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Deep-Sea Archaea Fix and Share Nitrogen in Methane-Consuming Microbial Consortia

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Science  16 Oct 2009:
Vol. 326, Issue 5951, pp. 422-426
DOI: 10.1126/science.1178223

Balancing the Nitrogen Budget

Setting the global budget for elements presents difficult challenges, such as accounting for possibly unknown sources or sinks. An unresolved imbalance in the oceanic nitrogen budget suggests that there may be additional sources of biological nitrogen fixation in the deep sea. Using high-resolution imaging techniques, Dekas et al. (p. 422; see the Perspective by Fulweiler) observed direct assimilation of isotopically labeled N2 by anaerobic methane-oxidizing archaea from deep marine sediment and the subsequent transfer of nitrogen to their sulfate-reducing bacterial symbionts. This slow and energetically costly conversion by archaea is dependent upon methane and requires physical contact with the associated bacterial partner. Such syntrophic consortia represent a potential source of nitrogen in the oceans and may help to balance the global nitrogen budget.

Abstract

Nitrogen-fixing (diazotrophic) microorganisms regulate productivity in diverse ecosystems; however, the identities of diazotrophs are unknown in many oceanic environments. Using single-cell–resolution nanometer secondary ion mass spectrometry images of 15N incorporation, we showed that deep-sea anaerobic methane-oxidizing archaea fix N2, as well as structurally similar CN, and share the products with sulfate-reducing bacterial symbionts. These archaeal/bacterial consortia are already recognized as the major sink of methane in benthic ecosystems, and we now identify them as a source of bioavailable nitrogen as well. The archaea maintain their methane oxidation rates while fixing N2 but reduce their growth, probably in compensation for the energetic burden of diazotrophy. This finding extends the demonstrated lower limits of respiratory energy capable of fueling N2 fixation and reveals a link between the global carbon, nitrogen, and sulfur cycles.

Nitrogen-fixing (diazotrophic) bacteria and archaea convert dinitrogen (N2) into ammonia (NH3) for assimilation. Biological N2 fixation counteracts the removal of bioavailable N by microbial processes such as denitrification and anaerobic ammonium oxidation (anammox) and provides a source of N to the majority of the biosphere that cannot directly assimilate N2. Many photosynthetic cyanobacteria fix N2 in ocean surface waters and have been the primary focus of studies on marine diazotrophy. Recently, a discrepancy between the calculated rates of oceanic denitrification and N2 fixation has suggested that other less well–studied or currently unknown diazotrophic microorganisms may exist and fix substantial amounts of N2 (15). Indeed, recent discoveries of new phylogenetically and physiologically diverse diazotrophs, including hyperthermophilic methanogens from hydrothermal vents (6), have shown that N2 fixation can occur in extreme environments and localized habitats of enhanced productivity in the deep sea (5, 7, 8).

Here we show that syntrophic aggregates of archaea (of the ANME-2 group) and bacteria [Desulfosarcina/Desulfococcus (DSS)] mediating sulfate-dependent anaerobic oxidation of methane (CH4) (AOM) in deep-sea sediments are capable of N2 fixation. The ANME-2/DSS consortia have been studied in recent years both because of their potentially critical role in marine carbon cycling and their enigmatic obligate syntrophy (9, 10). These consortia are most abundant in areas of high CH4 concentration, such as cold seeps, but are present throughout continental margin sediments [(9) and references therein]. They currently represent the main filter for oceanic CH4 release to the atmosphere, consuming up to 80% of naturally released CH4 in marine sediments (9); however, the specific mechanism(s) coupling the ANME-2 and DSS cells remains unclear. Recent metagenomic sequencing of the ANME-2/DSS consortia identified the presence of nitrogenase genes required for N2 fixation (nif genes) (11). This result, along with preliminary N isotope data, suggests that microbes within the consortia are able to fix N (11). We used submicron-scale ion imaging by nanometer secondary ion mass spectrometry (nanoSIMS) coupled to fluorescence in situ hybridization (FISH) to specifically identify the ANME-2 species as diazotrophs while detailing and quantifying patterns of N assimilation within the individual members of these metabolically interdependent consortia.

