cultures. To resolvethis conundrum, we present results from a numerical model ofNO3
− isotope dynamics, demonstrating that deviations in δ18ONO3
vs. δ15NNO3 from a trajectory of 1 expected for denitrification areexplained by isotopic over-printing from coincident NO3
− productionby nitrification and/or anammox. The analysis highlights two drivingparameters: (i) the δ18O of ambient water and (ii) the relative fluxof NO3
− production under net denitrifying conditions, whethercatalyzed aerobically or anaerobically. In agreement with existinganalyses, dual isotopic trajectories >1, characteristic of marinedenitrifying systems, arise predominantly under elevated rates ofNO2
− reoxidation relative to NO3− reduction (>50%) and in associa-
tion with the elevated δ18O of seawater. This result specifically im-plicates aerobic nitrification as the dominant NO3
− producing term inmarine denitrifying systems, as stoichiometric constraints indicateanammox-based NO3
− production cannot account for trajectories>1. In contrast, trajectories <1 comprise the majority of model solu-tions, with those representative of aquifer conditions requiringlower NO2
− reoxidation fluxes (<15%) and the influence of thelower δ18O of freshwater. Accordingly, we suggest that widely ob-served δ18ONO3 vs. δ15NNO3 trends in freshwater systems (<1) mustresult from concurrent NO3
− production by anammox in anoxic aqui-fers, a process that has been largely overlooked.
nitrate | nitrification | denitrification | isotopes | anammox
The advent of the Haber–Bosch process late in the 19th cen-tury initiated an unprecedented increase in anthropogenic
loading of reactive nitrogen (N) to the biosphere, setting intomotion cascades of environmental impacts, including eutrophica-tion and hypoxia, ecosystem acidification, and loss of biodiversity(1). This intensification of environmental N release from agricul-tural and industrial activities, power generation, municipal andseptic wastewater, and domestic fertilizer has tremendously al-tered the global N cycle, effectively doubling annual global Nturnover (1). In groundwater, the most common nitrogenouscontaminant is nitrate (NO3
−), with recognized and long-termeffects on both human and ecological health. Thus, control andelimination of NO3
− contamination are priorities of environ-mental and health agencies worldwide. Despite its significance toglobal health and ecosystem function, identifying sources of NO3
−,tracing its dispersal and attenuation, and gauging its ecologicalimpact remain challenging.Mitigation of NO3
− pollution has necessitated identificationof its sources and hydrologic flow paths to monitor the fate andnatural attenuation processes occurring in pollutant plumes. Tothis end, the natural abundance stable isotope ratios of nitro-gen (15N/14N) and oxygen (18O/16O) in NO3
− have provided an
invaluable tool to differentiate sources, track their distribution,and determine the biogeochemical transformations acting onNO3
−. By convention, isotope ratios are reported using δ notation,where δ15N = ([15N/14N]sample/[
15N/14N]air − 1) × 1,000 and δ18O =([18O/16O]sample/[
18O/16O]VSMOW − 1) × 1,000, in units of per mille(‰). Given two isotopic tracers for a single compound, thisapproach can be powerful, as each isotope system provides com-plementary information on sources and biogeochemical transfor-mations (2). Accurate interpretation of isotope distributions,however, strongly hinges on knowledge of the isotope com-position of source terms and on a rigorous understanding ofisotopic discrimination associated with biological transforma-tions of N pools. The isotopic discrimination associated withspecific N transformations is quantified by the isotope effect, e,where e (‰) = [(lightk/heavyk) − 1] × 1,000, and k refers to the re-spective specific reaction rate constants of light and heavy iso-topologues (3). Although many of the important source terms andisotope effects of the N cycle are constrained, some remain equiv-ocal. In particular, recent observations emerging from bacterial andarchaeal cultures and from incubations of environmental sampleshave uncovered isotopic discrimination trends for NO3
− isotopesthat appear at odds with trends typically ascribed to analogousbiological transformations in soils and aquifers (4–14). This de-velopment has led to conflicting environmental interpretations,reflecting a lack of consensus on fundamental isotope systematics ofthe processes driving the N cycle. Importantly, the discrepancies
Stable isotopes of nitrate have long provided a tool for track-ing environmental sources and biological transformations.However, divergent interpretations of fundamental nitrateisotope systematics exist among disciplinary divisions. In aneffort to transcend disciplinary boundaries of terrestrial andmarine biogeochemistry, we use a quantitative model forcoupled nitrogen and oxygen isotopes of nitrate founded onbenchmarks established from microbial cultures, to reconciledecades of nitrate isotopic measurements in freshwater andseawater and move toward a unified understanding of cyclingprocesses and isotope systematics. Our findings indicate thatdenitrification operates within the pervasive context of nitritereoxidation mechanisms, specifically highlighting the relativeimportance of nitrification in marine denitrifying systems andanammox in groundwater aquifers.
Author contributions: J.G. and S.D.W. designed research, performed research, analyzeddata, and wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.1Towhom correspondence may be addressed. Email: [email protected] or [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1601383113/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1601383113 PNAS | Published online October 4, 2016 | E6391–E6400
between isotopic trends in environmental systems and those fromculture-based observations raise the possibility that biogeochemicalN dynamics inferred from environmental NO3
− isotopic measure-ments reflect more complexity than previously realized.Conventional interpretation schemes for NO3
− isotopes differfrom culture observations with regard to the isotope systematic ofdenitrification, the stepwise reduction of NO3
− to NO2− (nitrite),
nitric oxide, nitrous oxide, and finally N2 by heterotrophic bacteria,which is the dominant loss term of reactive N from the biosphere.Early studies of NO3
− isotope dynamics in groundwater docu-mented parallel enrichment of δ15N and δ18O of NO3
− in associa-tion with NO3
− attenuation from denitrification, approximating alinear trajectory with a slope 0.5–0.8 (15–17). Indeed, this salienttrend has long been considered a unique diagnostic signal of de-nitrification (2). However, following the advent of the denitrifiermethod (18, 19), measurements in cultures of both freshwater andmarine denitrifying bacteria revealed dual isotope enrichments as-sociated with assimilatory and dissimilatory NO3
− consumptionsystematically following linear trajectories of ∼1 (9, 10, 12, 19, 20),contrasting with the lower values widely observed in freshwatersystems. This invariant coupling of N and O isotopic enrichmentshas been shown to originate from fractionation during enzymaticbond-breakage (8, 21, 22), confirmed directly from in vitro enzymestudies of eukaryotic assimilatory and prokaryotic dissimilatoryNO3
− reductases (11, 23).Interpretation schemes conventionally ascribed to nitrification
are also at odds with isotope systematics uncovered in cultureobservations. Nitrification refers to the sequential oxidation ofammonia (NH3) to NO2
− then NO3− by chemoautotrophic bac-
