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Keywords:

  • Juan de Fuca Ridge;
  • Northeast Pacific Ocean;
  • hydrothermal vent fluids;
  • nitrate and ammonium isotopes;
  • nitrogen cycle;
  • subsurface biosphere

Abstract

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Material and Methods
  5. 3. Results and Discussion
  6. 4. Summary and Concluding Remarks
  7. Acknowledgments
  8. References
  9. Supporting Information

[1] Little is known about dissolved inorganic nitrogen (DIN) transformations in hydrothermal vent (HV) fluids. Here, we present the first isotopic measurements of nitrate (δ15N and δ18O) and ammonium (δ15N) from three HV fields on the Juan de Fuca ridge (NE-Pacific). The dominant process that drives DIN concentration variations in low-T diffuse fluids is water mass mixing below the seafloor, with no effect on the DIN isotope ratios. Strong inter-site variations in the concentration andδ15N of NH4+in high-T fluids suggest different subsurface nitrogen (N) sources (deep-sea nitrate versus organic sediments) for hydrothermally discharged ammonium. Low NH4+ community N isotope effects (<3‰) for net NH4+consumption suggest an important contribution from gross ammonium regeneration in low-T fluids. Elevation of HV nitrate15N/14N and 18O/16O over deep-sea mean isotope values at some sites, concomitant with decreased nitrate concentrations, indicate assimilatory or dissimilatory nitrate consumption by bacteria in the subsurface, with relatively low community N isotope effects (15εk < 3‰). The low N isotope effects suggest that nitrate assimilation or denitrification occur in bacterial mats, and/or in situ production of low δ15N nitrate. A significantly stronger relative increase for nitrate δ18O than for δ15N was observed at many sites, resulting in marked deviations from the 1:1 relationship for nitrate δ15N versus δ18O that is expected for nitrate reduction in marine settings. Simple box-model calculation show that the observed un-coupling of N and O nitrate isotope ratios is consistent with nitrate regeneration by either nitrite reoxidation and/or partial nitrification of hydrothermal ammonium (possibly originating from N2 fixation). Our isotope data confirm the role of subsurface microbial communities in modulating hydrothermal fluxes to the deep ocean.

1. Introduction

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Material and Methods
  5. 3. Results and Discussion
  6. 4. Summary and Concluding Remarks
  7. Acknowledgments
  8. References
  9. Supporting Information

[2] The importance of low-temperature hydrothermal vents (HV) as a habitat for microbes [Jannasch and Mottl, 1985; Juniper et al., 1995; Huber et al., 2002, 2003; Butterfield et al., 2004; Mehta et al., 2003, 2005; Mehta and Baross, 2006] and macro-fauna [Tunnicliffe et al., 1985; Tunnicliffe, 1991] is well documented. These oases of life harbor high-biomass faunal communities that are sustained by chemoautotrophic bacteria (free-living or in symbiosis) at the base of the food chain, which gain metabolic energy from the oxidation of reducing substances in hydrothermal fluids, particularly hydrogen sulphide.

[3] Nitrogen (N) is an essential nutrient for all vent organisms, as a building block of proteins, but also as a possible energy source. Generally high levels of NH4+in high-T fluids (up to ∼350°C) and NO3in low-T diffuse fluids (<∼50°C) are discharged from HV, serving as potentially important substrate and N source for the chemolithotrophic bacteria [Butterfield et al., 1997; Lam et al., 2004]. Additional bioavailable N to fulfill the high N requirements of dense bacterial populations in HV systems may be procured through the fixation of molecular N even at temperatures close to the limits of life [Mehta et al., 2003, 2005; Mehta and Baross, 2006]. On the other hand, denitrification, i.e., the nitrate reduction by chemosynthetic CO2 fixing bacteria (or other suboxic modes of N2 production) may remove NOxfrom low-temperature diffuse fluids [Butterfield et al., 2004]. For example, it has been shown that sulphur-oxidizing bacteria can use nitrate as an electron acceptor for the oxidation of hydrogen sulphide in HV systems [e.g.,Hentschel and Felbeck, 1993; McHatton et al., 1996]. Moreover, Byrne et al. [2009] provided phylogenetic and biomarker evidence for the anaerobic oxidation of ammonium (anammox) to N2gas in deep-sea hydrothermal vents at the Mid-Atlantic Ridge. Anammox can account for a substantial loss of fixed N in various marine environments [e.g.,Thamdrup and Dalsgaard, 2002; Dalsgaard et al., 2003; Kuypers et al., 2003, 2005; Hamersley et al., 2007].

[4] The stable N isotope composition of vent organisms has previously been measured to study feeding ecology and trophic structure in HV systems. In earlier work, very low δ15N (for definition see section 2) values (∼−10 to +4‰) of HV organisms, for example, have been attributed to strong N isotope fractionation during chemosynthetic assimilation of ammonium or the local supply of 15N-depleted ammonium [Rau, 1981; Paull et al., 1985; Van Dover and Fry, 1994; Kennicutt and Burke, 1992; Van Dover, 2002].

[5] As a result of specific isotope effects associated with N cycle transformation, N (and nitrate O) isotope ratios represent a useful tool to assess nitrogen fluxes within the ocean. N2 fixation, the major input of bioavailable N to the ocean, adds new N with a δ15N of ∼−2–0‰ [Carpenter et al., 1997, 1999; Montoya et al., 2002]. The low-δ15N organic nitrogen is subsequently remineralized to dissolved inorganic N (DIN) through coupled ammonification and nitrification, thus leaving clear N-isotopic signatures in the DIN pool [Sigman et al., 2005; Knapp et al., 2008; Bourbonnais et al., 2009]. Water column denitrification in suboxic zones displays a strong kinetic N-isotope effect (ε) on the NO3 δ15N [Cline and Kaplan, 1975; Brandes et al., 1998b; Voss et al., 2001; Lehmann et al., 2003; Sigman et al., 2003, 2005], with values for εin the range of 15–30‰. Nitrate assimilation by algae or bacteria usually fractionates the N isotopes to a lesser extent, with most N-isotope effects reported for natural marine systems clustering around 5‰ [e.g.,Altabet, 2001]. While N isotopes alone can provide qualitative information on N-cycle transformations, their use is often limited because they do not allow for the quantitative separation of nitrate production and consumption. For example, the N isotope signals of N2 fixation and denitrification can negatively interfere with each other. The coupled measurement of the nitrate N and O isotopic composition has the potential to disentangle nitrate consumption and production processes in environments where they occur simultaneously (or where water masses with the respective N isotope signatures are mixed together). The separation of nitrate consumption from production is possible because, in contrast to denitrification and nitrate assimilation, with a constant ratio of N versus O isotope enrichment of 1:1 in marine settings [Granger et al., 2004, 2008; Lehmann et al., 2005; Sigman et al., 2005], nitrate production affects the δ15N and δ18O of new nitrate in fundamentally different ways [Lehmann et al., 2003, 2004; Sigman et al., 2005; Wankel et al., 2007; Bourbonnais et al., 2009]. The δ15N of new nitrate is determined by the δ15N of the organic matter being remineralized [Sigman and Casciotti, 2001]. According to current understanding, oxygen isotopes in nitrified nitrate or from nitrite re-oxidation are mostly derived from oxygen in ambient water (δ18O ∼ 0‰) and to a minor extent from dissolved oxygen (δ18O between 23.5‰ and 40‰ in the ocean interior [Bender, 1990; Levine et al., 2009]) [Andersson and Hooper, 1983; Kumar et al., 1983; DiSpirito and Hooper, 1986; Casciotti, 2002; Sigman et al., 2009]. Hence, the δ18O of newly generated nitrate is insensitive to the origin of the nitrogen being nitrified, be it from N2 fixing organisms, from other bacterial/algal biomass, or from denitrified N (nitrite). While observational data generally suggest that newly regenerated marine nitrate has a δ18O close to that of seawater (i.e., ∼1.4‰ [Sigman et al., 2009]), Casciotti et al. [2010] and Buchwald and Casciotti [2010] recently reported that oxygen atom incorporation during ammonium and nitrite oxidation occurs with significant isotope effects.

[6] As outlined above, work over the last two decades has provided (mostly qualitative) evidence for multiple N-cycle reactions in HV systems. Nevertheless, the fate of ammonium that is hydrothermally injected into the water column, and water column nitrate that is mixed into anaerobic hydrothermal vent fluids, is still uncertain. N isotope data for DIN in the high-T fluids barely exist, and the effects N-transformations in the diffuse fluids and HV plumes can have on the subsurface DINδ15N, prior to incorporation into the food chain, are unknown. HV ecosystems are likely to display unique DIN isotope signatures that may elucidate principal patterns of N (and O) isotope dynamics. In turn, the isotopic ratios of ammonium and nitrate should inform about the activity of ammonium-oxidizing and denitrifying organisms in this ecosystem. Also, in order to successfully use stable N isotopes for determining trophic structures in an ecosystem, it is crucial to know the base-lineδ15N, i.e., the δ15N of the inorganic substrate that becomes metabolized by the chemosynthetic bacteria at the base of the food chain.

