Impact of Bacterial NO₃⁻ Transport on Sediment Biogeochemistry

Abstract

Experiments demonstrated that Beggiatoa could induce a H₂S-depleted suboxic zone of more than 10 mm in marine sediments and cause a divergence in sediment NO₃⁻ reduction from denitrification to dissimilatory NO₃⁻ reduction to ammonium. pH, O₂, and H₂S profiles indicated that the bacteria oxidized H₂S with NO₃⁻ and transported S⁰ to the sediment surface for aerobic oxidation.

Large species of white sulfur bacteria in the family Beggiatoaceae may accumulate and transport NO₃⁻ at intracellular concentrations up to 500 mM (5, 10, 18, 19). Therefore, it has been suggested that anaerobic H₂S oxidation coupled with dissimilatory NO₃⁻ reduction to ammonium (DNRA) by these bacteria contributes to the formation of a suboxic zone. The suboxic zones are characterized by positive redox potential and only trace concentrations of free H₂S, in spite of high sulfate reduction rates (5-7, 10, 13). Calculations based on NO₃⁻ content, mobility, and biomass distributions of Beggiatoa or Thioploca in marine sediments have indicated that bacterial transport and reduction of NO₃⁻ may indeed match H₂S production and influence distribution of H₂S in the sediment (11, 18). The extent to which this potential is realized within marine sediments is, however, not clear.

Sulfide-depleted surface sediment is essential for survival of benthic infauna, and the pathway of benthic NO₃⁻ reduction is important for control of eutrophication in nitrogen-limited coastal waters. In this study, we wanted to get more direct measures of the environmental role of Beggiatoa spp. by measuring sediment profiles of O₂, pH, and S and turnover rates of N compounds in the presence and absence of natural Beggiatoa populations.

Sediment with Beggiatoa filaments was collected from Aarhus Bay, Denmark, in Plexiglas tubes (inside diameter, 54 mm). The effect of different NO₃⁻ or O₂ concentrations on pore water chemistry was investigated on intact sediment samples, while the impact of Beggiatoa filaments was studied in reconstructed samples prepared as follows. The upper 3 cm containing the bulk of Beggiatoa filaments was cut off and kept separately in beakers. The remaining sediment was homogenized, transferred to new Plexiglas tubes, and kept with H₂S-enriched anoxic bottom water for 24 h to kill the remaining Beggiatoa filaments. The effect was confirmed by microscopical inspection. The sediment cores were hereafter left for 6 h in aerated sulfide-free seawater. Beggiatoa filaments from beakers were then added to the surface of 10 cores with broad pipettes, the remaining 10 cores serving as controls. Filaments with a diameter of approximately 13 μm dominated the Beggiatoa community completely. All cores were kept at 16°C in aquariums with seawater renewed daily; Beggiatoa cores and control cores were incubated in separate aquariums. The O₂ concentration was maintained at 160 ± 10 μM. Half of the cores were incubated at a NO₃⁻ concentration of 47 ± 2 μM, while the other half was incubated at an in situ NO₃⁻ concentration (2 μM).

Pore water O₂, ΣH₂S (H₂S plus HS⁻ plus S²⁻), and pH profiles were measured with microsensors (8, 14, 15). Profiles of pore water and cellular NO₃⁻ were determined as described in reference 18, and NO₃ was measured according to Braman and Hendrix (1). For measurement of denitrification and DNRA, ¹⁵NO₃⁻ (50 μM, 98 atom%) was added to two aquariums containing reconstructed sediment cores with or without Beggiatoa filaments, respectively (n = 4 for each treatment). ¹⁵NO₃⁻ was renewed daily, and effluxes of ¹⁵N-labeled N₂ and NH₄⁺ were measured for 14 days according to Christensen et al. (3). ¹⁵N₂ and ¹⁵NH₄⁺ were determined according to Risgaard-Petersen et al. (16, 17).

