Interactive comment on “ Nitrogen cycling in the subsurface biosphere : nitrate isotopes in porewaters underlying the oligotrophic North Atlantic ”

Introduction Conclusions References


Introduction
Below the euphotic zone at the ocean surface, the dark ocean, including environments above and below the seafloor, hosts the largest habitable environment on the planet and is home to a wide range of globally relevant biogeochemical processes (Orcutt et al., 2011).While significant progress has been made in recent years toward characterizing the geological, chemical, and ecological composition of a variety of subsurface environments (Orcutt et al., 2011;Edwards et al., 2011Edwards et al., , 2012b)), the potential for impact of these systems on global biogeochemical cycles remains poorly understood.Most of our knowledge about subseafloor microbial activity stems from research focusing on productive continental margins, where relatively high fluxes of organic matter Figures

Back Close
Full idants of organic carbon, penetrate deeply into the sediment underlying oligotrophic ocean waters (D'Hondt et al., 2009;Murray and Grundmanis, 1980;Rutgers van der Loeff et al., 1990;Sachs et al., 2009;D'Hondt et al., 2015;Røy et al., 2012;Fischer et al., 2009).Furthermore, where underlain by relatively young and permeably ocean crust, O 2 and NO − 3 are also supplied via upward diffusion from oxic and nitrate-replete fluids flowing through basaltic basement as has been shown for the North Pond site, which is located on the western flank of the Mid-Atlantic Ridge (Orcutt et al., 2013;Ziebis et al., 2012).At North Pond, where the sediment cover is thin (<∼ 25 m), O 2 penetrates the entire sediment column; where sediment thickness is elevated, conditions become anoxic at mid-depths.Aerobic heterotrophic respiration likely dominates organic carbon oxidation in the upper sediment column.However, as organic carbon becomes limiting at greater depths, autotrophic processes (e.g., nitrification) are likely to gain relative importance.Further, there is evidence that the upward supply of oxidants from the basaltic basement supports increased microbial activity (Picard and Ferdelman, 2011).However, a fundamental understanding of the relative importance of specific metabolic activities that drive and sustain subsurface communities is lacking.
Because central ocean gyres cover roughly half of the global seafloor, understanding the nature of the biosphere hosted within these sediments may reveal important insights into its role in ocean scale dynamics of ocean nitrogen and carbon cycling.Here we focus specifically on elucidating subsurface nitrogen cycling and its role in supporting heterotrophic and autotrophic processes in oligotrophic deep-ocean sediments underlying the North Atlantic Gyre, at North Pond (22 • 45 N, 46 • 05 W).IODP Expedition 336 (Mid-Atlantic Ridge Microbiology, 16 September-16 November 2011) aimed to directly address the nature of microbial communities in both ocean crust and sediments at North Pond, a characteristic sediment-filled 70 km 2 depression surrounded by high relief topography common to the western flank of the Mid-Atlantic Ridge (Becker et al., 2001;Langseth et al., 1992).While a majority of seafloor subsurface biosphere research has focused on aspects of sediment carbon, sulfur and iron cycles, the potential role of N in supporting subsurface microbial activity has been Figures Back Close Full largely unexplored.Despite exceedingly oligotrophic conditions, life persists and evidence for active heterotrophic and autotrophic microbial communities in North Pond sediments is mounting (Ziebis et al., 2012;Picard and Ferdelman, 2011;Orcutt et al., 2013).
Nitrogen plays a central role as a limiting nutrient in many regions of the sunlit surface ocean (Rabalais, 2002), as nearly 90 % of the biologically available fixed N in the ocean resides below the euphotic zone in the deep ocean NO − 3 reservoir (Gruber, 2008).Globally, sediments (especially continental shelves) are considered a net sink of fixed nitrogen through reductive anaerobic processes including denitrification and anaerobic ammonium oxidation (Christensen et al., 1987;Devol, 1991;Prokopenko et al., 2013).
However, there is abundant evidence demonstrating the importance of both oxidative and reductive N cycling processes (and their tight coupling) operating in sediment environments.For example, coupled nitrification (the chemolithotrophic oxidation of NH + 4 to NO − 3 ) and denitrification (the generally heterotrophic reduction of NO − 3 to N 2 ) have been shown be important in N budgets in sediments of continental shelves/margins and estuaries (Risgaard-Petersen, 2003;Granger et al., 2011;Lehmann et al., 2004Lehmann et al., , 2005;;Wankel et al., 2009).In contrast to sediments on continental shelves, however, data from sediments underlying large swaths of the oligotrophic ocean suggest an entirely different framework.For example, NO Dual isotopes of NO − 3 represent a powerful tool for disentangling the combined activities of multiple N cycling processes (Casciotti et al., 2008;Lehmann et al., 2005;Sigman et al., 2005;Wankel et al., 2007;Marconi et al., 2015;Fawcett et al., 2015).Nitrate-removal processes (whether assimilatory or dissimilatory) have been shown to impart tightly coupled increases in both N and O isotope ratios of the remaining NO − 3 pool (Karsh et al., 2012;Kritee et al., 2012;Granger et al., 2008Granger et al., , 2004)).In contrast, however, nitrification, the two-step oxidation of NH + 4 to NO − 2 followed by NO − 2 oxidation to NO − 3 , represents a decoupling of the N and O isotope systems for nitrate (Casciotti and McIlvin, 2007;Buchwald and Casciotti, 2013;Wankel et al., 2007).Whereas the N atoms derive from the NH + 4 (which can be assumed to derive from the sedimentary organic nitrogen pool), the oxygen atoms derive, to varying degrees, from both water and dissolved O 2 (Buchwald and Casciotti, 2010;Buchwald et al., 2012;Casciotti et al., 2010).Thus, by combining isotope mass balances of both N and O in the NO − 3 system, along with our understanding of organism-level constraints on the isotope systematics of these transformations, we can deduce the relative roles of multiple N cycling processes (e.g., Wankel et al., 2009;Lehmann et al., 2004;Bourbonnais et al., 2009).
Here we use the dual isotopic composition of nitrate (N and O isotopes) as a record of microbial processes occurring in the low-energy sediments of North Pond underlying the oligotrophic North Atlantic gyre.By combining the N and O isotope mass balance constraints with an inverse reaction-diffusion model approach, we use these data to estimate rates of nitrification and denitrification, and to provide new constraints on some isotope parameters for these processes.