teria and archaea, coupled to aerobic respiration. Oxidation ofNO2
− to NO3− also occurs during the anaerobic oxidation of NH3
to N2 by anammox bacteria (24). Nitrification constitutes the solebiological production term for NO3
−. The NO3− δ18O values re-
portedly produced by nitrification in freshwater lakes and aquifersspan a contended range of 30‰, from −15‰ to 15‰ (2, 25),attributed to the origin of the oxygen atoms appended to NH3during the two-step process of nitrification: the biological oxida-tion of NH3 to NO2
− incorporates one oxygen atom from mo-lecular O2 and one from water (7, 26, 27); the subsequentoxidation of NO2
− to NO3− incorporates an O atom derived from
water (28). Recent work, however, has revealed kinetic isotopeeffects associated with enzymatic incorporation of each of thethree O atoms into the product NO3
− (5, 6), as well an inversekinetic isotope effect on the reactant NO2
− during oxidationto NO3
− (4) and the isotopic equilibration of O atoms betweenNO2
− and water (29, 30). These isotope effects have traditionallynot been considered in interpreting NO3
− isotope distributions,yet play a fundamental role in defining the isotopic compositionof nitrified NO3
− in tandem with compositional differences of theO atom sources (7, 13, 31). Thus, moving beyond early studies inwhich the δ18ONO3 from nitrification was interpreted as a three-part mixture of O atom sources, it is clear that consideration ofseveral isotope fractionation processes is required for accurateinterpretation of sources and cycling mechanisms.To date, observations in marine systems have revealed linear
trajectories of ∼1 and trajectories distinctly above the nominalvalue of 1 associated with water–column denitrification (32–37).Positive deviations from 1 are generally interpreted as reflectingthe isotopic imprints of biological NO2
− reoxidation superimposedon the trajectory of biological NO3
− consumption (34, 38). Infreshwater studies, however, this discrepancy between cultures andenvironment has been scantly acknowledged. Notably, someworkers have put forth a number of postulates to explain thisconundrum, detailed in S1. Postulates to Explain Deviations from 1in Δδ18O:Δδ15N Trajectories in Freshwater. Among these, the bi-ological production of NO3
− by nitrifiers occurring in tandem withdenitrification has been proposed to account for the apparentdifferences in δ18O vs. δ15N trajectories between cultures and
freshwater environments (39, 40), although this tenet has not beenexamined specifically. The potential for analogous biogeochemicaldynamics to affect isotope distributions in freshwater and marinesystems clearly merits exploring.To arrive at a shared understanding of environmental NO3
isotope systematics, we present the results of a multiprocess nu-merical model of dual NO3
− isotope dynamics parameterized onthe basis of fundamental features revealed from culture studies.From this improved understanding of isotopic fractionation duringredox cycling of N, we explore implications for NO3
− production –
by NO2− oxidizing bacteria and by anammox—occurring concur-
rently with denitrification, specifically focusing on resulting NO3− N
and O isotope trajectories. We use this framework to evaluate thepotential extent of processes other than unidirectional NO3
− con-sumption by denitrification, which may harbor the key for resolvingthe discrepancy between decades of groundwater NO3
− observa-tions and our physiological understanding of the isotope systematicsof microbial N cycling. The scenarios explored herein call attentionto the potential influence of N cycling dynamics that have beenlargely overlooked in aquatic environments and provide a unifiedframework for future investigations of N isotope biogeochemistry.
2. A Multiprocess Model of NO3− Dual Isotopes: Rationale
and AssumptionsTo evaluate the impact of nitrite reoxidation on coupled NO3
− Nand O isotope trajectories, termed Δδ18O:Δδ15N henceforth, wedevised a time-dependent one-box model simulating the evolutionof N and O isotopologue pools of NO3
− and NO2− in a closed
system during denitrification coincident with aerobic and anaerobicNO2
− oxidation by nitrifiers or by anammox bacteria, respectively(Fig. 1; terms defined in Table 1). The isotopologue-specific for-mulation of the model is described in S2. Equations Used in Time-Dependent NO3
− Isotope 1 Box Model.In brief, the simulated NO3
− pool is influenced by the dissimilativereduction of NO3
− to NO2− (NAR) and the concurrent production
of NO3− by NO2
− oxidation (NXR; Fig. 1). The NO2− pool, in turn,
reflects the balance of production by NAR and NH4+ oxidation
(AMO), as well as oxidation by NXR and reduction to nitric oxide(NIR). Both reductive processes, NAR and NIR, impart normal Nisotope effects, 15enar and
15enir, which are equivalent to their cor-responding O isotope effects, 18enar and
18enir (9, 34). During NAR, abranching isotope effect from O atom abstraction, 18enarBR (19, 29),also contributes to the δ18O of the NO2
− product. In turn, NXR ischaracterized by inverse kinetic isotope effects for NO2
− consump-tion, 15enxr and
18enxr (4, 5), and a normal isotope effect for O atomincorporation from water, 18enxr,H2O (5). AMO also has normalisotope effects for N, 15eamo (3, 41), and for O atom incorporationfrom H2O and molecular O2,
18eamo,H2O and 18eamo,O2, respectively(6). However, we assume no accumulation of ammonium (NH4
+)from the remineralization of organic material, such that there isno expression of the N isotope fractionation associated with AMO(15eamo), and the NO2
− produced by AMO adopts the δ15N ofammonium (δ15NNH4). Finally, NO2
− is subject to O isotope equil-ibration with water having an associated isotope effect, 18eeq (29, 30).We further consider the potential role of anaerobic NH3 ox-
idation (anammox), which stoichiometrically produces ∼0.26moles of NO3
− per mole of NH3 oxidized (or per 1.3 moles of NO2−
reduced) as a metabolic product from the NO2− pool (24, 42). The
model functions as described, except that anammox operates (Fig. 1,blue line) in lieu of aerobic nitrification (Fig. 1, red lines). Notably,unlike in AMO, the δ15N of the NH4
+ pool does not enter into theNO3
−mass balance during anammox. Similarly, the δ18O of NO3− is
not indirectly influenced by NO2− production from NH3, which only
occurs aerobically. NO2− oxidation by anammox is prescribed an
inverse N isotope effect, 15enxrAMX (Table 1) (43). Given no con-straint from culture work the corresponding O isotope effect,18enxrNO2AMX, it is assumed to be of similar magnitude to that forNO2
− oxidizers (Table 1). Model parameter ranges are listed in
E6392 | www.pnas.org/cgi/doi/10.1073/pnas.1601383113 Granger and Wankel
Table 1 and justified in Section 6. For brevity, we also established“standard” model conditions having generally midrange valuesfor isotope effects (Table 1).
3. Model ResultsWe discuss the contribution of distinct aspects of the reactionnetwork, highlighting those features exerting the most influenceon the isotope composition of the evolving NO3
− pool, namely,the isotope composition of ambient water and the NO2
− oxida-tion flux. We further explore features that exert moderate in-fluence on coupled NO3
− N and O isotope trajectories, includingisotope effect values, the isotope composition of initial pools,and NO2
− production via biological ammonia oxidation. Wethen consider scenarios specific to NO3
− production by anam-mox and finally examine the isotope composition of NO2
− thatemerges among model scenarios. Model results are comparedwith environmental observations in Section 4.
