Abiotic N_2 reduction in submarine hydrothermal systems could quickly fertilize prebiotic oceans

Domain: earth_science
Published: 2025-11-28
Editorial outcome: minor_revision

Abstract
NH3 or NH4+ is an essential component of the abiotic synthesis of organic compounds for the origin of life and an efficient greenhouse gas to address the faint young Sun paradox on the early Earth. Sustainable NH3 or NH4+ on the N2-dominated prebiotic Earth’s surface requires potent abiotic N2 reduction (ANR) in hydrothermal systems, which has not been detected in the geological record despite numerous laboratory demonstrations. Here we report high concentrations and extreme 15N depletions of NH4+ in hydrothermal veins in oceanic crusts drilled from the South China Sea basin. Our data indicate that abundant 15N-depleted NH4+ was produced by ANR in deep fluid but progressively overprinted by 15N-enriched biogenic NH4+ toward the surface. Modeling suggests that ANR could supply up to 9.0–10.8 × 1010 mol·year-1 NH4+ to global oceans, which is minor to the large nitrogen inventory in modern oceans, but could quickly fertilize the oceans and supply NH3 to the atmosphere in the prebiotic Earth.

Introduction
Given the important role of abiotic N2 reduction (ANR) in early Earth’s geological nitrogen (N) cycle, particularly in providing the essential compound of NH3 (or its dissolved equivalent NH4+) for the origin of life in submarine hydrothermal vents1–3 and for establishing a warm and habitable environment under dimmer solar luminosity (i.e., the faint young Sun paradox)4,5 on the N2-dominant early Earth surface6, this process has been intensively tested by laboratory experiments.

The experimental results demonstrated that ANR could be catalyzed by a variety of naturally occurring minerals and rocks (e.g., FeS, Fe-Ni alloy, green rust, magnetite, peridotite) under submarine hydrothermal conditions7–18. However, ANR has not been convincingly detected in field samples, particularly modern submarine hydrothermal vent fluids that have been collected for study. One of the major responsible factors is the overprinting by NH4+ generated in shallow hydrothermal systems from biologically processed N sources such as organic matter, dissolved NO3- and NH4+ in seawater, and dissolved organic N and NH4+ in pore water of sediment. For example, when a sediment cover exists near hydrothermal systems, elevated temperature (T) condition can induce decomposition of organic matter and/or NH4+ desorption from clays19. In addition, dissolved NO3- in seawater can be effectively reduced (even abiotically) in shallow hydrothermal systems as low as 24°C17,18. These processes can contribute remarkable amounts of NH4+ to increase the NH4+ concentrations of shallow hydrothermal fluids to more than an order of magnitude higher than the ambient seawater NH4+ concentration (~1 μM)20. Because these NH4+ components are all derived from surface N sources in shallow localities, they are referred to as surface NH4+ hereafter. Due to the similar N isotopic signatures of these surface N sources, e.g., +3‰ to +8‰ for dissolved NO3- in seawater21, +2‰ to +10‰ for marine organic matter/sediments22 and similar range (with high value up to +17‰) for dissolved organic N and NH4+ in interstitial water23, it is difficult to distinguish between NH4+ derived from these surface sources. Regardless, such 15N-enriched surface NH4+ can effectively overprint the 15N-depleted signal from deep source (e.g., –5‰ for the upper mantle)24,25 in shallow hydrothermal fluids.

A better geological proxy for detecting an ANR signal would be the overlooked hydrothermal veins deposited from focused flow of deep hydrothermal fluids, for two reasons. Firstly, although deep hydrothermal fluids could be derived from deeply circulated seawater, surface NH4+ could be progressively consumed (by NH4+ assimilation into alteration minerals in oceanic crust26–31) along seawater circulation pathway into depths. Consequently, the deep hydrothermal fluids should undergo minimal impact from surface NH4+ and have the best chance to expose NH4+ produced by ANR. Secondly, studies on field samples32,33, laboratory experiments34 and theoretical calculations35 suggest that NH4+ can substitute K+ and Na+ in silicate minerals. Thus NH4+ in deep fluids can partially partition into K+- and Na+-bearing vein minerals (e.g., plagioclase, epidote, chlorite) upon their deposition. Once fixed in the structure of vein minerals, NH4+ can be well protected from subsequent low-T disturbance. As a result, vein minerals deposited in the focused flow channel of deep fluids can best reveal the deep-fluid NH4+ signature and have the best chance to disclose the ANR signal (if there is any).

Despite recent advance in characterizing the NH4+ signature of hydrothermally altered oceanic crust (i.e., seafloor basalts, sheeted dikes and gabbros, and serpentinized peridotites)26–31, NH4+ in hydrothermal veins in oceanic crust has been rarely examined by far. International Ocean Discovery Program (IODP) Expeditions 367 and 368 drilled into the 16–32 million-year-old oceanic crusts in the South China Sea basin36 (Fig. 1; Supplementary Information). Recovered mid-ocean ridge basalts (MORB) from Hole U1502B show an E-MORB affinity (Supplementary Fig. 2). All these rocks have been altered at various degrees and contain abundant hydrothermal veins with thickness from submillimeter to a few millimeters (Supplementary Fig. 3). The mineral assemblage of hydrothermal veins is dominated by albite and quartz with variable amounts of Fe-Mg-Ca carbonates, chlorite, epidote, pyrite, and Fe–Mn hydroxides (“Methods”; Supplementary Data 1 and Supplementary Fig. 4), which were precipitated from relatively high-T fluids (200−300°C; Supplementary Information)36–38. The hydrothermal fluids, as determined from trace elements and radiogenic isotopes of vein minerals, were a mixture of modified seawater (after reaction with MORB) and deep magmatic fluid38. Here we report the N concentrations and isotope compositions of these vein samples, in comparison with those of their hosting altered MORB, to constrain the N cycle pathways and estimate the NH4+ flux in the deep oceanic hydrothermal systems.

Results
The altered MORB from Hole U1502B have bulk-rock N concentrations from 18 to 40 μg/g (Fig. 1), which are much higher than that of fresh MORB (<2 μg/g) but still fall in the upper end of the N concentration range of global altered MORB (2–48 μg/g)29, indicating that the alteration-induced N enrichment in these basalts is similar to those in global altered MORB. The δ15N values of the U1502B basalts (–7.6‰ to +0.2‰; Supplementary Data 2) are lower than the values of the surface source21–23 and thus suggest that the secondary N came from not only seawater/sedimentary source but also a 15N-depleted source. However, similar to the low-δ15N hydrothermally altered basalts from ODP Sites 801 and 114927 and DSDP Site 41729, no good correlation was observed between the bulk-rock concentrations of N and any other elements (Supplementary Data 2; Supplementary Fig. 5). This can be attributed to the variable hydrothermal conditions over their alteration history, e.g., NH4+ content, temperature, and secondary mineral assemblage (see detailed discussion in Yu et al.19). For example, high-T (>~300°C) alteration minerals (e.g., amphibole and albite) have lower NH4+-hosting capabilities than low-T (<~300°C) alteration minerals (e.g., clays)19,30. Some minerals (e.g., clays) even have multiple NH4+-hosting sites with high NH4+-hosting capacity in interlayer sites and low NH4+-hosting capacity in surface and edge sites19. Furthermore, oceanic basalts might experience low-T microbial alteration by lithochemotrophic organisms39 which could also contribute some biological N in rocks, but has not been quantified so far. All these complexities prevent further identification and quantification of the secondary N sources and its connection to ANR in altered MORB samples.

Veins from U1502B contain more abundant N (14–180 μg/g) with extremely low δ15N values from –3.8‰ to –20.4‰ (Fig. 1; Supplementary Data 2). Because the vein minerals (particularly the NH4+-bearing minerals, e.g., albite, epidote, and chlorite) were precipitated at relatively high temperatures (>200⁰C) that do not favor microbial activity, N contribution from microbial biomass to the samples can be excluded. The N concentrations of U1502B veins show good correlations with not only the modes of N-bearing secondary silicate minerals (Fig. 2), but also the concentrations of alkali elements Na and K and trace elements Rb and Cs (Fig. 2, Supplementary Fig. 5). These correlations verify that the N in the vein samples exists mainly in the form of NH4+ that substitutes Na+ and/or K+ in the vein minerals. The signatures of very low K2O concentrations (0.06±0.03 wt.%; 1SD; Supplementary Data 2) and high N/K molar ratios (0.51±0.25; 1SD), but high Na2O concentrations (0.55±0.58 wt.%; 1SD) and low Na/K molar ratios (0.05±0.03; 1SD) of the vein samples, which are similar to those of high-T altered oceanic crust (e.g., sheeted dikes and gabbros) in global oceans, further suggest that the secondary NH4+ in the U1502B veins is mainly hosted in the Na+ site rather than the K+ site30,40. Using N/Na molar ratio to remove the modal heterogeneity of NH4+-bearing minerals across samples, the U1502B vein samples still have higher N/Na molar ratios than not only their hosting altered basalts (Fig. 1) but also the high-T altered gabbroic sections in global oceanic basement (N/Na=0.0006–0.0022)30. This difference suggests that the NH4+ concentration was higher in the focused-flow hydrothermal fluids that formed the U1502B veins than in the diffusive fluids that dominantly altered the gabbros and basalts in global oceanic basements30,40.

It is interesting to observe that the veins display a persistent uphole increase in δ15N from –20.3‰ to –3.8‰ (Fig. 1) and a decrease in N/Na ratio from 0.084 to 0.018. Because the mineral assemblage and formation T of the veins did not change significantly throughout the core36–38, these gradual changes in N/Na ratio and δ15N value cannot be explained by temperature effect on elemental partitioning34 and N isotope fractionation35. Neither these changes can be explained by low-T organic contamination which would otherwise give higher N/Na ratios for shallower samples. These extremely low δ15N values can neither be attributed to a microbial source because the δ15N values of living microbial biomass in hydrothermal systems mostly fall in a range of –4‰ to +7‰41. Instead, these uphole changes are best explained by a two-component mixing model (Fig. 3a; “Methods”). The shallow component is characterized by low N/Na ratios (or low NH4+ concentrations) and positive δ15N values, consistent with a seawater-dominated fluid source containing 15N-enriched surface NH4+. The deep component is characterized by high N/Na ratios (or high NH4+ concentrations) and extreme 15N depletions (–12‰ to –21‰). Taking the isotope fractionation factor between mineral (e.g., albite) and aqueous NH4+ (~2‰ to 3‰ in the range of 200–300°C)35 into consideration, the δ15N value of NH4+ in the deep fluids is expected to vary from <–14‰ to –23‰.