Sediment and porewater collection
Sediment cores were collected at three sites in the North Pond Basin as part of the IODP Leg 336 expedition and have been 336-Scientists, 2012a).Four boreholes were drilled (U1382B, U1383D, U1383E and U1384A, referred to hereafter as "2B", "3D", "3E" and "4A").Sites 3D and 3E were next to each other and as drilling logs indicated that the core from 3E showed excessive signs of disturbance upon retrieval and potential contamination by seawater, it was not used further in this project.Sediment cores were retrieved using the Advanced Piston Corer, which penetrated the seafloor sediments until contact with basement, followed by extended core barrel coring of the upper section of basement rock.Site 2B (∼ 90 m sediment thickness, depth to basement) is located in the deeper part of the pond, approximately 25 m away from DSDP "legacy hole" 395A, which was instrumented as CORK observatory (Davis et al., 1992).Site 3D (∼ 42 m sediment thickness, depth to basement) lies in the northeastern region towards the edge of North Pond (∼ 5.9 km away from U1382A), whereas site 4A (∼ 95 m sediment thickness, depth to basement) is located on the northwest side in a deeper part of the basin, approximately 3.9 and 6.2 km distance from U1383 and U1382, respectively.All sediments were comprised of light-brown to brown nannofossil ooze with intercalations of foraminiferal sand.In the lowest few meters close to the sediment/basement contact, sediments exhibited a darker brown color and sometimes rust-colored clay-rich zones (Edwards et al., 2012a;Expedition-336-Scientists, 2012b).
Porewater samples for concentration and stable isotope analyses were collected either directly from cores on the shipboard catwalk during core retrieval (using methods described elsewhere Expedition-336-Scientists, 2012a) or from whole core rounds (∼ 10 cm core sections) that were preserved at −80 • C. Whole core rounds were transported frozen and thawed only during lab-based porewater extraction.Porewaters were extracted on the ship and in the lab using rhizon samplers (0.2 µm) (Seeberg-Elverfeldt et al., 2005).Approximately 40 mL of porewater was extracted from each 10 cm whole core round.Figures

Back Close
Full

Nitrate concentrations
Porewaters for nitrate samples were collected from three sources: (1) shipboard porewater extraction directly after core retrieval, (2) laboratory porewater extraction via rhizons from whole core rounds which had been frozen at −80 2 ) from group 1 were measured via ion chromatography ∼ 3 months after collection, while concentrations from groups 2 and 3 were measured by chemiluminescence after reduction in a hot acidic vanadyl sulfate solution on a NO x analyzer (Braman and Hendrix, 1989).Concentrations of NO − 2 were quantified by using the Griess-Ilosvay method followed by measuring absorption 540 nm or by chemiluminescence in a sodium iodide solution on a NO x analyzer (Braman and Hendrix, 1989), and NO − 3 was quantified by difference (Grasshoff et al., 2007).

Nitrate stable isotope composition
Nitrate N and O isotopic composition were measured using the denitrifier method (Casciotti et al., 2002;Sigman et al., 2001), in which sample NO − 3 is quantitatively converted to N 2 O using a lab-grown denitrifying bacterium before being extracted and purified on a purge and trap system similar to that previously described (McIlvin and Casciotti, 2010).Where detected, NO − 2 was removed by sulfamic acid addition (Granger and Sigman, 2009) prior to isotopic analysis of NO − 3 .Isotope ratios were measured on an IsoPrime100 (Elementar, Inc.) and corrections for drift, size and fractionation of O isotopes during bacterial conversion were carried out as previously described using NO

Dissolved oxygen profiles
Oxygen penetration depths, which have been discussed previously (Orcutt et al., 2013), vary distinctly among the three sites at North Pond indicating much greater respiratory consumption in 2B than in the profiles of other two sites, 3D and 4A (Fig. 1).In 2B, dissolved oxygen levels are drawn down to near detection by a depth of about 10 m (although low levels of dissolved O 2 seem to persist as deep as 30 m).In contrast, at site 3D, dissolved O 2 levels are drawn down to detection level for an interval of only ∼ 3 m between a depth of ∼ 30 to ∼ 33 m and in 4A, zero-level O 2 concentrations were observed over the interval between 32 and 54 m.At North Pond, O 2 (and NO − 3 ) is also supplied via diffusion from the underlying basaltic crustal aquifer (Fig. 1) (Orcutt et al., 2013;Ziebis et al., 2012).et al., 2012).At all depths in all three profiles, porewater NO  (Berelson et al., 1990;Goloway and Bender, 1982;Hammond et al., 1996;Jahnke et al., 1982;Grundmanis and Murray, 1982).More precisely, below the sedimentwater interface, NO − 3 concentrations increased significantly with depth (Fig. 1), before decreasing again with proximity to the basement/sediment contact.Mid-profile NO and 4A and was only detected at anoxic depths in site 2B (Fig. 1), where concentrations of up to 6.0 and 6.6 µM were observed at depths of 36 and 59 m, respectively.

NO − 3 N and O isotopic composition
Down-core changes in δ 15 N and δ 18 O varied markedly among the three cores (Fig. 1). .The relationship between δ 18 O NO 3 and δ 15 N NO 3 exhibited a slope of 1.8 for the upper portion of the 2B profile, 3.0 for the 3D profile and 2.4 for the 4A profile -consistently exceeding the 1 : 1 relationship expected from NO

Discussion
The distribution of porewater nitrate in deep-sea sediments is controlled by the combined influence of diffusion as well as several biologically catalyzed diagenetic processes including nitrification (ammonia and nitrite oxidation to nitrate) and denitrification (loss of N via nitrate reduction to gas phase products, NO, N 2 O or N 2 ).Here we use the concentration and dual N and O stable isotope composition of porewater NO − 3 to gain insight into the magnitude and distribution of N transformation processes.In comparison to models that predict the rates of these processes based solely on concentration profiles of NO − 3 and O 2 , for example, our approach estimates rates using the added constraints provided by recent studies of N and O isotope systematics of nitrification and denitrification (Granger et al., 2008;Buchwald and Casciotti, 2010;Casciotti et al., 2010;Buchwald et al., 2012).In particular, while there are strong and related N and O isotope effects during denitrification (Granger et al., 2008), the N and O source atoms during nitrate production are unrelated (Buchwald and Casciotti, 2010;Casciotti et al., 2010;Buchwald et al., 2012).Thus, changes in N and O isotopic composition between intervals of any one depth are the combined result of both diffusion of NO − 3 to/from overlying (and underlying) seawater, together with microbially mediated production and/or consumption of NO O source atoms to NO − 3 in these energy-lean systems.Specifically, we show below that the profiles of δ 15 N NO 3 and δ 18 O NO 3 can be explained by variations in the magnitude of nitrification and denitrification occurring throughout the sediment column, including substantial zones of overlap of these canonically aerobic/anaerobic processes.Finally, we use our model to predict the δ 18 O and δ 15 N stemming from nitrate production by nitrification, offering insights into both the nature of processes setting the O isotopic composition of oceanic NO − 3 , as well as the sources of N and/or the isotopic partitioning of available N sources in global ocean sediments.