[7] The main goal of this paper is to discuss, for the first time, the physical and biological processes that affect the concentrations and isotope composition of DIN (nitrate δ15N and δ18O and ammonium δ15N) in both high and low-T fluids at the Endeavor and Cobb Segments, and Axial Volcano on the Juan de Fuca Ridge (NE-Pacific). We see evidence for DIN isotope fractionation due to N consumption by assimilative N-uptake and denitrification in microbial mats/biofilms within the hydrothermal conduits. We also report on previously unseen nitrate N-to-O isotope anomalies in the HV fluids, and we present our results in light of a simple isotope box-model, which allows us to interpret the nitrate isotope anomalies as evidence for nitrate regeneration through a combination of nitrification and nitrite reoxidation processes.

2. Material and Methods

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Material and Methods
  5. 3. Results and Discussion
  6. 4. Summary and Concluding Remarks
  7. Acknowledgments
  8. References
  9. Supporting Information

2.1. Site Description and Sample Collection

[8] Hydrothermal vent fluids were sampled at different sites on Axial Volcano, and the Cobb and Endeavour Segments during five summer/autumn cruises between 2004 and 2009 onboard the R/V Thomas G. Thompson and R/V Atlantis, as part of the New Millennium Observatory (NEMO) and Endeavor-Axial Geochemistry and Ecology Research (EAGER) Projects (Figure 1a). The Endeavor and Cobb Segments are located at ∼48°N, 129°W and ∼46.7°N, 129.4°W, respectively (Figure 1a). Four different vent fields were visited at Endeavour Segment: Sasquatch, High Rise, Main Endeavour Segment (MEF) and Mothra (Figure 1b). The vents are located at a depth of ∼2200–2100 m. Even though Endeavour Segment is sediment-free, it is believed that high concentrations of methane and ammonium observed in hydrothermal fluids originate from the decomposition of organic matter buried at an early stage of the ridge segment's evolution [Lilley et al., 1993]. Cobb Segment was visited only in 2007 during an exploration dive. The temperature of high-T vent fluids at Cobb Segment is generally lower than at Endeavor, and venting is less vigorous. Axial Volcano (∼46°N, 130°W) rises 1100 m above the surrounding ocean floor and has a 3-sided caldera. Active vent sites are located along the caldera boundary fault and along the intersection of the north and south rift zone with the caldera boundary fault at depths of 1500 to 1540 m below sea level [Embley et al., 1990; Butterfield et al., 2004]. The latest eruptions occurred in the Southeast portion of the caldera in 1998 and more recently in 2011 (Figure 1c).

image

Figure 1. (a) Map of Axial Volcano and the Endeavor and Cobb Segments on the Juan de Fuca Ridge. (b) Endeavour Segment and the four major hydrothermal vent fields sampled (Sasquatch, High Rise, Main Endeavor Field and Mothra); (c) the summit caldera of Axial Volcano on the Juan de Fuca Ridge with enlarged view or the Southeast portion of the caldera, where an eruption occurred in 1998. The curved dashed line represents the caldera boundary fault where active vent sites are located (modified from Plate 1 in the study by Butterfield et al. [2004]). Black dots represent sampled vents. Only vents discussed in the text or in figures are labeled.

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[9] Low- and high-T fluids were collected using titanium syringe major samplers (760 ml), collapsible plastic bags with valves (up to 800 ml), and PVC piston samplers with Teflon spring seals on the Hydrothermal Fluid and Particle Sampler (HFPS), using the Remotely Operated Vehicles (ROV) ROPOS and JASON, and the Deep Submergence Vehicle (DSV) ALVIN. In situ temperature with HFPS was recorded at 1 Hz during sampling. The values reported here represent the average temperature during sample collection. In general, several sub-samples were collected at each site during each dive, and only the average values for the measured parameters are reported. Samples for nutrient concentration and isotopic measurements were transferred with a syringe into acid-washed and DI-rinsed 60-ml HDPE brown bottles. All fluid samples were purged with N2 gas for at least 10 min to remove H2S, which interferes with the colorimetric nutrient analysis. Water samples were kept frozen at −20°C until analysis. Additional sample aliquots were collected in 15 ml acid-washed and DI-rinsed HDPE bottles for onshore major element (i.e., Mg2+ in this study) analysis at the Pacific Marine Environmental Laboratory and the University of Washington.

2.2. Nutrient Concentrations

[10] Nitrate, nitrite, ammonium, and phosphate concentrations were analyzed onboard or onshore using standard colorimetric techniques. In addition, for many samples, NOx was measured by reduction to nitric oxide (NO) in a heated solution of acidic vanadium (III) and subsequent detection of the NO [Braman and Hendrix, 1989], with a precision for replicate analyses of ±0.2 μmol/L. The agreement between methods was excellent, and average concentrations are reported, where applicable. Mg2+concentration measurements were performed by ion-chromatography (IC) and by successive titration with EDTA and EGTA for [Mg2+] greater than ∼48 mmol/kg.

2.3. Nitrate N and O Isotope Ratios

[11] Nitrate N and O isotope ratios (denoted as δ15N and δ18O, with δ = (Rsample/Rstandard) − 1] × 1000, where R represents the ratio of 15N to 14N or 18O to 16O, respectively) for samples collected between 2006 and 2009 were measured using the “denitrifier method” [Sigman et al., 2001; Casciotti et al., 2002]. Samples collected in 2004, which were only analyzed two years after sample collection, are not considered, as we found clear evidence for biasing storage effects. The denitrifier method is based on the quantitative conversion of sample nitrate to N2O by cultured denitrifying bacteria that lack the active N2O-reductase enzyme. N2O gas was automatically extracted, purified and analyzed online using Tracegas-Isoprime or GasBench II preparation systems coupled to a continuous flow isotope ratio mass spectrometer (IRMS) (Micromass Isoprime Multiflow for samples from 2006 to 2007 and Thermo Finnigan DELTAplus XP for samples from 2008 to 2009). The general target sample size was 20 nmol N for samples with a [NO3] > 5 μmol/L, and 10 nmol N for samples with a [NO3] < 5 μmol/L. Pseudomonas chlororaphis (ATCC #43928) and P. chlororaphis (ATCC #13985) (formerly Pseudomonas aureofaciens) were used to measure δ15N and δ18O, respectively. N and O isotope ratios are reported in ‰ relative to atmospheric N2for N and V-SMOW for O isotopes, respectively. Isotope values were calibrated using an international KNO3reference material (IAEA-N3) with an assignedδ15N value of +4.7‰ (air) and a reported value of 25.6‰ for δ18O (V-SMOW) [Böhlke et al., 2003]. Replicate reproducibility for the method was generally better than ±0.2‰ for δ15N and ±0.4‰ for δ18O. For all samples collected before 2008, O isotope data were corrected for O isotope exchange with water during the reduction of nitrate to nitrous oxide, following the scheme described by Casciotti et al. [2002], using standards prepared from DI water with a δ18O of ∼865‰-V-SMOW. O-isotope exchange was always less than 5%. Blank contribution was generally below 0.5 nmol (i.e., 3–5% of the target sample size). Correction for O isotope scale contraction caused by O isotope exchange with water is more accurate when multiple nitrate isotope reference materials are used, i.e., IAEA-N3 and USGS-34 [Casciotti et al., 2007]. This standard bracketing correction has been applied to all 2008 and 2009 samples. Sigman et al. [2009] recently reported a systematic difference between the two correction schemes, where the older correction protocol resulted in a slight overestimation of the δ18O of deep-sea nitrate. Our measurements comply with this observation, and we corrected pre-2008 data accordingly.

[12] The presence of nitrite in mixed samples is known to interfere with the isotopic analysis of nitrate when using the denitrifier method [Granger et al., 2006; Casciotti and McIlvin, 2007; Casciotti et al., 2007; Granger and Sigman, 2009], because both nitrite and nitrate are converted to N2O. For mixed samples calibrated against nitrate isotope standards, the presence of nitrite leads to artificially low δ18O values because of the differential O isotope effects during nitrate versus nitrite reduction to N2O. According to Casciotti et al. [2007], the presence of 2% or more of nitrite in a sample can significantly modify the original δ18O. Nitrite concentrations were always lower than ∼2 μmol/L in our diffuse fluid samples (see below) and never higher than 10% of the sum of nitrate and nitrite. In 2008/2009 samples, where the NO2 concentrations exceeded 1%, the isotopic composition of nitrate was measured only after nitrite removal with sulfamic acid, following the procedure by Granger and Sigman [2009]. For the few samples that contained significant NO2 concentration (>2% of the total NOx) and that were analyzed prior to 2008 (and originally calibrated against nitrate isotope standards), we approximated the δ18ONO3+NO2 using the correction factors for NO2 on the nitrate calibration scale based on the analysis of pure nitrite standards [Casciotti and McIlvin, 2007; Casciotti et al., 2007].