Known N reservoirs that possibly contributed to the deep fluids, i.e., the mantle source of MORB (~ –5‰ for both N-MORB and E-MORB)24,25, dissolved atmospheric N2 (~0‰) and NO3- (+3‰ to +8‰)21 in seawater, and marine organic matter/sediments including dissolved organic N and NH4+ in interstitial water (mostly in the range of +2‰ to +10‰22, up to 17‰23), are all much more 15N-enriched. Thus, the extremely low δ15N values of NH4+ in the deep fluids have to be attributed to isotope fractionation associated with abiotic reactions in the deep hydrothermal system. In light of the N isotope fractionation factors determined by laboratory experiments17,18 and theoretical calculations35,42, only two abiotic processes can produce remarkably 15N-depleted NH4+ in submarine hydrothermal environments. One is partial reduction of NO3-. However, given that NO3- in bottom seawater has a δ15N value of about +5±2‰21, the lowest δ15N value of NH4+ product can be –10‰ (at 200°C) to –6‰ (at 300°C)18,35 (Fig. 3c), which are much less negative than the observed δ15N values of NH4+ in U1502B deep fluids. The other is partial reduction of N2. Although NH4+ is more enriched in 15N than N2 at isotopic equilibration42, experimental studies reveal that N2 is extremely difficult to reach isotope equilibration with other N species due to its strong triple bond as an energy barrier for isotope exchange even at high temperatures of 300–800°C43,44. As a result, abiotic reactions involving N2 generally produce large kinetic isotope fractionations, e.g., –18‰ to –16‰ at 600–800°C during NH3 decomposition43, a reverse process of ANR. From these values and the equilibrium isotope fractionation factors42, kinetic isotope enrichment factors of –13‰ to –16‰ can be deduced for ANR at 600–800°C (“Methods”). Accordingly, if the initial N2 is from the upper mantle source (–5‰), ANR-produced NH4+ at 600–800°C can have δ15N values as low as –21‰ (Fig. 3c). If ANR occurs at lower temperatures, which is highly likely for a submarine hydrothermal system, the magnitude of kinetic isotope fractionation should be larger (although not quantitatively constrained)43 and thus result in more negative δ15N values in the NH4+ product (Fig. 3c). N2 is commonly exsolved during mantle upwelling and partial melting beneath mid-ocean ridges24,25. The N2 outflux at global mid-ocean ridges (1.6×1010 mol·yr–1)45 is at the same order of magnitude with the N2 outflux at global arc volcanoes (2.0−6.7×1010 mol·yr–1)46, and thus can provide a sustainable source for ANR. Therefore, the extremely 15N-depleted NH4+ in the deep fluids, as recorded by the vein minerals, can be best explained by abiotic reduction of mantle N2 in the deep oceanic basement through the interaction between deep fluids and Fe2+-bearing minerals (Fig. 4). The relatively large δ15N range of the deep-fluid endmember in Fig. 3a, b may be attributed to different extents of ANR (Fig. 3c) and/or heterogeneous mixing with surface NH4+.

Given that the hydrothermal system below U1502B was sustained by heat flux from intrusive mafic dykes38, the most likely Fe2+-bearing minerals for ANR are pyroxene (ferrosilite as the Fe2+-rich endmember) and possibly olivine (fayalite as the Fe2+-rich endmember) in these rocks. The ANR can be expressed by Reactions (1)−(2) for ferrosilite or Reactions (3)−(4) for fayalite22:

9FeSiO3 + N2 + 3H2O + 2H+ ! 3Fe3O4 + 9SiO2 + 2NH4+ (1)

6FeSiO3 + N2 + 3H2O + 2H+ ! 3Fe2O3 + 6SiO2 + 2NH4+ (2)

9Fe2SiO4 + 2N2 + 6H2O + 4H+ ! 6Fe3O4 + 9SiO2 + 4NH4+ (3)

3Fe2SiO4 + N2 + 3H2O + 2H+ ! 3Fe2O3 + 3SiO2 + 2NH4+ (4)

The extreme 15N depletion in the vein samples progressively diminishes toward the surface, associated with a decrease in N/Na ratio (Fig. 1). This indicates the ANR-produced NH4+ in the deep fluid below U1502B has been progressively overprinted by surface NH4+ following enhanced mixing with seawater-derived fluids towards seafloor (Fig. 4). This mixing effect can also explain why the ANR signal is rarely recorded in global altered oceanic basalts, because the alteration fluids in the highly porous upper section of oceanic crust are dominated by relatively less evolved seawater that contains sediment-derived NH4+29. In contrast, in more evolved deep fluids, 15N-enriched surface NH4+ from seawater-dominated fluid could have been stripped away by oceanic basement rocks along the pathway that the shallow fluid was pumped down. Thus, the deep fluid can preserve the 15N-depleted signature of ANR, as manifested by the minerals deposited from the focused flow channel of these deep fluids. This mixing discrepancy is clearly shown by the difference in N concentration and isotope composition between veins and their direct wall-rock basalts in Hole U1502B (Figs. 1–2).

Nevertheless, the δ15N values of U1502B altered basalts are clearly lower than the range of surface NH4+ (+2‰ to +10‰) and extend towards the negative deep fluid values (Fig. 3b). This implies that the ANR signal could still have been partially implanted into altered basalts, but significantly weakened due to surface NH4+ overprinting. Significant 15N depletion (as low as –20‰) has also been observed in the hydrothermally altered oceanic basalts from the western Pacific (ODP Sites 1149 and 801 with ages of 130–170 Ma and fast half-spreading rates of 51−80 mm·yr–1)27 and the mid-Atlantic (DSDP Site 417 with an age of 120 Ma and a slow half-spreading rate of ~12.5 mm·yr–1)29, as well as their blueschist- to eclogite-facies metamorphic equivalents22,47. This may imply that ANR has occurred more commonly in worldwide submarine hydrothermal systems.

To assess the ANR contribution to the seawater NH4+ reservoir, we employed a commonly used model (“Methods”) to estimate the NH4+ concentration of the deep fluid, which gave a ballpark range of 12.5–15.0 mM. These values are higher than the few available NH4+ concentration data of high-T (300–380°C) hydrothermal fluids without sedimentary N contribution (e.g., <0.01 mM; Supplementary Data 4) but close to the NH4+ concentrations of the high-T (300–315°C) hydrothermal fluids in the Guaymas Basin (12.9–15.2 mM), which were speculated to originate from thermal degradation of organic matter48. Thus, the high NH4+ concentration of the deep fluid at U1502B may represent an upper end of ANR contribution to hydrothermal vents at a global scale.

Applying this concentration range and the flux (0.72×1013 kg·yr–1) of deep high-T hydrothermal fluids49, an upper limit of NH4+ flux of 9.0–10.8×1010 mol·yr–1 from high-T fluids into seawater can be obtained (“Methods”). The lower limit of ANR-generated NH4+ flux could be much smaller, but it cannot be quantified yet due to the lack of data. Nevertheless, this estimate provides a reference for a first-order understanding of the relative role of ANR in the geological N cycle.

The estimated NH4+ flux here is comparable to the outflux of N2 in mid-ocean ridges (1.6×1010 mol·yr–1)45 and arc volcanoes (2.0–6.7×1010 mol·yr–1)46, implying that ANR could have been an important mechanism to retain about half or more of the degassed mantle N2 into the oceans. The ANR-produced NH4+ can barely impact the modern marine N cycle given the large N reservoir (7.3×1017 mol) in global oceans50. However, ANR could have been a key process to supply NH4+ to Earth’s early oceans. Early Earth’s surface contained more ultramafic components with higher abundance of Fe2+-bearing minerals such as olivine and pyroxene which could have facilitated more intensive serpentinization (a hydrothermal alteration process of olivine and pyroxene) than in the Proterozoic-Phanerozoic oceans51. Since ANR can be closely associated with serpentinization16, ANR could be more productive in the Hadean and Archean oceans. NH4+ extracted from fluid inclusions in 3.5 Ga hydrothermal minerals show strong 15N depletion with δ15N values as low as –13‰52, which might imply an NH4+ source dominantly derived from ANR in the Archean submarine hydrothermal systems, although the ANR signal could be obscured in the altered basalt record by more rigorous biological N recycling in the Archean oceans53. Based on above estimated NH4+ production rate from ANR and the volume of Archean oceans (1.3–2.3 times of modern ocean volume54), the NH4+ concentration of the early oceans could reach the modern seawater N level (NO3-, NO2- and NH4+ all combined) within 0.24 million years, if no significant NH4+ sink is considered. If assume the Archean oceanic basement could assimilate NH4+ from seawater comparably to the modern oceanic basement (4.0×109 mol·yr–1; “Methods”), it would have taken 0.25 million years for the early oceans to accumulate NH4+ to the modern seawater N level. This suggests that NH4+ level could have been very quickly built up in the prebiotic ocean for the development of habitability and the origin of life, as well as supporting a microbial ecosystem (see Supplementary Information).

Methods
Sample Preparation
Altered basalt and vein samples were cleaned by removing surface materials and crushed into small size (1–2 mm) before further washed by deionized water. After drying at 50°C in an oven, the grains were ground to <200 mesh by hand in an agate mortar for further analyses.

Mineralogy
Mineralogical analysis of altered basalts and veins were performed on a D8 ADVANCE X-ray diffractometer (XRD) with a copper Kα tube at the State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanology, Chinese Academy of Sciences. Instrument settings were 40 mA, 40 kV, angle steps of 0.02°, a counting time per step of 0.15 s and scanning angles of 5–90°. The quantitative compositions of minerals were calculated using the K value method55 with DIFFRAC.EVA 5.1.0.5 software. The intensity ratio on the XRD curve was obtained from PDF 2–2004. The NIST corundum standard SRM 1976 was used for the correction of instrumental goniometers. The analytical error is 5%–10%.