Diffusion-reaction model
The diffusion-reaction inverse modeling approach used here is conceptually similar to other early diagenetic models that simulate porewater profiles of dissolved species through a sediment column harboring both oxic and anoxic organic matter remineralization (Christensen and Rowe, 1984;Goloway and Bender, 1982;Jahnke et al., 1982).It is an inverse modeling approach adapted to distinguish between heavy and light nitrate isotopologues (e.g., Lehmann et al., 2007).Specifically, we use the model to estimate rates of nitrification and denitrification required to fit the concentration profiles of each isotopologue, 14 NO − 3 , 15 NO − 3 , N 16 O − 3 , and N 18 O − 3 (and, thus, δ 15 N NO 3 and δ 18 O NO 3 values) under the assumption of steady-state diffusion and microbial production (by nitrification) and/or consumption (by denitrification).Rates of nitrification and denitrification in each porewater sampling interval (e.g., defined as the distance between the lower and upper midpoints between sampling depths) were estimated numerically by least squares fitting of the system of equations describing the distribution of each isotopologue (using a genetic algorithm included in the Solver package of Microsoft Excel 2011).This approach involves determination of a non-unique solution using numerical iteration and optimization, and is repeatedly iterated to evaluate robustness of model fits.Certain parameters are allowed to be optimizable by the model, including both the magnitude of, and connection between, the N and O isotope effects for denitrification Figures

Back Close
Full  1).Conditions at the uppermost part of the sediment column were constrained by measured concentrations and isotope ratios in bottom seawater.Measured concentrations of the NO − 3 isotopologues within each interval, together with the diffusive fluxes defined by the concentration gradients between the over/underlying intervals, were used for model fitting by least-squares optimization of microbial rates of nitrification and/or denitrification.
As measurable NH + 4 was not observed at any depths, it is not modeled as such.
NO − 3 is the only dissolved N species included in the model and we assume that all NH + 4 generated by remineralization is completely oxidized to NO − 3 (see below).To minimize complexity, other diagenetic reactions that may be important in many sedimentary environments, including anaerobic NH + 4 oxidation, removal of N species through interactions with Fe or Mn and the adsorption and retention of NH + 4 by clay minerals are not specifically addressed.We also neglect effects of compaction as well as potential changes in organic matter reactivity with depth.No difference in the diffusivity among NO − 3 isotopologues was included, since these differences are considered to be very small (Clark and Fritz, 1997).
Resolving the vertical dimension only, the mass balance differential equations are as follows: Tables Figures

Back Close
Full such that for denitrification (DNF): where D refers to the molecular diffusion coefficient for NO where f refers to the fractional abundance of a particular isotopologue and and and 15 R NTR and 18 R NTR are used to calculate the δ 15 N NTR and δ 18 O NTR , respectively.
For parameterizing diffusion, we use a porewater diffusion coefficient (D s ) based on the molecular diffusion coefficient (D m at 5 • C) for NO − 3 of 1.05 × 10 −5 cm 2 s −1 (Li and Gregory, 1974) adjusted for an average porosity (ϕ) of North Pond sediments of 64 % (Expedition-336-Scientists, 2012a), where D s = ϕ k D m and k is an empirically derived factor (we use 2.6) accounting for tortuosity of pore space (Hammond et al., 1996;McManus et al., 1995).
Compared with contemporaneous profiles of O 2 and Sr (Orcutt et al., 2013) and other dissolved ions, the NO − 3 concentration profiles suffer from some apparent analytical noise.The nature of the heterogeneity for NO − 3 concentration measurements was unclear.However, it is unlikely that this heterogeneity is environmental and we attribute it to small amounts of evaporation during freezer storage of the sediments, which is 13559 Introduction

Conclusions References
Tables Figures

Back Close
Full supported by the apparent smoothness of the isotopic measurements (evaporation would change the apparent concentrations without influencing the isotopic composition of solutes such as NO − 3 ).As such, for the purpose of the diffusion-reaction model, we applied a 5-point weighted triangular smoothing to the concentration data to eliminate outliers and unrealistically sharp gradients (Fig. 1).Given the relatively smooth and contiguous vertical profiles of δ 15 N NO 3 and δ 18 O NO 3 , only very minor smoothing to these data was required using a similar approach (Fig. 1).
Within this model architecture, we explore the influence of four key parameters that could affect the estimation of nitrification and denitrification rates by this isotope mass balance approach, specifically, 15  15 ε DNF value of 0.6 (Granger et al., 2008;Frey et al., 2014), could play a role in the dual isotope trajectory of NO − 3 consumption (Wenk et al., 2014).Further, in the absence of NH + 4 accumulation in these sediments, the δ 15 N NTR is equal to the source of NH  (Buchwald and Casciotti, 2010;Casciotti et al., 2010;Andersson and Hooper, 1983).Below, we use the model to optimize and predict these values and to explore the sensitivity of rate estimates to 15 ε DNF .
The model contains more parameters than can be explicitly estimated from the small number of data points measured.To minimize the number of variables as much as Introduction

Conclusions References
Tables Figures

Back Close
Full possible (and maximize the utility of the approach for constraining other variables), we adapt the model implementation for three different O 2 regimes: (1) "oxic intervals" where O 2 is poised as the more energy-yielding oxidant with respect to NO − 3 (here generally O 2 > ∼ 40µM, see below) and in which only nitrification is allowed to occur, (2) "transitional intervals" in which both denitrification and nitrification may occur (O 2 between ∼ 40 and 2 µM) and ( 3) "anoxic intervals" where O 2 is < 2µM and in which only denitrification is allowed to occur.In the oxic intervals -the model is used for parameter estimation of both δ 15 N NTR and δ 18 O NTR (in addition to nitrification rate), while in the anoxic intervals the model is used to estimate 15 ε DNF and 18 ε: 15 ε DNF (in addition to denitrification rate).In transitional intervals, 15 ε DNF and 18 ε: 15 ε DNF are held constant at 25 ‰ and 1, respectively, and the parameter δ 15 N NTR and δ 18 O NTR are estimated through model fitting, together with rates of both nitrification and denitrification.