2.4. Ammonium N Isotope Ratios

[13] The δ15N of ammonium was measured using a combination of the passive ammonia diffusion method [Sigman et al., 1997] with persulfate oxidation [Knapp et al., 2005] and the denitrifier method [Sigman et al., 2001] [also see Houlton et al., 2007]. Two to fifty ml of hydrothermal vent fluid (60–700 nmol sample ammonium) were pipetted into 30–60 ml glass media bottles. High-concentration samples were diluted with DI to yield at least 10 ml sample solution. A NH3(g) trap consisting of a 5 mm2, pre-combusted (500°C for 4 h) and acidified (∼5–10μL of 4 N H2SO4) GF/D glass fiber filter sandwiched between two sealed Teflon membranes (Millipore, LCWP01300) [Sigman et al., 1997], was added to the media bottles. Prior to closing the bottles with a Teflon septum and an aluminum seal, pH was raised above 9.2 by adding MgO (60 mg per ∼10 ml sample). Samples were agitated on an orbital shaker at room temperature for 7 days to warrant complete ammonium conversion to NH3(g) under basic conditions, and NH3 trapping in the diffusion packages. The NH3traps were then removed from the sample bottles, dipped into a 10% HCl solution, and, without any drying step, placed in 10-ml autoclave glass tubes with 6.5 ml of DI water. The test tubes were shaken vigorously in order to open the NH3traps and release the acidified disks, and the Teflon membranes were removed using clean dull-tipped forceps; these steps have proven to be important for 100% N recovery (without losing some (NH4)2SO4precipitate on the inner surface of the Teflon membranes). One ml of persulfate oxidizing reagent (POR), freshly made daily by dissolving 6 g of certified ACS-grade NaOH and 6 g of certified ACS-grade potassium persulfate (re-crystallized three times in 100 ml of DI water) [Knapp et al., 2005; Bourbonnais et al., 2009], was then added, and the closed tubes were autoclaved for at least one hour to allow complete ammonium oxidation to nitrate by the POR. After pH adjustment with ACS-grade HCl (pH ∼ 3–4), the N isotopic composition of the ammonium-derived nitrate was determined using the denitrifier method as described above.δ15N values were calibrated with NH4+ standards of known isotopic composition (IAEA N1, δ15N of NH4+ = 0.4‰ and IAEA N2, δ15N of NH4+ = 20.3‰), which were processed the same way as the samples, taking the procedural blank into consideration (generally less than 10%). For each standard/sample batch, DI additions and total N amount were adapted to the N content and volume of the samples. Multiple N isotope analyses of NH4+ standards showed that the passive ammonia diffusion/oxidation/denitrifier method produces accurate and reproducible (<±0.6‰) results. Although we did not observe a marked N isotope fractionation during incomplete N recovery, only the samples with a ∼80–100% yield were considered. Most 2008 and 2009 samples with [NH4+] higher than ∼100 μM were also analyzed using passive ammonia diffusion followed by direct combustion of the NH4+-loaded filters (sample requirement ∼5000 nmol N) in a Thermo Finnigan FlashEA 1112 series Elemental Analyzer coupled to an IRMS (Thermo Finnigan Delta Advantage). Results from both NH4+N-isotope techniques agreed well.

3. Results and Discussion

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Material and Methods
  5. 3. Results and Discussion
  6. 4. Summary and Concluding Remarks
  7. Acknowledgments
  8. References
  9. Supporting Information

3.1. Origin and Fate of Hydrothermally Discharged Ammonium

[14] During high-T hydrothermal circulation, Mg2+is almost completely removed from the aqueous solution through various water-rock reactions [Edmond et al., 1979; Seyfried, 1987]. At lower temperatures, mixing of hydrothermal fluids with Mg2+-rich and cold seawater elevates the [Mg2+] again. Hence, a linear (inverse) relationship between the temperature of the HV fluid and its Mg2+-content exists (r2 = 0.90, Figure 2a). [Mg2+] can thus be used as an indicator of mixing between zero-Mg2+pure high-T hydrothermal fluids and Mg2+-rich (52.9 mmol/kg) low-T crustal seawater.

image

Figure 2. Magnesium concentrations versus average fluid temperatures at Endeavour Segment (star = Sasquatch, down triangle = High Rise, square = Main endeavor Field, diamond = Mothra), Cobb Segment (triangle) and Axial Volcano (circle). Magnesium is almost completely removed from the hydrothermal vent fluids at higher temperatures and only slowly removed at lower temperatures. The colors represent the sampling years (yellow = 2004, purple = 2006, green = 2007, blue = 2008 and red = 2009). Standard deviation for sub-samples (n = 2 to 5) collected during the same dive and location is indicated.

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[15] Ammonium is the dominant DIN species under the highly reducing conditions encountered in high-T fluids (∼200°C to 350°C, or <10 mmol/kg Mg2+), with average concentrations decreasing from North to South along the Juan de Fuca Ridge: 1078 ± 4.7 μmol/L (n = 2) at Sasquatch, 863 ± 129 μmol/L (n = 11) at High Rise, 410 ± 53 μmol/L (n = 23) at MEF, and 396 ± 20 μmol/L (n = 5) at Mothra at the Endeavour Segment, 44 μmol/L (n = 1) at Cobb Segment, and 14 ± 3 μmol/L (n = 22) at Axial Volcano (Figures 3b–3d). NH4+ concentrations behave mostly conservatively with respect to Mg2+, indicating the dilution of high-NH4+hydrothermal fluids with zero-NH4+, oxygenated deep-seawater.

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Figure 3. (a) Nitrate (all sites) and ammonium concentrations at (b) Endeavour Segment, (c) Cobb Segment and (d) Axial Volcano versus [Mg2+] (note the different scales of the yaxis). In low-T vents, nitrate concentrations that fall below the mixing line between zero-nitrate, ammonium-rich, pure hot hydrothermal vents and nitrate-rich crustal seawater are indicative of microbial nitrate consumption. Ammonium concentration above ∼0μmol/L in low-T waters at Axial Volcano, occurring mainly at the site Marker 113 and its surrounding areas (black stars), is a sign of microbial ammonium production. Symbol and color scheme as inFigure 2legend. The seawater end-member is represented by a turquoise hexagon. Regression lines in Figure 3b: Sasquatch = solid line, High Rise = long dash, Main Endeavor Field = medium dash and Mothra = short dash). Standard deviations for sub-samples (n = 2 to 5) are indicated by the error bars.

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[16] The δ15N of ammonium in high-T fluids of the Endeavour Segment did not differ among years, but differs significantly among sites, with average values (all years combined) of 4.3 ± 0.7‰ (n = 3) at Sasquatch, 4.2 ± 0.5‰ (n = 11) at High Rise, 3.6 ± 0.4‰ (n = 19) at MEF and 3.1 ± 0.3‰ (n = 5) at Mothra (Figure 4a) (Kruskal-Wallis, p-value = 0.009). Since we were primarily interested in variations among vent fields, we grouped the values at the four Endeavour Segment sites for the subsequent discussion. It should be noted that the low average ammoniumδ15N in high-T vent fluids of the Endeavour Segment (3.7 ± 0.6‰ (n = 37)) does not closely match the only existing report on the N isotopic composition of NH4+ in hydrothermal fluids by Lilley et al. [1993], who measured a δ15N value of 12.4‰, but is consistent with a δ15N of 2.1‰ for an extinct sulfide chimney from Dante (at MEF) collected during the 2009 cruise (unpublished data).

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Figure 4. δ15N–NH4+ versus [Mg2+] and ln [NH4+] at (a, b) Endeavor and Cobb Segments and (c, d) Axial Volcano. The average δ15N-NH4+ in high temperature fluids is indicated by the horizontal lines (dashed line for Cobb Segment). Symbol and color scheme as in Figure 2legend. In Figures 4b and 4d, high-T end-members are encircled. The highest15N isotope enrichment was observed at Axial Volcano: Marker 113 (black stars), The Spot (circle), and CASM (down-triangle).

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[17] The average Endeavour Segment ammonium δ15N was significantly different from the δ15N of nitrate in Deep-Pacific seawater at ∼2100 m depth (5.6 ± 0.2‰ (n = 5), p-value = 0.0003). The averageδ15N of ammonium in high-T fluids of the Cobb Segment was 4.1 ± 0.1‰ (n = 3), i.e., not significantly different from theδ15N of ammonium at Endeavour Segment (Figure 4a). In contrast, the average δ15N of ammonium in high-T fluids at Axial Volcano was 6.7 ± 1.0‰ (n = 16) (Figure 4c), significantly greater than the δ15N observed at the Endeavor (p-value = 8 × 10−9) and Cobb (p-value = 0.007) segments, but not significantly different from theδ15N of deep-seawater nitrate in the ambient water column (6.4 ± 0.2‰, n = 4) (Figure 4c).

