Cycling and persistence of iron-bound organic carbon in subseafloor sediments

Domain: earth_science
Published: 2024-07-29
Editorial outcome: major_revision

Abstract
Reactive iron (FeR) serves as an important sink of organic carbon (OC) in marine surface sediments, which preserves approximately 20% of total OC (TOC) as reactive iron-bound OC (FeR-OC). However, the fate of FeR-OC in subseafloor sediments and its availability to microorganisms, remain undetermined. Here, we reconstructed continuous FeR-OC records in two sediment cores of the northern South China Sea encompassing the suboxic to methanic biogeochemical zones and reaching a maximum age of ~100 kyr. The downcore FeR-OC contributes a relatively stable proportion of 13.3 ± 3.2% to TOC. However, distinctly lower values of less than 5% of TOC, accompanied by notable 13C depletion of FeR-OC, are observed in the sulfate-methane transition zone (SMTZ). FeR-OC is suggested to be remobilized by microbially mediated reductive dissolution of FeR and subsequently remineralized, the flux of which is 18–30% of the methane consumption in the SMTZ. The global reservoir of FeR-OC in microbially active Quaternary marine sediments could be 19-46 times the size of the atmospheric carbon pool. Thus, the FeR-OC pool may support subseafloor microorganisms and contribute to regulating Earth’s carbon cycle.

Introduction
On geological timescales, the burial rate of sedimentary organic carbon (OC) exerts major control on the concentrations of atmospheric O2 and CO2 and thus substantially influences Earth’s environmental conditions. Reactive iron (FeR, defined as iron (oxyhydr)oxides that can be reductively dissolved by sodium dithionite) can promote the preservation of OC in a wide range of terrestrial and marine environments, as reactive iron-bound OC (FeR-OC) has long been believed to be more resistant to microbial degradation under aerobic conditions. It is estimated that ~20.2 ± 15.5% of the total OC (TOC) is directly bound to FeR in marine surface sediments, which corresponds to a global reservoir of 7–54 Pg of OC (roughly 1–6% of the amount of the atmospheric CO2), demonstrating its potentially important role in preserving the sedimentary OC. A comprehensive picture regarding the fate of FeR-OC on geological timescales is still lacking but several studies of sediment of Pleistocene as well as late Paleocene to early Eocene age suggest that the fraction of FeR-OC in TOC (fFeR-OC) remains relatively stable. However, the question to what degree the FeR-OC reservoir interacts with sedimentary biogeochemical processes, in particular those involving redox reactions of iron and sulfur, remains unresolved.

FeR has long been found to promote organic matter preservation in terrestrial soils as well, especially via adsorption. However, recent studies indicated the interactions between Fe and OC in soils are highly influenced by the redox oscillations during water-table fluctuatioins in terrestrial environments. Under anoxic conditions, Fe reduction releases FeR-OC and increases the anaerobic remineralization of soil organic matter. While under oxic conditions, newly-formed Fe promotes OC retention on mineral surfaces.

In anoxic marine sediments, FeR is one electron acceptor actively involved in biogeochemical processes. Both FeR reduction by microorganisms and biogenic sulfide produced during sulfate reduction could potentially weaken the association with OC. Indeed, recent lab incubations demonstrated that FeR-OC can be remobilized during microbial iron reduction and subsequently utilized as an electron donor and/or carbon source for microbial communities. Consequently, the remobilization of FeR-OC during early diagenesis may influence the relative size of the FeR-OC reservoir that is preserved in sediments for a long time. Additionally, the supply of FeR-OC is expected to depend on the hydrological conditions through the influence on continental weathering and FeR formation as well as on sea level through the influence on shelf topography and thus the transport of continental detritus into the deep sea; both factors are expected to vary periodically on glacial-interglacial timescales. To disentangle the effect of FeR-OC supply and early diagenetic reworking on sedimentary FeR-OC, downcore FeR-OC records need to be established and related to both geochemical zonation and sediment chronology.

In this study, we analyzed two gravity cores (QDN-G1 and QDN-14B, Fig. 1a) from the northern South China Sea in order to determine the fate of the sedimentary FeR-OC via its quantitative and isotopic analysis. The core QDN-G1 represents typical continental slope sediments (1478 m water depth), while core QDN-14B (1370 m water depth), around 35 km southwest from core QDN-G1, was influenced by nearby cold seeps expelling methane-rich fluids. Consequently, QDN-14B can be used to comparatively evaluate the influence of microbial activities on the potential remobilization of FeR-OC in the diagenetically active zones where sulfate reduction coupled to either organic matter remineralization or methane oxidation occurs. Meanwhile, the core QDN-G1 consisting of sediments with relatively low microbial activity is used for exploring the long-term preservation of FeR-OC on glacial-interglacial timescale with well established age model covering the past 97 kyr (see Methods and Supplementary Fig. 1 for detailed information).

To quantify the amount of FeR-OC, the citrate-bicarbonate-dithionite (CBD) method described in detail by Lalonde et al. and Salvadó et al. was applied in this study (see Methods). This method targets only reactive iron (oxyhydr)oxides, which are presumably accessible for microorganisms, and leaves unreactive phases such as iron-containing silicates untouched. By incorporating analyses of porewater geochemistry, including the concentrations of ferrous iron, sulfate, dissolved inorganic carbon (DIC), and the carbon isotope ratio of DIC (δ13CDIC), all of which are tied to microbially mediated processes, our study takes a critical step in assessing the stability of sedimentary FeR-OC in response to post-depositional microbial activities and sheds light on its fate in subseafloor sediments.

Results
The core recoveries of QDN-G1 and QDN-14B were 10.8 m and 8.4 m, respectively. These two cores penetrated sediments with distinct diagenetic activities. Microbially mediated early diagenetic redox reactions have resulted in distinct geochemical zonation, i.e., the iron reduction zone (IRZ), the sulfate reduction zone (SRZ), the sulfate-methane transition zone (SMTZ), and the methanogenic zone (MZ) (Fig. 1b). Despite similar TOC contents in sediments from the two cores (see below), the higher methane fluxes and the associated rapid depletion of sulfate via anaerobic oxidation of methane resulted in a shallower SMTZ in core QDN-14B (280–440 cmbsf) compared to core QDN-G1 (potentially around 720–960 cmbsf) (Fig. 1b). This difference in activity is supported by geochemical modeling (see Methods), which showed a more than two times higher net sulfate reduction rate in the SMTZ in core QDN-14B compared to core QDN-G1 (0.034 vs. 0.015 mol m−3 yr−1) (Supplementary Fig. 4).

The FeR contents in the two cores range from 0.7 to 5.7 mg g−1, which reaches the maximum at 200 cmbsf and 240 cmbsf in QDN-G1 and QDN-14B, respectively (Fig. 2a, f). TOC contents in the two cores range from 0.5 to 1.2% (Fig. 2b, g, black lines). The FeR-OC contents in the two cores range from 0.02 to 0.17% (Fig. 2b, g, red lines) and account for 3.2–20.5% of TOC (Fig. 2d, i). The downcore patterns of both TOC and FeR-OC content profiles are not consistent with that of FeR content. The presence of low levels of dissolved Fe(II) throughout the core QDN-G1 (3–35 μM, Fig. 1b) is consistent with some background activity of dissimilatory iron reduction, independent of the biogeochemical zone. However, there is no discernable imprint on the content of FeR-OC, except in the SMTZ (Fig. 2b). Interestingly, both the lowest FeR-OC content and the lowest fFeR-OC occurred in the SMTZ of each core. In QDN-G1 and QDN-14B, the average FeR-OC contents in non-SMTZ sediments are 0.12% and 0.10%, respectively, but drop to 0.06% and 0.03% in the SMTZ sediments, respectively (Table 1). Similarly, the average fFeR-OC in the non-SMTZ layers are 14.0% and 13.0%, repectively, but drop to 12.3% and 5.2% in the SMTZ sediments, respectively (Table 1).

The carbon isotope ratios of TOC (δ13CTOC) and FeR-OC (δ13CFeR-OC) provide insights into the sources of the corresponding OC pool. In QDN-G1 and QDN-14B, δ13CTOC varies from −22.5 to −19.7‰, and from −25.9 to −20.2‰, respectively (Fig. 2c, h, black lines); δ13CFeR-OC varies from −31.1 to −20.0‰, and from −25.2 to −17.2‰, respectively (Fig. 2c, h, red lines). Lower δ13CTOC and δ13CFeR-OC values are observed in SMTZ of both cores (Fig. 2c, h). The average δ13CTOC values of non-SMTZ sediments are −20.6 ± 0.7‰ and −21.5 ± 0.7‰, but 1.5‰ and 1.9‰ lower in SMTZ sediments in QDN-G1 and QDN-14B, respectively (Table 1). Similarly, the average δ13CFeR-OC values of non-SMTZ sediments are −21.8 ± 1.8‰ and −20.8 ± 1.4‰, but 3.1‰ and 2.8‰ lower in SMTZ sediments in QDN-G1 and QDN-14B, respectively (Table 1). All the δ13CTOC and δ13CFeR-OC values in non-SMTZ sediments indicate the accumulation of mainly marine-algal OC, which in the sampled area has an average δ13C value of −20.8 ± 0.4‰. However, terrestrial OC has much higher contribution in SMTZ sediments, where particulate terrestrial OC and riverine primary production have characteristic carbon isotope signatures of −24.1 ± 1.0‰ and −30.0 ± 2.6‰.

The cumulative evidence of lower FeR-OC content, lower fFeR-OC, and lower δ13CFeR-OC in SMTZ sediments, compared to relatively uniform records in non-SMTZ sediments (significance levels in Table 1, see Methods for more information) is suggestive of dynamic organic matter cycling in this sediment horizon. While we cannot entirely rule out that these distinct signals in SMTZs of both cores are related to the depositional history and coincidentally located in this horizon, we view this scenario as highly unlikely (see Supplementary Fig. 6 and Supplementary Discussion for details). Instead, we propose that the post-depositional microbial processes account for these distinctive characteristics of FeR-OC records.

Accordingly, two observations deserve attention: (i) the distinct depletions in 13C content and contents of FeR-OC within the SMTZ, and (ii) sediments below the SMTZ show similar features with respect to 13C content and contents of FeR-OC as sediments above the SMTZ. The relatively low content of FeR-OC strongly suggests that a substantial fraction of it has been remobilized and possibly remineralized. Two mechanisms could account for the low δ13CFeR-OC of the residual fraction: (a) remobilization/degradation of a 13C-enriched, weakly bound and/or more reactive fraction with the residual fraction being 13C-depleted; since the δ13C of the residual fraction is broadly consistent with an origin from terrestrial plants, the residual fraction could be tightly adsorbed terrestrial organic matter supplied together with the detrital minerals from land. (b) The alternative involves a gain remobilization of a large fraction of FeR-OC combined with adsorption of 13C depleted dissolved organic matter (DOM) produced in the course of anaerobic oxidation of methane; the extremely low molar ratios of FeR-OC to FeR below 1 in both SMTZs (Fig. 2e, j) indicate abundant binding sites available for DOM molecules. These two mechanisms are not mutually exclusive and could act in combination. In any case, both scenarios require remobilization and possibly degradation of a substantial fraction of FeR-OC. The fact that sediments below the SMTZ resemble those above the SMTZ is at first surprising if we assume that the more deeply buried sediments were at some point in the geologic past also situated in the SMTZ. In this case, re-adsorption of DOM produced during cycling of particulate organic matter of marine origin could be the cause for the similarity of FeR-OC related signals above and below the SMTZ. However, there is also evidence that SMTZs do not “move” continuously through the sediment column with increasing sedimentation but rather “oscillate” from one horizon to another where they then remain stagnant in position for extended periods.