Model estimated rates of nitrification and denitrification
Profiles of sedimentary porewater solutes reflect the combined influence of many processes including diagenetic reactions, which are intimately related to the availability, abundance and quality of organic carbon.In particular, the distribution of dissolved substrates that are available as electron acceptors for microbial respiration of organic carbon, generally reflect stepwise consumption by the most thermodynamically continental shelf sediments, dissolved O 2 and NO − 3 are typically consumed within a few cm or mm below the sediment-seawater interface, sediments underlying large areas of the oligotrophic ocean are characterized by very deep penetration of O 2 , in some cases even penetrating to the underlying ocean crust (D'Hondt et al., 2015(D'Hondt et al., , 2009;;Orcutt et al., 2013;Ziebis et al., 2012).In connection with this deep penetration of O 2 , deep-sea sediment porewaters also often exhibit extensive accumulation of NO − 3 above ambient seawater concentrations, associated with the oxidation of NH + 4 released by aerobic remineralization of sediment organic matter (and linked to the consumption of O 2 through Redfield stoichiometry) (Berelson et al., 1990;Christensen and Rowe, 1984;D'Hondt et al., 2009;Goloway and Bender, 1982;Seitzinger et al., 1984).In organic-rich sediments, NO − 3 concentration profiles may exhibit maxima only a few mm or cm below the sediment/water interface.In contrast, in the deep-sea sediments underlying the oligotrophic regions of the ocean, the sedimentary zone where NO − 3 accumulates to 10 to 30 µM above bottom seawater concentrations can extend over a much larger vertical extent and nitrate maxima can be found tens of meters below the seafloor.In effect, the redox zonation of O 2 respiration, NH isotopic composition and concentrations of NO − 3 in these porewaters reveals active nitrogen cycling processes within all three sites.
Using these changes in dual NO − 3 isotopic composition and concentration, we calculated the rates of nitrification and denitrification necessary to produce the observed patterns within each interval (in the transitional intervals, here we prescribe a value of 25 ‰ for 15 ε DNF , but explore the model sensitivity to this value later).Rates of nitrification and denitrification varied with depth, as well as across the three sites (Fig. 3; Table 1).Estimated rates of nitrification in the oxic and transitional intervals were up to 871 nmol cm −3 yr −1 , while rates of denitrification in the anoxic and transitional intervals reached up to 579 nmol cm −3 yr −1 (Fig. 3; Table 1).By comparison, maximum rates of nitrification estimated from O 2 and NO − 3 profiles in the South Pacific Gyre, perhaps the most organic matter depleted seafloor sediments in the world, were predicted to be only as high as 18-74 nmol cm −3 yr −1 (D' Hondt et al., 2009).In general nitrification rates in the oxic intervals near the seafloor were comparable to rates in the oxic layers near the underlying crust.The highest rates of denitrification typically coincided with depths having the lowest O 2 , with the exception of rather low rates near the central anoxic zone of core 2B.Nitrification, which requires O 2 , is observed as deep as 28 m in 2B and all the way to the underlying crust at the other two sites.Interestingly, modeled rates of nitrification were typically highest at comparatively low levels of dissolved O 2 (∼ 15µM in 2B, ∼ 10µM in 3D and ∼ 35µM in 4A) -suggesting an important role for micro-aerophilic nitrification.The depths of maximum denitrification rates generally coincided with the onset of O 2 levels below ∼ 2µM.In 2B, denitrification rates were highest at depths of ∼ 28 and ∼ 72 m (Fig. 3).In 3D, maximum denitrification rates were observed at 21 m -with similar rates between 20 and 25 m.Interestingly, the model indicated no denitrification within the 3 m anoxic zone of this core -suggesting limitation by organic substrate availability.In contrast, rates of denitrification were only estimated to occur within the anoxic zone of site 4A (e.g., not within the transitional intervals, although this was somewhat sensitive to prescribed 15 ε DNF , see below), with the highest rate of 24 nmol cm −3 yr −1 at a depth of 44 m.Overall, rates of both nitri-Figures

Back Close
Full 3 reduction (Fig. 1).Finally, the model suggests the co-occurrence of nitrification and denitrification (Fig. 3) in the transition zones of our model (depths at which O 2 is between 2 and 40 µM).Although denitrification rates generally did not exceed those of nitrification where the two processes are co-occurring (i.e., no net nitrate consumption), the increasing δ 15 N and δ 18 O of NO − 3 clearly reflects the influence of NO − 3 loss via denitrification.Indeed similar inferences have also been made about such overlap of nitrification and denitrification in the albeit much deeper and organic-rich hadopelagic sediments of the Ogasawara Trench (Nunoura et al., 2013).This observation illustrates the exceptionally extended vertical redox zonation of these sediments -and highlights the potential interaction between nitrogen transformations that are classically considered spatially explicit.

Model-predicted values of δ 15 N NTR : implications for N sources and processes in oligotrophic sediments
In general, where NH + 4 from remineralization does not accumulate -it is expected that the δ 15 N of NO than that expected based on the δ 15 N of sinking organic matter from the surface ocean of ∼ +3.7 ‰ (Altabet, 1988(Altabet, , 1989;;Knapp et al., 2005;Ren et al., 2012) Nitrogen isotope fractionation during organic matter remineralization has been reported (Altabet, 1988;Altabet et al., 1999;Estep and Macko, 1984;Lehmann et al., 2002), whereby the preferential remineralization of low δ 15 N organic matter leads to production of low δ 15 N NH + 4 (which could feed into the production of low δ 15 N NO − 3 ).The influence of this phenomenon is more likely, however, during remineralization of fresh organic matter and where the heterotrophic community has abundant access to highly labile proteinaceous organic matter (Altabet, 1988;Estep and Macko, 1984).
At North Pond, given the extremely low levels of organic material present in the sediments, it seems unlikely that preferential utilization of low δ 15 N organic material during diagenesis is responsible for the low δ 15 N NTR .
Competitive branching of NH + 4 supporting simultaneous nutritional supply (as an N source) and energy supply (via autotrophic ammonia oxidation) has been used to explain NO − 3 dual isotopic composition in N rich surface waters (Wankel et al., 2007).Because N isotope fractionation during ammonia oxidation was argued to be stronger than that of NH + 4 assimilation by phytoplankton under surface water conditions, it was argued that this competitive branching leads to a shunt of low δ 15 N into the NO − 3 pool via ammonia oxidation (Wankel et al., 2007).In contrast to the high-nutrient, sunlit, productive waters of Monterey Bay, however, under the energy-limited, and extremely low NH Although North Pond porewaters contain abundant NO − 3 , assimilation of NO − 3 as a nutritional N source requires an associated metabolic energy for reduction of NO − 3 (via an assimilatory nitrate reductase), a costly process in this energy poor environment.On the other hand, although NH + 4 is more easily assimilated by most microbes, its exceedingly low abundance in North Pond porewaters reflect its scarcity as source of N required for cell growth.Based on the estimated values of the N isotope effect for NO − 3 consumption (by denitrification) in the porewaters of 2B that average ∼ 20 ‰ (Table 2), there is little suggestion of NO − 3 assimilation, which would lead to much lower estimated values of 15 ε DNF (Granger et al., 2010).Thus, it is more likely that nutritional N originates from a reduced form such as NH + 4 and/or organic N. If the N isotope effect for ammonia oxidation is much larger than that of ammonia assimilation, then a competitive branching effect may also be contributing to a low δ 15 N NTR .However, given such low concentrations, it is likely that microbial acquisition of NH + 4 (whether for assimilation or for oxidation) is diffusion limited, conditions under which the N isotope effect is expected to be near 0 ‰ (Hoch et al., 1992).Under such conditions very little isotopic fractionation should be realized and competitive branching seems an unlikely explanation for the low δ 15 N NTR .
A final possibility for the low values of δ 15 N NTR could reflect the importance of benthic N-fixation operating at very low levels in North Pond sediments.Bacterial N-fixation is generally thought to result in biomass having a δ 15 N between −2 and 0 ‰ (Delwiche et al., 1979;Meador et al., 2007;Minigawa and Wada, 1986), which could effectively introduce new N and decrease bulk δ 15 N. Benthic N-fixation is not generally considered to be an important enough contribution to the total sediment organic matter to influence the bulk δ 15 N values of sediment organic matter.However, given the low organic matter flux to these sediments from the overlying oligotrophic surface waters, a proportionately smaller amount of N-fixation would be required to significantly impact the sediment organic δ 15 N value.While N-fixation is an energetically costly metabolism and might seem an unlikely strategy given the abundant porewater NO − 3 pool, it has been recently Introduction