[18] Assuming no secondary alterations due to microbial or thermic processes, the δ15N of ammonium in high-T fluids should reflect that of the original N source. During high-T subsurface hydrothermal circulation, under reducing conditions, nitrate from the deep sea can be abiotically converted to ammonium by transition metal oxides and sulfides as it infiltrates the subsurface through cracks and fissures [e.g.,Brandes et al., 2008]. By mass balance, if fixed N is neither consumed nor produced during high-T reactions, discharging hydrothermal waters should contain NH4+at concentrations (and N isotopic composition) that are equivalent to the nitrate of inflowing crustal waters with a deep-sea origin (i.e., ∼40μmol/L), at least at the un-sedimented (i.e., no apparent organic N source) hydrothermal systems of the Juan de Fuca Ridge. In this context, the elevated ammonium concentrations observed at the un-sedimented Endeavour Segment appear anomalous. These have been argued to originate from the decomposition of subsurface organic material in sediments buried at an earlier stage of the ridge formation. Pleistocene turbidite flows from deposits located about ∼40 km north of the Juan de Fuca Ridge might be the source of these sediments [Lilley et al., 1993]. If OM from these deposits was indeed the source for the ammonium at Endeavor, their 15N content must have been low compared to the mean δ15N-NO3of deep-seawater.

[19] Ammonium can, in theory, also be produced by abiotic reduction of N2by Fe-minerals in the subsurface high-T hydrothermal fluids, producing newly fixed NH4+ with a low δ15N [Brandes et al., 1998a; Schoonen and Xu, 2001; Dörr et al., 2003]. It is widely accepted that the δ15N of newly fixed N is close to the δ15N of the dissolved N2, which can either originate from a magmatic source (with a δ15N value of −5 ± 2‰ [Javoy and Pineau, 1991; Marty and Humbert, 1997]) or from circulating deep-seawater (with aδ15N value of ∼0‰). As pointed out by Lilley et al. [1993], it is unlikely that abiotic N2 fixation would occur to such extent only at the Endeavor and Cobb Segments. Moreover, it can be predicted that the average δ15N of hydrothermal ammonium would be even lower than 3.7‰, at least if high-temperature N2 fixation was the main ammonium source.

[20] At Axial Volcano, the similarity of the δ15N of ammonium in high-T fluids and deep-seawater nitrate (6.7‰ and 6.4‰, respectively) suggests that seawater nitrate that penetrates from the water column into the anaerobic subsurface through cracks and fissures represents the substrate for complete abiotic reduction to ammonium. However, the ammonium-N concentration in high-T fluids of Axial Volcano was lower than the nitrate-N concentration in the Deep-Pacific by ∼26μmol/L. This difference, indicating the net loss of fixed N in the high-T fluids, is likely caused by high-T ammonium ion substitution in secondary minerals during fluid interactions with basaltic rocks. For instance,Hall [1989] and Busigny et al. [2005] observed that N (mainly occurring as ammonium ions substituting for K+ and Na+/Ca2+ in minerals) indeed gets enriched in rocks during basalts alteration. Details of the actual N removal mechanism aside, the accordance of the δ15N of inflowing nitrate and of high-T end-member ammonium implies that the N scavenging in hot hydrothermal fluids must occur without significant N isotope fractionation. In the same line,Holloway et al. [2011] found that the δ15N of ammonium in hydrothermal waters of the Yellowstone National Park with a pH < 5 (the pH range for the high-T fluids in this study was ∼3.5 to 5) remained more or less unaffected by water rock interactions.

3.2. Biological Uptake and Isotopic Fractionation of Ammonium N in Low-T Fluids

[21] Ammonium in low-T fluids (<∼50°C) of the subsurface biosphere of hydrothermal vents can be produced either by organic matter remineralization (of both sedimentary and in situ produced organic N, e.g., by biotic N2 fixation [Mehta et al., 2003, 2005; Mehta and Baross, 2006]), and/or dissimilative nitrate reduction to ammonium (DNRA), which has been demonstrated to occur at temperatures up to 70°C [Vetriani et al., 2004; Voordeckers et al., 2005; Perez-Rodriguez et al., 2010]. On the other hand, ammonium can be consumed by biological processes in low-T fluids, displaying distinct N-isotope effects, such as ammonium assimilation, aerobic microbial ammonium oxidation [Lam et al., 2004, 2008], or anammox [Byrne et al., 2009].

[22] In most cases where ammonium concentration gradients appeared to behave conservatively with respect to Mg2+, the δ15N was invariant, confirming simple mixing of high-NH4+hydrothermal waters with zero-NH4+seawater in low-T hydrothermal fluids (Figures 3b–3d and Figure 4). However, at some sites of the Endeavour Segment and Axial Volcano, net ammonium production or consumption are evidenced by concentrations above or below those expected from conservative mixing in the low-T hydrothermal fluids (Figures 3b and 3d). Net ammonium production, either via partial DNRA, organic matter remineralization or the N2 fixation/remineralization cycle, corresponded with a decrease of δ15N-NH4+ with increasing [NH4+] in low-T fluids (i.e., Hulk at MEF and Hermosa, Vixen, Village and Escargo at AV,Figures 4b and 4d).

[23] Anomalously high ammonium concentrations were observed at Marker 113 diffuse vent fluids during all sampling campaigns (Figure 3d). Yet the δ15N-NH4+was either similar to the high-T end-member value or even greater (up to ∼10‰,Figures 4c and 4d), which appears inconsistent with ammonium production by N2 fixation or N isotope fractionation during partial DNRA (both processes would act to produce low δ15N-NH4+). Remineralization of high δ15N organic material to ammonium can also not account for the elevated δ15N-NH4+ values. Reported values of δ15N of hydrothermal vent fauna are generally low (∼−10 to +4‰ [Rau, 1981, and reference therein]), as was the δ15N of particulate material collected at Axial Volcano sites (4.6 ± 0.2‰ at Marker 33 and Gollum, 2009 cruise, unpublished data; 4.3 ± 1.2‰ [Levesque et al., 2005]). Therefore, we conclude that the elevated ammonium δ15N values reflect N-isotope fractionation during partial ammonium consumption by bacterial assimilation or nitrification occurring in tandem with ammonium production by processes mentioned earlier. That is, while net ammonium production is evidenced by non-conservative behavior of the ammonium concentration, theδ15N indicates that ammonium consumption occurs concurrently. The ammonium 15N enrichment was also observed at other sites, where NH4+ was clearly consumed relative to conservative mixing (up to ∼5.5‰ at CASM, Axial Volcano, Figures 4c and 4d).

[24] The net ammonium consumption N isotope effect can be estimated from the correlation between the natural logarithm of the ammonium concentration and the δ15N of the residual ammonium (closed-system Rayleigh Model [Mariotti et al., 1981]). However, open-system aspects and spatial/temporal variability in both the ammonium concentration andδ15N are likely to prevent any clear Rayleigh-type ammonium N isotope dynamics (Figures 3 and 4), and, even more importantly, in situ regeneration of NH4+ and the N isotope effects associated with this regeneration will bias estimates for εuptake for natural assemblages of bacteria in the diffuse HV fluids. Furthermore, the N isotope effects during ammonium oxidation (ε = +14 to +38‰ [Delwiche and Steyn, 1970; Mariotti et al., 1981; Yoshida, 1988; Casciotti et al., 2003]) and ammonium assimilation (ε = +14 to +27‰ [Hoch et al., 1992; Waser et al., 1998]) by microorganisms and algae in aquatic systems are highly variable and likely influenced by environmental conditions (e.g., substrate concentration and uptake rate), making it difficult to tell the two processes apart based solely on the degree of N-isotope enrichment. A plot ofδ15N-NH4+ versus the ln [NH4+] (Figures 4b and 4d) does not indicate obvious Rayleigh-type N-isotope dynamics. A significant relationship betweenδ15N-NH4+ and ln [NH4+] was only observed at Cobb Segment in 2007 (ε = 1.2‰, r2 = 0.7) and Axial Volcano in 2006 (ε = 2.6‰, r2= 0.3), although sample sizes for these data sets were limited. The computed ammonium N isotope effects can be taken as community N-isotope effects for net ammonium removal, and were much lower than both the N isotope effect expected for aerobic (and anaerobic) ammonium oxidation, as well as for ammonium assimilation at elevated NH4+ concentrations [Hoch et al., 1992]. Clearly, production of low-δ15N NH4+ occurs through gross ammonium regeneration.

[25] While the δ15N-NH4+ data alone do not allow us to constrain the actual ammonium removal pathway (uptake versus nitrification) the nitrate N and O isotopes in combination with elevated δ15N-NH4+ at Marker 113 suggest that partial nitrification of ammonium to nitrate must occur to some extent. The nitrate N and O anomalies that appear to be indicative of nitrate regeneration are discussed in detail below (see section 3.4).