The SMTZ as a discrete sedimentary horizon, where anaerobic oxidation of methane is coupled to sulfate reduction (S-AOM), ubiquitously occurs in organic-rich continental shelf and slope sediments. S-AOM is performed syntrophically by sulfate-reducing bacteria and methanotrophic archaea. In core QDN-14B, geochemical modeling reveals higher net sulfate reduction rates in the SMTZ stimulated by the methane-rich fluid in the core (Fig. 3b), which is further supported by the higher relative abundance and cell concentration of sulfate-reducing bacteria (Fig. 3e, f) and by elevated copy number of methyl-coenzyme M reductase subunit A (mcrA) gene (Fig. 3g), the key marker gene for both methane-producing and methane-consuming archaea. By contrast, in core QDN-G1, there is no evidence for elevated sulfate reduction activity in the SMTZ compared to the shallower sulfate reduction zone, based on modeled rates as well as relative and absolute abundance of sulfate-reducing bacteria (Supplementary Fig. 7b, e, f). This is consistent with the relatively moderate variation in fFeR-OC record in core QDN-G1 compared to core QDN-14B. Collectively, these lines of evidence suggest that due to the particularly high methane flux at core QDN-14B, the microbial activity in the SMTZ has stimulated the remobilization of FeR-OC.

A plausible mechanism for FeR-OC remobilization in the SMTZ is the occurrence of reductive dissolution of FeR by the biogenic hydrogen sulfide produced during sulfate reduction, and subsequent release of FeR-OC. Apart from chemical reduction of FeR, sulfate-reducing bacteria may also reduce FeR directly, with the rate of reduction being enhanced by sulfate reduction and the presence of biogenic sulfide. In addition, the process of AOM may be directly coupled with iron reduction (Fe-AOM) and facilitate FeR dissolution in this zone. In summary, the reduction of FeR could be enhanced by biological and chemical processes in the SMTZ, and result in the remobilization of FeR-OC.

Assuming that both TOC as well as FeR-OC lost in the SMTZ are remineralized by heterotrophic microorganisms, the remineralization fluxes of TOC and FeR-OC in the SMTZ can be estimated by the difference of their average accumulation rates between the sediment column above and within the SMTZ (see methods). In QDN-14B, the SMTZ is well defined by the depletion of sulfate, increasing DIC, and low δ13CDIC values (Fig. 1b). The TOC and FeR-OC remineralization fluxes in the SMTZ are estimated to be 15.8 and 5.8 mol m−2 kyr−1, respectively. FeR-OC remineralization contributes 36.7% to the TOC remineralization, which is disproportionally high compared to fFeR-OC (5.2 ± 1.8%) in this zone. Therefore, FeR-OC is preferentially remineralized compared to OC that is not bound to FeR (non-FeR-OC) and results in the low fFeR-OC. Stimulated by higher microbial activity, e.g., through S-AOM, Fe-AOM, and iron reduction, FeR-OC is remobilized and potentially remineralized in the SMTZ.

The estimation of the TOC and FeR-OC remineralization fluxes for core QDN-G1 is not as straightforward as for core QDN-14B due to the higher uncertainty of assigning the vertical extension of the SMTZ and the fact that contents of TOC and FeR-OC steadily decline already above the SMTZ within this core (Fig. 2b). This decline implies that in this core the remineralization fluxes are partially unrelated to processes within the SMTZ. To address this, we used an additional approach for estimating a lower flux boundary, which is only based on the four samples directly above the SMTZ as average (see methods). The resulting range of TOC and FeR-OC remineralization fluxes for core QDN-G1 are 21.6–32.8 mol m−2 kyr−1 and 3.3–5.0 mol m−2 kyr−1, respectively. FeR-OC remineralization contributes 10.1–23.1% to the TOC remineralization, which is comparable to the fFeR-OC (12.3 ± 2.6%) in this zone.

Altogether, the remineralization of FeR-OC may substantially contribute to TOC remineralization in the SMTZ. Moreover, since FeR-OC is likely enriched in labile organic matter, it has the potential to enhance the activity and growth of heterotrophic microbial communities in subseafloor sediments and be rapidly recycled upon remobilization. Considering that the SMTZ is ubiquitous in continental slope sediments, and assuming a FeR-OC remineralization flux of 3.3–5.8 mol m−2 kyr−1 as observed in the SMTZ of the two cores and a continental slope area of 3.01 × 107 km2, the global FeR-OC remineralization flux in the SMTZ of the continental slope area is estimated to be 0.10–0.17 Tmol yr−1, which is about 18-30% of the methane consumption flux in the SMTZ of continental slope sediments (0.56 Tmol yr−1) and 0.61% of the global sedimentary OC burial rate (13.3 Tmol yr−1). Using acetate as a proxy for OC, and the Gibbs energies of OC oxidation by sulfate of −81.5 KJ (per mol acetate) at 5°C and 100 bars of pressure (1000 m water depth), the remineralization of FeR-OC in the SMTZ of the continental slope can provide a power supply from 4.1 × 1015 to 6.9 × 1015 J yr−1. Assuming the cellular power demand for organisms oxidizing OC with sulfate of 7.7 × 10−14 W cell−1, the FeR-OC remineralization in the SMTZ of continental slope area could sustain a biomass from 1.7 × 1021 to 2.8 × 1021 microbial cells. Considering generally lower power demand of 10−20 to 10−16 W cell−1 for microorganisms in marine sediments, this amount of energy could potentially support an even larger population from 1.3 × 1024 to 2.2 × 1028 microbial cells. The average SMTZ depth in continental slope sediments is estimated to be 12.8 ± 12.1 m below seafloor, where the cell abundance typically ranges from 106 to 108 cells cm−3. Assuming the SMTZ depth interval of 1 m, the volume of global continental slope SMTZ sediments reaches 3 × 1013 m3, which harbors from 3 × 1025 to 3 × 1027 cells. Therefore, the remineralization of FeR-OC in continental slope SMTZ sediments could support a substantial fraction of subseafloor microbial life in this zone.

The seafloor receives FeR mainly from terrestrial input, which is typically associated with terrestrial OC. After entering the ocean, additional organic matter from the pool of marine dissolved organic carbon may be bound to FeR-bearing minerals by adsorption. Finally, after deposition in sediments, the upward diffusing Fe(II) can coprecipitate with marine OC into authigenic FeR after re-oxidation at the redox interface. The binding mechanism between FeR and OC in marine sediments has been inferred from the molar ratio of FeR-OC to FeR, with adsorption and coprecipitation characterized by ratios of <1 and 6–10, respectively. In the two cores studied here, the molar ratio of FeR-OC to FeR is always larger than 1, except in the SMTZ, suggestive of a mixed binding mechanism of adsorption and coprecipitation (Fig. 2e, j). Furthermore, δ13CFeR-OC values in both sediment cores are typical for marine-sourced OC and the high molar ratio of FeR-OC to FeR indicates coprecipitation as an important binding mechanism. All these clues suggest that a major proportion of FeR-OC in marine sediments is intrinsically labile marine OC.

The average fFeR-OC in the two cores studied, except within the SMTZ in QDN-14B, is 13.3 ± 3.2%. The relatively stable fFeR-OC in the two cores suggest a similar degradation rate of FeR-OC and non-FeR-OC on the timescales represented by these cores. The only exception is the active SMTZ in QDN-14B, where iron reduction is enhanced significantly by processes likely induced by sulfate-reducing bacteria, methanotrophic archaea and/or biogenic sulfide. The stimulated degradation of FeR-OC over non-FeR-OC leads to extremely low fFeR-OC in the SMTZ of QDN-14B. As these especially active SMTZs have limited distribution in global marine sediments and occupy narrow depth intervals in sediment columns, the overall impact on the FeR-OC reservoir in subseafloor sediments is small.

Taken together, the FeR-OC records in these two cores suggest that a stable proportion of TOC survives early diagenesis as FeR-OC and is sequestered in marine sediments on at least 100-kyr timescales. Considering previous studies showing high fFeR-OC in the samples even from late Paleocene to early Eocene, persistence of FeR-OC on even longer timescale is expected in marine sediments. This enables a rough estimation of the global FeR-OC reservoir by multiplying the TOC reservoir in global marine sediments from the Quaternary Period (0–2.59 Ma) storage with published fFeR-OC data in marine surface sediments. For deriving representative fFeR-OC values, all published fFeR-OC data in marine sediments using the CBD method were compiled and combined with our measured data and grouped into eight categories based on their study area information: delta and estuary, continental shelf, continental slope, deep sea, anoxic/sulfidic regions, wetland, mangrove and tephra (Fig. 4, Supplementary Table 3). One-way ANOVA and Tukey Honest Significant Difference (HSD) test revealed that significant difference (P < 0.05) in fFeR-OC values exist only between tephra-containing sediments and other environments (Supplementary Table 4). Considering TOC storage in the Quaternary sediments is estimated in the three domains shelf (water depth <200 m), margin (200 m < water depth <3500 m) and abyss (water depth >3500 m), delta/estuary and continental shelf were combined to represent shelf, continental slope was used to represent margin and deep sea was used to represent abyss. Combining the published TOC reservoir and the corresponding surface fFeR-OC values in these three domains, the global FeR-OC reservoir in Quaternary marine sediments was estimated to be 28,690 ± 12,165 Pg C (Supplementary Table 5), which is approximately 19–46 times the size of the atmospheric carbon pool.

Methods
Sediment core collection and age model establishment
The Qiongdongnan (QDN) basin lies in the rift belt of the northern continental margin of the South China Sea and receives terrestrial material mainly from the Indo-China Peninsula and Southeast China (especially Hainan Island) by river runoff and eolian dust. In addition, the material contribution of marine authigenic sources to the basin cannot be neglected.