Conclusions References
Tables Figures

Back Close
Full acknowledged that N-fixation in benthic environments may be widely underestimated, despite high levels of porewater DIN including NO − 3 and/or NH + 4 (Knapp, 2012).In fact, N-fixation could be ecologically favored in organic-lean sediments like those at North Pond owing to the formation of H 2 as an end product, which might afford an energetic advantage in sediment microbial communities.For example, H 2 production could help to fuel autotrophic metabolisms including both NO − 3 reduction (e.g., Nakagawa et al., 2005) or the Knallgas reaction (H 2 + O 2 ).Hydrogen-based metabolism has been proposed as a significant support for subsurface autotrophy underlying the oligotrophic South Pacific Gyre (D'Hondt et al., 2009).Although the involvement of so-called alternative nitrogenases (the Fe and V forms), which have been shown to display an even larger kinetic isotope effect (−6 to −7 ‰) than the Mo-bearing form (Zhang et al., 2014), could offer even greater leverage on lowering of the bulk δ 15 N (and source of N for nitrification), their involvement in non-sulfidic marine systems, where Mo is replete and soluble Fe and V is scarce, is expected to be minimal (Zhang et al., 2014).A similar argument was also made for the cryptic involvement of N fixation as source of low δ 15 N and explanation for dual NO − 3 isotopic patterns in the large oxygen minimum zone of eastern tropical North Pacific (Sigman et al., 2005).Thus, we conclude that the low predicted values of δ 15 N NTR provide compelling evidence for an important role of in situ N-fixation in these organic matter depleted sediments.
Finally, there is a conspicuous increase in the predicted δ 15 N NTR at sites 2B and 3D (up to +1.8 and +1.1 ‰, respectively) between 15-35 m.Although we have no compelling explanation for these observations, it is interesting that these values coincide with the transitional intervals over which δ 18 O NO 3 values increase much more rapidly than δ 15 N NO 3 .While it is possible that our model is insufficient for constraining the isotope dynamics at NP, it may also be that these suboxic depths support differential amounts of in situ nitrogen fixation leading to shifts in the bulk δ 15 N available for oxidation by nitrification.Introduction

Conclusions References
Tables Figures

Back Close
Full  3) the potential influence of oxygen isotope equilibration between water and NO − 2 (both abiotic and/or that catalyzed by activity of microbial nitrifying bacteria) (Casciotti et al., 2010).
In the upper profile of site 2B and throughout the profile of site 4A predicted values of δ 18 O NTR clustered between −2.8 and 0.0 % with no clear trends related to down core concentrations of O 2 or NO − 3 (Fig. 5).Although slightly lower, this range of values agrees remarkably well with values predicted by experiments using a co-culture of NH  (Buchwald et al., 2012;Casciotti et al., 2010).The resulting δ 18 O NTR can be described as: and NO − 2 oxidation to NO − 3 ( 18 ε H 2 O,2 ) (Buchwald et al., 2012).While the value of seawater δ 18 O can be considered to be relatively constant (∼ 0 ‰) in North Pond porewaters, the respiratory consumption of O 2 , as evident in the observed concentration profiles, imparts a relatively strong isotopic fractionation (Bender, 1990;Kroopnick and Craig, 1976)   than the time required for complete equilibration between NO − 2 and water (Buchwald and Casciotti, 2013).In contrast, the low O 2 interval from site 4A does not exhibit elevated δ 18 O NTR values near the oxygen minimum, perhaps suggesting that the turnover of NO − 2 here is slower (allowing complete equilibration) or that equilibration is biologically catalyzed.Although the concentrations of the NO − 2 pool were generally below detection, making the accurate determination of its turnover time impossible (via δ 18 O), the use of NO − 2 oxygen isotopes as an independent measure of metabolic processes where concentrations persist at measurable levels may be a potentially powerful indicator of biological turnover of NO − 2 (δ 15 N and δ 18 O of NO − 2 in 2B, where NO − 2 was detected at two depths, were not determined as part of this study).Future studies should target this pool as a complementary dimension for constraining subsurface biosphere metabolic rates.