3.1. Influence of δ18OH2O. Initial tests were parameterized usingstandard conditions (Table 1) to explore the sensitivity of theΔδ18O:Δδ15N trajectory to the δ18O of ambient water (from−20‰ to 0‰), for a range of relative fluxes of NO2
− oxidation(NXR/NAR from 0 to 0.9) and contrasting scenarios of NO2
equilibration with water. For simplicity, we predominantly con-sider the case of full equilibration of NO2
− oxygen isotopes withwater, which likely characterizes most freshwater systems, where
equilibration is probably rapid relative to biological cycling giventhe generally low pH (13, 14, 29, 30, 44). In marine denitrifyingregions, δ18ONO2 measurements also suggest full isotopic equil-ibration with water (34, 35), despite the higher pH of seawater(≤8.2). Results for simulations without NO2
− isotope equilibra-tion are otherwise presented in the supplements (S3. SimulationWithout Isotope Equilibration of NO2
− with Water and Fig. S1).Foremost, results reveal that the evolution of the Δδ18O:Δδ15N
trajectory during net denitrification can be substantially deflectedfrom a value of 1 by a co-occurring contribution of newly nitrifiedNO3
− (Fig. 2). Resulting Δδ18O:Δδ15N trajectories correspond to abroad range of solutions, from 0.1 to 3.2 (in terms of linear fitsrelating the dual isotopic composition of initial NO3
− to that of anyevolved modeled composition), of which a larger share yields slopesbelow the canonical value of 1.In all cases, the water δ18O emerges as a strong determinant of
the Δδ18O:Δδ15N trajectory: the lowest trajectories are associatedwith the lowest δ18OH2O and the highest trajectories with thehighest δ18OH2O (Fig. 2). Mechanistically, the δ18O of nitrifiedNO3
− is directly related to the water δ18OH2O through both Oisotope equilibration of intermediate NO2
− and the incorporationof an O atom from water during NO2
− oxidation. Under condi-tions of full isotopic equilibration of NO2
−, Δδ18O:Δδ15N trajec-tories are thereby insensitive to incorporation of O atomsfrom water or O2 during any oxidation of NH3 to NO2
−. TheΔδ18O:Δδ15N trajectories below 1 are associated with relativelylower δ18OH2O values that contribute to lower δ18O values for nitrifiedNO3
− (Fig. S2). By comparison, the few model solutions in whichΔδ18O:Δδ15N trajectories are above 1 at standard conditions are as-sociated with higher δ18OH2O values, characteristic of marine systems.Conversely, lower δ18OH2O values, characteristic of freshwater sys-tems, are largely associated with Δδ18O:Δδ15N trajectories below 1.
3.2. Importance of the NO2− Oxidation Flux.Given its importance in
linking the composition of water with that of the evolving NO3−
pool, the extent to which NO3− is produced relative to that re-
duced (expressed as NXR/NAR) is also clearly an influentialdriver of Δδ18O:Δδ15N trajectory. However, this flux is centralnot only because it governs the extent to which nitrified NO3
− isadded back to the NO3
− pool, but also because it modulates theδ15NNO3 returned to the NO3
− pool. The influence of NXR/NAR on the Δδ18O:Δδ15N trajectory can be summarized asfollows (Fig. 2): when NO2
− oxidation is nearly equal to NO3−
reduction (NXR/NAR ≥ 0.8), the δ15NNO3 produced by nitrifi-cation converges on that removed by denitrification (notwith-standing the contribution to the NO2
− pool from AMO; S4.Impact of NO3
− Production by AMO), thus nearly “restoring” theδ15N of the NO3
− pool to its original value. In turn, the δ18O ofnitrified NO3
− is insensitive to NXR/NAR, deriving primarilyfrom the δ18OH2O when NO2
− is fully equilibrated, such that itcan be either higher or lower than the δ18ONO3 removed bydenitrification depending on the δ18OH2O. Thus, the largestexcursions in Δδ18O:Δδ15N trajectories, both above and below 1,occur at the higher values of NXR/NAR. In this respect, trajec-tories above 1, characteristic of marine denitrifying systems, con-gruently arise at elevated δ18OH2O.When NXR/NAR ratios are low (Fig. 2A), the transient NO2
pool is mostly reduced to NO rather than oxidized to NO3−, ren-
dering the δ15N of the NO2− pool relatively more 15N-enriched due
to isotopic discrimination by 15enir. The NO3− produced from the
oxidation of NO2− is further 15N-enriched by the inverse effects of
15enxr. Therefore, the δ15N of nitrified NO3− will be greater than
that removed by denitrification, driving the Δδ18O:Δδ15N trajec-tories to values below 1. The corresponding δ18O of nitrified NO3
(again insensitive to the prescribed NXR/NAR ratio due to fullisotopic equilibration of NO2
−; Fig. S2) either counters orotherwise augments the negative offset in Δδ18O:Δδ15N from 1imposed by the δ15NNO3 of nitrified NO3
− depending on the δ18O
Fig. 1. Box model architecture showing N transformations and associatedisotope effects influencing (A) δ15NNO3 and (B) δ18ONO3. See text and Table 1for details and references.
Granger and Wankel PNAS | Published online October 4, 2016 | E6393
of this nitrified NO3− (and thus, the δ18O of water). Regardless, all
Δδ18O:Δδ15N trajectories remain below 1 at low NXR/NAR ratios(Fig. S3), even for δ18OH2O values characteristic of seawater.
3.3. Influence of N and O Isotope Effects. To investigate the leverageof specific isotope effects on the evolving Δδ18O:Δδ15N trajectories,we examine the response of NO3
− trajectories while holding allother parameters constant (standard conditions; Table 1), plottingthem against δ18OH2O as a master variable (Fig. 3), given its centralrole in the composition of nitrified NO3
− as described above.
Results indicate that the Δδ18O:Δδ15N trajectory increasesprogressively with increasing 15enar, the N isotope effect of nitratereduction (Fig. 3A). This dynamic is best understood by consid-ering the influence of 15enar on the composition of the transientNO2
− pool subject to reoxidation to NO3−. The δ15NNO2 returned
to the NO3− pool by nitrification is more 15N-depleted at higher
15enar values, tending to more elevated Δδ18O:Δδ15N trajectories.However, although trajectories above 1 emerge more readily athigher prescribed 15enar, the majority of these solutions are notviable, resulting in observed (or apparent) isotope effects for
Table 1. Ranges of parameter values explored in the finite-differencing model exercise, including values prescribed in “standard”model parameterizations
Initial conditions Description Range Standard Comment Reference
[NO3−]initial Initial [NO3
−] 100 μmol/L 100 μmol/L[NO3
−]initial Initial [NO2−] 0 μmol/L 0 μmol/L
δ15NNO3,initial Initial δ15NNO3 −10‰ to +15‰ +5‰δ18ONO3,initial Initial δ18ONO3 −10‰ to +15‰ +5‰δ15NNH4,initial Initial δ15NNH4 −20‰ to +15‰ +5‰δ18OH2O δ18O of ambient H2O −20‰ to 0‰15enar N isotope effect for NO3
− reduction +5‰ to +25‰ +15‰ Coupled to 18enar (9)18enar O isotope effect for NO3
− reduction +5‰ to +25‰ +15‰ Coupled to 15enar (9)18enarBR Branching O isotope effect for NO3
− reduction 25‰ 25‰ (19)15enir N isotope effect for NO2
− reduction 0‰ to +20‰ +5‰ Coupled to 18enir (19, 64)18enir O isotope effect for NO2
− reduction 0‰ to +20‰ 5‰ Coupled to 15enir15enxr N isotope effect for NO2
− oxidation by nitrifiers −35 to 0‰ −15‰ (4)18enxrNO2 O isotope effect for NO2
− oxidation by nitrifiersor anammox
−7‰ to −3‰ −4‰ (5)
15enxrAMX N isotope effect for NO2− oxidation by anammox −35‰ (43)
15enxrAMX’ NXR-enabled equilibrium isotope effect betweenNO2
− and NO3−
18enxrH2O O isotope effect to H2O incorporation by nitrifiersand anammox
+12‰ to +18‰ +14‰ (5)
18eeq Equilibrium isotope effect between NO2− and H2O 13.5‰ 13.5‰ (30)
15eamo N isotope effect for NH3 oxidation by aerobicammonia oxidation
+26‰* 26‰* (41)
18eamoH2O O isotope effect for H2O incorporation by aerobicammonia oxidation
+18‰ to +38‰† 14‰ (6)
18eamoO2 O isotope effect for O2 incorporation by aerobicammonia oxidation
+18‰ to +38‰† 14‰ (6)
NXR/NAR Ratio of NO2− oxidation by nitrifiers to NO3
− reductionby denitrifiers
AMO/NAR Ratio of NO2− production by aerobic ammonia
oxidation vs. by NO3− reduction
*Not expressed given prescription of complete consumption of the ammonium pool.†The isotope effects of O atom incorporation from O2 and H2O during NH3 oxidation to NO2
− have only been determined as a ‘combined’ isotope effectranging between +18‰ and +38‰ at this time (7). A value of 28‰ was chosen and equally partitioned (+14‰) between the H2O and O2 pools.