3.3. Nitrate Consumption and Associated N and O Isotope Effects in Hydrothermal Vent Fluids

[26] Nitrate δ15N and δ18O values close to the N and O isotopic composition of ambient seawater nitrate (∼6‰ and 2‰, respectively) (Figures 5a–5d) suggest that nitrate in both high and low-T (<∼50°C) HV fluids mainly originates from mixing with nitrate-replete crustal seawater (∼40μmol/L in the deep Northeast Pacific Ocean). At several low-T fluids sites, however, elevated nitrateδ15N and δ18O (i.e., high Mg2+) (Figure 5), concomitant with decreased [NO3] (Figure 3a), were observed. This clearly indicates a N and O isotope fractionating nitrate-consuming process in the low-T subsurface biosphere prior to venting. The15N and 18O enrichment in the HV fluid nitrate was greatest at Axial Volcano (up to ∼3‰ for δ15N and ∼11‰ for δ18O, Figures 5c and 5d) compared to the Endeavor and Cobb Segments (between ∼1.4‰ and ∼2‰ for δ15N and ∼3‰ and ∼5‰ for δ18O, respectively, Figures 5a and 5b). Moreover, we consistently observed higher relative isotope enrichments for 18O versus 15N (Figure 5). Nitrate δ15N and δ18O values in diffuse vent fluids sampled at the same locations fluctuated between years, with no clear temporal trend. In general, the 15N and 18O isotope enrichment was greater in 2006 and 2009, and lowest in 2008. In the subsequent discussion, we treat each data set separately.

image

Figure 5. Nitrate δ15N and δ18O versus [Mg2+] at (a, b) Endeavor and Cobb Segments and (c, d) Axial Volcano. Same symbol and color scheme as in Figure 2. The seawater (SW) δ15N end-member is indicated by the solid black lines. Error bars represent the standard deviation for sub-samples (n = 2 to 5) collected at the same time and location. Sites where the highest heavy-isotope enrichments were observed at Axial Volcano are indicated by crosses (Bag City), black stars (Marker 113) and black squares (Cloud). Black arrows indicate isotopic fractionation during nitrate consumption in diffuse fluids (seeFigure 3a).

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[27] The development of a nitrate 15N and 18O isotope enrichment in diffuse fluids (due to faster consumption of the lighter isotopologues during biological reactions) depends on (1) the isotope effect associated with the biologic reaction and (2) the degree to which the N-removal process has advanced. The latter is, in turn, a direct function of the nitrate removal rate and the fluid flow velocity (i.e., nitrate supply). Indeed, the greatest nitrate15N and 18O enrichments were generally observed at the same vent sites in all sampling years (e.g., Bag City, Marker 113 and Cloud at Axial Volcano, Figures 5c and 5d), suggesting some microbiological, chemical or physical characteristic unique to these sites that make them conducive to nitrate removal and the associated enrichment of 15N and 18O in the residual nitrate. However, no significant relationship was observed between temperature, and the apparent nitrate 15N and 18O isotope enrichment. In fact, highest nitrate δ15N and δ18O values at the respective sites and sampling events encompass a broad range of temperatures (at the low-T end of the spectrum): e.g., ranging from ∼6°C at Cloud (Axial Volcano) to 71°C at Milli-Q (Endeavour Segment).

[28] Both assimilative (nitrate uptake by vent organisms) and dissimilative (denitrification) nitrate reduction are likely candidate processes to remove nitrate from HV, as both are known to fractionate nitrate isotopes in other environments [e.g., Brandes et al., 1998b; Voss et al., 2001; Lehmann et al., 2003; Sigman et al., 2003]. Biogeochemical evidence for respiratory denitrification by autotrophic bacteria has been reported previously for HV systems. For example, it has been shown that bacterial symbionts within the vestimentiferan Riftia pachyptila at the Genesis vent site (East Pacific Rise) can facultatively use nitrate as an electron acceptor for the oxidation of hydrogen sulphide, with either nitrite or N2 gas being the metabolic N product [Hentschel and Felbeck, 1993]. Similarly, sulphur oxidizing bacteria (e.g., Beggiatoasp.), found at Guaymas Basin hydrothermal vents, can accumulate nitrate at concentrations that are at least 3,000-fold higher than ambient concentrations in their vacuoles, which they subsequently also use for the oxidation of hydrogen sulphide [McHatton et al., 1996]. In diffuse fluids at Axial Volcano Butterfield et al. [2004]observed comparatively high concentrations of nitrite and nitrous oxide (20 to 600 nmol/L), typical intermediates and byproducts of denitrification in intermittently anoxic aquatic environments. While these previous studies have highlighted that active denitrification by microbes is likely to occur in hydrothermal fluids, nitrate assimilation for bacterial growth is another important process to be considered. The observed heavy-isotope enrichments were mostly restricted to the lower-T fluids (<50°C), where microbial cell densities are by far the highest (i.e., up to ∼10 times more than in seawater [Butterfield et al., 2004]). While ammonium is generally the form of fixed N that is preferred during N assimilation, by both photosynthetic organisms and bacteria [Dortch, 1990; Dugdale et al., 2007], Lee and Childress [1994]showed that S-oxidizing bacteria that live in symbiosis with the HV tubewormRiftia pachyptila exclusively assimilate nitrate, even under ammonium–replete conditions.

[29] The degree of community N and O isotope fractionation can potentially help us elucidate the pathway of nitrate removal in the HV diffuse fluids. Denitrification in the environment generally occurs with a significant nitrate isotope effect (ε) for both N and O isotopes of ∼20 to 30‰ [Cline and Kaplan, 1975, and references therein]. In contrast, nitrate assimilation seems to be associated with a significantly lower N-isotope effect of ∼5‰ in laboratory cultures and the natural environment [Altabet, 2001; Granger et al., 2004]. Here we attempt to estimate, for the first time, the community N and O isotope effects for nitrate removal in HV fluids, with the goal of assessing nitrate removal pathways. Analogous to the approach used to estimate the ammonium consumption N isotope effect above, the nitrate removal N and O isotope effects can be approximated using a closed system (Rayleigh) model, where a closed nitrate pool is consumed with a constant isotope effect as described by the equation [Mariotti et al., 1981]

  • display math

where the initial nitrate concentration is calculated from the [Mg2+] content of the fluid, and assuming a strict linear mixing relationship between pure hydrothermal vent fluids and seawater (Figure 3a). As explained above, HV systems do not really behave as closed systems, so that the Rayleigh approach is likely to underestimate the community N isotope fractionation [Lehmann et al., 2003, 2007, 2009]. Alternatively, in an open steady state model, we assume that new seawater with a fixed δ15N for nitrate is constantly being mixed into the hydrothermal conduits, balancing the loss of nitrate by denitrification and/or N uptake so as to yield a steady state. The associated community N isotope effect is then calculated using the following equation [Altabet, 2001; Sigman et al., 2003]:

  • display math

(Figure 6). The results from both approaches are shown in Table 1. The highest isotope effects were obtained using the open system model at Axial Volcano in 2006 (1.9‰ for 15εk and 8.6‰ for 18εk). The lowest isotope effects were observed using a closed-system model at the Endeavour Segment in 2008 (0.4‰ for both15εk and 18εk). Overall, the differences between the respective models were not very large. Strictly speaking, both models (closed-system and steady state) may not be representative of the real situation, as during hydrothermal circulation mixing is not necessarily continuous but rather episodic. However, modeling efforts bySigman et al. [2003] demonstrated that for different mixing regimes between open steady state and closed systems (i.e., if there is sporadic mixing), the calculated isotope effect should fall between the two extremes.

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Figure 6. δ15N and δ18O versus f(open system “steady-state” model), wheref is the fraction of nitrate consumed ([NO3]initial − [NO3]measured); [NO3]initial is considered to be equal to [Mg2+]measured/[Mg2+] sw × [NO3]sw, which is the [NO3] for the corresponding [Mg2+] on the mixing line, i.e., ∼40 μM, in HV fluids near the seawater end-member (Figure 3a), at (a, b) Endeavor and Cobb Segments and (c, d) Axial Volcano. The isotope effects are estimated based on the slopes of the linear regression lines (dashed line for Cobb Segment). Symbol and color scheme as in Figure 2. Linear regressions for each year are shown. The colors of the lines correspond to the colors of the symbols (i.e., sampling years). See Table 1 for N isotope effect estimations and the r2 of the linear regressions.

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Table 1. Estimated Nitrate N and O Isotope Effects According to a “Rayleigh” Closed System and an Open “Steady-State” System Model at Endeavour Segment, Cobb Segment and Axial Volcano on the Juan de Fuca Ridge From 2006 to 2009a
 2009200820072006Average
15εk (‰)18εk (‰)15εk (‰)18εk (‰)15εk (‰)18εk (‰)15εk (‰)18εk (‰)15εk (‰)18εk (‰)
  • a

    The r2 values for each linear regression are shown in parentheses. Refer to Figure 6 for plots and linear regressions of δ15N and δ18O of nitrate versus f, the fraction of nitrate consumed (open “steady-state” model). p-values ≤ 0.05 (significant relationship) for the linear regressions are indicated by the asterisk next to the r2.