Major and trace elements
Major and trace elements of altered basalts were determined at the Wuhan Sample Solution Analytical Technology Co., Ltd. (Wuhan, China). Major elements were measured using a Rigaku Primus Ⅱ X-ray Fluorescence spectrometer (XRF). Altered basalt powders were fluxed with 6.0 g co-solvent (Li2B4O7: LiBO2: LiF=9:2:1) and 0.3 g oxidant (NH4NO3) at 1150°C to make homogeneous glass discs for the XRF analysis. The standards GBW07103, GBW07111, and GBW07114 were used for data calibration. Analytical error is better than 5% of the absolute concentrations. Trace elements of altered basalts were analyzed using an Agilent 7700e inductively coupled plasma mass spectrometer (ICP-MS) after acid digestion of samples in high-pressure Teflon vessels. The USGS reference standards BHVO, BCR-2, and RGM-2 were used for data calibration. The analytical error is better than 10% of the absolute concentrations.

Major and trace elements of veins were analyzed in the Laboratory of Ocean Lithosphere and Mantle Dynamics, the Institute of Oceanology, Chinese Academy of Sciences. Major elements were analyzed using an Agilent 5100 inductively coupled plasma optical emission (ultra) spectrometer (ICP-OES). About 50 mg of sample powders and 250 mg of lithium metaborate (LiBO2) were mixed in a platinum crucible and melted in a muffle furnace at 1050°C for 1 h. The melt was further heated and spun on a Bunsen burner at about 1000°C to ensure that all melts formed a coherent single drop, which was finally poured into 5% HNO3 for dissolution. Repeated analyses of USGS reference standards (BHVO-2 and BCR-2) gave an analytical error better than 5% of the absolute concentrations. Trace elements of veins were analyzed using an Agilent 7900 ICP-MS. About 50 mg of sample powders were dissolved in an acid mixture of HCl, HNO3, and HF in a high-pressure jacket equipped Teflon beaker for 15 h, and then re-dissolved with 20% HNO3 for 2 h until complete digestion. The reference materials BHVO-2, GSP-2 and W-2 were used for data calibration. The analytical error is better than 10% of the absolute concentrations.

Nitrogen concentration and isotope composition
Nitrogen concentration and isotope composition of altered basalts and veins were analyzed by the offline sealed-tube combustion and extraction methods coupled with continuous flow isotope ratio mass spectrometry56 at the Stable Isotope Geochemistry Laboratory, University of Alberta. About 100 mg sample powders, 400 mg CuO reagents and pre-cleaned (1200°C for 3 h) quartz wool were sequentially loaded into a pre-cleaned (1200°C for 3 h) one-end sealed quartz tube. The sample tube was attached on a custom-made metal manifold to pump overnight and then sealed under high vacuum. The entire sealed sample tube was put into a programmable muffle furnace to combust at 900°C for 8 h followed by 600°C for 2 h. The combusted tube was then loaded into a tube cracker attached on the metal manifold and cracked under high vacuum. The released N2 was purified using a liquid N2 trap and quantified by a capacitance manometer. Finally, the N2 gas was introduced by an ultra high-purity helium stream through a GasBench II interface into a Thermo Finnegan MAT 253 isotope ratio mass spectrometer for N isotopic measurement. All N isotope data were reported in the δ notation, which is defined as δ15N = (15N/14N)sample / (15N/14N)standard – 1, where the standard is atmospheric N2. Repeated analyses of certified reference material (the low-organic content soil standard and high-organic content sediment standard from Elemental Microanalysis) and samples gave an analytical error better than 6% (2SD) of the absolute value for N concentration and 0.2‰ (2SD) for δ15N value.

Data modeling
Nitrogen mixing modeling
(a) Hydrothermal veins. Since the vein minerals assimilated NH4+ directly from the hydrothermal fluid as they precipitated, the uphole gradual decrease in N/Na ratio and increase in δ15N value are therefore considered to reflect decreasing NH4+ concentration with increasing δ15N value of the hydrothermal fluids as they migrated upwards. This can be best attributed to gradually intensified mixing of another fluid (with a low N/Na ratio and a high δ15N value) from the top into the upflowing deep fluid. For quantitative modeling of this case scenario, N concentration variations caused by inter-sample modal heterogeneity of NH4+-bearing minerals have to be removed first. For this purpose, N/Na molar ratio (rather than N concentration) is used together with δ15N for the mixing modeling. Accordingly, this two-endmember mixing process can be described as:

(N/Na)Sample = (N/Na)TF × f + (N/Na)DF × (1–f) (5)

δ15N Sample = [(N/Na)TF × f × δ15N TF + (N/Na)DF × (1–f) × δ15N DF] / (N/Na)Sample (6)

in which TF denotes top fluid, DF denotes deep fluid, and f refers to the fraction of the top fluid in any hydrothermal fluid. It should be noted that, because the partition coefficient of NH4+ and Na+ between minerals (e.g., albite, epidote, chlorite) and the multiple N-bearing secondary minerals are not available, the N isotope fractionation factors between the hydrothermal fluids and minerals (which are relatively small35), were not taken into consideration for simplification. Therefore, (N/Na)TF, (N/Na)DF, δ15N TF and δ15N DF do not represent the N/Na ratio and the δ15N value of deep fluids but the values of secondary minerals precipitated from the top and deep fluids, respectively.

The modeling results (Fig. 3a) indicate that the lower end member has a relatively large δ15N range with high N/Na ratios, e.g., if using a N/Na ratio of 0.085 (close to the largest value of the veins), a δ15N range of –12‰ to –21‰ is required to cover the data. In contrast, the upper end member is characterized by much more narrow ranges with low N/Na ratios and high δ15N values, which are consistent with a seawater source.

(b) Altered basalts. Different to hydrothermal veins whose N was solely sourced from hydrothermal fluid, altered oceanic basalts contain a small amount of mantle-inherited N (up to 2 μg/g, δ15N=–5‰)24,25,27,29–31. Further addition of NH4+ into oceanic basalts can be induced by hydrothermal alteration mostly occurring at near seafloor with relatively low temperatures, in which K+-rich secondary minerals commonly precipitate and simultaneously assimilate NH4+ from the ambient fluid26–31. In this case scenario, because the NH4+ in hydrothermal fluid can be taken as an unlimited reservoir in relative to the inherited NH4+ in fresh basalts due to the high water/rock ratio near seafloor, the mixing model for addition of hydrothermal NH4+ into altered basalts is slightly different to the fluid mixing model above for hydrothermal veins, but instead can be described as27,29–31:

δ15N Sample = (N/K)FB × δ15N FB + [(N/K)Sample – (N/K)FB] × δ15N DF / (N/K)Sample (7)

in which FB denotes fresh basalts. N/K molar ratio is used to remove the effect of modal heterogeneity of K+-bearing minerals on N concentrations of altered basalts. The N/K ratio of fresh basalt is calculated from the N concentration29 and K concentration57 of fresh oceanic basalts. Again, for simplification, the partition coefficients of NH4+ and K+ and N isotope fractionation factors between hydrothermal fluid and the multiple K+-bearing secondary minerals were not taken into consideration. Therefore, (N/K)DF and δ15N DF do not represent the N/K ratio and the δ15N value of deep fluids but the values of secondary minerals precipitated from deep fluids.

In Fig. 3b, mixing curves were illustrated for NH4+ addition from seawater (δ15N=6‰, which is an average value of marine organic matter and sediments)22 and two variably 15N-depleted deep fluids (δ15N=–12‰ and –21‰, respectively). The results show that the N in U1502B basalts is dominated by secondary N from a mixture of seawater and 15N-depleted deep fluids.

N isotope fractionation during ANR. The N isotope effects associated with abiotic transformation between N2 and NH3 can be described as:

N2 + 3H2 "k1 k2 2NH3 (8)

in which k1 denotes the kinetic isotopic fractionation factor for abiotic N2 reduction and k2 denotes the kinetic isotopic fractionation for abiotic NH3 decomposition. Consequently, the equilibrium isotope fractionation factor between N2 and NH3 (αN2–NH3) can be described as:

αN2–NH3 = k1 / k2 (9)

Currently, the k2 values have been only determined for the temperature range of 600−800°C (0.9841 to 0.9823)43. Integrated with the αN2–NH3 values calculated at the temperature range of 600–800°C (0.9869 to 0.9840)42, which are equivalent to isotope enrichment factors of –16‰ to –13‰ for the temperature range of 600–800°C. The isotope fractionation factors at lower temperatures43, which would fit our data even better.

δ15N of accumulated NH4+ from ANR. Because of the strong triple bond of N2, the NH4+ product will not exchange N isotope compositions with remaining N2 after ANR. Therefore, the δ15N value of accumulated NH4+ product from a certain degree of ANR follow the traditional Rayleigh model, which can be described by the equation below43,58:

δ15N p = δ15N 0 – ε × (f × ln f) / (1–f) (10)

in which δ15N 0 and δ15N p refer to the N isotopic value of initial N species (+6‰ for marine NO3- or –5‰ for mantle N2) and the product (NH4+), respectively; ε denotes the isotopic enrichment factor between the product and initial N species; f denotes the fraction of remaining N after the reaction. Examples of the modeling are illustrated in Fig. 3c.

Ammonium productivity from ANR. The common method to derive the NH4+ concentration of deep hydrothermal fluids from the NH4+ concentrations of vein samples is to use the NH4+ partition coefficient between the vein sample and fluid, which can be described by the equation below:

C NH4+ fluid = C NH4+ vein / Dvein–fluid (11)

in which C is NH4+ concentration of a vein sample or fluid; Dvein-fluid is the total partition coefficient of NH4+ between vein and fluid, which can be calculated by the following equation:

Dvein–fluid = Σ ni × Kd mineral–fluid (12)

in which n is the fraction of individual NH4+-bearing minerals (albite, epidote and chlorite with augite in some samples in this study); Kd mineral–fluid is the partition coefficient between a NH4+-bearing vein mineral and fluid.