Sediment samples from an active CH4 seep in the Eel River Basin, California, USA, were collected and anaerobically incubated with CH4 and one of several 15N-labeled N sources (12) (table S1). Nitrogen fixation, as demonstrated by the assimilation of 15N from 15N2 in coaggregated ANME-2 and DSS cells, occurred in all AOM consortia measured after 6 months of incubation with CH4 (12) (Fig. 1, A and B, and Fig. 2A). 15N enrichment within the consortia was as high as 10.5 15N atom %, which is 26 times the highest value observed in unlabeled ANME-2/DSS consortia (ranging from 0.35 to 0.4 15N atom %). Inhibition of either CH4 oxidation (incubations lacking CH4) or sulfate reduction [incubations treated with the inhibitor sodium molybdate (Na2MoO4)] prevented 15N incorporation (Fig. 2), implying that N2 fixation requires a functioning symbiosis between the CH4-oxidizing ANME-2 and sulfate-reducing DSS partners. Other microbial cells from the 15N2 incubation were not enriched in 15N (maximum 0.38 15N atom %, n = 10 cells), suggesting that 15N2 incorporation was specific to the ANME-2/DSS consortia over the incubation period and not due to nonspecific cycling of reduced 15N after fixation by an unrelated group of organisms. 15N was also incorporated from 15N-labeled cyanide (C15N), a toxic molecule structurally similar to N2 and known to be detoxified and assimilated by some, but not all, diazotrophs (13) (Figs. 1D and 2A). The broad substrate recognition by nitrogenase is hypothesized to be a relict ability from when the protein first evolved, when it may have primarily catalyzed reactions other than N2 fixation [such as cyanide detoxification (12, 1416)].

Fig. 1

Magnitude and distribution of 15N incorporation in representative methanotrophic ANME-2/DSS consortia from sediments incubated with CH4 and different 15N-labeled N sources, as indicated. (A to E) FISH using probes targeting ANME-2 (Eel932) in red and Desulfobacteraceae (DSS658) in green. (I to V) Ion micrographs of 12C15N/12C14N ratios of the same microbial consortia imaged by FISH in (A) to (E), demonstrating the location of 15N incorporation. The scale range varies between ion micrographs, with the minimum consistently set to natural abundance 15N/14N.

Fig. 2

Effect of N source on rates of cellular growth and respiration. (A) 15N incorporation in ANME-2/DSS consortia from bulk sediment incubated with each of the indicated N sources and/or inhibitors. Each data point represents the average 15N (atom %) value of the most 15N-enriched ion image (depth plane) of a single aggregate. Symbol size and shape indicate the time the aggregate was incubated: small solid black circles, 1 month; small gray circles, 3 months; large gray circles, 4 months; large black circles, 6 months. The horizontal dashed line represents natural abundance 15N/14N. NM, not measured. (Inset) Change in abundance of aggregates (aggs) over time in the N2 (dashed line) and NH4+ (solid line) incubations determined by staining with 4′,6′-diamidino-2-phenylindole. Error bars represent 1 SD from the mean. (B) Sulfide production in the different 15N sediment incubations. Each data point (square symbol) represents the value for a single incubation bottle at the time sampled, following the same symbol size and color trend noted in (A). There was no inhibition by 2-bromoethanesulfonic acid (BES) (12).

N2 fixation appears to be primarily mediated by ANME-2, based on the distribution of 15N within the consortia. In aggregates of shelled morphology (an inner sphere of archaeal cells surrounded by an outer layer of bacterial cells, approximately 500 cells total; n = 5 aggregates), the 15N label was concentrated in the center of the aggregate, where the ANME-2 biomass was concentrated (Figs. 1A and 3). Additionally, ANME-2/DSS aggregates showed 15N enrichment colocalized with light δ13C biomass, a signal diagnostic of methanotrophic ANME-2 species (11, 17) (n = 6 aggregates; fig. S1). This differs from the variable pattern of 15N incorporation observed in the majority of aggregates from incubations amended with 15N-labeled ammonium (15NH4+) and nitrate (15NO3) and indicates that the elevated enrichment from 15N2 within the ANME-2 archaea is attributable to diazotrophic activity, not simply a varying rate of protein synthesis between species (Fig. 1).

Fig. 3

Serial FISH and 12C15N/12C14N ion images in a representative shelled ANME-2/DSS aggregate showing the distribution of 15N incorporation from 15N2 with depth (left panels). In the FISH series, red indicates archaeal cells and green indicates bacteria. Comparison of 15N incorporation on the inside of the aggregate (dominated by archaea) and the outside (dominated by bacteria) is shown on the right. Each gray data point represents the 15N/14N of a hand-drawn ROI, approximating the size of a cell (1 μm) (12). Box and whisker plots indicate 75%, median, and 25% values for all ROIs drawn in either the inside (ANME-2, red) or outside (DSS, green) at a particular depth in the aggregate. The inset at right shows an example of ROIs designated for interior and exterior portions of the cell aggregate from a single depth. All ion micrograph values are scaled from 0.0036 to 0.11.