It has long been recognized that oxidation of H₂S with O₂ in Beggiatoa mats on the sediment surface acts as an efficient filter and a final barrier to the release of the toxic H₂S to the water phase (4, 9). Previous descriptive studies have further indicated that oxidation of H₂S with NO₃⁻ in Beggiatoa- or Thioploca-colonized sediments can act in a similar manner (5, 11, 18). Here we provide experimental evidence for this hypothesis. Our data show directly that Beggiatoa can induce rapid movement of the ΣH₂S front (ΣH₂S > 0.5 μM) deeper into the sediment and that this is coupled to NO₃⁻ transport (Fig. 1). In contrast to pure chemical immobilization of H₂S (e.g., see reference 2), this process is independent of sediment resuspension and fauna-mediated bioturbation and is able to proceed as long as sufficient NO₃⁻ is available for the oxidation of H₂S. In the absence of infauna and with the next resuspension event possibly not occurring for a long time, the migrating Beggiatoa may thus serve to prepare the sediment for infaunal recolonization by creating a less hostile (i.e., H₂S free) environment.

FIG. 1.

Profiles of ΣH₂S (circles), cellular pool of NO₃⁻ (squares), and pH (triangles). (A to C) Profiles in reconstructed sediment cores inoculated with Beggiatoa (open symbols) and control cores without Beggiatoa filaments (closed symbols) after 1 and 4 days of incubation. (D to F) Profiles in natural Beggiatoa-colonized sediments exposed to an in situ NO₃⁻ concentration of 1 μM (closed symbols) and with NO₃⁻-enriched bottom water (50 μM, open symbols). Error bars = standard error (n = 3 for ΣH₂S and n = 2 for NO₃⁻). The pH profiles are averaged from three profiles. Oxygen penetrated less than 0.2 mm in all cores and is not shown.

In the presence of Beggiatoa, DNRA was the predominant benthic NO₃⁻ reduction pathway. In contrast, denitrification was the only detectable pathway in the absence of Beggiatoa (Fig. 2). In theory, other, smaller bacteria that accompanied the transposed Beggiatoa tufts might be involved, but the instant and persistent DNRA activity strongly suggests that the large biomass of Beggiatoa was the primary agent.

FIG. 2.

Production of ¹⁵N₂ (circles) and ¹⁵NH₄⁺ (squares) during a 14-day time course from reconstructed sediment cores in the presence (open symbols) and absence (i.e., control cores) of Beggiatoa (closed symbols). Error bars indicate standard error (n = 4).

Beggiatoa performs H₂S oxidation in two steps (12, 20). H₂S is first oxidized:

(1)

Then S⁰ is oxidized:

(2)

The first step increases pH, while the second step decrease pH, and together the two processes are pH neutral. The distinct sigmoid pH profile in the Beggiatoa-inhabited sediment cores (Fig. 1) thus indicates a spatial separation of the processes. The pH maximum right at the H₂S front clearly indicates the location of the first step (reaction 1). Further, the distinct pH minimum at the oxic-anoxic interface strongly suggests that the Beggiatoa filaments prefer the energetically more favorable and much more acidogenic aerobic oxidation of S⁰:

(3)

A rapid disappearance of the subsurface pH minimum after reducing the O₂ concentration in the overlying water of Beggiatoa-colonized sediment (Fig. 3) further supported the proposed combined anaerobic-aerobic H₂S oxidation mediated by NO₃⁻ and S⁰ transport (the combination of reactions 1 and 3). This mechanism also implies a fourfold better utilization of NO₃⁻, as the first step only accounts for one-fourth of the complete H₂S oxidation to SO₄².

FIG. 3.

Change in pH profiles in Beggiatoa-colonized sediment cores following an immediate oxygen reduction in the water column from 266 μM to 30 μM. The time zero pH profile (closed circles) was measured at 266 μM O₂ and is averaged from pH measurements at four independent sites. pH was subsequently measured at the same site 6, 16, and 27 min after the O₂ concentration was reduced to 30 μM (open symbols). O₂ penetrated to 0.25 ± 0.03 mm (n = 7, broken line) at the high O₂ concentration. A few minutes after O₂ was reduced to 30 μM in the bottom water, no O₂ was detectable in the sediment. ΣH₂S was detectable from a depth of approximately 5 mm.

In conclusion, this study has demonstrated that bacterial nitrate transport is yet another complex mechanism that really has to be taken into account to understand how sulfide-depleted sediment environments are sustained.