Model sensitivity to prescribed 15 ε DNF in transitional intervals
In the transitional intervals -where both nitrification and denitrification are allowed to co-occur, the model is underdetermined and requires some variables to be prescribed.
We chose to prescribe a value for the kinetic isotope effect of denitrification ( 15 ε DNF ) and here examine the sensitivity of estimated rates nitrification and denitrification, as well as predicted values of δ 15 N NTR and δ 18 O NTR .Given the rather tightly confined range of determined values for 15 ε DNF in the anoxic zone of site 2B, averaging 20 ± 1.8 ‰, for illustration, we bracket our prescribed 15 ε DNF in transitional intervals with values of 15 and 25 ‰ (rate estimates where the prescribed 15 ε DNF is as low as 5 ‰ are given in Table 1).Overall, the model predicted rates of nitrification and denitrification, as well as values of δ 15 N NTR and δ 18 O NTR were largely insensitive to changes in the prescribed strength of the isotope effect for denitrification ( 15 ε DNF ) (Fig. 3).Specifically, when the prescribed value of 15 ε DNF decreased from 25 to 15 ‰, changes in the predicted values of δ 15 N NTR and δ 18 O NTR were generally small (Figs.respectively.An exception to this are the intervals bracketing the anoxic zone of the profile at site 2B (at depths of 27.9 and 70.8, 72.9 m), which yielded predicted δ 15 N NTR that were either 2.1 ‰ lower (at 27.9 m) or ∼ 5 ‰ higher (at 70.8 and 72.9 m).Predicted δ 18 O NTR values were also quite sensitive to 15 ε DNF in this interval with values that were 3.8 ‰ (at 27.9 m) and 7.2-8.0‰ higher (at 70.8 and 72.9 m).While we cannot rule out the potential influence of changes in physiological expression of isotope effects, the sensitivity of δ 15 N NTR and δ 18 O NTR to 15 ε DNF at these depths may point to an unresolvable artifact of this model approach.(Granger et al., 2008;Wada et al., 1975).While we have no direct evidence that such low values would be relevant in our study, we report the sensitivity of rate estimates and δ 15 N NTR and δ 18 O NTR (Table 1).In short, a prescribed value for 15 ε DNF of 5 ‰ lead to increased estimates of δ 15 N NTR and δ 18 O NTR by an average of 2.3 and 1.4 ‰, respectively (Figs. 4 and 5).These higher estimates of δ 15 N NTR would implicitly require a lower contribution of N-fixation derived nitrogen as argued for above, though not eliminate its role completely, especially in 4A where δ 15 N NTR values remain between −1 to +1 ‰ (Fig. 4).While rates of nitrification were less sensitive (somewhat higher in the upper layers of 4A), this very low prescribed value of 15 ε DNF often lead to dramatically increased estimates of denitrification rates -in particular in the upper transitional layers of profiles at 2B and 3D where maximum denitrification rates increased ∼ 10-20 fold (Table 1).This In the anoxic intervals, estimated values of 18 ε: 15 ε DNF ranged from 0.83 to 1.11 with an average value of 0.99 ± 0.1 (Table 2), consistent with a prominent role of respiratory nitrate reductase (Nar), which imparts a 18 ε: 15 ε DNF of ∼ 0.96 ± 0.01 (Granger et al., 2008).Notably, however, the lower values of 0.86 and 0.83 observed near the top and the core of the anoxic zone in site 2B could suggest influence of nitrate reduction by periplasmic nitrate reductase (NAP) (Granger et al., 2008) or chemolithotrophic NO − 3 reduction (Frey et al., 2014;Wenk et al., 2014), which has been shown to impart a lower 18 ε: 15 ε DNF closer to 0.6.In this particular interval, this could suggest that as much as 43 % of nitrate reduction is chemolithotrophic and perhaps metabolically linked to the oxidation of inorganic substrates such as reduced iron or sulfur species.Although outside the scope of this study, interrogation of genetic markers of respiratory and periplasmic nitrate reductase could shed more light on the role nitrate use by subsurface microbial communities.
As discussed above, the model-estimated values of 15 ε DNF (averaging 20.0 ± 1.8 ‰; Table 2) at site 2B are quite consistent with values from a wide range of studies (Granger et al., 2008), and references therein).Notably however, a different pattern emerges from the two anoxic intervals of site 4A.Although model-estimated values of 15 ε DNF were unresolvable at 38.8 m (likely because the changes in δ 15 N and δ 18 O were too small for reliable model fits), estimated 15 ε DNF values at 44.1 m were ∼ 8.1 ± 0.4 ‰, much lower than observed in 2B.In general, the values observed in 2B are consistent with observations from other environments hosting denitrification (e.g., OMZs, soils, groundwater aquifers, etc.) (Granger et al., 2008), and references therein), and suggest that denitrifying organisms may be adapted to low levels of carbon and that their physiological poise may be similar to those found in other anaerobic environments (albeit adapted to grow at exceedingly slow nitrate reduction rates).However, the lower estimated 15 ε DNF values in 4A might also reflect something more.Given the apparent low reactivity of the sediments of site 4A, it is also possible that these particularly low Introduction

Conclusions References
Tables Figures

Back Close
Full While a number of studies have shown that the apparent N isotopic effect for nitrate reduction by denitrification can vary from 5 to 30 ‰ (e.g., Barford et al., 1999;Delwiche and Steyn, 1970;Granger et al., 2008), recent evidence suggests these variations are largely regulated by changes in the combination of cellular uptake and efflux of NO − 3 leading to the expression (or repression) of the enzyme level isotope effect outside the cell (Granger et al., 2008;Kritee et al., 2012;Needoba et al., 2004).For example, at low extracellular NO − 3 concentrations -low 15 ε DNF values suggest that nitrate transport (having a low 15 ε) becomes the rate-limiting step (Granger et al., 2008;Lehmann et al., 2007;Shearer et al., 1991).In North Pond porewaters, however, at depths where O 2 is low enough for denitrification to occur, NO − 3 concentrations remain well above 30 µM, a threshold well above the K m for NO − 3 transporters (2-18 µM; Parsonage et al., 1985;Murray et al., 1989;Zumft, 1997), suggesting that low 15 ε DNF due to transport limitation of NO − 3 reduction is unlikely.
In general, greater expression of the intrinsic enzymatic isotope effect (e.g., higher observed 15 ε DNF ) should occur under conditions in which there is a higher efflux of intracellular NO − 3 relative to NO − 3 uptake (Kritee et al., 2012).Interestingly, this efflux/uptake ratio appears to be linked to nitrate reduction rates in denitrifying bacteria, with lower cell-specific nitrate reduction rates leading to lower efflux/uptake ratios and lower observed cellular level 15 ε DNF (Kritee et al., 2012).Indeed, evidence seems to indicate that this efflux/uptake ratio in denitrifying bacteria is highly regulated and that NO − 3 uptake is sensitive to cellular level energy supply.For example, under conditions in which organisms are required to maintain a careful regulation of energetically costly metabolic processes, it is logical that there would be a lower density of NO − driven decrease in NO − 3 uptake, lower intracellular NO − 3 concentrations and a lower efflux/uptake ratio.
We suggest that the difference between the lower 15 ε DNF value estimated from the anoxic interval of 4A and the more "conventional" values from deeper within anoxic intervals of 2B could stem from physiological-level controls on the cellular level expression of 15 ε DNF .Specifically, as all porewater evidence from site 4A (O 2 , NO − 3 , N and O isotopes) indicates substantially lower levels of microbial activity, denitrification may actually be more energy-limited by carbon (compared to denitrification in the deeper intervals of 2B).This suggests that the operation of denitrification under extremely carbon-poor environments (4A) may lead to conditions where the enzyme-level N isotope fractionation of denitrification is under-expressed on the cellular, and hence ecosystem level, and 15 ε DNF values are much lower than commonly encountered under even just slightly more energy-replete conditions (e.g., 2B).

Summary
In summary, the porewater nitrate isotopic composition reflects the active redox cycling of nitrogen by the subsurface microbial community -including both oxidative and reductive transformations.The variations in reaction rates across and within the three North Pond sites are generally consistent with the distribution of dissolved oxygen, but not necessarily with the canonical view of how redox thresholds act to spatially separate nitrate regeneration from dissimilatory consumption (e.g., denitrification).The were elevated, suggesting incorporation of 18 O-enriched dissolved oxygen during the nitrification process, and implying relatively rapid rates of nitrite turnover in environments supporting nitrification.In contrast, the accumulation of NO − 2 under denitrifying conditions likely reflects limitation of NO − 2 by organic matter availability and generally low rates of N based heterotrophic respiration.Importantly, our findings indicate that the production of organic matter by in situ autotrophy (e.g., nitrification and nitrogen fixation) must supply a large fraction of the biomass and organic substrate for heterotrophy in these sediments, supplementing the small organic matter pool derived from the overlying euphotic zone.Thus, this work sheds new light on an active nitrogen cycle operating, despite exceedingly low carbon inputs, in the deep sedimentary biosphere underlying half of the global ocean.Introduction