Core QDN-14B was recovered from the QDN basin at a water depth of 1370 m in 2015, ~600 m east of ROV1, which is an active seep site of “Haima” cold seeps. Core QDN-G1 was recovered outside the area of “Haima” cold seeps around 35 km away from QDN-14B at a water depth of 1478 m in 2018 (Fig. 1a). Sediment samples in core QDN-G1 were subsampled every 40 cm and preserved at −80°C. Known volumes of sediments were taken using tip cut-off syringes and sealed in glass vials for further measurements of density and porosity. For core QDN-G1, the age model of the upper 4.0 m was based on 7 accelerator mass spectrometry 14C ages of planktonic foraminifera (G. ruber) performed at the Beta Analytic Laboratory (Florida, USA) (Supplementary Table 1). The age model of the lower part (4.0–10.8 m) was established by aligning the benthic foraminifera (C. wuellerstorfi) δ18O record to the global benthic δ18O stack LR04 using the software QAnalySeries (v. 1.4.2) (Supplementary Fig. 1).

Porewater sampling and analysis
Porewater samples were extracted immediately onboard using Rhizon samplers (0.22 μm filter) before the core was cut open for sediment subsampling. To prevent oxidation of Fe(II), an aliquot of porewater was added to the ferrozine solution. Porewater samples for DIC measurements were preserved in pre-vacuumed glass vials. Porewater samples for ion measurements were acidified with concentrated HNO3. The rest of the porewater samples were preserved in pre-combusted amber glass vials at −20°C for dissolved organic carbon measurements.

The Fe(II) concentration was determined by ferrozine assay using a spectrophotometer (DR5000, Hach, USA). The concentrations of major cations and anions were determined by ion chromatography (Dionex ICS-5000+, Thermo Scientific, USA). Dissolved inorganic carbon (DIC) was measured using a total carbon analyzer (Multi 3100, Analytik Jena, Germany).

Physical properties of bulk sediments
Known volumes of sediments were subsampled by tip cut-off syringes and preserved in sealed glass vials for the measurements of physical properties, such as porosity and dry bulk density. All the physical properties were only measured for QDN-G1 samples. The averaged porosity and dry bulk density were used to represent the general porosities of the two cores.

Porosity was calculated by the volume ratio of water and wet sediment, where the volume of water was calculated from the difference between the wet and dry sample weight. The dry bulk density was calculated by dividing the dry sample weight by the original wet sample volume.

FeR and FeR-OC extraction
FeR and FeR-OC were extracted using the citrate-bicarbonate-dithionite (CBD) method according to Mehra and Jackson (1960), as modified by Lalonde et al. and Salvadó et al. Both poorly crystalline and crystalline iron (oxyhydr)oxides can be reduced by this method, including mainly goethite and hematite, which are the major iron (oxyhydr)oxides in marine sediments. Briefly, samples were freeze-dried and homogenized using an agate mortar and pestle. Samples (0.5 g) were weighed carefully into 40 mL Teflon tubes and extracted in a 30 mL solution of sodium dithionite (Cat. No. 71699-50G, Sigma-Aldrich, Germany) and trisodium citrate (Cat. No. 1009408, Sinopharm, China) buffered with sodium bicarbonate (Cat. No. S837271-500G, Macklin, China) at 80°C for 15 min in a water bath. After the extraction, the suspensions were centrifuged for 10 min at 4000 × g and then rinsed 5 times with artificial seawater. To evaluate the amount of OC remobilized during the extraction that was not bound to FeR, another sample aliquot was extracted as a control under the same experimental conditions, but replacing sodium dithionite and trisodium citrate with sodium chloride (Cat. No. 10019308, Sinopharm, China) with equivalent ion strength. The residuals were dried overnight in a 50°C oven, carefully weighed and manually ground.

The supernatant and rinse water were combined, acidified to pH < 2 and filtered through 0.22 μm filters. The dissolved iron was determined using a ferrozine assay. The iron extracted in the control group were negligible; therefore, the amount of FeR was calculated based on the iron extracted in the dithionite reduction group without further correction.

FeR-OC quantification and carbon isotope analyzes
The OC content and carbon isotope ratio were determined for both untreated and treated samples using an elemental analyzer (Vario EL III, Elementar) coupled to an isotope ratio mass spectrometer (Isoprime, Elementar) at the instrumental analysis centre, Shanghai Jiao Tong University. The inorganic carbon was removed by acid fumigation before the analysis. The samples were measured in triplicate, and the standard deviation was <0.05% for TOC and <0.2‰ for δ13C. The analytical precision was <0.06% for TOC (standard deviation for repeated measurements of the low organic content soil standard; n = 3) and <0.09‰ for δ13C (standard deviation for repeated measurements of the USGS40 standard, n = 3).

Calculation of fFeR-OC and δ13CFeR-OC
The fraction of FeR-OC in TOC (fFeR-OC) was determined as the difference of TOC amount in the residuals between the CBD extraction and control extraction devided by the TOC amount in the untreated sample.

fFeR-OC = (TOCcontrol − TOCCBD) / TOCbulk (1)

in which,
TOCcontrol = total organic carbon (mg) left after the control extraction,
TOCCBD = total organic carbon (mg) left after the CBD extraction,
TOCbulk = total organic carbon (mg) in the untreated sample.

A binary mixing model was used to calculate the carbon isotope ratio of FeR-OC, where the OC pool in the residual of the control group (TOCcontrol) is comprised of FeR-OC and OC remaining untouched after CBD extraction (TOCCBD). The carbon isotope ratios of FeR-OC was calculated according to the following mass-balanced equations:

TOCCBD + FeR-OC = TOCcontrol (2)

fTOC_CBD × δ13CCBD + fFeR-OC × δ13CFeR-OC = δ13Ccontrol (3)

in which,
FeR-OC = the amount of organic carbon (mg) bound to FeR,
TOCCBD = total organic carbon (mg) left after the CBD extraction,
TOCcontrol = total organic carbon (mg) left after the control extraction,
fTOC_CBD = TOCCBD / TOCcontrol,
fFeR-OC = FeR-OC / TOCcontrol,
δ13CCBD = the measured carbon isotope signature of TOCCBD,
δ13Ccontrol = the measured carbon isotope signature of TOCcontrol,
δ13CFeR-OC = the carbon isotope signature of FeR-OC.

Geochemical modeling
Net reaction rates of sulfate were estimated using the MATLAB (2021b) script published in Wang et al., considering molecular diffusion, sediment burial, fluid advection, and reaction under the assumption that the sulfate concentration profiles represent steady-state conditions.

The mass balance of sulfate is expressed as:

∂ / ∂x (D(x) ∂C(x) / ∂x) + (φ(x)b(x) + φ(x)v(x)) ∂C(x) / ∂x + R(x) = 0 (4)

Where C(x) is sulfate concentration in the pore water, x is depth below the seafloor, φ(x) is porosity, D(x) is the molecular diffusion coefficient of sulfate, θ2(x) is tortuosity, b(x) is porewater burial velocity, v(x) is externally driven flow velocity, R(x) is the rate of diagenetic reaction per unit volume of sediment.

The model applied a 5-point Gaussian filter to the sulfate concentration profile, with the weighting on the 5 points of: [0.06, 0.24, 0.4, 0.24, 0.06]. All parameters used for modeling are listed in Supplementary Table 2. Porosity profile was measured using QDN-G1 samples. The averaged porosity was applied as the constant porosity value for both cores assuming similar sediment properties in the study area. Diffusion coefficients of sulfate in the two cores were obtained using R package marelac with the input of respective salinity, temperature and pressure values (Supplementary Table 2). The tortuosity θ2 was calculated by the relation, θ2 = 1/φ, where f is the formation factor, which can be calculated based on the empirical relationship f = 1:3 × φ−1:45. We used the sedimentation rate near the seafloor 1.2 × 10−4 m yr−1 as the porewater burial velocity for both cores, assuming a similar sedimentation rate in the study area, which is calculated by the radiocarbon dating ages of the first two sampling layers below the seafloor in QDN-G1. A constant external flow advection velocity near the sediment-water interface of 10−5 m yr−1 was used. A minimum of three measured concentration data points were used to determine each reaction zone. The model quantifies uncertainties in the rate estimates by using a Monte Carlo technique. The depth-integrated net reaction rate yielded the total flux at steady state for the sediment column.

Estimation of TOC and FeR-OC remineralization fluxes in the SMTZ
The remineralization fluxes of TOC and FeR-OC in the SMTZ can be estimated by the difference of their average accumulation rates between the sediment column above and within the SMTZ, assuming that all the TOC and FeR-OC lost in the SMTZ is remineralized by heterotrophic microorganisms as opposed to mobilized and moved into another organic carbon pool.

The accumulation rates of TOC and FeR-OC are calculated by multiplying their contents in sediments by the sediment mass accumulation rate:

TOC accumulation rate = LSR × DBD × TOC% (5)

FeR-OC accumulation rate = LSR × DBD × FeR-OC% (6)

where DBD is the dry sediment bulk density in g cm−3, LSR is the linear sedimentation rate in cm kyr−1, TOC% and FeR-OC% are the weight percentage of TOC and FeR-OC in sediments, respectively. The TOC and FeR-OC accumulation rates are expressed in mol m−2 kyr−1. The average sedimentation rate (11.1 cm kyr−1) and the average dry bulk density (0.9 g cm−3) of QDN-G1 was used for the calculation for core QDN-14B, assuming similar sedimentation rate and bulk sediment properties in the study area.

When assigning the background levels of TOC and FeR-OC accumulation rates, which are independent of remineralization within the SMTZ, the average accumulation rate in the sediment column above the SMTZ was used. As TOC and FeR-OC contents in core QDN-G1 begin to steadily decline above the SMTZ (Fig. 2b), this approach may lead to an overestimation of remineralization fluxes within the SMTZ. Therefore, for core QDN-G1, we estimated a lower boundary of background TOC and FeR-OC accumulation rates, represented by the four samples above the SMTZ (560–720 cmbsf) as average.

DNA extraction, qPCR and amplicon sequencing
The 16S rRNA gene data was obtained for core QDN-G1 according to the following procedures.

The DNA for qPCR and V4 region of 16S rRNA gene sequencing was extracted from ~0.25 g of sediments using DNeasy® PowerSoil® Pro Kit (Cat. No. 47014, Qiagen, Germany), according to the manufacturer’s instructions.

The extracted DNA was used as template for qPCR to determine the abundance of bacterial 16S rRNA gene with the primer set 331F (5ʹ-TCCTACGGGAGGCAGCAGT-3ʹ) / 797R (5ʹ-GGACTACCAGGGTATCTAATCCTGTT-3ʹ). Standard curves were constructed using a 10-fold series dilution of the plasmids for six gradients carrying the bacteria 16S rRNA gene. qPCR was carried out in a volume of 20 μL, including 10 μL 2 × PowerUp™ SYBR™ Green Master Mix (Cat. No. A25742, Thermo Scientific, USA), 1.6 μL each primer (10 μM), 2 μL template DNA and 4.8 μL sterile ddeionized water. The qPCR program consisted of an initial cycle of 95°C for 5 min; 40 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 80°C for 10 s, and the data was collected at the final step of each cycle. The melting curve was generated using default program. All samples were subject to qPCR measurement with three technical replicates. The bacterial cell number was evaluated based on the abundance of 16S rRNA gene, applying the average copy number of the 16S rRNA gene on genomes of Bacteria (5.3 copies/genome, rrnDB version 5.8).