Serial FISH and SIMS images collected through individual aggregates reveal the three-dimensional distribution of 15N assimilation from 15N2 within AOM consortia (Fig. 3). The difference in 15N atom % between the group of cells on the aggregate exterior (DSS-dominated) and the group in the interior (ANME-dominated) became greater with increasing penetration into the core of the aggregate, corresponding to an increasingly pure population of ANME in the interior (Fig. 3). Although the aggregate exterior averaged 31% less 15N enrichment than the interior, all of the DSS cells on the periphery of the aggregate were enriched in 15N relative to natural abundance {average 15N atom % = 3.47 exterior [n = 313 regions of interest (ROIs)] and 5.01% interior (n = 297 ROIs), Fig. 3}, suggesting a passage of reduced N from the ANME cells in the interior to the DSS-dominated exterior. The reduced 15N enrichment in the DSS cells relative to the ANME cells is consistent with the trend observed in 15N labeling studies of other symbioses, in which reduced N is shared between a diazotrophic and a nondiazotrophic partner (18, 19). Transfer of reduced N species between symbionts is common, often in exchange for energy-rich metabolites or structural protection (20). It is possible that inherent variations in metabolism and growth between the two partners may also lead to an offset in 15N enrichment (21), and the possibility of concurrent fixation by both syntrophic partners at differing rates cannot be excluded at this time. However, in the context of molecular data acquired in parallel, this scenario appears less likely.

The analysis of nif sequences recovered from the 15N2 sediment incubation was consistent with previous reports of a CH4 seep–specific nifH clade (fig. S2). The diverse nifH genes recovered clustered primarily within a divergent clade of sequences reported from geographically distant deep-sea CH4 seeps and whole-cell enrichments of ANME-2/DSS consortia from the Eel River Basin (11, 22) (fig. S2). The existence of this nifH clade highlights the strong similarities between putative diazotrophs at geographically distant CH4 seeps; however, its divergence from known diazotrophs has made previous attempts to assign the clade to either the Bacteria or Archaea specultative (22). We therefore collected and analyzed partial nif operons from the incubations and found that they contained the typical gene order (nifH, nifI1, nifI2, nifD, and nifK) of the C-type operon in methanogenic archaea and some nonproteobacterial anaerobic diazotrophs (23) (fig. S3). Additionally, the nifD phylotypes within these operons grouped within a well-supported clade containing sequences retrieved from other CH4 seep sediment samples, methanogenic archaea (Methanococcus, 49% similarity), and nonproteobacterial N-fixing lineages rarely found at CH4 seeps but which have been hypothesized to have undergone lateral gene transfer with archaea (such as Clostridia and Roseiflexus spp.) (23, 24) (Fig. 4). In the context of seep microorganisms, these data are most consistent with an archaeal origin for these operons. The nifH fragments of the partial operons cluster within the putatively seep-specific nifH clade, suggesting that this clade is archaeal, and supporting our designation of the ANME-2 archaea as the primary diazotrophic microorganism in the consortia.

Fig. 4

Unrooted neighbor-joining tree of translated nifD sequences after global alignment. Bootstrap values from 85 to 100% (solid circles) and from 70 to 85% (open circles) are indicated at the nodes. The scale bar represents changes per amino acid position. The sequences obtained in this study are shown in bold. Alternative nitrogenases are those that use V-Fe and Fe-Fe cofactors (vnfD and anfD, respectively). Roman numerals represent nitrogenase clusters as originally defined in (30). Names of sequences represented by numbers can be found in table S2.