While the partition coefficients of NH4+ between these silicate minerals and fluid have not been all determined, they can be calculated by the Blundy and Wood model59, which suggests a non-linear least-squares regression relationship between the partition coefficients and the radii of isovalent cations (Li+, Na+, K+, NH4+, Rb+, Cs+)60–62 (Supplementary Fig. 7). The calculation results (Supplementary Data 3) show that the mineral-fluid partition coefficients of NH4+ and Rb+ are almost identical, because of their similarity in cation radius. This conclusion of similar partitioning behavior of NH4+ and Rb+ in mineral-fluid system has been not only demonstrated by a previous study for phengite63, but also supported by the good correlation between the concentrations of NH4+ and Rb+ in the studied vein samples (R2=0.80; Supplementary Fig. 5).

Because the Dvein-fluid values of NH4+ and Rb+ are almost identical, the NH4+ concentrations of fluid can be simply calculated from the equation below:

C NH4+ fluid = C NH4+ vein × (C Rb+ fluid / C Rb+ vein) (13)

The advantage of Eq. (13) is that most of the uncertainties associated with Eqs. (11, 12), including (i) Kd determination for individual minerals by laboratory experiments and the interacting effect with other cations (e.g., Na+) during NH4+ partitioning34, and (ii) the fraction of each NH4+-bearing mineral in bulk rock, can be canceled out. It should be noted that Eq. (13) cannot be applied to altered basalts because the initial N and Rb in the basalts prior to alteration are decoupled from hydrothermal fluids.

This method using Eq. (13) requires to know the Rb+ concentration of fluid, which is difficult to constrain due to the lack of well-preserved fluid samples. Here, we use the concentrations of high-temperature (> 300°C) hydrothermal fluids in modern submarine hydrothermal systems as a reference. To obtain representative aqueous elemental data of deep fluids, we only used the fluid data in literature with [Mg2+]=0, which indicates the correction for seawater contamination. The filtered data (Supplementary Data 4) gave very consistent concentrations of univalent cations (e.g., Na+, K+, and Rb+) although the high-temperature fluids came from a variety of different submarine localities around the world64–69.

We use the samples with the lowest δ15N values in two veins (a silicate vein with high quartz content and a silicate vein with low quartz content; N=67.4–180.4 μg/g; Rb= 0.84–2.72 μg/g; Supplementary Data 2) to best represent the deep fluid with least seawater contamination (Rb=31 mM; Supplementary Data 4), from which NH4+ concentrations of 12.5–15.0 mM can be deduced for the deep fluids at U1502B. Applying this concentration range and the flux of deep high-T hydrothermal fluids (0.72×1013 kg·yr–1)49, an upper limit of NH4+ flux of 9.0–10.8×1010 mol·yr–1 from high-T fluids into seawater can be obtained.

NH4+ uptake by global altered basalts. Ammonium in seawater can be assimilated into oceanic basement rocks during seafloor alteration/weathering26–31. The NH4+ assimilation flux in modern oceans can be calculated using the following equation:

F = C × D × P (14)

in which F is NH4+ assimilation flux, C is the N concentration of altered basalts (global average: 9.9 μg/g)29, D is density of altered basalts (2.7 g·cm–3), P is oceanic basalt production rate (2.1 km3·yr–1)70. This gave an NH4+ assimilation flux of 4.0×109 mol·yr–1 in modern global oceans.