The V4 region of 16S rRNA gene was amplified using the primer set 515F (5ʹ-GTGYCAGCMGCCGCGGTAA-3ʹ) / 806R (5ʹ-GGACTACNVGGGTWTCTAAT-3ʹ). DNA was amplified using the following cycling conditions: 95°C, 5 min; 30 cycles (95°C, 30 s; 50°C, 30 s; 72°C, 30 s); 72°C, 7 min. The PCR products of samples were sent to Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China) for high-throughput sequencing of the 16S rRNA gene using the Illumina Novaseq PE250 platform.

Sequence analysis
The raw reads of 16S rRNA gene of both cores QDN-G1 and QDN-14B were processed and analyzed using the QIIME 2 platform (version 2020.11). The primers and adapters were first trimmed out using Cutadapt (version 3.1). Raw sequences were then processed using DADA2, including quality filtering, denoising, paired-end sequence merging, chimera filtering and producing amplicon sequence variants (ASVs) and ASV Table. Taxonomy was assigned using q2-feature-classifier (a scikit-learn naive Bayes machine-learning classifier) with Silva database release 138. Multiple sequence alignment and phylogenetic tree construction were performed using the QIIME 2 plugin q2-phylogeny (align-to-tree-mafft-iqtree). Unassigned sequences, singletons and sequences affiliated with eukaryotes were discarded. Eventually, to eliminate uneven sequencing depths, the ASV table was rarefied to 14935 and 71773 sequences per sample for QDN-14B and QDN-G1, respectively, determined by the sample with the fewest sequences (Supplementary Data 1 and 2).

Statistical analyzes
Statistical analyzes were carried out in R (version 4.1.3). The Wilcoxon rank sum test was applied to compare TOC and FeR-OC records between SMTZ and non-SMTZ sediments in two sediment cores studied, including TOC content, FeR-OC content, fFeR-OC, δ13CTOC, and δ13CFeR-OC.