N2 fixation in ANME-2/DSS consortia is intriguing from an energetic standpoint; its cost is one of the highest for any anabolic process, requiring an investment of up to 16 adenosine triphosphate molecules (equivalent to ~800 kJ) for each N2 molecule reduced (8). Moreover, AOM coupled to sulfate reduction is thought to be one of the least energetically productive metabolisms known (10). At CH4 seeps, coupled CH4 oxidation and sulfate reduction reactions yield a total of approximately 40 kJ/mol of CH4 (10) that must be shared between the two syntrophic partners. Although other energy-limited diazotrophic microorganisms exist (such as methanogens) none to our knowledge generate less energy per mole of substrate than the ANME-2 species. One possibility is that in unusual environments, such as the deep sea, structural or mechanistic differences in the N2 fixation machinery may alter the energetic burden. The low sequence similarity of the recovered nif genes to those previously described suggests some deviation from characterized N2-fixing systems (Fig. 4 and figs. S2 and S3).

Slowed growth is a common response to the energetic burden of N2 fixation in active diazotrophs, including methanogenic archaea (25). Accordingly, using 15N incorporation as a proxy for growth, the ANME-2/DSS consortia in this study actively fixing N grew approximately 20 times slower on average than aggregates grown in parallel with ammonium (Fig. 2). Although ANME-2/DSS growth rates are substantially affected by the available N source, the rate of AOM by the consortia [estimated by CH4-dependent sulfide production (12)] was similar during growth on either N2 or NH4 (Fig. 2B). Therefore, regardless of the exact amount of energy required to fix N2 in these organisms, the consortia appear to compensate for the energetic burden of N2 fixation by slowing growth while maintaining similar rates of respiration.

The maintenance of nif genes by the ANME-2 cells, and their consortial ability to fix N in the laboratory, imply that they do so in marine environments. Diazotrophy within deep-sea CH4 seeps has not been detected directly, but N2 fixation has been suggested at these locales, based on low δ15N values of seep sediment and fauna (26, 27). Why N2 fixation would occur in anoxic marine sediments, often replete with ammonium, warrants further consideration. One explanation is that the CH4 seep environment differs from typical anoxic sediment in that the main source of C (CH4) is unaccompanied by N, poising its consumers for N limitation, similar to photoautotrophs (8) and aerobic methanotrophs (28). Indeed, measurements of pore water ammonium from the Eel River Basin CH4 seeps were highly variable, ranging from 101 to 16 μM over a 6-cm sediment depth profile; these concentrations would not completely inhibit N2 fixation in cultured diazotrophic methanogens (such as Methanococcus maripaludis) (29). Even in ammonium-replete sediments, localized zones of N limitation may occur (for example, within densely packed microbial consortia). Although the loss of nitrate and ammonium from CH4 seep sediments by catabolic bacterial processes (such as denitrification or anammox) has not yet been determined, these sinks for fixed N may also promote enhanced diazotrophy by the in situ microbial assemblage (3). Additionally, the current discrepancy in the oceanic fixed N budget underscores the possibility of new sources of fixed N in nontraditional and potentially unexpected habitats (13, 7). The extent to which the ANME-2/DSS consortia contribute to the putatively missing fraction of global fixed N inputs is unknown, but their input is probably not the only missing term in the equation. N2 fixation in ANME-2, combined with the diversity of nifH genes recovered from marine sediments here and previously (5, 11, 22), suggests that our inventory of marine diazotrophs is incomplete and that we are only beginning to understand the extent and importance of benthic marine N2 fixation.

Supporting Online Material

www.sciencemag.org/cgi/content/full/326/5951/422/DC1

Materials and Methods

SOM Text

Figs. S1 to S3

Tables S1 to S4

References

References and Notes

  1. Information on materials and methods is available on Science Online.
  2. We thank C. House, A. Schmitt, K. McKeegan, Y. Guan, J. Eiler, and L. Remusat for assistance with the ion microprobe data collection; S. Joye, M. Boyles, M. Walton, J. Howard, N. Dalleska, O. Mason, A. Green, P. Tavormina, S. Goffredi, C. Gammon, and the shipboard party and crew of the R/V Atlantis and DSSV Alvin for support in the field and laboratory; and J. Howard, W. Fischer, J. Amend, C. Anderson, D. Fike, D. Sigman, V. Rich, J. Bailey, D. Newman, J. Grotzinger, T. Hoehler, J. Delacruz, and three anonymous reviewers for helpful suggestions regarding this manuscript. Funding was provided by NSF (grant MCB-0348492), the Gordon and Betty Moore Foundation, and an NSF Graduate Research Fellowship (A.E.D.). The Caltech Center for Microanalysis and nanoSIMS 50L are funded by the Gordon and Betty Moore Foundation, and the University of California Los Angeles ion microprobe is partially supported by the NSF Instrumentation and Facilities Program.
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