It should be noted that, the NH4+ assimilation rate of altered basalts is strongly dependent on the NH4+ availability in local environment (e.g., absence or occurrence of N-rich organics and sediments)29. Thus, the NH4+ assimilation flux in the early oceans was likely not as large as that in modern oceans.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review of: Abiotic nitrogen reduction in submarine hydrothermal system\nBy: Sun et al.\nRecommendation: Reconsider after revision.\nManuscript summary: The authors report on nitrogen isotopic variation in NH4+ in samples from an Oligocene mid-ocean\nridge hydrothermal system and conclude that the extremely 15N-depleted NH4+ observed in deep fluids, as recorded by\nvein minerals, can be best explained by abiotic reduction of mantle N2 in the deep oceanic basement through the interaction\nbetween the deep fluids and Fe(II)-bearing minerals (e.g., olivine, pyroxene). Upscaling this process, they argue that this\nammonium flux is small relative to the large nitrogen inventory in modern oceans, but could be an important contributor to\nquickly fertilize the early oceans and supply NH3 to the atmosphere on the prebiotic Earth.\nReview summary: Prior to the evolution of nitrogenases, there was essentially no direct pathway known to derive ammonium\nfrom N2 (assuming the early Earth atmosphere was ammonia-poor). The abiotic reduction of N2 through hydrothermal basalt\nhas been hypothesized but direct evidence has been lacking. The study by Sun et al. delivers this evidence and therefore\nadds an important piece to our picture of the early nitrogen cycle.\nI find the logical flow of the investigation sound, the selected methods are robust, and the extrapolation of the findings are\nadequate. There are two major shortcomings that should be remedied before this manuscript could be reconsidered:\n1) The writing has to be improved. In my specific comments I started out with language and grammar corrections but they\nbecame too extensive. Most notably, the last paragraph of the introduction reads like a Methods section. That last paragraph\nshould define the question to be answered as well as tell the objective of the study which is not clear at all after reading the\nintroduction. A more general reader might get highly confused. Moreover, the figures, especially the schematic, should be\npolished up.\n2) The mechanism and catalytic reaction of N2 reduction to ammonium by Fe(II)-bearing minerals has to be described more\nprecisely. The authors should think more about the molecular mechanism that drives the reaction and suggest, if not identify,\nthe Fe mineral serving as catalyst. Write out a proposed chemical reaction in a formula. The samples used are derived from\nan Oligocene bedrock, yet the authors draw conclusions to processes on the prebiotic Earth, well over 3 billion years ago. If\nthe Fe minerals involved are better constrained, it can be argued that these minerals were also present in similar rock\nformations on the prebiotic Earth – which would essentially strengthen the extrapolation in time.\nGiven the quality of the data and the logic that the authors applied to draw conclusions, I would recommend reconsidering\nthis work for publication in Nature Communications after these two main points and the following minor points were\nreworked.\nSpecific comments:\nTitle: The wording of the title is awkward. The title should be better fleshed out and point towards the significance of the\npaper. For example: “Evidence for hydrothermal N2 reduction to ammonium on the prebiotic Earth”.\nL14, Abstract: “.. an essential component of the abiotic synthesis…\nL15, Abstract: take out “arguably” – such fill words are unnecessary.\nL20, Abstract: name the nitrogen isotopes.\nAbstract, overall: I suggest including this key finding in the abstract. “Therefore, the extremely\n15N-depleted NH4+ in the deep fluids, as recorded by the vein minerals, can be best explained by abiotic reduction of\nmantle N2 in the deep oceanic basement through the interaction between the deep fluids and Fe(II)-bearing minerals (e.g.,\nolivine, pyroxene).”\nL30-36. Split this very long sentence into two. Suggestion: “…laboratory experiments. These demonstrated that..”\nL33: “early Earth surface”\nL34ff.: References 7-9 on abiotic nitrogen reduction reactions on the early Earth are important but outdated. Please include\nmore recent work:\nNishizawa, M. et al. Minerals 11, 321 (2021)\nBuessecker, S. et al. Nat. Geosci. https://doi.org/10.1038/s41561-022-01089-9 (2022)\nL40: “detecting an ANR signal…”\nL51-57: Consider moving this to the methods and definitely modify the end of the introduction (see major comments).\nL60-63: Clarify what N pool you are talking about – bulk, inorganic, organic?\nL99: use plural, “basements”\nL102, 103, and Fig. 1: Are these data (one data point) based on technical replicates? How was the heterogeneity in samples\nof the same core depth evaluated? The robustness especially of the measurement reporting –20.3‰ is important because it\nessentially creates the decreasing trend with depth. The authors should make sure this is not an outlier and a data point set\nof 3 such as for sample 21R would create more confidence.\nL117-143: I like the logical flow of this paragraph very much. I think the way the conclusion that 15N depletion in ammonium\noriginates from reduction of mantle N2 is derived is quite reasonable and sound.\nL134: Looking through the text, it is not explained where N2 in the upper mantly actually comes from. The question of the\nultimate source of N2 would be important to evaluate how sustainable this reduced N source to the early oceans could have\nbeen. Please go into more detail (here or in the introduction) how N2 in the upper mantle is thought to be derived and\nrecycled.\nL158: Provide very briefly more insight into the locations and formation history where else significant 15N depletion was\nreported from. This will demonstrate better that the proposed process could be a global phenomenon.\nL173: I may have missed it somewhere in the supplements, but what is the basis of this calculated value. I suggest adding a\nreference to the supplements.\nL183: How did serpentinization rate influence the GOE – please corroborate for readers not familiar with:\nLeong, J.A.M., Ely, T. & Shock, E.L. Decreasing extents of Archean serpentinization contributed to the rise of an oxidized\natmosphere. Nat Commun 12, 7341 (2021). https://doi.org/10.1038/s41467-021-27589-7\nL191: Could an early biosphere have lived off that ammonium flux? How much biomass may it have sustained?\nL331ff., Methods: Include what standard was used to determine instrument broadening.\nFigure 1: Consider adding Fig. S1 as a small inset because it provides a quick idea on the origin of the cores\ngeographically.\nFigure 2: Specify what form of nitrogen in the legend and x axis label (bulk, organic, inorganic).\nFigure 4: Adjust to “early Earth ocean” or “Hadean-Archean ocean”. Color the ocean in a brighter blue for better contrast (the\ncolor contrast is in general not optimal in this figure). “Along the path of seawater circulation into depths,..” – reword. Add\nmore detail to the Box on the abiotic N2 reduction.\nFigure S8: Consider moving it into the main text, perhaps as additional panel in Fig. 2 or 3.", "reviewer_2": "The manuscript describes N-bearing secondary minerals in a hydrothermally altered basalt core of Oligocene age that are\nunusually isotopically depleted. These observations are novel and noteworthy, because isotope variation in the N system\nusually falls within a tighter range. The authors argue that the isotopically depleted nitrogen signatures were produced by\nthe abiotic reduction of seawater-sourced N2 by Fe(II) from the basaltic oceanic crust. They then make an effort to calculate\nthe rate of reduction and its implications for transforming N2 into bioavailable nitrogen by releasing NH4+ into the Archean\nocean.\nIn hydrothermal systems literature, studies examining nitrogen cycling are under-represented with respect to other\nbiogeochemical systems (e.g., carbon and sulfur cycling are far better understood). These novel isotope data are supported\nby well-described abundance and mineralogical data. The methodological approach is strong. I do not doubt that these data\nwill be of interest within the community.\nHowever, the authors do not thoroughly evaluate other potential sources of nitrogen to the altered basalt core. In particular,\nthey do not acknowledge the important role of NO3- reduction to NH4+ in unsedimented seafloor hydrothermal systems. In\nnearly every modern study of NH4+ isotopes in modern vent systems today, the abundance of NH4+ expelled and its\nisotope signature can be fully attributed to the reduction of NO3- sourced from seawater. The authors miss an opportunity to\nacknowledge the existing literature on this topic, and they do not fully discount this pathway. We include more specific\nsuggestions in the annotated paper attached to this review.\nThe authors emphasize that NH4+ in modern vents is ‘contaminated’ by seawater-derived NH4+, obscuring\nabiotic nitrogen reduction signals. This is not true for two reasons. First, seawater NH4+ is quite low in abundance (~0.1 umol/kg) compared\nwith the range of NH4+ abudance in unsedimented vent fluids (~1-40 umol/kg). Seawater NH4+ is very low compared to\nsediment-influenced vent fluids (10s of mmol/kg). Further, even if seawater-sourced NH4+ was added to a fluid, it could be\neasily accounted for by isotope mass balance. It is well accepted that the majority of vent-sourced NH4+ can be attributed to\nNO3- reduction.\nAnother important detail that is missing is whether or not different marine nitrogen cycling was different in the Oligocene\nocean, which could contribute nitrogen sources that are isotopically distinct from modern N2 and NO3-, and therefore impact\ndiscussion of fractionation factors.\nFinally, we were not convinced by the significance of the study to the Archean NH4+ pool as presently described. The\nauthors describe evidence for the abiotic production of NH4+ that is co-precipitated with secondary hydrothermal minerals.\nThe problem with this final calculation is that there is no discussion of how the NH4+ would be released from secondary\nminerals into the Archaean ocean. If it is immobilized by high temperature hydrothermal mineralization deep in the oceanic\nsubsurface, then NH4+ will not be released to seawater, nor will it be available biologically.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We appreciate the endorsement of the novelty and high quality of our study. The revisions corresponding\nto R1’s comments (see details below) are highlighted in brown in the tracked version of the manuscript.\nI find the logical flow of the investigation sound, the selected methods are robust, and the extrapolation of\nthe findings are adequate. There are two major shortcomings that should be remedied before this\nmanuscript could be reconsidered:\n1) The writing has to be improved. In my specific comments I started out with language and grammar\ncorrections but they became too extensive. Most notably, the last paragraph of the introduction reads like\na Methods section. That last paragraph should define the question to be answered as well as tell the\nobjective of the study which is not clear at all after reading the introduction. A more general reader might\nget highly confused. Moreover, the figures, especially the schematic, should be polished up.\nReply:\nWe have added a few sentences at the beginning and the end of the last paragraph in the Introduction to\nclarify the objectives of this study. Please see L65-67, L77-80.\nWe have also further improved the diagrams as suggested and added more detailed description in the\ncaptions for easy understanding.\n2\n2) The mechanism and catalytic reaction of N2 reduction to ammonium by Fe(II)-bearing minerals has to\nbe described more precisely. The authors should think more about the molecular mechanism that drives\nthe reaction and suggest, if not identify, the Fe mineral serving as catalyst. Write out a proposed chemical\nreaction in a formula. The samples used are derived from an Oligocene bedrock, yet the authors draw\nconclusions to processes on the prebiotic Earth, well over 3 billion years ago. If the Fe minerals involved\nare better constrained, it can be argued that these minerals were also present in similar rock\nformations on the prebiotic Earth – which would essentially strengthen the extrapolation in time.\nReply:\n1. Given that the hydrothermal system below the drilling site was sustained by intrusive dykes (Chen\net al., 2023Ref 38), the most likely Fe(II)-bearing mineral for the abiotic N reduction is pyroxene\n2\n(the rock constituting mineral of sheeted dykes and gabbros in oceanic crust) and possibly olivine\n(which can occur in small amounts in mafic dykes as well). This discussion and the chemical\nreactions have been added in L172-180.