One-way analysis of variance (ANOVA) was used to determine if there are significant differences in fFeR-OC in surface sediments of different marine environments. Tukey Honest Significant Difference (HSD) test was used for performing multiple pairwise-comparison between the environments to determine which among them were significantly different.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "This work explored the persistency of reactive-iron associated organic carbon and its behavior in\ncontinental slope sediments, then updated the global budget of FeR-OC. This work is significant.\nSome statements, however, need more explanations or calculation to support. See the comments\nbelow for details.\nLine 23: a comma is missing.\nLine 29: use the right format of scientific notation here and throughout the text.\nLine 45: add “an” before “active electron acceptor”.\nLine 58: “Nevertheless” is odd here and can be removed.\nLine 109: Fig. 2a is not cited yet.\nLine 117: provide a number or a range to show how low it is.\nLine 123: the average content of FeR-OC of 0.03% is contradictory to the statement on Line 119\nwhere the minimum FeR-OC content is 0.04%.\nLine 125: the average FeR-OC/DOC of 5.2% is is contradictory to the statement on Line 120\nwhere the minimum FeR-OC/TOC is 10%.\nLine 141-146: I agree with the authors that microbial activities are the most likely reason for the\nphenomenon that in both cores, lower values are observed in SMTZ sediments. But I don’t\nfollow how a glacial-interglacial cycle has something to do with the reasons proposed for the\nlower values. More explanations are needed here.\nLine 244: what is non-FeR-OC? Why is FeR-OC with a percentage of 10-20% in TOC\npreferentially\nremineralized? I don’t get the point. More explanation is needed.\nLine 269-272: using the numbers provided here, “a substantial fraction” the authors claim is less\nthan 4%. Is this “substantial”?\nLine 288: Based on the molar ratio of FeR-OC to FeR and the two mechanisms, the proportion can\nbe estimated to support “a large proportion” the authors proposed. Otherwise, the proposal is\nweak.\nLine 329: “shelf” should be “slope”?\nLine 331: change “is” to “are”.\nLine 331-334: a conjunction is needed for the two sentences.\nLine 354 & 357: provide the number of data to be averaged.", "reviewer_3": "The manuscript investigated the long-term variations in content and isotope of TOC and FeR-OC,\nemphasizing the impact of SMTZ in FeR-OC preservation. Based on the basically unaltered FeR-\nOC/TOC ratio long-buried sediments, this study provided an estimation of global budget of FeR-OC\nin Quaternary marine sediments. The topic is new and innovative, and experimental and analytical\ndesigns are convincing. The results provide an important experimental basis for understanding the\nrole of sedimentary FeR-OC in regulating long-term carbon cycle.\n1. Title, the current title could not be completely reflect the topic and the present study, such as the\nitems of digenesis and microbial community.\n2. Before the process of SMTZ, how the FeR-OC change and/or response towards the reduction\nprocess of dissimilatory Fe and Mn reduction?\n3. The impact of SMTZ in FeR-OC preservation was more significant in the sediment core QDN-14B\nnearby cold seeps due to the more abundant microbial occurrence; however, this degradation\nmechanism could not be suitable for the slope sedimentary setting, thus is the microbial evidence\nalso be increased in the normal slope core with SMTZ section? In addition to the support from the\nremineralization of FeR-OC, what is the role of methane release for this specific microbial\ncommunity structure?\n4. Line 143-154. As the authors mentioned, the sediment provenance could alter the\ncharacteristics of FeR-OC and TOC. Although the impacts of provenance were not apparent in the\nstudied two cores located in continental slopes, provenance was non-negligible factor in the shelfs\ndue to exposure and submersion cycles on glacial/interglacial timescales. Therefore, is the\nextrapolation of the stable FeR-OC/TOC ratio to shelf and delta/estuary reasonable or rigorous?\n5. Lines 212-216. Re-adsorption of isotopically depleted DOC to Fe was considered to result in the\nnegative δ13CFeR-OC values and low FeR-OC content. From the perspective of geological\nevolution, the sediments below the SMTZ have experienced the Fe reduction and DOC re-\nadsorption before burial in present depth, but the FeR-OC content and isotope characteristics are\nsimilar to those of sediments above SMTZ. Did some isotopically enriched OC adsorb to the FeR\nafter passing through the SMTZ? If so, some of the FR-OC maybe not only formed at the redox\ninterface as proposed in lines 288-289.\n6. Line 293-319, 339-360, the global estimation of Fe-OC over a larger spatial and temporal scale\nshould be important, however, much of these explanation and discussion as mentioned here could\nbe shorten to make the draft more focusing.\n7. Table 1 present clear comparisons of content and carbon isotope between non-SMTZ and SMTZ.\nStatistical analyses (e.g. one-way ANOVA) are recommended to guarantee the reliability of the\ncomparison, just like you did in Figure 4.", "reviewer_4": "Chen et al. present a pair of detailed short-core datasets which they use to use to investigate the\ncoupling of FeR and OC in marine sediments. This is a nice study, and looks to be scientifically\nsound. The findings are generally quite interesting, especially the suggestion that microbial\nremobilisation of FeR occurs in the SMTZ. However, I feel other than these data, the manuscript\ndoes not represent enough of a step forward to warrant publication in Nature Communications in\nits current form. My reasons for this are as follows:\n1. The title, and the abstract discuss ‘geological timescales’, while the term ‘long-buried sediments’\nis used in line 103. However, a study which investigates 100 kyr does not fit the bill to me.\nPersistence of FeR -OC over much longer timescales (up to tens of millions of years) has now been\ndemonstrated in numerous publications (Faust et al., 2021; Longman et al., 2021, 2024). Some of\nthese studies have not been cited in this work, so emphasising this as an aspect of novelty seems\nwrong. I would adjust the title and the primary narrative of the manuscript to better reflect the more\ninteresting data – what goes on through the SMTZ and below, and interactions of FeR and OC during\nburial.\n2. The compilation of data for the section discussing the scale of the marine sedimentary FeR-OC\nsink is lacking a large number of studies. There was a recently published updated dataset of FeR-\nOC in such environments (Longman et al., 2022), which cites a lot of work not considered here, and\na lot of environments not considered. Furthermore, the existence of that study, which provides\nestimates of the amount of FeR-OC buried every year, means this study as presented does not\nrepresent something fully novel.\n3. What is novel is trying to estimate the total FeR-OC pool in quaternary sediments. However, I\ncannot see the reason behind making this calculation. The authors are aiming to estimate the\namount of sediment which is microbially active, but the only microbial activity they demonstrate to\nhave an impact on feR-OC is in the SMTZ. As they themselves show, this zone in limited, and does\nnot represent the entirety of quaternary marine sediment. The cores studied here have SMTZ\nthicknesses of only up to a metre. Even in this study they only represent around 15% of the\nsediment. Outside of this there is no evidence for variability in FeR-OC driven by microbes. I really\ndon’t think any sort of extrapolation of FeR-OC based on the LaRowe estimate of Quaternary\nsediments makes much sense when trying to estimate what is impacted by microbes in the SMTZ. I\nwould suggest a better way to investigate this would be to focus on the SMTZ – I am no expert on\nthis, but are there estimates of thickness available globally?\n4. However, even if the adjustment to reflect only the SMTZ is made, the fact that data show the\nFeR-OC values are similar above and below the SMTZ in one core (and likely in the other if\ndeeper samples were available) suggests nothing of much consequence is ongoing. Yes, there\nmay be some remineralisation, but overall if we are indeed concerned about the long term sink, it doesn’t\nlook like the SMTZ and microbes really affect this. This is definitely not an issue, but framing it as a\nfinding of importance makes little sense to me.\n5. I’m not sure where the links to ‘deep life’ come from. Yes, FeR-OC can be a large labile reservoir\nof OC, but what suggests deep life? I assume the microbiology results? If so, make the link here\nclearer. However, if it is the microbiology, I see no evidence for this – you see some really interesting\nmicrobial activity in the SMTZ, but nothing to suggest this goes on in the ‘deep’ subsurface. Also, I\ndon’t think 900 cm is really ‘deep’. There’s papers which discuss truly deep subsurface microbial\nlife (e.g. Inagaki et al., 2015), which show microbial activity down to more than 2.5km (250000 cm).\nI’m sorry to seem so negative about this. The study is certainly interesting and the combination of\nFeR-OC measurements with microbiology, and everything going on in the SMTZ is interesting.\nHowever, I think the novelty is not there for this journal – the scaling up is unsuitable and the regular\ndiscussion of geological timescales seems wrong to me."}, "author_response": "Response to reviewers’ comments on ‘Reactive iron as an important reservoir of\nmarine organic carbon over geological timescales.’\nWe would like to thank the editor and reviewers their helpful comments and suggestions,\nwhich helped improve the manuscript significantly. We have made considerable efforts to\naddress all the concerns raised.\nIn summary, we have refocused our manuscript and emphasized the remobilization of Fe -OC\nR\nin SMTZ sediments and its impact on subseafloor microorganisms with extra statistical analysis\nand calculations. The estimation of Fe -OC reservoir has been largely shortened, but the\nR\ndataset used has been updated and extra statistical analysis has been done. Please find below\nour responses to the specific comments. Line numbers refer to lines in the clean version.\nReviewer #2 (Remarks to the Author):\nThis work explored the persistency of reactive-iron associated organic carbon and its behavior\nin continental slope sediments, then updated the global budget of FeR-OC. This work is\nsignificant. Some statements, however, need more explanations or calculation to support. See\nthe comments below for details.\nResponse: Thank you for the overall positive evaluation of our manuscript. Please see below\nour responses to detailed comments.\nLine 23: a comma is missing.\nResponse: Added.\nLine 29: use the right format of scientific notation here and throughout the text.\nResponse: We have changed all the OC reservoir-related unit to Pg throughout the text.\nLine 45: add “an” before “active electron acceptor”.\nResponse: Added.\nLine 58: “Nevertheless” is odd here and can be removed.\nResponse: Removed.\nLine 109: Fig. 2a is not cited yet.\nResponse: Thank you for pointing this out. The description of Fe content record is now added\nR\nto the revised manuscript and the Fig. 2a is cited. (Lines 124-126)\nLine 117: provide a number or a range to show how low it is.\nResponse: The numbers are now provided in the revised manuscript. (Line 127)\nLine 123: the average content of FeR-OC of 0.03% is contradictory to the statement on Line\n119 where the minimum FeR-OC content is 0.04%.\nResponse: Thank you for noticing. The inconsistent data description has been corrected\nthroughout the text in the revised manuscript. (Lines 128-129)\nLine 125: the average FeR-OC/DOC of 5.2% is is contradictory to the statement on Line 120\nwhere the minimum FeR-OC/TOC is 10%.\nResponse: Thank you for noticing. The inconsistent data description has been corrected\nthroughout the text in the revised manuscript. (Lines 128-129)\nLine 141-146: I agree with the authors that microbial activities are the most likely reason for\nthe phenomenon that in both cores, lower values are observed in SMTZ sediments. But I don’t\nfollow how a glacial-interglacial cycle has something to do with the reasons proposed for the\nlower values. More explanations are needed here.\nResponse: We have added more information to the introduction, which explains how glacial-\ninterglacial cycles might influence sedimentary Fe -OC records:\nR\n“Additionally, the supply of Fe -OC is expected to depend on the hydrological conditions\nR\nthrough the influence on continental weathering and Fe formation as well as on sea\nR\nlevel through the influence on shelf topography and thus transport of continental\ndetritus into the deep sea25-27; both factors are expected to vary periodically on glacial-\ninterglacial timescales.” (Lines 65-69)\nAt the same time, we have de-emphasized the role of glacial-interglacial variations. Given that\nthe chance is extremely small that the lower Fe -OC concentrations in the SMTZ are controlled\nR\nby the depositional history and coincidentally located in both cores in the SMTZ horizon, we\nhave added the following explanatory statement to the revised manuscript:\n“While we cannot entirely rule out that these distinct signals in SMTZs of both cores are\nrelated to the depositional history and coincidentally located in this horizon, we view\nthis scenario as highly unlikely.” (Lines 179-182)\nLine 244: what is non-FeR-OC? Why is FeR-OC with a percentage of 10-20% in TOC\npreferentially remineralized? I don’t get the point. More explanation is needed.\nResponse: We have added the explanation of “non-Fe -OC” as “OC that is not bound to Fe ”\nR R\nin the revised manuscript (Line 269-270). We assume that the Fe -OC is preferentially\nR\nremineralized over OC that is not associated with Fe in SMTZ, because Fe -OC only accounts\nR R\nfor 10-20% of TOC but contributed 36.7% to the TOC mineralization flux.