Endeavour Segment          
Closed system1.3 (0.49*)2.6 (0.57*)0.41 (0.57*)0.41 (0.31*)1.0 (0.97*)2.3 (0.99*)1.7 (0.71*)4.3 (0.59*)1.1 ± 0.62.4 ± 1.6
Open system2.0 (0.56*)3.9 (0.64*)0.90 (0.64*)0.96 (0.41*)1.7 (0.94*)3.7 (0.98*)1.8 (0.71*)4.7 (0.59*)1.6 ± 0.53.3 ± 1.6
Cobb Segment          
Closed systemnananana1.4 (0.85*)3.5 (0.88*)nana1.43.5
Open systemnananana2.1 (0.76*)5.3 (0.80*)nana2.15.3
Axial Volcano          
Closed system0.69 (0.41*)3.2 (0.93*)1.9 (0.57*)3.0 (0.58*)0.28 (0.11)1.6 (0.63*)1.2 (0.84*)5.1 (0.89*)1.0 ± 0.73.2 ± 1.4
Open system1.5 (0.55*)5.9 (0.89*)2.9 (0.48*)4.6 (0.51*)0.90 (0.20*)3.8 (0.60*)1.9 (0.63*)8.6 (0.75*)1.8 ± 0.95.7 ± 2.1

[30] The herein derived nitrate isotope effects are significantly lower than the ∼20–30‰ isotope effect expected for canonical denitrification, and generally closer to the N and O isotope effect of 5‰ expected for nitrate assimilation only. Assuming that denitrification occurs with a N and O isotope fractionation at levels similar to those reported for ocean oxygen minimum zones, the nitrate isotope data suggest that denitrification can account only for a minor fraction of the total nitrate removal in the hydrothermal fluids. It is also possible, however, that the N and O isotope fractionation of HV denitrification is suppressed by diffusion limitation [Lehmann et al., 2007]. Cell growth in the sub-seafloor primarily occurs on fixed surfaces (e.g., microbial mats [Moyer et al., 1995]). Analogous to the N-isotope-effect suppression due to substrate limitation reported for denitrification in benthic marine environments [Brandes and Devol, 1997; Lehmann et al., 2004, 2007], the diffusive nitrate supply to the actual sites of denitrification in such mats may be limiting so that nitrate is completely consumed. As a consequence, the nitrate N and O isotope effects would be significantly reduced, possibly with an apparent isotope effect of less than 2‰ [Brandes and Devol, 2002]. Finally, analogous to our above considerations regarding net N isotope effects of ammonium consumption, NO3depletion is likely to be the net result of co-occurring nitrate consumption and production. Hence, here-reported N and O isotope effects represent community isotope effects that may partly be biased by the regeneration of nitrate and the isotope effects associated with the regeneration processes [e.g.,Lehmann et al., 2004]. At least for the nitrate N isotope ratios, the low apparent isotope effects may demonstrate the occurrence of gross nitrate production, by either nitrification or N2 fixation (and the subsequent remineralization/nitrification of newly fixed organic N to nitrate). Both processes would increase nitrate concentrations while decreasing nitrate δ15N values, and thus erase, or at least mask, any N isotope signals resulting from denitrification or nitrate assimilation. Figure 6 and Table 1 show that the community nitrate isotope effects are larger for 18O than for 15N, especially at Axial Volcano. A ratio of 18O versus 15N isotope enrichment >1 (between ∼1 to 4 (Figures 7a and 7c)) is atypical for stand-alone denitrification and/or nitrate assimilation in marine environments [Granger et al., 2004, 2008]. Given previous work [Lehmann et al., 2004; Sigman et al., 2005; Knapp et al., 2008; Bourbonnais et al., 2009], such a decoupling of the 15N versus 18O nitrate isotope enrichment is expected if quantitative N regeneration occurs simultaneously to net N consumption in the vent fluids. In the next section, we will discuss the observed δ15N-δ18O relationship in the context of possible N regeneration pathways within the HV fluids that can lead to the observed nitrate N-to-O isotope anomalies.

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Figure 7. δ18O-NO3 versus δ15N-NO3 and Δ (15,18) versus Mg2+ concentration at (a, b) Endeavor and Cobb Segments and (c, d) Axial Volcano. Symbol and color scheme as in Figure 2 legend. The ratio of nitrate 18O versus 15N enrichment are estimated from slopes of the linear regressions (dashed-line for Cobb Segment in Figure 7a).

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3.4. Nitrate N-to-O Isotope Anomalies: Possible Causes and Constraints on N Regeneration

[31] Analogous to the approach proposed by Sigman et al. [2005], we quantify the deviation of nitrate isotope values from a 1:1 N versus O isotope fractionation relationship expected for pure denitrification/assimilation, using Δ(15,18):

  • display math

where δ15Nm = ∼6‰ and δ18Om = ∼2‰ are the mean δ15N and δ18O of deep-seawater, respectively, and18ε/15ε is the ratio of N versus O isotope enrichment during denitrification, i.e., 1. An equal enrichment for N and O isotopes (Δ(15;18) = 0‰) can be assumed as a baseline characteristic for pure denitrification [Sigman et al., 2005; Granger et al., 2008] and NO3 assimilation [Granger et al., 2004; Lehmann et al., 2005]. From Figures 7b and 7d, it can be derived that the Δ(15,18) is negative in Mg2+-rich, low-T hydrothermal fluids, where denitrification (and possibly nitrate assimilation) results in the net consumption of nitrate. The Δ(15,18) was significantly higher (up to ∼−8.5‰) at Axial Volcano compared to the Endeavor and Cobb Segments (up to ∼3‰). The nitrate isotope anomalies also varied between years, (with generally higher anomalies in 2006, 2007, and 2009) although no general temporal pattern could be discerned.

[32] We argue that the Δ(15,18) anomaly observed at some sites is due to the gross production of nitrate coupled to microbial nitrate consumption (assimilation and/or denitrification). However, we currently have few quantitative constraints on the different N fluxes involved or the physical and chemical characteristic of the subsurface biosphere (e.g., average fluid residence time, volume of hydrothermal conduits, and timing of mixing), so it is impossible to derive rates of processes from our isotope data. Therefore, we will only qualitatively discuss possible causes for the observed nitrate N and O isotope anomalies below. The exact causes for generally larger nitrate isotope anomalies at Axial Volcano compared to the Endeavor and Cobb Segments are unclear, as are the causes of any inter-annual variations. However, supplementary data on bacterial cell densities suggest a link between bacterial growth/activity and the observed expression of the N-to-O isotope anomalies. In fact, we observed a significant relationship (r2= 0.61, p-value = 0.0001) between bacterial cell counts in diffuse fluids (data from J. A. Baross and J. F. Holden laboratories) and the N-to-O nitrate anomalies for the years where cell counts were available (2008 and 2009) at Axial Volcano (Figure 8). Yet, at Endeavour Segment, where maximum NH4+concentrations were observed in the subsurface fluids, the nitrate N- versus O isotope anomaly was rather moderate and there was no correlation with bacterial cell density. Therefore causal links between bacterial biomass, “anomalous” nitrate isotope behavior and, possibly, nitrate regeneration through either ammonium or nitrite oxidation remain uncertain.

image

Figure 8. Δ(15,18) versus cell count at Axial Volcano (symbol and color scheme as in other figures).

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3.4.1. Partial Nitrification and N2 Fixation

[33] Ammonium oxidation in deep-sea hydrothermal vent plumes of the Main Endeavor Field, Juan de Fuca Ridge, has been reported byLam et al. [2004, 2008]. Byrne et al. [2009]also documented the diversity and activity of anammox bacteria in hydrothermal vent chimney samples on the Mid-Atlantic Ridge. Here, the partial re-oxidation of ammonium at the seawater end of the redox gradient in low-T, aerobic vent fluids would leave the residual NH4+ enriched in 15N, while the nitrate produced would be depleted in 15N [Delwiche and Steyn, 1970, and references therein]. While the δ15N of newly nitrified nitrate is directly dependent on the δ15N of the ammonium pool (with an offset that corresponds to the nitrification N-isotope effect), nitrification represents an absolute source with regard to the O isotopes.

[34] The general understanding at the moment is that two-thirds of the O atoms in new nitrate are being derived from ambient water, and that, because of important isotope effects during oxygen atom incorporation during ammonium and nitrite oxidation, theδ18O of nitrified nitrate should be 0.7–8.3‰ lower than the δ18O of the seawater (which was measured to be ∼0‰ in our 2004 hydrothermal fluid samples, unpublished results) [Buchwald and Casciotti, 2010; Casciotti et al., 2010]. On the other hand, 16O is preferentially extracted during nitrate reduction, causing the δ18O of the eliminated NO3 to be even lower than the δ18O of the nitrified nitrate. Hence, while nitrification tends to counteract the denitrification/assimilation–driven enrichment of both the δ18O and δ15N, this balancing effect is generally more pronounced for N than for O so that it can produce nitrate N-vs-O anomalies (more precisely, Δ(15,18) minima) [Sigman et al., 2005]. The Δ(15,18) effect is most marked if ammonium oxidation is not complete, either in an ammonium-replete setting, or when ammonium uptake and nitrification occur simultaneously (i.e., ammonium branching, with the N isotope effect associated with ammonium assimilation being lower than the N isotope effect associated with nitrification) [e.g.,Wankel et al., 2007].

