Jiang et al. (Nature Communications, 2025) reported that deep-sea cold seep sediments support elevated nitrogen-loss rates driven by an unexpectedly broad diversity of microorganisms, and estimated a global flux of 4.96–7.63 Tg N yr⁻¹ from the top 5 cm of cold seep sediments. The estimate rests on potential rates measured in slurry incubations at atmospheric pressure, extrapolated over an imprecisely known global seep area, and the putative agents — anammox bacteria in phyla beyond Planctomycetota, newly expanded nitrous-oxide reducers, and nitric-oxide-dismutating organisms — are implicated only by gene presence, transcript abundance, and predicted protein structures. The open problem is to determine the actual magnitude and the microbial agents of cold seep nitrogen loss: quantifying in situ (or pressure- and temperature-faithful) nitrogen-loss rates, constraining the global cold seep area, confirming whether the novel lineages actively catalyze nitrogen loss, and establishing the ecological and evolutionary mechanisms — the roles of the new nitrous-oxide-reducing phyla, the contribution of horizontal gene transfer of nosZ via mobile genetic elements, and the functional significance of the structural adaptations of cold-seep hydrazine synthase.

Background

Cold seeps are seafloor regions where methane-rich fluids percolate through sediments, sustaining chemosynthetic microbial communities (microbes that derive energy from chemical compounds rather than sunlight) in the deep sea. Fixed nitrogen is removed from the ocean by two main microbial nitrogen-loss pathways: denitrification, the stepwise reduction of nitrate to dinitrogen gas (N2), and anammox, anaerobic ammonium oxidation, in which ammonium and nitrite are combined to N2 via the intermediate hydrazine. The diagnostic enzyme of anammox is hydrazine synthase, whose alpha subunit is encoded by the gene hzsA; nitrous-oxide reductase (encoded by nosZ) catalyzes the last step of denitrification, reducing the greenhouse gas N2O to N2; nitric oxide dismutase (encoded by nod) splits NO into N2 and O2 and enables 'oxygenic' denitrification. Classically, all known anammox bacteria belong to the phylum Planctomycetota, and N2O reducers were thought to fall into two nosZ clades. Measuring nitrogen loss in situ is difficult: the standard approach incubates homogenized sediment slurries with 15N-labeled tracers in the laboratory at atmospheric pressure, which may misrepresent rates under the high hydrostatic pressure (hundreds of atmospheres) and low temperature of the deep sea. Only in sediments of the hadal trenches (the deepest ocean zones, at 6–11 km depth) have in situ rates been obtained with autonomous benthic landers (instrumented seafloor platforms that carry out incubations at ambient pressure), where laboratory and in situ N2-production rates matched closely (Thamdrup et al., PNAS 2021, Atacama Trench), and separate work shows high hydrostatic pressure can stimulate microbial nitrate reduction (Yang et al., Nature Communications 2024). Against this backdrop, Jiang et al. (Nature Communications 16, 1646, 2025; first posted as bioRxiv 10.1101/2024.06.05.597523) combined porewater geochemistry, 15N isotope-tracing on slurry incubations, metagenomics, and metatranscriptomics across South China Sea cold seeps (Lingshui, Haima, Site F). They measured average potential denitrification rates of 2.30 ± 4.03 nmol N cm⁻³ h⁻¹ and anammox rates of 0.14 ± 0.21 nmol N cm⁻³ h⁻¹ (far above non-seep controls), analyzed a 147-million-gene catalog and 3,164 metagenome-assembled genomes (MAGs; genomes binned directly from environmental DNA), and reported that nitrous-oxide reducers expand by three phyla, nitric-oxide-dismutating organisms by one phylum and two orders, and hzsA-bearing organisms span ten phyla beyond Planctomycetota — several of them transcribing hzsA at high levels. Extrapolating these potential rates over a global cold seep area of 2.05–3.15 × 10⁵ km², they estimated a global nitrogen-loss flux of 4.96–7.63 Tg N yr⁻¹, i.e. 1.65–2.54% of the ~300 Tg N yr⁻¹ lost from all marine sediments. The authors themselves flag the fragility of this claim: the measured rates are potentials that 'might not capture exact in situ rates', the experiments 'did not replicate the high hydrostatic pressure found in natural settings', the 'extent of global cold seep areas remains imprecise', the horizontal transfer of nosZ via mobile genetic elements (MGEs; plasmids and other transmissible DNA elements) 'may play a significant role' in diversification, and the structural adaptations of the novel hydrazine synthases (N-terminal signal peptides, a C-terminal α-helix, fused β–γ subunits) 'may optimize these proteins for the unique conditions of cold seeps' — as yet only predictions from AlphaFold (a deep-learning protein-structure prediction method) structural modeling, and they conclude that 'future studies with in situ techniques and advanced modeling approaches will help refine these estimates'.

Problem Statement

Determine whether deep-sea cold seeps are in fact major sites of marine nitrogen loss, and establish the magnitude, agents, and origins of that loss. The source study's claim is unvalidated in four linked respects that jointly define the problem: (1) Rate fidelity — quantify actual nitrogen-loss rates (denitrification and anammox) in cold seep sediments under natural conditions, i.e. in situ or under the ambient hydrostatic pressure and temperature, replacing the atmospheric-pressure slurry-incubation potentials on which the current estimate rests; (2) Global extent — constrain the global cold seep area (currently an imprecise 2.05–3.15 × 10⁵ km²) well enough to make the extrapolated global flux of 4.96–7.63 Tg N yr⁻¹, and its share of the ~300 Tg N yr⁻¹ marine sediment nitrogen loss, defensible; (3) Agent confirmation — establish whether the hzsA-bearing organisms in phyla beyond Planctomycetota, the expanded nitrous-oxide reducers, and the nitric-oxide-dismutating lineages actually catalyze nitrogen loss; (4) Mechanism and origin — determine the ecological roles of the newly recognized N2O-reducing phyla and their interactions with other microbial groups in cold seep sediments, quantify the contribution of horizontal gene transfer of nosZ via mobile genetic elements to the distribution and evolution of nitrogen-loss genes, and establish experimentally whether the structural adaptations of the cold-seep hydrazine synthases (N-terminal signal peptides, C-terminal α-helices, fused β–γ subunits) confer fitness advantages under cold seep conditions. Components (1)–(3) jointly settle the central quantitative question — the true contribution of cold seeps to the global marine nitrogen budget — while component (4) supplies the mechanistic and evolutionary account of the diversity that components (1)–(3) quantify.

References

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