\n2. These minerals are common in the early-Earth oceanic crust which was dominated by more\n(ultra)mafic lithology and experienced more serpentinization (Leong et al., 2021Ref 51 and reference\ntherein), which could have induced more abiotic nitrogen reduction. This discussion has been\nadded in L226-231.\nGiven the quality of the data and the logic that the authors applied to draw conclusions, I would\nrecommend reconsidering this work for publication in Nature Communications after these two main\npoints and the following minor points were reworked.\nSpecific comments:\nTitle: The wording of the title is awkward. The title should be better fleshed out and point towards the\nsignificance of the paper. For example: “Evidence for hydrothermal N2 reduction to ammonium on the\nprebiotic Earth”.\nReply:\nWe appreciate for this appealing title. We have changed the title toward this direction as “Abiotic nitrogen\nreduction in submarine hydrothermal systems: implication to ammonium in prebiotic oceans”. See L2.\nWe did not directly adopt the suggested title because we try to avoid leading the readers to think we are\nstudying hydrothermal samples from the prebiotic Earth in this study.\nL14, Abstract: “... an essential component of the abiotic synthesis…\nReply: revised as suggested. See L15.\nL15, Abstract: take out “arguably” – such fill words are unnecessary.\nReply: revised as suggested.\nReply: added as suggested. See L20, 21, 22.\nAbstract, overall: I suggest including this key finding in the abstract. “Therefore, the extremely\n15N-depleted NH4+ in the deep fluids, as recorded by the vein minerals, can be best explained by abiotic\nreduction of mantle N2 in the deep oceanic basement through the interaction between the deep fluids and\nFe(II)-bearing minerals (e.g., olivine, pyroxene).”\nReply:\nwe would love to include this in the abstract. Unfortunately, due to the 150-word limit to the abstract of\nNature Communications articles, we have to shorten the abstract significantly and have no room for this\nsentence.\nL30-36. Split this very long sentence into two. Suggestion: “…laboratory experiments. These\ndemonstrated that..”\nReply: revised as suggested. See L32.\nL33: “early Earth surface”\nReply: revised as suggested. See L31.\nL34ff.: References 7-9 on abiotic nitrogen reduction reactions on the early Earth are important but\noutdated. Please include more recent work:\nNishizawa, M. et al. Minerals 11, 321 (2021)\nBuessecker, S. et al. Nat. Geosci. https://doi.org/10.1038/s41561-022-01089-9 (2022)\nReply: added as suggested. Ref 14, 15 in the manuscript.\nL40: “detecting an ANR signal…”\nReply: revised as suggested. See L52.\nL51-57: Consider moving this to the methods and definitely modify the end of the introduction (see major\ncomments).\nReply:\n(more detailed introduction was given in Methods and Supplementary Material), which are important\njustification for the representation and advantage of this set of samples to address the outlined outstanding\nquestions. Therefore, we prefer to keep this information here.\nFollowing the main comment above, we did add clear statement of goals of studying this set of samples\n(L65-67, 77-80), which should make the readers easier to follow.\nL60-63: Clarify what N pool you are talking about – bulk, inorganic, organic?\nReply: It is bulk-rock N. This has been clarified in L84.\nL99: use plural, “basements”\nReply: Revised as suggested. See L121.\nL102, 103, and Fig. 1: Are these data (one data point) based on technical replicates? How was the\nheterogeneity in samples of the same core depth evaluated? The robustness especially of the measurement\nreporting –20.3‰ is important because it essentially creates the decreasing trend with depth. The authors\nshould make sure this is not an outlier and a data point set of 3 such as for sample 21R would create more\nconfidence.\nReply:\nYes, we did make duplicated measurements to validate the extremely low 15N value: two measurements\ngave: (1) N = 179.0 ppm, 15N = –20.3‰; (2) N = 180.9, 15N = –20.5‰. We also did this practice of\ndata validation on some other samples which could provide enough material for duplicate measurements.\nThe duplicated data are now added to Supplementary Table 2.\nIt is worth clarifying that the 3 data from section 21R are 3 different samples rather than 3 repeated\nanalyses of one sample. We were able to obtain this because section 21R has more abundant thick veins\nthat allowed for sampling enough material for N isotope analysis. The sample availability in other\nsections is less mainly due to the veins in those sections are very thin so it is not feasible for sampling\nenough material for N isotope analysis.\nL117-143: I like the logical flow of this paragraph very much. I think the way the conclusion that 15N\ndepletion in ammonium originates from reduction of mantle N2 is derived is quite reasonable and sound.\nReply: Thanks again for the endorsement.\nL134: Looking through the text, it is not explained where N2 in the upper mantle actually comes from.\nThe question of the ultimate source of N2 would be important to evaluate how sustainable this reduced N\nsource to the early oceans could have been. Please go into more detail (here or in the introduction) how\nN2 in the upper mantle is thought to be derived and recycled.\nNitrogen is known as a trace element (< 2 ppm) in the depleted mantle (the source of the MORB).\nAlthough the exact nitrogen species in the upper mantle is not well constrained, it has been widely\nobserved that the N in the upper mantle is persistently exsolved and emitted as N in mid-ocean ridges,\n2\nwith a global output flux at the same order of magnitude with the N output flux at global arc volcanoes\n2\n(Hilton et al., 2002 Ref 45; Bekaert et al., 2021Ref 46). This huge amount of N can provide a sustainable\n2\nsource for ANR. This discussion has been added in L163-166.\nL158: Provide very briefly more insight into the locations and formation history where else significant\n15N depletion was reported from. This will demonstrate better that the proposed process could be a global\nphenomenon.\nReply: added as suggested. See L198-201.\nL173: I may have missed it somewhere in the supplements, but what is the basis of this calculated value. I\nsuggest adding a reference to the supplements.\nReply: added as suggested. See L215, 217.\nL183: How did serpentinization rate influence the GOE – please corroborate for readers not familiar with:\nLeong, J.A.M., Ely, T. & Shock, E.L. Decreasing extents of Archean serpentinization contributed to the\nrise of an oxidized atmosphere. Nat Commun 12, 7341 (2021). https://doi.org/10.1038/s41467-021-27589-7\nReply: added as suggested. See L226-231 and Ref 51.\nL191: Could an early biosphere have lived off that ammonium flux? How much biomass may it have\nsustained?\nReply: This is an intriguing question. Thinking about microbial system living on ammonium in the\nreducing early oceans, the mostly likely metabolic pathway would be sulfate-reducing anaerobic\nammonium oxidation. We can look into this question from two different approaches:\n1. In the laboratory microbe culture experiments carried out in anaerobic environment with NH + and\n4\nexperiments with pH = 510 (optimal at 8) and T = 2050 C (optimal at 30 C), which well bracket\nthe conditions of early-Earth oceans. The growth rate can be up to 7.3109 CFU/mL over 24-72 hour\nexperiments, which is equivalent to 3.651012 cells in the 500 ml flasks in their experiments.\nAlthough these experiments were carried out with a [SO 2-] (~ 750 M) slightly higher than the\n4\nproposed [SO 2-] in Archean oceans (2.5  200 M; Habicht et al., 2002; Crowe et al., 2014), these\n4\nexperimental results clearly demonstrated that the sulfate and ammonium in the early oceans can\nsupport a biosphere, likely with less biomass yielded in the experiments.\n6\n2. From Gibbs energy point of view, we did a more quantitative assessment. We followed the\ncalculation method of Schrum et al. (2009) for the sulfate-reducing ammonium oxidation process:\n8SO 2- + 3NH +  4N + 3HS- + 5H+ + 12H O\n4 4 2 2\n∆𝐺 = ∆𝐺0 +𝑅𝑇𝑙𝑛( 𝛾 𝑁2 4[𝑁 2 ]4 ×𝛾 𝐻𝑆− 3[𝐻𝑆−]3×𝛾 𝐻+ 5[𝐻+]5] )\n(𝑇,𝑃,𝑆) (𝑇,𝑃,𝑆) 𝛾 8[𝑁𝐻 +]8×𝛾 3[𝑆𝑂 2−]3\n𝑁𝐻4 + 4 𝑆𝑂4 2− 4\nIn which ΔG is a function of pressure (P), temperature (T) and salinity (S); ΔG0 is the standard\nreaction Gibbs energy;  is activity coefficient (see details in Schrum et al., 2009 and reference\ntherein).\nFor a back-of-an-envelop calculation, we set the condition at fixed P of 1 bar and S of seawater\nsalinity, but variable T of 15–70 C, pH of 6–8, [SO 2-] from 2.5–200 M (Habicht et al., 2002;\nreducing ammonium oxidation can release energy when [NH +] accumulates to ~2004000 M when\n4\n[SO 2-] is 200 M or ~100015000 M when [SO 2-] is 2.5 M. Such [NH +] is possible in our\n4 4 4\ncalculations since there was little sink for NH + in the prebiotic oceans. The energy yield enables to\n4\nsupport a biosphere. However, due to the large uncertainty on the energy yield which is highly\ndependant on [NH +] and pH, it is difficult to constrain the biomass at the moment.\nappropriate, we can add these results in the revision next round.\nReference:\n(1) Mohammed Madani, R. et al. Novel simultaneous removal of ammonium and sulfate by\nisolated Bacillus cereus strain from sewage treatment plant. Water Air Soil Pollut. 233, 185\n(2022).\n(2) Habicht, K.S., Gade, M., Thamdrup, B., Berg, P. & Canfield, D. E. Calibration of sulfate\nlevels in the Archean ocean. Science 298, 2372-2374 (2002).\n(3) Crowe, S. A. et al. Sulfate was a trace constituent of Archean seawater. Science 346, 735-739\n(2014).\n(4) Schrum, H. N., Spivack, A. J., Kastner, M. & D’Hondt, S. Sulfate-reducing ammonium\noxidation: A thermodynamically feasible metabolic pathway in subseafloor sediment.\nGeology 37, 939-942 (2009).\nL331ff., Methods: Include what standard was used to determine instrument broadening.\nReply: added as suggested. See L396-397.\nFigure 1: Consider adding Fig. S1 as a small inset because it provides a quick idea on the origin of the\ncores geographically.\nReply: added as suggested. See Fig. 1.\nFigure 2: Specify what form of nitrogen in the legend and x axis label (bulk, organic, inorganic).\nReply: revised as suggested. See Fig. 2.\nFigure 4: Adjust to “early Earth ocean” or “Hadean-Archean ocean”. Color the ocean in a brighter blue\nfor better contrast (the color contrast is in general not optimal in this figure). “Along the path of seawater\ncirculation into depths,..” – reword. Add more detail to the Box on the abiotic N2 reduction.\nReply:\n1. We have revised the color and added details as suggested.\n2. Figure caption has been re-worded.\n3. This model is applicable to not only the Hadean-Archean oceans but Phanerozoic oceans as well,\nas it is established on the Cenozoic samples. So we still leave it as unspecified oceans.\nFigure S8: Consider moving it into the main text, perhaps as additional panel in Fig. 2 or 3.\nReply: This is now added as Fig. 3c as suggested.\nThe manuscript describes N-bearing secondary minerals in a hydrothermally altered basalt core of\nOligocene age that are unusually isotopically depleted. These observations are novel and noteworthy,\nbecause isotope variation in the N system usually falls within a tighter range. The authors argue that the\nisotopically depleted nitrogen signatures were produced by the abiotic reduction of seawater-sourced N2\nby Fe(II) from the basaltic oceanic crust. They then make an effort to calculate the rate of reduction and\nits implications for transforming N2 into bioavailable nitrogen by releasing NH4+ into the Archean\nocean.\nIn hydrothermal systems literature, studies examining nitrogen cycling are under-represented with respect\nto other biogeochemical systems (e.g., carbon and sulfur cycling are far better understood). These novel\nisotope data are supported by well-described abundance and mineralogical data. The methodological\napproach is strong. I do not doubt that these data will be of interest within the community.\nReply: we appreciate the endorsement of the novelty of our study. The revisions corresponding to R2’s\ncomments (see details below) are highlighted in blue in the tracked version of the manuscript.\nHowever, the authors do not thoroughly evaluate other potential sources of nitrogen to the altered basalt\ncore. In particular, they do not acknowledge the important role of NO3- reduction to NH4+ in\nunsedimented seafloor hydrothermal systems. In nearly every modern study of NH4+ isotopes in modern\nvent systems today, the abundance of NH4+ expelled and its isotope signature can be fully attributed to\nthe reduction of NO3- sourced from seawater. The authors miss an opportunity to acknowledge the\nexisting literature on this topic, and they do not fully discount this pathway. We include more specific\nsuggestions in the annotated paper attached to this review.