Conclusions References
Tables Figures

Back Close
Full  Full Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

−
3 concentration data from North Pond demonstrate an accumulation of NO − 3 with depth (Ziebis et al., 2012) implicating the role of in situ productivity supported by the autotrophic oxidation of ammonium and nitrite (e.g., nitrification).To what degree this NO − 3 pool supports other subsurface microbial communities as an electron acceptor, however, remains unclear.In addition, the supply of dissolved substrates (O 2 , NO − 3 , DOC) from the underlying crustal aquifer may play a primary role in supporting these deep sediment communities.This geochemical exchange among crust, ocean and sediments across vast reaches of the seafloor, and its link to subsurface microbial activity, underscores its potential importance in global biogeochemical cyclesDiscussion Paper | Discussion Paper | Discussion Paper | described extensively elsewhere (Expedition-Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | reflecting the production of NO − 3 by nitrification and the net flux of NO − 3 to the overlying water from this site of ∼ 4.6 µmoles m −2 d −1 (Ziebis et al., 2012), consistent with other studies of NO − 3 fluxes in pelagic deep-sea sediments 38.2, 42.2 and 49.1 µM at depths of 19.1, 23.0 and 56.3 mbsf in the cores from sites 2B, 3D and 4A, respectively, -depths that generally coincided with O 2 concentrations below 10 µM.Nitrite was below detection at sites 3D Discussion Paper | Discussion Paper | Discussion Paper | In 2B, sampling points near the most O 2 depleted depths and the lower portion of the profile fell closer to the expected 1 : 1 line for NO − Discussion Paper | Discussion Paper | Discussion Paper |

−
3 within porewaters.Under low oxygen, NO − 3 respiration by denitrification leads to a well-characterized increase in both δ 15 N and δ 18 O in conjunction with decreasing NO − 3 concentrations.In contrast, nitrification produces NO − 3 with a δ 15 N equal to the starting NH + 4 (when accumulation of NH + 4 and NO − 2 is negligible), while the δ 18 O of the newly produced NO − 3 is related to the δ 18 O of ambient H 2 O and O 2 , as well as kinetic and equilibrium isotope effects associated with the stepwise oxidation of NH + Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ( 15 ε DNF and 18 ε DNF : 15 ε DNF , respectively), as well as the N and O isotopic composition of new NO − 3 produced by nitrification (δ 15 N NTR and δ 18 O NTR , respectively).Uncertainty in model-estimates is expressed as the standard error of 10 model-run estimates (Table Discussion Paper | Discussion Paper | Discussion Paper |

−
3 adjusted for porosity, DNF and NTR refers to the reaction rate of denitrification or nitrification, respectively (in mass volume −1 time −1 ), C refers to the concentration of each isotopologue (in mass volume −1 ) and k refers to the first order rate constant (time −1 ).The fractionation factor, α, is defined as the ratio of rate constants for the light isotope over the heavy isotope (e.g., 15 α= 14 k/ 15 k) for a given process, alternatively expressed in terms of epsilon where ε = (α − 1) × 1000 in units of permil ( ‰).The term 18 ε: 15 ε DNF refers to the degree of coupling between the N and O isotope fractionation during denitrification, with a value of 1 indicating the two isotope effects are identical.For the δ 15 N and δ 18 O of nitrification (NTR) NTR14 N = NTR × f14 NNTR (7a) NTR15 N = NTR × f15 NNTR (7b) NTR16 O = NTR × f16 ONTR (7c) NTR18 O = NTR × f18 ONTR (7dDiscussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

+
4 being oxidized to NO − 3 , which is related to the δ 15 N of the organic matter being remineralized.The δ 18 O NTR stems from a combination of factors including the δ 18 O of the water and dissolved O 2 as well as the expression of kinetic isotope effects associated with the incorporation of O atoms from these pools Discussion Paper | Discussion Paper | Discussion Paper | (and kinetically) favorable metabolic processes (e.g., O 2 consumption precedes NO Discussion Paper | Discussion Paper | Discussion Paper | depth ranges, and, depending on sediment thickness and organic carbon content -the redox state of these sediments may never reach the potential for NO − 3 reduction to play a role as a thermodynamically viable metabolic pathway.While it is not necessarily apparent whether any NO − 3 respiration is occurring based on concentration profiles alone, dramatic increases in the δ 15 N and δ 18 O of NO − 3 with depth into the anoxic sediment intervals were observed in both 2B and 3D (Fig.1)indicating isotope fractionation by denitrification.The highest δ 15 N and δ 18 O values in 2B (+22.2 and +21.8 ‰, respectively) generally coincide with the lowest dissolved O 2 , while highest δ 15 N and δ 18 O values in 3D (+11.8 and +19.7 ‰, respectively) fall just below the depth of lowest O 2 .In stark contrast, 4A porewaters exhibit only a minor increase in δ 18 O of ∼ 2.7 ‰ within the anoxic interval, while δ 15 N increased by only 0.8 ‰ (Fig. 1).The stark distinction between 2B/3D and 4A notwithstanding, the dual Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | fication and denitrification were highest at site 2B and lowest in 4A, consistent with the putatively greater amount of microbial activity revealed by the sharper O 2 profile 2B.In all three cores maximum rates of nitrification exceeded those of denitrification, consistent with the net accumulation of NO − 3 throughout the sediment column.Even in 2B, where O 2 is below 2 µM over an interval of ∼ 40 m, the NO − 3 concentration profile exhibits no obvious influence by NO − remineralization of organic nitrogen.The δ 15 N of organic N of the North Pond sediments was not quantified in this study (concentrations are extremely low).Yet, the average model predicted δ 15 N of newly produced NO − 3 (δ 15 N NTR ) (Fig. 4) ranged from −3.1 to +1.1 ‰ (standard error typically ±0.3 to 0.4 ‰), generally lower Discussion Paper | Discussion Paper | Discussion Paper | δ 15 N NTR > +1 ‰ were only observed just above the O 2 minimum at sites 2B and 3D (Fig. 4).Confronted with this difference, we turn to other factors that might play a role in setting the δ 15 N NTR .The lower δ 15 N values of new NO − 3 can potentially be explained by a number of possible processes including: (1) isotopic fractionation during remineralization, (2) competitive branching between NH + 4 oxidation (whether anaerobic or aerobic) and NH + 4 assimilation or (3) contribution of low δ 15 N through organic matter derived from sedimentary N fixation.
+ 4 production conditions in North Pond porewaters mechanisms of NH + 4 partitioning are likely operating quite differently.Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | predicted values of the δ 18 O of nitrification (δ 18 O NTR ) We also observed variation in estimated values of the δ 18 O of newly produced NO − 3 (δ 18 O NTR ), ranging from −2.8 to as high as +4.1 ‰ (at the O 2 minimum in 3D), which may offer some insight into the nature of nitrification in these sediments and the deep ocean in general.The oxygen isotope composition of newly produced NO − 3 reflects the combination of several complex factors including (1) the δ 18 O of the ambient water and dissolved O 2 , (2) kinetic isotope effects during the enzymatically catalyzed incorporation of O atoms during oxidation of NH + , which ranged from −1.5 to +1.3 ‰(Buchwald et al., 2012).In systems where NH + 4 and NO − 2 oxidizing bacteria co-exist and are not substrate-limited, NO − 2 does not generally accumulate and the importance of oxygen isotope equilibration between NO − 2 and water can be considered minor (∼ 3 %) (Buchwald et al., 2012).In this case, the δ 18 O NTR is primarily set by the δ 18 O of water (seawater δ 18 O ∼ 0 ‰) and dissolved O 2 (∼ +26.4 ‰ for the deep N. Atlantic; Kroopnick et al., 1972) and the three kinetic isotope effects during the sequential oxidation of NH + 4 to NO − 3 Discussion Paper | Discussion Paper | Discussion Paper | where 18 ε is the kinetic isotope effect of O atom incorporation from O 2 during NH + 4 oxidation to NH 2 OH ( 18 ε O 2 ), and from water during NH 2 OH oxidation to NO