\nWe have rephrased the sentences to address this point:\n“Fe -OC remineralization contributes 36.7% to the TOC remineralization, which is\nR\ndisproportionally high compared to the percentage of Fe -OC in TOC (5.2±1.8%) in this\nR\nzone. Therefore, Fe -OC is preferentially remineralized compared to OC that is not\nR\nbound to Fe (non-Fe -OC) in the SMTZ of QDN-14B and results in the low percentage\nR R\nof Fe -OC in TOC.” (Lines 267-271)\nR\nLine 269-272: using the numbers provided here, “a substantial fraction” the authors claim is\nless than 4%. Is this “substantial”?\nResponse: In previous manuscript, we compared the number of cells that can be supported by\nthe Fe -OC remineralization flux in continental slope SMTZ sediments to the microbial cell\nR\nabundance in global marine sediments, which underestimated the significance of Fe -OC\nR\nremineralization flux in continental slope SMTZ sediments in supporting subsurface\nmicroorganisms. In the revised manuscript, the microbial cell abundance in continental slope\nSMTZ sediments is estimated and used instead:\n“Considering generally lower power demand of 10-20 to 10-16 W cell1 for\nmicroorganisms in marine sediments51, this amount of energy could potentially support\nan even larger population of 1024 to 1028cells. The average SMTZ depth in continental\nslope sediments is estimated to be 12.8±12.1 meters below seafloor31, where the cell\nabundance typically ranges from 106 to 108 cells cm3 46. Assuming the SMTZ depth\ninterval of 1 m, the volume of global continental slope SMTZ sediments reaches 3×1013\nm3, which harbors about 3×1025 to 3×1027 cells. Therefore, the remineralization of Fe -\nR\nOC in continental slope SMTZ sediments could support a substantial fraction of\nsubsurface microbial life in this zone.” (Lines 293-302)\nLine 288: Based on the molar ratio of Fe -OC to Fe and the two mechanisms, the proportion\nR R\ncan be estimated to support “a large proportion” the authors proposed. Otherwise, the\nproposal is weak.\nResponse: Yes, in principle the relative contribution of Fe -OC associated with Fe by\nR R\nadsorption and co-precipitation can be estimated by the end-member Fe -OC to Fe molar\nR R\nratios of these two mechanisms and the observed Fe -OC to Fe molar ratios in sediment\nR R\nsamples following a simple mass balance equation. However, as the end-member Fe -OC to\nR\nFe molar ratios of two mechanisms have large ranges, so it is not possible to give an accurate\nR\nnumber on this proportion. We propose that “a major proportion of Fe -OC is formed at the\nR\nredox interface in marine environments” not only because Fe -OC to Fe molar ratios higher\nR R\nthan 1 indicating co-precipitation are observed in most layers of both sediment cores, but also\nbecause the carbon isotope ratios of Fe -OC indicate that marine OC consistently dominate\nR\nover terrestrial OC.\nConsidering the Fe -OC pool is exchangeable during the early diagenesis. The sentence has\nR\nbeen rephrased to avoid ambiguity:\n“All these clues suggest that a large proportion of terrestrial Fe -OC is replaced by\nR\nmarine OC when new Fe and OC associations are formed at the redox interface near\nR\nthe seafloor. This process could happen either autochthonously after deposition, or\nallochthonously in neighbouring surface sediments and transported to the core location\nas resuspended particulates58, 59.” (Lines 315-320)\nLine 329: “shelf” should be “slope”?\nResponse: Corrected.\nLine 331: change “is” to “are”.\nResponse: Corrected.\nLine 331-334: a conjunction is needed for the two sentences.\nResponse: Corrected.\nLine 354 & 357: provide the number of data to be averaged.\nResponse: Thank you for the suggestion. We have added the number of data to be averaged\nin Fig. 4 and Table S4 in the revised manuscript.\nReviewer #3 (Remarks to the Author):\nThe manuscript investigated the long-term variations in content and isotope of TOC and FeR-\nOC, emphasizing the impact of SMTZ in FeR-OC preservation. Based on the basically unaltered\nFeR-OC/TOC ratio long-buried sediments, this study provided an estimation of global budget\nof FeR-OC in Quaternary marine sediments. The topic is new and innovative, and experimental\nand analytical designs are convincing. The results provide an important experimental basis for\nunderstanding the role of sedimentary FeR-OC in regulating long-term carbon cycle.\nResponse: Thank you for your appreciation on our manuscript. Please see below our responses\nto detailed comments.\n1. Title, the current title could not be completely reflect the topic and the present study, such\nas the items of digenesis and microbial community.\nResponse: We have changed the title to “Cycling and persistence of iron-bound organic carbon\nin subseafloor sediments” to fully reflect the dynamic cycling of Fe -OC we found in SMTZ\nR\nsediments and its general stability in non-SMTZ sediments.\n2. Before the process of SMTZ, how the FeR-OC change and/or response towards the reduction\nprocess of dissimilatory Fe and Mn reduction?\nResponse: Thank you for this comment. We added some information regarding the effect of\ndissimilatory iron reduction on Fe -OC in the manuscript:\nR\n“The presence of low levels of dissolved iron throughout the core QDN-G1 (3-35 M,\nFig. 1b) is consistent with some background activity of dissimilatory iron reduction,\nindependent of the biogeochemical zone. However, there is no discernable imprint on\nthe content of Fe -OC, except in the SMTZ (Fig. 2b).” (Lines 131-134)\nR\nMn reduction may affect the organic carbon that associated with Mn oxides in a similar way\nas iron reduction on Fe -OC, but it is beyond the scope of our study.\nR\n3. The impact of SMTZ in FeR-OC preservation was more significant in the sediment core QDN-\n14B nearby cold seeps due to the more abundant microbial occurrence; however, this\ndegradation mechanism could not be suitable for the slope sedimentary setting, thus is the\nmicrobial evidence also be increased in the normal slope core with SMTZ section? In addition\nto the support from the remineralization of FeR-OC, what is the role of methane release for\nthis specific microbial community structure?\nResponse: The modeled sulfate reduction rates, relative abundance of Desulfobacterota in\nbacteria and cell number of Desulfobacterota of core QDN-G1 (normal slope core) are shown\nin Supplementary Fig. 5. Although there is no evidence indicating elevated sulfate reduction\nrates and increased sulfate reducing bacteria in the SMTZ of QDN-G1 (Lines 222-226), the\nincrease of the microbial activity is expected in the SMTZ of as methane could be utilized by\nmethane oxidizing archaea in concert with sulfate reducing bacteria, which was supported by\nthe decrease of TOC and Fe -OC contents in the SMTZ (Fig. 2b).\nR\nThe high methane flux in QDN-14B because of the nearby cold seep stimulates the sulfate\nreduction coupling with anaerobic oxidation of methane, which leads to the high relative\nabundance of sulfate-reducing bacteria in all bacteria and high relative abundance of methane\nmetabolizing archaea among all archaea. (Lines 217-222)\n4. Line 143-154. As the authors mentioned, the sediment provenance could alter the\ncharacteristics of FeR-OC and TOC. Although the impacts of provenance were not apparent in\nthe studied two cores located in continental slopes, provenance was non-negligible factor in\nthe shelfs due to exposure and submersion cycles on glacial/interglacial timescales. Therefore,\nis the extrapolation of the stable FeR-OC/TOC ratio to shelf and delta/estuary reasonable or\nrigorous?\nResponse: We have refocused our manuscript on the dynamic cycling of Fe -OC in SMTZ\nR\nsediments, and largely shortened the part on Fe -OC reservoir estimation by providing only a\nR\nrough estimation based on an updated dataset and extra statistical analyses:\n“Taken together, the Fe -OC records in these two cores suggest that a stable proportion\nR\nof TOC survives early diagenesis as Fe -OC and is sequestered in marine sediments on\nR\nat least 100-kyr timescales. This enables a rough estimation of the global Fe -OC\nR\nreservoir by multiplying the TOC reservoir in global marine sediments from the\nQuaternary Period (0-2.59 Ma) storage50 with published Fe -OC/TOC ratios in marine\nR\nsurface sediments. For deriving representative Fe -OC/TOC ratios, all published Fe -\nR R\nOC/TOC data in marine sediments using the CBD method were compiled and combined\nwith our measured data and grouped into eight categories based on their study area\ninformation: delta and estuary, continental shelf, continental slope, deep sea,\nanoxic/sulfidic regions, wetland, mangrove and tephra (Fig. 4, Supplementary Table 3).\nOne-way ANOVA and Tukey Honest Significant Difference (HSD) test revealed that\nsignificant difference (P<0.05) in Fe -OC/TOC values exists only between tephra-\nR\ncontaining sediments and other environments (Supplementary Table 4). Considering\nTOC storage in the Quaternary sediments is estimated in the three domains shelf (water\ndepth <200 m), margin (200 m < water depth <3500 m) and abyss (water depth >3500\nm) 50, delta/estuary and continental shelf were combined to represent shelf, continental\nslope was used to represent margin and deep sea was used to represent abyss.\nCombining the published TOC reservoir and the corresponding surface Fe -OC/TOC\nR\nratios in these three domains50, the global Fe -OC reservoir in Quaternary marine\nR\nsediments was estimated to be 28,550 ± 12,160 Pg C (Supplementary Table 5), which is\napproximately 19-46 times the size of the atmospheric carbon pool.” (Lines 335-356)\n5. Lines 212-216. Re-adsorption of isotopically depleted DOC to Fe was considered to result in\nthe negative δ13CFeR-OC values and low FeR-OC content. From the perspective of geological\nevolution, the sediments below the SMTZ have experienced the Fe reduction and DOC re-\nadsorption before burial in present depth, but the FeR-OC content and isotope characteristics\nare similar to those of sediments above SMTZ. Did some isotopically enriched OC adsorb to\nthe FeR after passing through the SMTZ? If so, some of the FeR-OC maybe not only formed at\nthe redox interface as proposed in lines 288-289.\nResponse: Thank you for this comment. The re-adsorption of someisotopically enriched DOC\nto the Fe cannot be ruled out. We added following information to explain similar Fe -OC\nR R\nabove and below the SMTZ:\n“The fact that sediments below the SMTZ resemble those above the SMTZ is at first\nsurprising if we assume that the more deeply buried sediments were at some point in\nthe geologic past also situated in the SMTZ. In this case, re-adsorption of DOM produced\nduring cycling of particulate organic matter of marine origin could be the cause for the\nsimilarity of Fe -OC related signals above and below the SMTZ. However, there is also\nR\nevidence that SMTZs do not “move” continuously through the sediment column with\nincreasing sedimentation but rather “oscillate” from one horizon to another where they\nthen remain stagnant in position for extended periods37, 38.” (Lines 203-211)\nWe also acknowledge that what we originally proposed in Lines 288-289 “We propose that a\nlarge proportion of Fe -OC is formed at the redox interface in marine environments” was not\nR\naccurate. Here we intended to describe the replacement of terrestrial OC to marine sourced\nOC near the seafloor. It has been rephrased to “All these clues suggested that a major\nproportion of terrestrial Fe -OC is replaced by marine-OC when new Fe and OC associations\nR R\nare formed at the redox interface near the seafloor.” (Lines 315-317)\n6. Line 293-319, 339-360, the global estimation of Fe-OC over a larger spatial and temporal\nscale should be important, however, much of these explanation and discussion as mentioned\nhere could be shorten to make the draft more focusing.\nResponse: Thank you so much for this suggestion. We have revised this part of the manuscript\nsubstantially to make it more straightforward. (Lines 323-356)\n7. Table 1 present clear comparisons of content and carbon isotope between non-SMTZ and\nSMTZ. Statistical analyses (e.g. one-way ANOVA) are recommended to guarantee the reliability\nof the comparison, just like you did in Figure 4.\nResponse: Thank you for the suggestion. The significance tests have been done and the results\nhave been added to Table 1 and the main text (Lines 176-179). Detailed information has been\nadded to methods (Lines 568-577).\nReviewer #4 (Remarks to the Author):\nChen et al. present a pair of detailed short-core datasets which they use to use to investigate\nthe coupling of FeR and OC in marine sediments. This is a nice study, and looks to be\nscientifically sound. The findings are generally quite interesting, especially the suggestion that\nmicrobial remobilisation of FeR occurs in the SMTZ. However, I feel other than these data, the\nmanuscript does not represent enough of a step forward to warrant publication in Nature\nCommunications in its current form. My reasons for this are as follows:\n1. The title, and the abstract discuss ‘geological timescales’, while the term ‘long-buried\nsediments’ is used in line 103. However, a study which investigates 100 kyr does not fit the bill\nto me. Persistence of FeR -OC over much longer timescales (up to tens of millions of years) has\nnow been demonstrated in numerous publications (Faust et al., 2021; Longman et al., 2021,\n2024). Some of these studies have not been cited in this work, so emphasising this as an aspect\nof novelty seems wrong. I would adjust the title and the primary narrative of the manuscript\nto better reflect the more interesting data – what goes on through the SMTZ and below, and\ninteractions of FeR and OC during burial.