\nReply:\nwe thank R2 to bring the PhD thesis of Charoenpong (2019) (now Ref 18 in our manuscript) to our\nattention. In this revision, we have added more discussions on (1) the diverse sources of hydrothermal\nfluid in the study site (Chen et al., 2023Ref 38) and (2) the potential contribution of NO - reduction to NH +\n3 4\nin shallow hydrothermal systems, which actually strengthens our conclusion that the NH + from abiotic\n2 4\norganic matter, desorption from clays in sediments, and/or reduction of NO - in seawater by biological or\n3\nhydrothermal processes. Please see more explanations in the point-by-point replies below.\nWe also adopted the revision suggestions in the annotated PDF attachment and also itemized our reply\nbelow.\nThe authors emphasize that NH + in modern vents is ‘contaminated’ by seawater-derived NH +, obscuring\n4 4\nabiotic nitrogen reduction signals. This is not true for two reasons. First, seawater NH + is quite low in\n4\nabundance (~0.1 umol/kg) compared with the range of NH + abudance in unsedimented vent fluids (~1-40\n4\numol/kg). Seawater NH + is very low compared to sediment-influenced vent fluids (10s of mmol/kg).\n4\nmass balance. It is well accepted that the majority of vent-sourced NH4+ can be attributed to NO -\n3\nreduction.\nReply*:\n4\nto refer to not just the low background NH + in seawater but the NH + derived from several nitrogen-\n4 4\nbearing compounds in seawater, including those from NO - reduction (Charoenpong, 2019 Ref 18), decay of\n3\norganic matter, and desorption from clay minerals in sediments.\nThis has now been clarified by re-wording “seawater-derived NH +” to “surface NH +” with a detailed\n4 4\nexplanation of this term and the contributions from the multiple sources outlined above. See L36-51.\nAnother important detail that is missing is whether or not different marine nitrogen cycling was different\nin the Oligocene ocean, which could contribute nitrogen sources that are isotopically distinct from\nmodern N2 and NO3-, and therefore impact discussion of fractionation factors.\nReply**:\nRecent studies indicate that the Oligocene-Miocene South China Sea had oxic bottom water which was\nwell connected with the Pacific deep water (Zhang and Sun, 2023). Therefore, the Oligocene-Miocene\nnitrogen cycling in the South China Sea is expected not too different to those in the Pacific Ocean; the\nlatter show a stable marine nitrogen cycle since the Oligocene as indicated by their nitrogen isotopic\nsignatures that fall into the normal range of global marine sediments (+2‰ to +10‰; Sadofsky and\nBebout, 2004; Kast et al., 2019).\nWe also obtained the bottom sediments from two IODP drill holes in the South China Sea, i.e., U1503A\n(~37 km south of our study site) and Site U1431E (~350 km southwest of our study site). They gave 15N\nvalues of +3.6‰ and +4.0‰, respectively, which also fall in the +2‰ to +10‰ range. Therefore, these\nsediment archives consistently show no significantly different marine nitrogen cycle in the Oligocene\nSouth China Sea.\nSo far, no evidence hints at significantly different marine nitrogen cycle since the Oligocence in the study\narea.\nReference:\n(1) Zhang, Z. & Sun, Z. The early-mid Miocene abyssal brown/green claystone from IODP Site\nU1503A in the northern South China Sea: Implications for paleoclimate and paleoceanography.\nGondwana Research 120, 286-303 (2023).\n(2) Sadofsky, S. J. & Bebout, G. E. Nitrogen geochemistry of subducting sediments: new results\nfrom the Izu-Bonin-Mariana margin and insights regarding global nitrogen subduction.\nGeochem. Geophys. Geosyst. 5, Q03I15.\n(3) Kast, E.R., et al. Nitrogen isotope evidence for expanded ocean suboxia in the early Cenozoic.\nScience 364, 386–389 (2019).\nFinally, we were not convinced by the significance of the study to the Archean NH4+ pool as presently\ndescribed. The authors describe evidence for the abiotic production of NH4+ that is co-precipitated with\nsecondary hydrothermal minerals. The problem with this final calculation is that there is no discussion of\nhow the NH4+ would be released from secondary minerals into the Archaean ocean. If it is immobilized\nby high temperature hydrothermal mineralization deep in the oceanic subsurface, then NH4+ will not be\nreleased to seawater, nor will it be available biologically.\nWe would like to clarify that, NH + is a hydrophilic cation, geochemically close to Rb+. While the vein\n4\nminerals can incorporate NH + from the fluid, it only accounts for a small fraction of the total NH + in\n4 4\nfluid. The majority of the ANR-derived NH + would have remained in the fluid and been discharged to\n4\nseawater when the deep fluid migrated to the surface. This can be more quantitatively demonstrated by\nthe partition coefficient (D) of NH + between minerals and fluid, e.g., D = 0.12 and D\n= 0.14 at 400 C (Pöter et al., 2004 Ref 32 in our manuscript). These D values decrease quickly with\ndecrease of temperature. Compared with muscovite and K-feldspar, the studied vein minerals here\n(plagiocalse, epidote, chlorite) have even less NH +-hosting capabilities. For example, D =\n0.10-0.67, D = 0.12-0.72 (Honma & Itihara, 1981 Ref 34). Accordingly, the NH + content of\nPlagioclase-Muscovite 4\nfluid can be calculated by the NH + content of vein minerals and partition coefficient between minerals\n4\nand fluid. The detailed calculation is given in Methods 2.4.\nL17: check grammar here\nReply: We have reworded ‘sustainable amount of NH /NH +’ to ‘sustainable NH /NH +’. See L17.\n3 4 3 4\nL18: Grammar\nReply: Revised by removing “however”. See L18.\nL33: Abiotic nitrogen reduction is a process, but I would not use the term reaction here because the authors\nhave not specified the reactants.\nReply: Revised as suggested: ‘reaction’ is replaced by ‘process’. See L31.\nL30-36: This sentence is very long. Suggest splitting it up to present each idea described.\nReply: Revised as suggested. The sentence is now split into two sentences. See L32.\nL37-39: I do not agree that contamination by biogenic NH4+ in seawater is the reason for the lack of\nevidence for ANR in vent fluids.\nSeawater NH4+ abundance is very low (~0.1 um) compared to NH4+ abundance in vent fluids (~1-30 um\nin unsedimented vents, ~10 mm in sedimented vents; see Supp Info in Reeves et al., PNAS, 2014 for a good\nlist of NH4+ abundance data across many geologic settings). Contamination from seawater-derived NH4+\nis a minor influence on the vent NH4+ pool.\nReply: Please see the Reply above marked by *, and the revision in L36-51.\ngeneral, particularly in terms of the number of studies that combine abundance measurements with stable\nisotopes.\nReply: we totally agree with this point and added it in L46-49.\nL42: Usually the assumption is that vent fluids are initially 100% deep seawater, rather than 'partially\ngenerated'\nReply:\nWe have removed “partially” to reduce confusion. Nevertheless, trace element and radiogenic isotope\nstudies on the vein minerals from the studied drill core indicate that the hydrothermal fluid in the study site\n(U1502) involved not only modified seawater, but also magmatic fluid from the depths (Chen et al., 2023\nRef 38). This has been added in L75-77.\nL45: This is an oversimplified statement. The earlier statement is true, that N4+ in the circulating fluid can\nbe incorporated into secondary minerals. However, the predominant thinking is that much of the NH4+\nthat vents at the seawater is derived from the reduction of seawater-sourced NO3-.\nThe authors must acknowledge the possibility of seawater-sourced NO3- reduction as a source of NH4+ to\ncirculating vent fluids, because NO3- is the second most abundant nitrogen species in seawater. it is\nparticuarly elevated in deep water.\nChapter 3 of the MIT/WHOI PhD thesis by Net Charoenpong has a great summary:\nhttps://darchive.mblwhoilibrary.org/bitstreams/20632fca-af49-5ee9-823b-0e4bc59e4a1c/download\nReply:\nAs pointed out in the Reply above marked by *, we have acknowledged the contribution of NO - in\n3\nhydrothermal fluids. It should be pointed out that those literature data were mostly obtained from samples\ncollected from hydrothermal vents, which belong to the ‘shallow hydrothermal fluid’ in our discussion and\ndistinct to the ‘deep hydrothermal fluid’ we referred to in our manuscript.\nL47: The mineral formulas do not contain NH4+. It would be worth a brief comment here to state that\nNH4+ can substitute for K+ (or other ions).\nReply: Revised as suggested. See L58-59.\nL52: Given that NH4+ in hydrothermal systems is thought to be generated by reduction of seawater-derived\nNO3-, it would be worth stating whether the d15N of seawater NO3- or dissolved N2 has remained constant\nsince the Oligocene. Did the downwelling seawater in the Oligocene have the same N isotope\ncharacteristics as today?\nhttps://www.science.org/doi/full/10.1126/science.aau5784?casa_token=WQhq182HUSEAAAAA%3Ai13\n4L3BBliNljY89TB8VQKvc3PNjOxrB84ZgHkcYjjfZU00OMr36gymvIKS2FO7Yd0kTWfKD_UsV-a5L\nReply: please see the Reply above marked by **.\nL67: Have these basalts also experienced hydrothermal alteration?\nReply: Yes, numerous studies in elemental geochemistry and stable and radiogenic isotopes have\ndemonstrated that these oceanic basalts have experienced hydrothermal alteration at various temperatures.\nThe detailed sample description and relevant discussions with references have been given in the papers\ncited in this sentence. In our revision, we added ‘hydrothermally altered basalts’ to highlight this point. See\nL90-91.\nL68: This ...observation?\nReply: We have rephrased this sentence for clarification. See L93-95.\nL70: How high is high? How low is low?\nReply: The temperature ranges have been added. See L95-98.\nL71: Can you split these ideas into shorter sentences and provide more specifics? Which type of mineral\nsuite hosts more NH4+? Briefly, why?\nReply: Revised as suggested. This information has also been incorporated in L93-98.\nL83-86: This section is explained nicely and supported by the figures.\nReply: thanks.\nL91-92: Check grammar here. Confusing phrasing.\nReply: the sentence has been rephrased; see L113-115.\nL106-108: The logic works here - I can follow the thought process to explain the observation.\nReply: We appreciate this positive comment.\nL109: By extremophiles, are you referring to living microbial biomass?\nL111-114: These are very interesting observations, despite my critique of the analysis.\nReply: We appreciate the positive comment.\nL119: Critically, the authors have not mentioned dissolved deepwater NO3-. This must be included as a\npossible (and plausible) source of nitrogen, along with DON.\nReply: added as suggested. See L141-143.\nL125: What if incomplete nitrate reduction to ammonium occured at <200°C in the shallow downwelling\nzone? It could then achieve more depleted (more negative values) and then be carried along with the fluid\nas it became progressing hotter, until being removed by secondary mineralization.\nChapter 2 of the MIT/WHOI PhD thesis by Net Charoenpong describes NO3- reduction experiments at\nhydrothermal conditions, including 150-200 degree experiments, that produce epsilon values between 11\nto 67 per mil:\nhttps://darchive.mblwhoilibrary.org/bitstreams/20632fca-af49-5ee9-823b-0e4bc59e4a1c/download\nReply:\n1. We have carefully checked the experimental data in the PhD thesis by Charoenpong (2019), which\nhas been referred in the discussion of N isotope fractionation factors in L150. We found that their\nresults are in good agreement with our theoretical calculation results (Li Y. et al., 2021 Ref 35) except\none point at a low temperature of 175 ºC (see Fig. R2 below).\n2. A detailed look at the data of the 175 ºC experiments by Charoenpong (2019) show incomplete\nnitrogen yields, i.e., produced NH + plus remaining NO - are less than 100% (see Fig. R3a below).\n4 3\nWe speculate that the loss of nitrogen in these experiments may indicate that the nitrate reduction\nprocess in these experiments may not be simple nitrate reduction to ammonium but involve other\ngaseous byproduct such as NO and N O, which have been observed in similar experiments by\n3. To further test this, we carried out abiotic nitrate reduction experiments in Fe2+-bearing solution at\nroom temperature. The results also show significant N loss based on the mass balance of recovered\nNH + product and remaining NO - (see Fig. R3c). Therefore, the anomalously large isotope\n4 3\nfractionations observed in both Charoenpong’s 175 ºC experiments and our room-T experiments (Fig.