NXR/NAR = 0.10
15N NO3- (‰ vs Air)
0 5 10 15 20-5 25 30
3- (‰ v
-5 ‰-10 ‰
A NXR/NAR = 0.50
15N NO3- (‰ vs Air)
0 5 10 15 20-5 25 30
-5 ‰-10 ‰
B NXR/NAR = 0.80
15N NO3- (‰ vs Air)
0 5 10 15 20-5 25 30
-5 ‰-10 ‰
Fig. 2. Predicted evolution of NO3− Δδ18ONO3 (δ18ONO3 – δ18ONO3,initial) plotted on the corresponding Δδ15NNO3 (δ15NNO3 – δ15NNO3,initial) associated with net
denitrification coincident with NO3− production by nitrification. Simulations derive from standard model conditions (Table 1) for incremental prescriptions of
NXR/NAR (A–C) and of δ18OH2O (colors), for full oxygen isotopic equilibration of NO2− with water.
E6394 | www.pnas.org/cgi/doi/10.1073/pnas.1601383113 Granger and Wankel
denitrification (15ednf, derived from the change in δ15NNO3 relativeto the fraction of NO3
− removed) substantially higher than theempirical maximum of ∼30‰ observed in the environment (Fig.S3). This dynamic arises because the N isotope effects for NO2
reduction (15enir), NO2− oxidation (15enxr), and the NXR/NAR
flux ratio amplify the 15N-enrichment of the NO3− pool relative
to its consumption beyond that imparted specifically by 15enar, thusincreasing apparent values of 15ednf. Thus, viable solutions (i.e.,15ednf ≤ 30‰) for Δδ18O:Δδ15N trajectories above 1 emergepredominantly at 15enar amplitudes of ≤15‰. Conversely, viablesolutions stemming from more elevated 15enar amplitudes are onlyproduced at relatively low NO3
− production (NXR/NAR), cor-responding to Δδ18O:Δδ15N trajectories close to 1. Therefore,Δδ18O:Δδ15N trajectories above 1, characteristic of marine deni-trifying systems, and of ∼0.6 for freshwater systems, do not resultat high 15enar values, suggesting that the organism-level isotopeeffect for denitrification (15enar) in environmental settings is gen-erally not as high as the 15enar of ∼25‰ often observed in cultureconditions (10) (Section 4).The amplitude of 15enir for the reduction of NO2
− also modu-lates the isotopic evolution of the NO3
−, with higher 15enir (coupledhere to 18enir) corresponding to lower Δδ18O:Δδ15N trajectories(Fig. 3B). Stronger 15N discrimination during NO2
− reduction toNO acts to increase the δ15N of the NO2
− pool, thereby increasingthe δ15N of NO3
− produced from any oxidation of NO2− and
lowering the Δδ18O:Δδ15N trajectory (Fig. S3).The amplitude of the inverse isotope effect prescribed to 15enxr
also has an important influence on the δ15NNO3 of newly nitrifiedNO3
−. Lower 15enxr values (i.e., more negative) give rise to lowerδ15NNO2, countered by production of correspondingly higherδ15NNO3, thereby acting to lower the Δδ18O:Δδ15N trajectory (Fig.3C). Conversely, less negative 15enxr amplitudes produce NO3
having a relatively lower δ15N, leading to higher Δδ18O:Δδ15Ntrajectories.In the model, 18enar is, by design, equivalent to the corresponding
15enar. As such, its influence on Δδ18O:Δδ15N trajectories appearsidentical to 15enar (Fig. S4A). This congruence, however, is onlyincidental because the δ18ONO2 produced from NO3
− reduction isentirely erased given full isotopic equilibration of NO2
− with water.Therefore, 18enar actually has no bearing on the δ18O of newlynitrified NO3
−. Similarly, the oxygen isotope effect for NO2−
reduction, 18enir, also has an apparent influence on the NO3−
Δδ18O:Δδ15N trajectory related to its coupling with the corresponding15enir (Fig. S4C). Here again, however, any increase in the δ18ONO2imposed by its reduction to NO is ultimately erased due to isotopicequilibration of the NO2
− with water, such that the value of 18eniris inconsequential to the Δδ18O:Δδ15N trajectory.In contrast, the amplitude of 18enxrNO2 does affect theΔδ18O:Δδ15N
trajectory because the influence of 18enxrNO2 occurs downstreamof NO2
− equilibration. Given this inverse isotope effect, lower(more negative) values of 18enxrNO2 are associated with an in-crease in the δ18O of newly nitrified NO3
−, and consequently, arelative increase in corresponding Δδ18O:Δδ15N trajectories(Fig. S4E). However, the influence of 18enxrNO2 on Δδ18O:Δδ15Ntrajectories is generally muted because 18enxrNO2 only affects two-thirds of the O atoms in the newly nitrified NO3
− and becausethe corresponding 15enxr has a greater amplitude than 18enxrNO2(Table 1) but opposing influence on Δδ18O:Δδ15N trajectories.Finally, the isotope effect associated with the incorporation of
an O atom from water during NO2− oxidation, 18enxrH2O, also
plays a role in determining the δ18O of nitrified NO3−. As pre-
scribed, the δ18O of the O atom incorporated from water duringNO2
− oxidation is ∼14‰ lower than that of ambient water, thuscontributing a lower δ18O than that from the two oxygen atomsin NO2
−. Because the amplitude of 18enxrH2O has so far provento be relatively invariant among cultures and experimentalstrains (5, 7), we do not consider variations of this value onΔδ18O:Δδ15N trajectories.