\nResponse: Thank you! We have revised the manuscript per your suggestions as follows:\n(1) We have changed the title to “Cycling and persistence of iron-bound organic carbon in\nsubseafloor sediments” to reflect the dynamic cycling of Fe -OC pool during early\nR\ndiagenesis. This new title also reflects the changed focus of the narrative.\n(2) We have emphasized the novelty of our study as exploring the fate of Fe -OC in\nR\nsubseafloor sediments during microbially mediated diagenetic processes in the\nintroduction:\n“A comprehensive picture regarding the fate of Fe -OC on geological timescales is still\nR\nlacking but several studies of sediments of Pleistocene age suggest that the Fe -OC\nR\nfraction remains relatively stable11-14. However, the question to what degree the Fe -OC\nR\nreservoir interacts with sedimentary biogeochemical processes, in particular those\ninvolving redox reactions of iron and sulfur, remains unresolved.” (Lines 44-49)\n(3) The potential effect of diagenetic redox reactions and depositional history on sedimentary\nFe -OC records has been added to the introduction as background information with\nR\nliterature review:\n“Fe has long been found to promote organic matter preservation in terrestrial soils as\nR\nwell, especially via adsorption5, 15. However, recent studies indicated the interactions\nbetween Fe and OC in soils are highly influenced by the redox oscillations during water-\nR\ntable fluctuatioins16-18 in terrestrial environments. Under anoxic conditions, Fe\nR\nreduction releases Fe -OC and increased the anaerobic mineralization of soil organic\nR\nmatter17, 18. While under oxic conditions, newly-formed Fe promotes OC retention on\nR\nmineral surfaces16. In anoxic marine sediments, Fe is one electron acceptor actively\nR\ninvolved in biogeochemical processes19-21. Both Fe reduction by microorganisms and\nR\nbiogenic sulfide produced during sulfate reduction could potentially weaken the\nassociation with OC. Indeed, recent lab incubations demonstrated that Fe -OC can be\nR\nremobilized during microbial iron reduction and subsequently utilized as electron donor\nand/or carbon source for microbial communities22-24. Consequently, the remobilization\nof Fe -OC during early diagenesis may influence the relative size of the Fe -OC reservoir\nR R\nthat preserved in sediments for long time. Additionally, the supply of Fe -OC is expected\nR\nto depend on the hydrological conditions through the influence on continental\nweathering and Fe formation as well as on sea level through the influence on shelf\nR\ntopography and thus transport of continental detritus into the deep sea25-27; both\nfactors are expected to vary periodically on glacial-interglacial timescales. To\ndisentangle the effect of Fe -OC supply and early diagenetic reworking on sedimentary\nR\nFe -OC, downcore Fe -OC records need to be established and related to both\nR R\ngeochemical zonation and sediment chronology.” (Lines 51-72)\n(4) We refocus the discussion on the dynamic cycling of Fe -OC in SMTZ sediments, providing\nR\nmore information on the most likely mechanism of Fe -OC remobilization and\nR\nremineralization, and the scenario of the interactions between Fe and OC beneath the\nR\nSMTZ:\n“Accordingly, two observations deserve attention: (i) the distinct depletions in 13C\ncontent and contents of Fe -OC within the SMTZ, and (ii) sediments below the SMTZ\nR\nshow similar features with respect to 13C content and contents of Fe -OC as sediments\nR\nabove the SMTZ. (i) The relatively low content of Fe -OC strongly suggests that a\nR\nsubstantial fraction of it has been remobilized and possibly remineralized. Two\nmechanisms could account for the low 13C of the residual fraction: (a)\nFeR-OC\nremobilization/degradation of a 13C-enriched, weakly bound and/or more reactive\nfraction34 with the residual fraction being 13C-depleted; since the 13C of the residual\nfraction is broadly consistent with an origin from terrestrial plants, the residual fraction\ncould be tightly adsorbed terrestrial organic matter supplied together with the detrital\nminerals from land. (b) The alternative involves again remobilization of a large fraction\nof Fe -OC combined with adsorption of 13C depleted dissolved organic matter (DOM)\nR\nproduced in the course of anaerobic oxidation of methane35; the extremely low molar\nratios of Fe -OC to Fe below 1 in both SMTZs (Fig. 2e and j) indicate abundant binding\nR R\nsites available for DOM molecules15, 36. These two mechanisms are not mutually\nexclusive and could act in combination. In any case, both scenarios require\nremobilization and possibly degradation of a substantial fraction of Fe -OC.\nR\n(ii) The fact\nthat sediments below the SMTZ resemble those above the SMTZ is at first surprising if\nwe assume that the more deeply buried sediments were at some point in the geologic\npast also situated in the SMTZ. In this case, re-adsorption of DOM produced during\ncycling of particulate organic matter of marine origin could be the cause for the\nsimilarity of Fe -OC related signals above and below the SMTZ. However, there is also\nR\nevidence that SMTZs do not “move” continuously through the sediment column with\nincreasing sedimentation but rather “oscillate” from one horizon to another where they\nthen remain stagnant in position for extended periods37, 38” (Lines 186-211)\n(5) We have largely shortened the last section on the persistence of Fe -OC on geological\nR\ntimescale, but only kept a rough estimation of Fe -OC reservoir in Quaternary sediments.\nR\n2. The compilation of data for the section discussing the scale of the marine sedimentary FeR-\nOC sink is lacking a large number of studies. There was a recently published updated dataset\nof FeR-OC in such environments (Longman et al., 2022), which cites a lot of work not\nconsidered here, and a lot of environments not considered. Furthermore, the existence of that\nstudy, which provides estimates of the amount of FeR-OC buried every year, means this study\nas presented does not represent something fully novel.\nResponse: Thank you so much for your comment and suggestions. We had not considered\nsome data from the mentioned environments because of their limited distribution in the\nprevious manuscript. However, we agree that all the published data should be used for\ncompilation and statistical analyses. In the revised manuscript, the most up-to-date dataset\nwas used and grouped into eight marine environments (Fig 4). The detailed information was\nupdated in Supplementary Table 3. We have put more information in the footnote of\nSupplementary Table 3 for clarification:\n“The most up-to date published dataset compiled by Longman et al., 2022 was used21,\nwith following exceptions: Ghaisas et al., 2021 was not included for different Fe -OC\nR\nextraction method22; Tao et al. 2017 and Longman et al., 2024, which were not included\nin Longman et al., 2022, were included here14, 20.”\n3. What is novel is trying to estimate the total FeR-OC pool in quaternary sediments. However,\nI cannot see the reason behind making this calculation. The authors are aiming to estimate the\namount of sediment which is microbially active, but the only microbial activity they\ndemonstrate to have an impact on feR-OC is in the SMTZ. As they themselves show, this zone\nin limited, and does not represent the entirety of quaternary marine sediment. The cores\nstudied here have SMTZ thicknesses of only up to a metre. Even in this study they only\nrepresent around 15% of the sediment. Outside of this there is no evidence for variability in\nFeR-OC driven by microbes. I really don’t think any sort of extrapolation of FeR-OC based on\nthe LaRowe estimate of Quaternary sediments makes much sense when trying to estimate\nwhat is impacted by microbes in the SMTZ. I would suggest a better way to investigate this\nwould be to focus on the SMTZ – I am no expert on this, but are there estimates of thickness\navailable globally?\nResponse: We estimated the Fe -OC reservoir in microbially active Quaternary sediments, but\nR\n“microbially active” here is not related to the specific microbial processes remobilizing Fe -OC\nR\nin SMTZs. Instead, it only means the sediments deposited since Pleistocene are not yet\naffected by severe high temperature and most of the microbial degradation of OC takes place\nin this portion of marine sediments. We put more information to clarify the different Fe -OC\nR\ndegradation patterns in general non-SMTZ and SMTZ sediments before we start with the\nestimation:\n“The average Fe -OC/TOC ratio in the two cores studied, except within the SMTZ in\nR\nQDN-14B, is 13.3±3.2%. The relative stable Fe -OC/TOC ratios in the two cores indicates\nR\na similar degradation rate of Fe -OC and non-Fe -OC. The only exception is the especially\nR R\nactive SMTZ in QDN-14B influenced by the methane-rich fluids, where iron reduction is\nenhanced significantly by processes likely induced by sulfate reducing bacteria,\nmethanotrophic archaea and/or biogenic sulfide. The stimulated degradation of Fe -OC\nR\nover non-Fe -OC leads to extremely low Fe -OC/TOC ratios in the SMTZ of QDN-14B. As\nR R\nthese especially active SMTZs have limited distribution in global marine sediments and\noccupy narrow depth intervals in sediment columns, the overall impact on the Fe -OC\nR\nreservoir in subseafloor sediments is small.” (Lines 323-333)\nIn SMTZ sediments, the Fe -OC records we observed represents the characteristic of residual\nR\nFe -OC after the remobilization and remineralization. Therefore, instead of the Fe -OC\nR R\nreservoir in the SMTZ, the remineralization flux of Fe -OC in the SMTZ of global continental\nR\nslope sediments was estimated. In the revised manuscript, the microbial cell abundance was\nestimated and compared with the biomass that can be sustained by the Fe -OC\nR\nremineralization flux in this zone:\n“Considering generally lower power demand of 1020 to 1016 W cell1 for microorganisms\nin marine sediments51, this amount of energy could potentially support an even larger\npopulation of 1024 to 1028cells. The average SMTZ depth in continental slope sediments\nis estimated to be 12.8±12.1 meters below seafloor31, where the cell abundance\ntypically ranges from 106 to 108 cells cm346. Assuming the SMTZ depth interval of 1 m,\nthe volume of global continental slope SMTZ sediments reaches 3×1013 m3, which\nharbors about 3×1025 to 3×1027 cells. Therefore, the remineralization of Fe -OC in\nR\ncontinental slope SMTZ sediments could support a substantial fraction of subsurface\nmicrobial life in this zone.” (Lines 293-302)\n4. However, even if the adjustment to reflect only the SMTZ is made, the fact that data show\nthe FeR-OC values are similar above and below the SMTZ in one core (and likely in the other if\ndeeper samples were available) suggests nothing of much consequence is ongoing. Yes, there\nmay be some remineralisation, but overall if we are indeed concerned about the long term\nsink, it doesn’t look like the SMTZ and microbes really affect this. This is definitely not an issue,\nbut framing it as a finding of importance makes little sense to me.\nResponse: Similar to the last comment, with the information we added to clarify the different\nFe -OC degradation patterns in general non-SMTZ and SMTZ sediments, this concern should\nR\nalso be addressed (Lines 323-333).\n5. I’m not sure where the links to ‘deep life’ come from. Yes, FeR-OC can be a large labile\nreservoir of OC, but what suggests deep life? I assume the microbiology results? If so, make\nthe link here clearer. However, if it is the microbiology, I see no evidence for this – you see\nsome really interesting microbial activity in the SMTZ, but nothing to suggest this goes on in\nthe ‘deep’ subsurface. Also, I don’t think 900 cm is really ‘deep’. There’s papers which discuss\ntruly deep subsurface microbial life (e.g. Inagaki et al., 2015), which show microbial activity\ndown to more than 2.5km (250000 cm).\nResponse: Thank you for pointing this out. Yes, the ‘deep life’ mentioned here refers to\nmicroorganisms living in the subsurface sediments. We agree that comparing to the truly deep\nsubsurface microbial life, we only discussed microbial life in relatively shallow sediments. We\nhave rephrased “deep life” as “subseafloor microorganisms” to avoid the misunderstanding\n(Line 31).\nI’m sorry to seem so negative about this. The study is certainly interesting and the combination\nof FeR-OC measurements with microbiology, and everything going on in the SMTZ is interesting.\nHowever, I think the novelty is not there for this journal – the scaling up is unsuitable and the regular\ndiscussion of geological timescales seems wrong to me.\nResponse: Thank you for your helpful comments and suggestions. Now we have substantially\nrevised the manuscript by reemphasizing the novelty of our study in the introduction with an\nupdated literature review, and reframed our discussion with more focus on dynamic cycling of\nFe -OC in SMTZ sediments and its impact on subseafloor microorganisms. We have largely\nR\nshortened the part on the estimation of Fe -OC reservoir in Quaternary sediments, but kept\nR\nthe rough estimation we made with updated dataset and extra statistical analyses.\nReferences.\nFaust, J. C., Tessin, A., Fisher, B. J., Zindorf, M., Papadaki, S., Hendry, K. R., Doyle, K. A., & März,\nC. (2021). Millennial scale persistence of organic carbon bound to iron in Arctic marine\nsediments. Nature Communications, 12(1), 1–9. https://doi.org/10.1038/s41467-020-20550-\nA., Imachi, H., Ito, M., Kaneko, M., Lever, M. A., Lin, Y.-S., Methé, B. A., Morita, S., Morono, Y.,\nTanikawa, W., Bihan, M., Bowden, S. A., … Yamada, Y. (2015). Exploring deep microbial life in\ncoal-bearing sediment down to ~2.5 km below the ocean floor. Science, 349(6246), 420–424.