Introduction

Nitrate is abundant (∼30–40 μM) in the deep, oxygenated bottom waters that overlie cold seeps, but no data describing dissimilatory nitrate reduction (hereafter DNF) or total nitrate removal rates in such environments are available. Most studies of nitrate cycling at cold seeps have focused on qualitative descriptions of vacuolate sulfide-oxidizing bacteria (Teske and Nelson, 2006 and references therein), which concentrate nitrate in their vacuoles and couple nitrate reduction to sulfide oxidation (McHatton et al., 1996, Sayama et al., 2005).

Gulf of Mexico (GOM) cold seeps are characterized by abundant stocks of reduced organic carbon in the form of liquid and gaseous hydrocarbons (Arvidson et al., 2004, Joye et al., 2004). Denitrifying bacteria have a metabolic flexibility similar to that of sulfate-reducing bacteria and can degrade liquid hydrocarbons (Widdel and Rabus, 2001). Considering the abundant carbon sources and relatively high concentrations of nitrate in overlying waters (Joye et al., 2004, 2010), heterotrophic DNF is a likely metabolic pathway at cold seeps.

Heterotrophic, or non-sulfide-based, DNF has not been measured at cold seeps, which is surprising, as cold seeps support extremely high rates of heterotrophic metabolism, mainly sulfate reduction (Arvidson et al., 2004; Bowles et al., in press). Hexadecane oxidation coupled to DNF, for example, is a highly exergonic process generating 983 kJ per mole N2 formed (Widdel and Rabus, 2001):

where activities were assumed to be 10−2, pH 7, and liquid n-hexadecane was considered. Here we present the first rate assays of heterotrophic potential denitrification from sediments from a northern GOM cold seep.

Materials and methods

Sediment covered by a Beggiatoa mat was collected from an active cold seep in the Northern GOM (lease block Mississippi Canyon 118; see Lapham et al., 2008 for details). Geochemistry (nitrate, nitrite, ammonium, sulfate and sulfide concentration determination) sampling and methods were as described in Joye et al. (2004). We measured heterotrophic potential denitrification rates in helium-purged, nitrate, nitrite, and ammonium-free, sulfide- and sulfate-free sediment slurries (2:1 artificial porewater (adapted from Weston and Joye, 2005) to sediment) from Beggiatoa-inhabited surface sediments (0–6 cm) and a deeper layer (6–12 cm) lacking visible filaments. Nitrate removal rates were determined from the linear decrease in nitrate concentration over time, while heterotrophic potential denitrification rates were estimated from the evolution of 29N2 and 30N2 over time (Kana et al., 1998). Briefly, ∼75 μM 15NO3− and 2 mM C (equimolar C from lactate and acetate) were added to headspace-free hungate tubes and tubes were sampled for dissolved constituents at multiple time points (Porubsky et al., 2009).

Results and discussion

The concentration of total dissolved inorganic nitrogen species (ammonium, 260 μM; nitrate, 180 μM; and nitrite, 8 μM) was highest just below the sediment surface (1.5 cm, Figure 1a). Pore water sulfate concentration profiles illustrated significant removal at depth and a concomitant increase in sulfide (Figure 1b). The observation of low sulfide accumulation and significant concentrations of total dissolved inorganic nitrogen in upper sediments may reflect nitrate storage and sulfide oxidation by Beggiatoa (Joye et al., 2004).

Figure 1
figure 1

(a) Geochemical constituent NH4+ (μM), NO3− (μM), NO2− (μM), SO42− (mM) and H2S (μM) versus depth (cm). (b) Average concentrations of reactant, NO3− (μM), and potential products, NO2−, 29N2 and 30N2 (μM) versus time (h) for slurried sediments from 0 to 6 cm, as well as (c) 6–12 cm horizons.

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Both the upper and lower sediments supported significant rates of heterotrophic potential denitrification. In the surface sediments, nitrate was depleted rapidly and the production of 15N-labeled dinitrogen products (17 μM 30N2 and <1 μM 29N2) was easily detectable after 4 h (Figure 1b). Linear substrate removal and product accumulation over time yielded estimates of nitrate removal and heterotrophic potential denitrification rates of 96 and 32 μM N day−1, respectively. In the deeper sediments, nitrate was also rapidly consumed (Figure 1c) and at the same time 5 μM of 30N2 formed; 29N2 generation was low (<1 μM). Rates of nitrate removal and heterotrophic potential denitrification were lower, being 52 and 11 μM N reduced day−1, respectively.

At GOM cold seeps, sulfate reduction is the only terminal metabolic process that has been quantified (Arvidson et al., 2004; Joye et al., 2004). We show that denitrification and nitrate removal are also important processes at these seeps. Denitrification is well documented in numerous other environments (Seitzinger, 1988 and references therein), and integrated rates typically range from 1 to >1000 μmol N m−2 h−1 in freshwater and marine systems. The depth-integrated potential denitrification rates for GOM seeps are 80 and 27 μmol N m−2 h−1 for upper and lower sediment horizons, respectively. These rates are comparable with the rates from coastal marine sediments (Seitzinger, 1988), and from moderately eutrophic to eutrophic freshwater environments. Nitrate removal could also reflect additional processes such as assimilation, vacuole storage, reduction to ammonium (DNRA), or nitrite production and removal via anaerobic ammonium oxidation (ANAMMOX). Previously published data would suggest that DNRA may be more important in sulfidic and carbon-rich cold seep sediments than ANAMMOX (Burgin and Hamilton, 2007).

The integrated heterotrophic potential denitrification rates result in an extremely fast turnover time (on the order of 2 days) for the nitrate pool in these seep sediments (Figure 2). The sulfate pool turnover time was 11–72 days (Bowles et al., in press; Figure 2). When factoring in total nitrate removal, the turnover time of the nitrate pool is faster, being 0.7 and 0.4 days in the upper and lower sediment horizons, respectively. These results lead us to postulate that (1) heterotrophic denitrification is a relevant and important component of C and N cycling in cold seeps and (2) dissimilatory nitrate reduction, nitrate storage in vacuoles and/or assimilation into biomass require tight coupling of nitrogen-cycling processes and raises the potential for internal nitrate sources, such as anaerobic nitrification (Luther et al., 1997), and/or downward advection of bottom water nitrate within these habitats.

Figure 2
figure 2

Turnover times (day) of nitrate and sulfate from MC118 (sulfate turnover time calculated from the data in Bowles et al., in press) due to total nitrate removal and heterotrophic potential denitrification, and SR, respectively.

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