3.4. Initial Isotope Composition of the NO3− Pool. The initial isotopic
composition of NO3− is also linked to the evolution of its dual iso-
topic trajectory during net consumption coupled with contempora-neous production. Under conditions of a fully equilibrated NO2
pool, this dependence stems chiefly from the difference between theδ18ONO3 and the δ18OH2O. Mechanistically, the difference in δ18Ovalues between NO3
− and water imposes a corresponding differencebetween the δ18ONO3 removed by NO3
− reduction and the δ18ONO3returned to the NO3
− pool by NO2− oxidation, in relation to the
δ15NNO3 concurrently being removed and returned.For nearly all combinations of model conditions, the δ15NNO3
added by nitrification is greater than that removed concurrentlyby denitrification, owing to the compounding influences of iso-
A B C
D E F
Fig. 3. Predicted NO3− Δδ18O:Δδ15N trajectories (represented as the apparent linear slope calculated with respect to the initial NO3
− isotope composition;color scale) associated with net denitrification and coincident NO3
− production for a range of δ18OH2O given full oxygen isotopic equilibration of NO2−,
plotted over (A) 15enar, (B)15enir, (C)
15enxr, and (D) δ15NNO3,initial. Trajectories plotted over 15enar for NO3− production by anammox only for (E) 30% of total N2
production by anammox and for (F) 50% N2 production by anammox, with 15enirAMX = 20‰, and 15enxrAMX = −35‰. Parameter values are otherwise anchoredat standard conditions (Table 1).
Granger and Wankel PNAS | Published online October 4, 2016 | E6395
tope effects associated with NO2− reduction (normal) and oxi-
dation (inverse), 15enir and15enxr. On its own, the higher δ15N of
nitrified NO3− thus tends to lower the Δδ18O:Δδ15N trajectory
from a value of 1 for most model conditions.The δ18ONO3 produced by nitrification either counters or
otherwise amplifies the negative deviations from a Δδ18O:Δδ15Ntrajectory of 1 imposed by the corresponding δ15NNO3. When theinitial δ18ONO3 is nearer to the δ18OH2O, the δ18ONO3 producedduring nitrification is higher than that removed by denitrifica-tion, such that the Δδ18O:Δδ15N trajectory increases (Fig. 3D).Conversely, when the initial δ18ONO3 is elevated relative to theδ18OH2O, denitrification removes a δ18ONO3 that is similar to, orgreater than, that added by nitrification, contributing further to adecrease in Δδ18O:Δδ15N trajectory from a value of 1 imposed bycorresponding δ15NNO3 dynamics.The difference in δ18O between water and NO3
− thus pro-vides a first order rule of thumb to explain the magnitude ofthe Δδ18O:Δδ15N trajectory. For instance, in marine systems, thedifference between the δ18O of subsurface NO3
− (≥2‰) and sea-water (∼0‰) is small. At any value of 18enar, the δ18O of nitrifiedNO3
− is greater than the δ18ONO3 removed by denitrification, thusgenerating Δδ18O:Δδ15N trajectories above 1 for numerous modelconditions (Fig. 3D and Fig. S5). In contrast, in freshwater systems,the difference between the δ18O of NO3
− and water can be large(e.g., δ18ONO3 of +20‰ and δ18OH2O of −10‰ would not beunusual), such that the δ18O of nitrified NO3
− tends to be similar to,or can be lower than, the δ18ONO3 removed by denitrification,promotingΔδ18O:Δδ15N trajectories below 1 under most conditions(Fig. 3D and Fig. S5). In this light, Δδ18O:Δδ15N trajectories abovea nominal value of 1 have an intrinsically greater likelihood ofemerging in marine systems, whereas trajectories below 1 aremore likely for freshwater systems.
3.5. Influence of NH3 Oxidation to NO2−. As parameterized, the pro-
duction of NO2− by AMO yields invalid 15ednf values (>30‰) at
elevated NO2− oxidation fluxes (NXR/NAR ratios ≥ 0.5) while
exerting little influence on Δδ18O:Δδ15N trajectories with lowerNO2
− oxidation (Fig. S6). For these reasons, we relegate detaileddiscussion of the impact of AMO to S4. Impact of NO3
Production by AMO.
3.6. NO3− Production by Anammox. To assess whether NO3
− pro-duction by anammox is potentially sufficient to induce departuresof Δδ18O:Δδ15N trajectories from a value of 1, we consider twoscenarios, the first in which anammox rates are bounded by theorganic matter remineralization stoichiometry of denitrification,and the second in which anammox rates contribute an equalproportion of the N2 flux relative to denitrification (45). In thefirst scenario, anammox rates are controlled by NH4
+ suppliedonly from the decomposition of organic material coupled withNO3
− respiration, wherein the release of 1 mol of NH4+ from
organic matter requires respiration of 5.9 mol of NO3− to N2 (46).
Each mole of NH4+ then reacts with 1 mol NO2
− (producedtransiently by denitrification) to form N2 (24, 42). Concurrently,an additional 0.3 mol NO2
− is returned to the NO3− pool (42). Of
the total N2 produced, 30% originates from anammox and the restfrom canonical denitrification. The oxidation of NO2
− by anam-mox therein corresponds to a relatively small NXR/NAR flux ratioof 0.05.The Δδ18O:Δδ15N trajectories generated from these model
conditions are consistently below 1, regardless of δ18OH2O (Fig. 3E).In this respect, trajectories as low as 0.6, characteristic of freshwatersystem, are only attained with prescriptions of relatively low 15enar of≤5‰. Thus, within this scenario, anammox can only explain ex-cursions in freshwater Δδ18O:Δδ15N trajectories assuming specifi-cally low values of 15enar.When the anammox rate is parameterized to account for 50%
of N2 production, the corresponding NXR/NAR flux ratio in-
creases to ∼0.1 and Δδ18O:Δδ15N trajectories are on the order of∼0.6 for a relatively broader ranges of 15enar amplitudes (Fig. 3F).Therefore, if anammox rates in freshwater systems are not strictlybounded by ammonification coupled to NO3
− respiration—namely,if NH4
+ is also released via de-sorption from clays (47, 48) or fromorganic matter degradation pathways fueled by fermentation orother oxidants [e.g., Fe(III), Mn(III/IV)]—the production of NO3
by anammox could easily account for Δδ18O:Δδ15N trajectories of0.6 commonly observed in freshwater systems.The amplitude of 15enxrAMX is subject to some uncertainty. A
recent study showed that enrichment cultures of anammox pro-duced NO3
− with a δ15NNO3 ∼61‰ greater than correspondingδ15NNO2 following initial resuspension of cells (interpreted as an“enzyme-catalyzed equilibrium isotope effect” between NO3
−), after which NO3− production was associated with a
15enxrAMX amplitude of −35‰ (43). Accordingly, if NO3− pro-
duced by anammox had a δ15N ∼61‰ greater than that of cor-responding NO2
−, then an even smaller contribution of NO3− from
anammox would be required to influence Δδ18O:Δδ15N trajecto-ries (Fig. S7). From any perspective, anammox clearly emergesas a compelling candidate to explain negative deviations fromΔδ18O:Δδ15N trajectories of 1 observed in freshwater systems.Notably, Δδ18O:Δδ15N trajectories remain below 1 regardless of
anammox rate or 15enxrAMX values, owing to the relatively smallNO2
− oxidation flux permitted by anammox stoichiometry. Byitself, anammox thus fails to reproduce Δδ18O:Δδ15N trajectoriesabove 1 observed in marine systems. In our model, even ifanammox accounts for 100% of N2 production, the correspondingNXR/NAR ratio amounts to a mere 0.23, a value insufficient togenerate Δδ18O:Δδ15N trajectories akin to those observed in ox-ygen deficient zones, regardless of prescribed 15enxrAMX ampli-tudes (Fig. S8). Thus a significant input of NO3
− from aerobicNO2
− oxidation (or NO2− oxidation coupled to other electron
acceptors) is required to explain Δδ18O:Δδ15N trajectories >1 inoceanic oxygen deficient zones (Section 4.1).