+4. 1
‰ at the four intervals coinciding with the maximum O 2 drawdown (Fig.5), and thus may point to incorporation of high-δ 18 O O 2 .For example, assuming a δ 18 O value of 0 ‰ for seawater, using Eq.(11) and a combined isotope effect of 18 ‰ for the twosteps of NH + 4 oxidation to NO − 2 ( 18 ε O 2 + 18 ε H 2 O,1 ; the two have not yet been resolved from one another; Casciotti et al., 2010) and a value of 15 ‰ for 18 ε H 2 O,2 (O atom incorporation during NO − 2 oxidation to NO − 3 , Buchwald et al., 2012), δ 18 O NTR values of −2, +2 or +6 ‰ would imply incorporation of O from an O 2 pool with a value of ∼ +45, +57 or +69 ‰, respectively.If these higher values are indeed the result of enriched O 2 incorporation, then they also provide indirect information on the degree of oxygen isotope equilibration occurring between NO − 2 and water.Specifically, if some proportion of an elevated δ 18 O O 2 signal is propagated into the NO − 3 pool, then this suggests that the intermediate NO − Discussion Paper | Discussion Paper | Discussion Paper | 4 and 5), varying by a maximum of 0.9 ‰ (average 0.5 ‰) and 2.3 ‰ (difference 0.6 ‰)Discussion Paper | Discussion Paper | Discussion Paper | sensitivity of denitrification to 15 ε DNF values makes intuitive sense, since more denitrification would be required to generate the strongly elevated δ 15 N and δ 18 O values observed in 2B and 3D.predicted values of 18 ε: 15 ε DNF and 15 ε DNF Discussion Paper | Discussion Paper | Discussion Paper | 15 ε DNF values stem from denitrification operating under extreme physiological energy limitation -as discussed below.
Discussion Paper | Discussion Paper | Discussion Paper | incorporation of nitrate dual isotopes into an inverse reaction-diffusion model provides evidence for extensive zones of overlap where O 2 and NO − 3 respiration (nitrification and denitrification) co-occur.The isotope modeling also yielded estimates for the δ 15 N and δ 18 O of newly produced nitrate (δ 15 N NTR and δ 18 O NTR ), as well as the isotope effect for denitrification ( 15 ε DNF ), parameters with high relevance to global ocean models of N cycling.Estimated values of δ 15 N NTR were generally lower than previously reported Introduction Discussion Paper | Discussion Paper | Discussion Paper | δ 15 N values for sinking PON in this region, suggesting the potential influence of sedimentary N-fixation and remineralization/oxidation of the newly fixed organic N. Model estimated values of δ 18 O NTR generally ranged between −2.8 and 0.0 ‰, consistent with lab studies of nitrifying bacteria cultures.Notably, however, some δ 18 O NTR values Discussion Paper | Discussion Paper | Discussion Paper | Biosphere Investigations (C-DEBI) to S. D. Wankel and W. Ziebis and a postdoc fellowship to C. Buchwald from C-DEBI.This is a contribution from the Center for Dark Energy Biosphere InvestigationsDiscussion Paper | Discussion Paper | Discussion Paper | Nunoura, T., Nishizawa, M., Kikuchi, T., Tsubouchi, T., Hirai, M., Koide, O., Miyazaki, J., Hirayama, H., Koba, K., and Takai, K.: Molecular biological and isotopic biogeochemical prognoses of the nitrification-driven dynamic microbial nitrogen cycle in hadopelagic sediments, Environ.Microbiol., 15, 3087-3107, doi:10.1111/1462-2920.12152,2013.Orcutt, B. N., Sylvan, J. B., Knab, N. J., and Edwards, K. J.: Microbial ecology of the dark ocean Discussion Paper | Discussion Paper | Discussion Paper | Ziebis, W., McManus, J., Ferdelman, T., Schmidt-Schierhorn, F., Bach, W., Muratli, J., Edwards, K. J., and Villinger, H.: Interstitial fluid chemistry of sediments underlying the North Atlantic Gyre and the influence of subsurface fluid flow, Earth Planet.Sc.Lett., 323-324, 79-91, 2012.Zumft, W. G.: Cell biology and molecular basis of denitrification, Microbiol Mol.Biol.R., 61Discussion Paper | Discussion Paper | Discussion Paper |

Figure 1 .
Figure 1.Depth profiles from IODP sites U1382B, U1383D and U1384A at North Pond of porewater concentrations of O 2 (from Orcutt et al., 2013) and NO − 3 as well as the N and O isotopic composition of NO − 3 .(δ 15 N NO 3 and δ 18 O − NO 3).The red circle at the top of the profiles

Figure 2 .Figure 3 .Figure 4 .Figure 5 .
Figure 2. Dual isotope plot illustrating the relationship between δ 15 N NO 3 and δ 18 O NO 3 in North Pond porewaters.The diagonal line, rooted at a value for bottom seawater (δ 15 N of +5.5 ‰ and δ 18 O of +1.8 ‰), depicts a 1 : 1 slope representative of the expected change in δ 15 N and δ 18 O by the process of denitrification alone.Trends falling well above this 1 : 1 line, together with the concentration profiles reflect the combined role of nitrification in these porewaters.

− 3 and NO − 2 concentration profiles
and will cause elevated δ 18 O O 2 values.Using a separate reaction-diffusion model (not shown) we estimated the δ 18 O O 2 to be as high as +70 ‰ where concentrations of O 2 have been drawn down > 95 % of the level found in bottom seawater.Incorporation of this highly 18 O-enriched O 2 by nitrification in these low O 2 intervals may contribute to observed increases in δ 18 O NTR predicted by our model.In particular, in the low O 2 intervals of 2B and 3D, δ 18 O NTR values as high as Further work being indicated, incorporation of dual nitrite isotopes could certainly aid in resolving this apparent sensitivity.However, this sensitivity was not observed in the other transitional intervals of 2B, 3D or 4A and conclusions regarding δ 15 N NTR and δ 18 O NTR still appear robust.