[35] The N isotope balance predicts that in the HV fluids where partial nitrification generates low-δ15N nitrate, it simultaneously leaves 15N-enriched ammonium substrate behind (according to the nitrification N isotope effects of 14–38‰ [Delwiche and Steyn, 1970, and references therein]). The observed increase of the δ15N-NH4+in the low-T fluids at some sites of the Endeavor and Cobb Segments, as well as Axial Volcano (Figure 4), hence, confirms that oxidation of ammonium is occurring to some extent. We would expect the nitrification-driven Δ(15,18) minimum to be most pronounced at Endeavour Segment and, to a lesser extent, Cobb Segment, where the measured ammonium concentrations in diffuse HV fluids (up to ∼65μmol/L and ∼10 μmol/L, respectively) were often higher than the nitrate concentrations. Furthermore, the average δ15N of the hydrothermally discharged ammonium was also significantly lower at these sites compared to Axial Volcano, further enhancing the expression of the nitrate isotope anomaly. As discussed above, there were indeed systematic inter-site variations with regard to the Δ(15,18). However, curiously, the lowest Δ(15,18) was observed at Axial Volcano, where the influence of incomplete ammonium oxidation is likely to be less important and where, as a consequence, the Δ(15,18) should be less pronounced ([NH4+] in diffuse fluids was close to 0 μmol/L at most sites).

[36] N2 fixation in hydrothermal vents has been previously documented by Mehta et al. [2003, 2005] and Mehta and Baross [2006] who found expressed nitrogenase genes (nifH) in anaerobic hydrothermal fluids at Axial Volcano and who were able to isolate a methanogenic archeaeon that can fix nitrogen at a temperature up to 92°C. N2 fixers are assumed to thrive in environments where fixed N forms that are energetically more favorable are scarce. Moreover, an anaerobic environment may be conducive to N2 fixation, as nitrogenase, the enzyme involved in N2-fixation, is inhibited by oxygen [e.g.,Berman-Frank et al., 2005]. N2 fixation may occur in microsites, e.g., microbial mats and particulate material, where O2and DIN concentrations may be low, providing an important source of bioavailable N for hydrothermal vent organisms in low-T fluids [e.g.,Proctor, 1997; Zehr et al., 2003]. N2 fixation produces organic material with a δ15N of ∼−2 to 0‰ [Carpenter et al., 1997, and references therein]. Upon ammonification of the N2-fixation-derived biomass and subsequent (incomplete) nitrification after mixing of the anaerobic vent fluids with oxic seawater, low-δ15N N can be transferred to the NO3 pool. Given the nitrate O isotope systematics described above, the δ18O, however, is rather insensitive toward this N2fixation as the incorporation of O atoms into the newly produced nitrate molecule does not discriminate between possible origins of the precursor N compounds. The above-described nitrification/denitrification mechanism to produce theδ15N-δ18O decoupling would, hence, be enhanced if newly produced nitrate is derived from the remineralization and nitrification of chemosynthetically fixed N2. And even in the case of complete nitrification of the ammonium, a negative Δ(15,18) can be expected [Bourbonnais et al., 2009].

3.4.2. Nitrate/Nitrite Redox Cycle

[37] Nitrate is not only regenerated by the oxidation of ammonium, it can also originate from the re-oxidation of nitrite. Under suboxic conditions in hydrothermal vent fluids, nitrate is reduced to nitrite. Along redox gradients or upon mixing of anaerobic and oxic waters, a large fraction of product nitrite may be re-oxidized to nitrate. We are only beginning to understand nitrite N and O isotope systematics in nature [Casciotti and McIlvin, 2007; Casciotti et al., 2010; Buchwald and Casciotti, 2010]. For example, factors that control observed offsets between the δ15N of nitrate and nitrite in the Eastern Tropical North Pacific OMZ (up to 30‰) are uncertain. However, previous work suggests that nitrite re-oxidation is an even more efficient mechanism for lowering the nitrate Δ(15,18) [Casciotti and McIlvin, 2007; Sigman et al., 2005], as it adds high δ18O to the nitrate pool. That is, by mass balance, nitrate reduction from a particular nitrate pool and subsequent (complete) nitrite re-oxidation should produce nitrate with a similarδ15N to that initially consumed. The δ18O of the re-oxidized nitrite, on the other hand, will likely be higher than theδ18O of the nitrate consumed, due to the “branching fractionation” (preferential extraction of 16O) during nitrate reduction (i.e., produced nitrite is enriched in 18O) and the incorporation of an O-atom with a relatively higherδ18O (compared to the δ18O of the O-atom lost during nitrate reduction) during re-oxidation of nitrite to nitrate. If portions of the nitrite are further reduced to gaseous forms of N, both the nitriteδ15N and the δ18O are increased in parallel. Therefore, while this redox cycle leaves the δ15N of nitrate essentially unchanged, it would act to increase the δ18O. If anything, we would expect nitrite oxidation to be the driver of N-to-O nitrate isotope anomalies particularly in higher-T diffuse fluids, where nitrate reduction to nitrite (favored by the more reducing conditions) and subsequent nitrite re-oxidation after mixing with oxygenated deep seawater, is most likely to occur. However, as mentioned earlier, the highest nitrate isotope anomalies were not observed in the highest T (generally < ∼50°C) diffuse fluids. In summary, several processes (partial nitrification, N2 fixation, and nitrite oxidation) can theoretically produce similar negative nitrate Δ(15,18) signatures [Sigman et al., 2005; Casciotti and McIlvin, 2007], just as observed at the Juan de Fuca Ridge vent sites. In the next section, we will present results from a simple isotope box model, which we used to assess the relative fluxes of the candidate processes that could explain the N-to-O nitrate isotope anomalies in diffuse vent fluids.

3.4.3. Estimates on the Relative Importance of the Possible Nitrate Regenerating Processes

[38] We attempt here to assess the role of ammonium oxidation, N2fixation or nitrite re-oxidation in the hydrothermal conduits of the Juan de Fuca Ridge, applying a simplified steady state one-box model (analogous to the one we used in previous work [Bourbonnais et al., 2009]) in order to calculate Δ(15,18) as a function of relative changes in potential N-regenerating and consuming processes (Figure 9). In this model, we included nitrate inputs through partial nitrification of hydrothermal ammonium, N2fixation and seawater mixing, and nitrate removal through bacterial uptake and/or denitrification. Finally the model includes the internal nitrite/nitrate cycle, where a portion of the nitrite from nitrate reduction is re-oxidized to nitrate. Theδ15N of ammonium-derived nitrate is calculated according toMariotti et al. [1981]:

  • display math

where f is the fraction of reactant remaining, δ15N-NH4+initial is the δ15N of the initial reactant pool, and εnit is the kinetic isotope effect of ammonium oxidation to nitrate. We used an average N isotope effect of 26‰ during ammonium oxidation [Delwiche and Steyn, 1970, and references therein]. N2 fixation (i.e., the remineralization of newly fixed OM) adds nitrate with δ15N of −1‰ (with insignificant isotopic fractionation) [Carpenter et al., 1997, and references therein]. We assumed that part of the assimilated nitrate would be returned following organic matter ammonification and nitrification (recycled production term in Table 2). Therefore, net nitrate uptake is the gross nitrate uptake minus the recycled production. Independent of the original N source (hydrothermal ammonium versus OM from N2 fixation or bacterial uptake), a δ18O of −3.8‰ is assumed for nitrified nitrate (mean value taken from Buchwald and Casciotti [2010]). With regard to the internal nitrite/nitrate cycling, complete nitrite oxidation returns nitrate with a δ15N equal to the original nitrate, and a δ18O of nitrate of 0‰, as also assumed in the study by Sigman et al. [2005]. Subsurface mixing with seawater adds nitrate with a δ15N of ∼6.0‰ and a δ18O of ∼2.0‰. Nitrate removal occurs either by assimilation or by denitrification. We used average isotope effects of 5‰ for nitrate assimilation [Altabet, 2001], 25‰ for denitrification [Cline and Kaplan, 1975, and references therein] and 1.5‰ [Brandes and Devol, 2002] for nitrate consumption occurring in bacterial mats. Figure 10 shows the model results for both Axial Volcano and Endeavour Segment. We simulated seven representative scenarios, in which we varied the relative importance of single N fluxes in order to gain information on their respective potentials for generating the observed Δ(15,18) minima in the diffuse vent fluids (see Table 2 and Figure 10 for more details on the parameters used for the different scenarios). In the first 3 scenarios, nitrate is produced only by the oxidation of hydrothermal ammonium and we varied the relative rates and extent of hydrothermal NH4+ oxidation relative to net nitrate uptake. In scenarios 4, 5, and 6, the oxidation of hydrothermal ammonium was suppressed and we varied the relative rate of nitrate production from N2fixation or nitrite re-oxidation relative to net nitrate uptake. Finally, in scenario 7, partial ammonium oxidation and nitrite re-oxidation were combined. Across all considered scenarios, we also varied the ratio of denitrification and N uptake, and differentiated between denitrification in the open HV conduits versus that by microbial mats and biofilms on conduit walls (seeTable 2for more detail). For all scenarios, decreasing the mixing with deep-seawater (while concomitantly increasing the input of nitrate from one of the three processes mentioned above), caused a decrease in the Δ(15,18).

image

Figure 9. Simplified steady state model used in section 3.4.3 (adapted from Sigman et al. [2005] and Bourbonnais et al. [2009]): (a) representation of the addition of nitrate from N2 fixation (with a δ15N of −1‰) or from hydrothermal-ammonium oxidation with aδ15N that corresponds to the integrated product of partial ammonium oxidation [Mariotti et al., 1981], and a δ18O of −3.8‰ (average value taken from Casciotti et al. [2010]); (b) the input of nitrate from mixing with deep-seawater with aδ15N of ∼6‰ and a δ18O of ∼2‰; (c) the gross nitrate removal by nitrate assimilation and/or denitrification; (d) the remineralization of newly biosynthesized organic N to ammonium, coupled to nitrification, returning nitrate with a δ18O of −3.8‰ (and not changing the nitrate δ15N); (e) representation of the internal cycle of NO3 reduction and NO2re-oxidation (see text for details).