3.7. NO2− Isotope Dynamics. Recent methodological advances en-
able the direct quantitation of NO2− δ15N and δ18O at environ-
mental concentrations (49, 50), providing a complementary tracerfrom which to diagnose environmental N cycling. Under condi-tions examined here, the predicted δ15N of the NO2
− pool rangesfrom 30‰ lower than the corresponding δ15NNO3 (Δδ15NNO2-NO3 =δ15NNO2 − δ15NNO3 = −30‰), to 10‰ greater than the corre-sponding δ15NNO3 (Fig. 4). In the context of observed Δδ18O:Δδ15Ntrajectories, δ15NNO2 emerges as sensitive to the prescribedNXR/NAR flux ratio (Fig. 4 and Fig. S3). Relatively large neg-ative Δδ15NNO2-NO3 offsets correspond to elevated Δδ18O:Δδ15Ntrajectories and elevated NXR/NAR amplitudes. Conversely, moremodest Δδ15NNO2-NO3 values are more prevalent at lower NXR/NARamplitudes (≤0.2) and Δδ18O:Δδ15N trajectories below 1. Theδ18ONO2, in turn, can provide affirmation that NO2
− is equil-ibrated with water, which we anticipate in freshwater systems, andwhich has been observed in most denitrifying marine systems (34,35). Therefore, if measured concurrently with those of NO3
− (andambient water), the δ15N and δ18O of NO2
− offer added quanti-tative constraints on the relative flux of NO2
− returned to NO3−
pool under denitrifying conditions (29, 30, 33, 34).
4. Links to Environmental Studies4.1. Marine Systems. Discrete lines of evidence suggest that asubstantial fraction of the NO2
− produced by denitrification inlow oxygen zones of the ocean is returned to the NO3
− pool byaerobic NO2
− oxidation (34, 51, 52). Mechanistically, the redoxpotential of waters bounding oxygen-deficient zones may bedynamic, responding to periodic advection of oxygenated watersnear redox boundaries (51, 53). Evidence that NO2
− reoxidationoccurs concurrently with denitrification originates in part fromobservations of Δδ18O:Δδ15N trajectories exceeding an expected
E6396 | www.pnas.org/cgi/doi/10.1073/pnas.1601383113 Granger and Wankel
value of 1. Measurements of the δ15N and δ18O of NO3− and
NO2− from the Eastern Tropical Pacific and the Peru Upwelling
region suggest that NO2− reoxidation is the more likely cause of
such patterns (32–34). Specifically, the δ15N of NO2− at the
subsurface is 30–40‰ lower than the coincident NO3− δ15N,
reaching δ15N values as much as 60‰ lower than the coexistingNO3
− (Δδ15NNO2-NO3 = −30 to −60‰). Best fit solutions to aninverse finite-difference model tracking the evolution of NO3
and NO2− isotopes along isopycnals indicated substantial NO2
reoxidation flux, with NXR/NAR ≥ 0.50 (34). Model fits furtherdiagnosed moderate 15enar values of ∼13‰, 15enxr values on theorder of −30‰, no fractionation during NO2
− reduction (15enir of
0‰), complete oxygen isotope equilibration of NO2− with sea-
water, and no significant input of NO2− from AMO.
The interpretations of recent observations in marine denitrify-ing waters generally corroborate the predictions from the modelpresented here. For illustration, we adapted the model architec-ture outlined above to use an inverse approach to numericallyoptimize NO3
− isotope measurements along a subsurface iso-pycnal at the Costa Rica Dome (35) (Fig. 5). The measurementsevidenced a progressive NO3
− decrease along the isopycnal sur-face with a concurrent increase in δ15N and δ18O, correspondingto a Δδ18O:Δδ15N trajectory >1 and an apparent isotope effect,15ednf, of 28‰. Values of 15enar,
15enir,15enxr, and NXR/NAR were
numerically optimized, iteratively finding the least squares fit tomeasured NO3
− concentration, δ15N and δ18O, assuming negligi-ble NO2
− production by AMO and full equilibration of NO2− with
water (S5. Model Inversion). Accordingly, the apparent Δδ18O:Δδ15N trajectory corresponds to an elevated NXR/NAR flux ratio of0.64 ± 0.07, and diagnoses of moderate values for 15enar,
15enxr,and 15enir of 14.1 ± 2.3‰, 10.9 ± 4.4‰, and −16.0 ± 4.5‰,respectively (Fig. 5).Interestingly, the curvature in Δδ18O:Δδ15N trajectories ap-
parent in our model results (Fig. 2) is substantiated by analogouscurvature in dual NO3
− isotope trajectories documented indenitrifying ocean waters (34–37) (Fig. 5). The increase of NO3
N and O isotope ratios associated with net denitrification alongisopycnals in the subsurface follows apparent Δδ18O:Δδ15N tra-jectories distinctly above a nominal slope of 1 that show clearcurvature at higher extents of net NO3
− consumption. In ourmodel, the degree of curvature increases in proportion to theNO2
− oxidation flux (Fig. 2), because the δ15N of nitrified NO3−
increases progressively as a function of net NO3− consumption,
whereas the corresponding δ18O of nitrified NO3− remains di-
rectly dependent on the δ18O of water (Fig. S2). The curvature inΔδ18O:Δδ15N trajectory in our model simulations thus appearvalidated by analogous patterns in NO3
− isotope distributions inmarine denitrifying environments.Anammox has been detected in oxygen minimum zones of the
ocean, where its potential contribution to the total N2 flux hasgenerated considerable debate (54–57), with estimates rangingfrom 30% to 100% of the total N2 flux. The model exercise heresuggests that NO3
− production by anammox cannot, by itself,explain Δδ18O:Δδ15N trajectories exceeding 1, given diagnoses ofNO2
− reoxidation to the NO3− pool that far exceed stoichiometric
Fig. 4. Predicted difference between δ15NNO2 and δ15NNO3 (Δδ15NNO2-NO3) as-sociated with net denitrification and coincident NO3
− production plotted againstthe NXR/NAR ratio, in relation to 15ednf (color scale). Discrete simulations derivefrom randomized parameter ranges (Table 1), with full oxygen isotopic exchangeof NO2
−, AMO/NAR = 0, δ18OH2O = -10‰, δ15NNO3,initial = 5‰, and δ18ONO3,intial =0‰. Open symbols correspond to model solutions where 15ednf > 30‰.