\nR3b, d) are superimposed effect of the isotope fractionation between NO - and NH + and the isotope\n3 4\nfractionation between NO - and NO or N O. A full understanding of this complexity requires much\n3 2\nmore experimental efforts which are beyond the scope of this study.\n4. Nevertheless, despite this complexity, the temperatures of these experiments with anomalously large\nN isotope fractionations, i.e., 175 ºC in Charoenpong (2019) or room-T in our new experiments, are\nall much lower than the formation T range of the studied vein samples (>200 ºC). Thus the\n14\nfractionation factor of -67‰ obtained from the experiments is not considered as a reference datum\nfor the discussion in the study.\nFig. R2. Comparison of N isotope fractionation factors between experimental results of Charoenpong\n(2019) and theoretical calculations of Li Y. et al. (2021).\nFig. R3. Comparison of N yield (a, c) and isotope fractionations (b, d) of abiotic nitrate reduction in\nlaboratory experiments at 175 ºC (Charoenpong, 2019) and at room T (21 ºC; our unpublished data).\nReply: “higher” has been replaced by “less negative”. See L150.\nL141: Is Fe(II) the only reductant? What about aqueous H2?\nReply:\nYes, H could also be a potential reductant contributing to the process. But in the deep hydrothermal system\n2\nin oceanic crust, H is mainly produced by interaction of water and Fe2+-bearing minerals. Thus it is already\n2\nincluded in the expression of “the interaction between the deep fluids and Fe2+-bearing minerals”.\nL143: See my earlier comment about the abundance of NH4+ being very low in seawater. A more plausible\nNH4+ source in the shallowest parts of the core could be biogenic marine sediment.\nReply:\nTo be consistent with the previous expression in the manuscript, it is now re-worded to ‘surface NH +’,\n4\nwhich includes NH + from a variety of surface processes, such as biogenic degradation, nitrate reduction,\n4\ndesorption from clay. See L171.\nL151-152: Check grammar here.\nReply: the sentence has been rephrased. See L189-191.\nL175: The flux is not into seawater, as the authors write, but rather into secondary minerals.\nReply: Please see the Reply above marked by ***.\nL190-191: The NH + will not reach the Archean ocean if it is fixed into secondary minerals deep in the\n4\nsubseafloor by the processes the authors put forward. It might then reach the ocean if the minerals are\nsubsequently weathered, but what if they are subducted instead? I don't buy this argument - how would the\nNH + be mobilized, exactly?\nL388: typo\nReply: corrected. See L448.\nL495: Fluid compositions with Mg2+ = 0 are the product of a mathematical regression that completely\nremoves seawater contamination. Not \"little contamination\"\nL501: What was the concentration of NH + and Rb in these two veins, and what average Rb fluid\n4\nconcentration was used, so that the reader can more easily follow the outcome of the calculation?\nReply: These values have been added. See L557-559.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #1 (Remarks to the Author):\nMy suggestions have been worked in and the manuscript presents itself significantly improved over last\nyear's version.\nJust some minor comments below. I can recommend this updated manuscript for publication in Nature\nCommunications.\nL20. \"at IODP Site U1502.\" Nature Communications has a very broad readership and so I suggest to\nchange this to the general field site or geographical name (e.g., oceanic crusts in the South China Sea\nbasin). The detail here is not needed in the abstract and virtually nobody will know what/where this is.\nReply: revised as suggested (see L22-23).\nL56/57. \"diminished surface NH4+ in depths\" – seems contradictory.. surface or deep NH4+ ?\nReply: the sentence has been rephrased for clarity (L59-60)\nL177-180. These chemical formulas are much appreciated!\nReply: Thanks; no revision is needed here.\nL230. just checking the grammar: \"..may have been more active and productive..\"?\nReply: “active and” is removed (see L234).\nReference 4. It is Science volume 177 and not volume 117.\nReply: Corrected (see L455).\nReference 15. Check first author last name.\nReply: Corrected (see L479).\nReference 21. Missing publication year.\nReply: I guess R1 meant Ref 19 & 20. The publication years have been added now. See L491, 493.\nFigure 3. fonts are a bit small but I think the journal will still format this anyways.\nReply: Text in the diagram have been enlarged.\nThe following suggestions aim to enhance the engagement of non-specialists and minimize potential\nmisunderstandings.\n1. The term “secondary silicate minerals” in Figure 2 may mislead some readers. Literally, quartz is a\ntypical alteration mineral in this study, but it is not included in the “Altered silicate mineral” listed in\nSupplementary Table 1. I suggest this term should be revised for clarity.\nReply: For clarification, the term “secondary silicate minerals” should be “nitrogen-bearing secondary\nsilicate minerals”. This has been corrected in both Figure 2 caption (L652) and Supplementary Table 1.\n2. As mentioned in the abstract of the manuscript, NH3 or NH4+ can promote prebiotic reactions and\nresolve the faint young sun paradox. But many readers may not be familiar with the background involved.\nI suggest adding a relevant discussion.\nReply: Added as suggested (see L33).\n3. I note that the other three reviewers provided numerous constructive comments, which have\nsignificantly contributed to improving the quality of this manuscript. I also find that the authors have\nprovided thorough and thoughtful responses to these reviewers’ suggestions. The authors have made\nmuch effort to improve the writing, incorporated additional thermodynamic calculations, and\nsupplemented experimental data. I suggest that the sections on the added thermodynamic calculations and\nexperimental supplements should be included in the supplementary information, which may help address\npotential questions from readers who have similar concerns.\nReply: A suggested, we have added the discussions based on thermodynamic calculations and incubation\nexperiments into the Supplementary Information (text S3).\n2"}, {"round": 2, "reviewer_comments": {"reviewer_1": "My suggestions have been worked in and the manuscript presents itself significantly improved over last year's version.\nJust some minor comments below. I can recommend this updated manuscript for publication in Nature Communications.\nL20. \"at IODP Site U1502.\" Nature Communications has a very broad readership and so I suggest to change this to the\ngeneral field site or geographical name (e.g., oceanic crusts in the South China Sea basin). The detail here is not needed in\nthe abstract and virtually nobody will know what/where this is.\nL56/57. \"diminished surface NH4+ in depths\" – seems contradictory.. surface or deep NH4+ ?\nL177-180. These chemical formulas are much appreciated!\nL230. just checking the grammar: \"..may have been more active and productive..\"?\nReference 4. It is Science volume 177 and not volume 117.\nReference 15. Check first author last name.\nReference 21. Missing publication year.\nFigure 3. fonts are a bit small but I think the journal will still format this anyways.", "reviewer_4": "Review of “Abiotic nitrogen reduction in submarine hydrothermal systems: Implication to ammonium in prebiotic oceans” by\nWD Sun et al.\nI am satisfied with this manuscript, with only several minor suggestions.\nThe authors conducted numerous analyses on altered basalts and hydrothermal veins from core U1502B, including\ninvestigations of mineral phases, analyses of major and trace elements, as well as measurements of N concentrations and\nisotopic ratios. The authors discovered that the hydrothermal veins have unusually high N concentrations and strongly 15N-\ndepleted isotopic signatures. The authors interpret the signatures as evidence of abiotic nitrogen reduction (ANC) from\nmantle-derived N2 and use these data to estimate the global N flux generated by ANC.\nOverall, this manuscript, including both experimental analyses and theoretical calculations, is robust, and the interpretation\nis reasonable. Also, the writing is reasonably good. Therefore, I recommend that this paper be accepted only with minor\nrevisions.\nThe following suggestions aim to enhance the engagement of non-specialists and minimize potential misunderstandings.\n1. The term “secondary silicate minerals” in Figure 2 may mislead some readers. Literally, quartz is a typical alteration\nmineral in this study, but it is not included in the “Altered silicate mineral” listed in Supplementary Table 1. I suggest this term\nshould be revised for clarity.\n2. As mentioned in the abstract of the manuscript, NH3 or NH4+ can promote prebiotic reactions and resolve the faint young\nsun paradox. But many readers may not be familiar with the background involved. I suggest adding a relevant discussion.\n3. I note that the other three reviewers provided numerous constructive comments, which have significantly contributed to\nimproving the quality of this manuscript. I also find that the authors have provided thorough and thoughtful responses to\nthese reviewers’ suggestions. The authors have made much effort to improve the writing, incorporated additional\nthermodynamic calculations, and supplemented experimental data. I suggest that the sections on the added thermodynamic\ncalculations and experimental supplements should be included in the supplementary information, which may help address\npotential questions from readers who have similar concerns."}, "author_response": "Response to Referees' comments\nReviewer #1 (Remarks to the Author):\nMy suggestions have been worked in and the manuscript presents itself significantly improved over last\nyear's version.\nJust some minor comments below. I can recommend this updated manuscript for publication in Nature\nCommunications.\nL20. \"at IODP Site U1502.\" Nature Communications has a very broad readership and so I suggest to\nchange this to the general field site or geographical name (e.g., oceanic crusts in the South China Sea\nbasin). The detail here is not needed in the abstract and virtually nobody will know what/where this is.\nReply: revised as suggested (see L22-23).\nL56/57. \"diminished surface NH4+ in depths\" – seems contradictory.. surface or deep NH4+ ?\nReply: the sentence has been rephrased for clarity (L59-60)\nL177-180. These chemical formulas are much appreciated!\nReply: Thanks; no revision is needed here.\nL230. just checking the grammar: \"..may have been more active and productive..\"?\nReply: “active and” is removed (see L234).\nReference 4. It is Science volume 177 and not volume 117.\nReply: Corrected (see L455).\nReference 15. Check first author last name.\nReply: Corrected (see L479).\nReference 21. Missing publication year.\nReply: I guess R1 meant Ref 19 & 20. The publication years have been added now. See L491, 493.\nFigure 3. fonts are a bit small but I think the journal will still format this anyways.\nReply: Text in the diagram have been enlarged.\nThe following suggestions aim to enhance the engagement of non-specialists and minimize potential\nmisunderstandings.\n1. The term “secondary silicate minerals” in Figure 2 may mislead some readers. Literally, quartz is a\ntypical alteration mineral in this study, but it is not included in the “Altered silicate mineral” listed in\nSupplementary Table 1. I suggest this term should be revised for clarity.\nReply: For clarification, the term “secondary silicate minerals” should be “nitrogen-bearing secondary\nsilicate minerals”. This has been corrected in both Figure 2 caption (L652) and Supplementary Table 1.\n2. As mentioned in the abstract of the manuscript, NH3 or NH4+ can promote prebiotic reactions and\nresolve the faint young sun paradox. But many readers may not be familiar with the background involved.\nI suggest adding a relevant discussion.\nReply: Added as suggested (see L33).\n3. I note that the other three reviewers provided numerous constructive comments, which have\nsignificantly contributed to improving the quality of this manuscript. I also find that the authors have\nprovided thorough and thoughtful responses to these reviewers’ suggestions. The authors have made\nmuch effort to improve the writing, incorporated additional thermodynamic calculations, and\nsupplemented experimental data. I suggest that the sections on the added thermodynamic calculations and\nexperimental supplements should be included in the supplementary information, which may help address\npotential questions from readers who have similar concerns.\nReply: A suggested, we have added the discussions based on thermodynamic calculations and incubation\nexperiments into the Supplementary Information (text S3).\n2"}]

Decision letter