\nhttps://doi.org/10.1126/science.aaa6882\nLongman, J., Faust, J. C., Bryce, C., Homoky, W. B., & März, C. (2022). Organic Carbon Burial\nWith Reactive Iron Across Global Environments. Global Biogeochemical Cycles, 36(11),\ne2022GB007447. https://doi.org/10.1029/2022GB007447\nLongman, J., Gernon, T. M., Palmer, M. R., & Manners, H. R. (2021). Tephra Deposition and\nBonding With Reactive Oxides Enhances Burial of Organic Carbon in the Bering Sea. Global\nBiogeochemical Cycles, 35(11), e2021GB007140. https://doi.org/10.1029/2021GB007140\nLongman, J., Manners, H. R., Gernon, T. M., McManus, J., Palmer, M. R., Rowland, S. J., & Sutton,\nP. A. (2024). Production and preservation of organic carbon in sub-seafloor tephra layers.\nMarine Chemistry, 258, 104334. https://doi.org/10.1016/j.marchem.2023.104334\nResponse: Thank you for the information. The papers studying the long-term preservation of\nFe -OC have been cited in the introduction.\nR"}, {"round": 2, "reviewer_comments": {"reviewer_2": "I have no more suggestions/comments.", "reviewer_3": "I have carefully reviewed your revised manuscript and am pleased to note that you have addressed\nthe issues raised during the previous round of reviews comprehensively. The revised discussions\nhave improved the coherence of the manuscript and greatly enhanced the quality of the paper.\nHowever, some of the newly added statements still need to be reconsidered.\nYou mentioned that the lower FeR-OC contents, lower FeR-OC/TOC values, and lower δ13CFeR-OC\nin SMTZ sediments are ascribed to the remobilization/degradation of FeR-OC (lines 201-203), which\nis caused by the biological and chemical reduction of FeR (lines 232-240). Further, the calculated\nFeR-OC remineralization fluxes in the SMTZ of core QDN-G1 and QDN-14B were similar (5.0 mol m-\n2 kyr-1 and 5.8 mol m-2 kyr-1, respectively). I am confused why sulfate reduction activity in the\nSMTZ of core QDN-G1 does not peak like that of core QDN-14B (lines 222-225). The cell number of\nDesulfobacterota in the SMTZ is even lower than that in non-SMTZ. Since I did not see any\ninterpretation of the FeR reduction mechanism of core QDN-G1, it seems that the authors\nconsidered it is caused by the biological process as core QDN-14B. But why?\nLines 315-317. “All these clues suggest… near the seafloor.” I do not think this is an evidence-based\nstatement, as even at the uppermost part of the core, the δ13CFeR-OC is highly enriched.\nMoreover, there exists the possibility that the terrestrial input FeR did not initially associate with\nterrestrial OC, but rather, it bonded with marine OC once entering the ocean.\nLines 324-325. “The relative stable FeR-OC/TOC ratios in the two cores indicates a similar\ndegradation rate of FeR-OC and non-FeR-OC”. This statemen seems against the general concept\nthat FeR would protect the OC from microbial degradation. If this is correct, one can suppose that\nthe FeR did not have any effect on the preservation of OC on the glacial-interglacial timescales in\nthe non-SMTZ sediments. I think this is not what the author intended to convey in this study.", "reviewer_4": "I am delighted to see Chen et al. have taken my comments on board, and the manuscript is\ngreatly improved in its current form. Very nice work! I would suggest with a few more changes\nit will be suitable for publication. My small suggestions are below (and my line numbers refer to the clean\nnew version of the manuscript):\nL17 onwards: I think using TOC and OC in the same sentence is confusing. As the standard in the\nfield is to use the term FeR-OC for the coupling, I would suggest sticking with OC rather than using\nTOC when describing organic carbon throughout.\nL22: Not sure how you come to the conclusion it’s marine sourced – we don’t know where the FeR\ncomes from do we? Could be weathered material from the land. Same for the OC – some\ncomponent of terrestrial OC is likely.\nL40: I would rephrase to start the sentence with ‘It is estimated ~20.5…’\nL46: Just for reference, the Longman et al. (2024) study (ref.13) shows some sediments of Eocene\nage containing high FeR-OC.\nL114-115: Can you include a reference for the marine sediment zonation?\nTable 1: I wonder if some of these data, which are crucial to the ms, could be presented in box plot\nform? For example, the FeR-OC values and the d13CFeR-OC would be nicely presented as a box\nplot.\nL238 ‘summary’ rather than ‘sum’\nL257: ‘The depth interval’ is an odd way to say it. Maybe simply say ‘The SMTZ…’\nL337: This could be expanded as an explanation for the samples of even older age which show\nFeR-OC coupling to be important.\nJack Longman"}, "author_response": "Response to reviewers’ comments on ‘Cycling and persistence of iron-bound organic\ncarbon in subseafloor sediments.’\nWe thank the editor and reviewers for re-considering our revised manuscript. We have\nfollowed the valuable advice and further revised the manuscript, including clarifying\nquestionable statements that were newly added to the manuscript during the first revision.\nWe have also improved our estimation on the Fe -OC remineralization flux in the SMTZ. Please\nR\nfind below our responses to the specific comments. Line numbers refer to lines in the clean\nversion.\nReviewer #2 (Remarks to the Author):\nI have no more suggestions/comments.\nResponse: We thank Reviewer 2 again for his/her efforts in reviewing our manuscript.\nReviewer #3 (Remarks to the Author):\nI have carefully reviewed your revised manuscript and am pleased to note that you have\naddressed the issues raised during the previous round of reviews comprehensively. The\nrevised discussions have improved the coherence of the manuscript and greatly enhanced the\nquality of the paper. However, some of the newly added statements still need to be\nreconsidered.\nResponse: We thank the reviewer for the supportive comments and recognition of our revision.\nWe have further improved our manuscript based on his/her suggestions. Please see below our\nresponses to detailed comments.\nYou mentioned that the lower FeR-OC contents, lower FeR-OC/TOC values, and lower\nδ13CFeR-OC in SMTZ sediments are ascribed to the remobilization/degradation of FeR-OC\n(lines 201-203), which is caused by the biological and chemical reduction of FeR (lines 232-\n240). Further, the calculated FeR-OC remineralization fluxes in the SMTZ of core QDN-G1 and\nQDN-14B were similar (5.0 mol m-2 kyr-1 and 5.8 mol m-2 kyr-1, respectively). I am confused\nwhy sulfate reduction activity in the SMTZ of core QDN-G1 does not peak like that of core\nQDN-14B (lines 222-225). The cell number of Desulfobacterota in the SMTZ is even lower than\nthat in non-SMTZ. Since I did not see any interpretation of the FeR reduction mechanism of\ncore QDN-G1, it seems that the authors considered it is caused by the biological process as\ncore QDN-14B. But why?\nResponse: The reviewer has a good point: despite multiple lines of evidence for higher sulfate-\nreducing activity in core QDN-14B, the fluxes of Fe -OC remineralization, as approximated by\nR\nour approach, appear rather similar.\nIn response to this valuable comment, we now more explicitly acknowledge that the estimate\nprovided for the less active core QDN-G1 is associated with a higher uncertainty due to the\nsteady decline in Fe -OC cand TOC ontent above the SMTZ. Consequently, we also used a\nR\nslightly modified approach to estimate the lower boundary of the flux. The resulting flux is\nmore consistent with the sulfate reduction rates. For global extrapolation, we now use the\nentire range of the two cores to express the uncertainty (see revised paragraph in lines 274 to\n284).\nWe additionally added information to the methods on the estimation of TOC and Fe -OC\nR\nremineralization fluxes in the SMTZ (Lines 515-541).\nLines 315-317. “All these clues suggest… near the seafloor.” I do not think this is an evidence-\nbased statement, as even at the uppermost part of the core, the δ13CFeR-OC is highly enriched.\nMoreover, there exists the possibility that the terrestrial input FeR did not initially associate\nwith terrestrial OC, but rather, it bonded with marine OC once entering the ocean.\nResponse: We thank the reviewer for pointing this out. We agree that the previous statement\nmay have been misleading. We have added the sentence “After entering the ocean, additional\norganic matter from the pool of marine dissolved organic carbon may be bound to Fe -bearing\nR\nminerals by adsorption59,60.” (Lines 316-318) and we clarified the text by deleting the\nstatement regarding the replacement of terrestrial organic matter.\nLines 324-325. “The relative stable FeR-OC/TOC ratios in the two cores indicates a similar\ndegradation rate of FeR-OC and non-FeR-OC”. This statemen seems against the general\nconcept that FeR would protect the OC from microbial degradation. If this is correct, one can\nsuppose that the FeR did not have any effect on the preservation of OC on the glacial-interglacial\ntimescales in the non-SMTZ sediments. I think this is not what the author intended to convey in\nthis study.\nResponse: The reviewer is correct that we did not intend to make the theme of “preferential\npreservation of Fe -OC relative to non-Fe -OC on geological timescales” the major focus of the\nR R\npaper. A more comprehensive study would be required to emphasize this point and generalize\nthis observation. Nevertheless, we feel that it is worth mentioning this observation. We slightly\nsoftened the statement by replacing “indicates” with “suggests” and by adding “on the\ntimescales represented by these cores” (Line 331-333).\nReviewer #4 (Remarks to the Author):\nI am delighted to see Chen et al. have taken my comments on board, and the manuscript is\ngreatly improved in its current form. Very nice work! I would suggest with a few more changes\nit will be suitable for publication. My small suggestions are below (and my line numbers refer\nto the clean new version of the manuscript):\nResponse: We are pleased to hear that our efforts and the revised manuscript are well received.\nWe would like to thank the reviewer again for all the constructive suggestions, which greatly\nimproved our manuscript. We have further improved our manuscript based on the reviewer’s\nsuggestions. Please see below our responses to detailed comments.\nL17 onwards: I think using TOC and OC in the same sentence is confusing. As the standard in\nthe field is to use the term FeR-OC for the coupling, I would suggest sticking with OC rather\nthan using TOC when describing organic carbon throughout.\nResponse: Thank the reviewer for this suggestion. We now use “fFe -OC” instead of “Fe -\nR R\nOC/TOC” to represent the fraction of Fe -OC in TOC throughout the text, which makes the\nR\nterminology simpler and consistent with other recent publications in the field. In other cases,\nTOC is used when we refer to the bulk organic carbon in the sediments, which includes both\nFe -OC and non-Fe -OC.\nR R\nL22: Not sure how you come to the conclusion it’s marine sourced – we don’t know where the\nFeR comes from do we? Could be weathered material from the land. Same for the OC – some\ncomponent of terrestrial OC is likely.\nResponse: Agreed. There is probably a fraction of Fe -OC coming from the land. The word\nR\n“marine-sourced” has been removed from the sentence.\nL40: I would rephrase to start the sentence with ‘It is estimated ~20.5…’\nResponse: Rephrased.\nL46: Just for reference, the Longman et al. (2024) study (ref.13) shows some sediments of\nEocene age containing high FeR-OC.\nResponse: The sentence has been changed to “A comprehensive picture regarding the fate of\nFeR-OC on geological timescales is still lacking but several studies of sediments of Pleistocene\nas well as late Paleocene to early Eocene age suggest that the fraction of Fe -OC in TOC (fFe -\nR R\nOC) remains relatively stable11-14.”(Line 47-50).\nL114-115: Can you include a reference for the marine sediment zonation?\nResponse: A classic paper from Canfield and Thamdrup (2009) discussing the geochemical\nzonation in marine sediments has been cited (Line 117).\nTable 1: I wonder if some of these data, which are crucial to the ms, could be presented in box\nplot\nform? For example, the FeR-OC values and the d13CFeR-OC would be nicely presented as\na box plot.\nResponse: We have put all the information presented in Table 1 into box plots as the new\nSupplementary Fig. 5.\nSupplementary Figure S5 Comparisons of the TOC and Fe -OC records between non-SMTZ and\nR\nSMTZ sediments in two cores, including TOC content (a), Fe -OC content (b), fFe -OC (c), Fe -\nR R R\nOC to Fe molar ratio (d), carbon isotope ratio of TOC (e), and carbon isotope ratio of Fe -OC\nR R\n(f). fFe -OC at 410 and 420 cmbsf in QDN-14B are biased by extremely low TOC and are not\nR\nincluded for analysis. Significant levels in Wilcoxon test are indicated (ns: P > 0.05、***: P <=\n0.001、****: P <= 0.0001). Box plots indicate mean (solid square), median (middle line), 25th,\n75th percentile (box) and 1.5 times interquartile range (whiskers) with data points (solid dots)\noverlapped on top.\nL238 ‘summary’ rather than ‘sum’\nResponse: Done.\nL257: ‘The depth interval’ is an odd way to say it. Maybe simply say ‘The SMTZ…’\nResponse: Done.\nL337: This could be expanded as an explanation for the samples of even older age which show\nFeR-OC coupling to be important.\nJack Longman\nResponse: We thank the reviewer for this suggestion. We have added the following sentence\nto the manuscript to expand this statement a little bit to the samples of older ages:\n“Considering previous studies showing high fFe -OC in the samples even from late Paleocene\nR\nto early Eocene12, persistence of Fe -OC on even longer timescales is expected in marine\nR\nsediments.” (Lines 343-345)"}, {"round": 3, "reviewer_comments": {"reviewer_3": "I am delighted to see Chen et al. have taken my comments, and the manuscript is greatly improved\nin its current form. I have no more suggestions/comments."}, "author_response": ""}]

Decision letter