Fig. 5. (A) Best inverse fit Δδ18O:Δδ15N trajectory describing NO3− δ15N and δ18O in an oceanic oxygen deficient zone in the Eastern Tropical North Pacific
(35). Initial NO3− concentrations were interpolated based on the “initial” profiles outside the oxygen deficient zone (35), δ18OH2O = 0‰, and the AMO flux is
assumed to be negligible. Best-fit estimates of 15enar,15enir, and
15enxr are +14.1 ± 2.3‰, +10.9 ± 4.4‰, and −16.0 ± 4.5‰, respectively, with a diagnosed NXR/NAR of 0.64 ± 0.07. Model-predicted Δδ15NNO2-NO3 values are between −20‰ and −22‰ and the apparent isotope effect, 15ednf, is 28‰. (B) Best inverse fitΔδ18O:Δδ15N trajectory for NO3
− δ15N and δ18O in a contaminated aquifer in Cape Cod, MA (54). Best-fit estimates of 15enar,15enir, and
15enxr for the ETNPare +10.2 ± 1.3‰, +9.6 ± 4.2‰, and −24.0 ± 7.7‰, respectively, with a diagnosed NXR/NAR of 0.31 ± 0.08, model-predicted Δδ15NNO2-NO3 values between −6‰and −7‰, and an apparent isotope effect, 15ednf, of 16‰.
Granger and Wankel PNAS | Published online October 4, 2016 | E6397
constraints of anammox. Even if anammox is assumed to accountfor 100% of N2 production, the corresponding NO2
− flux remainsinsufficient to generate Δδ18O:Δδ15N trajectories above 1.Accordingly, analyses of NO3
− and NO2− isotope ratios in other
denitrifying regions of the Pacific and Indian Oceans haveconverged on similar interpretations, ultimately diagnosing asubstantial return flux of NO2
− to the NO3− pool catalyzed
largely by aerobic nitrification (34–37).Despite general agreements between model and observations,
some aspects remain puzzling. For one, the δ15NNO2 in deni-trifying marine waters can be substantially lower than predictedby the current exercise, with Δδ15NNO2-NO3 values ranging be-tween −30‰ and −60‰ in situ (34, 35), compared with −20%and −30‰ in our model (Fig. 4). Indeed, the best-fit parametersto the Costa Rica Dome data predict corresponding Δδ15NNO3-NO2values on the order of −20% to −22‰ (Fig. 5) compared withmeasured Δδ15NNO3-NO2 values between −20‰ and −50‰(35). Lower Δδ15NNO3-NO2 values can only be generated in thecurrent model framework by assuming an elevated reoxidation toreduction flux (NXR/NAR), as well as large amplitude isotopeeffects for 15enar and
15enxr on the order of 30‰ and −30‰,respectively. However, while realizing Δδ15NNO2-NO3 values as lowas −60‰ and Δδ18O:Δδ15N trajectories above 1, such simulationsresult in apparent values of 15ednf that far exceed the empiricallimit of 30‰ (Fig. 4). Otherwise, 15enxr may be effectively sub-sumed into an equilibrium isotope effect of −61‰, the purportedenzyme-catalyzed equilibrium isotope effect between NO2
− (43), such that the net production of NO3− by the NO2
oxidoreductase enzyme can generate Δ15NNO2-NO3 values of∼−60‰, albeit at elevated NXR/NAR flux ratios. This scenario,however, still results in apparent 15ednf values that far exceed30‰, thus inconsistent with observations. Arguably, isotopicallycatalyzed enzymatic isotope equilibration by NO2
− oxidoreductaseneed not be associated with a net oxidative flux. Such a scenario isanalogous to simulations here where NXR is nearly equal to NAR(with NAR representing NO3
− reduction by NO2− oxidoreductase
in lieu of NO3− reductase). The equilibrium isotope effect
of −61‰ is then implicitly the sum of the oxidative and reductiveisotope effects with respect to NO2
−, −31‰ for NO2− oxidation
vs. −30‰ for NO2− production from NO3
−, wherein the resultingΔ15NNO2-NO3 of the NO2
− pool is ∼−60‰. Again, apparent val-ues of 15ednf emerging from such simulations are unrealistic (onthe order of 60‰). Thus, within the current model framework,the putative enzyme-catalyzed equilibrium isotope effect betweenNO2
− and NO3− does not appear to readily explain the very low
Δδ15NNO2-NO3 observed in marine denitrifying systems.The discrepancy between our modeled Δδ15NNO2-NO3 and
observations may otherwise derive in part from the constantN transformation rates in our simulations, as more negativeδ15NNO2 values could be generated within representative modelconditions given time/space-variable rates of NO2
− accumulationand depletion and/or isotope effects. Nevertheless, inverse fitsallowing for time-variable rates of N transformations still im-plicate 15enxr values on the order of −30‰ (34, 35), distinctlylower than values observed in nitrifier cultures of –15‰ (5, 7).Additionally, the inverse-model fits are contingent on the di-minished expression of 15enir (5, 7), a diagnosis that is also ech-oed in our model (Fig. S3). Such low 15enir amplitudes appearcontradictory to expectations from cultures and enzymaticstudies (19, 43, 58, 59) and are also inconsistent with some recentfield observations (36), where an increase in δ15NNO2 alongisopycnal surfaces in a denitrifying eddy at the Peru margin (inwhich no NO3
− remained) was associated with an apparent 15enirof 12‰. The very low δ15NNO2 observed in marine denitrifyingzones thus remains perplexing and merits further inquiry.
4.2. Freshwater Systems. As demonstrated here, the empiricalΔδ18O:Δδ15N trajectories between 0.5 and 0.8 recurrently ob-
served in freshwater systems can be explained by superimposingthe isotopic systematics of denitrification with those of oxidativeNO3
− production. This dynamic could arise on the premise thatredox conditions in aquifers may be dynamic, owing to in-tercalated microzonation within sediments (60) and/or peri-odic downward percolation of oxygenated waters. However,nitrifying organisms and their biogeochemical activity are rarelydetected at the heart of denitrifying aquifers (61, 62). Thus, in theabsence of any O2-requiring transformations, it is likely that anyreturn of NO2
− to the NO3− pool in anaerobic aquifers is asso-
ciated with anammox, the anaerobic oxidation of NH3 coupled toreduction of NO2
−, which yields NO3− (as well as N2) as a met-
abolic product (24, 42). Indeed, recent studies suggest that asubstantial fraction of N2 production in aquifers originates fromanammox (63–65), rivaling N2 production by canonical de-nitrification in some instances (45).We turn to a well-studied site of groundwater contamination
on Cape Cod, MA (47), to estimate isotope effects and relativeNO2
− oxidation rates that could explain NO3− isotope distribu-
tions therein. Within the contaminant plume, ∼180 μM NO3− de-
creased to ∼35 μM along an anoxic groundwater flow path.Concomitant with this decrease in NO3
− concentration, pro-nounced changes in δ15NNO3 and δ18ONO3 were also observed,increasing from ∼+15‰ to +45‰ and from −1‰ to +18‰,respectively, yielding a corresponding Δδ18O:Δδ15N trajectory of∼0.73 and an apparent N isotope effect for the observed de-nitrification, 15ednf, of ∼16‰ (47). We use the inverse approachoutlined above to numerically optimize values of 15enar,
and NXR/NAR, iteratively finding the least squares fit to measuredNO3
− concentration, δ15N and δ18O (S5. Model Inversion). We alsomake a simplifying assumption that AMO is negligible in the anoxicportion of the aquifer. Given the pH and δ18O of groundwater atthis site (∼6.5 and −6.5‰, respectively) (47), we also assume thatany NO2
− will be fully equilibrated with water.As expected, deviation of the dual isotopic composition from a
Δδ18O:Δδ15N trajectory of 1–0.73 can be accommodated by con-comitant production of NO3
− along this flow path (Fig. S8). Best-fit solutions yield estimates of 15enar,
15enir, and15enxr of 10.6 ±
0.2‰, 10.2 ± 3.9‰, and −31.3 ± 7.7‰, respectively. Consistentwith expectations, lower actual values of 15enar are diagnosed (i.e.,below the observed 15ednf of ∼16‰), as the increase in δ15NNO3 isalso influenced by NO3
− production with a relatively high δ15N viaNXR. Under these conditions, the required amount of NO3
production relative to its removal is on the order of 13 ± 0.3%(i.e., NXR/NAR = 0.13), much lower than that predicted by re-cent dual isotope models of ocean denitrifying zones in which thediagnosed NXR/NAR can be as high as 0.9 (34). Although notmeasured in the original aquifer study, these best-fit parametersalso allow us to predict the δ15N of coexisting NO2
−—for which wearrive at Δδ15NNO2-NO3 values between −6.1‰ and −4.4‰. Thisprediction of a relatively small δ15N difference between coexistingpools of NO3
− and NO2− under aquifer conditions provides a
target for future studies and a means for refining our estimates ofN cycling in groundwater and other environments.Notably, the estimated value for 15enxr of ∼−31‰ for the
aquifer is higher than that observed in cultures of NO2− oxidizing
bacteria (7), yet largely consistent with that for anammox (43),potentially implicating anammox as an important N removalprocess in this aquifer. The diagnosis of NXR/NAR ratio of 0.13corresponds to ∼65% of total N2 production in the aquiferfueled by anammox, a value also consistent with recent in-dependent estimates made in the same aquifer (45). Indeed,given the stoichiometric constraints of NO3
− production byanammox, we suggest these model results may prove diagnosticof the role of anammox in ground waters globally.