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image

Figure 10. Model results for Δ(15,18) for simulation scenarios presented in Table 2 for Axial Volcano (plain lines) and the Endeavour Segment (dashed lines): (a) partial ammonium oxidation, (b) N2fixation, and (c) nitrite re-oxidation (rates always relative to net nitrate uptake). Maximum Δ(15,18) anomalies observed at Axial Volcano (∼8.3‰) and Endeavour Segment (∼2.4‰) are indicated by horizontal black lines.

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Table 2. Model Simulation Scenarios Described in Section 3.4.3a
 Fractional Crustal NH4+ Oxidation (Figure 10a)N2 Fixation (Figure 10b)NO2Re-oxidation (Figure 10c)NO2Re-oxidation and Fractional NH4+ Oxidation (Figure 10c)
Scenario 1Scenario 2Scenario 3Scenario 4Scenario 5Scenario 6Scenario 7
  • a

    Corresponding values for Δ(15,18) for the various simulations are provided in Figure 10.

Fraction NH4+ consumed (%)255075nanana75
Denitrification/gross nitrate uptake0.250.50.250.50.250.40.4
% denitrification in microsites05025255000
Recycled production/NO3 assimilation0.50.750.50.750.50.2–0.50.2–0.5

[39] Our model simulation suggests that all 3 processes are capable of generating nitrate N and O isotope anomalies of a magnitude similar to that actually observed at the Endeavour Segment (minimum Δ(15,18) of −2.4‰). In our simplistic model, nitrite re-oxidation and partial ammonium oxidation can generate very low Δ(15,18) values (<−4.0‰), especially for a lower fraction of ammonium consumed, when N isotope fractionation is fully expressed, whereas N2 fixation has less potential to generate negative Δ(15,18) anomalies. Even at high rates of N2 fixation with no preformed NO3, the Δ(15,18) does not exceed ∼−3‰. Therefore, at least at Axial Volcano, it seems very unlikely that the entire nitrate isotope anomaly can be attributed to the remineralization of fixed N. Figure 10c(Scenario 7) shows that only a combination of processes (for a given ratio of 0.5 for nitrite re-oxidation/net nitrate uptake and for a 75% fractional ammonium consumption can generate a Δ(15,18) of <−8‰, as at Axial Volcano (−8.3‰). While it seems clear that nitrate production is important in the investigated diffuse hydrothermal fluids, too many unknowns preclude a more quantitative assessment of actual N regeneration pathways. The observed nitrate N and O isotope data are consistent with all of the above candidate processes, but without independent data on actual rates, the Δ(15,18) data do not allow us to reliably predict the relative importance of nitrite re-oxidation and nitrification of hydrothermal ammonium versus nitrification of N2-fixation derived ammonium, particularly at those sites where nitrification is incomplete [Wankel et al., 2007].

4. Summary and Concluding Remarks

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Material and Methods
  5. 3. Results and Discussion
  6. 4. Summary and Concluding Remarks
  7. Acknowledgments
  8. References
  9. Supporting Information

[40] First-time measurements of nitrateδ15N and δ18O and ammonium δ15N from vent fields of the Juan de Fuca Ridge (Northeast Pacific) reveal significant inter-field variations in the isotopic composition of DIN in HV diffuse waters. Highestδ15N of NH4+were observed at Axial Volcano in high-T fluids (6.7‰), indicating that deep-sea water nitrate (6.4‰) represents the original source of N for ammonium in the hot subsurface. The NH4+concentration in high-T fluids at Axial Volcano was lower than the NO3 in the seawater source, suggesting N enrichment in the rocks during basalt alteration (associated with no apparent isotope effect). The δ15N of ammonium in high-T fluids was significantly lower at both the Endeavor (3.7‰) and Cobb Segments (4.1‰), implying a low-δ15N sedimentary source at those sites. DIN concentration changes are mostly driven by advective mixing, but at some sites we have clear DIN isotopic evidence for biological N transformations in habitable lower temperature diffuse HV fluids. As for the actual N consuming and regenerating processes, our isotope data are ambivalent. Lowered NH4+ community N isotope effects (<3‰) for net NH4+consumption suggest an important contribution from gross ammonium regeneration. The generally reduced apparent nitrate N isotope fractionation suggests that fixed N uptake by bacteria is the most important N consuming process. Nitrification and denitrification are also likely to occur in microbial mats/biofilms, within the low-temperature HV conduits, where substrate diffusion limitation can lead to the underexpression of biological N isotope fractionation processes. We observed clear, and, at this magnitude, previously unseen nitrate N-to-O isotope anomalies in the HV fluids, varying strongly from site to site and from year to year, indicating spatial and temporal variations in relative rates of the nitrate regeneration versus consuming processes. We interpreted negative Δ(15,18) signatures as evidence for nitrate regeneration either by nitrite reoxidation, partial nitrification of hydrothermal ammonium and/or N2fixation and the remineralization/nitrification of the newly fixed N. Using a simple isotopic box model, we demonstrated that all three processes can produce negative Δ(15,18) at levels that were observed at the Endeavour Segment, but only a combination of processes (e.g., nitrite re-oxidation and partial NH4+ oxidation in Figure 10c, scenario 7) can generate Δ(15,18) values <−8‰, as observed at Axial Volcano. While we cannot provide conclusive evidence with regard to the actual nitrate regenerating pathways at work, it is striking that the largest observed Δ(15,18) correspond to highest bacterial cell densities at Axial Volcano, implying an important role of microorganisms in shaping DIN concentration and DIN isotope gradients in the subsurface and in diffuse HV fluids at this vent field. Here-presented DIN isotope data thus provide qualitative evidence that net loss of N from hydrothermal fluids can be attributed to microbial processes in diffuse fluids, highlighting the role of subsurface microbial communities in modulating hydrothermal geochemical fluxes to the deep ocean. Yet, limited knowledge of the physical characteristic of the subsurface biosphere of hydrothermal systems prevents more quantitative estimates on overall N elimination rates in HV. The strong variability of these systems over short time scales further complicates any modeling efforts. Time series sampling at shorter intervals could perhaps improve our knowledge of the processes that fractionate N isotopes in low-T vent fluids. Future studies of the microbial community mediating N-cycle processes, integrated with N isotope data, are required to gain an in-depth understanding of microbial pathways that turn over or produce fixed N in hydrothermal diffuse fluids. In the same vein, denitrification, anammox and DNRA rate measurements in discharging vent fluids are ongoing (A. Bourbonnais et al., manuscript in preparation, 2012), and should yield a more complete picture of N-elimination processes in the subsurface biosphere of hydrothermal systems.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Material and Methods
  5. 3. Results and Discussion
  6. 4. Summary and Concluding Remarks
  7. Acknowledgments
  8. References
  9. Supporting Information

[41] The authors wish to thank James Holden, Bill Chadwick, the officers and crew of the R/V Atlantis and R/V Thomas G. Thompson, and the ROPOS, Jason and Alvin submersible teams. Mark Rollog and Kevin Roe are thanked for laboratory assistance, and Rika Anderson (John Baross's lab, University of Washington) and Helene C. Ver Eecke (James Holden's lab, University of Massachusetts) for sharing bacterial-count data. Comments on earlier drafts of the paper by J. Granger, and three anonymous reviewers helped to improve the manuscript. This work was funded through an NSERC (Natural Sciences and Engineering Research Council of Canada) graduate fellowship to A.B., by NSERC Discovery and SNF (Swiss National Science Foundation) R'Equip funds granted to M.F.L., and by NSERC Discovery grant to S.K.J. This publication is partially funded by the Joint Institute for the Study of the Atmosphere and Ocean (JISAO) under NOAA Cooperative Agreement NA17RJ1232, contribution 1886. PMEL contribution number 3749.

References

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Material and Methods
  5. 3. Results and Discussion
  6. 4. Summary and Concluding Remarks
  7. Acknowledgments
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Material and Methods
  5. 3. Results and Discussion
  6. 4. Summary and Concluding Remarks
  7. Acknowledgments
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
ggge2114-sup-0001-t01.txtplain text document2KTab-delimited Table 1.
ggge2114-sup-0002-t02.txtplain text document1KTab-delimited Table 2.

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