E6398 | www.pnas.org/cgi/doi/10.1073/pnas.1601383113 Granger and Wankel
5. Summary and ConclusionsForemost, our results demonstrate that deviations from the ca-nonical Δδ18O:Δδ15N trajectory of 1 for denitrification mustemerge due to concurrent NO3
− production catalyzed by nitri-fication and/or anammox, not only in marine systems, but also infreshwater systems, where this tenet has been given limited con-sideration. Assuming that full O isotopic equilibration of NO2
− ispertinent to both freshwater and marine denitrifying systems, ourresults emphasize the sensitivity of the δ18O of newly producedNO3
− to the δ18O of ambient water and the isotope effects forO atom equilibration and incorporation. Trajectories above 1emerge specifically where the NXR/NAR flux ratio is high (≥0.5)and predominantly where the difference between the δ18O ofNO3
− and that of water is small. The diagnosis of high NO3−
production in the generation of Δδ18O:Δδ15N trajectories above 1disputes the notion that anammox could be the sole NO3
−-pro-ducing reaction in marine denitrifying systems, given stoichio-metric and biochemical limitations on the amount of NO2
− thatanammox can return to the NO3
− pool, and thus implicates asignificant contribution by aerobic NO2
− oxidation.Importantly, the majority of solutions to randomized combi-
nations of model conditions yield Δδ18O:Δδ15N trajectories <1for simulations using a fully equilibrated NO2
− pool. In this re-spect, our analysis of NO3
− isotopes from a representativeaquifer (47) suggests that the characteristic Δδ18O:Δδ15N tra-jectory therein (∼0.73) arises from relatively low NXR/NAR fluxratios (∼0.13), which is well aligned with the stoichiometric andbiochemical constraints of anammox. Thus, in addition to theleverage of distinctive δ18OH2O between freshwater and marinesystems, characteristic NO3
− isotope trends between denitrifyinggroundwater and marine systems also appear to reflect funda-mental differences in key N transformation pathways operativein these systems.Within this model framework, the δ15N of NO2
− provides anadditional diagnostic to estimate the relative contribution ofnitrification (and/or anammox) to the NO3
− pool. In particular,very deplete δ15NNO2 values relative to the NO3
− pool may becharacteristic of elevated NXR/NAR flux ratios: a dynamic thatappears corroborated by analogously low δ15NNO2 values inmarine denitrifying zones, albeit lower than those predicted byour model simulations.Some uncertainties also remain that require resolution to en-
sure accuracy of interpretations. For one, O isotope effects asso-ciated with NO3
− production by anammox remain undocumented;whereas dynamics may be similar to those for NO2
− oxidizingbacteria, this should be confirmed. Second, the nature and envi-
ronmental extent of the purported “enzyme-catalyzed equilibriumN isotope effect” between NO3
− and NO2− during anammox
should also be further explored. Diagnostic estimates of NXR/NAR flux ratios are sensitive to the broad potential range of Nisotope effects during NO2
− oxidation to NO3−. Third, the ap-
parent discrepancy between model and measured δ15NNO2 inmarine systems merits examination to assess the involvement ofNO2
− in potential abiotic/inorganic reactions. Finally, the premisethat NO2
− oxygen isotopes in marine and freshwater denitrifyingsystems are fully equilibrated with water should be further in-terrogated, as even partial O isotope disequilibrium could influ-ence diagnostics of N fluxes and associated isotope effects (S3.Simulation Without Isotope Equilibration of NO2
− with Water).Continued inquiry into isotope systematics of N cycling will lead toan increasingly robust framework from which to examine N cycledynamics across ecosystems.
6. Materials and MethodsGiven the number of variables involved in calculating the model solutions, wemade a number of decisions for model parameterizations to maintain clarity.First, we chose a range of isotope effects based on published studies using purecultures of representative organisms and/or purified enzymes when possible(Table 1). As studies of organism-level isotope effects have demonstratedbroad variability, whether by bacterial strain or growth conditions, for brevityand simplicity, we also established “standard” model conditions having gen-erally midrange values for isotope effects (Table 1). Second, a study publishedthis year demonstrates that the N and O isotope effects for NIR are coupleddifferently depending on the type NO2
− reductase involved (59). Our simula-tions, however, are based on parameterization of 15enir and
18enir coupled in aratio of 1:1, assumed before this insight. Regardless, this inaccuracy does notultimately impact the conclusions reached herein (Section 3.3 and S3.2. Influ-ence of 18«nar and
18«nir). Third, the N and O isotope effects for NXR in themodel parameterization remain uncoupled because studies of NO2
−-oxidizingorganisms have evidenced no connection in their magnitudes (5). Finally, weallow the initial N and O isotopic composition of NO3
− to vary across a rep-resentative range (−10‰ to +15‰ for both δ15N and δ18O) while using valuesof +5‰ for both as standard conditions.
To date, no information exists on O isotope systematics of anammox, suchthat we assumeO isotope effects similar to that of NO2
− oxidizing bacteria, asenzymatic pathways are analogous (66). However, the organism-level in-verse N isotope effect reported for anammox cultures for NO3
− productiondriven by NXR exhibits higher values (−35‰) (43) than those observed incultures of bacterial NO2
− oxidizing bacteria (−13‰) (4) and may also includea very large enzymatically driven isotope equilibrium between NO3
− of −61‰ (43).
ACKNOWLEDGMENTS. This manuscript benefited from comments by twoanonymous reviewers. This work was supported by National ScienceFoundation Grants EAR-1252089 (to J.G.) and EAR-1252161 (to S.D.W.).
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